Oncogene database

5292

PIM1

Pim-1 proto-oncogene, serine/threonine kinase;PIM1;Pim-1 proto-oncogene, serine/threonine kinase

Oncogene PIM1|pim-1 kinase 44 kDa isoform|pim-1 oncogene (proviral integration site 1)|proto-oncogene serine/threonine-protein kinase pim-1|serine/threonine-protein kinase pim-1

6p21.2

protein-coding

The proto-oncogene pim-1 is a serine/threonine kinase the over-expression of which promotes lymphoma formation. Neither the normal function of Pim-1 nor the biochemical mechanism for cancer development mediated by the gene has been delineated, although recent studies have provided compelling evidence that Pim-1 is involved in differentiation and cell survival. We now provide the first evidence that Pim-1 may be involved in the proliferative process. By confocal microscopy, we observed a dynamic redistribution of Pim-1 during the cell cycle, the protein moving from the nucleus and cytoplasm in interphase to the spindle poles during mitosis. From a computer search for putative substrates of Pim-1 that are located in the spindle poles, we discovered that the nuclear mitotic apparatus (NuMA) protein has two peptide sequences that contain preferred phosphorylation sites for Pim-1 kinase. Recombinant glutathione-S-transferase-Pim-1 also readily phosphorylates immunoprecipitated NuMA. By confocal microscopy and co-immunoprecipitation we showed the interaction of the Pim-1 and NuMA proteins in HeLa cells that had been arrested during mitosis with nocodazole. Pim-1 also appeared to interact with heterochromatin-associated protein 1beta (HP1beta) and the cytoplasmic proteins dynein and dynactin via complex formation with NuMA. In our studies, overexpressed wild-type-Pim-1-GFP (green fluorescent protein) fusion protein was found to co-localize in the spindle pole with NuMA during mitosis. In contrast, the kinase-dead mut-Pim-1-GFP fusion protein did not co-localize with NuMA, and appeared to promote apoptosis. Further evidence for apoptotic cell death was the observed blebbing and fragmentation of the chromosomes and a decrease in the level of NuMA protein detected by confocal microscopy. These results strongly suggest that Pim-1 kinase plays a role, most likely by phosphorylation, in promoting complex formation between NuMA, HP1beta, dynein and dynactin, a complex that is necessary for mitosis.

The human pim-1 proto-oncogene was expressed in Escherichia coli as a glutathione-S-transferase (GST)-fusion protein and the enzymatic properties of its kinase activity were characterized. Likewise, a Pim-1 mutant lacking intrinsic kinase activity was constructed by site-directed mutagenesis (Lys67 to Met) and expressed in E. coli. In vitro assays with the mutant Pim-1 kinase showed no contaminating kinase activity. The wild-type Pim-1 kinase-GST fusion protein showed a pH optimum of 7 to 7.5 and optimal activity was observed at either 10 mM MgCl2 or 5 mM MnCl2. Higher cation concentrations were inhibitory, as was the addition of NaCl to the assays. Previous work by this laboratory assaying several proteins and peptides showed histone H1 and the peptide Kemptide to be efficiently phosphorylated by recombinant Pim-1 kinase. Here we examine the substrate sequence specificity of Pim-1 kinase in detail. Comparison of different synthetic peptide substrates showed Pim-1 to have a strong substrate preference for the peptide Lys-Arg-Arg-Ala-Ser*-Gly-Pro with an almost sixfold higher specificity constant kcat/Km over that of the substrate Kemptide (Leu-Arg-Arg-Ala-Ser*-Leu-Gly). The presence of basic amino acid residues on the amino terminal side of the target Ser/Thr was shown to be essential for peptide substrate recognition. Furthermore, phosphopeptide analysis of calf thymus histone H1 phosphorylated in vitro by Pim-1 kinase resulted in fragments containing sequences similar to that of the preferred synthetic substrate peptide shown above. Therefore, under optimized in vitro conditions, the substrate recognition sequence for Pim-1 kinase is (Arg/Lys)3-X-Ser/Thr*-X , where X is likely neither a basic nor a large hydrophobic residue.

Peptide nucleic acids (PNA) are promising antisense molecule for blocking gene expression in cell culture or in vivo. Nevertheless because they are poor efficient to pass the cellular membrane, it is necessary to use a vectorisation agent to observe an inhibitory effect. We describe the coupling of the rhodamine labeled 17-mer antisense PNA to a fusogenic peptide from antenapedia via S-S linkage, the studies of the penetration of this complex into fibroblast cells and its inhibitory effect on pim1 targeted protononcogene.

Aneuploidy and chromosomal aberrations are hallmarks of most human epithelial malignancies. Here we show that overexpression of the oncogenic kinase Pim-1 in human prostate epithelial cells induces genomic instability by subverting the mitotic spindle checkpoint. Cells overexpressing Pim-1 have a defect in the mitotic spindle checkpoint, abnormal mitotic spindles, centrosome amplification, and chromosome missegregation. Polyploidy and aneuploidy ensue due to a delay in completing cytokinesis. These results define a novel role for elevated Pim-1 expression in promoting genomic instability in human prostate tumors.

The Pim-1 oncogene encodes a serine-threonine kinase that relays signals from cytokine receptors and contributes to the formation of lymphoid tumors when expressed at high levels. Here we show that the protein kinase Cdc25 C-associated kinase 1 (C-TAK1) is a binding partner and a substrate of Pim-1. A physical interaction of Pim-1 and C-TAK1 could be shown biochemically and in yeast two-hybrid assays. Immunofluorescence experiments suggested that Pim-1.C-TAK1 complexes are predominantly cytoplasmic. When transiently transfected, Pim-1 was also found in the nucleus and could recruit C-TAK1 to this compartment. Both Pim-1 and C-TAK1 underwent autophosphorylation, but only Pim-1 was able to phosphorylate C-TAK1 but not vice versa. Mass spectrometry analysis of C-TAK1 suggested that the sites of autophosphorylation and Pim-1-mediated phosphorylation are distinct and not overlapping. Phosphorylation by Pim-1 decreased C-TAK1 kinase activity significantly, in particular its ability to phosphorylate and inactivate Cdc25C, a protein that actively promotes cell cycle progression at the G(2)/M phase. Hence our findings directly suggest a novel role for Pim-1 as a positive regulator at the G(2)/M transition of the cell cycle.

Pim1, a serine/threonine kinase, is involved in several biological functions including cell survival, proliferation, and differentiation. While pim1 has been shown to be involved in several hematopoietic cancers, it was also recently identified as a target of aberrant somatic hypermutation in diffuse large cell lymphoma (DLCL), the most common form of non-Hodgkin s lymphoma. The crystal structures of Pim1 in apo form and bound with AMPPNP have been solved and several unique features of Pim1 were identified, including the presence of an extra beta-hairpin in the N-terminal lobe and an unusual conformation of the hinge connecting the two lobes of the enzyme. While the apo Pim1 structure is nearly identical with that reported recently, the structure of AMPPNP bound to Pim1 is significantly different. Pim1 is unique among protein kinases due to the presence of a proline residue at position 123 that precludes the formation of the canonical second hydrogen bond between the hinge backbone and the adenine moiety of ATP. One crystal structure reported here shows that changing P123 to methionine, a common residue that offers the backbone hydrogen bond to ATP, does not restore the ATP binding pocket of Pim1 to that of a typical kinase. These unique structural features in Pim1 result in novel binding modes of AMP and a known kinase inhibitor scaffold, as shown by co-crystallography. In addition, the kinase activities of five Pim1 mutants identified in DLCL patients have been determined. In each case, the observed effects on kinase activity are consistent with the predicted consequences of the mutation on the Pim1 structure. Finally, 70 co-crystal structures of low molecular mass, low-affinity compounds with Pim1 have been solved in order to identify novel chemical classes as potential Pim1 inhibitors. Based on the structural information, opportunities for optimization of one specific example are discussed.

