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At least 361 records · Page 20Linked to original sources
PubMed1987

Mammary preneoplasia and tumorigenesis in the BALB/c mouse: structure and modification of mouse mammary tumor virus DNA sequences.

Mouse mammary tumor virus (MMTV) DNA sequences were examined in mammary tissues from BALB/c mice, including both preneoplastic hyperplastic alveolar nodule (HAN) outgrowth lines, tumors derived from those preneoplastic tissues, and DMBA-induced mammary tumors. Over 60 different HAN and tumors samples were analyzed. The D2 HAN line contained one additional provirus, whereas Cv-2 and Cv-4 HAN lines that are infected with exogenous virus exhibited multiple virus integration events. D2 tumors showed the same provirus pattern as D2 HANs, whereas Cv-2 and Cv-4 tumors exhibited a subset of the acquired proviruses found in the parental HAN populations. Differential methylation patterns of virus-specific sequences were observed that resembled those described for other tissues in which viral DNA replication and integration has occurred, i.e., acquired proviruses were hypomethylated and endogenous proviruses were methylated. In tumors that arose from HAN lines and exhibited only endogenous proviruses, demethylation of the subgenomic Mtv-6 locus was observed. Demethylation of Mtv-6 was not detected in any of the preneoplastic tissues. Altered methylation of Mtv-8 and -9 was observed in both Cv-2 and Cv-4 tumors. Finally, mammary tumors induced by DMBA carried no acquired proviruses and demethylation of endogenous MMTV proviruses was demonstrated.

5-Methylcytosine↗
PubMed1989

Induction of multiple independent T-cell lymphomas in rats inoculated with MOloney murine leukemia virus.

Tumor cell DNA derived from different lymphoid organs of 30 rats serially inoculated at birth with Moloney murine leukemia virus (MoMuLV) was examined by Southern blot analysis and hybridization to the following DNA probes: MoMuLV long terminal repeat (LTR), Moloney leukemia virus integration regions 1, 2, 3, and 4 (Mlvi-1, Mlvi-2, Mlvi-3, and Mlvi-4), T-cell receptor beta locus, and immunoglobulin heavy chain locus. This analysis revealed that the tumors segregating in different lymphoid organs in 10% of the animals were clonally unrelated. These findings are consistent with the hypothesis that the MoMuLV-induced rat thymic lymphomas are polyclonal in origin. At least two factors may be responsible for this phenomenon: (i) increase in the number of the available target cells in virus-infected animals, and (ii) genetic instability associated with provirus integration in the developing premalignant clones.

Animals↗
PubMed1994

Immunodetection of the 33 K/92 K polymerase proteins in cymbidium ringspot virus-infected and in transgenic plant tissue extracts.

An antiserum was raised against the 33 K protein encoded by the 5' proximal gene of cymbidium ringspot tombusvirus RNA. This antiserum reacts specifically with the 33 K and 92 K proteins, which constitute the viral replicase, in CyRSV-infected Nicotiana benthamiana plants and in transgenic plants transformed with the full-length replicase gene. In inoculated leaves of infected plants, synthesis of the 33 K/92 K proteins stops ten days after inoculation, whereas in newly produced systemically infected leaves there was continuous production of these proteins. In transgenic plants, both proteins were detected showing that readthrough of the termination codon of the 33 K protein does not depend on the presence of the replicating virus. The subcellular localization of the 33 K/92 K proteins is similar in infected and transgenic plants. No correlation was found between the level of expression of integrated virus gene and level of resistance to the challenging virus.

Amino Acid Sequence↗
PubMed1984

Amplification and rearrangement in hepatoma cell DNA associated with integrated hepatitis B virus DNA.

DNA of hepatitis B virus is found to be integrated into the genome of infected human liver cells and may be related to the development of primary liver carcinoma. We have previously reported the cloning of cellular DNA with integrated HBV sequences from the PLC/PRF/5 cell line which derives from a human primary liver carcinoma. Two clones, designated as A-10.7 and A-10.5, and a third uncloned fragment are compared by restriction enzyme mapping, hybridization and nucleotide sequencing. The results indicate that amplification of integrated viral DNA and host flanking regions has occurred, followed by transposition and/or major deletions. The implications of these findings for the development of primary liver carcinoma are discussed.

Base Sequence↗
PubMed1985

Co-transcribed 3' host sequences augment expression of integrated hepatitis B virus DNA.

