Download Bleomycin, an Apoptosis-mimetic Drug That Induces Two Types of

(CANCER RESEARCH 53, 5462-5469,
November 15. 1993]
Bleomycin, an Apoptosis-mimetic Drug That Induces Two Types of Cell Death
Depending on the Number of Molecules Internalized1
Omar Tounekti, GéraldinePron, Jean Belehradek, Jr., and Lluis M. Mir
Laboratoire de Biochimie-Enzymologie
(U.K.A. 147 Ceñiré
National de la Recherche Scientifique:
ABSTRACT
Bleomycin i III M i. a compound currently used in anticancer therapy, is
unable to cross the plasma membrane efficiently. Electropermeabilization
allows a defined number of HI .M molecules to enter directly into the cell
cytoplasm. Such a procedure has revealed that BLM is intrinsicly highly
cytotoxic. Here we show that the mechanisms of the cell death caused by
BLM are closely related to the number of BLM molecules introduced into
the cell cytoplasm. When only a few thousand BLM molecules are inter
nalized, cells display an arrest in the (. .-M phase of the cell cycle and
become enlarged and polynucleated before dying. These observations par
allel the "mitotic death" seen with ionizing radiations. By contrast, when
several million molecules of BLM are internalized, morphological changes
identical to those usually associated with apoptosis are observed as well as
very rapid DNA fragmentation into oligonucleosomal-sized
fragments. We
demonstrate that this fragmentation, which occurs within a few seconds
after BLM internalization, is consistent with the direct internucleosomal
cleavage of chromatin by BLM. Our findings reinforce the importance of
DNA digestion as an early and essential step in the morphological changes
associated with apoptosis.
INTRODUCTION
BLM2 is a water-soluble
antibiotic
that was first isolated by
Umezawa et al. (1) in 1966. BLM cytotoxicity to mammalian cells is
considered related to its ability to induce single- and double-strand
DNA breaks (2) but other mechanisms, such as base propenal gen
eration, have also been proposed (3). BLM is also a good chelator of
several metals, e.g., iron and copper. Cobalt (4-6) and zinc (7) chelation by BLM gives rise to a noncytotoxic complex unable to gen
erate DNA breaks.
BLM cytotoxicity is considerably potentiated in vitro when cultured
cells are exposed to appropriate electric pulses (8, 9). The antitumor
effects of the compound are also highly increased in vivo by electric
pulses delivered locally to the tumor site (10-12). The plasma mem
brane is known to limit BLM uptake (9) and electropermeabilization
(13) is an efficient mean of circumventing this barrier, thus allowing
the direct internalization of BLM molecules into the cytoplasm (9).
Moreover, the number of internalized molecules is closely related to
the external concentration of BLM. With this procedure it is possible
to predetermine the average number of BLM molecules that will be
introduced into cells (9).
According to Wyllie et al. (14), cell death can be categorized as
either necrosis or apoptosis. At a morphological level, necrosis is
associated with cell swelling, the rupture of membranes, and the
dissolution of an organized structure. In contrast, apoptosis is char
acterized by cell shrinkage and chromatin condensation. At a bio
chemical level, necrosis results from the loss of osmoregulation, with
Received 4/26/93; accepted 9/13/93.
The cosls 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 work was supported by a grant from the Association pour la Recherche sur le
Cancer.
- The
Fe-BLM,
ethylene
buffered
abbreviations used are: BLM, bleomycin; Co-BLM. cobalt BLM complex:
iron BLM complex; ATA. aurintricarboxylic acid; CH. cycloheximide; EGTA.
glycol-his(ß-aminoethyl ether) MMW.W-tetraacetic
acid; PBS, phosphatesaline (0.2 g/liter KC1; 0.2 g/liter KH2PO4; 8 g/liter NaCI; 2.16 g/liler
); MEM, Eagle's minimum essential medium.
U. 140 INSERM) Institut Gustave Roussy, 94805 Vi/lejuif, France
random DNA degradation by lysosomal enzymes at a late stage.
During apoptosis, internucleosomal DNA digestion is caused by the
activation of an endogenous endonuclease, which is thought to play a
central role in apoptosis. A Ca2+/Mg2+-dependent endonuclease was
first implicated in apoptosis because it was present in nuclei prepared
from thymocytes undergoing apoptosis (15) and because Ca2+ chelators (e.g., EGTA) can inhibit apoptosis (16-18). DNA digestion and
death in cells induced to undergo apoptosis are also inhibited by the
endonuclease inhibitor ATA (19, 20) and by Zn2+ (15, 21). In some
but not all cases in which death occurs by apoptosis, the inhibition of
protein synthesis by CH prevents the appearance of the oligonucleosomal ladder (22); this suggests that apoptosis is often dependent upon
active metabolism and protein synthesis by the dying cell. The endo
nuclease can also be triggered by intracellular acidification alone (23).