BACKGROUND AND OBJECTIVES: Diamond Blackfan anemia (DBA) is a congenital disease characterized by defective erythroid progenitor maturation. Patients bone marrow progenitor cells do not respond to erythropoietic growth factors, such as erythropoietin. mutations in the gene encoding for ribosomal protein (RP) S19 account for 25% of cases of DBA. The link between defective erythropoiesis and RPS19 is still unclear. Two not mutually exclusive hypotheses have been proposed: altered protein synthesis and loss of unknown extraribosomal functions. DESIGN AND METHODS: We used yeast two-hybrid screening and a human liver cDNA library obtained at 19-24 weeks of gestation, when hepatic erythropoiesis is efficient, to search for proteins interacting with RPS19. RESULTS: We found that RPS19 binds PIM-1, an ubiquitous serine-threonine kinase whose expression can be induced in erythropoietic cells by several growth factors, such as erythropoietin. The PIM-1/RPS19 interaction was demonstrated both in vitro and in living cells and led to phosphorylation of RPS19 in an in vitro kinase assay. We also showed that in human 293T cells PIM-1 interacts with ribosomes and may be involved in translational control. Three DBA-associated RPS19 mutations alter the binding between RPS19 and PIM-1. INTERPRETATION AND CONCLUSIONS: A link between erythropoietic growth factor signaling and RPS19 has been identified for the first time.

The proto-oncogene Pim-1 encodes a serine-threonine kinase which is a downstream effector of cytokine signaling and can enhance cell cycle progression by altering the activity of several cell cycle regulators among them the G1 specific inhibitor p21(Waf), the phosphatase Cdc25A and the kinase C-TAK1. Here, we demonstrate by using biochemical assays that Pim-1 can interact with the phosphatase Cdc25C and is able to directly phosphorylate the N-terminal region of the protein. Cdc25C is functionally related to Cdc25A but acts specifically at the G2/M cell cycle transition point and can be inactivated by C-TAK1-mediated phosphorylation. Immuno-fluorescence experiments showed that Pim-1 and Cdc25C co-localize in the cytoplasm of both epithelial and myeloid cells. We find that phosphorylation by Pim-1 enhances the phosphatase activity of Cdc25C and in transfected cells that are arrested in G2/M by bleomycin, Pim-1 can enhance progression into G1. Therefore, we propose that Pim-1 activates Cdc25C by a direct phosphorylation and can thereby assume the function of a positive cell cycle regulator at the G2/M transition.

The Pim-1 proto-oncogene, encoding a serine-threonine kinase, has been found to play an important role in regulating apoptosis, differentiation, proliferation and tumourigenesis. The present study was conducted to assess the importance of Pim-1 in oral tumourigenesis in vivo. Reverse transcriptase-polymerase chain reaction and immunohistochemistry were used to study the expression of Pim-1 in oral squamous cell carcinoma (OSCC) and non-cancerous match tissue (NCMT) sampled from the periphery of the tumours. Pim-1 mRNA expression in OSCC was significantly higher than that in NCMT in 36 tissue pairs (1.33+/-0.41 versus 0.97+/-0.29, P=0.03). The percentage of OSCCs exhibiting strong cytoplasmic Pim-1 immunoreactivity was significantly higher than that of NCMT (60% versus 19%, P=0.007). Pim-1 immunoreactivity is higher in the more differentiated components of a tumour. In around 10% of OSCC cases, Pim-1 immunoreactivity was found in the nucleus as well. These results show novel findings of the up-regulation of Pim-1 expression from NCMT to OSCC. The pathogenetic role of Pim-1 expression in oral tumourigenesis deserves further investigation.

Pim-1 is an oncogene activated in mouse T-cell lymphomas induced by Moloney and AKR mink cell focus (MCF) viruses. Pim-1 was previously mapped to chromosome 17 by somatic cell hybrids, and subsequently to the region between the hemoglobin alpha-chain pseudogene 4 (Hba-4ps) and the alpha-crystalline gene (Crya-1) by Southern blot analysis of DNA obtained from panels of recombinant inbred strains. We have now mapped Pim-1 more accurately in t-haplotypes by analysis of recombinant t-chromosomes. The recombinants were derived from Tts6tf/t12 parents backcrossed to + tf/ + tf, and scored for recombination between the loci of T and tf. For simplicity ALL t-complex lethal genes properly named tcl-tx are shortened to tx. The Pim-1 gene was localized 0.6 cM proximal to the tw12 lethal gene, thus placing the Pim-1 gene 5.2 cM distal to the H-2 region in t-haplotypes. Once mapped, the Pim-1 gene was used as a marker for further genetic analysis of t-haplotypes. tw12 is so close to tf that even with a large number of recombinants it was not possible to determine whether it is proximal or distal to tf. Southern blot analysis of DNA from T-tf recombinants with a separation of tw12 and tf indicated that tw12 is proximal to tf. The mapping of two allelic t-lethals, t0 and t6 with respect to tw12 and tf has also been a problem.(ABSTRACT TRUNCATED AT 250 WORDS)FAU - Ark, B

The serine/threonine kinase human Pim1 (hereafter PIM1) cooperates with human c-Myc (hereafter MYC) in cell cycle progression and tumorigenesis. However, the nature of this cooperation is still unknown. Here we show that, after stimulation with growth factor, PIM1 forms a complex with the dimer of MYC with MAX (Myc-associated factor X) via the MYC BoxII (MBII) domain. MYC recruits PIM1 to the E boxes of the MYC-target genes FOSL1 (FRA-1) and ID2, and PIM1 phosphorylates serine 10 of histone H3 (H3S10) on the nucleosome at the MYC-binding sites, contributing to their transcriptional activation. MYC and PIM1 colocalize at sites of active transcription, and expression profile analysis revealed that PIM1 contributes to the regulation of 20% of the MYC-regulated genes. Moreover, PIM1-dependent H3S10 phosphorylation contributes to MYC transforming capacity. These results establish a new function for PIM1 as a MYC cofactor that phosphorylates the chromatin at MYC-target loci and suggest that nucleosome phosphorylation, at E boxes, contributes to MYC-dependent transcriptional activation and cellular transformation.

