SummaryThe human colon adenocarcinoma HT29-D4 cell line is an interesting model for studies on epithelial cell differentiation. Undifferentiated cells are malignant proliferating cells, whereas differentiated cells act like epithelial polarized cells. In the present study, we first characterized the action of taxoids on the microtubular network of HT29-D4 cells according to the state of differentiation. Microtubular bundles were found in undifferentiated cells but not in differentiated cells, even with 500-fold higher taxoid concentrations for 96 h. This finding led us to study changes in microtubules according to the polarity status of the cell. E-MAP-115 was expressed only in differentiated cells; expression of β-tubulin isotypes was altered in them relative to undifferentiated cells. Classes I, II, III, IVa and IVb isotypes were expressed in both phenotypes; however, differentiated epithelial cells displayed a specific increase in class III β-tubulin. Thus, the increase in expression of this β-tubulin isotype in differentiated cells is not restricted to neuronal cells. Moreover, these expression changes may reflect a higher stability of microtubular network in differentiated cells, which may explain the lower activity of anti-microtubule agents, independently of the mitotic process. These results indicate that the composition of microtubules should be considered as one of the criteria involved in the response of tumour cells to chemotherapy with anti-microtubule agents.

MDL 27048 [trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2- methyl-2-propen-1-one] fluoresces when bound to tubulin but not in solution. This effect has been investigated and found to be mimicked by viscous solvents. Therefore, MDL 27048 appears to be a fluorescent compound whose intramolecular rotational relaxation varies as a function of microenvironment viscosity. The binding parameters of MDL 27048 to tubulin have been firmly established by fluorescence of the ligand, quenching of the protein fluorescence, and gel equilibrium chromatography. The apparent binding equilibrium constant was (2.75 +/- 0.45) x 10(6)M-1, and the binding site number was 0.81 +/- 0.12 (10 mM sodium phosphate-0.1 mM GTP, pH 7.0, at 25 degrees C). The binding is exothermic. The binding of MDL 27048 overlaps the colchicine and podophyllotoxin binding sites. Binding of MDL 27048 to the colchicine site was also measured by competition with MTC [2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one] , a well-characterized reversibly binding probe of the colchicine site [Andreu et al. (1984) Biochemistry 23, 1742-1752; Bane et al., (1984) J. Biol. Chem. 259, 7391-7398]. In contrast with close analogues of colchicine, MDL 27048 and podophyllotoxin neither affected the far-ultraviolet circular dichroism spectrum of tubulin, within experimental error, nor induced tubulin GTPase activity. Like podophyllotoxin, an excess of MDL 27048 over tubulin induced no abnormal cooperative polymerization of tubulin, which is characteristic of colchicine binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Cryptophycin 52 (C52) is a new synthetic compound of the cryptophycin family of antitumor agents that is currently undergoing clinical evaluation for cancer chemotherapy. The cryptophycin class of compounds acts on microtubules. This report details the mechanism by which C52 substoichiometrically inhibits tubulin self-assembly into microtubules. The inhibition data were analyzed through a model described by Perez-Ramirez [Perez-Ramirez, B., Andreu, J. M., Gorbunoff, M. J., and Timasheff, S. N. (1996) Biochemistry 35, 3277-3285]. We thereby determined the values of the apparent binding constant of the tubulin-C52 complex to the end of a growing microtubule (K(i)) and the apparent binding constant of C52 to tubulin (K(b)). The binding of C52 depended on tubulin concentration, and binding induced changes in the sedimentation pattern of tubulin, which indicates that C52 induces the self-association of tubulin and tubulin aggregates other than microtubules. Using analytical ultracentrifugation and electron microscopy, we show that C52 induces tubulin to form ring-shaped oligomers (single rings). We also show that C52 inhibits the formation of double rings from either GTP- or GDP-tubulin. In addition, the advances made by electron crystallography in understanding the structure of the tubulin and the microtubule allowed us to visualize the putative binding site of C52 and to reconstruct C52-induced ring oligomers by molecular modeling.

Several fluorescence properties of two enantiomers, NSC 613862 (S)-(-) and NSC 613863 (R)-(+), have been compared. Even though the two isomers showed the same fluorescence behavior in solution in different solvents, drastic differences were observed after binding to purified calf brain tubulin. Binding measurements for the two compounds were performed both by fluorescence spectroscopy and by column gel permeation, a direct method of measurement. For both isomers, the binding was characterized by the presence of one high-affinity binding site with an apparent association constant of (3.2 f 0.5) X lo6 M-I and (4.1 f 0.9) X lo6 M-' for the Rand S-isomer, respectively, and by several low-affinity sites.Both isomers were also shown to induce GTPase activity in tubulin. The high-affinity binding site seems to be the same for the two isomers. Moreover, fluorescence competition experiments suggest at least a partial overlap of the colchicine and podophyllotoxin site. To explain the differences in fluorescence behavior after binding to tubulin, we hypothesize that the R-isomer is positioned differently in its binding locus as compared with the S-isomer.Initially synthesized by Elliott et al. (1968) to act as analogues of folic acid, a number of compounds that possess an aromatic nucleus with a l-deaza-7,8-dihydropteridine structure have been reported to inhibit mitosis and produce an accumulation of cells in metaphase (Wheeler et al., 1981). Among them, NSC 181928, ethyl (5-amino-1,2-dihydro-3-[ (N-methy1anilino)methyll pyrido [3,4-b]pyrazin-7-yl)carbamate has been studied in particular. Hamel and Lin (1982) demonstrated that this drug was an active antitubulin agent which inhibited both polymerization and binding of colchicine to tubulin and stimulated tubulin-dependent GTP hydrolysis.Bowdon et al. (1987) showed that another member of the same series, NSC 370147, ethyl (5-amino-1,2-dihydro-2methyl-3-phenylpyrido [ 3,4-b] pyrazin-7-yl)carbamate, was able to competitively inhibit the binding of [3H]c~lchicine and slightly enhance the binding of [3H]vincristine to purified tubulin.However, NSC 370147 is a racemic compound. Its two chiral isomers, NSC 613862 (S)-(-) and NSC 613863 (R)- (+) (see Chart I), have displayed significant differences in Abstract published in Advance ACS Abstracts, September 1, 1993. I Abbreviations: BSA, bovine serum albumin; PG buffer, 10 mM sodium phosphate and 0.1 mM GTP, pH 7.0; MDL 27048, trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylam~o)phenyl]-2-methyl-2-pro~n-1 -one; MTC, 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6cyclohep~~en-1-one; SDS, sodium dodecyl sulfate; TME, tropolone methyl ether; AS,,, difference between theapparent sedimentation coefficient of tubulin alone and liganded.