Inhibin/activin expression in human and rodent liver: subunits α and ßB as new players in human hepatocellular carcinoma?

BACKGROUND: Activins and inhibins belong to the TGFß-superfamily, which controls cell proliferation and differentiation in many organs. Activin A, the dimer of inhibin ßA subunit, acts strongly anti-proliferative in hepatocytes. Little is known on the other activin/inhibin subunits in human liver and hepatocellular carcinoma (HCC). METHODS: We studied the expression of the complete inhibin family α, ßA, ßB, ßC, ßE in normal liver, tumour-adjacent and HCC tissue, 12 additional organs and rodent liver. A total of 16 HCC and 10 disease-free livers were analysed. Expression of inhibin subunits was determined by qRT-PCR, normalised to RNA input and by geNorm algorithm, and confirmed by immunohistochemistry. RESULTS: Remarkably, ßA expression was not decreased in HCC. Similarly, ßC and ßE exhibited no major changes. In contrast, inhibin α, barely detectable in normal liver, was strongly increased in tumour-adjacent liver and dramatically enhanced in HCC. ßB was strongly enhanced in some HCC. At variance with human liver, rodent liver showed higher inhibin α and ßC expression, but ßA was somewhat, and ßB dramatically lower. CONCLUSIONS: Upregulation of inhibin α - and possibly of ßB - may shield HCC cells from anti-proliferative effects of activin A. Dramatic variations between humans and rodents may reflect different functions of some inhibins/activins.

New cellular tools reveal complex epithelial-mesenchymal interactions in hepatocarcinogenesis.

To enable detailed analyses of cell interactions in tumour development, new epithelial and mesenchymal cell lines were established from human hepatocellular carcinoma by spontaneous outgrowth in culture. We obtained several hepatocarcinoma (HCC)-, B-lymphoblastoid (BLC)-, and myofibroblastoid (MF)-lines from seven cases. In-depth characterisation included cell kinetics, genotype, tumourigenicity, expression of cell-type specific markers, and proteome patterns. Many functions of the cells of origin were found to be preserved. We studied the impact of the mesenchymal lines on hepatocarcinogenesis by in vitro assays. BLC- and MF-supernatants strongly increased the DNA replication of premalignant hepatocytes. The stimulation by MF-lines was mainly attributed to HGF secretion. In HCC-cells, MF-supernatant had only minor effects on cell growth but enhanced migration. MF-lines also stimulated neoangiogenesis through vEGF release. BLC-supernatant dramatically induced death of HCC-cells, which could be largely abrogated by preincubating the supernatant with TNFbeta-antiserum. Thus, the new cell lines reveal stage-specific stimulatory and inhibitory interactions between mesenchymal and epithelial tumour cells. In conclusion, the new cell lines provide unique tools to analyse essential components of the complex interplay between the microenvironment and the developing liver cancer, and to identify factors affecting proliferation, migration and death of tumour cells, neoangiogenesis, and outgrowth of additional malignancy.

Coffee consumption protects human lymphocytes against oxidative and 3-amino-1-methyl-5H-pyrido[4,3-b]indole acetate (Trp-P-2) induced DNA-damage: results of an experimental study with human volunteers.

Aim of the study was to investigate the impact of coffee on DNA-stability in humans. DNA-damage was monitored in lymphocytes of eight individuals with single cell gel electrophoresis assays before and after consumption of 600 ml coffee (400 ml paper filtered and 200 ml metal filtered/d) for five days. Under standard conditions, no alteration of DNA-migration was seen, but a strong reduction of DNA-migration attributable to endogenous formation of oxidised purines and pyrimidines was detected with restriction enzymes; furthermore DNA-damage caused by reactive oxygen radicals (H2O2 treatment) and by the heterocyclic aromatic amine 3-amino-1-methyl-5H-pyrido[4,3-b]indole-acetate was significantly reduced after coffee consumption by 17% and 35%, respectively. Also in in vitro experiments, inhibition of H2O2 induced DNA-damage was observed with coffee at low concentrations (

Inherent growth advantage of (pre)malignant hepatocytes associated with nuclear translocation of pro-transforming growth factor alpha.

