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Polymorphonuclear leukocytes (PMNs) from individuals carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase (G6PD) exhibit a great decrease in this enzymatic activity and in hexose monophosphate shunt (HMS). 12--tetradecanoylphorbol-13-acetate (TPA) greatly stimulates HMS of normal PMNs, while it does not affect that of the deficient PMNs. Similarly, the stimulation of HMS by methylene blue is largely reduced in G6PD-deficient PMNs. These changes are paralleled by a 58% decrease in TPA-stimulated superoxide radical (O) formation by the deficient PMNs. G6PD activity is not detectable in the deficient PMNs incubated with dehydroepiandrosterone, and these cells show a near complete inhibition of O production. It thus seems that the low ability of G6PD-deficient PMNs in the production of O depends on the low NADPH generation by HMS in these cells. The decrease in TPA-stimulated O production suggests a reduced response of G6PD-deficient cells to promoting agents. [ABSTRACT FROM PUBLISHER]

Mounting evidence underlines the role of inducible nitric oxide synthase (iNOS) in hepatocellular carcinoma (HCC) development, but its functional interactions with pathways involved in HCC progression remain uninvestigated. Here, we analyzed in preneoplastic and neoplastic livers from Fisher 344 and Brown Norway rats, possessing different genetic predisposition to HCC, in transforming growth factor-alpha (TGF-alpha) and c-Myc-TGF-alpha transgenic mice, characterized by different susceptibility to HCC, and in human HCC: (i) iNOS function and interactions with nuclear factor-kB (NF-kB) and Ha-RAS/extracellular signal-regulated kinase (ERK) during hepatocarcinogenesis; (ii) influence of genetic predisposition to liver cancer on these pathways and role of these cascades in determining a susceptible or resistant phenotype and (iii) iNOS prognostic value in human HCC. We found progressive iNos induction in rat and mouse liver lesions, always at higher levels in the most aggressive models represented by HCC of rats genetically susceptible to hepatocarcinogenesis and c-Myc-TGF-alpha transgenic mice. iNOS, inhibitor of kB kinase/NF-kB and RAS/ERK upregulation was significantly higher in HCC with poorer prognosis (as defined by patients' survival length) and positively correlated with tumor proliferation, genomic instability and microvascularization and negatively with apoptosis. Suppression of iNOS signaling by aminoguanidine led to decreased HCC growth and NF-kB and RAS/ERK expression and increased apoptosis both in vivo and in vitro. Conversely, block of NF-kB signaling by sulfasalazine or short interfering RNA (siRNA) or ERK signaling by UO126 caused iNOS downregulation in HCC cell lines. These findings indicate that iNOS cross talk with NF-kB and Ha-RAS/ERK cascades influences HCC growth and prognosis, suggesting that key component of iNOS signaling could represent important therapeutic targets for human HCC.

Sporadic colorectal cancer (CRC) is a major health concern worldwide. Epidemiologic evidence suggests a polygenic predisposition to CRC, but the genes responsible remain unknown. Here, we performed genome-wide scanning of male (ACI/SegHsd x Wistar-Furth)F2 (AWF2) rats to map susceptibility genes influencing the evolution of early colorectal lesions to adenocarcinoma following 1,2-dimethylhydrazine administration. Phenotypic analysis revealed higher incidence/multiplicity and lower size of adenomas in ACI/SegHsd (ACI) and (ACI/SegHsd x Wistar-Furth)F1 (AWF1) than Wistar-Furth (WF) rats and higher incidence/multiplicity of poorly differentiated adenocarcinomas in WF than ACI rats, with intermediate values in AWF1 rats. Linkage analysis of 138 AWF2 rats identified three loci on chromosomes 4, 15 and 18 in significant linkage with lesion multiplicity that were identified as rat Colon cancer resistance (rCcr) 1, rCcr2 and rCcr3, respectively. Seven other loci on chromosomes 5, 6, 15, 17, 18 and 20 were in suggestive linkage with adenoma/adenocarcinoma multiplicity/surface area. Six of them were identified as rCcr4-9 and a locus on chromosome 5 was identified as a susceptibility locus, rCcs1. Significant interactions between rCcr3 and rCcr6, rCcr6 and rCcr8 and rCcr5 and rCcr9, and four novel epistatic loci controlling multiplicity/size of colorectal lesions were discovered. Apc, located at rCcr3, did not show functional promoter polymorphisms. However, influence of susceptibility/resistance genes on Wnt/beta-catenin pathway was shown by defective beta-catenin inactivation in WF but not in ACI and AWF1 rat adenocarcinomas. These data indicate that inheritance of predisposition to CRC depends on interplays of several genetic factors, and suggest a possible mechanism of polygenic control of CRC progression.

