Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Oncogene (1999) 18, 6641 ± 6646 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc Mitochondrial DNA determines the cellular response to cancer therapeutic agents Keshav K Singh*,1, James Russell1, Barbara Sigala1, Yonggong Zhang1, Jerry Williams1 and Kylie F Keshav2 1 Johns Hopkins Oncology Center, 600 North Wolfe Street, Room 2-121, Baltimore, Maryland, MD 21287, USA; 2Department of Bioscience and Biotechnology, Drexel University, 32nd and Chestnut Streets, Philadelphia, Pennsylvania, PA 19104, USA Mutations in the mitochondrial genome leading to mitochondrial dysfunction have been reported in a variety of cancers. However, the potential implication of these ®ndings in the cellular response to cancer therapeutic agents is unclear. To examine the importance of mitochondrial DNA (mitDNA) encoded functions in cancer therapeutic response, we determined the clonogenic survival of HSL2 (Rho+, HeLa subline), and its derivative cell line lacking mitDNA (Rho0) after exposure to dierent anticancer agents. We found that isogenic Rho0 cells lacking mitDNA were extremely resistant to adriamycin and photodynamic therapy (PDT) induced cell death, whereas the Rho+ cell line was sensitive. However, there was no measurable dierence in the responses of these cell lines to either alkylating agent or g-radiation. We show that the development of resistance to adriamycin was not due to changes in apoptotic cell death, cell cycle response or to the uptake of adriamycin in isogenic Rho0 cells. We also demonstrate that exposure of HeLa cells to adriamycin leads to mutations in mitDNA. These studies provide direct evidence that mitDNA plays an important role in cellular sensitivity to cancer therapeutic agents. Keywords: mitochondria; mitochondrial DNA; cell death; adriamycin Introduction With the exception of erythrocytes, all human cells contain mitochondria (Schatz, 1995; Singh, 1998). Cells derive up to 95% of their energy through oxidative phosphorylation carried out in mitochondria. Mitochondria, therefore, are regarded as the powerhouse of the cell (Schatz, 1995; Singh, 1998). In addition to producing energy, mitochondria also support many essential cellular functions. These include intermediatory metabolism, ion homeostasis, synthesis of lipids, amino acids, and nucleotides, active transport processes, cell motility, cell proliferation and initiation of programmed cell death (apoptosis) (Schatz, 1995; Singh, 1998; Wallace et al., 1997; Petit and Kroemer, 1998). Thus mitochondria participate in numerous functions in the cell. *Correspondence: KK Singh Received 18 February 1999; revised 28 June 1999; accepted 6 July 1999 Interestingly, one of the most common and profound phenotypes of many cancer cells is their defective mitochondrial function (Pedersen, 1978, 1997; Rempel, 1996). Mitochondria occupy up to 50% of the cytoplasmic volume of most animal cells. However, most cancer cells have few mitochondria, less mitochondrial content and are highly glycolytic (Pedersen, 1978, 1997; Rempel et al., 1996). Cancer cell mitochondria have a characteristic shape and size (Pedersen, 1978, 1997; Rempel et al., 1996). Studies indicate that cancer cell mitochondria are small, have few cristae, and have altered membrane composition and altered membrane potential (Pedersen, 1978, 1997; Rempel, 1996). Studies also indicate that the commitment to apoptosis is coupled with an early drop in mitochondrial membrane potential, a drop in the rate of mitochondrial translation and defects in maturation of precursors of mitochondrial proteins (Petit and Kroemer, 1998). Mitochondria house approximately 1000 proteins that are encoded by nuclear DNA (Schatz, 1995; Singh, 1998). These include anti-apoptotic Bcl2 and apoptosis inducing factor (Petit and Kroemer, 1998). Mitochondria contain their own DNA (Schatz, 1995; Keshav and Yoshida, 1998). Mitochondrial DNA (mitDNA) encodes 13 proteins consisting of four enzyme complexes of the respiratory chain (Complex I, III, IV and ATP synthase) and