Positive Contribution of Pathogenic Mutations in the Mitochondrial Genome to the Promotion of Cancer by Prevention from Apoptosis

Research Article

Yujiro Shidara,1,3 Kumi Yamagata,3 Takashi Kanamori,3 Kazutoshi Nakano,2 Jennifer Q. Kwong,4 Giovanni Manfredi,4 Hideaki Oda,1 and Shigeo Ohta3

Departments of 1Pathology and 2Pediatrics, Tokyo Women’s Medical University, School of Medicine, Tokyo, Japan; 3Department of Biochemistry and Cell Biology, Institute of Development and Aging Sciences, Graduate School of Medicine, Nippon Medical School, Kawasaki, Japan; and 4Department of Neurology and Neuroscience, Weill Medical College of Cornell University, New York, New York

Abstract mitochondrial DNA (mtDNA) mutations and the rapid proliferation The role of mitochondrial dysfunction in cancer has been a of cancer cells with no physiologic advantage may account for the subject of great interest and much ongoing investigation. accumulation of somatic neutral mutations in mtDNA. In fact, Although most cancer cells harbor somatic mutations in extensive computer modeling suggests that if a mtDNA mutation mitochondrial DNA (mtDNA), the question of whether such occurs in a tumor progenitor cell, mtDNA homoplasmy (i.e., a pure mutations contribute to the promotion of carcinomas remains population of mutant mtDNA molecules) can be achieved entirely unsolved. Here we used trans-mitochondrial hybrids (cybrids) by chance through unbiased mtDNA replication and sorting during containing a common HeLa nucleus and mtDNA of interest to cell division without selection for physiologic advantage (9). This compare the role of mtDNA against the common nuclear model can explain the occurrence of homoplasmic neutral somatic background. We constructed cybrids with or without a mutations in mtDNA in cancers. homoplasmic pathogenic point mutation at nucleotide posi- On the other hand, comprehensive scanning of somatic mtDNA tion 8,993 or 9,176 in the mtDNA ATP synthase subunit 6 gene has recently revealed that functionally relevant point mutations in (MTATP6) derived from patients with mitochondrial encepha- mtDNA and polypeptide-encoding genes were detected in 50% of lomyopathy. When the cybrids were transplanted into nude patients (10). Thus, cancer cells seem to harbor pathogenic mice, the MTATP6 mutations conferred an advantage in the mutations in mtDNA as well as neutral ones. In addition, a transient early stage of tumor growth. The mutant cybrids also increased depletion of mtDNA causing oxidative phosphorylation dysfunction faster than wild type in culture. To complement the mtDNA was found to influence the expression of some nuclear genes mutations, we transfected a wild-type nuclear version of involved in tumorigenic and invasive phenotype (11). Furthermore, MTATP, whose codons were converted to the universal genetic inherited heterozygous mutations in the nuclear genes encoding for codes containing a mitochondrial target sequence, into the two ubiquitously expressed mitochondrial enzymes, succinate nucleus of cybrids carrying mutant MTATP6. The restoration of dehydrogenase and fumarate hydratase (fumarase), that catalyze MTATP slowed down the growth of tumor in transplantation. sequential steps in the Krebs cycle, have been shown to cause Conversely, expression of a mutant nuclear version of MTATP6 predisposition to two types of inherited neoplasia syndromes in the wild-type cybrids declined respiration and accelerated (12–15). These findings prompted us to investigate the relationship the tumor growth. These findings showed that the advantage in between mitochondrial dysfunction and carcinogenesis. tumor growth depended upon the MTATP6 function but was In the present study, we tried to characterize the role of not due to secondary nuclear mutations caused by the mutant pathogenic mtDNA mutations in the promotion of cancer using mitochondria. Because apoptosis occurred less frequently in two methods. First, we used trans–mitochondrial hybrid cells the mutant versus wild-type cybrids in cultures and tumors, the (cybrids). Cybrids were generated by repopulating HeLa cells pathogenic mtDNA mutations seem to promote tumors by devoid of mtDNA with mtDNA derived from enucleated cells of preventing apoptosis. (Cancer Res 2005; 65(5): 1655-63) patients harboring heteroplasmic mtDNA mutations in the mitochondrial ATP synthase subunit 6 gene (MTATP6) associated with neuropathy, ataxia, and retinitis pigmentosa (16) or mater- Introduction nally inherited Leigh syndrome (17). This technique allowed for the The role of mitochondrial dysfunction in carcinogenesis has isolation of cybrid clones containing either homoplasmic mutant been investigated extensively by various approaches. It has been or homoplasmic wild-type mtDNA against a common nuclear established that the majority of cancer cells harbor homoplasmic background (18, 19). Second, we transfected a nuclear version of somatic mutations in the mitochondrial genome (1–8). However, MTATP6, whose codons were converted to the universal genetic despite the close association between carcinogenesis and somatic codes containing a mitochondrial localization presequence, into mutations, it remains unclear whether these somatic mutations are the nucleus of mutant cybrids to complement and restore the contributors to the development of tumors. The high frequency of defect caused by the mtDNA mutations (20). Here, we show that two different pathogenic MTATP6 mutations contribute to promotion of tumors by prevention of apoptosis. Note: Supplementary data for this article are available at Cancer Research on-line (http://cancerres.aacrjournals.org/). Requests for reprints: Shigeo Ohta, Department of Biochemistry and Cell Biology, Materials and Methods Institute of Development and Aging Sciences, Graduate School of Medicine, Isolation and Culture of Cybrid Cell Lines and Their Culture. Primary Nippon Medical School, Kosugi-cho, Nakahara-ku, Kawasaki, 211-8533, Japan. Phone: 81-44-733-9267; Fax: 81-44-733-9268; E-mail: [email protected]. skin fibroblasts were obtained from a 9-month-old male who had clinical I2005 American Association for Cancer Research. characteristics of maternally inherited Leigh syndrome. His mtDNA www.aacrjournals.org 1655 Cancer Res 2005; 65: (5). March 1, 2005

Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Cancer Research contained a point mutation with a T-to-G transversion at nucleotide mouse Cy5 (Jackson Immunochemicals, West Grove, PA). We visualized position 8,993 in the ATP synthase subunit 6 gene (MTATP6; refs. 16, 17). selected digital images with different pseudocolors for Mitotracker or The fibroblasts were used to construct cybrids after enucleation as FLAG, as appropriate, and merged them in the RBG format for the described (19). Platelets were obtained from siblings, an 18-year-old female evaluation of colocalization with a Zeiss Confocal microscope (Zeiss, and a 13-year-old male, who also had clinical characteristics of maternally Oberkochen Germany). When necessary, H-1206 VECTASHIELD Mounting inherited Leigh syndrome but with a mild clinical form (21). They harbored Medium with 4V,6-diamidino-2-phenylindole was used as a counterstain. a T-to-C transition at nucleotide position 9,176 in the MTATP6 gene (21, 22). Tumors were fixed overnight in 10% formalin at 4jC embedded in The enucleated fibroblasts or the platelets were fused with EB8, a U0 HeLa paraffin, sectioned to 5 Am, immunostained with mouse monoclonal anti- derived line that are completely lacking mtDNA and resistant to 8- FLAG M2, anti Ki-67 antibodies or rabbit polyclonal anti-collagen type IV thioguanine (19). Cybrids were selected with 8-thioguanine and maintained antibody (Immunotech, Westbrook, ME), and incubated with secondary in a glucose-rich medium, DMEM/F-12 (Invitrogen Co., Carlsbad, CA) Biotinylated Link anti-mouse IgG or anti-rabit IgG for the LSAB2 System, containing glucose (3.2 mg/mL), pyruvate (0.6 mg/mL), uridine (50 Ag/mL), horseradish peroxidase (DAKO, Glostrup, Denmark), and third streptavidin and 10% fetal bovine serum. Mycoplasma contamination was checked peroxidase conjugated (DAKO). Hematoxylin was used as a counterstain. monthly. Detection of Apoptosis. Apoptotic cells were detected by the terminal Detection of Mutant mtDNA. Total DNA from cybrids or tumors was deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) method isolated using a DNeasy Tissue Kit (QIAGEN Sciences, Valencia, CA). To using the ApoTaq Peroxidase In situ Oligo Ligation Kit (Intergen, detect the T8,993G mutation, a 139-bp PCR fragment was amplified using Burlington, MA). The substrate for horseradish peroxidase, 3-amino-9- forward and reverse primers corresponding to mtDNA nucleotide positions ethylcarbazole, produces a red color in positive nuclei (23). Hematoxylin 8,917 to 8,935 and 9,043 to 9,055, respectively. PCR products were digested and methyl green were used as a counterstain. The percentage of with the restriction enzyme AvaI. The T8,993G mutation creates a novel positively stained cells was determined using at least six sections from AvaI site and thus the mutant PCR products are cleaved in 72- and 62-bp each tumor and cell clone. For each tumor or cell culture, the percentage fragments. To detect the T9,176C mutation, a 178-bp PCR fragment was of positively stained cells was calculated, and the numbers obtained were amplified using forward and reverse primers corresponding to mtDNA averaged to yield the mean F SD. nucleotide positions 9,025 to 9,046 and 9,177 to 9,203 respectively. PCR For detecting DNA ladders, DNA was isolated from three 2 106 cybrid products were digested with the restriction enzyme ScrFI. The T9,176C cells (semiconfluent on 100-mm-diameter dishes) using an Apoptotic DNA mutation creates a novel ScrFI site and thus the mutant PCR products are Ladder KIT (Roche, Indianapolis, IN) according to the manufacturer’s cleaved in 151- and 28-bp fragments. Digested PCR products were resolved directions. Samples were subjected to agarose gel electrophoresis and by agarose gel electrophoresis and the relative proportions of mutant and stained with ethidium-bromide (24). With this method, DNA from intact wild-type molecules were quantified by densitometry. For quantification nuclei was excluded, and thus the intact DNA did not disturb the pattern purposes, standards of mutant and wild-type mtDNAs were mixed at of electrophoresis. various ratios. No mtDNA fragments were amplified under the conditions To detect apoptotic cells by flow cytometry, cybrid cells were incubated from BALB/c nu/nu mouse mtDNA. with phenol red-free DMEM/F-12 containing 0.2% charcoal-stripped fetal Measurement of Oxygen Consumption. Semiconfluent cells were bovine serum 24 hours before analysis. Trypsinized cells were resuspended trypsinized and harvested. The cells were suspended at 0.7 to 1.5 107/mL in a hypotonic propidium iodide solution (50 Ag/mL) containing 0.1% in the culture medium and placed in a respiration chamber (50 AL) with an sodium citrate and 0.1% Triton X-100 for 30 minutes, and subjected to flow oxygen electrode. The rate of oxygen consumption was measured at 37jC cytometric analysis with a FACScan flow cytometer (Becton Dickinson, San using a Clark-type electrode (Strathkelvin Instruments, Glasgow, Scotland). Jose, CA; ref. 25). By this method, apoptotic cells were separated from intact Transplantation of Cybrids to Form Tumors in Nude Mice. Cybrid cells, regardless of amounts of nuclear DNA. cells suspended in Matrigel (5 106 cells per 0.2 mL) were injected s.c. at For judging apoptotic cells, the nuclei of living cells treated with cisplatin four positions into female athymic mice (4-week-old BALB/c nu/nu). were double-stained with propidium iodide and Hoechst 33342 (5 Amol/L At indicated times after transplantation, animals were killed and tumors each). Cells with propidium iodide–positive and fragmented nuclei were were dissected and analyzed. Tumor volume was calculated using the judged as dead and apoptotic, respectively. formula V =(A B2)/2, where V is volume (mm3), A is the long diameter Statistical Analysis. Differences between two groups were compared (mm), and B is the short diameter (mm). using the two-tailed unpaired Student’s t test. P < 0.05 was considered Histology. Tumors were removed 3 to 14 days after transplantation and statistically significant. fixed overnight in 10% formalin at 4jC. Fixed tissues were embedded in paraffin, sectioned at 5 Am, and stained with the H&E stain. Plasmid Construction and Transfection. The plasmid with a nuclear Results version of the MTATP6 gene (P1A6F, designated as NuATP6 in this article) was a kind gift from Prof. Eric A. Schon of the Department of Neurology, Characterization of MTATP6 Cybrids. To explore the role of Columbia University (20). In brief, it was constructed by converting codons pathogenic mitochondrial mutations on the development of from the mitochondrial to universal type and inserted behind the cancer, we constructed cybrids with the HeLa nucleus and mitochondrial targeting sequence derived from the P1 isoform of subunit c mtDNA with a T-to-G transversion at mtDNA nucleotide position of human ATP synthase. In addition, the gene contained a FLAG sequence at 8,993 (16, 17) or a T-to-C transition at nucleotide 9,176 (21) in the its 3V-end as an epitope tag. The gene was inserted into an expression vector, MTATP6 gene. The mutant mtDNAs were derived from patients pEF-BOS. A mutant version (muATP6), which contained the T8,993G point with maternally inherited Leigh syndrome (26). We selected wild- mutation was constructed from P1A6F by site-directed mutagenesis. The type and mutant homoplasmic cybrid clones (Fig. 1A). Cybrid plasmids containing NuATP6 or muATP6 were transfected using Polyfect clones 8W1, 8W2, and 8W3 were homoplasmic wild type; clones Transfection Reagent (QIAGEN Sciences) according to the manufacturer’s 8M7, 8M8, and 8M9 were homoplasmic for the T8,993G mutation; directions. Stable transfectants were selected in the presence of Geneticin clones 9W4, 9W5, and 9W6 were homoplasmic wild type; clones G418 and confirmed to express NuATP6 or muATP6 by anti-FLAG staining. Immunologic Techniques. For immunodetection of NuATP6 or 9M10 and 9M11 were homoplasmic for the T9,176C mutation. muATP6, cells were grown on 4-well plastic dishes (SonicSeal Slide, The homoplasmic mtDNA genotypes were confirmed periodically Nalge Nunc, Roskilde, Denmark), treated with MitoTracker Red to ensure that there was no drift towards heteroplasmy. (Molecular Probes, Eugene, OR; 10 nmol/L for 30 minutes), fixed, We assessed the respiratory function in the cybrid clones by immunostained with mouse monoclonal anti-FLAG M2 antibodies (Sigma measuring oxygen consumption rates with an oxygen electrode. Immunochemicals, St. Louis, MO), and incubated with secondary anti- As previously described (27), we found that mutant cybrid clones