In comparison, mitotic death (24), also termed delayed reproductive
death (25), corresponds to a less characterized cell death mechanism.
Mitotic death is generally a slow process that is dependent upon
mitotic activity during which cells will usually complete at least one
mitosis prior to their disintegration. Mitotic death was first described
in cells treated with low doses of ionizing radiations (24, 25) while
high doses of ionizing radiations induced apoptosis (26).
In this article, we demonstrate that BLM is able to induce two types
of cell death depending on the number of BLM molecules internalized
after electropermeabilization. At low concentrations of bleomycin,
cells arrest in the G2-M phase of the cell cycle and die after a time
period corresponding to three doubling times. At high concentrations,
bleomycin directly induces events analogous to those observed during
apoptosis and can be considered as an apoptosis mimetic.
MATERIALS
AND METHODS
Cells and Chemicals.
DC-3F cells, a Chinese hamster lung fibroblast line
(27), were maintained in previously described culture conditions (8). The
human head and neck carcinoma cell line A-253 (28) (kindly provided by
Professor J. Lazo, Pittsburgh, PA) were maintained in McCoy's 5A (modified)
medium (Gibco BRL), supplemented with 10% fetal bovine serum, penicillin,
and streptomycin. Lyophilized BLM (Roger Bellori) was dissolved in 0.9%
NaCI and stored at -20°C. Deoxyribonuclease-free
ribonuclease and proteinase K were purchased from Boehringer
Mannheim
(Mcylan, France). Cis-
platin, in the form of an injectable solution, was purchased from Lilly France
S. A. (Saint-Cloud, France); all other drugs, chemicals, and enzymes were
purchased from Sigma Chemical Co. (La Verpilliere, France). Regular MEM
and McCoy's 5A (modified) as well as S-MEM (calcium-free MEM) cell
culture medium and fetal calf serum were obtained from GIBCO Laboratories
(Cergy-Pontoise, France). Co-BLM was prepared according to the method of
Poddevin et al. (6) in which sodium bicarbonate is used to buffer the Co-BLM
at pH 7. To obtain Fe-BLM complex, BLM and FeCK were mixed at a 1/1
molar ratio and incubated for l h at room temperature.
Electric Shock Procedures. Cell electropermeabilization was performed
using the "electropulsator" PS 15, a square wave pulse generator commercially
available from Jouan (Saint-Herblain,
France). After trypsinization
of expo
nentially growing cells and inactivation of trypsin by complete medium, cells
were washed three times in 0.5 ITIMCa2+ supplemented S-MEM (without
serum). Cells were then resuspended in the same ice-cooled medium at a
density of 2.2 X IO7 cells/ml. Aliquots of 67.5 jxl of the monodispersed cell
suspension were mixed with 7.5 /xl of drug solutions at a 10-fold concentration.
Fifty fil of the mixture were immediately deposited between the two electrodes
(2 mm apart) and subjected to the electric treatment (8 pulses of 100 /is and
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BLEOMYCIN.
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1250 V/cm at a frequency of 1 Hz). After delivery of the electric pulses, cells
were kept for 5 min at 24°Cand then diluted in complete medium and seeded
DRUG
100
in triplicate in complete culture medium (500 cells/cell culture dish, 60 mm in
diameter) for colony inhibition assay or treated as shown below.
Morphological Analysis. Electropermeabilized cells were diluted in com
plete medium and seeded in a 24-well plate (Falcon) (IO5 cells/well). After
various incubation times, cells were harvested by trypsinization in culture
medium, stained by the addition of an equal volume of a trypan blue solution
(0.08% trypan blue and 0.005% p-hydroxybenzoic acid methyl, sodium salt in
PBS) and observed and counted in a hemocytometer under a phase-contrast
50
v
o
microscope using X160 magnification. Cells were assigned to different cat
egories according to their morphological aspect. For electron microscopy
observations, treated cells were fixed with 2% glutaraldehyde for 1 h, dehy
drated, processed, and observed using a Zeiss 902 electron microscope.
Biochemical Assays. DNA fragmentation was monitored by a gel electrophoresis method adapted from Smith et al. (29). Briefly, samples of K)6 cells
were incubated at 50°Cfor l h in 20 /A!of 10 HIMEDTA-50 HIMTris-HCl (pH
8.0) containing 0.5% (w/v) sodium lauryl sarkosinate and 0.5 mg/ml proteinase
K. Then, 10 ju.1of 0.5 mg/ml DNase-free RNase were added to each sample and
incubation continued at 50°Cfor 1 h. Samples were heated to 70°C,and 10 ¿il
Ja _
of 30% (w/v) glycerol, 0.25% (w/v) Bromophenol blue, and 0.25% (w/v)
xylene cyanol were mixed with each sample before loading them into the dry
wells of a 1.5% (w/v) agarose gel containing 0.1 ng/ml ethidium bromide.
