Quantification of hepatitis C virus in patients treated with peginterferon-alfa 2a plus ribavirin treatment by COBAS TaqMan HCV test.

Extremely low levels of serum hepatitis C virus (HCV) RNA can be detected by COBAS TaqMan HCV test. To investigate whether the COBAS TaqMan HCV test is useful for measuring rapid virological response (RVR) and early virological response (EVR) to predict sustained virological response (SVR), we compared the virological response to PEG-IFN-alfa 2a plus RBV in 76 patients infected with HCV genotype 1 when undetectable HCV RNA by the COBAS TaqMan HCV test was used, with those when below 1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test was used, which corresponded to the use of traditional methods. Among the 76 patients, 28 (36.8%) had SVR, 13 (17.1%) relapsed, 19 (25.0%) did not respond, and 16 (21.0%) discontinued the treatment due to side effects. The positive predictive values for SVR based on undetectable HCV RNA by COBAS TaqMan HCV test at 24 weeks after the end of treatment [10/10 (100%) at week 4, 21/23 (91.3%) at week 8 and 26/33 (78.7%) at week 12] were superior to those based on <1.7 log IU/mL HCV RNA [17/19 (89.4%) at week 4, 27/38 (71.0%) at week 8, and 27/43 (62.7%) at week 12]. The negative predictive values for SVR based on <1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test [46/57 (80.7%) at week 4, 37/38 (97.3%) at week 8, and 32/33 (96.9%) at week 12] were superior to those based on undetectable HCV RNA [48/66 (72.7%) at week 4, 46/53 (86.7%) at week 8, and 41/43 (95.3%) at week 12]. The utilization of both undetectable RNA and <1.7 log IU/mL HCV RNA by COBAS TaqMan HCV test is useful and could predict SVR and non-SVR patients with greater accuracy.

The PX domains of p47phox and p40phox bind to lipid products of PI(3)K.

PX domains are found in a variety of proteins that associate with cell membranes, but their molecular function has remained obscure. We show here that the PX domains in p47phox and p40phox subunits of the phagocyte NADPH oxidase bind to phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P(2)) and phosphatidylinositol-3-phosphate (PtdIns(3)P), respectively. We also show that an Arg-to-Gln mutation in the PX domain of p47phox, which is found in patients with chronic granulomatous disease, eliminates phosphoinositide binding, as does the analogous mutation in the PX domain of p40phox. The PX domain of p40phox localizes specifically to PtdIns(3)P-enriched early endosomes, and this localization is disrupted by inhibition of phosphoinositide-3-OH kinase (PI(3)K) or by the Arg-to-Gln point mutation. These findings provide a molecular foundation to understand the role of PI(3)K in regulating neutrophil function and inflammation, and to identify PX domains as specific phosphoinositide-binding modules involved in signal transduction events in eukaryotic cells.

Influence of anti-HBc seropositivity on the risk of hepatocellular carcinoma in HCV-infected patients after adjusting for confounding factors.

It is controversial whether past hepatitis B virus infection constitutes an additional risk of hepatocellular carcinoma (HCC) among patients with hepatitis C virus (HCV). The incidence of HCC between 1994 and 2004 was analysed among 1262 patients who were only positive for HCV. The cumulative incidence of HCC was assessed by Kaplan-Meier analysis and the difference between two groups was assessed by the log-rank test. The effect of anti-HBc positivity on the risk of HCC was assessed with multivariate Cox proportional analysis. Anti-HBc was positive in 522 (41.4%) patients. The proportion of male patients (56.7 vs 46.8%, P < 0.001) and mean age (60.8 vs 56.9 years, P < 0.001) were significantly higher in the anti-HBc positive group. HCC developed in 339 patients (mean follow-up 7.0 years), with cumulative incidence rates at 3, 5 and 10 years of 12.7, 24.5 and 41.9% in the anti-HBc positive group and 10.6, 17.7 and 33.4% in the negative group, respectively (P = 0.005). However, anti-HBc seropositivity did not reach statistical significance in multivariate analysis including age and gender (hazard ratio, 1.06; 95% CI, 0.85-1.31; P = 0.63). Anti-HBc positivity and HCC incidence were confounded by male gender and older age.

