Biomedical subjects

M Vuillaume

Publications and source records attributed to M Vuillaume.

At least 19 recordsLinked to original sources

PubMed2001

Characterization of the electrochemical oxidation of peroxynitrite: relevance to oxidative stress bursts measured at the single cell level.

The electrochemical signature of peroxynitrite oxidation is reported for the first time, and its mechanism discussed in the light of data obtained by steady-state and transient voltammetry at microelectrodes. Peroxynitrite is an important biological species generated by aerobic cells presumably via the near diffusion-limited coupling of nitric oxide and superoxide ion. Its production by living cells has been previously suspected during cellular oxidative bursts as well as in several human pathologies (arthritis, inflammation, apoptosis, ageing, carcinogenesis, Alzheimer disease, AIDS, etc.). However, this could only be inferred on the basis of characteristic patient metabolites or through indirect detection, or by observation of follow-up species resulting supposedly from its chemical reactions in vivo. In this work, thanks to the independent knowledge of the electrochemical characteristics of ONO2- oxidation, the kinetics and intensity of this species released by single human fibroblasts could be established directly and quantitatively based on the application of the artificial synapse method. It was then observed and established that fibroblasts submitted to mechanical stresses produce oxidative bursts, which involve the release within less than a tenth of a second of a complex cocktail composed of several femtomoles of peroxynitrite, hydrogen peroxide, nitric oxide, and nitrite ions.

Electrochemistry↗

PubMed2001

Relationship between posttranslational modification of transaldolase and catalase deficiency in UV-sensitive repair-deficient xeroderma pigmentosum fibroblasts and SV40-transformed human cells.

Xeroderma Pigmentosum (XP) is a rare recessively inherited human disease associated with a hypersensitivity to ultraviolet radiation. The ultraviolet component of sunlight can initiate and promote the formation of cutaneous tumors as seen in the skin cancer-prone XP patients. Previously, we have found that the low activity of the NADPH-dependent antioxydant enzyme, catalase, which we have observed in XP diploid fibroblasts and SV40-tranformed cells, could be restored by the addition of NADPH. Here we have analyzed transaldolase, which regulates NADPH levels produced by the pentose phosphate pathway in order to examine how it influences the catalase activity regulated in XP and SV40-transformed cells. We find that transaldolase activity is high in XP and SV40-transformed human fibroblasts, whereas transaldolase transcription is unchanged, suggesting that modification of transaldolase activity is due to a posttranslational modification of the protein. Two-dimensional electrophoresis analysis has allowed us to identify a complex set of transaldolase isoforms and to postulate that the phosphorylation of specific isoforms could be correlated with the different enzymatic activities seen. Our results show that high transaldolase activity corresponds to a low catalase activity in SV40-transformed cells and in fibroblasts from XP patients who have a high predisposition to develop skin cancer.

Acatalasia↗

PubMed2000

Analysis of individual biochemical events based on artificial synapses using ultramicroelectrodes: cellular oxidative burst.

Carbon fiber platinized ultramicroelectrodes placed within micrometres of a single living cell are used to monitor cellular events. This artificial synapse is used here to collect and examine the very nature of the massive oxidative bursts produced by human fibroblasts when their membrane is locally depolarized by a puncture made with a micrometre sized sealed pipette. The electrochemical analysis of the response indicates that oxidative bursts consist of a mixture of a few femtomoles of highly cytotoxic chemicals: hydrogen peroxide, nitrogen monoxide and peroxynitrite, together with nitrite ions, which may result from a partial spontaneous decomposition of peroxynitrite prior to its release by the cell.

Animals↗

PubMed2000

Abnormal expression of the ATM and TP53 genes in sporadic breast carcinomas.