Electrophoresis was carried out in 2 ITIMEDTA-89 IHMTris-borate (pH 8.0)
until the marker dye had migrated 3^4 cm.
Nuclei were prepared from cells cultured in a 75-cm2 flask that were washed
I
using a Potter dounce A (30 strokes). Unbroken cells and nuclei were separated
by centrifugation (500 g for 15 min at 4°C). Nuclei were incubated with
Fe-BLM and DNA fragmentation
was monitored as described for cells.
Flow Cytometry. Trypsinized cells were washed with cold PBS and fixed
in 80% ethanol in PBS at -20°C. After 12 h, samples were washed with cold
PBS and incubated in 200 fig/ml DNase-free RNase for 30 min at 37°C.
50
O)
o
twice with cold PBS and incubated with 1 ml of lysis solution (20 HIM
Tris-HCl; 1 HIMEDTA; 10 f¿Mpepstatin; 10 JIM leupeptin: 1 mM a-dithiothreitol; 0.4 mM phenyl-methyl-sulfonyl
fluoride) for 5 min at 4°C.Cells were
collected with a rubber policeman and plates were rinsed with another millilitcr
of lysis solution. After 25 min of incubation at 4°C,cells were homogenized
O
O
50
Time (h)
Fig. I. Evolution of the morphology of the A-253 cells and of their ability to exclude
the trypan blue after electropermeabilization in the presence of 10 nM (a) or 10 fiM (b)
BLM external concentrations. A, Cells with normal morphology and size (category I); O,
trypan blue-negative enlarged cells (category II); D, trypan blue-negative shrunken cells
showing membrane blebbing (category III); •¿.
trypan blue-positive cells (category IV).
No cells belonging to category II were detected in the presence of 10 JIM external BLM
and no cells belonging to category III were detected in the presence of 10 nM external
BLM.
Samples were then supplemented with 1 mg/ml propidium iodide, incubated at
37°Cfor 30 min, stored in the dark at 4°C,and analyzed within 24 h using a
Coulter Epics Profile flow cytometer.
RESULTS
DC-3F cells 28 h after the treatment confirmed these observations
(Fig. 2). These cells were enlarged compared to the untreated cells
(Fig. 2a), some were binucleated and showed micronuclei (Fig. 2c),
while others displayed an arrest in mitosis (Fig. 2b). These results
show signs of a highly disturbed cell cycle. That was confirmed by
flow cytometry analysis; within 8 h after the electropermeabilization
of DC-3F cells in the presence of 10 HM BLM, cells displayed a
marked arrest in the G2-M phase of the cell cycle (Fig. 3). In the
absence of BLM, normal fluorescence profiles were obtained 6 h (Fig.
3; control 0) as well as 5 min, 2, 4, and 24 h after the delivery of
electric pulses.
Exposure to High Concentrations of BLM. When a high BLM
external concentration (10 /J.M)was used, cells ceased to adhere to the
plastic support. They exhibited no increase in size but on the contrary
rapid, marked shrinkage and membrane blebbing (category III). The
maximal percentage of this type of cells was detected 6 h after the
treatment. Electron microscopy revealed a chromatin condensation
forming granular masses along the nuclear membrane (Fig. 2d). Thus,
electropermeabilized cells treated with high concentrations of bleomycin (10 JAM)displayed the morphological changes usually associ
ated with apoptosis. Flow cytometric analysis of DC-3F cells treated
with K) /J.MBLM (Fig. 4) revealed a subpopulation displaying prop
idium iodide fluorescence reduced compared to that of the G{i-Gt cell
cycle region, which can be considered as the A,, region described by
Telford et al. (30) in cell populations undergoing apoptosis. We also
analyzed the DNA of cells treated with different concentrations of
BLM 6 h after the delivery of electric pulses, the time at which the
maximal percentage of apoptotic cells was observed in our morpho-
Exposure to Low Concentrations of BLM. In preliminary ex
periments, A-253 or DC-3F cells showed wide morphological varia
tions after their electropermeabilization
in the presence of low
amounts of BLM (100 nM). Less than 0.001% cells survived among
those that were actually permeabilized (8. 9). We subdivided the
treated cells according to their appearance and we counted each cat
egory at different times after the electropermeabilization. These cat
egories were: (0) cells with a normal morphology and size (category
I); (b) trypan blue negative enlarged cells with a diameter more than
two fold the normal cell diameter (category II); (c) trypan blue nega
tive shrunken cells showing considerable reduction of the normal cell
size and displaying membrane blebbing (category III); and (d) trypan
blue positive normal sized and shrunken cells (category IV).
