The novel HBx mutation F30V correlates with hepatocellular carcinoma in vivo, reduces hepatitis B virus replicative efficiency and enhances anti-apoptotic activity of HBx N terminus in vitro.

OBJECTIVE: We aimed to investigate HBx genetic elements correlated with hepatitis B virus (HBV) -related hepatocellular carcinoma (HCC) and their impact on (a) HBV replicative efficiency, (b) HBx binding to circular covalently closed DNA (cccDNA), (c) apoptosis and cell-cycle progression, and (d) HBx structural stability. METHODS: This study included 123 individuals chronically infected with HBV: 27 with HCC (77.9% (21/27) genotype D; 22.1% (6/27) genotype A) and 96 without HCC (75% (72/96) genotype D; 25.0% (24/96) genotype A). HepG2 cells were transfected by wild-type or mutated linear HBV genome to assess pre-genomic RNA (pgRNA) and core-associated HBV-DNA levels, HBx-binding onto cccDNA by chromatin immunoprecipitation-based quantitative assay, and rate of apoptosis and cell-cycle progression by cytofluorimetry. RESULTS: F30V was the only HBx mutation correlated with HCC (18.5% (5/27) in HCC patients versus 1.0% (1/96) in non-HCC patients, p 0.002); a result confirmed by multivariate analysis. In vitro, F30V determined a 40% and 60% reduction in pgRNA and core-associated HBV-DNA compared with wild-type (p <0.05), in parallel with a significant decrease of HBx binding to cccDNA and decreased HBx stability. F30V also decreased the percentage of apoptotic cells compared with wild-type (14.8 ± 6.8% versus 19.1 ± 10.1%, p <0.01, without affecting cell-cycle progression) and increased the probability of HBx-Ser-31 being phosphorylated by PI3K-Akt kinase (known to promote anti-apoptotic activity). CONCLUSIONS: F30V was closely correlated with HBV-induced HCC in vivo, reduced HBV replicative efficiency by affecting HBx-binding to cccDNA and increased anti-apoptotic HBx activity in vitro. This suggests that F30V (although hampering HBV's replicative capacity) may promote hepatocyte survival, so potentially allowing persistent production of viral progeny and initiating HBV-driven hepatocarcinogenesis. Investigation of viral genetic markers associated with HCC is crucial to identify those patients at higher risk of HCC, who hence deserve intensive liver monitoring and/or early anti-HBV therapy.

Reciprocal antagonism between the netrin-1 receptor uncoordinated-phenotype-5A (UNC5A) and the hepatitis C virus.

Hepatitis C virus (HCV) infection is a leading cause of hepatocellular carcinoma (HCC), mainly through cirrhosis induction, spurring research for a deeper understanding of HCV versus host interactions in cirrhosis. The present study investigated crosstalks between HCV infection and UNC5A, a netrin-1 dependence receptor that is inactivated in cancer. UNC5A and HCV parameters were monitored in patients samples (n=550) as well as in in vitro. In patients, UNC5A mRNA expression is significantly decreased in clinical HCV(+) specimens irrespective of the viral genotype, but not in (HBV)(+) liver biopsies, as compared to uninfected samples. UNC5A mRNA is downregulated in F2 (3-fold; P=0.009), in F3 (10-fold, P=0.0004) and more dramatically so in F4/cirrhosis (44-fold; P<0.0001) histological stages of HCV(+) hepatic lesions compared to histologically matched HCV(-) tissues. UNC5A transcript was found strongly downregulated in HCC samples (33-fold; P<0.0001) as compared with non-HCC samples. In vivo, association of UNC5A transcripts with polyribosomes is decreased by 50% in HCV(+) livers. Consistent results were obtained in vitro showing HCV-dependent depletion of UNC5A in HCV-infected hepatocyte-like cells and in primary human hepatocytes. Using luciferase reporter constructs, HCV cumulatively decreased UNC5A transcription from the UNC5 promoter and translation in a UNC5A 5'UTR-dependent manner. Proximity ligation assays, kinase assays, as well as knockdown and forced expression experiments identified UNC5A as capable of impeding autophagy and promoting HCV restriction through specific impact on virion infectivity, in a cell death-independent and DAPK-related manner. In conclusion, while the UNC5A dependence receptor counteracts HCV persistence through regulation of autophagy in a DAPK-dependent manner, it is dramatically decreased in all instances in HCC samples, and specifically by HCV in cirrhosis. Such data argue for the evaluation of the implication of UNC5A in liver carcinogenesis.

