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

Haferkamp S, Tran SL, Becker TM, Scurr LL, Kefford RF, Rizos H - Aging (Albany NY) (2009)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Westmead Institute for Cancer Research and Melanoma Institute of Australia, University of Sydney at Westmead, Westmead NSW 2145, Australia.

ABSTRACT
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.

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Relative contributions of the p53 and pRb tumour suppressor pathways in N-RAS Q61K-induced                                            melanocyte senescence.                                    (A) Melanocytes were transduced with                                        lentiviruses containing the indicated shRNA constructs. Three days post                                        infection the cells were re-transduced with lentiviruses expressing N-RASQ61K                                        or copGFP, as shown. Representative examples at 15days after infection are                                        shown. Cell proliferation (Ki67), chromatin condensation (DAPI), and the                                        appearance of increased SA-β-Gal activity were analyzed and quantitated.                                        Percentage of cells positive for each indicated marker are shown in                                        histograms, which correspond to the mean ± s.d. of at least two independent                                        transduction experiments from a total of at least 300 cells. Cells enlarged                                        to show DAPI-stained chromatin foci are indicated with arrows (bar =10 μm). LM, light microscopy (bar=100μm).                                    (B) Expression of the indicated proteins                                    was determined by western blot analysis at 15 days after infection of human                                    epidermal melanocytes with the indicated shRNA constructs and either                                    lentivirus expressing N-RASQ61K or the copGFP                                    control.                                    (C)                                    The impact of pRb-silencing on the N-RASQ61K induced senescence                                    was determined by quantitating key senescence markers (Ki67 expression,                                    SAHF formation, SA-β-Gal activity) at 10 and 15 days post N-RAS                                    transduction. Percentage of cells positive for each indicated marker is shown                                    in histograms, which correspond to the mean ± s.d. of at least two                                    independent transduction experiments from a total of at least 300 cells.
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Figure 3: Relative contributions of the p53 and pRb tumour suppressor pathways in N-RAS Q61K-induced melanocyte senescence. (A) Melanocytes were transduced with lentiviruses containing the indicated shRNA constructs. Three days post infection the cells were re-transduced with lentiviruses expressing N-RASQ61K or copGFP, as shown. Representative examples at 15days after infection are shown. Cell proliferation (Ki67), chromatin condensation (DAPI), and the appearance of increased SA-β-Gal activity were analyzed and quantitated. Percentage of cells positive for each indicated marker are shown in histograms, which correspond to the mean ± s.d. of at least two independent transduction experiments from a total of at least 300 cells. Cells enlarged to show DAPI-stained chromatin foci are indicated with arrows (bar =10 μm). LM, light microscopy (bar=100μm). (B) Expression of the indicated proteins was determined by western blot analysis at 15 days after infection of human epidermal melanocytes with the indicated shRNA constructs and either lentivirus expressing N-RASQ61K or the copGFP control. (C) The impact of pRb-silencing on the N-RASQ61K induced senescence was determined by quantitating key senescence markers (Ki67 expression, SAHF formation, SA-β-Gal activity) at 10 and 15 days post N-RAS transduction. Percentage of cells positive for each indicated marker is shown in histograms, which correspond to the mean ± s.d. of at least two independent transduction experiments from a total of at least 300 cells.

Mentions: The inhibition of p53 expression did not alter the cell cycle arrest induced by oncogenic N-RASQ61K (15 days after infection only 5% of N-RASQ61K melanocytes showed positive Ki67 staining regardless of p53 expression and this can be compared to 23% Ki67 positive p53- melanocytes infected with the copGFP control; Figure 3A). Similarly, cellular senescence was initiated and maintained in the presence or absence of p53 expression; increased SA-β-Gal activity appeared in 48% of p53- cells compared to 38% in the p53-positive control cells, 15 days post transduction (Figure 3A) and the two different p53-specific shRNAs exerted similar effects (data not shown). In fact no markers of senescence, including cell morphology, SA-β-Gal activity, appearance of SAHF or Ki67 incorporation discriminated between p53-intact and p53- senescent melanocytes. It is important to note, however, that p21Waf1 expression was not induced by oncogenic N-RAS in p53-deficient melanocytes (Figure 3B).


