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
Secreted frizzled-related protein 1 wikipedia , lookup
Point mutation wikipedia , lookup
Deoxyribozyme wikipedia , lookup
Endogenous retrovirus wikipedia , lookup
Free-radical theory of aging wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Transformation (genetics) wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
(CANCER RESEARCH 35, 3332-3335, November 1975] Is There a Role for Mitochondrial Genes in Carcinogenesis?' Henry D. Hoberman Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 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 “the most striking 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,―at the 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 biochemical properties of the cancer cell, particularly in the Otto Warburg (33-35). Although much of what he held to 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, hard was struck by the extraordinary variation in number, May 19 to 22, 1975, Key Biscayne, Fla. Aided by USPHS Grant CA size, form, density and frequency of lesions that they pre 03651from the National CancerInstitute. Summary 3332 CANCER RESEARCH VOL. 35 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1975 American Association for Cancer Research. Mitochondria sent. He pointed to the necessity, therefore, of critical mor phological control of mitochondrial pellets prepared for biochemical studies. These observations as well as similar observations recorded by others (4, 23, 24) strongly suggest that procedures used to isolate mitochondria from normal tissues, when applied to tumor tissue, may eliminate those of greatest biochemical interest. In any case, when consid eration is given to the observed pleomorphism of tumor cell mitochondria and to the lesionsthat have beennoted, there would seem to be little reason to expect uniformity of en ergy metabolism among all types of neoplasms. As has already been indicated, 1 of Warburg's beliefs, namely, that mitochondria are autonomous organelles, de serves greater attention than it has received to date. War burg derived support for his view that mitochondria are heritable structures from plant genetics, a view that early on recognized the existence of cytoplasmic genes. Among the workers whom Warburg cited for special mention were M. W. Woods and H. G. DuBuy of the National Cancer Insti tute, who found, in leaves of a variegated species of Nepeta cataria (catnip), mixed cells containing multiple mitochon drial types (37). Most significantly, the mitochondrial phenotypes were transmitted to progeny by non-Mendelian inheritance. Borrowing heavily from Woods and DuBuy, who were themselves convinced of the pertinence of their studies to cancer ( I I ), Warburg proposed that, once a mitochondrion was damaged, it remained so, transmitting its defect to progeny (presumably mitochondria), just as would occur, he asserted, in the case of a damaged nuclear gene. Although there would seem to be reason to allow the speculation that cancer cells, through defects in mitochon drial structure, may have nonfunctioning respiratory chains, the question whether such defects are transmissible is as yet highly conjectural. My assignment as a participant in this meeting is specifically to consider whether, due to aberrations caused by a change of nutritional conditions, mitochondria can play a role in carcinogenesis. Without at this time speculat ing on that question, there can be no doubt that mitochon drial function per se is sensitive to the withdrawal from the diet of certain vitamins and trace elements. An extreme ex ample is the effect of a lack of dietary copper. In this condi tion the loss of activity of cytochrome oxidase is so severe that liver mitochondria of animals killed at the height of the deficiency are unable to respire (14). Although iron depri vation, because of the obligatory role of heme and non-heme forms of iron in electron transport, would also be expected to embarrass electron flow, dietary restriction of iron by curtailing the synthesis of hemoglobin rather than of iron-containing enzymes of the respiratory chain so limits the life-span of experimental animals that death occurs before loss of respiratory function is noted (5). Turning to the vitamins, the nature of experimentally induced abnormalities of mitochondrial metabolism is pre dictable from the known function of the coenzymes of which the vitamin is a precursor. Thus lack of riboflavin, a precur sor of flavin mono- and dinucleotides, has been shown to bring about a slowing of oxidation of NADH and succinate by mitochondria of deficient animals (7). A lack of dietary and Carcinogenesis pantothenate causes a deficiency of CoA so that, under standably, carbohydrate and fatty acid oxidation, as