Sensitive quantitative assay for point mutations in the rat H-ras gene based on single nucleotide primer extension.

Point mutations in oncogenes and tumor suppressor genes occur at early stages in the carcinogenic process. Point mutations in ras family oncogenes are the most common mutational events in several types of human cancer, and are available as molecular markers for the detection of cancer cells in carcinogenicity bioassay systems as well as in clinical samples. Although several techniques are utilized to detect point mutations in carcinogenicity bioassay systems, the sensitivity is too low to determine a small number of mutations. In order to overcome the disadvantage and to sensitively determine gene mutation rates for in vivo carcinogenicity bioassays of presumptive carcinogens, we established a Thermosequenase Cycle End Labeling (TCEL) method, a sensitive approach based on single nucleotide primer extension. One of the characteristics of the method is a high sensitivity of 1:100,000, ten times the sensitivity of the mutant allele-specific amplification now commonly employed. Using TCEL, we here quantified H-ras mutations in the livers of rats treated with a genotoxic carcinogen, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline. Our findings suggest that this method may be applied for many genetic targets as a component in vivo.

p16 promoter hypermethylation in human hepatocellular carcinoma with or without hepatitis virus infection.

BACKGROUND: Epigenetic alteration through methylation is one of the most important steps in carcinogenesis. However, the relation between hepatitis virus infection and epigenetic alterations is poorly understood. METHODS: Sixteen patients without hepatitis B virus (HBV) and hepatitis C virus (HCV) and 35 patients with HBV or HCV who underwent liver resection for hepatocellular carcinoma (HCC) were studied. Mutation of p53 was detected by direct sequencing. Methylation status of p16 was evaluated in tumor and noncancerous liver tissues by methylation-specific polymerase chain reaction. RESULTS: In HCC without HBV and HCV, p53 mutations were detected in 5 (31%) of 16 HCCs. Methylation of p16 promoter was detected in 2 (25%) of 8 moderately differentiated HCCs, 6 (75%) of 8 poorly differentiated HCCs, and none of 16 noncancerous tissue specimens. In HCC with HBV or HCV, p53 mutations were detected in 8 (23%) of 35 HCCs. Methylation of p16 promoter was detected in 2 (100%) of 2 well-differentiated HCCs, 13 (76%) of 17 moderately differentiated HCCs, 12 (75%) of 16 poorly differentiated HCCs, and 9 (26%) of 35 noncancerous liver tissue specimens. CONCLUSIONS: Our results suggest that hepatitis viruses might induce methylation of p16 promoter in liver with chronic inflammation, before appearance of HCC.

Peripheral blood mononuclear cells are possible extrahepatic replication sites for hepatitis C virus.

BACKGROUND/AIMS: Hepatitis C virus is a major causative agent of chronic liver disease and hepatocellular carcinoma and is considered to be a hepatotropic virus. It remains controversial whether hepatitis C virus exists in peripheral blood mononuclear cells and replicates there. In order to resolve this issue, we performed nested RT-PCR (reverse transcription polymerase chain reaction) and RT-PCR in situ hybridization in peripheral blood mononuclear cells of patients with chronic hepatitis C. METHODOLOGY: We collected peripheral blood mononuclear cells from patients with chronic hepatitis C, extracted total RNA from the samples, and performed nested RT-PCR to detect hepatitis C virus RNA in the peripheral blood mononuclear cells lysates. We also fixed peripheral blood mononuclear cells of the patients in 4% paraformaldehyde and performed RT-PCR in situ hybridization with a digoxigenin-labeled RNA probe to detect hepatitis C virus RNA in the cells. RESULTS: Using these methods, we detected both positive- and negative-stranded hepatitis C virus RNA in peripheral blood mononuclear cells of hepatitis C patients. To determine in which cell population of peripheral blood mononuclear cells hepatitis C virus is present, we performed PCR in situ hybridization after incubation with fluorescent latex microbeads which could be phagocytozed by monocytes. We obtained positive signals of the replicative hepatitis C virus genome not only in lymphocytes but also in monocytes. CONCLUSIONS: RT-PCR in situ hybridization with a nonradioactive probe was found to be useful for in situ detection of hepatitis C virus RNA. Our findings suggest that peripheral blood mononuclear cells may be extrahepatic replication sites for hepatitis C virus.

