Epidermal growth factor and proliferation in rat hepatocytes in primary culture isolated at different times after partial hepatectomy

Primary hepatocyte cultures have been prepared from normal adult rat liver and from rat liver at 4, 8, 12, 24, and 48 h following partial hepatectomy (removal of 70% of the liver). Cells were maintained in minimal essential medium alone or supplemented with hormones. Comparing DNA synthesis in normal adult rat hepatocytes with DNA synthesis in hepatocytes isolated from regenerating livers, we found with minimal essential medium alone little DNA synthesis in normal adult rat hepatocytes and in hepatocytes isolated 4, 8, or 12 h after 70% hepatectomy. In hepatocytes isolated 24 h after partial hepatectomy, however, the incorporation of [3H]thymidine was 3 times the rate of normal hepatocytes. The addition of insulin to minimal essential medium had minimal effect on DNA synthesis in all hepatocytes. Addition of epidermal growth factor alone or in combination with insulin resulted in a dramatic increase in DNA synthesis in hepatocytes from regenerating rat liver. Increased incorporation was detectable as early as 4 h after partial hepatectomy and reached a maximum at 24 h after the operation. Results obtained with [3H]thymidine incorporation were confirmed by autoradiography and by direct DNA determinations in hepatocyte cultures. Epidermal growth factor binding to the hepatocytes was determined and agreed with previously reported binding studies. Binding of epidermal growth factor in hepatocytes isolated at 4 h after partial hepatectomy was the same as in normal hepatocytes but was undetectable in hepatocytes isolated from rats at 12 and 24 h after partial hepatectomy.     

Cell specificity of rat cytokeratin p39 during azo dye-induced hepatocarcinogenesis

Monoclonal or affinity-purified antibodies specific to Novikoff hepatoma cytokeratin p39 were employed to study the origin and fate of p39-containing cell types during hepatocarcinogenesis induced with N,N-dimethyl-p(m-tolylazo)aniline. Frozen sections were obtained from the livers of animals autopsied temporally during carcinogen feeding and were assayed immunohistochemically. In normal, untreated liver or in liver from animals fed the hepatotoxin alpha-naphthyl-isothiocyanate, the localization of p39 was restricted to bile duct epithelial cells while hepatocytes were non-reactive. However, during carcinogen treatment we observed a sequential appearance of immunoreactive cells which were similar morphologically to the classical 'oval' cells of hepatocarcinogenesis; eventually these cell types were enriched around the preneoplastic hepatocyte nodules. Occasional transformed hepatocytes within the nodules exhibited strong immunoreactivity. In the later stages of hepatocarcinogenesis, these antibodies stained the epithelial cells in areas of severe adenosis as well as the neoplastic epithelial cells of cholangiomas and some, but not all, hepatocellular carcinomas. Our results document the presence of p39 in the 'oval' cells of hepatocarcinogenesis and indicate that some populations of transformed hepatocytes exhibit this cytokeratin after transformation.     

Inhibition of DNA synthesis by phenobarbital in primary cultures of hepatocytes from normal rat liver and from hepatic nodules.

One of the many hypotheses put forward to explain the mechanism by which phenobarbital (PB) promotes hepatocarcinogenesis is by differential mitoinhibition of surrounding hepatocytes while allowing the initiated hepatocytes to respond to growth stimuli and form foci and nodules. Given the similarity in structures between PB and orotic acid (OA), another rat liver tumor promoter, the present investigation was designed to determine (i) whether PB, like OA, exerts its mitoinhibitory effect at a site beyond the growth factor receptor and receptor mediated early events; and (ii) whether PB exerts a differential mitoinhibitory effect by selectively inhibiting the non-initiated hepatocytes but not the initiated hepatocytes in vitro. Our studies demonstrate that, like OA, PB also inhibits DNA synthesis in hepatocytes from normal rat liver in a dose dependent manner with 80-90% at a dose of 6 mM. One target site may lie beyond the growth factor receptor mediated early events because PB inhibited DNA synthesis in hepatocytes primed with the growth factor 24 h earlier. Interestingly, PB inhibited DNA synthesis not only in hepatocytes from non-nodular surrounding liver but also in hepatocytes from persistent hepatic nodules initiated with 1,2-dimethylhydrazine and promoted with OA. Therefore, our results suggest that although PB is a mitoinhibitor of DNA synthesis in hepatocytes, it does not appear to create as strong a differential mitoinhibition between non-nodular surrounding and initiated hepatocytes as is evident in the resistant hepatocyte and OA models. These results raise the question whether differential mitoinhibition is the major contributing factor in the PB mediated rat liver tumor promotion.     

