Depletion of mitochondrial DNA by ethidium bromide treatment inhibits the proliferation and tumorigenesis of T47D human breast cancer cells

Toxicology Letters 170 (2007) 83–93

Depletion of mitochondrial DNA by ethidium bromide treatment inhibits the proliferation and tumorigenesis of T47D human breast cancer cells Man Yu a,b,c , Yurong Shi a , Xiyin Wei a , Yi Yang a , Yunli Zhou a , Xishan Hao a , Ning Zhang a,d , Ruifang Niu a,∗ a

State Key Laboratory of Breast Cancer Prevention and Treatment, Tianjin Cancer Hospital and Institute, Tianjin 300060, PR China b Center for Advanced Research in Environmental Genomics (CAREG), University of Ottawa, 20 Marie Curie, Ottawa, Ontario K1N 6N5, Canada c Department of Cellular and Molecular Medicine, University of Ottawa, 451 Smyth Road, Ottawa, Ontario K1H 8M5, Canada d Department of Biological Sciences, 375 Dodge Hall, Oakland University, 2200 N. Squirrel Road, Rochester, MI 48309-4401, USA Received 28 November 2006; received in revised form 20 February 2007; accepted 23 February 2007 Available online 1 March 2007

Abstract In order to investigate the role of mitochondrial DNA (mtDNA) in human breast cancer cell proliferation and apoptosis, a mtDNA-deficient cell line, T47D ␳0 , was generated following a long-term exposure to ethidium bromide (EtBr). T47D ␳0 cells showed a marked decrease in mitochondrial membrane potential (Ψ m ). However, the apoptosis rate of T47D ␳0 cells was the same as that of their parental cells, suggesting that the change in Ψ m was insufficient to induce cell death. Electromicroscopy revealed a profound alteration of mitochondrial morphology, which was consistent with the loss of mtDNA and the decrease in Ψ m . Disruption of mtDNA resulted in a slower proliferation rate in tissue culture and a reduction in anchorage-independent growth. An in vivo assay revealed a severe impairment of tumorigenicity in T47D ␳0 cells, indicating the biological relevance of in vitro studies. Taken together, our results suggest that the integrity of mtDNA plays a critical role in human breast cancer cell proliferation and tumorigenesis. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Breast cancer; Mitochondrial DNA; ␳0 ; T47D

1. Introduction

Abbreviations: Cox-2, cytochrome c oxidase subunit II; DAB, 3,3 diaminobenzidine; DMSO, dimethyl sulfoxide; Ψ m , mitochondrial membrane potential; EtBr, ethidium bromide; FITC, fluorescein-5isothiocyanate; ROS, reactive oxygen species; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; PI, propidium iodide; SCID, severe combined immune deficiency ∗ Corresponding author. Tel.: +86 22 23340123x5225; fax: +86 22 23359984. E-mail address: [email protected] (R. Niu).

Mitochondria play an essential role in the maintenance of cellular homeostasis, including ATP synthesis, redox regulation, thermogenesis and production of secondary messengers (Chan, 2006; Singh, 2006). For example, oxidative phosphorylation in mitochondria provides cells with essential energy by utilizing the respiratory chain to establish an inner membrane potential, which drives ATP synthesis by the F1F0-ATPase (Chen and Butow, 2005). Recently, increasing evidence

