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Dequalinium Induces a Selective Depletion of Mitochondrial DNA from HeLa Human Cervical Carcinoma Cells
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
245, 137–145 (1998)
EX984236
Dequalinium Induces a Selective Depletion of Mitochondrial DNA from HeLa Human Cervical Carcinoma Cells1 Kristen R. Schneider Berlin, Chandramohan V. Ammini, and Thomas C. Rowe2 Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Florida 32610-0267
Treatment of cultured human cervical carcinoma cells with the anticancer drug dequalinium (DEQ) was found to cause a delayed inhibition of cell growth. This inhibition was preceded by a loss of mitochondrial DNA (mtDNA), a decrease in cytochrome c oxidase activity, and an increase in the level of lactate, indicating that growth inhibition was due to the loss of mtDNA-encoded functions. There was a progressive two-fold loss of mtDNA following each cell division in the presence of DEQ, suggesting that this drug was acting by inhibiting some aspect of mtDNA synthesis. Furthermore, cells became resistant to the growth inhibitory and cytotoxic affects of DEQ when they were grown under conditions that bypassed the need for mtDNA-encoded functions. Resistance was not associated with significant changes in drug accumulation. These results suggest that the DEQ-induced depletion of mtDNA plays an important role in drug cytotoxicity. © 1998 Academic Press
Key Words: dequalinium; DNA; mitochondria; cancer.
INTRODUCTION
Dequalinium chloride (DEQ) is a cationic, lipophilic compound with anticarcinoma activity [1] (see Fig. 1). Lipophilic, cationic compounds preferentially localize in the mitochondria of living cells due to the high negative transmembrane potential maintained by functional mitochondria and the net positive charge carried by these compounds (reviewed in [2]). Carcinoma cells have been shown to have a higher mitochondrial membrane potential than untransformed epithelial cells, resulting in an increased accumulation and retention of lipophilic cationic compounds, including DEQ [1, 3]. DEQ has been shown to prolong the life span of mice implanted with bladder carcinoma .190%, which was significantly greater than other anticancer drugs that 1 This work was supported by Grants GM47535 and CA 60158 from the National Institutes of Health. 2 To whom reprint requests should be addressed. Fax: (352) 3929696. E-mail: [email protected].
were tested (i.e., cisplatin, cytosine arabinoside, methotrexate, and cyclophosphamide) [1]. More recently, Koya et al. [4], have identified another lipophilic cation, MKT-077, that is also selectively accumulated in mitochondria of tumor cells. This drug demonstrated significant activity against human renal carcinoma, prostate carcinoma, and melanoma implanted in nude mice. The life span in mice implanted with human melanoma and treated with MKT-077 was prolonged .300%. Phase I clinical trials with MKT-077 have been initiated in patients with refractory carcinomas. So far, no significant toxicities, including myelosuppression or cardiac toxicity, have been observed at doses up to 48 mg/m2 [4]. Although the anticancer activity of DEQ is thought to be related to the preferential accumulation of this compound into carcinoma cells, the molecular target of this drug is unknown. Several hypotheses for the antiproliferative action of DEQ have been proposed, including the inhibition of calmodulin [5], the inhibition of the mitochondrial ATPase [6, 7], and the inhibition of protein kinase C [8]. Due to the selective accumulation of DEQ in mitochondria, we recently became interested in determining whether this drug might be acting by interfering with mitochondrial DNA (mtDNA) function. The results of these studies are presented in this paper. MATERIALS AND METHODS Chemicals and reagents. Minimal essential media (a modification), MEM vitamin solution, MEM sodium pyruvate solution, and MEM amino acids solution, fetal bovine serum (FBS), proteinase K, and 0.05% trypsin/0.53 mM EDTA were from Gibco BRL (Grand Island, NY). Dulbecco’s modified Eagle’s medium (DMEM) base, uridine, thymidine, phenylmethylsulfonyl fluoride (PMSF), glucose, sodium bicarbonate, Hepes free acid, L-glutamine, phenol red, phenol, ferrocytochrome C (type III, horse heart), salmon sperm DNA, Tris base, potassium ferricyanide, dimethyl sulfoxide (DMSO), and sodium ascorbate were from Sigma Chemical Co. (St. Louis, MO). Drugs. Dequalinium chloride and oligomycin were purchased from Sigma Chemical Co. Dequalinium (1 mM stock solution) was solubilized in water by probe sonication for 20 min using an Ultrasonics Model W-225 probe sonicator (50% duty cycle, output control of 3). The resulting drug solution was then filter-sterilized using a
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0014-4827/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1.
Structure of DEQ.
0.2-mm Acrodisc filter (Gelman Sciences, Ann Arbor, MI). Oligomycin (5 mg/ml stock solution) was solubilized in dimethylsulfoxide. Cell culture. All mammalian cell experiments in this project were performed using HeLa S3 cells. HeLa S3 cells are a human cervical carcinoma cell line with a doubling time of 24 h. These cells were obtained from Dr. Bert Flanagan (Department of Molecular Genetics and Microbiology, University of Florida). Cells were maintained as subconfluent monolayer cultures in minimal essential media (a modification) with 10% FBS (aMEM) at 37°C under a humidified atmosphere containing 5% CO2. To maintain logarithmic growth, cells were reseeded at a density of 5 3 105 in 75-cm3 tissue culture flasks every 2–3 days. In some experiments, cells were maintained in DMEM containing 10% FBS or DMEM media supplemented with 1 mM pyruvate, 40 mM uridine, 40 mM thymidine, and 10% FBS (DMEM-PUT). DNA probes. The human mtDNA clone pKB Mbo I 2.8 contains the 2.8-kb Mbo I fragment of mtDNA cloned into the Bam HI site of pBR322 and was obtained from Dr. David Clayton (Stanford University). Total cellular DNA was isolated from HeLa cells as described under “Isolation of Cellular DNA.” Labeling of pKB Mbo I 2.8 or total HeLa cell DNA was done using [a-32P]dATP (3000 Ci/mmol, ICN Biomedicals; Costa Mesa, CA) and the Prime-It II random prime labeling kit from Stratagene (La Jolla, CA). Isolation of cellular DNA. Total HeLa cell DNA was isolated using a modification of the protocol described by Sambrook et al. [9]. In general, HeLa cells (8 3 105 cells) were lysed in 2 mls of 20 mM Tris–HCl, pH 8.0, 20 mM EDTA, pH 8.0, 1% SDS, 1 mg/ml proteinase K, and incubated at 50°C overnight. The samples were then extracted (33) with equal volumes of TE-saturated phenol, pH 8.0, and the residual ether removed by extraction (32) with ethyl ether. The deproteinized samples were then treated with DNAse-free RNAse A at 37°C for 1 h. Samples were subsequently phenol (31)and ether (31)-extracted and the DNA ethanol was precipitated. The DNA precipitate was collected by centrifugation at 4°C in a Beckman JA20 rotor (13,000g) and solubilized in TE buffer, pH 8.0 (10 mM Tris–HCl, pH 8.0, 1 mM EDTA, pH 8.0), by incubating at 50°C for 2 h. Samples were then stored at 4°C. Quantification of DNA. DNA was quantified by fluorimetry as described by Labarca and Paigen [10]. Fluorescence was measured using a Perkin-Elmer LS-2 filter fluorimeter, using an excitation wavelength of 356 and an emission wavelength of 458. DNA samples were diluted into PSE buffer (50 mM NaPO4, pH 7.4, 2 M NaCl, 1 mM EDTA, pH 8.0) containing 1 mg/ml Hoechst 33258, and fluorescence was measured. The concentrations were based on a standard curve of salmon sperm DNA. Determination of mtDNA content by dot blotting. To determine the effects of DEQ and oligomycin on HeLa cell mtDNA content, HeLa cells were seeded in triplicate into 100-mm dishes (1.5 3 105 cells per dish) in a-MEM and allowed to attach overnight. The media was then removed and replaced with fresh media containing the appropriate concentrations of sterile drug. At various timepoints,
