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.

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.

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.

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.

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.

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.

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.

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.

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.

Expression of cytochrome P450 2A5 in preneoplastic and neoplastic mouse liver lesions.

Cytochrome P450 (CYP) 2A5 is involved in the metabolism of carcinogens like aflatoxin B1 and N-nitrosodiethylamine (NDEA), and CYP2A5 levels are increased in some pathological states of the liver (e.g., infectious hepatitis and porphyria). We analyzed the expression of CYP2A5 during experimental liver carcinogenesis in three different mouse strains (C3H/He, C57BL/6J, and B6C3F1) with immunohistochemical techniques and in situ hybridization. In normal liver, CYP2A5 protein and mRNA were detected in centrilobular hepatocytes only. Phenobarbital treatment increased the number of CYP2A5-positive centrilobular hepatocytes and the CYP2A5-positive areas were extended into the middle zone in all strains, but periportal hepatocytes remained negative. Fifty percent of the spontaneous foci in untreated mice, over 90% of the foci in mice treated with NDEA or phenobarbital and all of the hepatocellular adenomas and carcinomas displayed positive immunostaining and a strong CYP2A5 mRNA signal by in situ hybridization. In the liver tumors metastasized to the lung, expression of CYP2A5 had largely disappeared. CYP2A5 expression in neoplastic and putative preneoplastic lesions, although sometimes heterogeneous, was apparently independent of the typical zonal expression pattern in normal tissue. As expected, the C57BL/6J mice developed fewer foci and tumors than the C3H/He and B6C3F1 mice, but the phenotype of CYP2A5 overexpression was similar in all the strains. Our data suggest that the increased expression of CYP2A5 may play an important role in the development of liver cancer in mice and may be used as a novel marker for spontaneous and NDEA-induced mouse liver foci.

Levels of transforming growth factor beta and transforming growth factor beta receptors in rat liver during growth, regression by apoptosis and neoplasia.

Transforming growth factor beta1 (TGF-beta1) has been implicated as inhibitor of cell proliferation and a potent inducer of apoptosis in vitro and in vivo after the administration of high doses. To assess the role of endogenous TGF-beta1, we quantitated the cytokine and its receptors in rat liver during regenerative and hyperplastic growth, regression by apoptosis, and in hepatocellular carcinoma (HCC). This was accomplished by Northern blot analysis and by RNase protection assay of the messenger RNA (mRNA) of TGF-beta1 and TGF-beta receptors (TbetaR) types I to III and by an activity bioassay of the TGF-beta proteins. Untreated rat livers were found to contain 15.6 +/- 4.8 ng TGF-beta1 protein/g tissue; TGF-beta2 protein was not detected. To induce toxic cell death and subsequent regenerative DNA synthesis in the liver, rats were treated with a necrogenic dose of carbon tetrachloride (CCl4). After 24 and 48 hours, there was an upregulation of TGF-beta1 (mRNA, up to tenfold; protein, about twofold) and of TbetaRs (mRNA: two- to fourfold); that indicates an overall enhanced production of and sensitivity to TGF-beta1, which may serve to confine the regenerative response. Hyperplastic liver growth and regression of the hyperplasia were induced by treatment with cyproterone acetate (CPA) or nafenopin (NAF) followed by withdrawal; neither mRNAs of TGF-beta1 and TbetaR types I to III nor TGF-beta1 protein exhibited significant changes during the growth phase or during regression by apoptosis. We also studied neoplastic growth. HCC, obtained after long-term treatment with NAF, exhibited high rates of cell replication and apoptosis. The majority of lesions contained mRNA and protein of TGF-beta1 and mRNA of TbetaR types I to III at concentrations similar to those of the surrounding tissue. In conclusion, during liver regeneration there is a pronounced upregulation of expression of both TGF-beta1 and TbetaRs I to III, but not during mitogen-induced liver growth or regression. It appears that apoptosis is induced via altered local concentration of TGF-beta1, in a paracrine and/or autocrine way. By this mechanism the lethal effects of TGF-beta1 may be locally confined, and overshoots of apoptosis in the liver may be prevented.

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.

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.

Inherent increase of apoptosis in liver tumors: implications for carcinogenesis and tumor regression.

We quantitatively assessed rates of cell replication and of apoptosis during the development and regression of liver cancer. In rats, apoptotic activity gradually increased from normal liver to putative preneoplastic foci (PPF), to hepatocellular adenoma (HCA), and to hepatocellular carcinoma (HCC). At all stages, rates of cell replication were higher than of apoptosis, allowing a preferential net gain of (pre)neoplastic cells. As in rats, in human HCC, birth and death rates were increased manifold, indicating a species-independent phenomenon. Implications of the increasing cell turnover were studied in rats using the administration and withdrawal of nafenopin (NAF), a liver mitogen and nongenotoxic carcinogen. Prolonged NAF treatment enhanced cell number in normal liver by 25%, while PPF and liver tumors were amplified at least 100-fold. After stopping NAF treatment, cell replication ceased, while cell elimination by apoptosis was increased in normal and (pre)neoplastic liver. HCA and HCC showed the most pronounced shifts from replication toward apoptosis. As a result, 5 weeks after halting NAF, 20% of cells in normal liver, but about 85% of (pre)neoplastic lesions including HCC, were eliminated. The implications of these findings include that nongenotoxic carcinogens can act as survival factors even for malignant cells. Furthermore, tumor cells not only exhibit excessive proliferation, but also undergo apoptosis at rates that far exceed those in normal tissue. Therefore, inhibition of cell death by the survival activity of nongenotoxic carcinogens results in selective growth of (pre)neoplastic lesions. On the other hand, blockade of survival effects leads to excessive apoptosis in (pre)neoplasia and seems promising as a therapeutic concept for the selective elimination of (liver) cancer.

Concepts of cell death and application to carcinogenesis.

The occurrence of cell death as a physiologic event in multicellular organisms has been known for more than 150 yr. In 1972, the term apoptosis was introduced on morphological grounds. The hypothesis that all kinds of cell death can be categorized as either "apoptotic" or "necrotic" is not generally confirmed. Cells seem to use different pathways for suicide, as reflected by different morphology: condensation-prominent, Type I or apoptosis; autophagy-prominent, Type II; and so forth. Type II cell death was found in mammary tissue and mammary tumor cells and in a variety of other organs. For unequivocal identification of the various types of cell death, morphological, biochemical, and functional criteria may be used in combination. During tumor development in various organs of animals and humans, not only rates of cell proliferation but also rates of cell death may increase with increasing malignancy. Morphological and functional criteria (antipromotion, withdrawal of survival factors) indicate that cell death in tumors frequently is of an active nature.