Cell-cycle deregulation is an early event of hepatocarcinogenesis. We evaluated the role of changes in activity of nuclear factor kappaB (NF-kappaB) and some related pathways in this alteration, and the interference of N-(4-hydroxyphenyl)retinamide (HPR), a retinoid chemopreventive for various cancer types, with these molecular mechanisms and the evolution of preneoplastic liver to cancer. Male F344 rats, initiated according to the 'resistant hepatocyte' model of liver carcinogenesis, received weekly 840 nmol of liposomal HPR (SL-HPR)/100 g body wt or empty liposomes, between 5 and 25 weeks after initiation. Inhibition of DNA synthesis and induction of apoptosis occurred in pre-cancerous lesions, 7-147 days after starting SL-HPR, and a decrease in carcinoma incidence and multiplicity was observed 25 weeks after arresting treatment. An increase in NF-kappaB expression and binding activity, and under-expression of the inhibitor kappaB-alpha (IkappaB-alpha) were found in preneoplastic liver and neoplastic nodules, 5 and 25 weeks after initiation, respectively. These lesions also showed low expression of Mat1A and low activity of methionine adenosyltransferase I/III, whose reaction product, S-adenosyl-l-methionine, enhances IkappaB-alpha expression. SL-HPR prevented these changes and induced a decrease in expression of iNos, c-myc, cyclin D1 and Vegf-A genes, that were over-expressed in preneoplastic liver and nodules, and a decrease in Bcl-2/Bax, Bcl-2/Bad and Bcl-xL/Bax mRNA ratios with respect to the lesions of control rats. Liposomes alone did not influence the parameters tested. These results indicate that signal transduction pathways controlled by NF-kappaB, nitric oxide and S-adenosyl-l-methionine are deregulated in pre-cancerous lesions. Recovery from these alterations by SL-HPR is associated with chemoprevention of hepatocarcinogenesis. Overall, these studies elucidate some molecular changes, in early stages of hepatocarcinogenesis, and underline their pathogenetic role. Moreover, they demonstrate a partially new mechanism of HPR chemopreventive effect and indicate the potential clinical relevance of this compound for prevention of hepatocellular carcinoma.

A number of genetic interactions are involved in the control of cell cycle, but their role and nature have not been completely clarified. The knowledge of the behavior of these interactions in hepatocellular carcinoma, could optimize preventive and therapeutic strategies based on cell cycle restraint. We studied downstream events following c-MYC and CYCLIN D1 gene inhibition, by lipoplex-delivered MYC and CYCLIN D1 antisense oligodeoxy nucleotides (aODNM, aODND1), in in vitro cultured human HepG2 and rat Morris 5123 hepatoma cells. 0.5-20 micro M aODN(M) and aODND1 inhibited in vitro growth of both cell types. Scramble oligomer (SCR) and sense ODNs had no or relatively poor effect. Ten micromolar aODNM and aODND1, but not SCR, also induced a significant increase in the apoptotic index of HepG2 and 5123 cells, and inhibited colony formation in soft agar by HepG2 cells. Treatment of the cells with aODNM plus aODND1 had no additive effect on growth and apoptosis. aODNM and aODND1 induced >50% decrease in c-MYC and CYCLIN D1 gene expression, respectively, at both mRNA and protein level. The inhibition of gene expression by aODNs was highly specific, and SCR was without effect. The reduction in c-MYC and CYCLIN D1 expression by aODNs, was associated with a >50% decrease in E2F1 mRNA and protein production, without changes in CYCLIN A and CYCLIN E expression. These results suggest the involvement of both c-MYC and CYCLIN D1 on E2F1 gene function, and indicate that aODNM and aODND1 may inhibit hepatoma cell growth through down-regulation of the E2F1 gene. The inhibition of E2F1 gene expression by E2F1 aODN, was associated with strong growth restraint of HepG2 cells. Thus, interactions of c-MYC and CYCLIN D1 with E2F1 gene are essential for cell cycle activity in hepatoma cells, and their inhibition may have a therapeutic effect.

