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.

Fibroblast growth factor receptor 3-IIIc mediates colorectal cancer growth and migration.

BACKGROUND: Deregulation of fibroblast growth factor receptor 3 (FGFR3) is involved in several malignancies. Its role in colorectal cancer has not been assessed before. METHODS: Expression of FGFR3 in human colorectal tumour specimens was analysed using splice variant-specific real-time reverse transcriptase PCR assays. To analyse the impact of FGFR3-IIIc expression on tumour cell biology, colon cancer cell models overexpressing wild-type (WT-3b and WT3c) or dominant-negative FGFR3 variants (KD3c and KD3b) were generated by either plasmid transfection or adenoviral transduction. RESULTS: Although FGFR3 mRNA expression is downregulated in colorectal cancer, alterations mainly affected the FGFR3-IIIb splice variant, resulting in an increased IIIc/IIIb ratio predominantly in a subgroup of advanced tumours. Overexpression of WT3c increased proliferation, survival and colony formation in all colon cancer cell models tested, whereas WT3b had little activity. In addition, it conferred sensitivity to autocrine FGF18-mediated growth and migration signals in SW480 cells with low endogenous FGFR3-IIIc expression. Disruption of FGFR3-IIIc-dependent signalling by dominant-negative FGFR3-IIIc or small interfering RNA-mediated FGFR3-IIIc knockdown resulted in inhibition of cell growth and induction of apoptosis, which could not be observed when FGFR3-IIIb was blocked. In addition, KD3c expression blocked colony formation and migration and distinctly attenuated tumour growth in SCID mouse xenograft models. CONCLUSION: Our data show that FGFR3-IIIc exerts oncogenic functions by mediating FGF18 effects in colorectal cancer and may constitute a promising new target for therapeutic interventions.

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.

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.

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.

Role of active cell death (apoptosis) in multi-stage carcinogenesis.

Active cell death is a genetically encoded self-destruction of a cell. There occur morphologically different types of active cell death, e.g. apoptosis in the liver or autophagic cell death in human mammary carcinoma cells after tamoxifen treatment (Pre)neoplastic lesions in rat liver exhibit enhanced rates of apoptosis, which tend to increase with increasing malignancy. Tumor promoters and non-genotoxic carcinogens inhibit active cell death, thereby increasing the accumulation of (pre)neoplastic cells and accelerating the development of cancer. On the other hand promoter withdrawal, fasting or application of negative growth signals such as transforming growth factor beta 1 (TGF beta 1) enhance apoptosis and can lead to selective regression of preneoplastic lesions or tumors.

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.

Apoptosis and multistage carcinogenesis in rat liver.

Apoptosis is a type of active cell death. It is involved in the homeostasis of cell number in tissues and is controlled by the growth regulatory network in the organism. It is also involved in the active removal of damaged cells. We have studied the role of apoptosis in cancer pre-stages and overt cancer in vivo, using rat liver as our main model system. Quantitative determination of apoptosis in histological specimens revealed that the rate of apoptosis tends to increase from normal to (pre)neoplastic to malignant cells. Thereby active cell death largely counterbalances the increasing replicative activity in developing malignancy. Tumor promoters shift the balance in favor of cell replication, whereas promoter withdrawal, fasting or TGF-beta 1 favor apoptosis (anti-promotion). Preneoplastic cells are more susceptible than normal liver cells to stimulation of both cell replication or cell death. Consequentially (pre)neoplastic tissue may preferentially grow or die during the appropriate treatment. Regimens that favor apoptosis and lower cell replication are shown to result in the elimination of preneoplastic cell clones from the liver (anti-initiation) and to reduce the cancer risk of the animal.

FGF5 as an oncogenic factor in human glioblastoma multiforme: autocrine and paracrine activities.

Fibroblast growth factor 5 (FGF5) is widely expressed in embryonic but scarcely in adult tissues. Here we report simultaneous overexpression of FGF5 and its predominant high-affinity receptor (FGFR1 IIIc) in astrocytic brain tumour specimens (N=49) and cell cultures (N=49). The levels of both ligand and receptor increased with enhanced malignancy in vivo and in vitro. Furthermore, secreted FGF5 protein was generally present in the supernatants of glioblastoma (GBM) cells. siRNA-mediated FGF5 downmodulation reduced moderately but significantly GBM cell proliferation while recombinant FGF5 (rFGF5) increased this parameter preferentially in cell lines with low endogenous expression levels. Apoptosis induction by prolonged serum starvation was significantly prevented by rFGF5. Moreover, tumour cell migration was distinctly stimulated by rFGF5 but attenuated by FGF5 siRNA. Blockade of FGFR1-mediated signals by pharmacological FGFR inhibitors or a dominant-negative FGFR1 IIIc protein inhibited GBM cell proliferation and/or induced apoptotic cell death. Moreover, rFGF5 and supernatants of highly FGF5-positive GBM cell lines specifically stimulated proliferation, migration and tube formation of human umbilical vein endothelial cells. In summary, we demonstrate for the first time that FGF5 contributes to the malignant progression of human astrocytic brain tumours by both autocrine and paracrine effects.

