Oncogene database

1026

CDKN1A

cyclin-dependent kinase inhibitor 1A (p21, Cip1);CDKN1A;cyclin-dependent kinase inhibitor 1A (p21, Cip1)

CDK-interacting protein 1|CDK-interaction protein 1|DNA synthesis inhibitor|cyclin-dependent kinase inhibitor 1|melanoma differentiation associated protein 6|wild-type p53-activated fragment 1

6p21.2

protein-coding

p21(Waf1/Cip1/Sdi1) is best known as a broad-specificity inhibitor of cyclin/cyclin-dependent kinase complexes, but p21 also interacts with many other regulators of transcription or signal transduction. p21 induction, which is mediated by p53 and by p53-independent mechanisms, is essential for the onset of cell cycle arrest in damage response and cell senescence. The effects of p21 knockout in mice and its expression patterns in human cancer are consistent with a role for p21 as both a tumour suppressor and an oncogene. Several functions of p21 are likely to promote carcinogenesis and tumour progression. These include endoreduplication and abnormal mitosis that develop in tumour cells after release from p21-induced growth arrest, the ability of p21 to inhibit apoptosis through several different mechanisms, and its ability to stimulate transcription of secreted factors with mitogenic and anti-apoptotic activities. The latter effects of p21 show close resemblance to paracrine activities of senescent cells and to tumour-promoting functions of stromal fibroblasts. Therapeutic strategies targeting the oncogenic consequences of p21 expression may provide a new approach to chemoprevention and treatment of cancer.

We have previously found that the protein kinase C (PKC) inhibitor, CGP 41 251, blocks oncogenic ras-p21 protein- and beta-PKC-induced oocyte maturation, but only weakly inhibits insulin-induced oocyte maturation (which requires activation of wild-type endogenous ras-p21). Because the dose-response curves for inhibition of oncogenic p21- and beta-PKC-induced oocyte maturation by CGP 41 251 superimpose and because the ras-p21-inactivating antibody, Y13-259, does not inhibit beta-PKC-induced oocyte maturation, we concluded that the oncogenic, but not wild-type, protein requires beta-PKC as a downstream target. Because multiple isoforms of PKC exist and several of these, such as epsilon-PKC, have been found to be important on ras signal transduction pathways, we have investigated which PKC isoforms are critical to each ras protein. For this purpose, we used PKC-isoform-specific inhibitors, which have been shown to inhibit selectively the function and translocation of PKC isoforms in vitro and in vivo. Specifically, the peptides KLFIMN, QEVIRN, and EAVSLKPT each inhibit beta-1, beta-2, and epsilon-PKC, respectively, but do not cross-inhibit other PKC isoforms. We find that the epsilon-PKC inhibitory peptide strongly blocks insulin- but not oncogenic ras-p21-induced oocyte maturation whereas the beta-2 inhibitory peptide more strongly inhibits oncogenic ras-p21-induced oocyte maturation, corroborating our previous studies. The beta-1 inhibitory peptide has little effect on either protein. We conclude that selective inhibition of individual PKC isoforms permits the distinction between signal transduction initiated by oncogenic and activated wild-type p21 proteins and implicate different specific PKC isoforms in mitogenic signal transduction by each of these proteins. The ability to dissect the role of individual PKC isozymes in this regulation is of therapeutic significance.

We have employed a computational approach to design peptides, from known oncogenic proteins, that inhibit tumor growth. This approach has been applied to the ras-p21 protein that becomes oncogenic when single amino acid substitutions occur at critical positions in its polypeptide chain, such as at Gly 12 and Gln 61. In this approach, using two sampling methods, molecular dynamics and the electrostatically driven Monte Carlo (EDMC) method, we have computed the average structures of wild-type and oncogenic forms of ras-p21 alone and bound to a number of its target proteins, such as the ras-binding domain (RBD) of raf, guanine nucleotide exchange protein (GAP) and SOS guanine nucleotide exchange protein (GAP). By superimposing the average structures of the oncogenic forms on those of their wild-type counterparts, we have identified a number of domains that change conformation. These domains are potential effector domains that are involved uniquely in oncogenic ras-p21 signaling. We have therefore synthesized peptides corresponding to these domains and tested them in Xenopus laevis oocytes for their abilities to inhibit oncogenic ras-p21 selectively. Using this approach, we have identified three peptides from ras-p21 and one peptide each from the RBD of raf, GAP and SOS that selectively inhibit oncogenic but not insulin-activated wild-type ras-p21-induced oocyte maturation. We have synthesized the ras-p21 peptides attached to a penetratin leader sequence to enable cell membrane penetration and introduced these peptides into a ras-transformed pancreatic cancer cell line; these peptides, but not an unrelated negative control peptide, cause the cells to undergo complete phenotypic reversion. On the other hand, none of these peptides has any effect on the growth of untransformed pancreatic acinar cells in culture, further suggesting that they may not interfere with normal cell growth. Thus these peptides can be useful agents in the treatment of cancers. We have further used these peptides to demonstrate that oncogenic and wild-type ras-p21 proteins utilize different signal transduction pathways and to identify where these differences occur in cells.

