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J Samarut

Publications and source records attributed to J Samarut.

At least 73 records · Page 4Linked to original sources
PubMed1993

Importance of 3' non-coding sequences for efficient retrovirus-mediated gene transfer in avian cells revealed by self-inactivating vectors.

Avian leukosis virus-derived vectors were constructed with an internal transcriptional promoter and various 3' non-coding sequences. Deletions were introduced into the downstream U3 long terminal repeat (LTR) to obtain self-inactivation of LTR-mediated transcription after one round of replication. However, 3' non-coding sequences appeared to determine not only self-inactivation of the vectors but also gene transfer efficiency. Further analysis revealed the influence of these sequences on both internal gene expression and RNA packaging. One construct permitted gene transfer while inactivating 5' LTR-promoted transcription.

Animals↗
PubMed1993

Transforming growth factor beta 1-mediated growth inhibition in chick embryo fibroblasts: reversion by virally-expressed nuclear oncogenes.

Transforming growth factor beta 1 (TGF-beta 1) inhibits growth of primary cultures of chick embryo fibroblasts by affecting G1 and strongly increasing the generation time. This inhibition is reversed by the nuclear oncogenes v-jun, v-fos, v-myc, but not v-erbA and v-ets. It is also reversed by v-myb from either avian myeloblastosis virus or avian E26 retrovirus. Taken together, these results strongly suggest that independent, functional interferences may take place between the TGF-beta 1-induced growth inhibitory pathway and the oncogen-driven stimulatory pathway(s) at the level of the AP-1, Myc, and Myb transcription factors.

Animals↗
PubMed1993

The 12S adenoviral E1A protein immortalizes avian cells and interacts with the avian RB product.

Quail cells were immortalized for the first time by using retroviruses expressing the 12S adenoviral E1A gene. In these cells, interaction between the 12S E1A product and the quail RB protein was shown, suggesting that the 12S adenoviral E1A product works in avian cells through similar biochemical pathways as in mammalian cells by interacting and inactivating host cellular proteins, including the RB product. These results confirm that the RB product exhibits a universal function among higher vertebrates in controlling cellular growth and tumor progression.

Adenoviridae↗
PubMed1993

Leukemogenicity of v-myb-transformed monoblasts cells can be modulated by normal bone marrow environment.

The avian myeloblastosis virus (AMV) causes monoblastic leukemia in the chick. Two non-producer clones of AMV-transformed monoblasts, BM2/C3A and BM2L/A2B5, have been described (see Bottazzi et al., this issue). They differ in their growth requirements and in their ability to induce leukemia when injected into the chick embryo. We first genetically tagged these clones by retroviral infection with a vector expressing the bacterial lacZ gene. Then, we injected the lacZ-positive cells via the chorioallantoic vein into chick embryos. With BM2L/A2B5 cells, the bone marrow of the injected birds was rapidly invaded by lacZ-positive cells. In addition, these cells rapidly overgrew cultures of bone marrow cells derived from injected animals. Conversely, the growth of BM2/C3A was inhibited in the injected animals and only a few blue cells, with the morphology of macrophages, were detected in cultures of bone marrow cells. We developed an in vitro assay to mimic in vitro the differential growth of BM2/C3A and BM2L/A2B5 observed in vivo. These data strongly suggest that BM2/C3A cells retain their ability to differentiate into macrophages in the normal bone marrow environment and that BM2L/A2B5 cells differ from BMC/C3A in the loss of this capacity.

Animals↗
PubMed1992

v-erbB oncogene expression accounts for most variations in protein synthesis after avian erythroblastosis virus infection of chicken embryo fibroblasts: a two-dimensional electrophoresis study.

The effect of the v-erbA and/or v-erbB oncogenes on cellular gene expression was investigated after separation by two-dimensional polyacrylamide gel electrophoresis of [35S]methionine-labelled proteins from chicken embryo fibroblasts (CEF), infected by either the avian erythroblastosis virus (AEV) carrying both oncogenes, or by viruses carrying only one of them. We observed significant changes in the synthesis of 34 proteins in AEV-transformed CEF as compared with control cells. The synthesis of 24 of them was increased while the synthesis of the other 10 proteins was decreased. The expression of v-erbB alone is necessary and sufficient to induce changes in the synthesis of 27 proteins while the 7 remaining modifications are observed only in cells expressing v-erbB together with v-erbA. Moreover, the deregulation of protein synthesis by v-erbB-expressing viruses was correlated with the morphological transformation state of cells.

