In vivo studies of cell cycle regulating proteins in rats during liver regeneration and during promotion of liver carcinogenesis

University dissertation from Stockholm : Karolinska Instiutet

Abstract: There are more than one hundred different types of cancer in humans. However, the major platform for all types of cancer is automous and uncontrolled cell division and though most types of cancer are possible to treat and cure by surgery, chemical therapy or irradiation, more efficient and less toxic therapies are urgently needed. In the modern society we are exposed to more toxins than ever. The liver, as main detoxifier of our blood, is handling many hazardous compounds. Unfortunately, after being metabolised in the liver, many of these are able to act as promoters for one of the most lethal types of cancer, the hepatocellular carcinoma (HCC). Previous studies demonstrated that nuclear accumulation of p53 and other proteins is essential for efficient tumor suppression. Based on this, the aim of the study was to investigate whether altered localization and/or p53 protein expression is an early event in tumor development in liver that possibly contributes to the growth advantage of initiated hepatocytes. We also addressed the question of which impact 2-AAF exerts on cell cycle at early stages of tumor promotion. Preneoplastic foci were induced in rat liver by treatment with diethylnitroseamin (DEN), combined with either of the four tumor promoters, 2acetylaminofluorene, 17-alpha ethinylestradiol, choline-deficient diet or deoxycholic acid. This was combined with 2/3 partial hepatectomy as a proliferative stimulus. The immunohistochemical results showed that all four promoters decreased the focal expression of p53 and p21, two proteins known to inhibit replication upon DNA damage, in both nucleus and cytoplasm. In contrast, in surrounding tissue increased nuclear levels of p53 and p21 was observed. These data suggest that deregulation of p53 and p21 might be a common feature occurring early during promotion of HCC in vivo. We also found that treatment with 2-AAF prevented the induction of nuclear p53 in foci in response to gamma-irradiation in initiated male Wistar rats, which correlated with increased cytoplasmic expression of Mdm2 and Bcl-2 in foci. It is possible that complex formation with elevated Mdm2 together with increased Bcl-2 protein expression contributes to the sequestration and inactivation of p53 in cytoplasm. Growth signalling in regenerating liver was studied in non-initiated rats subjected to the tumor promoter 2-AAF. We found that 2-AAF exercise an almost complete mitoinhibitory effect in synchronised hepatocytes. Nuclear levels of cdk 4, cyclin D3, cyclin E, p53, p 130 and pRb were increased while the levels of PCNA, cdk 2, E2F- 1, -3 and -4 were decreased. Furthermore, both nuclear and cytoplasmic expression of cyclin A and - B proteins was lost. Finally, we found that p107 a member of the Rb-family and necessary for S-phase progression, but not pRB was increased in normal regenerating liver. In mitoinhibited liver the expression level of these two proteins was rather the opposit indicating slightly different roles for pRb and p107 during regeneration and cell cycle control, at least in this animal model.

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