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O Brison

Publications and source records attributed to O Brison.

At least 37 records · Page 2Linked to original sources
PubMed1993

Gene amplification and tumor progression.

Proto-oncogenes are the genes which are most frequently found amplified in human tumor cells. Acquisition of a drug-resistant phenotype by gene amplification is frequent for in-vitro cultured cells but is very rare in human tumors. Proto-oncogenes amplified in human tumors belong essentially to one of three families (erbB, ras, myc) or to the 11q13 locus. Amplification is always specific for the tumor cells and is not found in constitutional DNA of the patient, indicating that amplification of the gene is selected for during tumor growth. For genes of the first three families, amplification results in overexpression in most of the cases. These are strong arguments in favor of a role of this amplification in tumor progression. The gene whose overexpression is the driving force for the selection of the amplification of the 11q13 locus is not known. The prad1 gene is presently a good candidate. Amplification of one type of proto-oncogene is generally not restricted to one tumor type. However, the N-myc gene is amplified mainly in tumors of neuronal or neuroendocrine origin and L-myc amplification is restricted to lung carcinomas. To understand the role of proto-oncogene amplification and overexpression in tumor progression it is necessary to know the function of the corresponding protein in the cell. erbB proteins are transmembrane receptors for growth factors. ras genes encode small GTP-binding proteins which are possibly involved in signal transduction. The myc proteins are transcription factors. The expression of the c-myc gene is induced a few hours after cells of various types have been induced to proliferate. The genes of these three families therefore encode proteins which appear to be involved in signal transduction. It is possible that overexpression of one of them, as a result of gene amplification, makes the cell a better responder to low levels of growth stimuli. For several genes which are found amplified in human tumors, it was shown that overexpression of the normal protein could confer a transformed or tumorigenic phenotype to in-vitro cultured cells. In addition, several studies on animal and human tumor-derived cell lines with an amplified proto-oncogene have established a relationship between proto-oncogene amplification and the tumorigenic phenotype. In neuroblastomas, it was proposed that down-modulation of MHC Class I antigens is a consequence of N-myc amplification and that this could be important in the progression toward a metastatic phenotype.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Partial engraftment of donor bone marrow cells associated with long-term remission of haemophagocytic lymphohistiocytosis.

We used polymerase chain reaction amplification of minisatellite sequences or of a Y chromosome-specific sequence and Southern blotting to analyse long-term engraftment (12-82 months) after bone marrow transplantation (BMT) for familial haemophagocytic lymphohistiocytosis (FHL). Six children aged from 1 to 18 months were transplanted with bone marrow from an HLA-identical sibling in five cases and from an HLA-nonidentical related donor (one mismatched HLA antigen) in one. The conditioning regiment included VP 16-213 (900 mg/m2), busulfan (16 mg/kg), cyclophosphamide (200 mg/kg) and, in one case, aracytine (2 g/m2). Four patients are alive without therapy more than 3 years after BMT; the other two relapsed 1 year after BMT. DNA was extracted from separated polymorphonuclear cells and mononuclear cells, as well as from separated E+ and E- cells in one case and CD16+ (natural killer) and CD16- cells in two cases. Engraftment was partial in the four long-term survivors. Recipient cells were largely predominant in three of them as well as in one of the patients who relapsed (the donor also developed FHL 18 months after BMT). E+, E-, CD16+ and CD16- cells presented the same pattern of chimaerism. Engraftment failed to occur in the patient who received an HLA-nonidentical bone marrow. These results indicate that partial engraftment is compatible with long-term remission of FHL and that the presence of a small proportion of cells of donor origin can prevent FHL-related lymphocyte and macrophage activation.

Blotting, Southern↗
PubMed1992

TGF-alpha production correlates with tumorigenicity in clones of the SW613-S human colon carcinoma cell line.

