Institut für Tumorbiologie-Krebsforschung, University of Vienna, Borschkegasse 8a, A-1090 Vienna, Austria
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Abstract |
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Abbreviations: AB, apoptotic body; CPA, cyproterone acetate; G cells, hepatocytes negative for placental glutathione S-transferase; G+ cells, hepatocytes positive for placental glutathione S-transferase; GST, glutathione S-transferase; LI, labeling index; NNM, N-nitrosomorpholine; PB, phenobarbital; TGF-ß1, transforming growth factor ß1.
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Introduction |
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Rat liver offers a great advantage to study first events in carcinogenesis because early progeny of initiated cells have been known for several decades to be phenotypically altered foci (5); meanwhile their biological and molecular features have been well characterized (6,7). A further important step forward was the detection of the almost selective immunoreactivity of single, putatively initiated hepatocytes for placental glutathione S-transferase (GST; G+ cells) (8). We and others have found that a few days after treatment with various initiating genotoxic carcinogens, such as N-nitrosomorpholine (NNM) and other nitrosamines, aflatoxin B1 or methylazoxymethanol acetate, G+ single cells emerge, but not after application of promoters and other non-genotoxic agents (811). The number of cells increases with the dose of the initiator or drops when metabolic activation to the ultimate carcinogen is inhibited (8, 10, 11). We observed a consecutive development of G+ single cells to G+ (mini) foci suggesting that the expression of the G+ phenotype is persistent and heritable to daughter cells (9). In addition, we found that without tumor promotion ~30% of G+ single cells and with tumor promotion even more G+ single cells give rise to small preneoplasia consisting of at least 10 cells (B.Grasl-Kraupp, G.Luebeck, A.Wagner, A.Löw-Baselli, M.de Gunst, T.Waldhör, S.Moolgavkar and R.Schulte-Hermann, manuscript in preparation). Moreover, placental GST remains expressed in large foci and in hepatocellular adenomas and carcinomas, suggesting that this marker identifies all stages of carcinogenesis from the putatively initiated single cell through to frank malignancy (12,13). In conclusion, a great proportion of G+ cells can be considered to be `initiated' and to be capable of evolving into hepatic (pre)neoplasia. The number of these putatively initiated cells present in the liver may therefore be an important determinator of the carcinogenic risk.
Growth regulation has been studied in further advanced stages of rat hepatocarcinogenesis, such as foci, adenomas and carcinomas (1417). Rates of replication and death of cells, and thus overall cell-turnover, steadily increase during neoplastic development. Without treatment with a promoter, the high rate of cell proliferation is balanced by cell loss and little net growth of the lesion occurs. Tumor promoters such as phenobarbital (PB) and cyproterone acetate (CPA) have been shown to inhibit apoptosis and to prolong the lifespan preferentially of (pre)neoplastic liver cells, thereby leading to selective growth of (pre)neoplastic lesions (14,16). Conversely, (pre)neoplasia also shows excessive responses to death signals exerted by promoter withdrawal, food restriction or by transforming growth factor ß1 (TGF-ß1), which may lead to selective elimination of the lesion (14,1719). Thus, it appears as one of the main characteristics of liver (pre)neoplasia to `over-respond' towards stimulators and suppressors of growth. It is as yet unknown whether this property is acquired during initiation or at later stages of carcinogenesis.
We found recently that in intact rat liver G+ cells show rates of DNA replication and of apoptosis significantly higher than G hepatocytes (18). Considering the increased cell turnover from the beginning of carcinogenesis onwards, the question emerges whether initiation causes a direct change in the growth regulatory machinery of G+ cells. This direct change could result from altered uptake, production and/or processing of growth regulatory factors by G+ cells (15,20,21). Alternatively, the crucial event during initiation may cause resistance to cytotoxic or growth inhibitory signals, which would indirectly lead to preferential growth of initiated cells, while proliferation of normal cells is prevented (22). An impaired interaction between neighbouring G+ and G cells may also cause changes in growth control (23). To address these questions extended studies on initiated cells appear particularly worthwhile.
