We discovered that the hepatitis C virus (HCV) envelope glycoprotein E2 binds to human hepatoma cell lines independently of the previously proposed HCV receptor CD81. Comparative binding studies using recombinant E2 from the most prevalent 1a and 1b genotypes revealed that E2 recognition by hepatoma cells is independent from the viral isolate, while E2±CD81 interaction is isolate speci®c. Binding of soluble E2 to human hepatoma cells was impaired by deletion of the hypervariable region 1 (HVR1), but the wild-type phenotype was recovered by introducing a compensatory mutation reported previously to rescue infectivity of an HVR1-deleted HCV infectious clone. We have identi®ed the receptor responsible for E2 binding to human hepatic cells as the human scavenger receptor class B type I (SR-BI). E2±SR-BI interaction is very selective since neither mouse SR-BI nor the closely related human scavenger receptor CD36, were able to bind E2. Finally, E2 recognition by SR-BI was competed out in an isolate-speci®c manner both on the hepatoma cell line and on the human SR-BI-transfected cell line by an anti-HVR1 monoclonal antibody. Keywords: hepatitis C virus/hypervariable region 1/ scavenger receptor class B type I/second envelope glycoprotein

Histone deacetylases (HDACs) are a family of enzymes involved in the regulation of gene expression, DNA repair, and stress response. These processes often are altered in tumors, and HDAC inhibitors have had pronounced antitumor activity with promising results in clinical trials. Here, we report the crystal structure of human HDAC8 in complex with a hydroxamic acid inhibitor. Such a structure of a eukaryotic zinc-dependent HDAC has not be described previously. Similar to bacterial HDAC-like protein, HDAC8 folds in a single ␣͞␤ domain. The inhibitor and the zinc-binding sites are similar in both proteins. However, significant differences are observed in the length and structure of the loops surrounding the active site, including the presence of two potassium ions in HDAC8 structure, one of which interacts with key catalytic residues. CD data suggest a direct role of potassium in the fold stabilization of HDAC8. Knockdown of HDAC8 by RNA interference inhibits growth of human lung, colon, and cervical cancer cell lines, highlighting the importance of this HDAC subtype for tumor cell proliferation. Our findings open the way for the design and development of selective inhibitors of HDAC8 as possible antitumor agents.T he epigenetic control of gene expression is operated through a series of posttranslational modifications of chromatin that influence the electrostatics of DNA-protein interactions and generate docking sites for a large number of chromatininteracting proteins (1, 2). The acetylation status of lysine residues found in the accessible N termini of core histones is one of the posttranslational chromatin modifications that impinge on gene expression. Acetylation and deacetylation of histones are controlled by the enzymatic activity of histone acetyltransferases and histone deacetylases (HDACs) (3, 4). Alterations of gene expression are a hallmark of cancer, and mounting evidence suggests that at least a part of these alterations is mediated by epigenetic mechanisms (5, 6). Importantly, the aberrant recruitment of HDACs has been mechanistically linked to malignancy in leukemias and lymphomas (7,8), and small-molecule HDAC inhibitors show antitumor activity in preclinical models and in clinical trials and have the promise to become effective, new antineoplastic therapeutics (9).At least 18 HDAC subtypes exist, and they are subdivided into three classes (10): class I (HDACs 1-3 and 8), homologous to the yeast Rpd3 deacetylase; class II (HDACs 4-7, 9, and 10), related to the yeast Hda1 deacetylase; and class III proteins (Sirtuins 1-7), which are yeast Sir2 homologs. HDAC11 has homology to both class I and II enzymes but cannot unambiguously be assigned to either class. Class I and II HDACs, as well as HDAC11, are all zinc-dependent hydrolases. The therapeutically relevant HDAC inhibitors are thought to be nonselective or poorly selective inhibitors of all or most of class I and II enzymes but do not inhibit class III HDACs (9). It is not clear whether the antitumor properties of HDAC inhibitors are due to their l...