The BTB/POZ family of proteins has been implicated in multiple biological processes, including tumourigenesis, DNA damage responses and cell cycle progression and development. MIZ-1 (Myc-interacting zinc-finger protein 1) is known to activate transcription of CDKN1A. We recently found that a kidney cancer-related POK transcription factor, KR-POK, is highly expressed in kidney, brain and bone marrow cancer tissues and is a potential proto-oncoprotein. Mouse Kr-pok represses transcription of the CDKN1A by acting on the proximal promoter. The BiFC/FRET assay, co-immunoprecipitation and glutathione S-transferase-fusion protein pull-down assay indicate that MIZ-1 and Kr-pok interact via their POZ domains. Oligoucleotide pull-down assays and chromatin immunoprecipitation assays revealed that MIZ-1 binds to the proximal GC-box#3 (bp, -55 to -63) and the MIZ-1-binding elements, MRE-A (bp, -90 to -64) and MRE-B (bp, -27 to -17). Interestingly, MIZ-1 also binds to the distal p53-binding elements. Kr-pok binds to the proximal GC-box#1 (bp, -95 to -100) and #3 (bp, -55 to -63) relatively strongly. It also shows weak binding to the MREs and the distal p53-binding elements. Kr-pok competes with MIZ-1 in binding to these elements and represses transcription by inhibiting MIZ-1/p300 recruitment, which decreases the acetylation of histones H3 and H4. Our data indicate that Kr-pok stimulates cell proliferation by interfering with the function of MIZ-1 in CDKN1A gene transcription using a mechanism that is radically different from other MIZ-1-interacting proteins, such as B-cell lymphoma 6, c-Myc and Gfi-1.

We here establish a mouse cancer model called Multi-Hit that allows for the evaluation of oncogene cooperativities in tumor development. The model is based on the stochastic expression of oncogene combinations ( hits ) that are mediated by Cre in a given tissue. Cells with cooperating hits are positively selected and give rise to tumors. We used this approach to evaluate the requirement of Ras downstream effector pathways in tumorigenesis.

The tumour-suppressor gene CDKN1A (encoding p21Waf/Cip1) is thought to be epigenetically repressed in cancer cells. FBI-1 (ZBTB7A) is a proto-oncogenic transcription factor repressing the alternative reading frame and p21WAF/CDKN1A genes of the p53 pathway. FBI-1 interacts directly with MBD3 (methyl-CpG-binding domain protein 3) in the nucleus. We demonstrated that FBI-1 binds both non-methylated and methylated DNA and that MBD3 is recruited to the CDKN1A promoter through its interaction with FBI-1, where it enhances transcriptional repression by FBI-1. FBI-1 also interacts with the co-repressors nuclear receptor corepressor (NCoR), silencing mediator for retinoid and thyroid receptors (SMRT) and BCL-6 corepressor (BCoR) to repress transcription. MBD3 regulates a molecular interaction between the co-repressor and FBI-1. MBD3 decreases the interaction between FBI-1 and NCoR/SMRT but increases the interaction between FBI-1 and BCoR. Because MBD3 is a subunit of the Mi-2 autoantigen (Mi-2)/nucleosome remodelling and histone deacetylase (NuRD)-HDAC complex, FBI-1 recruits the Mi-2/NuRD-HDAC complex via MBD3. BCoR interacts with the Mi-2/NuRD-HDAC complex, DNMTs and HP1. MBD3 and BCoR play a significant role in the recruitment of the Mi-2/NuRD-HDAC complex- and the NuRD complex-associated proteins, DNMTs and HP. By recruiting DNMTs and HP1, Mi-2/NuRD-HDAC complex appears to play key roles in epigenetic repression of CDKN1A by DNA methylation.

An oncogenic 21-kDa (p21) protein (Harvey RAS protein with Val-12) has been covalently modified with a functional reagent that contains a photoactivatable aromatic azide group. This modified p21 protein has been introduced quantitatively into NIH 3T3 cells using an erythrocyte-mediated fusion technique. The introduced p21 protein was capable of inducing enhanced pinocytosis and DNA synthesis in the recipient cells. To identify the putative intracellular protein(s) that specifically interact with the modified p21 protein, the cells were pulsed with [35S]methionine at selected times after fusion and then UV-irradiated to activate the azide group. The resulting nitrene covalently binds to amino acid residues in adjacent proteins, thus linking the p21 protein to these proteins. The cells were then lysed, and the lysate was immunoprecipitated with the anti-p21 monoclonal antibody Y13-259. The immunoprecipitate was analyzed by SDS/PAGE to identify p21-protein complexes. By using this technique, we found that three protein complexes of 51, 64, and 82 kDa were labeled specifically and reproducibly. The most prominent band is the 64-kDa protein complex that shows a time-dependent rise and fall, peaking within a 5-hr period after introduction of the p21 protein into the cells. These studies provide evidence that in vitro the p21 protein becomes associated with a protein whose mass is about 43 kDa. We suggest that the formation of this complex may play a role in mediating early events involved with cell transformation induced by RAS oncogenes.