The V617F activating point mutation in Jak2 is associated with a proportion of myeloproliferative disorders. In normal hematopoietic cells, Jak2 signals only when associated with a growth factor receptor, such as the erythropoietin receptor (EpoR). We sought to identify the molecular requirements for activation of Jak2V617F by introducing a point mutation in the FERM domain (Y114A), required for receptor binding. Whereas BaF3.EpoR cells are readily transformed by Jak2V617F to Epo independence, we found that the addition of the FERM domain mutation blocked transformation and the induction of reactive oxygen species. Further, while cells expressing Jak2V617F had constitutive activation of STAT5, cells expressing Jak2V617F/Y114A did not, suggesting that signaling is defective at a very proximal level. In addition, expression of the Myc and Pim proto-oncogenes by Jak2V617F was found to be FERM domain dependent. An inducible constitutively active STAT5 mutant expressed in BaF3 cells was sufficient to induce Myc and Pim. Finally, the FERM domain in Jak2V617F was also required for abnormal hematopoiesis in transduced primary murine fetal liver cells. Overall, our results suggest that constitutive activation of Jak2 requires an intact FERM domain for a transforming phenotype, and is necessary for activation of the major target of Jak2, STAT5.

The pim-1 gene is frequently found activated by proviral insertion in murine T cell lymphomas. Overexpression of pim-1 in lymphoid cells by transgenesis formally proved its oncogenic potential. The pim-1 cDNA sequence predicts that both murine and human pim-1 encode a 34 kd protein with homology to protein kinases. In this study, we show that the murine pim-1 gene encodes a 44 kd protein in addition to the predicted 34 kd protein. The 44 kd protein is an amino-terminal extension of the 34 kd protein and is synthesized by alternative translation initiation at an upstream CUG codon. Contrary to previous findings by others, we provide evidence that both murine and human pim-1 gene products are protein-serine/threonine kinases. Murine 44 kd and 34 kd pim-1 proteins exhibit comparable in vitro kinase activity and are both mainly cytoplasmic, but they differ in in vivo association state and half-life.

mutation of the p53 gene is a common event during tumor pathogenesis. Other mechanisms, such as mdm2 amplification, provide alternative routes through which dysfunction of the p53 pathway is promoted. Here, we address the hypothesis that elevated expression of pim oncogenes might suppress p53 by regulating Mdm2. At a physiological level, we show that endogenous Pim-1 and Pim-2 interact with endogenous Mdm2. Additionally, the Pim kinases phosphorylate Mdm2 in vitro and in cultured cells at Ser(166) and Ser(186), two previously identified targets of other signaling pathways, including Akt. Surprisingly, at high levels of Pim expression, as would occur in tumors, active, but not inactive, Pim-1 or Pim-2 blocks the degradation of both p53 and Mdm2 in a manner that is independent of Mdm2 phosphorylation, leading to increased p53 levels and, proportionately, p53-dependent transactivation. Additionally, Pim-1 induces endogenous ARF, p53, Mdm2, and p21 in primary murine embryo fibroblasts and stimulates senescence-associated beta-galactosidase levels, consistent with the induction of senescence. Immunohistochemical analysis of a cohort of 33 human mantle cell lymphomas shows that elevated expression of Pim-1 occurs in 42% of cases, with elevated Pim-2 occurring in 9% of cases, ALL of which also express Pim-1. Notably, elevated Pim-1 correlates with elevated Mdm2 in MCL with a p value of 0.003. Taken together, our data are consistent with the idea that Pim normally interacts with the p53 pathway but, when expressed at pathological levels, behaves as a classic dominant oncogene that stimulates a protective response through induction of the p53 pathway.

mRNA expression of the serine/threonine kinase protooncogene Pim-1 was investigated in mitogen-treated normal bovine lymphocytes. After stimulation with Con A and phorbol ester (PMA), normal bovine PBMC exhibited a 3.5-fold induction of pim-1 mRNA within 4 h of stimulation. By 17 h poststimulation, however, the level of pim-1 mRNA had fallen to approximately 50%. Similar transient kinetics of pim-1 expression were also observed in mitogen-stimulated bovine lymph node lymphocytes and the amount of pim-1 mRNA induced was dependent on the type of mitogen stimulus. Typically, stimulation with Con A and PMA together acted synergistically resulting in a greatly increased amount of pim-1 mRNA induction compared to stimulation with either Con A or PMA alone. To determine if an increase in pim-1 mRNA stability contributed to the overall increase in pim-1 mRNA levels observed after mitogen stimulation, RNA half-life studies were performed. At 4 h poststimulation, pim-1 mRNA in bovine lymph node lymphocytes was relatively stable with a t1/2 of approximately 80 min; at 17 h poststimulation, however, the t1/2 (approximately 35 min) was markedly decreased. In addition, cyclohexamide treatment was found to markedly increase the stability of pim-1 transcripts in bovine PBMC suggesting that a protein synthesis-dependent posttranscriptional pim-1 mRNA degradation pathway may be involved in the regulation of pim-1 mRNA levels in lymphoid cells. To investigate the possible contribution of the destabilizing (UAUU)n motif to pim-1 mRNA stability, the t1/2 of the short 2.4-kb germ cell-specific pim-1 transcript found in rat testes was compared to that of the longer 2.8-kb somatic transcript expressed in stimulated rat lymphocytes. t1/2 determination experiments showed that the 2.4-kb testes-specific transcript, which is missing the destabilizing (UAUU)n sequence, was quite stable (t1/2 much greater than 6 h). In contrast, the t1/2 of the longer 2.8 kb somatic cell pim-1 transcript that contains this A/U-rich sequence motif was found to be much shorter (t1/2 approximately 130 min) in mitogen-activated rat lymphocytes. Together these findings indicate that the transient induction of pim-1 gene expression is associated with normal lymphocyte activation and that the stability of pim-1 transcripts is regulated in lymphocytes during the course of activation. In addition, the differences in pim-1 mRNA stability observed in germ cells and lymphocytes of the same animal are likely the result of molecular mechanisms involving differential regulation by the message destabilizing (UAUU)n motif.

BACKGROUND: PIM kinases are mediators of cytokine signalling pathways in hematopoietic cells and contribute to the progression of certain types of leukemia and solid tumor. Here the prognostic impact of proto-oncogene PIM-1 was analyzed in gastric carcinoma. PATIENTS AND METHODS: cancer tissues of 117 patients with potentially curative (R0) resections for gastric cancer were immunohistochemically stained for PIM-1. RESULTS: Cytoplasmic immunoreactivity for PIM-1 in tumor (46%) was higher (p=0.003) compared to that in gastric glands (16%) and foveolae (1%). PIM-1 immunoreactivity in gastric carcinoma correlated with tumor grading (p<0.05) and Lauren category (p<0.02). Overexpression of PIM-1 in gastric glands (p<0.001) was associated with formation of lymph node metastases (p=0.035) and survival (p=0.04). Multivariate analysis of PIM-1 expression in gastric glands confirmed its association with prognosis. CONCLUSION: Up-regulation of PIM-1 oncogene might be a tumor marker for gastric cancer. The correlation of PIM-1 overexpression in gastric glands with formation of lymph node metastases and survival proposes a prognostic role in gastric cancer.

