CHROMOSOMAL INSTABILITY IS CORRELATED WITH TELOMERE EROSION AND INACTIVATION OF G2 CHECKPOINT FUNCTION IN HUMAN FIBROBLASTS EXPRESSING HUMAN-PAPILLOMAVIRUS TYPE-16 E6 ONCOPROTEIN

Cell cycle checkpoints and tumor suppressor gene functions appear to b e required for the maintenance of a stable genome in proliferating cel ls. In this study chromosomal destabilization was monitored in relatio n 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-1 6) E6 oncoprotein to inactivate p53, Chromosome aberrations were enume rated during in vitro aging of isogenic control (F5neo) and HPV-16E6-e xpressing (F5E6) fibroblasts. We found that structural and numerical a berrations in chromosomes were significantly increased in F5E6 cells d uring aging in vitro and fluorescence in situ hybridization (FISH) ana lysis using chromosome-specific probes demonstrated the occurrence of rearrangements involving chromosome 4 and 6 in genetically unstable F5 E6 cells. Flow cytometry and karyotypic analyses revealed increased po lyploidy and aneuploidy in F5E6 cells only at passages >16, although t hese cells displayed defective mitotic spindle checkpoint function ass ociated with inactivation of p53 at passages 5 and 16, G2 checkpoint f unction was confirmed to be gradually but progressively inactivated du ring 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 di splayed extension of in vitro lifespan and did not induce beta-galacto sidase at high passage, Erosion of telomeres during in vitro aging of telomerase-negative F5neo cells was demonstrated by Southern hybridiza tion and by quantitative FISH analysis on an individual cell level. Te lomeric signals diminished continuously as F5neo cells aged in vitro b eing 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 stabilize d at passage 34 when telomerase was expressed. Chromosomal instability in E6-expressing cells was correlated (P < 0.05) with both loss of te lomeric signals and inactivation of C2 checkpoint function. The result s suggest that chromosomal stability depends upon a complex interactio n among the systems of telomere length maintenance and cell cycle chec kpoints.