Debi P. Sarkar

Sustained Activation of Mitogen-Activated Protein Kinases and Activator Protein 1 by the Hepatitis B Virus X Protein in Mouse Hepatocytes In Vivo

ABSTRACT Transcriptional activation of diverse cellular genes by the X protein (HBx) of hepatitis B virus (HBV) has been suggested as one of the mechanisms for HBV-associated hepatocellular carcinoma. However, such functions of HBx have been studied using transformed cells in culture and have not been examined in the normal adult hepatocytes, a natural host of HBV. Using an efficient hepatocyte-specific virus-based gene delivery system developed in our laboratory earlier, we studied the HBx action in vivo. We demonstrate that following virosome-mediated delivery of HBx DNA, a large population (>50%) of hepatocytes express the HBx protein in a dose-dependent manner, which induces a significant increase in the activity of extracellular signal-regulated kinases (ERKs) in the livers of HBx-transfected mice. Inhibition of HBx-induced ERK activation following intravenous administration of PD98059, a mitogen-activated protein kinase kinase kinase (MEK) inhibitor, confirmed the requirement for MEK in the activation of ERKs by HBx. Induction of ERK activity by HBx was sustained for up to 30 days. Interestingly, sustained activation of c-Jun N-terminal kinases (JNKs) for up to 30 days was also noted. Such constitutive ERK and JNK activation as a consequence of continued HBx expression also led to sustained stimulation of further downstream events, such as increased levels of c-Jun and c-Fos proteins along with the persistent induction of activator protein 1 binding activity. Taken together, our data suggest a critical role of these molecules in HBx-mediated cell transformation.

TNFα–308G/A Polymorphism as a Risk Factor for HPV Associated Cervical Cancer in Indian Population

Background: Investigation of the potential association of single nucleotide polymorphisms (SNPs) at –308 G/A and –238 G/A of Tumor necrosis factor α (TNFα) with susceptibility to HPV-16 associated cervical cancer in Indian women. Methods: The study included 165 histologically confirmed cases with 45 precancer and 120 cancer patients and an equal number (165) of healthy controls with normal cervical cytology. PCR-RFLP was employed to analyze TNFα promoter polymorphisms, which were confirmed by direct sequencing. Both patients and controls were screened for Human Papillomavirus (HPV) infection. Results: The frequency of –308 A allele in TNFα was significantly higher in cases compared with control subjects (21% in cases vs. 9% in controls; p < 0.01), with an odds ratio of 2.7 (95% CI = 1.41–5.15). Also, women carrying A allele for this locus presented 3 times increased susceptibility to HPV 16 infection as evident from carrier genotype distribution between HPV positive cases and control subjects (24% in HPV positive cases vs. 9% in controls; p < 0.01; OR = 3.1; 95% CI = 1.60–6.03). No such association was found for TNFα–238 (G/A) polymorphism with the risk of development of cervical cancer. Conclusion: It suggests that SNP at –308 (G/A) of TNFα promoter may represent an increased risk for HPV infection and development of cervical cancer in Indian women.

NOVEL GENE DELIVERY TO LIVER CELLS USING ENGINEERED VIROSOMES

© 1997 Federation of European Biochemical Societies.

Long‐term reduction of jaundice in Gunn rats by nonviral liver‐targeted delivery of Sleeping Beauty transposon

Asialoglycoprotein receptor (ASGPR)‐mediated endocytosis has been used to target genes to hepatocytes in vivo. However, the level and duration of transgene expression have been low because of lysosomal translocation and degradation of the DNA and lack of its integration into the host genome. In this study we packaged the DNA of interest in proteoliposomes containing the fusogenic galactose‐terminated F‐glycoprotein of the Sendai virus (FPL) for targeted delivery to hepatocytes. After the FPL binds to ASGPR on the hepatocyte surface, fusogenic activity of the F‐protein delivers the DNA into the cytosol, bypassing the endosomal pathway. For transgene integration we designed plasmids containing one transcription unit expressing the Sleeping Beauty transposase (SB) and another expressing human uridinediphosphoglucuronate glucuronosyltransferase‐1A1 (pSB‐hUGT1A1). The latter was flanked by inverted/direct repeats that are substrates of SB. In cell culture, FPL‐mediated delivery of the E. coli β‐galactosidase gene (LacZ) resulted in transduction of ASGPR‐positive cells (rat hepatocytes or Hepa1 cell line), but not of ASGPR‐negative 293 cells. Intravenous injection of the FPL‐entrapped pSB‐hUGT1A1 (4‐8 μg/day, 1‐4 doses) into UGT1A1‐deficient hyperbilirubinemic Gunn rats (model of Crigler‐Najjar syndrome type 1) resulted in hUGT1A1 expression in 5%‐10% of hepatocytes, but not in other cell types. Serum bilirubin levels declined by 30% ± 4% in 2 weeks and remained at that level throughout the 7‐month study duration. With histidine containing FPL, serum bilirubin was reduced by 40% ± 5%, and bilirubin glucuronides were excreted into bile. No antibodies were detectable in the recipient rats against the F‐protein or human UGT1A1. Conclusion: FPL is an efficient hepatocyte‐targeted gene delivery platform in vivo that warrants further exploration toward clinical application. (HEPATOLOGY 2009.)

