Studies were conducted to determine whether gene expression profiles following a single dose of radiation would yield equivalent profiles following fractionated radiation in different tumor cell lines. MCF7 (breast), DU145 (prostate), and SF539 (gliosarcoma) cells were exposed to a total radiation dose of 10 Gy administered as a single dose (SD) or by daily multifractions (MF) of 5 Â 2 Gy. Following radiation treatment, mRNA was isolated at 1, 4, 10, and 24 h and processed for cDNA microarray analysis. To determine the influence of the tumor microenvironment on gene expression, one cell type (DU145) was evaluated growing as a solid tumor in athymic nude mice for both radiation protocols. Unsupervised hierarchical cluster map analysis showed significant differences in gene expression profiles between SD and MF treatments for cells treated in vitro, with MF yielding a more robust induction compared with SD. Several genes were uniquely up-regulated by MF treatment, including multiple IFN-related genes (STAT1, G1P2, OAS1, OAS3, G1P3, IFITM1) and TGF-b-associated genes (EGR1, VEGF, THBS1, and TGFB2). DU145 cells grown in vivo exhibited a completely different set of genes induced by both SD and MF compared with the same cells exposed in vitro. The results of the study clearly show distinct differences in the molecular response of cells between SD and MF radiation exposures and show that the tumor microenvironment can significantly influence the pattern of gene expression after radiation exposures. [Cancer Res 2007;67(8):3845-52]

The X-ray crystal structure of epitope II on the E2 protein of hepatitis C virus, in complex with nonneutralizing antibody mAb#12, has been solved at 2.90-Å resolution. The spatial arrangement of the essential components of epitope II (ie, the C-terminal α-helix and the N-terminal loop) was found to deviate significantly from that observed in those corresponding complexes with neutralizing antibodies. The distinct conformations are mediated largely by the flexibility of a highly conserved glycine residue that connects these components. Thus, it is the particular tertiary structure of epitope II, which is presented in a spatial and temporal manner, that determines the specificity of antibody recognition and, consequently, the outcome of neutralization or nonneutralization.H epatitis C is a major public health problem worldwide. More than 170 million people are infected by the hepatitis C virus (HCV) (1). Approximately 70% of infected people fail to clear the virus during the acute phase of the disease and become chronic carriers (2). Liver cirrhosis, which develops in about 10-20% of chronically infected patients, is linked with a high risk for hepatocellular carcinoma in later life (2, 3). To date, there is neither an effective immune globulin for prophylaxis nor a vaccine for the prevention of hepatitis C. The development of a safe and effective HCV vaccine remains a top priority for the global control of HCV infections.The HCV envelope glycoprotein E2 has long been considered an important immunogenic target in efforts to develop an HCV vaccine candidate. This consideration is largely based on the role of the E2 protein in facilitating the entry of HCV into hepatocytes via interaction with the host entry factors (4-10). Recently, the crystal structure of the E2 core, in complex with a neutralizing antibody, was solved (11). The E2 core study described the interface crucial for host entry factor CD81-mediated entry, thus providing a site of vulnerability that can be exploited in immunogen design. The crystal structure also revealed that nearly 62% of the E2 core amino acid residues are either disordered or in loop structures, the overall effect of which indicates a striking flexibility in the E2 protein structures. Whether the intrinsic structural heterogeneity of the E2 protein is linked to the viral entry process or not is currently unknown.Epitope II resides on the E2 protein between residues 427 and 446, a location that places it in the vicinity of the described E2-CD81 interface in the flexible area of the E2 protein (11-13). Paradoxically, different antibodies are able to bind to a similar set of residues on epitope II; however, their interactions with these residues can lead to either HCV neutralization or nonneutralization, as defined in an in vitro HCV cell culture system (12, 13). In addition, some epitope II-specific nonneutralizing antibodies were shown to interfere with the neutralization by antibodies at epitope I, another epitope on the E2 protein between residues 412 and 426 (12). Furthermore, de...

Antibodies to epitopes in the E2 protein of hepatitis C virus (HCV) reduce the viral infectivity in vivo and in vitro. However, the virus can persist in patients in the presence of neutralizing antibodies. In this study, we generated a panel of monoclonal antibodies that bound specifically to the region between residues 427 and 446 of the E2 protein of HCV genotype 1a, and we examined their capacity to neutralize HCV in a cell culture system. Of the four monoclonal antibodies described here, two were able to neutralize the virus in a genotype 1a-specific manner. The other two failed to neutralize the virus. Moreover, one of the nonneutralizing antibodies could interfere with the neutralizing activity of a chimpanzee polyclonal antibody at E2 residues 412 to 426, as it did with an HCV-specific immune globulin preparation, which was derived from the pooled plasma of chronic hepatitis C patients. Mapping the epitope-paratope contact interfaces revealed that these functionally distinct antibodies shared binding specificity for key amino acid residues, including W 437 , L 438 , L 441 , and F 442 , within the same epitope of the E2 protein. These data suggest that the effectiveness of antibody-mediated neutralization of HCV could be deduced from the interplay between an antibody and a specific set of amino acid residues. Further understanding of the molecular mechanisms of antibody-mediated neutralization and nonneutralization should provide insights for designing a vaccine to control HCV infection in vivo.