Who is more likely to respond to dual treatment with pegylated-interferon and ribavirin for chronic hepatitis C? A gender-oriented analysis.

We assessed, in real-life practice, viral, demographic, genetic and metabolic factors influencing the sustained virologic response (SVR), with a gender-oriented analysis, in patients with chronic hepatitis C virus (HCV) treated with pegylated interferon and ribavirin. Six hundred and seventy naïve patients were treated with dual therapy and evaluated by gender and HCV genotype. Associations between baseline variables and SVR were assessed by multivariate logistic regression analysis. Among 362 genotype 1 patients, SVR was achieved in 158 patients (44%), and SVR was independently associated with age less than 50 years (OR 2.12; 95% CI 1.09-4.30; P=0.039) and C/C genotype rs12979860 SNP (OR 2.83; 1.19-6.74; P=0.002) in 163 females, while absence of visceral obesity (OR 2.491; 1.131-5.487; P=0.023), HCV-RNA lower than 400,000 IU/mL (OR 2.66; 1.273-5.558; P=0.009) and C/C genotype rs12979860 SNP (OR 4.969; 2.401-10.283; P<0.001) were independently associated with SVR in 199 males. Combining favourable baseline variables, the probability of obtaining SVR ranged from 27.6% to 84.2% in females, and from 14.3% to 85.7% in males. The rate of SVR was 81.1% in 175 genotype 2 patients, and 69% in 100 genotype 3 patients. Rapid virologic response was the only valid predictor of SVR regardless of other features. In conclusions, in the setting of HCV genotype 1, chronic hepatitis, combining rapid virologic response and predictive factors, which are different for females and males, allows clinicians to single out a group of patients whose likelihood of SVR exceeds 80%. For these patients, triple therapy with first-generation protease inhibitors may be unwarranted.

The sodium/iodide symporter NIS is a transcriptional target of the p53-family members in liver cancer cells.

Thyroid iodide accumulation via the sodium/iodide symporter (NIS; SLC5A5) has been the basis for the longtime use of radio-iodide in the diagnosis and treatment of thyroid cancers. NIS is also expressed, but poorly functional, in some non-thyroid human cancers. In particular, it is much more strongly expressed in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) cell lines than in primary human hepatocytes (PHH). The transcription factors and signaling pathways that control NIS overexpression in these cancers is largely unknown. We identified two putative regulatory clusters of p53-responsive elements (p53REs) in the NIS core promoter, and investigated the regulation of NIS transcription by p53-family members in liver cancer cells. NIS promoter activity and endogenous NIS mRNA expression are stimulated by exogenously expressed p53-family members and significantly reduced by member-specific siRNAs. Chromatin immunoprecipitation analysis shows that the p53-REs clusters in the NIS promoter are differentially occupied by the p53-family members to regulate basal and DNA damage-induced NIS transcription. Doxorubicin strongly induces p53 and p73 binding to the NIS promoter, leading to an increased expression of endogenous NIS mRNA and protein in HCC and CCA cells, but not in PHH. Silencing NIS expression reduced doxorubicin-induced apoptosis in HCC cells, pointing to a possible role of a p53-family-dependent expression of NIS in apoptotic cell death. Altogether, these results indicate that the NIS gene is a direct target of the p53 family and suggests that the modulation of NIS by DNA-damaging agents is potentially exploitable to boost NIS upregulation in vivo.

miRNA let-7c promotes granulocytic differentiation in acute myeloid leukemia.