The pro-peptide of transforming growth factor alpha (proTGFalpha) was recently found in hepatocyte nuclei preparing for DNA replication, which suggests a role of nuclear proTGFalpha for mitogenic signalling. This study investigates whether the nuclear occurrence of the pro-peptide is involved in the altered growth regulation of (pre)malignant hepatocytes. In human hepatocarcinogenesis, the incidence of proTGFalpha-positive and replicating nuclei gradually increased from normal liver, to dysplastic nodules, to hepatocellular carcinoma. ProTGFalpha-positive nuclei almost always were in DNA synthesis. Also, in rat hepatocarcinogenesis, proTGFalpha-positive nuclei occurred in (pre)malignant hepatocytes at significantly higher incidences than in unaltered hepatocytes. For functional studies unaltered (GSTp(-)) and premalignant (GSTp(+)) rat hepatocytes were isolated by collagenase perfusion and cultivated. Again, DNA synthesis occurred almost exclusively in proTGFalpha-positive nuclei. GSTp(+) hepatocytes showed an approximately 3-fold higher frequency of proTGFalpha-positive nuclei and DNA replication than GSTp(-) cells. Treatment of cultures with the mitogen cyproterone acetate (CPA) elevated the incidence of proTGFalpha-positive nuclei and DNA synthesis in parallel. Conversely, transforming growth factor beta1 (TGFbeta1) lowered both. These effects of CPA and TGFbeta1 were significantly more pronounced in GSTp(+) than in GSTp(-) hepatocytes. In conclusion, nuclear translocation of proTGFalpha increases in the course of hepatocarcinogenesis and appears to be involved in the inherent growth advantage of (pre)malignant hepatocytes.

Role of apoptosis for mouse liver growth regulation and tumor promotion: comparative analysis of mice with high (C3H/He) and low (C57Bl/6J) cancer susceptibility.

Apoptosis constitutes one of the organisms defense lines against cancer. We investigated whether failure of apoptosis may be concurrently causative for the high cancer susceptibility in C3H/He as compared to C57BL/6J mice (low cancer susceptibility). First, in short-term in vivo experiments (7-21 days), mouse liver growth (C3H/He, C57BL/6J) was induced by administration of phenobarbital (PB; 2 days 500 ppm + 5 days 750 ppm via the food) or nafenopin (NAF; 7 days 500 ppm via the food), cessation of PB or NAF treatment served to initiate liver involution. Liver weight, DNA content, hepatocyte ploidy and apoptotic activity were studied as endpoints. Secondly, in a long-term study liver carcinogenesis was initiated by a single dose of N-nitrosodiethylamine (NDEA, 90 mg/kg b.w.) to 5-weeks-old C57Bl/6J and C3H/He mice. After 2 weeks, mice received either standard diet or a diet containing phenobarbital (PB, 90 mg/kg b.w.) for up to 90 weeks. Cell proliferation and apoptosis in normal liver tissue and (pre)neoplastic tissue was quantitatively analysed by histological means. The short term studies revealed that PB and NAF-induced mouse liver growth is essentially due to cell enlargement (hypertrophy). A moderate increase of liver DNA content was brought about by hepatocellular polyploidization; C3H/He mice exhibited the most pronounced ploidy shift, corresponding to their high cancer susceptibility. Upon cessation of PB or NAF treatment, regression of liver mass was neither associated with a loss of DNA nor an increase in apoptoses in the liver of C3H/He and C57Bl/6J mice; food restriction did not enforce the occurrence of apoptosis. Thus, the mouse strains did not differ with respect to the occurrence of apoptosis. In the long-term study, PB promoted liver tumor formation in all strains, exhibiting quantitative differences in growth kinetics of preneoplasia rather than a specific biological quality. Quantitative analysis of apoptosis in normal and (pre)neoplastic liver tissue of C3H/He and C57BL/6J mice revealed no clue to explain their different cancer susceptibility. Rather, cell proliferation seems to be the prevailing determinant of tumor promotion in the liver of both mouse strains.

Impaired Ras membrane association and activation in PPARalpha knockout mice after partial hepatectomy.

