ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, June 1993, p. 1264-12690066-4804/93/061264-06$02.00/0Copyright X 1993, American Society for Microbiology

Bleomycin Affects Cell Wall Anchorage of Mannoproteinsin Saccharomyces cerevisiae

ROBERT BEAUDOUIN,' SEUNG T. LIM,' JEAN-ALEX STEIDE,2 MARIE POWELL,2JUDITH MC.KOY,2 A. J. PRAMANIK,2 ELMA JOHNSON,1 CAROL WOOD MOORE,2

AND PETER N. LIPKEl*

Department ofBiological Sciences and Institute for Biomolecular Structure and Function,Hunter College of the City University ofNew York, New York; New York 10021,1 and

Department ofMicrobiology, City University ofNew York Medical School andSophie Davis School ofBiomedical Education, New York, New York 100312

Received 16 December 1992/Accepted 7 April 1993

Bleomycin induces strand breakage in DNA through disruption of glycosidic linkages. We investigated theability of bleomycin to damage yeast cell walls, which are composed primarily of carbohydrate. Bleomycintreatment of intact yeast cells facilitated enzymatic conversion of yeasts to spheroplasts. Bleomycin treatmentalso altered anchorage of mannoproteins to the cell wall matrix in intact cells or isolated cell walls. Cell surfacemannoproteins were labelled with 12.5, and their solubilization was monitored. Seventeen hour treatments withbleomycin released some of the label directly into treatment supernatants and facilitated extraction ofmannoproteins 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 apparentmolecular weights of 96,000, 80,000, 61,000, 41,000,31,500, and 21,000 (determined after deglycosylation withendo-N-acetylglucosaminidase H). The similarity of results obtained in the presence and absence of cyclohex-imide, the appearance of cell wall effects after only 10 min of treatment, and the similarity of effects in intactcells and isolated cell walls are consistent with direct drug-induced damage and inconsistent with a mechanismdependent 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 glycosidiccross-linking structures in the cell wall.

Bleomycin, an antitumor drug, induces breaks in DNA inall tested organisms. Such breaks are due to oxidation ofdeoxyribose furan rings and breakage of the N-glycosidicbase linkages (3, 19, 24). In addition, we have noticed thatbleomycin-treated yeast cells are fragile and are much easierto spheroplast than cells not treated with the drug (14, 18).We surmised that the drug has effects on the structure of theyeast cell wall. This has been confirmed by a thin-sectionelectron microscopy study (18) demonstrating bleomycin-induced cell wall lesions and localization of a substantialfraction of the bleomycin to the cell wall. The susceptibilityto lytic enzymes and the bleomycin localization to the cellwall imply that bleomycin has biochemical effects on the cellwall matrix.

Fungal cell walls contain two major components: glyco-protein complexes with high carbohydrate contents andfibrous polysaccharides. These two classes of materials arepresent in roughly equal amounts and together account for80 to 90% of the total wall material (1, 2, 4). Minor compo-nents of yeast cell walls include lipids and other polysaccha-rides, including chitin (4, 5, 7). In Saccharomyces cerevi-siae, the major fibrous component is a 11 -- 3-linked glucanwith an average of 1,500 glucose units per molecule (5). Abranched 131 -- 6-, 131 -> 3-linked glucan is also present andmay anchor the exterior layer of mannoproteins in the cellwall matrix (5, 28). The amorphous component in yeastsconsists of some glucans and a large number of mannopro-teins containing 10 to 90% carbohydrate (2, 5). The manno-

* Corresponding author.

proteins can be subdivided into two classes. Those that are

covalently associated with the cell wall are an integral part ofthe cell wall matrix and contribute critically to its architec-ture and stability. This class has very slow turnover andtherefore appears to be a permanent part of the cell wall (22,27). A second class is associated with the cell wall througheither noncovalent bonds or physical entrapment within thecell wall matrix (27).

