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Biomedical subjects

R Bertrand

Publications and source records attributed to R Bertrand.

At least 37 records · Page 2Linked to original sources
PubMed1998

Bcl-xL modulates apoptosis induced by anticancer drugs and delays DEVDase and DNA fragmentation-promoting activities.

Using an episomal eucaryotic expression vector, we derived three stable transfected human leukemic U-937 variant lines showing differential expression of the Bcl-xL protein. Preventive effect of Bcl-xL on cell death induced by various concentrations of camptothecin (DNA topoisomerase I inhibitor; CPT) was observed in the three lines with most pronounced effect in cells containing the highest level of Bcl-xL expression. These results show that increased cell death protection by Bcl-xL is correlated with its level of expression. The extent of DNA strand break formation and DNA synthesis inhibition following CPT treatments was similar in control and transfected U-937 cells, suggesting that Bcl-xL acts downstream of CPT-DNA topoisomerase I-mediated DNA strand breaks. Modulation of cell death by Bcl-xL was also observed in cells treated with etoposide, vinblastine, paclitaxel, and cisplatinum (II) diammine dichloride. To define whether Bcl-xL functions downstream or upstream of apoptogenic proteolytic cascade activation, we compared kinetics of DNA fragmentation in treated cells with kinetics of caspase 1-like, caspase 3-like, and N-tosyl-L-phenylalanylchloromethyl ketone (TPCK)-sensitive activities. In CPT-treated U-937 cells, caspase 3-like and TPCK-sensitive activities promoting DNA fragmentation in a cell-free system were detected much more rapidly in extracts obtained from CPT-treated U-937 cells compared to those obtained from CPT-treated U-937-Bcl-xL variant cells. These results suggest that Bcl-xL delays their activation that correlates with the occurrence of DNA fragmentation. Addition of recombinant Bcl-xL in extracts containing DEVDase and TPCK-sensitive activities did not inhibit these activities, suggesting that Bcl-xL acts primarily upstream of their activation in the apoptotic process. Taken together, these results suggest that Bcl-xL is a primary checkpoint that can block or delay transmission of cell death signals emerging from DNA damage and prevents activation of an apoptogenic proteolytic cascade.

Antineoplastic Agents, Phytogenic↗
PubMed1998

Bax-alpha promotes apoptosis induced by cancer chemotherapy and accelerates the activation of caspase 3-like cysteine proteases in p53 double mutant B lymphoma Namalwa cells.

Stable transfected human p53 (mt/mt) B lymphoma Namalwa variant lines showing differential expression of the Bax-alpha protein were derived under hygromycin selection. Overexpression of Bax-alpha in these variant cells accelerates cell death induced by short or continuous treatments with various concentrations of camptothecin, etoposide, vinblastine and shows no accelerating cell death activity in cis-platinum and paclitaxel-treated cells. Activation of apoptosis and oligonucleosome-sized DNA fragmentation was observed in the variant lines with more pronounced effect in cells containing high level of Bax-alpha protein. These results suggest that increased cell death mediated by anticancer drugs correlates with Bax-alpha level of expression and that Bax-alpha sensitizes Namalwa cells treated at low drug concentrations. The extent of DNA synthesis inhibition following DNA topoisomerase inhibitor treatments was similar in control and all transfected Namalwa cells suggesting that Bax-alpha acts downstream of DNA topoisomerase-mediated DNA strand breaks. To define further the relation between Bax-alpha expression and apoptosis activation, kinetics of caspase activation was measured in drug-treated cells. Caspase activities were measured using specific fluorogenic peptide derivatives DABCYL-YVADAPV-EDANS and Ac-DEVD-AMC, substrates of the caspase 1-like and caspase 3-like families, respectively. In control and Bax-alpha transfected Namalwa cells no increase in caspase 1-like activity was detected following camptothecin and etoposide treatments. In contrast, a significant difference in Ac-DEVD-AMC hydrolysis activity was observed in Bax-alpha transfected Namalwa cells compared to that of control Namalwa cells after camptothecin and etoposide treatment. Increased caspase 3-like activity correlated also with poly(ADPribosyl) polymerase cleavage. Taken together, these results suggest that Bax-alpha sensitize B lymphoma cells to series of anticancer drugs and accelerates the activation of apoptotic protease cascade.