Liver regeneration after partial hepatectomy (PH) involves several signaling mechanisms including activation of the small GTPases Ras and RhoA in response to mitogens leading to DNA synthesis and cell proliferation. Peroxisome proliferator-activated receptor-alpha (PPARalpha) regulates the expression of several key enzymes in isoprenoid synthesis, which are key events for membrane association of Ras and RhoA. Thus the role of PPARalpha in cell proliferation after PH was tested. After PH, an increase in PPARalpha DNA binding was observed in wild-type mice, correlating with an increase in the PPARalpha-regulated enzyme acyl-CoA oxidase. In addition, the PPARalpha-regulated genes farnesyl pyrophosphate synthase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase were significantly increased in wild-type mice. However, these increases were not observed in PPARalpha knockout (PPARalpha -/-) mice. The peak in DNA synthesis observed 42 h after PH was reduced by approximately 60% in PPARalpha -/- mice, despite increases in TNF-alpha and IL-1. Also, under these conditions, membrane association of Ras was high in wild-type mice after PH but was impaired in PPARalpha -/- mice. Accordingly, Ras was significantly elevated in the cytosol in PPARalpha -/- mice. This observation correlated with lower levels of active GTP-bound Ras after PH in PPARalpha -/- mice compared with wild-type mice. Similar observations were made for RhoA. Moreover, deletion of PPARalpha blunted the activation of cyclin-dependent kinase (cdk)2/cyclin E and cdk4/cyclin D complexes. Collectively, these results support the hypothesis that PPARalpha is necessary for cell cycle progression in regenerating mouse liver via mechanisms involving prenylation of small GTPases Ras and RhoA.

Expression and dimerization of the rat activin subunits betaC and betaE: evidence for the ormation of novel activin dimers.

Activins are cytokines of the transforming growth factor beta family, which plays a central role in the determination of cell fate and the regulation of tissue balance. Family members are composed of two subunits and this dimerization is critical for liganding their cognate receptors and execution of proper functions. In the current study we focused on the localization of activin betaA, betaB, betaC and betaE subunits in the adult rat and analyzed the composition of putative activin beta dimers. By dissecting tissue distribution of various activins, we found that the liver, in particular the hepatocytes, is the major source for activin betaC and betaE transcripts, since other tissues almost failed to express these isoforms. In sharp contrast, the emergence of activin betaA and betaB appeared ubiquitous. Using a highly selective proteome approach, we were able to identify homo- as well as heterodimers of individual activin subunits, indicating a high redundancy of ligand composition. Certainly, this broad potential to homo- and heterodimerize has to be considered in future studies on activin function.

Role of transforming growth factor alpha and prostaglandins in preferential growth of preneoplastic rat hepatocytes.

The role of transforming growth factor alpha (TGFalpha) and prostaglandins (PGs) in the preferential growth of preneoplastic liver cells was studied. Rats received the genotoxic hepatocarcinogen N-nitrosomorpholine (NNM); placental glutathione S-transferase (GSTp) was used as a marker to identify preneoplastic foci. Preneoplastic foci expressing TGFalpha (TGFalpha(+)) grew more rapidly than TGFalpha negative (TGFalpha(-)) ones. Almost all tumours studied were positive for TGFalpha. The key enzymes of prostaglandin synthesis, cyclooxygenase I (Cox-1) and II (Cox-2), were present in all unaltered and preneoplastic cells and tended to decrease in the later stages of hepatocarcinogenesis. Immunostaining revealed that cultures of hepatocytes, isolated from NNM-treated livers by collagenase perfusion, contained 1-2% GSTp-positive (GSTp(+)) and 9% TGFalpha(+) hepatocytes; 0.6% of the cells were GSTp(+)/TGFalpha(+). Cox-1 and Cox-2 were present in all cells. DNA replication was almost exclusively associated with expression of TGFalpha. GSTp(+) hepatocytes showed a 3- to 4-fold higher probability of TGFalpha expression and of DNA synthesis than GSTp-negative (GSTp(-)) cells. PGE(2) or PGF(2alpha) increased expression of TGFalpha and DNA replication in GSTp(-) cells but not in GSTp(+) cells. PGA(2) and PGJ(2) decreased DNA synthesis in TGFalpha(+) cells without an obvious effect on the intracellular levels of TGFalpha. The Cox-2 inhibitor SC236 suppressed DNA replication preferentially in GSTp(+) cells; this inhibition was reversed by PGE(2)/F(2alpha). Indomethacin had no effect. These results suggest the following conclusions. (i) Growth regulation of preneoplastic GSTp(+) cells in culture exhibits distinct differences from GSTp(-) cells and elevated expression of TGFalpha contributes to their growth advantage. (ii) TGFalpha renders preneoplastic hepatocytes sensitive to suppression of DNA synthesis by PGA(2)/J(2). (iii) SC236, a Cox-2 inhibitor, may have preventive value in hepatocarcinogenesis.