Since only a small fraction of cell wall materials is solublein the absence of lytic agents, it is difficult to monitorwhether cell wall components are altered by drug treatmentsor other perturbations (5, 29). Furthermore, cell wall glyco-proteins are not well resolved by standard methods, sincethe carbohydrate is heterogeneous in both charge and sizeand alters binding of sodium dodecyl sulfate (SDS) toproteins as well. As an initial approach to analysis of cellwall effects of bleomycin, we assayed for bleomycin-medi-ated solubilization of cell wall mannoproteins. This class ofglycoprotein is characteristic of the cell wall, and somemannoproteins comprise the outermost layer of the cellsurface (1, 29). A large fraction of mannoprotein is co-valently linked to the cell wall matrix, but when liberated,this material is water soluble (22, 29). Although mannopro-teins are often not resolvable by gel electrophoresis in theirnative state, many of them form discernable but diffusebands following removal of the N-linked carbohydrate (8, 26,29). Such deglycosylation is easily accomplished by diges-tion with endo-N-acetylglucosaminidase H (endo H), whichcleaves the di-N-acetylchitobiose linkage in high-mannoseN-asparaginyl-linked glycoproteins (25). Therefore, endoH-induced molecular mass shifts from nonresolvable (>200

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kDa) sizes to resolvable bands are characteristic of cell wallmannoproteins. We reasoned that if bleomycin facilitatescell wall dissolution, as implied by electron micrographs anddata on spheroplast formation, such mannoproteins shouldbe released from the cell wall matrix and solubilized in theextracellular medium.

MATERIALS AND METHODS

Materials. Single clones of S. cerevisiae CM1069-40(MATa4 ade2-40 cycl-45 trpS-27 ilv1-92) were grown in liquidYPAD at 21°C overnight with extensive aeration as previ-ously described (15-17). Growth was suspended at 10 cellsper ml, and cells were centrifuged at 3,900 x g and 4°C,washed twice with deionized water, and resuspended at107/ml.Bleomycin was obtained as a gift from Bristol Laborato-

ries, Syracuse, N.Y., as sterile bleomycin sulfate. Concen-trations of stock solutions were monitored by A292. Treat-ment times and bleomycin concentrations for individualexperiments are described in Results.

Isotopic labelling. Two Iodobeads (Pierce Chemical Co.,Rockford, Ill.) were added to 100 ,ul of a cell suspensioncontaining 108 washed cells in 0.1 M Tris buffer, pH 7.2. Thecell suspension was incubated at room temperature for 5 minand then chilled to 0°C. Approximately 1.5 mCi of Na125Iwas added, and the cells were labelled for 2 min. Cells werethen harvested by centrifugation and washed two or moretimes with deionized water to remove excess label. Thisprocedure t8ypically incorporated 2 x 107 to 3 x 107 cpm of25I into 10 cells.Cell wall isolation. Isotopically labelled cells (5 x 107) were

suspended at 0°C in 0.02 M sodium acetate buffer, pH 5.5,containing 1 mM phenylmethylsulfonyl fluoride. Glass beadsprewashed with the same buffer were added to the suspen-sion. The suspension was agitated on a Vortex mixer atmaximum speed for 10 successive 2-min intervals with 1-minchilling intervals. The homogenate was centrifuged at 3,900x g for 8 min. The pellet was harvested and washed fourtimes with 0.02 M sodium acetate-0.4 M KCl-1 mMMgCl2-1 mM phenylmethylsulfonyl fluoride and once withdeionized water. Cell wall fragments were kept frozen indeionized water until use. Protein was determined by thebicinchoninic acid method (Pierce Chemical Co.).