Fig. 1 shows the evolution in the percentage of cells of each
category after exposure of the A-253 cells to only 10 nw BLM and to
the permeabilizing electric pulses. The maximal percentage (63%) of
enlarged cells (category II) appeared after a time period corresponding
roughly to three doubling times: 72 h for the A-253 (Fig. 1). Phase
contrast microscopy observations showed that these cells were multinucleated and also had micronuclei. These results were also found
after exposing DC-3F cells to BLM in the same experimental condi
tions: a maximum percentage (60%) of enlarged cells was detected
after 28 h (results not shown), i.e., again, after a period roughly
equivalent to three doubling times. Electron microscopy studies with
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BLEOMYCIN.
AN APOPTOSIS-MIMETIC
DRUG
•¿
H
Fig. 2. Electron microscopy of DC-3F cells treated with various concentrations of BLM. a, control electropermeabilized cells (28 h); b and c, cells electropermeahilized
presence of 10 n%iexternal BLM concentration (28 h); d, cells electropermeahilized in the presence of 10 ¿IM
external BLM concentration (6 h); X 13,000.
logical studies. We detected the oligonucleosomal ladder characteris
tic of apoptosis (Fig. 5A ). However, this oligonucleosomal ladder was
never obtained when BLM external concentrations were below 10 /AM
whatever the time (i.e., 6, 28, 48, and 72 h) after the delivery of
electric pulses (results not shown). The oligonucleosomal ladder was
also detected when the lysis buffer was added 5 min after electric
pulse delivery to DC-3F cells (Fig. 5ß),and to A-253 cells (results not
shown).
Generation of DNA Double-Strand Breaks by Internalized
BLM. To further investigate the direct effects of the internalized
BLM molecules, we used cobalt, a well known inhibitor of BLM
cytotoxicity. As expected, Co-BLM prepared 5 min in advance, so that
certainly all the BLM molecules had chelated the cobalt, was unable
in the
to cause DNA degradation and no nucleosomal ladder was observed
(Fig. 6A, Lane g). When BLM (10 /J.M)and CoCl2 (500 JÃœLM)
were
mixed only 30 s before their addition to the cells (that was almost
immediately followed by electric pulse delivery) (Fig. M, Lane f), or
even when CoQ2 was added to BLM immediately before using the
mixture (Fig. 6A, Lane e), the resulting chelation of BLM still com
pletely inhibited DNA degradation and the appearance of the nucleosome ladder. This demonstrates that chelation of BLM occurs very
rapidly in the presence of excess CoCU and prevents BLM activity.
Thus, by stopping BLM activity with the adjunction of an excess
amount of CoCl2 after electric pulse delivery, it seemed possible to
determine either the minimal period between BLM internalization and
the detection of DNA fragmentation, or, if the BLM action was im-
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BLEOMYCIN.
AN APOPTOSIS-M1MET1C
DRUG
provoked DNA fragmentation irrespective of the time when the ad
dition occurred: 30 s (Lane f)', 1 min (Lane g); 2 min (Lane h); 3 min
(Lane ;'); or 4 min (Lane j). When CoCl2 was added 5 min after the
electric pulses, i.e., together with the addition of the lysis buffer, we
tried to avoid an artefactual effect due to the presence in the lysis
buffer of EDTA, a potent chelator of divalent ions such as Co2"1".
Therefore, incomplete lysis buffer prepared without EDTA was first
included, and EDTA was added 1 min later (Fig. 6D, Lane c and
control Lane e). The point we wish to underscore is that DNA frag
mentation was still clearly detectable when CoCl2 was added only 30
s after the permeabilizing treatment. From the results shown in Fig. 6,
A-C, we can conclude that CoCU actually blocks BLM activity: 30 s
8
200
-(BO
200 400 ÃŒBO
sea 1000
460
KL3
Fig. 3. Flow cytometric DNA analysis of DC-3F cells al various times after electropermeabilization in the presence of 10 nM BLM. The number of cells is represented as a
function of fluorescence. Control (0) corresponds to the fluorescence profile of DNA
content of cells electropermeabilized in Ihe absence of BLM, 6 h after the electric pulse
delivery.
mediate, the time required to obtain detectable amounts of oligonucleosomal fragments. However, we needed to show that electropores were still present and able to allow rapid CoCl2 internalization
into the cells when CoQ2 was added. Fig. 65 demonstrates that BLM
was still able to enter the cells and provoke DNA fragmentation when
added to the cell suspension 30 s after the delivery of electric pulses.
