Biomedical subjects

C Briand

Publications and source records attributed to C Briand.

At least 37 records · Page 2Linked to original sources

PubMed1998

Heat-shock protein 90 (hsp90) binds in vitro to tubulin dimer and inhibits microtubule formation.

Hsp90 interacts with steroid hormone receptors, protein kinases, and cytoskeletal proteins. The mode of action of hsp90 on microtubules and tubulin has not been investigated. Using isolated purified hsp90 and isolated tubulin, we demonstrated in vitro by difference absorption and fluorescence spectroscopy that hsp90 bound to tubulin with an apparent affinity constant of 5 x 10(5) M-1, assuming an apparent stoichiometry of 1 at 25 degrees C. Using microcalorimetry, we found a delta H of -9.8 +/- 0.8 kJ.mol-1. The binding of hsp90 to tubulin was confirmed by a sedimentation assay. Moreover, we showed that hsp90 inhibited tubulin polymerisation.

Animals↗

PubMed1998

The two-state process of the heat shock protein 90 thermal denaturation: effect of calcium and magnesium.

Scanning microcalorimetry, native PAG electrophoresis, and circular dichroism were used to characterize thermal denaturation and oligomerization of heat shock protein 90 (hsp90) and the calcium and magnesium effect on these processes. The calorimetric curve of the hsp90 dimer consists of two transitions centered at 53.8 and 63.1 degrees C. Using specific ligand geldanamycin, we have found that N-terminal domains in the hsp90 dimer are melted independently in the lower-temperature peak, while the higher-temperature one comprises unfolding of two non-interacting parts of the middle domains and dimerization region. Unfolding of the N-terminal domain gives start to oligomerization of dimers; oligomers consist of dimers not dissociating upon denaturation. Calcium and magnesium strongly decrease the hsp90 thermostability and thereby cause oligomerization at lower temperature. We suggest that calcium affects the hsp90 oligomerization, known to be important for its chaperone activity, by shifting the unfolding temperature of the hsp90 N-terminal domain close to the heat shock temperature range.

Animals↗

PubMed1998

Effect of microtubule disruption on cell adhesion and spreading.

Microtubules have been involved in a variety of cellular processes. In this study, we examined the role of the microtubular system in the adhesion and spreading of the adenocarcinoma cell line HT29-D4. Disruption of microtubules by nocodazole or navelbine resulted in an increase in cell adhesion to purified ECM proteins. This enhanced cell adhesion is mediated by integrins, but is not attributable to quantitative changes in the number of integrin receptors at the cell surface, as determined by flow cytometric analysis. In contrast to attachment, spreading of HT29-D4 cells was reduced by nocodazole treatment in a dose-dependent manner. Thus, microtubule depolymerization appears to increase initial attachment of cells to extracellular matrix, while impeding subsequent cell spreading.

Adenocarcinoma↗

PubMed1998

Thermodynamic analysis of calcium and magnesium binding to calmodulin.

To elucidate some aspects still debated concerning the interaction of Ca2+ and Mg2+ with CaM, the thermodynamic binding parameters of Ca2+-CaM and Mg2+-CaM complexes were characterized by flow dialysis and isothermal microcalorimetry under different experimental conditions. In particular, the enthalpy and entropy changes associated with Ca2+ and Mg2+ binding to their sites were determined, allowing a better understanding of the mechanism underlying cation-CaM interactions. Ca2+-CaM interaction follows an enthalpy-entropy compensation relationship, suggesting that CaM explores a subspace of isoenergetical conformations which is modified by Ca2+ binding. This Ca2+-induced change in CaM dynamics is proposed to play a key role in CaM function, i.e. in its interaction with and/or activation of target proteins. Furthermore, data show that Mg2+ does not act as a direct competitor for Ca2+ binding on the four main Ca2+ binding sites, but rather as an allosteric effector. This implies that the four main Mg2+ binding sites are distinct from the EF-hand Ca2+ binding sites. Finally, Ca2+ is shown to interact with auxiliary binding sites on CaM. These weak affinity sites were thermodynamically characterized. The results presented here challenge the current accepted view of CaM ion binding.

