Oncogene database

3725

JUN

jun proto-oncogene;JUN;jun proto-oncogene

Jun activation domain binding protein|activator protein 1|enhancer-binding protein AP1|jun oncogene|p39|proto-oncogene c-Jun|transcription factor AP-1|v-jun avian sarcoma virus 17 oncogene homolog|v-jun sarcoma virus 17 oncogene homolog

1p32-p31

protein-coding

This paper is to investigate the expression of oncogene jun fos and myb mRNA in the lung of rats exposed to chronic hypoxia. 15 SD rats were put in low oxygen chamber (FiO2 = 0.1), 8 hrs daily for 1, 2 and 3 weeks. Five rats breathing room air served as control. oncogene expression in lung tissue assessed by the use of in situ hybridization. The results showed that (1) there was a slight expression of jun mRNA but not fos and myb mRNA in the control normoxic rats lung; (2) it was found that a less expression of jun mRNA in lung after 1 week hypoxia, but after 2 week hypoxia jun mRNA elevated again and significantly increased after 3 week hypoxia as compared with that in normoxia; (3) the oncogene myb mRNA expression showed significant increase in 1 and 2 week hypoxia and returned to normal status in 3 week hypoxia; (4) after 1 to 3 week hypoxia, a significant increased expression of fos mRNA was found as compared with that in normoxia. It is suggested hypoxia may induce increased expression of proto-oncogene jun myb and fos, which may be related to proliferation of pulmonary arterial smooth muscle cells and fibroblasts.

The proto-oncogene c-jun is involved in cellular proliferation by interfering with signals that lead to cellular differentiation. Moreover the induction of metalloproteinase gene appears to utilise the c-jun oncogene as intracellular messenger. The aims of this study were to evaluate a) the expression of c-jun oncogene in pancreatic cancer b) its relation with tumor histological features. Surgical specimens of pancreatic cancer were collected from 22 patients radically operated upon, and from 11 submitted to palliation. As a control group, 5 specimens of normal pancreas and 5 specimens of chronic pancreatitis were studied. C-jun staining was graded as follows: (-) positive cells < 10%, (+) from 10 to 30%, (+2), from 30 to 60%, (+3) from 60 to 90%. Glucagon and somatostatin staining was graded counting the positive cells of total numer of Langherans islet cells counted. c-Jun expression (low: < 30% and high: > 30%) was related to stage, architectural and cytological grading, vascular, lymph nodal, perineural invasion. Normal pancreas and chronic pancreatitis tissues appear to express the c-jun protein in less than 10% of ductal cells. The percentage of tumor cells stained for c-jun is increased as compared to the control group in 28/33 cases: in 13 (46%) it ranges from 10 to 30%; in 10 (36%) from 30 to 60% and in 5 (18%) from 60 to 90%. The frequency of high or low c-jun expression is not different in relation to the histological features of tumor. Moreover, c-jun protein is present in 40% of cells of Langherans islets in normal pancreas, chronic pancreatitis and pancreatic cancer. The Langherans islet cells stained for c-jun exhibit also a positivity for glucagon. In conclusion; a) in pancreatic cancer, the expression of c-jun is increased in tumour cells in majority of cases as compared to the control group, b) a c-jun positivity is also found in alpha cells with a pattern not different from control group, but the relation between the alpha cells and c-jun production is unknown, c) c-jun expression does not vary in relation to histological findings.

Jun is a transcription factor belonging to the activator protein 1 family. A mutated version of Jun (v-Jun) transduced by the avian retrovirus ASV17 induces oncogenic transformation in avian cell cultures and sarcomas in young galliform birds. The oncogenicity of Jun probably results from transcriptional deregulation of v-Jun-responsive target genes. Here we describe the identification and characterization of a growth-related v-Jun target, a homolog of heparin-binding epidermal growth factor-like growth factor (HB-EGF). HB-EGF is strongly expressed in chicken embryo fibroblasts (CEF) transformed by v-Jun. HB-EGF expression is not detectable or is marginal in nontransformed CEF. Using a hormone-inducible Jun-estrogen receptor chimera, we found that HB-EGF expression is correlated with v-Jun activity. In this system, induction of v-Jun is followed within 1 hr by elevated levels of HB-EGF. In CEF infected with various Jun mutants, HB-EGF expression is correlated with the oncogenic potency of the mutant. Constitutive expression of HB-EGF conveys to CEF the ability to grow in soft agar and to form multilayered foci of transformed cells on a solid substrate. These observations suggest that HB-EGF is an effector of Jun-induced oncogenic transformation.

The AP-1 transcription factor (the Jun and Fos proteins) is suspected of playing an important role in the biology of human cancer. Human epithelial ovarian tumors and cancer cell lines express the c-jun and jun-B proto-oncogenes at a high level, in contrast with the jun-D gene. We have investigated here the functional relevance of these observations for the growth of ovarian cancer cells. Transient constitutive expression of a dominant negative c-jun mutant (TAM67) in human AZ224, SKOV3 and OVCAR3 ovarian cancer cells inhibited the outgrowth of selection marker-resistant colonies by at least 75% as opposed to a control plasmid. Transfection of jun-B did not affect these cell lines, while jun-D transfection had a cell line-specific effect. In comparison, transfection of the tumor-suppressor gene p53 had a less important inhibitory effect on OVCAR3 cells and no effect on SKOV3 and AZ224 cells when compared to TAM67. Regulated TAM67 expression in AZ224 cells, from plasmids containing the mouse metallothionein or the MMTV promoter, suppressed cancer cell growth in vitro and in nude mice without evidence of increased cell death. Our observations support a role for the c-jun proto-oncogene as a positive mediator of human ovarian cancer cell growth and make it a potential therapeutic target.

N-Methyl-N -nitro-N-nitrosoguanidine (MNNG) is a potent inducer of cellular stress leading to chromosomal aberrations, point mutations, and cell death. To study the effect of capsaicin on c-jun expression when given with MNNG to rats, Fisher-344 rats that had been administered MNNG were treated with capsaicin in their diet and organs were removed for measuring c-jun transcripts. We show that pre- or post-treatment of capsaicin relative to MNNG administration up- or down-regulates (depending on the organ) c-jun expression in a consistent pattern in most organs. In fact, we found in this study that capsaicin inhibits c-jun induction, stimulated by MNNG, in the spleen, heart, stomach and lung. Since MNNG, a methylating agent, is a powerful carcinogen that is very effective in the induction of c-jun mRNA, the results suggest that capsaicin uptake in the diet could play a role in inhibition of tumorigenesis induced by MNNG.

Signal transducers and activators of transcription (STATs) are transcription factors that mediate normal biologic responses to cytokines and growth factors. However, abnormal activation of certain STAT family members, including Stat3, is increasingly associated with oncogenesis. In fibroblasts expressing the Src oncoprotein, activation of Stat3 induces specific gene expression and is required for cell transformation. Although the Src tyrosine kinase induces constitutive Stat3 phosphorylation on tyrosine, activation of Stat3-mediated gene regulation requires both tyrosine and serine phosphorylation of Stat3. We investigated the signaling pathways underlying the constitutive Stat3 activation in Src oncogenesis. expression of Ras or Rac1 dominant negative protein blocks Stat3-mediated gene regulation induced by Src in a manner consistent with dependence on p38 and c-Jun N-terminal kinase (JNK). Both of these serine/threonine kinases and Stat3 serine phosphorylation are constitutively induced in Src-transformed fibroblasts. Furthermore, inhibition of p38 and JNK activities suppresses constitutive Stat3 serine phosphorylation and Stat3-mediated gene regulation. In vitro kinase assays with purified full-length Stat3 as the substrate show that both JNK and p38 can phosphorylate Stat3 on serine. Moreover, inhibition of p38 activity and thus of Stat3 serine phosphorylation results in suppression of transformation by v-Src but not v-Ras, consistent with a requirement for Stat3 serine phosphorylation in Src transformation. Our results demonstrate that Ras- and Rac1-mediated p38 and JNK signals are required for Stat3 transcriptional activity induced by the Src oncoprotein. These findings delineate a network of tyrosine and serine/threonine kinase signaling pathways that converge on Stat3 in the context of oncogenesis.

Oncogenic (activated) Ras is a signal transducer that activates multiple effector-mediated signaling pathways leading to altered cell morphology, growth and differentiation, and neoplastic transformation. Activating mutations of Ras family genes have been detected in many types of human cancers, including lung cancer. However, the signaling mechanisms by which oncogenic Ras controls cancer cell growth is poorly characterized. This study evaluates the role of two specific signaling pathways, the c-Jun NH2-terminal kinase (JNK) pathway, and the extracellular signal-regulated kinase (ERK) pathway, in oncogenic Ras-induced morphological transformation of NCI-H82 human small cell lung cancer cells. In the NCI-H82 cell line, oncogenic Ras causes a marked and sustained activation of JNK but only has a modest effect on activation of the ERK pathway. The persistent JNK activation is associated with Ras-induced changes in cell morphology and enhanced transforming activity. Furthermore, JNK activation correlates with the induction of c-Jun expression, c-Jun phosphorylation on serines 63 and 73, and increased AP-1 activity. Deregulation of the JNK pathway using a dominant-negative mutant of JNK1, JNK1(APF), completely reverses the oncogenic Ras-induced transformed phenotype, including morphological reversion and inhibition of anchorage-independent growth and low-serum growth. Moreover, expression of JNK1(APF) leads to a decrease in c-Jun/AP-1 activity. In contrast, inhibition of ERK activation via a pharmacological approach using a mitogen-activated protein kinase/ERK kinase-specific inhibitor 2-(2 -amino-3 -methoxyphenyl)-oxanaphthalen-4-one is unable to reverse the Ras-induced transformed morphology and c-Jun/AP-1 induction. These results demonstrate that the JNK/c-Jun/AP-1 pathway plays an essential role in mediating oncogenic Ras function in lung carcinoma cells.

The serine/threonine kinase Cot is a member of the mitogen-activated protein kinase (MAPK) kinase kinase family implicated in cellular transformation. Enhanced expression of this protein has been shown to activate both the MAPK and the c-Jun N-terminal kinase (JNK) pathways and to stimulate the nuclear factor of activated T cells and NF-kappaB-dependent transcription. However, the nature of the normal functions of the Cot protein and the molecular mechanisms responsible for its oncogenic potential are still largely unknown. Here, we show that overexpression of the cot proto-oncogene is sufficient to stimulate the expression of c-jun and that, in turn, the activity of c-Jun is required for Cot-induced transformation. These observations prompted us to explore the molecular events by which Cot regulates c-jun expression. We found that Cot potently stimulates the activity of the c-jun promoter utilizing JNK-dependent and -independent pathways, the latter involving two novel members of the MAPK family, p38gamma (ERK6) and ERK5. Molecularly, this activity was found to be dependent on the ability of Cot to activate, in vivo, members of each class of the MAPK kinase superfamily, including MEK, SEK, MKK6, and MEK5. Furthermore, the use of dominant interfering molecules revealed that Cot requires JNK, p38s, and ERK5 to stimulate the c-jun promoter fully and to induce neoplastic transformation. These findings indicate that Cot represents the first example of a serine/threonine kinase acting simultaneously on ALL known MAPK cascades. Moreover, these observations strongly suggest that the transforming ability of Cot results from the coordinated activation of these pathways, which ultimately converge on the regulation of the expression and activity of the product of the c-jun proto-oncogene.

We have identified the intracellular detoxification enzyme, glutathione-S-transferase (GST), as a potent inhibitor of the activation of jun by its kinase, jun-N-terminal kinase (JNK), in vitro. ALL three major isozymes (alpha, mu, and pi) bind to JNK-jun complexes and inhibit activation of jun by JNK. We now find that GST inhibits JNK-induced oocyte maturation in vivo and strongly inhibits oocyte maturation induced by oncogenic ras-p21 protein, but not by insulin-activated normal cellular p21 protein. These results correlate with the finding that oncogenic, but not insulin-activated normal, p21 induces high levels of activated JNK. GST also strongly blocks induction of oocyte maturation by protein kinase C (PKC) which is a critical downstream target of oncogenic but not normal ras-p21. Thus, we have established a new function for GST as a potent physiological inhibitor of the ras-JNK-jun pathway.

The present study was conducted to delineate whether a possible mechanism for 13-(S)-hydroxyoctadecadienoic acid (13-HODE) and 15-hydroxyeicosatrienoic acid (15-HETrE) reversal of experimentally-induced skin hyperproliferation in guinea pig is via the modulation of epidermal nuclear mitogen activator protein (AP-1), a nuclear transcription factor associated with tissue turnover. The data revealed that topical application of 13-HODE and/or 15-HETrE on the induced hyperproliferative skin reversed the hyperproliferation and up-regulated the suppressed AP-1 expression. A further analysis of the two major subunits of AP-1 (c-fos and c-jun) revealed a selective up-regulation of c-fos. These results underscore the modulatory role of lipoxygenase-derived hydroxy fatty acids on nuclear transcription factors and explains, at least in part, the antiproliferative effects of 13-HODE and 15-HETrE.

The nuclear phosphoprotein c-Jun is a major component of the AP-1 transcription factor, whose activity is augmented by many oncogenes. An important mechanism to stimulate AP-1 function is N-terminal phosphorylation of c-Jun at the serine residues 63 and 73 by the c-JunN-terminal kinases (JNKs). Mice and cells harboring a mutant allele of c-jun, which has the JNK phosphoacceptor serines changed to alanines (junAA), were used to determine the function of c-Jun N-terminal phosphorylation (JNP) during oncogenic transformation in vitro and in vivo. JunAA immortalized fibroblasts expressing v-ras and v-fos showed reduced tumorigenicity in nude mice, but the efficiency of v-src transformation was unaffected by the lack of JNP. To assess the significance of JNP in tumour development in vivo, two transgenic mouse tumour models were employed. Skin tumour development caused by constitutive activation of the ras pathway by K5-SOS-F expression and c-fos-induced osteosarcoma formation were impaired in mice lacking JNP. Inhibition of JNP may, therefore, be a novel therapeutic strategy to inhibit tumour growth in vivo. oncogene (2000).

PURPOSE: We have previously found that microinjection of activated MEK (mitogen activated kinase kinase) and ERK (mitogen-activated protein; MAP kinase) fails to induce oocyte maturation, but that maturation, induced by oncogenic ras-p21 and insulin-activated cell ras-p21, is blocked by peptides from the ras-binding domain of raf. We also found that jun kinase (JNK), on the stress-activated protein (SAP) pathway, which is critical to the oncogenic ras-p21 signal transduction pathway, is a strong inducer of oocyte maturation. Our purpose in this study was to determine the role of the raf-MEK-MAP kinase pathway in oocyte maturation and how it interacts with JNK from the SAP pathway. METHODS: We microinjected raf dominant negative mutant mRNA (DN-raf) and the MEK-specific phosphatase, MKP-T4, either together with oncogenic p21 or c-raf mRNA, into oocytes or into oocytes incubated with insulin to determine the effects of these raf-MEK-MAP kinase pathway inhibitors. RESULTS: We found that oocyte maturation induced by both oncogenic and activated normal p21 is inhibited by both DN-raf and by MKP-T4. The latter more strongly blocks the oncogenic pathway. Also an mRNA encoding a constitutively activated MEK strongly induces oocyte maturation that is not inhibited by DN-raf or by MKP-T4. Surprisingly, we found that oocyte maturation induced by JNK is blocked both by DN-raf and MKP-T4. Furthermore, we discovered that c-raf induces oocyte maturation that is inhibited by glutathione-S-transferase (GST), which we have found to be a potent and selective inhibitor of JNK. CONCLUSION: We conclude that there is a strong reciprocal interaction between the SAP pathway involving JNK and the raf-MEK-MAP kinase pathway and that oncogenic ras-p21 can be preferentially inhibited by MEK inhibitors. The results imply that blockade of both MEK and JNK-oncogenic ras-p21 interactions may constitute selective synergistic combination chemotherapy against oncogenic ras-induced tumors.

Evi-1 encodes a nuclear protein involved in leukemic transformation of hematopoietic cells. Evi-1 possesses two sets of zinc finger motifs separated into two domains, and its characteristics as a transcriptional regulator have been described. Here we show that Evi-1 acts as an inhibitor of c-Jun N-terminal kinase (JNK), a class of mitogen-activated protein kinases implicated in stress responses of cells. Evi-1 physically interacts with JNK, although it does not affect its phosphorylation. This interaction is required for inhibition of JNK. Evi-1 protects cells from stress-induced cell death with dependence on the ability to inhibit JNK. These results reveal a novel function of Evi-1, which provides evidence for inhibition of JNK by a nuclear oncogene product. Evi-1 blocks cell death by selectively inhibiting JNK, thereby contributing to oncogenic transformation of cells.

Exposure of cells to ionizing radiation results in both activation of protein kinase C (PRKC, also known as PKC) and induction of transcription of the JUN proto-oncogene. PRKC plays a pivotal role in radiation-induced JUN expression, since inhibition of PRKC abrogates the JUN signal. However, the specific PRKC isoforms involved in radiation-induced elevation of JUN mRNA levels have not been identified. Here we demonstrate that in DT40 B-lineage lymphoid cells, the mu isoform of PRKC (PRKCM) is critical for the response of JUN to ionizing radiation. The zinc chelator, 1, 10-phenanthroline, abrogated induction of JUN after exposure to ionizing radiation, indicating that this PRKCM-mediated response is also dependent on zinc.

Overexpression of v-Jun in chicken embryo fibroblasts (CEF) leads to oncogenic transformation phenotypically characterized by anchorage independent growth and release from contact inhibition (focus formation). The mechanisms involved in this oncogenic conversion however, are not yet clear. Because Jun is a transcription factor, it has been assumed that oncogenic transformation results directly from deregulated AP-1 target gene expression. However, a number of experimental observations in avian cell culture models fail to correlate oncogenesis with AP-1 activity suggesting that transformation induced by v-Jun may occur through an indirect mechanism. To test this possibility, we introduced point mutations into the basic DNA binding domain of v-Jun and created mutants that exhibit altered binding specificity. When expressed in CEF, these mutants fail to deregulate three known v-Jun target genes (JTAP-1, apolipoprotein A1, c-Jun) thus demonstrating in vivo specificity changes. Each of the binding specificity mutants was also tested for its ability to induce oncogenic transformation. Interestingly, expression of these mutants in CEF results in a phenotype indistinguishable from the vector control with respect to growth rate, focus formation and the ability to form colonies in soft agar. These results are consistent with a model requiring direct AP-1 target deregulation as a prerequisite of v-Jun induced cell transformation. With this in mind, we generated a series of additional mutants that retain the ability to bind AP-1 sequence elements, but vary in their oncogenic potential. We demonstrate the use of these mutants to screen v-Jun induced gene targets for a functional role in cell transformation.

We investigated the role of endogenous AP-1 in human tumor cell lines by introducing SupJunD-1, a dominant-negative mutant of AP-1, using vesicular stomatitis virus G protein (VSV-G)-pseudotyped retrovirus vectors. Single inoculation of six human tumor cell lines, originating from osteosarcomas, non-small cell lung carcinomas or cervical carcinomas, with recombinant SupJunD-1 virus at a high multiplicity of infection readily inhibited colony formation in soft agar. We detected no significant changes in expression levels of AP-1 components c-Jun or Fra-1, adhesion molecules CD44 or E-cadherin, or cell cycle regulator p53, which are encoded by genes previously reported to be under the control of AP-1 in some mouse or human cell lines. By varying the dosage of VSV-G-pseudotyped retrovirus, we were able to change the proviral copy number of supjunD-1 from 1 to approximately 10 and monitor suppression of endogenous AP-1 function as assessed by growth characteristics of the tumor cell lines, we found a SupJunD-1 dosage which significantly suppressed anchorage-independent growth without affecting the cellular growth in monolayer cultures at all. We conclude that endogenous AP-1 levels necessary for oncogenic activity are much higher than those sufficient to support normal growth.

Human T cell leukemia virus type 1 (HTLV-1) Tax protein transforms primary human T cells in vitro. We previously showed that Tax induces the expression of various family members of the transcription factor AP-1 such as c-Jun, JunD, c-Fos, and Fra-1 at the mRNA level in T cells. In this study, we have examined the ability of Tax to activate transcription through the AP-1-binding site (AP-1 site). A transient transfection study showed that Tax can activate transcription through the AP-1-binding site in a human T cell line, whereas any combination of AP-1 proteins did so much less than Tax, indicating that the activation of the AP-1 site by Tax may require a mechanism other than the induction of AP-1 mRNA. Fresh peripheral blood leukemia cells of ALL surveyed ATL patients displayed constitutive AP-1 DNA-binding activity, whereas no normal individuals did. However, the HTLV-1 genes, including tax, are not significantly expressed in fresh leukemia cells from ATL patients. Our present results suggest that activation of AP-1 occurs through Tax-dependent and -independent mechanisms in HTLV-1-infected T cells, which may play some roles in dysregulated phenotypes of HTLV-1-infected cells.

Crystalline silica has been classified as a group 1 human carcinogen in the lung. However, its mechanisms of action on pulmonary epithelial cells which give rise to lung cancers are unclear. Using a nontransformed alveolar type II epithelial cell line (C10), we show that alpha-quartz silica causes persistent dose-related increases in phosphorylation of c-Jun-NH2-terminal amino kinases (JNKs) that are inhibited by antioxidants (P < or = 0.05). Increases in activator protein-1 (AP-1) binding to DNA and transactivation of AP-1-dependent gene expression by silica were accompanied by increases in steady-state mRNA levels of the AP-1 family members, c-jun, junB, fra-1, and c-fos at 8 h and elevated mRNA levels of fra-1 at 24 h (P < or = 0.05). Addition of tetramethylthiourea inhibited silica-associated increases infra-1 and proportions of cells in S-phase (P < or = .05). Our findings indicate that silica induces JNK activity, AP-1-dependent gene expression, ie., fra-1, and DNA synthesis via oxidative stress. Moreover, they suggest that silica may act mechanistically as a mitogen or tumor promoter, rather than a genotoxic carcinogen, in the development of lung cancers.

Cellular Jun (c-Jun) and viral Jun (v-Jun) can induce oncogenic transformation. For this activity, c-Jun requires an upstream signal, delivered by the Jun N-terminal kinase (JNK). v-Jun does not interact with JNK; it is autonomous and constitutively active. v-Jun and c-Jun address overlapping but not identical sets of genes. Whether ALL genes essential for transformation reside within the overlap of the v-Jun and c-Jun target spectra remains to be determined. The search for transformation-relevant targets of Jun is moving into a new stage with the application of DNA microarrays technology. Genetic screens and functional tests remain a necessity for the identification of genes that control the oncogenic phenotype.

Using subtractive hybridization techniques, we have isolated a gene termed JAC that is strongly and specifically activated in avian fibroblasts transformed by the v-jun oncogene of avian sarcoma virus 17 (ASV17), but not in cells transformed by other oncogenic agents. Furthermore, JAC is highly expressed in cell lines derived from jun-induced avian fibrosarcomas. Kinetic analysis using a doxycycline-controlled conditional cell transformation system showed that expression of the 0.8-kb JAC mRNA is induced rapidly upon activation of the oncogenic v-jun allele. Nucleotide sequence analysis and transcriptional mapping revealed that the JAC gene contains two exons, with the longest ORF confined to exon 2. The deduced 68-amino acid chicken JAC protein is rich in cysteine residues and displays 37% sequence identity to mammalian high-sulfur keratin-associated proteins. The promoter region of JAC contains a consensus (5 -TGACTCA-3 ) and a nonconsensus (5 -TGAGTAA-3 ) AP-1 binding site in tandem, which are both specifically bound by the Gag-Jun hybrid protein encoded by ASV17. mutational analysis revealed that the two AP-1 sites confer strong transcriptional activation by Gag-Jun in a synergistic manner. Ectopic expression of JAC in avian fibroblasts leads to anchorage-independent growth, strongly suggesting that deregulation of JAC is an essential event in jun-induced cell transformation and tumorigenesis.

The cytogenetic region 46C-F on the right arm of Drosophila chromosome 2, which contains the homolog of the human jun proto-oncogene, has been genetically mapped and characterized. This project led to the identification and characterization of a Jra (jun-related antigen) mutation, which has been described in detail elsewhere. Three mutagens, EMS, DEB and gamma-rays, were used to isolate 126 lethal lines for this interval. Complementation analysis of the 126 lethal lines identified 29 lethal complementation groups in the region; nine of which have now been correlated with known genes or phenotypes. The region has been subdivided into ten intervals using various small deletions, seven intervals in 46C/D and three intervals in 46E/F. Sixteen P-element lines have been mapped to this interval and are allelic to eight of our complementation groups. The remaining unidentified complementation groups have been analyzed for critical phase, which is when the first observable defect arises and/or when death occurs. There are twelve embryonic lethal groups and seven larval lethal groups. Three lines show visible abnormalities in gut and tracheal development prior to death.

Previous studies showed that antagonists of bombesin (BN)/gastrin-releasing peptide (GRP) inhibit the growth of various cancers by interfering with the growth-stimulatory effects of BN-like peptides and down-regulating epidermal growth factor receptors on tumors. Because the overexpression of the human epidermal growth factor receptor-2 (ErbB-2/HER-2/neu) oncogene plays a role in the progression of many breast cancers, we investigated whether BN/GRP antagonists can affect HER-2 in mammary tumors. Female nude mice bearing orthotopic xenografts of MDA-MB-435 human estrogen-independent breast cancers were treated daily with BN/GRP antagonists RC-3095 (20 microg) or RC-3940-II (10 microg) for 6 weeks. The expression of BN/GRP receptors on tumors was analyzed by reverse transcription-PCR and immunoblotting. We also evaluated whether the mRNA expression for the c-jun and c-fos oncogenes is affected by the therapy. Both BN/GRP antagonists significantly inhibited growth of MDA-MB-435 cancers; RC-3095 reduced tumor volume by 40% and RC-3940-II by 65%. The GRP receptors (subtype 1) were detected in MDA-MB-435 tumors, showing that they mediate the inhibitory effect of the antagonists. tumor inhibition was associated with a substantial reduction in the expression of mRNA and protein levels of the ErbB/HER receptor family as well as with a decrease in the expression of c-jun and c-fos oncogenes. BN/GRP antagonists RC-3940-II and RC-3095 could be considered for endocrine therapy of estrogen-independent breast cancers that express members of the ErbB/HER receptor family and the c-jun and c-fos oncogenes.

Osteopontin, a major non-collagenous bone matrix protein, is strikingly upregulated in various tissues under certain pathologic conditions, including cancer. However, the mechanism of upregulation of the osteopontin gene in tumor cells remains unclear. Okadaic acid, a strong non-phorbol ester tumor promoter, is known to stimulate the expression of osteopontin. The aim of the present study was to understand the mechanism by which okadaic acid regulates osteopontin gene expression. Okadaic acid stimulated osteopontin mRNA expression in several cell lines within 3 h, and the increase in osteopontin mRNA was sustained for 24 h. New protein synthesis was required for the okadaic acid-elicited increase in osteopontin mRNA expression. A serial promoter deletion study showed that the okadaic acid-response element is located between positions -265 and -73, a sequence that includes the Runx2, Ets-1, and AP-1 binding sequences. Okadaic acid increased the mRNA expression of AP-1 components but not of Runx2 or Ets-1. Site-directed mutagenesis and electrophoretic mobility shift assays confirmed that protein binding of the AP-1 consensus sequence is necessary for the okadaic acid-mediated osteopontin gene upregulation. These results indicate that de novo induction of the oncoprotein AP-1 is required for okadaic acid-stimulated osteopontin gene upregulation.

