The ISGLS definition of PHLF was associated with OS and RFS in HCC patients, and long-term survival will be improved by reducing the incidence of PHLF.

The N-terminal 198 residues of NS3 (NS3-N) of Hepatitis C virus (HCV) subtype 1b obtained from 29 patients, as well as full-length NS3 (NS3-Full), were analysed for their subcellular localization, interaction with the tumour suppressor p53 and serine protease activity in the presence and absence of the viral cofactor NS4A. Based on the subcellular-localization patterns in the absence of NS4A, NS3-N sequences were classified into three groups, with each group exhibiting either dot-like, diffuse or a mixed type of localization. Chimeric NS3-Full sequences, each consisting of an individual NS3-N and a shared C-terminal sequence, showed the same localization patterns as those of the respective NS3-N. Site-directed mutagenesis experiments revealed that a single or a few amino acid substitutions at a particular position(s) of NS3-N altered the localization pattern. Interestingly, NS3 of the dot-like type, either NS3-N or NS3-Full, interacted with p53 more strongly than that of the diffuse type, in both the presence and the absence of NS4A. Moreover, NS3-N of the dot-like type suppressed trans-activating activity of p53 more strongly than that of the diffuse type. Serine protease activity did not differ significantly between the two types of NS3. In HCV RNA replicon-harbouring cells, physical interaction between NS3 and p53 was observed consistently and p53-mediated transcriptional activation was suppressed significantly compared with HCV RNA-negative control cells. Our results collectively suggest the possibility that NS3 plays an important role in the hepatocarcinogenesis of HCV by interacting differentially with p53 in an NS3 sequence-dependent manner. INTRODUCTIONChronic, persistent infection with Hepatitis C virus (HCV) often leads to liver cirrhosis and hepatocellular carcinoma (HCC) (Saito et al., 1990). However, the exact mechanisms of HCV-associated pathogenesis and carcinogenesis are largely unknown.HCV possesses a single-stranded, positive-sense RNA genome of 9?6 kb, which encodes a polyprotein of approximately 3000 aa. The polyprotein is processed into at least 10 structural and non-structural (NS) viral proteins by cellular and viral proteases (Reed & Rice, 2000). One of the viral proteases, the NS3 serine protease, has become a research focus, as it is indispensable for virus replication and, therefore, would be a good target for antiviral drugs. The serine protease is encoded in the N-terminal portion of NS3 and is responsible for cleavage at the NS3/4A, NS4A/4B, NS4B/5A and NS5A/5B junctions. NS4A, a cofactor for NS3, stabilizes it to augment its serine protease activity, being virtually essential for complete cleavage of the HCV polyprotein (Reed & Rice, 2000). The C-terminal portion of NS3 possesses the NTPase/helicase activity (Kim et al., 1995), which is essential for viral RNA replication.In addition to its key role in the life cycle of HCV, possible involvement of NS3 in viral persistence and hepatocarcinogenesis has been studied. For example, NS3 was reported to transform NIH3T3 (Sakamuro et al.,...

Purpose: To show whether single nucleotide polymorphisms (SNP) of drug metabolic genes were associated with toxicity of 2′,2′-difluoro 2′-deoxycytidine (gemcitabine)-based chemoradiotherapy and overall survival (OS) of patients with pancreatic cancer.Experimental Design: We evaluated 17 SNPs of the CDA, dCK, DCTD, RRM1, hCNT1, hCNT2, hCNT3, and hENT1 genes in 154 patients with potentially resectable pancreatic adenocarcinoma who were enrolled in clinical trials at The University of Texas M.D. Anderson Cancer Center (Houston, TX) from February 1999 to January 2006, with follow-up until April 2009. Patients received neoadjuvant concurrent gemcitabine and radiation therapy with or without gemcitabine-cisplatin induction therapy. The association of genotypes with toxicity or OS was tested, respectively, by logistic regression and Cox regression analysis.Results: None of the 17 SNPs, individually, had a significant association with OS. A combined genotype effect of CDA A-76C, dCK C-1205T, DCTD T-47C, hCNT3 C-69T, hENT1 T-549C, and hENT1 C913T on OS was observed. Patients carrying 0 to 1 (n = 43), 2 to 3 (n = 77), or 4 to 6 (n = 30) variant alleles had median survival time of 31.5, 21.4, and 17.5 months, respectively. The hazard ratio of dying was 1.71 (95% confidence interval, 1.06-2.76) and 3.16 (95% confidence interval, 1.77-5.63) for patients carrying two to three or four to six at-risk genotypes (P = 0.028 and P < 0.001), respectively, after adjusting for clinical predictors. CDA C111T, dCK C-1205T, dCK A9846G, and hCNT3 A25G, individually and jointly, had a significant association with neutropenia toxicity.Conclusions: These observations suggest that polymorphic variations of drug metabolic genes were associated with toxicity of gemcitabine-based therapy and OS of patients with resectable pancreatic cancer. Clin Cancer Res; 16(1); 320-9. ©2010 AACR.2′,2′-Difluoro 2′-deoxycytidine (gemcitabine) is the standard first-line agent for treatment of pancreatic cancer. However, 75% of patients do not benefit from this therapy (1), and other than stage, it is not clear what factors predict clinical response to gemcitabine. A major dose-limiting side effect of gemcitabine is hematologic toxicity such as neutropenia and thrombocytopenia, which often result in dose reduction or longer intervals between gemcitabine administrations. However, there is no available biomarker that predicts the toxicity of gemcitabine.Gemcitabine is a nucleoside analogue and a prodrug that requires cellular uptake and intracellular phosphorylation ( Fig. 1; ref. 2). Five of the nucleotide transporters found in humans-human concentrative nucleotide transporter (hCNT) 1 to 3 (a.k.a. solute carrier family 28 A1-A3) and human equilibrative nucleotide transporter (hENT) 1 and 2 (solute carrier family 29)-seem to be responsible for cellular uptake of gemcitabine (2). Once inside the cell, gemcitabine is phosphorylated by deoxycytidine kinase (dCK) to its monophosphate form. This first stage of phosphorylation is the rate-limiting step for furth...