MicroRNAs (miRNAs), small non-coding RNAs that regulate gene expression post-transcriptionally, are involved in many complex cellular processes. Several miRNAs are differentially expressed in hematopoietic tissues and play important roles in normal differentiation, but, when aberrantly regulated, contribute to the abnormal proliferation and differentiation of leukemic cells. Recently, we reported that a small subset of miRNAs is differentially expressed in acute promyelocytic leukemia (APL) blasts and is modulated by treatment with all-trans-retinoic acid (ATRA). In particular, PML/RARα-positive blasts from APL patients display lower levels of miRNA let-7c, a member of the let-7 family, than normal promyelocytes and its expression increases after ATRA treatment. In this study, we investigated the effects of let-7c in acute myeloid leukemia (AML) cells. We found that ectopic expression of let-7c promotes granulocytic differentiation of AML cell lines and primary blasts. Moreover, we identified PBX2, a well-known homeodomain protein whose aberrant expression enhances HoxA9-dependent leukemogenesis, as a novel let-7c target that may contribute to the AML phenotype. Together, these studies raise the possibility that perturbation of the let-7c-PBX2 pathway may have a therapeutic value in AML.

p53-paralog DNp73 oncogene is repressed by IFNα/STAT2 through the recruitment of the Ezh2 polycomb group transcriptional repressor.

The DNp73 proteins act as trans-repressors of p53 and p73-dependent transcription and exert both anti-apoptotic activity and pro-proliferative activity. DNp73s are frequently up-regulated in a variety of human cancers, including human hepatocellular carcinomas (HCCs). Increased levels of DNp73 proteins confer to HCC cells resistance to apoptosis and, irrespective to p53 status, a chemoresistant phenotype. Here, we show that interferon (IFN)α down-regulates DNp73 expression in primary human hepatocytes (PHHs) and HCC cell lines. IFNα has been used as pro-apoptotic agent in the treatment of malignancies and there is increasing evidence of IFNα effectiveness in HCC treatment and prevention of recurrence. The precise mechanisms by which class I IFNs exert their anti-proliferative and anti-tumor activity remain unclear. IFNα binding to its receptor activates multiple intracellular signaling cascades regulating the transcription of numerous direct target genes through the recruitment of a complex comprising of STAT1, STAT2 and IFN regulatory factor (IRF)9 to their promoters. We found that, in response to IFNα, the P2p73 promoter undergoes substantial chromatin remodeling. Histone deacetylases (HDACs) replace histone acetyl transferases. STAT2 is recruited onto the endogenous P2p73 promoter together with the polycomb group protein Ezh2, leading to increased H3K27 methylation and transcriptional repression. The reduction of DNp73 levels by IFNα is paralleled by an increased susceptibility to IFNα-triggered apoptosis of Huh7 hepatoma cells. Our results show, for the first time, that IFN-stimulated gene factor 3 recruitment may serve both in activating and repressing gene expression and identify the down-regulation of DNp73 as an additional mechanism to counteract the chemoresistance of liver cancer cells.

A survey of chronic hepatitis B patient management practices in the European Union.

The current study sought to evaluate the characteristics of chronic hepatitis B virus (HBV) infection and current management practices in the European Union by surveying physician and patient records. A detailed survey of physician practices and management of patients with CHB was conducted between July and October 2006 in France, Germany, Italy and Spain. A total of 200 physicians participated in the survey, and data were collected from 2023 patients with chronic HBV infection. Most patients were men (69%), who had hepatitis B e antigen (HBeAg)-negative disease (64%), and demonstrated evidence of significant disease [53%; moderate fibrosis (35%), compensated cirrhosis (14%), or decompensated cirrhosis (4%)]. Among the 1665 HBV-monoinfected patients surveyed, 1184 (71%) were currently receiving treatment for chronic HBV infection. At treatment initiation, 70% of HBeAg-positive patients had both pretreatment serum HBV DNA levels or=2 x the upper limit of normal (ULN), and 81% of HBeAg-negative patients had HBV DNA levels of or=2 x ULN, while the HBeAg-negative patients had HBV DNA levels

A restricted signature of miRNAs distinguishes APL blasts from normal promyelocytes.