Human T-cell leukemia virus I is the etiologic agent of adult T-cell leukemia (ATL), an aggressive T-cell malignancy. The viral oncoprotein Tax, through the activation of nuclear factorkappaB (NF-kappaB), CCAAT-enhancer binding protein (CREB), and activated protein-1 (AP-1) pathways, is a transcriptional regulator of critical genes for T-cell homeostasis. In ATL cells, activated AP-1 complexes induce the production of transforming growth factor beta1 (TGF-beta1). TGF-beta1 is an inhibitor of T-cell proliferation and cytotoxicity. Here we show that, in contrast to normal peripheral T cells, ATL cells are resistant to TGF-beta1-induced growth inhibition. The retroviral transduction of the Tax protein in peripheral T cells resulted in the loss of TGF-beta1 sensitivity. Transient transfection of Tax in HepG2 cells specifically inhibited Smad/TGF-beta1 signaling in a dose-dependent manner. In the presence of Tax transfection, increasing amounts of Smad3 restored TGF-beta1 signaling. Tax mutants unable to activate NF-kappaB or CREB pathways were also able to repress Smad3 transcriptional activity. Next we have demonstrated that Tax inhibits TGF-beta1 signaling by reducing the Smad3 DNA binding activity. However, Tax did not decrease the expression and the nuclear translocation of Smad3 nor did it interact physically with Smad3. Rather, Tax induced c-Jun N-terminal kinase (JNK) activity and c-Jun phosphorylation, leading to the formation of Smad3/c-Jun complexes. Whereas c-Jun alone abrogates Smad3 DNA binding, cotransfection of Tax and of a dominant-negative form of JNK or a c-Jun antisense-restored Smad3 DNA binding activity and TGF-beta1 responsiveness. In ATL and in normal T cells transduced by Tax, c-Jun was constitutively phosphorylated. Thus, we describe a new function of Tax, as a repressor of TGF-beta1 signaling through JNK/c-Jun constitutive activation, which may play a critical role in ATL leukemogenesis.

Although epidemiological studies have long established that inorganic arsenic is a potent human carcinogen, the underlying mechanisms are still poorly understood. Recent studies suggest that inorganic arsenic may act as a tumor promoter by perturbing key signaling transduction pathways. We have shown previously that arsenite can potently activate the mitogen-activated protein kinase cascades and induce the expression of proliferation-associated genes, including proto-oncogenes c-jun and c-fos. In order to elucidate further the molecular mechanisms underlying its tumor-promoting properties, we investigated the signaling events involved in arsenite-mediated induction of c-fos and c-jun. We found that induction of both c-fos and c-jun by arsenite can be substantially inhibited by the MEK- selective inhibitor U0126, suggesting that the ERK pathway is critically involved in their up-regulation. Interestingly, arsenite dramatically induced the phosphorylation and acetylation of histone H3 preceding the induction of mRNAs encoding c-fos and c-jun. Finally, chromatin immunoprecipitation assays revealed that arsenite treatment markedly induced the phosphorylation/acetylation of histone H3 associated with the c-fos and c-jun genes through an ERK-dependent pathway. Our results strongly suggest that arsenic-triggered alterations in chromatin structure perturb specific gene transcription, including that of proto-oncogenes c-jun and c-fos, and may thereby contribute to the carcinogenic process.

The protein encoded by the v-Jun oncogene shows increased transforming activity compared to c-Jun, its normal cellular counterpart. One major determinant of this increased transforming activity is an in-frame deletion of a region near the amino-terminus of the protein. This region, referred to as the delta domain, functions as a docking site for Jun N-terminal kinase (JNK), the mitogen-activated protein (MAP) kinase that phosphorylates c-Jun to regulate its transcriptional properties. As a consequence of this deletion, v-Jun is unresponsive to JNK signaling, and it is widely believed that it is the uncoupling of v-Jun from JNK signaling that underlies the oncogenic effects of the delta-domain deletion; however, this idea has never been directly tested. Here we use JNK overexpression as well as alanine scanning mutagenesis to test this idea. Point mutants that are uncoupled from JNK signaling do not show enhanced transforming activity, suggesting that disruption of the Jun-JNK interaction is not the mechanism by which the delta-domain deletion enhances transforming activity. Consistent with this idea, we have generated a panel of point mutants that show markedly enhanced transforming activity, despite the fact that they do not perturb the ability of JNK to either dock with or phosphorylate c-Jun in vitro or in vivo. The fact that these mutants cluster in a small region suggests the existence of an additional regulator of Jun function whose activity is disrupted by mutations in this region.

The signal pathway mediating induction of p15(INK4b) and p16(INK4a) during HepG2 growth inhibition triggered by the phorbol ester tumor promoter TPA (12-O-tetradecanoylphorbol 13-acetate) and the Chinese herb Saikosaponin a was investigated. Western blot of three activated forms of mitogen-activated protein kinase (MAPK) (p-ERK, p-JNK and p-p38) demonstrated that phosphorylation of ERK is dramatically induced (11.6-fold ) by TPA during 15 min to 1 h and significantly induced (2.5-fold) by Saikosaponin alpha at 30 min, whereas phosphorylation of JNK was induced only by TPA during 30 min to 1 h. Phosphorylation of p38 was not induced by either drug. During this period, phosphorylation of one of the downstream transcriptional factors of MAPK cascade, ATF2, was 3.2- and 2.0-fold induced by TPA and Saikosaponin a, respectively, whereas that of another transcriptional factor, c-jun, was induced by TPA only. On the other hand, expressions of proto-oncogene c-jun, junB and c-fos were induced by TPA and Saikosaponin a during 30 min to 6 h of treatment. Pretreatment of 20 microg/ml PD98059, an inhibitor of MEK which is the upstream kinase of ERK, prevents the TPA- and Saikosaponin a-triggered HepG2 growth inhibition by 50 and 30%, respectively, accompanied by a 50 - 85% decrease of the p15(INK4b)/p16(INK4a) RNAs and proteins induced by both drugs. Inductions of c-fos RNA by both drugs and c-jun phosphorylation by TPA were also significantly reduced by PD98059 pretreatment. In addition, AP-1 DNA-binding assay using nonisotopic capillary electrophoresis and laser-induced fluorescence (CE/LIF) demonstrated that the AP-1-related DNA-binding activity was significantly induced by TPA and Saikosaponin a, which can be reduced by PD98059 pretreatment. These results suggested that activation of ERK together with its downstream transcriptional machinery mediated p15(INK4b) and p16(INK4a) expression that led to HepG2 growth inhibition.

Jun dimerization protein 2 (JDP2) was identified as a bZIP protein that forms dimers with Jun proteins. JDP2 represses transcriptional activation of reporter constructs containing 12-O-tetradecanoylphorbol 13-acetate (TPA)-responsive elements (TRE) or cyclic AMP responsive elements (CRE). JDP2, overexpressed by the avian retroviral vector RCAS, induces partial oncogenic transformation of chicken embryo fibroblasts. JDP2-expressing cells form multilayered foci in monolayer cultures but do not show anchorage-independent growth. Both the carboxyl and the amino terminus of JDP2 are required for the transforming activity. Chimeric constructs of JDP2 carrying the leucine zipper domain of Fos, GCN4 or EB1 fail to transform CEF. The leucine zipper of Fos mediates only heterodimerization; it cannot homodimerize. In contrast, the leucine zippers of GCN4 and of EB1 exclusively homodimerize and do not form dimers with other bZip proteins. The results with the JDP2 chimeras suggest that the JDP2 homodimer and the JDP2/Jun heterodimer (or other bZip heterodimers formed with the Fos leucine zipper) are nontransforming, leaving as possible transforming combination the JDP2/Fos heterodimer. The unexpected transforming activity of a negative regulator of TRE- and CRE-dependent transcription raises an important question concerning the mechanisms of transformation by the related bZIP proteins Jun and Fos that address the same target sequences.

The fos and jun proto-oncogenes are members of the set of genes known as cellular immediate-early genes. Their expression is induced transiently by a great variety of extracellular stimuli associated with mitogenesis, differentiation processes or depolarization of neurons. They encode DNA binding proteins that form dimeric complexes through a leucine zipper structure that function as transcription factors. Continuous overexpression of fos or jun causes transformation of fibroblasts. Because of their ubiquitous expression it is believed that the target genes regulated by Fos and Jun are different in the many circumstances in which they are expressed. Thus, their functional specificity is likely to be regulated at several levels. We have uncovered several potential mechanisms that could contribute to their regulation. These include formation of a large number of heterodimeric complexes, post-translational modification by phosphorylation and a novel reduction/oxidation (redox) mechanism, presence of both positive and negative transcriptional domains and the ability of Fos and Jun to induce distinct bends in DNA structure.

We determined the mRNA levels of c-jun and c-fos in the liver of C3H-H-2 degrees jvs mice. Both were higher in jvs mice than in normal mice. The level of c-jun mRNA increased gradually after birth, but in the control mice there was almost no change. In addition, alpha-fetoprotein and aldolase A mRNA levels were also higher than in normal littermates. These results suggest that the pattern of the gene expression in jvs mice partly resembles the one that occurs in undifferentiated hepatocytes and/or hepatocellular carcinoma.

55 patients suffering from stage III or IV carcinoma of cervix were treated with two pulses of neo-adjuvant chemotherapy prior to radical radiotherapy. 51% (26/51) had a partial response. The initial response to chemotherapy is associated with significantly better long-term survival. The 3-year survival of chemotherapy responders is 62% against 21% for non-responders (P = 0.009 log-rank test). To detect possible differences in oncogene expression in biopsy specimens taken from responding and non-responding patients, paraffin-fixed material was immunocytochemically stained for the expression of the protein products of ras, c-myc and c-jun proto-oncogenes. The frequency of oncogene expression was ras 80.4%, c-myc 45.1% and c-jun 39.2%. There was no statistically significant association between oncogene expression, time to local recurrence or development of metastases or survival.

To identify possible nuclear signals mediating long-term regulation of the pancreas by gastrointestinal hormones, the expression of c-fos, c-jun, and c-myc was investigated in rat pancreatic acini. Stimulation of the acini with cholecystokinin octapeptide (CCK-8, 100 pM), bombesin (10 nM), or carbachol (10 microM), but not gastrin (100 nM), secretin (100 nM), or vasoactive intestinal peptide (10 nM) induced an increase in oncogene mRNA expression. The percent increases of c-fos, c-jun, and c-myc mRNA were 207 +/- 40, 171 +/- 26, and 46 +/- 19 (n = 5) for CCK-8; 223 +/- 71, 159 +/- 31, and 43 +/- 21 (n = 5) for bombesin; and 125 +/- 51, 123 +/- 58, and 67 +/- 19 (n = 5) for carbachol, respectively. CCK-induced increases in oncogene mRNA were rapid and transient. c-fos and c-jun mRNA levels were increased after 30 min stimulation, peaked at 1 h, and returned to basal level in 2 h. Activation of c-myc was more prolonged with levels remaining elevated for at least 3 h. The effects of CCK-8 were concentration dependent. Detectable stimulation was seen at 10 pM; maximal stimulation occurred at 10 nM and was not affected by further increase in the concentration of CCK-8. JMV-180, a high-affinity site CCK receptor agonist and low-affinity site antagonist, alone did not stimulate c-fos mRNA expression but inhibited c-fos mRNA expression induced by CCK-8. These results suggest that the interaction between CCK and the low-affinity state of the CCK receptor is responsible for oncogene activation.

The mechanisms of action of Ewing s sarcoma (EWS) associated EWS-ETS oncoproteins have largely remained unresolved. Here, we analyzed how two EWS-ETS proteins, EWS-ER81 and EWS-Fli-1, in vitro activate the matrix metalloproteinase (MMP)-1 promoter that is upregulated in a subset of EWSs. EWS-ER81 and EWS-Fli-1 interact with and thereby activate the MMP-1 promoter, which is potentiated by the cofactor p300 and the proto-oncoprotein c-Jun. Further, EWS-ER81 binds to c-Jun in vitro and in vivo. The interaction between c-Jun, p300 and EWS-ER81 or EWS-Fli-1 may also be relevant to the regulation of other yet-to-be-identified genes that are responsible for EWS formation.

OBJECTIVE: To study the expression of oncogene C-jun in rat nasal mucosa with allergic rhinitis and the relevance with rhinorrhea. METHOD: By using EA to make animal model of allergic rhinitis successfully; the amount of rhinorrhea was calculated at 30 min. 4 h, 8 h. Immunohistochemical technique, computer image analysis technique and histologic examination were used to investigated the expression of C-jun in rat nasal mucosa at the same time. The relevance between them was used explored. RESULT: There was a lot of C-jun protein at 30 min. The difference of C-jun between 30 min group and control group was significant (P < 0.01) and the relevant coefficient was r = 0.784 (P < 0.05). The C-jun protein decreased at 4 h, but there was still significant difference (P < 0.05) and r = 0.626 (P < 0.05). The C-jun was as same as control group at 8 h, There was no difference between them. CONCLUSION: The expression of oncogene C-jun in inflammatory cells may be a marker in early stage of activation of inflammatory cells, and related to the producing and secreting of cytokines and inflammatory mediators in inflammatory cells of allergic rhinitis.

Anaplastic large-cell lymphoma is associated with a chromosomal translocation generating an oncogenic fusion protein: the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). We have generated several independent lines of human NPM-ALK transgenic mice using the haematopoietic cell-specific Vav promoter. Lymphomas develop in two transgenic lines in which the Vav promoter regulates NPM-ALK expression. The transgenic line with higher copy number displays an early-onset phenotype in which ALL mice succumb to aggressive lymph node tumours with intestinal involvement, whereas the second line displays late-onset tumour development in the spleen and/or liver. Lymphomas from both lines are phenotypically distinct and display B-lineage characteristics with aberrant coexpression of myeloid markers. The NPM-ALK kinase is active in primary tumour tissue and forms a multimeric complex with tyrosine-phosphorylated proteins, that is, Shc. Jun and ERK kinase activities in tumours are elevated by up to 30-fold and fivefold, respectively, in comparison with sIgM-stimulated primary B cells. The new transgenic models provide a system for investigating the oncogenic events mediated by NPM-ALK in situ and a physiologically relevant context for developing tyrosine kinase inhibitor therapies of potential use in the clinic.

The retinoblastoma protein (Rb) controls cell proliferation, differentiation, and senescence and provides an essential tumor suppressive function that cells must eliminate to attain unlimited proliferative potential. Elimination of the Rb pathway also results in apoptosis, however, thereby providing an efficient surveillance mechanism to sense the loss of Rb. To become tumorigenic cells must thus overcome not only Rb function but also the apoptotic response caused by the loss of Rb function. We show that oncogenic Ras (RasV12) potently blocks cell death in Rb family member knockout mouse embryo fibroblasts (TKO cells). Activation of phosphatidylinositol 3-kinase and Raf by oncogenic Ras mediated this protection, implying that multiple Ras effector pathways are required, in concert, for this pro-survival signal. Although activation of Raf by selective Ras mutants protected TKO cells from cell death, pharmacologic inhibition of MEK had little effect on RasV12 protection, suggesting that a Raf-dependent, MEK-independent pathway was important for this effect. We show that this Raf-dependent protection occurred through activation of c-Jun and thus AP-1 activation. These observations could account for the dependence of Ras transformation on c-Jun activity and for the roles of AP-1 in oncogenesis. Our results support the concept of two oncogenic events cooperating to achieve a balance between immortalization and survival.

Jun/JunD and Jun/GCN4 chimeras transform chicken embryo fibroblasts and activate the collagenase promoter in these same cells. Individual constructs differ widely in the two activities, and there is no correlation between transformation and transactivation. These results suggest that oncogenic transformation by Jun is not caused merely by an upregulation of AP-1 activity. Jun constructs with a modified dimerization domain allowing only homodimerization are active in transformation and transactivation in chicken embryo fibroblast cultures. Homodimers of Jun therefore transform and transactivate.

OBJECTIVE: To study the potential relationship between the expressions of c-jun and c-fos oncogenes and the prognosis of medulloblastoma. METHODS: The specimens from 70 cases of medulloblastoma of the posterior fossa and 10 cases of normal cerebellar tissues were collected to determine c-jun and c-fos expressions by immunohistochemical staining in formalin fixed paraffin-embedded sections. RESULTS: (1) It showed that c-fos and c-jun protein expression was negative in 10 normal cerebellar tissue, while positive c-fos, c-jun immunoreactivity was found in 70 medulloblastoma specimens. The positive rate of c-jun and c-fos was 80% and 77%, respectively. There was high expression of c-jun and c-fos protein in medulloblastoma tissues. (2) There were positive correlations and strong co-operativity between c-jun and c-fos expression (r = 0.493, P < 0.01). (3) Correlative analysis indicated that expression of c-jun, c-fos were significantly correlated with survival time (c-jun: r = -0.447, P < 0.01; c-fos: r = -0.590, P < 0.01). The higher the expression level of c-jun and c-fos protein was, the worse the prognosis was in medulloblastoma patients. CONCLUSIONS: High expression of c-jun and c-fos protein could be noted in medulloblastoma tissues. The two transcription factors show positive correlation and strong co-existence between c-jun and c-fos expressions. The expression levels of c-jun as well as c-fos are negatively correlated with the mortality rate and life expectancy of patients with medulloblastoma. In addition, the co-expression of c-jun and c-fos could serve as an indicator for judging the prognosis of medulloblastoma.

Kruppel-like transcription factors (KLFs) represent one of the most diverse set of regulators in vertebrate organisms. KLF family members are involved in cell proliferation and differentiation control in normal as well as in pathological situations. Here, we demonstrate that KLF6 behaves as a functional antagonist of the c-Jun proto-oncoprotein. Thus, KLF6 overexpression downregulated c-Jun-dependent transcription and a physical interaction between c-Jun and KLF6 was detected. Moreover, cell proliferation induced by c-Jun was significantly decreased by KLF6. The inhibition of c-Jun functions correlates directly with c-Jun protein degradation induced by KLF6. We also show that ALL KLF6 effects on c-Jun were largely dependent on phorbol ester (TPA/ionomycin) extracellular stimulation, which enhanced KLF6 nuclear translocation and transcriptional activity and modified its phosphorylation status. Our data are consistent with a novel mechanism of KLF6s role as an inhibitor of cell proliferation by counteracting the function of the c-Jun proto-oncoprotein involving enhanced c-Jun degradation by the proteasome-dependent pathway, and further reinforces KLF6 as a potential tumor suppressor gene product.

Treatment of quiescent Balb/c 3T3 cells with insulin-like growth factor I (IGF I) resulted in the stimulation of proto-oncogene c-jun transcription. Cells exposed to cycloheximide and IGF I together showed super-induction of c-jun transcripts. Nuclear run-off assay revealed that IGF I up-regulated c-jun only while cycloheximide was present. The stability of c-jun mRNA was markedly increased in the cells treated with IGF I. These results suggest that IGF I controls the expression of c-jun by increasing the transcriptional activity and stabilizing the existing transcripts.

FRA-1, a member of the FOS family of transcription factors, is overexpressed in a variety of human tumors, and contributes to tumor progression. In addition to mitogens, various toxicants and carcinogens persistently induce FRA-1 expression in vitro and in vivo. Although the mitogen induced expression of c-FOS is relatively well understood, it is poorly defined in the case of FRA-1. Our recent analysis of the FRA-1 promoter has shown a critical role for a TRE located at -318 in mediating the TPA-induced expression. The -379 to -283 bp promoter segment containing a critical TRE (-318), however, is insufficient for the induction of FRA-1 promoter. Here, we show that a 40-bp (-276/-237) segment, comprising a TCF binding site and the CArG box (collectively known as serum response element, SRE), and an ATF site, is also necessary for the FRA-1 induction by TPA and EGF. Interestingly, the -283 to +32 bp FRA-1 promoter fragment containing an SRE and an ATF site alone was also insufficient to confer TPA sensitivity to a reporter gene. However, in association with the -318 TRE, the SRE and ATF sites imparted a strong TPA-inducibility to the reporter. Similarly, EGF also required these motifs for the full induction of this gene. Using ChIP assays we show that, in contrast to c-Jun, SRF, Elk1, ATF1 and CREB proteins bind to SRE and ATF sites of the FRA-1 promoter, constitutively. RNAi-mediated knockdown of endogenous SRF, ELK1 and c-JUN protein expression significantly reduced TPA-stimulated FRA-1 promoter activity. Thus, a bipartite enhancer formed by an upstream TRE and the downstream SRE and ATF sites and the cognate factors is necessary and sufficient for the regulation of FRA-1 in response to mitogens.

The aryl hydrocarbon receptor (AhR) has a fundamental role during postnatal liver development and is essential for mediating dioxin toxicity. However, the genetic programs mediating, both, the toxic and physiological effects downstream of the transcription factor AhR are in major parts unknown. We have identified the proto-oncogene c-jun as a novel target gene of AhR. Induction of c-jun depends on activation of p38-mitogen-activated protein kinase (MAPK) by an AhR-dependent mechanism. None of the kinases that are known to phosphorylate p38-MAPK is activated by AhR. Neither the dephosphorylation rate of p38-MAPK is reduced. Furthermore, increased p38-MAPK phosphorylation in response to dioxins does not require ongoing transcription. These findings establish activating cross-talk with MAPK signaling as a novel principle of AhR action, which is apparently independent of the AhR s function as a DNA-binding transcriptional activator.

In previous studies we have found that oncogenic (Val 12)-ras-p21 induces Xenopus laevis oocyte maturation that is selectively blocked by two ras-p21 peptides, 35-47, also called PNC-7, that blocks its interaction with raf, and 96-110, also called PNC-2, that blocks its interaction with jun-N-terminal kinase (JNK). Each peptide blocks activation of both JNK and MAP kinase (MAPK or ERK) suggesting interaction between the raf-MEK-ERK and JNK-jun pathways. We further found that dominant negative raf blocks JNK induction of oocyte maturation, again suggesting cross-talk between pathways. In this study, we have undertaken to determine where these points of cross-talk occur. First, we have immunoprecipitated injected Val 12-Ha-ras-p21 from oocytes and found that a complex forms between ras-p21 raf, MEK, MAPK, and JNK. Co-injection of either peptide, but not a control peptide, causes diminished binding of ras-p21, raf, and JNK. Thus, one site of interaction is cooperative binding of Val 12-ras-p21 to raf and JNK. Second, we have injected JNK, c-raf, and MEK into oocytes alone and in the presence of raf and MEK inhibitors and found that JNK activation is independent of the raf-MEK-MAPK pathway but that activated JNK activates raf, allowing for activation of ERK. Furthermore, we have found that constitutively activated MEK activates JNK. We have corroborated these findings in studies with isolated protein components from a human astrocyte (U-251) cell line; that is, JNK phosphorylates raf but not the reverse; MEK phosphorylates JNK but not the reverse. We further have found that JNK does not phosphorylate MAPK and that MAPK does not phosphorylate JNK. The stress-inducing agent, anisomycin, causes activation of JNK, raf, MEK, and ERK in this cell line; activation of JNK is not inhibitable by the MEK inhibitor, U0126, while activation of raf, MEK, and ERK are blocked by this agent. These results suggest that activated JNK can, in turn, activate not only jun but also raf that, in turn, activates MEK that can then cross-activate JNK in a positive feedback loop.

In resting cells, c-Jun is phosphorylated on five sites. Three of these sites reside next to its DNA binding domain and negatively regulate DNA binding. In response to expression of oncogenic Ha-Ras, phosphorylation of these sites decreases, while phosphorylation of two other sites within c-Jun s activation domain is greatly enhanced. Phosphorylation of these residues, serines 63 and 73, stimulates the transactivation function of c-Jun and is required for oncogenic cooperation with Ha-Ras. We now show that the same changes in c-Jun phosphorylation are elicited by a variety of transforming oncoproteins with distinct biochemical activities. These oncoproteins, v-Sis, v-Src, Ha-Ras, and Raf-1, participate in a signal transduction pathway that leads to increased phosphorylation of serines 63 and 73 on c-Jun. While oncogenic Ha-Ras is a constitutive stimulator of c-Jun activity and phosphorylation, the normal c-Ha-Ras protein is a serum-dependent modulator of c-Jun s activity. c-Jun is therefore a downstream target for a phosphorylation cascade involved in cell proliferation and transformation.

OBJECTIVES: To investigate c-jun oncoprotein (JUN) expression in diseases of the human prostate gland at the tissue level and to determine its relationship to clinicopathologic variables. METHODS: The expression of JUN was studied using immunohistochemistry in archival tissue from benign prostatic hyperplasia (BPH) (n = 16) and prostate adenocarcinoma (PCa) (n = 36) specimens. RESULTS: JUN-specific positive nuclear immunostaining was observed in 13 (81.25%) of 16 BPH and 31 (86.1%) of 36 PCa specimens. JUN-specific immunostaining was significantly stronger in the PCa than in the BPH tissue (P = 0.006). In the PCa tissue, no significant correlation was found between JUN immunohistochemical expression and tumor histologic grade (Gleason score) or serum prostate-specific antigen level. CONCLUSIONS: JUN expression may play a role in normal cell function of the prostatic epithelium and is expressed in most BPH and PCa samples. The finding that JUN-specific immunostaining intensity was stronger in the vast majority of PCa than in the BPH samples implies that the role of c-jun may be enhanced during malignant transformation.

Chicken c-Jun proteins synthesized in vitro in reticulocyte extract consist of several electrophoretic isoforms resulting from phosphorylation which can be specifically reversed by purified protein phosphatase 2A (PP2A). Using the phosphatase inhibitors okadaic acid and microcystin-LR, we conclude that the isoforms seen in vitro represent a balance between the action of an unidentified kinase(s) which phosphorylates c-Jun and dephosphorylation by an endogenous PP2A-like phosphatase. c-Jun proteins are also subject to phosphorylation in vivo in chick embryo fibroblasts (CEF), which can be reversed by PP2A. In contrast, the viral Jun oncoprotein encoded by ASV17 is not subject to PP2A-sensitive phosphorylation in vitro and is hypophosphorylated in comparison with c-Jun in ASV17-transformed CEF. Hybrids between c-Jun and v-Jun demonstrate that differential phosphorylation in vitro is a consequence of deletion of 27 amino acids in the N-terminal third of v-Jun. The deletion is important for oncogenic activation and lies in a domain, termed delta, which regulates c-Jun transactivation function. PP2A-sensitive phosphorylation in vitro correlates with the differential responsiveness of c-Jun and v-Jun to a recently identified cell type-specific inhibitor of transactivation function.

