[CANCER RESEARCH 48, 6837-6843, December 1, 1988]
Internucleosomal Cleavage and Chromosomal Degradation by Bleomycin and
Phleomycin in Yeast1
C. W. Moore2
Department of Radiation Biology and Biophysics, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642; and Department of Microbiology,
Medical School, and Biochemistry and Biology Graduate Programs, City University of New York, New York, New York 10031
ABSTRACT
Native chromosomal structure, breakage, and overall degradation were
studied following the reaction of whole cells with the anticancer drug
bleomycin and structurally related phleomycin. Electrophoretic analyses
of cellular DNA established that phleomycin was more reactive with
DNA than equimolar bleomycin in the range of 0.67-33 x IO"*1M,
produced an optimally visible, though less-extended, oligonucleosomal
series at concentrations 12 to 35 times lower than bleomycin, and
degraded DNA within nucleosomes. Chromosomes were cleaved into
nucleosomes and degraded by phleomycin over substantially narrower
dose ranges (1 to 2 x 10"* M) than by bleomycin (about 1 to 17 x 10~*
M). Bleomycin exhibited higher specificity for internucleosomal cleavage
than phleomycin, and trimmed but did not degrade nucleosomes at <3 x
10~*M. Identical nucleosomal repeat sizes (166 ±3.8 base pairs) were
produced by the analogues. The higher reactivity of phleomycin does not
result solely from its higher rate of internucleosomal and intranucleosomal chromatin cleavage, since short phleomycin reactions always resulted
in more extensive chromatin cleavage than long bleomycin reactions at
low concentrations. In vivo (cellular) repair of chromatin damage was
comparable (approximately 90% in 1 h) after cells were exposed to low
drug concentrations which produced similar numbers of chromatin
breaks, and thus also does not account for the higher chromosomal
breakage caused by phleomycin than bleomycin at low doses. At high
doses, unrepaired breaks are substantially higher after phleomycin treat
ments than after bleomycin treatments, and thus contribute to the higher
lethal effects of phleomycin than bleomycin.
In spite of these similarities, the actual mechanism of the
DNA interaction with phleomycin in vitro appears to differ
from that with bleomycin (9, 10, 18, 19). The Cu(II)bleomycin
complex intercalates, while Cu(II)phleomycin does not (18). In
addition, bleomycin, but not phleomycin, degrades relaxed
DNA to a greater extent than either positively or negatively
superhelicalDNA(lO).
These differences between phleomycins and bleomycins could
have important consequences with respect to their mechanism
of action on genomic structures in cells. Chromosomal organi
zation in eukaryotic cells is important in the nature and extent
of its interaction with phleomycins and bleomycins. Unlike
prokaryotic organisms and purified DNAs, the fundamental
unit of organization of DNA in eukaryotic chromatin is the
nucleosome. Bleomycin releases nucleosomes from isolated
mammalian nuclei (20-23). In the current study, we have in
vestigated the reaction of both structural families with chro
mosomal DNA in the model eukaryote Saccharomyces cerevisiae. The study was designed to simultaneously compare cleavage
between and within nucleosomes, chromosomal repair, and
degradation of chromatin, to the reaction rates, concentrations,
and cytotoxicities of bleomycin and phleomycin. We judged it
crucial to determine these interrelationships because of the
importance of knowing how the in vivo (cellular) mechanism of
action on chromosomes relates to lethal properties of the anticancer glycopeptides.
INTRODUCTION
We have investigated differences between activities of the low
molecular weight anticancer bleomycins and structurally related
phleomycins which have important consequences with respect
to the mechanisms of action of this family of anticancer anti
biotics on genomic structures in cells. Phleomycins, which are
substantially more cytotoxic, differ structurally from their bleo
mycin analogues in the oxidation state of their sulfur heterocycles (1; Fig. 1). Similar activities in producing DNA breaks
have been reported for phleomycins and bleomycins (7, 8),
although phleomycin produces fewer double-strand breaks of
PM2 phage DNA than do bleomycins (9, 10). Intercalation of
the bithiazole in DNA is thought to be necessary for the
production of double-strand breaks but not single-strand breaks
in vitro (9). Cellular repair of chromosomal breaks produced by
phleomycin and bleomycin requires the function of the same
nuclear genes (11-15). Preferred cleavage sites and relative
frequencies of cleavages at these sequences in vitro are also
similar for phleomycin and bleomycin (16, 17).
