Lesions and preferential initial localization of [S-methyl-3H]bleomycin A2 on Saccharomyces cerevisiae cell walls and membranes

Abstract

Extensive lesions were produced in cell walls of Saccharomyces cerevisiae by the bleomycin family of anticancer antibiotics (30 min to 4 h). Electron micrographs revealed that the alterations were most frequently large breaks and small interruptions or holes in cell walls, which sometimes extended into cell membranes. Large portions of cell walls were sometimes lost. Cell walls were frequently ruptured in one or more positions. More than 75% of bud scar regions in single-plane sections and all bud scars in serial sections exhibited many interruptions and breaks after 3 or 4 h of treatment. The discovery of extensive damage to cell walls was consistent with the preferential (approximately 70%) association of radiolabeled bleomycin with cell walls and perimeters of bud scar regions after short exposures (30 min). After longer exposures, the distribution of silver grains changed from a predominant association with cell walls (30 min) to an increased association with the cell cytoplasm (1 to 4 h). This correlated with increased ultrastructural damage, since damage to cell walls was generally more frequent and more severe with increasing length of treatment (30 min to 4 h) or dose (25 to 100 micrograms/ml). Although DNA lesions are believed to be the lethal properties of bleomycins, the lesions produced in cell walls are also lethal properties of the antibiotics. The distributions of lesions on cell walls suggested a generalized interaction of the antibiotic with a cell wall component. These results led us to hypothesize a mechanism of effective antifungal action for the bleomycin family of antibiotics.

Bleomycin affects cell wall anchorage of mannoproteins in Saccharomyces cerevisiae

“…We surmised that the drug has effects on the structure of the yeast cell wall. This has been confirmed by a thin-section electron microscopy study (18) demonstrating bleomycininduced cell wall lesions and localization of a substantial fraction of the bleomycin to the cell wall. The susceptibility to lytic enzymes and the bleomycin localization to the cell wall imply that bleomycin has biochemical effects on the cell wall matrix.…”

“…Such breaks are due to oxidation of deoxyribose furan rings and breakage of the N-glycosidic base linkages (3,19,24). In addition, we have noticed that bleomycin-treated yeast cells are fragile and are much easier to spheroplast than cells not treated with the drug (14,18). We surmised that the drug has effects on the structure of the yeast cell wall.…”

“…Cells or cell walls were harvested, washed twice with deionized water, and treated with 20 mM dithiothreitol (DTT) at 30°C for 30 min unless otherwise described. The insoluble material was collected by centrifugation, washed once more with water, and incubated in 3 mg of Lyticase (Sigma) or Zymolyase (ICN) per ml in a buffer containing 0.9 M sorbitol, 0.1 M sodium citrate, 0.1 M EDTA, and 0.02 M DTJ, pH 5.8 (14,18). The pH of digestion was chosen to correlate with previous experiments in which a low pH was used to stop the action of the bleomycin (14,15,18).…”

“…The insoluble material was collected by centrifugation, washed once more with water, and incubated in 3 mg of Lyticase (Sigma) or Zymolyase (ICN) per ml in a buffer containing 0.9 M sorbitol, 0.1 M sodium citrate, 0.1 M EDTA, and 0.02 M DTJ, pH 5.8 (14,18). The pH of digestion was chosen to correlate with previous experiments in which a low pH was used to stop the action of the bleomycin (14,15,18). Lyticase and Zymolyase are partially purified enzyme mixtures containing endo-,B1,3 glucanase and proteases as the major lytic components (10,11,29).…”

Electromechanical effects on multilayered cells in nonuniform rotating fields

“…Although initially double-shelled models were used for theoretical calculations [41][42][43], only the extension to four shells rendered a good agreement with experimental ER spectra. In fact, electron micrographs of S. cerevisiae reveal a three-layered structure outside the plasma membrane [44][45][46]. Therefore in this work we use the complete spherical four-shelled model (cell interior, plasma membrane, thick inner wall, and thin outer wall) that best fits the experimental data [43].…”