The attenuation of the P53 response to DNA damage in rodent liver preneoplastic enzyme-altered foci

Sammanfattning: In connection with life-time bioassays in rodents, the liver is one of the organs most frequently affected. During the carcinogenic process, single preneoplastic hepatocytes develop into hepatocellular adenoma or carcinoma (HCC). Preneoplastic hepatocytes are identified on the basis of their overexpression of the inactivating phase 11 enzyme glutathione - S-transferase Pi (GST-P). In the presence of continuos exposure to carcinogen, initiated hepatocytes expand clonally to form hepatic enzyme-altered foci (EAF). Such development of EAF might be considered to be an adaptive response and their pattern of gene expression may provide mechanistic information concerning the action of a putative carcinogen. Our studies have focused on the response of the tumor suppressor p53 to DNA damage in EAR Activated by several types of cellular stress, the p53 protein is involved in regulating cell cycle arrest and apoptosis. The overall aim of our project was to characterize the attenuated p53 response to DNA damage in preneoplastic EAF lesions and the possible role of this attenuation in the development of EAF by diethylnitrosamine (DEN). To this end, after receiving an initiating neonatal dos of DEN female Sprague-Dawley rats were exposed to DEN or the non-genotoxic agent phenobarbital (PB), which induced the development of EAF in both cases. A challenging dose of DEN was also administered 24 hours prior to sacrifice to elicit a p53 response in these EAF. Only EAF arising from treatment with DEN exhibited an attenuated p53 response in comparison to that of surrounding, non-EAF tissue and PB-induced EAF. This attenuation was enhanced by prolonging the period of treatment, as well as in larger EAF. The attenuated p53 response to DEN-induced DNA damage was also present in primary co-cultures of hepatocytes isolated from EAF (GST -P -positive hepatocytes) and from non-EAF tissue (GST-P-negative hepatocytes). Treatment of such co-cultures with CoCl2, which mimics hypoxia, resulted in nuclear accumulation of p53 in the GST-P-positive cells. This finding demonstrates that a p53 response may be evoked by hypoxic stress, but not by genotoxic chemicals. Additional studies with the P13 kinase inhibitors caffeine and wortmannin, as well as with ATM antisense oligonucleotides indicated that ATM is involved in signalling to p53 following DEN-induced damage of DNA. Immunohistochemical analysis of the livers of DEN-treated rats and Western blotting of macroscopic EAF tissue revealed lowered expression of ATM in these tissues. Thus, down-regulation of ATM may to some extent explain the attenuated p53 response to DEN exhibited by EAT. Upon examining the combined p53-MDM2 response in rat liver at different time-points following a single injection of DEN, significant temporal and spatial variations were observed. Midzonal areas demonstrated a transient combined p53 - MDM2 response 6 - 24 hours after the DEN challenge, whereas in centrilobular areas this response culminated 24 - 72 hours after injection. MDM2 was constitutively expressed in midzonal areas. Furthermore, following repeated treatment with low doses of DEN, GST -P -positive EAF were found to be particularly prevalent in this same zone. Finally, the influence of the p53 gene dosage on the development of p53-negative preneoplastic lesions was investigated. Treatment of p53 (+/+) and (+/-) mice for 15 - 20 weeks with DEN revealed a genotype - dependent difference in the numbers of p53-negative preneoplastic hepatic lesions obtained, with p53 (+/-) mice developing significantly fewer p53 -negative lesions than p53 wild-type (+/+) mice. However, the total number and average size of all preneoplastic lesions were similar in these two types of mice. In conclusion, these findings indicate that an attenuated p53 response to DNA damage confers a growth advantage on preneoplastic focal lesions in the liver. The selective pressure for focal lesions exhibiting such p53 attenuation can be modulated by altering the p53 gene dosage or by exposure to xenobiotics. These observations indicate that the attenuated p53 response in preneoplastic lesions is an adaptive response to genotoxic stress.