Is There a Role for Mitochondrial Genes in Carcinogenesis?'

(CANCER RESEARCH 35, 3332-3335, November 1975]

Henry D. Hoberman

Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461

Summary development of cancer, is heritable, since the damaged respiratory apparatus is a part of the mitochondrion, which Although defective respriration is not characteristic of all is itself an autonomous organelle. (c) Dedifferentiation is tumors, recent comparative studies on the ultrastructure of the result of the replacement of respiration, which depends normal and tumor cell mitochondria indicate that in on the structural integrity of the mitochondrion, by fermen malignant cells mitochondria deviate from normal not only tation, the reactions of which are catalyzed by enzymes in in relative abundance but also in the size, form, density, and solution (state of disorganization). frequency of appearance of lesions. Normal and abnormal It does not do Warburg justice that most students of mitochondria may populate the same cell, suggesting that oncology have rejected all of his postulates because of their there may be a gradation in respiratory deficiency depend disagreement with 1 (the 1st). While perhaps based less on ing on the proportion of normal to abnormal forms. fact than on the innate suitability of the concept to his Recent advances in mitochondrial genetics suggest that overview of carcinogenesis, his idea of an association of a aberrant mitochondria may be formed as a result of the defective, heritable mitochondrion with cancer may, in due presence of an abnormal mitochondrial genome. In analogy time, turn out to be not entirely farfetched. with the petite mutant of certain strains of yeast, animal In regard to the question of energy metabolism of cells may be transformed by treatment with dyes that alter neoplastic as compared with normal cells, there persists an the structure of their mitochondrial DNA, so that their impression, despite recurrent controversy, of the existence mitochondria also become deficient in enzymes of the of a genuine difference of respiratory function between the 2 respiratory chain. Whether nutritional or other deficiencies kinds of tissues. In his comprehensive review of the are mutagenic with respect to mitochondrial DNA of glycolysis and respiration of tumors, Aisenberg summarizes animal cells is not known; nor is it known whether his views, in part, in the statement that â€œthemoststriking mitochondrial mutagenesis is causally involved in car property of neoplastic energy metabolism remains the high cinogenesis. New knowledge of cytoplasmic genetics and of glycolytic rates of slices of tumor tissue,â€•atthe same time mitochondrial DNA and membrane structure and dynamics recognizing that a high rate of glycolysis is not uniquely should encourage investigations aimed at examining the restricted to tumor tissue (1). possible role of mitochondrial genes in neoplastic transfor In a more recent survey of the same field, Wenner (36), mation. while emphasizing that the energy derived from glycolysis by minimal deviation tumors neither predominates nor even comprises an appreciable proportion of the total energy No one can give thought to the subject of mitochondrial generated by the cell, concluded that vehement glycolysis, metabolism in relation to cancer without recalling the aerobic as well as anaerobic, remains I of the striking theory of carcinogenesis proposed about 20 years ago by Otto Warburg (33-35). Although much of what he held to biochemical properties of the cancer cell, particularly in the rapidly growing tumor. be true is presently given little credence, one of his convic More recently, attention has been directed to properties tions, namely, that the respiration of tumor tissue is defec of tumor cell mitochondria that can be visualized under the tive, still remains viable, as judged by the number of re electron microscope rather than to their biochemical char ports, both pro- and con-Warburg, appearing in the cancer acteristics. Bernhard, in his review of this field of investiga literature. tion (3), although underlining the great variability of While mention of Warburg in relation to cancer usually mitochondria in tumor as compared with normal cells, calls to mind his concept of the energy metabolism of formed the general impression that cancer cells have fewer tumors, it should be noted that Warburg's belief that tumor mitochondria than do their normal counterparts, their respiration was defective was only 1 of 3 related ideas on number decreasing with development of the tumor. He carcinogenesis. I have taken the liberty of paraphrasing his also noted numerous swollen mitochondria. While recogniz main thoughts on this problem in the following sentences. ing that mitochondrial swelling might somehow be second (a) Due to damage to its respiratory apparatus, the tumor ary to intensive growth, Bernhard found the same lesions in cell adapts to the life of an anaerobic organism. (b) The cells that were well preserved in all respects and presumably defect in respiration, which is the specific stimulus to the were actively growing at the time of fixation. Summarizing his overall impressions of tumor cell mitochondria, Bern 1 Presented at the Conference on Nutrition in the Causation of Cancer, May 19 to 22, 1975, Key Biscayne, Fla. Aided by USPHS Grant CA hard was struck by the extraordinary variation in number, 03651from the National CancerInstitute. size, form, density and frequency of lesions that they pre