Co-cultured endothelial and Kupffer cells regulate hepatocyte replication

We studied the effect of co-culture with endothelial and/or Kupffer cells on hepatocyte replication in primary culture by means of [H-3]-thymidine incorporation. Hepatocytes, sinusoidal endothelial and Kupffer cells were extracted from rat liver by a two-step perfusion and subsequent two-step elutriation method. The replication was promoted in proportion with the densities of endothelial cells, but inversely suppressed by co-culture with Kupffer cells. The replicative effect of endothelial cells was not observed with the high number of hepatocytes cultured, however. The hepatocyte replication was also regulated in accordance with the ratios of endothelial/Kupffer cells. These results indicate that endothelial cells have a positive effect and Kupffer cells have a negative effect on hepatocyte replication, and that this replication may be associated with hepatocarcinogenesis and/or tumor-growth of hepatocellular carcinoma.     

Peroxisome proliferators do not increase DNA synthesis in purified rat hepatocytes

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

Induced drug resistance inhibits selection of initiated cells and cancer development

Compounds exerting a mitoinhibitory effect on normal hepatocytes are potent promoters in the resistant hepatocyte model of chemical carcinogenesis in combination with stimulation of regenerative growth by partial hepatectomy or treatment with carbon tetrachloride. 2- Acetylaminofluorene (2-AAF) almost completely inhibits liver cell regeneration after partial hepatectomy, allowing only resistant cells to participate in regenerative growth. After initiation by diethylnitrosamine and promotion with 2-AAF and partial hepatectomy (PH), focal growth of initiated cells generates liver lesions which occupy 40% of the hepatic volume three weeks after PH. In this work the mechanism for the anti promoting effects of phenobarbital and 3- methylcholantrene were investigated as well as their effects on the development of malignant hepatocellular carcinoma in the resistant hepatocyte model. Treatment with phenobarbital or, especially, 3- methylcholanthrene rendered normal rat hepatocytes resistant to the mitoinhibitory effect of 2-AAF. In combination with 2-AAF/PH, 3- methylcholanthrene shortened the regenerative growth period to less than one week. In the Solt-Farber protocol for experimental hepatocarcinogenesis, treatment with phenobarbital or 3- methylcholanthrene during promotion with 2-AAF/PH permitted hepatocytes surrounding the focal lesions to respond with regenerative growth. The foci and surrounding liver grew until the liver/body mass index reached the control value. With phenobarbital treatment the total focal volume was 20% of the liver volume three weeks after PH, whereas the corresponding value in the case of 3-methylcholanthrene was only 1%. Labelling index data supported the conclusion that growth of the liver lesions in the resistant hepatocyte model was dependent on differential inhibition of normal hepatocyte growth by the promoter and that the size of the foci obtained was related to the length of time after PH required to complete liver regeneration. 3-methylcholanthrene induced 2- AAF resistance prevented the development of large persistent nodules and hepatocellular carcinoma while phenobarbital delayed cancer development with several month. The data thus supports the idea that the degree of clonal expansion during promotion determines the size of the population at risk for malignant transformation, as well as the final frequency of carcinomas.      

