Conformational change induced by coenzyme binding to bovine liver dihydrofolate reductase: a spectrofluorimetric study.

When NADPH was added in excess to a bovine liver DHFR solution, a fluorescence peak due to an energy transfer mechanism was apparent at 450 nm. It did not vary over time. The intrinsic fluorescence peak of DHFR at 320 nm was quenched and this phenomenon increased over the time-course after NADPH addition. This result was ascribed to a slow DHFR conformational change induced by NADPH binding, which has never been previously described in such a long time scale (more than 30 min). A kinetic scheme accounting for this mechanism has been proposed. Furthermore, this interconversion between two protein conformers led to an increase in the initial apparent rate of the enzymatic reaction catalyzed by DHFR.

Comparative thermodynamic study of the interaction of some antifolates with dihydrofolate reductase.

The thermodynamic parameters of the binding of antifolate drugs to bovine liver dihydrofolate reductase (EC 1.5.1.3., 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase) have been measured with a flow microcalorimetric method. These parameters are greatly influenced by the structure of the inhibitor and/or by the presence of NADPH and above all by temperature. For all the compounds studied, binding at 37 degrees C is driven by favourable enthalpy variations, whereas entropy variations are unfavourable. At 10 degrees C, reactions are both enthalpically and entropically driven. These effects can be explained by a partial thermal denaturation of dihydrofolate reductase at 37 degrees C, which is restructured by NADPH and/or the antifolate. The refolding induced by the antifolate trimetrexate may explain its high association constant in the binary system (without NADPH), and the weaker cooperative effect of NADPH in the ternary system, as compared to methotrexate. In contrast, the poor affinity of trimethoprim for mammalian dihydrofolate reductase in binary and ternary systems at 37 degrees C is the result of a weaker stabilizing effect of this compound as regards temperature increase. Heat capacity variation linked to the complex formation reaction showed that this conformational transition is more pronounced between 25 and 37 degrees C than between 10 and 25 degrees C. Thus, the ability of the inhibitors to give to dihydrofolate reductase a more stable thermal behaviour at 37 degrees C is determinant in their binding.

Thermodynamic study of dihydrofolate reductase inhibitor selectivity.

The thermodynamic parameters of the binding of some folate analogues (methotrexate, trimetrexate and trimethoprim) to dihydrofolate reductases from different species have been measured with a flow microcalorimetric method at 37 degrees C. In the absence of NADPH, the three inhibitors exhibited a higher affinity for E. coli DHFR than for vertebrate DHFRs. This selectivity in favor of bacterial DHFR is entropy driven and is correlated with a weaker conformational change for bacterial DHFR than for vertebrate DHFRs, and with additional hydrophobic contacts, provided by this enzyme to the ligands. In presence of NADPH, as reported in the literature, trimetoprim shows a high selectivity in favor of bacterial DHFR, contrarily to methotrexate and trimetrexate, whose affinities are elevated and highly similar for mammalian and bacterial enzymes. The positive cooperative effect of NADPH, which has an enthalpic origin, fluctuates widely with inhibitor structure and with enzyme species. For trimethoprim, the cooperative effect is much more pronounced for bacterial DHFR than for vertebrate DHFRs. But the role of NADPH is not to induce a selectivity: it only increases the selectivity that trimethoprim already presented in absence of NADPH. Inversely, for methotrexate and trimetrexate, the cooperative effect is stronger for vertebrate enzymes than for the bacterial enzyme, and thus, NADPH cancels the selectivity the two antifolic compounds had, in the absence of NADPH, for the bacterial enzyme.

Opposite effects of antimicrotubule agents on c-myc oncogene expression depending on the cell lines used.

We investigated the expression of c-myc in HT29-D4, HBL100 and Caco-2 cells treated with microtubule stabilising (paclitaxel) or depolymerising agents (vinblastine, nocodazole). After induction by epidermal growth factor (EGF), c-myc expression decreased in HT29-D4 cells treated with all the antimicrotubule agents. In HBL100 and Caco-2, when microtubules were stabilised with paclitaxel, c-myc expression also decreased. In contrast, its expression increased after treatment with depolymerising agents. In both cell lines, we also observed that depolymerising agents alone induced c-myc expression whilst paclitaxel had no effect. This mRNA induction was confirmed at the protein level. In HT29-D4, no variation of c-myc expression was observed. Then, we showed that the increase of mRNA level was due to activation of gene transcription. These results indicate that modulation of c-myc expression varied depending on the cell lines used and the type of antimicrotubule agents. This work provides a potential link between the microtubular network and c-myc gene expression.

A novel fluorometric assay for quantitative analysis of dihydrofolate reductase activity in biological samples.

In an effort to study the level of dihydrofolate reductase (DHFR), the main molecular target of antifolate drugs, in healthy and malignant tissues of human origin, a new and convenient fluorometric enzymatic assay has been developed. The technique measures the overall decrease in fluorescence emission at 454 nm (lambda ex = 342 nm) due to the contributions from coenzyme oxidation and substrate reduction. This technique was developed by using an enzyme purified from beef liver. All criteria of quality were checked: sensitivity, reproducibility and specificity made it suitable for low activity measurements. It was successfully applied to human tissue crude extracts.

Multidrug-resistant phenotype influences the differentiation of a human colon carcinoma cell line.