Detection of serum hepatitis B virus DNA by real-time quantitative polymerase chain reaction (TaqMan PCR) during lamivudine treatment: comparison with three other assays.

Monitoring of hepatitis B virus (HBV) levels in serum plays an important role in the management of chronic hepatitis B in patients receiving lamivudine. We evaluated the usefulness of real-time quantitative polymerase chain reaction (TaqMan PCR) for the measurement of HBV DNA. The subjects were 22 patients with chronic hepatitis B treated with lamivudine for 4-12 months. HBV DNA was measured by TaqMan PCR. For comparison, HBV DNA was also measured in 88 sera by branched DNA (bDNA) assay, transcription-mediated amplification (TMA) assay, and Amplicor monitor test. Correlation was significant between the results of TaqMan PCR and those of the three other assays (r=0.630, 0.681, and 0.715, respectively; P<0.05). Of the 22 patients, HBV DNA was beneath the detection limit at the start of therapy in 4 (18%) on the bDNA assay, 3 (14%) on the TMA assay, 2 (9%) on the Amplicor test, and 0 (0%) on TaqMan PCR. Of the 19 patients for whom sera were available at 12 weeks of therapy, HBV DNA was not detected in 16 (84%) on the bDNA assay, 12 (63%) on the TMA assay, 6 (32%) on the Amplicor test, and 2 (11%) on TaqMan PCR. Tyrosine-methionine-aspartate-aspartate (YMDD) variants emerged in three patients; TaqMan PCR detected HBV DNA throughout treatment and revealed significantly increased viral loads before biochemical breakthrough. We conclude that monitoring of HBV by TaqMan PCR is useful for evaluating response to lamivudine treatment and for early detection of drug-resistant variants.

Sequencing of human-viral DNA junctions in hepatocellular carcinoma from patients with HCV and occult HBV infection.

DNA of free hepatitis B viruses (HBV) has been detected in the liver of patients infected with hepatitis C virus (HCV). It is unknown whether HBV DNA is integrated into such livers; if so, it may affect hepatocarcinogenesis. Hepatocellular carcinomas (HCCs) from 34 patients without HBV surface antigen (HBsAg) and with anti-HCV, and from 7 patients with HBsAg and without anti-HCV as controls, were examined, using the cassette-ligation-mediated polymerase chain reaction and primers based on HBV DNA sequence. In the controls, HBV DNA had been integrated into human DNA of all HCCs. On the basis of HBV DNA in tumor tissue, 23 of the 34 patients with anti-HCV had occult infection. Junctions between human DNA and HBV DNA were detected in 10 of the 34 patients without HBsAg and with anti-HCV. HBV DNA was integrated into chromosome 11q in 4 of the 10 HCCs with junctions. The DNA to either side of the human-viral junctions was sequenced. Clinically, the mean tumor size of these 10 HCCs was 39 mm; that of the 24 HCCs without integrated HBV was 25 mm. The surrounding tissue was cirrhotic in 2 of the 10 former HCCs and in 16 of the latter 24 HCCs. In conclusion, integrated HBV was detected in some patients with HCV infection; in these patients, the integrated DNA was associated with accelerated hepatocarcinogenesis.

Post-initiation inhibition of MeIQx hepatocarcinogenesis in rats by cysteine.

Rats were administered cysteine at a dose of 100 mg/kg b.w. 5 times per week after 2-amino-3, 8-dimethylimidazo [4,5-f] quinoxaline (MeIQx) treatment. Significant decrease in numbers and areas of glutathione S-transferase placental form (GST-P)-positive foci, putative preneoplastic lesions, and silver-stained nucleolar organizer regions were evident in the livers of rats treated with cysteine after MeIQx treatment. Morever, post-initiation stage cysteine treatment resulted in decreased hepatic insulin-like growth factor (IGF)-I mRNA expression. Thus post-initiation cysteine treatment may exert chemopreventive effect on MeIQx hepatocarcinogenesis.

