Multiple Signatures of the JC Polyomavirus in Paired Normal and Altered Colorectal Mucosa Indicate a Link with Human Colorectal Cancer, but Not with Cancer Progression.

The JC polyomavirus (JCV) has been repeatedly but discordantly detected in healthy colonic mucosa, adenomatous polyps, and colorectal cancer (CRC), and proposed to contribute to oncogenesis. The controversies may derive from differences in JCV targets, patient's cohorts, and methods. Studies of simultaneous detection, quantification, and characterization of JCV presence/expression in paired samples of normal/altered tissues of the same patient are lacking. Therefore, we simultaneously quantified JCV presence (DNA) and expression (mRNA and protein) of T-antigen (T-Ag), Viral Protein 1 (Vp1), and miR-J1-5p in paired normal/altered tissues of CRC or polyps, and from controls. JCV signatures were found in most samples. They increased in patients, but were higher in normal mucosa than in corresponding polyp or CRC lesions. JCV non-coding control region (NCCR) DNA rearrangements increased in CRC patients, also in normal mucosa, thus before the onset of the lesion. A new ∆98bp NCCR DNA rearrangement was detected. T-Ag levels were higher in normal mucosa than in adenoma and adenocarcinoma lesions, but decreased to levels of controls in established CRC lesions. In CRC, miR-J1-5p expression decreased with CRC progression. Vp1 expression was not detected. The data indicate a JCV link with the disease, but possible JCV contributes to oncogenesis should occur at pre-polyp stages.

Methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms in breast cancer: a Sardinian preliminary case-control study.

Two common polymorphisms in the MTHFR gene, C677T and A1298C, are associated with reduced enzyme activity and may be associated with breast cancer susceptibility. We performed a case-control study to investigate the association between the two SNPs in the MTHFR gene and risk of breast cancer. In total, 58 breast cancer patients and 58 unaffected controls were enrolled in the study. Polymerase chain reaction/restriction fragment length polymorphism technique (PCR-RFLP) was conducted to determine the genotypes. No significant differences were found in the genotypes of the two polymorphisms of the MTHFR gene between cases and controls. The OR and 95% CI for the 677CC, 677CT and 677TT genotypes were 1.00, 0.95 (0.39-2.31) and 0.87 (0.27-2.80), respectively; those of the 1298AA, 1298AC and 1298CC genotypes were 1.00, 0.59 (0.26-1.36) and 0.78 (1.32-4.66) respectively. Furthermore, it has been shown in patients with breast cancer a risk of presenting with an aggressive biophenotype about twice or three times higher in the presence of the C677T and A1298C polymorphisms, respectively. Finally, the A1298Cpolymorphism is significantly associated with increased recurrence risk of lymph node-positive breast cancer. Our study has not shown a significant association between MTHFR gene polymorphisms and breast cancer risk. However, it highlighted the key-role played by the presence of mutant alleles for both polymorphisms in increasing the risk of developing more aggressive phenotypes; moreover, specifically in A1298C, it might also lead to a higher risk of developing lymph node metastasis.

EGFR kinase-dependent and kinase-independent roles in clear cell renal cell carcinoma.

Epidermal growth factor receptor (EGFR) is associated with progression of many epithelial malignancies and represents a significant therapeutic target. Although clear cell renal cell carcinoma (CCRCC) has been widely investigated for EGFR molecular alterations, genetic evidences of EGFR gene activating mutations and/or gene amplification have been rarely confirmed in the literature. Therefore, until now EGFR-targeted therapies in clinical trials have been demonstrated unsuccessful. New evidence has been given about the interactions between EGFR and the sodium glucose co-transporter-1 (SGLT1) in maintaining the glucose basal intracellular level to favour cancer cell growth and survival; thus a new functional role may be attributed to EGFR, regardless of its kinase activity. To define the role of EGFR in CCRCC an extensive investigation of genetic changes and functional kinase activities was performed in a series of tumors by analyzing the EGFR mutational status and expression profile, together with the protein expression of downstream signaling pathways members. Furthermore, we investigated the co-expression of EGFR and SGLT1 proteins and their relationships with clinic-pathological features in CCRCC. EGFR protein expression was identified in 98.4% of CCRCC. Furthermore, it was described for the first time that SGLT1 is overexpressed in CCRCC (80.9%), and that co-expression with EGFR is appreciable in 79.4% of the tumours. Moreover, the activation of downstream EGFR pathways was found in about 79.4% of SGLT1-positive CCRCCs. The mutational status analysis of EGFR failed to demonstrate mutations on exons 18 to 24 and the presence of EGFR-variantIII (EGFRvIII) in all CCRCCs analyzed. FISH analysis revealed absence of EGFR amplification, and high polysomy of chromosome 7. Finally, the EGFR gene expression profile showed gene overexpression in 38.2% of CCRCCs. Our study contributes to define the complexity of EGFR role in CCRCC, identifying its bivalent kinase-dependent and kinase-independent functions, both potentially involved in CCRCC progression. These results might have important implications on therapeutic approaches to CCRCC, since the disruption of the interaction between EGFR/SGLT1, mediated by anti-EGFR antibodies and/or SGLT1 inhibitors, might constitute a novel therapeutic target for CCRCC treatment, and new clinical trials should be evaluated on the basis of this therapeutic proposal.

