Biomedical subjects

R B Ray

Publications and source records attributed to R B Ray.

At least 19 recordsLinked to original sources

PubMed2000

Hepatitis C virus core protein promotes immortalization of primary human hepatocytes.

Hepatitis C virus (HCV) core protein has many intriguing properties as a viral factor and is implicated in cell growth regulation. In this study, the cell growth regulation potential of HCV core protein was investigated by introduction of the core genomic region into primary human hepatocytes, a natural host for virus replication and tropism. Core-transfected primary human hepatocytes displayed altered cell morphology resembling that of low-differentiated epithelial cells. Those cells retained an immortalized phenotype and exhibited continuous growth after more than 50 passages over 2 years. Stable hepatocyte transfectants exhibited albumin secretion and HCV core protein expression. Telomerase activity, a characteristic of immortalized or transformed cells, was evident in the transfected hepatocytes immediately after senescence. Anchorage-independent growth of the immortalized hepatocytes provided further evidence for a transformed phenotype. Results from these studies suggest that the HCV core protein promotes primary human hepatocytes to an immortalized phenotype, which may predispose cells over an extended period of time to undergo a transforming event. Thus, HCV core protein appears to contribute to virus-mediated pathogenesis in a persistently infected host.

Adult↗

PubMed2000

Hepatitis C virus NS5A protein modulates transcription through a novel cellular transcription factor SRCAP.

Hepatitis C virus NS5A protein transcriptionally modulates cellular genes and promotes cell growth. NS5A is likely to exert its activity in concert with cellular factor(s). Using a yeast two-hybrid screen, we have demonstrated that NS5A interacts with the C-terminal end of a newly identified cellular transcription factor, SRCAP. The authenticity of this interaction was verified by a mammalian two-hybrid assay, in vitro pull-down experiment, and an in vivo coimmunoprecipitation assay in human hepatoma (HepG2) cells. An in vitro transient transfection assay demonstrated that SRCAP can efficiently activate transcription when recruited by the Gal4 DNA-binding domain to the promoter. However, down-regulation of p21 promoter activity by NS5A was enhanced following ectopic expression of SRCAP. Together these results suggest that the interaction of NS5A and SRCAP may be one of the mechanisms by which NS5A exerts its effect on cell growth regulation contributing to hepatitis C virus-mediated pathogenesis.

Adenosine Triphosphatases↗

PubMed2000

Hepatitis C virus NS5A protein protects against TNF-alpha mediated apoptotic cell death.

Hepatitis C virus (HCV) often causes a prolonged and persistent infection which may lead to hepatocellular carcinoma. We have previously reported that the nonstructural 5A (NS5A) protein of HCV promotes cell growth [Ghosh, A.K., Steele, R., Meyer, K., Ray, R., Ray, R.B., 1999. Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth. J. Gen. Virol. 80, 1179-1183]. In this study, we investigated the role of HCV NS5A (genotype 1a, strain H) in TNF-alpha induced apoptotic cell death. HepG2 cells expressing NS5A exhibited an inhibitory role in relation to TNF-alpha mediated apoptotic cell death. The NS5A protein blocked the activation of caspase-3 and inhibited proteolytic cleavage of the death substrate poly (ADP-ribose) polymerase in TNF-alpha induced cells. Together, these results suggest that HCV NS5A protein protects against TNF-alpha mediated apoptotic cell death.

Blotting, Western↗

PubMed1999

PTEN transcriptionally modulates c-myc gene expression in human breast carcinoma cells and is involved in cell growth regulation.

A novel tumor suppressor gene, PTEN, has recently been identified at chromosome 10q23, which is inactivated in a number of different tumor types including breast cancer. An investigation of the functional role suggested that PTEN transcriptionally represses both exogenous and endogenous c-myc expressions in human breast carcinoma cells. PTEN, when ectopically expressed in human breast carcinoma cells, exhibited an inhibition of phosphorylation of both activating residues of protein kinase B (PKB)/AKT at Ser-473 and Thr-308 without any significant alteration of AKT expression. Furthermore, introduction of PTEN into human breast carcinoma cells induced apoptotic cell death and inhibited cell growth and tumor formation in nude mice. Taken together, our data suggest that PTEN acts as a transcriptional repressor, inhibits the AKT-mediated cell survival signaling pathway, and negatively regulates human breast carcinoma cell growth. These results further emphasize the potential of PTEN as a gene therapeutic agent.

Animals↗

PubMed1999

MBP-1 physically associates with histone deacetylase for transcriptional repression.

