Pharmac. Ther. Vol. 54, pp. 217-230, 1992 Printed in Great Britain. All fights reserved

0163-7258/92 $15.00 © 1992 Pergamon Press Ltd

Associate Editor: J. S. LAZO

MITOCHONDRIAL D N A DAMAGE BY ANTICANCER AGENTS GURMIT SINGH,* SHEILA M . SHARKEY a n d ROGER MOOREHEAD OCF, Hamilton Regional Cancer Center and Department of Pathology, McMaster University, Hamilton, Ontario L8V IC3, Canada Abstraet--Mitochondrial DNA (mtDNA) is susceptible to damage by a number of anticancer agents either directly or indirectly. This damage is of little consequence if only a few of the mtDNA molecules are damaged. However, multiple drug treatments could result in a significant effect on a cell's ability to survive. The differential effect of anticancer agents on either organ specific toxicities or selective tumor kill can be partially accounted for by differential mtDNA content of cells and on the basis of differential protective mechanisms within mitochondria of various organs or tumor tissue. The concept of damage to mitochondria, especially its genome, is a subject of active investigation in various laboratories. This area of research may provide mechanism(s) by which organ specific toxicities or tumor specific toxicities may be elaborated. Also, the concept of targeting tumor specific mitochondria and/or mtDNA by anticancer agents is very attractive but has not come to fruition due to a lack of understanding of the regulation of the genome in tumor cells. Future investigations in this arena will enhance our knowledge on the interaction between anticancer agents and extranuclear DNA.

CONTENTS i. Introduction 2. Mitochondrial Genome 2.1. Structure 2.2. Replication 2.3. Repair 3. Mitochondria as a Drug Reservoir 4. Detoxification Systems in Mitochondria 4.1. Glutathione and superoxide dismutase 4.2. Heat shock proteins 5. Relationship between mtDNA Damage and Mitochondrial Function 5.1. Oxidative phosphorylation 5.2. Calcium homeostasis 6. Mitochondria in Tumor Cells 7. Mitochondrial Damage by Anticancer Agents 7.1. Toxicities 7.2. Antitumor effects 8. Conclusions Acknowledgements References

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1. I N T R O D U C T I O N Mitochondrial D N A ( m t D N A ) comprises a b o u t 0 . 1 - 1 % o f the total D N A in most m a m m a l i a n cells. The size o f the m t D N A ranges from approximately 16 K b in animals to more than 100 K b in plants (Wallace, 1982). It codes in part for several critical mitochondrial inner m e m b r a n e *Corresponding author.

Abbreviations: mtDNA, mitochondrial deoxyribonucleic acid; D-loop, displacement loop; L-strand, light strand; H-strand, heavy strand; ATP, adenosine triphosphatc; 5-FU, 5-fluorouracil; PDT, photodynamic therapy. 217