Ras-driven proliferation and apoptosis signaling during rat liver carcinogenesis is under genetic control.

Fast growth and deregulation of G1 and S phases characterize preneoplastic and neoplastic liver lesions of genetically susceptible F344 rats, whereas a G1-S block in lesions of resistant BN rats explains their low progression capacity. However, signal transduction pathways responsible for the different propensity of lesions from the 2 rat strains to evolve to malignancy remain unknown. Here, we comparatively investigated the role of Ras/Erk pathway inhibitors, involved in growth restraint and cell death, in the acquisition of a phenotype resistant or susceptible to hepatocarcinogenesis. Moderate activation of Ras, Raf-1 and Mek proteins was paralleled in both rat models by strong induction of Dab2 and Rkip inhibitors. Levels of Dusp1, a specific ERK inhibitor, increased only in BN rat lesions, leading to modest ERK activation, whereas a progressive Dusp1 decline occurred in corresponding lesions from F344 rats and was accompanied by elevated ERK activation. Furthermore, a gradual increase of Rassf1A/Nore1A/Mst1-driven apoptosis was detected in both rat strains, with highest levels in BN hepatocellular carcinoma (HCC), whereas loss of Dab2IP, a protein implicated in ASK1-dependent cell death, occurred only in F344 rat HCC, resulting in significantly higher apoptosis in BN than F344 HCC. Taken together, our results indicate a control of the Ras/Erk pathway and the pro-apoptotic Rassf1A/Nore1A and Dab2IP/Ask1 pathways by HCC susceptibility genes. Dusp1 possesses a prominent role in the acquisition of the phenotype resistant to HCC by BN rats, whereas late activation of RassF1A/Nore1A and Dab2IP/Ask1 axes is implicated in the highest apoptosis characteristic of BN HCC.

Dual-specificity phosphatase 1 ubiquitination in extracellular signal-regulated kinase-mediated control of growth in human hepatocellular carcinoma.

Sustained activation of extracellular signal-regulated kinase (ERK) has been detected previously in numerous tumors in the absence of RAS-activating mutations. However, the molecular mechanisms responsible for ERK-unrestrained activity independent of RAS mutations remain unknown. Here, we evaluated the effects of the functional interactions of ERK proteins with dual-specificity phosphatase 1 (DUSP1), a specific inhibitor of ERK, and S-phase kinase-associated protein 2 (SKP2)/CDC28 protein kinase 1b (CKS1) ubiquitin ligase complex in human hepatocellular carcinoma (HCC). Levels of DUSP1, as assessed by real-time reverse transcription-PCR and Western blot analysis, were significantly higher in tumors with better prognosis (as defined by the length of patients' survival) when compared with both normal and nontumorous surrounding livers, whereas DUSP1 protein expression sharply declined in all HCC with poorer prognosis. In the latter HCC subtype, DUSP1 inactivation was due to either ERK/SKP2/CKS1-dependent ubiquitination or promoter hypermethylation associated with loss of heterozygosity at the DUSP1 locus. Noticeably, expression levels of DUSP1 inversely correlated with those of activated ERK, as well as with proliferation index and microvessel density, and directly with apoptosis and survival rate. Subsequent functional studies revealed that DUSP1 reactivation led to suppression of ERK, CKS1, and SKP2 activity, inhibition of proliferation and induction of apoptosis in human hepatoma cell lines. Taken together, the present data indicate that ERK achieves unrestrained activity during HCC progression by triggering ubiquitin-mediated proteolysis of its specific inhibitor DUSP1. Thus, DUSP1 may represent a valuable prognostic marker and ERK, CKS1, or SKP2 potential therapeutic targets for human HCC.

Aberrant iNOS signaling is under genetic control in rodent liver cancer and potentially prognostic for the human disease.

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.

Altered methionine metabolism and global DNA methylation in liver cancer: relationship with genomic instability and prognosis.