MicroRNAs (miRNAs) are small non-coding RNAs involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation. A small set of miRNAs is differentially expressed in hematopoietic cells and seemingly has an important role in granulopoiesis and lineage differentiation. In this study, we analysed, using a quantitative real-time PCR approach, the expression of 12 granulocytic differentiation signature miRNAs in a cohort of acute promyelocytic leukemia (APL) patients. We found nine miRNAs overexpressed and three miRNAs (miR-107, -342 and let-7c) downregulated in APL blasts as compared with normal promyelocytes differentiated in vitro from CD34+ progenitors. Patients successfully treated with all-trans-retinoic acid (ATRA) and chemotherapy showed downregulation of miR-181b and upregulation of miR-15b, -16, -107, -223, -342 and let-7c. We further investigated whether the APL-associated oncogene, promyelocytic leukemia gene (PML)/retinoic acid receptor alpha (RARalpha), might be involved in the transcriptional repression of miR-107, -342 and let-7c. We found that PML/RARalpha binds the regulatory sequences of the intragenic miR-342 and let-7c. In addition, we observed, in response to ATRA, the release of PML/RARalpha paralleled by their transcriptional activation, together with their host genes, EVL and C21orf34alpha. In conclusion, we show that a small subset of miRNAs is differentially expressed in APL and modulated by ATRA-based treatment.

Initiation of hepatitis B virus genome replication and production of infectious virus following delivery in HepG2 cells by novel recombinant baculovirus vector.

One of the major problems in gaining further insight into hepatitis B virus (HBV)/host-cell interactions is to improve the existing cellular models for the study of HBV replication. The first objective of this study was to improve the system based on transduction of HepG2 cells with a recombinant baculovirus to study HBV replication. A new HBV recombinant baculovirus, Bac-HBV-1.1, in which the synthesis of pre-genomic RNA is driven by a strong mammalian promoter, was generated. Transduction with this new recombinant baculovirus led to higher levels of HBV replication in HepG2 cells compared with levels obtained with previously described baculovirus vectors. The initiation of a complete HBV DNA replication cycle in Bac-HBV-1.1-transduced HepG2 cells was shown by the presence of HBV replicative intermediates, including covalently closed circular DNA (cccDNA). Only low levels of cccDNA were detected in the nucleus of infected cells. Data showed that cccDNA resulted from the recycling of newly synthesized nucleocapsids and was bound to acetylated histones in a chromatin-like structure. HBV particles released into the supernatant of transduced HepG2 cells were infectious in differentiated HepaRG cells. Several Bac-HBV-1.1 baculoviruses containing HBV strains carrying mutations conferring resistance to lamivudine and/or adefovir were constructed. Phenotypic analysis of these mutants confirmed the results obtained with the transfection procedures. In conclusion, an improved cell-culture system was established for the transduction of replication-competent HBV genomes. This will be useful for future studies of the fitness of HBV mutants.

RelA/NF-kappaB recruitment on the bax gene promoter antagonizes p73-dependent apoptosis in costimulated T cells.

The balance between antiapoptotic and proapoptotic proteins of the Bcl-2 family is critical in determining the fate of T cells in response to death stimuli. Proapoptotic genes, such as bax, are generally regulated by the p53 family of transcription factors, whereas NF-kappaB subunits can activate the transcription of antiapoptotic Bcl-2 members. Here, we show that CD28 activation protects memory T cells from irradiation-induced apoptosis by both upregulating bcl-xL and inhibiting bax gene expression. We found that p73, but not p53, binds to and trans-activates the bax gene promoter in irradiated T cells. The activation of RelA/NF-kappaB subunit in CD28 costimulated T cells and its binding onto the bax gene promoter results in suppression of bax transcription and decrease in both p73 and RNA polymerase II recruitment in vivo. RelA recruitment on the bax gene promoter is also accompanied by the lost of p300 binding and the parallel appearance of histone deacetylase-1-containing complexes. These findings identify RelA/NF-kappaB as a critical regulator of T-cell survival by affecting the balance of Bcl-2 family members.

Bisperoxovanadium, a phospho-tyrosine phosphatase inhibitor, reprograms myogenic cells to acquire a pluripotent, circulating phenotype.