Khat (Catha edulis) consumption causes genotoxic effects in humans.

We used the micronucleus (MN) test to determine the genetic damage caused by khat, a widely consumed psychostimulant plant, in exfoliated cells of volunteers who chewed the drug on a regular basis. In the first study in which we compared the frequency of MN in buccal and bladder mucosa cells in 20 khat consumers (10-160 g/day) and 10 controls, a pronounced (8-fold) increase in micronucleated buccal mucosa cells was seen among khat consumers; khat consumption did not lead to a detectable elevation of micronucleated bladder mucosa cells. Among heavy khat chewers, 81% of the MN had a centromere signal indicating that khat is aneuploidogenic. To investigate the effect of simultaneous consumption of tobacco and alcoholic beverages, we compared the MN frequency in buccal cells of 25 khat consumers (20-85 g/day) who smoked cigarettes (15-60/day) and drank alcoholic beverages (15-80 g of pure ethanol/day) with a control group (control group I) of 25 individuals matched for age, body weight, tobacco and alcohol consumption and with another control group of 25 individuals (control group II) not consuming any of the drugs. The frequency of buccal mucosa cells with MN was higher in control group I than in group II and the effect of khat, tobacco and alcohol was found to be additive. A time-kinetics study on khat-induced MN showed that the highest frequency of MN was observed during the fourth week after consumption. In light of the large body of evidence on the close association between genetic damage and cancer, these results suggest that khat consumption, especially when accompanied by alcohol and tobacco consumption, might be a potential cause of oral malignancy.

Peroxisome proliferators do not increase DNA synthesis in purified rat hepatocytes.

There have been numerous reports that chemicals which induce peroxisomes in rodent liver increase DNA synthesis in isolated hepatic parenchymal cells, but not as well in vitro as in vivo. It is also known that tumour necrosis factor alpha (TNFalpha) is mitogenic in isolated hepatocytes. Since Kupffer cells are a major source of TNFalpha in the liver and have recently been shown to be activated by peroxisome proliferators, the possibility exists that the effect of peroxisome proliferators on DNA synthesis in parenchymal cells is via Kupffer cell contamination of isolated hepatocyte preparations. The purpose of this study was to evaluate this hypothesis by studying the effect of model peroxisome proliferators on purified hepatocyte preparations. Hepatocytes were prepared from rat liver by standard calcium-free and collagenase perfusion. Subsequently, cells were centrifuged through Percoll to remove contaminating non-parenchymal cells. Cells were at least 99.9% pure as assessed by cell counting using specific markers for hepatocytes (resorufin O-glucoside) and Kupffer cells (FITC-labelled latex beads). Hepatocytes were cultured in Williams medium + 10% fetal bovine serum for 24 h followed by culture for 48 h in Williams medium plus or minus drug or mitogen additions. Under these conditions epidermal growth factor stimulated DNA synthesis assessed by incorporation of [3H]thymidine approximately 5-fold over control levels. The peroxisome proliferators WY,14-643 and nafenopin, however, had no effect on DNA synthesis, although they did increase acyl-CoA oxidase as expected. In contrast, TNFalpha increased cell proliferation nearly 10-fold in purified hepatocytes, an effect nearly doubled by WY-14,643. Further, when conditioned medium from purified Kupffer cells incubated with WY-14,643 was added to pure hepatocytes, DNA synthesis was increased over 2-fold in a time-dependent manner. Collectively, these data support the hypothesis that peroxisome proliferators do not influence DNA synthesis in isolated hepatocytes per se. Rather, they stimulate cytokine production by Kupffer cells which in turn increases DNA synthesis in parenchymal cells. An increase in mitogenic cytokine production by Kupffer cells is necessary for stimulation of DNA synthesis in purified rat parenchymal cells.

Biology of transforming growth factor beta in hepatocarcinogenesis.