We have previously reported the cloning and structural analysis of integrated hepatitis B virus DNA copies from the human hepatoma cell line PLC/PRF/5. Here we show that the cloned DNA fragments of 10.7 kb and 10.5 kb contain intact coding sequences for HBsAg since Ltk- cells transfected with these DNAs secrete considerable amounts of HBsAg. We show for the 10.7-kb fragment that multiple readthrough messages composed of viral as well as cellular sequences are transcribed. These RNAs differ only in their 3' sequences. Furthermore, the 10.7-kb insert leads to a substantial increase in HBsAg produced compared with HBV DNA and with the 10.5-kb insert. We provide evidence that the different 3' sequences on the HBsAg transcripts account for the augmentation of expression.

Animals↗
PubMed1983

Excision of the DBA ecotropic provirus in dilute coat-color revertants of mice occurs by homologous recombination involving the viral LTRs.

The dilute (d) coat color mutation of DBA/2J mice is closely associated with the site of integration of an ecotropic murine leukemia provirus. In five independent dilute revertants (d+) examined, reversion to wild-type coat-color correlated with the loss of most of the ecotropic proviral genome, indicating that virus integration caused the mutation. Molecular cloning and restriction enzyme analysis of the viral preintegration site as well as two independent dilute revertants, showed that exactly one long terminal repeat (LTR) remained in each revertant DNA, suggesting that virus excision occurred by homologous recombination involving the viral LTRs.

Alleles↗
PubMed1993

Integration of simian virus 40 into cellular DNA occurs at or near topoisomerase II cleavage hot spots induced by VM-26 (teniposide).

Inhibition of DNA topoisomerase II in simian virus 40 (SV40)-infected BSC-1 cells with a topoisomerase II poison, VM-26 (teniposide), resulted in rapid conversion of a population of the SV40 DNA into a high-molecular-weight form. Characterization of this high-molecular-weight form of SV40 DNA suggests that it is linear, double stranded, and a recombinant with SV40 DNA sequences covalently joined to cellular DNA. The majority of the integrants contain fewer than two tandem copies of SV40 DNA. Neither DNA-damaging agents, such as mitomycin and UV, nor the topoisomerase I inhibitor camptothecin induced detectable integration in this system. In addition, the recombination junctions within the SV40 portion of the integrants correlate with VM-26-induced, topoisomerase II cleavage hot spots on SV40 DNA. These results suggest a direct and specific role for topoisomerase II and possibly the enzyme-inhibitor-DNA ternary cleavable complex in integration. The propensity of poisoned topoisomerase II to induce viral integration also suggests a role for topoisomerase II in a pathway of chromosomal DNA rearrangements.

Animals↗
PubMed1991

[Integration of carcinogenic viruses in the genome of in-vitro cultured human fibroblasts].

Avian sarcoma viruses are known for inducing no transformation of human diploid fibroblasts. Nevertheless, we show that the Rous sarcoma virus can infect and transform some human fibroblastic cell lines, replicate and express viral proteins, integrate into the host genome and prevent expression of MHC class I antigens on cell membranes. Cell transformation happens together with important and significant abnormalities of the cell karyotype and proviral integration is most often close to the c-src oncogene on chromosomes 1 and 20.

Avian Sarcoma Viruses↗
PubMed1988

Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line.

Here we describe development and application of highly sensitive fluorescence methodology for localization of single-copy sequences in interphase nuclei and metaphase chromosomes by nonisotopic in situ hybridization. Application of this methodology to the investigation of Epstein-Barr virus integration in the Namalwa lymphoma cell line has revealed two EBV genomes closely integrated at the known site on chromosome 1. Detecting sequences as small as 5 kb, we further demonstrate resolution within interphase nuclei of two fragments of the viral genome spaced only 130 kb apart. Results indicate that the viral genomes are in opposite orientations and separated by roughly 340 kb of cellular DNA. This work demonstrates the feasibility and resolving power of interphase chromatin mapping to assess the proximity of closely spaced DNA sequences. Implications for virology, gene mapping, and investigation of nuclear organization are discussed.

Base Sequence↗
PubMed2005

TFII-I regulates induction of chromosomally integrated human immunodeficiency virus type 1 long terminal repeat in cooperation with USF.

