The effects of the differentiation inducing agents (DIAS), sodium butyrate (NaBu), retinoic acid (RA), dimethylformamide (DMF), hexamethylene bisacetamide (HMBA), forskolin, and 12-O-tetradecanoylphorbol-13-acetate (TPA), on the growth, morphology, and estrogen receptor (ER) content and epithelial membrane antigen (EMA) expression on a serumless human breast cancer cell line (MCF-7) were compared. All these agents reversibly caused a concentration-dependent growth inhibition in monolayers and markedly reduced colony-forming efficiency in soft agar. A twofold increase in doubling time was obtained with RA (1 microM), but cell replication ceased with NaBu (1 mM), forskolin (50 microM), DMF (1%), HMBA (5 mM), and TPA (8 nM). Total growth arrest induced by these last compounds was preceded by an accumulation of cells in G0/G1 phase observed at 24 h by flow cytometry and accompanied by a change in cell morphology as seen by light and electronic microscopy. An increase in cell volume and the presence of lipid droplets was noted in treated cells that were spread out, as compared with controls. The acquisition of a more mature phenotype was confirmed by an increased expression of EMA monitored by flow cytometry. A specific reduction in the number of ER without any constant dissociation (Kd) modification was also observed after treatment with the 5 DIAs. No modification of morphological or biochemical characteristics, including EMA expression and ER binding, were observed for RA (1 microM)-treated cells. All these results suggest that induction of a more differentiated phenotype is associated with a block in G1 cell cycle phase, resulting in total growth arrest. Apparently, RA (1 microM)-treated cells did not fulfill these criteria, since only a slight accumulation in G1 and a slowed growth rate were evaluated.
Isoniazid (INH), one of the most effective antimycobacterial drugs, specifically inhibits, at an early stage of its action, the biosynthesis of mycolic acids, specific mycobacterial lipids which play a central role in the cell envelope architecture of mycobacteria. In the present study, the consequences of the action of INH on the cell morphology of Mycobacterium tuberculosis and Mycobacterium aurum were examined. Electron microscopy was used to observe bacilli which were previously treated with either subinhibitory concentrations of INH or the MIC of the drug, leading to a decrease of 20 to 35% (by weight) of their mycolic acid contents. The earlier effect of INH on the ultrastructure of mycobacteria, as revealed by negative staining of bacilli, was the alteration of the bacterial poles; this event was observed prior to the bacteriostatic action of the drug and was accompanied by a release of material from the poles into the extracellular medium. In a later stage of the drug's action, cell deformation occurred and more extracellular material was seen. The material released following the action of the drug on susceptible mycobacterial cells was identified as being almost exclusively composed of proteins. Labeling of amino acids with 35S prior to and during the action of INH on M. aurum and subsequent analysis of the labeled proteins led to the conclusion that they consisted of secreted proteins which were up to 20-fold oversecreted in the presence of the drug. Competitive enzyme-linked immunosorbent assay with the secreted 45/47-kDa antigen complex of M. tuberculosis demonstrated up to 20-fold oversecretion of these proteins. Taken together, the production of oversecreted proteins following the decrease of the cell envelope mycolate content by INH strongly suggests that mycolic acids may act as a barrier in the export of proteins secreted by mycobacteria.
The addition of D-arabinose, D-galactose, D-glucosamine, or D-mannose to the growth medium of Mycobacterium smegmatis suppressed the inhibitory effects of ethambutol both on acetate labeling of cell wall-linked mycolic acids and on the increase in the delipidated cell dry weight. The addition of D-glucose or D-fructose had no effect. It is proposed that ethambutol inhibits an early step of glucose conversion into the monosaccharides used for the biosynthesis of structurally and biologically important cell wall polysaccharides: arabinogalactan, arabinomannan, and peptidoglycan.Ethambutol (EMB) is an efficient antituberculosis molecule used in combination with other drugs (3). Moreover, there is renewed interest in EMB because of its activity against opportunistic pathogens, like Mycobacterium avium (4), which are resistant to conventional antituberculosis drugs and because of its ability to increase the susceptibility of this pathogen to other antimycobacterial drugs (5,7,13).The mode of action of EMB has not yet been elucidated (for reviews, see references 7 and 16). The early inhibitory effects described for EMB concerned the synthesis of phospholipids (1, 8), the transfer of mycolic acids to cell walllinked arabinogalactan (14), and the incorporation of labeled glucose into the arabinose-containing polysaccharides of the cell wall (15).Inhibition of arabinogalactan synthesis would explain the inhibition of its acylation by mycolic acids, but the corresponding experiments have been performed with intact cells (14) and could be a secondary effect resulting from inhibition of some central metabolism.Inhibition The effect of EMB on the arabinogalactan content of the cell wall was determined for cells that were delipidated with chloroform-methanol and then extracted with 70% ethanol to eliminate arabinomannan and mannan, which are not covalently linked to the wall (6). Cell residues were hydrolyzed (with 1 M trifluoroacetic acid at 110°C for 2 h), and the galactose content was determined by gas-liquid chromatography, with erythritol being added to the hydrolysis medium as a standard for quantification.The cell-free system that incorporates acetate into mycolic acids was prepared and used as described previously (12) by isolating at the tops of centrifuge tubes the fluffy layer of cells from an exponential-growth-phase culture disrupted in a French pressure cell. The protein content of the fluffy layer was determined by the Lowry procedure as described previously (12). The assay medium did not contain magnesium, an ion known to reduce the activity of EMB. To test the drug effect, the cell-free system was preincubated for 30 min in the presence of 25 ,ug of EMB per ml (5 x the MIC), and then [14C]acetate was added for 90 min of incubation. Lipids were extracted as indicated above; lipids and cell residues were saponified, and total acids were isolated. Aliquots were counted, and total acids were analyzed as methyl esters by thin-layer chromatography (the solvent was CH2Cl2); radioactivity was determined on thin-layer...
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