Decreased ornithine decarboxylase activity in the kidneys of Dahl salt-sensitive rats.

To assess the roles of polyamines (putrescine, spermidine, and spermine) and ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, in the development of salt-sensitive hypertension, we evaluated activity and expression of ODC, urinary polyamine excretion, and antizyme (endogenous ODC inhibitor protein) expression in Dahl salt-sensitive (SS) and salt-resistant (SR) rats after they were fed on a low (0.3%) or high (4%) salt diet for 4 weeks. We also examined the effects of spermidine and difluoromethylornithine (DFMO: a specific inhibitor of ODC) on the systolic blood pressure and ODC protein expression in SS rats fed a high salt diet. Renal ODC activity and urinary polyamine excretion in SS rats were lower than those in SR rats after 4 weeks treatment with a low or high salt diet. The renal ODC protein expression of SS rats was paradoxically increased as compared to the SR group. A high salt diet did not alter ODC activity but increased ODC protein only in SS rats. ODC mRNA and antizyme protein expressions were not significantly different among the four groups. Spermidine supplementation attenuated and DFMO exaggerated hypertension in SS rats fed a high salt diet. Spermidine down-regulated and DFMO up-regulated renal ODC protein in SS rats on a high salt diet. ODC activity was decreased but protein was paradoxically increased in kidneys of SS rats. ODC protein was suggested to increase in compensation for the inhibition of its activity. Impaired ODC activity and polyamine production in the kidney may exaggerate salt-sensitive hypertension in SS rats.

Changes in hypervariable region 1 in patients with chronic hepatitis C of genotype 1b with biochemical response to interferon.

BACKGROUND/AIMS: The outcome of interferon (IFN) therapy of hepatitis C virus (HCV) infection can be classified as a complete viral response (CR), biochemical response (BR), or no response (NR). Why alanine aminotransferase (ALT) activity decreases in patients with BR despite viral persistence is unknown. METHODS: Of 158 patients infected with HCV genotype 1b, all 20 patients with BR and 20 of the 114 patients with NR, matched for viral load to the BR group, were studied. We sequenced nucleotides in the hypervariable region (HVR) of serum HCV RNA, and analyzed quasispecies of this region by the polymerase chain reaction and single-strand conformation polymorphism (PCR-SSCP). RESULTS: In HVR 1, SSCP patterns differed after therapy; the major clone before therapy disappeared with therapy in eight of the 20 BR patients, but in none of the 20 NR patients (P=0.0033; Fisher's exact test). PCR products of HVR 1 from six patients were cloned before and after therapy, and 40 clones from each patient were sequenced each time. Results of cloning and sequencing were generally consistent with those of SSCP. For the six patients, a major clone could be identified both before and after therapy. In two patients with BR, there were many changes in the amino acid sequence of the major clone after IFN; in one patient with NR, mutations were not found. CONCLUSION: Changes in the major viral clone with IFN treatment may be related to the decrease in ALT activity in some patients, in spite of the continued presence of HCV RNA.

Thrombospondin-1 mediates smooth muscle cell proliferation induced by interaction with human platelets.

OBJECTIVES: Platelet adherence and activation are associated with smooth muscle cell (SMC) proliferation and arterial restenosis. This study examined platelet-SMC interaction on fibrillar type I collagen and analyzed the role of thrombospondin (TSP)-1 in platelet-induced SMC proliferation. METHODS AND RESULTS: When SMCs cultured on fibrillar collagen were treated with human platelets (5 preparations), 7.45+/-2.94% of the cells passed through S phase within 24 hours, as determined by bromodeoxyuridine nuclear labeling. The addition of platelets markedly induced SMC TSP-1 mRNA expression and cell surface protein accumulation, which colocalized with adhered platelets, as determined by alpha(IIb) integrin immunostaining. Direct interaction of platelets with SMCs was necessary for its effect on proliferation and TSP-1 accumulation, as determined in the transwell culture system. The anti-TSP-1 blocking antibody strongly inhibited platelet-induced SMC proliferation by approximately 60%. Analysis of the receptors for TSP-1 accumulation on the SMC surface revealed that beta1 integrins are mainly involved. The anti-beta1 integrin blocking antibody, which potently suppressed TSP-1 accumulation on SMCs, also markedly inhibited platelet-stimulated SMC proliferation. CONCLUSIONS: TSP-1 and beta1 integrin interaction is involved in platelet-stimulated SMC proliferation. This in vitro coculture system could prove useful for examining the molecular mechanism underlying platelet-induced vascular remodeling and for studying the mechanism of a tested drug for restenosis.