The human colon carcinoma cell line HT29-D4, which constitutively expresses a very low level of the MDR1 gene product, was made multidrug resistant by transfection with a human MDR1 cDNA from the pHaMDR1/A expression vector and selection by colchicine. Resistant clones were 3- to 15-fold resistant to colchicine and were cross-resistant to doxorubicin (3- to 4-fold). MDR1 gene expression was associated with the expression of functional P-glycoprotein (gp-170); the function was reversed by verapamil and cyclosporin A. HT29-D4 cells are able to differentiate in vitro by replacement of glucose by galactose in the culture medium and also to release the carcinoembryonic antigen (CEA). Under these culture conditions, MDR1 mRNA and gp-170 were always expressed and the protein remained functional. Upon galactose treatment, resistant clones were less differentiated since they showed a heterogeneous monolayer organization accompanied by heterogeneous staining of cell-surface CEA and a high decrease (60-90%) of CEA release.

Activity measurements in human tissues of the methotrexate molecular target: a novel fluorometric assay.

As DHFR is the main molecular target of MTX, a widely used anticancer drug, its level in human tissues is likely to be one of the factors determining tissue sensitivity towards this drug. Forty-one biopsies were analyzed for their DHFR activity by a convenient spectrofluorometric assay developed in our laboratory; this sensitive method proved to be suitable for measurements in very small human samples. Statistical analysis of the results showed that (i) DHFR activity is not an index of tumorogenicity, at least in the cases studied, (ii) tumorous extracts contain modulators of DHFR activity.

Follicle-like structures formed by intestinal cell lines derived from the HT29-D4 adenocarcinoma cell line: morphological and functional characterization.

When cultured in high glucose containing medium, the human colon carcinoma cell line HT29-D4 and a clone derived by transfection with the MDR1 cDNA (MDR31) form multilayers of unorganized cells which are not polarized and are linked by desmosomes. Within these multilayers appear spontaneously large multicellular follicle-like-structures (FLS) where polarized cells linked by tight junctional complexes surround a lumen. Electron microscopy showed that some FLS display well developed brush borders with densely packed microvilli. Others have irregularly oriented microvilli of various lengths or are even completely devoid of apical differentiation. The lumen contains a variable amount of amorphous osmiophilic material. The apical surface of FLS forming cells express dipeptidylpeptidase IV, carcinoembryonic antigen, the mucin MUC1 and for the transfected cells the gp-170 protein. The organic anion fluorescein is transported from the cell to the lumen of FLS. Rhodamine 123, a substrate of the gp-170 ABC transporter is also concentrated in the lumen formed by MDR31 cells. Verapamil and cyclosporine A inhibited this last transport. Cyclic AMP stimulates the formation of these structures since treatment of post-confluent multilayers dramatically increased the number of FLS in HT29-D4 and MDR31 cell cultures within 24 h. The spontaneous formation of these morphologically and functionally polarized structures appeared at random and might respond to the coincidence of fluctuating parameters of the regulatory pathways (cAMP, Ca2+).

Variations in fluorescence and enzymic properties of bovine dihydrofolate reductase.NADPH complex during the slow conformational change induced by coenzyme binding.

When NADPH was added in large excess to bovine dihydrofolate reductase (H2folate reductase), there was a slow isomerization process between two conformers of the binary complex (B1<-->B2), as shown by changes in the fluorescence properties. Thus, we monitored the time dependence of (a) the quenching of protein intrinsic fluorescence intensity, (b) the polarization state of the fluorescence light emitted by NADPH.H2folate reductase complexes and (c), from a more biological point of view, the enzymic activity of binary complex solutions. The kinetics for these three processes were in good agreement using the same temperature conditions. Furthermore, fluorescence studies provided information on the NADPH environment in the binary complex. As soon as NADPH bound to H2folate reductase, light emitted by the invariant Trp24 residue located within the coenzyme-binding site was quenched by an energy-transfer process. Moreover, Trp57 and/or Trp113 emissions were partially quenched. The subsequent NADPH-bound protein conformational change caused an additional quenching, probably of Trp57 and/or Trp113 emissions. Thus, NADPH.H2folate reductase conformation was modified but no change was observed at the coenzyme-binding site, at least in our fluorescence study. These results were confirmed by polarization measurements. The conformational change, as well as the instantaneous NADPH binding, resulted in a more rigid form of the protein, as shown by an increase in steady-state anisotropy values. Finally, the isomerization process led to a more active enzymic form.

Involvement of nuclear factor kappaB in c-Myc induction by tubulin polymerization inhibitors.

We showed previously that microtubule disassembly by vinblastine induces the proto-oncogene c-myc in epithelial mammary HBL100 cells. In this study, we demonstrate that vinblastine treatment in these cells, in contrast to what was observed with the colon adenocarcinoma cell line HT29-D4, activated the transcription factor NFkappaB, which has been involved in c-myc regulation. The microtubule disassembly also induced IkappaB degradation. Using transient transfection analysis, we show that the trans-activation of c-myc by vinblastine was decreased when NFkappaB binding sites on c-myc promoter were mutated. Additionally, we demonstrate that microtubule dissolution trans-activated a thymidine kinase-CAT construct containing an NFkappaB binding site at -1180 to -1080 bp relative to the P1 promoter. Thus, vinblastine up-regulates the enhancer activity of the NFkappaB binding site. These results suggest that microtubule disassembly induced by vinblastine can trans-activate the c-myc oncogene through NFkappaB. Taking into consideration the paradoxical roles of both c-myc and NFkappaB in proliferation or apoptosis, this data reveals the complex action mechanism of this microtubule interfering agent.