genes specifying 2 rRNA and 22 tRNA that are components of mitochondrial protein synthesizing system (Schatz, 1995; Singh, 1998). Changes in the mitochondrial genome have been reported in cancer cells (Welter et al., 1989; Boultwood et al., 1996; Liang, 1996), and are implicated in carcinogenesis and other diseases (Singh, 1998). Furthermore, microscopic study of oncocytic tumors revealed mitochondrial hyperplasia (Tallini, 1997), and dierential expression of mitochondrial cytochrome oxidase II in benign and malignant breast tissues (Sharp et al., 1992) and mitochondria dependent increased sensitivity to cis-platinum is reported (Liang and Ullyatt, 1998). Taken together, these studies underscore the importance of the mitochondrion and its genome in cancer and cancer therapy. In order to de®ne the role of mitochondria in cancer therapy, we have examined the response of a tumor cell line completely lacking mitDNA to dierent anticancer agents. Our results demonstrate that mitDNA is important for cell death induced by adriamycin and PDT but not by other anticancer agents. This study indicates that the mitochondrial genome may play an important role in the response of cancer cells to therapeutic agents. Mitochondrial DNA and cell death KK Singh et al 6642 Results Mitochondrial DNA is important for cellular sensitivity to cancer therapeutic agents Ethidium bromide is a known inhibitor of mitDNA replication and transcription because it selectively intercalates with mitDNA (Nass, 1972). As a result human cell lines devoid of mitDNA can be isolated (Morais et al., 1994). To analyse the importance of the mitDNA encoded functions in cellular response to dierent anticancer agents, HeLa and its mitDNAless derivative were exposed to various agents. Figure 1a,b demonstrates that both cell lines were equally sensitive to ionizing radiation and to a model alkylating agent, MNNG. However, the response of mitDNA-less cells to adriamycin was dierent (Figure 1c). HeLa cells lacking mitDNA were approximately twice as resistant to adriamycin compared to the parental cells. The doses required for 90% cell kill were 1.6 mM (Rho0) and 0.75 mM (HeLa parental cells). Mitochondria have been implicated as a primary target of photodynamic therapy (PDT) (Sharkey et al., 1993; Hilf et al., 1987; Woodburn et al., 1992). However, the importance of mitDNA in PDT is not clear. We assayed the cytotoxic response of HeLa cells to PDT after exposure to Mesoporphyrin IX (MPIX), Suramin and light (Figure 1d). Interestingly, Rho0 cells showed complete insensitivity to photosensitization by MPIX and Suramin. We conclude that adriamycin and PDT-induced cytotoxicity requires mitDNA-encoded functions. However, these functions are not required for cell death induced by ionizing radiation or by model alkylating agent MNNG. Resistance to anticancer agents is not due to inhibition of apoptosis in Rho0 cells Mitochondria play a central role in programmed cell death or apoptosis (Petit and Kroemer, 1998). Mitochondria can trigger cell death in a number of ways including a disruption in energy metabolism (Kiberstis, 1999). Because Rho0 cells have altered (a) (b) (c) (d) Figure 1 Eect of anticancer agents on cell survival. Exponentially growing parental HSL2 (Rho+) and its derivative cell line devoid of entire mitDNA (Rho0) were exposed to (a) g-radiation; (b) MNNG; (c) adriamycin; (d) PDT. For PDT treatment cells were exposed to indicated concentration of suramin, mesoporphyrin IX (MPIX), or suramin plus MPIX for 4 h. Cells were then irradiated for 3 min, washed immediately with PBS and plated. Intensity of light irradiation ranged from 2.4 ± 2.6 mW/cm26100 measured by Blak-Ray ultraviolet meter (Ultraviolet Products, Inc San Gabriel, CA, USA). The cell survival curve was plotted as described in Materials and methods. All dose points were measured in triplicate. Each point indicates the mean+s.d. of three independent experiments Mitochondrial DNA and cell death KK Singh et al mitochondrial functions and are resistant to adriamycin, we investigated