cells were lysed and total cellular DNA was isolated as described under “Isolation of Cellular DNA.” DNA was quantified, and an equal amount of DNA from each sample was denatured in a solution containing 0.4 M NaOH and 10 mM EDTA. The samples were then applied to a positively charged nylon membrane (Hybond-N1; Amersham, Arlington Heights, IL) using the Bio-Rad (Hercules, CA) Bio-Dot SF apparatus and a protocol adapted from Sambrook et al. [9]. The DNA was cross-linked to the membrane in a UV Stratalinker (Stratagene, La Jolla, CA). The mtDNA was then detected by hybridization to a 32P-labeled mitochondrial-specific probe (pKB Mbo I 2.8) and the signal quantitated using a Betascope 603 Blot Analyzer (Betagen Corp.). This instrument has an electronic sensor which continuously records and analyzes b-radiation directly from all portions of a labeled membrane. Variations in total amount of DNA in each sample were corrected for by removing the mtDNA probe and rehybridizing the blot to 32P-labeled total genomic HeLa DNA. Cell growth experiments. To determine the effects of dequalinium and oligomycin on HeLa cell growth, cells (2.75 3 104) were seeded in triplicate into 30-mm tissue culture dishes and cultured overnight to allow attachment. The media was then removed by vacuum aspiration and replaced with fresh media containing the appropriate concentration of drug. At various timepoints, the cells were detached using 0.05% trypsin/0.53 mM EDTA, and counted using a Coulter Counter. Clonogenic cytotoxicity experiments. To assess the antiproliferative effects of dequalinium, clonogenic cytotoxicity experiments were performed using a procedure similar to that described by Kroll et al. [11]. HeLa cells were seeded in triplicate into 30-mm wells (300 cells per well) in 3 ml DMEM or DMEM-PUT media supplemented with 10% fetal bovine serum. The cells were cultured overnight to allow the cells to adhere to the surface of the dish. The media was then removed and replaced with 3 ml of fresh media containing various concentrations of dequalinium. After 10 days, the colonies were detected by staining with crystal violet (2% w/v) and counting. The drug washout experiments were done as described previously [12]. In this case, the drug-containing media was removed and the cells were washed (33) with 1 ml of 37°C phosphate-buffered saline and then placed in 3 ml fresh drug-free media and colonies were counted after 10 days as described above. Measuring lactate accumulation. The level of glycolytic activity in HeLa cells was assessed by measuring the accumulation of lactate in the media of cultured cells. HeLa cells were seeded in triplicate into 100-mm dishes (3.0 3 105 cells per dish) in 10 ml a-MEM media and cultured overnight to allow attachment to the surface of the dishes. The media was then removed by vacuum aspiration and replaced with 10 ml fresh a-MEM media containing the appropriate concentration of dequalinium. At various times of drug treatment, the media was aspirated and saved. The cells were then trypsinized with 0.05% trypsin/0.53 mM EDTA and counted by Coulter Counter. The aspirated media was combined with 2 vol of 10% trichloroacetic acid to remove the protein. After 10 min on ice, the sample was clarified by centrifugation at 10,000g for 10 min. The clear supernatant was then collected and assayed for lactate by a colorimetric assay using a kit from Sigma Chemical Co., according to the manufacturer’s instructions. The level of lactate was calculated as mg/106 cells and the data expressed relative to non-drug-treated controls. Cytochrome c oxidase activity. To assess mitochondrial function in HeLa cells, cytochrome c oxidase (COX) activity was measured using a spectrophotometric assay as described previously [13, 14]. HeLa cells were counted by Coulter Counter and seeded at 3 3 105 cells/100-mm dish in triplicate in 10 mls a-MEM media. Cells were incubated overnight at 37°C to allow the cells to attach. Following this incubation, the media was removed by vacuum aspiration and replaced with 10 mls fresh media containing the appropriate concentration of dequalinium. At various timepoints, cells were scraped, diluted in Hematall isotonic diluent (Fisher Scientific) and counted by Coulter Counter. The cells were collected by centrifugation and
DEQUALINIUM AND MITOCHONDRIAL DNA washed twice with 0.25 M sucrose before resuspending in 1 ml of water. Cells were then disrupted by 20 strokes in a tight-fitting Dounce homogenizer. To initiate the assay, 200 ml of the resulting lysate was combined with 100 ml of 0.1 M potassium phosphate (pH 7.0), 70 ml of 1% (w/v) reduced ferrocytochrome C (ferrocytochrome c was reduced with 16 mM sodium ascorbate and purified over a Sephadex G50 column prior to use), and 630 ml water. The reactions were incubated at 37°C and the absorbance at 550 nm was measured over a 30-min period against a blank containing 1 ml of 0.01 M potassium phosphate (pH 7.0), 0.07% oxidized ferrocytochrome c (ferrocytochrome c was oxidized by including 1 mM potassium ferricyanide in the blank). Drug accumulation assay. Accumulation of DEQ by the cells was measured using a fluorescence spectroscopy assay similar to that used by Darzynkiewicz et al. [15]. Briefly, HeLa cells (1.8 3 106 in 10 mls DMEM or DMEM-PUT media) were seeded in 100-mm tissue culture dishes and incubated at 37°C overnight to allow the cells to adhere. DEQ [1 mM] was then added. After 3 h, the media was removed by aspiration, and the cells rinsed once with 10 mls of 4°C Azide-Free Isotonic Diluent (Fisher Scientific) before being scraped off of the plates into 1 ml ice-cold diluent. Cells were then transferred to 1.5-ml Eppendorf tubes and pelleted at 500g in a Beckman JA-18.1 rotor. The supernatant was removed and the cells were resuspended in 1 ml methanol (Fisher Scientific). The resulting cell lysate was clarified by spinning the samples at 13,000g in the Beckman JA-18.1 rotor. The supernatant was diluted 1:3 in methanol, and fluorescence was measured using the Perkin–Elmer LS-5 Fluorescence Spectrofluorometer using a 283-nm excitation wavelength and an emission wavelength of 380 nm. The data were corrected for background fluorescence by subtraction of the fluorescence values for control, non-drug-treated cells. DNA unwinding assay. The DNA unwinding assay was done as described previously [12]. Briefly, reactions (30 ml) containing 50 mM Tris–HCl, pH 7.5, 50 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.3 mg supercoiled Bluescript KS2 plasmid DNA (Stratagene), and 10 units wheat germ topoisomerase I (Promega) were incubated at 37°C to relax the DNA. After 10 min, DEQ was added to the reactions and the incubation continued for an additional 20 min. Reactions were then terminated by adding 10 ml of a solution containing 1% SDS, 40 mM EDTA, pH 8, and the samples were phenol extracted to remove drug and protein. The resulting aqueous phase was then combined with 5 ml of 50% sucrose and 0.01% bromphenol blue. The DNA samples were then resolved by gel electrophoresis through a 1.2% agarose gel containing 13 TBE at 40 V for 16 h. Gels were stained with ethidium bromide and the DNA visualized and photographed under UV light. Statistics. Where appropriate, experimental samples were performed in triplicate and the data presented as mean 6 standard error.
RESULTS
DEQ causes a delayed inhibition of cell growth. The affects of DEQ on the growth of cultured HeLa cervical carcinoma cells was investigated by monitoring the cell counts in cultures continuously exposed to various concentrations of DEQ over a 4-day period (Fig. 2). Concentrations of DEQ #0.3 mM did not cause any significant inhibition of cell growth during this time course. However at 0.65 mM, a delayed inhibition of growth occurred after 72 h with 30% inhibition being observed by 96 h. A delayed inhibition of growth also occurred at 1 mM drug although the onset of inhibition was earlier (48 h). By 96 h, growth was inhibited approximately
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FIG. 2. DEQ induces a delayed inhibition of HeLa cell growth. HeLa cells incubated in the presence or absence of DEQ (1 mM) were counted at the indicated timepoints as described under Materials and Methods. Samples were performed in triplicate and the data were plotted as mean cell number 6 standard error.
50% with no apparent loss in viability as judged by trypan blue exclusion. However, drug incubations that were longer than 96 h resulted in a significant loss in cell viability even at 0.3 mM (data not shown). DEQ induces a selective loss of mtDNA. The antitumor action of DEQ is thought to be related to the preferential accumulation of this drug in mitochondria [1]. DEQ has also been shown to inhibit the mitochondrial ATPase, suggesting that this may be the primary target of drug action [6, 7]. However, it is possible that other mitochondrial functions may also be affected by DEQ. Interestingly, drugs which inhibit mtDNA synthesis cause a delayed inhibition of cell growth similar to that seen with DEQ [13, 16]. To investigate this possibility, total cellular DNA isolated from DEQtreated HeLa cells was dot blotted onto a nylon membrane and hybridized to a mitochondrial-specific DNA probe. The radioactive signal in each sample was then quantitated using a Betascopt Blot analyzer. To correct for any differences in cellular DNA content, the mitochondrial probe was removed and the membrane hybridized to a total cellular DNA probe. Continuous exposure of cells to DEQ for 72 h resulted in a dosedependent loss in mtDNA (Fig. 3). The mtDNA content was decreased to 40% of control levels at 0.3 mM and .75% at 0.5 mM DEQ. No further decrease in mtDNA content was observed at concentrations .0.5 mM.
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FIG. 3. Dose-dependent effects of continuous DEQ exposure on HeLa cell mtDNA content. Total cellular DNA was isolated from HeLa cells continuously exposed to various concentrations of DEQ for 72 h and the mtDNA content determined by dot blotting as described under Materials and Methods. Samples were performed in triplicate and the mtDNA content was plotted as percentage of control 6 standard error.