Formation of spheroplasts. Bleomycin-treated and controlcells or isolated cell walls were successively digested in aprotocol derived from the standard procedures for preparingspheroplasts (Fig. 1; 23). All washes and incubation super-natants were kept for analysis of released radioactivity andprotein. Cells or cell walls were harvested, washed twicewith deionized water, and treated with 20 mM dithiothreitol(DTT) at 30°C for 30 min unless otherwise described. Theinsoluble material was collected by centrifugation, washedonce more with water, and incubated in 3 mg of Lyticase(Sigma) or Zymolyase (ICN) per ml in a buffer containing 0.9M sorbitol, 0.1 M sodium citrate, 0.1 M EDTA, and 0.02 MDTJ, pH 5.8 (14, 18). The pH of digestion was chosen tocorrelate with previous experiments in which a low pH wasused to stop the action of the bleomycin (14, 15, 18).Lyticase and Zymolyase are partially purified enzyme mix-tures containing endo-,B1,3 glucanase and proteases as themajor lytic components (10, 11, 29).

SDS-polyacrylamide gels and autoradiographic analyses.Unless otherwise stated, equal amounts of radioactivitywere analyzed for all samples. Samples were lyophilized andredissolved in 0.01 M sodium acetate buffer, pH 5.0. Sam-

Cells or Cell Walls

1ISupernatant 4 Centrifuge

Pellet

1I

± Bleomycin

Fraction I

± DTT

Supernatant 4 CentrifugeFraction 11 4

Pellet

4 + Glucanase

Supernatant 4- - CentrifugeFraction III4 7

PelletV

SDS ExtractFraction IV

FIG. 1. Flow chart for preparation of fractions for analysis of cellwall mannoproteins. Details are given in the text. Starting materialvaried as to label and whether intact cells or isolated cell walls wereused. The fractions analyzed in individual experiments are desig-nated with roman numerals as shown here.

ples were digested with 0.001 U of endo H (BoehringerMannheim) for 2 h at room temperature, and SDS-gel samplebuffer was added (12). Samples were loaded on SDS-10 or12% polyacrylamide gels and run for 2 h (12). Some gelswere then stained with Coomassie blue. All gels were thenimmersed in a soaking solution (60% methanol, 3% glycerol,37% H20) for 10 min, dried under vacuum for 1 to 1.5 h, andexposed to Kodak XARS autoradiographic film at -70°C.

RESULTS

Effects of bleomycin on rate of spheroplast formation. Weconfirmed the cell wall effects of bleomycin treatment byassessing rates of spheroplast formation. Cells were incu-bated overnight in growth medium in the presence or ab-sence of bleomycin (25 ,ug/ml), washed, and converted tospheroplasts. Drug-treated and control cells were harvested,washed, and treated with 10 mM DTT to increase cell wallpermeability or treated with water. The cells were washedagain and incubated with a lytic enzyme mixture containingp1 -- 3 glucanase (Lyticase) to disrupt the cell wall (10).Table 1 shows that incubation in the presence of the drugincreased the rate of spheroplast formation at least 10-fold.Bleomycin facilitation of spheroplast formation was alsoobserved when the cells were treated with bleomycin for 1 h

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TABLE 1. Rates of spheroplast formation for controland bleomycin-treated yeast ceilsa

Pretreatment Bleomycin Lysis timetreatment

Water 3-4 h+ 20-30 min

DTI- 3-4 h+ 15-25 min

a Cells were incubated for 17 h with or without bleomycin (25 ,ug/ml) inYPAD medium, washed, and incubated for 30 min in deionized water or 10mM DTT at 30'C. The cells were then washed and incubated with Lyticase.Formation of spheroplasts was monitored microscopically.

in the presence of the protein synthesis inhibitor cyclohex-imide (10 ,ug/ml; data not shown). This implies that bleomy-cin-induced facilitation of spheroplast formation is not de-pendent on de novo protein synthesis and therefore is notdependent on drug-induced DNA damage.

