Chem.-Biol. Interactions, 51 (1984) 233--246 233 Elsevier Scientific Publishers Ireland Ltd.

THE EFFECT OF BLEOMYCIN ON DNA IN ESCHERICHIA COLI K12 CELLS

KAZUO YAMAMOTO* and FRANKLIN HUTCHINSON**

Yale University, Department of Molecular Biophysics and Biochemistry, New Haven, CT (U.S.A.)

(Received December 5th, 1983) (Revision received June 14th, 1984) (Accepted June 16th, 1984)

SUMMARY

In Escherichia coli cells treated to reduce colony-forming ability to about 10%, bleomycin causes fewer than six randomly located DNA single-strand breaks or three double-strand breaks per genome. This is many fewer than produced by strand-breaking agents such as ionizing radiations in cells with similar loss of colony-forming ability. Bleomycin treatment to this level of colony-forming ability does affect the intracellular DNA, as shown by a change in the sedimentation rate of the chromosomal structure found in lysates made with sodium dodecyl sulfate. Bleomycin may act on only a limited part of the chromosome of such cells, perhaps the part associated with the outer cell membrane, or it may make strand breaks that are less repairable than those formed by ionizing radiations. Extensive DNA de- gradation in heavily treated cells (colony-forming ability 1% or less) could be from the action on DNA of bleomycin entering freely through membranes which are no longer intact, or from enzymatic degradation in heavily damaged cells.

Key words: Bleomycin -- DNA strand breaks --Escherichia coli

INTRODUCTION

The anti tumor drug bleomycin inhibits the ability of bacterial or cultured mammalian cells to form colonies. It has been reported frequently that the DNA is degraded in treated E. coli cells [1--3] and treated mammalian cells

*Permanent address: Kobe University School of Medicine, Department of Radiation Biophysics, Chuo-ku, Kobe 650, Japan. **To whom correspondence should be sent.

0009-2797/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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[1 ,4--14] . Commonly, it is presumed that this degradation causes the loss of colony-forming ability.

This presumption has led to extensive studies of the action of bleomycin on DNA in solution (reviews: Refs. 15--17). There are two predominant reactions. In one, an unaltered base is released, leaving an unbroken chain, but a site in the DNA which might be acted on by the same enzymes which act on AP (apurinic or apyrimidinic) sites created by depurination or glycosylases. In the other, the base released carries the sugar fragment - -CH=CH--CHO attached to the N1 position [18] ; in the DNA there is a single-strand break with a 5'-phosphate end group and glycolic acid, --CH2-- COOH, attached to the 3'-phosphate [19] . These two reactions occur with comparable frequency. About 10% of the time, single-strand breaks form in both strands of the double helix, producing a double-strand break [20 ,21] .

There are reports that bleomycin does not act on RNA in solution [22-- 25] . A few experiments show that high concentrations of bleomycin can affect specific enzyme reactions under certain conditions [26,27] ; however, these experiments really do not offer any support for the hypothesis that bleomycin could affect cells by direct action on enzymes.

All these results are in accord with the notion that bleomycin affects cells by its action on DNA. We were surprised, therefore, when preliminary experiments showed minimal DNA damage in E. coli cells treated with bleomycin to colony-forming levels of 10--20% of controls.

Our experiments differed from those of previous workers, who (except Refs. 10 and 28, see Discussion) measured strand breaks in heavily treated cells with greatly reduced ability to form colonies. They did this for under- standable reasons. Strand-breaking agents such as X-rays reduce cell colony- forming ability to low levels at exposures which form rather few strand breaks per genome. For cells treated to 10% colony-forming ability, special techniques must be used in the DNA extraction to avoid inducing more strand breaks by enzymatic action and by viscous shearing forces than the number made by the strand-breaking agent. Thus, most workers have used heavily treated cells and implicitly assumed that the number of breaks in less heavily treated cells would be proport ionally smaller.

In this paper are reported experiments which show, in E. coli cells treated with bleomycin to reduce colony-forming ability to 10%, much less randomly distributed DNA damage than implied by most previous work.

Sedimentation of large DNA The experiments described in this paper involve sedimentation of very

large DNA. To interpret the data, it is necessary to consider certain anomalies in the sedimentation properties of such large molecules.

Zimm and coworkers [29,30] have developed a theory for the change in shape of a very long linear flexible molecule caused to move through a viscous medium by centrifugal forces. If the molecule moves fast enough, the normal random coil form becomes distorted to a different shape with a smaller sedimentation coefficient. Thus, the coefficient decreases with increased centrifuge speed, as has been verified by experiment [31--33] .