Antineoplastic Agents↗
PubMed1997

Probing the hydrophobic interactions in the skeletal actomyosin subfragment 1 and its nucleotide complexes by zero-length cross-linking with a nickel-peptide chelate.

The complex of Ni(II) and the tripeptide Gly-Gly-His catalyzes, in the presence of monoperoxyphthalic acid, a zero-length protein-protein cross-linking via an oxidative radical pathway involving mainly aromatic amino acids and not at all nucleophilic residues [Brown, K. C., Yang, S.-H., and Kodadek, T. (1995) Biochemistry 34, 4733-4739]. We have taken advantage of this unprecedented cross-linking system to directly and selectively probe the solution structure and functioning of the hydrophobic interface between F-actin and skeletal myosin subfragment 1 (S-1) at the level of its aromatic components, in the absence and in the presence of nucleotides (ATP and ADP) or nucleotide analogs (AMPPNP, PPi, and ADP. AlF4). Following verification of the structure of the Ni(II)-peptide chelate and of its oxidized active form by electrospray mass spectrometry, complexes of F-actin and S-1 or proteolytic S-1 derivatives and complexes of S-1 and proteolytic F-actin derivatives were readily cross-linked under various controlled conditions without apparent alteration of the acto-S-1 recognition. The covalent adducts were identified on electrophoretic gels using specific protein labeling with the oxidation-resistant fluorophor, monobromobimane, combined with immunochemical staining. Two types of actin-heavy chain conjugates were produced. One, with a mass of 180 kDa, was formed in the rigor state or with ADP bound; the other one, with a mass of 200 kDa, was generated from the ternary complexes comprising a gamma-P-containing ligand. They were accumulated with an efficiency of 8 and 6%, respectively. For each reversible complex, the 180 kDa:200 kDa band ratio was essentially as predicted from the nucleotide-dependent A to R equilibrium mechanism of the acto-S-1 interaction in solution [Geeves, A. M., and Conibear, P. B. (1995) Biosphys. J. 68, 194s-201s]. Both covalent species resulted from the cross-linking of an actin monomer to the central 50 kDa segment, and their distinct mobilities reflect gamma-P-mediated structural changes at or near the actin-50 kDa fragment interface. Peptide mapping showed the cross-linking to take place between the 506-561 S-1 segment and the 48-113 actin stretch. The localization of these regions in the atomic F-actin-S-1 model implies that nucleotide-modulated close contacts, involving aromatic residues, are operating between the C-terminal helix of the hydrophobic strong actin-binding motif of S-1 bound to the primary actin monomer and the top portion of the adjacent lower actin subunit. The specificity of the nickel-peptide cross-linking, as assessed with the acto-S-1 complex, makes it a candidate for potential general use in investigations of the hydrophobic interactions within other protein motor-based assemblies.

Adenosine Diphosphate↗
PubMed1997

Cholera toxin triggers apoptosis in human lung cancer cell lines.

Cholera toxin (ChT) inhibits signals generated by multiple growth factors in human lung cancer cells, resulting in cell growth inhibition. We now report that ChT triggers apoptosis as shown by DNA fragmentation and activation of caspases cleaving poly(ADP-ribose) polymerase and lamin B. Apoptosis induced by ChT in a small cell lung cancer cell line is not affected by manipulations of intracellular cAMP through preincubation with isobutylmethylxanthine but can be modestly increased through inhibition of protein kinase C with chelerythrine. Thus, apoptosis is actively suppressed in lung cancer cells by a ChT-sensitive-growth regulatory pathway, and these observations may have significant implications in the development of novel strategies for lung cancer treatment.

1-Methyl-3-isobutylxanthine↗
PubMed1997

The Bcl-xL and Bax-alpha control points: modulation of apoptosis induced by cancer chemotherapy and relation to TPCK-sensitive protease and caspase activation.