well as turnover of the citrate cycle, are severely disrupted (22). Whether these or other impairments of mitochondrial function have a potential for carcinogenesis brings us back to the main question. Taking into account what has already been said about specific biochemical and ultrastructural features of tumor cell mitochondria, it would seem less probable that aberrant mitochondria are capable of stimu lating cell growth and division than that healthy mitochon dna normally exert a controlling influence on cell growth and division. Be that as it may, any theory of carcinogenesis that includes a role for mitochondria must take into consideration the possibility that, for cancer to develop as a result of mitochondrial impairment, the defect must be mutagenic for the mitochondrion. A changeof nutritional environment of a cell may reveal the existence of mutant forms of mitochondria. This is exemplified by the appearance of a population of mutant organisms, containing abnormal mitochondria, when cells of Saccharomyces cerevisiae, a facultative anaerobic strain of yeast, are changed from growth on a medium containing glycerol or ethanol as a carbon source to a medium containing glucose or other fermentable sugar. While growth of the organism on alcohol or glycerol is oxygen dependent, growth on glucose-containing media takes place at the expense of fermentative energy. When cells of S. cerevisiae are plated on a soft nutrient medium containing glucose, colonies are formed among which are some smaller than the majority. The smaller colonies (petites), on under going mitotic division, yield progeny having the same appearance as the parents, as well as other heritable properties, among which is a defect of respiratory function. The loss of respiratory function has been found to be due to mitochondria that lack the capacity to synthesize or assem ble polypeptides that are precursors of cytochrome oxidase, cytochrome b, cytochrome c1 , and the rutamycin-sensitive ATPase of oxidative phosphorylation. Without these con stituents of the respiratory chain, petite mutants do not respire or carry out oxidative phosphorylation (I 2, 17, 18). As detected simply by a change of one carbon source for another, the petite mutation is spontaneous yet occurs with a frequency that can be orders of magnitude greater than rates ofspontaneous nuclear mutations. Under certain other conditions the rate of mutation can reach 100%. Thus Ephrussi et a!. (13) found that acriflavin, an acridine dye, could transform an entire population of cells to the petite form. Of special significance was the observation that the yield of mutant colonies was the same whether the parent cells were of a haploid or diploid strain. This independence of the effect of the dye from gene dosage provided strong evidence from which it was concluded that the mutation was not that of a nuclear but of a cytoplasmic gene. In S. cerevisiae, cytoplasmic genes are contained in the mitochon drion, the genetic message being encoded in the mitochon drial DNA. The frequency of mitochondrial mutation can be in creased even when cells are metabolizing under aerobic con ditions. Mass formation of petite mutants of S. cerevisiae NOVEMBER 1975 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1975 American Association for Cancer Research. 3333 H. D. Hoberman was accomplished by preventing mitochondr'al synthesis of ATP by inhibiting respiration with cyanide or antimycin A, while simultaneously blocking uptake of glycolytically formed ATP with an appropriate inhibitor (bongkrekik acid). These results were interpreted to indicate that the constant presence of ATP within mitochondria is essential to normal replication of mitochondrial DNA (30). An alternative interpretation is that interference with energy utilization in mitochondria may be mutagenic for mitochon drial DNA. In the presence of ethidium bromide, a phenanthrid@ne dye that intercalates between complementary bases in du plex DNA, mitochondrial DNA synthesis is selectively in hibited and preexisting mitochondrial DNA is progressively degraded (15). The petite mutation is thereby enhanced to the extent that mass formation of the respiration-deficient mutant occurs. In a concentration that readily enhances production of mitochondrial mutants, ethidium bromide is without effect on the synthesis or degradation of nuclear DNA. This selective action of the dye has now been shown also to be expressed in animal cells. When mouse L-cells were exposed to a concentration of ethidium bromide, 1 @tg/ml, the cell content of cytochrome oxidase and cytochrome b declined (29). The mitochondria enlarged and were noted to have fewer cristae; those that remained appeared abnormally organized. Both normal and abnormal mitochondria were seen