A sample of a primary brain tumor of glial cell origin was surgically removed from a 7-year-old girl. The histopathological analysis showed a heterogeneous tumor containing highly cellular areas composed of small, poorly differentiated cells with frequent mitoses suggestive of a glioblastoma multiforme. There were also areas presenting as features of lower-grade astrocytoma. Strong immunohistochemical staining for glial fibrillar acidic protein was demonstrated, while vimentin, neurofilament, and S-100 protein were ALL positive in just the astrocytic part of the tumor. The DNA extracted from a fresh tumor sample at diagnosis was processed by Southern blot analysis and hybridized with a 2.0 kb N-myc oncogene probe recognizing the first intron and the second exon of the human gene. A 20-fold amplification of the oncogene was found. The possible role of such a molecular alteration is discussed in light of the clinical presentation and histopathological features.

By means of low-stringency cross-species hybridization to Southern DNA blots, human c-jun sequences were used to identify a unique Drosophila melanogaster locus (Djun). The predicted DJun protein is highly homologous to members of the mammalian Jun family in both the DNA binding and leucine zipper regions. Djun was mapped by in situ hybridization to position 46E of the second chromosome. It encodes a 1.7-kilobase transcript constitutively expressed at ALL developmental stages. Functionally, Djun in cooperation with mouse c-fos can trans-activate activator protein 1 DNA binding site when introduced into mammalian cells. Taken together, these data suggest that Djun, much like its mammalian homolog, may activate transcription of genes involved in regulation of cell growth, differentiation, and development. Furthermore, the identification of Djun allows one to exploit the genetics of Drosophila to identify genes in signal transduction pathways involving Djun and thus c-jun.

The constitutive activation of JNK has been implicated in Ras-induced cellular transformation and activated JNK is down-regulated by the tumor suppressor protein, RASSF1A. In this study, we examined whether RASSF1A blocked oncogenic Ras-induced JNK activation. Exogenous expression of H-RasG12V induced JNK phosphorylation and RASSF1A co-transfected with H-RasG12V efficiently suppressed Ras-triggered JNK activation in various cancer cell lines. RASSF1A expression revived the H-RasG12V-induced p27Kip1 down-regulation. JNK siRNA treatment also promoted recovery from the H-RasG12V-induced p27Kip1 down-regulation. These results demonstrate that RASSF1A inhibited H-RasG12V-induced JNK activation and JNK-mediated p27Kip1 down-regulation. From these results, we propose that RASSF1A exerts a tumor-suppressing effect by blocking oncogenic Ras-induced JNK activation.

OBJECTIVE: To study the rules of protein expression of proto-oncogene (c-Fos/c-Jun) in different brain areas and nucleus of psychological stressed mice and the regulatory effect of modified Xiaoyao Pill (SXP). METHODS: The mouse psychological stress model was established by electrical stimulation; SXP was administrated at the dose of 2 mg/g; the protein expression of c-Fos and c-Jun in different brain areas and nucleus, including hippocampus (CA1-4), central amygdaloid nucleus (CAN), paraventricular nucleus (PVN) and supraoptic nucleus (SON), were detected by immunohistochemical method. RESULTS: The protein expressions of c-Fos and c-Jun in ALL the tested brain areas of model mice 3 h after being stressed for 1, 3 or 5 times, were significantly higher than those of the normal mice (P < 0.01); After being stressed for one time, the expression at 1 h after stimulation was lower than that at 3 h after stimulation (P < 0.05), the expression in mice treated with SXP was lower than that in the untreated group, 3 h after three or five times of stress (P < 0.05 or P < 0.01). CONCLUSION: The protein expression of c-Fos and c-Jun began to rise 1 h after psychological stress and reached the peak at the 3rd h. That of c-Fos dropped to the normal level approximately after 6 h, but the dropping did not happen in expression of c-Jun, it remained on the high level unweakened after repeated stimulation; SXP can remarkably down-regulate the proto-oncogene (c-Fos and c-Jun) expressions after psychological stress in mice.

Herpesvirus saimiri (HVS), a member of the gamma-herpesvirus family, encodes an oncoprotein called Saimiri Transforming Protein (STP) which is required for lymphoma induction in non-human primates. However, a detailed mechanism of STP-A11-induced oncogenesis has not been revealed yet. We first report that STP-A11 oncoprotein interacts with TNF-alpha receptor-associated factor (TRAF) 6 in vivo and in vitro. Mutagenesis analysis of the TRAF6-binding motif (10)PQENDE(15) in STP-A11 reveals that Glu (E)(12) residue is critical for binding to TRAF6 and NF-kappaB activation. Interestingly, co-expression of E12A mutant, lack of TRAF6 binding, with cellular Src (Src) results in decreased transcriptional activity of Stat3 and AP-1, a novel target of STP-A11 compared to that of wild type. Furthermore, the presence of STP-A11 enhances the association of TRAF6 with Src and induces the translocation of both TRAF6 and Src to a nonionic detergent-insoluble fraction. Taken together, these studies suggest that STP-A11 oncoprotein up-regulates both NF-kappaB and AP-1 transcription activity through TRAF6, which would ultimately contribute cellular transformation.

Fos and Jun, the protein products of the nuclear proto-oncogenes c-fos and c-jun, associate preferentially to form a heterodimer that binds to DNA and modulates transcription of a wide variety of genes in response to mitogenic stimuli. Both Fos and Jun contain a single leucine zipper region. Previous studies have shown that the leucine zippers of Fos and Jun are necessary and sufficient to mediate preferential heterodimer formation. The leucine zipper regions from Fos and Jun are also known to fold autonomously, most likely as two-stranded, parallel coiled coils. We show here that 8 amino acids from Fos and from Jun are sufficient to mediate preferential heterodimer formation in a background of the GCN4 leucine zipper sequence. Using pH titration and amino acid replacements, we also show that destabilization of the Fos homodimer by acidic residues provides a major thermodynamic driving force for preferential heterodimer formation.

The human oncogene JUN encodes a component of the AP-1 complex and is consequently involved in a wide range of pivotal cellular processes, including cell proliferation, transformation, and apoptosis. Nevertheless, despite extensive analyses of its functions, it has never been directly involved in a human cancer. We demonstrate here that it is highly amplified and overexpressed in undifferentiated and aggressive human sarcomas, which are blocked at an early step of adipocyte differentiation. We confirm by cellular and xenograft mouse models recapitulating these sarcoma genetics that the failure to differentiate is dependent upon JUN amplification/overexpression.

Although activating protein-1 (AP-1) transcription factors play an important role in mediating metastasis for nasopharyngeal carcinoma (NPC), the biological and physiological functions of AP-1, in relation to the oncogenic phenotype of NPC, are not fully understood. Our previous study showed that the latent membrane protein 1 (LMP1) mediated a primary dimer form of c-jun and jun B. In this study, we used a NPC cell line that express a specific inhibitor of AP-1, a dominant-negative c-jun mutant (TAM67), to investigate the role of AP-1 in regulating the NPC oncogenic phenotype. First, we observed that TAM67 inhibited cell growth in vitro and in vivo. Next, with Western blotting, we discovered that TAM67 impaired the cyclin D1/cdk4 complex but had little effect on the cyclin E/cdk2 complex, concomitantly with inhibiting Rb phosphorylation. RT-PCR and luciferase assay results demonstrated that the levels of cyclin D1 mRNA and the promoter activity in TAM67 transfectants were reduced as compared with control cells. Thereby, we show that blockade of AP-1 transcriptional activity has a negative impact on cyclin D1 transcription. We obtained the first evidence that TAM67 prevented NPC growth both in vitro and in vivo. AP-1 appears to be a novel target for treating or preventing LMP1-positive NPC effectively.

Recent advances indicate a link between tumour promoters, transformation, and AP-1 activity. Protein kinase C activation increases AP-1 DNA-binding activity independently of new protein synthesis. AP-1 is also stimulated by transforming oncoproteins and growth factors. These proteins are thought to participate in a signalling cascade affecting the nuclear AP-1 complex composed of the Jun and Fos proteins. Because c-Jun is the most potent transactivator in the AP-1 complex and is elevated in Ha-ras-transformed cells, in which c-Fos is downregulated, we focused on it as a potential target. c-Jun could convert input from an oncogenic signalling cascade into changes in gene expression. Indeed, transformation of rat embryo fibroblasts by c-Jun requires an intact transcriptional activation domain and cooperation with oncogenic Ha-ras. expression of oncogenic Ha-ras augments transactivation by c-Jun and stimulates its phosphorylation. Here we describe the mapping of the Ha-ras-responsive phosphorylation sites to serines 63 and 73 of c-Jun. Site-directed mutagenesis indicates that phosphorylation of these serines is essential for stimulation of c-Jun activity and for cooperation with Ha-ras in ocogenic transformation.

The human PVT-1 gene is located on chromosome 8 telomeric to the c-Myc gene and it is frequently involved in the translocations occurring in variant Burkitt s lymphomas and murine plasmacytomas. It has been proposed that PVT-1 regulates c-Myc gene transcription over a long distance. To get new insights into the functional relationships between the two genes, we have investigated PVT-1 and c-Myc expression in normal human tissues and in transformed cells. Our findings indicate that PVT-1 expression is restricted to a relative low number of normal tissues compared to the wide distribution of c-Myc mRNA, whereas the gene is highly expressed in many transformed cell types including neuroblastoma cells that do not express c-Myc. Reporter gene assays were used to dissect the PVT-1 promoter and to identify the region responsible for the elevated expression observed in transformed cells. This region contains two putative binding sites for Myc proteins. The results of transfection experiments in RAT1-MycER cells and chromatin immunoprecipitation (ChIP) assays in proliferating and differentiated neuroblastoma cells indicate that PVT-1 is a downstream target of Myc proteins.

The activating protein-1 (AP-1) transcription factor transduces growth signals through signal transduction pathways to the nucleus, leading to the expression of genes involved in growth and malignant transformation in many cell types. We have previously shown that overexpression of a dominant negative form of the cJun proto-oncogene, a cJun dominant negative mutant (Tam67), blocks AP-1 transcriptional activity, induces a G(1) cell cycle block and inhibits breast cancer cell growth in vitro and in vivo. We found that AP-1 blockade by Tam67 in MCF-7 breast cancer cells downregulates cyclin D1 transcriptional activity by at least two mechanisms: by suppressing transcription at the known AP-1 binding site (-934/-928) and by suppressing growth factor-induced expression through suppressing E2F activation at the E2F-responsive site (-726/-719). AP-1 blockade also led to reduced expression of E2F1 and E2F2, but not E2F4, at the mRNA and protein levels. Chromatin immunoprecipitation and supershift assays demonstrated that AP-1 blockade caused decreased binding of E2F1 protein to the E2F site in the cyclin D1 promoter. We also found that Tam67 suppressed the expression of the E2F1 dimerizing partner, DP1 and E2F-upregulated cell cycle genes (cyclins E, A, B and D3) and enhanced the expression of E2F-downregulated cell cycle genes (cyclins G(2) and I). Reduced expression of other E2F-regulated genes was also seen with AP-1 blockade and E2F suppression. Thus, the AP-1 factor regulates the expression of cyclin D and E2F (the latter in turn regulates E2F-downstream genes), leading to cell cycle progression and breast cancer cell proliferation.

The effects of antibodies against sperm antigens and the c-myc proto-oncogene product on early embryonic development were investigated in mice. Affinity-purified Fab antibodies against lithium diiodosalicylate (LIS)-solubilized murine sperm extract and fertilization antigen (FA-1) reduced (p less than 0.01 to p less than 0.001) blastulation rates of in vitro cultured 2-cell murine embryos primarily because of an arrest of development at the morula stage. Similarly, the c-myc monoclonal antibody (mAb) affected early embryonic development in a dose-dependent manner. These effects were specific, since immunoabsorption, with its respective peptide, completely blocked the inhibitory effect of the c-myc mAb. Anti-LIS sperm Fab identified four protein bands (approx. 36, 29, 24.6, and 17.6 kDa) on Western blots of extracts from unfertilized and fertilized ova, one band (approx. 68 kDa) each on 4-8-cell embryo and morula extracts, and one band (approx. 53 kDa) on blastocyst extracts. Anti-FA-1 Fab did not react with unfertilized or fertilized ova, but specifically identified two protein bands (approx. 53 and 25.7 kDa) on blots of 2-cell-embryo extract, one band (approx. 25.7 kDa) on morula extract, and one band (approx. 53 kDa) on blastocyst extract. The c-myc mAb did not react with any band corresponding to the c-myc protein on blots of extracts from unfertilized or fertilized ova, 2-cell embryos, 4-8-cell embryos, morulae, or blastocysts. These results suggest that some of the cross-reacting sperm antigens that are expressed during early cleavages, and the product of the c-myc proto-oncogene may have a role in normal early embryonic development.

c-Myc induces widely varying cellular effects, including cell proliferation and cell death. These different cellular effects are determined, in part, by c-Myc protein expression levels, which are regulated through several transcriptional and post-transcriptional pathways. c-Myc transcripts can be detected in cells at ALL stages of B and T lymphocyte development. However, little is known about c-Myc protein expression, and how it varies, in developing lymphocytes. Here mice have been generated in which the endogenous c-Myc locus has been modified (c-Myc(G)) so that it encodes a GFP-c-Myc fusion protein. c-Myc(G/G) mice are viable, appear normal and exhibit grossly normal lymphocyte development. Flow cytometric analyses revealed significant heterogeneity in c-Myc protein expression levels in developing c-Myc(G/G) B and T lymphocytes. GFP-c-Myc expression levels were highest in proliferating lymphocytes, suggesting that c-Myc up-regulation is important for promoting lymphocyte cell division, and demonstrating that GFP-c-Myc expression is a marker of proliferating lymphocytes in vivo.

We show here that TPA treatment of MCF-7 cells represses estrogen receptor dependent transcriptional activity, while increasing the AP1 binding activity. These two events are probably linked, since the transcriptional activity of the estrogen receptor in these cells is repressed by overexpression of both cJun or cFos, the components of the AP1 transcripts factor. On the contrary no repression was observed after overexpression of another member of the jun family, the JunD. The repression caused by cJun or cFos may depend on partially different pathways. Our results suggest that the inhibition of TPA of the estrogen dependent growth of the MCF-7 cells is caused by over expression of cJun and cFos.

Homopurine.homopyrimidine (Pu.Py) tracts are likely to play important biological role in eukaryotes. Using circular dichroism, UV-thermal denaturation and gel electrophoresis, we have analyzed the structural polymorphism of a 21-bp Pu.Py DNA segment within human c-jun protooncogene 3 -region, a potential target for triplex formation. Results show that below physiological pH and in the presence of Na+/K+ with Mg2+ the duplex is destabilized/disproportionated, resulting in strand mediated structural transitions to the self-associated structures of G- and C-rich strands separately, identified as G-quadruplex and i-motif species. A significant differential behavior of the monovalent cations was observed, accordingly the presence of Na+ in acidic as well as neutral pH facilitated the duplex formation, while K+ favored the formation of self-associated structures. In Na+ and Mg2+, under acidic and neutral pH conditions, the duplex displayed triphasic and biphasic melting profiles, respectively. This self-association property of oligonucleotides might limit their use as duplex targets in triplex formation. Study is also relevant for understanding structural and biological properties of DNA sequence containing homopurine tracts.

In a study of interactions between the raf-MEK-MAPK (ERK) and JNK-jun pathways, we found previously that JNK can induce phosphorylation of raf but not vice versa. In this study, we investigate the nature of the JNK-induced phosphorylation of raf. In in vitro experiments in which immunobead-bound raf is phosphorylated by activated JNK, we find strong phosphorylation signals at raf-Ser259 and Ser338. The Ser259 phosphorylation is surprising since it is associated with inhibition of migration of raf to the cell membrane where it can interact with ras-p21. We also find that in oocytes induced to mature with oncogenic ras-p21, which induces high levels of phosphorylated JNK and MAPK, the same pattern of phosphorylation of raf occurs. In contrast, in oocytes induced to mature with insulin, which requires activation of wild-type ras-p21, phosphorylation of raf-Ser338 but not raf-Ser259 occurs. In oncogenic ras-transformed human pancreatic cancer MIA-PaCa-2 cells, phosphorylation of both raf serines occurs. Treatment of these cells with the ras peptide, PNC-2 attached to a penetrating sequence that blocks JNK and MAPK phosphorylation and induces tumor cell necrosis, results in a marked decrease in phosphorylation of raf-Ser259, but not that of raf-Ser338. These results suggest that oncogenic ras-p21 induces phosphorylation of both raf-Ser259 and Ser338 and that raf-Ser 259 phosphorylation may be effected by activated JNK.

CDCA4, a member of the TRIP-Br transcriptional co-factor family, has been shown to possess a unique role in regulating the transcriptional activities of p53 as well as E2F1 transcription factors. In this study, we aimed to identify a pivotal transcriptional target gene regulated by CDCA4, so we suppressed CDCA4 expression by CDCA4-specific short interference RNA (siRNA) in HeLa cells, and then performed a DNA microarray analysis. Among the identified genes, we focused on JUN, 14-3-3eta, and IL6ST (gp130) mRNAs which were up-regulated in CDCA4-specific siRNA-transfected cells compared to control siRNA-transfected cells. We confirmed that JUN, 14-3-3eta, and IL6ST proteins were up-regulated when cells were transfected with CDCA4-specific siRNA. 14-3-3eta and IL6ST protein levels were unchanged upon on transfection of cells with JUN-specific siRNA, indicating that 14-3-3eta and IL6ST genes are not a direct target of JUN. Serine 63 and 73 phosphorylation of JUN was unchanged when cells were transfected with CDCA4-specific siRNA. In addition, JUN-driven reporter activity was unaffected by CDCA4 co-transfection, suggesting that CDCA4 affects solely JUN mRNA expression. Finally, by preparing various JUN promoter reporter constructs, we minimized the JUN promoter sequence that was affected by CDCA4 co-expression. Together, these results add an important role of CDCA4 in the context of transcriptional regulation and cell fate determination through the JUN oncogene.

The TOJ3 gene was originally identified on the basis of its specific activation in avian fibroblasts transformed by the v-jun oncogene of avian sarcoma virus 17 (ASV17). Overexpression of TOJ3 induces cellular transformation of embryonic avian fibroblasts, revealing an intrinsic oncogenic potential. Transforming activity has also been demonstrated for MSP58, the human homolog of TOJ3, and oncogenic cell transformation by MSP58 is specifically inhibited by the tumor suppressor PTEN. To investigate the mechanism of aberrant TOJ3 gene activation in jun-transformed fibroblasts, the entire quail TOJ3 gene including 13 exons and the 5 regulatory region was isolated. Functional analyses of the promoter by transcriptional transactivation assays revealed that the specific induction of TOJ3 is mediated by a cluster of three noncanonical AP-1 binding motifs (5 -CAGCTCA-3 or 5 -CACCTCA-3 ) which share the 3 half-site with the consensus motif (5 -TGA(C)/(G)TCA-3 ). Electrophoretic mobility shift assays and chromatin immunoprecipitation analyses showed that Jun binds to these motifs with an affinity similar to that observed for binding to an AP-1 consensus site. Noncanonical binding sites are also present in the chicken and human TOJ3/MSP58 promoter regions. These results confirm and extend the previous observation that TOJ3 represents an immediate effector gene of Jun and may point to an essential role of TOJ3/MSP58 in carcinogenesis involving aberrant AP-1 expression.

Although the structure of several members of the GH receptor family has been defined, signal transduction following GH binding to its receptor has not been elucidated. Mouse osteoblasts were used to study the effect of GH on immediate early gene expression and, subsequently, the cellular signal(s) mediating this expression were analysed. GH rapidly and transiently induced the expression of c-jun and jun B in concert with the already reported expression of c-fos. The GH-induced expression of c-fos was completely blocked by the protein kinase inhibitors staurosporine and H7, indicating that the action of GH is mediated by one or several protein kinases. We next analysed the identity of the putative protein kinases in more detail by using a more specific protein kinase inhibitor, namely the ether-lipid 1-O-alkyl-2-O-methylglycerol, understood to be an inhibitor of protein kinase C (PKC). Data obtained from these studies revealed that GH-induced expression of c-fos is mediated by PKC. In addition, we observed a profound increase in formation of the PKC activator diacyglycerol upon addition of GH, a natural activator of PKC. In conclusion, upon binding of GH to mouse osteoblasts, the receptor-mediated cellular signal involves diacyglycerol formation and activation of PKC, leading to the induction of oncogene expression. Finally, the expression of c-fos, c-jun and jun B results in an increased binding of protein complexes to AP-1 binding sites.

Persistent activation of the Hedgehog (HH)/GLI signaling pathway has been implicated in the development of a number of human cancers. The GLI zinc finger transcription factors act at the end of the HH signaling cascade to control gene expression, and recent studies have shown that the activity of GLI proteins can be additionally modified by integration of distinct signals, such as the MEK/extracellular signal-regulated kinase (ERK) and phosphinositide-3 kinase (PI3K)/AKT pathway. However, little is known about the identity of the upstream activators of these HH/GLI interacting signaling pathways in cancer. Here, we provide evidence that integration of the HH/GLI and epidermal growth factor receptor (EGFR) pathway synergistically induces oncogenic transformation, which depends on EGFR-mediated activation of the RAS/RAF/MEK/ERK but not of the PI3K/AKT pathway. EGFR/MEK/ERK signaling induces JUN/activator protein 1 activation, which is essential for oncogenic transformation, in combination with the GLI activator forms GLI1 and GLI2. Furthermore, pharmacologic inhibition of EGFR and HH/GLI efficiently reduces growth of basal cell carcinoma (BCC) cell lines derived from mice with activated HH/GLI signaling. The results identify the synergistic integration of GLI activator function and EGFR signaling as a critical step in oncogenic transformation and provide a molecular basis for therapeutic opportunities relying on combined inhibition of the HH/GLI and EGFR/MEK/ERK/JUN pathway in BCC.

Loss of E-cadherin-mediated cell-cell contacts can elicit a signaling pathway that leads to acquisition of an invasive phenotype. Here, we show that at the receiving end of this pathway is the proto-oncogene c-Jun, a member of the activator protein-1 family of transcription factors that play a key role in stimulation of cell proliferation and tumor promotion. Cell separation or abrogation of E-cadherin-mediated cell-cell contacts both cause a dramatic increase in accumulation of the c-Jun protein. Unlike growth factors that enhance the expression of c-Jun by activating the transcription of the c-jun gene, the cell contact-dependent increase in c-Jun accumulation is not accompanied by a corresponding increase in c-Jun mRNA or c-Jun protein stability but rather in the translatability of the c-Jun transcript. Consistently, the increase in c-Jun accumulation is not dependent on activation of the mitogen-activated protein kinase or beta-catenin pathways but is mediated by signals triggered by the restructured cytoskeleton. Depolymerization of the cytoskeleton can mimic the effect of cell separation and cause a dramatic increase in c-Jun accumulation, whereas Taxol inhibits the cell contact-dependent increase. This novel mechanism of c-Jun regulation seems to underlie the robust overexpression of c-Jun in tumor cells of patients with colon carcinoma.

Infection of replicating quail myoblasts with avian sarcoma virus 17 (ASV-17) results in the inhibition of terminal differentiation into multinucleated myotubes and in the acquisition of anchorage-independent proliferation. expression of v-jun, the ASV-17 oncogene, concomitantly leads to the accumulation of the gag-jun polyprotein P65 in the nucleus and to the lack of expression of typical differentiation-specific genes such as myosin heavy chain (MHC) and alpha-actinin. Surprisingly, expression of desmin, the muscle-specific subunit of intermediate filaments, is conserved in ASV-17-transformed myoblasts. Analysis of clonal strains of transformed myoblasts suggests that (i) suppression of morphological and biochemical differentiation depends on the absence of muscle-specific gene transcripts; (ii) inhibition of muscle differentiation by v-jun does not depend on the transcriptional silencing of MyoD, a muscle-specific regulatory gene; (iii) expression of desmin is compatible with proliferation of ASV-17-transformed cells and is independent of v-jun and MyoD levels of expression. The present data suggest that nuclear localization of v-jun prevents terminal differentiation in myoblasts and selectively down-regulates muscle-specific genes in terminally differentiated myotubes. In this respect, the behaviour of v-jun is quite different from that of v-myc, thus suggesting that these two oncogenes, although both encoding nuclear proteins, may have different mechanisms of action.

The human papillomavirus (HPV) E6 and E7 oncoproteins play important roles in cervical carcinogenesis through multiple mechanisms, including upregulation of cyclooxygenase-2 (COX-2), which has been shown to be involved in both carcinogenesis and cancer progression. To explore the role of E5 in cervical carcinogenesis, we herein investigated the effect of HPV 16 E5 on COX-2 expression. Our results revealed that E5 induced COX-2 expression through the epidermal growth factor receptor-signaling pathway, with nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) acting as critical factors in E5-induced COX-2 expression. NF-kappaB inhibition blocked COX-2 expression more potently than inhibition of AP-1. Our findings collectively suggest that the HPV 16 E5 oncoprotein mediates cervical carcinogenesis at least in part via upregulation of COX-2 expression through NF-kappaB and AP-1, with NF-kappaB playing a larger role.

Psoriasis is a common, sometimes severe, non-malignant skin disease characterized by hyperproliferation and abnormal differentiation of keratinocytes. Because proto-oncogenes are implicated in both cell proliferation and differentiation, their expression could be modified in skin diseases such as psoriasis. The c-fos and c-jun proto-oncogenes, whose products associate to form a heterodimeric transcription factor, are among the first genes to be expressed when certain cells are stimulated to either proliferate or differentiate. Recent studies in our laboratory have shown that the c-fos proto-oncogene is highly expressed in normal human adult skin. In the present study, we used in situ hybridization with RNA to compare the expression and localization of c-fos and c-jun transcripts in 15 lesional and non-lesional psoriatic skin samples. Two clinical variants of psoriasis were studied: the most severe and chronic form or plaque-type psoriasis (N = 10) and rapidly resolutive guttate-type psoriasis (N = 5). Quantitative analysis was performed using a semi-automatic image analyzer and the "Starwise grain" software program. Our control samples included 10 normal skins and eight specimens from other benign hyperproliferative non-psoriatic skin diseases, consisting of three with inflammation (seborrheic dermatitis and atopic dermatitis), and 5 without inflammation (seborrheic keratoses). Control genes we used for in situ hybridization and RNA integrity were keratin 14, which is expressed in the epidermis and was normally expressed in ALL tissue analyzed, and ribosomal RNA. Our data showed that c-fos and c-jun were expressed to an equivalent extent, both spatially and quantitatively, in ALL specimens tested. expression was significantly decreased in plaque-type but not in guttate-type psoriasis. It was also decreased in the three other benign inflammatory cutaneous hyperproliferative disorders, but not in the five non-inflammatory cases. These results were surprising because hyperproliferation was here associated with a decrease in proto-oncogene expression, thus suggesting that c-fos and c-jun do not play a crucial role in the control of keratinocyte proliferation in vivo. However, their reduced expression in some abnormally differentiated skins indicates that both c-fos and c-jun proto-oncogenes may play a key role in keratinocyte differentiation. Their altered expression correlated with severity of the disease and the presence of an inflammatory infiltrate. These data offer a new insight into the role and regulation of these proto-oncogenes in vivo in humans.