Association between human leukocyte antigen class II alleles and human papillomavirus-mediated cervical cancer in Indian women.

We investigated the association of human leukocyte antigen (HLA) II (DRB1 and DQB1) alleles with susceptibility to human papillomavirus (HPV)-associated cervical precancer and cancer cases in a hospital-based case-control study in a northern Indian population. A total of 202 subjects, including 100 patients comprising 31 cervical precancer (cervical intraepithelial neoplasia [CIN] 2/3) and 69 invasive cervical cancer cases, and 102 healthy controls participated in the study. Both patients and controls were screened for HPV infection using a polymerase chain reaction (PCR-based approach. Low-resolution PCR-sequence specific priming (PCR-SSP) was used to genotype HLA II (DRB1 and DQB1). Our results demonstrate that the DRB1*15 allele/DRB1*15-DQB1*06 haplotype may have a predisposition for HPV infection (p(c) < 0.05) or cervical cancer/precancer (p(c) < 0.05) development, whereas the DRB1*04 allele/DRB1*04-DQB1*03 haplotype might exhibit susceptibility to cervical precancerous lesions (p(c) < 0.05). The DRB1*13 allele/DRB1*13-DQB1*06 haplotype was strongly protective against risk to HPV infection (p(c) < 0.002) as well as cervical cancer (p(c) 0.01). Therefore, we have demonstrated that HLA DR-DQ polymorphisms are involved in genetic susceptibility to cervical cancer or HPV infection in a northern Indian population.

Targeted cytosolic delivery of hydrogel nanoparticles into HepG2 cells through engineered Sendai viral envelopes.

Hydrogel nanoparticles of cross‐linked polyvinylpyrrolidone (PVP‐NP) (35–50 nm in diameter) containing fluoresceinated dextran (FITC‐Dx) were encapsulated in reconstituted Sendai viral envelopes containing only the fusion (F) protein (F‐virosomes1). Incubation of these loaded F‐virosomes with human hepatoblastoma cells (HepG2) in culture resulted in membrane‐fusion‐mediated delivery of NPs to the cell cytoplasm, as inferred from the ability of cells to internalize FITC‐Dx loaded PVP‐NP (PVPf‐NP) in the presence of azide (an inhibitor of the endocytotic process). Introduction of PVPf‐NP into the HepG2 cells was assured by selective accumulation of FITC fluorescence in the cytosolic compartment. The structural integrity of the internalized PVPf‐NP was also confirmed by fluorescence microscopy and ultracentrifugation analysis. The potential usefulness of PVP‐NP‐mediated cytosolic release of water soluble drugs both in vitro and in vivo has been established for the first time.

Targeted delivery of hepatitis C virus-specific short hairpin RNA in mouse liver using Sendai virosomes

Internal ribosome entry site (IRES)-mediated translation of input viral RNA is the initial required step for the replication of the positive-stranded genome of hepatitis C virus (HCV). We have shown previously the importance of the GCAC sequence near the initiator AUG within the stem and loop IV (SLIV) region in mediating ribosome assembly on HCV RNA. Here, we demonstrate selective inhibition of HCV-IRES-mediated translation using short hairpin (sh)RNA targeting the same site within the HCV IRES. sh-SLIV showed significant inhibition of viral RNA replication in a human hepatocellular carcinoma (Huh7) cell line harbouring a HCV monocistronic replicon. More importantly, co-transfection of infectious HCV-H77s RNA and sh-SLIV in Huh7.5 cells successfully demonstrated a significant decrease in viral RNA in HCV cell culture. Additionally, we report, for the first time, the targeted delivery of sh-SLIV RNA into mice liver using Sendai virosomes and demonstrate selective inhibition of HCV-IRES-mediated translation. Results provide the proof of concept that Sendai virosomes could be used for the efficient delivery of shRNAs into liver tissue to block HCV replication.

