title: Direct evidence from siRNA-directed "knock down" that p16(INK4a) is required for human fibroblast senescence and for limiting ras-induced epithelial cell proliferation.
abstract: The selective pressure for disruption of the cyclin-dependent kinase inhibitor p16(INK4a) in human cancer has been postulated to reflect its role in mediating growth arrest, both in response to telomere erosion (replicative senescence) and to oncogene-induced and other "stress" signals. Given the known species-specific differences in regulation of senescence, we have tested this hypothesis in human, as opposed to rodent, cells by designing a small interfering RNA (siRNA) to knock down p16(INK4a) expression. Transfection of this siRNA into late-passage normal human diploid fibroblasts allowed at least temporary escape from entry into replicative senescence. Furthermore, in our in vitro model of early-stage, RAS-induced thyroid tumorigenesis, sequential transfections with this siRNA allowed outgrowth of small clusters of proliferating epithelial cells, consistent with escape from the spontaneous "senescence", which normally curtails their proliferative response to mutant RAS. These data provide the first direct evidence that p16(INK4a) is necessary for the initiation of both telomere-dependent and telomere-independent senescence in human cells.
authors.affiliation: Department of Pathology, University of Wales College of Medicine, Cardiff CF14 4XN, UK.
authors.forename: Jane
authors.initials: J
authors.lastname: Bond
authors.forename: Christopher
authors.initials: C
authors.lastname: Jones
authors.forename: Michele
authors.initials: M
authors.lastname: Haughton
authors.forename: Catherine
authors.initials: C
authors.lastname: DeMicco
authors.forename: David
authors.initials: D
authors.lastname: Kipling
authors.forename: David
authors.initials: D
authors.lastname: Wynford-Thomas
mesh_terms: D002455:Cell Division; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D004171:Diploidy; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D005786:Gene Expression Regulation; D006801:Humans; D007150:Immunohistochemistry; D009154:Mutation; D034741:RNA, Small Interfering / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D013961:Thyroid Gland / Q000166:cytology; D014162:Transfection; D018631:ras Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D034741:RNA, Small Interfering; D004247:DNA; D018631:ras Proteins