Enhancement of the clonability of adult parenchymal hepatocytes with the liver tumor promoter phenobarbital

An in vivo clonogenic assay system was utilized to investigatethe effect of the tumor promoter, phenobarbital (PB), on adultparenchymal hepatocyte proliferation. Enzymatically dispersedhepatocytes from female Fischer 344 rats were injected intothe interscapular and mammary fat pads of isogeneic recipientanimals where they proliferate to form hepatocyte colonies within3 weeks. The number of hepatocytes required to form a colonyin 50% of the transplantation sites (LND₅₀) was 23 700 cellsand 520 cells when normal adult liver cells were injected intonon-hepatectomized and 2/3 hepatectom-ized normal recipientanimals, respectively. Thus, a partial hepatectomy increasedthe hepatocyte clonogenicity by a factor of 40. A 2-week pre-treatmentof both the donor and recipient animals with PB (0.1% in thedrinking water) significantly increased the clonogenicity ofthe liver cells when transplanted into non-hepatectomized (15-fold)and 2/3 hepatectomized (2-fold) animals. However, PB treatmentof the recipient animals was not required for the majority ofthis mitogenk effect since the clonability of PB-treated donorcells was increased (92% of the maximum stimulation observed)even when they were transplanted into untreated control animals.Furthermore, the PB-induced effect on hepatocyte clonabilitywas reversible since the removal of PB from the donor animals2 weeks prior to their use reduced the clonability of the hepatocytes(LND₅₀=20 500 cells) to that observed for cells which were neverexposed to PB. These results are consistent with the postulatethat rather than PB being directly mitogenic, it primarily increasesthe clonability of adult parenchymal hepatocytes by inducinga reversible cellular alteration which enhances their responsivenessto endogenous growth stimuli.     

Aberrant cell cycle checkpoint function in transformed hepatocytes and WB-F344 hepatic epithelial stem-like cells

Cell cycle checkpoints are barriers to carcinogenesis as they function to maintain genomic integrity. Attenuation or ablation of checkpoint function may enhance tumor formation by permitting outgrowth of unstable cells with damaged DNA. To examine the function of cell cycle checkpoints in rat hepatocarcinogenesis, we analyzed the responses of the G (1), G (2) and mitotic spindle assembly checkpoints in normal rat hepatocytes, hepatic epithelial stem-like cells (WB-F344) and transformed derivatives of both. Normal rat hepatocytes (NRH) displayed a 73% reduction in the fraction of nuclei in early S-phase 6-8 h following 8 Gy of ionizing radiation (IR) as a quantitative measure of G (1) checkpoint function. Chemically and virally transformed hepatocyte lines displayed significant attenuation of G (1) checkpoint function, ranging from partial to complete ablation. WB-F344 rat hepatic epithelial cell lines at low, mid and high passage levels expressed G (1) checkpoint function comparable with NRH. Only one of four malignantly transformed WB-F344 cell lines displayed significant attenuation of G (1) checkpoint function. Attenuation of G (1) checkpoint function in transformed hepatocytes and WB-F344 cells was associated with alterations in p53, ablated/attenuated induction of p21 (Waf1) by IR, as well as aberrant function of the spindle assembly checkpoint. NRH displayed 93% inhibition of mitosis 2 h after 1 Gy IR as a quantitative measure of G (2) checkpoint function. All transformed hepatocyte and WB-F344 cell lines displayed significant attenuation of the G (2) checkpoint. Moreover, the parental WB-F344 line displayed significant age-related attenuation of G (2) checkpoint function. Abnormalities in the function of cell cycle checkpoints were detected in transformed hepatocytes and WB-F344 cells at stages of hepatocarcinogenesis preceding tumorigenicity, sustaining a hypothesis that aberrant checkpoint function contributes to carcinogenesis.     

In vitro inhibition of tritiated thymidine uptake in Morris hepatoma cells by normal rat liver extract: a possible liver chalone.