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demonstrates that mitochondria are also responsible for mediating apoptosis (Hajra and Liu, 2004; H.C. Lee et al., 2004; M.S. Lee et al., 2004). Upon internal cellular damage, inhibition of Bcl-2 may induce the release of cytochrome C from mitochondria, initiating apoptotic cascade through Apaf-1 and Caspase9 (Orrenius et al., 2007). Enhanced energy consumption and reduced apoptosis are two key characteristics of cancer cells (Alirol and Martinou, 2006; Hajra and Liu, 2004). Mutation of mitochondrial DNA (mtDNA) is linked to tumorigenesis (Chatterjee et al., 2006). Although the majority of mitochondrial proteins are synthesized in the nucleus and imported into mitochondria, mitochondria possess their own genome, a ∼16.6 kb circular doublestranded molecule, which encodes 13 key subunits of the respiratory chain complexes, as well as 22 transfer RNAs and 2 ribosomal RNAs (Chan, 2006; Singh, 2006). Compared with nuclear DNA (nDNA), mtDNA is more vulnerable to damages due to the lack of protective histones and introns, and insufficient DNA repair capacity (Chen and Butow, 2005). In addition, mitochondria are a major source of oxygen-derived free radicals, collectively known as reactive oxygen species (ROS), the potent DNA mutagenes (Vergani et al., 2004). Thus, the frequency of somatic mutation in mtDNA is about 10–20 times higher than that in nDNA (Copeland et al., 2002). Numerous mtDNA mutations, including intragenic deletion, duplication, missense and chain-termination point mutation, have been identified in virtually all types of cancer examined (Chatterjee et al., 2006; Copeland et al., 2002). It has also been assumed that disruption of mtDNA could enhance in vivo apoptosis and inhibit tumorigenicity in some certain tumor cells (Cavalli et al., 1997; Wang et al., 2001). The effects of mitochondrial dysfunction may be particularly important in estrogen-inducible cancers, such as breast cancer, because the metabolism of estradiol through redox-cycling intermediates generates large quantities of local ROS in breasts and the oxidized metabolites of estrogen, E2,3,4-semi-quinones and E2,3,4-quinones, can directly bind to mtDNA to form adducts, both of which may facilitate neoplastic transformation (Yager, 2000; Yager and Davidson, 2006). Similar to the observations in other types of cancer, reduced mtDNA content has been widely detected in breast cancer, especially in estrogen-positive ones (H.C. Lee et al., 2004; M.S. Lee et al., 2004; Mambo et al., 2005; Tseng et al., 2006). Decrease in the copy number of mtDNA in breast cancer is presumed to result in mitochondrial dysfunction, and contribute to altered energy metabolism and decreased ROS; these, in turn, might degrade unconstrained tumor cell proliferation and inva-

sion (Gatenby and Gillies, 2004). To test this hypothesis, a novel mtDNA-depleted cell line was generated from human breast cancer T47D cells by long-term exposure to ethidium bromide (EtBr), designated as T47D ␳0 . Preliminary studies revealed that T47D ␳0 cells were defective in mitochondrial structure and function, leading to impairments in cell proliferation in vitro and tumorigenesis in vivo. 2. Materials and methods 2.1. Cell culture and isolation of ␳0 cell line T47D cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% FBS and 2 mM lglutamine at 37 ◦ C under a 5% CO2 atmosphere. To develop ␳0 cells, wild-type cells were chronically exposed to 50 ng/ml EtBr (Sigma) in medium additionally supplemented with 100 ␮g/ml pyruvate and 50 ␮g/ml uridine. After 40 days in culture, single cell clones were isolated by limiting dilution in a 96well plate. The mtDNA status of these clones was determined by DNA dot blot and PCR analysis. To confirm the absence of mtDNA, 1 × 104 cells from each potential ␳0 cell line were studied for the capacity to survive in “␳0 test medium”, which only consisted of DMEM, 10% dialyzed FBS (lacking the pyrimidines in regular FBS), and 2 mM l-glutamine. Viable cells were counted by 0.2% trypan blue exclusion method over 6 days. Positive T47D ␳0 cells did not survive in this medium because they were auxotrophic for pyrimidines and pyruvate. To standardize the cell culture condition, in all comparative studies between T47D and T47D ␳0 cells, wild-type cells were also cultured in medium supplied with 100 ␮g/ml pyruvate and 50 ␮g/ml uridine. All cell culture medium and supplements were purchased from Gibco/Invitrogen. 2.2. DNA dot blot Ten micrograms of total DNA prepared from wild-type and ␳0 cells were denatured by adding 0.5 M NaOH. Then, samples were loaded on a Bio-Dot® Microfiltration apparatus (Bio-Rad), transferred to the positively charged nylon membrane (Boehringer Mannheim) and crosslinked at 80 ◦ C for 2 h. Resultant DNA dots were hybridized with a 1284bp biotin-labeled mtDNA probe (nt 15,885–599) and a control 710-bp ␤-actin probe (nt 260–969), respectively. Biotin-mediated hybridization signals were detected using the 3,3 -diaminobenzidine (DAB, Sigma) colorimetric method. The sequence selected as an mtDNA probe spanned the fulllength of the D-loop region in mitochondrial genome. There was no nuclear sequence (pseudogene) equivalent to this portion in human mtDNA. 2.3. PCR analysis MtDNA was amplified in a volume of 50 ␮l containing 0.25 units of Taq polymerase (Promega) and 10 pmol of primer