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same volume. Upon microscopic inspection, individual cells in each tumor seemed to be similar in size irrespective of their mtDNA genotype. Moreover, cells at the maximum surface were enumerated to confirm that the larger volumes of tumors were due to substantial increases in cell number (Fig. 2E). Thus, differences in tumor size should be ascribed to an increase in cell number of the mutant cybrids. Each tumor showed nearly the same distribution pattern and content in collagen IV of the matrix (Supplementary Fig. S1). Hematoxylin stainings also suggested no marked difference in the extracellular matrix of the tumors (Fig. 2C). Moreover, to confirm that mutant cybrid tumors had faster growth, we transplanted a mixture (1:1) of mutant and wild-type cybrids into nude mice. The proportion of mutant mtDNA in the tumor increased progressively and eventually replaced entirely the wild-type mtDNA (Fig. 3A and B). Because mtDNA copy number per cell in wild type and mutant cybrids remained constant during tumorigenesis (data not shown), the progressive increase in the proportion of mutant mtDNA in the mixed tumors should be due to faster proliferation or more resistance against cell death in the mutant cybrids. When compared the growth rate of tumors (Fig. 2B), the wild-type mtDNA seemed to be selectively excluded in the mixture of the wild-type and mutant cybrids (Fig. 3A and B). Taken together, these findings suggest that mtDNA mutant cells have an apparent advantage in forming tumors as compared with wild-type cells especially in the early stage, providing an explanation for why homoplasmic mtDNA mutations are found in many tumors with mitochondrial dysfunction. Advantage in Increase of Cultured Mutant Cybrids. To study the faster growth of tumors derived from the mutant cybrids, we compared mutant and wild-type cybrids in terms of their increasing rates in culture. We did growth curve measurements Figure 1. mtDNA genotype and oxygen consumption in HeLa cybrids containing either wild type or mutant mtDNA. A, mtDNA genotyping in cybrid clones. mtDNA comparing mutant and wild-type cybrids and found that the amplified from clones derived from a patient with the T8,993G mutation were mutant cybrids increased faster than wild type, especially in the digested with AvaI(lanes 1-3 and 7-9). mtDNA amplified from clones derived from beginning of culture (Fig. 3C). Mutant cybrids started to increase a patient with the T9,176C mutation were digested with ScrFI (lanes 4-6 and 10-11). Lanes 1-11, digested PCR products from cybrid clones 8W1, 8W2, 8W3, without a lag time, whereas wild types started to increase at day 2. 9W4, 9W5, 9W6, 8M7, 8M8, 8M9, 9M10, and 9M11, respectively (agarose gel Although we examined the frequency of the Ki-67 positive cells as a electrophoresis). Densitometric analyses confirmed that all clones contained either homoplasmic wild type or homoplasmic mutant mtDNA (>98%). B, proliferation index, no difference among cybrid clones was seen representative traces of oxygen consumption. Semiconfluent cells of cybrid clones (Supplementary Fig. S2). Then, to confirm the advantage for 7 8M7 and 8W1 were trypsinized and 0.7 to 1.5 10 /mL cells were placed into a increasing mutant cybrids, once again, we mixed mutant and wild- respiration chamber (50 AL) equipped with a Clark-type oxygen electrode. The rate of oxygen consumption was slower in the 8M7 (T8,993G mutant) clone as type cybrids in a 1:1 ratio and followed the relative proportions of compared with the 8W1 (wild type) clone. C, average rates of oxygen consumption the mutant and wild-type mtDNAs in the mixed cultures over time. in mutant and wild-type cybrid clones. P < 0.01, 8993W versus 8993M and 9176W This experiment clearly recapitulated our findings in the mixed versus 9176M as determined by two-tailed unpaired Student’s t test. Columns, averages of three independent experiments; bars, FSD. tumors in the mice, because we observed that the relative content of mutant mtDNA progressively increased and eventually replaced the wild-type mtDNA (Fig. 3D). Because mutant and wild-type cells had decreased oxygen consumption as compared with wild type contained the same amount of mtDNA (see day 0 in Fig. 3D), this (Fig. 1B and C). in vitro experiment confirmed the advantage for increasing the Transplantation of Cybrids into Nude Mice. To compare mutant cybrids. parental U0 HeLa, mutant and wild-type cybrids in terms of their Expression of Wild Type and Mutant MTATP6 from the potential to form tumors, we injected 5 106 cybrid cells s.c. into Nucleus. It may be possible that mutant mtDNA induces nude mice. Settlement frequencies from the mutant cybrids were secondary mutations in nuclear genes that accelerate the higher than those from the wild type and settlement frequency proliferation of mutant cybrids. This might be one of the molecular from the parental U0 HeLa was very low (Fig. 2A). In this study, only mechanisms of how the mutant cybrids gained their apparent the settled tumors were considered for further examination. Mice advantage in the increase. A direct way to determine the role of were sacrificed and tumor volumes were measured. Tumors mtDNA mutations would be to manipulate the mtDNA. However, a derived from mutant cybrids seemed larger and grew faster than technology to transfect mtDNA into mammalian cells has yet to be those from wild-type cybrids (Fig. 2B, C, and D). All the examined established. Thus, alternatively, we transfected in mutant cybrids a mutant cybrid tumors showed larger volumes as compared with nuclear version of MTATP6 whose codons had been converted into wild type especially in the early stage (Fig. 2B and D). At day 14 universal ones (NuATP6; ref. 20). The gene contained an NH2- when no massive necrotic death was seen, each tumor attained the terminal presequence (from the P1 isoform of subunit c of human www.aacrjournals.org 1657 Cancer Res 2005; 65: (5). March 1, 2005