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proteins including subunits of apocytochrome oxidase and ATPase (Attardi and Schatz, 1988). The genomes of human (Anderson et al., 1981), murine (Bibb et ai., 1981), bovine (Anderson et al., 1982) and xenopus (Roe et al., 1985) mitochondria have been completely sequenced. There is direct evidence that mtDNA is 5- to 500-fold more sensitive than nuclear DNA to damage induced by several chemicals, with the highest differential relating to polycyclic hydrocarbons (Allen and Coombs, 1980). Potent carcinogens such as aflatoxin and benz(~)pyrene have been implicated to damage mtDNA (Niranjan et al., 1982; Backer and Weinstein, 1982). Damage to mtDNA contributes to the cytotoxic, mutagenic and carcinogenic potential of several drugs and environmental chemicals with reactive electrophilic metabolites, especially since 'activation' of foreign compounds can occur within or at the surface of mitochondria (Niranjan and Avadhani, 1980). Furthermore, mitochondria play a central role, not only in cellular respiration, but also in maintenance of intracellular pH and ion homeostasis (Zaccarato and Nicholls, 1982). mtDNA is particularly susceptible to damage from anticancer agents because: (a) the negative supercoiled structure of mtDNA makes it sensitive to damage; (b) unlike nuclear DNA, mtDNA is not associated with protective histone or non-histone proteins (Salazar et al., 1982); (c) besides the lack of protective proteins, mtDNA has limited repair capacity (Clayton et al., 1974; Singh and Maniccia-Bozzo, 1990); (d) it has low replication fidelity, that is only one origin of transcription (Bandy and Davidson, 1980); (e) since mtDNA is attached to the inner mitochondrial membrane it is susceptible to (i) mutagens activated by enzymes of the electron transport chain, also located in the inner membrane and (ii) electrophiles generated by these same enzymes (Singh et al., 1985; Wilkie et al., 1983; Shay and Werbin, 1987). 2. MITOCHONDRIAL GENOME 2.1. STRUCTURE The two strands of mtDNA are referred to as the heavy strand (H-strand) and light strand (L-strand). At the origin of synthesis of the H-strand there is a region known as the displacement loop (D-loop) wherein short daughter strands of the H-loop are stably associated with the parental strand (Billum and Clayton, 1978). It has been proposed but not established that the D-loop acts as a primer for H-strand synthesis although other functions have been postulated (Anderson et al., 1981; Clayton, 1982; Doda et al., 1981). This segment of the DNA is relatively relaxed and triple stranded. High levels of turnover in this area and the exposure of single stranded DNA here would seem to make this area one of increased sensitivity to DNA damaging agents, similar to nuclear DNA in S-phase. Unlike nuclear DNA, mtDNA is not associated with histone proteins and a secondary, chromatin-like structure is not present. Although mtDNA can be isolated in association with proteins it is unlikely that this association affords mtDNA protection through structural alterations or diminishing base exposure (Clayton, 1982). mtDNA contains the genetic information for encoding 13 polypeptides (all of which are parts of the respiratory chain), 2 ribosomal and 22 transfer RNAs (Anderson et al., 1981). Of the 13 polypeptides, seven are subunits of complex I, one is cytochrome b of complex III, three are subunits of complex IV and the remaining two are components of complex V. This genome is very ordered with only one origin of replication and one promoter for transcription (Berk and Clayton, 1974). Therefore point mutations in any of the mRNAs can result in altered subunits of complexes I, II, III, or IV while mutations in either rRNA or tRNA can affect the synthesis of all of these complexes. Nuclear-encoded components of the electron transport chain are imported into the mitochondria and combined with those subunits derived from mitochondrial DNA to produce a functional electron transport chain. The mammalian mitochondrial DNA has a remarkable degree of compactness. Unlike nuclear DNA or yeast mitochondrial genome, there are very few intergenic nucleotides and no intragenic sequences (Wallace, 1982). The tRNA genes are all interspersed between the coding regions. The mRNA's, rRNA's and tRNA's all appear to be products of precise endonucleolytic cleavage of large transcripts by a process whereby tRNA genes serve presumably as processing signals (Ojala et al., 1981). The mammalian mitochondrial genome also exhibits several other unique features.

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The genetic code for mitochondrial DNA differs from the 'Universal' genetic code in that UGA codes for tryptophan rather than a stop codon as in the universal code, AUA for methionine rather than arginine, AGA for termination rather than arginine (Barrell et al., 1980). Certain structural features found in all other tRNA's are lacking in mitochondrial tRNA's (de Bruijn et aL, 1980). 2.2. REPLICATION mtDNA exhibits a unique mode of replication in which the two DNA strands have a distinct origin of replication (Berk and Clayton, 1974). The process of replication is initiated at the H-strand origin and L-strand synthesis does not begin until the H-strand has been elongated approximately two-thirds around the molecule, mtDNA isolates have a high proportion that contain a third strand of DNA which is a replicative intermediate in which the newly synthesized H-strand (7S DNA) displaces a segment of the parental DNA creating a triple-stranded structure (D-loop) (Kasamatsu and Vinograd, 1974). This part of the genome would hypothetically be a 'hot-spot' for damage by anticancer agents because the structural geometry would be ideal for intercalation. Furthermore, damage at this location would disable the genome from further replication. The replication of mtDNA and the biogenesis of mitochondria is not synchronized with the mitotic cycle. The specific sequence of events in this process and control signals are yet unclear. However the number of mtDNA molecules in dividing tissue culture cells is relatively constant and thus it is believed that the replication process must be highly organized (Bogenhagen and Clayton, 1977). Since the mitochondrial genome does not contain any sequences for DNA polymerases, it is believed that the components of the mitochondrial replication, namely polymerases, must be of nuclear origin and thus must be imported into the mitochondria. The details of mitochondrial replication are beyond the scope of this review. 2.3. REPAIR