Continuous exposure of avian and mammalian cells to the DNA-intercalating compound ethidium bromide is known to induce a progressive dilution of mtDNA following each round of cell division due to an inhibition of mtDNA synthesis [16, 17]. If DEQ also acts by inhibiting mtDNA synthesis, a similar two-fold dilution of mtDNA should occur with each cell doubling. To determine if DEQ caused a two-fold loss of mtDNA with each cell division, the mtDNA content was measured at different times in HeLa cells continuously treated with DEQ (1 mM) and the data were plotted relative to the number of cell doublings (Fig. 4). Within the first cell doubling, DEQ reduced the mtDNA content to approximately 50% of control values. After two cell doublings, the mtDNA content was further reduced to approximately 25% of controls, indicating that similar to ethidium bromide, DEQ causes a two-fold reduction in mtDNA content with each cell doubling. Analysis of the mtDNA by gel electrophoresis and Southern blotting after treatment of cells for 96 h with DEQ did not reveal significant mtDNA degradation (data not shown), indicating that DEQ was probably acting by inhibiting mtDNA synthesis rather than inducing an active breakdown of the mtDNA. DEQ unwinds DNA. Several inhibitors of mtDNA synthesis (i.e., ethidium bromide and 4-quinolone
drugs) have been shown to induce DNA unwinding. It has been hypothesized that this interaction may interfere with the function of proteins that are required for mtDNA replication (i.e., polymerases, helicases, topoisomerases, etc.). To determine if DEQ is also a DNAunwinding compound, supercoiled Bluescript KS2 plasmid DNA was relaxed with wheat germ topoisomerase I. After 10 min, DEQ was added to the samples and the incubations were continued for an additional 20 min before terminating the reactions with the protein denaturant SDS. Drug and protein were then removed by phenol extraction and the DNA was analyzed by gel electrophoresis (Fig. 5). Untreated Bluescript plasmid DNA contains two bands representing supercoiled form I (faster migrating form) and nicked circular relaxed form II (slower migrating form) DNA, respectively (lane A). Following relaxation by topoisomerase I, supercoiled Bluescript DNA was converted to a relaxed closed circular form that comigrated with form II DNA (lane C). Addition of increasing concentrations of DEQ (8 –32 mM), after the initial incubation with topoisomerase I, caused an increase in the mobility of Bluescript DNA back towards highly supercoiled form I DNA, indicating that DEQ induces significant unwinding of duplex DNA (lanes D–F).
FIG. 4. DEQ induces a time-dependent progressive dilution of mtDNA content in HeLa cells. HeLa cells treated with DEQ (1 mM) for various lengths of time were trypsinized and counted, and the total cellular DNA was isolated as described under Materials and Methods. Equal amounts of total cellular DNA were then dot blotted onto a nylon membrane and the membrane was hybridized to a 32 P-labeled mtDNA-specific probe as described in the legend for Fig. 3.
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FIG. 5. DEQ unwinds DNA. Drug unwinding of circular Bluescipt KS2 DNA was assayed as described under Materials and Methods. Lanes C–F, Bluescript DNA incubated with topoisomerase I in the presence of 0, 8, 16, or 32 mM DEQ. Lanes A and B are no-enzyme controls containing Bluescript DNA incubated in the presence or absence of 32 mM DEQ, respectively. Arrows mark the positions of form I (highly supercoiled) and form II (nicked circular) forms of Bluescript KS2 plasmid DNA.
Effect of DEQ on cytochrome c oxidase activity. The DEQ-induced loss of mtDNA would be expected to cause a loss of mtDNA-encoded functions. MtDNA encodes for a number of proteins that are important in mitochondrial energy metabolism. Cytochrome c oxidase (COX) is a multisubunit lipoprotein that forms an important part of the mitochondrial electron transport chain [18]. Three subunits of this enzyme are coded for by mtDNA with the remaining subunits being coded for by nuclear DNA [19]. In addition to three subunits for COX, mtDNA contains genes for apocytochrome b, and subunits for cytochrome c, NADH dehydrogenase, and ATPase proteins [19]. A DEQ-induced depletion of mtDNA content would therefore be expected to affect the expression of a number of genes necessary for energy metabolism. Correspondingly, any decrease in respiration would likely be compensated for by an increase in glycolytic energy pathways and hence an increase in lactate, a by-product of glycolysis. To determine whether DEQ had an affect on the activity of any of the proteins involved in mitochondrial respiration, COX activity was measured in HeLa cells continuously treated with DEQ using a spectrophotometric assay [14]. DEQ (1 mM) induced a decrease in COX activity to 48 6 6% of control levels within one cell doubling. Following two cell doublings, the COX activity declined to 23 6 3% of control levels. These data correlate with the time course of DEQ-induced mtDNA loss shown in Fig. 4. We did not observe any inhibition of COX activity by DEQ in vitro, indicating that the progressive decrease in activity seen in drug-treated cells was not due to a direct inhibition of this enzyme by drug (data not shown). A drug-induced decrease in respiratory function would be expected to compromise ATP production and result in a compensatory increase in glycolytic activity in the cell. To determine the effects of continuous drug
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exposure on glycolysis, lactate accumulation was measured in HeLa cells continuously treated with 1 mM DEQ. In contrast to COX activity, there was no significant increase in lactate accumulation following one cell doubling, suggesting that there were still sufficient levels of ATP to sustain growth. However, by 1.25 cell doublings, lactate levels had rapidly increased three- to fourfold relative to controls. These data illustrate that DEQ induces a time-dependent, delayed increase in lactate accumulation in HeLa cells which inversely correlates with the DEQ-induced loss of respiratory function as measured by COX activity. Effect of culture medium on DEQ cytotoxicity. Mammalian cell lines can grow in the absence of mtDNA-encoded functions if the growth media is supplemented with pyrimidines, pyruvate, and high concentrations of glucose [12, 20, 21]. Cells lacking mtDNA are deficient in respiration and therefore rely on glycolysis for energy. These cells are also auxotrophic for pyrimidines since de novo pyrimidine synthesis is coupled to active mitochondrial electron transport. The requirement for pyruvate is not entirely clear but may relate to its function in the cellular synthesis of alanine and ketone bodies. To determine the effect of growth media on drug cytotoxicity, HeLa cells grown under respiration-dependent (DMEM) or respiration-independent (DMEMPUT) conditions were continuously exposed to DEQ for 1, 2, 3, 4, and 10 days and the viability was determined using a clonogenic assay. DMEM media contains glucose, but no pyruvate or pyrimidines and cells therefore require mitochondrial function for survival. DMEM-PUT media is identical except that it is supplemented with pyruvate and pyrimidines and therefore supports cell growth in the absence of mtDNAencoded functions. The viability of HeLa cells grown under respiration-dependent conditions (DMEM media) was decreased in both a dose- and time-dependent fashion with survival being ,1% at 1 mM DEQ at all times tested (Fig. 6A). In contrast, when cells were grown under respiration-independent conditions (DMEM-PUT), there was no significant decrease in cell survival following exposure of HeLa cells to DEQ (0.25 to 1 mM) for periods of time up to 2 days (Fig. 6B). Although cell viability was affected by longer drug exposures (3 to 10 days), it was modest in comparison to that observed in cells grown under respiration-dependent conditions. For example, viability following a 10 day exposure of HeLa cells to 250 mM DEQ under respiration-independent conditions was 50%, which contrasts with ,1% viability for cells grown under respiration-dependent conditions. Although the drug resistance observed in cells grown under respiration-independent conditions suggested that DEQ was acting by a mitochondrial mechanism,
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mM DEQ for 3 h. The results indicate that there is no significant difference in steady-state concentrations of DEQ in HeLa cells grown in DMEM or DMEM-PUT, suggesting that drug resistance was not due to alterations in drug accumulation (data not shown). DISCUSSION
FIG. 6. Cytotoxicity of DEQ on HeLa cells cultured under respiration-dependent or -independent conditions. HeLa cells were continuously treated with various concentrations of DEQ for 1, 2, 3, 4, or 10 days and cytotoxicity was assessed using a clonogenic assay as described under Materials and Methods. Cells were grown under respiration-dependent (DMEM media, A) or respiration-independent (DMEM-PUT media, B) conditions. Samples were done in triplicate and the data expressed as percentage of non-drug-treated controls 6 standard error.