The ataxia telangiectasia gene (ATM) has been implicated as a risk factor in the development of sporadic breast carcinomas. ATM protein expression was analyzed by immunohistochemistry in 17 breast carcinomas with two monoclonal antibodies whose immunohistochemical use was first validated by comparing the immunoreactivity observed in spleen samples from ataxia telangiectasia and trauma patients. In normal breast ducts, ATM showed nuclear expression in the epithelial but not in the myoepithelial cells. In contrast, this nuclear expression was absent or low in the epithelial cancer cells in 10 of 17 (59%) of the tumors studied. Allelic imbalance in the ATM gene was found in three of seven tumors examined. Two of these showed reduced ATM protein expression, but this did not correlate with the presence of ATM mutations in the tumor DNA detected by restriction endonuclease fingerprinting screening. These results suggest that the reduced ATM protein expression could be attributable, in certain tumors, to deletions or rearrangements within or close to the ATM gene. Positive p53 immunostaining was found in 10 tumors, with TP53 mutations detected in 8. Three tumors had both low ATM expression and mutated TP53. Our results indicate that in the majority (15 of 17) of the sporadic breast carcinomas examined, not only is the functionality of the ATM-p53-mediated DNA damage response compromised, but also other signaling pathways activated by these two multifunctional proteins are likely to be impaired, which could be a contributing factor to tumor development and progression.

Allelic Imbalance↗

PubMed1999

High incidence of cancer in a family segregating a mutation of the ATM gene: possible role of ATM heterozygosity in cancer.

ATM mutations predispose cells to malignancy by promoting chromosomal instability. We have identified a family with multiple cancers that segregates a mutant allele of ATM, IVS61+2insTA, which causes skipping of exon 61 in the mRNA, as well as a previously undescribed polymorphism, IVS61+104C(54):T(46). The mutation was inherited by two sisters, one who developed breast cancer at age 39 and the second at age 44, from their mother, who developed kidney cancer at age 67. Molecular studies were undertaken to determine the role of the ATM gene in the development of cancer in this family. Studies of irradiated lymphocytes from both sisters revealed elevated numbers of chromatid breaks, typical of A-T heterozygotes. Studies on lymphoblastoid cell lines established from these individuals revealed abnormal p53 induction and apoptosis after DNA damage. Loss of heterozygosity (LOH) in the ATM region of chromosome 11q23.1 showed that the normal ATM allele was lost in the breast tumor of the older sister. LOH was not seen at the BRCA1 or BRCA2 loci. BRCA2 is not likely to be a cancer-predisposing gene in this family because each sister inherited different chromosomes 13 from each parent. The sisters share their maternal BRCA1 allele, although no mutation in this gene was detected in the family. Our findings suggest that haploinsufficiency at ATM may promote tumorigenesis, even though LOH at the locus supports a more classic two-hit tumor suppressor gene model.

Adult↗

PubMed1999

Amplification of the inflammatory cellular redox state by human immunodeficiency virus type 1-immunosuppressive tat and gp160 proteins.

In the course of our studies on oxidative stress as a component of pathological processes in humans, we showed that microintrusion into cells with microcapillary and ultramicroelectrochemical detection could mimic many types of mechanical intrusion leading to an instant (0.1 s) and high (some femtomoles) burst release of H2O2. Specific inhibitors of NADPH enzymes seem to support the assumption that this enzyme is one of the main targets of our experiments. Also, human immunodeficiency virus type 1 (HIV-1) gp160 inhibits the cooperative response of uninfected T cells as well as Tat protein release by infected cells does. In this study, we analyzed in real time, lymphocyte per lymphocyte, the T-cell response following activation in relation to the redox state. We showed that the immunosuppressive effects of HIV-1 Tat and gp160 proteins and oxidative stress are correlated, since the native but not the inactivated Tat and gp160 proteins inhibit the cellular immune response and enhance oxidative stress. These results are consistent with a role of the membrane NADPH oxidase in the cellular response to immune activation.

Arsenicals↗

PubMed1998

Low catalase activity in xeroderma pigmentosum fibroblasts and SV40-transformed human cell lines is directly related to decreased intracellular levels of the cofactor, NADPH.

