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[CANCER RESEARCH 48, 4982-4992, September 1, 1988] Selective Loss of Mitochondrial DNA after Treatment of Cells with Ditercalinium (NSC 335153), an Antitumor Bis-intercalating Agent Evelyne Segal-Bendirdjian,1 Dominique Coulaud, Bernard P. Roques, and Jean-Bernard Le Pecq Unité de Physicochimie Macromoléculaire(CNRS UÀ147, INSERM U140) ¡E.S-B., J-B. L.] and Laboratoire de Microscopie Cellulaire et Moléculaire[D. C.J, Institut Gustave Roussy, 94805 Villejuif Cedex; and Départementde Chimie Organique (CNRS VA 498, INSERM U 266), UER des Sciences Pharmaceutiques et Biologiques, 4, Avenue de l'Observatoire, 75006 Paris; France ABSTRACT Ditercalinium (NSC 335153), a bifunctional intercalating molecule with antitumor activity, is found to express its toxicity through a mech anism of action completely different from that of other monointercalating agents. Electron microscopic observation of ditercalinium-treated cells shows a drastic alteration of mitochondria! structure. Cells deficient in mito chondria! respiration (GSK3 cells) isolated by A. Franchi et al. (Int. J. Cancer, 27: 819-827, 1981) are about 25-fold more resistant than cells deficient in glycolysis (DS7 cells) isolated by J. Pouysséguret al. (Proc. Nati. Acad. Sci. USA, 77: 2698-2701, 1980). Revenants have been isolated from GSK3 cells. In these cells, the sensitivity to ditercalinium has been recovered with mitochondria! respiration. Ditercalinium treat ment of 1.1210 leukemic mouse cells leads to a specific elimination of mitochondria! DNA detected by DNA-DNA hybridization. No measur able alteration of nuclear DNA is observed. In contrast, the monomeric analogue of ditercalinium only alters nuclear DNA and does not change the mitochondria! DNA content. The activity of cytochrome c oxidase, an enzyme which contains a subunit coded by the mitochondria! DNA, decreases exponentially in treated cells with a half-life of 24 h, corre sponding to the turnover of the enzyme. These results suggest that ditercalinium exerts a specific cytotoxic effect at the level of mitochondria! DNA. This action could account for the delayed cytotoxicity induced by this compound. INTRODUCTION In an effort to obtain new antitumoral agents with high binding affinity for DNA, various bifunctional intercalators have been synthesized (1-3). In the series of 7H-pyridocarbazole, dimerization yielded molecules which bis-intercalate into DNA, depending on the nature, the flexibility, and the inni/.ation state of the linking chain. Among these molecules, diter calinium, which bis-intercalates into DNA through the large groove (4), displays anticancer activity on a variety of animal tumor models (2, 5). Several observations suggested that the mechanism of action of mono- and bis-intercalators are different (6, 7). The dimers are about 10- to 40-fold more toxic than their respective monointercalating counterparts; they are character ized by a delayed cytotoxicity: treated cells continue growing for seven to eight generations before arrest; they do not provoke a block of cell cycle progression in the G2 + M phase of the cell cycle, as other intercalating agents. In a bacterial study, ditercalinium has also been shown to be cytotoxic on Escherichia coli polA mutants and not on polA uvrA double mutants, suggesting that this dimer which binds reversibly to DNA, is able to induce in vivo DNA conformational changes recognized by the uvr ABC repair system in E. coli. This recognition could lead to a futile and abortive DNA repair process (8). Received 1/11/88; revised 5/24/88; accepted 6/2/88. 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. 1To whom requests for reprints should be addressed, at the Institut Gustave Roussy, 39 rue Camille Desmoulins, 94805 Villejuif Cedex, France In addition, preliminary electron microscopy analyses re vealed that the cytotoxic effects of ditercalinium is accompanied with alterations of the mitochondria! morphology (9). Because recent reports underlined frequent alterations of mitochondria in carcinoma cells and had suggested that mito chondria could represent a specific target for antitumor drugs (10, 11), we have investigated the effect of ditercalinium on mitochondria. In this study, we present evidence that ditercalinium causes a selective degradation of mitochondrial DNA. MATERIALS AND METHODS Conditions of Cell Culture. The Chinese hamster lung fibroblast clone 023 and their derivatives DS7 and GSK3 were isolated by Pouysségur et al. and their properties are described in previous works (12, 13). Briefly, DS7 and GSK3 are derivatives of 023 cells after ethylmethanesulfonate mutagenesis and tritium suicide selection. The DS7 cell line has a total block in the glycolytic pathway. The GSK3 cell line maintains a permanent high rate of glycolysis. Those cells were grown in Dulbecco's modified Eagle medium (GIBCO) supplemented with 10% fetal calf serum and antibiotics (50 lU/ml penicillin