Biomedical subjects

J Feunteun

Publications and source records attributed to J Feunteun.

At least 73 records · Page 4Linked to original sources

PubMed1992

Transformation by hamster polyomavirus: identification and functional analysis of the early genes.

A strategy involving polymerase chain reaction amplification of cDNAs was designed to study the expression of the hamster polyomavirus (HaPV) early region in HaPV-transformed rat fibroblasts, productively HaPV-infected cells, and HaPV-induced lymphoma. We identified three mRNAs resulting from alternative splicing of open reading frames leading to coding capacities for three polypeptides with molecular weights similar to those of the murine polyomavirus large T, middle T (MT), and small T (ST) antigens. The corresponding intronless cDNAs direct the in vitro synthesis of polypeptides with the expected electrophoretic mobilities. The biological activities carried by the HaPV early genes were assayed by transfection of appropriate cell systems. The fragment of genomic viral DNA that encodes the three early antigens contains all of the genetic information necessary for immortalization of primary rat embryo fibroblasts and transformation of F111 rat cells. The large T antigen is sufficient for immortalization, although the MT and ST antigens stimulate the growth and modify the phenotype of immortal cell lines. A stringent cooperative effect was observed in the transformation of F111 cells, which requires the simultaneous presence of the MT and ST antigens, as opposed to the transformation by murine polyomavirus, which can be carried out by the MT antigen alone.

Animals↗

PubMed1992

Early gene expression in lymphoma-associated hamster polyomavirus viral genomes.

Hamster polyomavirus (HaPV) is the causal agent of hair follicle epithelioma in hamsters belonging to a colony bred in Berlin-Buch. These tumors shed virus particles that are assembled in the keratinized layer of the epidermis. By contrast, HaPV induces lymphomas after inoculation into newborn hamsters from a distinct colony bred in Potsdam. These lymphoid tumors accumulate massive amounts of episomal viral genomes characterized by deletions that alter specifically the regulatory and the late coding sequences. Assuming that these alterations of the regulatory region may affect the transcription of the viral oncogenes in the tumor cells, the transcriptional activity of the wild-type and deleted early promoters have been studied in vitro in transient chloramphenicol acetyltransferase (CAT) expression assays. These assays performed in various cell types demonstrate that both versions of the HaPV early promoter carry a weak constitutive activity. Simultaneous expression of the HaPV early gene products leads to a strong stimulation of CAT activity with a concomitant activation of the replication of the plasmid constructs. The results obtained with origin-defective CAT vectors indicate that the replication contributes significantly to the stimulating effect of the early gene products. Indeed, transfection of massive amounts of CAT vectors that are unable to replicate can simulate the dosage effect of replication and also leads to measurable CAT activities. Under these conditions, the wild-type promoter is more active than the deleted version, indicating that sequences within the deletion carry a distinct stimulatory effect on transcription. This conclusion is supported by the observation that the lymphoma cells contain a low level of early transcripts, indicating that the deleted episomal viral templates accumulated in these tumors carry a weak transcriptional activity.

Animals↗

PubMed1991

Familial breast-ovarian cancer locus on chromosome 17q12-q23.

Familial breast cancer has been linked to the D17S74 locus on chromosome 17q. To confirm this finding and to investigate whether ovarian cancer is also linked to this locus, five large families with a hereditary predisposition to cancer of the breast and ovary were investigated. Three families were positive for linkage. For the largest family the lod score was 2.72. These findings suggest that the chromosomal region 17q12-q23, previously shown to contain a gene for early-onset breast cancer, is also associated with a proportion of hereditary ovarian cancers.

Adult↗

PubMed1991

Stable cell line of T-SV40 immortalized human glomerular visceral epithelial cells.

