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 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.

Characterization of cell‐cycle progression and growth of WB‐F344 normal rat liver epithelial cells following gamma‐ray exposure

“…4B). A similar emptying of the S-phase compartment following irradiation of WB-F344 cells with 8 Gy of ␥-rays was observed by Kaufmann et al (6).…”

“…Trosko 4), and Trosko and Ruch (5), have reported on extensive studies of the capacity of these cells to communicate with one another via gap junctional intercellular communication (GJIC) and the impact of GJIC on carcinogenesis. Kaufmann et al (6) have examined the influence of the number of in vitro passages (generations) of WB-F344 cells on their G 1 and G 2 checkpoint function following exposure to ionizing radiation (IR). This cell line has also been used to examine effects imparted by irradiated cells on their unirradiated neighbors, also known as bystander cells.…”

“…It has been well documented that G 2 /M-delay and its duration depend on the type of cells, type of IR, absorbed dose, dose rate, and cell-cycle stage during which cells were irradiated (10 -22). Cell-cycle progression is controlled by a family of checkpoint genes modulated in response to IR and other DNA damaging agents (6,23,24). In this work, we used flow cytometry (FCM) and cell counting to monitor WB-F344 cell-cycle progression and growth after they were irradiated at plateau phase with various doses of ␥-rays (1-10 Gy), and were subsequently subcultured at low density in fresh growth medium.…”

Splenic Niche Cells from Young Heterochronic Parabionts Have Decreased Capability to Amplify T-cell Proliferation in Vitro

“…Cell lysates were prepared as described in [14]. T-cells after stimulation were incubated in the lysis buffer (150 mmol NaCl, 20 mmol TRIS, 10 % glycerol, 1 % Triton X-100, 5 mmol EDTA, 50 mmol NaF, 1mmol PMSF) for 30 min on ice.…”

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“…Prevention of telomere erosion by ectopic expression of the catalytic subunit of human telomerase (hTERT) has been shown to prevent crisis in cells expressing SV40 large T antigen or HPV16E6 oncoprotein [7-10]. Normal diploid human fibroblasts expressing hTERT have been reported to maintain a normal karyotype and preserve cell cycle checkpoint function for at least 200 population doublings [11,12], although others have suggested that otherwise normal telomerase-expressing human fibroblasts do display alterations in expression of tumor suppressor genes, growth characteristics, and transient genetic instability [13-16] These studies have failed to directly address the question as to whether cells can maintain a stable genome in the absence of a functional DNA damage induced G 1 checkpoint.…”