title: p16(Ink4a) in melanocyte senescence and differentiation.
abstract: BACKGROUND
  The Ink4a-Arf tumor suppressor locus encodes two growth inhibitors, p16 and Arf, both of which are also implicated as effectors in cellular senescence. Because human germline defects in the INK4A-ARF locus are associated with familial melanoma, melanocytes may have unusual INK4A-ARF functions or controls of cell senescence. Because senescence is believed to be an anticancer mechanism, we investigated the role of Ink4a-Arf and its individual components in melanocyte senescence.
  
  
  METHODS
  Melanocytes were cultured from littermate mice with zero, one, or two functional copies of the Ink4a-Arf locus. Senescence was evaluated by cumulative population doubling curves and by the assessment of acidic beta-galactosidase (an indicator of senescence) expression. Pigmentation and cell size were evaluated by spectrophotometry and microscopy. p16 and Arf expression in primary and spontaneously immortalized melanocyte or melanocyte precursor cell lines were evaluated by immunoblotting. Retroviral vectors containing normal p16 and Arf complementary DNAs were used to restore expression of these genes in Ink4a-Arf(-/-) melanocytes.
  
  
  RESULTS
  Wild-type melanocytes (i.e., Ink4a-Arf(+/+)) senesced within 4-5 weeks of culture. Ink4a-Arf(-/-) melanocytes did not senesce and readily became immortal. Ink4a-Arf(+/-) melanocytes showed defective senescence. Senescent Ink4a-Arf(+/+) melanocytes were heavily pigmented, but Ink4a-Arf(+/-) and Ink4a-Arf(-/-) melanocytes were less pigmented. All of six spontaneously immortalized melanocyte or melanocyte precursor lines from Ink4a-Arf(+/+) mice lacked p16 protein expression, although most retained Arf protein expression. After restoration of p16 but not Arf expression, Ink4a-Arf(-/-) melanocytes stopped growing, became highly melanized, and expressed acidic beta-galactosidase. By contrast, restoration of Arf but not p16 expression led to cell death without evidence of senescence.
  
  
  CONCLUSIONS
  Normal mouse melanocyte senescence and associated pigmentation require both copies of Ink4a-Arf and appear to depend more on p16 than on Arf function. Mutations of the INK4A-ARF locus may favor tumorigenesis from melanocytes by impairing senescence, cell differentiation, and (where ARF is disrupted) cell death.
authors.affiliation: Department of Anatomy and Developmental Biology, St. George's Hospital Medical School, Cranmer Terrace, London SW17 0RE, U.K.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Sviderskaya
authors.forename: Simon P
authors.initials: SP
authors.lastname: Hill
authors.forename: Tracy J
authors.initials: TJ
authors.lastname: Evans-Whipp
authors.forename: Lynda
authors.initials: L
authors.lastname: Chin
authors.forename: Seth J
authors.initials: SJ
authors.lastname: Orlow
authors.forename: David J
authors.initials: DJ
authors.lastname: Easty
authors.forename: Sok Ching
authors.initials: SC
authors.lastname: Cheong
authors.forename: David
authors.initials: D
authors.lastname: Beach
authors.forename: Ronald A
authors.initials: RA
authors.lastname: DePinho
authors.forename: Dorothy C
authors.initials: DC
authors.lastname: Bennett
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002478:Cells, Cultured; D016922:Cellular Senescence; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics / Q000502:physiology*; D008544:Melanocytes / Q000502:physiology*; D008545:Melanoma / Q000209:etiology / Q000235:genetics; D051379:Mice; D008810:Mice, Inbred C57BL; D010858:Pigmentation; D012190:Retroviridae / Q000235:genetics
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16