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At least 235 records · Page 13Linked to original sources
PubMed1976

Integrated simian virus 40 sequences in transformed cell DNA: analysis using restriction endonucleases.

The DNAs from five independent simian virus 40 (SV40) transformants of the BALB/c3T3 mouse cell line were digested with either the HpaII or the BamHI restriction endonuclease and the resulting fragments were fractionated by gel electrophoresis. The DNA fragments were denatured in situ in the gel and transferred to a membrane filter. Fragments containing viral DNA were detected by hybridization with high specific activity 32P-labeled SV40 complementary RNA (cRNA) synthesized in vitro. Each of the lines yielded a small number of fragments containing SV40 DNA and the fragments from each line were different. This observation shows that the structure of the integrated SV40 DNA and/or its location in the host DNA are different in each line.

Cell Line↗
PubMed2006

Significance of murine retroviral mutagenesis for identification of disease genes in human acute myeloid leukemia.

Retroviral insertion mutagenesis is considered a powerful tool to identify cancer genes in mice, but its significance for human cancer has remained elusive. Moreover, it has recently been debated whether common virus integrations are always a hallmark of tumor cells and contribute to the oncogenic process. Acute myeloid leukemia (AML) is a heterogeneous disease with a variable response to treatment. Recurrent cytogenetic defects and acquired mutations in regulatory genes are associated with AML subtypes and prognosis. Recently, gene expression profiling (GEP) has been applied to further risk stratify AML. Here, we show that mouse leukemia genes identified by retroviral insertion mutagenesis are more frequently differentially expressed in distinct subclasses of adult and pediatric AML than randomly selected genes or genes located more distantly from a virus integration site. The candidate proto-oncogenes showing discriminative expression in primary AML could be placed in regulatory networks mainly involved in signal transduction and transcriptional control. Our data support the validity of retroviral insertion mutagenesis in mice for human disease and indicate that combining these murine screens for potential proto-oncogenes with GEP in human AML may help to identify critical disease genes and novel pathogenetic networks in leukemia.

Acute Disease↗
PubMed1981

Titration of integrated simian virus 40 DNA sequences, using highly radioactive, single-stranded DNA probes.

Nick-translated simian virus 40 (SV40) [32P]DNA fragments (greater than 2 X 10(8) cpm/micrograms) were resolved into early- and late-strand nucleic acid sequences by hybridization with asymmetric SV40 complementary RNA. Both single-stranded DNA fractions contained less than 0.5% self-complementary sequences; both included [32P]-DNA sequences that derived from all regions of the SV40 genome. In contrast to asymmetric SV40 complementary RNA, both single-stranded [32P]DNAs annealed to viral [3H]DNA at a rate characteristic of SV40 DNA reassociation. Kinetics of reassociation between the single-stranded [32P]DNAs indicated that the two fractions contain greater than 90% of the total nucleotide sequences comprising the SV40 genome. These preparations were used as hybridization probes to detect small amounts of viral DNA integrated into the chromosomes of Chinese hamster cells transformed by SV40. Under the conditions used for hybridization titrations in solution (i.e., 10- to 50-fold excess of radioactive probe), as little as 1 pg of integrated SV40 DNA sequence was assayed quantitatively. Among the transformed cells analyzed, three clones contained approximately one viral genome equivalent of SV40 DNA per diploid cell DNA complement; three other clones contained between 1.2 and 1.6 viral genome equivalents of SV40 DNA; and one clone contained somewhat more than two viral genome equivalents of SV40 DNA. Preliminary restriction endonuclease maps of the integrated SV40 DNAs indicated that four clones contained viral DNA sequences located at a single, clone-specific chromosomal site. In three clones, the SV40 DNA sequences were located at two distinct chromosomal sites.

Animals↗
PubMed1993

Integration of murine leukemia virus DNA depends on mitosis.

In synchronized rat or mouse cells infected with Moloney murine leukemia virus (MLV), integration of viral DNA and production of viral proteins occur only after the cells traverse mitosis. Integration is blocked when cells are prevented from progressing through mitosis. Viral nucleoprotein complexes isolated from arrested cells contain full-length viral DNA and can integrate this viral DNA in vitro, showing that the block to integration in arrested cells is not due to a lack of mature integration machinery. When infected cells traverse mitosis, there is a sharp increase in nuclear accumulation of viral DNA. The dependence of integration on mitosis may therefore be due to a requirement for mitosis and nuclear envelope breakdown for entry of the viral integration complex into the nucleus.

Animals↗
PubMed1998

Hepatocellular carcinomas infected with the novel TT DNA virus lack viral integration.

A novel DNA virus designated TT virus (TTV) was cloned from a patient with posttransfusion hepatitis and is thought to be a new hepatitis virus. At present, hepatitis B virus (HBV) and hepatitis C virus (HCV) are known to induce hepatocellular carcinoma (HCC). But, actually, in Japan approximately 5 to 10% of HCCs are in HBV-negative and HCV-negative (NBNC) patients. In order to study the possible role of TTV in hepatocarcinogenesis, we investigated the frequency of the TTV genome in liver tissue of 20 HCC patients. As a result, 3 of 8 NBNC HCC patients and 5 of 12 HBV- or HCV-associated HCC patients were TTV positive, and TTV was shown not to be specific for NBNC HCC. For all TTV-positive patients, we also confirmed that the TTV genome was not integrated into host hepatocyte DNA.