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This distortion affects the dependence on DNA length of the sedimentation coefficient at a fixed centrifuge speed. For short molecules, the random coil shape in no t appreciably distorted at usual sedimentation rates. As the length increases, the molecule moves faster because the mass is greater, and the random coil is more easily distorted because it is more extended in space. The theory shows that the sedimentation coefficient first increases less with increasing length than expected from results with shorter molecules, and then reaches a limiting sedimentation rate which is never exceeded even for very long molecules.

The existence of this limit has been confirmed by experiment [34 ,35] . For single-strand DNA sedimented in a Spinco SW50. 1 rotor at 26 000 rev./min, the limiting coefficient is that which would be expected for a molecule of mol. wt. 5 × l 0 s centrifuged so slowly that it remains an un- distorted random coil. The more rigid double-strand DNA makes a more extended random coil in solution which distorts more readily, and its limiting sedimentation coefficient at 26 000 rev./min corresponds to DNA of mol. wt. 2.8 × 108 . The random coil is less distorted at very low centrifuge speeds, and the limiting sedimentation coefficient at 3700 rev./min corresponds to a molecular weight of 2 X 109 for double helical DNA. (This speed corresponds to a sedimentation time of about 48 h; slower speeds would require longer time, but for longer times diffusion causes unacceptably large changes in the sucrose gradients.)

The numerical values given apply only to linear DNA. DNA in a more compact form will have its shape distorted by motion through a viscous medium only at higher speeds.

MATERIALS AND METHODS

Bacterial strains The experiments were done with E. coil K12 AB2497 thyA arg his leu

pro thr.

Media K medium (M9 buffer + 1% glucose + decolorized, vitamin-free 1%

Casamino acids) containing 10 ug/ml thymine was used for cell growth. M9 buffer used in this work was 18.7 mM NH4C1, 41.5 mM Na2HPO4, 22 mM KH:PO4, 1 mM MgSO4.7 H20, 0.1 mM CaC12 and 0.1 ~g/ml thiamine. Lambda buffer was: 0.03 M Tris (hydroxymethyl) aminomethane-HCl, (pH 7.2) containing 0.05 M MgSO4.7 H20 and 0.25 g/1 gelatin. L agar plates contained: 1% tryptone, 0.5% yeast extract, 0.05% NaCl, adjusted to pH 7.0 and 1% Difco bactoagar.

Bleomycin treatment Bleomycin A2 (Lot 3~), the generous gift of the Developmental Thera-

peutics Program in Chemotherapy, NCI, was dissolved in double-distilled water (2 mg/ml) and kept frozen at - 2 0 ° C in small quantities until used. Cells to be tested were grown in K medium and, while still in exponential

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growth, were washed at 4°C in M9 buffer by low speed centrifugation. They were resuspended in K medium or M9 buffer and held on ice until used. Bleomycin was added to the desired level and the culture incubated 10 min at 37°C.

In some cases, the culture was then immediately diluted 10Ofold by pipetting into K medium (or M9 buffer); tests showed that this dilution was adequate to stop any further action of bleomycin in the experiments in which this technique was used. In certain experiments, (specified in the text) the cells were washed by three low-speed centrifugations in the cold with M9 buffer, and then resuspended in the appropriate medium.

Radioactive labeling Cell DNA was labeled radioactively by growing in K medium containing

either [methylA4C]thymine (New England Nuclear) at 3 ug/ml (0.5--1.2 uCi/ml) or [methyl-3H]thymine (New England Nuclear) at 5 ug/ml (12 u Ci/ml) unless otherwise stated. After labeling, the unincorporated label was washed out by three low-speed centrifugations in M9 buffer at 4°C. Radio- activity was determined by adding a 0.4-ml sample to 4 ml of toluene- based F963 scintillant (New England Nuclear) and counting in a liquid scintillation counter.