Received 1/11/88; revised 6/29/88. 8/25/88; accepted 8/31/88.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1This study was supported by the National Institutes of Health (Grant
CA25609, Department of Health and Human Services), United Cancer Council,
Inc. (Rochester), American Cancer Society, Inc. (IN18; New York, NY), contract
DE-AC02-76EVO3490 with the U.S. Department of Energy at the University of
Rochester Department of Radiation Biology and Biophysics, and The City Uni
versity of New York Medical School and City College.
2To whom requests for reprints should be addressed, at Department of
Microbiology, City University of New York Medical School, Science Building,
Room 910, Convent Avenue at 138th Street, New York, NY 10031.
MATERIALS
AND METHODS
Electrophoretic Analyses
Culturing Conditions. Diploid strain CM-1293 (Table 1) was routinely
grown at 23°Cwith aeration in supplemented synthetic minimal me
dium (wt/vol, 0.67% yeast nitrogen base without amino acids and
ammonium sulfate, 0.005% ammonium sulfate, 1% succinic acid, 0.6%
NaOH, 2% dextrose, 0.002% adenine, 0.003% isoleucine, 0.015%
valine, and 0.002% tryptophan (24-27) for 24 h (early stationary phase;
approximately 6.8 ±0.5 x IO7cells per ml) from starting inocula of 8
x IO6 cells per ml. Cells were harvested by centrifugation at 4'C in a
Dupont Servali RC-5B SS34 rotor at 2,500 x g (2 min) and washed
twice with deionized water.
Phleomycin and Bleomycin. Phleomycin and bleomycin were supplied
by Bristol Laboratories, Syracuse, NY, through the courtesy of Dr.
William Bradner. Each antibiotic was dissolved and diluted in deionized
water (pH 5.0) just prior to use, and its absorbance was monitored at
292 nM.
Reaction Conditions. For treatments, 1 x IO7washed cells per ml of
deionized water (pH 4.5 to 5.0) were incubated at 23°Cfor 60 min with
aeration. Throughout this incubation, the pH of the reaction was 5.0.
EDTA was added to 0.1 M at the end of treatment periods. Cells were
subsequently sedimented at 4°Cin an RC-5B Servali SS34 rotor at
3,000 x g, washed twice with 0.05 M EDTA, then immediately con
verted to spheroplasts and lyzed.
In order to optimize the validity of comparative measurements,
phleomycins and bleomycins were compared on the same cell popula
tion, in each of several replicated, independent experiments. Rigorous
consistency was followed in culturing conditions and drug lots, as well
as pH, cell density, and temperatures during and after drug treatments.
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CHROMOSOMAL CLEAVAGE IN YEAST BY BLEOMYCIN AND PHLEOMYCIN
Sedimentation Analysis
NHj
Radioisotopic Labeling. Nucleic acids were labeled by growing strain
A364A (Table 1) at 23°Cwith aeration for 15.5 h (late logarithmic
phase) from starting inocula of fresh cells (5 x IO6 cells per ml) in
supplemented synthetic minimal medium (given above); [6-3H]uracil
(specific activity, 20-30 Ci/mmol; New England Nuclear Corp., Bos
ton. MA) was added to 7 ¿iCi/ml.Cells were harvested by centrifugation
at 4°Cin an RC-5B Sorvall SS34 rotor at 3,000 x g, washed once in
BLEOMYCINS:
deionized water, chased in supplemented synthetic minimal medium
(containing 0.0017% uracil) without radiochemical for 60-90 min, and
washed twice with deionized water. RNA was hydrolyzed completely
as described below.
Treatment Conditions. Treatment conditions were identical to those
described above, except that exposure periods were reduced to 20 min
and EDTA was added to 0.05 M at the end of 20 min.