BACKGROUND: It has not been well established whether genetic variations can be biomarkers for clinical outcome of gemcitabine therapy. The purpose of this study was to identify single nucleotide polymorphisms (SNPs) of gemcitabine metabolic and transporter genes that are associated with toxicity and efficacy of gemcitabine-based therapy in patients with locally advanced pancreatic cancer. METHODS: The authors evaluated 17 SNPs of the CDA,dCK, DCTD, RRM1, hCNT1-3, and hENT1 genes in 149 patients with locally advanced pancreatic cancer who underwent gemcitabine-based chemoradiotherapy. The association of genotypes with neutropenia, tumor response to therapy, overall survival, and progression-free survival (PFS) was analyzed by logistic regression, log-rank test, Kaplan-Meier plot, and Cox proportional hazards regression. RESULTS: The CDA A-76C, dCK C-1205T, RRM1 A33G, and hENT1 C913T genotypes were significantly associated with grade 3 to 4 neutropenia (P ¼ .020, .015, .003, and .017, respectively).The CDA A-76C and hENT1 A-201G genotypes were significantly associated with tumor response to therapy (P ¼ .017 and P ¼ .019). A combined genotype effect of CDA A-76C, RRM1 A33G, RRM1 C-27A, and hENT1 A-201G on PFS was observed. Patients carrying 0 to 1 (n ¼ 64), 2 (n ¼ 50), or 3 to 4 (n ¼ 17) at-risk genotypes had median PFS times of 8.3, 6.0, and 4.2 months, respectively (P ¼ .002). CONCLUSIONS: The results indicated that some polymorphic variations of drug metabolic and transporter genes may be potential biomarkers for clinical outcome of gemcitabine-based therapy in patients with locally advanced pancreatic cancer. Cancer 2010;116:5325-35.

RNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA), an RNA duplex 21 to 23 nucleotides (nt) long, or short hairpin RNA (shRNA) resembling siRNA, mediates degradation of the target RNA molecule in a sequence‐specific manner. RNAi is now expected to be a useful therapeutic strategy for hepatitis C virus (HCV) infection. In the present study we compared the efficacy of a number of shRNAs directed against different target regions of the HCV genome, such as 5′‐untranslated region (5′UTR) (nt 286 to 304), Core (nt 371 to 389), NS3–1 (nt 2052 to 2060), NS3–2 (nt 2104 to 2122), and NS5B (nt 7326 to 7344), all of which except for NS5B are conserved among most, if not all, HCV subtype 1b (HCV‐1b) isolates in Japan. We utilized two methods to express shRNAs, one utilizing an expression plasmid (pAVU6+27) and the other utilizing a recombinant lentivirus harboring the pAVU6+27‐derived expression cassette. Although 5′UTR has been considered to be the most suitable region for therapeutic siRNA and/or shRNA because of its extremely high degree of sequence conservation, we observed only a faint suppression of an HCV subgenomic replicon by shRNA against 5′UTR. In both plasmid‐ and lentivirus‐mediated expression systems, shRNAs against NS3–1 and NS5B suppressed most efficiently the replication of the HCV replicon without suppressing host cellular gene expression. Synthetic siRNA against NS3–1 also inhibited replication of the HCV replicon in a dose‐dependent manner. Taken together, the present results imply the possibility that the recombinant lentivirus expressing shRNA against NS3–1 would be a useful tool to inhibit HCV‐1b infection.