Cell culture studies with initiated hepatocytes at very early stages of cancer development would enable investigations on the molecular and biological alterations associated with initiation. Previous in vitro studies used (pre)neoplastic cells from later stages (2428). In most cases these studies concentrated on cells expressing -glutamyltranspeptidase, a less reliable marker that detects only parts of the G+ cell population (13). The present paper describes a system that allows for the direct comparison of putatively initiated and unaltered hepatocytes in primary cultures. We found that cultured G+ cells show (i) an inherently higher DNA replication than unaltered G hepatocytes indicating a direct change in growth regulation and (ii) an over-response towards known growth stimulators or suppressors; these observations are nearly identical to data on further advanced liver (pre)neoplasia obtained from whole-animal experiments. Thus, this new ex vivo cell culture system offers novel possibilities to study basic defects in growth regulation of initiated cells and to develop and test strategies designed to eradicate the earliest stages of carcinogenesis.
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Materials and methods |
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[3H]thymidine was injected i.p. at a dose of 0.2 mCi/kg body wt at 1 p.m., 6 p.m. and 11 p.m. in order to label all cells replicating DNA on the day of killing. In animals kept under an inverted lightdark rhythm DNA synthesis occurs roughly between 5 p.m. and 11 p.m. (17,18); at 9 a.m. animals were killed by decapitation under CO2-narcosis. (For more detailed descriptions see refs 17,18.) The experiment was performed according to the Austrian guidelines for animal care and protection.
Studies on intact liver tissue
Histology.
Specimens of liver tissue were fixed in Carnoy's solution and processed as described (18). Two serial sections, 2 µm thick, were cut; one of the sections was stained with hematoxylin & eosin (H&E), the second one was stained for the placental GST.
Immunostaining for placental GST.
The polyclonal antiserum, raised against rat Yp-subunit of the enzyme, was obtained from Biotrin International (Dublin, Eire). The following schedule was used: hydrogen peroxide to block endogenous peroxidases (3%, 20 min, room temperature); 2.5% bovine serum albumin (BSA) in TBS (0.05 M Tris, 0.3 M NaCl pH 7.6; 30 min, room temperature); anti-Yp (1:5000 in 2.5% BSATBS, overnight, 4°C); rinsing with TBS; biotinylated goat-anti-rabbit IgG (1:600 in BSATBS; 90 min, room temperature; Dako, Glostrup, Denmark); rinsing with TBS; streptavidin (1:300 in TBS, 45 min, room temperature; Dakopatts); diaminobenzidine for colour development. The specifity of immunohistochemistry was confirmed by omitting the primary antibody.
Quantitative evaluation of G+ single cells and G+ foci.
G+ single cells and G+ multicellular foci were identified by the anti-placental GST stain. Their numbers were registered and calculated per cm2 evaluated tissue section (at least 1 cm2 per animal, measured by means of a semi-automatic image analyzer `VIDS IV' from Ai-Tektron GmbH, Meerbusch, Germany).
Determination of DNA synthesis and of apoptosis.
Immunohistochemically stained sections were coated with a solution of 1% gelatine (Bio-Rad, Richmond, CA) and 0.05% chromalaun (Merck, Darmstadt, Germany) and were air-dried. Autoradiography was performed as described (17). DNA synthesis was determined by counting the number of labeled hepatocytes per 100 nucleated hepatocytes [labeling index (LI)]. LIs were assayed on at least 2000 unaltered cells in each liver and in all nucleated G+ cells in culture.
The incidence of residues of hepatocytes undergoing death by apoptosis [apoptotic bodies (ABs)] were determined in H&E-stained serial sections for the G and G+ cell populations according to previously established criteria (29).
Because interindividual variations were small, LI and AI obtained from different livers of the same experimental group were pooled.
