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. H uman and rodent hepatocarcinogenesis is characterized by the progressive development of foci of altered hepatocytes (FAH), neoplastic nodules, and hepatocellular carcinoma (HCC). 1,2 Epidemiological evidence suggests a polygenic predisposition for human HCC. 2 Studies on rodents allowed mapping the loci responsible for the progression of preneoplastic hepatocytes to malignancy. [3][4][5][6][7] However, the nature and temporal occurrence of the events underlying resistance to HCC remain uncertain. The evaluation of these mechanisms could help in understanding the molecular pathways affected by susceptibility genes to evaluate cancer risk and identify potentially reversible phases of carcinogenesis.Previous research has shown cell cycle deregulation in neoplastic liver lesions induced by the "resistant hepatocyte" protocol in susceptible Fisher 344 (F344) rats. [8][9][10] Lower or no changes were found in resistant Brown Norway (BN) rats in which overexpression of p16 INK4A , a well-known inhibitor of the cyclin-dependent kinase (Cdk) 4/6, occurs. Forma- PH, partial hepatectomy; BrdU, siRNA, small interfering RNA. From the

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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.

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P revious studies on genetic predisposition to hepatocellular carcinoma (HCC) of rats led to identification of 4 hepatocarcinogenesis susceptibility (Hcs1-4) loci, and 7 resistance (Hcr1-7) loci 1,2 (De Miglio et al., unpublished data). Resistance alleles, dominantly transmitted to the progeny, 3,4 apparently modify the activity of susceptibility loci. Recent evidence suggests the presence of at least 3 oncosuppressor genes at Hcr1 locus. 4 Molecular mechanisms underlying these effects are unknown. Available evidence suggests the existence of a relatively stable genome in neoplastic lesions of resistant rats, as shown by the absence of c-myc amplification in the lesions of a resistant Wistar rat strain, which instead is present in susceptible Fisher 344 (F344) rats. 5,6 Moreover, allelic imbalance occurs, at several chromosomes, in HCCs of susceptible (F344 x Wistar Furth)F1 rats, 7 but not in those of resistant BFF1 rats. 8 HCCs induced in LFF1 rats, generated by crossing the susceptible F344 and Long-Evans strains, show allelic imbalance at Hcs1, Hcr1, and Hcr6. 4 c-myc is located at Hcs1 in a segment syntenic to human chromosomal regions in which frequent allelic gain occurs. 9 These observations suggest that mechanisms controlling cell growth are differently affected in neoplastic liver lesions of susceptible and resistant rats.Overexpression of c-myc in c-myc-and c-myc/Tgf-␣-transgenic mice is associated with deregulation of the pRb-E2F pathway. 10 Interaction of c-Myc with various cell cycle components occurs in in vitro growing cells. 11,12 Enzymes controlling cell cycle include cyclin-dependent kinases (CDKs), activated by binding to the cyclins. 13 Complexes of CDK4 and CDK6 with D-type cyclins are required for the G1 phase progression. Further progression through G1 requires cyclin E, and passage through