C. Balsano

Induction of the DNA-binding activity of c-jun/c-fos heterodimers by the hepatitis B virus transactivator pX.

The hepatitis B virus (HBV) X protein (pX) is capable of activating transcription regulated by viral and cellular promoters containing binding sites for different transcription factors, including AP1. In this study we have analyzed the mechanisms of AP1 induction by pX. The hepatitis B virus transactivator was able to activate TRE (12-O-tetradecanoylphorbol-13-acetate response element)-directed transcription in different cell lines, including HepG2, HeLa, CV1, and PLC/PRF/5 cells. pX-induced AP1 activation in HepG2 cells was associated with an increase in the DNA-binding activity of c-Jun/c-Fos heterodimers, which was not dependent either on an increase in the overall amount of c-Fos and c-Jun proteins in the cells or on formation of dimers between pX and the two proteins, thus suggesting the involvement of posttranslational modifications of the transcription factor. The observation that the overexpression of c-Jun and c-Fos in the cells results in a strong augmentation of the effect of pX on TRE-directed transcription is additional evidence indicating the involvement of posttranscriptional modifications of c-Jun/c-Fos heterodimers. The increased AP1 binding observed in the presence of pX was unaffected by the protein kinase C inhibitors calphostin C and sphingosine and by the protein kinase A inhibitor HA1004, while it was almost completely blocked by staurosporine, a potent and nonspecific protein kinase inhibitor, suggesting that protein kinase C- and A-independent phosphorylation events might play a role in the phenomenon. The ability of pX also to increase TRE-directed transcription in cell lines in which AP1-binding activity is not increased (i.e., HeLa, CV1, and PLC/PRF/5 cells) suggests that pX can activate canonical TRE sites by different mechanisms as well.

Full-length and truncated versions of the hepatitis B virus (HBV) X protein (pX) transactivate the cMYC protooncogene at the transcriptional level

The products of the human hepatitis B virus (HBV) and woodchuck hepatitis B virus X genes (pXs) transactivate homologous and heterologous genes including the HBV-X and core promoters, the human immunodeficiency viruses 1 (HIV-1) and 2 (HIV-2) long terminal repeats and the beta interferon regulatory sequences. We report here that pX is also able to influence the expression of both extrachromosomal transfected c-myc regulatory sequences and endogenous c-myc gene. pX acts by increasing transcription of the c-myc gene and do not affect c-myc mRNAs stability. The presence of the first AUG of the X-ORFs is indeed necessary for the production of an active pX. The very carboxyterminus of the pX protein is dispensable for this transactivating activity and at least one domain important for its action is located between aminoacids 103 and 117.

Antioxidant Effects of Natural Bioactive Compounds

Reactive oxygen species (ROS), as well as reactive nitrogen species (RNS) play either harmful or beneficial role in biological systems. Beneficial effects of ROS include physiological roles in cellular responses against infectious agents and in several cellular signalling pathways. Harmful effects are due to high concentrations of ROS, which can damage biomolecules, including lipids, proteins and nucleic acids. The harmful effects of ROS are counterbalanced by the antioxidant action of both antioxidant enzymes and non-enzymatic antioxidants; however, despite the presence of the cells antioxidant system, oxidative damage accumulates during the life cycle and has been proposed to play a pivotal role in the development of age-dependent diseases such as atherosclerosis, arthritis, neurodegenerative disorders and cancer. Numerous epidemiological studies indicate that a reduced risk of various lifestyle diseases, mainly cardiovascular diseases and cancer, as well as other disorders, is associated to a diet rich in fruits, vegetables and their products. The drive to enhance the consumption of fruits and vegetables in the human diet is linked with positive effects of beneficial antioxidants impacting on health promotion. In this review we present an outline of main roles of ROS in biological processes and diseases and how natural bioactive compounds of fruits and vegetables determine their health-promoting properties.