Tumor development and apoptosis.

Apoptosis or other types of active cell death may play a major role at various stages of carcinogenesis. Active cell death can be induced, by internal and exogenous signals, in preneoplastic, neoplastic and even malignant cells. It may reverse the effects of initiation and promotion and may lead to tumor regression. Conversely tumor promoters may inhibit active cell death in preneoplastic cells and thereby accelerate cancer development.

[Relevance of apoptosis for carcinogenesis]. / Bedeutung der Apoptose für die Tumorentstehung.

Apoptosis is a type of active, genetically programmed cell death. It occurs under specific conditions and is characterized by it's morphology. It is controlled by genes, hormones and other factors regulating the growth of organs and cells in the organism. In the liver and some other epithelial tissues transforming growth factor beta 1 and related peptides seem to be involved in the homeostasis of cell multiplication and cell death. In the course of carcinogenesis, initiated, preneoplastic and neoplastic cells and cell foci in the liver show enhanced DNA synthesis and also enhanced apoptosis. Tumor promoters inhibit apoptosis and increase cell replication and can thereby shift the balance between birth and death of cells accelerating tumor development. Fasting can have the opposite effect.

Hepatocarcinogenic potential of di(2-ethylhexyl)phthalate in rodents and its implications on human risk.

The plasticizer di(2-ethylhexyl) phthalate (DEHP), to which humans are extensively exposed, was found to be hepatocarcinogenic in rats and mice. DEHP is potentially set free from objects made of synthetic materials (e.g., those used in medicine). Chronically, the greatest amounts are transferred to persons undergoing hemodialysis (up to 3.1 mg/kg b.w. per day) who would thus be considered the individuals most endangered by tumorigenesis. Although toxicokinetics seem to play a certain unclear role in the course of DEHP-related toxicity, toxicodynamic factors appear more decisive. DEHP is a representative of "peroxisome proliferators" (PP), a distinct group of substances that, in rodents, do not only induce peroxisomes but also specific enzymes in other organelles, organ growth, and DNA synthesis. The cluster of the characteristic effects of PP is generally, although perhaps not quite appropriately summarized as "peroxisome proliferation," and is strongest in the liver. The lowest observed effect level (LOEL) and the no observed effect level (NOEL) of peroxisome proliferation in the rat, as determined by the induction of specific enzymes (peroxisomal beta-oxidation, carnitine-acetyl-transferase, cytochrome P-452), DNA synthesis, and hepatomegaly, may be assumed as 50 and 25 mg/kg b.w. per day, respectively. DEHP and other carcinogenic PP are neither genotoxic nor tumor initiators, but they appear to be tumor promoters, also implicating a threshold level for the carcinogenic effect. Although a causal relationship between a particular effect of peroxisome proliferation and hepatocarcinogenesis is as yet unknown, peroxisome proliferation as a whole phenomenon appears to be associated with the potential of tumor induction, as shown by comparison of the relative strength of individual PP and by comparison of species and organ specificities. Likewise, LOEL and NOEL of rodent carcinogenesis, that is, 300 and 50 to 100 mg/kg b.w. per day, respectively, are above but not too far from the corresponding values for the investigated parameters of peroxisome proliferation. Thus, with respect to dose alone, worst-case exposure in hemodialysis patients is at least 16-fold below the LOEL of any characterized PP-specific effect of DEHP and approximately 100-fold below that of DEHP-related tumorigenesis. Also, primates are less responsive to PP than rats with respect to the investigated biochemical and morphological parameters. If this lower primate responsiveness is extrapolated to estimate carcinogenicity in humans, we might thus arrive at an even larger safety margin than when based on exposure alone. Doses of PP hypolipidemics that had clearly induced several indicators of peroxisome proliferation in rats did not cause any clear-cut enhancements in the peroxisomes of patients, even though most of these hypolipidemics were considerably stronger PP than DEHP. Thus, an actual threat to humans by DEHP seems rather unlikely. Accordingly, hepatocarcinogenesis was neither enhanced in workers exposed to DEHP nor in patients treated with hypolipidemics.