Whether or not repair of mtDNA occurs is an issue on which consensus has not been reached. The high levels of mtDNA damage and mutation which occur after exposure to agents which interact with DNA have been attributed to either increased damage due to the previously mentioned structural differences in mtDNA vs nuclear DNA or to the lack of repair of incurred damage (Singh and Maniccia-Bozzo, 1990; Tomkinson et al., 1990). Clayton and colleagues have demonstrated that mitochondria are unable to repair pyrimidine dimers and mtDNA replication is inhibited after exposure to UV light (Clayton et al., 1974). Also, the lack of strand scission in mtDNA but not nuclear DNA after exposure to cisplatin indicates the absence of excision repair capabilities in mitochondria (Singh and Maniccia-Bozzo, 1990). The inability to demonstrate excision repair processes cannot be interpreted as the general absence of repair. Myers et al. (1988) showed that mitochondria repair O6-methylguanine residues and isolated from hepatic mitochondria a methyltransferase similar to but distinct from that found in the nucleus. In the same study these investigators again demonstrated lack of excision repair in that O6-butylguanine residues in mtDNA were removed very slowly compared to those in nuclear DNA. This slow decrease was attributed to mtDNA turnover while excision is the primary mode of nuclear repair. The isolation of mitochondrial forms of excision enzymes involved in the correction of oxidative damage, which are distinguishable from nuclear forms raises questions regarding the lack of this type of repair in these organelles (Tomkinson et aL, 1990). Mitochondrial uracil DNA glycosylase, apurinic/apyrimidinic endonucleases and UV endonucleases have been purified and characterized (Domena et al., 1988; Tomkinson et al., 1988, 1990). Interestingly, the mitochondrial form of UV endonuclease III isolated from xeroderma pigmentosum group D lymphoblasts is altered. In extracts from these cells this enzyme is absent or present in significantly reduced amounts implying a relationship between the two forms of this enzyme (Tomkinson et aL, 1990). Using a probe specific for mtDNA, Pettepher and colleagues have demonstrated time dependent decreases in alkali-labile sites in mtDNA after treatment with the

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nitrosourea Streptozotocin implying an excision repair mechanism. The specific steps involved in the removal of these N7-methylated guanine residues have yet to be defined (Pettepher et al., 1991). Despite the preceding indications of repair, many unsettled issues remain. The enzymes isolated have been proposed by some to be involved in the initiation of degradation of damaged mitochondrial genomes allowing only those which are intact to be replicated (Tomkinson et al., 1990). Also, mtDNA turnover as an explanation for decreases in altered bases and molecular forms of the genome plagues most studies done to date. A nicking-closing enzyme namely Type-I topoisomerase was isolated from rat liver and L-cell mitochondria (Fairfield et al., 1979). This topoisomerase is capable of generating swivels in closed circular DNA and is capable of generating catenanes, but only if one of the strands is nicked. However, Type-II DNA topoisomerases generate transient double-strand breaks and thus not only can it relax supercoiled DNA, but can reversibly generate catenanes and knots from closed circles (Castora et al., 1981; Liu, 1980). It has been shown that the mitochondrial enzyme is distinct and not a nuclear contaminant because the nuclear enzyme requires ATP and histone H1 whereas the mitochondrial enzyme does not. Since some of the antitumor agents like VP-16 may be operating through the inhibition of topoisomerases and the implication of induced topoisomerases in drug resistant cells, it is important to consider these enzymes if and when specific mtDNA damaging agents are investigated.