Bleomycin treatment of cell walls. If bleomycin acts di-rectly on cell walls, it should induce changes in wall struc-ture in a cell-free system. We therefore tested for bleomycin-mediated release of mannoproteins from isolated cell walls.Intact cells were surface labelled with 125I (29), and the cellwalls were isolated. The walls from 107 cells were treated for17 h with bleomycin (25 or 100 ,Ig/ml) in 1 ml of deionizedwater. After centrifugation, the supematants were saved foranalysis (fraction I of Fig. 1). The cells were then incubatedfor 30 min in DTT-EDTA buffer at pH 7.4, washed withsorbitol-citrate-EDTA buffer, and then incubated for 30 minat 30°C with Lyticase. Bleomycin caused the release of anincreased amount of labelled material in each of the treat-ment supernatants. The supernatants represent fractions I,II, and III of Fig. 1, as labelled (Table 2). Similar resultswere seen after bleomycin treatments of cell walls labelledwith [3H]leucine (data not shown).The wall material extracted by Lyticase digestion (fraction

III) was analyzed by gel electrophoresis (Fig. 2A). The samenumber of counts per minute was applied in each lane (theextract from cell walls not treated with bleomycin representsextract from 6.8 times more material; Table 1). No discretebands were visible from these untreated walls. Therefore,most of the 1"I in the sample from untreated walls must havebeen associated with material that did not enter the gel. Thisresult was consistently observed in several electrophoreses.The nature of this material released from control cells is not

TABLE 2. Release of 1"I-labelled material from controland bleomycin-treated cell wallsa

MaterialFraction Bleomycin releasedtreatment (10-5 cpm/cell)

Treatment supernatant (I) - 523+ 2,558

DTT-EDTA supernatant (II) - 650+ 1,118

Lyticase supernatant (III) - 3,504+ 23,756

a Cell walls were isolated from 11I-labelled cells and treated with orwithout bleomycin (25 ,ug/ml) for 17 h in deionized water at 107 cells per ml.The walls were then subjected to the protocol for spheroplast formationdescribed in Materials and Methods. The radioactivity released into eachsupeematant wash was determined.bRoman numerals refer to the fractions shown in Fig. 1.

A B

Bleomycin

110-84-

47-

33.-

24-

10MM$

FIG. 2. '25I-labelled material released from isolated cell walls."MI-labelled cell walls from 107 cells were treated with bleomycin at25 or 100 ,ug/ml for 17 h in 1 ml of deionized water, washed, treatedwith DTT, and digested with Lyticase. The Lyticase-solubilizedmaterial and the cell wall residue were analyzed by polyacrylamidegel electrophoresis. (A) The material solubilized by Lyticase (frac-tion III) was digested with endo H before loading of equal amountsof radioactivity on the gel. (B) Samples of the residues fromLyticase treatment (fraction IV; 50,000 cpm each) were extractedwith hot SDS, digested with endo H, and analyzed. Positions ofstandard proteins are marked in kilodaltons.

known. On the other hand, bleomycin-treated walls releasedsubstantial amounts of labelled material that formed diffusebands on the gel. Such heterogeneity is typical of cell wallmannoproteins (29). Therefore, bleomycin-treated cells musthave released 12I-labelled material in addition to that re-leased from controls.To confirm bleomycin-mediated changes in anchorage of

cell wall mannoproteins, we analyzed the cell wall residues.Control and bleomycin-treated, "sI-labelled cell walls thathad been treated with DTT and Lyticase were extracted withhot SDS (fraction IV of Fig. 1), and the released material wasrun on polyacrylamide gels. The cell wall samples containedequal amounts of radioactivity before extraction. The auto-radiographs of the gels revealed that bleomycin-treated cellwalls released highly heterogeneous material characteristicof heavily glycosylated cell wall mannoproteins. As in Fig.2A, little resolvable 1"I-labelled material was released fromcell walls not exposed to bleomycin (Fig. 2B). Therefore,analyses of both enzymatic extracts of cell walls and SDSextracts of the insoluble pellets showed that bleomycintreatment facilitated solubilization of cell wall glycoproteins.