M. Yu et al. / Toxicology Letters 170 (2007) 83–93

pair specific for the mtDNA D-loop region: forward 5 -CCT GTC CTT GTA GTA TAA AC-3 and reverse 5 -TTG AGG AGG TAA GCT ACA T-3 (1284-bp). The amplifications of cytochrome c oxidase subunit II (Cox-2) gene and control ␤actin gene were also conducted. The sequence of primers were Cox-2: forward 5 -TTC ATG ATC ACG CCC TCA TA-3 and reverse 5 -CGG GAA TTG CAT CTG TTT TTA-3 (462-bp); ␤-actin: forward 5 -ATC ATG TTT GAG ACC TTC AAC A-3 and reverse 5 -CAT CTC TTG CTC GAA GTC CA-3 (318bp). The PCR program included an initial denaturation of 5 min and 35 cycles as follows: 35 s of denaturation at 94 ◦ C, 35 s of annealing at 58 ◦ C and 50 s of extension at 72 ◦ C. 2.4. Cell growth parameters and cell cycle assay Briefly, wild-type and T47D ␳0 cells (1 × 104 per dish) were plated in triplicate and the number of cells were counted in six consecutive days. Doubling times were calculated using the formula: N/N0 = ekt , where N was the cell number for a cell line at a particular time point (t) and N0 was the corresponding cell number at time 0. The constant k was calculated for each line between 48 and 120 h, the period in which cell growth rate was linear. For cell cycle analysis, cells were trypsinized, washed with 1× PBS and fixed in 70% ethanol at 4 ◦ C for at least 18 h. After washing with 1× PBS, propidium iodide (PI, 20 ␮g/ml, Sigma) in buffer containing RNase A (50 ␮g/ml) was added to the cell pellets, and incubation was carried out for 30 min. DNA content was then measured by flow cytometry (Beckman Coulter EPICS-XL). More than 10,000 events were recorded in each case. 2.5. Colony formation in soft agar Two percent of gum agar (Sigma) was mixed with DMEM and FBS to give 0.8% agar and 10% FBS. Then, 1 ml of 0.8% agar was added to a six-well plate and allowed to set as the base layer. The top layer of agar was similarly prepared to give 0.4% agar and 10% FBS. 0.9 ml of 0.4% agar was mixed with 0.1 ml of cell suspension (containing 2000 cells from either ␳0 or control cell line) by vortexing the contents vigorously until the cells were evenly suspended. The cell-containing mixture was plated onto the base layer agar, followed by being overlaid with 1 ml of DMEM containing 100 ␮g/ml pyruvate and 50 ␮g/ml uridine. After 15 and 30 days of incubation at 37 ◦ C in 5% CO2 incubator, colonies >0.1 mm were counted using an ocular micrometer on an inverted microscope (Olympus). 2.6. Determination of mitochondrial membrane potential (Ψ m ) Rhodamine 123 uptake by mitochondria is directly proportional to its membrane potential. Wild-type, 20-day EtBr-treated and ␳0 cells (1–2 × 105 /ml) were stained with rhodamine 123 (10 ␮g/ml final concentration, Sigma) at 37 ◦ C for 30 min and were washed with PBS. The fluorescent intensities