A phosphate-buffered saline extract of normal rat liver tissue almost completely inhibited DNA synthesis in Buffalo rat Morris hepatoma 7777 cells in vitro. This effect was tissue-specific because it did not occur with extracts of kidney, spleen, heart, lung, muscle, and hepatoma. The liver extracts did not inhibit in vitro human prostate carcinoma or phytohemagglutinin-stimulated human and rat peripheral blood lymphocyte cultures. The addition of 10% fetal calf serum in the incubation medium had no effect on this inhibition. We determined that doses of liver extract that inhibit thymidine incorporation were not toxic inasmuch as treated cells remained viable, as indicated by trypan blue exclusion. The liver extract may thus contain a cell-specific mitotic inhibitor, a chalone for hepatoma cells.     

Testing for induction of DNA synthesis in human hepatocyte primary cultures by rat liver tumor promoters.

Parenchymal liver cells were isolated from human liver pieces of surgical waste as well as from rat livers. DNA synthetic activity was measured after different times in primary culture by [3H]thymidine incorporation and autoradiography. Labeling of control cultures of human hepatocytes at densities between 8,000 and 15,000 cells/cm2 was very low (0.4 to 1.3%). Human recombinant epidermal growth factor increased labeling 2- to 4-fold (P less than 0.01). Treatment with known inducers of liver growth in rats, namely, cyproterone acetate, alpha-hexachlorocyclohexane, nafenopin, phenobarbital, and rifampicin did not increase the number of labeled human liver cells. In some of the experiments, a 24-h exposure to the chemicals of rat or human hepatocytes was followed by a 24-h treatment with epidermal growth factor (EGF). In rat hepatocytes, incorporation rates were significantly increased. Cyproterone acetate and EGF acted in an additive manner, alpha-hexachlorocyclohexane and EGF were clearly overadditive, and phenobarbital had little effect. In human hepatocytes, little alteration in labeling indices was found; in some cases labeling was, rather, found to be lower than in cultures treated with EGF alone. These results show that human hepatocytes cultured in vitro are sensitive to stimulation of DNA synthesis by EGF; they differ from rat hepatocytes in their response to some drugs which show liver growth-promoting activity in rodents.     

Sequential alterations in growth control and cell dynamics of rat hepatocytes in early precancerous steps in hepatocarcinogenesis

This set of experiments is the second of a series designed to explore alterations in cell dynamics and growth control of new populations of hepatocytes that appear to play a role in the carcinogenic process induced in the liver by chemical carcinogens. This is part of an ongoing study of the biochemical and molecular basis for cancer development. A rat model for hepatocarcinogenesis, the resistant hepatocyte model, was chosen with its synchrony of several steps in the process. Carcinogenesis was initiated by the administration of a single necrogenic dose of diethylnitrosamine. Resistant hepatocytes so induced were stimulated to proliferate rapidly to form nodules by a mitogenic stimulus in the presence of a brief exposure to dietary 2-acetylaminofluorene sufficient to inhibit the proliferation of the majority of uninitiated hepatocytes, the nonresistant population. A small subset of these hepatocyte nodules, the persistent nodules, was examined at 2, 4, and 6 mo postinitiation. Duration of phases of the cell cycle, growth fraction, doubling time, cell death, and cell loss and the responses and subsequent recovery after the application of a strong mitogenic stimulus, partial hepatectomy, were measured. The first precancerous hepatocyte nodule, at 2 mo, showed a "normal" duration of phases of the cell cycle. The growth fractions were about 4,4, and 8% at 2, 4, and 6 mo, respectively, as compared to 0.4% in the surrounding hepatocytes. Accompanying the increased growth fractions were considerable levels of cell loss, measuring about 3% at 2 mo and 7% at 6 mo. At 6 mo, the hepatocyte nodule population, unlike the hepatocytes in the surrounding liver, shows a failure to return to its base-line level after stimulation of cell proliferation by partial hepatectomy. The results of this study have identified two new steps in the early precancerous phase of hepatocarcinogenesis relating to alterations in the control of cell proliferation and are consistent with the hypothesis that new and evolving cell populations may play an important role in the step-by-step carcinogenic process. These new populations appear to acquire alterations in growth control in a seriatim fashion, with retention of some "normal" properties.     