and 50 Mg/ml streptomycin). L1210 cells were main tained in culture in RPMI 1640 Medium (Flow Laboratories), supple mented with fetal calf serum (9%), penicillin (100 lU/ml), streptomycin (50 Mg/ml), and ,8-mercaptoethanol (60 MM).All cells were maintained at 37°Cin a 5% CO2 atmosphere. Drugs. Ditercalinium (2,2'-[4,4'-bipiperidine-l,l'-bis(ethane-l,2diyl)]bis( 10-methoxy-7H-pyrido[4,3-c]carbazolium d¡chloridedihydrochloride), a 7H-pyridocarbazole dimer (NSC 335153) was from the Roger Mellon Laboratory. The monomeric derivative, 2-[2-(Ar-piperidyl)ethyl]-10-methoxy-7H-pyrido[4,3-c]carbazole chloride hydrochloride, and the dina-rii- non-antitumor analogue with a longer linking chain between the two intercalating rings, l,3-bis|Ar-[2-(10-methoxy7H-pyrido[4,3-c]carbazole-2-yl)ethyl]-4-piperidyl)propane tetramesylate, were synthesized as previously described (1, 2). All drugs were dissolved in water, and sterilized by filtration through a 0.2-fim polycarbonate membrane (Nucleopore Corp., Peasanton, CA). Drug concentrations, in the sterile solutions, were determined by ab sorption spectroscopy. These solutions were stored at 4"C, in the dark. Immediately before use the solutions were serially diluted to the appro priate concentration in culture medium. Drug Exposure and Cell Survival. Throughout this study, the effects of the drugs were determined on exponentially growing cells. For drug treatment of the Chinese hamster lung fibroblasts, cells were seeded the day before in 35-mm diameter Petri dishes at a density of 5 x 10" cells/dish for the 023 and DS7 cell lines and 1 x 10s cells/ dish for the GSK cell line and left overnight for the attachment. The medium was then replaced either with 1 ml of fresh medium (controls), or with 1 ml of medium containing the drug at the indicated concentra tions. After 3 h of incubation at 37°C,the drug was washed off by rinsing the dishes twice with 3 ml of medium and trypsinized for the determination of the colony forming ability. About 500-1000 cells were plated in triplicate in 60-mm diameter Petri dishes containing 5 ml of medium. The colonies were counted about 1 week later. In these conditions, the cloning efficiency of the controls was about 40% for 023 and DS7 lines and 30% for GSK. For drug treatment of LI 210 cells, exponentially growing cells (1 x 10s cell/ml) were incubated in medium containing the drug for 24 h at 4982 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research. LOSS OF MITOCHONDRIA!. 37°C.The cells were then centrifugea and washed twice with phosphate buffered saline (2.7 mM KC1, 1.5 mM KH2PO4, 137 mM NaCl, 8.1 mM Na2HPO.i) and once with RPMI before dilution in fresh medium at the initial density. At different times, after washing, aliquots were removed for DNA analysis or cytochrome oxidase activity determination. The L1210 cells were treated either with 1.4 fiM of the monomeric analogue, or with 0.14 ^M of ditercalinium. These doses were known to be toxic doses giving less than 1% of surviving LI 210 cells (7). Total cell numbers in the different cultures were determined by means of a Coultronic Coulter Counter. Lactic Acid Determination in 023, DS7, GSK3 Cells. 72 h after treatment the cells were washed twice with DMEM2 without serum and incubated at zero time with DMEM2 supplemented with 10% dialyzed fetal calf-serum. At different times, the medium of four dishes (two control and two treated ones) was removed, filtered, and assayed for lactate content using the Boehringer lactic acid assay kit following the supplier's instructions. The cells in each dish were counted. The content of lactic acid was expressed in mg/106 cells. Electron Microscopy. For the ultrastructural studies, the cells were grown in 90-mm dishes. After 3 h of treatment, the drug was removed, the cells were washed twice and refed with fresh medium for 3 days. After this postincubation time, the cell monolayers were fixed at room temperature by adding 1.5% glutaraldehyde (6% in volume of 25% solution, from Merck) to the culture medium. In this solution, the cells were scraped off the plastic and centrifuged. The pellet was resuspended in new fixative containing Sifirensen phosphate buffer (0.66 mM, pH 7.4) with 1.5% glutaraldehyde for l h at 4°C.After 2-h washing with buffer, cells were postfixed in 2% OsO4 in the same buffer. Dehydration in graded ethanol and propylene oxide, Epon embedding, and uranyllead staining were classical. Observations were made in the Zeiss EM902; optimal contrast was obtained by selecting the elastic electrons with the slit of the spectrometer. Extraction of Total Cellular DNA from L1210 Cells. Cells (5 x IO7 to 1 x IO8cells) were washed twice with phosphate buffered saline and kept at —70°C until used. Cell pellets were then suspended in 5 volumes of 0.15 M NaCl-10 mM EDTA (pH 7.0) were lysed in 1% sodium dodecyl sulfate at 60°Cfor 15 min. The lysate was incubated for 2 h at 37°Cwith proteinase K (100 Mg/ml), deproteinized