Institutet för miljömedicin (IMM) / Institute of Enviromental Medicine
Abstract
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