Human subcultures (third passage) of glomerular visceral epithelial cells (VEC) isolated from one month old kidney were successfully transfected by two recombinant plasmids containing the cloned oncogenes from the simian virus 40 large T antigen and H-ras gene. One postcrisis cell clone (56/10 A1) was selected, propagated and characterized. One hundred percent of the 56/10 A1 cells (current passage greater than 100th; doubling time 30 hrs) expressed the nuclear T-SV40 antigen assayed by IF; the cells failed to express H-ras (RNA blot analysis). Immortalized cells were morphologically and phenotypically compared to parental cell type (third passage). Phenotypic characterization of the 56/10 A1 cells was achieved using indirect immunofluorescence (IF) and immunogold silver staining coupled to bright field and epipolarization microscopy. Both parental and 56/10 A1 cells displayed positivity for cytokeratin, CALLA and PHM5, whereas von Willebrand factor was not detected in the two cell types. Since we have previously shown that human glomerular epithelial cells in culture synthetize plaminogen activator (PA) related compounds, we investigated the secretion pattern of these products in parental and transfected cells. Zymographic analysis of secreted PA related compounds revealed production of free urokinase (u-PA) and type 1 plasminogen activator inhibitor (PAI-1) complexed to tissular plasminogen activator (t-PA). Finally, in the transfected cells, increased cGMP generation under atrial natriuretic factor (ANF) stimulation agreed with previous work performed on nontransfected human VEC. In conclusion, the establishment of a human permanent cell line which retains most of the phenotypic features of parental glomerular visceral epithelial cells should represent a new tool to study human glomerular cell functions.

Antigens, Viral, Tumor↗

PubMed1991

Identification and characterization of the hamster polyomavirus middle T antigen.

Hamster polyomavirus (HaPV) is associated with lymphoid and hair follicle tumors in Syrian hamsters. The early region of HaPV has the potential to encode three polypeptides (which are related to the mouse polyomavirus early proteins) and can transform fibroblasts in vitro. We identified the HaPV middle T antigen (HamT) as a 45-kDa protein. Like its murine counterpart, HamT was associated with serine/threonine phosphatase, phosphatidylinositol-3 kinase, and protein tyrosine kinase activities. However, whereas mouse middle T antigen associates predominantly with pp60c-src and pp62c-yes, HamT was associated with a different tyrosine kinase, p59fyn. The ability of HaPV to cause lymphoid tumors may therefore reside in its ability to associate with p59fyn, a potentially important tyrosine kinase in lymphocytes.

Amino Acid Sequence↗

PubMed1991

The gene for mannose-binding protein maps to chromosome 10 and is a marker for multiple endocrine neoplasia type 2.

Human mannose-binding lectin (MBL) is a serum protein which appears to function as an opsonin in first line host defense. In situ hybridization studies assign the human MBL gene to chromosome 10q11.2----q21. A restriction fragment length polymorphism (RFLP) was found using TaqI with a 0.8-kb cDNA probe for MBL (probe 48-11), yielding heterozysity in 34% of individuals tested. Using this biallelic RFLP, linkage analysis of 30 families confirms the assignment of MBL to the region of multiple endocrine neoplasia, type 2a (MEN2A) with a maximum lod score of 7.54 at a recombination fraction of 0.00 (males) and 0.097 (females). The presence of two crossovers between MEN2A and MBL in these families indicates that a defect of MBL itself is not the cause of the hereditary thyroid cancer syndrome. The addition of MBL to the genetic map of the pericentromeric region of chromosome 10 should prove useful for improved localization of the MEN2A mutation.

Biomarkers, Tumor↗

PubMed1990

Analysis of the hamster polyomavirus infection in vitro: host-restricted productive cycle.

In a search for a host fully permissive for the hamster polyomavirus (HaPV) productive cycle in cell culture, the replication of the viral genome has been assayed in a panel of murine and hamster cell types. These experiments led to the conclusion that hamster cells represent the most permissive host for HaPV DNA replication although some murine cells also permit replication of the viral DNA. A single burst of infectious particles is demonstrable in some replication-competent cells, but the outcome of the infection appears to be clearly host dependent. In one hamster cell line (GD36), the virus can be propagated by successive productive cycles. In other hamster cells, despite a successful initial virus burst following transfection of viral DNA, a block in the production of virus particles seems to prevent the spread of infection. This type of restriction level may play a role in the in vivo host range of HaPV.

Animals↗

PubMed1990

Transfer of immortality by transfection of genomic DNA from SV40 established cell lines into rat embryo fibroblasts.

The cellular immortalization activity of cloned genes can be identified either in a colony-forming assa of transfected primary rat embryo fibroblasts or in a co-operation assay together with ras. However the demonstration of immortalization activities carried by cellular genes has not been reported. Here we establish that SV40 early genes integrated in genomic DNAs can be stably transferred into rat embryo fibroblasts and selected via their immortalization activity. Attempts to extend this assay to the identification of dominant genes putatively involved in the immortality of several other immortal post-crisis or tumor cells have been unsuccessful suggesting that the immortal phenotype can be brought about through different pathways.

