Biomedical subjects

B Dutrillaux

Publications and source records attributed to B Dutrillaux.

At least 55 records · Page 3Linked to original sources

PubMed1999

DNA methylation and chromosome instability in breast cancer cell lines.

We show that in a series of eight breast cancer cell lines, a direct relationship exists between the overall DNA demethylation and the percentage of rearranged chromosomes, except for cell lines with a highly rearranged genome which can be weakly demethylated. A real time fluorescent detection method was used to quantify by reverse transcription-PCR the expression of the DNA methyltransferase 1 and of the newly discovered DNA demethylase. The overall DNA methylation status seems to result from a complex interplay between the expression of these two genes. Our results suggest that in these tumor cells, the overall DNA demethylation is implicated in one of the mechanisms at the origin of the genome instability and that besides the role of the DNA methyltransferase 1, that of the DNA demethylase may be essential in the control of DNA methylation.

Breast Neoplasms↗

PubMed1999

Evidence for in vitro selection during cell culturing of breast cancer: detection by flow and image cytometry.

Detailed studies of chromosome rearrangements within solid tumors require karyotype analysis after cell culturing. However, different cell subpopulations with various growth capacities within one tumor may introduce biases in karyotype analysis, known as the in vitro selection. In our laboratory, 22% of karyotypes from breast cancers established after short-term culture were normal. Using interphase fluorescence in situ hybridization (FISH) for the determination of chromosome 1 arm imbalances and flow cytometry measurements of ploidy, we demonstrated that at least 2/3 of these tumors were mainly composed of aneuploid cell populations. Thus, the incidence of normal or balanced karyotypes among breast cancers is probably below 7%. This is the first direct proof for the existence of an in vitro selection within breast cancer cultures, suggesting cautious interpretation of cytogenetic data.

Adult↗

PubMed1999

Cytogenetic study of eight new cases of radiation-induced solid tumors.

Radiation-induced tumors were selected according to the criteria defined by Cahan (1948) for sarcomas. Cell cultures and/or xenografts in nude mice were performed with biopsies obtained from second primary tumors. Karyotypes of eight tumors were established after R-banding. After comparison with literature data on 15 other cases, two distinct cytogenetic patterns could be distinguished. One was characterized by polyclonal karyotypes, of which a large proportion were simple and carriers of balanced translocations. Another one was characterized by monoclonal chromosome alterations observed in highly aneuploid and complex karyotypes, in which many deletions were observed. These two different patterns could be related to the modality of metaphase harvesting. Polyclonal karyotypes were preferentially observed after long-term cultures, and monoclonal karyotypes after short-term cultures or xenografts. The following scheme of radiation oncogenesis is proposed: a) induction of recessive gene mutations including that of tumor suppressor genes; b) accumulation of genomic alterations in the irradiated tissue with aging, including deletions or mutations of normal alleles from mutated tumor suppressor genes; and c) loss of tumor suppressor gene function and initiation of a multistage tumor development and progression. Polyclonal abnormalities are assumed to exist in noncancerous cells which acquired radiation-induced chromosome aberrations.

Aneuploidy↗

PubMed1999

Effect of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide (PK11195), a specific ligand of the peripheral benzodiazepine receptor, on the lipid fluidity of mitochondria in human glioma cells.

When human glioma cells were incubated for 24 hr in serum-free medium with nanomolar concentrations of 1-(2-chlorophenyl)-N-methyl-N(1-methylpropyl)-3-isoquinoline carboxamide (PK11195), a specific ligand of the peripheral benzodiazepine receptor (PBR), a significant increase in the membrane fluidity of mitochondria isolated from these cells was registered. These effects were not observed with a shorter incubation time (2 hr) of the cells with PK11195 nor in the presence of serum. Other significant associated changes were observed: a significant increase of 16+/-4% of [3H]thymidine incorporation into DNA was detected in cells in the presence of PK11195 in serum-free medium, and an increase of 33+/-5% as compared to controls in nonyl acridine orange uptake, as indicator of mitochondrial mass, was also registered in cells treated with 10 nM PK11195. [3H]PK11195 binding was decreased in cells incubated with PK11195; a 45% decrease compared to controls was obtained. In view of the effect of PBR ligands on DNA synthesis, changes in mitochondrial lipid metabolism through interaction with PBRs might lead to biogenesis of mitochondria to support the increased metabolic requirements for cell division, which is even higher in malignant cells.

Antineoplastic Agents↗

PubMed1999

Amplification and over-expression of OZF, a gene encoding a zinc finger protein, in human pancreatic carcinomas.

The OZF gene encodes a protein consisting of 10 zinc finger motifs and is located on chromosome 19q3.1. We report here the amplification and over-expression of the OZF gene in pancreatic carcinomas. Increased gene copy number was detected in 3 of 12 tumour cell lines and 2 of 12 primary pancreatic carcinomas. Expression was detected in all cell lines, and the gene was over-expressed in cell lines with OZF gene amplification. Five of 8 tumours, including 2 primary tumours with OZF gene amplification, displayed high levels of OZF protein, whereas normal pancreas expressed low levels. Immuno-histochemical analysis showed that expression was restricted to tumour cells. Thus, high-level expression of OZF is frequent in pancreatic carcinomas and may contribute to the development or progression of this tumour.

Blotting, Western↗

PubMed1999

Distinct chromosomal alterations associated with TP53 status of LoVo cells under PALA selective pressure: a parallel with cytogenetic pathways of colorectal cancers.

This study investigates the chromosomal alterations involved in the acquisition of PALA resistance of LoVo colorectal cancer cells homozygous for wild-type TP53 before and after transfection with a 143Ala-mutated TP53 gene. PALA resistance was always associated with an increased number of CAD gene copies, but gene amplification sensu stricto was rarely observed. Interestingly, distinct chromosome patterns were found in relation to the TP53 status of the cells. In parental LoVo cells, the CAD copy number was increased through gains of normal chromosome 2 whereas in transfectant clones, resistance mostly occurred through chromosome rearrangements. The relationship with the two different cytogenetic patterns described in colorectal tumors is discussed.

Aspartic Acid↗

PubMed1999

TP53 status and gene amplification in human colorectal carcinomas.

Gene amplification is one of the characteristics of cancer cells. In vitro studies suggested that alterations of the TP53 gene might be responsible for gene amplification. We have examined the presence of TP53 mutations and looked for cytogenetic evidence of gene amplification in a series of 79 primary colorectal carcinomas. Other parameters such as the pattern of cytogenetic alterations, microsatellite instability, tumor site, and histological staging were also considered. A multiparametric study supported by statistical analyses suggests the existence of two major pathways of colorectal carcinogenesis. No relationships could be established between the presence of TP53 alterations and gene amplification.

Aged↗

PubMed1999

Quantitative fluorescence in situ hybridization in lung cancer as a diagnostic marker.

The diagnosis of lung cancer is quite often hampered by the existence of various cell types within samples such as biopsies or pleural effusions. We have established a new marker for image cytometry of interphase tumor cells of the lung by using the most recurrent and early cytogenetic event in lung cancer, the loss of the short arm of chromosome 3. The method is based on the detection of the imbalance between the long and the short arms of chromosome 3 by performing two-color fluorescence in situ hybridization on both arms. Fourteen tumors were analyzed after short-term culture and compared with the corresponding cytogenetic data obtained from metaphase analysis. Results on interphase nuclei and control experiments on metaphases were the same, with imbalance ratios ranging from 1.0 to 2.0 (mean value 1.6, median 1.5). To assess the clinical significance of this approach, three pleural effusions were analyzed. Data showed that normal cells within the sample could have been distinguished from the tumor cells based on different imbalance values between the long and the short arms. Thus, our method allows refined detection of lung tumor cells within samples containing heterogeneous cell populations.

Biomarkers, Tumor↗

PubMed1999

Comparative karyotype of rat and mouse using bidirectional chromosome painting.

A comparative karyotype of rat (Rattus norvegicus) and mouse (Mus musculus) based on chromosome G-banding morphology, heterologous chromosome painting results and available gene mapping data is proposed. Whole chromosome painting probes from both species were generated by PARM-PCR amplification of flow sorted chromosomes. Bidirectional chromosome painting identifies 36 segments of syntenic homology and allows us to propose a nearly complete comparative karyotype of mouse and rat (except for RNO 13 p and RNO 19 p12-13). Seven segments completely covered the RNO chromosomes 3, 5, 8, 11, 12, 15 and 18. Eight segments completely covered the MMU chromosomes 3, 4, 6, 7, 9, 12, 18 and 19. The RNO chromosomes 5, 8, 18 show complete homology with the MMU chromosomes 4, 9 and 18, respectively. Bidirectional hybridization results clearly assign 16 segments to subchromosomal regions in both species. Interpretation of the results allows subchromosomal assignment of all the remaining segments apart from seven distributed on chromosomes MMU 15, MMU 10 B2-D3 and MMU 17 E3-E5. The proposed comparative karyotype shows overall agreement with available comparative mapping data. The proposed repartition of syntenic homologous segments between the two species provides useful data for gene-mapping studies. In addition, these results will enable the reconstruction of the karyotype of the Cricetidae and Muridae common ancestor and the definition of the precise rearrangements which have occurred in both mouse and rat lineages during evolution.

Animals↗

PubMed1999

Cloning, characterization, and chromosome assignment of Zfp146 the mouse ortholog of human ZNF146, a gene amplified and overexpressed in pancreatic cancer, and Zfp260 a closely related gene.

The human OZF gene (ZNF146), located in chromosome band 19q13.1, is amplified and overexpressed in pancreatic carcinomas. It encodes a protein consisting solely of ten Krüppel zinc finger motifs. We report here the isolation and the characterization of the murine OZF cDNA (Zfp146). Comparison of the deduced amino acid sequences between murine, human and bovine cDNAs revealed a strong identity (95%). A closely related gene, Zfp260, was also isolated and characterized. It encodes a putative protein consisting of three vestigial zinc finger motifs followed by ten Krüppel zinc fingers sharing 79% identity and no gap insertion with the Zfp146 zinc fingers. In vitro transcription/translation of both genes led to synthesis of proteins of the predicted size. Co-expression was observed at the mRNA level in eight adult mouse tissues. Two-color FISH revealed co-localization of both genes on mouse chromosome 7 (band B1-B3). The co-expression and co-localization of Zfp146 and Zfp260 together with the close similarity of their zinc finger domains, suggests that both participate in the same regulatory pathway.

Amino Acid Sequence↗

PubMed1999

Alterations of DNA methylation patterns in germ cells and Sertoli cells from developing mouse testis.

In situ alterations of DNA methylation were studied between 14 d postcoitum and 4 d postpartum in Sertoli cells and germ cells from mouse testis, using anti-5-methylcytosine antibodies. Compared to cultured fibroblasts, Sertoli cells display strongly methylated juxtacentromeric heterochromatin, but hypomethylated chromatids. Germ cells always possess hypomethylated heterochromatin, whereas their euchromatin passes from a demethylated to a strongly methylated status between days 16 and 17 postcoitum. This hypermethylation occurs in the absence of DNA replication, germ cells being blocked in the G(0)-G(1) phase from day 15 postcoitum to birth. The DNA hypermethylation of germ cells is maintained until birth and could be visualized on both chromatids of metaphase chromosomes at the first postpartum cell division. Subsequently, the DNA hypermethylation is lost semiconservatively, being replaced by a methylation pattern recalling the typical fibroblast pattern. These alterations of DNA methylation follow a strict chronology, are chromosome structure and cell-type dependent, and may underlie profound changes of genome function.

Animals↗

PubMed1999

A dual approach in the study of poly (ADP-ribose) polymerase: in vitro random mutagenesis and generation of deficient mice.

A dual approach to the study of poly (ADP-ribose)polymerase (PARP) in terms of its structure and function has been developed in our laboratory. Random mutagenesis of the DNA binding domain and catalytic domain of the human PARP, has allowed us to identify residues that are crucial for its enzymatic activity. In parallel PARP knock-out mice were generated by inactivation of both alleles by gene targeting. We showed that: (i) they are exquisitely sensitive to gamma-irradiation, (ii) they died rapidly from acute radiation toxicity to the small intestine, (iii) they displayed a high genomic instability to gamma-irradiation and MNU injection and, (iv) bone marrow cells rapidly underwent apoptosis following MNU treatment, demonstrating that PARP is a survival factor playing an essential and positive role during DNA damage recovery and survival.

Age Factors↗

PubMed1998

Light-induced photoactivation of hypericin affects the energy metabolism of human glioma cells by inhibiting hexokinase bound to mitochondria.

Glucose-dependent energy required for glioma metabolism depends on hexokinase, which is mainly bound to mitochondria. A decrease in intracellular pH leads to a release of hexokinase-binding, which in turn decreases glucose phosphorylation, ATP content, and cell proliferation. Thus, intracellular pH might be a target for therapy of gliomas, and a search for agents able to modulate intracellular pH was initiated. Hypericin, a natural photosensitizer, displays numerous biological activities when exposed to light. Its mechanism and site of action at the cellular level remain unclear, but it probably acts by a type II oxygen-dependent photosensitization mechanism producing singlet oxygen. Hypericin is also able to induce a photogenerated intracellular pH drop, which could constitute an alternative mechanism of hypericin action. In human glioma cells treated for 1 h with 2.5 microg/ml hypericin, light exposure induced a fall in intracellular pH. In these conditions, mitochondria-bound hexokinase was inhibited in a light- and dose-dependent manner, associated with a decreased ATP content, a decrease of mitochondrial transmembrane potential, and a depletion of intracellular glutathione. Hexokinase protein was effectively released from mitochondria, as measured by an ELISA using a specific anti-hexokinase antibody. In addition to decreased glutathione, a response to oxidative stress was confirmed by the concomitant increase in mRNA expression of gamma-glutamyl cysteine synthetase, which catalyzes the rate-limiting step in overall glutathione biosynthesis, and is subject to feedback regulation by glutathione. Hypericin also induced a dose- and light-dependent inhibition of [3H]thymidine uptake and induced apoptosis, as demonstrated by annexin V-FITC binding and cell morphology. This study confirmed the mitochondria as a primary target of photodynamic action. The multifaceted action of hypericin involves the alteration of mitochondria-bound hexokinase, initiating a cascade of events that converge to alter the energy metabolism of glioma cells and their survival. In view of the complex mechanism of action of hypericin, further exploration is warranted in a perspective of its clinical application as a potential phototoxic agent in the treatment of glioma tumors.

Anthracenes↗

PubMed1998

Fluorescence-based analysis of DNA ploidy and cell proliferation within fine-needle samplings of breast tumors: a new approach using automated image cytometry.

BACKGROUND: Automated image cytometry can allow concurrent quantification of several parameters in each individual cell within a population, opening new possibilities for diagnosis and prognosis. In this study, the authors investigated the capacity of this method for performing a bivariate analysis of DNA ploidy and synthesis in fine-needle samplings obtained without aspiration from breast tumors. METHODS: Samplings from 25 unselected cases of ductal infiltrative breast adenocarcinoma and 2 cases of fibroadenoma were analyzed. For each case, 3-5 slides (containing approximately 1000 cells each) were quantified to assess experimental precision. Ploidy was determined by fluorescent staining of DNA using 4,6-diamidino-2-phenylindole (DAPI). Contaminating lymphocytes were taken as internal controls to calculate DNA indices. DNA synthesis was analyzed by immunofluorescent detection of 5-bromodeoxyuridine (BrdU) incorporation. Measurements were compared with flow cytometric data obtained from the same patients. RESULTS: Relative error in determination of DNA indices was generally below 5%. Determination of proliferation indices were more variable, with a mean relative error of 25%. Two different populations of BrdU positive cells were detected systematically, one in the diploid and another in the aneuploid fraction. For both cytometric methods, DNA indices were similar in all 27 cases, whereas BrdU labeling indices showed no significant correlation in 13 cases. The remaining cases were not comparable due to lack of flow cytometric data. Labeling indices obtained by image cytometry did not reveal any significant correlation with Scarff-Bloom-Richardson grading or clinical staging. CONCLUSIONS: Automated image cytometry allows concurrent measurement of ploidy and cell proliferation within individual breast carcinoma cells. Statistical reliability can be reached with a relative small number of cells (1000), which is crucial for samples in which the cell number is too low for flow cytometry analysis. Visual control for artifact elimination and better characterization of cell populations makes this a powerful tool for tumor cell investigation. Automated image cytometry allows the obtainment of valuable prognostic parameters of traditional flow cytometry with the relatively small number of cells obtained in aspiration procedures.

Adult↗

PubMed1998

Amplification of DNA sequences from chromosome 19q13.1 in human pancreatic cell lines.

Conventional cytogenetics and comparative genomic hybridization (CGH) were utilized to identify recurrent chromosomal imbalances in 12 pancreatic adenocarcinoma cell lines. Multiple deletions and gains were observed in all cell lines. Losses affecting chromosomes or chromosome arms 9p, 13, 18q, 8p, 4, and 10p and gains involving chromosome arms or bands 19q13.1, 20q, 5p, 7p, 11q, 3q25-qter, 8q24, and 10q were commonly observed. Interestingly, 19 distinct sites of high-level amplification were found by CGH. Recurrent sites involved 19q13.1 (6 cases), 5p (3 cases), and 12p and 16p (2 cases). Amplification of KRAS2 was demonstrated in 2 cell lines and that of ERBB2 in another. To define the occurrence of chromosome 19 amplification further, two-dimensional analysis of NotI genomic restriction digests and fluorescence in situ hybridization using probes from band 19q13.1 were utilized. High-level amplification of overlapping sets of chromosome 19 NotI fragments was exhibited in 3 cell lines of which 2 showed amplification of both OZF and AKT2 genes and 1 that of AKT2 alone. In these 3 cell lines, amplification of chromosome 19 sequences was associated with the presence of a homogeneously staining region. Our results provide evidence of heterogeneity in the extent of chromosome 19 amplification and suggest the existence of yet unknown amplified genes that may play a role in pancreatic carcinogenesis.

Chromosome Aberrations↗

PubMed1998

Potentiation of lonidamine and diazepam, two agents acting on mitochondria, in human glioblastoma treatment.

BACKGROUND: Cellular metabolism in glioblastoma multiforme, the most common primary brain tumor in humans, is characterized by a high rate of aerobic glycolysis that is dependent on mitochondria-bound hexokinase. Moreover, high levels of glucose utilization and tumor aggressiveness in glioblastoma are associated with a high density of mitochondrial benzodiazepine receptors. We sought to inhibit glioblastoma metabolism by simultaneously inhibiting hexokinase with lonidamine and binding benzodiazepine receptors with diazepam. METHODS: Cellular glioblastoma metabolism in five glioblastoma cell lines was assessed in vitro by measuring cell proliferation (by use of a tetrazolium-based colorimetric assay, measurement of DNA synthesis, and assessment of cell cycle distribution), by measuring membrane fluidity (by fluorescence polarization measurement of cells stained with a fluorescent probe), and by measuring changes in intracellular pH. Immunodeficient nude mice bearing subcutaneous xenografts of human glioblastoma cells were used to assess the antitumor activities of lonidamine and diazepam; the mice were treated twice daily with lonidamine (total daily dose of 160 mg/kg body weight) and/or diazepam (total daily dose of 1 mg/kg body weight) for 10 consecutive days. RESULTS: When used in combination, the two drugs had a stronger effect on glioblastoma cell proliferation and metabolism in vitro than did either agent used alone. In vivo, the combination of lonidamine and diazepam was significantly more effective in reducing glioblastoma tumor growth than either drug alone (two-sided P<.01, Mann-Whitney U test, comparing growth of treated tumors with that of untreated tumors); this tumor growth retardation was maintained as long as treatment was given. CONCLUSION: The combination of lonidamine and diazepam--drugs that target two distinct mitochondrial sites involved in cellular energy metabolism--potentiates the effects of the individual drugs and may prove useful in the treatment of human glioblastomas.

Animals↗

PubMed1998

Analysis of distribution in the human, pig, and rat genomes points toward a general subtelomeric origin of minisatellite structures.

We have developed approaches for the cloning of minisatellites from total genomic libraries and applied these approaches to the human, rat, and pig genomes. The chromosomal distribution of minisatellites in the three genomes is strikingly different, with clustering at chromosome ends in human, a seemingly almost even distribution in rat, and an intermediate situation in pig. A closer analysis, however, reveals that interstitial sites in pig and rat often correspond to terminal cytogenetic bands in human. This observation suggests that minisatellites are created toward chromosome ends and their internalization represents secondary events resulting from rearrangements involving chromosome ends.

Animals↗

PubMed1998

Increased radiation-induced chromosome breakage after progesterone addition at the G1/S-phase transition.

Pregnant females appear to have an increased chromosomal sensitivity to gamma-irradiation. This hypersensitivity was found to parallel the increase of gestation hormone amounts [M. Ricoul, L. Sabatier, B. Dutrillaux, Increased chromosome radiosensitivity during pregnancy, Mutat. Res. 374(1997) 73-78]. An in vitro experiment was developed to study the effect of progesterone. We performed irradiations of whole blood from normal human donors and chromosome were analysed in first generation metaphases. By comparison to untreated controls, all cultures in which progesterone was added around the 24th h of culture exhibited an increased frequency of chromosome rearrangements, principally dicentrics and rings, which confirms the role of progesterone in the results of in vivo studies. BrdU incorporation studies suggested that progesterone was particularly efficient just before the entry into S-phase, which corresponds to the G1/S transition period. Cultures with an increased frequency of chromosome breakage had a slightly higher mitotic index than controls. It is suggested that progesterone may stimulate DNA repair in cells which reached the end of G1-phase with unrepaired breaks. This would allow the cells to enter the S-phase and survive, although some illegitimate repair leads to chromosome rearrangements, visible at the following metaphase.

Blood Cells↗