Figure 2. Transplantation of cybrids into nude mice and time course of mtDNA genotype shift during tumor growth. A, settlement efficiency of transplantation. Each cybrids clone (f5 106 cells) was injected hypodermically into nude mice. Ten days after transplantation, efficiency of tumor formation (% successful tumor implantation for each cybrids clone) was calculated from 12 to 24 experiments. Columns 0 and 1-11, parental U0 cell, cybrid clones 8W1, 8W2, 8W3, 9W4, 9W5, 9W6, 8M7, 8M8, 8M9, 9M10, and 9M11, respectively. B, time course of increase in volume of tumors. Tumors derived from wild type (8W1) versus tumors derived from T8,993G mutant (8M7; P < 0.001 at days 4, 5, and 6; P < 0.01 at day 7; P < 0.05 at days 3, 8, and 9). Tumors derived from the wild type (9W4) versus tumors derived from T9,176C mutant (9M10; P < 0.001 at days 4, 5, and 6; P < 0.01 at day 7; P < 0.05 at days 3, 8, and 9). No significance was found at day 14. Points, average values from three independent experiments; bars, SD. C, representative photographs of 6-day-old tumors derived from clones 8W1 or 8M7 stained with H&E. Bar, 1 mm. Tumor derived from the mutant cybrids seems larger. Top left, portion of magnification. Bar, 10 Am. D, columns, average volumes of the tumors 6 days after transplantation from three independent experiments; bars, SD. P < 0.001, group 8993W versus 8993M and group 9176W versus 9176M. E, average cell number in sections of the maximum surface of tumors 6 days after transplantation from three independent experiments. P < 0.001, group 8993W versus 8993M and group 9176W versus 9176M.