Animals↗

PubMed1990

The hamster polyomavirus--a brief review of recent knowledge.

The hamster polyomavirus was first isolated by Graffi et al. in Berlin-Buch from skin epithelioma arising spontaneously in the Buch Syrian hamster colony. Virus particles are assembled in the nuclei of keratinized cell layer. The genome organization is identical to the murine polyomavirus genetic map including, in particular, the existence of a coding capacity for an early gene product analogous to the middle T antigen. The virus and the cloned DNA can immortalize primary cells and transform established cell lines from rodent origin. The HaPV can also induce lymphoma and leukemia after inoculation into newborn animals from a Potsdam Syrian hamster colony geographically separated from the colony affected by the spontaneous epitheliomas. The tumor incidence is high (30-80%), the latency short (4-8 weeks). The lymphomas are virus free but contain large amounts of nonrandomly deleted viral genomes. Transgenic mice produced by microinjection of HaPV DNA into the pronucleus of fertilized eggs of Gat: NMRI mice develop both, epitheliomas and lymphomas. The mice tumors contain extrachromosomal viral DNA. A search for a cellular host fully permissive for HaPV productive cycle in vitro lead to the conclusion that the hamster cells represent the most permissive context for the HaPV genome replication; however, in only one cell line the virus can be propagated by successive productive cycles leading to the establishment of a persistent infection.

Animals↗

PubMed1990

SV40 induced cellular immortalization: phenotypic changes associated with the loss of proliferative capacity in a conditionally immortalized cell line.

Immortalization of rodent embryo fibroblasts by SV40 is dominantly maintained by the large T antigen. The aim of this work is to characterize some of the events associated with the loss of proliferative capacity in a rat cell line, called REtsAF, which is conditionally immortalized by the tsA58 allele of SV40 large T antigen. DNA replication is arrested less than 24 h after the shift to the restrictive temperature (39 degrees C). This arrest occurs without specificity relative to the cell cycle stage, which suggests that a function essential throughout the cell cycle is affected. A two-dimensional SDS polyacrylamide gel electrophoresis analysis of proteins shows that, although the global rate of protein synthesis is only slightly affected at 39 degrees C, the rate of accumulation of specific proteins is either increased or decreased. Finally we present biochemical and electron microscopy data showing that alterations of the mitochondria occur upon shift to 39 degrees C.

Animals↗

PubMed1988

An improved electrotransfection method using square shaped electric impulsions.

Transfection of DNA into non adherent cells can be achieved by electropermeation. Previously published results, partially successful, were obtained using exponential decaying electric impulsions. However, one limitation of this technique has been the damaging effect of this type of impulsions resulting in poor cell recovery. We report hereby the electropermeation of human lymphoblastoid cell lines using a commercially available electropulsator delivering repeated, short, high voltage, square shaped, electric pulses. The parameters of transfection have been optimized using the "Lucifer Yellow Permeation Assay". With the optimum electric parameters, virtually all the cells were permeated and at least 70% survived the shocking conditions. Both transient expression and permanent integration and expression of DNA was observed.

Cell Line↗

PubMed1988

Expression of histamine and vasoactive intestinal peptide (VIP) receptors in immortalized rat fetal intestinal cells.

Four intestinal cell lines derived from rat fetuses at 19 days of gestation were successfully propagated after electroporation in the presence of different recombinant DNAs containing the viral oncogenes E1A from Adenovirus 5 and large T from SV40 or Polyoma. These immortalized intestinal cells, designated SLC, possess several properties observed in the parent cells of this tissue, including the expression of cytoplasmic villin, enkephalinase and retention of VIP receptors. In contrast, histamine elevated cAMP levels in the SLC cell lines only. The data suggest that the transfection of fetal rat intestinal cells by E1A and large T is associated with the induction of functional histamine receptors coupled with the Gs/Gi regulatory proteins of adenylate cyclase.

Animals↗

PubMed1988

cis activation of the c-myc gene in bovine papilloma virus type 1/human c-myc hybrid plasmids.