Defective control of apoptosis appears to play a central role in the pathogenesis of human diseases including neoplasic, autoimmune, and neurodegenerative diseases. Conversely, cancer chemotherapy and ionizing radiation can induce cancer cell death by apoptosis, and deregulated apoptosis following cancer chemotherapy could define a new category of drug resistance mechanism. By understanding the role that some major regulators of apoptosis play either at the commitment or execution phases of cell death in a given tissue and pathology, we will be in a better position to design and explore new therapeutic modalities. The Ced-9 - Bcl-like and Ced-3 - Ice-like gene family products are intrinsic proteins regulating the decision of a cell to survive or die and executing part of the cell death process itself, respectively. Among the various Bcl-like proteins, the effects and functions of the Bcl-x and Bax proteins in controlling apoptosis induced by cancer chemotherapy have been studied recently. In human cancer variant cell lines showing differential expression of the Bcl-xL protein, a preventive effect of Bcl-xL on cell death induced by various cytotoxic drugs is observed, with greater effects in cells containing the highest level of Bcl-xL expression. Similarly, overexpression of Bax-alpha in cancer cell lines sensitizes these cells to some cancer chemotherapy compounds. Modulation of apoptosis either negatively by Bcl-xL or positively by Bax-alpha resides downstream of the primary mechanism of action of anticancer drugs, suggesting that they act primarily as intrinsic control points following cytotoxic drug injuries. An emerging family of Ced-3 - Ice like cysteine proteases (caspases) has been also identified and several studies have revealed their importance in executing the process of cell death. More recently, activation of a N-tosyl-L-phenylalanylchloromethyl ketone (TPCK)-sensitive pathway was also suggested to play an important role in apoptosis induction following cancer chemotherapy. Evidence obtained using a combination of assays including cell-free systems and enzyme activity assays now suggests that Bcl-xL and Bax-alpha control points function upstream of TPCK-sensitive protease and caspase activation. Bcl-xL delays and prevents activation of apoptotic protease cascades whereas Bax-alpha shows the opposite effect, accelerating their activation.

Animals↗
PubMed1997

The CrmA- and TPCK-sensitive pathways that trigger oligonucleosome-sized DNA fragmentation in camptothecin-induced apoptosis: relation to caspase activation and high molecular weight DNA fragmentation.

Abstract: In human B lymphoma Namalwa variant cells expressing the serpin-like CrmA protein, the kinetics of oligonucleosome-sized DNA fragmentation was retarded compared with that of control Namalwa cells following camptothecin treatment. However, no difference in the kinetics of high molecular weight DNA fragmentation was observed between the two lines after camptothecin treatment. Similar delay and inhibition of the oligonucleosome-sized DNA fragmentation was observed in human B lymphoma Namalwa and monocytic-like leukemia U-937 cells coincubated in the presence of various concentrations of N-tosyl-L-phenylalanyl chloromethylketone and camptothecin. The effect of N-tosyl-L-phenylalanyl chloromethylketone was similar to that of CrmA and did not prevent the appearance of high molecular weight DNA fragments. Similar suppression of camptothecin-induced internucleosomal DNA fragmentation was also observed in a cell-free system when cytosolic extracts obtained from camptothecin-treated Namalwa and U-937 cells were coincubated with untreated nuclei in the presence of N-tosyl-L-phenylalanyl chloromethylketone. Furthermore, N-tosyl-L-phenylalanyl chloromethylketone had no significant effects on caspase-3-like activities in camptothecin-treated Namalwa and U-937 cells. Hydrolysis of Ac-Asp-Glu-Val-Asp-amino-4-methylcoumarin, a fluorogenic substrate with caspase-3-like activities, was detected in extracts prepared from camptothecin-treated Namalwa and U-937 cells with no apparent difference in the time courses of caspase-3-like activation in the absence or presence of N-tosyl-L-phenylalanyl chloromethylketone. Similarly, N-tosyl-L-phenylalanyl chloromethylketone was a weak inhibitor of caspase-3-like activities in vitro. Taken together, these observations suggest that the pathway sensitive to N-tosyl-L-phenylalanyl chloromethylketone is involved in camptothecin-induced oligonucleosome-sized DNA fragmentation. Furthermore, inhibition of this pathway had no effect on caspase-3-like activation and on the occurrence of high molecular weight DNA fragmentation.