The c-myc gene is amplified and the c-Ki-ras gene is mutated in the SW613-S human colon carcinoma cell line. Two cell types with different levels of c-myc amplification are present in the SW613-S cell population and representative cell clones can be isolated. The clones with a high level of amplification and expression of the c-myc gene are tumorigenic in nude mice whereas those with a low level are not. The tumorigenic clones secrete transforming growth factor alpha (TGF-alpha) in the culture medium whereas the non-tumorigenic clones do not produce any detectable amount. Accordingly the level of TGF-alpha mRNA is higher and the transcription rate of the gene is increased in the tumorigenic clones. The acquisition of the tumorigenic phenotype by cells of non-tumorigenic clones, following introduction of c-myc gene copies by transfection, is accompanied by an increase in the steady-state level of TGF-alpha mRNA. These findings suggest a role for an elevated level of TGF-alpha production in the tumorigenic phenotype of SW613-S cells. The possibility that this role is indirect is discussed.

Animals↗
PubMed1992

Increased expression of cytokeratin and ferritin-H genes in tumorigenic clones of the SW 613-S human colon carcinoma cell line.

Subclones of the SW 613-S human colon carcinoma cell line differ by their ability to induce tumors in nude mice and by their level of amplification of the c-myc gene. Clones with a high level of amplification are tumorigenic in nude mice whereas those with a low level are not. Genes overexpressed in the tumorigenic clones as compared to the nontumorigenic ones were searched by differential screening of a cDNA library. Two cDNA clones corresponding to cytokeratin K18 and ferritin-H chain were isolated. The steady state level of the corresponding mRNAs is higher in cells of all tumorigenic clones. The level of cytokeratin K8 mRNA, the specific partner of cytokeratin K18 in intermediate filaments of epithelial cells, is also elevated in these cells. For all three genes, this is mainly due to an increase in the transcription rate, as shown by a nuclear run-on assay. Immunoblotting experiments showed that cytokeratins K8, K18, and K19 are more abundant in cells of tumorigenic clones. The mRNA of the other subunit of apo-ferritin (ferritin-L chain) is expressed at the same level in both types of clones. The mRNAs of cytokeratins K18 and K8 and of ferritin-H chain are also overexpressed in cells of nontumorigenic clones which have acquired a tumorigenic phenotype after transfection of c-myc gene copies.

Base Sequence↗
PubMed1992

Short-term study of chimaerism after bone marrow transplantation for severe aplastic anaemia.

Chimaerism was studied early (2 weeks to 3 months) during haematopoietic reconstitution after bone marrow transplantation in 18 severe aplastic anaemia patients (acquired SAA: 14 patients; Fanconi anaemia: four patients). Fourteen patients received marrow from an identical sibling donor, one from the phenoidentical father and three from a matched unrelated donor. Peripheral blood cell DNA was first analysed by Southern blotting with a multilocus minisatellite probe (33.6.3) or a Y chromosome specific probe (pHY2.1). For all 14 patients grafted with a genotypically identical sibling donor, the post-graft DNA profile strictly matched the respective donor profile (minisatellite probe) or disclosed the Y chromosome specific band in the case of female patients grafted with a male donor. In contrast, for the one patient grafted in a mismatched situation and for two out of three patients grafted with a matched unrelated donor, the results indicated autologous bone marrow recovery. This difference between patients grafted with an identical sibling donor and those grafted in other situations is statistically significant (P less than 0.01). The 15 patients with circulating cells of donor origin were then studied by polymerase chain reaction amplification of the DNA samples. The three male patients with a female donor were studied by amplification of a Y chromosome specific sequence (DYZ1), allowing the detection of one male cell in 10(4) female cells. In all three cases, residual male nucleated cells were detected. The analysis was performed by amplification of the 33.6.3 minisatellite sequence for the 12 remaining patients. No residual recipient cells were detected within the sensitivity limit of the method which is 1% in that case. This suggests that detection of residual host cells depends on the sensitivity of the technique used.

Adolescent↗
PubMed1989

Methylation of ribosomal cistrons in diploid and tetraploid Odontophrynus americanus (Amphibia, Anura).