Mounting evidence underlines the role of genomic hypomethylation in the generation of genomic instability (GI) and tumorigenesis, but whether DNA hypomethylation is required for hepatocellular carcinoma (HCC) development and progression remains unclear. We investigated the correlation between GI and DNA methylation, and influence of methionine metabolism deregulation on these parameters and hepatocarcinogenesis in c-Myc and c-Myc/Tgf-alpha transgenic mice and human HCCs. S-adenosyl-L-methionine/S-adenosylhomocysteine ratio and liver-specific methionine adenosyltransferase (MatI/III) progressively decreased in dysplastic and neoplastic liver lesions developed in c-Myc transgenic mice and in human HCC with better (HCCB) and poorer (HCCP) prognosis (based on patient's survival length). Deregulation of these parameters resulted in a rise of global DNA hypomethylation both in c-Myc and human liver lesions, positively correlated with GI levels in mice and humans, and inversely correlated with the length of survival of HCC patients. No changes in MATI/III and DNA methylation occurred in c-Myc/Tgf-alpha lesions and in a small human HCC subgroup with intermediate prognosis, where a proliferative activity similar to that of c-Myc HCC and HCCB was associated with low apoptosis. Upregulation of genes involved in polyamine synthesis, methionine salvage and downregulation of polyamine negative regulator OAZ1, was highest in c-Myc/Tgf-alpha HCCs and HCCP. Our results indicate that alterations in the activity of MAT/I/III, and extent of DNA hypomethylation and GI are prognostic markers for human HCC. However, a small human HCC subgroup, as c-Myc/Tgf-alpha tumors, may develop in the absence of alterations in DNA methylation.

Identification and chromosome mapping of loci predisposing to colorectal cancer that control Wnt/beta-catenin pathway and progression of early lesions in the rat.

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.

Mapping a sex hormone-sensitive gene determining female resistance to liver carcinogenesis in a congenic F344.BN-Hcs4 rat.

Hepatocellular carcinoma (HCC) is prevalent in human and rodent males. Hepatocarcinogenesis is controlled by various genes in susceptible F344 and resistant Brown Norway (BN) rats. B alleles at Hcs4 locus, on RNO16, control neoplastic nodule volume. We constructed the F344.BN-Hcs4 recombinant congenic strain (RCS) by introgressing a 4.41-cM portion of Hcs4 from BN strain in an isogenic F344 background. Preneoplastic and neoplastic lesions were induced by the "resistant hepatocyte" protocol. Eight weeks after initiation, lesion volume and positivity for proliferating cell nuclear antigen (PCNA) were much higher in lesions of F344 than BN rats of both sexes. These variables were lower in females than in males. Lesion volume and PCNA values of male RCS were similar to those of F344 rats, but in females corresponded to those of BN females. Carcinomatous nodules and HCC developed at 32 and 60 weeks, respectively, in male F344 and congenics and, rarely, in F344 females. BN and congenic females developed only eosinophilic/clear cells nodules. Gonadectomy of congenic males, followed by beta-estradiol administration, caused a decrease in Ar expression, an increase in Er-alpha expression, and development of preneoplastic lesions comparable to those from BN females. Administration of testosterone to gonadectomized females led to Ar increase and development of preneoplastic lesions as in F344 males. This indicates a role of homozygous B alleles at Hcs4 in the determination of phenotypic patterns of female RCS and presence at Hcs4 locus of a high penetrance gene(s), activated by estrogens and inhibited/unaffected by testosterone, conferring resistance to females in which the B alleles provide higher resistance.

Role of HSP90, CDC37, and CRM1 as modulators of P16(INK4A) activity in rat liver carcinogenesis and human liver cancer.

Current evidence indicates that neoplastic nodules induced in liver of Brown Norway (BN) rats genetically resistant to hepatocarcinogenesis are not prone to evolve into hepatocellular carcinoma. We show that BN rats subjected to diethylnitrosamine/2-acetylaminofluorene/partial hepatectomy treatment with a "resistant hepatocyte" protocol displayed higher number of glutathione-S-transferase 7-7(+) hepatocytes when compared with susceptible Fisher 344 (F344) rats, both during and at the end of 2-acetylaminofluorene treatment. However, DNA synthesis declined in BN but not F344 rats after completion of reparative growth. Upregulation of p16(INK4A), Hsp90, and Cdc37 genes; an increase in Cdc37-Cdk4 complexes; and a decrease in p16(INK4A)-Cdk4 complexes occurred in preneoplastic liver, nodules, and hepatocellular carcinoma of F344 rats. These parameters did not change significantly in BN rats. E2f4 was equally expressed in the lesions of both strains, but Crm1 expression and levels of E2f4-Crm1 complex were higher in F344 rats. Marked upregulation of P16(INK4A) was associated with moderate overexpression of HSP90, CDC37, E2F4, and CRM1 in human hepatocellular carcinomas with a better prognosis. In contrast, strong induction of HSP90, CDC37, and E2F4 was paralleled by P16(INK4A) downregulation and high levels of HSP90-CDK4 and CDC37-CDK4 complexes in hepatocellular carcinomas with poorer prognosis. CDC37 downregulation by small interfering RNA inhibited in vitro growth of HepG2 cells. In conclusion, our findings underline the role of Hsp90/Cdc37 and E2f4/Crm1 systems in the acquisition of a susceptible or resistant carcinogenic phenotype. The results also suggest that protection by CDC37 and CRM1 against growth restraint by P16(INK4A) influences the prognosis of human hepatocellular carcinoma.