Posttreatment Incubation. After EDTA was added to the reaction
mixture, cells were immediately pelleted by centrifugation at 4°Cin an
RC-5B Sorvall SS34 rotor at 3,000 x g, washed twice at 4°Cwith
R,= |CH2]2
EDTA (0.05 M) and suspended in buffered medium (1% yeast extract;
(Difco Laboratories, Detroit, Michigan), 2% peptone (Difco), 2% dex
trose, 0.008% adenine, and 0.05 M phosphate; pH 5) at 37°Cfor aerobic
PHLEOMYCINS:
R|=(CH2)2
S
R2 = terminal amines
Fig. 1. Chemical structures of bleomycins and phleomyeins (2-6).
Spheroplast Conversion. Cells (at IO8 per ml) were converted to
spheroplasts in 1 mg zymolyase 5000 (Kirin Brewery Co., Ltd., Takasaki, Japan) per ml of 25 HIM dithiothreitol, 1.2 M sorbito!, 20 IHM
KH2PO4,0.5 mM CaCl2, and 10 mM EDTA at 32°C.Spheroplasts were
then pelleted at 1500 x g in a Servali RC-5B refrigerated centrifuge
with an SS34 rotor.
DNA Extraction and Purification; Gel Electrophoresis. Sphleroplasts
were incubated at 60°Cfor 30 min in lysis buffer (2% SDS, 20 mM
EDTA, 200 mM NaCl, and 100 mM Tris-base) equal to the volume of
the pellet. One-fifth volume of 5 M potassium acetate was added and
the mixture was held at 4°Cfor 1 h, spun down at 12,800 x g for 10
min, then resuspended in 100 n\ of 10 mM Tris-base and 1 HIMtransl,2-diaminocyclohexane-./VyVVV'JV'-tetraacetic acid (Sigma). RNase A
(Sigma) was added to a concentration of 100 Mg/ml and incubated at
30°Cfor 2 h. Proteinase K (EM Biochemicals) was then added to a
posttreatment incubation. The control (untreated) and treated cells
were incubated identically.
Spheroplast Formation, Velocity Sedimentation, and Molecular
Weight Determinations. Each of these was carried out as previously
described (24). Nucleic acids were sedimented at 8,000 rpm in an
SW50.1 rotor (Beckman Instruments, Inc., Fullerton, CA) at 20°Cas
described previously (24, 29-31). The length of the ultracentrifugation
was dependent upon the particular experiment. The gradient system
was precalibrated with phage markers to establish the relationship of
sedimentation distance, time and force to molecular weight. This rela
tionship was used to compute weight-average molecular weights from
the amount of radioactivity sedimenting in different positions through
out the gradient. With T4 and T7 DNAs as standards, the gradient
system was determined to be isokinetic, and the constant, A, and
exponent, a, were experimentally determined for the Studier equation
5 = kM". With the aid of a computer program, weight-average molec
ular weights (.1A.) were calculated from distributions of radioactivity in
gradients and related to number-average molecular weights (24, 2932). From the distributions of radiolabeled DNA under the established
sedimentation conditions, the numbers of single-strand breaks relative
to the untreated control were calculated as follows:
concentration of 100 Mg/ml and the solution was incubated for l h at
37°C.The DNA was purified by extraction with phenol/chloroform
(28), followed by three extractions with ethyl ether to remove residual
phenol. To the resulting aqueous layer 1/10 volume of 3 M sodium
acetate and approximately 3 x volume 100% cold ethanol was routinely
added and held at -20°Cfor at least overnight to precipitate the DNA.