Binding Sites↗

PubMed1998

Convergent effects of growth factors, hormones, and fibronectin are necessary for the enterocyte differentiation of a colon adenocarcinoma cell line (HT29-D4).

The aim of this work was to show in serum-free medium a convergent effect of physiological factors and extracellular matrix proteins on the differentiation process of enterocytes by taking as a model the HT29-D4 clone that has the feature of differentiating when subcultured in fetal bovine serum glucose-free medium. We show that triiodothyronine (T3) as well as insulin promotes limited cell growth and differentiation, whereas fibronectin or bovine serum albumin (BSA) induces cell growth and a low level of differentiation. However, insulin, T3, fibronectin, and BSA together with epidermal growth factor and transferrin promoted satisfactory growth and enterocyte morphology with epithelial electrophysiological properties in HT29-D4 cells. With these factors adequate protein targeting was achieved since cells apically expressed the carcinoembryonic antigen, and basolaterally transferrin and insulin receptors, beta 1 and alpha v beta 6 integrins, talin, vinculin, and focal adhesion kinase (FAK). Talin, vinculin, FAK, and alpha v beta 6 integrin, the fibronectin receptor, were clustered in focal contacts, which agrees with a possible role of fibronectin in final cell growth, the latter process mediating the final phase of differentiation. This level of differentiation can be maintained for a long time. Thus HT29-D4 cells appear to be a suitable model to study the implication of integrins in the differentiation process of human enterocytes.

Adenocarcinoma↗

PubMed1998

The effect of combining antitubulin agents on differentiated and undifferentiated human colon cancer cells.

The cytotoxicity of sequential combinations of a taxoid [paclitaxel (TAX) or docetaxel (TXT)] with a vinca alkaloid [vinorelbine (NVB)] was compared in differentiated and undifferentiated HT29-D4 cells. Agents were titrated from low doses inducing no modification of microtubule network to high doses corresponding to the clinically relevant concentrations that block mitosis. For undifferentiated cells, the sequential combination NVB/TAX was more efficient than TAX/NVB (22% cell survival versus 37% for 5 nM TAX and NVB). Surprisingly, we successively obtained synergism for low doses of both compounds [NVB (1-5 nM) and TAX (1-15 nM)], then additivity and finally antagonism when one of the compounds was at the concentration inducing mitotic block. The three patterns of results were also obtained with NVB/TXT combinations. For the synergistic combinations at the lowest concentrations, cytotoxicity occurred by apoptosis following mitosis. For differentiated cells, the most cytotoxic combinations were 1 microM TAX or TXT for 3 days followed by 1 microM NVB for 3 days, and 0.75 nM TAX or TXT for 9 days followed by 1 microM NVB for 3 days, the latter producing synergistic effects. Cytotoxicity occurred by apoptosis for the two states of differentiation. Major differences depending on cell phenotype were demonstrated: low sensitivity of differentiated cells to antitubulin agents and the difference in apoptotic pathways since mitosis is not involved in differentiated cells.

Antineoplastic Agents, Phytogenic↗

PubMed1997

Conformation and thermal denaturation of apocalmodulin: role of electrostatic mutations.

Scanning microcalorimetry and circular dichroism were used to study conformational state and heat denaturation of Ca2+-free synthetic calmodulin (SynCaM) and three charge reversal mutants. We produced evidence for the major role of the electrostatic potential in the stability and flexibility of SynCaM. The substitution of 118DEE120 by 118KKK120 (SynCaM12A) does not influence the flexibility of the protein; the replacement of 82EEE84 by 82KKK84 (SynCaM8) decreases its level, while the combination of these two mutations in SynCaM18A significantly increases the flexibility. The heat denaturation of apoSynCaM and its mutants is well approximated by two two-state transitions with the lower-temperature transition corresponding to C-terminal lobe melting and the higher-temperature one to N-terminal lobe melting. The difference in transition temperatures for the two lobes decreases in SynCaM8 and increases in SynCaM18A, suggesting a modification in the influence of one lobe to the other. The electrostatic mutations change the parameters of thermal denaturation of SynCaM lobes in a similar way as pH conditions affect thermal transition parameters of multidomain proteins, leading to a linear temperature dependence of transition enthalpy. One domain of the N-terminal lobe in apoSynCaM18A is unfolded in the native state. Near-UV CD spectra point out the invariability of the local structure of aromatic residues upon mutations, although the secondary structure undergoes striking transformations. Cacodylate ions strongly and specifically alter the helical content of SynCaM. Our data unambiguously demonstrate that the two lobes are not independent, and interactions between the lobes are mediated by the electrostatic potential of the molecule.