Studies on primary hepatocyte cultures
Isolation of hepatocytes.
Rat liver cells were isolated by collagenase perfusion according to the technique of Seglen (30) with modifications as described by Parzefall et al. (31). In brief, animals were narcotized with a mixture of isofluranedinitrogenoxideoxygen at flow rates of 60 (3%)/1200/800 ml/min (Forane; Abbot Laboratories, Kent, UK). Perfusion commenced with buffer A at a flow rate of 10 ml/min and reached 50 ml/min after 10 min. During this time span the liver was freed of blood. Thereafter the processus papilliformus caudatus of the liver was tied and cut off for histology (see above for details). Collagenase perfusion started with collagenase buffer B at a flow rate of 50 ml/min for 1 min and was continued with a rate of 20 ml/min under recirculation. On average after 10 min liver tissue softening appeared to be sufficient. Thereafter the liver was placed in a beaker with washing medium supplemented with 2 U/ml Trasylol (Bayer, Leverkusen, Germany). The specimen was freed from undigested parts, and cells were released by cutting the tissue with scissors and by shaking the cells into the medium. Then, a 150 µm nylon mesh (type Scrynel NY; Zürcher Beuteltuchfabrik, Switzerland) was used for cell sieving. Purification of parenchymal cells by four cycles of low-speed centrifugation (30 g, 5 min) and determinations of viabilities and cell yields were performed as described (31).
Media and buffers.
Pre-perfusion buffer A contained per litre: 6.8 g NaCl, 0.4 g KCl, 2.2 g NaHCO3, 1 g glucose (all obtained from Merck), 1 g HEPES (USB, Cleveland, OH), 60 mg penicillin (170 µM; Biochrom, Berlin, Germany), 100 mg streptomycin (70 µM; Biochrom), 12.4 mg HeparinNa (Serva) in distilled water, pH 7.4. Collagenase buffer B: 680 mg NaCl, 40 mg KCl, 220 mg NaHCO3, 100 mg glucose, 100 mg HEPES, 6 mg penicillin, 10 mg streptomycin, 30 mg CaCl2·2H2O (Merck) and 20 mg collagenase type IV (Sigma, St Louis, MO) in 100 ml distilled water, pH 7.4. Buffers A and B were used at 37°C and aerated by carbogen.
Washing medium consisted of minimal essential medium (MEM; Life Technologies, Paisley, UK). Medium for plating and culture was Williams' medium E (WE; Seromed, Vienna, Austria). Both media types were supplemented with 20 mM HEPES, 10 µg/ml gentamycin, 6.7 nM insulin, 0.7 nM glucagon, 10 nM triiodothyronine, 100 nM dexamethasone, 150 µM ascorbic acid, 1 mM pyruvate (all obtained from Merck).
Culture and treatment.
Petri dishes (3.5 cm diameter; NUNC, Roskilde, Denmark) were coated with diluted collagen (~0.1 mg/ml in distilled water), that was prepared according to the method of Ehrmann and Gey (32). Cells were seeded at a density of 30 000 viable cells/cm2 in 2 ml WE2 plus 10% fetal calf serum and were incubated at 37°C in an incubator with 5% CO2 in air at 98% relative humidity. After an attachment period of 1 h, the monolayers were rinsed with WE2, refed with 1.5 ml of WE2 only and left for 23 h to recover from the isolation stress. Media were changed again after 20 h and every 24 h thereafter. If not stated otherwise, treatment commenced 4 h after plating (time point 0 in the experimental protocol) and was renewed with every medium change.
PB (Fluka, Buchs, Switzerland) was prepared as a stock of 100 mM in distilled water and was added as aliquot for a final concentration of 1 mM in the medium. CPA, a gift from Schering AG (Berlin, Germany) was dissolved in dimethyl sulfoxide. In all experiments the final concentration of CPA and solvent in the medium was 10 µM and 0.2%, respectively. Recombinant mature TGF-ß1 synthesized by chinese hamster ovary transfectants was supplied generously by Bristol-Myers Squibb (Seattle, WA). TGF-ß1 was dissolved in 4 mM HCl in PBS containing 1 mg/ml BSA.