Visceral fat accumulation is an independent risk factor for hepatocellular carcinoma recurrence after curative treatment in patients with suspected NASH.

BACKGROUND AND AIMS: Visceral fat accumulation reportedly increases the risk of hepatocellular carcinoma (HCC) development in patients with chronic liver disease. However, it has not been fully elucidated whether visceral fat accumulation increases the risk of HCC recurrence after curative treatment in patients with suspected non-alcoholic steatohepatitis (NASH). Therefore this was investigated in the current study. METHODS: 62 patients with naive HCC with suspected NASH were enrolled. All were curatively treated with percutaneous radiofrequency ablation between 1999 and 2006. The visceral fat area (VFA) was determined in each patient from CT images, taken at the time of HCC diagnosis. Patients were divided into two groups based on VFA: the high VFA group (>130 cm(2) in males, >90 cm(2) in females, n = 27) and the others (n = 35). The effects of VFA on HCC recurrence were analysed together with other factors including patients' background, tumour-related factors and liver function-related factors. RESULTS: The cumulative recurrence rates differed significantly between the two groups; 15.9, 56.5 and 75.1% at 1, 2 and 3 years, respectively, in the high VFA group, and 9.7, 31.1 and 43.1%, respectively, in the controls (p = 0.018). Multivariate analysis indicated visceral fat accumulation (risk ratio 1.08, per 10 cm(2), p = 0.046) and older age (risk ratio 1.06 per 1 year, p = 0.04) as independent risk factors of HCC recurrence. CONCLUSIONS: Visceral fat accumulation is an independent risk factor of HCC recurrence after curative treatment in patients with suspected NASH.

Absence of PIK3CA hotspot mutations in hepatocellular carcinoma in Japanese patients.

A recent study revealed that the p110alpha (PIK3CA), catalytic subunit of phosphatidylinositol 3-kinase (PI3K), is somatically mutated in many types of cancer. For example, PIK3CA is mutated in an estimated 35.6% of hepatocellular carcinoma (HCC) cases. To measure the frequency of PIK3CA hotspot mutations in Japanese HCC patients, exons 9 and 20 of the PIK3CA gene were sequenced in 47 clinical HCC samples. Contrary to expectations, no hotspot mutations were found any of the HCC samples. In addition, we found abnormally migrating waves near the end of exon 9 in the PCR chromatograms from 13 of the 47 samples. PCR amplification and subsequent cloning and sequencing revealed that these chromatograms contained two distinct sequences, the wild-type p110alpha sequence and a different sequence found on human chromosome 22q11.2, the Cat Eye Syndrome region, which contains a putative pseudogene of PIK3CA. These abnormally migrating waves were also found in noncancerous liver tissue, indicating that this was not a result of HCC-associated mutations. Therefore, it is likely that the percentage of hotspot mutations in the PIK3CA gene of Japanese HCC patients is lower than was previously reported.

The hepatitis B virus X protein enhances AP-1 activation through interaction with Jab1.

Hepatitis B virus X protein (HBx) has many cellular functions and is a major factor in hepatitis and hepatocellular carcinoma caused by HBV infection. A proteomic approach was used to search for HBx-interacting proteins in order to elucidate the molecular mechanism of hepatocarcinogenesis. HBx was attached to myc and flag tags (MEF tags) and expressed in 293T cells; the protein complex formed within the cells was purified and characterized by mass spectrometry. COP9 signalosome (CSN) subunits 3 and 4 were subsequently identified as HBx-interacting proteins. In addition, CSN subunit 5, Jun activation domain-binding protein 1 (Jab1), was shown to be a novel cellular target of HBx. In vivo and in vitro interactions between HBx and Jab1 were confirmed by standard immunoprecipitation and GST pull-down assays. An analysis of HBx deletion constructs showed that amino acids 30-125 of HBx were responsible for binding to Jab1. Confocal laser microscopy demonstrated that HBx was mainly localized in the cytoplasm, while Jab1 was found mainly in the nucleus and partially in the cytoplasm, and that the two proteins colocalized in the cytoplasm. The cotransfection of HBx and Jab1 resulted in substantial activator protein 1 (AP-1) activation and knockdown of endogenous Jab1 attenuated AP-1 activation caused by HBx. In addition, the coexpression of HBx and Jab1 potentiated phosphorylation of JNK, leading to the subsequent phosphorylation of c-Jun, whereas the level of c-Jun and JNK phosphorylation induced by HBx was decreased in Jab1 knockdown cells. These results suggest that the interaction between HBx and Jab1 enhances HBx-mediated AP-1 activation.