Cell wall effects of bleomycin treatment of intact cells. Todetermine whether bleomycin affects the walls of intactcells, experiments similar to those done with isolated cellwalls were carried out. Intact cells were incubated in growthmedium with or without bleomycin (25 ,ug/ml, 17 h) andsubjected to a standard protocol for making spheroplasts.

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TABLE 3. Protein released from bleomycin-treatedand control cells

Solubiization Bleomycin Protein releasedmediuma treatment (10-12 g/cell)

DTT (II)b 0.03+ 0.48

Zymolyase (III) 0.34c+ 0.55

a Cells were grown in YPAD medium for 17 h with or without bleomycin (25SLg/ml), washed, and successively incubated with 20mM DTI and Zymolyase(3 mg/ml). The protein contents of triplicate samples of the supernatantsolutions from the incubations were determined with bicinchoninic acidreagent.

b Roman numerals refer to the fractions shown in Fig. 1.c Digested with Zymolyase for 3.5 h.d Digested with Zymolyase for 25 min.

The cells were digested with Zymolyase until spheroplastswere produced. The supematants from the DTT treatments(fraction II of Fig. 1) and the Zymolyase digestions (fractionIII) were analyzed for proteins. Bleomycin-treated cellsreleased 16 times as much protein into the DTT solution asdid untreated cells (Table 3, top). Zymolyase solubilized lessmaterial from control cells than from bleomycin-treatedcells. This difference in the amount of material releasedoccurred despite a period of enzyme digestion of untreatedcells eight times as long as that for bleomycin-treated cells.Note that glucanases do not dissolve the entire cell wall butdigest holes big enough for the spheroplast to escape (1).Therefore, the enzymes normally do not solubilize all of themannoproteins in the cell wall.The material released into the growth medium (fraction I

in Fig. 1) was analyzed by SDS-gel electrophoresis andstained with periodic acid dansyl Schiff reagent, which isspecific for saccharides containing cis-hydroxyls (6). Man-noproteins contain cis-hydroxyl groups in the mannoseresidues and are found primarily in cell walls (2, 5). Whilemannoproteins were evident in the supernatants from bleo-mycin-treated cells, no staining was visible in supernatantsfrom cells that had not been treated with bleomycin (data notshown).While the enhanced release of proteins and mannoproteins

was consistent with bleomycin-mediated cell wall damage,the release could also be due to drug-induced membranedamage and leakage of intracellular proteins. We thereforemonitored for bleomycin-mediated release of cell wall man-noproteins. Yeast cells were grown to the mid-logarithmicphase, washed, and surface labelled with "2I (29). Thelabelled cells were then treated with or without bleomycinunder nongrowing conditions (25 Lg/ml, 17 h at 30°C indeionized water at 107 cells per ml in the presence or absenceof cycloheximide [10 ,ug/ml]) and treated with DTT andLyticase as before. Polyacrylamide gel analysis of the SDS-extractable materials remaining in the cell wall after Lyticasedigestion revealed label in poorly resolved bands, which ischaracteristic of cell wall mannoproteins (Fig. 3, four leftlanes). In contrast, proteins that stained with Coomassieblue showed sharp bands, demonstrating that other proteinswere not labelled and that the gel had resolved proteins well(right lanes). The Coomassie blue staining pattern was notvisibly different in cellular extracts treated with or withoutendo H (data not shown). Densitometry of the gel revealedconsistent differences in the pattern of "2I-labelled bandsbetween the control and bleomycin-treated lanes (Fig. 4).Specifically, walls from bleomycin-treated cells showed sub-

BleomycinCycloheximide

110-84-

47.-

33-

24-

18

-- + + -

+ - + -

- + +

+ - +

Autoradiogram Coomassie BlueFIG. 3. Polyacrylamide gel analysis of SDS-soluble proteins

from Lyticase-digested cell pellets (fraction IV). Surface '21I-la-belled cells (107/ml) were treated with or without bleomycin (25,ug/ml, 17 h), and the cell walls were digested with DTT and Lyticasebefore SDS extraction of the proteins and deglycosylation with endoH. The four left-hand lanes show the autoradiogram, and the four onthe right show Coomassie blue staining of the same gel. Positions ofstandard proteins are marked in kilodaltons.

