Regulation of p53 and Susceptibility to Cell Death in Chemically-Induced Preneoplastic Hepatocytes

Induction of preneoplastic lesions, termed enzyme-altered foci (EAF), is considered to be an early step in the development of liver cancer. In view of the relatively homogenous properties of the majority of EAF, these lesions are suggested to arise as an adaptive response to toxic stress. An overall goal of this thesis was to study the role of adaptations to genotoxic stress in EAF development and how resistance to toxicity in EAF was affected. In association with carcinogenesis much focus has been on the tumor suppressor gene p53. This gene is commonly mutated in cancer cells, thus indicating a critical role in preventing cancer development. Previously, it has been found that EAF lesions from rats treated with the genotoxic agent diethylnitrosamine (DEN) display an attenuated p53 response to genotoxic stress, compared to normal liver tissue. In the first study the underlying mechanisms of the attenuated p53 response was examined in EAF hepatocytes. We found that in contrast to genotoxic agents, treatment of primary hepatocytes with the hypoxiamimicking agent CoCl2 induced a p53 response in both EAF and normal cells. In addition, we further found decreased levels of serine-15 phosphorylated p53 in EAF tissue upon toxic stress. The kinase ATM is involved in the phosphorylation of p53 after DNA damage, specifically at serine-15, and analysis of EAF tissue, employing immunohistochemistry and western blot analysis, revealed reduced levels of ATM compared to normal tissue. We hypothesized that an attenuated p53 response in EAF hepatocytes reflects an adaptation to acute cytotoxic and genotoxic stress seen at high doses. In the second study, rats received relatively low doses of DEN for 10 or 20 weeks, and their livers were subsequently analyzed. All doses induced EAF lesions and a majority of these demonstrated a relatively low p53 response after DEN treatment, as compared to surrounding tissue. Compared to rats receiving DEN at high dose rates, rats receiving the same cumulative dose at a low dose rate generally possessed larger preneoplastic lesions that were more “p53-negative”. These data argue against our hypothesis and suggest subtler genotoxic effects behind the alteration of the p53 response. EAF hepatocytes have been found to be relatively resistant to toxicological stress and apoptosis induced by xenobiotics. However, in the third study, in vitro treatment with different sphingolipids, such as ceramide and sphingosine, was found to induce cell death, predominantly in EAF hepatocytes. TLC analysis and immunohistochemistry demonstrated altered levels of sphingosine, sphingosine-1-phosphate and glucosylated ceramide in EAF tissue. In in vivo experiments, administration of sphingomyelin-supplemented diet to DENtreated rats was found to reduce the number of preneoplastic lesions. Sphingolipids have been suggested to exert their apoptotic effects by decreasing levels of phosphorylated Akt (pAkt). In the last study, we found that a fraction of EAF displayed higher immunohistochemical staining of ceramide compared to the surrounding tissue, which correlated with lower staining of pAkt. Western blot analysis revealed that sphingosine was able to inhibit insulin-induced Akt phosphorylation in vitro. Furthermore, we found that the levels of p27 were increased in EAF and that these cells also were more sensitive to treatment with rapamycin, an inhibitor of mTOR. In conclusion, our results suggest that the DNA damage signaling pathway, including ATM and p53, is downregulated in EAF hepatocytes. This may result in a resistance to cell death in EAF hepatocytes and confer a growth advantage to EAF in a toxic environment. Furthermore, the sphingolipid levels and the pAkt-mTOR signaling pathway seem to be altered in many EAF cells. This may render these cells more susceptible to cell death induced by sphingolipids. List of original articles I. Silins I, Finnberg N, Ståhl A, Högberg J and Stenius U. Reduced ATM kinase activity and an attenuated p53 response to DNA damage in carcinogen-induced preneoplastic hepatic lesions in the rat. Carcinogenesis, 22, 2023-2031, 2001. II. Silins I, Nordstrand M, Högberg J and Stenius U. Sphingolipids suppress preneoplastic rat hepatocytes in vitro and in vivo. Carcinogenesis, 24, 1077-1083, 2003. III. Silins I, Stenius U, Högberg J Induction of preneoplastic rat liver lesions with an attenuated p53 response by low doses of diethylnitrosamine. Manuscript, 2003. IV. Silins I, Högberg J, Stenius U. Susceptibility to cell death in preneoplastic hepatocytes; the role of Akt kinase. Manuscript, 2003.