Reduced ATM kinase activity and an attenuated p53 response to DNA damage in carcinogen-induced preneoplastic hepatic lesions in the rat.

In previous studies we have demonstrated that the p53 response to DNA damage in preneoplastic liver lesions, referred to as enzyme-altered foci (EAF), is attenuated. In the present investigation comparative quantitative RT-PCR revealed no major difference in the p53 mRNA levels in EAF and non-EAF tissue. When CoCl(2) was employed to induce hypoxia-inducible factor (HIF-1alpha), both non-EAF and EAF hepatocytes readily accumulated p53, whereas EAF hepatocytes did not accumulate p53 upon treatment with diethylnitrosamine (DEN). The p53 response was also induced in EAF hepatocytes by the inhibitor of nuclear export, leptomycin B. An inhibitor of DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM), wortmannin, blocked the DEN-induced p53 response in non-EAF hepatocytes. Assay of kinase activity in immunoprecipitated material from EAF and non-EAF tissue revealed attenuated ATM activity in EAF. Immunohistological and western blot analysis of the level of ATM protein was in agreement with the activity measurements and no phosphorylation of Ser15 in p53 was detected in EAF tissue 24 h after a challenging dose of DEN. Taken together with previously published data, these data indicate selective attenuation of the DNA damage pathway in EAF hepatocytes. Down-regulation of DNA damage-induced and ATM-mediated phosphorylation of p53 may confer a growth advantage on EAF hepatocytes.

Xenobiotics modulate the p53 response to DNA damage in preneoplastic enzyme-altered foci in rat liver; effects of diethylnitrosamine and phenobarbital.

Enzyme-altered foci (EAF) develop in rat liver in response to carcinogen treatment. Our hypothesis is that EAF adapt to genotoxic stimuli by lowering their expression of p53 and that such decreased p53 expression confers a growth advantage on the hepatocytes present in EAF. After a single neonatal dose of diethylnitrosamine (DEN), rats were treated with either 2 - 12 additional doses of DEN or phenobarbital (PB) for 3 - 14 months. Twenty-four hours prior to sacrifice, all rats also received a challenging dose of DEN. The numbers of p53-positive hepatocytes (demonstrating immunohistological staining in the nucleus) in EAF and surrounding tissue were subsequently determined. In DEN-treated rats, p53 expression was attenuated in EAF compared to surrounding tissue. The longer the period of treatment and the larger the size of the EAF, the fewer the p53-positive hepatocytes/mm2 were observed in these lesions. These data were confirmed by Western blot analysis. PB-treated rats did not demonstrate this effect seen in DEN-treated rats. In this case, the expression of p53 was not related to size of EAF or length of treatment. Many EAF in PB-treated animals contained very large numbers of p53-positive cells. Upon staining for terminal deoxynucleotidyl transferase-mediated X-dUTP nick-end labeling (the TUNEL procedure), many apoptotic hepatocytes were also seen in EAF. These data indicate that the p53 response to DNA damage can be modulated by xenobiotics. This can be explained as an adaptive alteration in the p53 response.

Monoclonal antibody probes for p21WAF1/CIP1 and the INK4 family of cyclin-dependent kinase inhibitors.

Inhibition of cyclin dependent kinases (cdk) by proteins of two families of cdk inhibitors (CKIs) represents one of the key modes of cell-cycle control. Although not fully understood at present, the functions of the individual members of the Cip/Kip and INK4 families of CKIs have been implicated in fundamental biological processes as diverse as cellular proliferation, responses to genotoxic stress, regulation of cellular differentiation, and senescence. In addition, the seven currently known CKIs qualify as either established or candidate tumor suppressors whose loss or inactivation contribute to molecular pathogenesis of a wide range of tumor types. In this study, we report the isolation and characterization of a panel of 10 mouse monoclonal antibodies (MAbs) that specifically recognize p21WAF1/CIP1 (p21) or the individual members of the INK4 family of CKIs: p15INK4b (p15), p16INK4a (p16), p18INK4c (p18), or p19INK4d (p19). These antibodies are proving to be invaluable molecular probes for analyses of protein abundance, subcellular localization, interacting cellular proteins, and ultimately the function(s) of these cell cycle regulators. Epitopes targeted by the antibodies were mapped by peptide enzyme-linked immunoadsorbent assay (ELISA), and performance of the MAbs assessed in a range of immunochemical techniques. Individual MAbs of our series recognize distinct pools of the respective CKIs, a feature reflected by their differential applicability in immunoblotting, immunoprecipitation, and immunostaining including immunohistochemistry on archival paraffin-embedded tissue sections. Together, these antibodies represent useful reagents to study CKIs in cells and tissues, a set of tools that should help elucidate the physiological roles played by the individual CKIs, and better understand the molecular mechanisms of loss or inactivation of these (candidate) tumor suppressors in human malignancies.