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1975 American Association for Cancer Research. Mitochondria and Carcinogenesis sent. He pointed to the necessity, therefore, of critical mor pantothenate causes a deficiency of CoA so that, under phological control of mitochondrial pellets prepared for standably, carbohydrate and fatty acid oxidation, as well as biochemical studies. These observations as well as similar turnover of the citrate cycle, are severely disrupted (22). observations recorded by others (4, 23, 24) strongly suggest Whether these or other impairments of mitochondrial that procedures used to isolate mitochondria from normal function have a potential for carcinogenesis brings us back tissues, when applied to tumor tissue, may eliminate those to the main question. Taking into account what has already of greatest biochemical interest. In any case, when consid been said about specific biochemical and ultrastructural eration is given to the observed pleomorphism of tumor cell features of tumor cell mitochondria, it would seem less mitochondria and to the lesionsthat havebeennoted, there probable that aberrant mitochondria are capable of stimu would seem to be little reason to expect uniformity of en lating cell growth and division than that healthy mitochon ergy metabolism among all types of neoplasms. dna normally exert a controlling influence on cell growth As has already been indicated, 1 of Warburg's beliefs, and division. Be that as it may, any theory of carcinogenesis namely, that mitochondria are autonomous organelles, de that includes a role for mitochondria must take into serves greater attention than it has received to date. War consideration the possibility that, for cancer to develop as a burg derived support for his view that mitochondria are result of mitochondrial impairment, the defect must be heritable structures from plant genetics, a view that early on mutagenic for the mitochondrion. recognized the existence of cytoplasmic genes. Among the A changeof nutritional environment of a cell may reveal workers whom Warburg cited for special mention were M. the existence of mutant forms of mitochondria. This is W. Woods and H. G. DuBuy of the National Cancer Insti exemplified by the appearance of a population of mutant tute, who found, in leaves of a variegated species of Nepeta organisms, containing abnormal mitochondria, when cells cataria (catnip), mixed cells containing multiple mitochon of Saccharomyces cerevisiae, a facultative anaerobic strain drial types (37). Most significantly, the mitochondrial of yeast, are changed from growth on a medium containing phenotypes were transmitted to progeny by non-Mendelian glycerol or ethanol as a carbon source to a medium inheritance. Borrowing heavily from Woods and DuBuy, containing glucose or other fermentable sugar. While who were themselves convinced of the pertinence of their growth of the organism on alcohol or glycerol is oxygen studies to cancer ( I I ), Warburg proposed that, once a dependent, growth on glucose-containing media takes place mitochondrion was damaged, it remained so, transmitting at the expense of fermentative energy. When cells of S. its defect to progeny (presumably mitochondria), just as cerevisiae are plated on a soft nutrient medium containing would occur, he asserted, in the case of a damaged nuclear glucose, colonies are formed among which are some