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O Brison

Publications and source records attributed to O Brison.

At least 19 recordsLinked to original sources
PubMed1996

Characterization of new human c-myc mRNA species produced by alternative splicing.

Characterization of two human c-myc cDNAs corresponding to the mRNAs 2.5 and 3.1 kb in length transcribed from P0 previously demonstrated the existence of alternative acceptor sites at the end of intron 1 and intron 2, respectively [Bentley, D.L. and Groudine, M. (1986) Mol. Cell. Biol. 6, 3481-3489]. We investigated the use of these alternative acceptor sites in each c-myc mRNA species. We characterized cDNAs corresponding to c-myc mRNAs transcribed in the SW613-S human carcinoma cell line. The use of the alternative acceptor site at the end of intron 1 was demonstrated in two out of 10 cDNAs corresponding to the 3.1-kb mRNA transcribed from P0 and in three out of 10 cDNAs corresponding to the mRNAs transcribed from P1 or P2. The use of this acceptor site is therefore not restricted to the 2.5-kb mRNA transcribed from P0. The mRNAs resulting from the use of this acceptor site is therefore not restricted to the 2.5-kb mRNA transcribed from P0. The mRNAs resulting from the use of this acceptor site would encode for a variant form of the p67 polypeptide lacking one amino-acid residue. Conversely, the use of the alternative acceptor site at the end of intron 2 was not found in any of the cDNAs corresponding to the mRNAs transcribed from P0 (0/10), from P1 or P2 (0/10) and from P3 (0/10). In the course of this study, we isolated a cDNA corresponding to another new c-myc mRNA species. This mRNA is produced by alternative splicing within intron 1 and encodes only for p64.

Alternative Splicing↗
PubMed1995

The proto-oncogene FGF-3 is constitutively expressed in tumorigenic, but not in non-tumorigenic, clones of a human colon carcinoma cell line.

The human colon carcinoma cell line, SW613-S, is composed of cells with a high-level amplification of the MYC proto-oncogene that are tumorigenic in nude mice and of cells with a low-level amplification of MYC that are not tumorigenic. Transcripts from FGF-3, a member of the fibroblast growth factor gene family, accumulate in cells from tumorigenic clones, but are undetectable in those from non-tumorigenic clones. Nuclear run-on analyses indicate that this differential FGF-3 expression is regulated at the level of transcription initiation. Determination of the structure of the FGF-3 transcripts indicates that they are generated by splicing of the three exons and termination at the single polyadenylation site predicted from the genomic sequence. Their size heterogeneity is due to multiple initiation sites spanning a 700 base-pair long promoter region. FGF-3 is activated in tumors induced in nude mice by MYC-transfected cells from non-tumorigenic clones. However, in most of the cell lines established from these tumors, FGF-3 expression tends to be lost upon in vitro propagation. Thus, in these transfectant cell lines, the presence of exogenous MYC gene copies is not sufficient to activate FGF-3 expression and in vivo growth is also required.

Actins↗
PubMed1995

IGF-2 autocrine stimulation in tumorigenic clones of a human colon-carcinoma cell line.

Clonal analysis has shown that the SW613-S human colon-carcinoma cell line is heterogeneous: some cell clones display a high level of amplification of the c-myc gene and are tumorigenic in nude mice, whereas others have a small number of copies of this gene and are non-tumorigenic. Tumorigenic clones can proliferate in a chemically defined serum-free medium, whereas non-tumorigenic clones cannot. Suramin, like anti-insulin-like growth factor (IGF) or anti-IGF-I receptor antibodies, efficiently inhibits the growth of tumorigenic clones in defined medium. Inhibition by suramin or by anti-IGF antibodies can be reversed by pure IGF-I or IGF-2. Pure IGF-1 or IGF-2 or culture medium conditioned by tumorigenic clones can stimulate DNA synthesis in cells of non-tumorigenic clones. Co-culture with cells of tumorigenic clones sustains the growth of non-tumorigenic clones in defined medium. Cells of both tumorigenic and non-tumorigenic clones express high-affinity IGF-1 receptors at their surface but tumorigenic clones produce on average 5 times more IGF-1 and 25 times more IGF-2 than non-tumorigenic ones. These results indicate that autocrine growth stimulation of tumorigenic clones by IGFs through the IGF-1 receptor is essential for their ability to grow in defined medium. Since cells of tumorigenic clones produce IGF-2 at levels 80 times higher than IGF-1 and since an antibody strictly specific for IGF-1 has no effect on DNA synthesis in cells of tumorigenic clones grown in defined medium, IGF-2 is very likely the main effector in the autocrine loop.

