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Suppression of human tumor cell proliferation through mitochondrial targeting

Division of Cardiovascular Diseases, Departments of Medicine, Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Mayo Foundation, Rochester, Minnesota, USA; and
^* Section of Clinical Pharmacology, Department of Medicine, Dartmouth Medical School and Dartmouth Hitchkock Medical Center, Lebanon, New Hampshire, USA

¹Correspondence: Guggenheim 7, Mayo Clinic, Rochester, MN, 55905, USA. E-mail: terzic.andre{at}mayo.edu

	ABSTRACT

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Intracellular calcium signaling plays a central role in cell proliferation. In leukemic cells, the calcium release-activated calcium channels provide a major pathway for calcium entry (I_CRAC) perpetuating progression through the cell cycle. Although I_CRAC is under mitochondrial regulation, targeting mitochondrial function has not been exploited to control malignant cell growth. The benzothiadiazine diazoxide, which depolarized respiration-dependent mitochondrial membrane potential, reduced the rate of proliferation and arrested human acute leukemic T cells in the G0/G1 phase. Diazoxide did not alter cellular energetics, but rather inhibited the mitochondria-controlled I_CRAC and reduced calcium influx into tumor cells. The antiproliferative action of diazoxide was mimicked by removal of extracellular calcium or by the tyrphostin A9, an I_CRAC inhibitor. Deletion of the mitochondrial genome, which encodes essential respiratory chain enzyme subunits, attenuated the inhibitory effect of diazoxide on I_CRAC-mediated calcium influx and cell proliferation. Thus, manipulation of mitochondrial function and associated calcium signaling provides a basis for a novel anticancer strategy.—Holmuhamedov, E., Lewis, L., Bienengraeber, M., Holmuhamedova, M., Jahangir, A., Terzic, A. Suppression of human tumor cell proliferation through mitochondrial targeting.

Key Words: mitochondrial DNA • calcium release-activated calcium channels • diazoxide • cancer

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
PROLIFERATION OF LEUKEMIC cells is under control of intracellular Ca²⁺ signaling (1) . Prolonged elevation of cytosolic Ca²⁺ regulates nucleocytoplasmic communication, increases the efficiency and specificity of gene transcription, and drives cells through Ca²⁺-sensitive checkpoints of the cell cycle (2 3 4 5 6) . It has become increasingly evident that mitochondria play a central role in shaping the pattern of intracellular Ca²⁺ dynamics (7 8 9) . In T lymphocytic leukemia cells, gating of the Ca²⁺ release-activated Ca²⁺ channels (CRAC), as well as the rate and magnitude of Ca²⁺ influx (I_CRAC) through these store-operated Ca²⁺ channels, are determined by the mitochondrial status, underscoring the significance of mitochondria in defining cell cycle progression (10 11 12 13 14 15 16) .

A potential dependent Ca²⁺ uptake into energized mitochondria reduces cytosolic Ca²⁺ in the proximity of CRAC channels, promoting channel opening (11 , 13 , 15) . Conversely, in cells with de-energized mitochondria and reduced mitochondrial Ca²⁺ uptake, the elevated free Ca²⁺ concentration maintains store-operated channels closed in accord with the Ca²⁺-dependent inactivation of I_CRAC (14 15 16) . Although mitochondria have the potential to regulate intracellular Ca²⁺ signaling and determine the transcriptional potential of a cell (15) , targeting mitochondria has not been exploited to control I_CRAC-mediated Ca²⁺ influx and associated cell proliferation.

Prototypic drugs such as diazoxide, which display a predilection for the inner mitochondrial membrane, were recently recognized for their ability to oxidize components of the respiratory chain and reduce mitochondrial membrane potential (17 18 19) . We demonstrate that diazoxide suppresses proliferation of human acute T lymphocytic leukemia cells through mitochondrial targeting. Diazoxide depolarized mitochondria and inhibited mitochondria-dependent Ca²⁺ influx, arresting cell cycle progression without disturbing cell energetics. The antiproliferative action of diazoxide was lost in cells with deleted mitochondrial DNA lacking functional mitochondria. These findings indicate that manipulation of functional mitochondria is an effective strategy of disrupting intracellular Ca²⁺ signaling and cell proliferation.