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were immediately measured in FL1 channel by flow cytometry (Beckman Coulter EPICS-XL). 2.7. Annexin V-FITC assay Early apoptotic changes for wild-type and ␳0 cells were evaluated using an Annexin V-FITC Apoptosis Detection kit (BD Bioscience). Samples were washed with cold PBS and resuspended in binding buffer at a concentration of 1 × 106 cells/ml. Then, 100 ␮l of this solution (1 × 105 cells) were transferred to a 5 ml culture tube and mixed with 5 ␮l of Annexin V-FITC and 5 ␮l of PI. After incubating in the dark for 15 min, 400 ␮l of binding buffer were added and the samples were analyzed by flow cytometry within 1 h. At least 1 × 104 events were collected on each sample. 2.8. Mitochondrial protein detection by Western blotting In brief, as to Bcl-2, Bax and procaspase 9, cells were washed with PBS and lysed in a buffer containing 100 mM NaCl, 10 mM Tris–HCl, pH 7.6, 1 mM EDTA, 1 mM PMSF, and 1% NP-40. For cytosolic cytochrome C determination, lysis of PBS-washed cells was performed in isotonic buffer (10 mM HEPES pH 6.9, 200 mM mannitol, 70 mM sucrose and 1 mM EGTA) with protease inhibitors by Dounce homogenization. Unbroken cells, nuclei, and heavy membranes were pellet at 1000 × g and discarded. The cytosolic fraction was then collected by the high-speed centrifugation as previously described (Khaled et al., 1999). After the addition of a loading buffer containing 50 mM Tris–HCl, pH 6.8, 2% SDS, 8% glycerol, and 0.4% ␤-mercaptoethanol, an equal amount of protein (30 ␮g) for each sample was separated by 12% SDS-PAGE and transferred to a PVDF membrane (Pall). Incubation was conducted in appropriately diluted primary antibodies to Bcl-2, Bax, procaspase 9, and cytochrome C (Santa Cruz) at 4 ◦ C overnight. Then, membranes were probed with HRP-conjugated secondary antibody (Dako) and signals were visualized using an enhanced chemiluminescence reagent (Amersham-Pharmacia). 2.9. Transmission electron microscopic observation Electron photomicrographs for wild-type and ␳0 cells were prepared as described (Fukuyama et al., 2002). In brief, cells were washed with PBS and fixed in 4% glutaraldehyde/1% paraformaldehyde for 2 h. Then, cell pellets were postfixed with 1% OsO4, dehydrated through ethanol, and embedded in LX112. Ultrathin sections were stained with uranyl acetate and lead citrate and then were examined for mitochondrial morphology on a H7000 electron microscope operating at 80 kV (Hitachi). 2.10. In vivo tumorigenicity study To determine in vivo tumorigenicity, we used 6-week-old female severe combined immune deficiency (SCID) mice as

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animal models (purchased from Laboratory Animal Research Center, Beijing University). T47D cells are estrogen-receptor positive and require the presence of estrogen for optimal growth in vivo. A 60-day release pellets containing 1.5 mg of 17␤estradiol (Innovative Research of America) were implanted subcutaneously (s.c.) into each animal at the lateral side of neck. Three days later, wild-type and ␳0 cells (1 × 107 cells) suspended in 100 ␮l PBS were inoculated s.c. into the left and right flanks of the mice (n = 5 for each group). Tumor growth was monitored assuming spherical growth of tumor volumes. When a tumor mass was visually detectable, its maximum (a) and minimum (b) diameters were recorded every 3 days using a caliper. The tumor volume (V) was calculated according to the formula: V = 1/2 × (a2 × b). On day 51, mice were sacrificed and individual tumor weight was determined. All experiments were approved by the Institutional Animal Care and Use Committee. 2.11. Statistical methods Data were presented as means ± S.D. Comparisons of quantitative measures between two cell lines were done using a two-tailed unpaired Student’s t-test. P < 0.05 was considered to be statistically significant.

3. Results 3.1. Generation and verification of T47D ␳0 cell line We selectively depleted mtDNA from T47D cell line by culturing cells with EtBr for 40 days and isolating clones by limited dilution. MtDNA levels of ␳0 clones were assessed by DNA dot blot and PCR. As shown in Fig. 1A, a mtDNA probe failed to hybridize to total DNA isolated from T47D ␳0 cells, indicative of mtDNA depletion. In control, a probe revealed the existence of ␤-actin gene in both cells, suggesting that nDNA in our ␳0 cells was intact. A PCR analysis was used to further confirm the depletion of mtDNA. The amplification of the mtDNA-encoded D-loop region and the Cox-2 gene were missing in ␳0 cells, while parental cells yielded strong PCR products at predicted size (Fig. 1B). Taken together, our results suggested that we were able to specifically deplete mtDNA from T47D cells. 3.2. Attenuated Ψ m in ␳0 cells Mitochondria are key regulators of cell death following alterations in the Ψ m in response to various triggers. In many cell types, mitochondrial membrane permeability is considered as the point of non-return in the cell suicide. To determine the Ψ m after mtDNA

