title: p16/cyclin-dependent kinase inhibitor 2A deficiency in human melanocyte senescence, apoptosis, and immortalization: possible implications for melanoma progression.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Basic Medical Sciences, St. George's Hospital Medical School, London, UK.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Sviderskaya
authors.forename: Vanessa C
authors.initials: VC
authors.lastname: Gray-Schopfer
authors.forename: Simon P
authors.initials: SP
authors.lastname: Hill
authors.forename: Nico P
authors.initials: NP
authors.lastname: Smit
authors.forename: Tracy J
authors.initials: TJ
authors.lastname: Evans-Whipp
authors.forename: Jane
authors.initials: J
authors.lastname: Bond
authors.forename: Lucy
authors.initials: L
authors.lastname: Hill
authors.forename: Veronique
authors.initials: V
authors.lastname: Bataille
authors.forename: Gordon
authors.initials: G
authors.lastname: Peters
authors.forename: David
authors.initials: D
authors.lastname: Kipling
authors.forename: David
authors.initials: D
authors.lastname: Wynford-Thomas
authors.forename: Dorothy C
authors.initials: DC
authors.lastname: Bennett
mesh_terms: D017209:Apoptosis; D002461:Cell Line, Transformed; D016922:Cellular Senescence; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000172:deficiency* / Q000378:metabolism*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004268:DNA-Binding Proteins; D018450:Disease Progression; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015151:Immunoblotting; D007621:Karyotyping; D007700:Kinetics; D008544:Melanocytes / Q000378:metabolism*; D008545:Melanoma / Q000378:metabolism*; D010641:Phenotype; D019098:Telomerase / Q000378:metabolism*
chemical_list: C493956:CDKN1A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D019098:Telomerase