Calcium↗

PubMed1997

Pretreatment by tubulin agents decreases C-MYC induction in human colon carcinoma cell line HT29-D4.

For HT29-D4 cell line, we confirmed the interaction between c-Myc protein and microtubules by immunoprecipitation. We then studied the effect of antimitotic agents, nocodazole and the taxoids [paclitaxel (taxol) and docetaxel (taxotere)] on c-myc oncogene expression. The expression was analyzed by RT-PCR and Western blot. Taxol (1 microM), taxotere (1 microM) and nocodazole (3 microM) inhibited by 30-50% the c-myc induction produced by growth factors in culture medium. According to the flow cytometry analysis, the inhibition is not linked to the mitotic block. These results observed for both stabilizing and depolymerizing agents suggest that microtubular system is involved in c-myc expression more through its dynamic properties which influence signal transduction and intracellular transports than through its direct interaction with c-Myc protein.

Antineoplastic Agents, Phytogenic↗

PubMed1996

Crystal structure of the RNA binding ribosomal protein L1 from Thermus thermophilus.

L1 has a dual function as a ribosomal protein binding rRNA and as a translational repressor binding mRNA. The crystal structure of L1 from Thermus thermophilus has been determined at 1.85 angstroms resolution. The protein is composed of two domains with the N- and C-termini in domain I. The eight N-terminal residues are very flexible, as the quality of electron density map shows. Proteolysis experiments have shown that the N-terminal tail is accessible and important for 23S rRNA binding. Most of the conserved amino acids are situated at the interface between the two domains. They probably form the specific RNA binding site of L1. Limited non-covalent contacts between the domains indicate an unstable domain interaction in the present conformation. Domain flexibility and RNA binding by induced fit seems plausible.

Amino Acid Sequence↗

PubMed1996

[Modification of the response to paclitaxel of human differentiated colon cancer line after long-term treatment at very low dose].

Taxoids (paclitaxel, Taxol and docetaxel, Taxotere), drugs which stabilize microtubules, demonstrate marked activity against ovarian, brain and lung cancer. We investigated, on the human colon adenocarcinoma HT29-D4 cell line, firstly the effects of taxoids in function of the differentiation state and secondly the effects of a low dose of paclitaxel on the differentiation process. The cellular parameters studied were microtubular network, cell cycle and cell tubulin content. Undifferentiated cells treated with paclitaxel or docetaxel (10-100 nM) classically induced bundles in interphasic cells and pseudoasters in mitotic cells, arrest in cell cycle in G2M and increase in tubulin content. Inversely, no modification was observed in differentiated cells after similar taxoid treatment. Long-term low-dose pretreatment of differentiated cells restitutes some sensitivity to taxoids since these cells become responsive to further taxoid treatment, as reflected by modifications of the microtubule network.

Adenocarcinoma↗

PubMed1995

Differential effects of ethyl 5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl carbamate analogs modified at position C2 on tubulin polymerization, binding, and conformational changes.