Alpharetrovirus↗
PubMed1992

BTG1, a member of a new family of antiproliferative genes.

The BTG1 gene locus has been shown to be involved in a t(8;12)(q24;q22) chromosomal translocation in a case of B-cell chronic lymphocytic leukemia. We report here the cloning and sequencing of the human BTG1 cDNA and establish the genomic organization of this gene. The full-length cDNA isolated from a lymphoblastoid cell line contains an open reading frame of 171 amino acids. BTG1 expression is maximal in the G0/G1 phases of the cell cycle and is down-regulated when cells progress throughout G1. Furthermore, transfection experiments of NIH3T3 cells indicate that BTG1 negatively regulates cell proliferation. The BTG1 open reading frame is 60% homologous to PC3, an immediate early gene induced by nerve growth factor in rat PC12 cells. Sequence and Northern blot analyses indicate that BTG1 and PC3 are not cognate genes. We then postulate that these two genes are the first members of a new family of antiproliferative genes.

3T3 Cells↗
PubMed1992

The various domains of v-myb and v-ets oncogenes of E26 retrovirus contribute differently, but cooperatively, in transformation of hematopoietic lineages.

The genome of the avian leukemia virus E26 is a unique example of association between two transcription factors which appear as a fused composite nuclear oncoprotein, P135gag-myb-ets. Previous studies with E26 have shown that v-myb and v-ets must cooperate to fully transform both erythrocytic and myelomonocytic precursor cells in vivo and in vitro. To analyse further the contribution of the individual domains involved in the transformation of various hematopoietic lineages, we have constructed several mutant viruses expressing a fusion protein with deletions in either v-myb or v-ets. We show here that integrity of the v-ets oncogene is necessary for transformation of the erythrocytic cells but that neither the DNA-binding domain nor the trans-activating domain of v-myb is required for this transformation. The DNA-binding domain of v-ets is necessary to transform myelomonocytic cells. Furthermore, we show that E26 onco-protein also transforms granulocytic cells. The v-ets DNA-binding domain is not necessary to transform them, whereas deleting the v-myb DNA-binding domain strongly reduces transformation of these cells. These data show that the v-myb and v-ets DNA-binding domains provide quite different contributions to the transformation of various hematopoietic lineages by E26.

Animals↗
PubMed1991

A novel mechanism of action for v-ErbA: abrogation of the inactivation of transcription factor AP-1 by retinoic acid and thyroid hormone receptors.

Ligand-activated retinoic acid receptor alpha (RAR alpha) and c-ErbA alpha repress the AP-1-mediated transcriptional activation of the interstitial collagenase gene promoter by specifically decreasing the activity of the AP-1 transcription factor. On the other hand, the v-ErbA oncoprotein fails to repress the AP-1 activity and acts as a dominant negative oncoprotein by overcoming the repression of the AP-1 activity induced by RAR alpha and c-ErbA alpha. This maintenance by v-ErbA of a fully active AP-1 complex is correlated with the abrogation by this same oncogene product of the growth-inhibitory response of chicken embryo fibroblasts to retinoic acid treatment. This new mechanism of action of v-ErbA together with its previously discovered dominant repressor effect on transcription of thyroid hormone-activated target genes may explain the contribution of the v-erbA oncogene to sarcomatogenic and leukemogenic transformation.

Carrier Proteins↗
PubMed1991

A chromosome 12 coding region is juxtaposed to the MYC protooncogene locus in a t(8;12)(q24;q22) translocation in a case of B-cell chronic lymphocytic leukemia.

We performed molecular cloning and sequencing of the breakpoints of a new chromosomal translocation involving the MYC protooncogene locus. This secondary t(8;12)(q24;q22) was associated with a primary t(11;14)(q13;q32) translocation in a case of B-cell chronic lymphocytic leukemia (CLL) in blastic transformation. In this leukemia, Northern blot and nuclease analyses SI showed that MYC was strongly expressed with initiation of the transcription at both the 5' and 3' promoters as observed in Burkitt's lymphomas; no coding change was observed in MYC putative regulatory sequences. The breakpoint on chromosome 8 mapped to the 3' end of the MYC locus, in a region containing a potential Z-DNA tract, and where we identified two DNase 1 hypersensitive sites. A rearranged MYC gene fragment was cloned and shown to contain chromosome 12 information by Southern blot analysis and by in situ hybridization. A genomic probe subcloned from the isolated region of the chromosome 12 recognized a 1.8 kb transcript in virtually all the tissues tested but a preferential expression of this new gene, which we termed BTG1 (for B-cell translocation gene 1) was observed in the CLL cells and in tissues of lymphoid origin. This chromosome 12 coding sequence is conserved in evolution and a transcript of similar size is present in murine tissues.