Cell Division↗
PubMed1995

Constitutive or inducible overexpression of the IGF-2 gene in cells of a human colon carcinoma cell line.

Two types of clones have been isolated from the SW613-S human colon carcinoma cell line. Clones with a high level of amplification of the c-myc gene are tumorigenic in nude mice and can proliferate in chemically defined, serum-free medium, whereas clones with a low level of amplification are nontumorigenic and cannot multiply in defined medium. The expression level of the insulin-like growth factor type 1 (IGF-1) gene is low in tumorigenic clones and undetectable in nontumorigenic clones. Tumorigenic clones produce high levels of IGF-2 (and IGF-binding proteins), compared to nontumorigenic clones. This is the consequence of a differential transcriptional regulation of the IGF-2 gene between the two types of clones. This regulation consists of a modulation of the activity of promoters P3 and P4. Overexpression of the IGF-2 gene is constitutive in tumorigenic clones: it is stably maintained during in vitro propagation of the cells. Tumorigenic cell lines obtained after transfer of c-myc gene copies into the cells of nontumorigenic clones exhibit a high level of expression of the IGF-2 gene when they are grown in vivo, as subcutaneous tumors in nude mice. This high level of expression is lost in most of these cell lines when they are returned to in vitro culture conditions indicating that, in these cells, IGF-2 overexpression is not constitutive but inducible by in vivo growth conditions. We had previously shown that tumorigenic clones use the overproduced IGF-2 as an autocrine growth factor. The results reported here suggest than IGF-2 overexpression has an important role in the tumorigenic phenotype of these cells.

Animals↗
PubMed1995

Studies on hemopoietic chimerism following allogeneic bone marrow transplantation in the molecular biology era.

Donor hematopoiesis or donor chimerism in the host following allogeneic bone marrow transplantation (BMT) has appeared crucial to the engraftment process. However, as molecular techniques exploiting neutral variation in human genetic material have been used in the study of chimerism, the detection of residual host cells or mixed hemopoietic chimerism has indicated that donor chimerism is not obligatory following BMT. This review focuses on the detection and significance of mixed chimerism (MC) in patients transplanted for both malignant and non-malignant hemopoietic disease and attempts to tease out the contribution of MC to engraftment, leukemia relapse, graft rejection and long-term disease-free survival.

Anemia, Aplastic↗
PubMed1995

An Sp1 binding site and the minimal promoter contribute to overexpression of the cytokeratin 18 gene in tumorigenic clones relative to that in nontumorigenic clones of a human carcinoma cell line.

Clones of cells tumorigenic or nontumorigenic in nude mice have been previously isolated from the SW613-S human colon carcinoma cell line. We have already reported that tumorigenic cells overexpress the cytokeratin 18 (K18) gene in comparison with nontumorigenic cells and that this difference is mainly due to a transcriptional regulation. We now report that a 2,532-bp cloned human K18 gene promoter drives the differential expression of a reporter gene in a transient assay. A 62-bp minimal K18 promoter (TATA box and initiation site) has a low but differential activity. Analysis of deletion and substitution mutants as well as hybrid SV40-K18 promoters and reconstructed K18 promoters indicated that an important element for the activity of the K18 promoter is a high-affinity binding site for transcription factor Sp1 located just upstream of the TATA box. This Sp1 binding element, as well as the intron 1 enhancer element, stimulates the basal activity of the minimal promoter through mechanisms that maintain the differential activity. Gel shift assays and the use of an anti-Sp1 antibody have shown that both tumorigenic and nontumorigenic SW613-S cells contain three factors able to bind to the Sp1 binding element site and that one of them is Sp1. A hybrid GAL4-Sp1 protein transactivated to comparable extents in tumorigenic and nontumorigenic cells a reconstructed K18 promoter containing GAL4 binding sites and therefore without altering its differential behavior. These results indicate that the Sp1 transcription factor is involved in the overexpression of the K18 gene in tumorigenic SW613-S cells through its interaction with a component of the basal transcription machinery.

