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 ®broblasts 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) ®broblasts. We found that structural and numerical aberrations in chromosomes were signi®cantly increased in F5E6 cells during aging in vitro and¯uorescence in situ hybridization (FISH) analysis using chromosome-speci®c 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 416, although these cells displayed defective mitotic spindle checkpoint function associated with inactivation of p53 at passages 5 and 16. G2 checkpoint function was con®rmed to be gradually but progressively inactivated during in vitro aging of E6-expressing cells. Aging of F5neo ®broblasts was documented during in vitro passaging by induction of a senescence-associated marker, pH 6.0 lysosomal b-galactosidase. F5E6 cells displayed extension of in vitro lifespan and did not induce b-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 telomerasenegative F5E6 cells, but telomeres appeared to be stabilized at passage 34 when telomerase was expressed. Chromosomal instability in E6-expressing cells was correlated (P50.05) 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.

Cell cycle checkpoints are barriers to carcinogenesis as they function to maintain genomic integrity. Attenuation or ablation of checkpoint function may enhance tumor formation by permitting outgrowth of unstable cells with damaged DNA. To examine the function of cell cycle checkpoints in rat hepatocarcinogenesis, we analyzed the responses of the G (1), G (2) and mitotic spindle assembly checkpoints in normal rat hepatocytes, hepatic epithelial stem-like cells (WB-F344) and transformed derivatives of both. Normal rat hepatocytes (NRH) displayed a 73% reduction in the fraction of nuclei in early S-phase 6-8 h following 8 Gy of ionizing radiation (IR) as a quantitative measure of G (1) checkpoint function. Chemically and virally transformed hepatocyte lines displayed significant attenuation of G (1) checkpoint function, ranging from partial to complete ablation. WB-F344 rat hepatic epithelial cell lines at low, mid and high passage levels expressed G (1) checkpoint function comparable with NRH. Only one of four malignantly transformed WB-F344 cell lines displayed significant attenuation of G (1) checkpoint function. Attenuation of G (1) checkpoint function in transformed hepatocytes and WB-F344 cells was associated with alterations in p53, ablated/attenuated induction of p21 (Waf1) by IR, as well as aberrant function of the spindle assembly checkpoint. NRH displayed 93% inhibition of mitosis 2 h after 1 Gy IR as a quantitative measure of G (2) checkpoint function. All transformed hepatocyte and WB-F344 cell lines displayed significant attenuation of the G (2) checkpoint. Moreover, the parental WB-F344 line displayed significant age-related attenuation of G (2) checkpoint function. Abnormalities in the function of cell cycle checkpoints were detected in transformed hepatocytes and WB-F344 cells at stages of hepatocarcinogenesis preceding tumorigenicity, sustaining a hypothesis that aberrant checkpoint function contributes to carcinogenesis.

A series of promoting and non-promoting barbiturates and hydantoins were examined for their ability to sustain the growth of a phenobarbital (PB)-dependent hepatocyte line in cell culture. The effective liver tumor promoters, pentobarbital, allobarbital and 5-ethyl-5-phenylhydantoin, replaced PB and supported 6/27C1 hepatocyte colony formation in vitro at 52-87% of the level induced by PB. The weak promoters secobarbital and amobarbital supported colony formation at only 11-19% of the PB control. A significant correlation was observed for in vivo and in vitro promotion activities of barbiturates and hydantoins, indicating that clonal expansion by 6/27C1 hepatocytes was promoter-dependent. Cell density also appeared to influence hepatocyte growth in vitro. Hepatocyte colonies acquired the ability to grow in the absence of PB, such that after 10 days incubation with PB, approximately 50% of colonies continued to grow in the absence of promoter. This phenomenon of clone-size-dependent hepatocyte growth suggested the operation of an autocrine growth factor pathway. Addition of the hepatocyte mitogen and autocrine growth factor, transforming growth factor-alpha (TGF-alpha), to culture medium lacking PB induced a dose-dependent increase in 6/27C1 hepatocyte colony formation. At the optimal concentration of 3 ng/ml, TGF-alpha sustained hepatocyte clonal expansion at 84% of the level induced by 2 mM PB. Individual 6/27C1 colonies that grew from single cells in the presence of TGF-alpha were tested for promoter-dependent colony formation. Either PB or TGF-alpha supported colony formation by these cells at similar levels and when combined at optimal concentrations, the response appeared to be saturated. When these factors were tested in combination at suboptimal concentrations, the two compounds were additive for supporting colony formation by the parental 6/27C1 line. The ability of TGF-alpha to replace PB and sustain hepatocyte clonal expansion was confirmed with the tumorigenic 6/15 hepatocyte line. These results suggest that TGF-alpha and PB may promote hepatocarcinogenesis by stimulating a common signal transduction pathway.

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