NSC 613863 (R)-(+) and NSC 613862 (S)-(-) (CI980) are two chiral isomers of ethyl 5-amino 2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl carbamate which have potent antitubulin activity. The S-isomer is a more potent antimitotic compound than the R-isomer, and the two isomers differ markedly in binding to tubulin [Leynadier, D., Peyrot, V., Sarrazin, M., Briand, C., Andreu, J. M., Rener, G. A., & Temple, C., Jr. (1993) Biochemistry 32, 10675-10682]. To understand the origin of such differences, we studied the interactions of three R- and S-isomer structural analogs which differ in C2 (the chiral carbon), i.e., C179, NSC 337238, and NSC 330770. C179 is a methylated dehydrogenated achiral compound. It bound to tubulin with an apparent affinity Ka of (2.29 +/- 0.17) x 10(4) M-1, inhibited tubulin polymerization in vitro at a half-inhibitory concentration (IC50) of 100 microM, and presented no GTPase activity. The substitution of -CH3 by -H leads to the NSC 337238 compound. It bound to tubulin with a higher affinity [Ka = (2.62 +/- 0.35) x 10(5) M-1] and inhibited tubulin polymerization at a lower concentration (IC50 = 14 microM). It presented no GTPase activity and induced the formation of abnormal polymers at a protein critical concentration (Cr) of 2 mg mL-1. NSC 330770, a demethylated hydrogenated molecule, interacted strongly with tubulin [Ka = (3.30 +/- 0.56) x 10(6) M-1].(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Molecular interaction of tubulin with 1-deaza-7,8-dihydropteridines: a comparative study of enantiomers NSC 613862 (S) and NSC 613863 (R) by Raman and Fourier transform infrared spectroscopy.

Pre-resonance Raman spectroscopy has been applied to compare the vibrational modes of the R and S chiral isomers of 1-deaza-7,8-dihydropteridine when they are bound to tubulin. The main Raman bands are due to the chromophore and are coupled with the pi-pi electronic transition of C = C and C = N vibrational stretching. On binding to tubulin, the Raman spectra of both isomers are modified. However, the modifications induced are different for each isomer. The Raman bands due to C = C stretching from the phenyl ring are more strongly modified for the bound R isomer than for the S isomer. This leads us to suggest that R and S isomers differ in terms of their orientation in front of the binding locus of tubulin. In fact, with respect to the orientation of the bulky methyl group, the chromophore of the R isomer is more likely to be positioned against the external surface of either tubulin or GTPase proteins, while that of the S isomer is likely to be positioned away from the surface. The conformational changes induced in tubulin by R and S isomers have also been studied by Fourier transform infrared spectroscopy and by the analysis of amide I and II absorption bands. Both enantiomers induce similar minor changes to the tubulin secondary structure, corresponding to a decrease in the disordered alpha-helical content and accompanied by an increase in the undefined conformation content.

Animals↗

PubMed1995

Thermal denaturation of bacterial and bovine dihydrofolate reductases and their complexes with NADPH, trimethoprim and methotrexate.

Scanning microcalorimetry was used for the study of thermal denaturation of E.coli and bovine liver dihydrofolate reductases (cDHFR and bDHFR, respectively) and their complexes with NADPH, trimethoprim (TMP) and methotrexate (MTX) at pH 6.8. It was shown that the denaturation temperature of bDHFR is 7.2 degrees C less than that of cDHFR and that ionic strength is equally important for the thermostability and cooperativity of the denaturation process of the two proteins. Binding of antifolate compounds significantly stabilizes DHFR against heat denaturation. The stabilizing effect and the transition cooperativity depend on the nature of the inhibitor, the presence of NADPH and the origin of the enzyme. The dependence of calorimetric denaturation enthalpy (calculated per gram of protein) on denaturation temperature for DHFRs, their complexes with NADPH and binary/ternary complexes with TMP/MTX fits to the same straight line with the slope of 0.66 J/Kg. This relatively high value indicates an essential role of hydrophobic contacts in the stabilization of DHFR structure. The change of denaturation temperatures in binary complexes with MTX/TMP (in comparison with the free enzymes) is as much as 14.2 degrees C/8.5 degrees C and 13.3 degrees C/3.2 degrees C for cDHFR and bDHFR, respectively. The same change in ternary complexes with MTX/TMP is much more pronounced and equals to 21.9 degrees C/16.8 degrees C and 29.0 degrees C/16.4 degrees C. The vast difference of binary and ternary complexes thermostability demonstrates the important role of cofactor in the stabilization of enzyme. Moving from binary to ternary systems causes a significant increase in denaturation temperatures, even when corresponding association constants do not change (cDHFR binary/ternary complexes with MTX) or increases very slightly (bDHFR binary/ternary complexes with TMP). In all other cases the increase of denaturation temperature for each protein in complex with ligands correlates with the association constant for the corresponding complex.