We have previously shown that fibroblasts from ultra-violet (UV) hypersensitive xeroderma pigmentosum patients (XP) are markedly deficient in catalase activity resulting in high intracellular levels of hydrogen peroxide (H2O2) following UV irradiation. No direct correlation between catalase activity and repair ability was found since XP variant cells which are proficient in nucleotide excision repair (NER) showed activities as low as those found in NER deficient classical XP groups A and D. However, in contrast to the skin cancer prone XP patients, another NER deficient syndrome, trichothiodystrophy (TTD), which does not exhibit any cancer predisposition, was found to present normal catalase activity. Moreover, it was found that a variety of SV40 transformed human cell lines also showed decreased catalase activities. Our previous data showed that a molecular analysis of the normal, XP, TTD or transformed human fibroblast cell lines did not reveal any differences in levels of catalase transcription or amount of catalase protein subunits. These results incited us to examine the structure/function relationship of the tetrameric active enzyme form of catalase (which is the only one able to carry out H2O2 dismutation) with its cofactor NADPH. In the present study, we have measured the effects on catalase activity after adding NADPH either to acellular extracts or during cell culture of the different cell types. The NADPH levels were also quantified directly in intact cells using flow cytometry. Our results show a clear relationship between low catalase activity and striking decrease in intracellular NADPH levels.

Catalase↗

PubMed1998

The p53-mediated DNA damage response to ionizing radiation in fibroblasts from ataxia-without-telangiectasia patients.

PURPOSE: To assess the functionality of the p53-mediated pathway, activated by the ataxia-telangiectasia gene product (ATM) in response to ionizing radiation, in cells derived from four ataxia-without-telangiectasia patients. These patients exhibit cerebellar ataxia and cellular abnormalities that are compatible with the diagnosis of ataxia-telangiectasia (AT), but the telangiectasias normally seen in AT patients are absent. MATERIALS AND METHOD: Protein and RNA extracts were prepared from primary fibroblast cultures non- or exposed to 5 Gy of ionizing radiation in order to monitor the modulation in p53 and ATM protein levels by immunologic techniques and WAF1/Cip1(p21) mRNA by Northern blotting. RESULTS: A sub-optimal response in terms of increased levels of p53 and the transcriptional activation of WAF1/Cip1(p21) was see in the ataxia-without-telangiectasia fibroblast cultures examined over a 4 h period post-irradiation when compared with normal fibroblast cultures. The ATM protein was expressed at much reduced levels in the ataxia-without-telangiectasia and the classical AT fibroblast cultures examined when compared with normal fibroblast cultures. CONCLUSIONS: Despite the milder clinical phenotypes observed in these ataxia-without-telangiectasia patients and the presence of low levels of ATM protein in the fibroblast cultures, their response to ionizing radiation quantitatively resembles that reported in fibroblast cultures established from classical AT patients.

Ataxia↗

PubMed1997

Phenylarsine oxide inhibits ex vivo HIV-1 expression.

Phenylarsine oxide (PAO), which is described as an inhibitor of tyrosine phosphatase activity, inhibits H2O2 release from human peripheral blood mononuclear cells (PBMCs) as measured by electrochemistry. Since human immunodeficiency virus type 1 (HIV-1) replication is known to be favored under oxidative stress conditions, ex vivo experiments using uninfected PBMCs, primary monocytes or a latently infected promonocytic U1 cell line show that HIV-1 replication and reactivation, monitored by p24 antigen measurement, are inhibited by PAO in a time- and concentration-dependent manner. These observations can be linked with the inhibition of NF-kappa B activation when uninfected monocytes are induced by either tumor necrosis factor alpha (TNF-alpha) phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide (LPS).

Arsenicals↗

PubMed1997

Retroviral-mediated correction of DNA repair defect in xeroderma pigmentosum cells is associated with recovery of catalase activity.

Xeroderma pigmentosum (XP) is a rare inherited disease associated with photosensitivity, a very high susceptibility to develop neoplasm on sun-exposed skin and neurological abnormalities for some patients. We previously reported that diploid cell lines established from XP skin biopsies present an abnormal low level of catalase activity, which is involved in the defense against oxygen free radicals. This biochemical dysfunction, probably involved in the skin cancer formation, has been difficult to be directly related to the nucleotide excision repair (NER) defect in XP. In this paper we report that the retroviral-mediated transduction of XP diploid cells by the XPC and XPD/ERCC2 cDNAs fully and stably corrects the NER defect in terms of survival and unscheduled DNA synthesis (UDS) after ultraviolet (UV) irradiation. The catalase activity in transduced cells was recovered up to normal levels only in cells transduced with repair genes correcting the repair defect. These results imply that: (i) the reduced catalase activity in XP, which might result from cellular depletion of its NADPH cofactor, is directly related to impaired DNA repair, and (ii) this depletion might be one of the multiple cellular consequences of XP inborn defect.