once with phenol, once with phenol-chloroform-isoamylalcohol (25:24:1), and once with chloroform-isoamylalcohol (24:1). The DNA extract was then dialyzed overnight against Tris-HCl IO"2 M, EDTA IO"3 M (pH 7.5). Then the DNA extract was incubated with 200 Me of heat treated RNase A/ml (15 min at 100°C)for 2 h at 37'C. After deproteination, as indicated above, NaCl was added (100 mM) and the preparation was precipitated overnight at —20°C with 2 volumes of 95% ethanol. The precipitate was then solubilized in Tris-HCl 10~3 M, EDTA 10"3M (pH7.5). DNA formamide, with nick-translated 32P-labeled heat-denatured mouse mt DNA probe (0.2 Mg;5-9 x IO7cpm/Mg). The filters were washed several times at room temperature in 2x SSC - 0.1% sodium dodecyl sulfate and several times at 62°Cin the same buffer. Filters were then autoradiographed at —70°C with Kodak X-Omat RP film and a Dupont Cronex Lightning-Plus intensifying screen. Probes. The mitochondria! DNA probe (a gift from Dr. D. A. Clayton, Stanford University, CA), consisted of the entire mouse mi tochondria! DNA genome with plasmid pACYC 177 as the vector cloned into Escherìchiacoli HB101. The nuclear DNA probe (pMRB 1.1), generously given by Dr. M. Meunier-Rotival, UnitéINSERM 56, Hôpital de Kremlin Bicêtre, France, contained interspersed repeats of mouse DNA (16). Cytochrome Oxidase and Protein Determination. For cytochrome oxidase activity, cultures were centrifuged, washed twice with 0.25 M sucrose, and resuspended in water. The cell suspension was transferred to a Dounce homogenizer and a few up-and-down strokes of the piston were sufficient to break all the cells. The homogenates obtained were used as source of enzyme. Cytochrome oxidase was measured in the following incubation me dium: 0.1 ml of 0.1 M potassium phosphate buffer (pH 7.0); 0.07 ml of reduced ferrocytochrome c 1% (type III, Sigma). The blank cuvet was oxidized with 0.01 ml potassium ferricyanide 0.1 M. Immediately before the assay, 0.2 ml of a suitable amount of homogenate was added to the sample cuvet to initiate the reaction. The volume of the incubation medium was adjusted to 1.0 ml with distilled water. Temperature of the reaction was 38°C.The decrease in absorbancy was measured at 550 nm during 30 min. The concentration of protein was determined by the method of Bradford using bovine serum albumin as reference standard (17). RESULTS Comparison of Ditercalinium Cytotoxicity on Cells Deficient in Glycolytic or Respiration Pathway. Pouysséguret al. (12, 13) recently characterized cell lines of Chinese hamster lung fibroblasts deficient in either the glycolytic or the respiration path way. The DS7 cell line is deficient in the phosphoglucoisomerase enzyme and derives all its energy from mitochondria! res piration. The GSK3 cell line is deficient in respiration and derives all its energy from glycolysis. These cells were used to estimate the role of mitochondria in controlling drug cytotoxicity. Fig. 1 shows the cytotoxicity of ditercalinium of both these cell lines compared to the parental line 023. 100 Southern Blot Hybridization. Hindlll restriction enzyme was pur chased from Amersham. DNAs were digested for 3 h with Hindlll according to the supplier's recommendations. Digests were analyzed by gel electrophoresis on 0.8% agarose gel in Tris-borate EDTA (pH 8.0) containing 0.5 Mfi/mlof ethidium bromide. Electrophoresis was carried out in a horizontal slab gel apparatus at 30 V for 18 h. After electro phoresis, the DNA fragments were acid depurinated in 0.25 M HC1 twice for 15 min, then denaturateli by soaking the gel in several volumes of 0.4 M NaOH for 30 min. After neutralization of the gel in several volumes of a 0.5 M Tris-hydrochloride (pH 7.4)-3 M NaCl solution for 30 min, the gel was blotted onto nitrocellulose filter by the method of Southern (14). The nitrocellulose filters were moistened with 5x SSC (Ix SSC is 0.15 M NaCl plus 0.015 M sodium citrate), and prehybridized for 2 to 4 h at 42°Cin solution containing 5x SSC, 5x Denhardt io GSK3 O) C C o solution (15), 0.1% sodium dodecyl sulfate, salmon sperm DNA (0.35 mg/ml), and 50% formamide. Hybridization was carried out overnight at 42°Cin solution containing 5x SSC; Ix Denhardt solution, 0.1% 30 sodium dodecyl sulfate, salmon sperm DNA (0.1 mg/ml), and 50% 2The abbreviations used are: DMEM, Dulbecco's modified Eagle's medium; mt DNA, mitochondrial DNA; <T;,-, thèdose required to inhibit the cloning efficiency to a factor of 0.37. Fig. 1. Effects of ditercalinium on cell survival as a function of drug concen tration. After 3 h of drug treatment, the cell cloning efficiency were measured as described in "Materials and Methods." Insert, effect of ditercalinium on the DS7 cell survival. Curve, average of three independent experiments. 