ATP synthase) to target the protein to mitochondria and a FLAG consumption (Fig. 4A). The incomplete rescue of oxygen con- epitope for ease of immunodetection. It was shown that in sumption by NuATP6 in mutant cybrids and the partial decrease by homoplasmic mutant cybrids this gene product partially restores muATP6 in wild-type cybrids are explained by the persistence of normal mitochondrial oxidative phosphorylation (20). We isolated endogenous mutant and wild-type MTATP6, respectively. stable transfectants with NuATP6 and confirmed the expression of Increase Rates of Cybrids Modulated by Transfection with NuATP6 by immunostaining with anti-FLAG antibodies (Supple- Nuclear Versions of MTATP6. Increase rates of cybrid trans- mentary Fig. S3). Conversely, we transfected a nuclear version of fectants were examined in vitro culture. When NuATP6 was MTATP6 containing the T8,993G mutation (muATP6) into wild- transfected into mutant cybrid 8M7, resulting transfectants (8M7 type cybrids. As expected, stable transfections of NuATP6 in + NuATP6) grew significantly slower than control mutant cybrids mutant cybrids partially restored oxygen consumption, whereas (8M7m) mock transfected with an empty plasmid (Fig. 4B). introduction of muATP6 into wild-type cybrids decreased oxygen Conversely, when muATP6 was transfected into 8W1 wild-type

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Promotion of Cancer by Pathogenic mtDNA Mutations cybrids (8W1 + muATP6), resultant transfectants increased significantly faster than control wild-type cybrids (8W1m) mock transfected with an empty vector. Moreover, to confirm the effect by NuATP6 on the increase of the transfectants, we mixed 8M7 + NuATP6 and mock transfectants 8M7m in a 1:1 ratio. We also measured the increase rates of 8W1m mock and muATP6 transfected cybrids mixed in a 1:1 ratio. Because both muATP6 and NuATP6 were tagged with FLAG, we were able to identify transfected cells throughout the duration of the experiment. After 6 days, there was a decrease in the number of NuATP6 FLAG-positive cybrids relative to mock transfected (Fig. 4C and Supplementary Fig. S4, left). Conversely, there was an increase in the number of muATP6 FLAG-positive cybrids as compared with the mock transfectants in the mixed cultures (Fig. 4C and Supplementary Fig. S4, right). These findings indicate that the modulation of mitochondrial ATP synthase activity via expression of mutant or wild-type MTATP6 from the nucleus can affect cell proliferation and override the effect of the mtDNA genotype. Next, we transplanted the 8M7 + NuATP6 or 8W1 + muATP6 cybrids into nude mice and followed tumor growth. The results indicated that the expression of a functional MTATP6 in 8M7 + NuATP6 cybrids slowed down tumor growth as compared with tumors derived from mock transfected 8M7m; whereas a defective MTATP6 in 8W1 + muATP6 cybrids promoted tumor growth as compared with tumors derived from mock transfected 8W1m cybrids (Fig. 4D). To recapitulate in vivo the growth advantage conferred by muATP6 to wild-type cybrids, we mixed in a 1:1 ratio 8M7 + NuATP6 cybrids with mock 8M7m ones, or conversely, 8W1 + muATP6 cybrids with mock 8W1m, and transplanted the mixtures into nude mice. Confirming the results in cell culture, FLAG- positive mutant cybrids expressing NuATP6 resulted in smaller tumors, indicating that functional MTATP6 conferred a disadvan- tage in tumor growth (Supplementary Fig. S5, left) as compared with expression of defective MTATP6 (Supplementary Fig. S5, right). A quantitative analysis of FLAG-positive cybrids using several transfected cybrid clones confirmed the reproducibility of these observations in forming tumors derived from cybrids (Fig. 4E). Taken together, these observations consistently showed that the increase advantage in culture and the growth advantage in vivo tumor depends upon the decline in MTATP6 function, and not upon secondary effects from the nucleus. Lower Frequency of Apoptosis of Mutant Cybrids In vitro and In vivo. To explore the molecular mechanism underlying the growth advantage in tumors, we hypothesized that the mutant mtDNA may protect cells from apoptosis, and therefore growth advantage of mutant cybrids may derive from increased survival. To test this Figure 3. mtDNA genotype shift in cotransplantation and coculture of wild-type hypothesis, apoptosis was examined by three independent methods. cybrids with mutants. A, cybrid cells from T8,993G wild-type clone 8W1 and mutant clone 8M7 were mixed (1:1) and transplanted into nude mice. Total DNA First, we looked at TUNEL staining during the course of 8 days of was isolated from tumors at indicated days after implantation and the ratio of continuous culture. Although TUNEL-positive cells were rare, they mutant to wild-type mtDNA was estimated by RFLP/PCR. Representative patterns of agarose gel electrophoresis of digested PCR products showing the were more abundant in wild-type cybrids than in mutant cybrids mtDNA genotype shift. B, cybrid cells from wild-type 8W1 and mutant 8M7 or from throughout the duration of culture (Fig. 5A and Supplementary wild-type 9W4 and mutant 9M10 were mixed (1:1) and transplanted into nude mice. Points, relative proportions of wild type and mutant mtDNA (expressed as Fig. S6). Especially at day 1, TUNEL-positive cells were more in wild- % total mtDNA) from three independent experiments; bars, SD. P < 0.001, 8W1 type cybrids, which agrees with the finding of the lag time as seen in versus 8M7 and 9W4 versus 9M10 on each day. C, cells were seeded onto a Fig. 3C. Second, DNA fragmentation was more extensive in the wild culture dish after trypsinization, continued to culture for the indicated periods and then enumerated after suspension under a microscope. Points, average cell type than in the mutant cybrids (Fig. 5B). Third, flow cytometric numbers from three independent experiments after continuous culture on each analysis revealed that the population of apoptotic cells belonging to day; bars, SD. P < 0.001, 8M7 versus wild type and 9M10 versus wild type on f days 3, 4, 6, and 8. D, cybrid cells from T8993G wild-type clone 8W1 and mutant sub-G1 phase was more abundant in the mutant ( 8% of total cells) clone 8M7 or from T9,176C wild-type clone 9W4 and mutant clone 9M10 were than in the wild-type cybrids (f2% of total cells; Fig. 5C and D). mixed (1:1) and continuously cultured for the indicated days. Points, relative proportions of wild type and mutant mtDNA (expressed as % total mtDNA) in Thus, all three methods confirmed that wild-type cybrids undergo average values from three independent experiments; bars, SD. P < 0.001, 8W1 spontaneous apoptosis more frequently than mutants. In addition, versus 8M7 and 9W4 versus 9M10 on each day. www.aacrjournals.org 1659 Cancer Res 2005; 65: (5). March 1, 2005