Many essential biological pathways, including cell growth, development, and metabolism, are regulated by thyroid hormones (THs). TH action is mediated by intracellular receptors that belong to a large family of ligand-dependent transcription factors, including the steroid hormone and retinoic acid receptors. So far it has been assumed that TH receptors (TRs) regulate gene transcription only through the classical protein-DNA interaction mechanism. Here we provide evidence for a regulatory pathway that allows cross-talk between TRs and the signal transduction pathway used by many growth factors, oncogenes, and tumor promoters. In transient transfection studies, we observed that the oncogenes c-jun and c-fos inhibit TR activities, while TRs inhibit induction of the c-fos promoter and repress AP-1 site-dependent gene activation. A truncated TR that lacks only 17 amino acids from the carboxy terminus can no longer antagonize AP-1 activity. The cross-regulation between TRs and the signal transduction pathway appears to be based on the ability of TRs to inhibit DNA binding of the transcription factor AP-1 in the presence of THs. The constituents of AP-1, c-Jun, and c-Fos, vice versa, can inhibit TR-induced gene activation in vivo, and c-Jun inhibits TR DNA binding in vitro. This novel regulatory pathway is likely to play a major role in growth control and differentiation by THs.

The nuclear phosphoprotein c-Jun, encoded by the proto-oncogene c-jun, is a major component of the AP-1 complex. A potent transcriptional regulator, c-jun is also able to transform normal rat embryo cells in cooperation with an activated c-Ha-ras gene. By deletion analysis, we identified the regions of c-Jun encoding transformation and transactivation functions. Our studies indicate that there is a direct correlation between the ability of the c-Jun protein to activate transcription and cotransform rat embryo cells. The regions involved in these functions include the conserved leucine zipper/DNA binding domain and an effector domain near its N terminus. This N-terminal region spans amino acids 61 to 146 of the c-Jun protein and is highly conserved among ALL Jun family members. These results support the hypothesis that c-Jun transforms cells by stimulating the expression of transformation-mediating genes.

Genomic amplification of c-Jun and its upstream kinases have been implicated as a mechanism of progression from well-differentiated to dedifferentiated liposarcoma. To further define the role of c-Jun in liposarcoma progression, we performed immunohistochemistry for c-Jun and its activating kinase ASK1 on a series of liposarcomas (n = 81). We correlated the results with fluorescence in situ hybridization to detect c-Jun amplification. We also derived new cell lines from dedifferentiated liposarcomas with c-Jun amplification. c-Jun protein is expressed in the majority of dedifferentiated liposarcomas (91%) and their well-differentiated components (59%), but only in the minority of pure well-differentiated liposarcomas (27%). When c-Jun is amplified in dedifferentiated liposarcoma, it is interspersed with amplified MDM2 on ring and giant marker chromosomes. MDM2 amplification is one of the earliest events in liposarcoma development, and these results suggest that c-Jun was amplified at a similar time in the evolution of the tumour. In addition, shRNA to c-Jun in c-Jun-amplified liposarcoma cells reduces cell number in vitro and inhibits tumour formation in vivo without an observable effect on the differentiation state of the liposarcoma cells. Thus, c-Jun amplification is oncogenic in liposarcomas but not always sufficient for inhibition of adipocytic differentiation.

Human papillomaviruses (HPV) are the main etiological factor for cervical carcinoma. HPV-16 is the most prevalent high-risk HPV-genotype found in HPV-associated cancers. We studied the effect of HPV-16 E7 oncoprotein on cadherin-mediated cell adhesion. The expression of E7 strongly suppressed the cadherin-mediated cell adhesion in the rat fibroblast cell line 3Y1. This suppression was associated with the decreased expression of N-cadherin at the transcriptional level. The treatment of 3Y1 cells that express E7 (E7-3Y1) with MEK inhibitor recovered the cadherin-mediated cell adhesion together with the accumulation of N-cadherin at the cell-cell contact site. Moreover, the suppression of c-Jun, which is the element of AP-1 transcriptional factor, leads to the recovery of N-cadherin expression and cadherin-mediated cell adhesion in E7-3Y1 cells. Taken together, our results demonstrate that E7 regulates cadherin-mediated cell adhesion through the modulation of cadherin expression via the MEK-ERK and AP-1 signaling pathway.

Cells avert the development of malignancy in response to deregulated oncogene expression by activating a regulated cell death pathway. However, the molecular mechanism underlying this oncogene-induced cellular death process remains unclear. Here, we show that retroviral expression of oncogenic H-ras induced cell death in a caspase-independent manner in normal cells. Inhibition of c-Jun NH2-terminal kinase (JNK) by pretreatment with SP600125 or a dominant-negative form of JNK blocked cell death. Rac1 and phosphoinositide 3-kinase (PI3K) were activated in cells overexpressing oncogenic H-ras. Inhibition of Rac1 with RacN17, a dominant-negative form of Rac1, attenuated oncogenic H-ras-induced JNK activation and subsequent cell death. Interestingly, inhibition of PI3K with LY294002 or by small interfering RNA-mediated knockdown of PI3K p85 or p110 subunits also clearly attenuated JNK activation and cell death. No cross talk was observed between Rac1 and PI3K, indicating that these pathways operate in parallel. Our findings show that JNK is necessary for oncogenic H-ras-induced, caspase-independent cell death, and that both PI3K and Rac1 activities are required for JNK activation and cell death. Determining the molecular mechanisms that mediate cell death responses to deregulated oncogenes provides a more refined understanding of cellular disposal processes in normal cells and increases our appreciation of these events as a mechanism for protecting against malignant progression.

A cancer/testis antigen, CAGE, is widely expressed in various cancer tissues and cancer cell lines but not in normal tissues except the testis. In the present study, ectopic expression of CAGE in fibroblast cells resulted in foci formation, suggesting its cell-transforming ability. Using stable HeLa transfectant clones with the tetracycline-inducible CAGE gene, we found that CAGE overexpression stimulated both anchorage-dependent and -independent cell growth in vitro and promoted tumor growth in a xenograft mouse model. Cell cycle analysis showed that CAGE augments the levels of cyclin D1 and E, thereby activating cyclin-associated cyclin-dependent kinases and subsequently accelerating the G(1) to S progression. Moreover, increased cyclin D1 and E levels in CAGE-overexpressing cells were observed even in a growth arrested state, indicating a direct effect of CAGE on G(1) cyclin expression. CAGE-induced expression of cyclins D1 and E was found to be mediated by AP-1 and E2F-1 transcription factors, and among the AP-1 members, c-Jun and JunD appeared to participate in CAGE-mediated up-regulation of cyclin D1. CAGE overexpression also enhanced retinoblastoma phosphorylation and subsequent E2F-1 nuclear translocation. In contrast, small interfering RNA-mediated knockdown of CAGE suppressed the expression of G(1) cyclins, activation of AP-1 and E2F-1, and cell proliferation in both HeLa cervical cancer cells and Malme-3M melanoma cells. These results suggest that the cancer/testis antigen CAGE possesses oncogenic potential and promotes cell cycle progression by inducing AP-1- and E2F-dependent expression of cyclins D1 and E.

OBJECTIVE: To investigate the cell apoptosis and expressions of c-fos and c-jun proteins induced by acrylamide (AA) in L-02 human liver embryo cell. METHODS: The cell survival rate, cell apoptosis and protein expressions of c-fos and c-jun were measured respectively after L-02 cell were exposed to different final concentration of AA (low: 0.01, 0.1 mmol/l; middle: 0.1, 0.5, 2.5 mmol/l; high: 5.0, 7.5 mmol/l) for 12 h and 24 h, respectively. RESULTS: The cell survival rate in the low dose AA-treated group was higher than that in the control group, but the high dose AA-treated groups demonstrated a significantly lower cell survival rate than the control group (P < 0.05). The percentages of apoptosis were significantly higher than that of the control group in the high dose AA-treated groups (P < 0.05). The expressions of c-fos and c-jun of AA-treated group were higher than that in the control group (P < 0.05), and the expressions increased first and then decreased with the AA exaltation; furthermore, the expressions also raised along with the exprosure time (P < 0.05). CONCLUSION: AA can induce the apoptosis of L-02 cell and high protein expressions of c-fos and c-jun proto-oncogenes.

Multiple tumorigenic pathways converge on the activating protein-1 (AP-1) family of dimeric transcription complexes by affecting transcription, mRNA decay, posttranslational modifications, as well as stability of its JUN and FOS components. Several mechanisms have been implicated in the phosphorylation- and ubiquitylation-dependent control of c-Jun protein stability. Although its dimer composition has a major role in the regulation of AP-1, little is known about the influence of heterodimerization partners on the half-life of c-Jun. The FOS family member Fra-1 is overexpressed in various tumors and cancer cell lines wherein it controls motility, invasiveness, cell survival and cell division. oncogene-induced accumulation of Fra-1 results from both increased transcription and phosphorylation-dependent stabilization of the protein. In this report, we describe a novel role of Fra-1 as a posttranslational regulator of c-Jun. By using both constitutively and inducible transformed rat thyroid cell lines, we found that c-Jun is stabilized in response to RAS oncoprotein expression. This stabilization requires the activity of the extracellular signal-related kinase (ERK) pathway, along with c-Jun heterodimerization with Fra-1. In particular, heterodimerization with Fra-1 inhibits c-Jun breakdown by a mechanism dependent on the phosphorylation of the Fra-1 C-terminal domain that positively controls the stability of the protein in response to ERK signaling. Therefore, Fra-1 modulates AP-1 dimer composition by promoting the accumulation of c-Jun in response to oncogenic RAS signaling.

The JNKs (c-Jun N-terminal kinases) are stress-activated serine/threonine kinases that can regulate both cell death and cell proliferation. We have developed a cell system to control JNK re-expression at physiological levels in JNK1/2-null MEFs (murine embryonic fibroblasts). JNK re-expression restored basal and stress-activated phosphorylation of the c-Jun transcription factor and attenuated cellular proliferation with increased cells in G1/S-phase of the cell cycle. To explore JNK actions to regulate cell proliferation, we evaluated a role for the cytosolic protein, STMN (stathmin)/Op18 (oncoprotein 18). STMN, up-regulated in a range of cancer types, plays a crucial role in the control of cell division through its regulation of microtubule dynamics of the mitotic spindle. In JNK1/2-null or c-Jun-null MEFs or cells treated with c-Jun siRNA (small interfering RNA), STMN levels were significantly increased. Furthermore, a requirement for JNK/cJun signalling was demonstrated by expression of wild-type c-Jun, but not a phosphorylation-defective c-Jun mutant, being sufficient to down-regulate STMN. Critically, shRNA (small hairpin RNA)-directed STMN down-regulation in JNK1/2-null MEFs attenuated proliferation. Thus JNK/c-Jun regulation of STMN levels provides a novel pathway in regulation of cell proliferation with important implications for understanding the actions of JNK as a physiological regulator of the cell cycle and tumour suppressor protein.

Type beta transforming growth factor (TGF-beta) is a multifunctional regulator of cell growth and differentiation. In the BC3H1 muscle cell line, TGF-beta blocks the onset of differentiation when added to undifferentiated myoblasts and causes dedifferentiation when added to fully differentiated myocytes. The goal of the present study was to determine whether TGF-beta-dependent repression of muscle-specific genes was preceded by modulation in expression of members of the jun proto-oncogene family, which function as growth factor-inducible transcription factors. junB mRNA was expressed at a basal level in differentiated BC3H1 myocytes. Within 15 min following exposure of myocytes to TGF-beta, junB mRNA began to accumulate; a peak of expression 20-fold above basal levels was observed after 2 h with a gradual decline thereafter. Nuclear run-on transcription assays showed that induction of junB by TGF-beta occurred at the level of transcription through a mechanism independent of protein synthesis. junB was also induced by 20% fetal bovine serum, platelet-derived growth factor, and insulin, but the maximal level of expression in response to these growth factors was lower and less sustained than in the presence of TGF-beta. In contrast to the dramatic effects of TGF-beta on junB expression, c-jun showed only a 2.5-fold increase in expression in response to TGF-beta. In an effort to identify additional members of the jun family which might be regulated by TGF-beta, a cDNA library was prepared from the poly(A)+ mRNA of TGF-beta-stimulated BC3H1 myocytes and was screened under conditions of reduced stringency with a v-jun DNA probe. From this screen, a new jun-related gene product was identified which shared a high degree of homology with regions of c-jun and junB which have been implicated in transcriptional activation, dimerization, and DNA binding. The transcript for this jun-related gene was expressed constitutively in BC3H1 cells and was not regulated by TGF-beta. Three members of the jun family thus exhibit distinct responses to TGF-beta in BC3H1 cells. The rapid transcriptional induction of junB is among the earliest and most dramatic responses to TGF-beta yet described and suggests that junB may mediate certain of the diverse biological effects of this growth factor.

The three members of the jun proto-oncogene family c-jun, jun b and jun D were mapped on the mouse chromosome by in situ hybridization. The c-jun locus is on chromosome 4 subregion C5----C7, whereas jun B and jun D are co-localized on chromosome 8 subregion C. RFLP analysis of interspecific hybrids confirmed the mapping of jun B and D and showed that they are situated about 7.3 +/- 3.5 cM apart. Thus despite their possible origin from a single ancestral gene they are not closely linked on the chromosome. Using the same probes, we showed that the human genome also contains sequences homologous to the mouse jun B and jun D. They are located on human chromosome 19 p13.2, a region that may be involved in chromosomal translocation in acute lymphocytic leukemia (ALL), acute nonlymphocytic leukemia (ANLL) and malignant melanoma (MEL). Finally, the present data identify a new segmental homology between mouse and human chromosomes.

The time course and signal-transduction requirements for proto-oncogene c-jun expression in T-cells were investigated. expression of c-jun mRNA was evident at 30 min after stimulation. Both the activation of Ca2+/phospholipid-dependent kinase as well as an increased intracellular free Ca2+ concentration were necessary for the maximal induction of c-jun mRNA and synthesis of Jun protein 1 h after stimulation.

The expression of the proto-oncogene c-jun in response to estradiol treatment in immature and mature rat uterine tissue was measured using a cDNA encoding the mouse c-jun proto-oncogene. This probe hybridized to a major RNA band of 2.7 kb and a minor 3.2 kb band. In Northern blots of total RNA from both immature and mature rat uteri, estradiol treatment resulted in at least a 3 fold increase in expression of the 2.7 kb band over control levels by 3 hr post injection. By 12 hr post injection, expression of c-jun mRNA had returned to control levels. A strong induction (greater than 5 fold) of c-jun mRNA expression was also observed in stroma-myometrial tissue isolated from mature rats approximately 3 hours after treatment with estradiol. The similar kinetics of induction of c-fos and c-jun emphasizes the functional significance of the fos/jun heterodimer in control of uterine cell proliferation.

In the TT cell line of human medullary thyroid carcinoma, the viral Harvey ras (v-rasH) oncogene induces differentiation, marked by morphological changes, diminution of growth, and increased expression of the calcitonin gene. Here, we show that the transcriptional factor c-jun is increased during v-rasH induced differentiation of TT cells both at the mRNA and functional protein levels. In contrast, nuclear proteins with binding activities related to AP2, AP3, NF1/CTF, and Sp1 were unchanged in v-rasH differentiated TT cells.

Angiotensin (Ang) II causes hypertrophy of rat aortic smooth muscle cells in culture and results in the rapid activation of c-fos. This study demonstrated that Ang II also activated c-jun and, in addition, could activate the AP-1 enhancer element. These data add support for a role of Ang II as an important mediator of vascular smooth muscle cell growth.

mutations in codon 61 of the Ha-ras proto-oncogene have been shown to occur with a high frequency in both spontaneous and carcinogen-induced liver tumours of the B6C3F1 mouse. In the present study we analysed the frequency and pattern of Ha-ras mutations in liver tumours in a different strain of mice, namely the CF1 mouse. These liver tumours occurred spontaneously or after administration of either aflatoxin B1, phenobarbital or dieldrin. mutation analysis was performed by in vitro amplification of DNA using the polymerase chain reaction combined with selective oligonucleotide hybridization. Our results demonstrate the presence of mutations in the Ha-ras gene in liver tumours of the CF1 strain. In total, 6 out of 35 liver tumours of male CF1 mice, two of them occurring after treatment with the tumour promoter phenobarbital solely, contained mutations at either the first or second base of codon 61 of the Ha-ras gene. The types of mutations found in liver tumours of the CF1 mouse were very similar to those described in the B6C3F1 mouse, indicating that mutations in the Ha-ras proto-oncogene may represent a critical event in mouse hepatocarcinogenesis.

The c-jun proto-oncogene codes for an important component of the AP-1 transcription factor complex which regulates the expression of many genes. This paper demonstrates that the steady state c-jun mRNA level in the avian oviduct is markedly decreased to 50% of control values within 30 minutes after estrogen injection into the animals. In the avian liver, the level is rapidly increased over 10 fold. Nuclear run-off transcription analysis demonstrates that the decrease in the c-jun mRNA levels in the avian oviduct occurs at least in part at the level of transcription. These changes are reproducible in repeat experiments. This response in the avian oviduct is unique since 1) it is the first demonstration of a steroid effect on c-jun expression in any animal system, 2) the changes in c-jun mRNA occur much more rapidly than most steroid responsive genes, and 3) it is the rare demonstration of an estrogen inhibition of the expression of a gene. A role for the c-jun proto-oncogene as an early regulatory gene in the cascade model for steroid action is proposed.

MicroRNAs (miRNAs) are potent negative regulators of gene expression involved in ALL aspects of cell biology. They finely modulate virtually ALL physiological pathways in metazoans, and are deeply implicated in ALL main pathologies, among which cancer. Mir-221 and miR-222, two closely related miRNAs encoded in cluster from a genomic region on chromosome X, are strongly upregulated in several forms of human tumours. In this work, we report that the ectopic modulation of NF-kB modifies miR-221/222 expression in prostate carcinoma and glioblastoma cell lines, where we had previously shown their oncogenic activity. We identify two separate distal regions upstream of miR-221/222 promoter which are bound by the NF-kB subunit p65 and drive efficient transcription in luciferase reporter assays; consistently, the site-directed mutagenesis disrupting p65 binding sites or the ectopical inhibition of NF-kB activity significantly reduce luciferase activity. In the most distal enhancer region, we also define a binding site for c-Jun, and we show that the binding of this factor cooperates with that of p65, fully accounting for the observed upregulation of miR-221/222. Thus our work uncovers an additional mechanism through which NF-kB and c-Jun, two transcription factors deeply involved in cancer onset and progression, contribute to oncogenesis, by inducing miR-221/222 transcription.

An activator protein-1 (AP-1) based bioassay-guided phytochemical investigation on Podocarpus latifolius led to the isolation of three new sempervirol-type diterpenes, cycloinumakiol (1), inumakal (2), and inumakoic acid (3), along with three known norditerpenes (4-6). Compounds 4 and 6 were responsible for the observed bioactivity.

Modulation of endogenous gene function, through sequence-specific recognition of double helical DNA via oligonucleotide-directed triplex formation, is a promising approach. Compared to the formation of pyrimidine motif triplexes, which require relatively low pH, purine motif appears to be the most gifted for their stability under physiological conditions. Our previous work has demonstrated formation of magnesium-ion dependent highly stable intermolecular triplexes using a purine third strand of varied lengths, at the purine*pyrimidine (Pu*Py) targets of SIV/HIV-2 (vpx) genes (Svinarchuk, F., Monnot, M., Merle, A., Malvy, C., and Fermandjian, S. (1995) Nucleic Acids Res. 23, 3831-3836). Herein, we show that a designed intramolecular version of the 11-bp core sequence of the said targets, which also constitutes an integral, short, and symmetrical segment (G(2)AG(5)AG(2))*(C(2)TC(5)TC(2)) of human c-jun protooncogene forms a stable triplex, even in the absence of magnesium. The sequence d-C(2)TC(5)TC(2)T(5)G(2)AG(5)AG(2)T(5)G(2)AG(5)AG(2) (I-Pu) folds back twice onto itself to form an intramolecular triple helix via a double hairpin formation. The design ensures that the orientation of the intact third strand is antiparallel with respect to the oligopurine strand of the duplex. The triple helix formation has been revealed by non-denaturating gel assays, UV-thermal denaturation, and circular dichroism (CD) spectroscopy. The monophasic melting curve, recorded in the presence of sodium, represented the dissociation of intramolecular triplex to single strand in one step; however, the addition of magnesium bestowed thermal stability to the triplex. Formation of intramolecular triple helix at neutral pH in sodium, with or without magnesium cations, was also confirmed by gel electrophoresis. The triplex, mediated by sodium alone, destabilizes in the presence of 5 -C(2)TC(5)TC(2)-3 , an oligonucleotide complementary to the 3 -oligopurine segments of I-Pu, whereas in the presence of magnesium the triplex remained impervious. CD spectra showed the signatures of triplex structure with A-like DNA conformation. We suggest that the possible formation of pH and magnesium-independent purine-motif triplexes at genomic Pu*Py sequences may be pertinent to gene regulation.

The Mos protein is a serine/threonine protein kinase that is likely to be a part of signal transduction pathways that regulate cell growth. We show here that expression of the v-Mos protein leads to a transient transcriptional activation of the c-fos and the c-jun proto-oncogenes in NIH 3T3 cells. Different cis-acting promoter elements are responsible for this effect. In the c-fos promoter the dyad symmetry element (DSE, also known as serum response element, SRE) is sufficient to confer responsiveness to the v-Mos protein. In the c-jun promoter the 12-0-tetradecanoyl-phorbol-13-acetate (TPA) response element (TRE) mediates this effect. Various Mos mutants with decreased transforming activity have diminished trans-acting activity on the c-fos and c-jun promoters. These results suggest that the highly transforming v-Mos protein exerts at least some of its effects through the induction of expression of the c-fos and c-jun protooncogenes.

Azatyrosine [L-beta-(5-hydroxy-2-pyridyl)alanine] is known to convert ras- or raf- transformed NIH3T3 cells to a normal phenotype. We report herein that azatyrosine also inhibits the growth of c-erbB-2-transformed cells and induces normal morphology. We attempted to identify the signal-transduction process triggered by oncogenic c-ErbB-2 that was inhibited by azatyrosine. Azatyrosine neither affected the phosphorylation of the introduced oncogenic c-ErbB-2 nor did it suppress activation of Ras induced by introduction of c-ErbB-2. Thus, azatyrosine did not inhibit the signal transduction from oncogenic c-ErbB-2 to Ras. However, azatyrosine inhibited increases in phosphorylation of c-Raf-1 and c-Jun induced by oncogenic c-ErbB-2. Furthermore, azatyrosine inhibited activation of the 12-O-tetradecanoylphorbol-13-acetate (TPA) response element in response to stimulation by oncogenic c-ErbB-2. These results suggest that azatyrosine acts downstream of Ras in signal transduction from oncogenic c-ErbB-2 to nuclear factors.

Primary lung cancer specimens of the non-small cell (NSC) types were examined for expression of the c-jun gene at the protein level by immunocytochemistry using the polyclonal antibody c-jun 890. The results obtained revealed that the c-jun p39 protein was expressed in 82% (18/22) of squamous cell carcinomas, 77% (10/15) of adenocarcinomas and 40% (2/5) of large cell anaplastic carcinomas, but no staining was seen in the non-cancer cases. Positive immunostaining was also seen in 16.7% (3/18) of the nonneoplastic bronchial cells of positively stained squamous cell carcinomas and in 40% (4/10) of adenocarcinomas. We suggest that elevated expression df c-jun p39 protein which was seen in a total of 75% (30/40) of the NSCLC, plays a role in lung cancer.

PEA3 is a transcription factor which binds to the polyoma virus enhancer and whose activity is regulated by the expression of a number of oncogenes. We show here that PEA3 also binds specifically to the collagenase and fos cellular promoters. On the collagenase promoter, PEA3 acts synergistically with AP-1 to achieve maximum levels of transcription activation by 12-O-tetradecanoylphorbol-13-acetate (TPA), and non-nuclear oncoproteins, thereby defining a TPA- and oncogene-responsive unit (TORU). From a comparative study of the collagenase TORU and the analogous polyoma virus TORU, we conclude that both the binding affinity of the PEA3 motif and the spacing between PEA3 and AP-1 modulate transcription activation induced by oncogene expression.

We present evidence that the glucocorticoid receptor (GR) and transcription factor Jun/AP-1 can reciprocally repress one another s transcriptional activation by a novel mechanism that is independent of DNA binding. Overexpression of c-Jun prevents the glucocorticoid-induced activation of genes carrying a functional glucocorticoid response element (GRE). Conversely, GR is able to repress AP-1-mediated transcriptional activation. Mutant analysis reveals that the ligand binding and DNA binding domains of GR and the region including the leucine zipper of c-Jun are required for repression. Gel retardation analysis demonstrates that bacterially expressed c-Jun disrupts GR-GRE complexes. These data indicate that members of two distinct classes of transcription factors can oppose one another s activity through a mechanism likely involving protein-protein interactions.

The adenovirus early region 1A (E1A) oncogene interferes with the expression level and activity of the AP-1 transcription factor family. E1A abolished the transactivating function of AP-1 (Jun/Fos), which binds to the 12-O-tetradecanoylphorbol-13-acetate-responsive element of the collagenase gene (collTRE). In contrast, the activity of another member of the AP-1 family that binds to the c-junTRE was not repressed. The mRNA expression of the c-jun gene was, in fact, strongly elevated in various cell types expressing the E1A gene of either adenovirus type 5 (Ad5) or Ad12. The regulation of the junB gene by adenovirus E1A, on the other hand, depended both on the cell type and on the transforming adenovirus serotype. The fact that E1A-induced alterations in the repertoire of AP-1 transcription factors depend on its transforming domain in conserved region 1 suggests that the effects are relevant for the transformation process.

A surface-enhanced Raman scattering (SERS)-based sensor was developed for the detection of the oncoprotein c-Jun at nanomolar levels. c-Jun is a member of the bZIP (basic zipper) family of dimeric transcriptional activators, and its overexpression has been associated with carcinogenic mechanisms in several human cancers. For our sensing purpose, we exploited the ability of c-Jun to heterodimerize with its native protein partner, c-Fos, and therefore designed a c-Fos peptide receptor chemically modified to incorporate a thiophenol (TP) group at the N-terminal site. The TP functionality anchors the c-Fos protein onto the metal substrate and works as an effective SERS probe to sense the structural rearrangements associated with the c-Fos/c-Jun heterodimerization.

The transcription factor activator protein-1 (AP-1) participates in many aspects of cell physiology, such as cellular proliferation, transformation, and death. AP-1 is a dimeric complex that primarily contains Jun and Fos family members. Here, we report that JUN is a target of heat shock transcription factor HSF1. HSF1 is the master regulator of genes encoding molecular chaperones, and is involved in cellular processes such as the stress response, cell differentiation, aging and carcinogenesis. In HeLa cells, JUN transcription was rapidly induced by heat treatment. We found that HSF1 bound to the JUN promoter and was necessary for its efficient response to heat shock. In heat-shocked cells, c-Jun-mediated gene expression was induced slowly following accumulation of c-Jun protein. Forced expression of active HSF1 in cells resulted in an increase in c-Jun expression and activation of c-Jun target genes. These results show that HSF1 regulates JUN expression, thereby modulating AP-1 activity.