Accurate prediction of response to interferon therapy by repeated measurement of hepatitis C virus core protein in patients with chronic hepatitis C.

OBJECTIVE AND METHODS: Serum levels of hepatitis C virus (HCV), a predictor of the response to interferon (IFN) therapy, can fluctuate widely in patients with chronic hepatitis C, even without antiviral therapy. In order to increase the accuracy of predicting the response to therapy, serum samples from 134 patients with chronic hepatitis C were collected twice: 1.0-4.5 months before and just before the start of IFN therapy, and were tested for HCV core protein by a fluorescent enzyme immunoassay. RESULTS: Forty-one (31%) patients had a complete response to IFN and 93 (69%) had no response. The most useful cutoff value between high and low viral loads for predicting the response to therapy was 40 pg/ml of HCV core protein. A complete response was obtained more frequently in 32 of 45 patients with persistently low viral loads than in those with persistently high viral loads (7 of 71) (p < 0.0001). In 2 of 18 patients with a low viral load at one time point but a high viral load at another, the rate of complete response was similar to that in patients with persistently high viral loads. CONCLUSION: Prediction of the response to IFN therapy based on HCV core protein measurement at two time points before therapy is more reliable than that based on HCV core protein measurement at only one time point.

Erythrocyte-binding polyamine as a tumor growth marker for human hepatocellular carcinoma.

BACKGROUND/AIMS: Polyamines are essential for cell proliferation, differentiation, and transformation. Concentrations of polyamines are higher in some cancer tissue than in normal tissue. We examined erythrocyte-binding polyamines to evaluate the usefulness of polyamines as pathophysiological markers for hepatocellular carcinoma. METHODOLOGY: We measured erythrocyte-binding polyamine levels in peripheral blood samples obtained from 51 normal adult controls, 136 patients with chronic viral hepatitis, 104 patients with viral hepatic cirrhosis, and 130 patients with hepatocellular carcinoma. RESULTS: We defined the concentration of spermidine plus spermine as the erythrocyte polyamine level, and designated the cut-off level for normal as the mean erythrocyte polyamine level +/- 2 SD in control. The erythrocyte polyamine level was abnormally elevated (positive) in 56 (43%) of 130 patients with hepatocellular carcinoma, 11 (8%) of 136 patients with chronic hepatitis, and 13 (13%) of 108 patients with cirrhosis. The level was higher in patients with a short tumor doubling time. In 27 patients with tumors, there was negative correlation between tumor doubling time and erythrocyte polyamine level (r = -0.46; P = 0.0147). CONCLUSIONS: We conclude that erythrocyte polyamine may be a useful tumor growth marker in patients with hepatocellular carcinoma.

Dynamics of hepatitis C virus monitored by real-time quantitative polymerase chain reaction during first 2 weeks of IFN-beta treatment are predictive of long-term therapeutic response.