Many malignancies harbor mutated ras proto-oncogenes encoding 21 kDa proteins with single amino acid substitutions. Previous studies have shown that the aberrant p21ras proteins are potential tumor-specific antigens in that CD4+ class II major histocompatibility complex-restricted T cells specific for the mutated segment of various oncogenic p21ras proteins can be elicited by immunization in vivo with synthetic peptides corresponding to the mutated segment. T-cell recognition of an antigenic peptide within a protein may be influenced substantially, either positively or negatively, by flanking amino acid sequences as well as by more distal immunogenic or tolerogenic epitopes within the same protein. This study examined whether T cells specific for the mutated segment of an oncogenic p21ras protein can be elicited by immunization in vivo with the protein. The results showed that p21ras protein bearing the transforming single amino acid substitution of leucine for glutamine at residue 61 could elicit T cells specifically reactive to the mutated region of the protein in C3H/HeN mice. Thus, an abnormal p21ras protein specifically associated with malignant transformation can be immunogenic in vivo. These results predict that in some circumstances, mutated p21ras proteins expressed by malignancies might elicit detectable mutation-specific T-cell responses.

Correct post-translational modifications of the ras proteins are essential for their membrane localisation and functioning. The flat revertant cell lines 1aCB and 8b, derived from the human bladder carcinoma cell line EJ, contain the transforming gene V12Ha-ras and are resistant to retransformation by ras protein or DNA, but still do require the presence of ras for proliferation. Both revertant cell lines demonstrated reduced levels of membrane associated p21ras when compared to their parental EJ cell lines. This reduced level in 1aCB was reflected by an increase in nuclear associated p21ras, as seen by immunofluorescence of endogenous and introduced ras. In addition, 1aCB had a reduced ratio of ras in the detergent to aqueous phases after triton X114 partitioning, suggesting a defect in Step I processing of the p21ras in the cell line. This was not however due to defects in the Step I enzymes farnesyltransferase or carboxymethyltransferase whose activities were not reduced.

In higher eukaryotes, the Ras and Raf-1 proto-oncoproteins transduce growth and differentiation signals initiated by tyrosine kinases. The Ras polypeptide and the amino-terminal regulatory domain of Raf-1 (residues 1-257) are shown to interact, directly in vitro and in a yeast expression system. Raf-1 (1-257) binds GTP-Ras in preference to GDP-Ras, and inhibits Ras-GAP activity. mutations in and around the Ras effector domain impair Ras binding to Raf-1 (1-257) and Ras transforming activity in parallel.

p74raf-1, a serine/threonine kinase, is structurally related to the protein kinase C (PKC) family and contains a cysteine motif in its N-terminal domain, which is essential for its regulation. It has been shown that p74raf-1 functions upstream of mitogen-activated protein (MAP) kinase kinase. We have constructed a p74raf-1 mutant (N delta raf) that only contains the N-terminal regulatory domain. When transiently expressed in COS-M6 cells, N delta raf efficiently blocked the activation of the MAP extracellular signal regulated kinase (ERK2), induced by either epidermal growth factor, phorbol ester, serum, or oncogenic p21ras. Similar constructs with the cysteine motifs from either PKC-alpha or diacylglycerol kinase did not inhibit activation of ERK2. Overexpression of full-length p74raf-1 rescued the inhibition of ERK2 by N delta raf in a stimulus dependent manner, indicating that N delta raf acts as a competitive inhibitor of wild-type p74raf-1. In contrast, overexpression of either PKC-alpha, -epsilon, or -zeta in N delta raf-containing cells could not rescue the inhibition of ERK2. We conclude that p74raf-1 is an essential mediator of epidermal growth factor- and phorbol ester-induced ERK2 activation and that the MAP kinase kinase activity of p74raf-1 cannot be substituted with either PKC-alpha, -epsilon or -zeta.

Frequencies of HPV type 16 and 18, evaluated by in situ hybridization technique in uterine cervix carcinomas of the IIIrd stage of clinical advancement, were 54% and 36.5% respectively. Presence of p 21 protein was detected in 85.3% cases of the cancers and showed no relation to HPV infection.