Enhancing effect of ethanol on aflatoxin b1-induced DNA-damage in glutathione-depleted rat hepatocytes

The effect of reduced glutathione (GSH) and ethanol on aflatoxin B1 (AFB1)-induced DNA single strand breaks was studied in primary cultured hepatocytes. Buthionine sulfoximine (BSO) which decreased intracellular GSH to 13% of those of the control levels increased DNA fragmentation of AFB1-treated hepatocytes by over 17% of those without BSO. Thus, a decrease in hepatocyte GSH levels increased AFB1-induced DNA damage. Although ethanol in itself did not induce DNA damage, a combination of BSO and ethanol increased the percentage by over 23% of that with BSO only. Ethanol did not affect the amount of GSH, total cytochrome P-450 (P450), glutathione S-transferase (GST) and epoxide hydrolase (EHase) in cultured hepatocytes. However, GSH-depleted rat hepatocytes exposed to ethanol significantly increased the level of P450IIIA, which activates AFB1. The enhancing effects of ethanol in the presence of BSO are probably due to the induction of this isozyme in rat hepatocytes. The GSH-depleted hepatocytes are more susceptible to chemical carcinogens in the presence of ethanol.     

Differential induction of gamma-glutamyl transpeptidase in primary cultures of rat and mouse hepatocytes parallels induction during hepatocarcinogenesis

In carcinogen-treated rats, gamma-glutamyl transpeptidase (GGT) is induced in preneoplastic liver lesions and liver tumors. However, in mice, GGT is rarely detected during hepatocarcinogenesis. Data in this study reveal that GGT is not induced in mouse hepatocytes when they are maintained in vitro under the same conditions that induce GGT activity in primary cultures of rat hepatocytes. GGT activity in rat hepatocytes increased 20-fold during the first 7 days in culture, but there was no induction of GGT in primary cultures of mouse hepatocytes. Comparison of intracellular glutathione levels in rat and mouse liver cells showed that the glutathione level was higher in the mouse liver cells than the rat. Blocking glutathione synthesis with buthionine sulfoximine reduced the intracellular glutathione concentration in mouse liver cells but did not trigger an induction of GGT. Analysis of the GGT mRNA in primary cultures of rat hepatocytes showed that only GGT mRNA(III) is induced. This is the same GGT mRNA species present in preneoplastic hepatic lesions and liver tumors in the rat (1-3). Therefore activation of promoter III in the GGT gene is responsible for induction of GGT in both hepatocytes in vitro and liver tumors in vivo. These data show that primary cultures of rat and mouse hepatocytes provide a model system with which to study interspecies differences in the regulation of this enzyme and to better understand the role of GGT in normal and neoplastic processes.      

Phenobarbital reduces EGF receptors and the ability of physiological concentrations of calcium to suppress hepatocyte proliferation

Optimal proliferation of cultured hepatocytes from normal ratsoccurs in response to epidermal growth factor (EGF) at an extracellularcalcium concentration of 0.4 mM, whereas physiological concentrationsof calcium have been shown to decrease hepatocyte proliferation.Exposure of hepatocytes in vivo to phenobarbital (PB, 0.1% inthe drinking water) reduced significantly the ability of physiologicallevels of calcium to suppress hepatocyte proliferation. An increasedrelative ability of hepatocytes to proliferate at physiologicalcalciumoncentrations versus that at 0.4 mM calcium was firstseen after 3 days of in vivo PB treatment and this effect wasmaintained during 2 months of exposure. Hepatocytes from short-termPB-exposed animals (i.e. 3–28 days) proliferated at physiologicalCa²⁺ concentrations 2–3 times better than those from controlanimals. However, after 2 months of continuous PB exposure,cell growth was reduced significantly at all extracellular Ca²⁺concentrations investigated. EGF binding studies demonstratedthatthe biphasic kinetic effect of PB on hepatocyte proliferationwas not correlated directly with the steady decline In EGF receptornumber or the lack of significant change in receptor bindingaffinitywith duration of PB exposure. These results imply that the effectivenessof PB in tumourpromotion may result from its ability to reducethe absolute magnitude of normal hepatocyte proliferation andto alter the growth regulatory effect of extracellular calcium.Further, the results argue that PB effects on hepatocyte proliferationare not mediated simply through regulation of EGF surface receptornumber or binding affinity. Additional events of the EGF-inducedcascade necessary for hepatocyte proliferation appear to bemodified by PB.     