Glutathione S-transferase isoenzyme patterns in different subtypes of enzyme-altered rat liver foci treated with the peroxisome proliferator nafenopin or with phenobarbital.

It is known that phenobarbital (PB) and the peroxisome proliferator (PP) nafenopin (NAF) promote tumor formation by stimulating selective growth of different subtypes of liver foci. While PB enhanced the gamma-glutamyltranspeptidase (GGT)-positive eosinophilic-clear cell foci (ECF), NAF amplified the GGT-negative weakly basophilic foci (WBF). These findings provide the possibility of using the occurrence of these foci subtypes as early indicators for the carcinogenic potential of PB- and PP-type promoters. In order to improve the methods for the discrimination between ECF and WBF we studied further differences in their phenotype, as determined by the expression pattern of glutathione S-transferase (GST) subunits. GST subunits of the alpha (Ya, Yc), mu (Yb1, Yb2) and pi family (Yp), which compose different GST isoenzymes, were demonstrated by immunohistochemical methods. ECF were the only foci subpopulation that expressed GST subunit Yp, while this subunit was always absent in WBF and in another focus subtype, the tigroid foci (TF). Neither PB nor NAF changed this pattern. Thus Yp expression was rather a function of the focus type than of the promoter used. Upon PB treatment expression of the GST subunits Yb1 and Yb2 was frequently elevated in ECF, while Ya and Yc remained more or less unchanged. In NAF-treated livers large WBF, however, showed diminished expression of all investigated GST subunits of the alpha and mu family. In conclusion, PB seems to promote mostly ECF with elevated levels of mu and pi class GSTs, while low levels or absence of all GSTs tested may be associated with growth selection of WBF through the PP NAF.

Peroxisomal enzyme induction uncoupled from enhanced DNA synthesis in putative preneoplastic liver foci of rats treated with a single dose of the peroxisome proliferator nafenopin.

Putative preneoplastic foci of spontaneous origin could be detected in the livers of 2 year old, untreated male Wistar rats. The unaltered and preneoplastic hepatocytes showed an identical expression of the peroxisomal marker enzyme acyl-CoA oxidase, as determined by immunohistochemical staining. A single dose of the peroxisome proliferator (PP) nafenopin (NAF) induced the enzyme predominantly in hepatocytes around the central venules and cell replication mainly in the periportal areas. However, upon one NAF application almost all of the preneoplastic foci showed a considerably weaker immunoreaction for peroxisomal acyl-CoA oxidase than the surrounding tissue. Concomitantly NAF elevated replicative DNA synthesis index in foci up to approximately 40%, while replication of hepatocytes in the unaltered portion of the livers increased only slightly to moderately. In conclusion, NAF-induced peroxisomal acyl-CoA oxidase and replicative DNA synthesis seem not to be necessarily coupled within the same liver cell. Furthermore, preneoplastic foci responded rather to the cell replicative than to the peroxisomal effects of NAF, suggesting that the PP-induced growth stimulus is of particular significance for the carcinogenic action of this class of compounds.

Enhancement of peroxisomal enzymes, cytochrome P-452 and DNA synthesis in putative preneoplastic foci of rat liver treated with the peroxisome proliferator nafenopin.

The peroxisome proliferator (PP) nafenopin (NAF) enhanced tumor development in rat liver through promotion of a subtype of putative preneoplastic cell foci, characterized by weak cytoplasmic basophilia. In order to elucidate the selective growth advantage of these weakly basophilic foci (WBF) we investigated the effects of NAF on their metabolic phenotype and DNA synthesis. In WBF, as well as in other foci subpopulations and in hepatocellular carcinomas the occurrence of five NAF-inducible enzymes, i.e. of peroxisomal beta-oxidation (acyl-CoA oxidase, bifunctional protein and thiolase), catalase and cytochrome P-452 was studied by immunohistochemical methods. In untreated livers almost all foci were stained with the same intensity as the surrounding tissue. When NAF was applied, most of the liver foci showed considerably less staining than the non-focal parenchyma in which pronounced enzyme induction had occurred. However, the subpopulation of WBF showed a more heterogeneous pattern of enzyme expression varying from less to even more than in the adjacent tissue. A similarly broad range of expression of peroxisomal enzymes was found in hepatocellular carcinomas. On average, however, the tumors exhibited less staining and lower activity of peroxisomal beta-oxidation than the surrounding parenchyma. WBF always showed higher rates of DNA synthesis than other foci subtypes and unaltered liver. In approximately one-third of these foci DNA synthesis was found to be enhanced concomitantly with elevated expression of peroxisomal beta-oxidation enzymes. In conclusion, WBF may have a selective growth advantage as they 'overrespond' to the inducing effects of NAF on DNA synthesis and peroxisomal enzymes.