Newly synthesized DNA was labeled with [3H]thymidine (sp. act. 6080 Ci/mmol; ARC, St Louis, MO) which was added at 0.5 µCi/ml medium 24 h before harvesting.
Determination of DNA replication and apoptosis in cultures.
Hepatocytes were fixed for 90 min at room temperature with 4% buffered formalin according to Lillie and were then kept in distilled water at 4°C until immunostaining for placental GST (see above). The stained plates were coated with 1% gelatine/0.05% chromalaun, air-dried and were then dipped into photo-emulsion (Ilford K5, Dreieich, Germany); time of exposure was, on average, 48 h. Immediately after autoradiography plates were stained for 5 min with 8 µg/ml Hoechst 33258 (Riedel de Haen, Seelze, Germany) in PBS, followed by two washes with distilled water. The plates were dried at room temperature and mounted in Kayser's glycerine gelatine (Merck).
Besides parenchymal cells the cultures contained a variable number of non-parenchymal cells. The typical hepatocyte morphology was used for distinction (33). Only areas of average cell densities on the culture plates were used for the following evaluations: to determine DNA synthesis a total of 1000 G and 300400 G+ hepatocyte nuclei were evaluated per dish. LI was calculated as percentages of labeled hepatocyte nuclei per total number of hepatocyte nuclei counted. For the evaluation of apoptosis at least 1000 hepatocyte nuclei per plate were scored for apoptotic morphology (condensed and fragmented nuclei) under the fluorescence microscope as described (34); the percentage of apoptotic incidence was calculated (apoptosis index, AI).
The incidence of apoptosis of cultured G+ hepatocytes cannot yet be determined due to unspecific immunostaining of apoptotic cells. As a consequence almost all apoptoses are false positive for the placental GST. Possibly, proteolytic degradation in dying cells yields reactive groups to which antisera bind unspecifically. This phenomenon was observed with other primary or secondary antibodies as well (unpublished observation).
Statistics
For studies in intact liver, at least five animals were evaluated per time point and treatment group. Wherever indicated, incidences are given and CI were calculated for P < 0.05. For in vitro studies, routinely four to six equally treated hepatocyte cultures per rat were run and harvested in parallel. If not stated otherwise, the means ± SD of three cultures from at least three different donor rats are given. The significance of differences of means was tested either by Student's t-test or KruskalWallis test.
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Results |
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Correlation of G+ hepatocytes in intact liver and in culture: stability of G+ phenotype
When the frequency of G+ cells was maximal, i.e. at days 2022 post-NNM treatment, livers were perfused with collagenase and isolated hepatocytes were cultivated. For each individual donor rat the percentage of G+ cells was determined in tissue sections of an intact, non-perfused liver lobule (see above) and, after 48 h in culture, nearly identical results were obtained with both approaches (Figure 3). This indicates that no selective loss or enrichment of G+ cells occurred during cell isolation.
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At 24 h in culture 90.79 ± 1.29% of the G+ cells were single and only a minor fraction occurred as small clusters of two G+ cells (6.44 ± 0.25%) or more than two G+ cells (2.77 ± 1.19). The percentage of single G+ cells remained stable (48 h, 88.5 ± 1.7%; 72 h, 87.7 ± 2.2%). This implies that, in case of intercellular communication, most of the G+ cells were surrounded by G cells only.
DNA synthesis and apoptosis in G and G+ hepatocytes in the intact liver and in culture
In the intact liver of the donor rats at day 21 post-NNM treatment, G+ cells showed significantly higher DNA replication rates and apoptotic activity than G cells (Figure 4). Thus, the putatively initiated cell compartment exhibited an enhanced cell-turnover as shown previously for further advanced preneoplasia (17,18).