Animals↗
PubMed1991

Inhibition of proliferation of primary avian fibroblasts through expression of histone H5 depends on the degree of phosphorylation of the protein.

To obtain stable and constitutive expression of histone H5 at levels comparable to those observed in normal chicken erythrocytes, an avian self-inactivating retroviral vector was used to transfer the H5 gene into cells which do not express this protein. The vector, pDAH5, was obtained by removing the CAAT and TATA boxes of the 3'LTR of the avian leukosis virus RAV-2 and inserting the H5 sequence. Infection of QT6 quail cells with the recombinant virus (DAH5) led to the stable integration of the foreign H5 gene at low copy number, to the formation of correctly initiated mRNA transcripts and to the production of H5 protein. The amount of H5 expressed was equivalent to that of a mature chicken erythrocyte. Expression of histone H5 in DAH5 transformed cells, such as QT6 or AEV-ES4, transformed chicken embryo fibroblasts had only slight effects on the growth rate and did not inhibit cell replication. Conversely, the effect of H5 expression on normal quail and chicken fibroblasts was dramatic: cells acquired the aspect of quiescent fibroblasts, grew very slowly, and nuclei looked compacted, often extruded from the cell. The H5 histone produced in QT6-transformed cells was found to be phosphorylated while in normal chicken fibroblasts the protein lacked this posttranslational modification. It is proposed that the chromatin-condensing role of histone H5 is inhibited by its phosphorylation.

Animals↗
PubMed1991

v-myb and v-ets cooperate for the mitogenic stimulation of primary fibroblasts by avian E26 retrovirus.

By using a series of deletion mutants, we have shown that the stimulation of fibroblast growth by E26 requires the cooperation of the two oncogenes, v-myb and v-ets, fused in the nuclear viral product. Of the two DNA-binding domains, only one must be present to promote anchorage-independent growth, whereas that of v-myb is required to allow growth in low serum medium. Furthermore, the v-ets oncogene comprises multifunctional domains.

Animals↗
PubMed1991

[V-erbA oncogene, model of oncogenic activation of hormone receptor].

The identification of the viral oncogene v-erbA carried by an avian leukemia retrovirus has directly demonstrated the involvement of hormone receptors in neoplastic transformation. v-erbA represents an altered form of a nuclear receptor of the thyroid hormone T3. It blocks the differentiation of chicken erythrocyte progenitor cells and contributes to sarcoma transformation in association with other oncogenes. The protein encoded by v-erbA behaves as an antagonist against the normal T3 receptors and retinoic acid receptors. The primary effects of the protein result in altering the transcription of genes normally under control of the intact receptors. Presumably among these target genes are to be found genes which control cell differentiation and proliferation.

Animals↗
PubMed1991

EGF promotes in vivo tumorigenic growth of primary chicken embryo fibroblasts expressing v-myc and enhances in vitro transformation by the v-erbA oncogene.

We report that the activation of the endogenous chicken EGF receptor leads to the tumorigenic growth in vivo of early passage chicken embryo fibroblasts (CEFs) that express a nonsarcomagenic oncogene, v-myc. To provide a continuous paracrine source of this growth factor in vivo, we employed irradiated Rat-1 cells which had been stably transfected with a synthetic cDNA to human EGF. Expression of another non-sarcomagenic nuclear oncogene, v-erbA, prones the CEFs to in vitro transformation by EGF, but does not cause EGF dependent tumorigenicity in vivo. The short period of incubation in the in vivo assay employed by our study (10 days), together with the genetic stability of primary chicken embryo fibroblasts, make it very likely that the reported alterations in cellular behaviour are a direct and primary effect of the expression of the relevant oncogenes and their cooperation with the EGF induced response. Dose response and ligand binding assays suggest that the EGF response is transmitted via the chicken c-erbB molecule, which by virtue of its preference for TGF-alfa is distinct from the mammalian EGF receptors studied so far. The level of expression of the endogenous chicken EGF receptor is within the same range as that reported for primary human fibroblasts (5-7 x 10(3) per cell). The cooperative effect of v-myc with chicken c-erbB probably takes place at a post receptor level, as its expression did not affect the steady state level or affinity for ligand of the chicken EGF receptor.

Animals↗
PubMed1991

v-erbA oncogene abrogates growth inhibition of chicken embryo fibroblasts induced by retinoic acid.