	MATERIALS AND METHODS

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Tumor cells with and without mitochondrial DNA
Human T leukemic Jurkat and Molt-4 cell lines (American Type Culture Collection, Rockville, MD) were cultured at an initial density of 5·10⁴ cells/mL in RPMI 1640 (Bio-Whittaker, Walkersville, MD) supplemented with 10% fetal bovine serum and 2 mM L-glutamine (37°C, 5% CO₂/95% air). To obtain cells devoid of functional mitochondria, mitochondrial DNA was depleted by long-term treatment of Molt-4 cells with ethidium bromide, an inhibitor of mitochondrial DNA replication (20 , 21) . After 26 days (equaling 14 doubling times for Molt-4 cells) in ethidium bromide (50 ng/mL), pyruvate (100 µg/mL), and uridine (50 µg/mL), parental cells were transformed into mitochondrial DNA-deficient rho⁰ cells. Mitochondria-deficient clones were selected for their pyrimidine dependence. Absence of mitochondrial DNA was confirmed by quantitative PCR using mitochondrial DNA specific primers (forward: 5'-ACAATAGCTAAGACCCAAACTGGG-3' and reverse: 5'-GCCCATGAGGTGGCAAGAAATGGG-3'). The concentration of total cellular DNA, extracted from 5·10⁶ Molt-4 and rho⁰ cells (DNeasy Tissue Kit, Qiagen, Chatsworth, CA), was determined using the PicoGreen dsDNA quantification reagent (Molecular Probes, Eugene, OR). Starting with the same amount of total DNA (50 ng), a 320 bp fragment of mitochondrial DNA was amplified in the linear range of the PCR using Taq polymerase. After 16 cycles, mitochondrial DNA was visualized by agarose gel electrophoresis using SYBR-Gold (Molecular Probes). Depletion of mitochondrial genes encoding respiratory chain components was demonstrated by Southern blot. Total cellular DNA digested with EcoRI was separated by agarose gel electrophoresis, transferred to nylon membranes, and probed with ³²P-labeled cDNAs encoding mitochondrial cytochrome c oxidase subunit II or nuclear ß-actin. Signals were analyzed using a PhosphorImager 445SI and quantified with the ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Cell cycle distribution
Cell number was determined using a hemocytometer (Bright Line, Hausser Scientific, Horsham, PA), and cell cycle distribution analyzed with a fluorescent activated cell sorter (Vantage Cytofluorimeter, Becton Dickinson, Rutherford, NJ). For flow cytometry, cells fixed in 70% ethanol and washed with Ca²⁺/Mg²⁺-free phosphate buffer were treated with Rnase, and nuclear DNA was labeled with 50 µg/mL propidium iodide. After data acquisition, the CellQuest software (Becton Dickinson) was used to analyze nuclear DNA distribution (22) .

Confocal microscopy and spectrofluorometry
Cells were labeled with mitochondria-specific fluorescent dyes Mito-Tracker Green-FM and 10-nonyl acridine Orange (Molecular Probes), and the intracellular distribution of mitochondria was examined by laser scanning confocal microscopy (LSM 510, Carl Zeiss) as described (9 , 19) . Cytoplasmic Ca²⁺ was imaged in separate experiments in cells loaded with Fluo-3/AM, and intracellular Ca²⁺ levels determined off-line using the image analysis software ANALYZE (Mayo Clinic, Rochester, MN). The rate of Ca²⁺ influx was determined from changes in the concentration of intracellular Ca²⁺ monitored by Indo-1 (Molecular Probes) (16) , using a spectrofluorometer (SPEX Fluorolog, SPEX Industry).

Electron microscopy
Cells (2·10⁶) were fixed using Trump’s buffer (1% glutaraldehyde, 4% formaldehyde, 0.1 M phosphate buffer, pH 7.2), rinsed, and postfixed in phosphate-buffered 1% osmium tetroxide. Samples were stained en bloc with 2% uranyl acetate for 30 min at 60°C, rinsed, dehydrated, and embedded in Spurr’s resin. Thin sections were cut on an Ultracut E ultramicrotome (Reichert-Jung, Wien, Austria), placed on copper grids, stained with lead citrate, and micrographed with an EM-1200 EX II electron microscope (Jeol, Peabody, MA) as described (23 , 24) .

Cellular respiration and ATP content
The rate of oxygen consumption was determined with an oxygen-sensitive electrode at 37°C in cells incubated (in mM: KCl 110, NaCl 10, KH₂PO₄ 2, MgSO₄ 1, pyruvate 5, malate 5, HEPES 20, pH 7.15) in the presence of saponin (0.005%) to provide access for mitochondrial substrates (19) . Cellular ATP was determined by high-pressure liquid chromatography (Hewlett Packard, Palo Alto, CA) of the acid-soluble cellular extract neutralized with K₂CO₃-HEPES (2.5 M/1 M) as described (23 , 25) .