The DNA was then pelleted 30 min, washed with 70% ethanol, held at
-80'C for 20 min to 2 h, and pelleted again (30 min). The ethanol was
removed, and the pellet dryed for 5 min in a Servali Speed-Vac at
45°C.The pellet was then resuspended in Tris-EDTA buffer. Each
sample (30 ^g) was loaded onto a 1.5% agarose gel made in 90 HIM
Tris base, 90 mM boric acid, and 20 mM EDTA and run vertically in
this buffer at approximately 2.5 mA for about 18 h. Larger than usual
quantities of yeast and standard DNAs were loaded in order to visualize,
respectively, the highest and lowest molecular-weight bands. Gels were
stained in the dark with ethidium bromide (0.5 ng,/m\; 30 min), destained 90 min in deionized water, and photographed on an ultraviolet
light box.
Strain
CM-1293
Ploidy
Diploid
10" x
(M,, untreated/A/» treated) - 1
A/, untreated/2
The A/«for the untreated, unincubated and the untreated, incubated
control cells were indistinguishable. In the interest of simplicity in
comparing bleomycin and phleomycin, certain assumptions were made
and are inherent in using this method to approximate numbers of
single-strand breaks; however, any error associated with the assump
tions should be equivalent for both congeners.
RESULTS
Electrophoretic Analyses of in Vivo (Cellular) Chromatin
Cleavage. Chromatin was cleaved in a dose-dependent manner
over a very wide range of bleomycin concentrations (6.7 x 10~7
to 3 x 10~3 M). A representative electrophoretic analysis is
shown in Fig. 2 (6.7 x 10~7M to 3.3 x 10~5M; >3.3 x 10~5M,
Table 1 Strains
Genotype"
MAT a ade2-119 trpSa ilvl-92 cycl-131
MAT o ade2-40 trpSb ilvl-92 cycl-45
MAT a adel ade2 ural his? Iys2 tyrl gall
A364A
Haploid
' Does not confer altered sensitivity to chromatin cleavage or killing by phleomycin or bleomycin.
Source (reference)
This laboratory (13)
Dr. Rochelle Esposito (24)
6838
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CHROMOSOMAL
CLEAVAGE IN YEAST BY BLEOMYCIN AND PHLEOMYCIN
of bands produced after these phleomycin treatments (Figs. 2
and 3) was 171.5 ±4 base pairs (Table 2). An oligomeric series
also was not visible at higher concentrations of 3.3 x IO"6 M
(Fig. 2, Lane f. Fig. 4, Lane </), and 5-33 x 10~" M (Fig. 4)
b c
phleomycin where very little DNA was actually retained. A
dose-dependent reduction in what remains of the chromosomal
band at the top of the gel was observed as a function of
increasing phleomycin concentration from 3.3 to 33 x 10~6M
phleomycin (Fig. 4).
More extended oligonucleosomal series of seven and eight
bands were visible after exposures to bleomycin. Banding series
were routinely observed up to 2 x 10~4.M,in the range of 100fold higher than for phleomycin. The intensity of these bands
increased from 10 to 23 x IO"6 M bleomycin (Fig. 2, Lanes g
1353
1078
872
Fig. 2. Agarose gel electrophoresis of DNA extracted and purified from
bleomycin- and phleomycin-treated (60 min. pH 5.0, 23'C) cells. Procedures are
described in detail under "Materials and Methods." The lengths of each major
band were calculated and are presented in Table 2. The data are representative of
five independent experiments. Lanes: a, 6.7 x 10~7M bleomycin: b. 6.7 x 10"7M
phleomycin; c, 2 x IO"6M bleomycin; d, 2 x 10"' M phleomycin; e. 3.3 x 10~"M
bleomycin;/, 3.3 x 10"'M phleomycin; g-j, 1.0 x 10~!M; 1.3 x 10"'M, 2.3 x
10~*M, 3.3 x 10~'.M bleomycin; and k, PhiX174 RF DNA //adii fragments
(Bethesda Research Laboratories); labeled in base pairs.
through /), and were superimposed against the pattern of non
specific dose-dependent degradation. The intensities of both
the multimeric bands and background DNA decreased signifi
cantly at >3.3 x IO"5 M (Lane j; higher concentrations, data
not shown). The width of the mononucleosomal band also
appears decreased in Lane j, possibly indicating the trimming
of nucleotide tails at the highest bleomycin test dose. The mean
multiplicity calculated for the nucleosomal bands cleaved in
bleomycin treatments was 174 ±7 base pairs in Fig. 2 and 166
±4 base pairs in ten independent experiments (Table 2).