smaller gene. Although there would seem to be reason to allow the than the majority. The smaller colonies (petites), on under speculation that cancer cells, through defects in mitochon going mitotic division, yield progeny having the same drial structure, may have nonfunctioning respiratory chains, appearance as the parents, as well as other heritable the question whether such defects are transmissible is as properties, among which is a defect of respiratory function. yet highly conjectural. The loss of respiratory function has been found to be due to My assignment as a participant in this meeting is mitochondria that lack the capacity to synthesize or assem specifically to consider whether, due to aberrations caused ble polypeptides that are precursors of cytochrome oxidase, by a change of nutritional conditions, mitochondria can cytochrome b, cytochrome c1 , and the rutamycin-sensitive play a role in carcinogenesis. Without at this time speculat ATPase of oxidative phosphorylation. Without these con ing on that question, there can be no doubt that mitochon stituents of the respiratory chain, petite mutants do not drial function per se is sensitive to the withdrawal from the respire or carry out oxidative phosphorylation (I 2, 17, 18). diet of certain vitamins and trace elements. An extreme ex As detected simply by a change of one carbon source for ample is the effect of a lack of dietary copper. In this condi another, the petite mutation is spontaneous yet occurs with tion the loss of activity of cytochrome oxidase is so severe a frequency that can be orders of magnitude greater than that liver mitochondria of animals killed at the height of the rates ofspontaneous nuclear mutations. Under certain other deficiency are unable to respire (14). Although iron depri conditions the rate of mutation can reach 100%. Thus vation, because of the obligatory role of heme and non-heme Ephrussi et a!. (13) found that acriflavin, an acridine dye, forms of iron in electron transport, would also be expected could transform an entire population of cells to the petite to embarrass electron flow, dietary restriction of iron by form. Of special significance was the observation that the curtailing the synthesis of hemoglobin rather than of yield of mutant colonies was the same whether the parent iron-containing enzymes of the respiratory chain so limits cells were of a haploid or diploid strain. This independence the life-span of experimental animals that death occurs of the effect of the dye from gene dosage provided strong before loss of respiratory function is noted (5). evidence from which it was concluded that the mutation was Turning to the vitamins, the nature of experimentally not that of a nuclear but of a cytoplasmic gene. In S. induced abnormalities of mitochondrial metabolism is pre cerevisiae, cytoplasmic genes are contained in the mitochon dictable from the known function of the coenzymes of which drion, the genetic message being encoded in the mitochon the vitamin is a precursor. Thus lack of riboflavin, a precur drial DNA. sor of flavin mono- and dinucleotides, has been shown to The frequency of mitochondrial mutation can be in bring about a slowing of oxidation of NADH and succinate creased even when cells are metabolizing under aerobic con by mitochondria of deficient animals (7). A lack of dietary ditions. Mass formation of petite mutants of S. cerevisiae