The stearoyl-CoA desaturase 1 (Scd1) gene is involved in the synthesis and regulation of unsaturated fatty acids. Its expression is increased by several treatments/conditions that are associated with hepatocarcinogenesis (peroxisome proliferators, iron overload, dichloroacetic acid). We found that the Scd1 gene is differentially expressed, showing >10-fold higher mRNA levels in the normal liver tissue of C3H/He mice, which are genetically susceptible to hepatocarcinogenesis, than of BALB/c mice, which are resistant. Similarly, Scd1 mRNA expression was approximately 4-fold higher in the normal liver of F344 rats, which are susceptible to hepatocarcinogenesis, than in Brown Norway (BN) rats, which are resistant. The chromosomal location of the Scd1 locus, both in mice and rats, excludes Scd1 candidacy as a hepatocellular tumor-modifier gene, as the Scd1 locus did not show allele-specific effects in a BALB/cxC3H/He intercross or in a BNxF344 backcross and intercross. No Scd1 coding polymorphisms were detected in the mouse and the rat strains showing elevated Scd1 expression. These results suggest that the Scd1 gene represents a downstream target of hepatocellular tumor-modifier loci in two rodent species.

Previous work has demonstrated that dehydroepiandrosterone (DHEA) strongly inhibits growth and de novo cholesterol (CH) biosynthesis in preneoplastic rat liver. Administration of a mixture of 4 ribo- or deoxyribonucleosides of adenine, guanine, cytosine and uracil/thymine, prevents growth inhibition but not inhibition of CH synthesis. The purpose of this paper was to identify the site of inhibition of CH synthesis by DHEA. Persistent nodules (PNs) were induced, in diethylnitrosamine-initiated male F344 rats, by 'resistant hepatocyte' protocol. Fifteen weeks after initiation, nodule bearing rats and normal controls received a diet containing 0.6% DHEA for 3 weeks. They were then killed. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) activity and mRNA levels were 18- and 14-fold higher, respectively in nodules than in normal liver. DHEA strongly inhibited HMGR activity in both tissues in vivo, but had a slight effect on HMGR activity, when added in vitro to the reaction mixture for determination of this activity. In vivo DHEA treatment caused a 65% decrease in the level of HMGR mRNA in PNs, which, however, does not seem to completely account for the decrease in HMGR activity (83%). Low density lipoprotein receptor (LDL-R) mRNA level underwent a slight decrease in PNs, with respect to control liver, which did not lead to a significant decrease in 125I-LDL binding to LDL-R. DHEA treatment caused 30% and 24% increases in LDL-R expression and 125I-LDL binding, respectively, in nodules. These observations indicate that in addition to HMGR gene expression, increased influx of LDL into preneoplastic cells may contribute to the deregulation of mevalonate synthesis by DHEA. The observation that HMGR activity and gene expression were still 3- to 5-fold higher in PNs of DHEA-treated rats than in control liver, and previous findings of preneoplastic liver cell growth in the presence of relatively low CH synthesis, suggest that even relatively low levels of mevalonate are sufficient for the growth of preneoplastic liver cells.

Liver nodules and carcinomas, developing in F344 rats initiated with diethylnitrosamine, exhibit high ornithine decarboxylase (ODC) activity and DNA synthesis. ODC-related RNAs of 1.8, 2.1 and 2.6 kb are produced by normal rat liver. Early preneoplastic nodules, developing 10 weeks after initiation, showed overproduction of 1.8 and 2.1 kb RNAs, while the 2.6 kb RNA was barely detectable. Rises in the 1.8, 2.1 and 2.6 kb RNAs occur in late nodules (30 weeks after initiation) and in carcinomas. The comparison of different tissues for relative increase in ODC activity, RNA levels and DNA synthesis showed that these parameters behaved in the same way: highest increases occurred in early nodules and carcinomas. These observations suggest that overexpression of ODC gene and alterations in regulatory mechanisms of ODC gene expression may be implicated in the progression of preneoplastic lesions to malignancy. Southern blot analysis of PstI DNA digests revealed the presence of ODC gene rearrangement in two carcinomas and in one late nodule. However, the role of this phenomenon in the progression of preneoplastic lesions is unclear, due to the possibility that ODC pseudogenes are involved instead of or in addition to ODC gene.