Apoptosis↗
PubMed1996

Decreased Fas antigen receptor expression in testicular tumor cell lines derived from polyomavirus large T-antigen transgenic mice.

MT-PVLT-10 transgenic mice express large T-antigen of polyomavirus under the control of the mouse metallothionein-1 promoter and mates of this transgenic line develop testicular tumors at advanced ages. The differential display technique was employed to compare mRNA expression from immortalized cell lines derived from normal or adenomatous testis from MT-PVLT-10 transgenic males. Using this technique, a complementary DNA fragment corresponding to the mouse Fas antigen receptor was recovered from normal testicular cells but not from tumor cells. RNAse protection assays with the Fas antigen specific fragment confirmed its differential expression. Normal testicular cells from the transgenic animals responded to treatment of interferon-gamma by increasing the expression of Fas antigen specific mRNA and were sensitive to the proliferative inhibitory effect of anti-Fas antibody in vitro. This proliferative inhibition was characterized by an accumulation of cells in S phase of the cell cycle. In contrast, the testicular tumor cells did not respond to either interferon-gamma or to anti-Fas antibody in vitro. These results suggest that the toss of proliferative inhibitory effect mediated by the Fas antigen pathway in tumor cells may be an important step in testicular tumor progression in the MT-PVLT-10 transgenic mice.

Animals↗
PubMed1996

Biochemical and molecular studies of human methenyltetrahydrofolate synthetase.

5-FormylH4folate is administered clinically under the name Leucovorin in association with the antineoplastic agent 5-fluorouracil (5-FU) to enhance the cytotoxic effects of 5-FU. The combination has been shown to be superior to 5-FU alone in the treatment of patients with metastatic colorectal carcinoma. Methenyltetrahydrofolate synthetase (MTHFS) catalyzes the transformation of 5-formyl-tetrahydrofolate to methenylH4folate, which is the obligatory initial metabolic step prior to the intracellular conversion of 5-formylH4folate to other reduced folates and the increase in intracellular folate pools required for 5-FU potentiation. In the following paper, we will summarize results of biochemical and molecular studies of human MTHFS.

Animals↗
PubMed1996

Biochemical and Molecular Studies of Human Methenyltetrahydrofolate Synthetase.

5-FormylH(4)folate is administered clinically under the name Leucovorintrade mark in association with the antineoplastic agent 5-fluorouracil (5-FU) to enhance the cytotoxic effects of 5-FU. The combination has been shown to be superior to 5-FU alone in the treatment of patients with metastatic colorectal carcinoma. Methenyltetrahydrofolate synthetase (MTHFS) catalyzes the transformation of 5-formyltetrahydrofolate to methenylH(4)folate, which is the obligatory initial metabolic step prior to the intracellular conversion of 5-formylH(4)folate to other reduced folates and the increase in intracellular folate pools required for 5-FU potentiation. In the following paper, we will summarize results of biochemical and molecular studies of human MTHFS.

Journal Article↗
PubMed1996

[Apoptosis of human leukemic cells induced by topoisomerase I and II inhibitors].

Comparison between five human leukemic lines (BV173, HL60, U937, K562, KCL22) suggest that the main determinant of their sensitivity to topoisomerase I (camptothecin) and II (VP-16) inhibitors is their ability to regulate cell cycle progression in response to specific DNA damage, then to die through apoptosis: the more the cells inhibit cell cycle progression, the less sensitive they are. The final pathway of apoptosis induction involves a cytoplasmic signal, active at neutral pH, needing magnesium, sensitive to various protease inhibitors and activated directly by staurosporine. Modulators of intracellular signaling (calcium chelators, calmodulin inhibitors, PKC modulators, kinase and phosphatase inhibitors) have no significant influence upon apoptosis induction. Conversely, apoptosis induction pathway is modified during monocytic differentiation of HL60 cells induced by phorbol esters. Lastly, poly(ADP-ribosyl)ation and chromatine structure should regulate apoptotic DNA fragmentation that is prevented by 3-aminobenzamide and spermine, respectively.