whether adriamycin resistance was due to inhibition of apoptosis. HeLa cells with functional mitochondria underwent apoptosis after treatment with adriamycin (1.5 mM, 45 min). When Rho0 cells were treated with the same drug apoptosis was also observed. The number of apoptotic cells was counted microscopically as described in Materials and methods. Table 1 indicates that there was no signi®cant reduction in number of cells undergoing apoptosis either after 24 or 48 h adriamycin treatment of Rho0 cells. We conclude that removal of mitochondrial function does not prevent apoptotic death and it does not contribute to resistance of Rho0 HeLa cells to adriamycin. Loss of mitDNA does not alter cell cycle response Adriamycin inhibits DNA replication because it directly intercalates between the bases and blocks DNA topoisomerase II function (Singhal et al., 1997) resulting in S phase block. This suggests that resistance to cytotoxicity could arise due to an altered cell cycle in Rho0 cells as opposed to Rho+ cells. As cells in S phase are more sensitive to adriamycin toxicity (Tooli et al., 1996; Nass, 1972; Singhal et al., 1997), we reasoned that dierences in cell cycle distribution between the two cell lines may account for their dierent drug sensitivities. To test this possibility we measured the cell cycle response of HeLa and the Table 1 Per cent apoptosis in Rho+ and Rho0 HeLa cells after adriamycin treatment. Cells were exposed to 1.5 mM adriamycin for 45 min and apoptosis was measured as described in Materials and methods Cell line Rho+ Untreated control 24 h after treatment 48 h after treatment Rho0 Untreated control 24 h after treatment 48 h after treatment Apoptotic total Apoptotic fraction (%) 5/209 6/214 14/223 2.4 2.8 6.3 1/210 2/217 4/215 0.5 0.9 1.8 isogenic Rho0 cells. The S phase fraction for the HeLa parental cells was approximately 0.3, and for the Rho0 cells 0.28 (Figure 2). This slight dierence does not seem to explain the observed twofold dierence in drug sensitivity between the cell lines. Adriamycin treatment causes cells to accumulate in S phase. After a short exposure (45 min) to 1.25 mM adriamycin, the S phase fraction of Rho+ cells increased by a factor of 2.5; for Rho0 cells, the S phase fraction doubled (data not shown). Because the cell cycle distributions are so similar, we conclude that mitDNA does not govern the cell cycle response to adriamycin. Adriamycin uptake is not altered in cells lacking mitDNA The high level of adriamycin resistance observed in Rho0 cells suggests that adriamycin cytotoxicity is dependent on mitDNA-encoded functions. However, it is possible that resistance may arise because of decreased uptake of adriamycin. To test this possibility, HeLa cells and its Rho0 derivative were grown in DMEM and exposed to 1.2 mM adriamycin for 45 min. The level of adriamycin uptake was quantitated by FACS analysis as described previously. Figure 3 demonstrates that level of adriamycin uptake was independent of presence or absence of mitDNA (mean peak value 173.2 for Rho+ versus the 178.5 for Rho0). We conclude that loss of mitDNA-encoded functions do not aect the level of uptake of adriamycin. Adriamycin induces damage to mitDNA Several studies have suggested that adriamycin-induced cardiomyopathy may be due to increased oxidative damage (Singhal et al., 1997; Palmeira et al., 1997). Adriamycin undergoes redox cycling between quinone and semiquinone which, when reoxidized in the presence of O2, generates highly reactive oxygen species, ROS (Singh, 1998; Kule et al., 1994). Human mitDNA has no protective histones and is continuously exposed to ROS generated endogenously during normal oxidative phosphorylation in mitochondria (Singh, 1998). We therefore envisioned that mitDNA may be susceptible to damage by adriamycin. To test Figure 2 Flow cytometry analysis of parental HSL2 (Rho+) and its derivative cell line devoid of entire mitDNA (Rho0). Cell lines were grown to exponential phase and analysed for cell cycle distribution as described in Materials and methods 6643 