Catalase↗

PubMed1997

Activation of the NADPH oxidase in human fibroblasts by mechanical intrusion of a single cell with an ultramicroelectrode.

We present here a real-time and single cell study of an oxidative stress mechanism in human fibroblasts. Hydrogen peroxide released by a single normal or SV40-transformed human fibroblast was detected at the surface of an ultramicroelectrode while puncturing the cell membrane with the ultramicroelectrode tip itself or with a micropipette. This mechanical intrusion induced the emission of large quantities (10(-15)-10(-14) mol) of H2O2 by the cell with a very short time delay (<0.5 s). We show that this H2O2 production was an active neo-production by fibroblasts when the membrane was stressed by the cellular puncture and is a model which could mimic similar effects as particle (virus, bacteria, etc.) intrusion into the cell. Cell incubations in the presence of some inhibitors of the different NADPH oxidase enzymes, using ultramicroelectrode measurements of the short time effects (<20 min) let us believe that an NADPH oxidase-like enzyme may be implicated in this induced-H2O2 generation. Phenylarsine oxide (PAO), a specific NADPH oxidase inhibitor, at concentrations between 0.5-50 microM seemed to quickly kill the transformed cells preferentially to the normal cells, pointing out for the future a possible anti-cancerous chemotherapic use.

Antioxidants↗

PubMed1997

Nijmegen breakage syndrome cells fail to induce the p53-mediated DNA damage response following exposure to ionizing radiation.

The functionality of the p53-mediated pathway, activated in response to DNA damage, has been assessed in primary fibroblast cell cultures and Epstein-Barr virus-transformed lymphoblastoid cell lines derived from Nijmegen breakage syndrome (NBS) patients. This autosomal recessive disease is characterized by microcephaly, growth and mental retardation, chromosomal instability, radiosensitivity, and high cancer incidence. The recent mapping of the NBS gene to chromosome 8q21 demonstrates that NBS is genetically distinct from ataxia telangiectasia (AT). Changes in p53 protein levels were significantly reduced and delayed in all the NBS fibroblast cell cultures and lymphoblastoid cell lines examined compared to normal cultures over a 4-h period postirradiation (5 Gy). The transcriptional activation of p21(WAF1/CIP1) mRNA was also lower in 12 NBS fibroblast cultures examined. In agreement with an abrogated p53 function, NBS cells exposed to ionizing radiation show an abnormal cell cycle arrest at G1-S and a prolonged accumulation of cells in the G2 phase. In contrast, exposure to the alkylating agent methyl methanesulfonate results in similar increases of p53 and p21(WAF1/CIP1) mRNA in both cell types. The ATM gene transcript was found to be expressed at similar levels in NBS and normal cells, whereas it was strongly reduced in the AT homozygote cells examined. These results suggest that the ATM gene product cannot substitute for that of the NBS gene in the signaling of cellular damage produced by ionizing radiation and that both are involved in the activation of p53. The suboptimal p53-mediated response could contribute to the high cancer risk and radiosensitivity seen in NBS patients.

Ataxia Telangiectasia↗

PubMed1996

The role of Ataxia telangiectasia and the DNA-dependent protein kinase in the p53-mediated cellular response to ionising radiation.