Satellite cells are the main source of myogenic progenitors in postnatal skeletal muscle, but their use in cell therapy for muscle disorders is limited because these cells cannot be delivered through circulation and they are rapidly exhausted in severe myopathies. The search for alternative donor cells is ongoing, but none of the candidates so far show all the features required for successful colonization and repair of diseased muscle. In this study, we show that bisperoxovanadium, a phospho-tyrosine phosphatase inhibitor, induces myogenic cells to acquire a gene expression profile and a differentiation potential consistent with the phenotype of a circulating precursors, while maintaining their myogenic potential. These effects are mediated, at least in part, by NF-kappaB activation through the Tyr42-IkappaB-alpha phosphorylation, as shown by the expression of the dominant negative mutant form of the p50 NF-kappaB subunit. Moreover, when bisperoxovanadium-treated cells are injected into the femoral artery of alpha-sarcoglican null dystrophic mice, they are able to circulate and to reach muscle tissue; importantly, they contribute to muscle regeneration, as shown by the expression of alpha-sarcoglican in some fibers. Our observations indicate that bisperoxovanadium, or similar compounds, may prove very valuable to obtain and to expand, from committed cells, multipotent cell populations suitable for gene-cell therapy applications and may help to understand the molecular basis of genome reprogramming and "stem-ness."

Viral hepatitis and liver cancer: the case of hepatitis C.

Chronic infection with the hepatitis C virus (HCV) is a major risk factor for the development of hepatocellular carcinoma (HCC) worldwide. The pathogenesis of HCC in HCV infection has extensively been analysed. Hepatitis C virus-induced chronic inflammation and the effects of cytokines in the development of fibrosis and liver cell proliferation are considered as one of the major pathogenic mechanisms. Increasing experimental evidence suggests that HCV contributes to HCC by directly modulating pathways that promote the malignant transformation of hepatocytes. Hepatitis C virus is an RNA virus that does not integrate into the host genome but HCV proteins interact with many host-cell factors well beyond their roles in the viral life cycle and are involved in a wide range of activities, including cell signaling, transcription, cell proliferation, apoptosis, membrane rearrangements, vesicular trafficking and translational regulation. At least four of the HCV gene products, namely HCV core, NS3, NS4B and NS5A, have been shown to exhibit transformation potential in tissue culture and several potentially oncogenic pathways have been shown to be altered by the expression of HCV proteins. Both HCV core and NS5A induce the accumulation of wild-type beta-catenin and the Wnt-beta-catenin pathway emerges as a common target for HCV (and HBV) in human HCCs, also independently from axin/beta-catenin gene mutations. Induction of both endoplasmic reticulum stress and oxidative stress by HCV proteins might also contribute to HCV transformation. Most of the putative transforming functions of the HCV proteins have been defined in artificial cellular systems, which may not be applicable to HCV infection in vivo, and still need to be established in relevant infection and disease models.

DNp73alpha protects myogenic cells from apoptosis.

The P73 gene is transcribed from two promoters, P1 and P2, that direct the expression of multiple transactivation competent (TA) and dominant negative (DN) isoforms. TAp73 transcription factors mediate cell cycle arrest and/or apoptosis in response to DNA damage and are involved in developmental processes. P73 mRNA levels increase and the P1p73 promoter is upregulated during myogenic differentiation of C2C12 skeletal muscle satellite cells. The DNp73 proteins act as trans-repressors of p53- and p73-dependent transcription, and possess both antiapoptotic and pro-proliferative potential. Here, we show that DNp73alpha is expressed in proliferating C2C12 myoblasts, rapidly accumulates in differentiating myocytes and remains elevated in C2C12 myotubes. By combining transactivation assays and chromatin immunoprecipitation analysis, we could show that the upregulation of the P2p73 promoter during myogenic differentiation is mediated by the coordinated recruitment and activity of MyoD and p53/p73. Abrogation of DNp73 expression by specific siRNA led to a strong potentiation of the spontaneous apoptosis of C2C12 myoblasts induced to differentiate. Finally, unlike TAp73 that contributes to DNA damage-induced apoptosis of myotubes, endogenous DNp73 mediates the relative resistance of differentiated myotubes to DNA damage. Altogether, our findings identify DNp73alpha as an important target in designing strategies aimed at the potentiation of the regenerative potential of skeletal satellite cells.