4983 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research. LOSS OF MITOCHONDRIAL The DS7 cell line appears extremely sensitive to ditercalinium. The CE37, after only 3 h of treatment, was found to be 0.26 (¿M. In the same conditions, the GSK3 cells were 25-fold less sensitive than DS7 (CE37 = 6.24 UM). The response of the 023 parental cell line is close to that of the DS7 cells (CE37 = 0.52 /IM). As a control of the specificity of this observation, the cytotoxicities of the monomeric analog and the dimeric nonanalog antitumor were measured. Values of CE37 are presented in Table 1. Interestingly, the three cell lines elicit similar sensitivity either for the monomeric derivative or for the dimeric analog of ditercalinium. In addition, the ditercalinium analog and the monomeric derivative are much less toxic than diter calinium on the two lines 023 and DS7. In addition the GSK3 cell line does not appear to be more resistant than the 023 and DS7 lines to these two analogs. A delayed arrest of cellular growth in observed after ditercal inium treatment with all these cell lines. This is well indicated by the appearance of small size colonies as previously observed (6, 7). Even the most sensitive cell line which is unable to make glycolysis displays such a delayed cytotoxicity effect. Measurement of Glycolysis after Treatment. Mitochondria! inactivation was expected to induce a cellular adaptive response leading to a stimulation of glycolysis. Therefore, the kinetics of lactate formation, a product of anaerobic glycolysis, were meas ured on the three cell lines after ditercalinium treatment, at a toxic (1.4 A<M)and a nontoxic dose (0.14 ¿tM). Cells were treated for 3 h with ditercalinium. The ditercali nium was then washed out and the accumulation of lactate measured in the medium as a function of time. Fig. 2 shows that after ditercalinium treatment, an accumulation of lactic acid is observed in the parental 023 cell culture medium. This indicates that there is a stimulation of glycolysis in the parental cells. However, this stimulation does not occur in DS7 cells which are deficient in the glycolytic pathway, nor in GSK3 cells which maintain a permanently high glycolysis rate because of a deficiency in the respiration pathway. The kinetics of lactate formation were also measured 3 days after treatment of the 023 cell line as shown in Fig. 3/4. Glycolysis is clearly stimulated but interestingly, the glycolysis stimulation is also observed after treatment with ditercalinium at the nontoxic dose. Con trastingly, the monomeric derivative has no effect on glycolysis, even at toxic doses (Fig. 3B). Ultrastructural Studies. Mitochondria! ultrastructure of ditercalinium-treated cells substantiates further the biochemical data. Fig. 4, A and B, and Fig. 5, A and B, show the ultrastructure of 023 and DS7 control cells, respectively. Mitochondria gen erally appeared oblong with cristae that traversed the entire width of the organelle. The density of the mitochondria! matrix was greater than that of the surrounding cytoplasm. Treatment with ditercalinium induced important structural changes of 023 cell mitochondria (Fig. 4, C and D). Most of them appeared enlarged and swollen and cristae were lacking or segregated at the periphery of a low density mitochondrial matrix. A few mitochondria contained small electron dense particles. The effect of ditercalinium on the very sensitive DS7 cells was more puzzling (Fig. 5, C-E). Mitochondria looked to be less modified than those of 023-treated cells in their shape, size, cristae content, and matrix density, although annular-shaped electron-dense bodies were frequently observed (Fig. 5Z>).Rare swollen mitochondria with a clear matrix were found beside normal-looking ones (Fig. 5/:"). In addition, treated cells pos sessed a quantity of lysozome-like Table 1 Cloning efficiency (CE31) of the parental O23 and the two variant DSF and GSK3 sublines after treatment with ditercalinium and its monomeric and dimeric analogs Ditercalinium Monomeric derivative Dimeric analog 1Means ±SE. 6 023 (MM) 0.52 ±0.05° 21.5 ±0.02 5.5 ±0.5 DS7 (MM) 0.26 ±0.03 18.3 ±2.0 2.34 ±0.2 DNA Õ OS e m 04 £ o GSK3 (MM) 6.24 ±0.06 11.7 ±0.1 6.5 ±0.6 < <X2 TIME - l "s \ large cavities containing (h) B 4 - GSK 0.4 023 < 0.2 01 24 48 TIME 72 96 23456 TIME (h) ih) Fig. 3. Rates of glycolysis of Chinese hamster fibroblasts 023. 3 h after Fig. 2. Lactate accumulation in Chinese hamster fibroblasts medium with (•, treatment, the cells were washed and grown in DMEM supplemented with 10% •,A) and without (O, D, A) ditercalinium treatment. 023 (•,O); GSK3 (•.D); fetal calf serum. 3 days later, cells were washed and changed to fresh medium DS7 (A, A). 3 h after treatment, the cells were washed and grown in DMEM containing 10% of dialysed fetal calf serum at time 0. Lactic acid secreted into medium was measured as described in "Materials and Methods." A, ditercalinium: supplemented with 10% fetal calf serum. Lactic acid secreted into medium was measured as described in "Materials and Methods." Each point was carried out •,0.14MM;O, 1.4 MM.fi, monomeric derivative: A, 2.7 MM;A 27 MM.Untreated in duplicate. cells: x. Each point was carried out in duplicate. 