Elevated serum levels of 90K/MAC-2 BP predict unresponsiveness to α-interferon therapy in chronic HCV hepatitis patients

BACKGROUNDnThe clinical outcome of hepatitis virus infections is though to depend on the complex interplay between the host immune response profile and virus factors. 90K/MAC-2 BP is a novel member of the Scavenger Receptor Cysteine Rich protein superfamily that functions as a molecular alarm signal for the cellular immune system against both cancer cells and virus infections.nnnMETHODSnTo assess the significance and the potential clinical usefulness of testing for serum levels of 90K/MAC-2 BP in chronic viral hepatitis patients we studied 115 consecutive patients with chronic HCV hepatitis, 28 HBsAg chronic hepatitis patients, 12 asymptomatic HCV carriers and 11 asymptomatic HBV carriers. 103 out of the 115 HCV patients have been treated with recombinant alpha 2a-interferon at the dose of 3 Mega Units (MU) t.i.w. for 6 months followed by 1.5 MU t.i.w. for 6 months, and have been followed up for a further 12 months. Serum levels of 90K/MAC-2 BP were measured by an immunoradiometric assay based on the specific SP-2 monoclonal antibody.nnnRESULTS AND CONCLUSIONSnSerum 90K/MAC-2 BP levels are increased in chronic viral hepatitis patients, being significantly higher in HCV than in HBV patients. In chronic HCV hepatitis, serum 90K/MAC-2 BP levels are related to both the degree of disease severity and duration of infection. Moreover, elevated 90K/MAC-2 BP serum levels are an independent predictor of failure to respond to alpha-interferon treatment in a cohort of community-acquired chronic hepatitis C patients.

DN-p73 is activated after DNA damage in a p53-dependent manner to regulate p53-induced cell cycle arrest

p53 and p73 genes are both activated in response to DNA damage to induce either cell cycle arrest or apoptosis, depending on the strength and the quality of the damaging stimulus. p53/p73 transcriptional activity must be tightly regulated to ensure that the appropriate biological response is achieved and to allow the cell to re-enter into the cell cycle after the damage has been repaired. In addition to multiple transcriptionally active (TA) isoforms, dominant negative (DN) variants, that lack the amino-terminal transactivation domain and function as trans-repressors of p53, p63 and p73, are expressed from a second internal promoter (P2-p73Pr). Here we show that, in response to a non apoptotic DNA damage induced by low doses of doxorubicin, p53 binds in vivo, as detected by a p53-specific chromatin immunoprecipitation assay, and activates the P2-p73 promoter. DN-p73α protein accumulates under the same conditions and exogenously expressed DN-p73α is able to counteract the p53-induced activation of the P2-p73Pr. These results suggest that DN-p73 may contribute to the autoregulatory loops responsible for the termination of p53/p73 responses in cells that do not undergo apoptosis. Accordingly, the activation of the P2-p73Pr is markedly enhanced in both p73−/− murine fibroblasts and in human cells in which p73 transcripts are selectively knocked-out by p73-specific small interfering RNAs.

Physical and functional interaction between HCV core protein and the different p73 isoforms

Hepatitis C virus (HCV) core protein is a structural viral protein that packages the viral genomic RNA. In addition to this function, HCV core also modulates a number of cellular regulatory functions. In fact, HCV core protein has been found to modulate the expression of the cyclin-dependent inhibitor p21WAF1/CIP1 and to promote both apoptosis and cell proliferation through its physical interaction with p53. Here, we studied the ability of HCV core to bind the p53-related p73 protein, its isoforms and its deletion mutants. We found that HCV core co-immunoprecipitated with p73 in HepG2 and SAOS-2 cells. Deletion mutational analysis of p73 indicates that the domain involved in HCV core binding is located between amino-acid residues 321–353. We also demonstrate that p73/core interaction results in the nuclear translocation of HCV core protein either in the presence of the p73 α or p73 β tumor-suppressor proteins. In addition, the interaction with HCV core protein prevents p73 α, but not p73 β dependent cell growth arrest in a p53-dependent manner. Our findings demonstrate that HCV core protein may directly influence the various p73 functions, thus playing a role in HCV pathogenesis.

Antibodies to hepatitis C virus in patients with hepatocellular carcinoma

To study the potential relationship between the hepatitis C virus (HCV), the major etiologic agent of parenterally transmitted non-A, non-B hepatitis, and the development of hepatocellular carcinoma (HCC), we tested the presence of anti-HCV antibodies in sera from a large panel of HCC patients of different racial and geographical origins. Anti-HCV antibodies were detected in 82 out of 114 (71.9%) HBsAg-negative HCC patients and in 15 out of 53 (28.3%) HBsAg-positive patients. No significant difference in the prevalence of anti-HCV antibodies was found in the HBsAg-negative HCC patients when they were divided according to presence of anti-HBs and/or anti-HBc antibodies, or absence of all hepatitis B virus (HBV) serological markers. The prevalence of anti-HCV antibodies was similar in HCC patients of Caucasian and African origin. No differences were noted when the patients were grouped according to sex. The mechanisms by which HCV might contribute to the development of HCC need to be further investigated. As for HBV infection, the necro-inflammation associated with HCV infection may induce cirrhosis, regeneration and eventually malignant transformation. The finding that few anti-HCV patients had HCC which is not superimposed on cirrhosis suggests that HCV could, however, exert some direct effect on the development of HCC.

