Tissue culture cells from several mammalian species, including three primate lines, were transfected with recombinant vectors carrying Escherichia coli xanthine-guanine phosphoribosyltransferase or Tn5 aminoglycoside phosphotransferase dominant selectable markers. Human HeLa and SV40-transformed xeroderma pigmentosum cells exhibited stable transformation frequencies of at least 10(-3) (0.1 percent). CV-1, an African green monkey kidney cell line, could be stably transformed with the exceptionally high frequency of 6 X 10(-2) (6 percent).

Although in vitro models have been a cornerstone of anti-cancer drug development, their direct applicability to clinical cancer research has been uncertain. Using a state-of-the-art Taqman-based quantitative RT-PCR assay, we investigated the multidrug resistance (MDR) transcriptome of six cancer types, in established cancer cell lines (grown in monolayer, 3D scaffold, or in xenograft) and clinical samples, either containing >75% tumor cells or microdissected. The MDR transcriptome was determined a priori based on an extensive curation of the literature published during the last three decades, which led to the enumeration of 380 genes. No correlation was found between clinical samples and established cancer cell lines. As expected, we found up-regulation of genes that would facilitate survival across all cultured cancer cell lines evaluated. More troubling, however, were data showing that all of the cell lines, grown either in vitro or in vivo, bear more resemblance to each other, regardless of the tissue of origin, than to the clinical samples they are supposed to model. Although cultured cells can be used to study many aspects of cancer biology and response of cells to drugs, this study emphasizes the necessity for new in vitro cancer models and the use of primary tumor models in which gene expression can be manipulated and small molecules tested in a setting that more closely mimics the in vivo cancer microenvironment so as to avoid radical changes in gene expression profiles brought on by extended periods of cell culture.

Dengue virus type 2, a member of the family Flaviviridae, encodes a single polyprotein precursor consisting of 3391 amino acids residues that is processed to at least 10 mature proteins by host and viral proteases. The NS3 protein contains a domain commonly found in cellular serine proteinases that in cooperation with NS2B is involved in polyprotein processing. In addition, NS3 and NS5 proteins contain conserved motifs found in several RNA helicases and RNA-dependent RNA polymerases, respectively. Both enzymatic activities have been suggested to be involved in viral RNA replication. In this report, we demonstrate that the NS3 and NS5 proteins interact in vivo in dengue virus type 2-infected monkey kidney (CV-1) cells and in HeLa cells coinfected with recombinant vaccinia viruses encoding these proteins as shown by coimmunoprecipitations and immunoblotting methods. We also show by immunofluorescence, metabolic labeling, and two-dimensional peptide mapping that NS5 is a nuclear phosphoprotein and that phosphorylation occurs on serine residues at multiple sites. Furthermore, NS5 exists in differentially phosphorylated states in the nuclear and the cytoplasmic fractions, and only the cytoplasmic form of NS5 is found to coimmunoprecipitate with NS3, suggesting that differential phosphorylation may control the interaction between these proteins and its function in the viral RNA replicase.

Viral replicases of many positive-strand RNA viruses are membrane-bound complexes of cellular and viral proteins that include viral RNA-dependent RNA polymerase (RdRP). The in vitro RdRP assay system that utilizes cytoplasmic extracts from dengue viral-infected cells and exogenous RNA templates was developed to understand the mechanism of viral replication in vivo. Our results indicated that in vitro RNA synthesis at the 3-untranslated region (UTR) required the presence of the 5-terminal region (TR) and the two cyclization (CYC) motifs suggesting a functional interaction between the TRs. In this study, using a psoralen-UV crosslinking method and an in vitro RdRP assay, we analyzed structural determinants for physical and functional interactions. Exogenous RNA templates that were used in the assays contained deletion mutations in the 5-TR and substitution mutations in the 3-stem-loop structure including those that would disrupt the predicted pseudoknot structure. Our results indicate that there is physical interaction between the 5-TR and 3-UTR that requires only the CYC motifs. RNA synthesis at the 3-UTR, however, requires long range interactions involving the 5-UTR, CYC motifs, and the 3-stem-loop region that includes the tertiary pseudoknot structure.Dengue viruses type 1-4 are members of the flavivirus family of positive-strand RNA viruses. The diseases caused by dengue viruses range from a simple form of dengue fever to a more complex form of dengue hemorrhagic fever/shock syndrome, which exhibits considerable morbidity and mortality, especially among children in the tropical and subtropical regions of the world (for reviews see Refs. 1-3). It is currently estimated that about 40% of the world's population living in these areas is at risk for dengue viral diseases, and about 5% of about one million dengue hemorrhagic/dengue shock syndrome cases are fatal (1). Dengue virus type 2 (DEN2) 1 is the most prevalent serotype identified over a 10-year period in epidemiological studies. DEN2 has a single-stranded RNA genome consisting of 10,723 nt (in New Guinea-C strain) (4) which encodes a single polyprotein arranged in the order, NH 2 -CprM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-COOH. The RNA genome contains a type I cap at the 5Ј-end but lacks a poly(A) tail at the 3Ј-end (5). The polyprotein precursor is processed in the endoplasmic reticulum, where the action of the cellular signal peptidase results in three structural proteins, the components of the virion, the capsid (C), precursor membrane protein (prM), and the envelope (E) protein (6 -8). The remainder of the polyprotein codes for at least seven nonstructural proteins that are generated by the combination of both the cellular signal peptidase and the virally encoded, twocomponent serine protease, NS2B/NS3. The NS3 protein of flaviviruses has a serine protease catalytic triad within the N-terminal region of 180 amino acid residues (9, 10) that requires NS2B for protease activity (11)(12)(13)(14)(15)(16)(17)(18)(19)(20). The conserved motifs found in the NTP-bind...