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.     

Expression of a hepatocyte membrane antigen during hepatocarcinogenesis and in the developing liver of the rat

Changes in the expression of a cell membrane antigen during hepatocarcinogenesis and in the developing liver were analyzed by HAM.4, a monoclonal antibody (MAb) against a membrane glycoprotein of normal rat hepatocyte. Of the precancerous lesions observed during hepatocarcinogenesis induced by diethylnitrosamine, 2-acetylaminofluorene and partial hepatectomy, early neoplastic foci were uniformly stained by HAM.4. In contrast, some cells in the neoplastic nodules at the late stage did not express HAM.4 antigen on the cell surface. Of the cancer tissues, well-differentiated hepatocellular carcinomas were stained by HAM.4 whereas poorly differentiated carcinomas did not bind HAM.4 In developing rat liver, HAM.4 antigen was first expressed on fetal hepatocytes at the 18th day of gestation. It gradually increased until 4 weeks after birth when the intensity of the stain was almost the same as in adult rat liver. These results suggest that the expression of a membrane antigen defined by HAM.4 is closely associated with the differentiation of bile canalicular face and that HAM.4 might be useful in characterizing differentiation of cells during malignant transformation of hepatocytes.     

Effect of antisense oligomer in controlling c-raf.1 overexpression during diethylnitrosamine-induced hepatocarcinogenesis in rat

Purpose

In ras-mediated signal transduction pathway, c-raf.1 is believed to have predominant oncogenic potential and has been found to be highly expressed in certain human and animal malignancies including hepatocellular carcinoma. In the present study, anticancer efficacy of antisense c-raf.1 oligomer on the inhibition of c-raf.1 mRNA overexpression during hepatocarcinogenesis was determined.

Methods

Initially antiproliferating effect of the antisense oligomers was studied in vitro by measuring the rate of tritiated thymidine incorporation into DNA in rat hepatocellular carcinoma cells in culture medium. Based on the findings, the antisense treatment was carried out in rat hepatocarcinogenesis model-initiated with diethylnitrosamine and promoted using 2-acetylaminoflourene. Different drug-metabolizing enzymes, lipid peroxidation, liver morphology and histopathological studies along with c-raf.1 gene expression by in situ hybridization were performed.

Results

c-raf.1 antisense oligomers exhibited an inhibitory effect (~68%) on cancer cell proliferation in vitro. Gross and microscopic examination of liver showed fewer (29%) and smaller hyperplastic nodules and preneoplastic lesions (30%) in carcinogen and antisense oligomer-treated group as compared with carcinogen control group. Treatment of antisense c-raf.1 oligomers enhanced cytochrome P-450 content (81%) and reduced glutathione S-transferase activity (33%), UDP glucuronosyltransferase activity (74%) and MDA concentration (30%) in carcinogen and antisense oligomer-treated group as compared with carcinogen control animals. The oligomer treatment also resulted in less expression in terms of c-raf.1 expressed lesion count as compared to carcinogen control group.

Conclusion

The study demonstrates that the antisense oligomer targeted against c-raf.1 mRNA inhibits the overexpression of c-raf.1 gene during hepatocellular carcinoma in rats.     