4984 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research. LOSS OF MITOCHONDRIA!. DNA Fig. 4. Electron micrographs of 023 cell line: control cells (. I and B); ditercalinium treated cells (C and D); monomeric derivative treated cells (/•.'). Cells were treated 3 h either with ditercalinium (1.4 /IM) or with the monomeric derivative (27 //M), then they were washed and refed with fresh medium for 72 h. After ditercalinium treatment, markedly can /:') be and identified by theand persisting peripheral membranes; treatment with the monomeric analogue does not induce alterationstheofmitochondria mitochondria are(/;'). Bars, 1swollen imi (.I. and C, and O.S ;<n>(B /'). 4985 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research. LOSS OF MITOCHONDRIA!, > • . • ^. t DNA .-, '" i 3'Ä *» •\ Jt • m •:•&' .Ã->'¿*ág3Õ W %*» Fig. 5. Electron micrographs of DS7 cell line: control cells (A and fi); ditercalinium treated cells (C, D, and E); monomeric derivative treated cells (F). The conditions of treatment were the same as in Fig. 4. After ditercalinium treatment, some mitochondria with normal structure remain (E). Dense annular bodies are frequent in the matrix. The large vesicles, filled with irregular dense material, are much more larger and numerous in treated cells (C, D, and E). No alterations are induced by the monomeric analogue (F). Bars, I pm (A, C, and F) and 0.5 urn (B, D, and E). 4986 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research. LOSS OF MITOCHONDRIAL filamentous material very similar to that found in a swollen mitochondria, as well as very dense irregular bodies (Fig. 5C), and membraneous residues either isolated, or connected to the membrane limiting the vesicle. Such single-membrane-limited vesicles were also present in the control cells, although rare and small. Their identification as strongly altered mitochondria is possible but uncertain. Those alterations were not seen after treatment of cells with equitoxic doses of the corresponding monomer (Fig. 4E and Fig. 5F). In control GSK3 cells, inactivation of the respiration pathway was associated with an alteration of the structure of mitochon dria. Many mitochondria appeared devoid of cristae (Fig. 6/1). So, no additional modification of the ultrastructure of mito chondria could be seen after treatment of those cells with ditercalinium (Fig. 6B). To further establish the specific relation between the diter calinium resistance of GSK3 cell lines and its mitochondria! deficiency, revenant cells were isolated. Cells were regularly reseeded. After 95 passages a beginning of reversion was ob served as glycolytic activity decreased. After 117 passages (about 14 months), a complete reversion of the GSK3 phenotype was observed. The growth rate and glycolytic activity could not be distinguished from that of control cells. In addition, the normal appearance of mitochondria under electron microscope was recovered (Fig. 6C). This reversion was accompanied by an increase in ditercalinium sensitivity. Revenant cells became as ditercalinium sensitive as the reference cell line 023 (Fig. 7/4). In addition glycolysis could be stimulated in these revenant cells by ditercalinium treatment (Fig. IB). Mitochondria! DNA Content of 1.1210 Cells Treated with Ditercalinium. The fate of mt DNA after ditercalinium treat ment was investigated on the murine leukemic LI210 cell line using a DNA probe containing the entire mouse mt DNA. Total cellular DNA was extracted during and at different times fol lowing ditercalinium treatment. DNA was treated with Hindlll restriction enzyme and analyzed using agarose gel electrophoresis. After transfer to nitrocellulose filter according to the Southern technique (14), DNA was hybridized with either a mt DNA probe or with a nuclear DNA probe. Hindlll digestion gave a single major band for mt DNA. We selected as a nuclear probe a cloned interspersed repeti tive sequence. This allowed us to analyze a global effect on nuclear DNA rather than a specific effect on a given gene. In that case a smear was observed on the autoradiogram, as previously shown (16). Results of these experiments are shown in Fig. 8, A and B. During the first 24 h of treatment with ditercalinium (Fig. 8Ä), no significant variation of mt DNA was observed. However 24 h after washing out ditercalinium, the mt DNA band almost completely disappeared. No reappearance of mt DNA was observed even in cells maintained in culture for 6 days. Con trastingly, the autoradiogram obtained with the nuclear probe did not reveal any alteration of nuclear DNA after ditercalinium treatment. Treatment with the ditercalinium monomeric analog showed a very different effect on mt DNA as well as on nuclear DNA (Fig. 8/1). No variation was observed in the content of mt DNA after treatment. But a clear modification of nuclear DNA was observed after treatment with the monomer analog. The