3. MITOCHONDRIA AS A DRUG RESERVOIR The electrochemical gradient in mitochondria is composed of two components: a pH gradient (concentration gradient of protons) and a membrane potential. The two mechanisms that generate an electrochemical gradients are: (l) active pumping of protons across a membrane and (2) the movement of the protons coupled to electron transfer (Ferguson and Sorgato, 1982). The action of the electron transport chain can produce both the membrane potential and pH gradient. The distribution of permeant ions between the inner and outer aqueous phases is used for measuring membrane potential. Rhodamine 123 (Rhl23) is a fluorescent lipophilic cation that accumulates in mitochondria specifically due to the membrane potential. Experiments using ionophores and nigericin have demonstrated that Rhl23 will not accumulate in the absence of a membrane potential and that it accumulates proportionally to the membrane potential. There will be some Rh123 accumulation as a result of the membrane potential of the cell membrane, but this is insignificant compared to the concentration of Rhl23 inside mitochondria. Thus Rhl23 staining and the use of either fluorescent microscopy or flow cytometry can determine membrane potential of mitochondria within a cell. Generally in mammalian cells the membrane potential accounts for 180mV and the pH gradient 60mV. Under steady state conditions the movement of protons out of the mitochondrial inner membrane equals the flux of proton back in and thus the electrochemical gradient does not fluctuate. Also, all the mitochondria within a cell have similar electrochemical gradients (Chen, 1988). There is an approximately 60 mV difference in mitochondriai membrane potential between normal epithelial cells and adenocarcinoma-derived MCF-7 cells (Modica-Napolitano and Aprille, 1987; Davis et al., 1985). Higher membrane potential is also observed in v-fos oncogene transformed fibroblasts compared to their untransformed counterparts (Zarbl et al., 1987). According to the Nernst equation, if the plasma membrane potential is 60 mV and mitochondrial membrane potential 180 mV, then the lipophilic cations will concentrate in the mitochondria by at least 10,000-fold greater than in the medium at equilibrium (Chen, 1988). Furthermore, retention of lipophilic cations in tumor cells as compared to normal cells provides selectivity of agents like Rh123 in some tumor cells. The retention in these tumor cells may be accorded by a higher mitochondrial membrane potential in certain tumor mitochondria and is also due to fewer mitochondria in tumor cells compared with normal cells (Singh and Shaughnessy, 1988; Pedersen, 1978). Thus cationic agents provide a potentially new target for the treatment of carcinomas aimed at the mitochondria.

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4. DETOXIFICATION SYSTEMS IN MITOCHONDRIA 4.1. GLUTATHIONE AND SUPEROXIDE DISMUTASE Under normal conditions, respiring mitochondria produce small, but significant quantities of reactive oxygen molecules, primarily superoxide anion (O~-) and hydrogen peroxide (H202). These oxygen molecules can damage membranes, nucleic acids and proteins, unless controlled (Reed, 1990). There are two primary antioxidant systems localized in the mitochondria that control reactive oxygen damage; superoxide dismutase (SOD) and glutathione (GSH). There are three types of SOD's: two cytosolic and one mitochondrial. Manganese superoxide dismutase (Mn-SOD) is encoded by the nucleus (Harris et al., 1991), synthesized in the cytosol and located in the mitochondrial matrix (Grace, 1990; Warner et al., 1991). Mn-SOD converts 02 to H202 and thus is important in protecting the mitochondria from reactive oxygen molecules. Levels of Mn-SOD appear to be induced by oxygen and several cytokines (Gardner and Fridovich, 1987; Lavelle et al., 1977). During an immune response, certain cytokines such as TNF, induce macrophages and neutrophils to produce O~-. TNF and other cytokines (IL-1 and INF) seem able to increase Mn-SOD expression. This may assist in selectively destroying infected or damaged cells while minimizing damage to host cells. The ability of Mn-SOD to be enhanced might be important in protecting cells from certain cytotoxic agents (Harris et al., 1991; Warner et al., 1991). H202, produced as a result of Mn-SOD activity, can cause harmful effects because there is no catalase in mitochondria to inactivate it. However, mitochondrial glutathione is present which safely reduces mitochondrial generated H202 (Meister, 1991). There are two main glutathione pools within the cell; the largest in the cytosol and a smaller portion (10-15%) in mitochondria (Reed, 1990). Mitochondria do not have the ability to produce GSH as they lack ?-glutamylcysteine or GSH synthase and thus must depend on the cytosol as a source of GSH. GSH uptake by the mitochondria is an energy-dependent process and appears to depend on: (a) high concentration of extramitochondrial GSH, (b) proton gradient and (c) state IV respiration (Kurosawa et al., 1990). In contrast, low cytosolic GSH levels do not promote the efflux of mitochondrial GSH; mitochondrial transport serves to conserve mitochondrial GSH at the expense of cytosolic GSH (M~trtensson et al., 1990). This phenomenon is important, especially in experiments that attempt to vary cell survival by depleting GSH with buthionine sulfoximine (BSO). BSO decreases cytosolic GSH with little effect on mitochondrial GSH. GSH also has the ability to effectively remove certain chemotherapeutic agents such as cisplatin and inhibit their interactions with mtDNA (Hamilton et al., 1985). 4.2. HEAT SHOCK PROTEINS

Heat shock proteins (hsp) or 'stress proteins' appear to contribute to a cell's defense through two mechanisms; they preserve certain essential components in a protected state and quickly reactivate cellular functions following stress (Kochevar et al., 1991). Hsp60 which is located in the mitochondria is constitutively expressed and is involved in protein import and folding (Lindquist and Craig, 1988). Its expression is enhanced during hyperthermia and possibly other stresses. The importance of Hsp60 has been demonstrated in yeast where inactivation of mitochondrial Hsp60 is lethal (Baker and Schatz, 1991).