Odontophyrynus americanus (Amphibia, Anura) genomic DNA from diploid and tetraploid specimens was treated with restriction enzymes sensitive to cytosine and adenine methylation (5 meC and 6 meA). In both diploids and tetraploids a high proportion of the total DNA was not cleaved by 5 meC-sensitive enzymes as observed on agarose gels stained with ethidium bromide. The DNAs were transferred to nitrocellulose filters and hybridized with cloned fragments containing sequences of Xenopus laevis 28S and 18S ribosomal DNA (rDNA). A high level of methylation of the ribosomal repeat units was revealed by 5 meC-sensitive enzymes in blood, liver, kidney and testis tissues. Adenine was methylated to a lesser degree and similarly in the rDNA from both germinative and somatic tissues. Comparison of the results obtained with DNA of diploids and tetraploids showed that methylation of ribosomal genes was increased in tetraploid genomes of adult frogs, but exact quantitative determinations could not be performed by this methodology. Cloning of the 28S region of the rDNA repeat unit was performed in the lambda gtWES lambda C vector. Restriction patterns obtained with methylation-sensitive enzymes using diploid and tetraploid derived clones confirmed the high level of methylation of the corresponding region of the ribosomal repeat unit in genomic DNAs. The implications of these results in the regulation of expression of the ribosomal genes in diploids and tetraploids are discussed.

Adenine↗
PubMed1989

Selection of cells with different chromosomal localizations of the amplified c-myc gene during in vivo and in vitro growth of the breast carcinoma cell line SW 613-S.

The c-myc gene is amplified in the human breast carcinoma cell line SW 613-S. At early in vitro passages, the extra copies of the gene were mainly localized in double minute chromosomes (DMs), as shown by in situ hybridization with a biotinylated c-myc probe. However, cells without DMs were also present in which the c-myc genes were found integrated into any of several distinct chromosomes (mainly 7q+, 4 and 4q+, and 1). When this cell line was propagated in vitro, the level of c-myc amplification decreased because cells with DMs and a high amplification level were lost and replaced by cells without DMs and having a low amplification level. On the contrary, when early passage SW 613-S cells were grown in vivo, as subcutaneous tumours in nude mice, cells with numerous DMs and a high level of c-myc amplification were selected for. In one cell line (SW 613-Tu1) established from such a tumour, the DM-containing cells were substituted at late passages for cells with a high number of c-myc copies integrated within an abnormally banded region, at band 17q24 of a 17q+ chromosome. When only cells with integrated genes were present, this cell line was still highly tumorigenic indicating that the localization of the c-myc genes in DMs was not required for these cells to be tumorigenic in nude mice. Furthermore, cells of the secondary tumours induced by SW 613-Tu1 did not contain any DMs showing that in vivo growth did not promote the release of integrated c-myc copies into DMs.

Blotting, Southern↗
PubMed1989

Structure of four amplified DNA novel joints.

The structures of four novel joints present in the amplified DNA of a Syrian hamster cell line highly resistant to N-(phosphonacetyl)-L-aspartate were analyzed. Novel joints J1, J2, and J4 were formed by recombination between two regions of wild-type DNA, whereas joint J3 is the end point of an inverted duplication. A fraction of the J3 copies displays a cruciform structure in the purified genomic DNA. The formation of J1 and J2 apparently involved a simple breakage and joining of the two wild-type sequences, whereas extra nucleotides are present at the junction point of J3 and J4. The two regions of the wild-type DNA which have recombined to form J1, J2, and J4 show few sequence similarities, indicating that these joints probably resulted from nonhomologous recombination. AT-rich regions are present in the vicinity of the breakpoint for the four joints and eight of 10 crossover points could be associated with putative topoisomerase I cleavage sites. Our results indicate that different types of novel joints are present in the amplified DNA of this cell line, which was isolated after several steps of selection.

Animals↗
PubMed1989

Long-term study of chimaerism in bone marrow transplantation recipients for severe aplastic anaemia.