Base Sequence↗
PubMed1995

[Value of the analysis of cellular chimerism following bone marrow graft by amplification of minisatellite sequences or specific of the Y chromosome].

We used polymerase chain reaction amplification of minisatellites sequences (33.6.3, G3, PMS51, YNZ22) and of a Y chromosome specific sequence (DYZ1) to document chimaerism in 50 patients. Indications for bone marrow transplantation (BMT) was acute leukemia (n = 23). Aplastic anemia (n = 18) and inherited newborn disease, SCID and familial hemophagocytic lymphohistiocytosis (n = 9). The method was informative in all cases. We were able to demonstrate engraftment in 23/23 leukemia patients, 15/18 in aplastic patients and 8/9 in the last group of patients. HLA mismatching BMT was significantly less efficient. Analysis with DYZ1 probe in case of sex mismatched. BMT revealed the frequency of host residual cells after Bott. Their detection in a 0.01-1% range does not seem to influence the outcome of the transplantation procedure.

Base Sequence↗
PubMed1995

A highly sensitive polymerase chain reaction method reveals the ubiquitous presence of maternal cells in human umbilical cord blood.

Umbilical cord blood is considered an alternate source of hematopoietic stem cells in bone marrow transplantation. However, its use might be hampered by contamination of neonatal blood with maternal cells, which could contribute unacceptably to graft-vs.-host disease (GVHD) after transplant. In a previous study (Socié et al., Blood 83:340, 1994), we used polymerase chain reaction (PCR) amplification of minisatellite sequences (sensitivity 1-0.1%) to address the question of this contamination. In a single case among 47 analyzed, we were able to detect a maternal-specific allele in the cord blood sample. We have now studied the same cord samples using a highly sensitive, allele-specific PCR amplification method. A maternal allele could be discriminated from neonate alleles in 10 cases and maternal cells were detected in all 10 cord blood samples. These cells amounted to 10(-4) to 10(-5) of cord blood nucleated cells. In three cases, cord blood separated cell subpopulations could be analyzed and were found to contain maternal cells at about the same level. The presence of maternal cells at such a low level in cord blood samples probably would have no effect on GVHD in a clinical setting of transplantation but raises interesting questions in terms of materno-fetal immune tolerance and transmission of viruses (in particular human immunodeficiency virus) from infected mother to child.

Alleles↗
PubMed1994

Highly sensitive polymerase chain reaction methods show the frequent survival of residual recipient multipotent progenitors after non-T-cell-depleted bone marrow transplantation.

Twenty-four male patients grafted for various pathologies with the marrow of a female donor and presenting a complete donor-type hematopoiesis when analyzed by polymerase chain reaction (PCR) amplification of minisatellite sequences 33.6.3 and MS51 (0.1% to 1% sensitivity) were studied by the highly sensitive technique of PCR amplification of the Y-chromosome-specific DYZ1 sequence (0.01% sensitivity). Residual recipient male cells were detected in all peripheral blood samples collected within 1 year posttransplantation. These residual cells were present in both the lymphocyte and polymorphonuclear cell fractions when such a separation was performed by Ficoll gradient centrifugation and, for samples of 13 of 15 patients, at comparable levels in both fractions. In 3 samples collected from 3 patients 4 months or more posttransplantation, residual recipient cells were detected in the polymorphonuclear cell fraction but were present at a lower level or were undetectable in the lymphocyte fraction. These cells are of hematopoietic origin because they were detected at equivalent levels in whole blood and in B and T lymphocytes sorted with antibody-coated magnetic beads. They were not detected in samples collected more than 15 months posttransplantation for 6 of 7 patients. The persistence of residual recipient cells within 1 year posttransplantation is not restricted to male patients receiving a transplant from a female donor because they were also detected in 2 female patients using an allele-specific amplification method for the thyroid peroxydase gene that also has a high sensitivity (0.01%). Our results indicate that at least residual recipient myeloid progenitors and possibly totipotent hematopoietic stem cells may survive intensive pretransplant conditioning regimen and support a transient residual hematopoiesis of the host posttransplantation.

