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(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 5462 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research. BLEOMYCIN. AN APOPTOSIS-MIMET1C 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 5463 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research. 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- 5464 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research. 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. 5465 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research. BLEOMYCIN, hg AN APOPTOSIS-MIMETIC DRUG 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 . 5466 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research. Ill lOMYCIN. b A c AN APOPTOSIS-MIMET1C DRUG 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 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research. 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. 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Cancer Res.,31: 2004— 2007, 1971. 49. Paika. K. D., and Krishan. A. Bleomycin-induced chromosomal aberrations in cul tured mammalian cells. Cancer Res.. 33: 961-965, 1973. 5469 Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1993 American Association for Cancer Research. 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. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/53/22/5462 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. 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