We used minisatellite probes to analyse by DNA fingerprints the long-term engraftment (median 4.3 years, range 1-2) of 21 bone marrow transplantation recipients for severe aplastic anaemia. Patients received their graft from histocompatible siblings. They were conditioned with cyclophosphamide (150 mg/kg) and a 6GY thoracoabdominal irradiation and did not have ex-vivo T cell depletion of marrow donor. DNA was extracted peripheral mononuclear cells and analysed by Southern blotting with 32P-labelled single-stranded RNA probes. Seven out of 21 donor-recipient pairs were sex-mismatched and additionally studied with a probe detecting a male specific repeated sequence on the Y chromosome. Red cell surface phenotype was also used as marker of engraftment in most cases. Long-term engraftment appeared complete for all patients studied with respect to the three methods.

Adolescent↗
PubMed1988

Plasmid vectors with multiple cloning sites and cat-reporter gene for promoter cloning and analysis in animal cells.

We have constructed two vectors, pGCAT-A and pGCAT-C, designed to facilitate the construction of recombinant plasmids containing the bacterial gene (cat) coding for chloramphenicol acetyltransferase (CAT) under the control of eukaryotic promoter and/or enhancer elements. The cat gene was inserted downstream from a multiple cloning site (MCS) region with eleven unique restriction sites. The MCS region is in opposite orientation in the two vectors. The CAT activity of control extracts from cells transfected with pGCAT-A or pGCAT-C is very low. Insertion of the viral SV40 early promoter into one of the sites of the MCS region of pGCAT-A or pGCAT-C results in a 30- to 400-fold stimulation of the CAT activity.

Acetyltransferases↗
PubMed1988

Both N-ras and c-myc are activated in the SHAC human stomach fibrosarcoma cell line.

A transforming N-ras gene was isolated from the SHAC human stomach fibrosarcoma cell line. A single-point mutation resulting in the substitution of histidine for glutamine at codon 61 was found in the SHAC transforming allele. The N-ras gene is overexpressed in the tumor cells and transformant cells. The N-ras p21 product was studied by immunoprecipitation and showed no alteration in mobility as compared to the normal p21 protein. The c-myc gene is amplified and overexpressed in these cells. This report gives evidence that an amplified c-myc and a mutated N-ras gene are both present in this tumor cell line and provides support for the idea that co-operation of at least 2 activated cellular oncogenes is required for carcinogenesis.

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

High c-myc amplification level contributes to the tumorigenic phenotype of the human breast carcinoma cell line SW 613-S.

The c-myc gene is amplified in the SW 613-S cell line which was established from a human breast carcinoma. This line is heterogeneous: it contains cells with a high level of amplification and carrying the extra copies of the c-myc gene in double minute chromosomes (DMs) and cells with few c-myc genes integrated into chromosomes. Clones with different levels of amplification and different cytological localization of the c-myc copies were isolated from the SW 613-S cell population. Those with a high level of amplification and expression of the c-myc gene were highly tumorigenic in nude mice whereas those with a low level were not. Introduction of c-myc gene copies by transfection into the cells of several non-tumorigenic clones restored the tumorigenic phenotype. Our results indicate that a high level of amplification of the c-myc gene is a requirement for the tumorigenicity of SW 613-S cells in animals.

Animals↗
PubMed1986

An Epstein-Barr virus transcription unit is at least 84 kilobases long.

We have studied the structure of the Epstein-Barr virus mRNAs expressed in B95-8, a productively-infected Marmoset cell line established from in vitro-infected B-lymphocytes. We constructed a cDNA library from the cytoplasmic polyadenylated RNAs of B95-8 in the lambda gt10 bacteriophage. We present here the analysis of a 3.5 kbp cDNA containing exons transcribed from the US, IR and UL regions of the viral genome. The corresponding transcription unit is at least 84 kbp long. Two exons are transcribed from the US region, five from the IR region and two from the UL region. The exons from the IR region consist of two tandem repeats of a unit containing two exons, 66 and 132 nucleotides, and of a third copy of the 66 nucleotide exon. The exons from the UL region contain an open reading frame coding for a 944 amino acid polypeptide. The C-terminal end of this polypeptide harbors three types of repeated sequences. The corresponding mRNA is the second described of a family of mRNAs produced by alternative splicing of exons transcribed from the US, IR and UL regions.