Oncogenic Pim-1 kinase is upregulated in multiple solid cancers, including human pancreatic ductal adenocarcinoma (PDAC), a highly lethal disease with few useful treatment options. Pim-1 is also transcriptionally induced upon oncogenic K-Ras-mediated transformation of the human pancreatic ductal epithelial (HPDE) cell model of PDAC. Given the near ubiquitous presence of mutant K-Ras in PDAC and its critical role in this disease, we wished to study the effects of oncogenic K-Ras signaling on Pim-1 expression, as well as the role of Pim-1 in growth transformation of PDAC cells. Pim-1 protein levels were upregulated in both PDAC cell lines and patient tumor tissues. Furthermore, ectopic oncogenic K-Ras increased Pim-1 expression in human pancreatic nestin-expressing (HPNE) cells, a distinct immortalized cell model of PDAC. Conversely, shRNA-mediated suppression of oncogenic K-Ras decreased Pim-1 protein in PDAC cell lines. These results indicate that oncogenic K-Ras regulates Pim-1 expression. The kinase activity of Pim-1 is constitutively active. Accordingly, shRNA-mediated suppression of Pim-1 in K-Ras-dependent PDAC cell lines decreased Pim-1 activity, as measured by decreased phosphorylation of the pro-apoptotic protein Bad and increased expression of the cyclin-dependent kinase inhibitor p27Kip1. Biological consequences of inhibiting Pim-1 expression included decreases in both anchorage-dependent and -independent cell growth, invasion through Matrigel and radioresistance as measured by standard clonogenic assays. These results indicate that Pim-1 is required for PDAC cell growth, invasion and radioresistance downstream of oncogenic K-Ras. Overall, our studies help to elucidate the role of Pim-1 in PDAC growth transformation and validate Pim-1 kinase as a potential molecular marker for mutated K-Ras activity.

The Class III beta-tubulin isotype (betaIII-tubulin) is a predictive biomarker in ovarian cancer and other solid tumor malignancies. We discovered that betaIII-tubulin function is linked to two GTPases: guanylate-binding protein 1 (GBP1), which activates its function, and GNAI1, which inhibits it. This finding was demonstrated in a panel of ovarian cancer cells resistant to several chemotherapeutic agents. Using a protein microarray, we identified PIM1 as the downstream partner of GBP1, recruited into the cytoskeleton under hypoxic conditions. The clinical value of these observations was tested by performing an archive study of 98 ovarian cancer patients, which demonstrated that the betaIII-tubulin -/PIM1- cohort responded to treatment, exhibiting long overall survival (OS), while betaIII-tubulin +/PIM+ patients experienced poor outcomes and OS times similar to patients receiving palliation alone. betaIII-tubulin expression is commonly believed responsible for paclitaxel resistance due to its enhancement of the dynamic instability of microtubules, which counteracts the activity of taxanes. In contrast, our research reveals that betaIII-tubulin behaves as a gateway for prosurvival signals, such as PIM1, to move into the cytoskeleton. When cells are exposed to microenvironmental stressors, they activate this pathway by telling the cytoskeleton to incorporate PIM1 through GBP1 and betaIII-tubulin, which ultimately leads to drug resistance. This discovery reveals that betaIII-tubulin does not act alone but requires partners to play its role. The discovery of such protein:protein interactions underlying this prosurvival cascade makes feasible the development of therapeutic approaches using novel compounds that are capable of inhibiting the transmission of prosurvival signals into the cytoskeleton.

Resistance to chemotherapeutic drugs is a significant clinical problem for the treatment of cancer patients and has been linked to the activation of survival pathways and expression of multidrug efflux transporters. Thus inhibition of these survival pathways or efflux transporter expression may increase the efficacy of drug treatment. Here we review the role of the oncogenic PIM kinase family in regulating important proliferation and survival pathways in cancer cells and the involvement of PIM kinases in the expression and activity of MDR-1 and BCRP, two of the most important drug efflux transporters. PIM kinases are over expressed in various types of tumors and regulate the activation of signaling pathways that are important for tumor cell proliferation, survival and expression of drug efflux proteins. This makes PIM kinases attractive targets for the development of anti-cancer chemotherapeutic drugs. Focussing mainly on solid tumors, we provide an update on the literature describing the tumorigenic functions of PIM kinases. Also we provide an overview of the development of selective small molecule PIM kinase inhibitors. Because of the intense effort by pharmaceutical companies and academia it is reasonable to expect that PIM kinase inhibitors will enter the clinic in the foreseeable future. We therefore finish this review with a discussion on the most efficient application of these PIM inhibitors. This includes a consideration of which tumor type is the most appropriate target for treatment, how to select the patient population that stands to gain the most from treatment with PIM inhibitors, which molecular markers are suitable to follow the course of treatment and whether PIM kinase inhibitors should be used as monotherapy or in combination with other cytotoxic agents.

OBJECTIVE: Vascular remodeling diseases (VRD) are mainly characterized by inflammation and a vascular smooth muscle cells (VSMCs) proproliferative and anti-apoptotic phenotype. Recently, the activation of the advanced glycation endproducts receptor (RAGE) has been shown to promote VSMC proliferation and resistance to apoptosis in VRD in a signal transducer and activator of transcription (STAT)3-dependant manner. Interestingly, we previously described in both cancer and VRD that the sustainability of this proproliferative and antiapoptotic phenotype requires activation of the transcription factor NFAT (nuclear factor of activated T-cells). In cancer, NFAT activation is dependent of the oncoprotein provirus integration site for Moloney murine leukemia virus (Pim1), which is regulated by STAT3 and activated in VRD. Therefore, we hypothesized that RAGE/STAT3 activation in VSMC activates Pim1, promoting NFAT and thus VSMC proliferation and resistance to apoptosis. Methods/Results- In vitro, freshly isolated human carotid VSMCs exposed to RAGE activator Nepsilon-(carboxymethyl)lysine (CML) for 48 hours had (1) activated STAT3 (increased P-STAT3/STAT3 ratio and P-STAT3 nuclear translocation); (2) increased STAT3-dependent Pim1 expression resulting in NFATc1 activation; and (3) increased Pim1/NFAT-dependent VSMC proliferation (PCNA, Ki67) and resistance to mitochondrial-dependent apoptosis (TMRM, Annexin V, TUNEL). Similarly to RAGE inhibition (small interfering RNA [siRNA]), Pim1, STAT3 and NFATc1 inhibition (siRNA) reversed these abnormalities in human carotid VSMC. Moreover, carotid artery VSMCs isolated from Pim1 knockout mice were resistant to CML-induced VSMC proliferation and resistance to apoptosis. In vivo, RAGE inhibition decreases STAT3/Pim1/NFAT activation, reversing vascular remodeling in the rat carotid artery-injured model. CONCLUSIONS: RAGE activation accounts for many features of VRD including VSMC proliferation and resistance to apoptosis by the activation of STAT3/Pim1/NFAT axis. Molecules aimed to inhibit RAGE could be of a great therapeutic interest for the treatment of VRD.

We have identified a conserved G-quadruplex forming sequence in the 3 untranslated region (UTR) of the proto-oncogene PIM1. Circular dichroism and thermal denaturation studies revealed that the PIM1 mRNA guanine-rich sequence forms a highly stable intramolecular G-quadruplex structure. Reporter gene assay demonstrated that the PIM1 RNA G-quadruplex represses translation. It is the first experimental evidence of an RNA G-quadruplex structure located in the 3 UTR that may act as posttranscriptional regulator.