Mitochondrial DNA and cell death KK Singh et al 6644 Figure 3 Uptake of adriamycin: parental HSL2 (Rho+) and its derivative cell line devoid of entire mitDNA (Rho0) was grown to exponential phase and exposed to adriamycin (1.25 mM) for 45 min. Cells were washed and analysed as described in Materials and methods this possibility, we isolated total DNA from Rho0 cells treated with adriamycin (0.75 mM) for 45 min and from untreated control cells. Total DNA was digested with PvuII, which gives rise to a single 16.6 Kb fragment of mitDNA. Southern blot analysis using a mitDNA speci®c probe revealed a 16.6 Kb fragment in the untreated control (Figure 4). Figure 4 shows that exposure to adriamycin causes damage to mitDNA (lane 3) resulting in deletion of mitDNA (smaller mitDNA fragments generated after treatment). Figure 4 demonstrates that the level of wild-type mitDNA (16.6 Kb fragment) was noticeably less after 24 h and a new smaller fragment was generated 48 h after short exposure to adriamycin. We did not ®nd any quantitative dierences in DNA loading between the treated and untreated control as judged by ethidium bromide staining. We conclude that adriamycin causes damage to mitDNA. Discussion Mutations in the mitochondrial genome occur in a variety of cancer cells (Polyak et al., 1998; Liang, 1996; Habano et al., 1998). However, the potential implication of mitDNA mutations in cancer therapeutic response has until now not been evident. Our study demonstrates that functions encoded by the mitochondrial genome are important in cancer therapeutic response. Cell death induced by adriamycin in Rho0 cells was signi®cantly less when compared to cells Figure 4 Adriamycin-induced damage to mitochondrial DNA: parental HSL2 cells were grown to exponential phase and exposed to adriamycin (1.25 mM) for 45 min. Cells were then washed and grown to indicated time. Total DNA was extracted (as described in Materials and methods), digested with PvuII and fractionated by 1% agarose gel electrophoresis. Southern blot analysis was performed using the mitochondrial DNA probe as described in Materials and methods. Marker; Lambda phage DNA digested with HindIII and EcoRI containing mitDNA. Results of this study also demonstrate the importance of mitDNA in PDT response because Rho0 cells show resistance to PDT (Figure 1d). We have demonstrated that the dierence in cell death between Rho+ and Rho0 cells was not due to a dierence in the cell cycle response or dierences in drug uptake. Rather the Rho0 derivative, like the parental HeLa cells, arrests in the S phase of cell cycle in response to adriamycin (Tooli et al., 1996; Singhal et al., 1997) and both of these cell lines also show equal uptake of adriamycin into the cell. Notably, the mitochondrial genome does not play an important role in cell death induced by g-radiation or model alkylating agent MNNG because the presence or absence of mitDNA did not aect the level of cell survival (see Figure 1). These results suggest that responses to these agents are independent of functions encoded by the mitochondrial genome. Rho0 cells possess all the essential structures involved in apoptosis, i.e. they contain (a) a normal mitochondrial transmembrane potential, (b) are capable of pre-apoptotic disruption of mitochondrial transmembrane potential, (c) contain mitochondrial apoptosis inducing factor(s) and (d) notably are competent in apoptosis (Marchetti et al., 1996; Petit and Kroemer, 1998). Consistent with the observations of Marchetti et al. (1996) and Petit and Kroemer (1998), we found no signi®cant dierences between the Mitochondrial DNA and cell death KK Singh et al number of Rho+ or Rho0 cells undergoing apoptosis after adriamycin treatment (Table 1). Our data therefore con®rms that removal of mitDNA-encoded functions does not aect apoptosis and indicates that apoptosis does not play a role in resistance to adriamycin. Furthermore, Eguchi et al. (1997) have demonstrated that necrotic death in HeLa cells is independent of mitochondrial function. Together, these studies