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 induces strand breakage in DNA through disruption of glycosidic linkages. We investigated the ability of bleomycin to damage yeast cell walls, which are composed primarily of carbohydrate. Bleomycin treatment of intact yeast cells facilitated enzymatic conversion of yeasts to spheroplasts. Bleomycin treatment also altered anchorage of mannoproteins to the cell wall matrix in intact cells or isolated cell walls. Cell surface mannoproteins were labelled with 12.5, and their solubilization was monitored. Seventeen hour treatments with bleomycin released some of the label directly into treatment supernatants and facilitated extraction of mannoproteins by dithiothreitol and lytic enzymes. Bleomycin treatments as short as 10 min caused changes in extraction of mannoproteins from intact cells. Specifically, cell wall anchorage of several mannoproteins was affected by the drug. There were drug-induced changes in extractability of mannoproteins with apparent molecular weights of 96,000, 80,000, 61,000, 41,000,31,500, and 21,000 (determined after deglycosylation with endo-N-acetylglucosaminidase H). The similarity of results obtained in the presence and absence of cycloheximide, the appearance of cell wall effects after only 10 min of treatment, and the similarity of effects in intact cells and isolated cell walls are consistent with direct drug-induced damage and inconsistent with a mechanism dependent on expression of bleomycin-damaged genes or other intracellular mediators. The results are consistent with bleomycin-mediated increases in cell wall permeability through disruption of glycosidic cross-linking structures in the cell wall.

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