The response of a cell to mitogens and differentiation agents involves the transcriptional induction of several cellular genes. Prominent among these so-called immediate early or competence genes are the nuclear oncogenes fos and myc. Although the precise function of these early response genes in growth control is not understood, it is likely that many of them are involved in the transition from G0 to G1 in the cell cycle. The findings that the products of nuclear proto-oncogenes jun and erbA are transcriptional factors supports the notion of the role of the nuclear oncoproteins in the regulation of gene expression. Recently, it has been reported that the FOS protein is associated in transcriptional complexes with the product of the jun oncogene, the transcription factor AP-1. As the fos gene is induced in response to mitogens during initiation of cell growth, we investigated whether expression of the nuclear transcription factor AP-1 is also inducible. We report that mouse c-jun gene transcription is rapidly induced by serum and phorbol-ester 12-o-tetradecanoyl phorbol 13-acetate (TPA). Furthermore, induction is transient and the mRNA is superinduced by inhibitors of protein synthesis.

The promoter region of a replication-dependent histone H3.2 gene contains a putative DNA binding site for the Jun oncoprotein within a 32-nucleotide regulatory domain. The hamster sequence differs by one nucleotide from the AP-1 consensus sequence found in several promoters responsive to phorbol 12-myristate 13-acetate. We have identified the factors interacting with this region as 42- and 45-kDa proteins by DNA affinity chromatography, immunoblotting, and UV crosslinking. These proteins, which are candidates for conferring high-level expression to the histone promoter, share an antigenic epitope with the DNA-binding domain of Jun but diverge from it at the amino terminus. The interaction of these proteins with the promoter of a replication-dependent cellular gene provides evidence that members of the Jun oncoprotein family may play specific roles in the expression of genes essential for progression of the cell cycle.

The products of the proto-oncogenes c-jun and c-fos are known to form a complex in vivo. Complex formation appears to stabilize protein-DNA interactions and is thought to play an important functional role in transcriptional regulation. Here we show that the viral Jun oncoprotein, which differs structurally from cellular Jun, is also capable of complex formation with Fos. Thus the oncogenic potency of viral Jun is unlikely to be due to an altered affinity for Fos. We have also defined, by deletion analysis, the domain of v-Jun responsible for complex formation to reside in the carboxy terminus encompassing the leucine zipper motif. We find that complex formation with c-Fos does not occur with v-Jun deletions affecting one or more leucine residues in the zipper domain. Our results are consistent with the hypothesis that the leucine zipper mediates Jun-Fos interaction.

We have studied the effects of c-Ha-ras oncogene in mouse NIH 3T3 fibroblasts by DNA transfection and analysis of gene expression at the mRNA and protein level in a heat- and heavy metal-inducible model system. The human c-Ha-ras proto-oncogene and oncogene were cloned under the hsp70 heat-shock promoter. Clonal lines of cells with negligible basal expression of the hsp-c-Ha-ras oncogene construct were chosen on the basis of the inducibility of p21c-Ha-ras protein and several transformation parameters. We demonstrate that the expression of ornithine decarboxylase (ODC) mRNA is enhanced approximately 4-6 h after the induction of the p21c-Ha-ras oncoprotein. This increase was reversible upon cessation of c-Ha-ras mRNA and protein synthesis, while constitutively elevated ODC was characteristic for stably c-Ha-ras-transformed cells. The high-level expression of ODC in ras-transformed cells was insensitive to tumour promoter stimulation. A similar mRNA induction by c-Ha-rasVal-12 was also observed for two other serum- and tumour promoter-regulated genes associated with the transformed phenotype: transin (stromelysin) and the glucose transporter. This prompted us to examine also potential changes in the expression of the serum- and tumour promoter-induced transcription factor genes junB and c-jun after induction of the hsp--c-Ha-ras construct. The junB mRNA was enhanced approximately 10-fold and the c-jun oncogene mRNA to a lesser degree in the hsp--c-Ha-ras-transfected cells after zinc activation of the hsp70 promoter. These effects were not seen in similarly treated control cells.(ABSTRACT TRUNCATED AT 250 WORDS)FAU - Sistonen, L

expression of the pS2 gene which is transcriptionally controlled by oestrogens in the breast cancer cell line MCF-7 is oestrogen independent in stomach mucosa. We show here that the level of MCF-7 cell pS2 mRNA can also be increased by the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). We further demonstrate, using transient transfection assays, that the -428 to -332 5 flanking sequence of the pS2 gene contains DNA enhancer elements responsive to oestrogens, TPA, EGF, the c-Ha-ras oncoprotein and the c-jun protein.

Human transforming growth factor-alpha (TGF-alpha, MW 5547) initiates a mitogenic program in "quiescent" 11-to 13-day-old primary cultures of adult rat hepatocytes. Using validated growth reinitiation assays and chemically defined conditions (Koch and Leffert, 1979a) that simulate proto-oncogene expression in regenerating liver (Kruijer et al., 1986), we find that 5.4 nM TGF-alpha stimulates: (i) increases in rates of amiloride-sensitive 22Na+ uptake; (ii) a transient induction in steady-state mRNA levels of proto-oncogene c-jun; (iii) specific increases in hepatocyte nuclear [3H]dT labeling indices, augmented synergistically by insulin and glucagon; and (iv) increases in rates of S-phase entry. Comparative studies indicate that TGF-alpha is a more effective hepatocyte growth promoter than mouse epidermal growth factor. These observations, and published reports linking normal and cancerous liver as biosynthetic sources of TGF-alpha, suggest an autocrine or paracrine role for TGF-alpha in the control of hepatic growth, regeneration, and gene expression.

A structural motif for DNA-binding proteins, the leucine zipper , has been proposed for the jun, fos and myc gene products, the yeast transcriptional activator GCN4, and the C/EBP enhancer-binding protein. These proteins ALL contain a region with four or five leucine residues spaced exactly seven amino acid residues apart whose sequence is consistent with the formation of an amphipathic alpha-helix. It has been proposed that the leucine zipper consists of two interdigitated alpha-helices, one from each monomer, that constitute the dimerization function necessary for high-affinity binding to DNA; an adjacent region of basic residues is thought to be responsible for specific protein-DNA contacts. In support of this model, substitution of the leucine residues within the motif can abolish dimerization and DNA-binding, and a synthetic peptide corresponding to the GCN4 leucine zipper forms alpha-helical dimers. Despite the conserved leucine residues, however, each protein has a distinct dimerization specificity. Specifically, GCN4 homodimer, Jun homodimer and Fos-Jun heterodimer proteins bind to the same DNA site, whereas Fos is unable to form homodimers, bind DNA, or interact with GCN4 (refs 8-14). Here, we alter the dimerization specificity of Fos by precisely replacing its leucine zipper with that from GCN4. This Fos-GCN4 chimaeric protein is able to bind to the target site in the absence of Jun, and can form DNA-binding heterodimers with GCN4 but not with Jun. These results indicate that the leucine zipper is sufficient to confer dimerization specificity and strongly suggest that Fos contacts DNA directly.

tumor necrosis factor alpha (TNF) mediates its in vivo antineoplastic effect primarily through its action on the endothelial surfaces of tumor vessels. Among other changes, endothelial cells increase the expression of surface procoagulant molecules which allow for the genesis of microthrombi and the eventual anoxic killing of the tumor tissue. TNF is also a potent antimitotic factor for endothelial cells. We investigated the molecular basis for these diverse actions of TNF by differential screening of a cDNA library prepared from TNF and cycloheximide-treated human umbilical vein endothelial (HUVE) cells. One of the cDNA clones identified encodes the transcription factor and proto-oncogene AP-1/c-jun. The expression of AP-1/c-jun following TNF treatment is mediated largely at the transcriptional level. Neither c-myc nor c-fos transcripts were induced by TNF. Since AP-1/c-jun has been previously identified as one of the earliest transcripts expressed in quiescent cells that have been stimulated by growth factors, our results suggest that mitogenic and antimitogenic stimuli evoke distinct yet overlapping sets of molecular responses and that the coordinate expression of AP-1/c-jun and c-fos is not mandatory. AP-1/c-jun transcript induction is thus one of the initial events mediated by TNF treatment of HUVE cells.

NANOG plays important roles in neoplastic processes. However, the molecular mechanism of NANOG in tumorigenesis remains to be elucidated. In this report, we demonstrated that forced expression of NANOG in 293 cells and cancer cells led to increased c-Jun expression, whereas downregulation of endogenous NANOG significantly reduced c-Jun expression in cancer cells. Dual luciferase reporter assays demonstrated that NANOG binds the c-Jun proximal promoter and transactivates the c-Jun gene. An ATTA consensus motif between the -160 and -268 region of the c-Jun promoter was identified as the NANOG-responsive element. Electromobility shift assay and chromatin immunoprecipitation results confirmed the direct binding of NANOG protein to the c-Jun promoter in vitro and in vivo. NANOG directly bound c-Jun protein as shown by GST pulldown and immunoprecipitation assays. Taking these findings together, we conclude that c-Jun is a direct target gene of NANOG and that c-Jun protein may be a novel co-activator of NANOG in cancer cells. We suggest the possibility that NANOG may play a significant role in carcinogenesis via its activation of c-Jun expression.

In the world, hepatocellular carcinoma (HCC) is one of the most common and most lethal cancers. Currently, standard therapy for unresectable HCC is a local-regional therapy with transarterial chemoembolisation (TACE). In this study, we sought to assess whether plasma circulating microRNAs (miRNAs) can be used to predict the prognosis of HCC patients receiving the TACE treatment. Firstly, we systematically examined TACE therapeutic effectiveness-related circulating miRNAs through miRNA Profiling Chips. As a result, we identified 19 circulating miRNAs to be significantly differentially expressed between the TACE-response group and the TACE-nonresponse group. In the second stage, we performed quantitative analyses of these candidate miRNAs in additional HCC patients treated with TACE and validated two of the aforementioned 19 miRNAs (miR-1285-3p and miR-4741) as candidate biomarkers for predicting prognosis of TACE. Interestingly, we found that miR-1285-3p could directly repress JUN oncogene expression in HCC cells, indicating miR-1285-3p could act as a potential tumor suppressor. In conclusion, our data indicate that circulating miR-1285-3p and miR-4741 was predictive of response to TACE therapy in HCC.

Nerve growth factor (NGF) induces the rapid but transient expression of two transcripts with homology to the proto-oncogene c-jun (AP-1) in PC12 cells. The c-jun transcripts increase within 15 min after NGF addition, peak at 1 h, and decline to basal level by 4 h. Actinomycin D inhibits the increase, and cycloheximide prevents the decrease of c-jun mRNA. Cyclic AMP induces a similar transient rise in c-jun transcripts in PC12 cells. The human melanoma cell line A875 that also expresses the NGF receptor expresses c-jun RNA constitutively, and the level of c-jun RNA is not affected significantly by NGF. NGF does not alter the RNA level of transcription factor SP-1 in PC12 cells. The results suggest that activation of the putative transcription factor c-jun is one of the early cellular responses of PC12 cells to NGF and that c-jun mRNA expression is dependent on cellular transcription and regulated by stabilization of c-jun mRNA.

We describe a ds-oligonucleotide-peptide conjugate that is able to efficiently dismount preformed DNA complexes of the bZIP regions of oncoproteins c-Fos and c-Jun (AP-1), and therefore might be useful as disrupters of AP-1-mediated gene expression pathways.

The proto-oncogene c-Jun plays crucial roles in tumorigenesis, and its aberrant expression has been implicated in many cancers. Previous studies have shown that the c-Jun gene is positively autoregulated by its product. Notably, it has also been reported that c-Jun proteins are enriched in its gene body region. However, the role of c-Jun proteins in its gene body region has yet to be uncovered. HP1a is an evolutionarily conserved heterochromatin-associated protein, which plays an essential role in heterochromatin-mediated gene silencing. Interestingly, accumulating evidence shows that HP1a is also localized to euchromatic regions to positively regulate gene transcription. However, the underlying mechanism has not been defined. In this study, we demonstrate that HP1a is involved in the positive autoregulatory loop of the Jra gene, the c-Jun homolog in Drosophila. Jra recruits the HP1a/KDM4A complex to its gene body region upon osmotic stress to reduce H3K36 methylation levels and disrupt H3K36 methylation-dependent histone deacetylation, resulting in high levels of histone acetylation in the Jra gene body region, thus promoting gene transcription. These results not only expand our knowledge toward the mechanism of c-Jun regulation, but also reveal the mechanism by which HP1a exerts its positive regulatory function in gene expression.

YAP/TAZ are nuclear effectors of the Hippo pathway regulating organ growth and tumorigenesis. Yet, their function as transcriptional regulators remains underinvestigated. By ChIP-seq analyses in breast cancer cells, we discovered that the YAP/TAZ transcriptional response is pervasively mediated by a dual element: TEAD factors, through which YAP/TAZ bind to DNA, co-occupying chromatin with activator protein-1 (AP-1, dimer of JUN and FOS proteins) at composite cis-regulatory elements harbouring both TEAD and AP-1 motifs. YAP/TAZ/TEAD and AP-1 form a complex that synergistically activates target genes directly involved in the control of S-phase entry and mitosis. This control occurs almost exclusively from distal enhancers that contact target promoters through chromatin looping. YAP/TAZ-induced oncogenic growth is strongly enhanced by gain of AP-1 and severely blunted by its loss. Conversely, AP-1-promoted skin tumorigenesis is prevented in YAP/TAZ conditional knockout mice. This work highlights a new layer of signalling integration, feeding on YAP/TAZ function at the chromatin level.

The membrane-bound mucinMUC4 is a high molecularweight glycoprotein frequently deregulated in cancer. In pancreatic cancer, one of the most deadly cancers in occidental countries, MUC4 is neo-expressed in the preneoplastic stages and thereafter is involved in cancer cell properties leading to cancer progression and chemoresistance. K-ras oncogene is a small GTPase of the RAS superfamily, highly implicated in cancer. K-ras mutations are considered as an initiating event of pancreatic carcinogenesis and K-ras oncogenic activities are necessary components of cancer progression. However, K-ras remains clinically undruggable. Targeting early downstream K-ras signaling in cancer may thus appear as an interesting strategy and MUC4 regulation by K-ras in pancreatic carcinogenesis remains unknown. Using the Pdx1-Cre; LStopL-K-rasG12D mouse model of pancreatic carcinogenesis, we show that the in vivo early neo-expression of the mucin Muc4 in pancreatic intraepithelial neoplastic lesions (PanINs) induced by mutated K-ras is correlated with the activation of ERK, JNK and NF-kappaB signaling pathways. In vitro, transfection of constitutively activated K-rasG12V in pancreatic cancer cells led to the transcriptional upregulation of MUC4. This activation was found to be mediated at the transcriptional level by AP-1 and NF-kappaB transcription factors via MAPK, JNK and NF-kappaB pathways and at the posttranscriptional level by a mechanism involving the RalB GTPase. Altogether, these results identify MUC4 as a transcriptional and post-transcriptional target of K-ras in pancreatic cancer. This opens avenues in developing new approaches to target the early steps of this deadly cancer.

We have employed a short term transfection assay to examine the response of HIV-1 LTR to transformation by the human normal and mutant T24 H-ras1 genes. The plasmid pBC12HIVCAT which carries the HIV-1 LTR sequences linked to the reporter gene chloramphenicol acetyl transferase (CAT) was transfected into rat 208F fibroblasts and their derivatives RFHO6N1-1 and RFHO6T1-1 transfectants. RFHO6N1-1 and RFHO6T1-1 express an exogenous human normal or mutant T24 H-ras1 gene respectively. expression of the mutant T24 but not the normal H-ras1 gene resulted in increased levels of HIV-1 LTR driven CAT activity. We have noted four motifs in the HIV-1 LTR region which resemble TPA-inducible and AP-1 binding consensus sequences. Since H-ras1 fos and jun/AP-1 respond to TPA and T24 H-ras1 is known to induce both fos and jun/AP-1 nuclear transcriptional factors, it is possible that the latter genes play a role in HIV-1 transcription. Moreover, H-ras1 oncogene activation may play an important role in HIV gene expression and in the activation of latent HIV.

Nuclear oncogene products have the potential to induce alterations in gene regulation leading to the genesis of cancer. The biochemical mechanisms by which nuclear oncoproteins act remain unknown. Recently, an oncogene, v-jun, was found to share homology with the DNA binding domain of a yeast transcription factor, GCN4. Furthermore, GCN4 and the phorbol ester-inducible enhancer binding protein, AP-1, recognize very similar DNA sequences. The human proto-oncogene c-jun has now been isolated, and the deduced amino acid sequence indicates more than 80 percent identity with v-jun. expression of cloned c-jun in bacteria produced a protein with sequence-specific DNA binding properties identical to AP-1. Antibodies raised against two distinct peptides derived from v-jun reacted specifically with human AP-1. In addition, partial amino acid sequence of purified AP-1 revealed tryptic peptides in common with the c-jun protein. The structural and functional similarities between the c-jun product and the enhancer binding protein suggest that AP-1 may be encoded by c-jun. These findings demonstrate that the proto-oncogene product of c-jun interacts directly with specific target DNA sequences to regulate gene expression, and therefore it may now be possible to identify genes under the control of c-jun that affect cell growth and neoplasia.

expression of the gene cpc-1 is required for cross-pathway-mediated regulation of amino acid-biosynthetic genes in Neurospora crassa. We have cloned cpc-1 and present an analysis of its structure and regulation. The cpc-1-encoded transcript contains three open reading frames, two of which are located in the 720-nucleotide leader segment preceding the cpc-1 coding region. The two leader open reading frames, if translated, would produce peptides 20 and 41 residues in length. The deduced amino acid sequence of the cpc-1 polypeptide, CPC1, contains segments similar to the DNA-binding and transcriptional activation domains of GCN4, the major cross-pathway regulatory protein of yeast. The structural and functional similarities of CPC1 and GCN4 proteins suggest that cpc-1 encodes the analogous transcriptional activator of N. crassa. Messenger RNA measurements indicate that cpc-1 is transcriptionally regulated in response to amino acid starvation. The segment of CPC1 similar to the DNA-binding domain of GCN4 also is similar to the DNA-binding domains of the avian sarcoma virus oncogene-encoded v-JUN protein and human c-JUN protein.

Biologically active molecular clones of avian sarcoma virus 17 (ASV 17) contain a replication-defective proviral genome of 3.5 kilobases (kb). The genome retains partial gag and env sequences, which flank a cell-derived putative oncogene of 0.93 kb, termed jun. The jun gene lacks preserved coding domains of tyrosine-specific protein kinases. It also shows no significant nucleic acid homology with other known oncogenes. The probable transformation-specific protein in ASV 17-transformed cells is a 55-kDa gag-jun fusion product.

The jun oncoprotein, which causes sarcomas in chickens, and the DNA-binding domain of yeast GCN4, which coordinately regulates the expression of amino acid biosynthetic genes, show significant homology. In yeast cells deleted for the GCN4 gene, GCN4 function can be conferred by a hybrid protein in which the GCN4 DNA-binding domain is replaced by the homologous region of jun. Moreover, in strains containing various mutations of the GCN4 binding site in the HIS3 promoter, HIS3 expression is affected similarly by the hybrid protein and by GCN4. These results indicate that the jun oncoprotein binds the same DNA sequences as GCN4, and strongly suggest that jun is derived from a normal cellular transcription factor (possibly AP-1, which recognizes similar sequences). This provides direct evidence for the idea that alterations in the machinery for proper gene expression can lead to the oncogenic state.

Transcriptional activation of RNA polymerase II in eukaryotic organisms ranging from yeasts to mammals has many common features such as enhancer elements, TATA elements, and activator proteins that bind specifically to promoter DNA. The JUN oncoprotein, which causes sarcomas in chickens, shows significant homology to the DNA-binding domain of GCN4, a yeast protein that stimulates transcription of the amino acid biosynthetic genes. The GCN4 and JUN proteins bind the same DNA sequences, consensus ATGA(C/G)TCAT, even though the DNA-binding domains are only 45% identical in amino acid sequence. The JUN protein almost certainly represents the oncogenic version of the normal AP-1 transcription factor, suggesting an evolutionary relationship between yeast and vertebrate activator proteins. Here, I demonstrate that JUN efficiently activates transcription in yeast either through its own or a heterologous DNA-binding domain. As is the case for yeast activator proteins, transcriptional stimulation by JUN requires an acidic activation region distinct from the DNA-binding domain. The functional interchangeability between yeast and vertebrate transcription factors strongly suggests a basic similarity in the molecular mechanism of eukaryotic transcriptional activation.

The Fos protein complex and several Fos-related antigens (FRA) bind specifically to a sequence element referred to as the HeLa cell activator protein 1 (AP-1) binding site. A combination of structural and immunological comparisons has identified the Fos-associated protein (p39) as the protein product of the jun proto-oncogene (c-Jun). The p39/Jun protein is one of the major polypeptides identified in AP-1 oligonucleotide affinity chromatography extracts of cellular proteins. These preparations of AP-1 also contain Fos and several FRA s. Some of these proteins bind to the AP-1 site directly whereas others, like Fos, appear to bind indirectly via protein-protein interactions. Cell-surface stimulation results in an increase in c-fos and c-jun products. Thus, the products of two protooncogenes (and several related proteins), induced by extracellular stimuli, form a complex that associates with transcriptional control elements containing AP-1 sites, thereby potentially mediating the long-term responses to signals that regulate growth control and development.

Some growth factor-induced genes, such as the c-fos gene, are activated rapidly and transiently without intervening protein synthesis. Others, like the rat transin gene, are activated more slowly but more durably and their activation requires prior protein synthesis. It is tempting to speculate that certain rapidly-activated genes code for transcription factors which interact directly with promoter regions of genes like the transin gene to trigger their expression. Unfortunately, little is known about the majority of primary response genes to support this hypothesis. The proto-oncogene c-jun codes for the transcription factor AP-1 or a closely related protein. We show that epidermal growth factor stimulates transcription of the c-jun gene in fibroblasts as a primary response. This supports the notion that increased expression of genes encoding transcription factors is an important element of the signal transduction mechanism, assuring the long-term transcriptional response of cells to growth factors.

Binding of the human transcription factor Jun/AP-1 to a conserved 8 bp nucleotide sequence (TRE) is responsible for increased transcription of different cellular genes in response to tumor promoters, such as TPA, and serum factors. Enhanced Jun/AP-1 activity in TPA-stimulated cells is regulated by two different mechanisms: a posttranslational event acting on pre-existing Jun/AP-1 molecules, and transcriptional activation of jun gene expression leading to an increase in the total amount of Jun/AP-1. Induction of jun transcription in response to TPA is mediated by binding of Jun/AP-1 to a high-affinity AP-1 binding site in the jun promoter region. Site-specific mutagenesis of this binding site prevents TPA induction and trans-activation by Jun/AP-1. These results clearly demonstrate that jun transcription is directly stimulated by its own gene product. This positive regulatory loop is likely to be responsible for prolonging the transient signals generated by activation of protein kinase C.

The requirements for activation of the transformation potential of the human c-fos proto-oncogene were investigated. Recombinant plasmids containing the Moloney murine leukemia virus long terminal repeat directing transcription of the c-fos coding region and either the authentic c-fos 3 untranslated region (UTR) or the 3 UTR from human c-myc were inefficient at inducing transformation. In contrast, a recombinant that substituted most of the c-fos 3 UTR with the 3 portion of the simian virus 40 T-antigen gene transformed cells well. This difference in transformation efficiency appeared to be due to significantly higher levels of fos mRNA and protein expressed from the transforming recombinant. This, in turn, was due to the much greater stability of its mRNA compared with those from the poorly transforming recombinants containing the c-fos or c-myc 3 UTR. Thus, the 3 UTR of the human c-fos mRNA is responsible for its rapid degradation and limits the steady-state levels of transcript and protein. Cells transformed by the activated human c-fos plasmids contained increased amounts of partially modified c-fos protein (c-Fos). This form of c-Fos turned over much more rapidly than the highly modified form of c-Fos induced by serum stimulation.

Proto-oncogenes encode proteins with three main sites of action: the cell-surface membrane, the cytoplasm and the nucleus. Although the exact biochemical function of most proto-oncogene products is not understood, several of them are known to be involved in signal transduction. A role in gene regulation through DNA binding has been suggested for a recently isolated member of the group of oncogenes acting at the nucleus, v-jun. The C-terminus of the putative v-jun-encoded protein is similar in sequence to the C-terminus of the yeast transcriptional activator GCN4 (refs 8, 9), which forms its minimal DNA-binding domain. GCN4 binds to specific sites whose consensus sequence is highly similar to the recognition sequence of the mammalian transcriptional activator AP-1 (refs 12, 13). Like GCN4, AP-1 binds to promoter elements of specific genes and activates their transcription. Because of the similarity between the recognition sites for GCN4 and AP-1, we examined the possibility that AP-1 could be the product of the c-jun proto-oncogene. The experimental results reported here indicate that the JUN oncoprotein is a sequence-specific transcriptional activator similar to AP-1.

We have recently identified by cDNA cloning a set of genes that are rapidly activated in cultured mouse cells by protein growth factors. Here we report that the nucleotide sequence of a cDNA (clone 465) derived from one of these immediate early genes (hereafter called jun-B) encodes a protein homologous to that encoded by the avian sarcoma virus 17 oncogene v-jun. Homology between the jun-B and v-jun proteins is in two regions: one near the N terminus and the other at the C terminus. The latter sequence was shown by Vogt et al. [Vogt, P. K., Bos, T. J. & Doolittle, R. F. (1987) Proc. Natl. Acad. Sci. USA 84, 3316-3319] to have regions of sequence similarity to the DNA-binding domain of the yeast transcriptional regulatory protein GCN4 and to the oncogenic protein fos. Southern blots of human, mouse, and chicken DNA demonstrate that jun-B and c-jun are different genes and that there may be other vertebrate genes related to jun-B and c-jun. These findings suggest that there is a jun family of genes encoding related transcriptional regulatory proteins. The jun-B protein, and perhaps other members of the jun family, may play a role in regulating the genomic response to growth factors.

The effects of a phorbol ester (TPA) and of members of the Jun and Fos oncoprotein family on the activity of the rat alpha-fetoprotein (AFP) promoter were checked by using transient expression experiments in HepG2 hepatoma cells. TPA blocked the activity of the rat AFP promoter in a dose-dependent manner. Overexpression of c-Jun specifically repressed the rat AFP promoter but not the albumin promoter. JunB and JunD were poorer inhibitors. c-Fos expression did not potentiate the negative effect of Jun. The Jun-induced repression does not require binding of c-Jun to the AFP promoter. DNase 1 footprinting experiments did not display any high affinity binding site for Jun on the AFP promoter. Integrity of the c-Jun DNA binding domain is not required for the c-Jun protein to block the AFP promoter. The N-terminal part of Jun, which contains the activating domain, is responsible for the repression as shown by using Jun-Gal4 chimera. Jun likely exerts its negative control on the AFP promoter via protein-protein interactions with a not yet identified trans-activating factor within the -134 to +6 region or with a component of the general machinery of transcription. Jun proteins can thus be key intermediates in regulatory cascades which result in the differential modulation of the AFP and albumin gene expression in the course of liver development and carcinogenesis.