Human immunodeficiency virus type 1 (HIV-1) replication is coupled to T-cell activation through its dependence on host cell transcription factors. Despite the enormous sequence variability of these factors, several cis elements for host factors are highly conserved within the 5' long terminal repeats (LTRs) of viruses from AIDS patients; among these is the RBEIII upstream element for the Ras response element binding factor 2 (RBF-2). Here we show that RBF-2 is comprised of a USF1/USF2 heterodimer and TFII-I, which bind cooperatively to RBEIII. Recombinant USF1/USF2 binds to the RBEIII core sequence 160-fold less efficiently than it binds to an E box element, but the interaction with RBEIII is stimulated by TFII-I. Chromosomally integrated HIV-1 LTRs bearing an RBEIII mutation have slightly elevated basal transcription in unstimulated Jurkat cells but are unresponsive to cross-linking of the T-cell receptor or stimulation with phorbol myristate acetate (PMA) and ionomycin. Induction is inhibited by dominant interfering USF and TFII-I but not by the dominant negative I-kappaB protein. USF1, USF2, and TFII-I bind to the integrated wild-type LTR in unstimulated cells and become phosphorylated during the induction of transcription upon stimulation with PMA. These results demonstrate that USF1/USF2 and TFII-I interact cooperatively at the upstream RBEIII element and are necessary for the induction of latent HIV-1 in response to T-cell activation signals.

Animals↗
PubMed1980

Arrangement of integrated avian sarcoma virus DNA sequences within the cellular genomes of transformed and revertant mammalian cells.

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.

Animals↗
PubMed1989

Instability of integrated hepatitis B virus DNA with inverted repeat structure in a transgenic mouse.

We established four cell lines, from the liver cells of a transgenic mouse, constructed with hepatitis B virus DNA that had an inverted repeat structure. The integrated DNA patterns of the four established cell lines were different from one another and from the original pattern. These data show that the instability of integrated hepatitis B virus DNA would also occur in somatic cells during replication, apart from meiosis, which was previously reported.

Animals↗
PubMed1997

Directed integration of minute virus of mice DNA into episomes.

Recent studies with adeno-associated virus (AAV) have shown that site-specific integration is directed by DNA sequence motifs that are present in both the viral replication origin and the chromosomal preintegration DNA and that specify binding and nicking sites for the viral regulatory Rep protein. This finding raised the question as to whether other parvovirus regulatory proteins might direct site-specific recombination with DNA targets that contain origin sequences functionally equivalent to those described for AAV. To investigate this question, active and inactive forms of the minute virus of mice (MVM) 3' replication origin, derived from a replicative-form dimer-bridge intermediate, were propagated in an Epstein-Barr virus-based shuttle vector which replicates as an episome in a cell-cycle-dependent manner in mammalian cells. Upon MVM infection of these cells, the infecting genome integrated into episomes containing the active-origin sequence reported to be efficiently nicked by the MVM regulatory protein NS1. In contrast, MVM did not integrate into episomes containing either the inactive form of the origin sequence reported to be inefficiently nicked by NS1 or the active form from which the NS1 consensus nick site had been deleted. The structure of the cloned MVM episomal recombinants displayed several features previously described for AAV episomal and chromosomal recombinants. The findings indicate that the rules which govern AAV site-specific recombination also apply to MVM and suggest that site-specific chromosomal insertions may be achievable with different autonomous parvovirus replicator proteins which recognize binding and nicking sites on the target DNA.

Animals↗
PubMed1991

Episomal and integrated human papillomavirus in cervical neoplasia shown by non-isotopic in situ hybridisation.

It was postulated that non-isotopic in situ hybridisation (NISH) signal types 1-3 for human papillomavirus in cervical biopsy specimens represent episomal or integrated virus. The aim of this study was to validate this hypothesis by independent molecular techniques. Fresh cervical intraepithelial neoplasia (CIN) and squamous cell cancer (SCC) tissue were examined for NISH signal pattern by hybridising with digoxigenin labelled HPV 16. DNA was extracted from the same samples and analysed by restriction endonuclease digestion and Southern blotting to determine the physical state of the viral genome. Six CIN biopsy specimens showed a type 1 NISH signal for HPV 16. On Southern analysis these biopsy specimens contained only episomal HPV 16. Three SCC with a type 2 NISH signal contained integrated HPV 16 by Southern analysis. Two specimens, a CIN 3 and an SCC with a type 3 NISH signal for HPV 16, showed the presence of both episomal and integrated HPV 16 with conventional Southern analysis and two dimensional gel electrophoresis. These results show that episomal HPV can be reliably determined by NISH type 1 signal, integrated HPV by type 2, and a combination of both episomal and integrated HPV, by a type 3 signal in archival paraffin wax embedded cervical biopsy specimens. This will add another variable to the epidemiological studies of HPV infection. In particular, it will now allow retrospective studies to be done to define the role of episomal and integrated HPV in the evolution of cervical intraepithelial neoplasia and other cervical disease associated with this virus.