Resistance of Copenhagen rats to chemical induction of glutathione S- transferase 7-7-positive liver foci

Copenhagen (Cop) rats are completely resistant to the chemical induction of mammary adenocarcinomas, but their susceptibility to hepatocarcinogenesis is virtually unknown. Rat liver is a well- characterized and easily manipulated tissue in which to study carcinogenesis. Therefore, if Cop rats are resistant to hepatocarcinogenesis, studies into resistance mechanisms may be feasible. Male Cop and F344 rats, 7-8 weeks old, were initiated using either N-nitrosodiethylamine (DEN) (200 mg/kg, i.p.) or a two-thirds partial hepatectomy (PH) followed by N-methyl-N-nitrosourea (MNU) (60 mg/kg, i.p.). The rats were then promoted using a modified resistant hepatocyte (RH) protocol (a combination of four doses of 2- acetylaminofluorene (2-AAF) and a single dose of CCl4 that provides a selective mitotic stimulus for initiated cells). Six weeks after initiation the rats were killed and liver sections were stained for glutathione S-transferase 7-7 (GST 7-7), a marker for putative preneoplastic hepatocytes. Cop rats were found to be highly resistant, having a approximately 9- and approximately 27-fold smaller percentage of liver area occupied by GST 7-7-positive foci than susceptible F344 rats following initiation by DEN and MNU respectively. Furthermore, gross liver nodules did not form in any of the Cop rats, whereas all F344 rat livers contained nodules. Hepatic necrosis caused by DEN during initiation, and CCl4 during promotion is necessary to stimulate compensatory hepatocyte division. We demonstrated that these agents do indeed increase serum transaminase levels and produce histologic evidence of necrosis in Cop rats. In order for liver foci to grow rapidly in the RH protocol, the surrounding normal hepatocytes must be mito-inhibited by 2-AAF. We found that the degree of mito-inhibition of normal hepatocytes by 2-AAF is the same in Cop and F344 rats. These results show that the Cop rat is highly resistant to the chemical induction of putative preneoplastic liver foci and nodules.      

Selection Pressure and Altered Hepatocellular Islands after a Single Injection of Aflatoxin B1

Development of glutathione S-transferase placental form (GST-P)-positive focal populations was investigated subsequent to single injections of the hepatocarcinogens aflatoxin B1 (AfB1), dimethylnitrosamine (DMN) and diethylnitrosamine (DEN). While DEN proved far more potent at inducing putative initiated hepatocytes, the AfB1 treatment was associated with a very rapid (3 weeks) development of lesions approaching nodular proportions. Autoradiographic investigation revealed selective incorporation of label into GST-P-positive hepatocytes and oval cells at the day 7 time point following AfB1 treatment. Administration of butylated hydroxyanisole (BHA) subsequent to carcinogen injection was associated with a decrease in the final yield of lesions and increased tritiated thymidine incorporation in perivenular zone 3 background hepatocytes. The results suggest that 'selection pressure', resulting in rapid growth and development of putative preneoplastic lesions, is inherent in a single injection of the mycotoxin and indicate that variations of the present short-term model may be useful for elucidating the mechanisms underlying AfB1-induced hepatocarcinogenesis.     

Selection pressure and altered hepatocellular islands after a single injection of aflatoxin B1

Development of glutathione S-transferase placental form (GST-P)-positive focal populations was investigated subsequent to single injections of the hepatocarcinogens aflatoxin B1 (AfB1), dimethylnitrosamine (DMN) and diethylnitrosamine (DEN). While DEN proved far more potent at inducing putative initiated hepatocytes, the AfB1 treatment was associated with a very rapid (3 weeks) development of lesions approaching nodular proportions. Autoradiographic investigation revealed selective incorporation of label into GST-P-positive hepatocytes and oval cells at the day 7 time point following AfB1 treatment. Administration of butylated hydroxyanisole (BHA) subsequent to carcinogen injection was associated with a decrease in the final yield of lesions and increased tritiated thymidine incorporation in perivenular zone 3 background hepatocytes. The results suggest that 'selection pressure', resulting in rapid growth and development of putative preneoplastic lesions, is inherent in a single injection of the mycotoxin and indicate that variations of the present short-term model may be useful for elucidating the mechanisms underlying AfB1-induced hepatocarcinogenesis.