The E5a gene of human papillomavirus type 11 (HPV-11) is a transforming oncogene. In this study, we investigated the mechanism of E5a induced transformation. Our results show that the expression of c-jun and junB, but not junD, was activated by HPV-11 E5a in NIH 3T3 cells and human epidermal keratinocytes. However, the expression of c-fos was activated by E5a in NIH 3T3 cells, but not in keratinocytes. We further investigated the mechanism of c-jun and junB induction by E5a. The amount of c-jun and junB RNAs correlated with the amount of E5a RNA in the heavy metal inducible system. E5a constitutively activated the expression of c-jun and junB at the initiation of transcription level. In addition, analyses of the effect of serum on c-jun expression in E5a transformed human epidermal keratinocytes show that EGF might have a stimulatory effect on c-jun gene expression in E5a expressing keratinocytes.

The structural properties of the basic subdomain of the basic zipper (bZIP) protein c-Jun were examined by joint means of 1H-NMR, CD and Fourier-transform infrared (FTIR) spectroscopies. The basic subdomain (residues 252-281 in c-Jun) is responsible for sequence-specific recognition of DNA. A modified basic subdomain bSD (residues 1-35) and its N-terminal part and C-terminal part fragments (NP, residues 1-19; and, CP, residues 16-35) were prepared by solid-phase synthesis and purified by HPLC. In aqueous solution, in the absence of DNA, bSD behaved mostly as an unstructured peptide characterized by only 5% alpha helix. However, upon mixing bSD and a specific DNA fragment, i.e. a CRE(cAMP-responsive element)-containing hexadecanucleotide, the alpha helix was stabilized to an extent of 20% at 20 degrees C or 35% at 2 degrees C. At the same time, no significant change could be detected in the DNA spectra. Addition of trifluoroethanol to an aqueous bSD sample resulted in an increase of the alpha-helix content so that about 60% of alpha helix was found at a ratio of 75% trifluoroethanol (20 degrees C). These effects were reflected in both CD and FTIR measurements. Changes shown by the CD spectra during the process suggested a mechanism dominated by a two-state helix/unordered transition. NMR data, namely alpha H chemical shifts, NOE cross-peaks and NH temperature coefficients provided indications for extended or nascent helix structures within four short stretches dispersed along the sequence for c-Jun bSD, contrasting with the unique and continuous stretch reported for Gcn4 (yeast general control protein 4) bSD in aqueous solution. Trifluoroethanol stabilized the alpha-helix structure mainly at these four sites. The malleability of the basic subdomain of c-Jun was emphasized in relation to its ability to fit the DNA helix in adopting an alpha-helix structure. The complex formation apparently requires substantial conformational change from the peptide and only little from the oligonucleotide.

The proteins encoded by early region 1 A (E1A) of human adenoviruses (Ad) modulate the expression of both adenovirus genes and various host cell genes. With these transcription-regulating properties the E1A proteins redirect the cell s metabolism, which enables them to induce oncogenic transformation in rodent cells. The E1A proteins modulate transcription by interacting both with gene-specific and general cellular transcription factors. Various members of the AP-1 and ATF/CREB families of transcription factors are targets for E1A-dependent regulation, including cJun, the protein product of the c-jun proto-oncogene. The E1A proteins modulate cJun-dependent transcription both positively and negatively, and affect the activity as well as the expression levels of cJun. By increasing the phosphorylation status of cJun, E1A can stimulate transcription regulated by cJun/ATF2 heterodimers. In contrast, E1A inhibits the expression of various metalloproteases by interfering with the DNA-binding capacity of cJun/cJun and cJun/cFos dimers, which might involve the association of E1A with the putative transcriptional coactivator p300. Since the ability of E1A to alter cJun-dependent transcription correlates with its transforming capacity, interference with cJun-dependent transcription may be an essential step in E1A-induced transformation.

E5a of HPV-11 is a transforming oncogene. Previously, we have shown that E5a constitutively activates the expression of protooncogene c-jun by transcriptional regulation through the AP-1 binding site in the c-jun promoter. In the present study, we used two different types of cells: the E5a transfected NIH 3T3 cells and human epidermal keratinocytes, and selectively inhibited different signal transduction pathways to investigate effects of E5a on c-jun expression. We find that protein kinase C and ras-dependent pathways are important for the c-jun induction by E5a, but not the cAMP-dependent pathway.

Paraquat (methyl viologen, PQ) is a widely used herbicide that produces oxygen-derived free radicals and severely injures human lungs. In this study we examined the effects of PQ on the protein kinase C (PKC), ornithine decarboxylase (ODC) and c-jun oncogene expression in WI-38 human lung cells. Exposure of cells to 25-200 microM PQ resulted in an increase of [3H]phorbol dibutyrate (PDBu) binding and PKC redistribution in a dose-dependent manner. Interestingly, a superoxide dismutase mimic, 4-hydroxyl-2,2,6,6-tetramethyl-piperidine-1-oxyl (Tempol, 2.5 mM) and catalase (400 micrograms/ml) could significantly reduce the PQ-stimulated increase of phorbol ester binding and particular PKC phosphorylating activity, but dimethylsulfoxide (DMSO, 1.5%), an effective .OH trapping agent, failed to prevent this stimulation. In addition, an endogenous substrate of PKC, 80 kDa protein, was found to be highly phosphorylated in intact WI-38 cells treated with 50 microM PQ. The increase of phosphorylated proteins could be completely or partly abolished by Tempol or catalase, but only the phosphorylation of 80 kDa protein was diminished by protein kinase C inhibitor, 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7). A maximal peak of ODC activity was observed at 6 h of treatment with 50 microM PQ. PQ induced activity was reduced at the following rates, Tempol 85%, DMSO 80% and catalase 45%, but H-7 failed to do so. Furthermore, we found that the level of c-jun mRNA was transiently increased by PQ and the peak appeared at 1 h of treatment. When correlated with the PKC result, Tempol, catalase and H-7 ALL effectively blocked PQ-elicited c-jun transcript expression, but DMSO only exhibited a weakly inhibitory effect. We therefore propose that superoxide anion (O2- and H2O2 generated by PQ could activate PKC and lead to induction of c-jun gene expression; on the other hand, O2- and .OH might trigger other kinase pathways to elevate ODC activity. Finally, the sequential expression of c-jun oncogene and ODC may cooperate to relieve the oxidative damages elicited by PQ.

Since the expression of glutathione S-transferase P-form (GST-P) has been suggested from in vitro studies to be partly regulated by the oncogene product, c-Jun and c-Fos, their distributions were compared in normal rat tissues and preneoplastic hepatic lesions induced by the Solt-Farber protocol. Immunohistochemically demonstrated GST-P protein was positively correlated with expression of both c-Jun and c-Fos in the epidermis of the skin and the smooth muscle of adult lung and with either c-Jun or c-Fos respectively in the bile ducts and bronchial epithelium. However, GST-P expression was also observed in proximal and distal straight segments of the kidney and other tissues negative for c-Jun and c-Fos and both c-Jun and c-Fos were present in the renal proximal and distal convoluted tubules, where GST-P was lacking. Thus, the localization of GST-P was in some cases clearly separable from those of c-Jun or c-Fos. GST-P was found to be focally expressed from an early stage of hepatocarcinogenesis, when c-Jun was not detectable. At later stages, this oncogene product was stained in 35.7% of GST-P-positive foci, with a clear relation to the degree of GST-P staining. Since GST-P is not always accompanied by appreciable c-Jun or c-Fos, these oncogene products are apparently not prerequisites for its expression. However, c-Jun may be partly responsible for maintaining high levels of GST-P in hepatic foci at later stages of hepatocarcinogenesis.

Asbestos fibers cause dose-dependent, persistent increases in mRNA levels of c-jun and c-fos proto-oncogenes in rat pleural mesothelial (RPM) cells, the progenitor cells of asbestos-induced mesothelioma (N. Heintz, Y. M. W. Janssen, and B. T. Mossman. Proc. Natl. Acad. Sci. USA, 90: 3299-3303, 1993). Here we report that addition of N-acetyl-L-cysteine decreases asbestos-mediated induction of c-fos and c-jun mRNA levels in a dose-dependent fashion. Exposure of RPM cells to asbestos causes depletion of total cellular glutathione, a response that can be abolished by pretreatment with N-acetyl-L-cysteine. Pretreatment of cells with buthionine sulfoximine, an agent which diminishes glutathione pools, increases the magnitude of induction of c-fos and c-jun mRNA by asbestos. To determine whether asbestos-induced effects on proto-oncogene expression could be attributed to extracellular generation of active oxygen species (AOS), RPM cells were exposed to H2O2 or xanthine and xanthine oxidase, a generating system of AOS. These oxidant stresses did not decrease cellular glutathione levels nor alter mRNA levels of c-fos or c-jun. However, increased mRNA levels of manganese-containing superoxide dismutase and heme oxygenase were observed, indicating that RPM cells respond to AOS by increased expression of genes encoding antioxidant enzymes. These data indicate that the signaling pathways leading to c-fos/c-jun proto-oncogene induction by asbestos are not triggered directly by formation of extracellular AOS. However, intracellular thiol levels appear to influence the expression of c-fos and c-jun, suggesting a redox-sensitive component in the signaling cascade which modulates gene expression of c-fos and c-jun by asbestos.

Asbestos causes persistent increases in c-jun mRNA and AP-1 DNA binding activity in hamster tracheal epithelial (HTE) cells, the progenitor cell type of asbestos-induced bronchogenic carcinoma. Studies here were designed to determine mechanisms of c-jun induction by asbestos and the phenotypic consequences of Jun expression in HTE cells. To examine whether asbestos or H2O2 induced transcription of c-jun, we transiently transfected HTE cells with a plasmid containing a fragment of the c-jun promoter coupled to a luciferase reporter gene. In addition, c-jun was overexpressed in cells using a full-length human c-jun construct, and effects on proliferation and transformation were examined. HTE cells transfected with the jun-luciferase construct showed increased luciferase activity when exposed to crocidolite asbestos or H2O2. These results demonstrate that asbestos and H2O2 activate AP-1-dependent gene transcription. Overexpression of c-jun led to increased proliferation and enhanced ability of HTE cells to grow in soft agar, an indication of cellular transformation. Data suggest that overexpression of c-jun may contribute to asbestos and oxidant-induced proliferation and carcinogenesis.

Previous studies have demonstrated that when cells of the mouse osteoblastic cell line MC3T3-E1 are exposed to IGF-I and IGF-II they exhibit rapid and transient induction of the transcript from the proto-oncogene c-fos [8]. To clarify the relationship between induction of cell proliferation and proto-oncogene expression in MC3T3-E1 cells, the acute affects of IGF-I and IGF-II, growth factors that stimulate cell proliferation, and of TGF-beta 1, which inhibits cell proliferation, northern analyses with cDNA-derived probes for the proto-oncogenes c-jun, jun-B, and jun-D were undertaken. Concurrent northern analyses with a probe for c-fos extended our previous results to include the effect of TGF-beta 1 on c-fos. IGF-I does not induce the c-jun, jun-B, or jun-D transcripts, the former and latter being produced at detectable levels constitutively. After 1 hour of exposure to IGF-II the c-jun transcript response ranges from onefold to 13-fold and the jun-D transcript response ranges around two-fold. After 1 hour of exposure to TGF-beta 1, the jun-B transcript response ranges from eightfold to 24-fold, the c-fos transcript response ranges between sixfold and sevenfold. The differences observed in the magnitude and kinetics of the induction provoked by these growth factors is consistent with the presence of a regulatory circuit acting through the Jun family members which may act to stimulate transcription differentially when bound to DNA either as homodimers or, with Fos family proteins, as heterodimers.

The cellular proto-oncogenes are important for proliferation, differentiation and transformation. To study growth regulation of bovine corneal endothelium (BCE), the authors examined the effect of serum on the expression of the c-jun proto-oncogene in these cells. Increased expression of both c-jun protein and c-jun mRNA was found. This response was transient. While the mRNA reached maximum levels at 45 min, c-jun protein levels peaked around 2 hr and declined thereafter. The peptide growth factors, basic fibroblast growth factor (FGF-2), insulin and transforming growth factor beta 1 (TGF beta 1), which are mitogenic in BCE, were found to effect c-jun protein expression differentially. Basic fibroblast growth factor and 20% fetal calf serum were very effective in inducing c-jun protein expression (in 81% +/- 5.2, 73% +/- 3.4 of cells, respectively), while insulin and TGF beta 1 were less effective (47% +/- 8.0, 32% +/- 4.3, respectively). Since c-jun protein has been shown to be important for cell cycle progression in fibroblasts, our data indicate that it may play a similar role in corneal endothelial cells. Thus, c-jun can be used as an indicator of growth regulatory activity in corneal endothelial cells and further analysis of its regulation will contribute to our understanding of the underlying control mechanisms in these cells.

OBJECTIVE: To investigate c-jun oncoprotein expression in transitional cell carcinomas (TCCs) of the urinary bladder and to determine its relationship to tumour grade and stage, and to the expression of epidermal growth factor receptor (EGFR), c-erbB-2 and p53 oncoproteins. MATERIALS AND METHODS: The expression of c-jun was studied using immunohistochemistry in a series of 48 TCCs of known EGFR, c-erbB-2 and p53 status. RESULTS: Forty-four of 48 (92%) tumours showed c-jun specific nuclear immunoreactivity of variable intensity. The intensity of c-jun immunostaining was significantly related to tumour stage (P = 0.009) and EGFR status (P = 0.01). There was no correlation between c-jun oncoprotein expression and c-erbB-2 or p53 immunoreactivity. c-jun expression was not related to clinical outcome in terms of patient survival or rate of tumour recurrence. CONCLUSION: The c-jun oncoprotein is expressed in the majority of TCCs of the urinary bladder. There is a positive association between intense c-jun immunoreactivity and muscle invasive growth, and EGFR positivity in TCCs.

We have already shown that alkylcatechol markedly enhances synthesis/secretion of nerve growth factor (NGF) in cultured mouse fibroblasts and astroglial cells through immediate accumulation of NGF mRNA and that the stimulatory effect of alkylcatechol on NGF synthesis/secretion is synergistically enhanced by the coadministration of phorbol 12-myristate 13-acetate (PMA). The stimulatory effect on NGF mRNA expression of astroglial cells in culture by 4-methylcatechol (MC), an alkylcatechol, and/or PMA was blocked by treatment of the cells with cycloheximide, suggesting de novo synthesis of some cellular protein(s) is essential for the observed increase in the NGF mRNA level. The exposure to MC and/or PMA caused a rapid increase in c-fos mRNA content, which was immediately followed by an increase in c-jun mRNA, prior to NGF mRNA elevation. The expression of c-fos mRNA was transiently enhanced in ALL cases of the treatment with MC and/or PMA. The c-jun mRNA expression was also observed transiently when the cells were treated with PMA alone, while the expression of c-jun mRNA was pronounced and long-lasting after the treatment with MC, which was much further enhanced by the coadministration of PMA. The result that the profile of the change in c-jun mRNA expression resembled that in NGF mRNA expression suggests that the increase in c-jun mRNA is responsible for the subsequent increase in NGF mRNA after MC treatment. The contransfection of mouse astroglial cells with expression plasmids of c-fos and/or c-jun and NGF promoter gene showed that simultaneous expression of both c-fos and c-jun genes was necessary to enhance NGF promoter activity.(ABSTRACT TRUNCATED AT 250 WORDS)FAU - Omae, E

A chimeric construct (VCD) consisting of parts from viral jun, chicken c-jun, and chicken junD was cloned into the replication-competent retroviral RCAS vector. This construct, RCAS-VCD, was found to have a higher focus forming potential in quail fibroblasts than the equivalent construct RCAS-VJ-1, expressing viral jun. DNAs from RCAS-VCD and RCAS-VJ-1 were transfected into primary quail embryo fibroblasts. Cells derived from one RCAS-VCD-induced focus survived cell crisis, which became manifest after 15 passages, and could be expanded into a long-term culture. This cell line, termed VCD, has been passaged for over 100 times so far. The cells grow to very high densities and then pile up into clumps of rounded cells. The culture releases a transforming virus with a titer of 10(5) FFU/ml, as tested on primary quail embryo fibroblasts. The transformed phenotype of VCD cells was verified by agar colony formation. VCD cells are capable of anchorage-independent growth with a cloning efficiency of 10%. Southern blot analysis of genomic DNA from VCD cells showed proviral integration of the RCAS construct without detectable rearrangements. Northern and Western blot analyses confirmed correct expression from integrated RCAS-VCD of predicted RNAs and of the chimeric Jun(VCD) protein. jun(VCD)-transformed cells provide a constant source of homogeneous cellular material for the investigation of the molecular mechanisms of jun-induced cell transformation and for the identification of direct and indirect targets of Jun protein function.

A new monoclonal antibody to human c-jun oncoprotein, designated NCL-DK4, has been produced. NCL-DK4 has been proved to be highly effective for use on formalin-fixed, paraffin-embedded tissues, enabling the study of c-jun expression at a cellular level in both normal and neoplastic human tissues. The expression of c-jun oncogene has been examined in normal, benign, and malignant breast tissues, and c-jun-specific immunoreactivity in carcinomas has been related to histological type, tumour grade, c-erbB-2, oestrogen receptor, progesterone receptor, and epidermal growth factor receptor expression. Normal and benign breast tissues showed c-jun-specific immunostaining, which was weaker and in fewer cells compared with the c-jun immunoreactivity observed in breast carcinomas. No relationship was found between the degree of immunostaining and the extent of proliferative changes in benign breast tissues. Ninety per cent of ALL breast carcinomas studied showed c-jun-specific nuclear staining. There were no statistically significant differences in the intensity of c-jun immunoreactivity among grade I, II, and III infiltrating ductal carcinomas. There was no significant relationship between c-jun oncoprotein expression and c-erbB-2, oestrogen, progesterone, and epidermal growth factor receptor immunoreactivity.

We here report the spontaneous loss of both homologues of the c-jun gene in two cell lines, isolated after transfection of rat embryo fibroblasts with single ras-oncogenes. These cells lines (designated A14 and B25) grow rapidly in vitro, have transformed morphologies and are invasive through reconstituted basal membranes. Both c-jun defective cell lines were found to be tumorigenic and metastatic in athymic mice. Loss of c-jun was paralleled by a dramatic decrease in interstitial collagenase expression, whereas stromelysin mRNA expression in c-jun- A14 and B25 cells was similar to that observed in c-jun+ transformed cell lines. Transient transfection experiments using reporter plasmids showed that stromelysin promoter activity in A14 cells was severely impaired by a point mutation in the -71 to -65 AP-1 motif, and was inhibited by a Jun dominant negative mutant. Gel mobility shift studies demonstrated the presence of a factor in A14 nuclear extracts capable of binding the stromelysin TRE. This factor bound JunB, JunD and Fos antibodies. Our findings suggest that c-Jun is not required for the tumorigenic and metastatic potential of ras-transformed fibrosarcoma cells, and that AP-1 protein(s) lacking c-Jun are capable of activating the stromelysin gene promoter.

Sequencing analysis of a 275-nt cDNA fragment encoding the DNA-binding region at the carboxyl terminal of the Xenopus c-jun protein product shows that this region exhibits 77-79% nucleotide homology and 93% amino acid homology with the DNA-binding region of mouse and human c-jun, and contains an intact leucine zipper motif and a basic DNA-binding region. Xenopus c-jun mRNA is induced in quiescent Xenopus A6 kidney cells by 12-O-tetradecanoylphorbol-13-acetate, PDGF and cycloheximide. This immediate early response suggests that Xenopus c-jun may be similar in both structure and function to that observed for its mammalian counterpart.

The sequence-specific transcription factor c-Jun displays oncogenic potential in mammalian cells either in cooperation with activated Ras in primary embryonic fibroblasts or alone in established cell lines. Although pathways for signal transduction leading to activation of c-Jun proteins have been extensively studied, little is known about the events downstream of c-Jun stimulation. We isolated cellular genes that are targets of c-Jun by differential screening of a cDNA library from primary rat embryo fibroblasts. Two transcripts with sequences similar to known genes were repressed following transitory expression of a c-Jun-encoding vector. They correspond to the SPARC and thrombospondin 1 (TS1) genes, encoding extracellular matrix proteins. These genes are tightly regulated during embryogenesis and in adult tissues and are involved in the control of cell growth. c-Jun transitory repression of these two genes was demonstrated both in primary cells and in FR3T3, an established fibroblast cell line. The repression was also detected in FR3T3 derivatives stably transformed by c-Jun or Ras. Although c-Jun regulation of the TS1 gene was found at the promoter level, preliminary results strongly suggest that repression of SPARC and TS1 gene expression are mediated by a secreted factor. In contrast, expression of these genes was unaffected by transformation with oncogenes from DNA viruses. Our results identify new, specific, probably indirect c-Jun target genes and suggest previously unsuspected regulatory roles for SPARC and thrombospondin in the control of cell growth.

c-Jun transcriptional activity is augmented by expression of oncogenic Ras and Raf proteins. This study demonstrates a direct correlation between Ras transforming activity and c-Jun activation, supporting an important role for c-Jun in transformation by Ras. Since we observed that Ras activated c-Jun transcriptional activity by increasing phosphorylation of the c-Jun activation domain at residues Ser-63/Ser-73 and that oncogenic Ras proteins activated extracellular signal-regulated protein kinases (ERK1 and ERK2) (also known as mitogen-activated protein kinases), we evaluated the possibility that ERKs were directly responsible for c-Jun activation. Coexpression of wild-type ERKs with oncogenic Ras proteins potentiated, while kinase-defective ERKs inhibited, Ras-induced transcriptional activation from the Ras-responsive element (Ets-1/AP-1) present in the NVL-3 enhancer and the serum-response element in the c-fos promoter. In contrast, coexpression of either wild-type or kinase-defective ERKs inhibited Ras and Raf activation of c-Jun transcriptional activity. Thus, although activation of both ERK and c-Jun are downstream consequences of activation of the Ras signal transduction pathway, our results suggest that Ras-induced c-Jun phosphorylation and transcriptional activation are not a direct consequence of ERK1 and ERK2 activation.

Growth factors, phorbol esters, and oncogenes such as ras, src, and sis are believed to stimulate c-Jun transcriptional activation by inducing increased phosphorylation at two serine residues (S63 and S73) within the N-terminal transactivation domain. Although S63 and S73 are conserved in the mutant v-Jun oncoprotein, they are not phosphorylated by two enzymes which target the corresponding residues in c-Jun in vitro; namely a partially purified c-Jun kinase from TPA-stimulated U937 cells and purified p54 mitogen activated protein (MAP) kinase. In addition, v-Jun activates transcription more strongly than c-Jun when fused to the Gal4 DNA-binding domain, and transcriptional activation by Gal4-v-Jun is unaffected when S63, S73, or both, are replaced with non-phosphorylatable alanine residues, amino acid substitutions which severely impair transcriptional activation by Gal4-c-Jun. The novel biochemical and transcriptional properties of v-Jun result from deletion of a 27 amino acid segment, termed delta, which is important for transforming activity. On the basis of these results we propose that unlike c-Jun, v-Jun transcriptional activation is independent of positive regulatory phosphorylation and that this may contribute to oncogenesis by v-Jun.

The expression of the oncogenes c-fos, c-jun, c-myc, c-erbB-1 and c-erbB-2 at the protein level was analyzed in squamous cell lung carcinomas of 121 patients by means of immunohistochemistry. Patients with overexpression of proteins encoded by the oncogenes c-fos, c-jun and c-erbB-1 had significantly shorter survival times than these without overexpression of these oncogene products (c-fos: p = 0.009; c-jun: p = 0.029; c-erbB-1: p = 0.018). No significant correlations were found between the expression of c-myc and c-erbB-2 products and the survival of the patients. In addition to the univariate analyses (Kaplan-Meier-estimates) multivariate analyses (Cox-regression-model) revealed that protein expression of the oncogenes c-fos, c-jun and c-erbB-1 are significant prognostic factors in addition to staging.

Using specific cDNAs isolated from mouse fibroblasts we determined tissue-specific expression of different matrix metalloproteinase genes: both stromelysin-1 and collagenase IV are highly expressed in heart and lung, whereas collagenase I is expressed most abundantly in skeletal muscle, kidney, and bone. High basal level expression of stromelysin-2 is found in heart and kidney. Like in man and rat, the expressions of collagenase I, stromelysin-1, and stromelysin-2 are regulated by the tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate and by UV irradiation, but not by cAMP. In contrast, the expression of the 72-kDa collagenase IV is not affected by either stimuli. We and others have shown previously that under cell culture conditions, the regulation of human collagenase I is regulated by the transcription factor Fos/Jun (AP-1). Here we show that in c-fos transgenic mice transcription of collagenase I is induced in thymus, spleen, and, most dominantly, in bone upon overexpression of Fos. Neither collagenase IV nor stromelysin-1 or stromelysin-2 expression is affected by c-Fos. The sites of induced collagenase I expression correlate with the sites of Fos-induced long-term cellular alterations in transgenic mice including bone remodeling and T cell development. In fact, in the developing bone tumors strongly enhanced levels of collagenase I transcripts were detectable. These results identify collagenase I as a Fos-regulated gene in vivo and suggest a possible role for Fos/Jun heterodimers in establishing the pathological phenotype of c-fos transgenic mice.

Jun and Fos are major components of the transcriptional complex AP-1 (Activator Protein-1), a collection of dimeric transcriptional activators composed of members of the Jun and Fos family of bZIP proteins, that bind to a common site known as TRE (TPA Responsive Element) or the AP-1 site. Transcription of c-jun is rapidly induced by exposure to different extra-cellular signals like growth factors, cytokines, tumor promoters (TPA), UV and other DNA-damaging agents. Transcriptional activation of c-jun is a two step mechanism. First, the pre-existing c-Jun protein is activated by posttranscriptional modifications, and second, modified c-Jun activates its own transcription, and the expression of AP-1-dependent genes. Modifications of c-Jun include dephosphorylations, phosphorylations and oxydo-reduction. The transcriptional activation by c-Jun is modulated by heterodimerization with other members of the bZIP family of proteins, and by transcriptional interference with other transcription factors like some members of the hormone nuclear receptors, or MyoD. AP-1 is tightly associated to both the control of cell proliferation and the oncogenic process. Constitutive activation of AP-1 leads to cell transformation in vitro, probably due to the accumulation of homodimeric c-Jun:c-Jun complexes. This hypothesis has been directly confirmed by constructing c-Jun hybrid proteins capable to form only homodimers. Deregulated expression of such proteins efficiently transforms primary cells in culture. These hybrid proteins constitute a powerful tool in order to identify new cellular functions AP-1-dependent, involved in the control of cell proliferation.

The activity of c-Jun is regulated by phosphorylation. Various stimuli including transforming oncogenes and UV light, induce phosphorylation of serines 63 and 73 in the amino-terminal activation domain of c-Jun and thereby potentiate its trans-activation function. We identified a serine/threonine kinase whose activity is stimulated by the same signals that stimulate the amino-terminal phosphorylation of c-Jun. This novel c-Jun amino-terminal kinase (JNK), whose major form is 46 kD, binds to a specific region within the c-Jun trans-activation domain and phosphorylates serines 63 and 73. Phosphorylation results in dissociation of the c-Jun-JNK complex. mutations that disrupt the kinase-binding site attenuate the response of c-Jun to Ha-Ras and UV. Therefore the binding of JNK to c-Jun is of regulatory importance and suggests a mechanism through which protein kinase cascades can specifically modulate the activity of distinct nuclear targets.