Reaction Rates of Chromatin Cleavage. The very' different
concentrations of bleomycin and phleomycin required to pro
duce approximately equivalent chromatin cleavage could result
from differing reaction rates of the congeners. We tested this
hypothesis systematically by varying the treatment times for a
range of concentrations of bleomycin and phleomycin from 30
s to 36 h. In no case, however, did a prolonged reaction with
bleomycin result in extents of chromatin cleavage similar to
that produced by the same concentration of phleomycin during
a short treatment period. After the chromatin damage and
degradation which occurs early during the reaction period, the
rate of increase in chromatin degradation slows considerably as
a function of increasing reaction time.
Fig. 5 illustrates the reaction of late stationary-phase cells
with 2 x IO"6M bleomycin for 20 min to 15 h, and that relatively
little additional chromatin degradation occurs with increased
treatment periods from 1 to 15 h. Nucleosomal patterns also
are not observed as a result of extended reaction periods. In
contrast, extensive chromatin degradation and banding patterns
are produced after short 2 x 10~6 M phleomycin treatments,
and dose-dependent increases in chromatin degradation were
always observed as a function of increased treatment times.
M»after Posttreatment Incubation. The widely differing ac
tivities of bleomycin and phleomycin could relate to a cell's
data not shown). In contrast, phleomycin produced dose-de
pendent DNA degradation over a very narrow range o! concen
trations (Fig. 2).
Very little chromatin was degraded at the lowest test concen
trations of bleomycin (0.67. 2 and 3.3 x 10~6 M). Equimolar
phleomycin, on the other hand, resulted in extensive chromatin
degradation, particularly at >2 x 10~2M. At 3.3 x 10~6 M,
almost no DNA larger than approximately 118 base pairs was
detectable.
A multimene series of DNA bands appears superimposed
upon the background of degraded DNA (Fig. 2, Lanes d and g
through j). Phleomycin (2 x 10~6 M) produced the largest
number of bands; five bands are visible in a "ladder" (Lane d).
At the lower concentration (6.7 x 10~7M; Lane b), three bands
are faintly visible. The mean band lengths were calculated to be
160 (monomer), 335 (dimer), 495 (trimer), 675 (tetramer), and
835 (pentamer) base pairs, with a multiplicity of approximately
166 ±3 base pairs (Table 2). A more extended oligonucleosomal
series was not observed with additional test concentrations of
phleomycin in this dose range (Fig. 3). The mean multiplicity
capacity to recognize and rejoin chemical interruptions in chro
matin, and it was reasoned that more unjoined breaks may
remain after phleomycin treatments than after bleomycin treat
ments. To test this hypothesis, DNA breaks were assayed before
and after a limited postexposure incubation under conditions
where the continuity of prelabeled DNA could be restored.
Concentrations of bleomycin and phleomycin were selected
which produced approximately similar numbers of breaks dur
ing treatment periods. This was important because at concen
trations producing dissimilar numbers of total breaks, it would
be difficult to determine whether unrejoined breaks resulted
simply from the increased chromatin disruptions and killing
caused by phleomycin.
Single-strand breaks introduced into parental (prelabeled)
[6-3H]DNA were assayed and quantitated by velocity sedimen
tation of DNA through precalibrated, isokinetic alkaline su-
6839
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CHROMOSOMAL CLEAVAGE IN YEAST BY BLEOMYCIN AND PHLEOMYCIN
Table 2 \umber of base pairs in D.Y.-fbands
Mean repeat and monomer lengths were calculated from digests in Figs. 2 and 3. and in all (10) independent experiments.
pairsMonomerDimerTrimerTetramerPentamerHexamerLength160335495675835Fig.
Phleomycin. base
pairsFig.