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It should be noted that the cytotoxic/necrogenic effects of NNM and the subsequent regeneration increase cell turnover in the liver, which remains elevated for at least 1 month (37). This effect was also obvious in the present study at day 21 in vivo and probably continued in culture. As a result, the frequency of G cells undergoing DNA replication was higher than usually observed in primary hepatocyte cultures isolated from rats without NNM pre-treatment (33).
Effect of the hepatomitogens PB and CPA on DNA synthesis in G and G+ hepatocytes in culture
Next we investigated whether G+ hepatocytes in culture show an over-response towards growth stimuli as known from animal experiments with large G+ preneoplastic foci (14,38). We chose two compounds with presumably different modes of action. In vivo, PB is considerably less effective than CPA as a hepatomitogen, and exerts its effect on foci growth mostly via suppression of apoptosis (14,16). In culture, a relatively high concentration of PB in the medium for 48 h induced an insignificant increase in DNA replication in the G and G+ cells (Figure 5). In contrast, 48 h of CPA treatment raised the LI in both populations ~2-fold (Figure 5
). Every fifth G+ cell was stimulated to DNA replication by CPA, while only every tenth G cell was recruited to the pool of replicating G hepatocytes. As a result, DNA replication in G+ cells was elevated to ~40% indicating a preferential response of cultivated G+ cells towards the hepatomitogen.
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Discussion |
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Firstly, it has been shown that placental GST is expressed in ~6080% of all putatively initiated/(pre)neoplastic cells (13,40). Since we were able to transfer G+ cells to culture without any selective loss or enrichment, it may be deduced that ~6080% of all subtypes of initiated cells were subject to our analysis. Previous studies used cells deriving from further advanced (pre)neoplasia that were often separated from normal liver cells by using differences in size and/or surface properties, such as receptors or membrane enzyme patterns. Due to heterogeneity in these properties it seems likely that only subpopulations of (pre)neoplastic cells were cultivated (2426,41).
For the analysis of initiated cells we decided on primary hepatocyte cultures of G cells together with G+ cells. This co-cultivation allows for immediate comparison of the two cell populations under identical conditions, a major advantage when differences are expected to be subtle. Supplements to the media and their concentrations were as close to the in vivo situation as possible (31). Then, hepatocytes in primary culture are known to retain their specific phenotype and metabolic competence for several days (31,42,43). Non-specific induction of the placental GST and other signs of de-differentiation did not appear for the first 3 days.
Secondly, we found recently that in the intact liver G+ single cells exhibit a low propensity to proliferate but that G+ lesions, consisting of at least two cells, show rates of DNA replication and of apoptosis ~4-fold higher than unaltered hepatocytes (36). When combining the data of single cells and mini lesions, as done in the present study, G+ hepatocytes altogether exhibit in vivo 2-fold elevated rates of cell replication and of cell death by apoptosis. In the course of hepatocarcinogenesis the defect in growth control increases further to 510-fold elevated levels over normal (17).
When cultured, the population of G+ cells retained the 2-fold increased replication. This property suggests a direct defect in growth regulation, since it persists under culture conditions and appears to be independent of intercellular contacts within the intact organ, and of cytokines, growth factors or hormones circulating in the whole body. In subsequent investigations we found that considerably more G+ cells synthesize transforming growth factor than G cells, which provides a reasonable explanation for this intrinsic growth advantage (B.Grasl-Kraupp, K.Hufnagl, E.Schausberger, W.Parzefall, A.Löw-Baselli, R.Schulte-Hermann, manuscript in preparation).
Thirdly, treatment of cultures with 1 mM PB caused insignificant stimulation of DNA synthesis to an approximately similar extent in both G and G+ cells. The results obtained are consistent with previous studies in which slight stimulation of DNA replication occurred at 1.5 mM and pronounced mitoinhibition at 3 mM (44). Also, in the intact liver of the rat strain currently used the effect of PB on DNA replication is marginal.