BACKGROUND: Recent evidence suggests that progressive stages of colorectal tumorigenesis can be defined by a sequence of genetic events characterized by deletion and expression of certain genes and appearance of several oncoproteins. Although the significance of these events is not entirely clear, oncoprotein expression may be directly involved in the tumorigenic mechanism. EXPERIMENTAL DESIGN: We examined the expression of two nuclear oncoproteins, JUN and FOS (Abbreviations used in this paper are lower case letters for oncogenes and upper case letters for oncoproteins), that have not been previously associated with development of colorectal cancer. This study involved detecting p39 c-JUN and p55 c-FOS, as well as two oncoproteins previously known to be expressed during colorectal tumorigenesis, p21 RAS and the tumor suppressor protein, p53. expression was detected with immunohistochemical methods on formalin-fixed, paraffin-embedded sections of normal human colons, tubular adenomas, tubulovillous adenomas, and adenocarcinomas. Either single oncoprotein expression or coexpression of four selected pairs; RAS/JUN, RAS/FOS, RAS/p53, or JUN/FOS were evaluated. RESULTS: We found that the expression of ALL four single oncoproteins and oncoprotein pairs were detected in very few normal colon specimens or tubular adenomas. However, ALL four oncoproteins and the four oncoprotein pairs were expressed significantly more often in adenocarcinomas. oncogene expression in tubulovillous adenomas varied with lesion size. The number of lesions expressing any of the four oncoproteins or pairs was not significantly different than normal colon in small (< 1 cm diameter) tubulovillous adenomas. Significantly more large lesions (> 1 cm. diameter) expressed the single oncoproteins RAS, JUN, and FOS than normal colon. Of the four oncoprotein pairs, only RAS/JUN was significantly coexpressed in large tubulovillous adenomas. CONCLUSIONS: We conclude that independent expression of RAS, JUN, and FOS occurred significantly more often in large tubulovillous adenomas and adenocarcinomas while p53 expression occurred primarily in adenocarcinomas. Also, although ALL four oncoprotein pairs were expressed significantly more often in adenocarcinomas, only RAS/JUN was significantly coexpressed in the large tubulovillous adenomas. These results are comparable with the observed sequential activation of Ki-ras and p53 described by others and suggests that the expression of the nuclear oncogenes, JUN and/or FOS, are also important events in colorectal tumorigenesis.

The v-maf oncogene, identified from AS42 avian retrovirus, encodes a nuclear bZip protein. To elucidate the molecular mechanism of cell transformation induced by this oncogene, we determined the specific binding sequences of its product. Maf protein recognized two types of relatively long palindromic consensus sequences, TGCTGACTCAGCA and TGCTGACGTCAGCA, at roughly equal efficiency. The middle parts of these Maf-binding sequences completely match with two binding sequences for AP-1 transcription factor, i.e., phorbol 12-O-tetradecanoate-13-acetate (TPA)-responsive element (TRE) and cyclic AMP responsive element, suggesting partial overlapping of the target genes for Maf and AP-1. Furthermore, Maf efficiently formed heterodimers with the components of AP-1, Fos and Jun, through their leucine zipper structures, and these heterodimers show binding specificities distinct from those for Maf-Maf and Jun-Jun homodimers. Thus, a multiple combination of the dimers should generate a greatly expanded repertoire of transcriptional regulatory potential. DNA data base search for the Maf-binding consensus sequences suggested that some of the TRE-like cis elements reported previously may actually be the targets for Maf family proteins or their heterodimers with other bZip proteins.

The presence and possible role of products of nuclear (c-fos and c-jun) and c-ras proto-oncogenes were investigated in preimplantation embryonic development in mice. Polyclonal antibodies to c-fos or c-jun proto-oncogene products did not affect development of in vitro-cultured embryos from two-cell to morula or from morula to late blastocyst stages. However, v-H-ras monoclonal antibody (mAb) to c-ras protein (p21), although it did not inhibit the development of in vitro-cultured embryos from two-cell to morula stages, it significantly (P < .001-.005) inhibited the development of morula to late blastocyst stages in a dose-dependent manner. The effects of v-H-ras mAb were specific, since immunoabsorption with synthetic ras peptide completely blocked inhibitory effects of v-H-ras mAb. Neither c-fos nor c-jun antibodies reacted with specific proteins corresponding to c-fos (62 kDa) and c-jun (39 kDa) products on the Western blots of various murine ova/embryos extracts. However, the c-fos and c-jun antibodies reacted with 62 and 39 kDa protein bands, respectively, on the blot of NIH 3T3 cells extract. The v-H-ras mAb specifically identified 21 +/- 3 kDa protein corresponding to c-ras p21 on the blots of early as well as late blastocyst extracts. The rat control ascites IgG1 did not react with any protein band on the blots of various ova/embryo extracts. The reactions of v-H-ras mAb on the Western blots of blastocyst extracts were specific, since immunoabsorbed antibody was unable to react with any specific band on blots of early or late blastocyst extract. These results were further confirmed by immunoprecipitation procedure utilizing v-H-ras mAb. Again, the v-H-ras mAb immunoprecipitated a 21 kDa band from early as well as late blastocyst extracts. The rat control ascites IgG1 did not react with any band corresponding to p21 in the immunoprecipitation procedure. These results suggest that the specific products of nuclear proto-oncogenes, the c-fos and c-jun, are not detected in murine ova and preimplantation embryos, and the respective antibodies do not inhibit embryogenesis, indicating that they may not play a major role in early embryonic development. On the other hand, the product of c-ras proto-oncogene is specifically expressed in the blastocyst-stage embryos and may have a possible role in preimplantation embryonic development in mice.

The viral jun (v-jun) oncogene encodes a transcription factor that can participate in the transactivation of genes through the AP-1 complex. Evidence indicates that the ability of v-jun to transform cells and stimulate transcription depends on the cell type. We have asked whether expression of the v-jun gene in benign tumor forming mouse keratinocytes that already express an activated c-rasHa oncogene would cause malignant progression. Our results showed that the v-jun transfection did not result in malignant progression; instead, we made the unexpected observation that the ability of these cells to invade reconstituted basement membrane matrix (in vitro) in response to the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate, was suppressed. This phenomenon could, in part, be explained by the suppression of the induction by phorbol ester of expression of the metalloproteinase, stromelysin (transin). Of interest was the finding that 12-O-tetradecanoylphorbol-13-acetate induction of other cellular genes known to be regulated by AP-1 was not inhibited in the benign tumor cells expressing v-jun.

The trans-acting factor AP-1 is a heterodimeric complex composed of c-Jun and c-Fos family proteins which bind and regulate genes containing a TPA responsive enhancer element. Although AP-1 binding sites have been identified within the regulatory region of major histocompatibility complex (MHC) class I genes in vitro, the role of AP-1 in regulating MHC class I transcription in vivo has not been investigated previously. The present study demonstrates that expression of c-Jun results in decreased MHC class I promoter activity as determined in cotransfection assays of an MHC class I reporter construct with a c-Jun expression construct. The c-Jun responsive element is located between bp -440 and -431 upstream of initiation of transcription as determined both functionally and by direct binding of purified c-Jun. Furthermore, over-expression of c-Jun reduced the steady state levels of endogenous MHC class I RNA in murine L cells by approximately 10-fold. These data indicate that c-Jun/AP-1 acts as a negative trans-acting factor that down-regulates MHC class I gene expression.

The c-fos and c-jun proto-oncogenes encode components of the transcription factor AP-1. To determine whether transformation by the v-fos or v-jun oncogene results in alterations in the level or regulation of this factor, we have characterized AP-1 DNA-binding activity in nuclear extracts prepared from v-fos- and c-fos-transformed rat fibroblast cell lines and v-jun-transformed chicken embryo fibroblasts under various growth conditions. During proliferation, the level of AP-1 DNA-binding activity does not differ among the v-fos, c-fos, or v-jun-transformed cells and their normal progenitors, despite constitutive overexpression of the corresponding oncoproteins. Therefore, although necessary, it is not likely that an increase in DNA binding is sufficient for fos or jun transformation. Normal rat and chicken fibroblasts demonstrate very low levels of AP-1 DNA-binding activity when quiescent, and upon serum stimulation a biphasic increase is observed. A similar cyclical pattern is seen in v-fos-transformed cells, but in v-jun-transformed cells AP-1 DNA-binding activity does not fluctuate in response to serum stimulation, which suggests that this level of control may be exerted through the Jun component of the AP-1 complex.

Interaction between protein kinase C (PKC)- and glucocorticoid receptor (GR)-mediated signaling is suggested by the ability of the PKC activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to inhibit GR-dependent transcription of the mouse mammary tumor virus (MMTV) long terminal repeat (LTR). Here we report that this interference is cell specific, as TPA augmented dexamethasone-induced transcriptional activation of the MMTV LTR in several T cell lines but was inhibitory in NIH-3T3 fibroblasts. TPA-GR synergism was determined to have occurred at the GR-responsive element (GRE) level by functional analysis of deletion mutants or synthetic GRE oligonucleotides driving chloramphenicol acetyl-transferase expression. Synergism required an intact GR DNA-binding domain, whereas amino- or carboxyl-terminal domains were dispensable. The effect was abrogated by the PKC inhibitor staurosporine, suggesting a role for PKC. Increased c-jun, jun-B, and jun-D expression above basal levels and increased transcriptional activity of AP-1/TPA responsive elements fused to chloramphenicol acetyl-transferase vectors were observed in T cells treated with TPA alone or in combination with dexamethasone. The ability of Jun proteins to cooperate with GR in T cells has been investigated after transfection of c-jun, jun-B, or jun-D expression vectors, which augmented GR-dependent transcription from either MMTV LTR or GRE. Conversely, c-jun and jun-B transfection blunted GR-dependent transcription in HeLa cells. The presence of c-fos had a negative influence on GR function and correlated with the cell-specific synergistic or antagonistic activity of Jun with respect to GR; high basal expression of c-fos as well as AP-1 DNA binding and transcriptional activity were observed in HeLa cells, but not in T cells. Furthermore overexpression of exogenous c-fos has an inhibitory effect on GR-dependent transcription from GRE in T cells. We propose that Jun plays a bifunctional role on GR-dependent transcriptional activation of GRE, selecting either synergistic or antagonistic activity depending on the cell-specific microenvironment. In this regard, intracellular levels of c-fos appear to be influential.

The present work demonstrates that ethanol induces expression of the c-jun proto-oncogene in human keratinocytes. Increased c-jun mRNA levels were detectable at 1 hr of exposure to 1% ethanol and at 24 hr remained above that in control keratinocytes. An increase in c-jun expression was also detectable at ethanol concentrations of 0.1 and 0.5%. Similar findings were obtained for the related jun-B and c-fos early response genes. The results also demonstrate that ethanol exposure is associated with increases in protein kinase C activity in both the cytosol and membrane fractions. This increase was detectable at 5 min and maximal at 30-60 min. The finding that induction of c-jun expression by ethanol was inhibited by the isoquinolinesulfonamide derivative H7, but not by HA1004, suggested that this effect is mediated by protein kinase C. Furthermore, down-regulation of protein kinase C by prolonged exposure to 12-O-tetradecanoylphorbol-13-acetate was associated with a block in ethanol-induced c-jun expression. We also demonstrated that ethanol exposure is associated with rapid (5-30 min) increases in intracellular levels of diradylglycerol. Taken together, these findings demonstrate that the exposure of keratinocytes to ethanol results in the activation of protein kinase C and c-jun expression.

Jun and Fos proteins are DNA-binding proteins that are involved in the control of gene expression through transcriptional regulation. We have made a deletion mutant of the c-jun gene that lacks amino acids 3-122 of c-jun, and thus is missing the major transactivation domain of c-jun, but retains the DNA-binding and leucine zipper domains. Unlike c-Jun, the mutant protein is unable to stimulate the transcription of an AP-1 responsive gene, and unlike c-jun this mutant gene is unable to transform rat embryo cells in cooperation with an activated ras gene. However, this mutant protein blocks in vitro DNA binding of Jun-Jun homodimers and Jun-Fos heterodimers, transcriptional activation induced by c-jun or c-fos and transformation of rat embryo cells induced by an activated ras gene and a deregulated c-jun or c-fos gene. In addition, transformation of rat embryo cells induced by an activated ras gene in the presence of the tumor promoter 12-O-tetradecanoyl phorbol 13-acetate (TPA) or by ras plus SV40 large T antigen is also inhibited by this dominant-negative mutant, suggesting that a member of the jun or fos family is involved in the pathways leading to transformation in these systems as well. The possible molecular mechanisms by which this dominant-negative mutant of c-jun blocks the functions of wild-type jun and fos family members are discussed.

Proton NMR studies have been performed on a 9.8-kDa synthetic fragment comprising the homodimeric leucine zipper domain of the human oncoprotein Jun to ascertain its conformation in aqueous solution. Analysis of two-dimensional scalar and dipolar-coupling experiments enabled almost ALL proton resonances to be sequence-specifically assigned and further revealed that the Jun leucine zipper forms a completely symmetric dimer in solution, consistent with the formation of a coiled-coil arrangement of parallel alpha-helical strands. The rates of exchange of individual amide protons with solvent, as well as hydrogen-bond lengths predicted from amide proton chemical shifts, are shown to correlate with residue position in the coiled-coil. A subset of 209 unambiguous distance constraints was compiled using rules recently formulated for interpreting the NOESY spectra of symmetric coiled-coils, and these were used in combination with experimentally determined hydrogen bond and dihedral angle constraints to compute a solution structure for the Jun leucine zipper domain.

The leukemogenic tyrosine kinase fusion protein Bcr-Abl activates a Ras-dependent pathway required for transformation. To examine subsequent signal transduction events we measured the effect of Bcr-Abl on two mitogen-activated protein kinase (MAPK) cascades--the extracellular signal-regulated kinase (ERK) pathway and the Jun N-terminal kinase (JNK) pathway. We find that Bcr-Abl primarily activates JNK in fibroblasts and hematopoietic cells. Bcr-Abl enhances JNK function as measured by transcription from Jun responsive promoters and requires Ras, MEK kinase (MAPK/ERK kinase kinase), and JNK to do so. Dominant-negative mutants of c-Jun, which inhibit the endpoint of the JNK pathway, impair Bcr-Abl transforming activity. These findings implicate the JNK pathway in transformation by a human leukemia oncogene.

We investigated the inhibitory activity of nerve growth factor (NGF) on apoptosis of rat peritoneal mast cells (PMCs) and compared it with that of recombinant stem cell factor (rSCF), which is a mast cell growth factor. When PMCs were incubated up to 72 hours in the presence of control medium, internucleosomal fragmentation of DNA indicating apoptosis was detected by agarose gel electrophoresis and flow cytometry. The aged PMCs showed morphological changes typical for apoptosis, such as chromatin condensation and loss of microvilli of the cell membrane. Addition of NGF or rSCF prevented development of the characteristic DNA fragmentation and decreased the proportion of apoptotic cells with low DNA content values in a dose-dependent manner. Polyclonal antibody to NGF completely abolished the inhibitory activity of NGF but not of rSCF. NGF-induced PMCs were in the G0/G1 phase of the cell cycle, but rSCF transited them from the G0/G1 phase to the S/G2M phase, suggesting that NGF, unlike rSCF, may have no proliferation activity to PMCs. By flow cytometric analysis with antibodies to NGF receptors p75LNGFR and p140trk, we defined that PMCs expressed p140trk but not p75LNGFR. Addition of herbimycin A or K-252a, tyrosine kinase inhibitors, to NGF resulted in blockage of the NGF-induced p140trk phosphorylation and restriction of the inhibitory activity of NGF on apoptosis of PMCs. These results indicated that NGF suppressed apoptosis of rat PMCs through the p140trk tyrosine phosphorylation and possessed no proliferative activity. Thus, NGF may act as a key factor to promote survival of connective tissue-type mast cells.

The v-maf oncogene encodes a nuclear bZip protein which specifically recognizes relatively long palindromic sequences related to an AP-1 site. In this study, we investigated the relationship of transactivation and transformation activity of Maf. The amino-terminal two thirds of the molecule were dispensable for its DNA-binding activity but conferred its transactivation potential. Transactivation activities of a set of deletion mutants correlated well with their cell transforming abilities. However, a point mutant associated with enhanced oncogenic activity was not more effective in transactivation than the wild type, suggesting that some other function(s) of Maf is also important for its transforming ability. We also examined the effect of other bZip proteins on the transactivation activity of Maf. Three small Maf family proteins (MafK, MafF and MafG), which are missing the transactivation domain of v-Maf, competitively inhibited transactivation by Maf. Co-expression of Jun or Fos also affected the transactivation potential of Maf by forming Maf/Jun or Maf/Fos heterodimers of distinct DNA-binding specificities. In addition to these factors, we noticed the presence of a strong endogenous transactivating activity associated with a sequence related to an NF-E2 site rather than the typical AP-1 site in fibroblast cells. These results indicate that AP-1 site-like cis-regulatory elements of eukaryotic genes are regulated by multiple sets of bZip dimers with different DNA-binding and transactivation properties.

The c-jun proto-oncogene plays a vital role in the carcinogenic process. Although numerous works have extensively investigated the induction mechanisms of c-jun by UV, hydrogen peroxide or 12-O-tetradecanoylphorbol-13-acetate, the mechanism induced by alkylating agents has received little attention. In this study, NIH 3T3 cells were exposed to methylmethanesulfonate (MMS), revealing that the agent clearly induced c-jun expression with a peak at 2 h. Pretreatment of cells with various kinase inhibitors, e.g. H7, genistein, herbimycin A and tyrphostin, did not show any significant effects on the MMS-induced c-jun expression. Benzamide, an inhibitor of poly(ADP)-ribosylation, enhanced the MMS-induced DNA breakages, but did not potentiate that agent which elicited c-jun expression. Another experiment showed that this agent transfected and overexpressed an activated v-H-ras gene in NIH 3T3 cells, which became more resistant to MMS-induced DNA damage but expressed the same level of c-jun transcript as compared with NIH 3T3 cells in response to MMS. If intracellular glutathione (GSH) was completely depleted by buthionine sulfoximine (BSO), the MMS-elicited c-jun expression was blocked. Subsequently, re-elevating intracellular GSH by washing off BSO caused the expression of c-jun by MMS to increase proportionately. Based on these findings, we can conclude that the mechanism by which MMS induced c-jun expression does not occur through activation of protein tyrosine kinases or initiation of DNA damage, but is closely related to the intracellular GSH.

Overexpression of Neu (ErbB-2/HER2) is found in approximately 20% of breast tumors. Activation of Neu by a point mutation (NeuT) causes constitutive tyrosine kinase activity of this transmembrane receptor and transforming activity in fibroblasts. To identify downstream targets of Neu, we have analyzed the ability of Neu to activate gene expression. expression of NeuT, but not normal Neu, caused transcriptional activation of Ets, AP-1, or NF-kappaB-dependent reporter genes. Dominant inhibitory Ras or Raf mutants blocked the Neu-mediated transcriptional activation, confirming that Ras signaling pathways were required for this activation. Analysis with Ets2 mutants indicated that activation of Ets2 transcriptional activity mediated by NeuT or oncogenic Ras required phosphorylation of the same Ets2 residue, threonine 72. Cotransfection of dominant inhibitory Ets2 mutants specifically blocked NeuT-mediated activation of Ets-dependent reporter genes. Furthermore, in focus formation assays using NIH 3T3 cells, the transforming activity of NeuT was inhibited 5-fold when NeuT was cotransfected with a dominant negative Ets2 mutant. However, parallel colony formation assays showed that the Ets2 dominant negative mutant did not inhibit the growth of normal cells. Together, these data show that NeuT activates a variety of transcription factor families via the Ras signaling pathway and that Ets activation is required for NeuT-mediated cellular transformation. Thus, downstream targets of Neu, including Ets transcription factors, may be useful points for therapeutic intervention in Neu/ErbB-2-associated cancers.

We have investigated the expression of Jun family proteins and composition of AP-1 in chicken embryo fibroblasts before and after transformation by the v-Jun oncoprotein of ASV17. We show that p39 c-Jun is the predominant Jun family protein expressed in normal fibroblasts, and that heterodimers of c-Jun and Fos-related partners (Fra s) account for the majority of the AP-1 DNA binding activity. Unexpectedly, because ASV17-transformed fibroblasts do not express p39 c-Jun, v-Jun replaces c-Jun as the predominant AP-1 constituent in association with similar or identical Fra s. This substitution has little effect on the overall level of TRE-specific DNA binding activity, however it results in a profound reduction in TRE-dependent transcriptional activity and a striking defect in signal-regulated phosphorylation of the Jun component of AP-1; whilst agonists of SAPK/JNK kinases trigger transient N-terminal phosphorylation of c-Jun in normal fibroblasts, no corresponding modification of v-Jun occurs in ASV17-transformed cells. Because SAPK/JNK-mediated phosphorylation is thought to regulate c-Jun transcriptional activity and thereby cellular gene expression in response to extracellular signals, we propose that subversion of this signal transduction process by v-Jun is likely to contribute to oncogenesis by ASV17.

In a previous paper, we reported on the structural properties of a 35-residue peptide corresponding to a modified basic subdomain (bSD) of the basic zipper protein c-Jun (residues 252-281) as determined by combined use of 1H-NMR, circular dichroism (CD) and Fourier transform infrared (FT-IR) spectroscopies [Krebs, D., Dahmani, B., El Antri, S., Monnot, M., Convert, O,. Mauffret, O., Troalen, F. & Fermandjian, S. (1995) Eur. J. Biochem. 231, 370-380]. The fragments NP and CP (the N-terminal residues 1-19 and C-terminal residues 16-35 of bSD, respectively) proved to be particularly useful for the assignment of the 1H-NMR spectra of the full-length bSD, which has been achieved completely in aqueous solution and partially in trifluoroethanol. Here, we report on the structural properties of NP and CP in aqueous solution and under varying H2O/trifluoroethanol conditions, again using 1H-NMR, CD and FT-IR experiments. Both CD and FT-IR results established that the fragments are weakly structured in aqueous solution. Addition of trifluoroethanol to aqueous solutions of the peptides produced their stabilization into helix, following a profile sigmoidal for NP and nearly linear for CP. Quantitative NOEs, secondary Halpha chemical shifts, NH temperature coefficients and 3JalphaN coupling constants for the peptides in aqueous solutions provided indications for weak helix features (nascent helices) manifested within two sites (continuous dNN NOEs) in both NP and CP. For each peptide, an excellent agreement was observed between experiments and predictions with the AGADIR program for the location of these nascent helices in the sequences. Trifluoroethanol provoked both the alpha-helix stabilization within these sites and the alpha-helix propagation to adjacent amino acid residues. Finally, our results reflected the high flexibility and helix potential of the NP and CP fragments, these two properties seeming crucial for the accommodation of c-Jun to its specific DNA targets. The results demonstrated also the fragmentation s benefits in dissecting a protein or a complex peptide into smaller fragments and analyzing their structure individually.

AU-rich RNA-destabilizing elements (AREs) found in the 3 untranslated regions of many labile mRNAs encoding proto-oncoproteins and cytokines generally contain (i) one or more copies of the AUUUA pentanucleotide and (ii) a high content of uridylate and sometimes also adenylate residues. Recently, we have identified a potent ARE from the 3 untranslated region of c-jun proto-oncogene mRNA that does not contain the AUUUA motif. In an attempt to further our understanding of the general principles underlying mechanisms by which AREs direct rapid and selective mRNA degradation, in this study we have characterized the functionally important structural features and properties of this non-AUUUA ARE. Like AUUUA-containing AREs, this non-AUUUA ARE directs rapid shortening of the poly(A) tail as a necessary first step for mRNA degradation. It can be further dissected into three structurally and functionally distinct regions, designated domains I, II, and III. None of three domains alone is able to significantly destabilize the stable beta-globin mRNA. The two unlinked domains, I and III, together are necessary and sufficient for specifying the full destabilizing function of this non-AUUUA ARE. Domain II appears functionally dispensable but can partially substitute for domain I. Domain swaps made between the c-jun non-AUUUA and the c-fos AUUUA-containing AREs reveal that the two AREs, while sharing no sequence homology, appear to contain sequence domains that are structurally distinct but functionally overlapping and exchangeable. These data support the idea that the ultimate destabilizing function of an individual ARE is determined by its own unique combination of structurally distinct and functionally interdependent domains. Our polysome profile studies show tha the destabilizing function of the c-jun non-AUUUA ARE does not require any active transit by ribosomes of the mRNA bearing it, further corroborating that the destabilizing function of AREs is not tightly coupled to ongoing translation by ribosomes. Moreover, unlike AUUUA-containing AREs, the c-jun ARE is insensitive to blockage of its effects by addition of transcription inhibitors. Thus, our data provide further evidence for the existence of a novel class of ARE with unique properties.

The transcription factor v-Rel is a transforming protein of the reticuloendotheliosis virus. We found that v-Rel activates the promoter of the proto-oncogene c-jun. Two elements in the c-jun promoter were required for the activation by v-Rel. One was a kB-site (v-Rel binding site), and the other was a c-jun promoter region between -52 and +148 (c-jun promoter (-52/+148)). Two promoters with the kB-site(s), those of human immunodeficiency virus (HIV) and SV40, were not activated by v-Rel, but their kB-sites were activated when introduced upstream of the c-jun promoter (-52/+148). Thus, the c-jun promoter (-52/+148) had information for the selective activation of the c-jun promoter by v-Rel. v-Rel bound to the c-jun kB-site with the higher affinity than c-Rel, thereby activating the c-jun promoter more efficiently than c-Rel. Moreover, the activity of v-Rel mutants upon the c-jun promoter correlates with their transforming activity. Thus, the c-jun promoter activation by v-Rel may play a role in the transformation caused by v-Rel.

In this study, we show that HPV-16 E5 induced anchorage-independent growth in immortalized human epidermal keratinocytes and that HPV-16 E5 in human keratinocytes had higher expression of c-jun and junB; also, we investigated the role of transcriptional initiation pathways in the expression elevation. In addition, Ras-dependent pathway, as well as PKC-dependent pathway, leads to HPV-16 E5-induced c-jun gene expression.