nuclear DNA probe revealed the appearance of low molecular DNA fragments. A reduction of the bulk DNA molecular weight was also observed on the gel after ethidium staining, with appearance of a pattern characteristic of subnucleosomal material (18, 19). DNA Effects on Cytochrome c Oxidase Activity of LI210 Cells. Because of the observed disappearance of mt DNA after diter calinium treatment, we measured the activity of cytochrome c oxidase which contains a subunit coded by mt DNA as shown in Fig. 9. Cytochrome c oxidase activity decreased exponentially im mediately after treatment with an half-life of 24 h. A small and significant decrease was also observed after monomer treatment but, after washing out the compound, the activity of the enzyme returned to that of control cells 24 h after the wash. DISCUSSION Ultrastructural studies of 023 cells treated with ditercalinium reveal a striking alteration of mitochondria with complete loss of cristae. The effect is less clearly interpretable in DS7 cells. There were less altered mitochondria in those cells than in 023 ones. Probably DS7 cells, in which metabolism depends only on mitochondria! respiration, are more sensitive than 023 ones to fewer mitochondria! alterations. Similar structural changes have already been observed after treatment with ditercalinium of another cell line in culture (9) and they looked similar to those which have been observed after long term treatment with ethidium bromide or chloramphenicol (20-22). However, contrarily to what was observed after ethidium bromide treatment, the mitochondria! structural changes that resulted from diter calinium treatment were irreversible even after removal of the drug. In the case of ethidium bromide, the morphological alterations showed a gradual return to almost normal mito chondria! ultrastructure after the removal of the drug. Contrast ingly, the monomeric analog of ditercalinium does not induce such an effect. These mitochondria! alterations are accom panied with a significant stimulation of glycolysis of ditercali nium treated cells, which is not observed with the monomeric analog. The mitochondrial alterations are associated with an almost complete loss of the mitochondrial DNA. As expected the loss of mitochondrial DNA is accompanied with the disappearance of the activity of the cytochrome c oxidase, an enzyme which contains a subunit coded by mt DNA. Interestingly, the rate at which cytochrome c oxidase activity disappears (ii/2 = 24 h) is close to that reported for the turnover of this enzyme, measured after chloramphenical treatment (21). One must also underline that the loss of cytochrome c oxidase activity is irreversible. In contrast, when cells are treated with the monomeric analog of ditercalinium (our results), with chloramphenicol or ethidium bromide (21, 23), the cytochrome c oxidase activity is fully recovered after removal of the drugs. After ditercalinium treatment, the loss of mt DNA is observed in the absence of detectable nuclear DNA alterations. However, treatment of cells with the monomeric analog results in altera tion of nuclear DNA. After treatment with the monomeric derivative, subnucleosomal bands of DNA corresponding to mono- di- and oligo-nucleosomes appear on agarose gel, sug gesting that selective degradation at the level of internucleosomal DNA occurs (18, 19). Such results are consistent with the observations of Markovits et al. (24) which showed that diter calinium did not cause any DNA protein-associated breaks in the nuclear DNA. Such breaks were clearly observed with antitumor monointercalating agents (25) and the monomeric analog of ditercalinium.3 It is therefore very likely that the primary effect of ditercali3J. Markovits, personal communication. 4987 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research. LOSS OF MITOCHONDRIA!. DNA iT 9'T ^ ' f- cf \ m^i *^ A =- A* _ -' K. fc »• ' « •".-•-. • :: ' • " ^*" •%»:.: i? ..X* i?*"02 '_v ' A» • ": :.#ÃŒP T:V,;;-- ^^ •• ' -:- '"' *1 :<r4A Fig. 6. Electron micrographs of GSK3 cell lines:. I. GSK3 in the 27th passage; B, GSK3 in the 27th passage treated with 1.4 ¿IM ditcrcalinium (the conditions of treatment were the same as in Fig. 4); C, GSK3 in the 117th passage, after the reversion of the phenotype. Mitochondria of GSK3 cells at early passages are swollen and have short, round cristae. Treatment with ditercalinium does not modify their morphology. Mitochondria of GSK3 cells revertants appear similar to those in control 023 parental cells. Bars, 1 //in. 4988 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research. LOSS OF MITOCHONDRIAL leads to a futile and abortive repair cycle (8). It is possible, therefore, to speculate that the DNA structural alteration may also be recognized in mitochondria. The existence of a DNA repair process at the level of mt DNA has been investigated (27, 28). This question is of impor tance because several compounds and carcinogens react pref erentially