2Multiplicity(160)168165169167x,
3Multiplicity(195)ISO172171169i,
3Length177.5
Figs. 2 and
base
2Multiplicity(185)173173171167*5=
experimentsLength169
10
±25347.5
18505.0±
14680.0±
±7.1840.0+7.1Multiplicity(176)174168170168x,
17338
±
22506
±
25667±
35818
±
23957±
±11Multiplicity(169)169169167164160xt=
= 171.5±4.0Length185345520685835Bleomycin.
174±6.7Mean,
166±3.8
= 165.7±3.4Length195360515685845Fig.
=177.4±
10.7Mean,
abed
310
234
194
118
Fig. 3. Agarose gel electrophoresis of DNA extracted and purified from
phleomycin-treated (60 min. pH 5.0. 23'C) cells. Procedures are described in
detail under "Materials and Methods." The data are representative of four
independent experiments. The lengths calculated for each major band are pre
sented in Table 2. Lanes: a, control (no phleomycin): b, 3.3 x IO"7 M; c. 6.7 x
IO-7M;d, 1 x 10~'M; e, 1.3 x 10~6M;/ 1.7 x \0'*M:g, 2 x 10~6M; h, 2.3 x
10"'M; /, 2.7 x lO-*M;j. 3.3 x 10~'M phleomycin; *. PhiX174 RF DNA HaeUl
fragments.
erose gradients (24). The size of native yeast DNA (A/wapprox
Fig. 4. Dose-dependent degradation of chromatin from cells exposed to phleo
imately 3 x IO8) is ideal for velocity sedimentation through
mycin, including high concentrations. Lanes: a, control (no phleomycin); b, 6.7
sucrose, particularly at low centrifugal speeds. "Single-strand
x IO-'M; c, 2 x IQ-'M; d. 3.3 x IO-'M; e. PhiX174 RF DNA Haelll fragments
(arrow, position of 310 base pair band);/ 5 X 10"' M; g, 6.7 x 10"'M; h, 1.3 X
breaks" in this assay include all lesions which ultimately result
10-'M;/, 2 x 10-'M;./, 2.7 x 10"'M;*, 3.3 x 10-'M.
in DNA breaks, including double-strand breaks and alkali-labile
sites, under the described velocity sedimentation conditions in
alkali; free base release by bleomycin and phleomycin (e.g., 33)
leaves DNA alkali-labile (34). Moreover, this assay is quite
6840
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CHROMOSOMAL CLEAVAGE IN YEAST BY BLEOMYCIN AND PHLEOMYCIN
10"
12-
i7T^
fi
A
t\Ã-
iséLp^
"tf^cw^na
'
1.0 0.8
0.6
0.4
0.2
O
1.0
08
06
I
I
04
I
'
'
'
I
0.2
'
'
'
I
O
DISTANCE SEDIMENTED
Fig. 6. Effect of aerobic posttreatment incubation on sedimentation rates of
DNA from phleomycin-treated, late logarithmic-phase A364A cells. After 20min exposures to phleomycin (23°C),cells were incubated for l h (A) or 2 h (Ä)
at 37'C in buffered medium (pH 7). In A: •¿.
A/, = 2 x 10", untreated cells; D.
A/, = 7.1 x IO7, 3.3 x 10~7M phleomycin; A, A/»= 1.2 x 10", cells incubated 1
h in medium after 3.3 x 10~7M phleomycin treatment. In B: •¿
M, = 2 x 10".
untreated cells; Ü.A/, = 9.9 x 10*. 6.7 x 1(T7M phleomycin treatment; A, A/. =
8.2 x 10~7M, cells incubated 2 h in medium after 6.7 x 10~7 M phleomycin
treatment.
weight as a result of the posttreatment incubation. The A/»
values calculated for DNAs after treatments with 3.3 x 10~7M
phleomycin and 6.7 x 10~7M bleomycin were very' similar [7.2
±0.2 x 10~7(from data as illustrated for representative exper
iments, Fig. 6/1) and 9.0 ±0.3 x IO7 (sedimentation profiles
not shown), respectively]. From the A/„calculated before and
after posttreatment incubation, the numbers of single-strand
breaks remaining after l h were determined for both phleomycin
and bleomycin (Table 3). The reductions from 1.8 ±0.05 to
0.17 ±0.03 (phleomycin, 3.3 x 10~7) and from 1.2 ±0.04 to
0.12 + 0.01 (bleomycin, 6.7 x 10"7M) single-strand breaks per
IO8 daltons represented approximately 90% of total breaks in
both cases.