The DNA-dependent protein kinase (DNA-PK), whose catalytic subunit shows structural similarities to the Ataxia telangiectasia (AT) gene product (ATM), has also been implicated in the p53-mediated signal transduction pathway that activates the cellular response to DNA damage produced by ionizing radiation. DNA-PK activity however was not found to be related to the transcriptional induction of WAFl/CIP1(p2l) in AT lymphoblastoid cell lines, following treatment with ionizing radiation. Normal protein and transcription levels of Ku70 and Ku80, as well as DNA-PK activity, were found in six different AT cell lines, 1-4 h following exposure to ionizing radiation, timepoints where reduced and delayed transcriptional induction of WAF1/CIP1 (p21) was observed. WAF1/CIP1 (p21) was found to be transcriptionally induced by p53 in normal cell lines over this same time period following exposure to ionizing radiation. These results suggest that despite the findings that in vitro DNA-PK may phosphorylate p53, in vivo it would not appear to play a central role in the activation of p53 as a transcription factor nor can it substitute for the ATM gene product in the cellular response following exposure to ionizing radiation.

Animals↗

PubMed1995

The role of the Ataxia telangiectasia gene in the p53, WAF1/CIP1(p21)- and GADD45-mediated response to DNA damage produced by ionising radiation.

The inducible response of the tumour suppressor gene p53 has been examined following exposure to DNA-damaging agents in Ataxia telangiectasia (AT) cell lines, an autosomal recessive disorder with multiple clinical and biological abnormalities including sensitivity to ionising radiation. The p53 induction was significantly delayed and reduced in the 8 AT cell lines examined over the 6 h following irradiation with no dose response in p53 induction being observed compared to control cells. The increase of WAF1/CIP1(p21) and GADD45 mRNA, two genes transcriptionally activated by p53, was also reduced in the AT cell lines after such treatment. In contrast, the increase in p53 protein, WAF1/CIP1(p21) and GADD45 mRNA expression following exposure to the alkylating agent methylmethane sulphonate (25 and 100 micrograms ml-1) was similar in both cell types. No alterations in the expression of EBNA-5, an EBV-encoded nuclear antigen which has been shown to bind p53 or mutations in the p53 gene (exons 4 to 8) were found in the AT cell lines studied. The AT gene product would thus appear to be involved upstream of p53, GADD45 and WAF1/CIP1 (p21) in the signalling of the presence of strand breaks produced by ionising radiation, with this defect in response contributing to the high cancer risk and radiosensitivity observed in this disorder.

Alkylating Agents↗

PubMed1995

Monitoring an oxidative stress mechanism at a single human fibroblast.

Easily oxidizable substances inside human diploid fibroblast cell strains were monitored amperometrically with a platinized carbon-fiber microelectrode. The experiment involved positioning a microelectrode over a single biological cell, forcing the electrode tip into the cell via micromanipulator control, and measuring the transient current corresponding to the complete electrolysis of electroactive species released by the cell. A second series of experiments involved puncturing a hole into the cell with a micropipet and measuring the transient current corresponding to the complete electrolysis of electroactive species emitted by the cell with an electrode positioned above the cell. The selectivity of both amperometric measurements was demonstrated through the use of known hydrogen peroxide scavengers (added catalase or intracellular peroxidase + added o-dianisidine) to the media bathing the cells. The abolition of the amperometric signal under these conditions suggested that hydrogen peroxide was the primary substance detected. The magnitude and the time course of the transient current measured implied that the hydrogen peroxide detected was not only that initially present in the cell before its membrane was pierced but represented mostly an oxidative stress response of the cell to its injury.

Cells, Cultured↗

PubMed1994

Rearrangement of CCND1 (BCL1/PRAD1) 3' untranslated region in mantle-cell lymphomas and t(11q13)-associated leukemias.

Rearrangement and overexpression of CCND1 (BCL1/PRAD1), a member of the cyclin G1 gene family, are consistent features of t(11q13)-bearing B-lymphoid tumors (particularly mantle-cell lymphoma [MCL]). Its deregulation is thought to perturb the G1-S transition of the cell cycle and thereby to contribute to tumor development. As suggested by previously published studies, rearrangement of the 3' untranslated region (3' UTR) of CCND1 may contribute to its activation in some lymphoid tumors. To define further the prevalence of such rearrangements, we report here the result of the molecular study of 34 MCL and six t(11q13)-associated leukemias using a set of probes specific to the different parts of the CCND1 transcript. We also sequenced the entire cDNA of the overexpressed CCND1 transcripts in a t(11q13)-associated leukemia. DNA from four of these 40 patients showed rearrangement of the 3' UTR of CCND1 coexisting with major translocation cluster (MTC) rearrangement. Southern blot and sequence analyses showed that, as a result of these rearrangements, the 3' AU-rich region containing sequences involved in mRNA stability and in translational control is eliminated. Moreover, the finding that the CCND1 mRNA half-life was greater than 3 hours (normal tissues, 0.5 hours) in three t(11q13)-associated cell lines stresses the importance of posttranscriptional derangement in the activation of CCND1. Finally, we did not observe any mutation in the coding frame of the CCND1 cDNA analyzed.