In these conditions, cobalt was actually internalized and thus available
for BLM inactivation. Indeed, cells preloaded with cobalt either by
electropermeabilization
in the presence of 500 /XMCoCl2 (Fig. 6C,
Lane b) or by addition of 500 /U.MCoCl2 30 s after the electroperme
abilization (Fig. 6C, Lane c), and then washed three times with
S-MEM medium before a second exposure to electric pulses in the
presence of 10 /J.MBLM were resistant to DNA cleavage. After three
washes, cells preloaded with cobalt were free of external cobalt be
cause BLM diluted in the supernatant sampled from the third washing
was not impeded to provoke DNA fragmentation (Fig. 6C, Lanes d
and e).
The effects of BLM occurred almost immediately after its intro
duction into DC-3F cells subjected to the permeabilizing electric
pulses. Fig. 6D shows that CoCl2, added after the electric pulses
delivered in the presence of BLM, was unable to inhibit the BLM-
after cell electropermeabilization under our experimental conditions,
cobalt enters the cells and immediately blocks BLM activity. Thus
BLM created DNA double-strand breaks in less than 30 s after its
internalization.
Our results suggest that the direct nuclease activity of BLM was
sufficient to explain DNA fragmentation into oligonucleosomal frag
ments. However, we decided to study the effect of CH and endonuclease inhibitors on the process following BLM internalization. DC-3F
cells were incubated for 24 h with 0.8 jug/ml CH to arrest protein
synthesis (31). Cells were then electropermeabilized in the presence of
10 JU.Mexternal BLM. DNA analysis performed 5 min after electric
pulse delivery shows that CH did not inhibit DNA degradation fol
lowing BLM internalization (Fig. 7/1). In parallel control experiments
we investigated the effect of CH on cisplatin and serum deprivationinduced apoptosis in DC-3F cells. Electrophoretic analysis showed
that DNA fragmentation was detectable after 48 h of culture following
a 2 h incubation with 20 fig/ml cisplatin (Fig. IB), and after 24 h of
culture in conditions of serum deprivation (Fig. 1C). On the contrary,
when CH (0.8 /xg/ml) was added to the culture medium after the 2 h
exposure to cisplatin (Fig. 75), or when CH was added at the same
concentration to the serum-free medium (Fig. 1C), no DNA fragmen
tation was detectable.
We also tested on the DC-3F cells the effect of known inhibitors of
the endonucleases implicated in apoptosis, but neither ATA nor EGTA
was able to inhibit DNA degradation induced by serum deprivation or
by cisplatin (results not shown). Similarly, the addition of EGTA
(1.5-10 HIM)and of ATA (0.1-1 ITIM)to the electropermeabilization
medium did not result in inhibition of the DNA degradation after BLM
treatment (results not shown). Furthermore, the use of zinc ions pro
vided results similar to those obtained with cobalt. Indeed, zinc BLM
1201
40
FL3
80
120
160
200
24O
FL3
Fig. 4. Flow cytometric DNA analysis of DC-3F cells 6 h after the electric pulse delivery. The number of cells is represented as a function of fluorescence, a, cells electroperme
abilized in the presence of Kl /J.MBLM. Notice the A,, peak (fluorescence 42.2) characteristic of apoptotic cells, b, cells electropermeabilized in the absence of BLM display normal
profile with a peak at 71.2 units of fluorescence representing cells in GC,-G, and a peak at 137.2 units of fluorescence representing cells in G2-M.
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BLEOMYCIN,
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AN APOPTOSIS-MIMETIC
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b
fedcba
c
d
e
f
g
Fig. 5. Electrophoretic analysis of the size of
DNA from DC-3F cells. A, DNA analysis 6 h after
electropermeabilization of cells in the presence of
various external BLM concentrations. Lane a, PhiX
114/HaeUl; Lane b. nonelectropcrmeahilized cells;
Lanes c-h. cells electropcrmeabilized in the pres
ence of: no BLM (c); 1(1nMBLM (d); 100 nw BLM
(<•);
l »¿M
BLM (/): 10 ^M BLM U); 100 UM BLM
(h). B, DNA analysis at various times after cell
clectropermeabilization
in the presence of 10 /IM
external BLM. Lane a, nonelectropermeabilized
cells; Lane b, electropermcabilized cells; Lanes c-g,
lysis buffer was added various times after elec
tropermeabilization: 5 min (c); 30 min (d); l h (e);
2 h (/); 4 h (g): PhiX 174/Wat-IlI (h).