Figure 4. Transfection of cybrids with nuclear versions of the ATP synthase subunit 6 gene and the growth characteristics of the transfectants. Cybrids were transfected with FLAG tagged nuclear versions of the ATP synthase subunit 6 gene, NuATP6 (wild type) or muATP6 (mutant). A, columns, average values of oxygen consumption in transfected cybrids obtained from three independent experiments. 8M7m, 8W1m, 9M10m, and 9W5m are mock transfectants with an empty plasmid of cybrid clones 8M7, 8W1, 9M10, and 9W5, respectively. (8M7 + NuATP6), (9M10 + NuATP6), and (8W1 + muATP6) are transfections with NuATP6 of 8M7, 9M10, and 8W1 cybrids, respectively. P < 0.01, 8M7m versus (8M7 + NuATP6), 8W1m versus (7 + NuATP6) and each other mock transfectant versus each corresponding transfectant with NuATP6 or muATP6. B, growth curves. 8W1m and 8M7m are control cybrids mock (m) transfected with an empty vector, whereas (8M7 + NuATP6) and (8W1 + muATP6) are cybrids stably expressing the NuATP6 and muATP6, respectively. P < 0.01, mock transfected controls versus transfectants with NuATP6 and mock transfected controls versus transfectants with muATP6 on each day of culture in three independent experiments. C, mixture at a ratio of 1:1 of (8M7 + NuATP6) and 8M7m or (8W1 + muATP6) and 8W1m was cultured for 6 days and immunostained with anti-FLAG antibodies. Proportion of FLAG-positive cells (expressed as % total cells) in mixed cultures on day 6. Columns 8M7, 8M8, or 8M9, proportion of FLAG-positive cells in the mixture of (8M7 + NuATP6) and 8M7m, (8M8 + NuATP6) and 8M8m, or (8M9 + NuATP6) and 8M9m, respectively. Columns 8W1, 8W2, or 8W3, proportion of FLAG-positive cells in the mixture of (8W1+muATP6) and 8W1m, (8W2 + muATP6) and 8W2m, or (8W3+muATP6) and 8W3m, respectively. Columns, averages of three independent experiments; bars, FSD. P < 0.001, group 8M versus group 8W. D, time course of tumor growth. Transfected cybrids were transplanted into nude mice and tumor size was measured at the time points indicated. The average sizes of tumors derived from 8M7 expressing NuATP6 (8M7+NuATP6) as compared with 8M7 transfected with empty vector (8M7m), and of 8W1 cybrids expressing muATP6 (8W1+muATP6) as compared with 8W1 cybrids transfected with empty vector (8W1m) were both significant (P < 0.001). Points, average values from three independent experiments; bars, SD. E, proportions of transfectants expressing FLAG on day 6 after transplantation into nude mice. 8M7m and 8M7+NuATP6 or 8W1m and 8W1+muATP6 were mixed 1:1 and transplanted into nude mice. After 6 days, tumors were fixed and immunostained with anti-FLAG antibodies and FLAG-positive and FLAG-negative cells were counted. Average proportions of FLAG-positive were obtained from three independent experiments. 8M7, proportion of FLAG-positive cells in a 1:1 mixture of 8M7m and 8M7+NuATP6. 8W1, proportion of FLAG-positive cells in a 1:1 mixture of 8W1m and 8W1+muATP6. Columns, average values from three independent experiments; bars, SD. profiles for the cell size (data not shown) and the cell cycle obtained apoptosis when cells were exposed against an apoptosis-inducing by flow cytometry did not markedly differ between the mutant and stimulus. When cybrids were treated with cisplatin, an apoptosis wild-type cybrids (Fig. 5C), which agrees with the Ki-67 staining as inducer, mutant cybrids presented less dead (propidium iodide seen in Supplementary Fig. S2. Then, we compared sensitivities to positive) and apoptotic with fragmented nuclei as judged by