with mt DNA, probably because mt DNA is not associated with proteins (29-32). For instance, it was reported that the reaction of dihydrodiol-epoxide derivative of benzo(a)pyrene with mt DNA is 40 to 90 times greater than with nuclear DNA (29). Several authors have reported that there is no DNA repair at the level of mt DNA (27). It was suggested that damaged mt DNA is degraded and leads to a preferential replication of nondamaged DNA. Replacement of mt DNA instead of its repair would represent an alternative defense mechanism.5 If 100 GSK3 O C .5 u 10 O) O "3 ditercalinium mt DNA complexes were recognized as a defect, as in bacteria, one could understand how such a compound leads to rapid mt DNA elimination. Such a process is observed with many compounds leading to the "petite" mutation induc B 95 th co 0.8u"b 3 06O)LU \ /• ^/_..,„ 0.4 .<§0,m- 123456 123456 TIME DNA 1 23456 (h) Fig. 7. A, effects of ditercalinium on GSK3 and GSK3 revenant cell lines survival. (Il) GSK3 cells were treated during their 27th passage (about 3 months after thawing) and (O) during their 117th passage (about 14 months after). (+) cell survival of treated 023 cells. Each curve represents an average of three independent experiments. H. rates of glycolysis of GSK3 cells. (D) GSK3 control cells, (•)3 h ditercalinium treated GSK3 cells ( 1.4 MM),(x) 023 control cells, (+) ditercalinium treated 023 cells (1.4 MM).Each point was carried out in duplicate. nium is at the level of mt DNA and that the irreversible mitochondria! alterations result from the irreversible loss of mt DNA. A compound like chloramphenicol which specifically prevents the synthesis of mitochondrial proteins causes mor phological, as well as metabolic, alterations very similar to that of ditercalinium, but these alterations are readily reversible (21, 22). Interestingly, under continuous exposure to chloramphen icol, a delayed toxicity is observed which resembles the one observed after ditercalinium treatment. Induction of a delayed cytotoxicity might represent a common feature of drugs acting on mitochondria. This delayed cytotoxicity was also observed in vivo. In tumor-bearing mice an unusual delayed cytotoxicity appeared. In Phase I clinical trials, ditercalinium caused lethaldelayed hepatotoxicity at therapeutic antineoplastic doses (26). The causes of this hepatotoxicity are under investigation. Some preliminary results4 suggest that this toxicity is related to the pharmacokinetic characteristics of the molecule and its concen tration in liver. Presently the mechanisms leading to mt DNA loss after ditercalinium treatment are unknown. However, it has been shown that ditercalinium induces a structural deformation on DNA which is recognized in bacteria by the repair system and 4 R. Fellous, D. Coulaud, I. El Abed, B. P. Roques, J. B. Le Pecq, E. Detain, and A. Gouyette, manuscript submitted for publication. tion in yeast (33). Comparison of the activity of ditercalinium and other drugs on cells deficient either in mitochondrial respiration or in glycolysis is of importance in studying the role of mitochondria in determining cytotoxicity. Inactivation of mitochondrial me tabolism in GSK3 cells results in increased resistance to diter calinium. This resistance appears correlated with mitochondrial deficiency since reversion of the mitochondrial defect in the GSK3 cells is associated with a parallel increase of ditercali nium cytotoxicity. In contrast, the lack of glycolysis in respi ration proficient cells (DS7) does not cause a significant in crease of ditercalinium toxicity. Furthermore, ditercalinium toxicity on DS7 cells still appears delayed although these cells are unable to produce energy through glycolysis. This observa tion suggests that the primary effect of ditercalinium must be mainly at the mt DNA level and not directly on the enzymes of the respiratory chain. If ditercalinium was able to directly inactivate the respiration chain, DS7 cells would be expected to stop growth immediately. Indeed, when DS7 cells have been treated with oligomycin, a specific inhibitor of mitochondrial ATPase, the DS7 cells die within l h (13). However, if mt DNA is the major toxic event after ditercalinium treatment, mito chondrial respiration could be expected to decline at a rate corresponding to the turnover of the respiratory enzyme units coded by mt DNA. These variant cell lines would therefore be extremely useful in demonstrating the role of mitochondrial functions in drug action. Chen et al. (34) have proposed that the membrane potential of mitochondria in some tumor cells might be higher than in normal cells. The mitochondria of tumor cells could therefore concentrate lipophilic cations such as rhodamine 123 and dequalinium (10, 11). Like dequalinium, ditercalinium, which bears at least two positive charges on its quaternary ammonium groups, because of the membrane potential, could also be con centrated by mitochondria. However, it cannot be excluded that ditercalinium is also able to induce some defects at another level, for instance on nuclear DNA. Tráganos et al. (35) have found an increase of denaturated DNA in nucleus after diter calinium treatment and Markovits et al. (24) have found a condensation of chromatine which was not observed in resistant cells. Clearly, the cellular action of ditercalinium is different from * E. T. Snow, S. Mita, and L. A. Loeb, Congress Abstract Mittelwhir, 1985. 