Isolated mitochondria
Mitochondria were isolated after cell homogenization with a Dounce homogenizer in buffer containing (in mM: mannitol 220, sucrose 50, MOPS 10, PMSF 1, and EGTA 1, pH 7.3) using differential centrifugation (26) . Mitochondria washed once with isolation buffer (without EGTA) were used immediately or stored with 1 mM PMSF/1 mM DTT in liquid nitrogen until processed. Mitochondrial cytochrome c and citrate synthase activities were measured from the oxidation of cytochrome c (550 nm) and deacylation of acetyl-CoA (412 nm) using a DU-7400 spectrophotometer (26) .

Experimental compounds
Diazoxide, thapsigargin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and the tyrphostin A9 (Sigma, St. Louis, MO) were dissolved in dimethyl sulfoxide (Sigma). The maximal concentration of solvent in the incubation medium was under 0.5%.

Statistical analysis
Data are expressed as mean ± SE. Where appropriate, Student’s t test or analysis of variance was performed for comparison between groups using a statistical analysis software (JMP, SAS Institute, Cary, NC). A value of P < 0.01 was considered statistically significant.

	RESULTS

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Suppression of cell proliferation by diazoxide
Human leukemic Jurkat cells proliferated vigorously at a rate of 7600 ± 840 cells/h (n=10; Fig. 1 A, B). Typical for asynchronous growth, Jurkat cells displayed a broad cell cycle distribution, with 52 ± 3%, 32 ± 2% and 17 ± 3% (n=9) of the cell population in the G0/G1, S and G2/M phase, respectively (Fig. 1C , inset). Diazoxide (100 µM) dramatically reduced the proliferation rate to 2650 ± 330 cells/h (n=10), resulting in a threefold decline in growth compared to untreated cells (P<0.01; Fig. 1A, B ). In fact, diazoxide (100 µM) arrested Jurkat cells in the G0/G1 phase away from the S phase (Fig. 1C ). On average, diazoxide increased the percentage of cells in the G0/G1 phase to 71 ± 2% (n=9), a 36% increase compared to untreated cells (P<0.01). In parallel, diazoxide decreased the number of cells in the S phase to 20 ± 2% (n=9), a 38% reduction (P<0.01; Fig. 1C ), without significantly affecting the G2/M phase (n=9; Fig. 1C ). Upon removal of diazoxide, Jurkat cells resumed their initial rate of proliferation and continued to grow at 7920 ± 880 cells/h, a value not significantly different from that of untreated cells (P=0.17; Fig. 1A, B ). Thus, diazoxide suppressed proliferation of acute leukemic T cells associated with reversible inhibition of cell growth.

View larger version (30K): [in this window] [in a new window]   Figure 1. Diazoxide suppresses proliferation of human T leukemic Jurkat cells. A) Time course of proliferation in cells initially plated at 5·104/mL in the absence (open circles) or presence (filled diamonds) of 100 µM diazoxide. Washout of diazoxide (arrow) restored the original rate of cell growth (open diamonds). B) Average rate of cell proliferation in the absence (open bar), presence (filled bar) and after washout (w/o; hatched bar) of 100 µM diazoxide. C) Cell cycle distribution in the absence (open bar) or presence (filled bar) of 100 µM diazoxide. The number of cells in each phase (G0/G1, S, and G2/M) is expressed as percent of total number of cells (20,000) in each condition. Asterisk indicates significant difference between groups in the absence and presence of drug.