p53 expression and TGF-alpha-induced replication of hepatocytes isolated from rats exposed to the carcinogen diethylnitrosamine.

Previous reports indicate that the expression of transforming growth factor alpha (TGF-alpha) is increased in enzyme-altered foci (EAF) arising in livers of rats treated with a carcinogen. Here we have investigated the effects of TGF-alpha on EAF cells in vitro. Hepatocytes were isolated from rats that had received repeated treatment with diethylnitrosamine (DEN) and whose livers contained glutathione S-transferase P (GST-P)-positive EAF. Primary cultures of GST-P-positive and GST-P-negative hepatocytes were exposed to TGF-alpha. TGF-alpha (20-40 ng/ml) increased DNA replication in the GST-P-negative, but not in the GST-P-positive cells. Furthermore, it was shown that this effect on GSTP-negative cells could be blocked by p53 antisense oligonucleotides. We conclude that EAF hepatocytes do not respond to TGF-alpha in vitro. This lack of response may reflect the attenuated expression of p53 in these cells. These data corroborate previous findings that, in response to DNA damage, many EAF hepatocytes do not accumulate p53.

Wild-type p53 expression in liver tissue and in enzyme-altered foci: an in vivo investigation on diethylnitrosamine-treated rats.

Previous reports have documented an attenuated p53 response to DNA damage in hepatocytes isolated from enzyme-altered foci (EAF). Here, we have studied this p53 response in vivo in rats with EAF. These animals received repeated doses of diethylnitrosamine (DEN) for 6 weeks and a challenging dose 24 h before death. Liver sections were then analysed using an immunohistological procedure for p53, or a double-staining procedure for p53 and glutathione-S-transferase pi (GST-P). In control rats or rats with EAF not given the challenging dose of DEN, there was no p53 staining. In control rats, only given the challenging dose of DEN, there was a centrilobular p53 nuclear staining that co-localized with TUNEL staining. In an experiment involving four rats with EAF 389 +/- 39 hepatocytes/mm2 of non-EAF tissue stained positively for p53, while the corresponding value for EAF tissue was 27.6 +/- 7.5. Thus, p53-positive cells were 14.6-fold more frequent in non-EAF than in EAF tissue. In many EAF no p53-positive cells were seen at all and 83% of the EAF demonstrated <20% of the number of p53-positive cells seen in non-EAF tissue. Very few EAF had as high a proportion of p53-positive cells as did the average non-EAF tissue. EAF >0.06 mm2 had significantly fewer p53-positive cells than smaller EAF. The ratio of p53 expression in non-EAF tissue and large EAF was 32.6. In a control experiment, four EAF-bearing rats were used as donors to prepare primary cultures of hepatocytes. After 24 h of exposure to DEN, many of the cultured cells became p53-positive. Among GST-P-negative hepatocytes, 12.8% were p53-positive, whereas only 0.25% of the GST-P-positive hepatocytes were p53-positive. Literature data suggest that the altered xenobiotic metabolism in EAF may give rise to a 3-4-fold difference in DNA damage between non-EAF and EAF tissues. It is concluded that GST-P-positive EAF hepatocytes have an attenuated p53 response to DNA damage. This attenuated response may facilitate clonal expansion of EAF under stress induced by DNA-damaging chemicals.