An internal segment (residues 58–119) of the hepatitis B virus X protein is sufficient to activate MAP kinase pathways in mouse liver

The human hepatitis B virus X protein (HBx) is known as a dual‐specificity transactivator stimulating the transcriptional machinery in the nucleus and signal transduction pathways in the cytoplasm. HBx‐induced activation of mitogen‐activated protein kinase (MAPK) signaling cascades is considered to play an important role in hepatitis B virus‐mediated hepatocarcinogenesis. Herein, we have identified the regions of HBx that are crucial for activating such signaling cascades in vivo. A truncated mutant incorporating regions C–E (amino acids 58–140) was as effective as the full‐length HBx in activating MAPKs and enhancing activator protein‐1 binding activity. While deletion of region C (amino acids 58–84) or D (amino acids 85–119) led to a drastic loss of function, region E (amino acids 120–140) was dispensable for the activation of signaling cascades. Overall, these findings provide the first evidence for the requirement of domain 58–119 of HBx in transmitting mitogenic signals to the nucleus in vivo.

Reconstituted Sendai virus envelopes as biological carriers: dual role of F protein in binding and fusion with liver cells☆

We have assessed the potential of reconstituted Sendai viral envelopes containing only the fusion protein (F-virosomes) as biological carriers for the delivery of drugs and macromolecules. [125I]lysozyme entrapped in F-virosome is used to study its distribution in various organs of Balb/c mouse in vivo as a function of dose and time. F-virosomes injected intravenously are rapidly cleared from circulation. A major percentage (55-60%) of vesicle contents is delivered to liver at 15 min after injection, showing thereby the liver to be the major site for the accumulation of vesicles. Uptake of virosomes by liver is found to reach a near saturation level at a dose of 0.5 mg F-protein associated with virosomes. In competition studies, the inhibitory effect of asialofetuin on the uptake of F-virosomes suggests the involvement of asialoglycoprotein receptor in its recognition by hepatic parenchymal cells. Incorporation of asialoganglioside-GM1 in the F-virosomes enhanced the uptake by about 1.6-fold. The observed specific interaction of hepatic receptor with F-protein containing a terminal galactose moiety is further supported by degalactosylation of F-virosomes with hard-shelled clam exoglycosidase. The uptake of degalactosylated F-virosomes by liver is found to be significantly reduced. The subcellular radioactivity profile in liver cells exhibits a considerable decrease in cytosolic localisation of the degalactosylated F-virosomal contents with a concomitant increase in their accumulation in lysosomal/mitochondrial fraction as compared to the untreated virosomes. Trypsinized and heat-treated F-virosomes also reflect similar subcellular distribution profile as that of degalactosylated virosomes. Moreover, F-virosomes are able to interact and deliver [125I]lysozyme to the HepG2 cells in culture in the presence of a potent inhibitor of endocytotic process. These results indicate the involvement of specific binding of F-proteins with hepatic receptors followed by their fusion with the membrane of liver cells in the delivery of [125I]lysozyme. The findings reported here open up the possibility of using F-virosomes with defined specificity as fusogenic vehicles for efficient delivery of drugs and biologically active macromolecules both in vivo and in vitro.

Targeted delivery of hygromycin B using reconstituted Sendai viral envelopes lacking hemagglutinin-neuraminidase

Hygromycin B was encapsulated in reconstituted Sendai viral envelopes containing only the fusion (F) protein (F‐virosomes). Incubation of loaded F‐virosomes with cultured HepG2 cells resulted in fusion mediated delivery of hygromycin B to the cell cytoplasm, as was inferred from inhibition of DNA synthesis. Binding of the F‐virosomes to HepG2 cells was mediated by the interaction of terminal β‐galactose residues of fusion protein with asialoglycoprotein receptor on HepG2 cells, subsequently leading to fusion between the two membranes. The cytotoxic effect of hygromycin B enclosed in F‐virosomes was comparable with that of F,HN‐virosomes containing both hemagglutinin‐neuraminidase (HN) and F protein and F,HNred‐virosomes containing HN whose disulfide bonds were irreversibly reduced (HNred). Hygromycin B loaded fusogenic liposomes were prepared by coreconstituting the viral envelope containing only fusion protein with exogenous lipids. These fusogenic liposomes were found to be more active than F‐virosomes at the same fusion protein concentrations.