Loss of nuclear p53 protein in preneoplastic rat hepatocytes is accompanied by Mdm2 and Bcl-2 overexpression and by defective response to DNA damage in vivo

Abstract

Previous studies have indicated that isolated preneoplastic rat hepatocytes in vitro fail to induce nuclear p53 protein and fail to block replication in response to genotoxic compounds. This suggests that defects in the protection of genomic integrity are part of their premalignant character. In the present study, we have investigated if similar defects occur in vivo. Preneoplastic glutathione-S-transferase (GST) 7-7-positive foci were induced in male Wistar rats by diethylnitrosamine (DEN) initiation and promotion with 2-acetylaminofluorene (2-AAF)/partial hepatectomy (PH). The response to genotoxic damage was studied by X-irradiation. p53 protein was moderately expressed in nuclei in surrounding hepatocytes. This nuclear p53 staining had decreased 2 weeks after 2-AAF treatment. In foci, the protein was detected in the cytoplasm whereas the nuclei were negative. Levels of p21 waf1/cip1 protein were high in nuclei and cytoplasm of surrounding hepatocytes, whereas the expression in foci was low. A low level of Mdm2 in nuclei was observed in surrounding liver, while both Mdm2 and Bcl-2 protein were strongly expressed in the cytoplasm in foci. X-ray exposure further induced nuclear expression of p53, p21 waf1/cip1 , and Mdm2 in surrounding hepatocytes, but focal nuclei were still negative. DNA replication was strongly reduced by X-irradiation in surrounding hepatocytes, but only partially reduced in the foci. These results indicate that the p53 pathway of response to genomic stress is impaired in preneoplastic Over the last decades the process of experimental hepatocarcinogenesis in rats has been intensively studied (reviewed in Schulte-Hermann 1 and Pitot 2 ). Early in the process, preneoplastic cells emerge, which are able to proliferate during exposure to cytotoxic/genotoxic tumor promoters 3 while phenotypically normal cells are blocked in their growth. 4 Changes in expression of several proteins have been related to the premalignant nature of the preneoplastic cells, such as increased expression of glutathione-S-transferase (GST) 7-7, p-glycoprotein, and ␥-glutamyltranspeptidase, and decreased expression of a sulfotransferase and alterations in cytochrome P450s and c-myc expression. [5][6][7][8][9][10][11][12] The molecular mechanisms of their escape from growth inhibition as normally seen in hepatocytes after exposure to genotoxic compounds are, however, not clear.The tumor suppressor p53 plays an important role in maintaining the integrity of the genome. 13 Wild-type p53 protein blocks the cell cycle in G1 after DNA damage and may stimulate DNA repair. [13][14][15][16] The p53 protein is mutated in over 50% of the human tumors indicating that loss of p53 is an important event in tumor development. 13,14 We have shown earlier that compounds with hepatic tumor promoting activity are able to induce p53 in rat liver in vivo, 17,18 and that cells from preneoplastic foci in vitro and in vivo express less nuclear p53 compared with surrounding hepatocytes during treatment with tumor promoters 19 (Ohlson et al., subm...

Persistent and remodeling hepatic preneoplastic lesions present differences in cell proliferation and apoptosis, as well as in p53, Bcl‐2 and NF‐κB pathways

“…It was suggested that this may facilitate clonal expansion of PNL under stress induced by DNA-damaging chemicals [Lennartsson et al, 1988]. In rats submitted to the RH model X-ray-induced genotoxic stress resulted in p53 accumulation in the cytoplasm of GST 7-7-positive PNL hepatocytes and in the nucleus of surrounding hepatocytes [Van Gijssel et al, 2000]. However, in this study the authors did not distinguish pPNL from rPNL regarding cytoplasmic p53 accumulation in contrast to our study.…”

“…The intensity of staining in the cytoplasm (p53 and Bcl-2) or whole cell (p65) of preneoplastic cells was classified as higher than (positive p53, Bcl-2, or p65 staining PNL) or equal to (negative p53, Bcl-2, or p65 staining PNL) the reference staining in the cytoplasm or whole cell of surrounding hepatocytes. A PNL was classified as positive for p53, Bcl-2, or p65 staining if 50% or more of the cells concerned were stained more intensively than the staining in surrounding hepatocytes [Van Gijssel et al, 2000]. To verify if the PNL were pPNL or rPNL, all the immunohistochemical evaluation analyses of p53, Bcl-2, and p65 staining were made comparing the serial histological sections with those marked for GST-P.…”

“…p53 accumulation in the cytoplasm was related to the incapacity of hepatic PNL of rats submitted to the RH model to respond to genomic stress [Van Gijssel et al, 2000], and to an increase in malignancy of hepatocarcinomas [Heinze et al, 1999] and other neoplasias [Nikolaev et al, 2003]. Increase in Bcl-2 expression in basophilic PNL, considered to be aggressive ] was observed in mouse hepatocarcinogenesis [Christensen et al, 1999].…”

“…The pPNL phenotype could be related to alterations in p53 translocation to the nucleus. This could be related to an increased genomic instability early in hepatocarcinogenesis, which not only sustains clonal expansion, but also increases the probability of further genetic changes leading towards malignant transformation [Van Gijssel et al, 2000]. The rPNL phenotype could be related to a normalization of the p53 pathway.…”

Cirrhotic livers reveal genetic changes in the MDM2-P14ARF system of cell cycle regulators

“…One of the early events in liver carcinogenesis is p53 overexpression, which has been detected in nodules of non-tumourous liver cirrhosis and which is considered to indicate a cellular stress response preceding manifestation of HCC (Livni et al, 1995). P53 is bound by oncoprotein MDM2 which may also contribute to hepatocarcinogenesis since MDM2 overexpression as a potential tumorigenic event was demonstrated in preneoplastic rat hepatocytes as well as in human HCC (Endo et al, 2000;Van Gijssel et al, 2000). The present study proves that MDM2 accumulation is also a feature of some regenerative nodules in human cirrhotic livers.…”