Changes in liver fatty acid-binding protein in rat enzyme-altered foci.

The level of liver fatty acid-binding protein (L-FABP) was analyzed in enzyme-altered foci (EAF) positive for GST-P, or after classification of foci into different subclasses by haematoxylin and eosin staining. Rats were treated with either an initiating single dose of diethylnitrosamine (DEN) followed by no treatment, treatment with phenobarbital, PCB, nafenopin or repeated injections of DEN, or alternatively non-treated or treated with nafenopin alone. Changes in the level of L-FABP were detected in the majority of EAF and both L-FABP-positive and -negative foci were seen. However, in rats initiated with DEN, EAF were almost exclusively L-FABP-negative. The fraction of L-FABP-negative foci increased with increasing foci size, while the time of treatment or the dose of the promoter did not seem to have any effect. It was also found that treatment with DEN gave a higher fraction of L-FABP-negative foci as compared to treatment with phenobarbital or PCB, indicating a specific effect of DEN. These data together with previously published findings suggest that L-FABP expression in EAF is determined by the initiating carcinogenic regimen and that it might be possible to use the expression of L-FABP in tumours to differentiate initiating chemicals.

Phenobarbital-induced cytoplasmic accumulation of beta1-integrin in rat liver enzyme altered foci; an immunohistological study.

In this immunohistological study we investigated integrin expression in EAF in female rats treated with diethylnitrosamine (DEN) as initiator and phenobarbital (PB) as promotor (DEN-PB treatment) for up to 32 weeks. Using a beta1-integrin antibody, there was an increased cytoplasmic staining and a decreased sinusoidal staining in EAF, as compared to non-EAF areas. The majority of small EAF and all larger EAF exhibited this altered distribution of beta1-integrin. The increased cytoplasmic staining was not found in EAF after a 10 week treatment-free period. In periportal areas in partial hepatectomized control rats a similar increase in cytoplasmic staining was seen. EAF in DEN-initiated and DEN-promoted rats (DEN-DEN treatment) were also studied. This protocol induced rapidly growing EAF. Most lesions did not show the increased cytoplasmic staining. However, after partial hepatectomy of DEN-DEN-treated rats, a cytoplasmic staining was seen in EAF. It is concluded that PB induced a reversible cytoplasmic beta1-integrin expression in many EAF and in all larger EAF. It is suggested that the alteration constitutes part of hepatocyte resistance to toxicological stress and apoptosis in EAF.

In vitro studies on non-genotoxic carcinogens: resistance to DNA synthesis inhibition in GST-P-positive hepatocytes isolated from enzyme-altered foci-bearing rats.

Hepatocytes were isolated from enzyme-altered foci (EAF)-bearing rats. Carcinogen-induced effects on DNA synthesis were studied in primary cultures of isolated cells. The test substance was added at the start and at 24hr. At 48hr the cultures were stained with antibodies against glutathione S-transferase P (GST-P) and DNA synthesis was estimated by thymidine incorporation. It was found that four of nine carcinogens induced a clear selective inhibition of DNA synthesis in GST-P-negative cells. Two of the four inhibiting carcinogens induced p53 in GST-P-negative cells, but did not in GST-P-positive cells. Of four tested non-carcinogens, none induced a selective inhibition of DNA synthesis in GST-P-negative hepatocytes. These findings suggest that this model system is useful when characterizing liver carcinogens and their carcinogenic mechanisms.

Low expression of the WAF1/CIP1 gene product, p21, in enzyme-altered foci induced in rat liver by diethylnitrosamine or phenobarbital.

The expression of the WAF1/CIP1 gene product, p21, in enzyme-altered foci (EAF) induced by diethylnitrosamine (DEN) and phenobarbital (PB) was examined. p21 expression in the nucleus of hepatocytes in EAF was decreased compared to surrounding tissue. Fifty-eight percent of all GST-P-positive EAF induced by DEN and 79% of the EAF induced by PB were p21-negative. The proportion of p21-negative EAF increased with the size of the foci and more than 90% of the largest EAF were p21-negative. p21 is a mediator of p53 signals leading to block of the cell cycle. In conjunction with previous data indicating that p53 is not induced in GST-P-positive hepatocytes isolated from EAF-bearing rats, the results of this study suggest a role for altered signaling in the G1-S check point in rat hepatocarcinogenesis.