Alkaloids↗
PubMed1995

Cloning and characterization of the human 5,10-methenyltetrahydrofolate synthetase-encoding cDNA.

Methenyltetrahydrofolate synthetase (MTHFS) catalyses the obligatory initial metabolic step in the intracellular conversion of 5-formyltetrahydrofolate to other reduced folates. We have isolated and sequenced a human MTHFS cDNA which is 872-bp long and codes for a 203-amino-acid protein of 23,229 Da. Escherichia coli BL21(DE3), transfected with pET11c plasmids containing an open reading frame encoding MTHFS, showed a 100-fold increase in MTHFS activity in bacterial extracts after IPTG induction. Northern blot studies of human tissues determined that the MTHFS mRNA was expressed preferentially in the liver and Southern blot analysis of human genomic DNA suggested the presence of a single-copy gene.

Amino Acid Sequence↗
PubMed1995

Production and properties of skeletal myosin subfragment 1 selectively labeled with fluorescein at lysine-553 proximal to the strong actin-binding site.

We describe, for the first time, the reaction of skeletal myosin subfragment 1 (S-1) with the succinimido ester of 6-[fluorescein-5(and 6)-carboxamido]hexanoic acid (FHS), which takes place at pH 7.0, 20 degrees C, within a 15 min period, in the presence of 1.5-1.8-fold molar excess of reagent over protein. As a result, 0.9-1.0 mol of fluorescyl group/mol of S-1 was covalently incorporated exclusively into the 95 kDa heavy chain as monitored by spectroscopic measurements. The central 50 kDa segment included the main site of fluorescence attachment as assessed by gel electrophoresis. The extent of S-1--FHS conjugation is strongly sensitive to F-actin binding but not to the interaction of nucleotides. The formation of the rigor F-actin--S-1 complex decreased the level of S-1 labeling to 20% without any competition between actin and S-1 for FHS binding. The derivatization of S-1 did not alter the K(+)-ATPase activity, but it enhanced the Ca(2+)-ATPase and Mg(2+)-ATPase to 150% and 225%, respectively, whereas it lowered the actin-activated ATPase to only 75% of the original activity. A double-reciprocal plot of the ATPase rate against actin concentration indicated a 2-fold decrease of the Vmax value for modified S-1, while the Km for actin was unchanged. Cosedimentation experiments did not reveal disruption of the rigor acto-S-1 interaction by the bound fluorophore. The labeled S-1 heavy chain was isolated, and its total tryptic digest was fractionated by reverse-phase HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1995

Identification and characterization of human mitochondrial methenyltetrahydrofolate synthetase activity.

We present evidence for the presence of the folate metabolism enzyme methenyltetrahydrofolate synthetase (MTHFS) in mitochondria. MTHFS activity was identified in the matrix of mitochondria purified from human liver biopsies. Mitochondrial and cytoplasmic MTHFS specific activities are similar, 85% of the total cellular MTHFS activity is in the cytoplasm and both native enzymes have similar molecular weights (approximately 25 kDa). Studies using purified mitochondrial MTHFS from CA46 human Burkitt lymphoma cells reveal that mitochondrial MTHFS behaves kinetically like the cytoplasmic enzyme with Km values of 4.7, 0.8 and 22 microM respectively for (6R,S)-5-formyltetrahydrofolate monoglutamate, (6S)-5-formyltetrahydrofolate pentaglutamate and ATP. This finding adds to previous observations that various folate-dependent enzymes reside in the mitochondria of eucaryotic cells. Intracellular tetrahydrofolate metabolism is highly compartmentalized and mitochondrial MTHFS activity is necessary for the entry of mitochondrial 5-formyltetrahydrofolate into the mitochondrial folate pool.

Burkitt Lymphoma↗
PubMed1995

Comparing times and performances of French- and English-speaking candidates taking a national examination of clinical decision-making skills.