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1975 American Association for Cancer Research. H. D. Hoberman was accomplished by preventing mitochondr'al synthesis mitochondrial DNA consist of circular, double-stranded of ATP by inhibiting respiration with cyanide or antimycin light and heavy chains of supercoiled molecules having a A, while simultaneously blocking uptake of glycolytically molecular weight of 9 to 10 million daltons (19, 28, 32). It formed ATP with an appropriate inhibitor (bongkrekik has recently been estimated that, in the HeLa cell, genes so acid). These results were interpreted to indicate that the far identified on mitochondrial DNA account for about 25% constant presence of ATP within mitochondria is essential of the potential information contained in the 5-nm-long to normal replication of mitochondrial DNA (30). An DNA molecule (2). Of singular interest is the fact that, on alternative interpretation is that interference with energy animal cell and yeast mitochondrial DNA, genes have been utilization in mitochondria may be mutagenic for mitochon identified that encode for tRNA's that almost without drial DNA. exception are specific for the hydrophobic amino acids (9). In the presence of ethidium bromide, a phenanthrid@ne It is, therefore, no wonder that all proteins synthesized on dye that intercalates between complementary bases in du mitochondrial ribosomes have so far been found to belong plex DNA, mitochondrial DNA synthesis is selectively in to the class of hydrophobic proteins associated with the hibited and preexisting mitochondrial DNA is progressively inner mitochondrial membrane (6). degraded (15). The petite mutation is thereby enhanced to In petite mutants that have grossly altered or no mito the extent that mass formation of the respiration-deficient chondrial DNA, it is found that mitochondria-like struc mutant occurs. In a concentration that readily enhances tures are formed that contain an outer membrane, an production of mitochondrial mutants, ethidium bromide is abnormal inner membrane with poorly developed cristae, without effect on the synthesis or degradation of nuclear Krebs cycle enzymes, and an incomplete respiratory chain DNA. This selective action of the dye has now been shown (6). These observations are consistent with amino acid also to be expressed in animal cells. incorporation studies (3 1) that suggest that mitochondrial When mouse L-cells were exposed to a concentration of DNA encodes for the synthesis ofhydrophobic polypeptides ethidium bromide, 1 @tg/ml, the cell content of cytochrome that are essential for complete assembly of functional inner oxidase and cytochrome b declined (29). The mitochondria mitochondrial membranes. enlarged and were noted to have fewer cristae; those that As has been indicated above, the presence in cells of remained appeared abnormally organized. Both normal and altered mitochondrial DNA results in the synthesis of abnormal mitochondria were seen in the same cell, suggest aberrant forms of mitochondrial structures. What then is ing not only a difference among mitochondria in their the case in malignant cells, in many of which mitochondrial sensitivity to the dye but also suggesting that mitochondrial DNA is different from normal? In malignant cells the mutation is an effect on individual organelles rather than concentration of mitochondrial DNA is usually several on the cell as a whole. The latter inference would also be times greater than in normal cells, resembling the concen consistent with the observation that the loss of mitochon tration found in embryonic cells (20). In mouse ascites cells, drial cytochromes is partial and for the fact that the action in which many damaged mitochondria appear, the mito of ethidium bromide on mouse L-cells is reversible. Thus chondrial DNA has an abnormal topography (2 1). For a while damaged mitochondria could not replicate, those recent review of mitochondrial DNA in malignant cells, see that were undamaged would be capable of doing so after re Ref. 24. moval of the dye. This uneven susceptibility of mitochon Great interest has been shown in a unique unicircular dna to a mutagen may be an explanation for the survival DNA dimer peculiar to mitochondria of human leukemic of animal cells that have been exposed to such agents. Un leukocytes. The frequency of such molecules in cases of like the petite mutant that, when its mitochondria are no chronic myelogenous leukemia was found to be proportion longer functional, readily adapts to an anaerobic life, ani ately related to the severity ofthe disease, while in remission mal cells may survive only when loss of mitochondrial the dimer content declined (8). Dimers of this kind are not, viability is incomplete. In the sensethat Warburg intended, however, limited to human tumors (25) but are found also in there would seem to be no facultative anaerobes among the normal human thyroid. animal cells. At presentat least there is no evidencethat the informa Effects of ethidium bromide on 1-leLa cells (26), Chang tion content of the abnormal dimer molecules of mitochon liver cells (16), regenerating liver (10), and the like are drial DNA of human leukemic cells is different from that similar to those to which attention has already been called, of the normal monomer (24). Thus at present the connec i.e., confirmatory of changesin the concentration of mito tion between abnormal mitochondrial DNA of human and chondrial cytochromes following from mutagenic effects of animal tumors and abnormal mitochondria is purely a cir the dye. In all instances, effects of ethidium bromide on cumstantial one. Mitochondrial damage and abnormalities mitochondrial but not nuclear DNA were observed. of mitochondrial DNA are common f'indings in malignant In the biogenesis of mitochondria, 2 separate and distinct cells. Mitochondrial mutagenesis, induced by intercalating genetic systems are involved in the synthesis and assembly dyes such as ethidium bromide, produces forms that re of polypeptide constituents of the membranes and enzyme semble those seen in tumor cells. Changes in mitochondrial systems, namely, that of the nucleus (cytoplasmic system) structure and in the concentration of mitochondrial cyto and that of the mitochondrion itself. Which mitochondrial chromes correlate with informational content of mitochon components are encoded in nuclear DNA and which in drial cytochromes correlate with informational content nitochondrial DNA is presently uncertain. In animal cells, of mitochondrial DNA, and in vitro abnormal changes in