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Promotion of Cancer by Pathogenic mtDNA Mutations morphology (Fig. 5E and F). Thus, the mutant cybrids are more biochemical hallmark of mitochondrial diseases and it is widely resistant against apoptosis than the wild type. used in the diagnosis of mitochondrial encephalomyopathies Finally, we compared the frequency of apoptosis and prolifer- (26, 30). ation rate in tumors derived from cybrid transplantation. Although it has been shown that a majority of cancer cell lines We counted the number of Ki-67- and TUNEL-positive cells in harbor mutant mtDNA, it has not yet been determined whether tumors. Although TUNEL-positive cells were rare, they were mtDNA mutations precede and lead to carcinogenesis. In light of significantly more numerous in tumors derived from wild type Warburg’s theory, it would be especially interesting to better than from mutant cybrids (Fig. 6A and Supplementary Fig. S7A). understand the role of mutant mtDNA associated with dysfunction On the other hand, there was no marked difference in Ki-67 of mitochondria in carcinogenesis. Furthermore, the high fre- positive cells (a proliferation marker; 28) between tumors (Fig. 6B quency of mtDNA alterations in cancer and their presence in the and Supplementary Fig. S7B). These results indicate that the early stages of disease could perhaps be exploited as clinical markers mutant mtDNAs suppress apoptosis in tumors as well as in for early cancer detection (3, 31–33). The mtDNA alterations cultured cybrids. detected in cerebrospinal fluid may be used as sensitive markers to monitor disease progression and predict relapse (34). The transplantation of cybrids into nude mice has been Discussion employed previously to investigate the role of mtDNA in Mitochondrial defects have long been suspected to play an tumorigenesis. Hayashi et al. (35) transplanted cybrid cells and important role in the development of cancer. Fifty years ago, U0 cells lacking mtDNA into nude mice and reported that tumors Warburg pioneered the research on the involvement of mito- were formed by the cybrids containing wild-type mtDNA but not by chondrial respiratory defects in cancer, and proposed a mecha- U0 cells devoid of mtDNA. In contrast, Morais et al. (36) reported nism to explain how these defects evolve during carcinogenesis. that U0 cells could form tumor. Because HeLa U0 cells did form Warburg hypothesized that a key event in carcinogenesis involved tumors but at a very low frequency in our study, the potential for the development of an injury to the mitochondrial respiratory forming tumors may depend upon the viability of U0 cells. In their machinery, resulting in a compensatory increase in glycolysis, experiments, they have not compared cybrids with wild type or leading to lactic acidosis (29). Lactic acidosis is also a typical mutant mtDNA. This is a very relevant issue because mtDNA

Figure 5. Lower frequency of apoptosis in cultured mutant cybrids. A, time-independent apoptosis in cybrids. After plating, cybrids were cultured for the indicated periods, then fixed and stained by the TUNEL method. Points, average values from three independent experiments; bars, SD. P < 0.001, 8W1 versus 8M7 or 9W4 versus 9M10 on each day. B, representative patterns of DNA fragmentation cybrid clones. DNA isolated from semiconfluent cells was subjected to 1.8% agarose gel electrophoresis. M r: 100 bp DNA ladder. Note that intact nuclei could be removed, so the intact DNA was not contaminated by this method (24). C, Representative flow cytometry profiles. Cybrids were trypsinized, stained with propidium iodide (PI) as described in Materials and Methods and subjected to flow cytometry to detect apoptotic cells (sub-G1 phase). Arrows, sub-G1, apoptotic cells. This method is designed to detect apoptotic cells as a single peak (25). D, quantification of apoptotic cells detected by flow cytometry. The frequency of apoptosis (expressed as % apoptotic cells over total number of cells) was obtained from three independent experiments for each cybrid clone. Columns 1-11, cybrid clones 8W1, 8W2, 8W3, 9W4, 9W5, 9W6, 8M7, 8M8, 8M9, 9M10, and 9M11, respectively. The frequency of apoptosis was significantly lower in groups 1 to 6 than in groups 7 to 11 (P < 0.001). Columns, average values from three independent experiments; bars, SD. E, sensitivity of cybrids against cell death induced with cisplatin, an apoptosis inducer. Cells were treated with 10 Amol/L cisplatin for 24 hours and double stained with propidium iodide and Hoechst 33342 (5 Amol/L each). Propidium iodide–positive cells were enumerated under a fluorescent microscope. F, sensitivity of cybrids against apoptosis induced with cisplatin. Fragmented nuclei stained with Hoechst 33342 were morphologically judged under the fluorescent microscope after the treatment as described in E.