The constitutively active serine/threonine kinase Pim-1 is upregulated in different cancer types, mainly based on the action of several interleukines and growth factors at the transcriptional level. So far, a regulation of oncogenic Pim-1 by microRNAs (miRNAs) has not been reported. Here, we newly establish miR-33a as a miRNA with potential tumor suppressor activity, acting through inhibition of Pim-1. A screen for miRNA expression in K562 lymphoma, LS174T colon carcinoma and several other cell lines revealed generally low endogenous miR-33a levels relative to other miRNAs. Transfection of K562 and LS174T cells with a miR-33a mimic reduced Pim-1 levels substantially. In contrast, the cell-cycle regulator cyclin-dependent kinase 6 predicted to be a conserved miR-33a target, was not downregulated by the miR-33a mimic. Seed mutagenesis of the Pim-1 3-untranslated region in a luciferase reporter construct and in a Pim-1 cDNA expressed in Pim-1-deficient Skov-3 cells demonstrated specific and direct downregulation of Pim-1 by the miR-33a mimic. The persistence of this effect was comparable to that of a small interfering RNA-mediated knockdown of Pim-1, resulting in decelerated cell proliferation. In conclusion, we demonstrate the potential of miR-33a to act as a tumor suppressor miRNA, which suggests miR-33a replacement therapy through delivery of miR mimics as a novel therapeutic strategy.

A full-length (6.1-kb) human genomic pim-1 gene, together with its immediate 5 -upstream promoter sequence (Ppim) was isolated and sequenced. The human pim-1 gene shares an overall nucleotide (nt) sequence identity of 53% with the previously reported murine pim-1 gene. It consists of six exons and five introns and contains a protein-coding region that is identical in nt sequence to a full-length human pim-1 cDNA. The gene codes for a predicted Pim-1 protein of 313 amino acids (aa) with an Mr of 35,690 and a pI of 5.7. The deduced aa sequence of the human Pim-1 has 94% identity with the murine Pim-1 whereas the nt sequences of the two genes are 88% identical. ALL of the conserved aa residues of the mouse pim-1 gene, which are homologous to known protein kinases are conserved in the predicted human protein. The human Ppim region is very G + C-rich (69%) and shares greater than 80% identity with the murine Ppim. The Ppim has no TATA- or CAAT-box sequences but does contain a number of nt sequences similar to the putative binding sites of several presumptive transcription factors.

The serine/threonine kinase Pim-1 directs selected signaling events that promote cell growth and survival and is overexpressed in diverse human cancers. Pim-1 expression is tightly controlled through multiple mechanisms, including regulation of mRNA turnover. In several cultured cell models, mitogenic stimulation rapidly induced and stabilized PIM1 mRNA, however, vigorous destabilization 4-6 hours later helped restore basal expression levels. Acceleration of PIM1 mRNA turnover coincided with accumulation of tristetraprolin (TTP), an mRNA-destabilizing protein that targets transcripts containing AU-rich elements. TTP binds PIM1 mRNA in cells, and suppresses its expression by accelerating mRNA decay. Reporter mRNA decay assays localized the TTP-regulated mRNA decay element to a discrete AU-rich sequence in the distal 3 -untranslated region that binds TTP. These data suggest that coordinated stimulation of TTP and PIM1 expression limits the magnitude and duration of PIM1 mRNA accumulation by accelerating its degradation as TTP protein levels increase. Consistent with this model, PIM1 and TTP mRNA levels were well correlated across selected human tissue panels, and PIM1 mRNA was induced to significantly higher levels in mitogen-stimulated fibroblasts from TTP-deficient mice. Together, these data support a model whereby induction of TTP mediates a negative feedback circuit to limit expression of selected mitogen-activated genes.

Specific genes can be inactivated or mutated in the mouse germ line. The phenotypic consequences of the mutation can provide pivotal information on the function of the gene in development and maintenance of the mammalian organism. The procedure entails homologous recombination in embryonic stem cells, which, on fusion to recipient blastocysts, give rise to chimaeric mice that can transmit the mutant gene to their offspring. Inbreeding can then yield mice carrying the mutation in both alleles allowing the phenotypic analysis of recessive mutations. In addition to mice lacking a particular gene function, cell lines carrying null alleles of normally expressed genes can be instrumental in assessing the function of the gene. These cell lines can either be obtained from homozygous animals or, should the mutation be lethal early in embryonic development, be generated by consecutive inactivation of both alleles by homologous recombination in cultured cells. Here we illustrate the feasibility of this latter approach by the efficient consecutive inactivation of both alleles of the pim-1 proto-oncogene in embryonic stem cells.

A new chemical series, triazolo[4,5-b]pyridines, has been identified as an inhibitor of PIM-1 by a chemotype hopping strategy based on a chemically feasible fragment database. In this case, structure-based virtual screening and in silico chemogenomics provide added value to the previously reported strategy of prioritizing among proposed novel scaffolds. Pairwise comparison between compound 3, recently discontinued from Phase I clinical trials, and molecule 8, bearing the selected novel scaffold, shows that the primary activities are similar (IC(50) in the 20 to 150 nM range). At the same time, some ADME properties (for example, an increase of more than 45% in metabolic stability in human liver microsomes) and the off-target selectivity (for example, an increase of more than 2 log units in IC(50)vs. FLT3) are improved, and the intellectual property (IP) position is enhanced. The discovery of a reliable starting point that fulfills critical criteria for a plausible medicinal chemistry project is demonstrated in this prospective study.

Loss of NKX3.1 is an early and consistent event in prostate cancer and is associated with increased proliferation of prostate epithelial cells and poor prognosis. NKX3.1 stability is regulated post-translationally through phosphorylation at multiple sites by several protein kinases. Here, we report the paradoxical stabilization of the prostate-specific tumor suppressor NKX3.1 by the oncogenic protein kinase Pim-1 in prostate cancer cells. Pharmacologic Pim-1 inhibition using the small molecule inhibitor CX-6258 decreased steady state levels and half-life of NKX3.1 protein but mRNA was not affected. This effect was reversed by inhibition of the 26S-proteasome, demonstrating that Pim-1 protects NKX3.1 from proteasome-mediated degradation. Mass spectrometric analyses revealed Thr89, Ser185, Ser186, Ser195, and Ser196 as Pim-1 phospho-acceptor sites on NKX3.1. Through mutational analysis, we determined that NKX3.1 phosphorylation at Ser185, Ser186, and within the N-terminal PEST domain is essential for Pim-1-mediated stabilization. Further, we also identified Lys182 as a critical residue for NKX3.1 stabilization by Pim-1. Pim-1-mediated NKX3.1 stabilization may be important in maintaining normal cellular homeostasis in normal prostate epithelial cells, and may maintain basal NKX3.1 protein levels in prostate cancer cells.