A transforming growth factor beta type II receptor gene mutation common in sporadic cecum cancer with microsatellite instability.

Mismatch repair genes are the responsible genes for hereditary non-polyposis colon cancer, and mutation of these genes causes replication error (RER). In several RER-positive colon cancer cell lines, mutations of repetitive sequences of transforming growth factor beta (TGF-beta) type II receptor (RII) gene have been reported. Since TGF-beta inhibits cell proliferation, loss of response to TGF-beta is an important tumor progression step. In this study, the relationship between RER status and mutation of the RII gene was analyzed in 112 cases of various types of sporadic gastrointestinal and hepatobiliary cancer (41 with gastric, 49 with colorectal, 5 with gallbladder, and 17 with hepatic cancers). RER was found in 17 cases (4 with gastric, 12 with colorectal, and 1 with gallbladder cancer), and 10 of those (3 with gastric and 7 with colorectal cancer) showed mutations of the RII gene. Of interest was that in all seven cases with colorectal cancer, tumors were located at the cecum. These data indicate that mutation of the RII gene, presumably caused by abnormality of repair gene, play an important role in carcinogenesis of sporadic gastrointestinal cancer, especially at the cecum.

In vivo gene therapy for alpha-fetoprotein-producing hepatocellular carcinoma by adenovirus-mediated transfer of cytosine deaminase gene.

The alpha-fetoprotein (AFP) gene is normally expressed in fetal liver and is transcriptionally silent in adult liver but overexpressed in human hepatocellular carcinoma (HCC). Here, we demonstrate that replication defective recombinant adenoviral vectors, containing the human AFP promoter/enhancer, can be used to express the Escherichia coli cytosine deaminase (CD) gene (AdAFPCD) and the beta-galactosidase gene (AdAF-PlacZ) in AFP-producing HCC cell lines. Expression of the CD gene by adenovirus from the AFP promoter/enhancer (AdAFPCD) induced cells sensitive to 5-fluorocytosine (5FC) in the AFP-producing cells but not in the AFP-nonproducing cells. Transduction by an adenoviral vector harboring an ubiquitous strong promoter and CD gene showed enzymatic activity and 5FC killing in all cell lines. When AdAFPlacZ was injected into the s.c. established hepatoma in vivo, expression of the beta-galactosidase gene was confined to AFP-producing HCC xenografts. Moreover, HCC xenografts regressed by transduction with AdAFPCD and subsequently with 5FC treatment in vivo. These findings suggest that utilization of the AFP promoter/enhancer in an adenoviral vector can confer selective expression of a heterologous suicide gene in hepatocellular carcinoma cells in vitro and in vivo.

In vivo selective gene expression and therapy mediated by adenoviral vectors for human carcinoembryonic antigen-producing gastric carcinoma.