B
complex was unable to cause DNA fragmentation when ZnCl2 (500
P.M)was added to BLM prior to or immediately before the use of the
mixture. By contrast, DNA fragmentation was still clearly detectable
when ZnCl2 was added only 30 s after BLM internalization (results
a
b
c
d
e
abc
g
f
e
not shown). In another control experiment, dimethyl sulfoxyde, scav
enger of free radical species, added at a concentration of 1 or 3 M
either at the time of the BLM internalization or l h or 30 min before
or after BLM internalization, did not impede the BLM-induced DNA
d
A
b a
abc
D
Fig. 6. Time course of DNA fragmentation in DC-3F cells. A, Cobalt prevention of DNA fragmentation by BLM in DC-3F cells. BLM, Co-BLM, or CoCl2 were added to the cell
suspension just before the electric pulse delivery. Lane a, PhiX \14IHae\\\\ Lane b, electropermeabilized cells; Lane c, cells electropermeabilized in the presence of 500 JIM CoCl2;
Lane d, cells electropermeabilized in the presence of 10 /AMBLM; Lane e, cells electropermeabilized in the presence of 10 UM BLM and 500 /IM CoCl2 added immediately after BLM;
Lanes fand g, cells electropermeabilized in the presence of 10 /AMBLM and 500 /IM CoCN mixed together either 30 s or 5 min before the addition to the cells and the electric pulse
delivery. B, BLM ability to reach the cytosol 30 s after cell electropermeabilization. Lysis buffer was added 5 min after electric pulse delivery. Lane a, BLM ( 10 LAM)added to the cells
30 s after electric pulse delivery; Lane b, BLM (10 /AM)present at the time of the electric pulse delivery (usual conditions); Lane c, PhiX I14/Hae\\\. In C, cells preloaded with cobalt
and free of external CoCl2 were resistant to DNA cleavage by subsequently added BLM. Lane a, electropermeabilized cells; Lane b, cells electroloaded wilh 500 LAM
CoCl2, washed
3 times, and then electrorjorated again with 10 /AMBLM; Lane c, addition of 500 /AMCoCl2 30 s after cell electropermeabilization.
incubation for 5 min, then 3 washes and second
electropermeabilization in the presence of 10 LAMBLM; Lane d, cells electropermeabilized in the presence of 10 /AMBLM diluted in the supernatant sampled from the third washing
of the CoCl2-preloaded cells; Lane e, cells electropermeabilizcd in the presence of 10 LAMBLM diluted in the supernatant sampled from the third washing of the cells preloaded with
CoCl2 added 30 s after electric pulse delivery; Lane f, cells electropermeabilized in the presence of 10 /AMBLM; Lane g, PhiX \14lHae\\\. D, DNA fragmentation by BLM ( 10 LAM)
introduced in electropermeabilized DC-3F cells and inactivated at fixed times after its introduction by the addition of 500 /AMCoCl2. Times are given as periods between electric pulse
delivery and the addition of CoCI2 (500 LAM).Lane a, DC-3F cells electropermeabilized in the absence of BLM; Lane b-j. DC-3F cells electropermeabilized in the presence of 10 JAM
BLM; Lane 6, CoCI^ added at 5 min; Lane c, CoClj added at 5 min in the absence of EDTA in the lysis buffer: Lanes d and e, no CoCN added; Lane f. Cod? added at 30 s; Lane
g, CoCl2 added at 1 min; Lane h, CoCl2 added at 2 min; Lane i, CoCl2 added at 3 min; Lane j, CoCl2 added at 4 min; Lane k, PhiX \14jHae\\\. All samples were treated with the
lysis buffer 5 min after the electric pulse delivery (and just after the addition of CoCI2 in Lanes b and c). In Lanes c and e, lysis buffer was prepared without EDTA, and EDTA was
added separately 1 min after the addition of lysis buffer .
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Ill lOMYCIN.
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A
c
AN APOPTOSIS-MIMET1C
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tropermeabilization (13) allows detection of BLM cytotoxicity at con
centrations as low as the nanomolar range (8, 9). We have previously
shown that BLM has two particular characteristics: (a) BLM is unable
to cross the plasma membrane by free diffusion; and (b) BLM is very
highly cytotoxic once in the cytoplasm (9). Indeed, BLM cytotoxicity
to electropermeabilized cells is detected when only a few hundred
BLM molecules reach the cytoplasm.
When cells are electropermeabilized in the presence of 10 /J.M
external BLM, 3 X 10'' molecules of BLM are found in the cell
abc
B
Fig. 7. Effects of Ihc protein synthesis inhibitor CH on DNA fragmentation. A, effect
of CH on BLM-induced DNA fragmentation. Lane a. cells cultured in 0.8 /xg/ml CH for
24 h then electropermcahilized in the presence of 10 JIM BLM: Lane b, cells cultured in
0.8 /ig/ml CH for 24 h then electropermcabilized in the absence of BLM; Lane c. PhiX
174/WiH'IU. B, effect of CH on cisplatin-induced DNA fragmentation. Lane a, PhiX
174/WuclII; Lane h, cells treated with 20 /ig/ml cisplatin for 2 h, washed, and cultured lor
48 h in 0.8 ng/ml CH: Lane c. cells treated with 20 /ig/ml cisplatin for 2 h. washed, and
cultured for 48 h. C. effect of CH on serum deprivation-induced DNA fragmentation. Lane
a, serum-deprived cells cultured in 0.8 fig/ml CH for 24 h; Lane h. scrum-deprived cells
cultured for 24 h: Lane i: PhiX 174/Hui-lII.