The relation between the change in hepatitis C virus (HCV) RNA levels at the start of interferon-beta (IFN-beta) treatment and the long-term therapeutic response remains poorly defined. In 20 patients with chronic hepatitis C who received IFN-beta (total dose 126-756 MU), the changes in serum HCV RNA during the first 2 weeks of therapy were monitored by real-time quantitative polymerase chain reaction (PCR). The serum HCV RNA level decreased rapidly during the first 24 h of therapy (first phase) and more slowly thereafter (second phase), with a mean exponential decay rate of 1.17 log10/day and 0.37 log10/day, respectively. Three patients had a sustained virologic response, 10 patients had a transient response, and 7 patients had no response. The differences in the rate of first-phase viral decline among the three groups were not significant (p = 0.21), but the differences in the rate of second-phase viral decline were significant (p = 0.0021). The mean decay rate between the end of the first 24 h and day 14 was 0.96 +/- 0.43 log10/day in sustained responders, 0.39 +/- 0.30 log10/day in transient responders, and 0.13 +/- 0.09 log10/day in nonresponders. We conclude that during the first 2 weeks of therapy, changes in serum HCV RNA levels as monitored by real-time quantitative PCR can be used to predict the long-term response to treatment with IFN-beta.

Cellular thiol status-dependent inhibition of tumor cell growth via modulation of retinoblastoma protein phosphorylation by (-)-epigallocatechin.

Tea polyphenols have been shown to inhibit tumor cell growth, but there is limited information on their effects on cell signaling and cell cycle control pathways. We have shown the involvement of such mechanisms as activation of mitogenic activated protein kinases, decreases in ornithine decarboxylase activity and in cellular thiol levels, elicitation of mitochondrial cytochrome c release, and activation of caspases by the green tea galloyl polyphenol, epigallocatechin (EGC). In the current study, we sought to determine how EGC alters cell cycle and its related control factors in its growth inhibitory effect in Ehrlich ascites tumor cells. The significant finding here is that EGC caused a dose-dependent accumulation of cells in the G1 phase and a decrease in the phosphorylation of the retinoblastoma (Rb) protein, which was also in a cellular thiol-dependent manner. The involvement of a cellular thiol-dependent modulation in Rb phosphorylation leading to the regulation of tumor cell growth by a green tea polyphenol is a novel observation, to the best of our knowledge.

Changes in serum levels of hepatitis C virus genotype 1b monitored by real-time quantitative polymerase chain reaction as a predictor of long term response to interferon-alpha treatment.

OBJECTIVE: The aim of this study was to find whether there is a relationship between the changes in the amounts of hepatitis C virus (HCV) at the start of interferon treatment and the long term response to therapy. METHODS: In 20 patients with HCV genotype 1b each given 880 MU of interferon-alpha, the changes in serum HCV RNA during the first 2 wk of therapy were monitored by real-time quantitative polymerase chain reaction (PCR). RESULTS: Real-time quantitative PCR detected HCV RNA at 10(1)-10(8) copies/ml. Serum HCV RNA decreased rapidly between 8 and 24 h after the first administration (first phase) and more slowly thereafter (second phase), with median exponential decays of 2.14 and 0.11 log10/day, respectively. Four patients had sustained virological responses, nine patients had transient responses, and seven patients had no responses. The differences in the rate of first-phase viral decline among the three groups were not significant (p = 0.34), but the differences in the rate of second-phase viral decline were significant (p = 0.0004); the median viral decline (interquartile range) in the second phase was 0.48 (0.42-0.50) log10/day in patients with sustained responses, 0.16 (0.10-0.19) log10/day in patients with transient responses, and 0.026 (0.017-0.040) log10/day in patients with no responses. CONCLUSIONS: Changes in serum levels of HCV genotype 1b in the first 2 wk of interferon-alpha treatment, monitored by real-time quantitative PCR, can be used for prediction of the long term therapeutic response.

Involvement of protein tyrosine phosphorylation and reduction of cellular sulfhydryl groups in cell death induced by 1' -acetoxychavicol acetate in Ehrlich ascites tumor cells.