Animals↗

PubMed1995

Simultaneous combination of microtubule depolymerizing and stabilizing agents acts at low doses.

The combined activity of a stabilizing and a depolymerizing agent was studied on microtubule formation in vitro and on cellular parameters related to the cytoskeleton. New compounds in each class of microtubular drugs, docetaxel, as stabilizing agent, and CI 980, as colchicine analog, currently in clinical trials, were tested at high and low concentrations. Simultaneous combination of docetaxel and CI 980, both in vitro and in cell lines, induced microtubule structures resulting from the association of the effects of the two drugs: short and numerous microtubules in vitro, abnormal asters or short bundles in cells depolymerized in their periphery. Moreover, combining the two drugs at low concentrations inducing neither modification of the microtubular network nor variation in cell polymerized tubulin content showed synergistic effects in mitotic cell block and, at lower concentrations, in inhibition of proliferation in the KB 3-1 cell line. Similar qualitative results were obtained with paclitaxel and colcemid, used in the place of docetaxel and CI 980, respectively. At low doses, the taxoid/colchine analog combination seems to induce some degree of mitotic inhibition, resulting from a subtle effect on the inhibition of microtubule dynamics.

Antineoplastic Agents↗

PubMed1994

Solution structure of Taxotere-induced microtubules to 3-nm resolution. The change in protofilament number is linked to the binding of the taxol side chain.

The synchrotron x-ray solution scattering profiles of microtubules assembled from purified GDP- or GTP-tubulin with the antitumor drug docetaxel (Taxotere) are consistent with identical non-globular alpha and beta-tubulin monomers ordered within the known surface lattice of microtubules, with a center to center lateral spacing of 5.7 +/- 0.1 nm. The higher angle part of the scattering profile, and therefore the substructure of the microtubule wall is identical in Taxotere- and Taxol-induced microtubules, to the resolution of the measurements. However, Taxotere-induced microtubules have a mean diameter of 24.2 +/- 0.4 nm, which is 1.12 +/- 0.01 times larger than that of paclitaxel (Taxol) induced microtubules. The population of Taxotere microtubules has on average 13.4 protofilaments, which is similar to control microtubules assembled with glycerol but is in marked contrast with Taxol-induced microtubules, which have on average 12 protofilaments under identical solution conditions. Model populations of Taxotere and Taxol microtubules with the distributions of protofilament numbers determined by electron microscopy reproduce the positions and approximate intensities of the experimental x-ray scattering data. Comparison of the structures and activities of both taxoids strongly suggests that the change of the more frequent lateral bond angle between tubulin molecules from 152.3 degrees (13-protofilament microtubules) to 150 degrees (12-protofilament microtubules) is linked to the binding of the side chain of Taxol. Optimal microtubule formation is obtained with unitary Taxotere to tubulin heterodimer ratio; however, ligand molecules in excess over tubulin dimers cause a loss of cylindrical scattering features, consistent with microtubule opening. The results are compatible with the observed biochemical and thermodynamic properties of this ligand-induced microtubule assembly system and also with the simple working hypothesis that taxoids would bind between adjacent microtubule protofilaments.

Docetaxel↗

PubMed1994

Wild-type and E106Q mutant carbonic anhydrase complexed with acetate.

The molecular structures of the acetate complexes of wild-type human carbonic anhydrase II (HCAII) and of E106Q mutant human carbonic anhydrase II were solved with high completeness (89-91%) to 2.1 and 1.9 A resolution, respectively. Both wild-type and mutant enzyme crystallize in space group P2(1) with cell dimensions a = 42.7, b = 41.7, c = 73.0 A and beta = 104.6 degrees. The altered active-site hydrogen-bond network caused by the mutation results in a different binding of the inhibitor in the two complexes. In the mutant, but not in the wild-type complex, a carboxylate O atom is within hydrogen-bond distance of Thr199 Ogamma1. In the wild-type enzyme ligand hydrogen bonding to this atom is normally only found for hydrogen-bond donors. The importance of this discrimination on catalysis by the enzyme is discussed briefly.