MBP-1, a c-myc promoter binding protein, is a mammalian transcription factor with intriguing properties including transcriptional repression of cellular genes. Recently, we have identified and characterized two different repressor domains of MBP-1. In this report, we have demonstrated that MBP-1 physically associates with histone deacetylase (HDAC), thus promoting formation of neucleosomes that inhibit transcription. Trichostatin A, an inhibitor of histone deacetylase, significantly reduces MBP-1-mediated transcriptional repression. However, MBP-1-mediated repression on c-myc promoter is resistant to histone deacetylase activity. Our results suggest that MBP-1 represses transcription by recruiting histone deacetylase as one of the mechanisms, whereas the other mechanism is resistant to HDAC activity and probably related to direct binding of promoter sequences or interaction through yet unidentified factor.

3T3 Cells↗

PubMed1999

Functional analysis of a transrepressor domain in the hepatitis C virus core protein.

Hepatitis C virus (HCV) is one of the major causative agents of chronic liver disease with the potential for development of hepatocellular carcinoma. The putative core protein of the virus has many intriguing properties, including transcriptional regulation of cellular and unrelated viral promoters. To further characterize the transregulatory function, a number of chimeric constructs were made by fusion of the core gene to the DNA binding domain of the yeast transactivator factor GAL4. The fusion protein exhibited a repressor activity on the herpes simplex virus thymidine kinase promoter via the upstream GAL4 DNA binding sites. A structure /function analysis of HCV core mutants in the context of the GAL4 DNA binding domain revealed that the transcriptional repressor activity was located near the N-terminus (amino acids 26 85). Transcription was strongly inhibited upon transfer of this repressor domain to a heterologous activation domain, (3CGln) of Epstein Barr virus transcription factor EBNA3C. Results from this study suggest that the HCV core protein contains an overall repressor activity, and that the repressor domain is located near the N-terminus.

3T3 Cells↗

PubMed1999

Hepatitis C virus NS5A protein modulates cell cycle regulatory genes and promotes cell growth.

The phosphoprotein NS5A of hepatitis C virus has recently been suggested to control PKR protein kinase for resistance to interferon. To investigate other functions of NS5A, studies were initiated on the regulation of transcription of important cellular genes and of cell growth by this protein. The results suggested that NS5A protein represses transcription of the cell cycle regulatory gene p21WAF1, while it activates the human proliferating cell nuclear antigen gene in murine fibroblasts and human hepatoma cells. Furthermore, introduction of NS5A into murine fibroblasts (NIH3T3) promoted anchorage-independent growth and tumour formation in nude mice. Thus, NS5A appears to exhibit a role in cell growth regulation.

Animals↗

PubMed1999

Functional domains of c-myc promoter binding protein 1 involved in transcriptional repression and cell growth regulation.

We initially identified c-myc promoter binding protein 1 (MBP-1), which negatively regulates c-myc promoter activity, from a human cervical carcinoma cell expression library. Subsequent studies on the biological role of MBP-1 demonstrated induction of cell death in fibroblasts and loss of anchorage-independent growth, reduced invasive ability, and tumorigenicity of human breast carcinoma cells. To investigate the potential role of MBP-1 as a transcriptional regulator, a chimeric protein containing MBP-1 fused to the DNA binding domain of the yeast transactivator factor GAL4 was constructed. This fusion protein exhibited repressor activity on the herpes simplex virus thymidine kinase promoter via upstream GAL4 DNA binding sites. Structure-function analysis of mutant MBP-1 in the context of the GAL4 DNA binding domain revealed that MBP-1 transcriptional repressor domains are located in the N terminus (amino acids 1 to 47) and C terminus (amino acids 232 to 338), whereas the activation domain lies in the middle (amino acids 140 to 244). The N-terminal domain exhibited stronger transcriptional repressor activity than the C-terminal region. When the N-terminal repressor domain was transferred to a potent activator, transcription was strongly inhibited. Both of the repressor domains contained hydrophobic regions and had an LXVXL motif in common. Site-directed mutagenesis in the repressor domains indicated that the leucine residues in the LXVXL motif are required for transcriptional repression. Mutation of the leucine residues in the common motif of MBP-1 also abrogated the repressor activity on the c-myc promoter. In addition, the leucine mutant forms of MBP-1 failed to suppress cell growth in fibroblasts like wild-type MBP-1. Taken together, our results indicate that MBP-1 is a complex cellular factor containing multiple transcriptional regulatory domains that play an important role in cell growth regulation.

3T3 Cells↗

PubMed1998

Hepatitis C virus core protein represses p21WAF1/Cip1/Sid1 promoter activity.