Alkaline sucrose gradient sedimentation Linear 5--20% (w/v) sucrose gradients were prepared, containing 0.8 M

NaC1, 0.2 M NaOH, 4.5 mM EDTA, total volume 4.8 ml. A 0.1-ml volume containing 0.5 M NaOH, 0.5% (w/v) Sarkosyl (Sigma), 10 mM EDTA, was layered on, followed by 0.05 ml of cells at 2 × 107/ml which had been washed (to reduce the bleomycin concentrat ion) by low-speed centrifugation and resuspended in 50 mM Tris, 0.2 mM EDTA (pH 7.6). The gradients were held 30 min at room temperature (about 20°C), then centrifuged for 2 h at 26 000 rev./min in a Beckman SW50.1 rotor at 20°C. About 30 equal volume fractions per gradient were collected, through a hole pierced in the bo t tom of the centrifuge tube, into a minivial containing 0.3 ml acid. Liquid scintillant was added and the samples counted.

Bleomycin must effectively be removed from the medium before lysis; if it is not, erratic DNA degradation is seen which apparently is caused by the action of bleomycin on DNA during the lysis process. Bleomycin is known to be active at quite alkaline pH [36] .

Neutral sucrose gradient sedimentation The technique has been described previously in detail [37] . Briefly, the

treated cells were washed to reduce the bleomycin concentrat ion and at 107/ml were converted to spheroplasts by incubating 10 min at ice temper- ature in 50 mM Tris, 2 mM EDTA, 200 ug/ml egg white lysozyme (Cal- biochem). A volume of 0.1 ml was gently layered on a 5--20% (w/v) sucrose gradient (total volume 4.8 ml) containing 5 mM Tris, 1 mM sodium citrate, 10 mM NaC1, 1 mM EDTA, 0.5% (w/v) sodium dodecyl sulfate and a satura-

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tion level (at room temperature) of chloroform. After a waiting period of 90 min at room temperature, the gradients were centrifuged at 20°C in an SW50.1 rotor. The gradients were fractionated and counted as described for alkaline gradients. DNA mass M was estimated assuming its sedimentation coefficient varies as M ~'3~ and taking T2 DNA (used as a marker) to have a mol. wt. of 110 × 106. The ability of these gradients to free the DNA from cellular debris is discussed in detail elsewhere [37 ].

RESULTS

DNA single-strand breaks DNA from cells treated for 10 min with bleomycin to 10--20% colony-

forming ability sediments on alkaline sucrose gradients (filled circles, Fig. 1) in a way indistinguishable from DNA from untreated cells (open circles). The sharp peak near fraction 10 is the limiting sedimentation rate for linear single-strand DNA of molecular weight 5 X l 0 s or greater (see section on

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Froction Fig. 1. Alkaline sucrose sedimentation in an SW50. I rotor of DNA from E. coli AB2497 cells treated with bleomycin for 10 min at 37°C. Sedimentation was at 26 000 rev./min for 2 h at 20°C. ~ ~, untreated controls;• • , cells treated with 2 ~g/ml bleomycin, colony forming ability 20%; +--- - - -+, cells treated with 10 ~g/ml bleomycin, colony- forming ability 3 % ; - - --, expected sedimentation pattern for DNA from E. coil AB2497 cells given 22 kilorads of gamma rays, colony-forming ability 20% of controls, interpolated from previous results [37]. The sharp asymmetrical peak at fraction 10 is an artifact caused by very large DNA (see text), and contains single-strand DNA ofmol , wt. > 5 × 108.

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DNA sedimentation). Thus, fewer than six random single-strand breaks (and alkali-labile bonds) per genome of 2.5 × 109 are produced by bleomycin t reatment which reduces colony-forming ability 10-fold. In contrast, another agent known to produce random single-strand breaks, gamma rays, induces many breaks for the same survival (Fig. 1, dashed line). It could be asked if bleomycin acts slowly and produces strand breaks of alkali-labile bonds after the 10 rain t reatment period. In one set of experiments, the treatment period was increased to 25 min, only a little less than the cell doubling time of 30--35 min. As long as the cell colony-forming ability after treat- ment was greater than about 10%, the DNA sedimented in alkaline conditions in a way indistinguishable from untreated controls (data not shown).

In other experiments, cells were treated for 10 min, then the bleomycin concentration was greatly reduced either by centrifugation or by Millipore filtration or by massive dilution. After 15--60 min of further incubation, the cells were lysed and sedimented on alkaline sucrose gradients as in Fig. 1. Some rather variable degradation was found, with the time course depending, for example, on the method by which the bleomycin was removed (data not shown). It could not be established whether the degradation was from delayed action of bleomycin, or from various metabolic effects occurring in cells which had already lost the ability to form colonies.

When cells were treated with bleomycin to 1% or less colony-forming ability, large numbers of DNA single-strand breaks were found (crosses in Fig. i) .