indicate neither apoptosis nor necrosis is likely to play a role in the observed resistance to adriamycin. p53 was recently shown to control the expression of several genes involved in protecting cells from mitochondria mediated apoptosis (Polyak et al., 1997). Since HeLa cells are de®cient in p53 function (Isaacs et al., 1997), our results also suggest that resistance to adriamycin and PDT is independent of p53 function. These results are further supported by Fisher et al. (1999) who recently provided direct evidence that tumor cell sensitivity to PDT is independent of p53. Adriamycin activation in mitochondria requires oneelectron reduction of the quinone to semiquinone free radicals by Complex I. The semiquinone is subsequently reoxidized in mitochondria to reactive oxygen species (ROS) that at suciently high levels results in cell death (Singh, 1998). It is conceivable that the removal of mitochondrial function leads to the inability of Rho0 cells to both activate adriamycin and to produce signi®cant amounts of ROS necessary for cell death. Reactive oxygen species produced by PDT is also required for cytotoxic eects of PDT (Munday et al., 1996; Fisher et al., 1999). Consistent with these observations, our results (Figure 1d) and those of Munday et al. (1996) show that cells lacking mitochondrial function are resistant to cytotoxic eects of PDT. These studies indicate that ROS produced by mitochondria must play a critical role in cell death induced by both adriamycin and PDT. Development of drug resistance to chemotherapeutic agents is a major clinical problem. Several underlying mechanisms have been suggested to contribute to drug resistance. These include: (1) ampli®cation or overexpression of the P-glycoprotein family of membrane transporters, (2) changes in cellular proteins involved in detoxi®cation or activation, (3) changes in proteins involved in DNA repair and (4) activation of oncogenes such as Her-2/neu, bcl-2 and p53 (ElDeiry, 1997). In this study, we provide evidence that mitDNA also plays an important role in development of drug resistance in cancer cells. We report that damage to mitDNA by adriamycin leads to deletion mutations in mitDNA (Figure 4). The mitochondrial genome appears to be a vulnerable target of adriamycin because it lacks protective histones and is continuously exposed to ROS produced within the mitochondria (Richter, 1988; Singh, 1998). ROS produced by adriamycin may make mitDNA even more vulnerable to damage. ROS produced by adriamycin modi®es bases in DNA and lead to preferential accumulation of 8-hydrooxyguanosine (8Oxo-G) lesions in mitDNA of adult rats (Palmeira et al., 1997). Repeated repair of 8-Oxo-G base by 8-OxoG DNA glycosylase (hOGG1) may cause strand breaks in mitDNA resulting in deletions in mitDNA (Figure 4, and Takao et al., 1998). Increased damage to mitDNA by other anticancer agents has also been reported (Segal-Bendirjan et al., 1988; Modica-Napolitano et al., 1996). Our system utilizing Rho0 cancer cells should serve as a tool for elucidating the mechanism of cytotoxicity of potential anticancer agents. Adriamycin, a member of the anthracycline class of drugs is one of the most eective anticancer agents and is widely used in therapy of childhood leukemias, lymphomas and solid tumors (Ito et al., 1990; Von Ho et al., 1979). However, as cumulative dose increases, the incidence of congestive heart failure increases rapidly (Von Ho et al., 1979; Shan et al., 1996). Recent evidence suggests that anthracycline therapy also leads to arrythmias, pericarditis-myocarditis syndrome, ventricular dysfunction and cardiomyopathies, sometimes 2 ± 15 years after treatment has ceased (Ito et al., 1990; Von Ho et al., 1979; Shan, 1996). Studies reported in this paper suggest that adriamycin-induced mutation in mitDNA may accumulate and contribute to cardiac pathology observed in cancer patients. Current studies are aimed at testing this possibility. Materials and methods Cell culture and clonogenic assays HSL2, (Rho+, a HeLa subline) and its derivative cell line