Hepatitis B virus (HBV) X gene expression in human cells and anti-HBx antibodies detection in chronic HBV infection

All mammalian hepatitis B virus genomes contain an open reading frame X (X-ORF) of unknown function which could encode a protein of 17 kDa. Using a plasmid containing the entire X-ORF preceded by the adenovirus type 2 major late promoter and its tripartite leader sequence efficient expression of the HBV X-gene was achieved. The X protein of 17 kDa was characterized by immunoblotting and immunoprecipitated with an antiserum prepared against a X fusion protein produced in E. coli. By cell fractionation and indirect immunofluorescence the X-protein was found at least in part associated with nuclei. Human cell extracts containing the X protein have been used to screen human sera for anti-HBx antibodies. Such antibodies were detected in sera from patients with active chronic hepatitis with ongoing viral replication. The efficient expression of the HBV X protein obtained will facilitate its functional analysis.

Reactive Oxygen Intermediates (ROIs) Are Involved in the Intracellular Transduction of Angiotensin II Signal in C2C12 Cells

Increasing evidence suggests that angiotensin II may act as a growth factor for several muscle cell types. Angiotensin II stimulation activates many immediate early response genes like c-Fos, c-Jun, c-Myc and Egr-1 in both vascular smooth muscle cells and cardiomyocytes, independently of whether a hyperplastic or hypertrophic response is taking place. In this study we report that angiotensin II significantly stimulates AP1-driven transcription in mouse skeletal muscle cells C2C12 stably transfected with a TRE-tk-CAT plasmid in a dose-dependent manner (peak stimulation at 10(-5) M of angiotensin II). Moreover, angiotensin II increases the binding of the AP1 complex to its DNA target in both quiescent C2C12 myoblasts and in differentiated C2C12 myotubes. Most of the TRE-bound complexes in both unstimulated and angiotensin II-treated cells consist of c-jun/c-fos heterodimers. Using a set of different protein kinase inhibitors, including HA1004, H7, tyrphostin, genistein and staurosporine, we could demonstrate that the angiotensin II-induced AP1 binding increase is not mediated by the cAMP-dependent pathway and that protein kinase C and tyrosine kinases are involved. Treatment of C2C12 cells with H2O2 induces a dose-dependent increase in c-jun/c-fos heterodimer binding, specifically reverted by the cysteine derivative and glutathione precursor N-acetyl-L-cysteine (NAC). The observation that the induction by angiotensin II of both the AP1 DNA binding activity and DNA synthesis in quiescent C2C12 myoblasts is abolished by NAC strongly suggests a role for reactive oxygen intermediates (ROIs) in the intracellular transduction of angiotensin II signals for immediate early gene induction and for cell proliferation.

Liver Fibrosis and Therapeutic Strategies: The Goal for Improving Metabolism

PURPOSE OF REVIEWnThis review summarizes the current state of knowledge on non-alcoholic fatty liver disease (NAFLD) and the hepatitis C virus (HCV)-associated liver fibrosis, and provides insight into the role of dysmetabolism in hepatic fibrogenesis. Clinical relevance of drugs correcting these metabolic disturbances in the reversion of liver fibrosis will also be discussed.nnnRECENT FINDINGSnLiver fibrosis affects more than ten millions of people worldwide and may lead to cirrhosis, liver failure, and death. Recent epidemiological data indicate that the incidence of liver fibrosis is expected to triple during the next 10 to 15 years as a result of the HCV infection and NAFLD escalation. In accordance with the modern view of liver fibrogenesis, the pathways involved in the pathogenesis of hepatic fibrosis appear to be broadly similar regardless of the etiology.nnnSUMMARYnSome features of metabolic syndrome, including obesity, insulin resistance, and type 2 diabetes represent a strong risk factor in development and progression of hepatic fibrosis. However, whatever the cause, fibrosis culminates in cirrhosis and results in liver failure, thus, a potent anti-fibrotic therapy is urgently needed to reverse scarring and eliminate progression to cirrhosis.