Apoptosis in the liver and its role in hepatocarcinogenesis.

Apoptosis seems to be the predominant type of active cell death in the liver (type I), while in other tissues cells may die via biochemically and morphologically different pathways (type II, type III). Active cell death is under the control of growth factors and death signals. In the liver, endogenous factors, such as transforming growth factor beta 1 (TGF-beta 1), activin A, CD95 ligand, and tumor necrosis factor (TNF) may be involved in induction of apoptosis. Release and action of these death factors seems to be triggered by exogenous signals such as withdrawal of hepato-mitogens, food restriction, etc. During stages of hepatocarcinogenesis, not only DNA synthesis but also apoptosis gradually increase from normal to preneoplastic to adenoma and carcinoma tissue. Also, in human carcinomas, birth and death rates of cells are several times higher than in surrounding liver. (Pre)neoplastic liver cells are more susceptible than normal hepatocytes to stimulation of cell replication and of cell death. Consequently, tumor promoters may act as survival factors, i.e., inhibit apoptosis preferentially in preneoplastic and even in malignant liver cells, thereby stimulating selective growth of (pre)neoplastic lesions. On the other hand, regimens favoring apoptosis and lowering cell replication may result in selective elimination of (pre)neoplastic cell clones from the liver. Finally, we have studied the first stage of carcinogenesis, namely the appearance of putatively initiated cells after a single dose of the genotoxic carcinogen N-nitrosomorpholine (NNM). Most of these cells were found to be eliminated by apoptosis, suggesting that initiation, at the organ level, can be reversed at least partially by preferential elimination of initiated cells. These events may be regulated by autocrine or paracrine actions of survival factors.

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.

Transforming growth factor beta 1-induced cell death in preneoplastic foci of rat liver and sensitization by the antiestrogen tamoxifen.

Previous studies have shown 5- to 10-fold higher rates of apoptosis in prestages of liver cancer (putative preneoplastic cell foci [PPF]) than in unaltered liver; fasting or withdrawal of tumor promoters enhanced apoptosis even further. We studied whether transforming growth factor beta 1 (TGF-beta 1), an inducer of apoptosis in normal liver, might be involved in induction of apoptosis in PPF. PPF were produced in 7-week-old female Sprague-Dawley rats with a single oral dose of the genotoxic carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). At 24 weeks of age, TGF-beta 1 was injected into animals (40 micro g/kg intravenously) either once and they were killed 4 hours later (single-dose experiment) or eight times at 24-hour intervals and they were killed 24 hours after the last administration (multiple-dose experiment). Further subgroups received daily subcutaneous injections of tamoxifen (TAM) (8 mg/kg) for 4 consecutive weeks before TGF-beta 1 treatment. In normal liver, the apoptosis incidence was low in solvent- and TAM-only-treated animals, in the single- as well as the multiple-dose experiment. TGF-beta 1, increased the apoptosis incidence severalfold, and the combined administration of TGF-beta 1 with TAM caused a further strong increase. The already-elevated basal apoptotic incidence in PPF was further increased by TGF-beta 1, and particularly by TGF-beta 1 plus TAM treatments, which resulted in a reduction of foci number and size. In summary, these results show that TGF-beta 1 can induce apoptosis in PPF. This apoptosis-inducing activity is strongly enhanced by the additional treatment with the antiestrogen TAM, which by itself does not have any cell death-inducing effect in the liver or PPF. The elevated apoptotic activity of PPF in response to TGF-beta 1 can lead to a selective reduction of the liver load with preneoplastic cells.

Cleavage of poly(ADP-ribose) transferase during p53-independent apoptosis in rat liver after treatment with N-nitrosomorpholine and cyproterone acetate.