we could not exclude the possibility that this result was simply due to differences in drug accumulation when cells were grown under these two conditions. To investigate this possibility, the steady-state concentration of DEQ was determined in HeLa cells grown in either DMEM or DMEM-PUT media using a fluorescence assay similar to that described by Darzynkiewicz et al. [15]. Time-course studies in our lab found that drug levels in HeLa cells reach steady state by 1 h following exposure to 1 mM DEQ (data not shown). Based on this finding, drug accumulation in DMEM and DMEM-PUT was determined following exposure of HeLa cells to 1
The data presented here demonstrate that DEQ, a cationic lipophilic compound, induces a delayed inhibition of cell growth in cultured human cervical carcinoma cells. DEQ also causes a selective loss of mtDNA from these cells, which was accompanied by a decrease in COX activity and a delayed increase in lactate accumulation. Furthermore, cells grown under respiratory-independent conditions are resistant to the cytotoxic effects of DEQ. These studies raise the interesting possibility that DEQ may exert its antiproliferative effects by depleting carcinoma cells of their mtDNA. In mammalian cells, mtDNA is present at a copy number of approximately 1000 –2000 copies per cell, comprising ,1% of the total cellular DNA (reviewed in [22]). Inhibition of one or more processes involved in mtDNA synthesis would be expected to cause a successive dilution of mtDNA with each cell division. If mtDNA synthesis were completely blocked, this would result in a two-fold loss of mtDNA following every cell division. Concentrations of DEQ that caused maximal loss of mtDNA induced a two-fold reduction of mtDNA from HeLa cells following each cell division, suggesting that this drug is blocking one or more critical activities (i.e., polymerases, helicases, topoisomerases, etc) required for mtDNA replication. Other compounds have also been shown to cause a selective loss of mammalian cell mtDNA, including the 4-quinolone DNA gyrase-inhibiting antibiotics nalidixic acid and ciprofloxacin [13], ethidium bromide [16], the polyamine analogues MGBG [23] and N1,N12bis(ethyl)spermine [24], 29,39-dideoxycytidine (a putative inhibitor of mitochondrial g DNA polymerase; [25]) and ditercalinium [14]. DEQ is structurally different from these compounds. However, DEQ does induce DNA unwinding, a property which is shared by several of the drugs that induce mtDNA loss (i.e., ethidium bromide, ditercalinium, and 4-quinolone drugs) [12, 14, 16]. Our in vitro studies indicate that unwinding of plasmid DNA occurs at concentrations $ 8 mM which are considerably higher (approximately 25-fold) than those required to inhibit cell growth and cause a loss of mtDNA. However, because lipophilic cations such as DEQ can theoretically accummulate to as much as 10,000-fold within mammalian mitochondria [2, 26], it is likely that drug concentrations which were found to induce inhibition of cell growth and loss of mtDNA result in intramitochondrial concentrations sufficient to cause extensive unwinding of mtDNA. The finding
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that DEQ induces DNA unwinding is consistent with previous DNA ultracentrifugation analyses by Wright et al. [27], which indicated that DEQ significantly altered the sedimentation coefficient of closed circular PM2 DNA. Based on helix unwinding angle calculations, these investigators concluded that DEQ was probably acting as a monofunctional DNA-intercalating agent. Therefore, although the structure of DEQ is significantly different from the DNA-intercalating compounds ethidium bromide and ditercalinium, it also appears to induce DNA unwinding by an intercalative mechanism. This contrasts with 4-quinolone drugs which mediate DNA unwinding by interacting with single-stranded regions in DNA [28]. DEQ caused a delayed inhibition of HeLa cell growth. This delayed inhibition of cell growth correlated well with the time- and dose-dependent decrease in mtDNA content. In mammalian cells, mtDNA is present in a copy number greatly in excess of that necessary to maintain cell viability. Wiseman and Attardi [16] observed a mtDNA threshold of 10%, below which mammalian cells were unable to support the energy requirements needed for growth or viability. The data presented here indicate that DEQ caused HeLa growth inhibition at concentrations that induced a greater than an 80% loss of mtDNA ($0.5 mM), which is consistent with results for other drugs which interfere with mtDNA synthesis [13, 16]. Mammalian cell mtDNA encodes protein components of the electron transport chain that are required for mitochondrial respiration (i.e., COX). It is therefore probable that the DEQ-induced depletion of mtDNA is a significant factor contributing to the observed decrease in mitochondrial COX activity due to the presence of fewer mtDNA templates available for transcription. However, we cannot rule out that DEQ may also act by directly interfering with mtDNA transcription or some posttranscriptional process. A third possibility is that DEQ might directly inhibit or inactivate COX. However, this last explanation is unlikely since DEQ was not found to have an affect on COX activity in HeLa cell extracts. It is not surprising that the DEQ-induced decrease in respiratory function would be associated with a compensatory increase in glycolysis. DEQ induced a 3- to 4-fold delayed increase in lactic acid accumulation which occurred between 1 and 2 cell doublings. The rise in lactate succeeds the decrease in COX activity. These results are consistent with the need for the cells to use an alternative, albeit less efficient, pathway for obtaining energy due to a loss in respiratory function. Cell lines that lack mtDNA have been generated [20, 21]. These cell lines are able to grow in the absence of mtDNA-encoded function because their requirement for respiratory function has been bypassed by supplementing the growth media with glucose, pyruvate, and
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pyrimidines. The studies presented here illustrate that HeLa cells grown under respiratory-independent conditions (DMEM-PUT media) were relatively resistant to DEQ cytotoxicity. Resistance could not be attributed to altered drug uptake and this suggests that DEQ exerts its cytotoxic effects by interfering with a critical mitochondrial function(s). Other mechanisms for the anticarcinoma effects of DEQ have been proposed. Bodden et al. [5] proposed that DEQ might block cellular proliferation by inhibiting calmodulin. Calmodulin is a multifunctional calcium-binding protein primarily localized in the cytosol. This protein regulates numerous cellular events including chromosome disjunction during mitosis [29]. In the yeast Saccharomyces cerevisiae, deletion of the calmodulin gene is a lethal mutation [30]. Yeast mutants temperature sensitive for calmodulin were found to lose viability when grown at a nonpermissive temperature. The loss in viability appeared to be related to an inability of the cells to complete mitosis due to defects in chromosome segregation, spindle pole body functions, and cytokinesis [29, 31]. Similarly, treatment of mammalian cells with calmodulin antagonists has been shown to inhibit cell proliferation and cause a rapid loss in cell viability [32]. This contrasts with the delayed loss of cell proliferation that is seen with DEQ, suggesting that calmodulin is probably not the primary target for this drug. Another hypothesized mechanism of action for DEQ is the inhibition of protein kinase C [8]. DEQ inhibits isolated protein kinase C-b1 with an IC50 of 8 –15 mM. The concentrations at which we observe depletion of mtDNA are 10- to 20-fold lower than those at which protein kinase C is inhibited (0.5–1.0 mM versus 8 –15 mM for protein kinase C-b1 inhibition). Also, similar to calmodulin, inhibition of protein kinase C affects a variety of critical cellular functions including nuclear DNA replication and it is therefore improbable that DEQ is mediating its antiproliferative effects via protein kinase C since since DEQ had no direct and immediate affects on nuclear DNA synthesis. DEQ has also been demonstrated to inhibit the in vitro activity of mitochondrial F1-ATPase from bovine heart with an IC50 of 8 –12 mM, suggesting that this enzyme may be an in vivo target of drug action [6, 7]. Due to the selective accumulation of DEQ in mitochondria, it is very likely that cytotoxic concentrations of drug probably cause at least some inhibition of the mitochondrial ATPase. Is it possible that DEQ inhibition of the mitochondrial ATPase might also be responsible for the depletion of mtDNA? Perhaps inhibition of the mitochondrial ATPase enzyme causes an indirect inhibition of mtDNA synthesis due to a decrease in the levels of mitochondrial ATP. Studies in S. cerevisiae indicate that oligomycin, a specific inhibitor of the mitochondrial ATPase, does not induce the formation of
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mitochondrial petite mutants (cells having mutations in mtDNA or that completely lack any mtDNA sequences) [33]. In contrast, DEQ was found to be a potent inducer of rho0 petites (cells completely lacking mtDNA), suggesting that the DEQ-induced loss in mtDNA is not due to inibition of the mitochondrial ATPase [34]. The intriguing discovery by Chen and co-workers that liphophilic cationic compounds such as DEQ accumulate to a much greater extent in the mitochondria of carcinoma cells relative to normal epithelial cells has led to the identification of a number of novel compounds possessing significant anticarcinoma activity [1, 4, 35]. Although these compounds are all lipophilic cations, their structures vary significantly and the molecular target of these drugs remains unclear. One of the most promising lipophilic cations with anticarcinoma activity is the rhodacyanine dye MKT-077. This drug has been shown to possess significant activity against human renal cell carcinoma, prostate carcinoma, and melanoma tumors implanted in nude mice [4]. Initial findings from Phase I clinical trials in patients with refractory carcinomas indicate that MKT077 does not exert any significant side effects, including cardiac toxicity or myelosuppression [4]. Prelimminary evidence also suggests that like DEQ, MKT-077 also causes a selective loss of mtDNA in cultured carcinoma cells [36]. Whether or not these two structurally different lipophilic cations exert their anticarcinoma affects through a common mechanism awaits further investigation. The authors thank David Gill, Amy Jennings, Alan Klopp, Mark Mainwaring, and Volkmar Weissig for their assistance and encouragement during the course of these studies.