4989 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research. LOSS OF MITOCHONDRIA!. DNA TI after wash C 244872 TIME (h) after wash TIME (h) after wash ME Ch! C 24 48 72 C 24 48 72 kb — 23.1 _ 94 _ 65 . 43 23 2.0 — 1.0 — 0.5 electrophoresis hybridization nuclear B ME(h)withdrug3 TI 6 9 24afterwash62448 probe ME(h)withdrug3 TI 6 924afterwash62448 mt probe TIME(h)withdrug3 2448 6 9 24afterwash6 _ 1 .0 _ 0.5 hybridization electrophoresis nuclear probe mt probe Fig. 8. Electrophoresis and hybridizations with either a mt DNA probe, or a nuclear DNA probe to the Hindlll treated cellular DNA prepared from I.I 2 Id cells. A, cells were treated 24 h with 1.4 >iMof the monomeric derivative. DNA extractions were made 24, 48, and 72 h after removing of the drug. Lane C, DNA from nontreated cells. The DNAs were treated for 3 h with Hindlll. Digests were analyzed by gel electrophoresis. The gel was then blotted onto nitrocellulose niter and hybridized with the 32P-labeled mt DNA probe and autoradiographed during 6 days. The niter was then dehybridized and rehybridized with a "P-labeled DNA nuclear probe containing repetitive sequences. The filter was then autoradiographed. B, cells were treated 24 h with 0.14 pM of ditercalinium. DNA extractions were made either during (3, 6, 9, 24 h) the treatment or 6, 24, and 48 h after the removing of the drug. The DNAs were then treated as above. Molecular weight markers were A phage DNA digested by Hindlll and 1-kilobase ladder fragments (Bethesda Research Laboratories). Only the first major band has been shown because it corresponds to approximately 90% of the mt DNA. 4990 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research. LOSS OF MITOCHONDRIA!. 100 0 24 48 72 96 TIMEIh) Fig. 9. Effect of treatments on cytochrome c oxidase activity of L1210 cells. Cells were treated 24 h either with 0.14 »Mditercalinium (•),or with 1.4 ^M of the monomeric derivative (+). Arrow, time at which the cells were treated. The activity is expressed as the percentage of control. Each point was carried in triplicate. that of its monomeric derivative and other monointercalating agents. Ditercalinium's effect is mainly at the level of mt DNA whereas monointercalating agents act at the level of nuclear DNA and DNA topoisomerase II (25). Furthermore, Lim and Neims (36) reported that Adriamycin had no effect on mt DNA and that bleomycin induced short breaks in mt DNA but at a dose much larger than that inducing nuclear DNA breaks. However, it is not clearly stated that the effect of ditercalinium is related to mt DNA elimination alone. The viability of a cell like GSK3, as well as other respiration deficient cells (37), with no measurable respiration and clear morphological alteration of the mitochondria, suggests that a mammalian cell can derive all its energy from glycolysis without need of a functional respiratory chain. Recently, Desjardins et al. (23, 38) have shown that chick embryo cells completely devoid of mt DNA are still viable. If this result could be extended to cells of other species, it would suggest that elimination of mt DNA alone is not sufficient to lead to cell death. These results extend previous observations (6-9) suggesting that ditercalinium, a bifunctional DNA intercalator, elicits antitumor activity by a mechanism of action completely different from that of monointeracalating antitumor drugs. Ditercali nium appears to have a selective action on mt DNA which may be responsible for the delayed cytotoxicity which has also been observed with other ant ¡mitochondria! agents. But it is not clear that such an effect is sufficient to account for cytotoxicity and antitumor action of ditercalinium. Additional effects, for in stance at the level of nuclear DNA, may also contribute to cell toxicity. Acknowledgments The authors are very grateful to Dr. A. Jacquemin-Sablon E. Delain for fruitful discussions and suggestions. 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Genet., 12: 133-139, 1986. 4992 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research. Selective Loss of Mitochondrial DNA after Treatment of Cells with Ditercalinium (NSC 335153), an Antitumor Bis-intercalating Agent Evelyne Segal-Bendirdjian, Dominique Coulaud, Bernard P. Roques, et al. Cancer Res 1988;48:4982-4992. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/48/17/4982 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]. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1988 American Association for Cancer Research.