Chemopreventive N-(4-hydroxyphenyl)retinamide (fenretinide) targets deregulated NF-{kappa}B and Mat1A genes in the early stages of rat liver carcinogenesis.

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.

Polygenic control of hepatocarcinogenesis in Copenhagen x F344 rats.

Cop and CFF1 rats exhibit resistance to hepatocarcinogenesis, associated with high rates of remodeling of neoplastic lesions. We have mapped hepatocarcinogenesis susceptibility, resistance and remodeling loci affecting the number, volume and volume fraction of neoplastic nodules induced by the "resistant hepatocyte" model in male CFF2 rats. Three loci in significant linkage with the number or volume of nonremodeling lesions were identified on chromosomes 1, 4 and 18. Suggestive linkage with number or volume fraction of total, nonremodeling or remodeling lesions was found for 7 loci on chromosomes 1, 2, 13, 14 and 15. All of these loci showed significant allele-specific effects on the phenotypic traits. We also detected by analysis of variance 19 2-way interactions inducing phenotypic effects not predictable on the basis of the sum of separate effects. These novel epistatic loci were in significant linkage with the number and/or volume of total, nonremodeling or remodeling nodules. These data indicate that susceptibility to hepatocarcinogenesis in Cop rats is controlled by a complex array of genes with several gene-gene interactions and that different genetic mechanisms control remodeling and nonremodeling liver nodules. Frequent deregulation in human liver cancer of genes positioned in chromosomal segments syntenic to rat susceptibility/resistance loci suggests some similarities between the genetic mechanisms involved in hepatocarcinogenesis in rats and humans.

Down-regulation of c-myc and Cyclin D1 genes by antisense oligodeoxy nucleotides inhibits the expression of E2F1 and in vitro growth of HepG2 and Morris 5123 liver cancer cells.

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.

Phenotypic reversion of rat neoplastic liver nodules is under genetic control.

Low DNA synthesis and high redifferentiation (remodeling) characterize neoplastic nodules induced by chemical carcinogens in hybrid BFF1 rats, generated by crossing the susceptible F344 and resistant BN strains. We performed whole-genome scanning of BFF2 rats to identify loci controlling remodeling of nodules induced, 32 weeks after initiation with diethylnitrosamine, by the RH protocol. Remodeling nodules were identified as areas lacking uniformity of GST-P immunostaining and with irregular margins. Two loci in suggestive linkage with the percentage of remodeling nodules were identified on chromosomes 7 and 1 (LOD scores 3.85 and 2.9 at D7Rat25 and D1Mgh14). Significant dosage-negative effect of the B allele on remodeling and additive interaction between these loci were found. Significant epistatic interactions, showing a recessive, remodeling-enhancing effect of B alleles, occurred between D1Mit3 and D11Rat11 (corrected p = 0.0013) and between D6Rat14 and D8Rat46 (corrected p = 0.028). These data show that remodeling of neoplastic nodules during rat hepatocarcinogenesis is under genetic control. Loci affecting remodeling map to chromosomal regions syntenic to chromosomal segments of human HCC showing structural abnormalities.

Stearoyl-CoA desaturase 1 (Scd1) gene overexpression is associated with genetic predisposition to hepatocarcinogenesis in mice and rats.

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.

Chromosome mapping of multiple loci affecting the genetic predisposition to rat liver carcinogenesis.