Hepatitis C virus (HCV) often causes a prolonged and persistent infection, and an association between hepatocellular carcinoma (HCC) and HCV infection has been noted. Recent experimental evidence using a cloned genomic region suggests that the putative core protein of HCV has numerous biological properties and is implicated as a viral factor for HCV mediated pathogenesis. WAF1/Cip1/Sid1 (p21) is the prototype of a family of proteins that inhibit cyclin-dependent kinases (CDK) and regulate cell cycle progression in eukaryotic cells. In this study, we have observed that the HCV core protein represses the transcriptional activity of the p21 promoter when tested separately by an in-vitro transient expression assay using murine fibroblasts (NIH3T3), human hepatocellular carcinoma (HepG2), and human cervical carcinoma (HeLa) cells. A deletion analysis of the p21 promoter suggested that the HCV core responsive region is located downstream of the p53 binding site. A gel mobility shift analysis showed that the HCV core protein does not bind directly to p21 regulatory sequences. Thus, the HCV core protein appears to act as an effector in the promotion of cell growth by repressing p21 transcription through unknown cellular factor(s).

Animals↗

PubMed1998

Inhibition of tumor necrosis factor (TNF-alpha)-mediated apoptosis by hepatitis C virus core protein.

Hepatitis C virus (HCV) putative core protein has displayed many intriguing biological properties. Since tumor necrosis factor (TNF) plays an important role in controlling viral infection, in this study the effect of the core protein was investigated on the TNF-alpha induced apoptosis of human breast carcinoma cells (MCF7). HCV core protein when expressed inhibited TNF-alpha-induced apoptotic cell death unlike the control MCF7 cells, as determined by cell viability and DNA fragmentation analysis. Additionally, HCV core protein blocked the TNF-induced proteolytic cleavage of the death substrate poly(ADP-ribose) polymerase from its native 116-kDa protein to the characteristic 85-kDa polypeptide. Results from this study suggest that the HCV core protein plays a role in the inhibition of TNF-alpha-mediated cell death. Thus, the ability of core protein to inhibit the TNF-mediated apoptotic signaling pathway may provide a selective advantage for HCV replication, allowing for evasion of host antiviral defense mechanisms.

Apoptosis↗

PubMed1997

Transcriptional repression of p53 promoter by hepatitis C virus core protein.

Our previous results have suggested that the putative core protein of hepatitis C virus (HCV) transcriptionally regulates cellular and viral genes, inhibits cisplatin and c-myc-mediated apoptotic cell death under certain conditions, and transforms primary rat embryo fibroblast cells with a cooperative oncogene. Because HCV appears to cause hepatocellular carcinoma, we evaluated the regulatory role of the HCV core protein on p53, a well known tumor suppressor gene, by an in vitro transfection assay. HCV core protein repressed transcriptional activity of the p53 promoter when tested separately in COS7 and HeLa cells. Deletion mutational analysis of the HCV core gene indicated that the regulatory domain involved in the repression of p53 transcriptional activity is located around amino acid residues 80-122 encompassing a putative DNA binding motif and two major phosphorylation sites. Results from this study suggest that the putative core protein may have an important biological role in the promotion of cell growth by repressing p53 transcription, and this appears to be consistent with certain earlier observations about HCV core moving into the nucleus.

Animals↗

PubMed1997

Inhibition of human immunodeficiency virus type 1 replication by a cellular transcriptional factor MBP-1.

A cellular transcriptional factor initially identified as the c-myc promoter binding protein (MBP-1) was subsequently characterized as a cell regulatory protein with multifunctional activities. In this study, the role of MBP-1 on human immunodeficiency virus type-1 (HIV-1) transcriptional activity was investigated. MBP-1 showed inhibition of HIV-1 long terminal repeat (LTR)-directed chloramphenicol acetyl transferase (CAT) activity in a transient cotransfection assay. Deletion of upstream elements of the HIV-1 LTR, including the nuclear factor kappa B (NF-kappa B) and Sp1 binding sites, did not affect the MBP-1 mediated suppression of HIV-1 LTR. The core promoter of the HIV-1 appeared to be the primary sequence involved in MBP-1 mediated inhibition. In the presence of HIV-1 TAR sequence and Tat protein, MBP-1 did not inhibit the viral promoter activity. In addition, cotransfection experiments with HIV-1 LTR and deletion mutants of MBP-1 suggested that the carboxyl terminal half of MBP-1 suppresses the HIV-1 promoter activity. Exogenous expression of MBP-1 showed suppression of HIV-1 replication in acutely infected cells and in cells cotransfected with a molecular clone of HIV-1. These results suggest that exogenous expression of MBP-1 plays an important role in the regulation of HIV-1 replication in infected cells.

DNA-Binding Proteins↗

PubMed1997

Separate domains of MBP-1 involved in c-myc promoter binding and growth suppressive activity.