cytoplasm (9). In nonpermeabilized cells, even after very long incu
bation times in the presence of high external BLM concentrations,
internalization of a similar number of BLM molecules cannot be
achieved. It is therefore probable that the results obtained with 10 JAM
BLM on electropermeabilized cells in this study have never been
observed before. The introduction of high amounts of BLM molecules
into the cell cytoplasm by electropermeabilization gives rise to all the
characteristics of apoptosis. This situation is somehow reminiscent of
an apoptosis-like cell death with BLM presumably playing the role of
the relevant endonuclease.
Many facts support the hypothesis that BLM acts directly as an
endonuclease and can be considered as an apoptosis-mimetic drug, at
high concentrations.
(a) As indicated by Kuo (32), Sidik and Smerdon (33), and our
work with isolated nuclei (Fig. 8), BLM cuts chromatin preferentially
between nucleosomes. This is further confirmed by the fact that BLMtreated naked DNA does not show the nucleosomal ladder; only a
smear, resulting from random DNA degradation, can be detected if
DNA is exposed to very high doses of Fe-BLM (100 /J.M)for long
incubation times (1 h) (data not shown).
(b) Cobalt, an inhibitor of BLM activity, prevented DNA degrada
tion when added to BLM just prior to electric pulse delivery or when
preloaded into the cells. On the contrary, cycloheximide, a protein
synthesis inhibitor, was unable to prevent the BLM-induced DNA
fragmentation, whereas it inhibits the endonuclease-operated
DNA
abed
fragmentation (data not shown). To avoid the possibility that, in our
conditions, iron-catalyzed generation of reactive oxygen species
might lead to DNA damage by itself, we electroporated DC-3F cells
in the presence of 10 /XMFeCl2 or of 10 JAMFeCl.v No sign of
oligonucleosomal ladder was detected (data not shown).
The last control experiment was the direct exposure of chromatin to
BLM without any membrane crossing restriction. Isolated nuclei were
prepared from the DC-3F cells and incubated in the presence of 10 JU.M
Fe-BLM. Again, internucleosomal DNA degradation was detected
after incubations of 2 h (Fig. 8, Lane a) or even after only 5 min (Fig.
8, Lane b).
DISCUSSION
This study shows that BLM induces two distinct modes of cell
death. Cells electropermeabilized in the presence of low external
BLM concentration, i.e. 10 nM, became enlarged, multinucleated,
developed micronuclei, and displayed an arrest in G2-M. By contrast,
when electropermeabilized in the presence of a high external BLM
concentration, i.e. 10 JAM,they apparently exhibited all the morpho
logical and biochemical changes associated with apoptosis: cell
shrinkage; membrane blebbing; reduced cell fluorescence; and inter
nucleosomal DNA fragmentation. Until now, most of the studies car
ried out with BLM have been performed using nonelectropermeabilized cells treated with BLM concentrations in the micromolar range,
level at which the drug begins to be toxic in these conditions. Elec-
Fig. 8. Agarose gel analysis of DNA of DC-3F nuclei treated with H) /AMBLM. Lane
a, nuclei treated with 10 LAMBLM for 2 h; Lane b, nuclei treated with H) /AMBLM for 5
min: Lane c, untreated nuclei; Lane d, PhiX \14/Hac\\\.
5467
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HI I-OMYCIN,
AN APOPTOSIS-MIMETIC
fragmentation induced in the same cells by exposure to cisplatin or by
serum deprivation.
(c) The DNA fragmentation observed in our experiments is a very
rapid phenomenon: in as few as 30 s, the time at which excess cobalt
was added in order to prevent subsequent BLM activity, DNA frag
mentation was already achieved. Even in the case of the type III
apoptosis (34), induced by the rapid activation of a preexisting endonuclease, DNA fragmentation requires longer times.
(d) In contrast with results obtained with X-radiation (26), we found
that the radical scavenger dimethyl sulfoxide present before, during or
after BLM treatment did not impede the BLM-generated DNA frag
mentation (data not shown). Thus, the formation of free radical spe
cies was not implied in the BLM-induced apoptosis-like cell death,
supporting a direct effect of BLM itself.
(e) We made it sure that the endonuclease triggered by intracellular
pH lowering is not implicated in BLM-induced apoptosis-like cell
death. Indeed, the pH of Fe-BLM as well as the pH of the mixture
(S-MEM plus BLM) were brought to physiological pH = 7 and did
not deviate from control medium pH across the range of BLM con
centrations tested. Furthermore, Zn++ ions, known to inhibit the
apoptosis-relevant endonuclease (21), were unable to prevent DNA
fragmentation when introduced 30 s after the BLM into the electropermeabilized cells. The endonuclease-operated fragmentation would not
be that rapid, supporting again a direct action of BLM.