5. RELATIONSHIP BETWEEN mtDNA DAMAGE AND MITOCHONDRIAL FUNCTION 5.1. OXIDATIVEPHOSPHORYLATION

Cells utilize the electrochemical gradient, generated by the electron transport chain in mitochondria to synthesize ATP (reviewed by Hatefi, 1985; Sherratt and Turnbull, 1990). The proteins of the electron transport chain, namely those in complexes I, III and IV, have some of their subunits encoded by the mitochondrial genome (Anderson et al., 1981). Therefore, damage to the mitochondrial genome could influence the extent to which protons can be translocated and thus JPT 54/2~

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the size of the electrochemical gradient. A decrease in the electrochemical gradient would reduce the force which drives protons through the FoFi-ATPase and therefore hinder ATP production. Furthermore, complexes I and III are involved in stabilizing reduced intermediates during electron transfer. A loss of this function in the electron transport chain would render these reduced intermediates susceptible to attack by oxygen or electrophiles, possibly leading to the generation of reactive oxygen molecules. Cytochrome b, which is also encoded by mtDNA, is necessary for incorporation of iron-sulphur components into complex III (Japa and Beattie, 1988). The iron-sulphur components are required for proper electron transfer and an alteration in their function leaves electrons available for reactions with oxygen. Complex IV stabilizes oxygen as it is used as the final electron acceptor by the respiratory chain. One of the important functions of this complex is to stabilize oxygen during its reduction until it can safely be released as water. Therefore, damage to subunits of this complex could destabilize and release the oxygen radical before it can be reduced to water. Loss of function of complex V may prevent the ATPase complex from dissipating the electrochemical gradient by preventing the flow of protons and ATP production. The lack of ATP would increase mitochondrial respiration as is observed when mitochondria are treated with uncouplers. Thus an increase in the rate of electron transfer and a concomitant increase in the amount of free radicals would be generated (Bandy and Davidson, 1990). It should also be recognized that production of reactive oxygen can inhibit several electron transport chain enzymes such as N A D H dehydrogenase, N A D H oxidase, succinate dehydrogenase, succinate oxygenase and ATPase activity (Zhang et al., 1990). Loss of respiratory chain activity may prevent re-oxidation of N A D H and FADH2 resulting in high mitochondrial concentrations of these two co-factors. Elevated levels of N A D H inhibits enzymes such as pyruvate dehydrogenase (Reed and Yeaman, 1987) and those involved in fatty-acid oxidation (Latipfifi et al., 1986).

5.2. CALCIUMHOMEOSTASIS The importance of mitochondria in regulating cytosolic calcium under normal conditions is probably minor since the affinity for calcium and its uptake rate are significantly greater in other organelles such as the endoplasmic and sarcoplasmic reticutum (Carafoli, 1987). But, under stressful conditions, where cytosolic calcium levels are increased due to an influx of calcium across the plasma membrane, mitochondria have the capability to sequester large amounts of calcium, in bound and precipitated forms, with little effect on their function (Fiskum, 1985). There are two conditions that can alter calcium homeostasis in the mitochondria: loss of the electrochemical gradient and oxidative stress. Both of these conditions can arise as a result of damage to mtDNA. As outlined earlier, damage to m t D N A can lead to impaired function of the respiratory chain enzymes. Loss of this enzyme system diminishes or possibly depletes mitochondria of its electrochemical gradient. Since calcium is taken up through a uniport that is dependent on the membrane potential, a decrease in the membrane potential would hinder the rate and extent of calcium accumulation. Also, the rate and extent of calcium accumulation is dependent on intramitochondrial concentration of inorganic phosphate. Since the electrochemical gradient is used to power the exchange of ATP for ADP and Pi, the loss of the membrane potential could limit intramitochondrial inorganic phosphate levels (Sherratt and Turnbull, 1990). Oxidative stress can also influence mitochondrial calcium homeostasis through oxidation of pyridine nucleotides (Richter and Kass, 1991; Richter and Frei, 1988). To alleviate an oxidative stress encountered by the cell, GSH is oxidized by glutathione peroxidase with the concomitant reduction of the reactive oxygen species. Reduction of the active oxygen usually results in a nonreactive molecule such as water. In order to regenerate GSH supplies, pyridine nucleotides are oxidized and studies indicate that it is this process that is involved in calcium release (Richter and Kass, 1991; Bellomo et al., 1984). Studies by Richter and Frei (1988) indicate that oxidized pyridine nucleotides are hydrolyzed at the bond between nicotinamide and ADP-ribosylation. This ADP-ribosylation is considered to control calcium release either through a pore or alteration in membrane permeability (Crompton et al., 1988). ADP-ribosylation returns to normal as calcium ion release diminishes.