Elucidation of the mechanisms underlying potential anticancer drugs continues and unraveling these mechanisms would not only provide a conceptual framework for drug design but also promote use of natural products for chemotherapy. To further evaluate the efficacy of the anticancer activity of 1'-acetoxychavicol acetate (ACA), this study investigates the underlying mechanisms by which ACA induces death of Ehrlich ascites tumor cells. ACA treatment induced loss of cell viability, and Western blotting analysis revealed that the compound stimulated tyrosine phosphorylation of several proteins with 27 and 70 kDa proteins being regulated in both dose- and time-dependent manner prior to loss of viability. Protein tyrosine kinase inhibitor herbimycin A moderately protected cells from ACA-induced toxicity. In addition, cellular glutathione and protein sulfydryl groups were also significantly reduced both dose- and time-dependently during evidence of cell death. Replenishing thiol levels by antioxidant, N-acetylcysteine (NAC), an excellent supplier of glutathione and precursor of glutathione, substantially recovered the viability loss, but the recovery being time-dependent, as late addition of NAC (at least 30 min after ACA addition to cultures) was, however, ineffective. Addition of NAC to ACA treated cultures also abolished tyrosine phosphorylation of the 27 kDa protein. These results, at least partly, identify cellular sulfhydryl groups and protein tyrosine phosphorylation as targets of ACA cytotoxicity in tumor cells.

GB virus C and TT virus infections in Japanese patients with autoimmune hepatitis.

The association of the newly identified viruses, GB virus C (GBV-C) and TT virus (TTV), with autoimmune hepatitis remains to be elucidated. Sera from 20 Japanese patients with autoimmune hepatitis and 50 volunteer blood donors were assayed for GBV-C RNA, antibodies to the GBV-C second envelope protein (E2), and TTV DNA. GBV-C RNA was examined by reverse-transcription polymerase chain reaction (PCR). Anti-GBV-C E2 (a marker of past infection) was tested by an enzyme-linked immunosorbent assay. TTV DNA was amplified by PCR using two different sets of primers: one derived from the original N22 sequence (Set A) and the other from the untranslated region (Set B). None of the patients or controls had GBV-C RNA. Anti-GBV-C E2 was found significantly more often in patients with autoimmune hepatitis (3/20) than in controls (1/50; P = 0.034). The prevalence of TTV DNA detected by primers Set A and that detected with either Set A or B were similar among patients with autoimmune hepatitis (4/20 and 16/20, respectively) and controls (9/50 and 40/50, respectively). Clinical characteristics did not differ in association with any of these viral markers. Of the 13 TTV isolates amplified with Set A, seven were classified as genotype 1a, four as genotype 1b, and 2 as genotype 3; no particular strain was associated with autoimmune hepatitis. These findings provide no compelling evidence that GBV-C or TTV has a pathogenic role in autoimmune hepatitis.

Modulation of ornithine decarboxylase activity and mitogen-activated protein kinases in protocatechualdehyde-induced apoptosis of CTLL-2 cells.

Protocatechualdehyde (PA, a dihydroxybenzene derivative) has previously been shown to induce apoptotic cell death in cytotoxic T cells (CTLL-2). However, the molecular mechanisms by which PA regulates apoptosis are still unclear. In this study, the possible roles of ornithine decarboxylase activity (ODC) and mitogen-activated protein kinases (MAPKs) in the PA-induced apoptosis process were further investigated. We demonstrated that PA inhibited ODC activity induced by IL2 in a time- and dose-dependent manner. Furthermore, the expression of ODC mRNA stimulated by IL2 was also effectively suppressed. 0.12 mM PA inhibited the activation of ERK1/2 induced by IL2 and enhanced the activation of JNK, which was abrogated by IL2. No alteration in the effect of p38 MAPK on the apoptosis process was observed in the CTLL-2 cells. PD98059 (a specific ERK1/2 inhibitor) inhibited cell growth, led to cell apoptotic death and effectively decreased ODC activity and suppressed ERK1/2 activation induced by IL2. These data indicate that PA induced apoptosis in CTLL-2 cells by two mechanisms; either via inhibiting ODC induction or interfering with MAPK signaling pathways.

Time Course of Change in Glutathione S-Transferase Positive Foci and Ornithine Decarboxylase Activity after Cessation of Long-term Alcohol Administration in Rats.