The aim of this work was to study the role of the tumor suppressor p53 and of poly(ADP-ribose) transferase (pADPRT) in the control of hepatocyte apoptosis in two different in vivo models, i.e., during the process of tumor initiation by the genotoxin and cytotoxin N-nitrosomorpholine (NNM) and after withdrawal of the hepatomitogen cyproterone acetate (CPA). Treatment with NNM induces apoptosis followed by necrosis and regenerative DNA synthesis. At the first wave of apoptosis 12 h after NNM application, no p53 expression could be detected by immunohistochemical analysis and immunoblotting. However, 24 h after treatment, numerous p53-positive hepatocyte nuclei were detected, whereas hepatocytes in early and later stages of apoptosis were always negative. Simultaneously with the increased p53 levels, p21 protein was induced. This was accompanied by a block in replicative DNA synthesis, as detected by proliferating-cell nuclear antigen immunostaining. Concomitantly with the increase in apoptosis, dramatic degradation of the nuclear enzyme pADPRT was observed, as evidenced by immunoblotting and activity blotting. The decrease in pADPRT enzymatic activity observed 12 h after treatment coincided with the greatest extent of pADPRT cleavage. One prominent cleavage product was 64 kDa, suggesting that granzyme B was involved in pADPRT degradation. In the second in vivo model we used, i.e., withdrawal of treatment with the hepatomitogen CPA, apoptosis of excessive hepatocytes but no necrosis occurs. Again, no induction of p53 expression could be detected in the liver even at the maximum level of apoptosis, whereas a strong correlation between induction of apoptosis and cleavage of pADPRT to a 64-kDa fragment was observed. These results from whole-animal experiments strongly suggest that the induction of apoptosis in rat liver after genotoxic and cytotoxic damage and during regression of hyperplasia is driven by a p53-independent pathway but is accompanied by cleavage of pADPRT.

Active cell death (apoptosis) and cellular proliferation as indicators of exposure to carcinogens.

An excess of cell multiplication over cell death is a crucial characteristic of preneoplastic and neoplastic cell populations. In many tumours, the rates of both cell birth and cell death are increased over those in the tissue of origin. Cell death may occur through active mechanisms: cellular suicide or active or programmed cell death, often referred to as apoptosis. The term 'apoptosis' was originally used on morphological grounds for a type of cell death characterized by condensation and fragmentation of cytoplasm and chromatin; however, other types of active cell death exist, in which cytoplasmic degradation by lysosomal, autophagic or proteasomal mechanisms may dominate, e.g. in some experimental mammary tumours and mammary tumour cell lines. Morphological and biochemical differences between the types of programmed cell death should be considered when selecting markers for identification and quantification of cell death. There is still a paucity of specific, efficient methods to assay active cell death, and unequivocal differentiation from degenerative necrosis, especially in tumours, may be difficult or impossible. Active cell death is regulated by a complex network of survival factors and death signals. Many mitogens of exogenous or endogenous origin not only stimulate cell birth but at the same time may inhibit cell death, i.e. increase survival. Endogenous factors also exist which induce active cell death; these include transforming growth factor beta1, CD95 or Fas ligand and tumour necrosis factor. Signal pathways leading to birth or death of cells appear to be interconnected to allow for the fine tuning of cell numbers in tissues. Active cell death can be triggered in two principal ways: by toxic chemicals or injury leading to damage of DNA or of other important cellular targets, and activation or inactivation of receptors by growth-regulating signal factors in the organism. Increases in cell proliferation or in cell survival induced by a chemical do not necessarily lead to cancer, but may indicate carcinogenic potential. Chemicals can affect the balance between replication and death of cells in a number of ways. Firstly, genotoxic carcinogens induce genetic damage which subsequently leads to activation of the suicide machinery, involving genes such as p53. As a result, cells with promutational lesions and mutations are eliminated, thereby providing protection from potentially initiated cells. Secondly, toxic doses of genotoxic or nongenotoxic agents induce acute or chronic injury, leading to cell death and subsequent regenerative proliferation. Thirdly, nongenotoxic carcinogens which are primary mitogens may increase the birth and/or inhibit the death of cells by direct interference with growth signalling pathways. This group of agents includes several trophic hormones; e.g. oestradiol stimulates both the replication and survival of mammary tumour cells. As demonstrated in the rat liver model, preneoplastic and neoplastic cells may be over-responsive to mitogenic or survival signals and thereby undergo selective growth. Conversely, preneoplastic clones and even malignant tumours may still depend on the survival effect of mitogens and regress upon withdrawal of the agent. This indicates that the mitogenic action of the agent is reversible and underlines the principal difference between genotoxic and nongenotoxic carcinogens. In conclusion, studies on cell proliferation and cell death are useful as adjuncts to carcinogenicity assays, and the results may facilitate the interpretation of effects. In conjunction with other biological data, this information may provide an indication of potential carcinogenicity.