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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1975 American Association for Cancer Research. Mitochondria and Carcinogenesis mitochondrial DNA are accompanied by changes in the DNA During Induction of Petites with Ethidium Bromide. J. Mol. morphology and cytochrome content of mitochondria. Biol.,52:323â€”335,1970. 16. Koch, J. The Cytoplasmic DNAs ofCultured Human Cells. Effects of In the light of the role of mitochondrial genes in directing Ethidium Bromide on Their Replication and Maintenance. European the synthesis of key components of the mitochondrion, J. Biochem., 30: 53-59, 1972. numerous observations of tumor-associated disturbances of 17. Mackler, B., Douglas, H. C., Will, S., Hawthorne, D. C., and Mahler, mitochondrial function cannot be ignored. To quote an H. R. BiochemicalCorrelatesofRespiratoryDeficiency.IV.Compo outstanding authority in the field of cytoplasmic genetics: sition and Properties of Respiratory Particles from Mutant Yeasts. â€œInthe past only nuclear genes were taken into considera Biochemistry,4: 2016-2020, 1965. tion in planning and evaluation of cancer research studies. 18. Mahler, H. R., Mackler, B., Grandchamp, S., and Slonimski, P. P. With the development of our knowledge about cytoplasmic BiochemicalCorrelatesof Respiratory Deficiency. I. The Isolation of genetics, it would be useful to reconsider past studies and to a Respiratory Particle. Biochemistry. 3: 668-677, 1964. design new investigations aimed specifically at examining 19. Nass, M. M. K. The Circularity of Mitochondrial DNA. Proc. NatI. Acad.Sci.U.S.,56:1215-1222,1966. the possible role of cytoplasmic genes in neoplastic transfor 20. Nass, M. M. K. Structure, Synthesis, and Transcription of Mitochon mationâ€• (27). drial DNA in Normal, Malignant. and Drug-treated Cells. in: K. W. McKerns (ed.), Hormones and Cancer, pp. 261-307. New York: Aca References demic Press, Inc., 1974. I. Aisenberg, A. C. The Glycolysis and Respiration of Tumors. New 21. Nass, S., and Nass, M. M. K. Intramitochondrial Fibers with York: Academic Press,Inc., 1961. Deoxyribonucleic Acid Characteristics: Observations of Ehrlich As 2. Attardi, G., Constantino, P., and Ojala, D. Molecular Approaches to citesTumor Cells. J. NatI. Cancer Inst., 33: 777-798, 1964. the Dissectionof the Mitochondrial Genomein HeLa Cells. In A. M. 22. Olsen, R. E., and Kaplan, N. 0. The Effect of Pantothenic Acid Kroon and C. Saccone (eds.), The Biogenesis of Mitochondria, pp. Deficiency Upon the Coenzyme A Content and Pyruvate Utilization of 9-29. New York: Academic Press, Inc., 1974. Rat and Duck Tissues. J. Biol. Chem., 175: 515-529, 1948. 3. Bernhard, W. Electron Microscopy of Tumor Cells and Tumor 23. Paoletti, C. A., and Riou, G. Le DNA Mitochondrial des Cellules Viruses, a Review. Cancer Res., 28: 491-509, 1968. Malignes. Bull. Cancer,57: 301-308, 1970. 4. Bernhard, W., and Tournier, P. Modification Persistante des Mito 24. Paoletti, C. A., and Riou, G. The Mitochondrial DNA of Malignant chondries dans des Cellules Tumorales de Hamster TransformÃ©epar Cells. in F. E. Hahn (ed.), Progress in Molecular and Subcellular L'Adenovirus 12. Intern. J. Cancer, 1: 61â€”80,1966. Biology, pp. 203â€”248.Berlin:Springer Verlag, 1973. 5. Beutler, E., and Blaisdell, R. K. Iron Enzymes in Iron Deficiency. J. 25. Paoletti, C. A., Riou, G., and Pairault, J. Circular Oligomers in Lab. Clin. Med., 52: 694-699, 1958. Mitochondrial DNA of Human and Beef Nonmalignant Thyroid 6. Borst, P. Mitochondrial Nucleic Acids. Ann. Rev. Biochem., 41: Glands. Proc. NatI. Acad. Sci. U.S., 69: 847-850, 1972. 