Animals↗
PubMed1986

New RNA species is produced by alternate polyadenylation following rearrangement associated with CAD gene amplification.

Mammalian cells selected to resist N-(phosphonacetyl)-L-aspartate (PALA) contain amplified copies of the CAD gene. While a single 7.9-kb mRNA species is detected in PALA-sensitive and most PALA-resistant cell lines, two RNA species (7.9 and 10.2 kb) are detected in two related drug-resistant mutants presumably derived from the same parental cell. In this report we show that the 10.2-kb RNA is produced as a direct consequence of a sequence rearrangement adjacent to the 3' end of the CAD gene in these cell lines. A CAD gene containing the sequence rearrangement was cloned from one of these lines and found to produce both RNA species when transfected into CAD-deficient cells. DNA sequencing and S1 analysis demonstrate that the 10.2-kb RNA is produced by alternative polyadenylation rather than by alternative splicing. Sequence analysis also reveals that several consensus poly(A) addition signals (AATAAA) were brought into close proximity to the CAD gene by virtue of the rearrangement. While sequences adjacent to each of the polyadenylation signals contain additional features postulated to be important for the selection of the site of poly(A) addition, S1 mapping analysis indicates that only one of the polyadenylation signals is used. A comparison of all of these sites suggests that multiple sequence motifs are required to form a functional polyadenylation and cleavage signal.

Animals↗
PubMed1986

Interferon modulation of c-myc expression in cloned Daudi cells: relationship to the phenotype of interferon resistance.

Treatment of interferon-sensitive Daudi cell with electrophoretically pure human interferon alpha markedly reduced the level of c-myc mRNA, increased the level of class I histocompatibility antigen (HLA) mRNA, and did not affect the level of actin mRNA within the same cells. In contrast, the level of c-myc mRNA or HLA mRNA did not change significantly following interferon treatment in different clones of Daudi cells selected for resistance to the antiproliferative action of interferon. These cells possessed interferon receptors, however, and responded to interferon modulation of other genes, including 2',5' oligoisoadenylate synthetase (M. G. Tovey, M. Dron, K. E. Mogensen, B. Lebleu, N. Metchi, and J. Begon-Lours, Guymarho, J. Gen. Virol., 64:2649-2653, 1983; M. Dron, M. G. Tovey, and P. Eid, J. Gen. Virol., 66:787-795, 1985). A clone of interferon-resistant Daudi cells which had reverted to almost complete sensitivity to both the antiproliferative action of interferon and the interferon-enhanced expression of HLA mRNA remained refractory, however, to interferon modulation of c-myc expression, suggesting that a reduced level of c-myc mRNA may not be a prerequisite for inhibition of cell proliferation in interferon-treated cells. Our results do not exclude the possibility, however, that posttranscriptional modification(s) of c-myc expression may precede an inhibition of cell proliferation in interferon-treated cells.

Cell Line↗
PubMed1985

Increased level of amplification of the c-myc oncogene in tumors induced in nude mice by a human breast carcinoma cell line.

Cell line SW 613-S, derived from a human breast carcinoma, contained double minute chromosomes (DMs) but lost them progressively upon in vitro cultivation. These cells were tumorigenic in nude mice. Cell lines were derived from the tumors and were found to have a high DM content. In three such cell lines, DMs were stably maintained upon in vitro cultivation, whereas in another they were progressively lost. We found that the c-myc oncogene is amplified 5- to 10-fold in SW 613-S and 20- to 90-fold in the different cell lines derived from the tumors. At least part of the additional c-myc copies were found associated with a purified DM fraction. In cell lines which lost the DMs during in vitro passages, the level of amplification was maintained. In situ hybridization experiments indicated that this loss was compensated by the acquisition of copies of the c-myc gene integrated into a chromosome. No major rearrangement of the amplified c-myc gene was detected. The amount of c-myc messenger RNAs is roughly proportional to the level of amplification. Our results indicate that growth of SW 613-S cells as tumors in nude mice selected cells with an increased level of amplification and expression of the c-myc oncogene.

Animals↗