TGF-beta is an important factor in the regulation of liver growth. It is an inhibitor of hepatocyte DNA synthesis and may induce active cell death, e.g., to remove excessive tissue mass. Studies using transgenic mice suggest that expression in the resting liver has to be well balanced; either under- or overexpression appear to cause an increased turnover of hepatocytes and to predispose to hepatocarcinogenesis. TGF-beta overexpression is frequently observed in human hepatocellular carcinomas, probably as a late event in tumor development. In men and mice, TGF-beta overexpression appears to be associated with loss of TGF-beta responsiveness often by disruption of TGF-beta signaling. However, mechanisms as mutations in TGF-beta receptor II or Smad2 and 4 genes, frequently observed in other human cancers, have only rarely been observed in hepatocellular carcinomas. Further studies may clarify the mechanisms by which hepatocellular tumors escape TGF-beta growth control, as well as analyze possible roles of TGF-beta overexpression in immunosuppression and angiogenesis.

Failure to demonstrate chemoprevention by the monoterpene perillyl alcohol during early rat hepatocarcinogenesis: a cautionary note.

The monoterpene perillyl alcohol (PA) is being considered as a useful chemopreventive and therapeutic agent against human cancers. However, no data are available on the effects of PA in the first stages of hepatocarcinogenesis. To study such effects, putatively initiated cells and preneoplastic foci in hepatocarcinogenesis were used as a model. Male Wistar rats were treated with a single dose of N:-nitrosomorpholine (NNM). Between days 4 and 91 after NNM, subgroups of rats received either PA (1 g/kg body wt/day) or phenobarbital (PB) (50 mg/kg body wt/day) in the diet. Since PA treatment reduced food intake, one control group was fed ad libitum, while a second control was pair fed between days 4 and 91. In order to enhance any treatment effects, all groups, including the controls, were treated with the potent tumor promoter PB after day 91 until the end of the experiment at day 266. Rats were killed at multiple time points and putatively initiated cells and preneoplastic foci were identified by staining positively for placental glutathione S-transferase (G+). The following results were obtained. (i) A few days after NNM treatment single G+ cells emerged; a considerable portion of which developed into foci. (ii) Treatment with PB resulted in an increase in number and size of G+ foci. (iii) PA treatment failed to reduce the number of G+ cells; it somewhat lowered rates of apoptosis in G+ foci and clearly increased their average size. (iv) Eighty-seven days of PA revealed no protective effect on day 266, but, similar to PB treatment, increased the growth of foci. In conclusion, PA exerted no detectable chemopreventive effect in the early stages of rat hepatocarcinogenesis. It rather exerted a PB-like tumor promoting activity. These data argue against a recommendation of PA as a chemopreventive agent for healthy humans.

The Fas-induced apoptosis analyzed by high throughput proteome analysis.

The fate of cytosolic proteins was studied during Fas-induced cell death of Jurkat T-lymphocytes by proteome analysis. Among 1000 spots resolved in two-dimensional gels, comparison of control versus apoptotic cells revealed that the signal intensity of 19 spots decreased or even disappeared, whereas 38 novel spots emerged. These proteins were further analyzed with respect to de novo protein synthesis, phosphorylation status, and intracellular localization by metabolic labeling and analysis of subcellular protein fractions in combination with two-dimensional Western blots and mass spectrometry analysis of tryptic digests. We found that e.g. hsp27, hsp70B, calmodulin, and H-ras synthesis was induced upon Fas signaling. 34 proteins were affected by dephosphorylation (e.g. endoplasmin) and phosphorylation (e.g. hsc70, hsp57, and hsp90). Nuclear annexin IV translocated to the cytosol, whereas decreasing cytosolic TCP-1alpha became detectable in the nucleus. In addition, degradation of 12 proteins was observed; among them myosin heavy chain was identified as a novel caspase target. Fas-induced proteome alterations were compared with those of other cell death inducers, indicating specific physiological characteristics of different cell death mechanisms, consequent to as well as independent of caspase activation. Characteristic proteome alterations of apoptotic cells at early time points were found reminiscent of those of malignant cells in vivo.

Quantitative analysis of tumor initiation in rat liver: role of cell replication and cell death (apoptosis).