Aged↗
PubMed2002

Constitutive activation of Stat5a by retrovirus integration in early pre-B lymphomas of SL/Kh strain mice.

We found that the second intron of Stat5a was one of the common integration sites of the endogenous ecotropic murine leukemia virus, i.e., SL/Kh virus integration-1 (Svi1), in early pre-B lymphomas in SL/Kh mice. The high expression of STAT5A induced by Svi1 integration and activation accelerated the transcription of its target genes such as c-Myc. Transfection of the constitutively active Stat5a mutant cDNA, but not of the wild-type cDNA, to the bone marrow cells induced colony formation of pre-B cells in a methylcellulose medium and escaped from dependence on IL-7. Such growth depended on a genetic factor in the SL/Kh strain. Consitutively high expression of Stat5a either by retrovirus integration or transfection of active mutant cDNA can be lymphomagenic to early pre-B cells in collaboration with a certain genetic background factor of mice.

3' Untranslated Regions↗
PubMed2006

Adeno-associated virus rep protein-mediated inhibition of transcription of the adenovirus major late promoter in vitro.

Adeno-associated virus (AAV) is a human parvovirus that normally requires a helper virus such as adenovirus (Ad) for replication. The four AAV replication proteins (Rep78, Rep68, Rep52, and Rep40) are pleiotropic effectors of virus integration, replication, transcription, and virion assembly. These proteins exert effects on Ad gene expression and replication. In transient plasmid transfection assays, Rep proteins inhibit gene expression from a variety of transcription promoters. We have examined Rep protein-mediated inhibition of transcription of the Ad major late transcription promoter (AdMLP) in vitro. Rep78/68 are the strongest transcription suppressors and the purine nucleotide binding site in the Rep proteins, and by implication, the ATPase activity or conformational change induced by nucleotide binding is required for full repression. Rep52 has modest effects, and Rep40 exerts no significant effect on transcription. Rep78/68 and their N-terminal 225-residue domain bind to a 55-bp AdMLP DNA fragment in gel shift assays, suggesting that protein-DNA interactions are required for inhibition. This interaction was confirmed in DNase I protection assays and maps to a region extending from the TATA box to the transcription initiation site. Gel shift, DNase I, and chemical cross-linking assays with TATA box-binding protein (TBP) and Rep68 indicate that both proteins interact with each other and with the promoter at adjacent sites. The demonstration of Rep interaction with TBP and the AdMLP suggests that Rep78/68 alter the preinitiation complex of RNA polymerase II transcription. These observations provide new insight into the mechanism of Rep-mediated inhibition of gene expression.

Adenosine Triphosphatases↗
PubMed2002

Simultaneous mapping of human papillomavirus integration sites and molecular karyotyping in short-term cultures of cervical carcinomas by using 49-color combined binary ratio labeling fluorescence in situ hybridization.

Infection with high-risk type human papillomavirus (HPV) is a necessary causal factor in the pathogenesis of cervical carcinoma. In most invasive cervical cancers, HPV is integrated in the host cell genome, and additional genetic aberrations are observed among which are chromosomal aberrations. To analyze in detail such often complex chromosomal changes and simultaneously map HPV integration sites, we extended the multiplicity of the combined binary ratio labeling fluorescence in situ hybridization (COBRA-FISH) technique to 49 by inclusion of a large Stokes' shift fluorochrome as the third binary label. The technique allows mapping of the integrated HPV genome in the context of p- and q-arm COBRA-FISH, with a sensitivity of one copy of the HPV genome as tested for HPV 16 in SiHa cells. We investigated the molecular karyotypes and integration patterns of HPV types 16 and 18 in metaphase spreads from short-term cultures of primary cervical carcinomas (n=5). Of the tested cervical carcinomas, two contained integrated HPV at 8q24, one of which in addition harbored the integrated virus near a translocation breakpoint. Two carcinomas had integrated HPV at 17q21 through 23 in a morphologically normal chromosome 17. One carcinoma contained HPV at 1q42 in a morphologically normal chromosome 1. Our data illustrate the efficacy of 49-color COBRA-FISH to resolve complex karyotypes and simultaneously map specific sequences in metaphases obtained from short-term solid tumor cultures.

Color↗
PubMed2004

Association of the gene encoding wingless-type mammary tumor virus integration-site family member 5B (WNT5B) with type 2 diabetes.