DISCUSSION
Phleomycin was substantially more reactive than bleomycin
in cleaving and degrading intracellular chromosomes, including
nucleosomes. Chromatin became digested as a function of
Fig. 5. Typical results from the reactions of late stationary-phase cells with 2
increased concentrations of bleomycin or phleomycin; prefer
x 10~6Mbleomycin for 20 min to 15 h. Lanes: a and /. Phi.\174 RF DNA Haelll
ential
internucleosomal cleavage became apparent and could be
fragments (arrow, position of 310 base pair band): b. control (no bleomycin); c,
visualized as oligonucleosomal series. The DNA repeat lengths
20 min; d, 60 min; e. 90 min:/. 2 h: g. 3 h; h. 4 h; i, 5 h;j, 6 h; /. ¡5h. DNAs
exhibit a very small fraction of nonspecific chromatin degradation which is not
cleaved by phleomycin were indistinguishable from those
detected in electrophoretic analyses of DNAs from cells treated during earlier
cleaved by bleomycin (Figs. 2 and 3; Table 2); the mean DNA
phases of growth.
length of 166 ±3.8 base pairs calculated in this report (Table
2) agrees closely with the nucleosomal size of approximately
sensitive under limited reaction conditions. DNAs from yeast
165 base pairs previously determined after enzymatic treat
exposed to equimolar concentrations of either analog were ments of isolated yeast nuclei or chromatin (35-42). Eventually,
sedimented with control DNA (from untreated cells) in the
however, the oligomeric series becomes lower order as a func
same rotor.
tion of increasing concentration of either congener. Finally, as
Preliminary experiments3 established that the DNA sedimen
tation profiles and molecular weights for strain CM-1293 were the high molecular weight chromatin is digested further, neither
the banding patterns nor mononucleosomes are visualized. At
indistinguishable from those for strain A364A (Table 1). How
high test concentrations of phleomycin (Fig. 4) or bleomycin
ever, both the spheroplast conversion and lysis steps for A364A
cells were faster than for CM-1293 cells. Since these were (data not shown), little, and eventually no, DNA can be visu
alized in the gels, except for a minor fraction remaining at the
crucial in order to gently lyse and rapidly release DNAs after
top of the gels. It is thus concluded that chromatin can be
drug treatments, we used A364A cells for determining molec
completely or nearly completely digested in this reaction. It is
ular weights of DNAs after limited digestion.
also concluded that phleomycin (Figs. 2 and 3) possesses re
As illustrated in Fig. 6, a fraction of the low molecular weight
duced specificity for internucleosomal cleavage, since phleo
chromatin clearly becomes two to eight times higher molecular
mycin produces a less discrete oligonucleosomal series than
' Unpublished.
bleomycin and the retention of DNAs is very low after cellular
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CHROMOSOMAL CLEAVAGE IN YEAST BY BLEOMYCIN AND PHLEOMYCIN
Table 3 Mwbefore and after posttreatment incubation
Posttreatment incubation (1 h) and calculations of A/„
and DNA breaks were carried out as described in "Materials and Methods" and "Results." Standard errors
were calculated from determinations in independent experiments.
10')7.2
Phleomycin, 3.3 x 10~7M
Bleomycin, 6.7 X 1(T7 MM.(x
breaks
(perdallons)1.8
10s
10')1.2
±0.2
±0.05
9.0 ±0.3BeforeSingle-strand
1.2 ±0.04M.(x
reactions with only approximately >2.3 x 10 6M phleomycin.