stantially more material soluble in hot SDS buffer than didthe controls for bands with apparent molecular weights of80,000 (B), 31,500 (E), and 21,000 (F). Control cells releasedmore material in bands with apparent molecular weights of96,000 (A), 61,000 (C), and 41,000 (D). In contrast, thedifferences in the high-molecular-weight region (>100,000)were not reproducible. The differences in peaks A through Fwere similar when the cells had been treated with bleomycinin the presence of cycloheximide (10 ,ug/ml) (compare lanes2 and 4 of Fig. 3 with lanes 1 and 3; also, Fig. 4). Compar-isons of solubilization at different times of digestion withLyticase showed that bleomycin-treated cells released sur-face-labelled proteins more rapidly than did untreated cells(data not shown). Thus, there appeared to be quantitativedifferences in the anchorage of several species of cell wallmannoproteins in bleomycin-treated cells compared withcontrols. Similar changes in the abundance of these labelledproteins were seen in two other independent labelling andtreatment experiments (data not shown).These changes in abundance of Lyticase-released cell wall

mannoproteins appear to be due to bleomycin facilitation ofrelease of these species of mannoproteins into the treatmentsupernatants. This interpretation was confirmed in an exper-iment which demonstrated DTT-mediated release of radio-labelled cell wall proteins. Cells were surface labelled with1"I (29) and treated for 10 min with bleomycin at 0, 5, 15, or50 ,ug/ml. The cells were then subjected to the same protocolas before the spheroplast formation. SDS-extractable la-belled proteins remaining in the cell pellets after Lyticasedigestion were analyzed (fraction IV). This experiment wasused to confirm that the labelled proteins were mannopro-teins, since they were susceptible to digestion with endo H(Fig. 5, lanes 1 and 2 versus 3 and 4). Even the brief (10-min)exposure to bleomycin apparently caused release of the

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FIG. 4. Densitometer traces of the autoradiogram shown in Fig.3. Peaks with significant, reproducible differences are marked A

through F. Molecular masses are marked in kilodaltons.

61-kDa mannoprotein into the DTT fraction (Fig. 5, lanes 4

to 6). The 61-kDa mannoprotein remained wall associated inthe control incubated without bleomycin (lane 3) and waspresent in the residue of bleomycin-treated cells that werenot pretreated with DTT solution (lanes 8 to 10). Therefore,bleomycin facilitated DTI-mediated release of this cell wallmannoprotein.

DISCUSSION

We have demonstrated that bleomycin affects yeast cellwalls in a consistent and reproducible manner. The effectson the cell wall compromise the integrity of the walls, asshown by the ease of spheroplast formation and the presenceof visible lesions in thin-section electron micrographs (18).

Bleomycin - 50 0 50 15 5 0 50 15 5DTT - + +++ +

1

Endo H - - + + + + + + +

205--l|ii

116 -_ __- i97__*l-

66- III*_

45>tS-III*

FIG. 5. Mannoproteins from Lyticase extracts of cells following10 min of treatment with bleomycin. Surface 12 I-labelled cells(1071ml) were treated briefly with bleomycin at the indicated con-centrations (in micrograms per milliliter) and digested for 30 minwith Lyticase. The samples in lanes 2 to 6 from the left wereincubated with DTT to release disulfide-bonded and entrappedmannoproteins before Lyticase treatment. The insoluble pelletswere extracted with SDS (fraction IV) and treated with or withoutendo H, as indicated, and 20,000 cpm was applied to each lane onthe gel. Molecular masses are marked in kilodaltons.