Amino Acid Sequence↗

PubMed1994

Breakpoints of Burkitt's lymphoma t(8;22) translocations map within a distance of 300 kb downstream of MYC.

The variant translocation t(8;22) in Burkitt's lymphoma (BL) cells joins band q24 of chromosome 8 distal to the proto-oncogene MYC to the immunoglobulin lambda locus. The distribution of breakpoints on chromosome 8 of 11 cell lines with t(8;22) has been investigated by in situ fluorescence hybridization and pulsed-field gel electrophoresis. We show that these chromosomal breakpoints generally fall within a region of about 300 kb 3' of MYC and that at least 8 out of 11 affect the previously characterized transcriptional unit PVT1. Comparable results were obtained in earlier experiments analyzing the variant t(2;8). Recently, in a series of BL cells carrying t(8;14), breakpoints upstream of MYC have been described at a similar distance. Therefore, our results suggest that deregulation of MYC by the immunoglobulin loci can occur at a distance of up to about 350 kbp of MYC.

Burkitt Lymphoma↗

PubMed1994

Immunophenotypic and genotypic analysis of acquired immunodeficiency syndrome-related non-Hodgkin's lymphomas. Correlation with histologic features in 36 cases. French Study Group of Pathology for HIV-Associated Tumors.

High-grade B-cell-type non-Hodgkin's lymphomas are observed in 5% to 8% of patients positive for the human immunodeficiency virus. Nearly all cases belong to one of the three major histologic types: centroblastic or large noncleaved cell, immunoblastic and Burkitt's lymphoma, or small noncleaved cell. Some cases that are polymorphic are termed high-grade B-cell, not otherwise specified (NOS). The authors determined the immunophenotype of each histologic category of acquired immunodeficiency syndrome (AIDS)-related non-Hodgkins' lymphoma and sought a relationship with the presence of the Epstein-Barr virus (EBV). B-cell differentiation antigens, activation marker expression (human leukocyte antigen-DR, CD10, CD19, CD20, CD21, CD22, CD23, CD25, CD30, CD38), and epithelial membrane antigen were analyzed. The clonality was determined by the detection of cytoplasmic immunoglobulin, surface immunoglobulin, and the analysis of joining region (JH) immunoglobulin gene configuration by Southern blot. Epstein-Barr virus was detected either by Southern blot analysis using BamHI W probe fragment or by in situ hybridization with EBV-encoded RNA transcripts-1 specific probe. The immunophenotypic and genotypic results were compared with the morphology results and with the presence or absence of EBV. Burkitt's lymphomas were associated with EBV in 50% of cases, were monoclonal, and expressed mostly immunoglobulin (Ig) MK, CD10, CD19, CD20, CD22, and CD38. This immunophenotypic profile closely resembled those of the centroblastic cases (large noncleaved cell), in which EBV was absent. Epstein-Barr virus was associated with 90% of immunoblastic cases, and only CD10, CD20, and CD38 were expressed. CD71 was expressed in all categories of non-Hodgkin's lymphoma, and CD21 and CD23 were rarely expressed. Two cases of immunoblastic lymphoma and one case of high-grade B-NOS were polyclonal regarding JH rearrangement, but EBV tested with 1.9-Kb Xhol fragment was clonal. No significant immunophenotypic changes were noted in relation to the presence of EBV. Such studies comparing morphology, immunophenotype, and genotype could help classify and better understand the pathogenesis of AIDS-related non-Hodgkin's lymphoma.

Adult↗