Chromatin modifications such as acetylation, methylation, phosphorylation, ubiquitination, and sumoylation of histones play an important role in the regulation of transcription (1-5). Together with DNA methylation, these modifications are part of a broad and multifaceted strategy whereby eukaryotes regulate gene expression at the epigenetic level (6 -8).Acetylation of histones is tightly controlled by the activity of histone acetyltransferases and histone deacetylases (HDACs) 2 (9, 10). Histone deacetylation may repress transcription by different mechanisms. On the one hand, this process increases the charge density on the N termini of the core histones thereby strengthening histone tail-DNA interactions and blocking access of the transcriptional machinery to the DNA template. In addition, histone modifications are specifically recognized by chromatin-interacting proteins thus favoring the formation of higher order chromatin structures (heterochromatin).There is ample evidence that aberrations in the epigenetic regulation of gene expression at the levels of DNA methylation and histone acetylation or histone methylation are an important component in the process of malignant transformation of human cells (11). A number of histone acetyltransferase mutations were identified in cancer, and HDACs were found to be overexpressed or aberrantly recruited in several human malignancies (12). Small molecule HDAC inhibitors induce cell cycle arrest, differentiation, and apoptosis in cancer cells with a significant window over normal cells, and some molecules have already entered clinical trials and show promising results (13).Higher organisms have evolved a considerable complexity in the histone deacetylase family; thus, in mammals, 17 different HDAC subtypes were identified, which were grouped into three classes according to their sequence homologies with the yeast proteins Rpd3 (class I), HDA1 (class II), and Sir (class III) (14 -16). Class I and class II proteins are evolutionarily related and share a common enzymatic mechanism, the Zn-catalyzed hydrolysis of the acetyl-lysine amide bond. The HDAC inhibitors that are presently in clinical trials are rather nonselective and are thought to inhibit most or many of the class I and II proteins. Class III proteins are evolutionarily unrelated to class I or class II and catalyze the transfer of the acetyl group onto the sugar moiety of NAD (16

The stem cell-associated transcription co-factor ZNF521 has been implicated in the control of hematopoietic, osteo-adipogenic and neural progenitor cells. ZNF521 is highly expressed in cerebellum and in particular in the neonatal external granule layer that contains candidate medulloblastoma cells-of-origin, and in the majority of human medulloblastomas. Here we have explored its involvement in the control of human and murine medulloblastoma cells.The effect of ZNF521 on growth and tumorigenic potential of human medulloblastoma cell lines as well as primary Ptc1−/+ mouse medulloblastoma cells was investigated in a variety of in vitro and in vivo assays, by modulating its expression using lentiviral vectors carrying the ZNF521 cDNA, or shRNAs that silence its expression.Enforced overexpression of ZNF521 in DAOY medulloblastoma cells significantly increased their proliferation, growth as spheroids and ability to generate clones in single-cell cultures and semisolid media, and enhanced their migratory ability in wound-healing assays. Importantly, ZNF521-expressing cells displayed a greatly enhanced tumorigenic potential in nude mice. All these activities required the ZNF521 N-terminal motif that recruits the nucleosome remodeling and histone deacetylase complex, which might therefore represent an appealing therapeutic target. Conversely, silencing of ZNF521 in human UW228 medulloblastoma cells that display high baseline expression decreased their proliferation, clonogenicity, sphere formation and wound-healing ability. Similarly, Zfp521 silencing in mouse Ptc1−/+ medulloblastoma cells drastically reduced their growth and tumorigenic potential.Our data strongly support the notion that ZNF521, through the recruitment of the NuRD complex, contributes to the clonogenic growth, migration and tumorigenicity of medulloblastoma cells.

We investigated the role of histone deacetylase 4 (HDAC4) using RNA interference (RNAi) and knockout cells to specifically address its role in cell cycle progression in tumor and normal cells. Ablation of HDAC4 led to growth inhibition in human tumor cells but not to detectable effects in normal human dermal fibroblasts (NHDF) or myelopoietic progenitors. HDAC4À/+ or HDAC4À/À murine embryonic fibroblasts showed no detectable growth defects. On the other hand, HDAC4 RNAi in HeLa cells produced mitotic arrest followed by caspase-dependent apoptosis. Mitotically arrested cells showed chromosome segregation defects. Even though the growth of both p53-wild-type and p53-null tumor cells were affected by HDAC4 ablation, segregation defects were observed only in p53-null cells. HDAC4 associates with the PP2A-B56 regulatory subunit, which is known to be involved in chromosome segregation, and RNAi of either the structural subunit A or the regulatory subunit B56 of PP2A also caused chromosome segregation defects. We conclude that HDAC4 is required for cell cycle progression of tumor cells by multiple mechanisms, one of which seems to be specific to p53-deficient cells through chromosome segregation defects. On the contrary, HDAC4 is not required for the progression of NHDF. We therefore suggest that systemic selective interference with the expression or function of HDAC4 is expected to have a significant therapeutic window, in particular, for p53-deficient tumors. [Cancer Res 2009;69(15):6074-82]