RESUMO
Cell cycle checkpoints and tumor suppressor gene functions appear to be required for the maintenance of a stable genome in proliferating cells. In this study chromosomal destabilization was monitored in relation to telomere structure, lifespan control and G2 checkpoint function. Replicative senescence was inactivated in secondary cultures of human skin fibroblasts by expressing the human papillomavirus type 16 (HPV-16) E6 oncoprotein to inactivate p53. Chromosome aberrations were enumerated during in vitro aging of isogenic control (F5neo) and HPV-16E6-expressing (F5E6) fibroblasts. We found that structural and numerical aberrations in chromosomes were significantly increased in F5E6 cells during aging in vitro and fluorescence in situ hybridization (FISH) analysis using chromosome-specific probes demonstrated the occurrence of rearrangements involving chromosome 4 and 6 in genetically unstable F5E6 cells. Flow cytometry and karyotypic analyses revealed increased polyploidy and aneuploidy in F5E6 cells only at passages > 16, although these cells displayed defective mitotic spindle checkpoint function associated with inactivation of p53 at passages 5 and 16. G2 checkpoint function was confirmed to be gradually but progressively inactivated during in vitro aging of E6-expressing cells. Aging of F5neo fibroblasts was documented during in vitro passaging by induction of a senescence-associated marker, pH 6.0 lysosomal beta-galactosidase. F5E6 cells displayed extension of in vitro lifespan and did not induce beta-galactosidase at high passage. Erosion of telomeres during in vitro aging of telomerase-negative F5neo cells was demonstrated by Southern hybridization and by quantitative FISH analysis on an individual cell level. Telomeric signals diminished continuously as F5neo cells aged in vitro being reduced by 80% near the time of replicative senescence. Telomeric signals detected by FISH also decreased continuously during aging of telomerase-negative F5E6 cells, but telomeres appeared to be stabilized at passage 34 when telomerase was expressed. Chromosomal instability in E6-expressing cells was correlated (P < 0.05) with both loss of telomeric signals and inactivation of G2 checkpoint function. The results suggest that chromosomal stability depends upon a complex interaction among the systems of telomere length maintenance and cell cycle checkpoints.
Assuntos
Aberrações Cromossômicas/genética , Fase G2/genética , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Proteínas Repressoras , Telômero/genética , Linhagem Celular , Senescência Celular/genética , Cromossomos Humanos Par 4 , Cromossomos Humanos Par 6 , Fibroblastos/enzimologia , Fibroblastos/metabolismo , Humanos , Hibridização in Situ Fluorescente , Proteínas Oncogênicas Virais/biossíntese , Polimorfismo de Fragmento de Restrição , Fuso Acromático/genética , Telomerase/biossíntese , Telômero/patologia , Translocação Genética/genética , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/análiseRESUMO
Chromosomal stability was linked to G2 checkpoint function in human fibroblasts expressing the human papillomavirus type 16 E6 oncoprotein. Soon after expression of E6, cells displayed an undamaged, diploid karyotype and normal mitotic delay after gamma-irradiation. As the E6-expressing cells aged through their in vitro life span, G2 checkpoint function diminished progressively. After 30-70 population doublings, 60-86% of the E6 cells displayed defective G2 checkpoint response. This attenuation of G2 checkpoint function was also associated with radiation-resistant cyclin B1/CDK1 protein kinase activity. Numerical and structural abnormalities of chromosomes developed in unirradiated E6 cells with kinetics that mirrored the loss of G2 checkpoint function. A significant correlation between inactivation of the G2 checkpoint and acquisition of chromosomal abnormalities was found, suggesting that the G2 checkpoint represents a barrier to genetic instability in cells lacking G1 checkpoint function.
Assuntos
Quebra Cromossômica/fisiologia , Fase G2/fisiologia , Fase G2/efeitos da radiação , Proteínas Oncogênicas Virais/metabolismo , Proteína Quinase CDC2/análise , Técnicas de Cultura de Células/métodos , Células Cultivadas , Senescência Celular , Ciclina B/análise , Ciclina B1 , DNA/análise , DNA/efeitos dos fármacos , Relação Dose-Resposta à Radiação , Fibroblastos/metabolismo , Fibroblastos/efeitos da radiação , Citometria de Fluxo , Fase G1/fisiologia , Fase G1/efeitos da radiação , Genes p53/fisiologia , Humanos , Fator Promotor de Maturação/efeitos da radiação , Microscopia de Fluorescência , Ploidias , Propídio/farmacologia , Proteínas Repressoras/metabolismo , Fase S , Fuso Acromático/fisiologiaRESUMO
In response to DNA damage, cells transduce a signal that leads to accumulation and activation of p53 protein, transcriptional induction of several genes, including p21, gadd45, and gadd153, and cell cycle arrest. One hypothesis is that the signal is mediated by DNA-dependent protein kinase (DNA-PK), which consists of a catalytic subunit (DNA-PKcs) and a regulatory subunit (Ku). DNA-PK has several characteristics that support this hypothesis: Ku binds to DNA damaged by nicks or double-strand breaks, DNA-PKcs is activated when Ku binds to DNA, DNA-PK will phosphorylate p53 and other cell cycle regulatory proteins in vitro, and DNA-PKcs shares homology with ATM, which is mutated in ataxia telangiectasia and involved in signaling the p53 response to ionizing radiation. The hypothesis was tested by analyzing early passage fibroblasts from severe combined immunodeficient mice, which are deficient in DNA-PK. After exposure to ionizing radiation, UV radiation, or methyl methane-sulfonate, severe combined immunodeficient and wild-type cells were indistinguishable in their response. The accumulation of p53, induction of p21, gadd45, and gadd153, and arrest of the cell cycle in G1 and G2 occurred normally. Therefore, DNA-PK is not required for the p53 response or cell cycle arrest after DNA damage.