Fig. 1. Isolation and verification of T47D ␳0 cells. (A) DNA dot blot of EtBr-treated and wild-type cells to determine mtDNA level. No mtDNA hybridization signal was detected in ␳0 cells; while ␤-actin signal was present with both cell types. (B) MtDNA and ␤-actin gene were amplified by PCR. The PCR products were visualized on 1% agarose gel by EtBr staining, showing the absence of mitochondrial genome in ␳0 cells. M indicates the DL2000 ladder, each band representing 100, 250, 500, 750, 1000, and 2000 bp. D-lp and actin indicate the mtDNA D-loop region and ␤-actin gene, respectively.

depletion, we first stained cells with rhodamine 123, a potential specific dye. Flow cytometric measurement exhibited a progressive decline in Ψ m from T47D, 20day EtBr-treated, to ␳0 cells (Fig. 2A). Compared with controls, a 30.4 ± 5.5% reduction in average fluorescent intensity incorporated into mitochondria was detected in T47D ␳0 cells (␳0 = 12.6 ± 5.1 versus wt = 42.7 ± 6.0, P < 0.05, Fig. 2B). Consistent results were also obtained from other cancer mutant ␳0 cells (Armand et al., 2004; Appleby et al., 1999). We next measured the retention ability of rhodamine 123 in wild-type and ␳0 cells by fluorescence microscopy. Although both wild-type and ␳0 cells showed bright mitochondrial stain being treated with rhodamine 123 at 10 ␮g/ml for 10 min, most of ␳0 cells lost the dye after 4 h in drug-free medium, while wild-type cells remained relatively bright (data not shown). 3.3. No significant apoptotic changes in ␳0 cells The early apoptotic change in wild-type and ␳0 cells was also measured using the Annexin V-FITC/PI double staining assay. We found that around 1.78% cells

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in wild-type group appeared early apoptotic whereas approximately 2.17% of ␳0 cell population underwent apoptosis (Fig. 3A and B). Consequently, no statistically significant difference of apoptotic changes were detected in ␳0 cells (Table 1). To confirm this observation, we subsequently compared the expression of Bcl-2, Bax, procaspase 9 and cytosolic cytochrome C proteins, several classic markers of apoptotic pathways, in these two groups of cells. As expected, no detectable difference in expression was identified in the Western blot analysis (Fig. 3C). 3.4. Disrupted mitochondrial morphology in ␳0 cells Loss of mtDNA can lead to distinct morphological changes in the internal structure of mitochondria. As displayed in Fig. 4A, wild-type T47D cells possessed a typical elongated mitochondrial appearance with parallel cristae and evenly distributed matrix density. In contrast, mitochondria in ␳0 cells exhibited aberrant phenotypes in many aspects, which mainly included either partial or even complete loss of regular cristae patterns, disorganized swollen appearance, condensate of multilamellar membrane structure, and increased matrix density (Fig. 4B–D). T47D ␳0 cells also contained abnormal cytoplasmic vacuoles that were present increasingly in number, size, and density and scattered in the vicinity of mitochondria (Fig. 4D). 3.5. Decrease in cell proliferation in ␳0 cells Fig. 2. Flow cytometric analysis of Ψ m in T47D wild-type, EtBrtreated 20d and ␳0 cells. (A) A progressive decline in Ψ m was observed in T47D cells treated with EtBr for 20 days and ␳0 cells, compared with wild-type cells. The figure is representative of three independent experiments. (B) The rhodamine 123 fluorescence intensity in ␳0 cells was three- to four-fold lower than that in parental cells. Data shown are mean ± S.D. from three independent experiments. *P < 0.05.