DNA sedimentation in neutral gradients Figure 2 shows neutral sedimentation of DNA from E. coli cells, with and

wi thout bleomycin treatment, which have been lysed with 0.5% sodium dodecyl sulfate. The peak at about fraction 16 is the limiting sedimentation coefficient for linear DNA corresponding to DNA of mol. wt. 2 × 109 (see section on DNA sedimentation). The effect of bleomycin t reatment is to reduce the quanti ty of fast-sedimenting DNA in some form more compact than a long flexible chain, and increase the amount sedimenting at the limit for linear DNA.

A similar e f fec t has beeen seen by Pellon et al. [28] for DNA from cells treated with 1 ug/ml bleomycin. (From their data it is not possible to deter- mine the colony-forming ability of the cells, but it certainly must have been >10%.) They extracted nucleoids from the treated cells, then unfolded the nucleoids by t reatment with RNase. Such unfolded nucleoids would be expected to sediment much as the structures in Fig. 2 from cells lysed with sodium dodecyl sulfate. Pellon et al. [28] only give mean sedimentation coefficients, but the decrease of 15--20% with bleomycin t reatment is consisitent with the data in Fig. 2. Thus, both Fig. 2 and Pellon et al. [28] show a change in the sedimentation of a DNA structure which is caused by treatment of E. coli cells with bleomycin.

A study of the same strain o fE . coli cells irradiated with gamma rays [37] showed that doses making two DNA double-strand breaks per genome cause

239

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Fraction Fig. 2. Ten to 25% neu t ra l sucrose grad ien ts of DNA f rom E. coli A B 2 4 9 7 cells t r ea ted wi th b l e o m y c i n for 10 min at 37°C. The gradients were cen t r i fuged in an SW50. 1 ro to r for 43 h at 3700 rev . /min, at 20°C. : , c, u n t r e a t e d con t ro l s ; * *, t r e a t ed wi th 5 ug /ml b leomyc in , 10% co lony- fo rming abi l i ty ; + +, t r e a t ed wi th 10 ug /ml bleo- mycin , 1% co lony - fo rming abi l i ty ; . . . . , e x p e c t e d s e d i m e n t a t i o n p a t t e r n of DNA f rom cells given 30 krad of gamma rays (10% co lony- fo rming abi l i ty) , i n t e r p o l a t e d f rom previous resul ts [ 3 7 ] . The a r row shows the s e d i m e n t a t i o n pos i t ion for l inear DNA of mol . wt. > 2 × 109 (see text ) .

a disappearance of the fast-sedimenting component ; with higher levels of gamma irradiation, the peak at fraction 10 (Fig. 2) breaks up in the way expected of linear DNA of mass approx. 2 × 109 [37] . This would suggest that the DNA from cells treated with bleomycin to 10% colony-forming ability (filled circles, Fig. 2) had received less than three random double- strand breaks per genome. It could be argued that ionizing radiation might break up crosslinks in the fast-sedimenting structures which would not be affected by bleomycin. Thus, the DNA at fraction 10 in Fig. 2 might not be the linear molecules deduced from previous work [37] , but more com- plicated structures with broken DNA pieces held together. However, this does not satisfactorily explain the small number of single-strand breaks (Fig. 2), since there should be two single-strand breaks for each double- strand break.

Note that large numbers of double-strand breaks are found in cells with 1% colony-forming ability or less (crosses, Fig. 2).

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Cells treated so that colony-forming ability is between 1% and 10% sometimes showed DNA with extensive breakage in both alkaline and neutral sedimentation {i.e., crosses Fig. 1), sometimes DNA with little degradation, and sometimes bimodal distributions with some degraded and some undegraded DNA (data not shown). Tests showed that the strand breaks observed were not the results of bleomycin activity in the lysate after washing the cells {Materials and Methods). Rather, it is thought that the variability is related to the presence of differing levels of bleomycin- resistant cells in cultures, as shown by the flattening of the curves of log (colony-forming ability) vs. bleomycin concentration [3 ,38] . For example, in a culture with a high level of resistant cells, bleomycin treatment to a low level of colony-forming ability would amount to heavy t reatment of the sensitive cells, presumably with degradation of the DNA in those cells.