We searched ERalpha cistromes of MCF-7 breast cancer cells for previously unrecognized ERalpha targets and identified proto-oncogene PIM-1 as a novel potential target gene. We show that the expression of PIM-1 is induced in response to estradiol in MCF-7 cells and that the induction is mediated by ERalpha-regulated enhancers located distally upstream from the gene. In keeping with the growth-promoting role of the PIM-1, depletion of the PIM-1 attenuated the proliferation of the MCF-7 cells, which was paralleled with up-regulation of cyclin-dependent protein kinase inhibitor CDKN1A and CDKN2B expression. Analysis of PIM-1 expression between invasive breast tumors and benign breast tissue samples showed that elevated PIM-1 expression is associated with malignancy and a higher tumor grade. In sum, identification of PIM-1 as an ERalpha target gene adds a novel potential mechanism by which estrogens can contribute to breast cancer cell proliferation and carcinogenesis.

Our objective was to profile genetic pathways whose differential expression correlates with maturation of visual function in zebrafish. Bioinformatic analysis of transcriptomic data revealed Jak-Stat signalling as the pathway most enriched in the eye, as visual function develops. Real-time PCR, western blotting, immunohistochemistry and in situ hybridization data confirm that multiple Jak-Stat pathway genes are up-regulated in the zebrafish eye between 3-5 days post-fertilisation, times associated with significant maturation of vision. One of the most up-regulated Jak-Stat genes is the proto-oncogene Pim1 kinase, previously associated with haematological malignancies and cancer. Loss of function experiments using Pim1 morpholinos or Pim1 inhibitors result in significant diminishment of visual behaviour and function. In summary, we have identified that enhanced expression of Jak-Stat pathway genes correlates with maturation of visual function and that the Pim1 oncogene is required for normal visual function.

U1 small nuclear interference (U1i) has recently been described as a novel gene silencing mechanism. U1i employs short oligonucleotides, so-called U1 adaptors, for specific gene knockdown, expanding the field of current silencing strategies that are primarily based on RNA interference (RNAi) or antisense. Despite the potential of U1 adaptors as therapeutic agents, their in vivo application has not yet been studied. Here we explore U1i by analyzing U1 adaptor-mediated silencing of the oncogene Pim-1 in glioblastoma cells. We have generated Pim-1-specific U1 adaptors comprising DNA, locked nucleic acids (LNA), and 2 -O-Methyl RNA and demonstrate their ability to induce a Pim-1 knockdown, leading to antiproliferative and pro-apoptotic effects. For the therapeutic in vivo application of U1 adaptors, we establish their complexation with branched low molecular weight polyethylenimine (PEI). Upon injection of nanoscale PEI/adaptor complexes into subcutaneous glioblastoma xenografts in mice, we observed the knockdown of Pim-1 that resulted in the suppression of tumor growth. The absence of hepatotoxicity and immune stimulation also demonstrates the biocompatibility of PEI/adaptor complexes. We conclude that U1i represents an alternative to RNAi for the therapeutic silencing of pathologically upregulated genes and demonstrate the functional relevance of Pim-1 oncogene knockdown in glioblastoma. We furthermore introduce nanoscale PEI/adaptor complexes as efficient and safe for in vivo application, thus offering novel therapeutic approaches based on U1i-mediated gene knockdown.

A new method is applied to synthesize hispidulin, a natural flavone with a broad spectrum of biological activities. Hispidulin exhibits inhibitory activity against the oncogenic protein kinase Pim-1. Crystallographic analysis of Pim-1 bound to hispidulin reveals a binding mode distinct from that of quercetin, suggesting that the binding potency of flavonoids is determined by their hydrogen-bonding interactions with the hinge region of the kinase. Overall, this work may facilitate construction of a library of hispidulin-derived compounds for investigating the structure-activity relationship of flavone-based Pim-1 inhibitors.

Previously, it has been shown that pancreatic ductal adenocarcinoma (PDA) tumors exhibit high levels of hypoxia, characterized by low oxygen pressure (pO2) and decreased O2 intracellular perfusion. Chronic hypoxia is strongly associated with resistance to cytotoxic chemotherapy and chemoradiation in an understudied phenomenon known as hypoxia-induced chemoresistance. The hypoxia-inducible, pro-oncogenic, serine-threonine kinase PIM1 (Proviral Integration site for Moloney murine leukemia virus 1) has emerged as a key regulator of hypoxia-induced chemoresistance in PDA and other cancers. Although its role in therapeutic resistance has been described previously, the molecular mechanism behind PIM1 overexpression in PDA is unknown. Here, we demonstrate that cis-acting AU-rich elements (ARE) present within a 38-base pair region of the PIM1 mRNA 3 -untranslated region mediate a regulatory interaction with the mRNA stability factor HuR (Hu antigen R) in the context of tumor hypoxia. Predominantly expressed in the nucleus in PDA cells, HuR translocates to the cytoplasm in response to hypoxic stress and stabilizes the PIM1 mRNA transcript, resulting in PIM1 protein overexpression. A reverse-phase protein array revealed that HuR-mediated regulation of PIM1 protects cells from hypoxic stress through phosphorylation and inactivation of the apoptotic effector BAD and activation of MEK1/2. Importantly, pharmacological inhibition of HuR by MS-444 inhibits HuR homodimerization and its cytoplasmic translocation, abrogates hypoxia-induced PIM1 overexpression and markedly enhances PDA cell sensitivity to oxaliplatin and 5-fluorouracil under physiologic low oxygen conditions. Taken together, these results support the notion that HuR has prosurvival properties in PDA cells by enabling them with growth advantages in stressful tumor microenvironment niches. Accordingly, these studies provide evidence that therapeutic disruption of HuR s regulation of PIM1 may be a key strategy in breaking an elusive chemotherapeutic resistance mechanism acquired by PDA cells that reside in hypoxic PDA microenvironments.Oncogene advance online publication, 21 September 2015; doi:10.1038/onc.2015.325.

Proviral integration near the Pim-1 gene is frequently observed in murine leukemia virus induced T-cell lymphomas in mice. Integration in the Pim-1 domain is associated with the presence of enhanced levels of a Pim-1 mRNA, which is normally expressed as a predominant 2.8 kb species at low levels in lymphoid tissues. The majority of integrations occurred in the 3 region of the Pim-1 transcription unit. This resulted in transcripts ranging in size from 2.0 to 2.6 kb, which were terminated in the 5 proviral LTR. Dependent on the site of integration up to 1300 bases of Pim-1 specific sequences were missing from the modified Pim-1 mRNA in these lymphomas.

Pim-1 is a putative oncogene activated in T-cell lymphomas induced by Moloney and AKR mink cell focus forming (MCF) viruses. We have determined the chromosomal localization of the Pim-1 gene in mice by Southern blot analysis of DNAs obtained from a panel of mouse-Chinese hamster somatic cell hybrids. The Pim-1 gene was localized on chromosome 17, a chromosome frequently aberrant in T-cell lymphomas. Two chromosomal regions, containing sequences homologous to regions within the Pim-1 locus, were localized on chromosome 6 and 16.