333â€”376,1972. 26. Radsack, K., Kato, K., Sato, N., and Kaprowski, H. Effect of 7. Burch, H. B., Hunter, F. E., Combs, A. M., and Schutz, B. A. Ethidium Bromide on Mitochondrial and Cytochrome Synthesisin Oxidative Enzymes and Phosphorylation in Hepatic Mitochondria HeLa Cells. Exptl. Cell Res., 66: 410-416, 1971. from Riboflavin-deficient Rats. J. Biol. Chem., 235: 1540-1544, 27. Sager, R. Cytoplasmic Genes and OrgandIes. New York: Academic 1960. Press, Inc., 1972. 8. Clayton, D. A., and Vinograd, J. Complex Mitochondrial DNA in 28. Sinclair, J. H., and Stevens, D. J. Circular DNA Filaments from Leukemic and Normal Human Myeloid Cells. Proc. NatI. Acad. Sci. Mouse Mitochondria. Proc. NatI. Acad. Sci. U. S., 56: 508-514, 1966. U. S., 62: 1077-1084, 1969. 29. Soslau, G., and Nass, M. M. K. Effects of Ethidium Bromide on the 9. Constantino, P., and Attardi, G. Atypical Pattern of Utilization of Cytochrome Content and Ultrastructure of L Cell Mitochondria. J. Amino Acids for Mitochondrial Protein Synthesis. Proc. NatI. Acad. Cell.Biol.,5!:514-524,1971. Sci. U. S., 70: 1490-1494, 1973. 30. Subik, J., Kolarov, J., and Kovac, L. Obligatory Requirement of 10. DeVries, H., and Kroon, A. M. Euflavin and Ethidium Bromide: Intramitochondrial ATP for Normal Functioning of the Eukaryotic Inhibitors of Mitochondriogenesis in Regenerating Rat Liver. Federa Cell. Biochem. Biophys.Res.Commun., 49: 192-198, 1972. tion European Biochem. Soc. Letters, 7: 347-350, 1970. 31. Tzagaloff, A., and Akai, A. Assembly of the Mitochondrial Mem II. DuBuy, H. G., andWoods,M. W. A PossibleCommonMitochondrial brane System. VIII. Properties of the Products of Mitochondrial Origin of the Variegational and Virus Diseases in Plants and Cancers Protein Synthesis in Yeast. J. Biol. Chem., 247: 6517-6523, 1972. in Animals. American Association for the Advancement of Science 32. Van Bruggen, E. F. J., Borst, P., Ruttenberg, G. J. C. M., Gruber, M., Conferenceon Cancer, pp. 162â€”169.Lancaster,Pa.: SciencePress, and Kroon, A. M. Circular Mitochondrial DNA. Biochim. Biophys. 1945. Acta, 119:437-439, 1966. 12. Ephrussi, B. The Interplay of Heredity and Environment in the 33. Warburg, 0., Posener, K., and Negelein, E. Uber den Stoffwechsel der Synthesis of Respiratory Enzymes in Yeast. Harvey Lectures, 46: 45â€” Carcinomzelle. Biochem. Z., 152: 309-344, 1924. 76, 1950. 34. Warburg, 0. On the Origin of Cancer Cells, Science, 123: 309-314, 13. Ephrussi, B., Hottinguer, H., and Chimines, A. M. Action de L'Acri 1956. flavine sur Levures.I. La Mutation â€œpetitecolonie.â€•Ann.Inst. Pas 35. Warburg, 0. On Respiratory Impairment in Cancer Cells. Science, teur,76:351â€”367,1949. 124:267-270,1956. 14. Gallagher, C. H., Judah, J. D., and Rees,K. R. The Biochemistryof 36. Wenner,C. E. Progressin Tumor Enzymology.Advan. Enzymol.,29: Copper Deficiency. Proc. Roy. Soc. London 5cr. B, 145: 134-150, 321-390,1967. 1956. 37. Woods, M. W., and DuBuy, H. G. Hereditary and Pathogenic Nature 15. Goldring, E. S., Grossman, L. I., Krupnick, D., Cryer, D., and of Mutant Mitochondria in Nepeta. J. Nail. Cancer Inst., 11: 1105- Marmur, J, The Petite Mutation in Yeast: Loss of Mitochondrial 1152,1951.

NOVEMBER 1975 3335

Henry D. Hoberman

Cancer Res 1975;35:3332-3335.

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