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Control of intramitochondrial calcium levels is important since three mitochondrial dehydrogenases, pyruvate dehydrogenase, NAD-dependent isocitric dehydrogenase and oxoglutarate dehydrogenase, are regulated by fluctuations in matrix calcium content (Carafoli, 1987; McCormack, 1985; Denton et al., 1972, 1978; McCormack and Denton, 1979). These three dehydrogenases are important in the TCA cycle and may affect the production of substrates (NADH or FADH2) available to the enzyme complexes of the electron transport chain. Activation of these dehydrogenases by calcium may augment rates of both the electron transport and ADP phosphorylation, possibly affecting ATP production (Gunter and Pfeiffer, 1990; Crompton et al., 1988). Also, growth factors such as epidermal growth factor and platelet-derived growth factor regulate cell proliferation through an alteration in intracellular calcium levels. Calcium, in conjunction with oxidative stress, has been associated with increased transcription rates of c-myc, c-fos and/~-actin gene which are important in cell differentiation (Birky, 1983; Bernardi, 1979). Thus, calcium and oxidative stress, to some extent, control cell proliferation and could possibly lead to altered growth rates (Cerutti, 1985). Since calcium is taken up through a uniport that is dependent on the membrane potential, a decrease in the membrane potential would hinder the rate and extent of calcium accumulation (Nicholls and Akerman, 1982). If this membrane potential falls below 130mV not only is calcium not accumulated, but it is also released from mitochondria. Also, the rate and extent of calcium accumulation is dependent on intramitochondrial concentration of inorganic phosphate. Since the electrochemical gradient is used to power the exchange of ATP for ADP and Pi, the loss of the membrane potential will limit intramitochondrial inorganic phosphate levels (Richter and Kass, 1991; Carafoli, 1987; Crompton et al., 1988).

6. MITOCHONDRIA IN TUMOR CELLS Altered mitochondrial functioning as an integral component of the oncogenic process was first proposed by Otto Warburg (1967) over 40 years ago. For some time the interpretation of his proposal was limited to 'tumor cells are more glycolytic'. More recently, interest has grown regarding the specific changes which occur in the mitochondria of neoplastic cells. Both structural and functional alterations have been determined. In the majority of tumors the number of mitochondria is reduced when compared to the tissue of origin, frequently by as much as 50% (Pedersen et al., 1970). Those present display altered shape, cristae structure, more dilute matrix and inclusions not seen in normal cell mitochondria. These differences become more pronounced with loss of differentiation and increased growth rate of tumors. Rapidly growing tumor cells contain mitochondria which are smaller and contain fewer cristae (Hurban et al., 1966, 1973). In terms of functional differences and the highly glycolytic nature of rapidly growing tumor cells, much attention has focused on hexokinase. This enzyme is responsible for committing glucose to the glycolytic pathway and is elevated in tumors with a large proportion (40-60%) bound to the mitochondrial membrane of these tumor mitochondria. This association enhances the stability of the enzyme and appears to alter its normal inhibition by glucose-6-phosphate. It has been determined that the ATP generated by oxidative phosphorylation is preferentially used by mitochondrially bound hexokinase to maintain glycolytic activity (Arora et al., 1991; Nakashima et al., 1986; Bessman and Geiger, 1980). The dynamics of this altered pathway differ between tumors with different growth rates. Generally, as tumor growth rate increases, the amount of cellular ATP generated by glycolytic reactions increases (Pedersen, 1978). The body of literature available regarding the physiology of bioenergetics in tumor mitochondria is enormous and a comprehensive review is beyond the scope of this review. Numerous differences have been demonstrated including alterations in respiration and oxidative phosphorylation levels, membrane proteins, lipid composition, transport systems and various enzyme activities (for reviews see Pedersen, 1978; Chen and Rivers, 1990). Pertinent to the present issue are the findings of increased Rh123 uptake by tumor cells. This is a cationic dye which accumulates in mitochondria apparently due to their membrane potential,