Furthermore, bleomycin is efficiently localized within thecell wall matrix within minutes after cells are exposed to thedrug (18). The present study complements and extends theelectron microscopic findings. Bleomycin-mediated damagewas seen at lower bleomycin doses and shorter exposuretimes, and we have identified mannoproteins as cell wallcomponents affected by the drug. Furthermore, we foundthat the cell wall effects of bleomycin occur in isolated cellwalls, so that drug-mediated wall damage is independent ofDNA damage and genetic effects.Bleomycin-induced cell wall changes appear to be medi-

ated by action of the drug directly on cell wall polymers oron the extracellular enzymes of cell wall metabolism. Thesimilarity of results in the presence and absence of cyclo-heximide, the appearance of cell wall effects after only 10min of treatment, and the similarity of effects in intact cellsand isolated cell walls are consistent with direct drug-induced damage and inconsistent with a mechanism depen-dent on expression of bleomycin-damaged genes or otherintracellular mediators. The cell wall activity ofbleomycin isnot dependent on the action of added lytic enzymes, sincetreatment with the drug alone increased solubilization ofhI-labelled cell wall mannoproteins (Table 2, fraction I) andof [ sH]Leu-labelled cell wall components.The cell wall changes due to bleomycin appear to include

release of cell wall mannoproteins during bleomycin treat-ment as well as with chemical and enzymatic digestion.These findings imply that damage is accompanied bychanges in anchorage of cell wall mannoproteins. An outerlayer of mannoproteins is the major permeability barrier inthe cell wall and is covalently bonded to the glucan matrix ofthe wall (4, 5, 28, 29). Bleomycin-induced release of manno-proteins would lead to increases in cell wall permeability byremoving some of the outer layer of the cell wall (29) andexposing the cell wall matrix of mannoproteins and glucans.Critical disulfides and glycosidic bonds would therefore bemore accessible to reduction by DTT and digestion withglucanase, respectively (Table 2 and Fig. 2 to 5). Such anincrease in permeability would also facilitate glucanase

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digestion of the cell walls (29) and lead to the observedquantitative differences in enzymic release of cell wallcomponents (Table 2 and Fig. 2) (4, 20, 21, 27). Suchbleomycin-facilitated solubilization of mannoproteins an-chored to the outer layer of the wall would also explain theresults shown in Fig. 2 to 5 and Tables 2 and 3. IneAperiments on cells or walls not treated with bleomycin,1"I-labelled mannoproteins remained associated with theinsoluble matrix of the cell wall, even after digestion withLyticase or Zymolyase. In contrast, some of the mannopro-teins were found in treatment supernatants (fractions I, II,and III) of bleomycin-treated cells or walls.The mode of action of bleomycin on DNA suggests that

bleomycin might cause breaks in cell wall polysaccharidesthat anchor external layer mannoproteins (4, 5, 28, 29). Suchbreaks would be analogous to the strand breaks that the drugcauses in DNA by oxidizing the deoxyribose residues (3, 24).It is consistent with such a mechanism that phosphorylatedcompounds are reported to potentiate bleomycin-inducedbreakage ofDNA in vitro. Like DNA, cell wall mannans arehighly phosphorylated (1) and the phosphoryl sugars may beimportant for structural integrity of the wall (9, 13).These results suggest that bleomycin represents a previ-

ously unknown class of potential antifungal agents. We arenot aware of any other drug or treatment regimen that hassimilar effects on the rate of spheroplast formation in S.cerevisiae or cell wall integrity. The drug doses and treat-ment times necessary to induce these cell wall changes are inthe same range as those needed to induce DNA damage andare within the therapeutically useful range for bleomycin asan antitumor drug (14).

ACKNOWLEDGMENTS

We thank Robert Del Vallee for advice and assistance.Support for these studies came from the Minority Access to

Research Careers program of NIGMS (R.B.), a Eugene LangFellowship (S.T.L.), and the Minority Biomedical Research Pro-gram of the NIH (J.-A.S. and E.J.). Additional support from theAaron Diamond Foundation and the NSF to C.W.M. and from theNIGMS and the Professional Staff Congress of CUNY to P.L. isgratefully acknowledged.

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