We have shown previously that the putative oncogene pim-1 is frequently activated by provirus insertion in murine leukemia virus-induced T cell lymphomas. Here we describe the structure of the pim-1 gene as determined by sequencing genomic and cDNA clones. The gene has an open reading frame, encoding a protein of 313 amino acids, extending over six exons and preceded and followed by stop codons in ALL reading frames. Proviruses always integrate outside the protein-encoding domain, showing a high preference for a small region in the 3 -terminal exon; integration in the 3 exon results in relatively high levels of pim-1 mRNA. Computer search reveals homology between pim-1 and protein kinases: ALL the domains characteristic of protein kinases are conserved in the pim-1 amino acid sequence. The highest homologies were observed with the protein-serine kinases.

The mouse PIM-1 gene is involved in the pathogenesis of virally-induced mouse lymphomas. We have cloned and analyzed the human homologue of the mouse PIM-1 gene to investigate its role in human lymphoma and leukemia. Overlapping cDNA clones from a K562 (human erythroleukemia cell line) library were isolated and sequenced. The deduced amino acid sequence showed significant homology to a number of the protein kinases but did not have a transmembrane region. Genomic clones from the 380 cell line (human B cell leukemia) were analyzed. The PIM-1 transcript was found to derive from 5 Kb of genomic DNA. Six exons and five introns were identified. The promoter region has no TATA or CAAT boxes, but did have multiple potential Sp1 binding sites (CCGCCC). Studies of expression of this gene using Northern blots of human cell lines showed it to be transcribed primarily in B lymphoid and myeloid cell lines. The characterization of the human PIM-1 gene will allow the definition of its role in hemopoietic malignancies and in hematolymphoid differentiation.

The mouse PIM-1 gene has been implicated in the evolution of retrovirus-associated mouse lymphomas. We have initiated a study of the human PIM-1 gene because of its potential importance as a human oncogene. We have isolated genomic and cDNA clones for this gene and characterized this locus in detail. The predicted PIM-1 protein is 313 amino acids in length. It has homology to a number of the protein kinases but does not have a transmembrane region. The amino acid corresponding to tyrosine-416 of pp60v-src is a tyrosine (position 198), which is consistent with the hypothesis that PIM-1 is a tyrosine kinase rather than a serine-threonine kinase. The PIM-1 gene was found to have six exons and five introns derived from 5 kb of genomic DNA. The site of transcription initiation was localized by S1 nuclease protection studies which indicated that the mature PIM-1 mRNA was approximately 2.7 kb in length. The promotor of this gene had no TATA or CAAT box but did have multiple GC boxes (CCGCCC) that might bind the Sp1 protein. The PIM-1 gene was expressed in myeloid and B lymphoid cell lines, but not in T lymphoid and nonhemopoietic lines. This initial characterization of PIM-1 will allow us to define its role in normal and malignant hematolymphoid differentiation.

The human homolog, hpim, of the murine pim-1 gene, which is activated in murine T-cell lymphomas by insertion of retrovirus proviral genomes in the pim-1 region, has been molecularly cloned; the cloned probe has been used to map the hpim locus to human chromosome region 6p21 by somatic cell hybrid analysis and chromosomal in situ hybridization. The hpim gene is expressed as a 3.2-kilobase mRNA in various human cell lines of hematopoietic lineage, most dramatically in the K562 erythroleukemia cell line, which contains a cytogenetically demonstrable rearrangement in the 6p21 region. A characteristic chromosome anomaly, a reciprocal translocation t(6;9)(p21;q33), has been described in myeloid leukemias and could involve the hpim gene.

The putative oncogene pim-1 is frequently activated by provirus insertion in MuLV-induced T cell lymphomas in mice. By analogy with other cellular oncogenes that are similarly activated (e.g., myc, myb, erbB) it is possible that pim-1 may also be involved in some human tumors. To study human pim-1 we cloned and sequenced human pim-1 cDNA clones. The human pim-1 codes for a protein of 313 amino acids (aa) which is highly homologous (94%) to the deduced amino acid sequence of mouse pim-1. ALL the mouse pim-1 residues which are homologous to protein kinases are conserved in the human pim-1. We also isolated the human pim-1 gene and mapped it relative to the cDNA. The RNA transcript of human pim-1 is approx. 3.0 kb and it is highly expressed in the erythroleukemia cell line K562.

Pim-1, a putative oncogene involved in T-cell lymphomagenesis, was mapped between the pseudo-alpha globin gene Hba-4ps and the alpha-crystallin gene Crya-1 on mouse chromosome 17 and therefore within the t complex. Pim-1 restriction fragment variants were identified among t haplotypes. Analysis of restriction fragment sizes obtained with 12 endonucleases demonstrated that the Pim-1 genes in some t haplotypes were indistinguishable from the sizes for the Pim-1b allele in BALB/c inbred mice. There are now three genes, Pim-1, Crya-1 and H-2 I-E, that vary among independently derived t haplotypes and that have indistinguishable alleles in t haplotypes and inbred strains. These genes are closely linked within the distal inversion of the t complex. Because it is unlikely that these variants arose independently in t haplotypes and their wild-type homologues, we propose that an exchange of chromosomal segments, probably through double crossingover, was responsible for indistinguishable Pim-1 genes shared by certain t haplotypes and their wild-type homologues. There was, however, no apparent association between variant alleles of these three genes among t haplotypes as would be expected if a single exchange introduced these alleles into t haplotypes. If these variant alleles can be shown to be identical to the wild-type allele, then lack of association suggests that multiple exchanges have occurred during the evolution of the t complex.

The proto-oncogene pim-1 was expressed in nine human tissues (including epidermis) examined by Northern blotting. expression of pim-1 was also observed in a number of carcinoma-derived keratinocyte lines in addition to strains derived from normal epidermis. With the exception of a squamous carcinoma line that exhibits little differentiated character in culture (SCC-4), where it was not detected, pim-1 expression was substantially higher after confluence than during log-phase growth in each case. The differentiation marker keratinocyte transglutaminase showed the same pattern of expression as pim-1 in relation to confluence in each of the cell lines and strains studied. The influences on pim-1 mRNA levels of several known effectors of keratinocyte differentiation were studied in the squamous carcinoma line SCC-9. pim-1 mRNA was stimulated by hydrocortisone and suppressed by the tumor promoter tetradecanoyl phorbol acetate. pim-1 mRNA was also regulated by calcium ion concentration in the culture medium, with expression being threefold higher in 0.15 mM than in 0.03 mM calcium ion. Keratinocyte transglutaminase was regulated similarly by these effectors. Thus pim-1 expression was associated with keratinocyte differentiation in these cultured cells.

The Pim-1 proto-oncogene is one of the most potent collaborators of the myc proto-oncogenes in inducing lymphomagenesis in mice. Contrary to the profound effects when overexpressed in vivo, Pim-1-deficient mice showed only subtle phenotypic alterations, which could indicate the presence of redundantly acting genes. In line with this, a PCR-based screen has led to the identification of a closely homologous gene, Pim-2. The X-linked Pim-2 gene is 53% identical to Pim-1 at the amino acid level and shares substrate preference and the usage of non-AUG initiation codons with Pim-1. We have used these data to test whether the strong synergistic interaction between Pim-1 and c-myc can be utilized to gain access to Pim-1 compensatory pathways. We reasoned that, upon proviral tagging in compound mutant mice (E mu-myc/Pim-1-/- mice), the selective advantage of cells carrying provirally activated genes, that act downstream from or parallel to Pim-1, would increase. We show here that this is the case. A dramatic increase (from 15 to 80%) was found in the frequency of proviral activation of the Pim-2 gene. These data show that the described strategy of complementation tagging represents a powerful new tool to identify components of pathways involved in processes as complex as multistep tumorigenesis.