Unlike PB, CPA is a potent hepatomitogen in rats; it exerts strong tumor promoting effects leading to tumor formation due to pronounced stimulation of replication and suppression of apoptosis preferentially in liver (pre)neoplasia (16, 38). In culture, G+ cells were much more stimulated to DNA replication by CPA than G cells. As a result, ~38% of G+ cells were cycling which appears remarkably high for non-synchronized primary hepatocyte cultures. Thus, we could show that also in culture the propensity towards increased replication causes a preferential response of G+ cells to certain growth stimuli.
Similar observations were reported for cultured hepatocytes from rats that had been treated with diethylnitrosamine and with PB; cells from further advanced preneoplasia were identified in culture by staining for -glutamyltranspeptidase, which detects only a subfraction of preneoplastic cells and additionally some unaltered cells. With CPA treatment, DNA synthesis in
-glutamyltranspeptidase-positive hepatocytes was stimulated to a higher extent than in enzyme-negative hepatocytes (27).
Fourthly, TGF-ß1 is one of the strongest growth suppressors and apoptosis inducers for hepatocytes in vivo and in culture (34,45,46). In analogy to initiated/(pre)neoplastic cells in intact liver, we observed that cultured G+ cells exhibited more pronounced decreases in DNA replication after TGF-ß1 than G cells. Likewise, cultured (pre)neoplastic cells isolated from rat liver or transformed hepatocyte lines were reported to be highly sensitive towards the growth inhibitory effect of TGF-ß1, although these studies did not allow for immediate comparison of unaltered and (pre)neoplastic cells (47,48). One report on co-cultivation of unaltered and (pre)neoplastic cells showed failure of TGF-ß to suppress DNA replication in the latter population, which may be largely due to de-differentiation of the cells and to culture conditions (28).
TGF-ß1 exerts growth-inhibition in the G1 phase of the cell cycle via affecting cyclin-D1-dependent kinase activity (39). Thus, the more pronounced response of G+ cells to TGF-ß1 may be due to their higher probability to be in G1. When cultures were pre-treated with CPA, an even higher proportion of G+ hepatocytes was shifted to G1. Thereby CPA pre-treatment amplified the difference in susceptibility of G+ and G cells towards TGF-ß1. Thus, the propensity for enhanced growth of G+ cells may be the cause of the elevated sensitivity towards growth inhibition by TGF-ß1.
Finally, liver cancer is among the eight leading causes of cancer death worldwide with a clear tendency to increase further; therapeutic possibilities are very limited and prognosis is usually poor. Various genotoxic and non-genotoxic noxes, such as aflatoxin B1 and infection with hepatitis virus type B or C, are responsible for the high incidence of this disease in some areas of Africa and Southeast Asia (49); it may be assumed that a considerable proportion of the local population carries initiated cells in the liver. Early cancer prophylaxis would offer the major advantage that potentially harmful clones, consisting of only a few initiated cells, should be eliminated more easily than large (pre)neoplasia. Most therapeutic strategies against cancer and its pre-stages require an accelerated cell turnover and over-response of the target cell towards growth suppressors/apoptosis inducers. Our study suggests that initiated liver cells may meet this criterion, at least in rats. Moreover, our new model may help to identify the basic molecular defects of this disease and to develop and test strategies for eradication of cancer prestages. Anticipating a high degree of analogy between rat and human hepatocarcinogenesis, much of the knowledge should then be valid and applicable for prophylaxis against human liver cancer.
In conclusion, the present work suggests that the inherent defect in growth regulation, a hallmark of (pre)neoplastic cells, occurs already in initiated cells. A novel ex vivo culture system is introduced that allows for studies on the basic defects in hepatocarcinogenesis and the development of cancer preventive strategies.
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Acknowledgments |
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Notes |
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References |
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