devoid of mitDNA (Rho0) were maintained as described previously (Morais et al., 1994). Cells were grown in humidi®ed incubators at 378C, 5% CO2. Culture medium consisted of high-glucose DMEM supplemented with FBS (10%), L-glutamine (2 mM), sodium pyruvate (110 mg/ml) and uridine (4 mg/ml) (Morais et al., 1994). Cells were subcultured weekly. Rho0 cells were refed twice weekly. Clonogenic assays were performed using exponentially growing cells. Three days prior to assay, 56105 cells were seeded into 75 cm2 ¯asks. Cells were trypsinized and resuspended in medium containing drug, at a ®nal cell concentration of 26104/ml. Exposure lasted for 45 min, after which cells were spun down, rinsed twice with PBS and plated out. For colony formation, cells were seeded at densities ranging from 100 ± 10 000/plate (Rho+) and 200 ± 20 000/plate (Rho0). Radiation experiments were performed on cells that had been plated out 18 h before exposure to radiation. Irradiation was performed using a 137 Cs source (Gammacell, Atomic Energy of Canada) at a dose rate of 0.8 Gy/min. Suramin was obtained from Dr Steve Howard (Mobay Chemical Corporation, Germany). Mesophyrin IX was from Sigma Chemicals (USA). After 10 ± 14 days, plates were stained, and colonies counted. Colonies were de®ned as containing at least 50 cells. For each experiment, all dose points were measured in triplicate, and survival curves represent data from three independent experiments. Survival was ®tted using SPSS software. Measurement of apoptosis For determination of apoptosis in response to adriamycin (1.5 mM) exponentially growing cells were treated for 45 min. Medium was removed and saved. Cells were trypsinized and added back to the medium they had grown in. Cells were spun down and resuspended in PBS. An equal volume of icecold methanol/acetic acid was added. Cells were spun down and resuspended in methanol/acetic acid. Cells were held on ice 10 min, spun down, resuspended in 100 ml methanol/acetic acid and dropped onto slide. After the ®xative evaporated, slides were immersed in Acridine Orange (6 mg/ml in PBS) for 5 min. Slides were washed in PBS, mounted with ddH2O, and apoptotic cells were counted on UV microscope. 6645 Mitochondrial DNA and cell death KK Singh et al 6646 Flow cytometry analysis Exponentially growing cells were trypsinized and ®xed in 70% alcohol for 10 min. For cell cycle analysis, cells were resuspended in propium iodide (40 mg/ml) and RNase (Sigma, St. Louis, MO, USA), (100 mg/ml) for 30 min at room temperature. Cells were spun down, resuspended in PBS and analysed on Coulter Epics ¯ow cytometer, (Coulter Electronics, Hialeah, FL, USA) with excitation at 488 nm. Cell cycle distribution was analysed using Multicycle software (Phoenix Flowsystems, San Diego, CA, USA). FACS analysis was also used to quantitate adriamycin uptake (Chevillard et al., 1990; Tooli et al., 1996). Exponentially growing cells were exposed to 1.25 mM drug for 45 min. The drug was rinsed o, cells were trypsinized, ®xed and analysed as above. Southern blot analysis Total DNA from untreated and adriamycin treated HeLa cells was isolated as described by Modica-Napolitano et al. (1996) and Tanaka et al. (1996). The DNA was digested with PvuII restriction enzyme which linearizes the mitDNA resulting in a single 16.6 Kb fragment (Modica-Napolitano et al., 1996). PvuII-digested DNA was size fractionated by gel electrophoresis on 1% agarose gel in Tris-borate EDTA (pH 8.0). Equal loading was visualized by ethidium bromide staining. The DNA was transferred overnight to a nylon membrane and probed with 32P-labeled mitDNA fragment spanning the region 7441 ± 8287 of mitDNA (King and Attardi, 1988). Hybridization, washing and labeling of probe was carried out as described (Modica-Napolitano et al., 1996). Acknowledgments This work was supported by grants from Alexander and Margaret Stewart Trust, CA/ES 66204, ES 09714-01 and American Heart Association 9930223N to KK Singh and the Plotkin ± Katz Foundation to KF Keshav. We thank Dr Rejean Morais for the kind gift of