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EX984236
Dequalinium Induces a Selective Depletion of Mitochondrial DNA from HeLa Human Cervical Carcinoma Cells1 Kristen R. Schneider Berlin, Chandramohan V. Ammini, and Thomas C. Rowe2 Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville, Florida 32610-0267
Treatment of cultured human cervical carcinoma cells with the anticancer drug dequalinium (DEQ) was found to cause a delayed inhibition of cell growth. This inhibition was preceded by a loss of mitochondrial DNA (mtDNA), a decrease in cytochrome c oxidase activity, and an increase in the level of lactate, indicating that growth inhibition was due to the loss of mtDNA-encoded functions. There was a progressive two-fold loss of mtDNA following each cell division in the presence of DEQ, suggesting that this drug was acting by inhibiting some aspect of mtDNA synthesis. Furthermore, cells became resistant to the growth inhibitory and cytotoxic affects of DEQ when they were grown under conditions that bypassed the need for mtDNA-encoded functions. Resistance was not associated with significant changes in drug accumulation. These results suggest that the DEQ-induced depletion of mtDNA plays an important role in drug cytotoxicity. © 1998 Academic Press
Key Words: dequalinium; DNA; mitochondria; cancer.
INTRODUCTION
Dequalinium chloride (DEQ) is a cationic, lipophilic compound with anticarcinoma activity [1] (see Fig. 1). Lipophilic, cationic compounds preferentially localize in the mitochondria of living cells due to the high negative transmembrane potential maintained by functional mitochondria and the net positive charge carried by these compounds (reviewed in [2]). Carcinoma cells have been shown to have a higher mitochondrial membrane potential than untransformed epithelial cells, resulting in an increased accumulation and retention of lipophilic cationic compounds, including DEQ [1, 3]. DEQ has been shown to prolong the life span of mice implanted with bladder carcinoma .190%, which was significantly greater than other anticancer drugs that 1 This work was supported by Grants GM47535 and CA 60158 from the National Institutes of Health. 2 To whom reprint requests should be addressed. Fax: (352) 3929696. E-mail: [email protected].
were tested (i.e., cisplatin, cytosine arabinoside, methotrexate, and cyclophosphamide) [1]. More recently, Koya et al. [4], have identified another lipophilic cation, MKT-077, that is also selectively accumulated in mitochondria of tumor cells. This drug demonstrated significant activity against human renal carcinoma, prostate carcinoma, and melanoma implanted in nude mice. The life span in mice implanted with human melanoma and treated with MKT-077 was prolonged .300%. Phase I clinical trials with MKT-077 have been initiated in patients with refractory carcinomas. So far, no significant toxicities, including myelosuppression or cardiac toxicity, have been observed at doses up to 48 mg/m2 [4]. Although the anticancer activity of DEQ is thought to be related to the preferential accumulation of this compound into carcinoma cells, the molecular target of this drug is unknown. Several hypotheses for the antiproliferative action of DEQ have been proposed, including the inhibition of calmodulin [5], the inhibition of the mitochondrial ATPase [6, 7], and the inhibition of protein kinase C [8]. Due to the selective accumulation of DEQ in mitochondria, we recently became interested in determining whether this drug might be acting by interfering with mitochondrial DNA (mtDNA) function. The results of these studies are presented in this paper. MATERIALS AND METHODS Chemicals and reagents. Minimal essential media (a modification), MEM vitamin solution, MEM sodium pyruvate solution, and MEM amino acids solution, fetal bovine serum (FBS), proteinase K, and 0.05% trypsin/0.53 mM EDTA were from Gibco BRL (Grand Island, NY). Dulbecco’s modified Eagle’s medium (DMEM) base, uridine, thymidine, phenylmethylsulfonyl fluoride (PMSF), glucose, sodium bicarbonate, Hepes free acid, L-glutamine, phenol red, phenol, ferrocytochrome C (type III, horse heart), salmon sperm DNA, Tris base, potassium ferricyanide, dimethyl sulfoxide (DMSO), and sodium ascorbate were from Sigma Chemical Co. (St. Louis, MO). Drugs. Dequalinium chloride and oligomycin were purchased from Sigma Chemical Co. Dequalinium (1 mM stock solution) was solubilized in water by probe sonication for 20 min using an Ultrasonics Model W-225 probe sonicator (50% duty cycle, output control of 3). The resulting drug solution was then filter-sterilized using a
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FIG. 1.
Structure of DEQ.
0.2-mm Acrodisc filter (Gelman Sciences, Ann Arbor, MI). Oligomycin (5 mg/ml stock solution) was solubilized in dimethylsulfoxide. Cell culture. All mammalian cell experiments in this project were performed using HeLa S3 cells. HeLa S3 cells are a human cervical carcinoma cell line with a doubling time of 24 h. These cells were obtained from Dr. Bert Flanagan (Department of Molecular Genetics and Microbiology, University of Florida). Cells were maintained as subconfluent monolayer cultures in minimal essential media (a modification) with 10% FBS (aMEM) at 37°C under a humidified atmosphere containing 5% CO2. To maintain logarithmic growth, cells were reseeded at a density of 5 3 105 in 75-cm3 tissue culture flasks every 2–3 days. In some experiments, cells were maintained in DMEM containing 10% FBS or DMEM media supplemented with 1 mM pyruvate, 40 mM uridine, 40 mM thymidine, and 10% FBS (DMEM-PUT). DNA probes. The human mtDNA clone pKB Mbo I 2.8 contains the 2.8-kb Mbo I fragment of mtDNA cloned into the Bam HI site of pBR322 and was obtained from Dr. David Clayton (Stanford University). Total cellular DNA was isolated from HeLa cells as described under “Isolation of Cellular DNA.” Labeling of pKB Mbo I 2.8 or total HeLa cell DNA was done using [a-32P]dATP (3000 Ci/mmol, ICN Biomedicals; Costa Mesa, CA) and the Prime-It II random prime labeling kit from Stratagene (La Jolla, CA). Isolation of cellular DNA. Total HeLa cell DNA was isolated using a modification of the protocol described by Sambrook et al. [9]. In general, HeLa cells (8 3 105 cells) were lysed in 2 mls of 20 mM Tris–HCl, pH 8.0, 20 mM EDTA, pH 8.0, 1% SDS, 1 mg/ml proteinase K, and incubated at 50°C overnight. The samples were then extracted (33) with equal volumes of TE-saturated phenol, pH 8.0, and the residual ether removed by extraction (32) with ethyl ether. The deproteinized samples were then treated with DNAse-free RNAse A at 37°C for 1 h. Samples were subsequently phenol (31)and ether (31)-extracted and the DNA ethanol was precipitated. The DNA precipitate was collected by centrifugation at 4°C in a Beckman JA20 rotor (13,000g) and solubilized in TE buffer, pH 8.0 (10 mM Tris–HCl, pH 8.0, 1 mM EDTA, pH 8.0), by incubating at 50°C for 2 h. Samples were then stored at 4°C. Quantification of DNA. DNA was quantified by fluorimetry as described by Labarca and Paigen [10]. Fluorescence was measured using a Perkin-Elmer LS-2 filter fluorimeter, using an excitation wavelength of 356 and an emission wavelength of 458. DNA samples were diluted into PSE buffer (50 mM NaPO4, pH 7.4, 2 M NaCl, 1 mM EDTA, pH 8.0) containing 1 mg/ml Hoechst 33258, and fluorescence was measured. The concentrations were based on a standard curve of salmon sperm DNA. Determination of mtDNA content by dot blotting. To determine the effects of DEQ and oligomycin on HeLa cell mtDNA content, HeLa cells were seeded in triplicate into 100-mm dishes (1.5 3 105 cells per dish) in a-MEM and allowed to attach overnight. The media was then removed and replaced with fresh media containing the appropriate concentrations of sterile drug. At various timepoints,