Journal Article↗

PubMed1994

Inhibition of microtubules and cell cycle arrest by a new 1-deaza-7,8-dihydropteridine antitumor drug, CI 980, and by its chiral isomer, NSC 613863.

CI 980 (NSC 613862; [S-(-)]) and NSC 613863 [R-(+)] are the two chiral isomers of ethyl 5-amino 1,2-dihydro-2-methyl-3-phenylpyrido[3,4-b]pyrazin-7-ylcar bamate (NSC 370147), which is a mitotic inhibitor with in vivo and in vitro activity against murine multidrug-resistant sublines. We have characterized the inhibition of in vitro microtubule assembly by the S (CI 980) and R (NSC 613863) enantiomers, their actions on the cytoplasmic microtubule network of epithelial-like PtK2 cells, and on the cell cycle of different human and murine leukemias and PtK2 cells. Assembly of purified tubulin, or tubulin plus microtubule-associated proteins, into microtubules was substoichiometrically inhibited by both compounds, which also induced a slow depolymerization of preassembled microtubules. Half inhibitory concentrations were 0.4-0.7 microM and 1.6-2.1 microM for the S and R isomers, respectively. Excess of both drugs induced polymerization of liganded tubulin into abnormal polymers similar to colchicine. The cytoplasmic microtubules of PtK2 cells were disrupted by both compounds in a concentration- and time-dependent manner, which was observed by indirect immunofluorescence microscopy and quantified by an enzyme-linked immunoassay of cytoskeletal tubulin. Half inhibitory concentrations were 6 nM S isomer, 100 nM R isomer, and 1 microM colchicine. Twenty nM S isomer or 500-700 nM R isomer gave nearly maximal effect. At these concentrations, half maximal microtubule depolymerization took place after 2 h of treatment. After drug removal, slow microtubule assembly and nearly complete reorganization of the cytoplasmic microtubules of PtK2 cells were observed (24 h). One nM S enantiomer or 25 nM R enantiomer induced mitotic arrest in 8 h in U937, HL60, and EL4 leukemias. PtK2 cells also stopped in mitosis after a 24-h incubation with 50 nM R isomer or 5 nM S isomer. The inhibition of cell division was irreversible in the leukemic cells, while PtK2 cells partially resumed growth. Although the interactions of CI 980 with microtubules in vitro are not very different from other drugs, it is a most potent cellular microtubule and mitotic inhibitor.

Animals↗

PubMed1994

Comparative effects of taxol and Taxotere on two different human carcinoma cell lines.

The effects of taxoids (taxol and Taxotere) were followed on two human cancerous cell lines (bladder carcinoma J82 cells and epidermoid carcinoma KB 3-1 cells). Three cellular parameters were studied, viz., the qualitative effect on cellular microtubules, the quantitation of tubulin, and the antimitotic action, using two-parametric flow-cytometric analyses in treated cells. In both of the cell lines the tubulin content increased after taxoid treatment before the accumulation of cells in the G2/M phase. The effects of taxoids on tubulin appeared at about a 10-fold lower concentration on KB cells than on J82 cells. After drug exposure, the microtubule network showed a striking difference between the two cell lines: microtubule bundles were predominant in the J82 cell line, whereas multiple asters were prevalent in the KB cell line. The formation of these structures was dose- and time-dependent. Asters were observed in mitotic cells and bundles were seen in interphase cells. The reversibility of these structures in both cell lines varied with the duration of exposure to drug. Some differences were shown between taxol and Taxotere: the effects of Taxotere as compared with taxol appeared at a 2-fold lower concentration and their reversibility was slower.

Antineoplastic Agents, Phytogenic↗