HSL2 and its mitDNA-less derivative cell lines. We also thank Dr Michael King for the mitDNA probe used in this study. References Boultwood J, Fidler C, Mills KI, Frodsham PM, Kusec R, Gaiger A, Gale RE, Linch DC, Littlewood TJ and Moss PA. (1996). Br. J. Haematol., 95, 426 ± 431. Chevillard S, Vielh P, Bastian G and Coppey J. (1990). J. Cancer Res. Clin. Oncol., 116, 633 ± 638. Eguchi Y, Shimizu S and Tsujimoto Y. (1997). Cancer Res., 57, 1835 ± 1840. El-Deiry WS. (1997). Curr. Opin. Onc., 9, 79 ± 87. Fisher AMR, Ferrario A, Rucker N, Zhang S and Gomer CJ. (1999). Cancer Res., 59, 331 ± 335. Habano W, Nakamura S and Sugi T. (1998). Oncogene, 17, 1931 ± 1937. Hilf R, Gibson SL, Penney DP, Ceckler TL and Bryant RG. (1987). Photochem. Photobiol., 46, 806 ± 817. Isaacs JS, Shen P, Garza A, Hansen MF, Barrett JC and Weissman BE. (1997). Oncogene, 14, 1669 ± 1678. Ito H, Miller SC, Billingham ME, Akimoto H, Torti SV, Wade R, Gahlmann R, Lyons G, Kedes L and Torti FM. (1990). Proc. Natl. Acad. Sci. USA, 87, 4275 ± 4279. Keshav KF and Yoshida S. (1998). Mitochondrial DNA replication. In: Singh KK. (ed.). Mitochondrial DNA Mutations in Aging, Disease and Cancer. Springer: New York, NY, USA. Kiberstis PA. (1999). Science, 283, 1475. King MP and Attardi G. (1988). Cell, 52, 811 ± 819. Kule C, Ondrejickova O and Verner K. (1994). Mol. Pharmacol., 46, 1234 ± 1240. Liang BC. (1996). Mut. Res., 354, 27 ± 33. Liang BC and Ullyatt E. (1998). Cell Growth Dier., 5, 694 ± 701. Marchetti P, Susin SA, Decaudin D, Gamen S, Castedo M, Hirsch T, Zamzami N, Naval J, Senik A and Kroemer G. (1996). Cancer Res., 56, 2033 ± 2038. Modica-Napolitano JS, Koya K, Weisberg E, Brunelli BT, Li Y and Chen LB. (1996). Cancer Res., 56, 544 ± 550. Morais R, Zinkewich-Peotti K, Parent M, Wang H, Babai F and Zollinger M. (1994). Cancer Res., 54, 3889 ± 3896. Munday AD, Sriratana A, Hill JS, Kahl SB and Nagley P. (1996). Biochim. Biophys. Acta, 1311, 1 ± 4. Nass MM. (1972). Exp. Cell. Res., 72, 211 ± 222. Palmeira CM, Serrano J, Kuehl DW and Wallace KB. (1997). Biochim. Biophys. Acta., 1321, 101 ± 106. Pedersen PL. (1978). Prog. Exp. Tumor. Res., 22, 190 ± 274. Pedersen PL. (1997). J. Bioenerg. Biomembrane, 29, 301 ± 302. Petit PX and Kroemer G. (1998). Mitochondrial regulation of apoptosis. In: Singh KK. (ed.). Mitochondrial DNA Mutations in Aging, Disease and Cancer. Springer: New York, NY, USA. Polyak K, Li Y, Lengauer C, Wilson JKV, Markowitz SD, Trush MA, Kinzler KW and Vogelstein B. (1998). Nature Genetics, 20, 291 ± 293. Polyak K, Xia Y, Zweier JL, Kinzler KW and Vogelstein B. (1997). Nature, 389, 300 ± 305. Rempel A, Mathupala SP, Grin CA, Hawkins AL and Pedersen PL. (1996). Cancer Res., 56, 2468 ± 2471. Richter C. (1988). FEBS Lett., 241, 1 ± 5. Schatz G. (1995). Biochim. Biophys. Acta., 1271, 123 ± 126. Segal-Bendirjan E, Coulaud D, Roques BP and Le Pecq JB. (1988). Cancer Res., 48, 4982 ± 4992. Shan K, Linco MA and Young JB. (1996). Intern Med., 125, 47 ± 58. Sharkey SM, Wilson BC, Moorehead R and Singh G. (1993). Cancer Res., 53, 4994 ± 4999. Sharp MGF, Adams SM, Walker RA, Brammer WJ and Varley JM. (1992). J. Pathology, 168, 163 ± 168. Singh KK. (1998). Mitochondrial DNA Mutations in Aging, Disease and Cancer. Springer: New York, NY, USA. Singhal PK, Iliskovic N, Li T and Kumar D. (1997). FASEB J., 11, 931 ± 936. Takao M, Aburatani H, Kobayashi K and Yasui A. (1998). Nucleic Acids Res., 26, 2917 ± 2922. Tallini G. (1997). Virchow Arch., 433, 5 ± 12. Tanaka M, Hayakawa M and Ozawa T. (1996). Methods Enzymol., 264, 407 ± 420. Tooli G, Corona G, Gigante M and Boiocchi M. (1996). Eur. J. Cancer, 32A, 1591 ± 1597. Von Ho DD, Layard MW, Basa P, Davis Jr HL, Von Ho AL, Rozencweig M and Muggia FM. (1979). Ann. Int. Med., 91, 710 ± 717. Wallace KB, Eells JT, Madeira VMC, Cortopassi G and Jones DP. (1997). Fundamental Appl. Tox., 38, 23 ± 37. Welter C, Kovacs G, Seitz G and Blin N. (1989). Genes, Chromosomes and Cancer, 1, 79 ± 82. Woodburn KW, Vardaxis NJ, Kaye AA, Reiss JA and Phillips DR. (1992). Photochem. Photobiol., 55, 697 ± 704.