Previously, we reported that adenoviral vectors carrying the carcinoembryonic antigen (CEA) promoter sequences to direct the Echerichia coli beta-galactosidase gene (AdCEA-lacZ) or cytosine deaminase (CD) gene (AdCEA-CD) confer selective gene expression on a CEA-positive gastric cancer cell line (MKN45) in vitro. Here, adenovirus-mediated tumor-specific gene therapy for CEA-positive gastric carcinoma in vivo was investigated. Using an animal model with i.p. disseminated MKN45 tumors, adenovirus-mediated tumor-specific transgene expression and therapeutic efficacy were analyzed. After an i.p. injection of AdCEA-lacZ, beta-galactosidase activity was confined to tumor xenografts. Moreover, CD mRNA was expressed exclusively in MKN45 tumor xenografts after infection with AdCEA-CD, despite the fact that an adenovirus-mediated transfer of CD DNA was detected in all tissues tested. In contrast, CD mRNA was detected not only in tumor xenografts but also in other organs of mice infected with AdCA-CD, in which CD gene expression is governed by an ubiquitous promoter. Suppression of tumor growth and prolongation of survival were noted in tumor-bearing mice treated with AdCEA-CD and 5-fluorocytosine (5FC) without observable adverse effects. In contrast, significant hepatic toxicity was noted in animals treated with AdCA-CD. These results reveal that the CEA promoter restricts CD gene expression to CEA-positive tumor cells in the adenoviral context in vivo, along with the beneficial therapeutic effects of 5FC treatment, suggesting the i.p. AdCEA-CD/5FC system may provide a novel approach to treatment of i.p. disseminated gastric cancer.

Adenovirus-mediated transduction of Escherichia coli uracil phosphoribosyltransferase gene sensitizes cancer cells to low concentrations of 5-fluorouracil.

5-fluorouracil (5-FU), although a widely used chemotherapeutic agent, has a limited effect in the treatment of human solid tumors due to their resistance to the cytotoxic effects of 5-FU. Escherichia coli uracil phosphoribosyltransferase (UPRT) is a pyrimidine salvage enzyme that catalyzes the synthesis of UMP from uracil and 5-phosphoribosyl-alpha-1-diphosphate. The present study demonstrates that adenovirus-mediated transduction of E. coli UPRT gene results in marked sensitization of colon, gastric, liver, and pancreas cancer cell lines to low concentration of 5-FU in vitro. The in vitro bystander effect was observed when only 10% of the hepatoma Hep3B cells were infected with UPRT-expressing adenovirus. In addition, 5-FU treatment of human hepatoma or gastric cancer xenografts in nude mice transduced with UPRT was demonstrated to result in significant in vivo antitumor effects. The adenovirus vector transduction of the UPRT gene followed by 5-FU administration is representative of a new chemosensitization strategy for cancer gene therapy.

Adenovirus-mediated prodrug gene therapy for carcinoembryonic antigen-producing human gastric carcinoma cells in vitro.

We analyzed the ability of a recombinant replication-defective adenovirus vector with the carcinoembryonic antigen (CEA) promotor to transfer the thymidine kinase gene of herpes simplex virus (HSVtk) into gastric cancer cells to confer sensitivity to ganciclovir (GCV). CEA-producing gastric cancer cell lines (MKN28 and MKN45), a CEA-nonproducing gastric cancer cell line (MKN1), and a human uterine cervical cancer cell line (HeLa) were infected with a recombinant adenovirus carrying lacZ reporter gene coupled to the CEA promoter (AdCEAlacZ). The efficiency of AdCEAlacZ-mediated gene transfer was correlated with the amount of CEA produced by each cell line. Furthermore, the 50% growth inhibitory concentrations (IC50) of GCV were 21 and 5.8 microm for MKN28 and MKN45, respectively, when infected with a recombinant adenovirus carrying the HSVtk gene coupled to the CEA promoter (AdCEAtk). However, MKN1 and HeLa cells infected with AdCEAtk remained resistant to GCV (IC50 > 300 microm of GCV). In addition, a bystander killing effect was demonstrated against MKN45 cells when only 20% of AdCEAtk-infected cells were mixed with uninfected cells. These data indicate the potential for targeted gene therapy using the cell type-specific promotor of the CEA gene against gastric cancers that produce CEA.