Certain proto-oncogenes transfer growth regulatory signals from the cell surface to the nucleus. These genes often show activation soon after cells are exposed to mitogenic stimulation but can also be activated as a nonmitogenic stress response. Cadmium (Cd) is a carcinogenic metal in humans and rodents and, though its mechanism of action is unknown, it could involve activation of such proto-oncogenes. Metallothionein (MT), a metal-inducible protein that binds Cd, can protect against many aspects of Cd toxicity, including genotoxicity and possibly carcinogenesis. Thus, the effects of Cd on expression of c-myc and c-jun in rat L6 myoblasts, and the effect of preactivation of the MT gene by Zn treatment on such oncogene expression, were studied. MT protein levels were determined by the Cd-heme assay, and MT, c-myc, and c-jun mRNA levels were measured using oligonucleotide hybridization and standardized to beta-actin levels. Cd (5 microM CdCl2, 0-30 h) stimulated both c-myc and c-jun mRNA expression. An initial peak of activation of c-myc expression occurred 2 h after initiation of Cd exposure, and levels remained elevated throughout the assessment period. Zn pretreatment markedly reduced the activation of c-myc expression by Cd compared to cells not receiving Zn pretreatment. Cd treatment increased c-jun mRNA levels by up to 3.5-fold. Again, Zn pretreatment markedly reduced Cd-induced activation of c-jun expression as minimal increases occurred with Cd exposures of < or = 1 h, but otherwise the Zn pretreatment prevented activation of c-jun. The Zn pretreatment elevated MT protein levels > 5-fold over control at the point of Cd exposure, but Cd exposure did not further elevate these Zn-induced MT levels. Similarly, Zn pretreatment did not result in increased relative MT mRNA levels above Cd exposure alone at various time points after Cd exposure. Therefore, Zn pretreatment, possibly by providing elevated MT protein levels at the point of Cd exposure, inhibited the Cd-induced c-myc and c-jun proto-oncogene expression. The extent of Cd-induced proto-oncogene activation thus may be limited by the presence of cellular MT.

Infection with different types of human papillomavirus (HPV) is associated with neoplasia at different anatomic sites. The "low-risk" HPVs (LR-HPV) are responsible for benign genital lesions such as condyloma acuminata. In order to clarify the tumorigenic mechanism of LR-HPV, the HPV infection status was investigated and the expression of the c-jun proto-oncogene in different HPV-related skin and genital lesions analyzed. Of the 17 condyloma specimens analyzed by Western blotting, 13 cases (76.5%) exhibited overexpression of the c-jun gene. ALL 13 cases harbored high copy numbers of the LR-HPV genome with an average of 926 copies per cell, whereas the other four cases had an average of 12 copies of LR-HPV per cell (P < 0.001). Further typing of HPV by Southern blotting revealed that HPV-6 and HPV-11 infections predominated in c-jun positive cases. The c-jun protein was detected much less frequently in cervical cancers (three of 29, or 10.3%) and skin warts (one of 10), and was not detected in five genital polyps or in five normal cervical tissues. These findings suggest a type 6/11-specific induction of c-jun gene expression in HPV-related neoplastic lesions.

rap-1A is a membrane-bound G-protein in the ras superfamily that, like the ras-p21 protein, is activated by binding GTP in place of GDP. When activated, however, this protein inhibits the action of ras-p21, which is to induce mitogenesis in cells A chimeric protein containing RAS-p21 residues 1-65 and rap-1A residues 66-184 becomes ras-p21-like in its activity. The critical changes in sequence that result in this transformation are G26N, 127H, E30D, K31E, and E45V. ALL of these substitutions occur in or around a critical effector domain of p21 that is involved in interacting with GTPase activating protein (GAP), raf-p74 protein and inositol-3-hydroxy kinase. Using molecular dynamics, we have computed the average low energy structures for each of the three proteins, ras-p21, rap-1A and mutant rap1A, called rap-M, that contains these critical amino acid substitutions. We find that rap-M more closely superimposes on ras-p21 (rms deviation 1.9 A) than on wild-type rap-1A (rms deviation 3.4 A). In particular, the amino terminal domains (residues 3-59) of both RAS-p21 and rap-M are superimposable while they deviate when the average structures of these two proteins are superimposed on that of wild-type rap-1A. We have identified Pro 34 as a critical residue which may determine if the protein transforms cells or inhibits cell transformation. In addition, we have found that ras-p21 and rap-M proteins are superimposable in the region 96-110 except at Asp 105. The 96-110 domain of ras-p21 has been found to be involved in the binding of this protein to the nuclear transcription protein, jun and its kinase, jun kinase, JNK. Both segments differ in structure from that of the rap-1A segment at Asp 108, implicating this residue as also being important in determining the activity of the protein. Overall, the oncogenic substitutions introduced into the rap-1A protein cause it to adopt a conformation that is very similar to that of ras-p21 rather than wild-type rap-1A.

Activation of the tyrosine kinase of a temperature-sensitive mutant v-Src oncoprotein in quiescent Rat-1 cells leads to passage through the cell cycle. Temperature shift experiments show that v-Src is needed to leave G0, to pass a relatively stable G1 "pause" point, and to pass a later G1 point committing cells to S phase. Classic immediate early responses that activate both AP-1 DNA binding and mitogen-activated protein (MAP) kinase are induced at G0 exit, but unexpectedly they rise again in mid-G1 and before the onset of S phase, fluctuations that parallel the need for v-Src. An estrogen-inducible mutant c-Raf-1 renders these cells susceptible to mitogenic stimulation by beta-estradiol, without v-Src activity, but greatly inhibits the ability of v-Src to induce DNA synthesis and MAP kinase, probably because v-Src physically associates with inactive c-Raf-1 at permissive but not restrictive temperature. This implicates c-Raf-1 association with enzymically active v-Src and consequent activation of the MAP kinase pathway in v-Src mitogenesis. Furthermore, temperature shift experiments indicate that the mid-G1 peak of MAP kinase activity is associated with cells reaching the G1 pause point, while the pre-S phase peak is needed for DNA synthesis. In contrast, cell transformation by v-Src does not require enhanced MAP kinase activity at any stage of the cell cycle.

The AP-1 transcription factor family is subject to sophisticated regulation in response to cell growth and stress stimuli. We show here that the transcriptional activity of c-Jun, a key AP-1 component, is stimulated by overexpression of the c-Mos proto-oncogene product in mammalian cells. This stimulation requires serines 63 and 73 of c-Jun, indicating that it is likely to be mediated by proline-directed kinase(s). Co-transfection of MKP-1, a specific MAP kinase antagonist, blocks the stimulation of c-Jun by c-Mos, while co-transfection of a dominant negative form of c-Raf-1 does not. Conditioned medium from c-Mos transfected cells fails to activate c-Jun in recipient cells, arguing against the involvement of a diffusible mitogen. These data suggest that c-Mos exerts its effect on c-Jun directly through a MAP kinase, acting downstream of c-Raf-1.

Since the discovery of v-jun as the transforming protein of the sarcoma virus 17 three mammalian homologues of v-jun have been isolated. ALL three jun genes respond to a multitude of agents, and the encoded proteins in turn bind to and regulate, positively or negatively, the transcription of dependent genes, thereby influencing cellular fate. In addition, through transcription factor "cross-talk" Jun influences the transcription of genes regulated by different classes of transcription factors, such as steroid hormone receptors. Although the role of Jun proteins in transcriptional regulation has been thoroughly analyzed in recent years, the role of Jun proteins in oncogenesis is still poorly understood.

Pituitary adenylate-cyclase-activating polypeptide (PACAP) has been shown to possess mitogenic activity in various tumor cells. The present study was designed to investigate signal transduction mechanisms and expression of the proto-oncogenes c-fos and c-jun linked to the mitogenic effect of PACAP in the pancreatic carcinoma cell line AR4-2J. PACAP-(1-27)-peptide and PACAP-(1-38)-peptide, but not the structurally related vasoactive intestinal polypeptide (VIP), potently stimulated [3H]thymidine incorporation and cell number at doses of 0.1-10 nM. Both molecular forms of PACAP strongly increased formation of cAMP and inositol trisphosphate, elevated cytosolic Ca2+ levels and induced mitogen-activated protein (MAP) kinase activity. Quantitative reverse-transcription PCR revealed that PACAP-(1-27)-peptide and PACAP-(1-38)-peptide elevated c-fos mRNA levels 50-100-fold, whereas c-jun mRNA levels increased only moderately (2-3-fold). The effect of PACAP on c-fos and c-jun expression in AR4-2J cells was rapid (20 min), transient (1-2 h), dose-dependent IC50, 0.5 nM) and was abolished by the specific PACAP receptor antagonist PACAP-(6-38)-peptide or inhibitors of protein kinase C or tyrosine kinases. Compared with PACAP, epidermal growth factor and gastrin equipotently stimulated c-fos transcription whereas VIP, secretin, forskolin or phorbolester showed only marginal effects. Both PACAP (1-27)-peptide and PACAP-(1-38)-peptide strongly increased the DNA binding activity of the c-fos/ c-jun heterodimer transcription factor AP-1 at 10 nM and also stimulated AP-1 transcriptional activity up to 20-fold in AR4-2J cells. These findings indicate that the mitogenic effect of PACAP mediated via activation of the GTP-binding protein coupled PACAP/VIP-1 (PV1) receptor is linked to the MAP kinase cascade, increased expression of the proto-oncogenes c-fos and c-jun and activation of the heterodimeric transcription factor AP-1.

Mining and mineral processing are important industries in the United States. A large number of workers are potentially exposed to silica during mining and to glass fibers during manufacturing. There is a concern regarding lung cancer risk among workers exposed to silica and glass fibers. Our previous studies showed that both glass fibers and silica induced transformation of BALB/c-3T3 cells. In order to explore the relationship between silica and glass fiber-induced cell transformation and oncoprotein expression, the protein products of seven proto-oncogenes (c-K-ras, c-H-ras, c-sis, c-myc, c-myb, c-erb B1 and c-jun) and one tumor suppressor gene (p53) were examined in BALB/c-3T3 cells transformed by glass fibers or silica using immunoblotting with specific monoclonal or polyclonal antibodies. The results showed that ALL transformants, including eight induced by glass fibers and eight by silica (Min-U-Sil 5), were positive for c-jun protein expression; the level of c-jun protein was elevated 8-21-fold in these transformants. Other protooncogene proteins in transformed cells were either not detectable or not different from non-transformed cells. These results suggest that the overexpression of c-jun is common in BALB/c-3T3 transformed cells induced by glass fibers or silica. It seems, therefore, that the expression of c-jun may play an important role in the transformation process.

The expression of glutathione S-transferase (GST)-pi and four oncogene products, c-Jun, c-Fos, c-H-Ras, and c-Myc, in human squamous cell carcinomas of the head and neck was investigated immunohistochemically before and after radiation therapy, to examine whether these oncogene products might be involved in GST-pi expression, and also to examine the relationship between their expression and therapeutic response. Clinical response to radiation was evaluated in terms of both tumor regression and relapse over two-year follow-up periods. The overall positive rates in 83 carcinoma specimens before therapy were 60.2% for GST-pi and 28.9-51.8% for the individual oncogene products, the positive rates for the oncogene products being higher in GST-pi-positive than in GST-pi-negative cancers. c-Jun was most highly correlated with GST-pi expression. Following radiation, the expression of GST-pi and the oncogene products was altered in about a half of 30 patients. Eleven of the 18 patients who exhibited prior positivity for GST-pi showed negative conversion, while 4 of the 12 patients with prior negativity demonstrated positive conversion. In most cases, changes in c-Jun staining coincided with those in GST-pi. Regarding clinical response to radiation therapy, the positive rates for GST-pi and c-Jun before radiation were higher in the residual cancer or relapse cases than in the group showing complete response without relapse. Examination of 26 patients with laryngeal cancer revealed that relapse occurred more frequently in cases exhibiting positive reactions for GST-pi, c-Jun, or c-H-Ras. These results suggest a direct link between c-Jun and GST-pi in head and neck cancers before and after radiation. Although GST-pi and the oncogene products can be influenced by radiation, GST-pi and c-H-Ras expression may be a risk factor for relapse of laryngeal cancer.

v-Rel is a transforming protein of the reticuloendotheliosis virus, and is a transcription factor regulating various cellular genes. We found that v-Rel activates the promoter of the proto-oncogene c-jun in a transient transfection assay system. Moreover, the expression of endogenous c-jun was augmented in cells expressing exogenous v-Rel, but not c-Rel. The transcriptional activities of v-Rel to the tested promoters containing the kB-site are lower than that of c-Rel, but that to the c-jun promoter was much higher than that of c-Rel. The N-terminal DNA binding domain of v-Rel, which is responsible for its high transforming activity of v-Rel was also responsible for the high transcriptional activity to the c-jun promoter. Thus, the activity of v-Rel upon the c-jun promoter correlates well with its transforming ability. Since c-Jun plays pivotal roles on cell proliferation in various types of cells, the activation of c-jun expression by v-Rel may be an essential step for the oncogenic transformation caused by v-Rel.

Asbestos and the phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), increase c-fos and c-jun mRNA levels and AP-1 DNA binding activity in rat pleural mesothelial (RPM) cells, a target cell of asbestos-induced mesotheliomas (N. H. Heintz et al., Proc. Natl. Acad. Sci. USA, 90: 3299-3303, 1993). Because protein kinase C (PKC) is the intracellular receptor of phorbol ester tumor promoters and asbestos is a putative tumor promoter in the respiratory tract, we hypothesized that PKC might play a critical role in asbestos-induced cell signaling pathways associated with regulation of proto-oncogenes. Using a panel of PKC antibodies, we identified PKC alpha as the major PKC isozyme in RPM cells. We then pretreated cells with phorbol ester dibutyrate to down-modulate PKC or with calphostin C, a specific PKC inhibitor, to determine if depletion of PKC alpha could block asbestos-induced c-fos/c-jun expression. Quantitation of Northern blots showed that fiber-associated c-fos/c-jun mRNA levels were significantly lower either after PKC alpha down-modulation or pretreatment with calphostin C. In addition, to determine whether tyrosine kinases also were involved in proto-oncogene activation by asbestos, tyrphostin AG82 or herbimycin A was added to RPM cells before exposure to asbestos. These inhibitors decreased crocidolite-induced c-fos but not c-jun levels, suggesting that tyrosine kinases have different regulatory roles in asbestos-induced c-fos versus c-jun signaling pathways. The ability to block induction of asbestos-induced proto-oncogene expression using pharmacological intervention may be important in prevention and treatment of asbestos-induced proliferative diseases including lung cancers, mesothelioma, and pulmonary fibrosis.

The cyclin kinase inhibitor p16, encoded by the CDKN2A gene, suppresses the transformation of mouse embryonic fibroblasts by oncogenic RAS. In contrast, the c-JUN transcription factor (a major component of AP-1) has been suggested to be required for RAS transformation of rodent fibroblasts. The CDKN2A gene and the JUN proto-oncogene have both been mapped to rat chromosome band 5q31-33. We here show that both copies of the CDKN2A gene are deleted in four of eight transformed cell lines derived from the transfection of rat embryo fibroblasts (REF) with HRASVAL12. In two cell lines, the homozygous deletions involved a larger area on 5q31-33, which included the JUN proto-oncogene. JUN-defective cells showed high AP-1 binding activity. Both AP-1 binding activity and stromelysin (transin) mRNA expression were found to be RAS-dependent in one of the JUN-defective cell lines. The finding of deletions of the CDKN2A gene in RAS-transformed REF cell lines is consistent with the concept that CDKN2A suppresses transformation by RAS. The occasional concomitant loss of the adjacent JUN proto-oncogene does not prevent establishment of transformed and tumorigenic cell lines.

The expression of the transcription factor c-JUN was investigated in the rat fascia dentata under normal conditions and after entorhinal cortex lesion. As shown by immunocytochemistry and in situ hybridization histochemistry c-JUN and its messenger RNA are present in the principal cell layers of the dentate gyrus and Ammon s horn (except hippocampal region CA2). Pre-embedding immunogold electron microscopy revealed an almost exclusive nuclear localization of c-JUN, where it is associated with chromatin. In addition, double immunolabelling for c-JUN and parvalbumin demonstrated that c-JUN immunoreactivity is primarily found in principal neurons since GABAergic parvalbumin-positive interneurons did not express c-JUN. After unilateral electrolytic lesion of the entorhinal cortex c-JUN was strongly up-regulated in the ipsilateral dentate gyrus within 2 h postlesion. This up-regulation was also present in the contralateral fascia dentata 12 h after entorhinal cortex lesion and returned to control levels on both sides 24 h postlesion. The cellular distribution of c-JUN did not change after entorhinal cortex lesion: parvalbumin-positive interneurons never contained c-JUN. These results point to a specific role of c-JUN in the granule cells of the fascia dentata in the normal animal and in rats with entorhinal cortex lesions. The selective induction of c-JUN after entorhinal lesion could be one of the first molecular steps that regulate transneuronal changes within granule cells after their denervation. A different mechanism has to be assumed for GABAergic interneurons known to receive an entorhinal innervation as well.

Stimulation of c-Jun transcriptional activity via phosphorylation mediated by the stress-activated or c-Jun amino-terminal (SAPK/JNK) subgroup of mitogen-activated protein kinases (MAP kinases) is thought to depend on a kinase-docking site (the delta region) within the amino-terminal activation domain, which is deleted from the oncogenic derivative, v-Jun [1] [2] [3]. This mutation markedly enhances v-Jun oncogenicity [4] [5]; however, its transcriptional consequences have not been resolved. In part, this reflects uncertainty as to whether binding of SAPK/JNK inhibits c-Jun function directly [6] [7] or, alternatively, serves to facilitate and maintain the specificity of positive regulatory phosphorylation [8]. Using a two-hybrid approach, we show that SAPK/JNK stimulates c-Jun transactivation in yeast and that this depends on both catalytic activity and physical interaction between the kinase and its substrate. Furthermore, c-Jun is active when tethered to DNA via SAPK/JNK, demonstrating that kinase binding does not preclude transactivation. Taken together, these results suggest that SAPK/JNK acts primarily as a positive regulator of c-Jun transactivation in situ, and that loss of the docking site physically uncouples v-Jun from this control. This loss-of-function model accounts for the deficit of v-Jun regulatory phosphorylation and repression of TPA response element (TRE)-dependent transcription observed in v-Jun-transformed cells and predicts that an important property of the oncoprotein is to antagonise SAPK/JNK-dependent gene expression.

v-rel is the oncogenic member of the Rel/NF-kappaB family of transcription factors. The mechanism by which v-Rel induces transformation of avian lymphoid cells and fibroblasts is not precisely known. However, most models propose that v-rel disrupts the normal transcriptional regulatory network. In this study we evaluated the role of AP-1 family members in v-Rel-mediated transformation. The overexpression of v-Rel, c-Rel, and c-Rel delta resulted in a prolonged elevation of c-fos and c-jun expression and in a sustained repression of fra-2 at both the mRNA and protein levels in fibroblasts and lymphoid cells. Moreover, the transforming abilities of these Rel proteins correlated with their ability to alter the expression of these AP-1 factors. v-Rel exhibited the most pronounced effect, whereas c-Rel, with poor transforming ability, elicited only moderate changes in AP-1 levels. Furthermore, c-Rel delta, which exhibits enhanced transforming potential relative to c-Rel, induced intermediate changes in AP-1 expression. To directly evaluate the role of AP-1 family members in the v-Rel transformation process, a supjun-1 transdominant mutant was used. The supjun-1 mutant functions as a general inhibitor of AP-1 activity by inhibiting AP-1-mediated transactivation and by reducing AP-1 DNA-binding activity. Coinfection or sequential infection of fibroblasts or lymphoid cells with viruses carrying rel oncogenes and supjun-1 resulted in a reduction of the transformation efficiency of the Rel proteins. The expression of supjun-1 inhibited the ability of v-Rel transformed lymphoid cells and fibroblasts to form colonies in soft agar by over 70%. Furthermore, the expression of supjun-1 strongly interfered with the ability of v-Rel to morphologically transform avian fibroblasts. This is the first report showing that v-Rel might execute its oncogenic potential through modulating the activity of early response genes.

OBJECTIVE: To examine the relationship between the expressions of glutathione S-transferase pi (GST-pi) and four oncogene products, c-Jun, c-Fos, c-H-Ras, and c-Myc, and clinicopathological prognostic factors and patients prognosis in endometrial carcinomas, and to assess their prognostic value in endometrial carcinomas. METHODS: Specimens of endometrial carcinoma obtained from 63 patients were investigated immunohistochemically using respective specific antibodies. RESULTS: The overall positive rates in 63 carcinoma specimens were 34.9% for GST-pi, 44.4% for c-Jun, 34.9% for c-Fos, 47.6% for c-H-Ras, and 54.0% for c-Myc. Multivariate analysis revealed that GST-pi expression correlated independently with paraaortic lymph node (PAN) metastasis, and c-Jun expression was independently related to pelvic lymph node (PLN) and PAN metastasis. The prognosis of patients with a GST-pi-positive tumor was significantly poorer than that of those with a GST-pi-negative tumor (P < 0.05). The patients with c-Jun-positive tumor also had a significantly worse prognosis than those with c-Jun-negative tumor (P < 0.05). No significant relationship between the expressions of the remaining three oncogene products, c-Fos, c-H-Ras, and c-Myc, and the examined prognostic factors and clinical outcome was apparent. CONCLUSION: These results suggest that the expressions of GST-pi and c-Jun may reflect the metastatic potential of endometrial carcinomas and that their expressions of endometrial carcinoma may be useful as a prognostic indicator for predictive testing.

The present work was designed (a) to study comparatively the effect of 2Hz and 100Hz electroacupuncture (EA) on the expression of oncogene c-fos/c-jun and three opioid (preproenkephalin-PPE; preprodynorphin-PPD; proopiomelanocortin-POMC) genes in the rat brain; (b) to clarify the role of Fos/Jun (AP-1) on opioid genes expression induced by EA stimulation through specific blockade of EA-induced Fos/Jun expression using antisense oligodeoxynucleotides (ODNs) of c-fos/c-jun. The results were: (a) 2Hz and 100Hz EA induced differential Fos expression in different brain areas; (b) EA of both frequencies accelerated PPE gene transcription, but 2Hz EA was more effective than 100Hz EA; (c) PPD expression was accelerated by 100Hz EA, but not by 2Hz EA; (d) the blockade of Fos/Jun expression by c-fos/c-jun antisense ODNs prevented EA from accelerating PPD but not PPE mRNA expression.

Exposure of B-lineage lymphoid cells to ionizing radiation induces an elevation of c-jun proto-oncogene mRNA levels. This signal is abrogated by protein-tyrosine kinase (PTK) inhibitors, indicating that activation of an as yet unidentified PTK is mandatory for radiation-induced c-jun expression. Here, we provide experimental evidence that the cytoplasmic tyrosine kinases BTK, SYK, and LYN are not required for this signal. Lymphoma B-cells rendered deficient for LYN, SYK, or both by targeted gene disruption showed increased c-jun expression levels after radiation exposure, but the magnitude of the stimulation was lower than in wild-type cells. Thus, these PTKs may participate in the generation of an optimal signal. Notably, an inhibitor of JAK-3 (Janus family kinase-3) abrogated radiation-induced c-jun activation, prompting the hypothesis that a chicken homologue of JAK-3 may play a key role in initiation of the radiation-induced c-jun signal in B-lineage lymphoid cells.

Erionite, a naturally occurring fibrous zeolite, is associated with the development of nonmalignant and malignant lung diseases and is more carcinogenic than asbestos fibers in man and rodent inhalation models of disease. To investigate the possible molecular mechanisms of erionite-induced toxicity and carcinogenesis and whether cationic content of erionite fibers was important, we examined c-fos and c-jun mRNA levels, activator protein-1 (AP-1) binding to DNA, and changes in cell proliferation and apoptosis in rat pleural mesothelial (RPM) cells exposed to different cation-substituted erionite fibers or crocidolite asbestos at various concentrations (1, 5, or 10 microg/cm2 dish) at time periods from 8 to 48 h after addition of minerals. c-fos mRNA levels in cells exposed to equal weight concentrations of various erionites and crocidolite fibers were increased comparably. When compared to other fibers, Na-erionite caused significantly increased levels of c-jun mRNA at lower mass concentrations (1 and 5 microg/cm2) than crocidolite asbestos, but comparable AP-1 binding to DNA. In comparison to untreated controls, numbers of RPM cells incorporating 5 -bromodeoxyuridine (BrdU) were increased dramatically after exposure to asbestos or Na-erionite at 5 and 10 microg/cm2. Significant dose-dependent increases in apoptosis were observed with asbestos at ALL time points, whereas erionites failed to induce apoptosis at 8 or 24 h, with minimal induction at higher concentrations than asbestos at 48 h. These data suggest that erionite increases the balance between cell proliferation (and/or abnormal DNA repair) and apoptosis, a normal mechanism of elimination of transformed or proliferating cells.

c-jun proto-oncogene expression is extinguished in cells transformed by v-Jun; however, the mechanistic basis of this phenomenon has not been elucidated. c-jun mRNA levels are greatly reduced in v-Jun-transformed cells, and we show that this reduction is associated with a similar decrease in the rate of c-jun transcription. Transcriptional down-regulation was also evident in functional assays in which the c-jun gene promoter was approximately 10-fold less active in v-Jun-transformed cells than it was in normal cells. This reduction was largely attributable to a conserved 12-O-tetradecanoylphorbol-13-acetate-responsive element (TRE)-like motif at position -72 (the proximal junTRE) that was essential for efficient basal expression in normal cells but that conferred little, if any, detectable transcriptional activity in v-Jun-transformed cells. DNA-binding analysis showed that this element was recognized by a mixture of c-Jun/Fra and cyclic AMP-responsive element-binding protein/activating transcription factor-like complexes in normal cells but that v-Jun/Fra heterodimers predominated in v-Jun-transformed cells. Furthermore, ectopic expression of v-Jun repressed c-jun promoter activity in normal cells through the proximal junTRE. Thus, the deficit in transcription mediated by the junTRE correlates with and is most likely attributable to binding of v-Jun to this element in vivo. We also find that the c-jun promoter is refractory to induction via the stress-activated protein kinase/c-jun NH2-terminal kinase pathway in v-Jun-transformed cells, suggesting that v-Jun interferes with signal-regulated gene expression. Therefore, c-jun is an example of a cellular gene, the transcription of which is regulated negatively by v-Jun in vivo.

The Cbl proto-oncogene product is a complex adapter protein that functions as a negative regulator of protein tyrosine kinases. It is rapidly tyrosine-phosphorylated and associates with Crk(L) and p85 phosphatidylinositol 3-kinase (PI3K) upon engagement of numerous receptors linked to tyrosine kinases. Elucidation of the mechanism(s) underlying Cbl deregulation is therefore of considerable interest. The 70Z Cbl oncoprotein shows increased baseline tyrosine phosphorylation in fibroblasts and enhances nuclear factor of activated T cells (NFAT) activity in Jurkat T cells. Its transforming ability has been proposed to relate to its increased phosphotyrosine content. We demonstrate that 70Z Cbl shows increased basal and activation-induced tyrosine phosphorylation and association with Crk(L) and p85 PI3K in Jurkat T cells. 70Z Cbl, however, retains the ability to enhance NFAT and activating protein 1 (AP1) activity in the absence of Crk(L)/p85 PI3K association. In contrast, the G306E mutation, which inactivates the phosphotyrosine binding domain of Cbl, blocks NFAT/AP1 activation by 70Z Cbl. We conclude that 70Z Cbl-induced NFAT/AP1 activation requires the phosphotyrosine binding domain but not Crk(L)/p85 PI3K association. We hypothesize that 70Z Cbl acts as a dominant negative by blocking the negative regulatory function of the Cbl phosphotyrosine binding domain on protein-tyrosine kinases.