Previous studies from our laboratory have shown that dehydroepiandrosterone (DHEA), an inhibitor of glucose-6-phosphate dehydrogenase (G6PD), prevents the development of gamma-glutamyltranspeptidase (GGT)-positive foci in the early stages of hepatocarcinogenesis in rats. Since high rates of DNA and cholesterol (CH) synthesis are observed during promotion of carcinogenesis, and mevalonate (MVA), or some other intermediates of CH synthesis, could be mediators of DNA synthesis, we investigated the effect of DHEA on CH synthesis in rat liver during the development of GGT-positive foci. Hepatocarcinogenesis was induced by diethylnitrosamine in female Wistar rats by the Solt-Farber protocol (initiation/selection) with and without phenobarbital treatment. A 15 day treatment with DHEA (0.6% in the diet), started after selection, caused a great fall in labeling and mitotic indices of GGT-positive foci, which was prevented by the simultaneous administration of a mixture of four deoxyribonucleosides (DRNs) of adenine, guanine, cytosine and thymine or four ribonucleosides (RNs) of adenine, guanine, cytosine and uridine, but not by the corresponding bases. DHEA greatly inhibited G6PD activity and the production of ribulose-5-phosphate, without affecting NADPH levels, due to the compensatory increase in malic enzyme and isocitric dehydrogenase activities. Serum lecithin/cholesterol acyltransferase activity underwent a reduction in conditions allowing a rapid growth of GGT-positive tissue (absence of DHEA or presence of DHEA plus DRNs or RNs). Liver slices isolated from DHEA-treated rats showed a rise in CH content, coupled with a 80% fall in the incorporation of labeled acetate, but not of labeled MVA, into CH. A 25 day treatment of rats subjected to initiation/selection, started after the appearance of persistent nodules, caused a 36 and 78% fall in the incorporation, in vivo, of 3H2O into nodular and surrounding liver CH respectively. DRN did not counteract DHEA-induced inhibition on CH synthesis. Thus DHEA inhibits the CH biosynthetic pathway before MVA synthesis, in conditions (presence of DHEA plus DRN/RN) allowing rapid growth of preneoplastic lesions. Therefore, the development of these lesions does not need the synthesis of large amounts of CH and CH metabolites. Thus, the antipromotion effect of DHEA may depend on a decreased availability of pentose phosphates for DNA synthesis.

A decrease of S-adenosyl-L-methionine liver content was observed between the 14th and the 35th day after the start of 2-acetylaminofluorene feeding in diethylnitrosamine-initiated rats according to the 'resistant-hepatocyte' model of hepatocarcinogenesis. The decrease was enhanced by phenobarbital given to the animals after the end of 2-acetylaminofluorene feeding. These changes were associated with an increase in ornithine decarboxylase activity and the spermidine:spermine ratio. S-adenosyl-L-methionine administration to rats caused a great fall in the percentage of gamma-glutamyltranspeptidase-positive liver as well as in polyamine synthesis. An increase in ornithine decarboxylase activity, associated with a decrease in the liver S-adenosyl-L-methionine pool, also occurred in normal animals on the first day following a partial hepatectomy and was enhanced by phenobarbital. The association of 2-acetylaminofluorene feeding with partial hepatectomy resulted in a slower liver regeneration, while the decrease in S-adenosyl-L-methionine level and the increase in polyamine synthesis were observed over a longer period of time after partial hepatectomy. These changes were further prolonged in diethylnitrosamine-initiated rats in which gamma-glutamyltranspeptidase-positive foci developed. In these animals a high level of polyamine synthesis was still present when liver regeneration was complete. At this stage of the observation period the labeling index was very low in surrounding liver, but still high in the gamma-glutamyltranspeptidase-positive areas. Phenobarbital stimulated polyamine synthesis and cell growth and further prolonged the period of time during which a high ornithine decarboxylase activity and labeling index were present. These results indicate that the liver lipotrope content could be a rate-limiting factor for cell growth and liver neoplasia promotion and this could depend on the modulation of polyamine biosynthesis.