cells were lysed and total cellular DNA was isolated as described under “Isolation of Cellular DNA.” DNA was quantified, and an equal amount of DNA from each sample was denatured in a solution containing 0.4 M NaOH and 10 mM EDTA. The samples were then applied to a positively charged nylon membrane (Hybond-N1; Amersham, Arlington Heights, IL) using the Bio-Rad (Hercules, CA) Bio-Dot SF apparatus and a protocol adapted from Sambrook et al. [9]. The DNA was cross-linked to the membrane in a UV Stratalinker (Stratagene, La Jolla, CA). The mtDNA was then detected by hybridization to a 32P-labeled mitochondrial-specific probe (pKB Mbo I 2.8) and the signal quantitated using a Betascope 603 Blot Analyzer (Betagen Corp.). This instrument has an electronic sensor which continuously records and analyzes b-radiation directly from all portions of a labeled membrane. Variations in total amount of DNA in each sample were corrected for by removing the mtDNA probe and rehybridizing the blot to 32P-labeled total genomic HeLa DNA. Cell growth experiments. To determine the effects of dequalinium and oligomycin on HeLa cell growth, cells (2.75 3 104) were seeded in triplicate into 30-mm tissue culture dishes and cultured overnight to allow attachment. The media was then removed by vacuum aspiration and replaced with fresh media containing the appropriate concentration of drug. At various timepoints, the cells were detached using 0.05% trypsin/0.53 mM EDTA, and counted using a Coulter Counter. Clonogenic cytotoxicity experiments. To assess the antiproliferative effects of dequalinium, clonogenic cytotoxicity experiments were performed using a procedure similar to that described by Kroll et al. [11]. HeLa cells were seeded in triplicate into 30-mm wells (300 cells per well) in 3 ml DMEM or DMEM-PUT media supplemented with 10% fetal bovine serum. The cells were cultured overnight to allow the cells to adhere to the surface of the dish. The media was then removed and replaced with 3 ml of fresh media containing various concentrations of dequalinium. After 10 days, the colonies were detected by staining with crystal violet (2% w/v) and counting. The drug washout experiments were done as described previously [12]. In this case, the drug-containing media was removed and the cells were washed (33) with 1 ml of 37°C phosphate-buffered saline and then placed in 3 ml fresh drug-free media and colonies were counted after 10 days as described above. Measuring lactate accumulation. The level of glycolytic activity in HeLa cells was assessed by measuring the accumulation of lactate in the media of cultured cells. HeLa cells were seeded in triplicate into 100-mm dishes (3.0 3 105 cells per dish) in 10 ml a-MEM media and cultured overnight to allow attachment to the surface of the dishes. The media was then removed by vacuum aspiration and replaced with 10 ml fresh a-MEM media containing the appropriate concentration of dequalinium. At various times of drug treatment, the media was aspirated and saved. The cells were then trypsinized with 0.05% trypsin/0.53 mM EDTA and counted by Coulter Counter. The aspirated media was combined with 2 vol of 10% trichloroacetic acid to remove the protein. After 10 min on ice, the sample was clarified by centrifugation at 10,000g for 10 min. The clear supernatant was then collected and assayed for lactate by a colorimetric assay using a kit from Sigma Chemical Co., according to the manufacturer’s instructions. The level of lactate was calculated as mg/106 cells and the data expressed relative to non-drug-treated controls. Cytochrome c oxidase activity. To assess mitochondrial function in HeLa cells, cytochrome c oxidase (COX) activity was measured using a spectrophotometric assay as described previously [13, 14]. HeLa cells were counted by Coulter Counter and seeded at 3 3 105 cells/100-mm dish in triplicate in 10 mls a-MEM media. Cells were incubated overnight at 37°C to allow the cells to attach. Following this incubation, the media was removed by vacuum aspiration and replaced with 10 mls fresh media containing the appropriate concentration of dequalinium. At various timepoints, cells were scraped, diluted in Hematall isotonic diluent (Fisher Scientific) and counted by Coulter Counter. The cells were collected by centrifugation and
DEQUALINIUM AND MITOCHONDRIAL DNA washed twice with 0.25 M sucrose before resuspending in 1 ml of water. Cells were then disrupted by 20 strokes in a tight-fitting Dounce homogenizer. To initiate the assay, 200 ml of the resulting lysate was combined with 100 ml of 0.1 M potassium phosphate (pH 7.0), 70 ml of 1% (w/v) reduced ferrocytochrome C (ferrocytochrome c was reduced with 16 mM sodium ascorbate and purified over a Sephadex G50 column prior to use), and 630 ml water. The reactions were incubated at 37°C and the absorbance at 550 nm was measured over a 30-min period against a blank containing 1 ml of 0.01 M potassium phosphate (pH 7.0), 0.07% oxidized ferrocytochrome c (ferrocytochrome c was oxidized by including 1 mM potassium ferricyanide in the blank). Drug accumulation assay. Accumulation of DEQ by the cells was measured using a fluorescence spectroscopy assay similar to that used by Darzynkiewicz et al. [15]. Briefly, HeLa cells (1.8 3 106 in 10 mls DMEM or DMEM-PUT media) were seeded in 100-mm tissue culture dishes and incubated at 37°C overnight to allow the cells to adhere. DEQ [1 mM] was then added. After 3 h, the media was removed by aspiration, and the cells rinsed once with 10 mls of 4°C Azide-Free Isotonic Diluent (Fisher Scientific) before being scraped off of the plates into 1 ml ice-cold diluent. Cells were then transferred to 1.5-ml Eppendorf tubes and pelleted at 500g in a Beckman JA-18.1 rotor. The supernatant was removed and the cells were resuspended in 1 ml methanol (Fisher Scientific). The resulting cell lysate was clarified by spinning the samples at 13,000g in the Beckman JA-18.1 rotor. The supernatant was diluted 1:3 in methanol, and fluorescence was measured using the Perkin–Elmer LS-5 Fluorescence Spectrofluorometer using a 283-nm excitation wavelength and an emission wavelength of 380 nm. The data were corrected for background fluorescence by subtraction of the fluorescence values for control, non-drug-treated cells. DNA unwinding assay. The DNA unwinding assay was done as described previously [12]. Briefly, reactions (30 ml) containing 50 mM Tris–HCl, pH 7.5, 50 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 0.3 mg supercoiled Bluescript KS2 plasmid DNA (Stratagene), and 10 units wheat germ topoisomerase I (Promega) were incubated at 37°C to relax the DNA. After 10 min, DEQ was added to the reactions and the incubation continued for an additional 20 min. Reactions were then terminated by adding 10 ml of a solution containing 1% SDS, 40 mM EDTA, pH 8, and the samples were phenol extracted to remove drug and protein. The resulting aqueous phase was then combined with 5 ml of 50% sucrose and 0.01% bromphenol blue. The DNA samples were then resolved by gel electrophoresis through a 1.2% agarose gel containing 13 TBE at 40 V for 16 h. Gels were stained with ethidium bromide and the DNA visualized and photographed under UV light. Statistics. Where appropriate, experimental samples were performed in triplicate and the data presented as mean 6 standard error.
RESULTS
DEQ causes a delayed inhibition of cell growth. The affects of DEQ on the growth of cultured HeLa cervical carcinoma cells was investigated by monitoring the cell counts in cultures continuously exposed to various concentrations of DEQ over a 4-day period (Fig. 2). Concentrations of DEQ #0.3 mM did not cause any significant inhibition of cell growth during this time course. However at 0.65 mM, a delayed inhibition of growth occurred after 72 h with 30% inhibition being observed by 96 h. A delayed inhibition of growth also occurred at 1 mM drug although the onset of inhibition was earlier (48 h). By 96 h, growth was inhibited approximately
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FIG. 2. DEQ induces a delayed inhibition of HeLa cell growth. HeLa cells incubated in the presence or absence of DEQ (1 mM) were counted at the indicated timepoints as described under Materials and Methods. Samples were performed in triplicate and the data were plotted as mean cell number 6 standard error.
50% with no apparent loss in viability as judged by trypan blue exclusion. However, drug incubations that were longer than 96 h resulted in a significant loss in cell viability even at 0.3 mM (data not shown). DEQ induces a selective loss of mtDNA. The antitumor action of DEQ is thought to be related to the preferential accumulation of this drug in mitochondria [1]. DEQ has also been shown to inhibit the mitochondrial ATPase, suggesting that this may be the primary target of drug action [6, 7]. However, it is possible that other mitochondrial functions may also be affected by DEQ. Interestingly, drugs which inhibit mtDNA synthesis cause a delayed inhibition of cell growth similar to that seen with DEQ [13, 16]. To investigate this possibility, total cellular DNA isolated from DEQtreated HeLa cells was dot blotted onto a nylon membrane and hybridized to a mitochondrial-specific DNA probe. The radioactive signal in each sample was then quantitated using a Betascopt Blot analyzer. To correct for any differences in cellular DNA content, the mitochondrial probe was removed and the membrane hybridized to a total cellular DNA probe. Continuous exposure of cells to DEQ for 72 h resulted in a dosedependent loss in mtDNA (Fig. 3). The mtDNA content was decreased to 40% of control levels at 0.3 mM and .75% at 0.5 mM DEQ. No further decrease in mtDNA content was observed at concentrations .0.5 mM.
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FIG. 3. Dose-dependent effects of continuous DEQ exposure on HeLa cell mtDNA content. Total cellular DNA was isolated from HeLa cells continuously exposed to various concentrations of DEQ for 72 h and the mtDNA content determined by dot blotting as described under Materials and Methods. Samples were performed in triplicate and the mtDNA content was plotted as percentage of control 6 standard error.