French-speaking candidates taking the translated version of the Medical Council of Canada's (MCC's) Qualifying Examination in Medicine often complain that poor performance could be due to translation defects. The purposes of this 1991 study were to determine (1) whether French-speaking candidates spend the same time as do English-speaking candidates in answering the questions in the fourth and last booklet of Part 1 of the MCC's Qualifying Exam, and (2) for items where the French-speaking candidates have more difficulty, whether these differences are within normal limits, and if not, could they be attributed to faults in the translation? Two versions of the fourth booklet, one in English and the other a translation into French, were administered to 229 randomly selected candidates (98 French speakers and 131 English speakers). The booklets contained 19 clinical problems and a total of 44 key features; performance was measured by the number of key features the examinees correctly responded to. (Key features are the critical or essential steps needed to resolve a clinical problem.) The French text was 16% longer, and the French candidates took longer to complete the two-hour examination (a mean of 116.31 minutes versus 107.84 minutes for the English speakers, p = .000). However, there was no direct relationship between the time spent on a section of the examination and the number of words it contained. The French candidates' overall scores did not differ from those of the English candidates (59.76% versus 61.33%, p = .11).(ABSTRACT TRUNCATED AT 250 WORDS)

Canada↗
PubMed1994

The covalent maleimidobenzoyl-actin-myosin head complex. Cross-linking of the 50 kDa heavy chain region to actin subdomain-2.

We have identified the region of actin involved in the covalent coupling of maleimidobenzoyl-G-actin to the central 50 kDa segment of the myosin-S-1 heavy chain by analyzing the structure of the maleimidobenzoyl-G-actin-S-1 conjugate using selective proteolytic digestions, amino acid sequence determinations and novel cross-linking reactions between S-1 and different maleimidobenzoyl-G-actin derivatives. The cross-linking is shown to occur only on the stretch of residues 48-67 in actin subdomain-2 with Lys-50, residing on the outer part of the DNase-I-binding loop, as the most likely site of cross-linking. Because the maleimidobenzoyl-F-actin-S-1 complex undergoes the same coupling process, the data provide experimental evidence in favor of the recent model of the rigor F-actin-S-1 complex suggesting a close contact between structural elements of the lower domain of the 50 kDa fragment and the top of actin subdomain-2.

Actins↗
PubMed1994

Induction of a common pathway of apoptosis by staurosporine.

The present observations show that staurosporine can rapidly trigger both the morphological changes and intranucleosomal DNA fragmentation typical of apoptosis. This occurred in a number of cell lines from various origins regardless of the state of differentiation and cell cycle phase, suggesting the presence of a common inducible suicide pathway. The broad apoptotic activity of staurosporine appears to be unique among other protein kinase or phosphatase inhibitors we tested. Results obtained in a cell-free assay suggest that cytoplasmic proteins directly modulated by staurosporine may be involved in a ubiquitous signal for the induction of DNA fragmentation and apoptosis.

Alkaloids↗
PubMed1994

Apoptosis induced by DNA topoisomerase I and II inhibitors in human leukemic HL-60 cells.

The induction of apoptosis following topoisomerase inhibitors proceeds in at least three distinct steps: (1) induction of cleavable complexes (potentially lethal damage), (2) topoisomerase-induced DNA damage, and (3) a presently unknown sequence of events that must either lead to cell cycle arrest (G2-block, differentiation) or apoptosis. DNA degradation provides a convenient way to quantify apoptosis in HL-60 cells. Extensive apoptosis can be induced rapidly in undifferentiated HL-60 cells without prevention by cycloheximide or actinomycin D. Therefore, HL-60 cells appear to express constitutively the apoptotic machinery that may be kept under control of a yet unknown repressor. The absence of the tumor suppressor p53 and the presence of bcl-2 are in contrast with the sensitivity of these cells to apoptosis. Agents that modify chromatin structure (zinc, poly[ADPribose] inhibitors, spermine) can block DNA fragmentation without affecting cell survival. By contrast macrophage-like differentiation by phorbol esters suppresses apoptosis without affecting topoisomerase-induced DNA damage. Better understanding of the apoptotic regulation in the widely used and characterized HL-60 cell line should allow the identification of new mechanisms and parameters of cellular sensitivity and resistance to the cytotoxic activity of anticancer agents.

Antineoplastic Agents↗