S-adenosylmethionine:S-adenosylhomocysteine (SAM/SAH) ratio, 5-methylcytosine (5mC) DNA content, and methylation and expression of c-myc, c-Ha-ras and c-Ki-ras have been studied in liver nodules, induced by diethylnitrosamine according to the 'resistant hepatocyte' model, and in regenerating liver (RL) between 0.5 and 72 h after partial hepatectomy (PH). Nodules, 11, 13 and 21 weeks after initiation, grew actively, showed a low tendency to remodel (persistent nodules), and did not exhibit carcinomatous changes. They underwent extensive remodeling after a 1-week SAM treatment (64 mumol/kg/day), and decreased in size and number after a 3-11-week treatment. A low SAM/SAH ratio was coupled, in nodules, with a high labeling index (LI), 2-fold fall in 5mC DNA content, increase in c-myc, c-Ha-ras and c-Ki-ras expression and hypomethylation of CCGG sequences in the DNA hybridizing with the three protooncogenes. In RL a low SAM/SAH ratio, overall DNA hypomethylation and enhanced c-myc expression were first observed 0.5 h after PH, reached a peak at 5 h and progressively returned to pre-PH levels later on. Maximum expression of c-Ha-ras and c-Ki-ras occurred 24-30 h after PH, roughly coincident with the LI peak. However, no great modifications of the methylation pattern of protooncogene CCGG sequence occurred at any time after PH, indicating the presence of hypomethylated genes and/or DNA sequences different from those investigated in this paper. SAM injection to nodule-bearing rats, for 1-11 weeks before killing, and to hepatectomized rats, 2 days before PH and then up to killing, largely prevented decrease in the SAM/SAH ratio and overall DNA methylation and inhibited LI and protooncogene expression. In nodules these effects were proportional to the treatment length and coupled with methylation of CpG residues in the CCGG sequence of the three protooncogenes studied. SAM treatment left the methylation pattern of these genes unchanged in RL. Kinetics of increase in protooncogene expression suggest a role in the regulation of cell cycle in RL. However, decrease in the SAM/SAH ratio, protooncogene hypomethylation and enhanced expression are apparently stable in nodules 11-21 weeks after initiation and could be implicated in continuous nodule growth and progression. Control of DNA methylation and gene expression by exogenous SAM could be a mechanism of the SAM anti-progression effect.

Liver ornithine decarboxylase (ODC) activity and content of S-adenosyl-L-methionine (SAM) and its catabolite 5'-methylthioadenosine (5'-MTA) were determined in the late stages of hepatocarcinogenesis. Wistar rats received one diethylnitrosamine dose, followed by a partial hepatectomy at the midpoint of a 15-day treatment with 2-acetylaminofluorene (2-AAF), and then by an 18-week phenobarbital (PB) treatment. Thirty-eight per cent of liver was gamma-glutamyltranspeptidase (GGT)-positive and no visible nodules and hepatocellular carcinomas developed 16 weeks after starting 2-AAF feeding. Hyperplastic nodules and hepatocellular carcinomas were found on weeks 24 and 56 respectively. On weeks 24 and 56 only approximately 10% of liver was occupied by GGT-positive foci. At all times studied the foci exhibited a low labeling index (LI), and liver ODC activity was near control values. By contrast, a high ODC activity and LI and a low SAM and 5'-MTA levels were found in hyperplastic nodules and neoplasia. These tissues exhibited a high 5'-MTA phosphorylase activity. SAM administration during PB treatment, caused a 25-36% fall of GGT-positive liver and prevented the development of hyperplastic nodules and hepatocellular carcinomas. This was coupled to a sharp increase of SAM and 5'-MTA liver contents. SAM and 5'-MTA inhibited hepatocyte DNA synthesis in vitro. The addition of 5'-MTA to the reaction mixture for the ODC assay strongly inhibited ODC activity. However, the preincubation of SAM with liver homogenates or hepatocytes, used to prepare crude ODC, was necessary to inhibit ODC activity by SAM. Adenine, an inhibitor of 5'-MTA-phosphorylase, enhanced inhibition of DNA synthesis and ODC activity by SAM and 5'-MTA. Thus, during a prolonged promoting treatment a selected population of GGT-positive foci appears to acquire a stable phenotype characterized by a high DNA and polyamine synthesis. The development of nodules and carcinomas is associated with low SAM and 5'-MTA contents and high ODC activity and LI. 5'-MTA accumulation, during SAM administration, is probably responsible for the inhibition of promotion by SAM.