Site-directed mutagenesis studies on the iron-binding domain and the determinant for the substrate oxygenation site of porcine leukocyte arachidonate 12-lipoxygenase.

cDNA for arachidonate 12-lipoxygenase of porcine leukocytes was expressed in Escherichia coli. The recombinant 12-lipoxygenase was purified by immunoaffinity chromatography to near homogeneity with a specific activity of about 1.5 mumol/min per mg protein. Each of eight histidine residues, which were well-conserved among various mammalian lipoxygenases and presumed as ligands for non-heme iron, was substituted with leucine by site-directed mutagenesis. Each mutant enzyme was immunoaffinity-purified to near homogeneity. Mutations of His-361, -366 and -541 caused a total loss of enzyme activity, and the iron content was much lower (0.10, 0.06 and 0.06 g atom/mol protein) than that of the wild-type enzyme (0.53). Mutations of His-128 and -356 gave 159% and 162% specific activity of the wild-type enzyme, and the iron contents were 0.55 and 0.52 g atom/mol protein. Substitution of His-426 decreased the activity to 5%, but the iron content was 0.4 g atom/mol protein. The expression level of mutants at His-384 and -393 was too low to precisely determine the iron content. Taken together, His-361, -366 and -541 may play important roles for iron-binding in catalytically active 12-lipoxygenase. Since a high homology of amino acid sequence was known between porcine leukocyte 12-lipoxygenase and mammalian 15-lipoxygenases, we attempted to convert the 12-lipoxygenase to a 15-lipoxygenase. A double mutation of Val-418 and -419 to Ile and Met increased the ratio of 15- and 12-lipoxygenase activities from 0.1 to 5.7.

Frequency of mutations of insulin receptor gene in Japanese patients with NIDDM.

To examine the prevalence of abnormalities in the insulin receptor structure gene in Japanese with non-insulin-dependent diabetes mellitus (NIDDM), a population of 51 patients with NIDDM was screened for mutations in this gene. Patient genomic DNAs of both alleles corresponding to 22 exons of the gene were amplified by polymerase chain reaction (PCR). The PCR products on pUC19 were sequenced. Three patients with heterozygous missense mutation Thr831-->Ala831 in exon 13 and one patient with heterozygous missense mutation Tyr1334-->Cys1334 in exon 22 of the beta-subunits were identified. Linkage analysis of one of the families plus statistical studies showed that the mutation Thr831-->Ala831 is possibly responsible for the onset of NIDDM. In COS cells transiently expressing both mutant receptor cDNAs and a cDNA of a M(r) 85,000 regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), the mutation Tyr1334-->Cys1334 impaired binding of the receptor with the M(r) 85,000 subunit of PI 3-kinase, but linkage analysis of the family showed that the mutation did not cosegregate with NIDDM in the pedigree. Therefore, one missense mutation (Thr831-->Ala831) in the insulin receptor, as found in three patients, is possibly involved in the etiology of a subset of the 51 NIDDM patients.

Transcriptional targeted gene therapy for hepatocellular carcinoma by adenovirus vector.

Hepatocellular carcinoma (HCC) is one of the most common malignancies with poor prognosis and is highly amenable to the development of novel therapeutic strategy. The human alpha-fetoprotein (AFP) gene is normally expressed in fetal liver and is transcriptionally silent in adult liver but overexpressed in HCC. In order to destroy AFP-producing HCC specifically, replication defective adenoviral vectors containing the transcriptional control elements of the AFP gene were designed. Expression of suicide genes by the AFP promoter/enhancer induced prodrug sensitivity in AFP (+) cells but not AFP (-) cells. The expression of suicide genes by ubiquitous promoter, however, showed no selectivity after prodrug treatment. Adenoviral vector transduced genes efficiently not only in vitro but also in vivo, and AFP-producing HCC xenografts regressed by transduction with transcriptionally targeted vectors and subsequent systemic administration of prodrug in animal model. Utilization of the transcriptional regulatory element to drive drug sensitive genes can be a promising strategy for cancer specific therapy.

Adenovirus-mediated drug-sensitivity gene therapy for hepatocellular carcinoma.