Blotting, Southern↗
PubMed1994

Analysis of integrated hepatitis B virus DNA and flanking cellular sequences in a childhood hepatocellular carcinoma.

The DNA of tumor tissue K1 obtained at autopsy from a case of hepatocellular carcinoma (HCC) in a 9-year-old boy contained integrated hepatitis B virus (HBV) DNA at a single site in the chromosome (case 2, Chang et al.: Hepatology 13:316-320, 1991). To characterize further the integrated viral DNA sequences, a genomic library of the K1 DNA was constructed in the lambda L47.1 vector. One phage clone, designated KTM-1, containing integrated HBV DNA and cellular flanking sequences was obtained from this library. The restriction map and DNA sequence of this clone showed that the integrated HBV DNA was partially deleted and rearranged. The most conserved viral DNA sequences were surface and X genes and arranged in the opposite orientation. The viral core gene was not present. Using chloramphenicol acetyltransferase (CAT) assay, the C-terminal truncated X open reading frame was demonstrated to retain its trans-activating ability. The result suggested that the functional integrated X gene may play a role in hepatocarcinogenesis. The study also showed that the right cellular flanking sequences were human alphoid repetitive sequences.

Base Sequence↗
PubMed2005

Impact of gene-modified T cells on HIV infection dynamics.

Previous clinical trials in HIV-infected patients involving infusion of T cells protected by an antiviral gene have failed to show any therapeutic benefit. The value of such a treatment approach is thus still highly controversial. In this study, the anticipated effects of gene-modified cells on virus and T-cell kinetics are analysed by mathematical modeling. Because technically only a small fraction of all T cells in a patient can be manipulated ex vivo, therapeutic success will depend on the accumulation of gene-modified cells after infusion into the patient by in vivo selection. Our simulations predict that a significant therapeutic benefit is conferred only by antiviral genes that inhibit HIV replication before virus integration (class I genes). Genes that inhibit viral protein expression (class II, used in previous clinical trials), require a much higher inhibitory activity than class I genes to promote the regeneration of T cells and reduce the viral load. Inhibition of virus assembly and release alone (class III) confers no selective advantage to the T cell and is therefore ineffective unless combined with class I (or, possibly, class II) genes. Also crucial in determining the clinical outcome are the regenerative capacity of the gene-modified cells and the level of HIV replication in the patient. These results can be important for guiding future strategies in the field of gene therapy for HIV infection.

Adoptive Transfer↗
PubMed1996

Overexpression of int-5/aromatase in mammary glands of transgenic mice results in the induction of hyperplasia and nuclear abnormalities.

Our recent studies have shown that the cellular gene at the mouse mammary tumor virus integration site in the int-5 locus is aromatase. To study the role of int-5/aromatase in normal mammary development and mammary neoplasia, we have generated transgenic mice that overexpress int-5/aromatase under the control of mouse mammary tumor virus enhancer/promoter. All the transgenic virgin (n = 10) and postlactational (n = 15) females that overexpress int-5/aromatase show various histological abnormalities. Overexpression of int-5/aromatase in mammary glands of virgin females leads to the enlargement of 40% of ducts, of which 65% had hyperplastic lesions, 20% had dysplastic lesions, and 15% had fibroadenoma lesions. Overexpression of int-5/aromatase in involuted mammary glands of transgenic females induces hyperplasia in 75-80% of ducts and glands that exhibit a range of morphological abnormalities, including formation of hyperplastic alveolar nodule (10%), ductal and glandular hyperplasia (70-80%), ductal and lobular dysplasia (15%), and nuclear abnormalities (2-5%) such as multinucleation and karyomegaly, which are all indicative of preneoplastic changes. Our results show that early exposure of mammary epithelium to in situ estrogen and continued exposure to in situ estrogen as a result of overexpression of int-5/aromatase appears to predispose mammary tissue to preneoplastic changes, which may, in turn, increase the risk of developing neoplasia and increase susceptibility to environmental carcinogens. These findings support clinical and experimental data that suggest that early estrogen exposure increases breast cancer risk.

Animals↗