Continuous exposure of avian and mammalian cells to the DNA-intercalating compound ethidium bromide is known to induce a progressive dilution of mtDNA following each round of cell division due to an inhibition of mtDNA synthesis [16, 17]. If DEQ also acts by inhibiting mtDNA synthesis, a similar two-fold dilution of mtDNA should occur with each cell doubling. To determine if DEQ caused a two-fold loss of mtDNA with each cell division, the mtDNA content was measured at different times in HeLa cells continuously treated with DEQ (1 mM) and the data were plotted relative to the number of cell doublings (Fig. 4). Within the first cell doubling, DEQ reduced the mtDNA content to approximately 50% of control values. After two cell doublings, the mtDNA content was further reduced to approximately 25% of controls, indicating that similar to ethidium bromide, DEQ causes a two-fold reduction in mtDNA content with each cell doubling. Analysis of the mtDNA by gel electrophoresis and Southern blotting after treatment of cells for 96 h with DEQ did not reveal significant mtDNA degradation (data not shown), indicating that DEQ was probably acting by inhibiting mtDNA synthesis rather than inducing an active breakdown of the mtDNA. DEQ unwinds DNA. Several inhibitors of mtDNA synthesis (i.e., ethidium bromide and 4-quinolone
drugs) have been shown to induce DNA unwinding. It has been hypothesized that this interaction may interfere with the function of proteins that are required for mtDNA replication (i.e., polymerases, helicases, topoisomerases, etc.). To determine if DEQ is also a DNAunwinding compound, supercoiled Bluescript KS2 plasmid DNA was relaxed with wheat germ topoisomerase I. After 10 min, DEQ was added to the samples and the incubations were continued for an additional 20 min before terminating the reactions with the protein denaturant SDS. Drug and protein were then removed by phenol extraction and the DNA was analyzed by gel electrophoresis (Fig. 5). Untreated Bluescript plasmid DNA contains two bands representing supercoiled form I (faster migrating form) and nicked circular relaxed form II (slower migrating form) DNA, respectively (lane A). Following relaxation by topoisomerase I, supercoiled Bluescript DNA was converted to a relaxed closed circular form that comigrated with form II DNA (lane C). Addition of increasing concentrations of DEQ (8 –32 mM), after the initial incubation with topoisomerase I, caused an increase in the mobility of Bluescript DNA back towards highly supercoiled form I DNA, indicating that DEQ induces significant unwinding of duplex DNA (lanes D–F).
FIG. 4. DEQ induces a time-dependent progressive dilution of mtDNA content in HeLa cells. HeLa cells treated with DEQ (1 mM) for various lengths of time were trypsinized and counted, and the total cellular DNA was isolated as described under Materials and Methods. Equal amounts of total cellular DNA were then dot blotted onto a nylon membrane and the membrane was hybridized to a 32 P-labeled mtDNA-specific probe as described in the legend for Fig. 3.
DEQUALINIUM AND MITOCHONDRIAL DNA
FIG. 5. DEQ unwinds DNA. Drug unwinding of circular Bluescipt KS2 DNA was assayed as described under Materials and Methods. Lanes C–F, Bluescript DNA incubated with topoisomerase I in the presence of 0, 8, 16, or 32 mM DEQ. Lanes A and B are no-enzyme controls containing Bluescript DNA incubated in the presence or absence of 32 mM DEQ, respectively. Arrows mark the positions of form I (highly supercoiled) and form II (nicked circular) forms of Bluescript KS2 plasmid DNA.
Effect of DEQ on cytochrome c oxidase activity. The DEQ-induced loss of mtDNA would be expected to cause a loss of mtDNA-encoded functions. MtDNA encodes for a number of proteins that are important in mitochondrial energy metabolism. Cytochrome c oxidase (COX) is a multisubunit lipoprotein that forms an important part of the mitochondrial electron transport chain [18]. Three subunits of this enzyme are coded for by mtDNA with the remaining subunits being coded for by nuclear DNA [19]. In addition to three subunits for COX, mtDNA contains genes for apocytochrome b, and subunits for cytochrome c, NADH dehydrogenase, and ATPase proteins [19]. A DEQ-induced depletion of mtDNA content would therefore be expected to affect the expression of a number of genes necessary for energy metabolism. Correspondingly, any decrease in respiration would likely be compensated for by an increase in glycolytic energy pathways and hence an increase in lactate, a by-product of glycolysis. To determine whether DEQ had an affect on the activity of any of the proteins involved in mitochondrial respiration, COX activity was measured in HeLa cells continuously treated with DEQ using a spectrophotometric assay [14]. DEQ (1 mM) induced a decrease in COX activity to 48 6 6% of control levels within one cell doubling. Following two cell doublings, the COX activity declined to 23 6 3% of control levels. These data correlate with the time course of DEQ-induced mtDNA loss shown in Fig. 4. We did not observe any inhibition of COX activity by DEQ in vitro, indicating that the progressive decrease in activity seen in drug-treated cells was not due to a direct inhibition of this enzyme by drug (data not shown). A drug-induced decrease in respiratory function would be expected to compromise ATP production and result in a compensatory increase in glycolytic activity in the cell. To determine the effects of continuous drug
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exposure on glycolysis, lactate accumulation was measured in HeLa cells continuously treated with 1 mM DEQ. In contrast to COX activity, there was no significant increase in lactate accumulation following one cell doubling, suggesting that there were still sufficient levels of ATP to sustain growth. However, by 1.25 cell doublings, lactate levels had rapidly increased three- to fourfold relative to controls. These data illustrate that DEQ induces a time-dependent, delayed increase in lactate accumulation in HeLa cells which inversely correlates with the DEQ-induced loss of respiratory function as measured by COX activity. Effect of culture medium on DEQ cytotoxicity. Mammalian cell lines can grow in the absence of mtDNA-encoded functions if the growth media is supplemented with pyrimidines, pyruvate, and high concentrations of glucose [12, 20, 21]. Cells lacking mtDNA are deficient in respiration and therefore rely on glycolysis for energy. These cells are also auxotrophic for pyrimidines since de novo pyrimidine synthesis is coupled to active mitochondrial electron transport. The requirement for pyruvate is not entirely clear but may relate to its function in the cellular synthesis of alanine and ketone bodies. To determine the effect of growth media on drug cytotoxicity, HeLa cells grown under respiration-dependent (DMEM) or respiration-independent (DMEMPUT) conditions were continuously exposed to DEQ for 1, 2, 3, 4, and 10 days and the viability was determined using a clonogenic assay. DMEM media contains glucose, but no pyruvate or pyrimidines and cells therefore require mitochondrial function for survival. DMEM-PUT media is identical except that it is supplemented with pyruvate and pyrimidines and therefore supports cell growth in the absence of mtDNAencoded functions. The viability of HeLa cells grown under respiration-dependent conditions (DMEM media) was decreased in both a dose- and time-dependent fashion with survival being ,1% at 1 mM DEQ at all times tested (Fig. 6A). In contrast, when cells were grown under respiration-independent conditions (DMEM-PUT), there was no significant decrease in cell survival following exposure of HeLa cells to DEQ (0.25 to 1 mM) for periods of time up to 2 days (Fig. 6B). Although cell viability was affected by longer drug exposures (3 to 10 days), it was modest in comparison to that observed in cells grown under respiration-dependent conditions. For example, viability following a 10 day exposure of HeLa cells to 250 mM DEQ under respiration-independent conditions was 50%, which contrasts with ,1% viability for cells grown under respiration-dependent conditions. Although the drug resistance observed in cells grown under respiration-independent conditions suggested that DEQ was acting by a mitochondrial mechanism,
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mM DEQ for 3 h. The results indicate that there is no significant difference in steady-state concentrations of DEQ in HeLa cells grown in DMEM or DMEM-PUT, suggesting that drug resistance was not due to alterations in drug accumulation (data not shown). DISCUSSION
FIG. 6. Cytotoxicity of DEQ on HeLa cells cultured under respiration-dependent or -independent conditions. HeLa cells were continuously treated with various concentrations of DEQ for 1, 2, 3, 4, or 10 days and cytotoxicity was assessed using a clonogenic assay as described under Materials and Methods. Cells were grown under respiration-dependent (DMEM media, A) or respiration-independent (DMEM-PUT media, B) conditions. Samples were done in triplicate and the data expressed as percentage of non-drug-treated controls 6 standard error.