The formation and development of initiated cells has been studied at the beginning of hepatocarcinogenesis. Rats received the genotoxic carcinogen N-nitrosomorpholine (NNM); placental glutathione S-transferase was used as a marker of initiated cells (G+ cells). Single G+ cells appeared within 24 h after NNM; their frequency increased steeply for approximately 2 weeks, then decreased and finally remained constant. G+ foci consisting of >/=2 G+ cells appeared successively after the single cells. Histological determination of DNA replication and apoptosis revealed that: the formation of single G+ cells may not depend on DNA replication of precursor cells; single G+ cells showed considerably lower DNA replication than G- normal hepatocytes; from the 2-cell stage onwards G+ foci displayed enhanced DNA replication and apoptosis. Data from histological sections were transformed into the third dimension by a new stereological method which considers the non-spherical shape of many G+ lesions. Rates of division and death of G+ cells and of formation and growth of G+ foci were estimated by a stochastic model: initially G+ clones appeared at a rate of 12 000 per day and liver until a maximal number of 176 000 (phase I) was reached; thereafter they declined to 134 000 (phase II); they then remained constant (phase III). Estimated division rates of G+ cells decreased from phase I to phase III, while the death rate increased in phase II, when every third G+ clone disappeared. As a result, at day 50 after NNM only 0.3% of G+ single cells had formed a clone containing >/=5 cells. In conclusion, experimental and computed parameters provide direct evidence that hepatocarcinogenesis evolves clonally and that initiated hepatocytes have a selective proliferation advantage, associated with an enhanced potential to undergo apoptosis. Thereby, depending on the conditions, initiated clones expand or become extinct. Extinction may lead to reversion of the biological effects of initiation.

Search for compounds that inhibit the genotoxic and carcinogenic effects of heterocyclic aromatic amines.

Over the last 30 years approximately 160 reports have been published on dietary compounds that protect from the mutagenic and carcinogenic effects of heterocyclic aromatic amines (HAAs). In the first section of this review, the current state of knowledge is briefly summarized. Based on the evaluation of the available data, various protective mechanisms are described, and the use of different methodologies for the detection of protective effects is critically discussed. In most antimutagenicity studies (>70%) bacterial indicators (predominantly Salmonella strain TA98) were used, and about 600 individual compounds and complex mixtures have been identified that attenuate the effects of HAAs. The most frequently used in vivo method to detect protective effects are adduct measurements; anticarcinogenic dietary factors were identified by aberrant crypt foci assays and liver foci tests with rats. The mechanisms of protection include inactivation of HAAs and their metabolites by direct binding, inhibition of enzymes involved in the metabolic activation of the amines, induction of detoxifying enzymes, and interaction with DNA repair processes. The detection spectrum of conventional in vitro mutagenicity assays with metabolically incompetent indicator cells is limited. These procedures reflect only simple mechanisms such as direct binding of the HAAs to pyrroles and fibers. It has been shown that these compounds are also effective in rodents. More complex mechanisms, namely, interactions with metabolic activation reactions are not adequately represented in in vitro assays with exogenous enzyme homogenates, and false-negative as well as false-positive results may be obtained. More appropriate approaches for the detection of protective effects are recently developed test systems with metabolically competent cells such as the human Hep G2 line or primary hepatocytes. SCGE tests and DNA adduct measurements with laboratory rodents enable the detection of antigenotoxic effects in different organs, including those that are targets for tumor induction by the amines. Medium term assays based on aberrant crypt foci in colon and liver foci tests have been used to prove that certain compounds that prevented DNA damage by HAAs also reduced their carcinogenic effects. These experiments are costly and time consuming and, due to the weak induction capacity of the amines, only pronounced anticarcinogenic effects can be detected. Over the years, a large bulk of data on HAA protective compounds has accumulated, but only for a few (e.g., fibers, pyrroles, constituents of teas, and lactic acid bacteria) is there sufficient evidence to support the assumption that they are protective in humans as well.

Suppression of apoptosis in C3H mouse liver tumors by activated Ha-ras oncogene.

Liver tumors were induced in male C3H mice by a single injection of N-nitrosodiethylamine and characterized with respect to the presence of base substitutions in the hot-spot position at codon 61 of the Ha-ras proto-oncogene. An increase in Ha-ras mutation prevalence was found with time after induction of tumors, suggesting that the activated ras gene provides a selective growth advantage. However, no significant differences in 5-bromodeoxyuridine labeling indices were evident between ras mutated and ras wild-type tumors, demonstrating that cell division rates in the two tumor populations were very similar. Apoptotic indices were determined by counting eosinophilic apoptotic bodies. The frequency of occurrence of apoptotic bodies was found to be approximately five times lower in tumors with Ha-ras mutations when compared with tumors not showing the mutation. This demonstrates that the activated p21(Ras) protein has anti-apoptotic activity in transformed mouse hepatocytes in vivo and suggests that the preferential outgrowth of Ha-ras-mutated hepatoma cells is mediated by suppression of apoptosis rather than by stimulation of cell division.