GST-P-positive hepatocytes isolated from rats bearing enzyme-altered foci show no signs of p53 protein induction and replicate even when their DNA contains strand breaks.

Hepatocytes were isolated from rats with enzyme-altered foci (EAF) in their livers and were studied in primary cultures. Cultures were treated with two doses of 0.6 mM diethylnitrosamine (DEN) at 1.5 and 24 h. At 48 h the cultures were double stained with antibodies against glutathione S-transferase P (GST-P) and p53 protein. Ten percent of the GST-P-immunonegative cells were p53-immunopositive. Thymidine incorporation was blocked in these cells. Both p53 expression and the block in thymidine incorporation could be eliminated by p53 antisense oligonucleotides. Less than 1% of the GST-P-positive cells in the same cultures were p53-immunopositive. Thymidine incorporation was less affected than in GST-P-negative cells. DNA strand breaks were also monitored by an immunological technique. Twenty-three percent of the GST-P-negative cells and 7% of the GST-P-positive cells were positive for this marker. Seven percent of the GST-P-positive cells with DNA strand breaks incorporated thymidine. Virtually none of the GST-P-negative cells with DNA strand breaks demonstrated thymidine incorporation. We suggest that GST-P-positive cells lack functional p53 protein and that this permits cells with damaged DNA to replicate.

Resistance against ethacrynic acid in glutathione transferase 7-7 (GST-P)-positive hepatocytes isolated from carcinogen-treated rats: the role of cytoskeletal changes and ATP depletion.

Ethacrynic acid (Ea) is a substrate for glutathione transferase 7-7 (GST-P) in rat. Toxic effects of Ea have been related to its metabolism and GSH depletion, but resistance conferred by GSTP1-1 (the human homologue) has also been reported. Hepatocytes from enzyme altered foci (EAF) express GST-P, and a model for selection of resistant EAF cells has been developed using Ea as a toxic agent. In the present study the effects of Ea in this model have been characterized. Hepatocytes from foci-bearing rats were isolated. Isolated cells were exposed to Ea for 1-4 hr in suspension. They were then allowed to attach to collagen-coated plates in a serum-containing medium. Preferentially GST-P-positive cells attached after Ea treatment, thus increasing the number of positive cells per attached cells (GST-P-%). Extracellular GSH, as well as alpha-tocopherol, did not influence the Ea effect. However, the effect of Ea was counteracted by inhibitors of glutathione transferase activity. Taxol, a microtubule stabilizing agent, also counteracted the effect of Ea on GST-P-%. 1,2-Dichloro-4-nitrobenzene (DCNB, 0.4 mM), which is a substrate for other glutathione transferase isoenzymes than GST-P, also increased the GST-P-%. However, the effect of DCNB was not inhibited by taxol. It was also found that Ea induced a drop in ATP levels, but this effect, as well as cell leakage, came later than the loss of attachment. The data suggest that the critical effect of Ea was cytoskeletal changes, and that GST-P conferred resistance by detoxification of Ea.

Isolation of glutathione S-transferase P-positive hepatocytes from carcinogen treated rats by use of ethacrynic acid as selecting agent.

Rat liver responds to carcinogen treatment with growth of glutathione S-transferase P (GST-P)-positive enzyme-altered foci. In this paper a method is described where GST-P-positive hepatocytes are isolated from carcinogen-treated rats. The method utilizes ethacrynic acid, which is a good substrate for GST-P, and which induces toxicity mainly in GST-P-negative cells. The toxicity results in a loss of attachment to collagen. The method gives a 70% pure population of GST-P-positive cells attached to collagen-coated plates. Use of additional markers supports the conclusion that the GST-P-positive cells were derived from foci. Isolated GST-P-positive hepatocytes spread out and formed primary cultures of normal appearance. It was also shown that they synthesized DNA and did not respond to transforming growth factor beta 1. It is concluded that isolated GST-P-positive hepatocytes can be used for studies on alterations in enzyme-altered foci that cannot be done with in situ immunohistochemistry or in situ hybridization.