When E. coli spheroplasts are gently lysed with Brij 58, the DNA is released in compact nucleoids containing 70% DNA, 20% messenger RNA, and 10% protein {review: Ref. 39). Nucleoids from cells treated with bleomycin to 9% colony-forming ability sediment on neutral sucrose gradients in the same way as nucleoids from untreated cells (Fig. 3). By contrast, nucleoids from

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Fig. 3. Neutral sedimentation of nucleoids. E. coli AB2497 cells were treated with bleo- mycin for 10 rain at 37°C. Spheroplasts were lysed in the presence o f 0.4% sodium deoxychola te , 1% Brij 58 (Atlas Chemical Industries, Inc.) and 2% Sarkosyl, then sedi- m e n t e d in 10--30% neutral sucrose gradients. The procedure was that of Ulmer et al. [41 ], as modi f ied f rom the original of S tonington and Pet t i john [57 ]. The gradients were cent r i fuged in an SW50.1 rotor at 2600 rev. /min for 17 h at 4°C. z, un t rea ted contro ls ; • , cells t rea ted with 5 ug/ml bleomycin, 20% colony- forming abil i ty; +, cells t rea ted with 20 ug/ml b leomycin , 9% colony- forming abili ty.

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cells irradiated with X-rays to a comparable level of colony-forming ability sediment much less rapidly, presumably because of single-strand and double- strand breaks in the DNA 146,411.

6

Time after adding bleomycin, min

Fig. 4. DNA synthesis in E. coli after treatment with bleomycin. (A) AB2497 cells were grown exponentially for several generations in K medium plus 5 pg/ml [)H]thymine, 3 pCi/ml, at 37°C (doubling time, 35-40 min). At 4 x 10’ cells/ml, the culture was divided, bleomycin added to aliquots as indicated and colony-forming ability determined 10 min later. At various times, 0.2-ml samples from each aliquot were added to 0.2 ml 10% TCA in an ice bath. After 30 min, each sample was poured on a glass fiber filter, which was washed twice with 5 ml of cold 5% TCA and once with 5 ml of cold water, then soaked in 95% ethanol, dried and counted. 3-z untreated control; colony- forming ability at 10 min, 5.0 X lO’/ml (100%); l -, i rg/ml bleomycin added to culture, colony-forming ability at 10 min, 2.04 x 107/ml (41%); +- +, 50 pg/ml bleomycin added to culture; colony-forming ability at 10 min, 4.1 x 106/ml (8%). (B) AB2497 cells were grown in medium with no radioactivity, then starved for amino acids for 80 min to allow completion of a round of DNA synthesis. The culture was divided, and each aliquot incubated 25 min in K medium with amino acids (to permit protein synthesis), but no thymine. Bleomycin was added to one aliquot and after 5 min incu- bation, [‘Hlthymine at 5 Icg/ml, 3 rCi/ml, was added to each aliquot. At various times, 0.4 ml samples were taken and treated as for curve A. o- o, untreated controls. Colony-forming ability at 125 min, 4.5 x lO*/ml (100%); l -, 5 ug/ml bleomycin added to culture; colony-forming ability at 125 min, 1.7 X lO*/ml(38%)

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Other results The small amount of detectable DNA damage in cells treated so as to

reduce colony-forming ability to 10% was surprising, in light of the generally held view that bleomycin affects cells by acting on DNA. Therefore, the amount of DNA synthesis was measured as a function of time in cells treated with bleomycin A2. The rationale was that practically all types of DNA damage reduce the rate at which a polymerase copies a template. However, the results (Fig. 4) show no detectable differences in total DNA synthesis between cells whose colony-forming ability had been reduced to 10% and untreated controls. Cohen and I [42] have reported similar results for E. coli cells treated with bleomycin A5.

The prompt expression of some action of bleomycin is shown by the time course of the induction of lambda prophage. The ability of bleomycin to induce prophage has previously been cited as a reason for believing that bleomycin acts on intracellular DNA in E. coli [43,44].

AB2497 (~) lysogens treated with 5/~g/ml bleomycin for 10 min (colony- forming ability 29%) or with 40 J /m: of 254 nm light (colony-forming ability 24%) produced comparable numbers of phage bursts 45--55 min after the end of either t reatment (data not shown). This is the burst time for AB2497 cells infected with the same phage and incubated under the same conditions. Thus, whatever bleomycin does to induce prophage must occur shortly after the 10-min t reatment at the latest.

DISCUSSION

Action of bleomycin on E. coli cells with 10--100% colony-forming ability The results given in Fig. 2 and those of Pellon et al. [28] , both show that

bleomycin affects some structure in E. coli cells which contains DNA. The chief surprise is that the effect on DNA seems very small compared to that induced by other agents, e.g. X-rays, which also induce DNA lesions such as strand breaks and AP sites.