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implying that neoplastic cells have a relatively higher membrane potential than their normal counterparts. Both the uptake and retention of this compound are increased. It is of interest to note that this is also the case for Ca 2* accumulation in tumor cells. Although increased cation uptake may be attributed to an increased electrochemical gradient, the question remains as to why these compounds are not released (even over extended time periods) as they are from normal cells. The selective toxicity of Rh123 in tumor cells has led to proposals for the targeting of neoplastic cells through exploiting this differential uptake by employing lipophilic cations which are toxic or using them as vehicles to deliver drugs whose uptake is not linked to membrane potentials (Chen, 1988). Several changes have also been noted in the mitochondrial genome of tumor cells; these include deletions and duplications (Ebner et al., 1991). Elevated mtDNA has been demonstrated in a number of tumors when compared to the tissue of origin (Pedersen, 1978). Structurally the DNA of these organdies have been found to differ with a high proportion forming circular dimers rather than monomers. The significance of the above findings in terms of mitochondrial function are unknown at present, although increased DNA content has been related by some to inhibited degradation of mitochondria in tumor versus normal cells. Less is known regarding possible differences in repair capabilities; such information awaits further determinations of these activities in normal cells.

7. MITOCHONDRIAL DAMAGE BY ANTICANCER AGENTS 7.1. TOXICITIES Various organ specific toxicities induced by anticancer agents may be related to damage of mtDNA. Cisplatin induced nephrotoxicity has been postulated to be induced by damage of mtDNA (Singh, 1989). Furthermore, preferential binding of cisplatin to mtDNA has also been observed (Murata et al., t990). Differential effects of the cisplatin on hepatic and renal mtDNA have been reported (Maniccia-Bozzo et aI., 1990). Bleomycin induced pulmonary toxicity may be partially related to damage of the mitochondrial genome. This anticancer agent has been shown to cause single stranded breaks of mtDNA (Osieka et al., 1976; Lim and Neims, 1987). Other chemotherapeutic agents such as 5-FU and methotrexate which inhibit the enzyme thymidylate synthetase also affect the synthesis of mtDNA but to a much lower extent than the effect on nuclear DNA. Thus the acute effect of either 5-FU or methotrexate is minimal; long term or chronic use may indeed have adverse effects on mitochondria. Indirect effects on mitochondria and/or mtDNA by other chemotherapeutic agents such as adriamycin and methylglyoxal bi(guanylhydrazone) (MGBG) have also been reported (Doroshow and Davies, 1986; Loesberg et al., 1991; Ellis et al., 1987; Vertino et al., 1991). Cardiac toxicity induced by adriamycin has been associated with its binding to cardiolipin, in cardiac mitochondria. AIDS patients treated with zidovudine demonstrate a destructive mitochondrial myopathy (Arnaudo et al., 1991). This effect is believed to be caused by the inhibition of mtDNA replication by zidovudine (Dalakas et al., 1990). The replication of DNA is altered by dideoxy nucleoside triphosphates such as zidovudine which serve as substrates for the enzyme DNA polymerase gamma responsible for the replication of mtDNA (Zimmerman et al., 1980). 7.2. ANTITUMOR EFFECTS Mitochondria and/or mtDNA has been postulated to be the site of various anticancer agents. Drugs such as MGBG have been recognized as potent antiproliferative agents. Clinical studies have shown that MGBG induces remissions in patients with acute myelocytic leukemia, malignant lymphoma and other neoplasms. It has been shown that mitochondrial ultrastructure is altered following MGBG treatment of cells (Porter et al., 1979). Effect of MGBG on mtDNA synthesis has also been demonstrated (Feurestein et al., 1979). Selective inhibition of mtDNA replication is shown prior to significant inhibition of nuclear DNA synthesis (Nass, 1984). Wilkie et aL (1983) have shown in a yeast model that the antileprosy drug clofazimine and