Prevalence and genomic variability of transfusion transmitted virus in Italian cryptogenic chronic liver disease and healthy blood donors.

BACKGROUND: Infection with transfusion transmitted virus, a new member of the Parvoviridae family, has been found in patients both with chronic and fulminant post-transfusion cryptogenic hepatitis. AIM: To evaluate the prevalence and clinical impact of transfusion transmitted virus infection in Italy. PATIENTS AND METHODS: Studies were carried out on 256 patients and control subjects from three centres from Northern, Central and Southern Italy (92 nonA-nonC chronic hepatitis, 10 acute non fulminant cryptogenic hepatitis, 41 hepatitis C virus-related chronic hepatitis and 113 blood donors). Serum transfusion transmitted virus was detected by nested polymerase chain reaction using two overlapping sets of primers. RESULTS: A total of 52 of the 92 patients (54.3%) with chronic cryptogenic liver disease and 17 of the 41 hepatitis C virus chronic hepatitis patients (41.4%) were transfusion transmitted virus-DNA positive. Transfusion transmitted virus co-infection in hepatitis C virus patients was not associated with either a higher severity of liver histology or higher alanine transaminase levels or signs of cholestasis, transfusion transmitted virus was found in 48 out of 113 (42.4%) blood donors. In the majority of samples, transfusion transmitted virus DNA was detected with only one of the two sets of primers used. Genotyping and phylogenetic analysis performed on 21 randomly selected viral isolates showed the presence of both type 1 and type 2 transfusion transmitted virus and allowed identification of two isolates with high homology to genotype 6, described, so far, mostly in Japan. CONCLUSIONS: Transfusion transmitted virus type 1 and 2 infection is common among blood donors and patients with liver disease in Italy. The pathogenic potential of transfusion transmitted virus type 1 and 2 in nonA-nonC hepatitis patients is unlikely but further studies are needed to evaluate the epidemiological and clinical impact of other transfusion transmitted virus subtypes.

Immunotherapy with interferon-alpha in patients affected by chronic hepatitis C induces an intercorrelated stimulation of the cytokine network and an increase in depressive and anxiety symptoms.

Immunotherapy with interferon-alpha (IFNalpha) may induce depressive symptoms, anxiety and major depression when administered for at least 1-3 months at a dose of 3-10 MUI daily, twice or three times a week. Previously, it has been shown that immunotherapy with interleukin-2 (IL-2) significantly induces the cytokine network, as measured by increases in serum IL-6, IL-10 and the IL-2 receptor (IL-2R), and that the immunotherapy-induced changes in the cytokine network are significantly correlated with the increases in depression ratings. The main aim of this study was to examine the effects of immunotherapy with IFNalpha on the cytokine network in relation to changes in depression and anxiety ratings. Fourteen patients, affected by chronic active C-hepatitis, were treated with IFNalpha (3-6 MUI s.c. three/six times a week for 6 months) and had measurements of serum IFN-gamma (IFNgamma), IL-2, IL-6, IL-6R, IL-8 and IL-10 before starting therapy and 2, 4, 16 and 24 weeks after immunotherapy with IFNalpha. Severity of depression and anxiety were measured with the Montgomery-Asberg Depression Rating Scale (MADRS) and the Hamilton Anxiety Rating Scale (HAM-A), respectively. Repeated measure (RM) design ANOVAs showed significantly higher MADRS and HAM-A scores 2-4 weeks and 4-6 months after starting IFNalpha-based immunotherapy than at baseline. RM design ANOVAs showed significantly higher serum IL-6 and IL-8 levels 2-4 weeks after starting IFNalpha-based immunotherapy and higher serum IL-10 levels 2-4 weeks and 4-6 months after starting therapy than at baseline. There were significant relationships between the IFNalpha-induced changes in serum IL-6 or IL-8 and the depression and anxiety scores. The findings show that IFNalpha-based immunotherapy induces the cytokine network and that IFNalpha-induced increases in IL-6 predicts the development of depressive symptoms. Depressive symptoms following IFNalpha treatment may be secondary to cytokine induction, including that of IL-6.