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

Haferkamp S, Tran SL, Becker TM, Scurr LL, Kefford RF, Rizos H - Aging (Albany NY) (2009)

Relative contributions of the p53 and pRb tumour suppressor pathways in N-RAS Q61K-induced                                            melanocyte senescence.                                    (A) Melanocytes were transduced with                                        lentiviruses containing the indicated shRNA constructs. Three days post                                        infection the cells were re-transduced with lentiviruses expressing N-RASQ61K                                        or copGFP, as shown. Representative examples at 15days after infection are                                        shown. Cell proliferation (Ki67), chromatin condensation (DAPI), and the                                        appearance of increased SA-β-Gal activity were analyzed and quantitated.                                        Percentage of cells positive for each indicated marker are shown in                                        histograms, which correspond to the mean ± s.d. of at least two independent                                        transduction experiments from a total of at least 300 cells. Cells enlarged                                        to show DAPI-stained chromatin foci are indicated with arrows (bar =10 μm). LM, light microscopy (bar=100μm).                                    (B) Expression of the indicated proteins                                    was determined by western blot analysis at 15 days after infection of human                                    epidermal melanocytes with the indicated shRNA constructs and either                                    lentivirus expressing N-RASQ61K or the copGFP                                    control.                                    (C)                                    The impact of pRb-silencing on the N-RASQ61K induced senescence                                    was determined by quantitating key senescence markers (Ki67 expression,                                    SAHF formation, SA-β-Gal activity) at 10 and 15 days post N-RAS                                    transduction. Percentage of cells positive for each indicated marker is shown                                    in histograms, which correspond to the mean ± s.d. of at least two                                    independent transduction experiments from a total of at least 300 cells.
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Figure 3: Relative contributions of the p53 and pRb tumour suppressor pathways in N-RAS Q61K-induced melanocyte senescence. (A) Melanocytes were transduced with lentiviruses containing the indicated shRNA constructs. Three days post infection the cells were re-transduced with lentiviruses expressing N-RASQ61K or copGFP, as shown. Representative examples at 15days after infection are shown. Cell proliferation (Ki67), chromatin condensation (DAPI), and the appearance of increased SA-β-Gal activity were analyzed and quantitated. Percentage of cells positive for each indicated marker are shown in histograms, which correspond to the mean ± s.d. of at least two independent transduction experiments from a total of at least 300 cells. Cells enlarged to show DAPI-stained chromatin foci are indicated with arrows (bar =10 μm). LM, light microscopy (bar=100μm). (B) Expression of the indicated proteins was determined by western blot analysis at 15 days after infection of human epidermal melanocytes with the indicated shRNA constructs and either lentivirus expressing N-RASQ61K or the copGFP control. (C) The impact of pRb-silencing on the N-RASQ61K induced senescence was determined by quantitating key senescence markers (Ki67 expression, SAHF formation, SA-β-Gal activity) at 10 and 15 days post N-RAS transduction. Percentage of cells positive for each indicated marker is shown in histograms, which correspond to the mean ± s.d. of at least two independent transduction experiments from a total of at least 300 cells.
Mentions: The inhibition of p53 expression did not alter the cell cycle arrest induced by oncogenic N-RASQ61K (15 days after infection only 5% of N-RASQ61K melanocytes showed positive Ki67 staining regardless of p53 expression and this can be compared to 23% Ki67 positive p53- melanocytes infected with the copGFP control; Figure 3A). Similarly, cellular senescence was initiated and maintained in the presence or absence of p53 expression; increased SA-β-Gal activity appeared in 48% of p53- cells compared to 38% in the p53-positive control cells, 15 days post transduction (Figure 3A) and the two different p53-specific shRNAs exerted similar effects (data not shown). In fact no markers of senescence, including cell morphology, SA-β-Gal activity, appearance of SAHF or Ki67 incorporation discriminated between p53-intact and p53- senescent melanocytes. It is important to note, however, that p21Waf1 expression was not induced by oncogenic N-RAS in p53-deficient melanocytes (Figure 3B).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Westmead Institute for Cancer Research and Melanoma Institute of Australia, University of Sydney at Westmead, Westmead NSW 2145, Australia.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus