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Biomedical subjects

Laurent Corcos

Publications and source records attributed to Laurent Corcos.

12 recordsLinked to original sources
PubMed2008

TAp73beta and DNp73beta activate the expression of the pro-survival caspase-2S.

p73, the p53 homologue, exists as a transactivation-domain-proficient TAp73 or deficient deltaN(DN)p73 form. Expectedly, the oncogenic DNp73 that is capable of inactivating both TAp73 and p53 function, is over-expressed in cancers. However, the role of TAp73, which exhibits tumour-suppressive properties in gain or loss of function models, in human cancers where it is hyper-expressed is unclear. We demonstrate here that both TAp73 and DNp73 are able to specifically transactivate the expression of the anti-apoptotic member of the caspase family, caspase-2(S). Neither p53 nor TAp63 has this property, and only the p73beta form, but not the p73alpha form, has this competency. Caspase-2 promoter analysis revealed that a non-canonical, 18 bp GC-rich Sp-1-binding site-containing region is essential for p73beta-mediated activation. However, mutating the Sp-1-binding site or silencing Sp-1 expression did not affect p73beta's transactivation ability. In vitro DNA binding and in vivo chromatin immunoprecipitation assays indicated that p73beta is capable of directly binding to this region, and consistently, DNA binding p73 mutant was unable to transactivate caspase-2(S). Finally, DNp73beta over-expression in neuroblastoma cells led to resistance to cell death, and concomitantly to elevated levels of caspase-2(S.) Silencing p73 expression in these cells led to reduction of caspase-2(S) expression and increased cell death. Together, the data identifies caspase-2(S) as a novel transcriptional target common to both TAp73 and DNp73, and raises the possibility that TAp73 may be over-expressed in cancers to promote survival.

Binding Sites↗
PubMed2006

Posterior maxillary segmental osteotomy for mandibular implants placement: case report.

The loss of mandibular molars can result in a maxillary dentoalveolar extrusion, leading to an insufficient interarch space. In severe cases, this space must be regained before the prosthetic reconstruction of the opposite edentulous area. The posterior maxillary segmental osteotomy (PMSO) is a simple but precision technique to manage this problem; without which one can achieve a good surgical outcome but a poor final occlusion. The purpose of this paper was to present a case of PMSO for mandibular implant placement and to discuss the important steps.

Adult↗
PubMed2006

CYP4A11 is repressed by retinoic acid in human liver cells.

CYP4A11, the major fatty acid omega-hydroxylase in human liver is involved in the balance of lipids, but its role and regulation are both poorly understood. We studied the effects of retinoids on the regulation of CYP4A11 in the human hepatoma cell line HepaRG. Treatment of HepaRG cells with all-trans-retinoic acid resulted in a strong decrease in CYP4A11 gene expression and apoprotein content and, furthermore, was associated with a 50% decrease in the microsomal lauric acid hydroxylation activity. Such a strong suppression of CYP4A11 expression by retinoids could have a major impact on fatty acid metabolism in the liver.

Carcinoma, Hepatocellular↗
PubMed2006

Determination of polyunsaturated fatty acid monoepoxides by high performance liquid chromatography-mass spectrometry.

Despite the implication of polyunsaturated fatty acid monoepoxides in a large panel of biological effects, few methods allowing their separation in a single run are available. We describe here a simple method based on reversed-phase ion-pair high-performance liquid chromatography (RP-HPLC) and developed to successfully separate the various monoepoxides of eicosatrienoic, arachidonic, eicosapentaenoic and docosahexaenoic acids. These compounds were easily identified by liquid chromatography-mass spectrometry (LC-MS) with atmospheric pressure chemical ionisation owing to the volatility of counter-ion species. Compared to established methods, this new protocol proved its ability to totally resolve, in a single run, all of the different regioisomeric epoxides. In the long run, this method will demonstrate its efficacy to give insights into the cytochrome P450-dependent metabolism of polyunsaturated fatty acids (PUFAs) and the generation of physiologically active epoxy-derivatives.

Aryl Hydrocarbon Hydroxylases↗
PubMed2005

A virus-directed enzyme prodrug therapy (VDEPT) strategy for lung cancer using a CYP2B6/NADPH-cytochrome P450 reductase fusion protein.

Virus-directed enzyme prodrug therapy (VDEPT) is an emerging strategy against cancer. Our approach is a P450-based VDEPT that consists of using cyclophosphamide (CPA) as a prodrug and a Cytochrome P450 2B6/NADPH cytochrome P450 reductase fusion protein (CYP2B6/RED) as a prodrug-activating enzyme. Due to the heterogenous expression of proteins in tumor cells, basal reductase activity may not be sufficient to supply CYP2B6 with electrons, the fusion protein should enable the expression of both proteins at high levels in tumor cells. CYP/RED fusion proteins have never been previously expressed in mammalian cells, to enable expression the fusion protein was cloned into an adenoviral vector and subsequently several pulmonary tumor cell lines were infected. The CYP2B6/RED fusion protein was detected by Western blot, its mRNA by Northern blot, and its heme incorporation into an active form by spectral analysis. Infection with the fusion gene increased RED activity in microsomes by a factor of 3 compared to the control. After infection and treatment with CPA, in cell lines with low endogenous RED, the fusion protein mediated significantly higher CPA-induced cytotoxicity compared to cells expressing solely CYP2B6. In conclusion, the fusion protein is functional for VDEPT by providing one protein for higher levels of CPA metabolism.

Adenoviridae↗
PubMed2005

[Alternative mRNA splicing, pathology and molecular therapeutics].

Pre-mRNA splicing operates towards at least 95 % of the transcript pool. It is subjected to a large number of variations, collectively regrouped under the term of alternative mRNA splicing, which occurs, on average, 6 to 8 times per pre-mRNA molecule. Consequently, many more proteins may be encoded from a single gene, which may satisfy a physiological need, or mark a pathological adaptation. The identification of mutations in sequences required for splicing, both constitutive and alternative, or for their control, has permitted to determine the causes of qualitative or quantitative variations in transcript levels associated with inherited diseases or cancer development. A number of molecular approaches have been undertaken to try to compensate for the effect of deleterious splicing mutations and to restore, at least in part, sufficient amounts of either the normal or a surrogate transcript. These include overexpression of splicing proteins, improvement of their activity by post-translational modification, splice-site increased or decreased usage, and RNA-mediated trans-splicing. Using such approaches, phenotypic improvements have been obtained in animal models, carrying new hopes for the development of therapeutic strategies aimed at correcting both inherited and acquired diseases that involve pre-mRNA splicing defects.

Alternative Splicing↗
PubMed2004

Topoisomerase I and II inhibitors control caspase-2 pre-messenger RNA splicing in human cells.

We have recently shown that the topoisomerase II inhibitor, etoposide (VP16), could trigger caspase-2 pre-mRNA splicing in human leukemic cell lines. This leads to increased inclusion of exon 9, which is specifically inserted into the short caspase-2S isoform mRNA and absent from the long caspase-2L isoform mRNA. One of the consequences of this alternative splicing is a decrease in the total amount of the mature form of caspase-2L mRNA and protein. In this study, we analyzed the effects of several representative molecules of various classes of cytotoxic agents on caspase-2 pre-mRNA splicing in both U937 leukemic cells and in HeLa cervix carcinoma cells. Very strikingly, both topoisomerase I (camptothecin and homocamptothecin derivatives) and II (VP16, amsacrine, doxorubicin, mitoxantrone) inhibitors induced exon 9 inclusion. DNA intercalating glycosyl indolocarbazole derivatives as well as DNA alkylating agents, such as cisplatin and melphalan, antimetabolites like 5-fluorouracil, and mitotic spindle poisons like vinblastine had no effect. Therefore, both classes of DNA topoisomerases can control pre-mRNA splicing of the caspase-2 transcript. In addition, the splicing reaction brought about by camptothecin was hampered in human CEM/C2 and in murine P388-45R leukemic deficient in topoisomerase I activity. Conversely, VP16 did not trigger caspase-2 alternative splicing in human HL60/MX2 leukemic cells harboring a mutant topoisomerase II. Minigene transfection analysis revealed that topoisomerase inhibitors did not change the splicing profile when cis-acting elements in intron-9, reported to control exon 9 inclusion independently of drug treatment, were removed. Rather, our experiments suggest that exon 9 inclusion induced by topoisomerase inhibitors reflects the activity exerted by topoisomerase I or II on proteins that control splicing reactions, or their direct involvement in pre-mRNA splicing.

Alternative Splicing↗
PubMed2003

The human caspase-2 gene: alternative promoters, pre-mRNA splicing and AUG usage direct isoform-specific expression.

Caspases have been shown to play important roles in apoptotic cell death, cytokine maturation and cell differentiation. However, the transcriptional regulation of the corresponding CASP genes remains poorly known. We describe a 5.1 kb fragment located upstream of the first translated exon in the human CASP-2 gene, which is known to encode caspase-2L and -2S protein isoforms. Transient transfection experiments, together with transcription start site mapping and transcript analysis, demonstrate that each caspase mRNA is initiated from separate promoter regions, and produced from alternative splicing events in these regions. The CASP-2L promoter is much stronger than the CASP-2S promoter, in good agreement with the respective transcript levels of the two caspases. In addition, several in-frame translational start sites can be identified for each isoform, one of which is common to both, present in the second common exon, and used efficiently. Surprisingly, the short isoform may also be initiated at a downstream AUG codon within the same exon. Thus, promoter strength, alternative transcriptional initiation and 5'-splicing events regulate the expression of the main caspase-2 isoforms that may be translated from alternative translation initiation codons.

5' Flanking Region↗
PubMed2003

Mitochondria as a target for inducing death of malignant hematopoietic cells.

Mitochondria plays a central role in apoptotic cell death. The intermembrane space of mitochondria contains a number of soluble molecules whose release from the organelle to the cytosol or the nucleus induces cell death. Thus, molecules that directly trigger mitochondria membrane permeabilisation are efficient cytotoxic drugs. Mitochondria is one of the cellular targets for commonly used epipodophyllotoxins, adenine deoxynucleoside analogs and taxanes as well as recently developped agents such as the pentacyclic triterpene betulinic acid and the lymphotoxic agent FTY720. Most informations on anthracyclines point to the mitochondrial membrane as the main target of cardiotoxicity. Mitochondria is also a target for arsenite trioxide, an old cytotoxic agent recently used for treating acute promyelocytic leukemia, lonidamine, a dichlorinated derivative of indazole-3-carboxylic acid developped as a chemosensitizer, the retinoic acid receptor gamma activator CD437 and nitric oxide (NO). Recently, cytotoxic drugs have been specifically designed to directly affect the mitochondrial function. These include the positively charged alpha-helical peptides, which are attracted to and disrupt the negatively charged mitochondrial membrane, thus inducing mammalian cell apoptosis when targeted intracellularly. Various strategies have been proposed also to directly inhibit Bcl-2 and related anti-apoptotic proteins, including antisense oligonucleotides (e.g. Genasense, currently tested in phase III trials), small molecules that mimic the BH3 dimerization domain of these proteins and kinase inhibitors. Ligands of the mitochondrial benzodiazepine receptor such as the isoquinolone carboxamide derivative PK11195 also overcome the membrane-stabilizing effect of Bcl-2, whereas the adenosine nucleotide translocator (ANT) and the mitochondrial DNA are two other potential cellular targets for cytotoxic agents. Potentially, new compounds directly targeting the mitochondria may be useful in treating hematological malignancies. The challenge is now to selectively target these mitochondria permeabilizing agents to malignant cells. This review briefly summarizes the role of the mitochondria in cell death and describes these various strategies for targeting the mitochondria to induce apoptosis.

Adenosine Triphosphate↗
PubMed2003

Comparative studies on the cytochrome p450-associated metabolism and interaction potential of selegiline between human liver-derived in vitro systems.

Selegiline was used as a model compound in a project aimed at comparing, evaluating, and integrating different in vitro approaches for the prediction of cytochrome p450 (p450)-catalyzed hepatic drug metabolism in humans (EUROCYP). Metabolic predictions were generated using homology modeling, cDNA-expressed p450 enzymes, human liver microsomes, primary cultured human hepatocytes, and precision-cut human liver slices. All of the in vitro systems correctly indicated the formation of two dealkylated metabolites, desmethylselegiline and methamphetamine. The metabolic instability of selegiline was demonstrated by all of the in vitro systems studied. Estimates of clearance varied from 16 l/h to 223 l/h. With the exception of one approach, all systems underpredicted the in vivo clearance in humans (236 l/h). Despite this, all approaches successfully classified selegiline as a high clearance compound. Homology modeling suggested the participation of CYP2B6 in the demethylation of selegiline and of CYP2D6 in the depropargylation of the drug. Studies with recombinant expressed enzymes and with human hepatic microsomal fraction supported the involvement of CYP2B6 but not of CYP2D6. These techniques also suggested the involvement of CYP1A2, CYP2C8, and CYP2C19 in the biotransformation of selegiline. In vitro, CYP2B6 was the most active form of p450 involved in selegiline metabolism. Metabolism by several enzymes operating in parallel implies a low interaction potential for the drug. None of the techniques alone was able to predict all aspects of the metabolic and kinetic behavior of selegiline in vivo. However, when used as an integrated package, all significant characteristics were predictable.

Cells, Cultured↗
PubMed2002

Differential influence of etoposide on two caspase-2 mRNA isoforms in leukemic cells.

Etoposide (VP-16) is an anticancer agent that induces apoptosis in human leukemic cell lines such as U937 and HL60. We performed RNase protection assays, with two distinct cRNA panels covering most of caspase and BCL-2-related genes, using total RNA from cell lines exposed to various concentrations of the drug. Our results show that VP-16 down-regulates expression of most surveyed genes with the noticeable exception of casp-2S mRNA that is up regulated whereas casp-2L mRNA is decreased. Since these mRNAs are produced by the alternative splicing of exon 9, we devised a reverse transcriptase-polymerase chain reaction method using primers from exons 8 and 10 to demonstrate that VP-16 stimulates the production of exon 9-containing sequences, irrespective of active transcription. However, this effect is specific of the 3'-end of the CASP-2 gene since no difference in the relative amounts of the 5'-end of the mRNA species is detected. Nevertheless, the level of full-length casp-2L mRNA together with that of procaspase-2L protein, which is pro-apoptotic, are decreased under VP-16 treatment, suggesting that an early cell response to treatment by cytotoxic agents is to down-regulate expression of selected pro-apoptotic proteins.

Alternative Splicing↗