Hepatitis C virus (HCV) RNA determination after two weeks of induction interferon treatment is an accurate predictor of nonresponse: comparison of two treatment schedules.

The aim of this study was to analyze of HCV kinetics during interferon treatment administered daily or three times weekly. Seventy-seven naive patients were randomized to two treatment courses starting with four weeks of high-dose interferon administered daily or three times weekly. Twenty-two patients (28.6%) achieved end-of-treatment response and nine (11.7%, four of whom received daily induction) sustained response. The initial decline of viral load was sharper in patients receiving daily induction, but the rates of early RNA clearance were independent of treatment schedule, being higher in patients with genotype non-1. Detectable HCV RNA during treatment predicted nonresponse more significantly than high pretreatment viral load or genotype 1. HCV RNA at week 2 was the best predictor (100% sensitivity in patients receiving daily induction). In conclusion, daily induction increased the HCV decline slope, but not the rate of virological response. HCV RNA at week 2 reliably identified nonresponders.

Sustained activation of the Raf/MEK/Erk pathway in response to EGF in stable cell lines expressing the Hepatitis C Virus (HCV) core protein.

Chronic hepatitis C virus (HCV) infection is a leading cause of liver cirrhosis and hepatocellular carcinoma (HCC) worldwide. The HCV capside core is a multifunctional protein with regulatory functions that affects transcription and cell growth in vitro and in vivo. Here, we show that both HCV genotype 1a and 3 core proteins activate MEK1 and Erk1/2 MAP kinases and that the costitutive expression of the HCV core results in a high basal activity of Raf1 and MAP/kinase/kinase, as determined by endogenous Raf1 in vitro kinase assay and immunodetection of hyperphosphorylated Erk1 and Erk2 even after a serum starvation. Moreover, the activation of both Erk1/2 and the downstream transcription factor Elk-1 in response to the mitogenic stimulus EGF is significantly prolonged. The sustained response to EGF in cells expressing the HCV core occurs despite a normal induction of the MAP phosphatases MKP regulatory feedback and is likely due to the costitutive activation of Raf-1 activity. The ability of HCV core proteins to directly activate the MAP kinase cascade and to prolong its activity in response to mitogenic stimuli may contribute to the neoplastic transformation of HCV infected liver cells.

Induction of gene expression via activator protein-1 in the ascorbate protection against UV-induced damage.

UV irradiation is a major insult to the skin. We have shown previously that exogenous vitamin C (ascorbate) accumulates in HaCaT keratinocytes, thus conferring the ability to prevent radical formation and cell death elicited by UV-B. Here, we have investigated the potential mechanisms accounting for the cytoprotective effects exerted by this antioxidant. Using a cDNA microarray hybridization, we identified several genes whose expression was up-regulated by ascorbate. We focused on the fra-1 gene, a member of the Fos family of transcription factors that down-regulates activator protein-1 (AP-1) target genes. Both in HaCaT and in normal human epidermal keratinocytes, we found Fra-1 mRNA induction as early as 2 h after ascorbate loading. Electrophoretic mobility-shift assay and antibody supershift analysis revealed that ascorbate modulates AP-1 DNA-binding activity and that Fra-1 is in AP-1 complexes in treated cells. Furthermore, transient-transfection studies, using an AP-1 reporter construct, showed that ascorbate was able to inhibit both basal and UV-B-induced AP-1-dependent transcription. Ascorbate also modulates UV-B-induced AP-1 activity by preventing the phosphorylation and activation of the upstream c-Jun N-terminal kinase (JNK), thus inhibiting phosphorylation of the endogenous c-Jun protein. These data suggest that ascorbate mediates cellular responses aimed at counteracting UV-mediated cell damage and cell death by interfering at multiple levels with the activity of the JNK/AP-1 pathway and modulating the expression of AP-1-regulated genes.