Aberrant cell cycle checkpoint function in transformed hepatocytes and WB-F344 hepatic epithelial stem-like cells

Abstract

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 G1, G2 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 G1 checkpoint function. Chemically and virally transformed hepatocyte lines displayed significant attenuation of G1 checkpoint function, ranging from partial to complete ablation. WB-F344 rat hepatic epithelial cell lines at low, mid and high passage levels expressed G1 checkpoint function comparable with NRH. Only one of four malignantly transformed WB-F344 cell lines displayed significant attenuation of G1 checkpoint function. Attenuation of G1 checkpoint function in transformed hepatocytes and WB-F344 cells was associated with alterations in p53, ablated/ attenuated induction of p21Waf1 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 G2 checkpoint function. All transformed hepatocyte and WB-F344 cell lines displayed significant attenuation of the G2 checkpoint. Moreover, the parental WB-F344 line displayed significant age-related attenuation of G2 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.