Auxotrophic reliance of EtBr-treated cells on pyrurate and uridine is a unique signature of ␳0 cells and is an effective way to determine the respiratory status and thus mtDNA content (King and Attardi, 1996). T47D ␳0 cells were incapable to grow in “␳0 test medium” that lacked pyruvate and uridine. Following a short initial growth lag, ␳0 cells displayed a gradual decrease in number and loss of viability within 6 days, indicating that

Table 1 Comparison of some properties between T47D and T47D ␳0 cells Property

Units

T47D cells

Doubling time Numbers of colonies (15 days) Numbers of colonies (30 days) Percent of early apoptosis Latent period of in vivo tumorigenicity Tumor weight

h

26.7 59.4 106.6 1.78 6.6 761.9

% day mg

± ± ± ± ± ±

1.9 (3) 7.0 (5) 10.5 (5) 0.16 (3) 1.3 (10) 195.4 (10)

T47D ␳0 cells 47.9 4.2 7.8 2.17 13.3 153.6

± ± ± ± ± ±

3.1* (3) 1.5* (5) 2.6* (5) 0.21 (3) 1.6* (9) 64.2* (9)

Data are shown as the mean ± S.D. The numbers of replicate experiments are indicated in parentheses. Each parameter was compared using a two-tailed Student’s t-test. *P < 0.05.

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Fig. 3. Determination of apoptotic alterations in T47D ␳0 cells. No significant change in early apoptosis was detected between the wild-type (A) and T47D ␳0 cells (B). The lower right quadrant represented the early apoptotic cells, positive for Annexin V-FITC and negative for PI. Data are representative results of three independently performed experiments. (C) Expression levels of several key apoptosis-related proteins (Bcl-2, Bax, procaspase 9 and cytosolic cytochrome C). The antibody against ␤-actin was utilized to confirm the equal loading of the samples. Cyt C represents cytochrome C.

depletion of mtDNA impaired mitochondrial function (Fig. 5A). We further assessed the proliferation of T47D ␳0 cells in complete medium. ␳0 cells exhibited a reduced growth rate with a doubling time of 47.9 ± 3.1 h, which was significantly prolonged compared to that of parental cells 26.7 ± 1.9 h (Fig. 5A, Table 1). Cell cycle analysis was indicative of G1 phase arrest in ␳0 cells (Fig. 5B). Cells were also evaluated for anchorage-independent colony formation ability, before and after mtDNA depletion. Two thousand cells were plated in soft agar and observed for the growth characteristics over 15 days. Expectedly, there was an obvious difference between the numbers of colonies found in wild-type cells compared to the ␳0 cells: 59.4 ± 7.0 versus 4.2 ± 1.5. Incubation for an

additional 15 days resulted in >100 colonies in the control group, whereas an average of only three additional colonies were seen in ␳0 cells. The differences were found to be statistically significant, both at days 15 and 30 (Table 1). 3.6. Reduced in vivo tumorigenicity in ␳0 cells Finally, we examined the tumorigenic properties of ␳0 and control cells in vivo. Our experiments showed that almost all mice injected with either wild-type or ␳0 cells developed tumors, with 100% (10/10) incidence in wild-type and 90% (9/10) in the ␳0 group. Tumors in the control group became visible on average 6 days after injection, while tumors in ␳0 group exhibited delayed

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Fig. 4. A transmission electron microscopic comparison of T47D (A) and T47D ␳0 cells (B–D). (A) 13,500× magnification of wild-type cells showing elongated mitochondria with parallel cristae and normal electron density (arrows). (B) Mitochondria in ␳0 cells were irregularly enlarged with almost complete or partial dissolution of the internal cristae structural pattern (13,500×, arrowheads). (C) 13,500× magnification of ␳0 cells showed that a mitochondrion with disorganized swollen appearance with only some protein remnants inside and some mitochondria with condensate of multilamellar-like membrane structure (arrowheads). (D) Cytoplamic vacuoles were enlarged and appeared more numerous in ␳0 cells. Abnormally increased matrix electron density in the cytoplasm was also observed (13,500×).

tumor visualization (approximately 13 days, Table 1). In vivo T47D ␳0 cells recapitulated their in vitro growth characteristics growing more slowly than the parental line, suggesting that ␳0 cells were much less tumorigenic than their wild-type counterparts (Fig. 6). In addition, tumor average weight of the ␳0 group was lower than that of control group, with an average 80% decrease (Table 1). Overall, our results revealed that T47D cells lacking mtDNA still possessed tumorigenenicty in vivo, even though their ability was impaired to some extent. It is reasonable to infer that the tumorigenicity of T47D cancer cells may be modulated by the mitochondrial genome. 4. Discussion Low concentration of EtBr has been long known as a mutagen to dramatically inhibit the mtDNA synthesis. In this study, we successfully established a novel T47D ␳0 breast cancer cell line after 40-day continuous treatment