we could not exclude the possibility that this result was simply due to differences in drug accumulation when cells were grown under these two conditions. To investigate this possibility, the steady-state concentration of DEQ was determined in HeLa cells grown in either DMEM or DMEM-PUT media using a fluorescence assay similar to that described by Darzynkiewicz et al. [15]. Time-course studies in our lab found that drug levels in HeLa cells reach steady state by 1 h following exposure to 1 mM DEQ (data not shown). Based on this finding, drug accumulation in DMEM and DMEM-PUT was determined following exposure of HeLa cells to 1
The data presented here demonstrate that DEQ, a cationic lipophilic compound, induces a delayed inhibition of cell growth in cultured human cervical carcinoma cells. DEQ also causes a selective loss of mtDNA from these cells, which was accompanied by a decrease in COX activity and a delayed increase in lactate accumulation. Furthermore, cells grown under respiratory-independent conditions are resistant to the cytotoxic effects of DEQ. These studies raise the interesting possibility that DEQ may exert its antiproliferative effects by depleting carcinoma cells of their mtDNA. In mammalian cells, mtDNA is present at a copy number of approximately 1000 –2000 copies per cell, comprising ,1% of the total cellular DNA (reviewed in [22]). Inhibition of one or more processes involved in mtDNA synthesis would be expected to cause a successive dilution of mtDNA with each cell division. If mtDNA synthesis were completely blocked, this would result in a two-fold loss of mtDNA following every cell division. Concentrations of DEQ that caused maximal loss of mtDNA induced a two-fold reduction of mtDNA from HeLa cells following each cell division, suggesting that this drug is blocking one or more critical activities (i.e., polymerases, helicases, topoisomerases, etc) required for mtDNA replication. Other compounds have also been shown to cause a selective loss of mammalian cell mtDNA, including the 4-quinolone DNA gyrase-inhibiting antibiotics nalidixic acid and ciprofloxacin [13], ethidium bromide [16], the polyamine analogues MGBG [23] and N1,N12bis(ethyl)spermine [24], 29,39-dideoxycytidine (a putative inhibitor of mitochondrial g DNA polymerase; [25]) and ditercalinium [14]. DEQ is structurally different from these compounds. However, DEQ does induce DNA unwinding, a property which is shared by several of the drugs that induce mtDNA loss (i.e., ethidium bromide, ditercalinium, and 4-quinolone drugs) [12, 14, 16]. Our in vitro studies indicate that unwinding of plasmid DNA occurs at concentrations $ 8 mM which are considerably higher (approximately 25-fold) than those required to inhibit cell growth and cause a loss of mtDNA. However, because lipophilic cations such as DEQ can theoretically accummulate to as much as 10,000-fold within mammalian mitochondria [2, 26], it is likely that drug concentrations which were found to induce inhibition of cell growth and loss of mtDNA result in intramitochondrial concentrations sufficient to cause extensive unwinding of mtDNA. The finding
DEQUALINIUM AND MITOCHONDRIAL DNA
that DEQ induces DNA unwinding is consistent with previous DNA ultracentrifugation analyses by Wright et al. [27], which indicated that DEQ significantly altered the sedimentation coefficient of closed circular PM2 DNA. Based on helix unwinding angle calculations, these investigators concluded that DEQ was probably acting as a monofunctional DNA-intercalating agent. Therefore, although the structure of DEQ is significantly different from the DNA-intercalating compounds ethidium bromide and ditercalinium, it also appears to induce DNA unwinding by an intercalative mechanism. This contrasts with 4-quinolone drugs which mediate DNA unwinding by interacting with single-stranded regions in DNA [28]. DEQ caused a delayed inhibition of HeLa cell growth. This delayed inhibition of cell growth correlated well with the time- and dose-dependent decrease in mtDNA content. In mammalian cells, mtDNA is present in a copy number greatly in excess of that necessary to maintain cell viability. Wiseman and Attardi [16] observed a mtDNA threshold of 10%, below which mammalian cells were unable to support the energy requirements needed for growth or viability. The data presented here indicate that DEQ caused HeLa growth inhibition at concentrations that induced a greater than an 80% loss of mtDNA ($0.5 mM), which is consistent with results for other drugs which interfere with mtDNA synthesis [13, 16]. Mammalian cell mtDNA encodes protein components of the electron transport chain that are required for mitochondrial respiration (i.e., COX). It is therefore probable that the DEQ-induced depletion of mtDNA is a significant factor contributing to the observed decrease in mitochondrial COX activity due to the presence of fewer mtDNA templates available for transcription. However, we cannot rule out that DEQ may also act by directly interfering with mtDNA transcription or some posttranscriptional process. A third possibility is that DEQ might directly inhibit or inactivate COX. However, this last explanation is unlikely since DEQ was not found to have an affect on COX activity in HeLa cell extracts. It is not surprising that the DEQ-induced decrease in respiratory function would be associated with a compensatory increase in glycolysis. DEQ induced a 3- to 4-fold delayed increase in lactic acid accumulation which occurred between 1 and 2 cell doublings. The rise in lactate succeeds the decrease in COX activity. These results are consistent with the need for the cells to use an alternative, albeit less efficient, pathway for obtaining energy due to a loss in respiratory function. Cell lines that lack mtDNA have been generated [20, 21]. These cell lines are able to grow in the absence of mtDNA-encoded function because their requirement for respiratory function has been bypassed by supplementing the growth media with glucose, pyruvate, and
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pyrimidines. The studies presented here illustrate that HeLa cells grown under respiratory-independent conditions (DMEM-PUT media) were relatively resistant to DEQ cytotoxicity. Resistance could not be attributed to altered drug uptake and this suggests that DEQ exerts its cytotoxic effects by interfering with a critical mitochondrial function(s). Other mechanisms for the anticarcinoma effects of DEQ have been proposed. Bodden et al. [5] proposed that DEQ might block cellular proliferation by inhibiting calmodulin. Calmodulin is a multifunctional calcium-binding protein primarily localized in the cytosol. This protein regulates numerous cellular events including chromosome disjunction during mitosis [29]. In the yeast Saccharomyces cerevisiae, deletion of the calmodulin gene is a lethal mutation [30]. Yeast mutants temperature sensitive for calmodulin were found to lose viability when grown at a nonpermissive temperature. The loss in viability appeared to be related to an inability of the cells to complete mitosis due to defects in chromosome segregation, spindle pole body functions, and cytokinesis [29, 31]. Similarly, treatment of mammalian cells with calmodulin antagonists has been shown to inhibit cell proliferation and cause a rapid loss in cell viability [32]. This contrasts with the delayed loss of cell proliferation that is seen with DEQ, suggesting that calmodulin is probably not the primary target for this drug. Another hypothesized mechanism of action for DEQ is the inhibition of protein kinase C [8]. DEQ inhibits isolated protein kinase C-b1 with an IC50 of 8 –15 mM. The concentrations at which we observe depletion of mtDNA are 10- to 20-fold lower than those at which protein kinase C is inhibited (0.5–1.0 mM versus 8 –15 mM for protein kinase C-b1 inhibition). Also, similar to calmodulin, inhibition of protein kinase C affects a variety of critical cellular functions including nuclear DNA replication and it is therefore improbable that DEQ is mediating its antiproliferative effects via protein kinase C since since DEQ had no direct and immediate affects on nuclear DNA synthesis. DEQ has also been demonstrated to inhibit the in vitro activity of mitochondrial F1-ATPase from bovine heart with an IC50 of 8 –12 mM, suggesting that this enzyme may be an in vivo target of drug action [6, 7]. Due to the selective accumulation of DEQ in mitochondria, it is very likely that cytotoxic concentrations of drug probably cause at least some inhibition of the mitochondrial ATPase. Is it possible that DEQ inhibition of the mitochondrial ATPase might also be responsible for the depletion of mtDNA? Perhaps inhibition of the mitochondrial ATPase enzyme causes an indirect inhibition of mtDNA synthesis due to a decrease in the levels of mitochondrial ATP. Studies in S. cerevisiae indicate that oligomycin, a specific inhibitor of the mitochondrial ATPase, does not induce the formation of
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mitochondrial petite mutants (cells having mutations in mtDNA or that completely lack any mtDNA sequences) [33]. In contrast, DEQ was found to be a potent inducer of rho0 petites (cells completely lacking mtDNA), suggesting that the DEQ-induced loss in mtDNA is not due to inibition of the mitochondrial ATPase [34]. The intriguing discovery by Chen and co-workers that liphophilic cationic compounds such as DEQ accumulate to a much greater extent in the mitochondria of carcinoma cells relative to normal epithelial cells has led to the identification of a number of novel compounds possessing significant anticarcinoma activity [1, 4, 35]. Although these compounds are all lipophilic cations, their structures vary significantly and the molecular target of these drugs remains unclear. One of the most promising lipophilic cations with anticarcinoma activity is the rhodacyanine dye MKT-077. This drug has been shown to possess significant activity against human renal cell carcinoma, prostate carcinoma, and melanoma tumors implanted in nude mice [4]. Initial findings from Phase I clinical trials in patients with refractory carcinomas indicate that MKT077 does not exert any significant side effects, including cardiac toxicity or myelosuppression [4]. Prelimminary evidence also suggests that like DEQ, MKT-077 also causes a selective loss of mtDNA in cultured carcinoma cells [36]. Whether or not these two structurally different lipophilic cations exert their anticarcinoma affects through a common mechanism awaits further investigation. The authors thank David Gill, Amy Jennings, Alan Klopp, Mark Mainwaring, and Volkmar Weissig for their assistance and encouragement during the course of these studies.
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