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this study does not despise the predominant role of the nucleus (35) and does not exclude the possibility that mutant mtDNA may influence the expression of genes involved in cell proliferation, for example by affecting the mitochondrial regulation of intracellular Ca2+ levels (40). Our study shows that cybrids harboring mutant mtDNA increase faster in culture and are more efficient at promoting tumorigenesis, as compared with cybrids harboring wild-type mtDNA, especially in the early stage when no marked massive necrotic cell death was seen. In addition, the concept that mutant mtDNA can overcome wild-type mtDNA within one cell is suggested by previous experiments where cells with and without mutant mtDNA were fused to each other (2). Furthermore, it has been shown that the proportion of certain pathogenic mutant mtDNAs tends to expand over time in muscle or brain of patients with mitochondrial encephalomyopathy (41). Thus, it is no surprise that mtDNA molecules harboring certain somatic mutations may quickly become the dominant species or even become entirely homoplasmic in cancer cells. The molecular mechanism by which mutants faster than wild-type cybrids remains to be fully clarified. Because we could see no marked difference in the proliferation index of the Ki-67 antigen in vitro and in vivo, one potentially important information that emerged from our studies, which may explain the different growth between mutant and wild-type cybrids, is the extent of apoptosis. Because apoptosis plays a critical role in cancer development and in the cellular response to anticancer agents (42, 43), the effect of mtDNA mutations on apoptosis in cancer cells is obviously an important area of investigation. The effect of mtDNA mutations on apoptosis is still unclear. In some experiments, U0 cells lacking mtDNA have been Figure 6. Lower frequency of apoptosis in mutant cybrids upon transplantation used as a model for defective mtDNA. For instance, it was reported 0 into nude mice. A, time course of an increase in TUNEL-positive cells. After that U cells were more resistant to a reagent that induces apoptosis transplantation, tumors are sectioned and subjected to TUNEL staining. Number than parental cells (44). On the other hand, U0 cells were shown to be of TUNEL-positive cells on a total of 200 cells counted on each indicated day + after transplantation. P < 0.001, 8W1 versus 8M7 and 9W4 versus 9M10 on each more sensitive to apoptosis than their U parental cells in culture (45) day. Points, average values from three independent experiments; bars, SD. and in vivo (46). One study showed that cytochrome c release and B, Ki-67-positive cells per 100 cells counted on day 6 after transplantation. caspase activation in response to staurosporine treatment were Columns, values from three independent experiments; bars, SD. maintained in U0 cells (47). However, few experiments have compared cybrids with and without mutant mtDNA. Therefore, mutations arise spontaneously in somatic cells at a relative high the exact role of pathogenic mtDNA mutations in the cellular rate. Thus, the potential role of mtDNA mutations in cancer apoptotic response remains to be defined. development, genetic instability, and disease progression warrants Although our study suggests that loss of apoptosis may have an a careful and comprehensive investigation. important role in fostering growth in tumors derived from mutant In the present study, as a model, we used cybrids harboring cybrids, it is not clear whether the difference in the frequency of mtDNA with or without pathogenic mutations at nucleotide apoptosis can fully explain the difference in tumor growth or positions 8,993 or 9,176 in the MTATP6 gene. This cybrid system growth rate in culture. The frequency of TUNEL-positive cells in has an advantage that the mtDNA sequences are identical except tumors from mutant cybrids was rather small, but because TUNEL- for a single pathogenic mutation between mutant and wild-type positive apoptotic cells seem transiently and continuously during clones, because both the mutant and wild-type mtDNAs are the life of the tumor, dead cells may should be more than the derived from the same subject (37). In contrast, cancer cells tend to TUNEL-positive cells. Thus, estimating the frequency of apoptotic have multiple mutations in mtDNA (38) making it difficult to cell death in tumors just by TUNEL staining may lead to correlate any phenotypic differences with a specific mutation. We underestimation. In fact, a flow cytometric analysis of apoptotic chose to use cybrids harboring the MTATP6 gene mutations for two features in cultured cells revealed a greater proportion of apoptotic reasons. First, cancer cells often contain mutations in the MTATP6 cell death than TUNEL staining. Additionally, because wild-type gene (6–8) and, in addition, most renal carcinomas lose ATP mtDNA markedly decreased in the genetic drift experiments using synthase activity (39). Second, we took an advantage of the the mixture of wild-type and mutant cybrids, the wild-type cybrids possibility of manipulating the content of mutant or wild-type themselves seemed to be eliminated (Fig. 3A, B, and D). Moreover, MTATP6 in cells by a previously established technique of nuclear wild-type cybrids in culture began to increase with a lag time gene transfer of MTATP6 (20). The ability to manipulate the growth accompanying apoptosis (Figs. 3C and 5A). Because settlement phenotypes of cancer cells expressing exogenous MTATP6 allowed frequencies from wild-type cybrids were lower than those from us to confirm that these phenotypes were directly due to mtDNA mutants, the early events may be crucial in the settlement and mutations and not to secondary nuclear modifications. However, promotion of tumorigenesis (Fig. 2A).

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Our findings seem to be inconsistent with a report (48) that encephalomyopathy. Because the physiologic advantage of two fibroblasts harboring the 8,993 MTATP6 mutation were more pathogenic mutant mtDNAs in tumorigenesis was revealed for the sensitive to metabolic stress and apoptosis than wild-type first time, investigations on mtDNA will be more important in fibroblasts. However, this discrepancy is likely to be due to the conquering cancers. difference between primary fibroblasts and cancer cells such as HeLa cybrids, for example, the glycolytic pathway in HeLa cells is Acknowledgments more active than that in fibroblasts. Received 6/8/2004; revised 11/14/2004; accepted 12/15/2004. Here we presented positive contributions of two pathogenic The costs of publication of this article were defrayed in part by the payment of page mutations of mtDNA to the promotion of tumorigenesis. However, charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. it will be required to use mutant mtDNAs derived from actual cancer We thank Prof. Eric A. Schon of the Department of Neurology and Genetics, tissues instead of mtDNA from patients with mitochondrial Columbia University, New York, NY for providing P1A6F (NuATP6).

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Downloaded from cancerres.aacrjournals.org on September 23, 2021. © 2005 American Association for Cancer Research. Positive Contribution of Pathogenic Mutations in the Mitochondrial Genome to the Promotion of Cancer by Prevention from Apoptosis

Yujiro Shidara, Kumi Yamagata, Takashi Kanamori, et al.

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