with EtBr. ␳0 status was verified by lack of detectable mtDNA on DNA dot blot and by PCR amplification. The resultant T47D ␳0 cells displayed dependence upon uridine and pyruvate for growth, a common feature for mtDNA-less cell lines. Uridine-dependence is the result of inactive dihydro-orotate dehydrogenase, which itself requires interaction with an electron donor from the respiratory chain for recycling FAD (King and Attardi, 1996). The dependence of extrinsic pyruvate is due to excessive cytosolic NADH gradually accumulation in ␳0 cells (King and Attardi, 1996; Malik et al., 2004). Sufficient supply of pyruvate may assist mtDNA-deficient cells to utilize excess NADH via the enzyme lactate dehydrogenase catalyzing the conversion of pyruvate to lactate. The results from cell proliferation, cell cycle distribution, and clonogenecity assays showed that T47D ␳0 cells had a slower growth rate than parental cells, a finding similar to that obtained with MOLT-4 and HeLa ␳0 cells (Armand et al., 2004; Schauen et al., 2006). We also

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Fig. 6. In vivo growth characteristics of wild-type and ␳0 tumors in SCID mice. Suppression of tumorigenicity was observed with the T47D ␳0 cells. Tumor volumes were bi-dimensionally determined every 3 days for 51 days. Values are presented as mean tumor volume ± S.D. (n = 10).

observed a certain amount of bi-nucleated proliferating T47D ␳0 cells, but it was much less than that in wild-type cells (data not shown), confirming the proliferative ability of our ␳0 cells was indeed impaired. Due to the low levels of intracellular ATP and G6PDH, which are determinants of NADPH production and are essential for cell growth (Appleby et al., 1999), it may be expected that ␳0 cells will proliferate more slowly. However, growth retardation was not identified in all mtDNA-depleted cells, such as the growth rate of 143B osteosarcoma ␳0 cells was normal (King and Attardi, 1989) and the hepatoma Hepa1-6 ␳0 cells even grew twice as fast as wild-type cells (Haque et al., 2006), suggesting that different types of cells may exhibit diverse sensitivities to the intracellular changes elicited by mtDNA depletion. The reasons why various types of cells react differently to the loss of mtDNA integrity still need further investigation. The Ψ m of our T47D ␳0 cells was reduced by approximately 30–40% compared to the parental cells, concurring with the findings in osteosarcoma 143B and leukemia MOLT-4 ␳0 cells (Armand et al., 2004; Appleby et al., 1999). It was noteworthy that the pathological mitochondria found in our ␳0 cells still can take up rhodamine 123, and hold a significant, albeit lowered, membrane potential in the absence of a functional system of oxidative phosphorylation, probably because

Fig. 5. Retarded growth of T47D ␳0 cells due to G1 phase arrest in the cell cycle. (A) T47D ␳0 cells exhibited profoundly delayed growth kinetics compared to their parental cells. In vitro growth of ␳0 cells showed auxotrophic dependence on pyruvate and uridine. Progressive loss of cell viability was noted when medium was not supplemented

with pyruvate and uridine. Each of the data points represents the mean of three separate dishes of cells, ±S.D. Note that, to standardize the cell culture condition, medium in wild-type group was also supplied with 100 ␮g/ml pyruvate and 50 ␮g/ml uridine. (B) DNA contents of exponentially growing cells were measured by flow cytometry using PI stain. Cell cycle profiles indicated G1 phase arrest in ␳0 cells. Numbers indicate percentage of cells in various phases. Note the different y axes for the two groups.

