Hepatocyte apoptotic bodies encasing nonstructural HCV proteins amplify hepatic stellate cell activation: implications for chronic hepatitis C.

Chronic hepatitis C infection leads to increased hepatocyte apoptosis. Because engulfment of apoptotic bodies (ABs) by hepatic stellate cells (HSC) is profibrogenic, we compared the effects of ABs derived from hepatitis C virus (HCV)-negative vs HCV-infected (Con1+) Huh7 hepatoblastoma cells on fibrogenic and activation-related mRNA expression by a human HSC line (LX2). Uptake of Huh7(Con1+) ABs by LX2 cells dose dependently upregulated profibrotic genes (COL1A1, TGFB1; TIMP1; TIMP2). When normalized to the apoptotic cytokeratin-18 M30 neoepitope, HCV(+) ABs exhibited a more pronounced effect than HCV(-) ABs. In contrast, neither noningested ABs nor nucleic acids obtained from Huh7, Huh7(Con1+) or HepG2 cells triggered those AB-dependent effects. Both the engulfment of Huh7(Con1+) ABs and their effects were partially blocked by masking of phosphatidylserine with annexin V and completely inhibited by the class-A scavenger receptor ligand, polyinosinic acid. Our findings demonstrate that AB uptake stimulates HSCs and indicate that HCV infection leads to amplified fibrogenic mRNA expression and enhanced HSC activation.

DC-SIGN-specific liposomal targeting and selective intracellular compound delivery to human myeloid dendritic cells: implications for HIV disease.

Myeloid dendritic cells (MyDCs), prime stimulators of antigen-specific immunity, can serve as one of the major reservoirs for human immunodeficiency virus type-1 (HIV-1). Utilizing mature monocyte-derived MyDCs generated with granulocyte/macrophage colony-stimulating factor, interleukin-4, and tumour necrosis factor-alpha as an in vitro model, we here present the first proof of concept for liposomal compound delivery to these cells by specifically addressing CD209, i.e. DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN), a MyDC-associated C-type lectin implicated in the transmission of HIV-1 to T helper cells. By employing a liposomally entrapped tracer, calcein, we demonstrate by flow cytometry and mathematics a superior targeting efficacy for DC-SIGN, as compared with select other MyDC markers (CD1a, CD4, CD45R0, and CD83). Fluorescence microscopy reveals time-dependent surface binding and intracellular uptake of DC-SIGN-specific liposomes by both immature and mature MyDCs. This pilot study implies that liposomal targeting to CD209 and related C-type lectins may afford therapeutic intracellular drug delivery to MyDCs and other reservoir and nonreservoir cells susceptible to infection with HIV-1.

Identification of the full-length huntingtin- interacting protein p231HBP/HYPB as a DNA-binding factor.

Neurodegeneration in Huntington's disease (HD) is associated with an elongated glutamine tract in the widely expressed huntingtin protein. Although the pathogenic mechanisms are still unknown, the distinct physical properties of mutant huntingtin in the brain suggest that other factors including huntingtin-interacting proteins might play a specific role. We have previously identified a DNA-binding motif in the proximal E1A promoter of adenovirus serotype 12 as responsible for E1A autoregulation. Here, we identified the p231HBP protein as a DNA-binding factor, the C-terminal portion of which has recently been characterized as the huntingtin-interacting protein HYPB of unknown function. We have determined the full-length cDNA sequence, identified several domains supporting its gene regulatory functions, and mapped the HBP231 gene to chromosome 3p21.2-p21.3. Our results provide an interesting molecular link between huntingtin and a DNA-binding factor, implicating that this interaction might result in the alteration of cellular gene expression involved in HD pathogenesis.

Differential susceptibility of renal carcinoma cell lines to tumor suppression by exogenous Fhit expression.

Hemizygous deletions of the fragile histidine triad (FHIT) gene at human chromosome band 3p14.2 and down-regulation of its gene product are found in the majority of renal cell carcinomas (RCCs). Functional tumor suppressive activity of Fhit in renal cancer cells previously was observed in RCC cell line RC48, which lacks endogenous Fhit expression. To further investigate the potential role of FHIT as a tumor suppressor gene in RCC, we transfected FHIT cDNA expression constructs into RCC cell lines RCC-1 and SN12C, which show low-level expression of endogenous Fhit and reveal an intact von Hippel-Lindau (VHL) gene. Stable transfectants of both cell lines showed no alterations of cell morphology, proliferation kinetics, or cell cycle parameters in vitro. The FHIT gene transfer rate, however, was significantly lower in RCC-1 cells compared with SN12C cells, suggesting a selection against exogenous Fhit expression. In addition, in nude mouse assays, a significant delay of tumor formation was observed for FHIT-transfected RCC-1 cell lines, with outgrowing tumors demonstrating loss of Fhit expression in the majority of cells. In contrast, tumorigenicity of FHIT-transfected SN12C cell clones was not suppressed, despite stable transgene expression. In conclusion, our results demonstrate a selective tumor suppressive activity of Fhit in RCC cells in vivo and suggest that the susceptibility to suppression is not restricted to cancer cells with complete loss of Fhit expression.

Novel tumor suppressor locus in human chromosome region 3p14.2.

BACKGROUND: Alterations of chromosome region 3p14 are observed in numerous human malignancies. Because the pattern of allelic losses suggests the existence of at least one tumor suppressor gene within this region, we established a library of yeast artificial chromosomes (YACs) containing contiguous human 3p14 sequences to permit a search for tumor suppressor loci within the 3p14 region by use of functional complementation. METHODS: YACs specific for human chromosome region 3p14 were transduced by spheroplast fusion into cells of the human nonpapillary renal carcinoma cell line RCC-1, which shows a cytogenetically detectable 3p deletion and is tumorigenic in nude mice. RESULTS: We identified a 3p14.2-specific YAC clone, located in the vicinity of the fragile histidine triad (FHIT) gene (but toward the telomere), that is capable of inducing sustained suppression of tumorigenicity in nude mice and of activating cellular senescence in vitro. Among 23 mice given injections of RCC-1 cells containing this YAC, 16 (70%) remained tumor free for at least 6 months, whereas tumor formation occurred after a median of 6 weeks in control mice given injections of either RCC-1 parental cells or a revertant cell line (in which the YAC had lost all human sequences) or RCC-1 parental cells containing other, unrelated YACs. Similar results were obtained following microcell-mediated transfer of the entire human chromosome 3. CONCLUSION: These data provide strong evidence for the existence of a novel tumor suppressor locus adjacent to the previously identified candidate tumor suppressor gene, FHIT, in 3p14.2. Positional cloning of the novel suppressor element within the 3p14.2-specific YAC and the sequence's molecular and functional characterization should add to the understanding of the pathogenesis of renal cell carcinoma and other human tumors that exhibit 3p14 aberrations.