Dose-response and threshold effects in cytotoxicity and apoptosis.

Cell death can occur as an active, programmed event in response to cytotoxic injury or to endogenous growth limiting factors; the latter serve to maintain homeostasis of cell number in tissues. Cells seem to use different pathways for programmed death, as reflected by their different morphology and different biochemistry. Severe cell damage leading to incapacitation of essential cell functions such as ATP synthesis or the maintenance of membrane potential may lead to "necrosis". In any event, the incidence and rate of cell death increase with increasing signal intensity. Cytotoxic injury requires a certain number of primary insults; cell death will therefore occur only beyond a definable threshold. Growth factor control of cell death is receptor-mediated with dose-response relations including threshold phenomena follow the general principles of receptor kinetics. The occurrence of programmed cell death during the stages of carcinogenesis introduces a reversible component into this disease. Therefore, there may exist thresholds of dose or durations of exposure to certain carcinogens below which irreversible disease is not generated.

Initiated rat hepatocytes in primary culture: a novel tool to study alterations in growth control during the first stage of carcinogenesis.

To study growth regulation in the beginning of carcinogenesis, we established a novel ex vivo model for co-cultivation of normal and putatively initiated hepatocytes. Rats received the genotoxic hepatocarcinogen N-nitrosomorpholine (NNM). This led to the appearance of hepatocytes expressing placental glutathione S-transferase (G(+) cells). These cells exhibited elevated rates of cell replication and apoptosis, as known from further advanced preneoplasia; G(+) cells were considered initiated. At days 20-22 post-NNM treatment their frequency was maximal (1-2%); approximately 40% were still single and 60% were arranged in mini foci. At this time-point liver cells were isolated by collagenase perfusion and cultivated. G(+) cells, identified by immunostaining of the culture-plates, were present at the same percentage as in vivo, excluding selective loss, enrichment or spontaneous expression of the G(+) phenotype. In untreated cultures G(+) hepatocytes showed significantly higher rates of replicative DNA synthesis than normal G(-) cells. Application of the hepatomitogen cyproterone acetate (CPA) elevated DNA replication preferentially in G(+) cells. Transforming growth factor beta1 (TGF-beta1) suppressed replicative DNA synthesis which was more pronounced in G(+) than in G(-) hepatocytes. Combined treatment with CPA and TGF-beta1 had no effect on G- cells, but considerably inhibited DNA replication in G(+) cells. This suggests that the effects of TGF-beta1 predominated in G(+) hepatocytes. We conclude that putatively initiated G(+) hepatocytes, both in vivo and in culture, exhibit higher basal rates of DNA replication than normal G(-) hepatocytes and an over-response to mitogens and growth inhibitors. Therefore, G(+) cells show (i) nearly identical behaviour in intact liver and in primary culture and (ii) inherent defects in growth control that are principally similar although somewhat less pronounced than in later stages of carcinogenesis. The present ex vivo system thus provides a novel and useful tool to elucidate biological and molecular changes during initiation of carcinogenesis.

Quantitative structure-activity relationships in a series of endogenous and synthetic steroids exhibiting induction of CYP3A activity and hepatomegaly associated with increased DNA synthesis.

The results of a quantitative structure-activity relationship (QSAR) study on a total of 14 steroids exhibiting induction of a CYP3A-associated activity and increase in liver weight/DNA synthesis is reported. It is found that different, but related, structural descriptors correlate with increase in ethylmorphine N-demethylase activity (r=0.92) and with the increase in liver weight (r=0.78) and DNA synthesis (r=0.78). Although there is a strong correlation between increase in liver weight and DNA content (r=0.999), neither of these correlated with ethylmorphine N-demethylase activity. These findings are discussed in the light of CYP3A induction, substrate specificity and inhibition; a proposed model of human CYP3A4 based on sequence homology with CYP102, a bacterial P450 of known crystal structure, demonstrates the possible mode of interaction between substrates and inhibitors within the putative active site.