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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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Oncogenic                                                N-RASQ61K induces proliferative arrest and senescence of human                                                melanocytes.                                        (A) Human melanocytes were transduced with                                        lentiviruses expressing N-RASQ61K or copGFP control. The                                        efficiency of transduction was controlled with the co-expression of copGFP                                        and was consistently above 90%. Cell proliferation (Ki67), chromatin                                        condensation (DAPI), and the appearance of increased SA-β-Gal activity were                                        analyzed and quantitated 15 days after infection. Percentage of cells                                        positive for the 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. Cells enlarged to show DAPI-stained                                        chromatin foci are indicated with arrows (bar =10 μm). LM, light                                        microscopy (bar=100μm). (B) Human                                        epidermal melanocytes infected with lentiviruses expressing N-RASQ61K                                        or copGFP were stained with DAPI and antibodies to H3K9Me, 15 days post                                        transduction (bar =10 μm). (C)                                        Expression of the indicated proteins was determined by western blot                                        analysis 15 days after infection of human epidermal melanocytes with                                        lentiviruses expressing N-RASQ61K or copGFP control.
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Figure 1: Oncogenic N-RASQ61K induces proliferative arrest and senescence of human melanocytes. (A) Human melanocytes were transduced with lentiviruses expressing N-RASQ61K or copGFP control. The efficiency of transduction was controlled with the co-expression of copGFP and was consistently above 90%. Cell proliferation (Ki67), chromatin condensation (DAPI), and the appearance of increased SA-β-Gal activity were analyzed and quantitated 15 days after infection. Percentage of cells positive for the 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. Cells enlarged to show DAPI-stained chromatin foci are indicated with arrows (bar =10 μm). LM, light microscopy (bar=100μm). (B) Human epidermal melanocytes infected with lentiviruses expressing N-RASQ61K or copGFP were stained with DAPI and antibodies to H3K9Me, 15 days post transduction (bar =10 μm). (C) Expression of the indicated proteins was determined by western blot analysis 15 days after infection of human epidermal melanocytes with lentiviruses expressing N-RASQ61K or copGFP control.

Mentions: The response of primary human melanocytes to the oncogenic, melanoma-associated N-RASQ61K mutant was evaluated by stably transducing N-RASQ61K into human epidermal melanocytes. Accumulation of N-RASQ61K was detected three days post-transduction and the impact of N-RAS on melanocyte proliferation was monitored over 15 days. As expected, 15 days post-transduction the majority of N-RASQ61K transduced melanocytes displayed several markers of oncogene-driven senescence, namely cell flattening, increase in cellular size, significantly reduced Ki67 expression, increased SA-β-Gal activity and the formation of SAHF (Figure 1A). As expected these foci were enriched for histone H3 methylated at lysine 9 (H3K9Me), a common marker of heterochromatin [24] (Figure 1B). In contrast, melanocytes accumulating the co-expressed Copepod GFP (copGFP) did not arrest, showed no evidence of chromatin condensation nor increased SA-β-Gal activity (Figure 1A).


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)

Oncogenic                                                N-RASQ61K induces proliferative arrest and senescence of human                                                melanocytes.                                        (A) Human melanocytes were transduced with                                        lentiviruses expressing N-RASQ61K or copGFP control. The                                        efficiency of transduction was controlled with the co-expression of copGFP                                        and was consistently above 90%. Cell proliferation (Ki67), chromatin                                        condensation (DAPI), and the appearance of increased SA-β-Gal activity were                                        analyzed and quantitated 15 days after infection. Percentage of cells                                        positive for the 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. Cells enlarged to show DAPI-stained                                        chromatin foci are indicated with arrows (bar =10 μm). LM, light                                        microscopy (bar=100μm). (B) Human                                        epidermal melanocytes infected with lentiviruses expressing N-RASQ61K                                        or copGFP were stained with DAPI and antibodies to H3K9Me, 15 days post                                        transduction (bar =10 μm). (C)                                        Expression of the indicated proteins was determined by western blot                                        analysis 15 days after infection of human epidermal melanocytes with                                        lentiviruses expressing N-RASQ61K or copGFP control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Oncogenic N-RASQ61K induces proliferative arrest and senescence of human melanocytes. (A) Human melanocytes were transduced with lentiviruses expressing N-RASQ61K or copGFP control. The efficiency of transduction was controlled with the co-expression of copGFP and was consistently above 90%. Cell proliferation (Ki67), chromatin condensation (DAPI), and the appearance of increased SA-β-Gal activity were analyzed and quantitated 15 days after infection. Percentage of cells positive for the 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. Cells enlarged to show DAPI-stained chromatin foci are indicated with arrows (bar =10 μm). LM, light microscopy (bar=100μm). (B) Human epidermal melanocytes infected with lentiviruses expressing N-RASQ61K or copGFP were stained with DAPI and antibodies to H3K9Me, 15 days post transduction (bar =10 μm). (C) Expression of the indicated proteins was determined by western blot analysis 15 days after infection of human epidermal melanocytes with lentiviruses expressing N-RASQ61K or copGFP control.
Mentions: The response of primary human melanocytes to the oncogenic, melanoma-associated N-RASQ61K mutant was evaluated by stably transducing N-RASQ61K into human epidermal melanocytes. Accumulation of N-RASQ61K was detected three days post-transduction and the impact of N-RAS on melanocyte proliferation was monitored over 15 days. As expected, 15 days post-transduction the majority of N-RASQ61K transduced melanocytes displayed several markers of oncogene-driven senescence, namely cell flattening, increase in cellular size, significantly reduced Ki67 expression, increased SA-β-Gal activity and the formation of SAHF (Figure 1A). As expected these foci were enriched for histone H3 methylated at lysine 9 (H3K9Me), a common marker of heterochromatin [24] (Figure 1B). In contrast, melanocytes accumulating the co-expressed Copepod GFP (copGFP) did not arrest, showed no evidence of chromatin condensation nor increased SA-β-Gal activity (Figure 1A).

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