Selective gene therapy represents a potent approach in cancer treatment that utilizes a cell's own nontoxic suicide genes. Currently, the suicide genes under investigation mediate sensitivity by encoding viral or bacterial enzymes that convert inactive prodrug into toxic antimetabolites that inhibit nucleic acid synthesis (1,2).

Vanoxonin, a new inhibitor of thymidylate synthetase.

Vanoxonin, a new inhibitor of thymidylate synthetase, was found in cultured broths of the strain MG245-CF2 classified as Saccharopolyspora hirsuta. Vanoxonin, C18H25N3O9, was obtained as colorless powder. Vanoxonin forms a vanadium complex which exhibits a strong inhibition against thymidylate synthetase. The concentration for 50% inhibition of the enzyme (IC50) was 0.7 micrograms/ml.

Vanoxonin, a new inhibitor of thymidylate synthetase. II. Structure determination and total synthesis.

Acid hydrolysis of vanoxonin yielded one mol each of 2,3-dihydroxybenzoic acid, L-threonine, L-N omega-hydroxyornithine. Presence of acetyl group in vanoxonin was suggested by the 1H NMR. Periodate oxidation of vanoxonin liberated one mol of acetic acid suggesting that the acetyl group bound to the omega-nitrogen of N omega-hydroxyornithine. The sequence of three components was determined to be L-N-(2,3-dihydroxybenzoyl)threonyl-L-(N omega-acetyl-N omega-hydroxy)ornithine by mass spectrometric analysis. This structure was confirmed by the total synthesis of vanoxonin.

Vanoxonin, a new inhibitor of thymidylate synthetase. III. Inhibition of thymidylate synthetase by vanoxonin-vanadium complex.

Quinquevalent vanadium complex with two mol of vanoxonin ligated by the two catechols was shown to be the active structure for inhibition of thymidylate synthetase. The catechol group of vanoxonin as the essential moiety for the inhibition of enzyme was further confirmed by studies of structure-activity relationships using the enzyme obtained from Ehrlich ascites carcinoma cells of mice. Vanoxonin-vanadium complex showed competitive inhibition with respect to deoxyuridylic acid but uncompetitive to 5,10-methylenetetrahydrofolate.

Anticoagulation during use of a left ventricular assist device.

Thirty-six mongrel dogs underwent 24hr left ventricular assist. The VAD was placed between the left atrium and the descending aorta, and the dogs were divided into four groups according to type of anticoagulation: no anticoagulation, argatroban, nafamostat mesylate, and nafamostat mesylate + prostacyclin analog. Results of this animal experiment revealed that a newly developed synthetic thrombin inhibitor argatroban can prevent activation of the intrinsic coagulation pathway. Argatroban is efficient under any blood coagulative condition, even lack of anti-thrombin III, because of its direct inhibitory effect on thrombin, making argatroban more useful than heparin as an anticoagulant for LVAD. Argatroban, as well as heparin, provides marked and significant prolongation of the prothrombin time from early assisted circulation, but produces a bleeding tendency. Nafamostat mesylate can maintain blood coagulation parameters within the acceptable range. Combined administration of nafamostat mesylate and a prostacyclin analog cause the least decrease in fibrinogen and alpha2-plasmin inhibitor among the four groups and causes no significant prolongation of prothrombin time.

A thromboxane A2 synthetase inhibitor as an anticoagulant for left ventricular assist devices.

A comparative study to establish more adequate anticoagulation therapy for left ventricular assist devices was done by administering various anticoagulants: heparin, argatroban (a pure thrombin inhibitor), a thromboxane A2 synthetase inhibitor, and protease inhibitor. Results of the investigation revealed that use of no anticoagulation activates the intrinsic pathway of blood coagulation and causes severe coagulopathy, heparin or argatroban causes bleeding tendency, and a thromboxane A2 synthetase inhibitor can maintain blood coagulation within the acceptable range, but not completely. Combined administration of a thromboxane A2 synthetase inhibitor and protease inhibitor was found to be the best anticoagulation therapy in this study.