Targeting mitochondria reduces cellular Ca²⁺ influx required for proliferation
Growth and cell cycle progression depend on intracellular energy supply and calcium signaling (1) . In leukemic cells, mitochondria are central in both processes by providing ATP through oxidative phosphorylation and by regulating store-operated Ca²⁺ influx (I_CRAC) through membrane potential-dependent Ca²⁺ buffering (1 , 12 13 14 15 16) . Proliferating Jurkat cells demonstrated abundance of energized mitochondria distributed throughout the cytosol (Fig. 2 A, B), with a capability to avidly consume oxygen (Fig. 2C ). Diazoxide (100 µM) significantly increased the rate of oxygen consumption (Fig. 2C, D ) and depolarized mitochondrial membrane (Fig. 2E, F ). Intracellular ATP concentration was, however, maintained at 1.48 ± 0.11 and 1.29 ± 0.09 nmol ATP/mg protein (n=3; P=0.15) in the absence and presence of diazoxide (100 µM), indicating that diazoxide-induced mitochondrial depolarization was insufficient to interrupt cellular energy metabolism (Fig. 3 A). Yet diazoxide significantly reduced cytosolic Ca²⁺, by 66 ± 7%, from 30854 ± 5287 to 10365 ± 2213 pixels of Fluo-3 fluorescence per image in untreated vs. diazoxide-treated cells (n=27; Fig. 3B ). In lymphocytic T cells, intracellular Ca²⁺ refills primarily through I_CRAC, the mitochondria-dependent store-operated Ca²⁺ influx channel (12 , 13 , 16) . In Jurkat cells with intact mitochondria, depletion of intracellular Ca²⁺ stores with thapsigargin (1 µM), a blocker of the endoplasmic reticulum Ca²⁺-ATPase (16) promoted vigorous Ca²⁺ influx (Fig. 3C ) sensitive to the tyrphostin A9 (1 µM), an inhibitor of I_CRAC (27 ; Fig. 3C ). Depolarization of mitochondria with diazoxide (100 µM) significantly reduced store-operated Ca²⁺ influx (Fig. 3C ), from 19.5 ± 2.8 to 14.8 ± 1.8 relative fluorescent units/min/10⁶cells (n=9, P<0.01). Store-operated Ca²⁺ influx was abolished in the presence of 1 µg/mL CCCP, a powerful mitochondrial uncoupler (Fig. 3C ). In turn, reduction of Ca²⁺ influx by removal of extracellular Ca²⁺ or by direct inhibition of I_CRAC had a profound inhibitory effect on cell growth, reducing the rate of proliferation by 62 ± 5% and 92 ± 7%, respectively (Fig. 3D ). Thus, by targeting mitochondria, diazoxide inhibits I_CRAC in Jurkat cells, thereby reducing the intracellular Ca²⁺ influx required for cell proliferation.

View larger version (30K): [in this window] [in a new window]   Figure 2. Diazoxide regulates mitochondrial function in Jurkat cells. A, B) Laser confocal microscopy of cells stained with specific mitochondrial fluorescent dyes (MitoTracker in panel A and 10-nonyl acridine orange in panel B) demonstrating abundant mitochondrial distribution throughout the cytosol. C) Diazoxide (100 µM) accelerates the rate of oxygen consumption in permeabilized cells in the presence of mitochondrial substrates, pyruvate (2.5 mM) and malate (2.5 mM). D) Average cellular respiration in the absence (open bar) and presence (closed bar) of 100 µM diazoxide. E) Diazoxide-induced leftward shift in the fluorescent distribution of the mitochondrial-potential sensitive dye, TMRM, indicating mitochondrial depolarization. F) Average mitochondrial depolarization in the absence (open bar) and presence (closed bar) of 100 µM diazoxide. In panels D, F, asterisk indicates significant difference between groups in the absence and presence of drug.

View larger version (43K): [in this window] [in a new window]   Figure 3. Diazoxide reduces cytosolic Ca2+ and inhibits ICRAC, required for proliferation of Jurkat cells. A) Intracellular ATP levels are maintained in the presence of diazoxide (100 µM), which B) significantly reduced cytosolic Ca2+. A total of 2·106 cells were used for the ATP assay, while Ca2+ was monitored in Fluo-3 AM loaded cells using scanning confocal microscopy. C) Emptying of intracellular Ca2+ stores with thapsigargin (1 µM) in Ca2+-free medium, activates Ca2+ influx through ICRAC monitored in 5 µM Indo-1 AM loaded cells on addition of 3 mM of extracellular CaCl2 (arrow). Ca2+ influx is inhibited by 1 µM A9, an inhibitor of ICRAC, or 0.1 µM CCCP, an uncoupler of mitochondrial oxidative phosphorylation, and reduced by diazoxide (100 µM). D) Cell growth was obtained in the presence of 335 µM Ca(NO3)2 (open bar), and was inhibited by removal of Ca2+ (closed bar) or by addition of 1 µM A9 (hatched bar). Asterisk indicates significant difference with control.