Genetic polymorphism of cytochrome P4502E1 in a Swedish population. Relationship to incidence of lung cancer.

Genetic polymorphism of CYP2E1 was investigated among 195 Swedish patients with lung cancer and 206 controls. Three different polymorphic sites were found, all in introns, using RFLP and the restriction enzymes DraI, RsaI and TaqI. The frequencies of the rare alleles were 0.08-0.18 and much lower than previously described among Japanese. No significant difference in distribution of the polymorphic alleles between controls and lung cancer patients was evident, in contrast to results of a previous Japanese study. However, examination of a polymorphic site in the 5'-flanking region, within a putative binding motif for the hepatic transcription factor HNF-1, revealed a significantly less frequent distribution of the mutated allele (c2) among the lung cancer patients as compared to controls. It is concluded that major interethnic differences exist in the genetic polymorphism of CYP2E1 and that people carrying the c2 allele might be at lower risk for developing lung cancer.

Selective toxicity in putative preneoplastic hepatocytes: a comparison of hydroquinone and duroquinone.

In this paper data is presented suggesting selective toxicity towards enzyme altered hepatocytes. Hydroquinone (HQ) treatment 24 or 48 h after diethylnitrosamine (DEN) initiation reduced the number of glutathione S-transferase-P (GST-P)-positive hepatocytes in situ. Furthermore, in experiments on primary cultures of hepatocytes from control rats a synergism in cell killing between DEN and HQ was observed. In another in vitro system the effect of HQ and duroquinone (DQ) on GGT-positive and -negative hepatocytes was investigated. DQ was shown to affect the GGT-positive cells, while HQ mainly affected GGT-negative cells. These results suggest that HQ can reduce the population of enzyme altered foci (EAF) precursor cells by synergistic interactions with DEN, but provide no support for the notion that HQ selectively damage cells in developed EAF. This conclusion is supported by previously published data on effects of HQ on the development of EAF.

Peroxisome proliferation and resistance to hydrogen peroxide in rat hepatocytes: is development of resistance an adaptation to cytotoxicity?

In this work the resistance of peroxisome-proliferated hepatocytes to hydrogen peroxide (H2O2) has been studied. The question has been raised as to whether this resistance is a response to cytotoxicity. In an initial series of experiments, hepatocytes were isolated from rats that had been treated with nafenopin (NAF-hepatocytes). Isolated cells were exposed to a H2O2-generating system or to H2O2 in pulses. The ability to attach to collagen was used as a toxicological endpoint. Loss of attachment was found to be correlated to glutathione (GSH) depletion, and NAF-hepatocytes were more resistant to GSH depletion and to loss of attachment induced by H2O2 than were control hepatocytes. NAF-hepatocytes were not resistant to hydroquinone or to adriamycin. It was also indicated that this resistance was related to an altered metabolism of H2O2, less dependent on GSH. In a second series of experiments, hepatocytes from altered hepatic foci-bearing rats, treated with nafenopin or di(2-ethylhexyl)phthalate (DEHP), were used. This model was used in an attempt to monitor the development of resistance in different subpopulations of hepatocytes. It was found that the majority of hepatocytes developed resistance towards H2O2, and that, for example, foci marker-positive hepatocytes were as resistant as marker-negative cells. In control experiments with this model, it was found that marker-positive cells were more resistant towards diethyl maleate (DEM) or phorone than were marker-negative cells. In addition to demonstrating the validity of the model, these control experiments indicate an increased steady-state level of H2O2 in cells from peroxisome proliferator-treated rats. Other control experiments suggested that a low GSH-peroxidase activity protected from, rather than aggravated, the effect of peroxisome proliferation on marker-negative and GSH-depleted cells. It is concluded that H2O2 metabolism may affect the function of collagen receptors, but that a shift in H2O2 metabolism, so that it becomes less dependent on GSH, conferred resistance to this effect. The apparent non-focal induction of resistance to peroxisome proliferators, as opposed to the focal induction of resistance induced by most liver carcinogens, may explain the lack of development of gamma-glutamyltranspeptidase-positive foci in peroxisome proliferator-treated rats.