Adolescent↗
PubMed1994

Cloning and sequence analysis of the beta subunit of the human translocon-associated protein.

A cDNA corresponding to the beta subunit of the human translocon-associated protein was cloned and sequenced. The polypeptide is 183 amino acids long and 96% homologous to its canine counterpart. Both polypeptides contain a cleavable signal sequence, an NH2-terminal domain extruding in the endoplasmic reticulum lumen, a transmembrane domain and a COOH-terminal domain located in the cytoplasm.

Amino Acid Sequence↗
PubMed1994

Search for maternal cells in human umbilical cord blood by polymerase chain reaction amplification of two minisatellite sequences.

Since our first report in 1989, 26 transplants by means of umbilical cord blood have been reported. Furthermore, systematic studies of the feasibility of using banked placental blood for bone marrow reconstitution of unrelated recipients on a large scale are in progress worldwide. However, already by 1989, it was pointed out that the use of cord blood might be hampered by contamination of neonatal blood with maternal cells contributing unacceptably to graft-versus-host disease (GVHD). In the present study, we used the polymerase chain reaction (PCR) amplification of 2 minisatellite sequences (33.6 and MS 51) to address this question. The sensitivity of PCR amplification of minisatellite sequences is known to be of 1% to 0.1%. This sensitivity has been confirmed in the present study, in which a dilution analysis was performed for each experiment in which cell separation was performed. The inclusion of the dilution experiment in these analyses allowed us to estimate the relative amount of contaminating maternal cells, if any. Among 47 cases (31 whole blood analyses, 10 gradient separations, and 6 subpopulation separations), the coamplification of the 2 minisatellites sequences allowed the discrimination of maternal and neonate alleles in 42 cases (89%). In 1 case, we were able to detect a child-specific allele in a mother's whole blood sample, thus validating our approach to search for maternal cells in cord blood. In a single other case, we were able to detect a maternal-specific allele in the cord blood sample. This maternal specific allele was detected in the whole blood, polymorphonuclear cell, and lymphocyte fractions. Comparison of the signal intensity obtained with these 3 cord blood samples to the result of the dilution experiment performed in the same analysis led to an estimate of 1 to 5% maternal cells in the polymorphonuclear cell fraction and 0.1% to 1% maternal cells in the whole blood and lymphocyte cell fractions. In conclusion, our study indicates that maternal cells are very rarely present in the cord blood collected at birth because we detected them in only 1 of 47 cases. More importantly, when detected, they were present at very low level in the lymphocyte cell fraction (0.1% to 1%). However, although small, this amount of cells may result in GVHD in a susceptible recipient. Because the method we used allows the detection of maternal cells within cord blood from 10(4) nucleated cells, it would thus be of interest in a cord blood banking perspective.

Alleles↗
PubMed1994

Efficient second-strand cDNA synthesis using T7 DNA polymerase.

We present a second-strand cDNA synthesis method that takes advantage of both the very high processivity and the very high 3' exonuclease activity of T7 DNA polymerase. The first strand is synthesized with reverse transcriptase using oligo(dT) as a primer. After alkaline hydrolysis of the mRNA template, a tract of dT residues is synthesized with terminal transferase at the 3' end of the first strand. The second strand is synthesized using oligo(dA) as a primer. Several oligo(dA) molecules probably anneal to the poly(dT) tract. Because the 3' exonuclease activity of T7 DNA polymerase is very high, the region of the tract annealed to these oligo(dA) molecules is digested. However, the region of the tract annealed to the very oligo(dA) molecule used as a primer for second-strand synthesis is protected. The resulting cDNA molecules could be cloned with a high efficiency. The size distribution of cloned c-myc DNAs was estimated by Southern blot analysis of phage DNA prepared from the amplified library and by analysis of isolated clones. The results indicate that this method allows to obtain full-length cDNA clones with a high efficiency.