Blunted suppression of proliferation in mitochondria-deficient cells
Mitochondrial biogenesis critically depends on mitochondrial DNA-encoded polypeptides of the respiratory chain (20 , 21 , 28) . To determine the contribution of mitochondria in the antiproliferative effect of diazoxide, cells void of functional mitochondria (rho⁰) were generated by depletion of mitochondrial DNA with ethidium bromide, a potent inhibitor of mitochondrial DNA replication and transcription (20 , 21 , 29) . The parental malignant lymphoid cell line Molt-4 (WT) possessed intact mitochondrial membranes and typical cristae, a signature of functional mitochondria (Fig. 4 A and inset). In contrast, rho⁰ cells displayed an aberrant mitochondrial phenotype (‘mitochondrial ghost’) with grossly distorted cristae and essentially an empty matrix (Fig. 4B and inset). Loss of mitochondrial DNA in rho⁰ cells, confirmed by quantitative PCR amplification (Fig. 4C ), was associated with reduced expression of the mitochondrial gene-encoded subunit II of cytochrome c oxidase (Fig. 4D , upper panel). The activity of cytochrome c oxidase was 25 ± 3 vs. 3 ± 1 nmol cytochrome c/min/10⁶ cells in Molt-4 and rho⁰ cells, respectively (n=4, P<0.01; Fig. 4D , lower panel, F). Rho⁰ cells did, however, maintain an intact nuclear genome, with comparable expression of the ß-actin gene in wild-type and mitochondrial DNA-deficient cells (Fig. 4E , upper panel) and unchanged activity of citrate synthase, a marker of a mitochondrial enzyme encoded by nuclear DNA (Fig. 4E , lower panel). Loss of functional mitochondria resulted in defective responsiveness of rho⁰ cells to diazoxide (Fig. 5 ). Whereas diazoxide (100 µM) essentially halved cytoplasmic Ca²⁺ concentration in parental Molt-4 cells (Fig. 5A , top panel), the mitochondrial potassium channel opener did not significantly modulate Ca²⁺ levels in rho⁰ cells (Fig. 5A , lower panel). Indeed, the diazoxide-induced reduction in Ca²⁺ concentration was 41 ± 3% vs. 1 ± 1%, respectively (P<0.01). Diazoxide was a less effective inhibitor of store-operated Ca²⁺ influx in mitochondrial-deficient compared to parental cells (Fig. 5B ). On average, diazoxide (100 µM) reduced the rate of thapsigargin-activated Ca²⁺ influx into wild-type parental cells from 8.5 ± 0.5 to 5.9 ± 0.9 RFU min/10⁶cells, a 41 ± 3% inhibition of Ca²⁺ influx (n=6, P<0.01). In contrast, in rho⁰ cells, diazoxide did not significantly affect the rates of Ca²⁺ influx, which were 3.2 ± 0.2 and 2.9 ± 0.3 RFU/min/10⁶cells in the absence and presence of the drug (n=6; P=0.25). Accordingly, in parental cells, diazoxide (100 µM) impaired the rate of proliferation, by 43 ± 5% (n=3), but this antiproliferative effect was significantly attenuated (15±6%, n=3) in mitochondrial DNA-deficient rho⁰ cells (Fig. 5C ; P<0.01). Thus, deletion of the mitochondrial genome, encoding essential enzyme subunits of the respiratory chain, reduced diazoxide-mediated inhibition of store-operated calcium influx and tumor cell growth.

View larger version (55K): [in this window] [in a new window]   Figure 4. Mitochondrial structure and function in wild-type parental Molt-4 and mitochondrial DNA-depleted rho0 cells. A) Electron micrographs of a wild-type parental (WT) T lymphocytic Molt-4 cell with typical intramitochondrial organization, dense matrix and abundant cristae (A, inset). B) A mitochondrial DNA-devoid rho0 cell with a ‘mitochondrial ghost’ appearance due to absence of electron-dense mitochondrial matrix and disorganized cristae (B, inset). C) Mitochondrial DNA (mtDNA) content in parental Molt-4 (WT) and rho0 cells measured by quantitative PCR (upper panel). Average mitochondrial DNA content in WT (open bar) and rho0 (closed bar) cells (lower panel). D) Southern blot analysis (upper panel) and activity (lower panel) of the mitochondrial genome-encoded subunit II of cytochrome c oxidase (COX II) in parental (WT) and mtDNA-deficient rho0 cells. C, D) Asterisk indicates significant difference between parental and mtDNA-deficient cells. E) Respective Southern blot analysis and activity of the nuclear genome encoded ß-actin (upper panel) and citrate synthase (lower panel) in parental (WT) and mtDNA-deficient (rho0 cells. F) Reduced cytochrome c oxidation in mtDNA-deficient (rho0) vs. parental (WT) cells.