We previously demonstrated that exogenous expression of MBP-1 induces rapid cell death in murine fibroblasts, and alters loss of anchorage-independent growth and tumorigenicity in human breast carcinoma cells. Here, we investigated the functional role of two different domains of MBP-1. A DNA-protein interaction study suggested that the amino-terminal half (amino acids 1-178) of MBP-1 possesses the c-myc P2 promoter binding activity. The same domain of MBP-1 also showed transcriptional repressor activity on c-myc promoter by in vitro transient expression assay. On the other hand, the carboxy terminal half (amino acids 190-335) of MBP-1 induced cell death in murine fibroblasts similar to full length MBP-1. Furthermore, exogenous protein expression from the carboxy terminal half of MBP-1 in human breast carcinoma (MCF-7) cells showed suppression of colony formation and loss of anchorage-independent growth. Results from this study suggest that MBP-1 exerts its biological effect through different functional domains.

3T3 Cells↗

PubMed1997

Chromosomal localization of the human gene encoding c-myc promoter-binding protein (MPB1) to chromosome 1p35-pter.

We report the mapping of the human gene MPB1 (c-myc promoter binding protein), a recently identified gene regulatory protein. MPB1 binds to the c-myc P2 promoter and exerts a negative regulatory role on c-myc transcription. Since exogenous expression from transfection of the MPB1 gene suppresses the tumorigenic property of breast cancer cells, there was interest in determining the chromosomal location of this gene. The human MPB1 gene was assigned to human chromosome 1p35-pter using Southern blot analyses of genomic DNAs from rodent-human somatic hybrid cell lines. A specific human genomic fragment was observed only in the somatic cell lines containing human chromosome 1 or the p35-pter region of the chromosome.

Animals↗

PubMed1997

Interrogations.

Explore the source record for details and available documents.

Female↗

PubMed1996

Suppression of apoptotic cell death by hepatitis C virus core protein.

We have previously demonstrated the role of hepatitis C virus (HCV) core protein in the transcriptional regulation of cellular and unrelated viral promoters. Furthermore, the core protein in cooperation with H-ras oncogene transforms primary rat embryo fibroblast cells to the tumorigenic phenotype. In the present study, the functional role of HCV core protein was investigated to determine its potential to inhibit the onset of apoptotic cell death. Expression of HCV core protein inhibited cisplatin mediated apoptosis in human cervical epithelial cells, and apoptosis induced by the overexpression of c-myc in Chinese hamster ovarian cells. Results from these studies suggest that the core protein may have a biological implication in the pathogenesis of HCV infection.

Animals↗

PubMed1996

Hepatitis C virus core protein inhibits human immunodeficiency virus type 1 replication.

We previously demonstrated that hepatitis C virus (HCV) core protein is a strong repressor of human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) basal transcription. In this study, we have localized the HCV core protein-response domain to a region between nucleotides -65 and +3 within the HIV-LTR. Thus, neither the upstream negative regulatory elements, or binding sites for various transcription factors (e.g. NF-kappa B, USF-1, IL2/IL-2R) nor the downstream TAR regions were involved in HCV core-mediated repression. HCV core protein mediated repression of the basal transcriptional activity of HIV-1 LTR was abrogated by the Tat protein. Furthermore, HeLa-T4 cells expressing HCV core protein showed inhibition of HIV-1 replication after acute infection with cell-free HIV. A similar observation was also noted in CD4+ and CD4-lymphocytic cell lines cotransfected with an infectious molecular clone of HIV-1 and the HCV core protein expression vector. Thus, a repression of basal transcription prior to the accumulation of threshold levels of Tat protein appears to restrict HIV-1 transcription and modulate viral replication.

Anti-HIV Agents↗

PubMed1996

Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype.

We have previously demonstrated that hepatitis C virus (HCV) core protein regulates cellular protooncogenes at the transcriptional level; this observation implicates core protein in the alteration of normal hepatocyte growth. In the present study, the transforming potential of the HCV core gene was investigated by using primary rat embryo fibroblast (REF) cells which were transfected with or without cooperative oncogenes. Integration of the HCV core gene resulted in expression of the viral protein in REF stable transformants. REF cells cotransfected with HCV core and H-ras genes became transformed and exhibited rapid proliferation, anchor-independent growth, and tumor formation in athymic nude mice. Results from these studies suggest that the core protein plays an important role in the regulation of HCV-infected cell growth and in the transformation to tumorigenic phenotype. These observations suggest a possible mechanism for this viral protein in the pathogenesis of hepatocellular carcinoma in HCV-infected humans.

Animals↗