(/) It is known that one molecule of BLM can carry out 10 cycles
of BLM-catalyzed DNA cleavage (35) and that the ratio of doublestrand breaks versus single-strand breaks is one-sixth in the case of
DNA showing a nucleosomal structure (36, 37). According to these in
vitro data one can postulate that 3 X IO6 molecules of BLM can
generate 5 X 10'' double-strand breaks. A mammalian cell contains
approximately 20 X IO6 nucleosomes. The ratio of the number of
nucleosomes on the number of double-strand breaks theoretically
generated is compatible with the detection of the nucleosomal ladder
in our experiments.
Thus, beyond 3 X IO6 internalized BLM molecules (corresponding
to 10 ¡J.M
external BLM concentration), cells undergo an apoptosislike cell death with BLM acting instead of the relevant endonuclease.
This particular form of apoptosis obtained with high doses of BLM is
very interesting because our results show that (a) BLM actually
creates double-stranded DNA breaks in the cells; therefore, BLM
actually acts like a "mininuclease" inside the cell, (b) The morpho
logical characteristics of apoptosis are a consequence of DNA degra
dation, as shown in the Chinese hamster DC-3F cells and in the human
A-253 cells. These results are in agreement with those of Arends et al.
(38) who demonstrated that nuclei incubated with an exogenous en
donuclease show the major nuclear morphological changes associated
with apoptosis. (c) DNA digestion seems to play an integral role in
causing cell death rather than just being a response to the apoptotic
cell death, (d) Finally, since the same BLM concentration leads to the
production of the oligonucleosomal ladder with nuclei and permeabilized cells, the hypothesis suggesting that the plasma membrane acts
as a barrier against BLM entry into cells is greatly reinforced.
When cells were electropermeabilized in the presence of 10 HM
external BLM, 3 X IO3 molecules of BLM were introduced into the
cell cytoplasm (9). Treated cells displayed an arrest in the G2-M phase
of the cell cycle, became enlarged, binucleated, and showed micronuclei. These results are in agreement with previous studies showing
that nonpermeabilized BLM-treated cells became enlarged and polynucleated (39^1) and displayed a G2-M blockage (42^16). Some
authors consider that repair systems cannot maintain genome integrity
above a critical threshold of lethal BLM-induced damage on DNA. In
these conditions, cells are unable to carry out a normal cell cycle (46).
This situation is reminiscent ofthat observed in cells treated with low
DRUG
doses of ionizing radiations which display mitotic death and die after
abnormal divisions (24, 25).
Since BLM is already known to induce chromosomal aberrations
both in vitro and in vivo (46-49), we suppose that G2-M blockage and
cell cycle abnormalities seen with cells electropermeabilized in the
presence of low doses of BLM are the result of the accumulation of a
critical number of double-strand breaks generated by BLM beyond the
efficiency of the repair systems. Taking into account the same in vitro
data as here above, one can postulate that the internalization of 3000
molecules of BLM would generate a maximum of 5000 double-strand
breaks in the cell genome.
We have recently demonstrated that, in the absence of cell electropermeabilization, low amounts of BLM reach the cytoplasm within
a wide range of BLM concentrations.3 Thus, in these conditions, it is
possible to obtain the biochemical and morphological changes shown
with 10 nin BLM and electropermeabilization.
Until our present study, the relationship between the number of
internalized BLM molecules and their biological effects had never
been studied. Considering the small number of BLM molecules re
quired to produce the observed effects, our results strongly support the
hypothesis that only the BLM-generated DNA double-strand breaks
are responsible for BLM cytotoxicity. Moreover, we demonstrate that
a unique BLM activity, i.e., its endonuclease ability, results in two
mechanisms of cell death which are closely related to the number of
BLM molecules present inside the cell and, in fact, to the number of
DNA double-strand breaks generated.
ACKNOWLEDGMENTS
The authors would like to thank Bernadette Léonfor the excellent technical
assistance, Dominique Coulaud for help in electron microscopy, Zohair Mishal
and Sophie Lafosse (Service commun de cytométrie,UPS 47, Villejuif) for
assistance with flow cytometry assays, and Lorna Saint-Ange for linguistic
revision of the manuscript. We also thank Professor John S. Lazo for a critical
reading of the manuscript.
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Bleomycin, an Apoptosis-mimetic Drug That Induces Two Types
of Cell Death Depending on the Number of Molecules
Internalized
Omar Tounekti, Géraldine Pron, Jean Belehradek, Jr., et al.
Cancer Res 1993;53:5462-5469.
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