This trimming of DNA linker regions and extensive chromatin degradation was not observed by Kuo and coworkers
(20-23) after bleomycin treatment of isolated mammalian nu
clei at concentrations both comparable to and considerably
higher than those we used. Kuo and Hsu (23) determined that
monosomes isolated from bleomycin-treated nuclei and re
treated in a limited digestion with bleomycin yielded a reduction
in the total quantity of monosomes (core and linker containing
180 base pairs) detectable after gel electrophoresis, but con
cluded bleomycin did not cleave within monosomes since their
size before and after retreatment was the same. We suggest,
however, that the degradation of isolated monosomes in the
bleomycin reaction required Fe(II), like other isolated DNAs
[e.g., review by Burger et al. (43)]. Isolated yeast DNA is
resistant to degradation by both bleomycin and phleomycin
unless ferrous ion is added to the reaction, and degradation of
yeast chromatin in isolated nuclei is partially dependent upon
exogenous Fe(II).4
We conclude that the higher reactivity of phleomycin than
bleomycin does not result from differences in rates of chromatin
cleavage and release of nucleosomes from chromatin in cells,
since even our shortest phleomycin reactions always resulted in
more extensive chromatin cleavage than long bleomycin reac
tions at low concentrations. With increased treatment time,
relatively little increased chromatin degradation occurs. Chro
matin cleavage by both analogues was compared after reaction
times from 30 s to several hours, and it has always been our
experience that extended treatments at doses below optimum
for visualization of nucleosomal bands do not produce the
banding patterns. This is consistent with the very rapid rates of
phleomycin and bleomycin reactions within the first few sec
onds, the reduced rates up to about 60 min, and the much
slower rates at later times (14-15, 27, 44-45). Phleomycin is
also substantially more cytotoxic than equimolar bleomycin
(13) when short phleomycin reactions are compared with long
bleomycin reactions.4 Because the lesions produced in cellular
chromatin are believed to be a principal cause of the lethal
properties of the bleomycins (5, 46-47), we routinely deter
mined survival in the same experiments and on the same cell
populations in which chromatin cleavage and single-strand
breaks were measured (12-15, 27, 44-45). In all experiments,
reactions with phleomycin resulted in 1.5 to 450 times higher
cytotoxicities than bleomycin (Reference 13 and Footnote 5).
It is also concluded that the lesions produced by low doses of
phleomycin and bleomycin are approximately equally reparable.
Reductions from 1.8 ±0.05 to 0.17 ±0.03 (after phleomycin
treatment) and 1.2 ±0.04 to 0.12 ±0.01 (after bleomycin
treatment) breaks per 10a dallons represented removal of ap
proximately 90% of the total breaks in l h (Table 3). Repair of
chromatin is significantly higher after bleomycin treatments
than after phleomycin treatments at high concentrations (data
* Unpublished experiments.
5 Unpublished studies.
breaks
(per
dallons)0.17
10"
+ 0.03
±0.2
1.7 ±0.2AfterSingle-strand
0.12 ±0.01Fraction
remaining0.093
0.096
not shown), and thus undoubtedly accounts for the dramatically
higher DNA breakage after phleomycin treatments than after
bleomycin treatments at high doses.
ACKNOWLEDGMENTS
We thank Dr. William Bradner (Bristol-Myers Company, Pharma
ceutical Research and Development Division, Syracuse) for providing
phleomycin and bleomycin for these experiments; Drs. Richard Rey
nolds (Harvard School of Public Health; Los Alamos National Labo
ratory) and Donald Morken (University of Rochester) for their assist
ance in preparing a computer program for calculating molecular
weights; Elizabeth Goldstein, Cheryl S. Jones, Susan Mancuso, Perrin
Pleninger, and Laurel Wall for carrying out experiments and analyses
of data; Drs. Abraham Worcel, Richard Borch, Helen Eberle, Don
Holtzu, Bret Jessee, Dennis Lohr, Court Saunders, and Shane Weber
for their critical assistance and encouragement; and Mark Pezzano and
Dr. Ajay Pramanik for helpful discussions.
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Internucleosomal Cleavage and Chromosomal Degradation by
Bleomycin and Phleomycin in Yeast
C. W. Moore
Cancer Res 1988;48:6837-6843.
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