A homopurine-homopyrimidine region of murine c-pim-1 proto-oncogene was chosen as a target for triple-helix-forming oligonucleotide. Oligonucleotide 5 -GGG-GAGGGGGAGG-3 was shown to bind to its target sequence in the presence of 50 mM Na+ or K+, 10 mM MgCl2 and 20 mM Tris-acetate, pH 7.5. This oligonucleotide is bound in an antiparallel orientation with respect to the homopurine sequence. As was shown by co-migration assay the triplex is stable up to 65 degrees C. At 37 degrees C it was practically irreversible: after 24 hours of co-migration assay there was no traces of triplex dissociation. The rate of triplex formation was highly accelerated with increase of temperature and Mg2+ concentration. This rate was higher for superhelical DNA when compared to the linear and circular ones and the preference was dependent from temperature and Mg2+ concentration. The precision of this interaction is extremely high: sequences in c-pim-1 promoter region with only one substitution when compared to the target gave negligible triplex formation under investigated conditions. These data suppose that natural triplex structures could play an important role in eukaryotic gene regulation and/or chromatin structure formation.

We have studied pim-1 proto-oncogene expression in human T cell responses to Ag receptor-generated signals. The pim-1 gene encodes a serine/threonine protein kinase that is expressed primarily in cells of hematopoietic lineage and is implicated in the intracellular signaling processes accompanying lymphocyte activation. We show here that pim-1 mRNA expression is rapidly induced after receptor cross-linking with anti-CD3 Abs. We examined the linkage of pim-1 expression to known signaling pathways generated through the T cell Ag receptor. pim-1 mRNA was not substantially induced after elevation of intracellular free Ca2+. In contrast, PMA, which directly activates PKC, induced rapid pim-1 expression. Further, anti-CD3- or PMA-induced pim-1 expression was markedly reduced by various PKC inhibitors and by deficiency of the PKC epsilon isoform in a mutant T cell line. Thus, T cell Ag receptor-linked pim-1 expression appears to be coupled to the PKC component of transmembrane signaling. Because the activation of protein kinase C has been shown to activate Raf-1 kinase activity, the involvement of Raf-1 in pim-1 expression was also investigated using a human T cell line stably transfected with an inducible Raf expression vector. Although the overexpression of activated Raf was shown to cause a substantial increase in IL-2 expression, no discernible effects on pim-1 were apparent. In addition, we examined transcriptional and post-transcriptional mechanisms involved in PKC-mediated pim-1 expression and observed that both transcriptional and post-transcriptional mechanisms are coordinately involved in the up-regulation of the pim-1 proto-oncogene.

In our previous work we have shown that the oligonucleotide 5 -GGGGAGGGGGAGG-3 gives a very stable and specific triplex with the promoter of the murine c-pim-1 proto-oncogene in vitro[Svinarchuk, F., Bertrand, J.-R. and Malvy, C.(1994)Nucleic Acids Res., 22, 3742-3747]. In the present work, we have tested triplex formation with some derivatives of this oligonucleotide which are designed to be degradation-resistant inside the cells, and we show that phosphorothioate and the oligonucleotide with a 3 terminal amino group are still able to form triplexes. Moreover these oligonucleotides, like the 13mer oligonucleotide of similar composition [Svinarchuk, F., Paoletti, J., and Malvy, C. (1995) J. Biol. Chem., 270, 14068-14071], are able to stabilize the targeted duplex. In vivo DMS footprint analysis after electroporation of the pre-formed triplex into the cell have shown the presence of the triple helix inside the cells. This triplex structure partially blocks c-pim-1 promotor activity as shown by transient assay with a c-pim-1 promoter-luciferase gene construct. To our knowledge these data are the first direct evidence that conditions inside cells are favorable for triplex stability with non-modified oligonucleotides. However we were unable to show triplex formation inside living cells using various methods of oligonucleotide delivery. We suppose that this may be due to the oligonucleotide being sequestered by cellular processes or proteins. Further work is needed to find oligonucleotide derivatives and ways of their delivery to overcome the problem of triplex formation inside the cells.

Bovine leukemia virus (BLV) induces a non-malignant, polyclonal, persistent lymphocytosis (PL) of circulating, CD5 B lymphocytes in cattle, with variable progression to CD5 B cell leukemia or lymphoma. We analyzed the expression of two proto-oncogenes, pim-1 and c-myc, proto-oncogenes deregulated in some human B cell leukemias and lymphomas, in peripheral blood mononuclear leukocytes (PBML) from BLV-infected PL cows. Results demonstrate that pim-1 and c-myc mRNA levels are elevated in unfractionated stimulated PBML from a sample of PL cows naturally infected with BLV. Results confirm that pim-1 is constitutively expressed, but not inducible in normal bovine peripheral blood B lymphocytes, but can be induced in the predominantly CD5 B lymphocytes from BLV-infected PL cows. Results further demonstrate that c-myc is inducible in bovine B and T lymphocytes regardless of BLV status, but the amount of induction is greater in B lymphocytes from BLV-infected PL cows than in B lymphocytes from noninfected control cows. These results suggest that pim-1 and c-myc are upregulated in B lymphocytes from BLV-infected PL cows and that deregulation of proto-oncogene expression is not limited to completely transformed cells, but can also characterize a naturally occurring, pre-neoplastic lymphocytic state.

Pim-1 is an oncogene-encoded serine/threonine kinase expressed primarily in cells of the hematopoietic and germ line lineages. Previously identified only in mammals, pim-1 cDNA was cloned and sequenced from the African clawed frog Xenopus laevis. The coding region of Xenopus pim-1 encoded a protein of 324 residues, which exhibited 64% amino acid identity with the full-length human cognate. Xenopus Pim-1 was expressed in bacteria as a glutathione S-transferase (GST) fusion protein and in COS cells. Phosphoamino acid analysis revealed that recombinant Pim-1 autophosphorylated on serine and threonine and to a more limited extent on tyrosine. Electrospray ionization mass spectroscopy was undertaken to locate these phosphorylation sites, and the primary autophosphorylation site of GST-Pim-1 was identified as Ser-190 with Thr-205 and Ser-4 being minor sites. Ser-190, which immediately follows the high conserved Asp-Phe-Gly motif in catalytic subdomain VII, is also featured in more than 20 other protein kinases. To evaluate the importance of the Ser-190 site on the phosphotransferase activity of Pim-1, Ser-190 was mutated to either alanine or glutamic acid, and the constructs were expressed in bacteria as GST fusion proteins and in COS cells. These mutants confirmed that Ser-190 is a major autophosphorylation site of Pim-1 and indicated that phosphorylation of Pim-1 on the Ser-190 residue may serve to activate this kinase.