Cloning, Molecular↗
PubMed1994

Detection of recipient cells after non T-cell depleted bone marrow transplantation for leukemia by PCR amplification of minisatellites or of a Y chromosome marker has a different prognostic value.

We used polymerase chain reaction amplification of minisatellite sequences (33.6.3, MS51, YNZ22) and of a Y chromosome-specific sequence (DYZ1) to document prospectively chimerism in 23 leukemia patients grafted with non T-cell depleted marrows from HLA-identical sibling donors. Twenty-two patients had a complete hematopoietic chimerism (within the sensitivity limit of the method used: 1%) early (about 1 month) after transplantation and one had detectable residual host cells (partial chimerism). These cells were still present after 8 months, and this patient relapsed 16 months after transplantation. Two patients with early complete chimerism relapsed 16 and 17 months after transplantation. Seven patients died from toxicity or infections and 13 are in clinical remission with a follow-up of 16 to 48 months. Nine male patients grafted with the marrow from a female donor were also studied by amplification of the DYZ1 marker (0.01% sensitivity). In all nine cases, residual male nucleated cells were detected early and up to 1 year after transplantation. These results suggest that the detection of persistent residual recipient cells above a 1% level might be predictive of relapse but that the detection of such cells in a 0.01-1% range is probably unrelated to relapse and does not seem to influence the outcome of the transplantation procedure.

Bone Marrow Transplantation↗
PubMed1994

Donor B cell lymphoma of the brain after allogeneic bone marrow transplantation for acute myeloid leukemia.

We present a case of EBV-induced cerebral B cell lymphoproliferation of donor origin after HLA-matched allogeneic BMT for AML. The presentation was note-worthy as this 4-year-old girl was grafted after a conditioning regimen without irradiation, with an unmanipulated, HLA-matched graft. Furthermore, the cerebral mass developed long after reduction of immunosuppressive therapy following improvement of severe GVHD. To our knowledge, such cerebral lymphoproliferation, under these transplant conditions, has never been described.

Blotting, Southern↗
PubMed1993

Bone marrow transplantation for Fanconi anemia using low-dose cyclophosphamide/thoracoabdominal irradiation as conditioning regimen: chimerism study by the polymerase chain reaction.

Since 1976, patients grafted at the Hôpital Saint-Louis for Fanconi anemia (FA) without evidence of leukemic transformation have been given a uniform conditioning regimen that consisted of low-dose cyclophosphamide (Cy) and thoracoabdominal irradiation (TAI). The use of low-dose Cy raised the question of whether it is sufficient for the establishment of a complete hematopoietic chimerism in all patients. We therefore initiated a study of chimerism early during hematopoietic reconstitution after bone marrow transplantation (BMT) and thereafter in transplanted FA patients. Minisatellite probes were used after DNA amplification by the polymerase chain reaction (PCR). From July 1989 to October 1992, 24 consecutive patients underwent BMT for FA, 19 of whom were assessable for chimerism. Our results using this sensitive technique showed that, among these 19 patients, all but one successfully engrafted. Engraftment was complete early after BMT in 12. The persistence of a small proportion of recipient's cells was detected in six. This partial hematopoietic chimerism was demonstrated to be only transient in at least five of the six patients. The one patient who failed to engraft showed a recipient-type profile for circulating cells early posttransplantation, indicating autologous bone marrow recovery. A second graft in this patient was also rejected. For both transplantations, the patient was grafted from a matched, unrelated donor. Therefore, 17 of 17 patients successfully grafted and with complete follow up data presented complete hematopoietic chimerism, within the sensitivity limit of the method used. In conclusion, lowering the Cy dose in the conditioning regimen of patients with FA could still allow complete engraftment to occur, at least in patients with an identical sibling donor.

Bone Marrow Transplantation↗
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↗