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the F1-ATPase and the adenine nucleotide translocater of mitochondria in T47D ␳0 cells were still functional, as previously identified in a few other ␳0 cell models (Appleby et al., 1999; Buchet and Godinot, 1998). Yet the precise mechanism to clarify how ATP produced by glycolysis is able to maintain the relatively low Ψ m in these derivative cells remains unraveled. In T47D ␳0 cells, early apoptosis was not induced to any significant degree and the cells managed to resist cell death even though they were surviving with low levels of Ψ m . The levels of several key proteins relevant to the apoptotic pathways also remained unchanged. A novel hypothesis concerning the mitochondria damage checkpoint (mitocheckpoint) in eukaryotic cells might explain our results (Singh, 2006). It is proposed that damages to mitochondria including mtDNA deletion may first activate mitocheckpoint, which permits cells to arrest in the cell cycle in order to repair some mitochondrial function and avoid the trigger of apoptosis. However, if such kinds of damages are severe, mitocheckpoint will fail, then leading to cellular senescence and consequently to programmed cell death (Singh, 2006). Indeed, Park et al. (2004) found that ␳0 cells possessed some features simulating aged cells, such as increased senescence-associated ␤-Gal activity, lipofuscin pigment, and plasminogen activator inhibitor-1 expression. Abnormalities in mitochondrial ultrastructure were identified in T47D ␳0 mutant cells. These included enlargements of the mitochondria with partial or almost total absence of the normal organization of cristae, increased matrix density, abnormal swollen appearance, and condensate of multilamellar membrane structure, similar to those observed in conditions associated with mitochondrial dysfunction, such as genetic mitochondrial disorders or exposure to mitochondrial toxins (Trimmer et al., 2000; Autunes et al., 2002). In this study, we did not examine the overall number of mitochondria in T47D ␳0 cells. However, parallel studies (Holmuhamedov et al., 2003; Wochna et al., 2005) found that the average number of dysfunctional mitochondrial scaffolds was not significantly different in ␳0 cells from that in wild-type counterparts, indicating that mitochondrial biogenesis proceeded independently of mtDNA. During tumorigenesis, the delicate balance between survival and cell death is altered. As a result, cancer cells are able to survive under adverse conditions that normally would trigger apoptosis such as hypoxia, low glucose, and lack of attachment. In this study, the in vivo tumorigenic phenotype of T47D cells was altered after depletion of mtDNA, as reflected in a prolonged latent period, a reduced proliferate rate, and a lowered tumor

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weight. Since we did not identify any apoptosis-related changes, it would indicate that the apoptotic pathways were not involved in triggering the reduced tumorigenicity in our ␳0 cells. Some other mechanisms must exist that modulate this process, probably through mitochondriato-nucleus interactions (Delsite et al., 2002; Singh et al., 2005; Singh, 2006). The importance of nucleus-encoded genes for maintaining the malignancy of tumor cells has already been addressed (Akimoto et al., 2005). It was found that genome chimera cells carrying nDNA from tumor cells and mtDNA from normal cells showed tumorigenicity, whereas those carrying nDNA from normal cells and mtDNA from tumor cells did not. In addition, there was accumulating evidence from in vitro experiments demonstrating that the mitochondrial genomic dysfunction may lead to impaired oxidative DNA repair in nucleus or/and chromosomal instability (Singh et al., 2005). Therefore, it is likely that the decreased tumorigenicity of our ␳0 cells is also mediated by influencing the normal function of nDNA. Besides the membrane potential and morphological changes, the bioenergetic function of mitochondria in T47 ␳0 cells was also damaged. For instance, recent work in our lab revealed a lowered production of ROS including O2 − and H2 O2 in these mutant cells (Yu et al., unpublished data). The decrease in ROS was thought to be able to retard the tumor progression, which might hold the reason for the proliferative defects and reduced tumorigenecity observed with our ␳0 cells (SanchezPino et al., 2007). Considering that T47D cell line is ER-positive and the mitochondrial genome contains potentially estrogen-responsive sequence, we are applying real-time RT-PCR to detect the mRNA expression of several ER-related genes (such as NCOR1 and aromatase gene) in T47D ␳0 cells (Demonacos et al., 1996). This work may be able to help us elucidate the possible existence of a correlation between mtDNA depletion and estrogen levels. In addition, it will be very interesting to determine whether the mitochondrial electron transfer velocity is affected as a result of mtDNA deletion since the damage in mtDNA could produce alterations in the expression of the mtDNA-encoded polypeptides required for electron transfer and ATP synthesis (Passos and Von Zglinicki, 2006). In conclusion, we have successfully established a mtDNA-depleted human breast cancer cell line. Our preliminary studies suggest that mtDNA plays an important role in human breast cancer cell proliferation and tumorigenesis, but not apoptosis. This cell line enables the future mechanistic investigations to reveal the molecular basis of mtDNA’s involvement in human breast cancer cell proliferation.

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