We will not consider here the possibility that bleomycin acts on a cell component other than DNA, for reasons given in the Introduction.

If bleomycin can act only on a very limited part of the genome, the large DNA molecules from treated cells shown in Figs. 1 and 2 could be readily explained and the unaltered sedimentation of the intact nucleoid (Fig. 3) would be plausible. For example, bleomycin might act only on that part of the genome associated with the outer cell membrane of E. coli [45--47]. At the limited sites of attack, damage to the DNA could be con- siderable, but still give DNA which would sediment as shown in Figs. 1 -3 . This assumption is attractive because it explains how bleomycin could affect intracellular DNA without involving a mechanism to get the bulky drug molecule into the bacterial cell.

Some support for the concept that bleomycin has limited access to the bacterial genome can be derived from data on the mutagenic effect of bleomycin on bacteria. Bleomycin does not mutate several his loci on the

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chromosome of Salmonella thyphimurium, a bacterium closely related to E. coli that is used in the Ames test for mutagenic compounds [48--50]; bleomycin does mutate his G428 on a multicopy plasmid, but not if his G428 is on the chromosome [51]. From our point of view, extensive but localized damage to chromosomal DNA results in loss of colony-forming ability in most cells in which the his locus has been affected; only for a locus on a non-essential plasmid is a detectable mutat ion formed. Mutagenesis by bleo- mycin of the Salmonella trp E8 locus (presumably on the chromosome) has been reported [52] , but only for mutagenesis of cells on a agar surface; no mutagenesis by bleomycin could be detected for cells treated in liquid medium.

Strand breaks induced by bleomycin that are unusually difficult to repair also could explain the data in Figs. 1--3 and the mutagenesis results stated in the previous paragraph. The typical bleomycin-induced single-strand break comes from oxidative attack on a sugar [19,53] and appears very similar to the single-strand breaks induced by ionizing radiations [54,55] which are known to be readily repairable. A more likely candidate would be the bleomycin-induced double-strand break, for which the structure is not known.

Intracellular strand breakage induced by bleomycin may require exposure to the drug for times longer than 25 min; however, this is not true for breaks induced in DNA in solution, and the prompt induction of lambda prophage shows that at least one cellular effect of bleomycin is expressed within minutes.

The lack of an effect of bleomycin on DNA synthesis {Fig. 4 and Ref. 42) does not follow in any simple way from the assumption of bleomycin attack on only a limited part of the chromosome, or of a non-repairable strand break. While ad hoc assumptions can explain the resuts -- e.g., continual reinitiation of DNA synthesis at the origin of replication -- the lack of an effect on DNA synthesis must be considered a puzzle at present.

DNA degradation in heavily treated cells Our results here are in agreement with those of other workers [1--14].

There is no evidence as to whether the degradation is the result of entry of bleomycin into cells through membranes which are no longer intact, from the action of nucleases in nonreproducing cells, or from other causes. It is not known whether this degradation is a consequence of the loss of colony- forming ability or vice versa.

Relevance to the effect of bleomycin on mammalian cells Iqbal et al. [10] measured DNA damage by the sensitive alkaline elution

technique in bleomycin-treated mammalian cells with 10--20% colony- forming ability. They concluded that their results were best interpreted as showing extensive DNA degradation in some cells, but no measureable degradation in the DNA from other cells; the apparent number of cells with undegraded DNA was much larger than the number able to form colonies, so some cells which had lost the ability to form colonies also contained DNA

244

without extensive degradation. Thus, their results for mammalian cells are comparable with ours for E. coli cells.

Fujimoto, using radioactive bleomycin and radioautography, found that the drug bound to the nuclear membrane in mammalian cells [56]. There were no detectable levels within the nucleoplasm [56], suggesting that bleo- mycin might not penetrate the nuclear membrane.

ACKNOWLEDGEMENTS

The authors acknowledge with gratitude the expert technical assistance and advice of Ms. Judith Stein, the assistance of Mr. Amitabh Chak who did the experiments shown in Fig. 2, the gift of bleomycin A2 by the Develop- mental Therapeutics Program in Chemotherapy, N.C.I. and the help of Mrs. Estelle Mackinnon in preparing the manuscript. This research was supported by Grant CA 17838 from the National Cancer Institute, Department of Health and Human Services.

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