In our previous experiments, we showed that cessation of long-term alcohol administration enhances hepatocarcinogenesis in the rat. In the present study, we examined the time course of hepatocarcinogenesis induced by diethylnitrosamine (DEN) after cessation of alcohol using numbers and areas of glutathione S transferase placental form (GST-P)-positive foci and the activity of ornithine decarboxylase (ODC) in males of the Wistar strain. Fifty six rats were given a single i.p. injection of DEN (200 mg/kg body weight), maintained on basal solid diet for two weeks, then maintained on liquid diet in which 36% of total calories were provided by ethanol (Al diet) for 12 weeks, and then eight rats were killed. The remaining rats were divided into 6 groups. Three alcohol cessation groups were maintained on control liquid diet (C diet) instead of Al diet for 3, 6 and 12 weeks, respectively. The others, as reference groups were maintained on the Al diet continuously for the same periods, respectively. The numbers of GST-P-positive foci per unit area of the liver were not markedly changed after cessation of alcohol. However, their areas were increased with time, so that values in the alcohol cessation groups at 3 and 12 weeks were significantly higher than those in the reference groups at the same points, respectively. Furthermore, ODC activity was significantly elevated in the alcohol cessation groups at 3 and 6 weeks compared to reference groups, but not at 12 weeks when reduction was rather observed. These results suggest that cessation of long-term alcohol administration enhances hepatocarcinogenesis and this effect may be closely related to activation of cell proliferation due to the interruption of alcohol insult.

Cessation of Long-term Alcohol Administration and Two-day Cycling of Exposure Respectively Promote and Inhibit Hepatocarcinogenesis in Rats.

The effects of different patterns of alcohol administration on hepatocarcinogenesis induced by diethylnitrosamine (DEN) in male Wistar rats were assessed using a modified Ito's medium-term bioassay system. Carcinogenic potential was scored by comparing numbers and areas of glutathione S transferase placental form (GST-P)-positive foci. The activity of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine synthesis, was also measured as a parameter of cell proliferation. Rats were given a single i.p. injection of DEN (200 mg/kg body weight), maintained on basal solid diet for two weeks, then divided into five groups: group A maintained on liquid diet in which 36% of total calories were provided by ethanol (diet Al) for 24 weeks; group B maintained on diet Al for 12 weeks and subsequently on control diet (diet C) for 12 weeks; group C maintained on diet C for 24 weeks; group D maintained on a cycle of two days on diet Al followed by two days on diet C; group E maintained on another liquid diet in which 18% of total calories were provided by ethanol for 24 weeks. The numbers and areas per cm2 of GST-P positive foci in group B were highest and in group D were the lowest among the five groups. ODC activities in groups A and E were significantly lower than in group C, that for group B was intermediate. These results suggest that the intermittent intake of alcohol exerts preventive potential on hepatocellular lesion development, and that interruption of long-term alcohol administration enhances hepatocarcinogenesis.

Farnesol-induced growth inhibition in Saccharomyces cerevisiae by a cell cycle mechanism.

The growth of budding yeast, Saccharomyces cerevisiae, was inhibited in medium containing 25 microM farnesol (FOH). The FOH-treated cells were still viable, and were characterized by a transition from budded to unbudded phase as well as a significant loss of intracellular diacylglycerol (DAG). FOH-induced growth inhibition could be effectively prevented by the coaddition of a membrane-permeable DAG analogue which can activate yeast protein kinase C (PKC). However, yeast cell growth was not initiated upon addition of the PKC activator when the cells had been pretreated with FOH for 20 min. The failure in cell growth recovery was believed to be due to a signalling-mediated cell cycle arrest in FOH-pretreated cells. Differential display analysis demonstrated that the expression of cell cycle genes encoding DNA ligase (CDC9) and histone acetyltransferase (HAT2) was strongly repressed in FOH-treated cells. Repression of the expression of these genes was effectively cancelled when cells were grown in medium supplemented with DAG. The authors propose an interference with a phosphatidylinositol-type signalling which is involved in cell cycle progression as a cause of FOH-induced growth inhibition in yeast cells.