Oncogenic B-RAFV600E Promotes Anchorage-Independent Survival of Human Melanocytes

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Selective Loss of Wild-Type p16INK4a Expression in Human Nevi

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P53 in human melanoma fails to regulate target genes associated with apoptosis and the cell cycle and may contribute to proliferation

<p>Abstract</p> <p>Background</p> <p>Metastatic melanoma represents a major clinical problem. Its incidence continues to rise in western countries and there are currently no curative treatments. While mutation of the <it>P53 </it>tumour suppressor gene is a common feature of many types of cancer, mutational inactivation of <it>P53 </it>in melanoma is uncommon; however, its function often appears abnormal.</p> <p>Methods</p> <p>In this study whole genome bead arrays were used to examine the transcript expression of P53 target genes in extracts from 82 melanoma metastases and 6 melanoma cell lines, to provide a global assessment of aberrant P53 function. The expression of these genes was also examined in extracts derived from diploid human melanocytes and fibroblasts.</p> <p>Results</p> <p>The results indicated that P53 target transcripts involved in apoptosis were under-expressed in melanoma metastases and melanoma cell lines, while those involved in the cell cycle were over-expressed in melanoma cell lines. There was little difference in the transcript expression of P53 target genes between cell lines with null/mutant <it>P53 </it>compared to those with wild-type <it>P53</it>, suggesting that altered expression in melanoma was not related to <it>P53 </it>status. Similarly, down-regulation of P53 by short-hairpin RNA (shRNA) had limited effect on P53 target gene expression in melanoma cells, whereas there were a large number of P53 target genes whose mRNA expression was significantly altered by P53 inhibition in melanocytes. Analysis of whole genome gene expression profiles indicated that the ability of P53 to regulate genes involved in the cell cycle was significantly reduced in melanoma cells. Moreover, inhibition of P53 in melanocytes induced changes in gene expression profiles that were characteristic of melanoma cells and resulted in increased proliferation. Conversely, knockdown of P53 in melanoma cells resulted in decreased proliferation.</p> <p>Conclusions</p> <p>These results indicate that P53 target genes involved in apoptosis and cell cycle regulation are aberrantly expressed in melanoma and that this aberrant functional activity of P53 may contribute to the proliferation of melanoma.</p

Senescence

Cellular senescence is a program initiated by many stress signals including aberrant activation of oncogenes, DNA damage, oxidative lesions and telomere attrition. Once engaged senescence irreversibly limits cellular proliferation and can potently prevent tumour formation in vivo. The precise mechanisms driving senescence are still not completely defined, although the pRb and p53 tumour suppressor pathways are critical effectors. Senescent cells also develop aberrant gene expression profiles and acquire proinflammatory behaviour that may contribute to organismal ageing and age-related diseases, including cancer. It is not yet clear whether the pro-ageing actions of senescent cells can be minimised in vivo, but the therapeutic potential of this stress-induced program may depend on establishing a new equilibrium that favours tumour suppressor activity.20 page(s

The Role of sumoylation in senescence

Cellular senescence is a program initiated by many stress signals including aberrant activation of oncogenes, DNA damage, oxidative lesions and telomere attrition. Once engaged senescence irreversibly limits cellular proliferation and potently prevents tumor formation in vivo. The precise mechanisms driving the onset of senescence are still not completely defined, although the pRb and p53 tumor suppressor pathways converge with the SUMO cascade to regulate cellular senescence. Sumoylation translocates p53 to PML nuclear bodies where it can co-operate with many sumoylated co-factors in a program that activates pRb and favors senescence. Once activated pRb integrates various proteins, many of them sumoylated, into a repressor complex that inhibits the transcription of proliferation-promoting genes and initiates chromatin condensation. Sumoylation is required for heterochromatin formation during senescence and may act as a scaffold to stabilize the pRb repressor complex. Thus, SUMO is a critical component of a tumor-suppressor network that limits aberrant cell proliferation and tumorigenesis.12 page(s

The Role of sumoylation in senescence

Cellular senescence is a program initiated by many stress signals including aberrant activation of oncogenes, DNA damage, oxidative lesions and telomere attrition. Once engaged senescence irreversibly limits cellular proliferation and potently prevents tumor formation in vivo. The precise mechanisms driving the onset of senescence are still not completely defined, although the pRb and p53 tumor suppressor pathways converge with the SUMO cascade to regulate cellular senescence. Sumoylation translocates p53 to PML nuclear bodies where it can co-operate with many sumoylated co-factors in a program that activates pRb and favors senescence. Once activated pRb integrates various proteins, many of them sumoylated, into a repressor complex that inhibits the transcription of proliferation-promoting genes and initiates chromatin condensation. Sumoylation is required for heterochromatin formation during senescence and may act as a scaffold to stabilize the pRb repressor complex. Thus, SUMO is a critical component of a tumor-suppressor network that limits aberrant cell proliferation and tumorigenesis.16 page(s

Wild type and melanoma-associated mutant p16INK4a proteins do not oligomerize in vivo

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The Relative contributions of the p53 and pRb pathways in oncogene-induced melanocyte senescence

Oncogene-induced senescence acts as a barrier against tumour formation and has been implicated as the mechanism preventing the transformation of benign melanocytic lesions that frequently harbour oncogenic B-RAF or N-RAS mutations. In the present study we systematically assessed the relative importance of the tumour suppressor proteins p53, p21(Waf1), pRb and p16(INK4a) in mediating oncogene-induced senescence in human melanocytes. We now show that oncogenic N-RAS induced senescence in melanocytes is associated with DNA damage, a potent DNA damage response and the activation of both the p16(INK4a)/pRb and p53/p21(Waf1) tumour suppressor pathways. Surprisingly neither the pharmacological inhibition of the DNA damage response pathway nor silencing of p53 expression had any detectable impact on oncogene-induced senescence in human melanocytes. Our data indicate that the pRb pathway is the dominant effector of senescence in these cells, as its specific inactivation delays the onset of senescence and weakens oncogene-induced proliferative arrest. Furthermore, we show that although both p16(INK4a) and p21(Waf1) are upregulated in response to N-RAS(Q61K), the activities of these CDK inhibitors are clearly distinct and only the loss of p16(INK4a) weakens senescence. We propose that the ability of p16(INK4a) to inhibit the cyclin D-dependent kinases and DNA replication, functions not shared by p21(Waf1), contribute to its role in senescence. Thus, in melanocytes with oncogenic signalling only p16(INK4a) can fully engage the pRb pathway to alter chromatin structure and silence the genes that are required for proliferation.15 page(s