The c-myc gene amplification observed in human tumors is likely to represent an activation mechanism aiming at an increased transcription level. In order to evaluate the biological significance of this amplification in the malignant transformation we have designed an experimental model that could possibly mimic this situation in vitro. We have constructed a series of plasmids which physically link the human c-myc gene to the bovine papilloma virus type 1 genome (BPV1) and therefore should be maintained as amplified episomes upon transformation of rodent cells. Anticipating that the high copy number will bring about the immortalizing capacity of the c-myc gene, the constructions were introduced into primary rat embryo cells. Immortal cell lines were established by transfection of the hybrid plasmids carrying either the complete BPV1 genome or the transforming region of the viral genome. The BPV1 DNA alone or the c-myc gene alone has no activity in this assay. The analysis of the established cell lines demonstrates that the transfected plasmids are present not as free copies as anticipated but rather integrated as tandem repeats. We present data which strongly suggest that the immortalization capacity of the hybrid plasmids reflects the activation of the c-myc gene by the transactivable BPV1 enhancer. Although both the BPV1 early genes and the c-myc gene are actively transcribed, most of the cell lines do not display a transformed phenotype.

Animals↗

PubMed1988

Analysis of hamster lymphomas for the presence of hamster papovavirus DNA.

The hamster papovavirus (HaPV) is a polyomavirus isolated from skin epitheliomas arising spontaneously in young Syrian hamsters. It can induce lymphomas and leukaemias in newborn hamsters. Although no virus particles are detectable by electron microscopy, high amounts of monomeric and oligomeric forms of extrachromosomal HaPV DNA molecules are found in the lymphoma cells. These molecules display deletions of about 300 nucleotides in length. Their role in the lymphoma induction is discussed.

Animals↗

PubMed1988

The hamster polyomavirus transforming properties.

The hamster papovavirus (HaPV) is a polyomavirus isolated from hair follicle tumor arising spontaneously in newborn hamsters which can also induce lymphoma and leukemia. This tissue specificity displayed in vivo can be bypassed in vitro since HaPV carries the full transforming properties of a polyomavirus (immortalization and transformation). We report here the phenotypic characterization of cells that were selected as immortalized or transformed and express constitutively the HaPV early genes. The viral genome is integrated in the host DNA and the early region is actively transcribed and translated.

Animals↗

PubMed1987

Properties of simian virus 40 mutants lacking the Asp4-Glu-Asp stretch at the carboxyl-terminus of large T antigen.

The biological activity carried by the carboxy-terminal domain of SV40 large T antigen has been investigated by isolating mutants deleted for a stretch of six acidic residues which by analogy with polyoma middle T antigen might be essential for the activity of the protein. We have constructed an "in-phase" deletion of 37 residues that includes the complete acid residues cluster. In order to parallel the polyoma hr-t mutants genotype, the deletion was introduced in virus strains either competent or defective for the small t antigen. We conclude from these experiments that the deletion of this unusual sequence does not affect per se any of the known biological properties of the virus.

Amino Acid Sequence↗

PubMed1987

Induction of lymphomas by the hamster papovavirus correlates with massive replication of nonrandomly deleted extrachromosomal viral genomes.

The hamster papovavirus isolated from skin epithelioma can induce lymphomas and leukemias after subcutaneous inoculation into newborn hamsters. The lymphoma cells are virus free but contain large amounts of extrachromosomal hamster papovavirus DNA. We have cloned and partly sequenced some of these DNA molecules from independent tumors. These genomes displayed overlapping deletions consistently sharing a common end within the noncoding regulatory sequences; the other end was variable but always extended into the sequence coding for the N-terminal part of the viral capsid VP2. This unique in vivo interaction between a polyomavirus and its cellular host, the genesis of these variant molecules, and their role in the lymphoma formation are discussed.

Animals↗

PubMed1987

[Mechanisms of malignant transformation].

A meeting sponsored by the Société Française du Cancer has been held on October 3rd 1986 in Villejuif. Some of the most recent developments in the study of malignant transformation were presented. The molecular cloning of the gene altered in the human retinoblastoma, and the role of the myc gene in the shut-off of the immune response illustrated the cellular oncogenes field. The implication of viruses like the HTLV1, the papillomaviruses or the hepatitis B virus in human cancer was largely documented. The hamster papovavirus and some avian leukemogenic viruses were presented as models of animal oncogenic viruses. Finally the biochemical properties of growth factors like the interleukin II or the platelet-derived growth factor (PDGF) were described.

Animals↗