Recent reports have suggested that WNT signaling is an important regulator for adipogenesis or insulin secretion and might be involved in the pathogenesis of type 2 diabetes. To investigate possible roles of the WNT genes in conferring susceptibility to type 2 diabetes, we examined the association of the genes that encode members of the WNT family with type 2 diabetes in the Japanese population. First, 40 single-nucleotide polymorphism (SNP) loci within 11 WNT genes were analyzed in 188 subjects with type 2 diabetes (case-1) and 564 controls (control-1). Among them, six SNP loci exhibited a significant difference (P<.05) in the allele and/or genotype distributions between case and control subjects. These SNP loci were further analyzed in another set of case (case-2; n=733) and control (control-2; n=375) subjects to confirm their statistical significance. As a result, one SNP locus in the WNT5B gene was strongly associated with type 2 diabetes ( chi 2=15.6; P=.00008; odds ratio=1.74; 95% confidence interval 1.32-2.29). Expression of the WNT5B gene was detectable in several tissues, including adipose, pancreas, and liver. Subsequent in vitro experiments identified the fact that expression of the Wnt5b gene was increased at an early phase of adipocyte differentiation in mouse 3T3-L1 cells. Furthermore, overexpression of the Wnt5b gene in preadipocytes resulted in the promotion of adipogenesis and the enhancement of adipocytokine-gene expression. These results indicate that the WNT5B gene may contribute to conferring susceptibility to type 2 diabetes and may be involved in the pathogenesis of this disease through the regulation of adipocyte function.

3T3 Cells↗
PubMed1988

Human bronchial epithelial cells with integrated SV40 virus T antigen genes retain the ability to undergo squamous differentiation.

Human bronchial epithelial cells transformed by either DNA virus infection (SV40 or Adenovirus 12-SV40 hybrid virus) or transfection with the SV40 large T antigen gene were studied for their ability to undergo squamous differentiation when exposed to 12-O-tetradecanoylphorbol-13 acetate (TPA), transforming growth factor-beta 1 (TGF-beta 1), or fetal bovine serum (FBS), agents that induce the squamous differentiation of normal human bronchial epithelial cells. Squamous differentiation occurred in all ten T-antigen-positive cell cultures when they were exposed to either FBS or TGF-beta 1, but none differentiated when exposed to TPA. From one cell line, designated BEAS-2B, two subclones were isolated, one of which was induced to undergo squamous differentiation by FBS, and a second that failed to undergo squamous differentiation and was mitogenically stimulated when exposed to serum. These phenotypically different subclones provide a new in vitro cellular system for delineating the mechanism(s) of human bronchial epithelial cell squamous differentiation in response to FBS or TGF-beta 1.

Antigens, Viral↗
PubMed1983

Evidence of transcription from the late region of the integrated simian virus 40 genome in transformed cells: location of the 5' ends of late transcripts in cells abortively infected and in cells transformed by simian virus 40.

By means of S1 mapping, we observed that spliced 16S and 19S viral late mRNAs--in addition to early mRNAs--were present in cytoplasmic polyadenylated RNA preparations from simian virus 40-transformed cell lines of rat or mouse origin containing no detectable amount of free viral DNA. The amounts of early and late virus-specific mRNAs in these lines were quantified by hybridization of radioactive cytoplasmic polyadenylated RNA with cloned region-specific restriction fragments. The relative amount of late viral mRNA produced in these transformed cells was found to be of the same order as that produced in simian virus 40-infected, nonpermissive baby mouse kidney cells. Moreover, by using the S1 nuclease protection method, we compared the 5' ends of late mRNAs produced (i) in transformed cells, (ii) in abortively infected mouse cells, and (iii) in the late phase of the lytic cycle. The 5' ends of late mRNAs both in abortively infected and in transformed cells were less heterogeneous than the 5' ends of late mRNAs produced during the lytic cycle; however, they were a subset of the 5' ends of late transcripts produced in the lytic cycle.

Animals↗
PubMed1995

HPV DNA positivity and natural killer cell activity in the clinical outcome of mild cervical dysplasia: integration between virus and immune system.

The objective was to examine the prevalence of human papillomavirus (HPV) DNA infection in mild cervical dysplasia and to evaluate longitudinally the persistence of HPV DNA positivity in an observational study, aiming at identifying the role of peripheral blood lymphocyte natural killer activity in the natural history of dysplastic disease. Twenty-three patients with histologically proven mild cervical dysplasia were selected. The HPV DNA positivity, determined by polymerase chain reaction, and cervical dysplasia were monitored cytologically and colposcopically at the 3rd (time 1), 6th (time 2) and 12th months (time 3), and defined by biopsies for routine histology taken at times 2 and 3. For each patient included in the study, the immune reactivity was evaluated at the time of diagnosis and afterwards, longitudinally during the follow-up. The immune status analysis included T lymphocyte subsets (CD3, CD4, CD8, CD56, CD16 monoclonal antibodies by Beckton Dickinson, Mountain View, Calif., USA) and determinations of natural killer cell activity (against the sensitive cell line K 562). Eighteen out of the 23 women with mild cervical dysplasia (78.3%) were found positive for HPV DNA, with a significantly high representation of HPV DNA type 16 (55.6% of cases). At the end of the study, 12 out of 18 HPV-DNA-positive women became negative (defined by two or more negative tests) for the original HPV DNA type, with 66.7% of spontaneous HPV DNA negativization rate (p = 0.6).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