View larger version (41K): [in this window] [in a new window]   Figure 5. Blunted effect of diazoxide on cytosolic Ca2+ and cell growth in mitochondrial DNA-deficient cells. A) Diazoxide reduces intracellular Ca2+ in parental Molt-4 (WT), but not mitochondrial DNA-deficient (rho0) cells loaded with Fluo-3 AM. B, C) Reduced inhibition by diazoxide (100 µM), in rho0 compared to parental (WT) cells, of thapsigargin-mediated Ca2+ influx through ICRAC (B) and of the rate of proliferation (C). B, C) Asterisk indicates significant difference between parental (WT) and mtDNA-deficient cells.

	DISCUSSION

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Traditionally, mitochondria have been recognized as the main source of intracellular energy production securing energetic homeostasis of the cell through oxidative phosphorylation (30) . More recently it has become apparent that mitochondria are also central in cellular Ca²⁺ signaling and contribute to intracellular Ca²⁺ dynamics by virtue of their membrane potential-dependent Ca²⁺ buffering capacity (1 , 7 8 9 , 13) and proximity to specialized cellular Ca²⁺ compartments (31) . Mitochondria are critical in the regulation of vital Ca²⁺-dependent processes, including activation of transcription factors and gene expression (3 , 5) . Commitment to and completion of the cell division cycle in cells with a high metabolic drive, such as fast growing cancer cells, are processes particularly sensitive to inhibition of mitochondrial function (32) . Here, we exploited mitochondrial control over intracellular Ca²⁺ and demonstrate that depolarization of mitochondrial membrane potential down-regulates intracellular Ca²⁺, suppressing proliferation in human leukemia cell lines. Thus, targeting mitochondria to disrupt communication between mitochondria and Ca²⁺ signaling pathways emerges as an efficient approach in control of malignant cell growth.

In human leukemia cells such as T lymphocyte-derived Jurkat and Molt-4 cells used here, the mitochondrial functional status determines Ca²⁺-dependent gating of the store-operated Ca²⁺ current I_CRAC, a key pathway for sustained Ca²⁺ entry (1 , 13) . High mitochondrial membrane potential promotes mitochondrial Ca²⁺ uptake and activates I_CRAC, leading to transcriptional activation and progression through cell cycle (15 , 32) . We demonstrate that diazoxide, which targets the inner mitochondrial membrane and oxidizes the respiratory chain (17 , 18) , inhibited I_CRAC and suppressed cell proliferation. The effects of diazoxide on Ca²⁺ flux and cell proliferation were attenuated in respiratory-deficient mitochondrial DNA-depleted cells, underscoring the requirement for a functional electron transport chain in mediating the action of diazoxide. Previous reports identify mitochondrial succinate dehydrogenase (33 , 34) and a mitochondrial K⁺ conductance (17 , 18 , 35) as molecular targets for diazoxide. Indeed, inhibition of a key component in the respiratory chain and/or activation of potassium influx would result in mitochondrial depolarization (36 , 37) , in accord with the reduction in membrane potential induced here by diazoxide. A surge in mitochondrial membrane potential during the G0/G1 phase of the cell cycle is a prerequisite for proliferating cells to engage into the S phase (32) . Clamping mitochondrial membrane potential at a depolarized level by diazoxide provides a mechanistic basis for the antiproliferative action and cell arrest in the G0/G1 phase away from the S phase. Such a property could be explored to develop novel adjunctive therapy in the treatment of proliferative disorders.

In summary, this study demonstrates a novel antiproliferative mechanism through regulation of mitochondria-dependent intracellular Ca²⁺ homeostasis. Specifically, we show suppression of leukemic cell proliferation by the benzothiadiazine diazoxide through depolarization of the mitochondrial membrane and disruption of intracellular Ca²⁺ dynamics. Deletion of mitochondrial DNA attenuated the antiproliferative action of diazoxide, underscoring the critical role of functional mitochondria and associated Ca²⁺ signaling in cell cycle progression. In this way, mitochondria by orchestrating intracellular Ca²⁺ homeostasis emerge as unique targets for regulation of malignant growth and proliferation.

	ACKNOWLEDGMENTS

 
We thank Drs. I. Ovsyannikova, S. Trushin, C. Ozcan, and P. P. Dzeja (Mayo Clinic) for input in cell preparation and helpful suggestions. This work was supported by the National Institutes of Health and the American Heart Association. L.D.L. was supported in part by the O’Haus Family Foundation. A.T. is an Established Investigator of the American Heart Association.

Received for publication January 3, 2002. Revision received February 26, 2002.

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