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the antidepressant chloripramine cause inhibition of growth and affect mtDNA (Wilkie and Fearon, 1985). It has also been shown that various chemical carcinogens selectively attack mtDNA and in yeast produce a mitochondrial mutation (petite) (Ferguson and Turner, 1988). This preferential attack on mtDNA compared to nuclear DNA is believed to be related to the 'naked' condition of mtDNA which is devoid of the proteins that are intimately associated with nuclear DNA. Newer modalities of cancer therapy, namely photodynamic therapy (PDT), appear to target mitochondria. Specifically photosensitizers such as photofrin have been implicated in causing mitochondrial damage (Singh et al., 1987; Gibson and Hilf, 1985). Recently it has been shown that resistance to PDT can be induced (Singh et al., 1991). Preliminary data obtained from our laboratory has indicated that this resistance may be due to morphological alteration of mitochondria. Furthermore it is curious that the PDT resistant cells are cross resistant to cisplatin but not to other chemotherapeutic agents such as adriamycin. The mechanism of this unique mode of resistance is under active investigation in our laboratory. Mitochondria-specific dyes such as Rh123 have been reported to inhibit the growth of tumor cells with no effect on normal cells. This antitumor effect is thought to be selective due to preferential accumulation and retention of cationic dyes in carcinoma cell mitochondria to a much greater extent than most normal cells (Bernal et al., 1983). Since these dyes are also photoactive, they can potentially be used for selective photochemotherapy (Oseroff et al., 1986). The effect of these agents on mitochondrial DNA have not been investigated yet. It is recognized that rapidly proliferating tumor cells are preferentially inhibited by tetracyclines while their effect on normal cells is limited. They are known to inhibit mitochondrial protein synthesis and thereby achieve antiproliferative activity (Kroon and Van Den Bogert, 1985). It has also been suggested that deprivation of energy-generating capacity may hinder repair of damage induced by radiation on DNA, since their repair processes are energy dependent (Ebringer, 1990).

8. CONCLUSIONS Damage to mitochondria by various drugs and toxicants has been recognized and observed morphologically for a very long time. However it was only in 1966, that the first vertebrate mitochondrial DNA was isolated and characterized. Since that time effort has been concentrated on investigating the role of mtDNA and its interactions with nuclear DNA. The organelle DNA has been used to exploit the evolutionary aspects of the origin of mitochondria. The importance

ANTI CANCER

AGENTS

Z~mtDNA coded mRNA Z~rntDNA coded Proteins

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\ Amt

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CELL DEATH

FIG. I.

Co++

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of mitochondria in cellular function is best described by a quote from the lives of a cell by Lewis Thomas, that is: " I t is a good thing for the entire enterprise that mitochondria and chloroplasts have remained small, conservative and stable, since these two organelles are, in a fundamental sense, the most important living things on earth. Between them they produce the oxygen and arrange for its use. In effect, they run the place". The importance of damage induced on m t D N A by various carcinogens and anticancer agents has been established recently by a number of independent investigators. The overall cascade of events are appropriately depicted in Fig. 1. This figure summarizes the various subtle changes that occur in mitochondria following damage induced by anticancer agents. The type of damage or interaction of anticancer agents with m t D N A is irrelevant. Since the genome is replicated or transcribed in its entirety, any sort of drug intercalation or genome damage may result in profound effects on mitochondrial function. The electrochemical gradient that exists in the mitochondria may be particularly vulnerable as compounds are electrophoresed into the organelle and thus make it a storage depot for extremely toxic compounds. Nature has provided various systems to m o p up such toxic compounds. The large copy number of m t D N A also gives the mitochondria ability to overcome damage induced on a few copies of m t D N A . However, when specific agents such as anticancer agents are used to damage D N A and stop tumor cells from proliferating, m t D N A is also affected in the process. In some instances such as cisplatin-induced nephrotoxicity or adriamycin-induced cardiotoxicity the culprit may be m t D N A . Acknowledgements--Financial support in the form of an operating grant from the Medical Research Council

of Canada (Grant # MA-8509) and a career scientist award from the Ontario Cancer Foundation to G. Singh is gratefully acknowledged. Studentships for S. M. Sharkey from PMAC/MRC(C) and R. Moorehead from McMaster University are also acknowledged. I would also like to thank colleagues around the world who have provided reprints. This information was invaluable in the preparation of this review. I would like to apologize to all those whose contribution to this topic could not be cited within the confines of this review or simply due to an oversight.

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