Selenium metabolism in isolated hepatocytes: inhibition of incorporation in proteins by mono(2-ethylhexyl)phthalate, a metabolite of the peroxisome proliferator di(2-ethylhexyl)phthalate.

Di(2-ethylhexyl)phthalate (DEHP), a rat liver carcinogen, induces peroxisomal proliferation and a concomitant oxidative stress, but decreases liver glutathione peroxidase (GSH-Px) activity. This enzyme is a selenoprotein and we have investigated the influence of mono(2-ethylhexyl)phthalate (MEHP), a major metabolite of DEHP, on selenium incorporation in hepatocellular proteins. [75Se]Selenious acid (6 nM) was added to primary cultures of rat hepatocytes and protein incorporation was assessed by SDS-PAGE and autoradiography. High concentrations of MEHP (1.0-3.0 mM) inhibited selenium labeling of all major selenoproteins in 3-24 h experiments, but also inhibited protein synthesis as assessed by leucine incorporation. The protein synthesis inhibition was reversible. Lower concentrations of MEHP (0.3-0.5 mM) did not decrease the 75Se-labeling in 24 h experiments and did not inhibit leucine incorporation. However, conditions that significantly induced peroxisomal proliferation also affected the 75Se-labeling. Thus in 72 h experiments, 0.05-0.25 mM MEHP increased the labeling of a 58 kd protein, decreased the labeling of a 23 kd protein (with the same mol. wt as GSH-Px), had no effect on a 20 kd protein and decreased the labeling of a 15 kd protein (as compared to MEHP-free control plates). The pattern of changes associated with peroxisomal proliferation mimicked that seen in livers from selenium-deficient animals, as reported by others. These data indicate that the bioavailability of selenium is decreased by DEHP. This effect may relate to a transient inhibition of protein synthesis, but also to the DEHP-induced peroxisomal proliferation.

gamma-Glutamyltranspeptidase-positive rat hepatocytes are protected from GSH depletion, oxidative stress and reversible alterations of collagen receptors.

The aim of this study has been to define cytotoxic mechanisms that may cause clonal expansion in the liver of pre-carcinogenic cells. An in vitro model, which has been described previously, was used. Hepatocytes were isolated from carcinogen-treated rats and a high proportion of the cells were gamma-glutamyltranspeptidase (GGT)-positive. The cells were incubated in suspension and exposed to toxic agents in concentrations that induced a moderate increase in cellular leakage within 3 h. Samples were withdrawn and sampled cells were then allowed to attach to collagen-coated plates. Attached cells were stained and the ratio of GGT-positive/GGT-negative cells (GGT-ratio) was determined. The initial GGT-ratio was 10.4 +/- 4.7% and an increased ratio was taken as a sign of toxicity that resulted in a selection of GGT-positive cells. In a first series of experiments it was shown that hydroquinone and menadione increase the GGT-ratio, while diquat, sodium selenite, diethyl maleate or phorone do not. However, diethyl maleate in combination with diquat increased the GGT-ratio. Hydrogen peroxide (5 mM) increased the GGT-ratio as effectively as hydroquinone (0.3 mM). Lower concentrations of H2O2 (0.05 mM) increased the GGT-ratio in GSH-depleted cells. The changes induced by hydroquinone and H2O2 in low concentration were reversible. In another series of experiments, plates coated with antibodies against beta 1-integrin were used. An increase in the GGT-ratio was obtained with anti beta 1-integrin, but not with broad spectrum anti-rat hepatocyte or anti-rat beta 2-microglobulin antibodies as substrata. These data suggested an involvement of the beta 1-integrin in the selection. Taken together, these data indicate that GGT-positive hepatocytes are protected against GSH depletion and oxidative stress that may result in reversible receptor alterations.