Retinoic acid inhibits chicken embryo fibroblast (CEF) proliferation by altering the G1 phase of the cell cycle with induction of a strong increase in the generation time. This growth-inhibitory response to retinoic acid is abrogated by expression of the v-erbA oncogene, suggesting an interference between retinoic acid receptors and the v-ErbA oncoprotein. Moreover, CEF expressing either the v-src, v-jun or v-fos oncogenes are also insensitive to retinoic acid treatment. In contrast, CEF expressing either the v-myc, v-myb-ets, v-mil, v-sea or v-erbB oncogenes are still sensitive to retinoic acid. These data strongly suggest functional interferences between the retinoic acid receptors and the AP-1 transcription factor complex in the control of expression of genes involved in CEF proliferation.

Animals↗
PubMed1990

Cooperation of v-jun and v-erbB oncogenes in embryo fibroblast transformation in vitro and in vivo.

Retroviral vectors carrying either the v-jun and v-erbB sequences or the v-jun gene linked to the neomycin resistance gene were constructed on the basis of the structural genome organization of avian erythroblastosis virus (AEV). These viruses, called JB and JN, respectively, were rescued as Rous-associated virus-1 pseudotypes, and they were shown to successfully transform chicken embryo fibroblasts in vitro. However, in agar, colonies developed from JB-infected fibroblasts were three to five times larger than those obtained after infection with JN or with AEV Pst124 carrying only a functional v-erbB gene. In vivo, on chorioallantoic membrane (CAM) assays, JB produced fibrosarcomas that were more rapidly growing and much larger than those induced by JN or AEV Pst124. Moreover, in chickens infected in ovo with JB, multiple fibrosarcomas arose in different organs a few days after birth, whereas no tumor could be detected in parallel experiments in either JN- or AEV Pst124-infected animals. These results demonstrate that in embryo fibroblast cells, v-jun and v-erbB can act synergistically to enhance the transformation potential of either oncogene alone both in vitro and in vivo.

Alpharetrovirus↗
PubMed1990

A new avian leukosis virus-based packaging cell line that uses two separate transcomplementing helper genomes.

An avian leukosis virus-based packaging cell line was constructed from the genome of the Rous-associated virus type 1. The gag, pol, and env genes were separated on two different plasmids; the packaging signal and the 3' long terminal repeat were removed. On a plasmid expressing the gag and pol genes, the env gene was replaced by the hygromycin resistance gene. The phleomycin resistance gene was inserted in the place of the gag-pol genes on a plasmid expressing the env gene. The plasmid containing the gag, pol, and Hygror genes was transfected into QT6 cells. Clones that produced high levels of p27gag were transfected with the plasmid containing the Phleor and env genes. Clones that produced high levels of env protein (as measured by an interference assay) were tested for their ability to package NeoR-expressing replication-defective vectors (TXN3'). One of the clones (Isolde) was able to transfer the Neo+ phenotype to recipient cells at a titer of 10(5) resistance focus-forming units per ml. Titers of supernatants of cells infected with Rous-associated virus type 1 prior to transfection by Neor vectors were similar. Tests for recombination events that might result in intact helper virus showed no evidence for the generation of replication-competent virus. The use of selectable genes inserted next to the viral genes to generate high-producer packaging cell lines is discussed.

Animals↗
PubMed1990

Overexpression of avian or mouse c-jun in primary chick embryo fibroblasts confers a partially transformed phenotype.

The coding sequences of avian (quail) or murine c-jun proto-oncogenes were introduced into a non-defective retroviral vector derived from Rous sarcoma virus (RSV) in which c-jun replaces v-src. Primary avian fibroblasts chronically infected with either one of these viruses exhibit some phenotypic traits characteristic of RSV-transformed cells, including sustained growth in low serum medium and ability to develop colonies from single cells in agar, even though they are still of normal morphology and contact inhibited. This altered growth control correlates with enhanced AP1-specific DNA binding activity as well as with higher levels of c-Jun products. Unexpectedly, repression of the endogenous c-Jun product is observed in cells overexpressing murine c-Jun. Cells expressing the avian and the murine c-Jun products display qualitatively similar phenotypes; nevertheless, for every transformed trait considered, the murine c-jun seemed more potent than its quail homologue. These data suggest that the avian or murine c-jun proto-oncogenes may trigger a subset of the 'transforming functions' normally induced by v-src, and which are more specifically related to growth in low serum and in the absence of solid support.

Amino Acid Sequence↗