Previous studies on (BNxF344)F1 (BFF1) rat model of genetic predisposition to hepatocarcinogenesis led to the identification, in BFF1xF344 backcross progeny, of two hepatocarcinogenesis susceptibility (Hcs) and three resistance (Hcr) loci affecting the progression of neoplastic liver nodules. To evaluate the presence of other hepatocarcinogenesis-related loci in the BFF1 genome, nodule induction by resistant hepatocyte model in 116 male BFF2 rats 32 weeks after initiation with diethylnitrosamine was subjected to quantitative trait loci analysis. The rats were typed with 179 genetic markers, and linkage analysis identified three loci on chromosomes 1, 16, and 6, in significant linkage with nodule mean volume (V), volume fraction, and number, respectively, and two loci on chromosomes 4 and 8 in suggestive linkage with V. These loci were differently positioned with respect to Hcs and Hcr loci mapped previously in backcross rats. On the basis of phenotypic and allele distribution patterns of BFF2 rats, loci on chromosomes 1 and 16 were identified as Hcs3 and Hcs4, and loci on chromosomes 4, 8, and 6 as Hcr4, Hcr5, and Hcr6. Additive interactions occurred between Hcs3 and Hcs4, and Hcr4 and a locus on chromosome 3 with less than suggestive linkage with V. All of the loci were in chromosomal regions syntenic to mouse and/or human chromosomal segments showing allelic gain or loss in hepatocellular carcinomas. These data indicate that inheritance of predisposition to rat liver tumor is characterized by the interplay of several genetic factors and suggest some possible mechanisms of polygenic control of human liver cancer.

Chemoprevention of hepatocarcinogenesis: S-adenosyl-L-methionine.

Accumulation of genetic changes characterizes the progression of cells, initiated by carcinogens, to full malignancy. Various epigenetic mechanisms, such as high polyamine synthesis, aberrant DNA methylation, and production of reactive oxygen species, may favor this process by stimulating growth and inducing DNA damage. We observed a decrease in S-adenosyl-L-methionine (SAM) content in the liver, associated with DNA hypomethylation in rat liver, during the development of preneoplastic foci, and in neoplastic nodules and hepatocellular carcinomas, induced in diethylnitrosamine-initiated rats by "resistant hepatocyte" (RH) protocol. Reconstitution of the methyl donor level in the liver by SAM administration inhibits growth and induces phenotypic reversion and apoptosis of preneoplastic cells. A 6-month SAM treatment results in a sharp and persistent decrease in development of neoplastic nodules, suggesting a long duration of SAM chemopreventive effect. Various observations support the suggestion of a role of DNA methylation in chemoprevention by SAM: (1) Exogenous SAM reconstitutes the SAM pool in preneoplastic and neoplastic liver lesions. (2) DNA methylation is positively correlated with SAM:S-adenosylhomocysteine (SAH) ratio in these lesions. (3) 5-Azacytidine, a DNA methyltransferase inhibitor, inhibits chemoprevention by SAM. (4) c-Ha-ras, c-Ki-ras, and c-myc are hypomethylated and overexpressed in preneoplastic liver. Their expression is inversely correlated with SAM:SAH ratio in SAM-treated rats. (5) S-Adenosyl-L-methionine treatment results in overall DNA methylation and partial methylation of these genes. Other possible mechanisms of SAM treatment include inhibition of polyamine synthesis, linked to partial transformation of SAM into 5'-methylthioadenosine (MTA), and antioxidant and antifibrogenic activities of both SAM and MTA.

Cell cycle deregulation in liver lesions of rats with and without genetic predisposition to hepatocarcinogenesis.

Preneoplastic and neoplastic hepatocytes undergo c-Myc up-regulation and overgrowth in rats genetically susceptible to hepatocarcinogenesis, but not in resistant rats. Because c-Myc regulates the pRb-E2F pathway, we evaluated cell cycle gene expression in neoplastic nodules and hepatocellular carcinomas (HCCs), induced by initiation/selection (IS) protocols 40 and 70 weeks after diethylnitrosamine treatment, in susceptible Fisher 344 (F344) rats, and resistant Wistar and Brown Norway (BN) rats. No interstrain differences in gene expression occurred in normal liver. Overexpression of c-myc, Cyclins D1, E, and A, and E2F1 genes, at messenger RNA (mRNA) and protein levels, rise in Cyclin D1-CDK4, Cyclin E-CDK2, and E2F1-DP1 complexes, and pRb hyperphosphorylation occurred in nodules and HCCs of F344 rats. Expression of Cdk4, Cdk2, p16(INK4A), and p27(KIP1) did not change. In nodules and/or HCCs of Wistar and BN rats, low or no increases in c-myc, Cyclins D1, E, and A, and E2F1 expression, and Cyclin-CDKs complex formation were associated with p16(INK4A) overexpression and pRb hypophosphorylation. In conclusion, these results suggest deregulation of G1 and S phases in liver lesions of susceptible rats and block of G1-S transition in lesions of resistant strains, which explains their low progression capacity.