Sviderskaya Elena V, Gray-Schopfer Vanessa C, Hill Simon P, Smit Nico P, Evans-Whipp Tracy J, Bond Jane, Hill Lucy, Bataille Veronique, Peters Gordon, Kipling David, Wynford-Thomas David, Bennett Dorothy C
Department of Basic Medical Sciences, St. George's Hospital Medical School, London, UK.
J Natl Cancer Inst. 2003 May 21;95(10):723-32. doi: 10.1093/jnci/95.10.723.
The melanoma susceptibility locus cyclin-dependent kinase inhibitor 2A encodes two unrelated cell growth inhibitors, p16 and alternative reading frame (ARF). In fibroblasts, both proteins are implicated in cellular senescence, a key barrier to tumor development. The p16 coding sequence is more often mutated in melanoma families than is the ARF sequence. To investigate the role of p16 in melanocytes, we assessed aspects of growth, apoptosis, and immortalization in melanocytes cultured from two melanoma patients, both of whom had two inactive p16 alleles but functional ARF.
Growth and senescence were evaluated by cumulative population-doubling curves, and apoptosis by terminal deoxytransferase labeling. Expression of p53 and p21, which are associated with fibroblast senescence, was assessed by immunoblotting. Amphotropic retroviruses were used to transfer exogenous gene sequences into the melanocytes.
Both melanocyte cultures showed high rates of apoptosis, which were reduced when the cells were grown in the presence of keratinocyte feeder cells or human stem cell factor plus endothelin 1. With these growth factors, both cultures proliferated for 45-55 net population doublings, markedly longer than the maximum of 10 net population doublings of normal adult human melanocytes in similar media, indicating impaired senescence. One of the cultures developed chromosomal aberrations, with numerous dicentric chromosomes at senescence, consistent with telomere dysfunction. p53 and p21 levels were not elevated in senescent normal melanocytes but were elevated in senescent p16-deficient melanocytes. Interference with p53 function by transfer of human papillomavirus 16-E6 further extended the lifespan of p16-deficient melanocytes. Human telomerase reverse transcriptase was sufficient to immortalize both these cell strains but not normal melanocytes.
Normal senescence in human melanocytes requires p16 activity. p53 contributes to a delayed form of senescence that requires telomere shortening, in p16-deficient melanocytes. These findings provide some basis for the role of p16 in melanoma susceptibility.
黑色素瘤易感基因座细胞周期蛋白依赖性激酶抑制剂2A编码两种不相关的细胞生长抑制剂，即p16和可变阅读框（ARF）。在成纤维细胞中，这两种蛋白质都与细胞衰老有关，而细胞衰老是肿瘤发展的关键障碍。在黑色素瘤家族中，p16编码序列比ARF序列更常发生突变。为了研究p16在黑素细胞中的作用，我们评估了从两名黑色素瘤患者培养的黑素细胞的生长、凋亡和永生化情况，这两名患者都有两个无活性的p16等位基因，但ARF功能正常。
通过累积群体倍增曲线评估生长和衰老情况，通过末端脱氧核苷酸转移酶标记评估凋亡情况。通过免疫印迹法评估与成纤维细胞衰老相关的p53和p21的表达。使用嗜性逆转录病毒将外源基因序列导入黑素细胞。
两种黑素细胞培养物均显示出高凋亡率，当细胞在角质形成细胞饲养层细胞或人干细胞因子加内皮素1存在的情况下生长时，凋亡率降低。在这些生长因子的作用下，两种培养物均增殖了45 - 55次净群体倍增，明显长于正常成人黑素细胞在类似培养基中最多10次净群体倍增的情况，表明衰老受损。其中一种培养物出现了染色体畸变，衰老时出现大量双着丝粒染色体，这与端粒功能障碍一致。衰老的正常黑素细胞中p53和p21水平未升高，但衰老的p16缺陷黑素细胞中p53和p21水平升高。通过转染人乳头瘤病毒16 - E6干扰p53功能进一步延长了p16缺陷黑素细胞的寿命。人端粒酶逆转录酶足以使这两种细胞系永生化，但不能使正常黑素细胞永生化。
人黑素细胞的正常衰老需要p16活性。在p16缺陷的黑素细胞中，p53导致一种延迟形式的衰老，这种衰老需要端粒缩短。这些发现为p16在黑色素瘤易感性中的作用提供了一些依据。