in the same cell, suggest ing not only a difference among mitochondria in their sensitivity to the dye but also suggesting that mitochondrial mutation is an effect on individual organelles rather than on the cell as a whole. The latter inference would also be consistent with the observation that the loss of mitochon drial cytochromes is partial and for the fact that the action of ethidium bromide on mouse L-cells is reversible. Thus while damaged mitochondria could not replicate, those that were undamaged would be capable of doing so after re moval of the dye. This uneven susceptibility of mitochon dna to a mutagen may be an explanation for the survival of animal cells that have been exposed to such agents. Un like the petite mutant that, when its mitochondria are no longer functional, readily adapts to an anaerobic life, ani mal cells may survive only when loss of mitochondrial viability is incomplete. In the sense that Warburg intended, there would seem to be no facultative anaerobes among animal cells. Effects of ethidium bromide on 1-leLa cells (26), Chang liver cells (16), regenerating liver (10), and the like are similar to those to which attention has already been called, i.e., confirmatory of changesin the concentration of mito chondrial cytochromes following from mutagenic effects of the dye. In all instances, effects of ethidium bromide on mitochondrial but not nuclear DNA were observed. In the biogenesis of mitochondria, 2 separate and distinct genetic systems are involved in the synthesis and assembly of polypeptide constituents of the membranes and enzyme systems, namely, that of the nucleus (cytoplasmic system) and that of the mitochondrion itself. Which mitochondrial components are encoded in nuclear DNA and which in nitochondrial DNA is presently uncertain. In animal cells, 3334 mitochondrial DNA consist of circular, double-stranded light and heavy chains of supercoiled molecules having a molecular weight of 9 to 10 million daltons (19, 28, 32). It has recently been estimated that, in the HeLa cell, genes so far identified on mitochondrial DNA account for about 25% of the potential information contained in the 5-nm-long DNA molecule (2). Of singular interest is the fact that, on animal cell and yeast mitochondrial DNA, genes have been identified that encode for tRNA's that almost without exception are specific for the hydrophobic amino acids (9). It is, therefore, no wonder that all proteins synthesized on mitochondrial ribosomes have so far been found to belong to the class of hydrophobic proteins associated with the inner mitochondrial membrane (6). In petite mutants that have grossly altered or no mito chondrial DNA, it is found that mitochondria-like struc tures are formed that contain an outer membrane, an abnormal inner membrane with poorly developed cristae, Krebs cycle enzymes, and an incomplete respiratory chain (6). These observations are consistent with amino acid incorporation studies (3 1) that suggest that mitochondrial DNA encodes for the synthesis ofhydrophobic polypeptides that are essential for complete assembly of functional inner mitochondrial membranes. As has been indicated above, the presence in cells of altered mitochondrial DNA results in the synthesis of aberrant forms of mitochondrial structures. What then is the case in malignant cells, in many of which mitochondrial DNA is different from normal? In malignant cells the concentration of mitochondrial DNA is usually several times greater than in normal cells, resembling the concen tration found in embryonic cells (20). In mouse ascites cells, in which many damaged mitochondria appear, the mito chondrial DNA has an abnormal topography (2 1). For a recent review of mitochondrial DNA in malignant cells, see Ref. 24. Great interest has been shown in a unique unicircular DNA dimer peculiar to mitochondria of human leukemic leukocytes. The frequency of such molecules in cases of chronic myelogenous leukemia was found to be proportion ately related to the severity ofthe disease, while in remission the dimer content declined (8). Dimers of this kind are not, however, limited to human tumors (25) but are found also in the normal human thyroid. At present at least there is no evidencethat the informa tion content of the abnormal dimer molecules of mitochon drial DNA of human leukemic cells is different from that of the normal monomer (24). Thus at present the connec tion between abnormal mitochondrial DNA of human and animal tumors and abnormal mitochondria is purely a cir cumstantial one. Mitochondrial damage and abnormalities of mitochondrial DNA are common f'indings in malignant cells. Mitochondrial mutagenesis, induced by intercalating dyes such as ethidium bromide, produces forms that re semble those seen in tumor cells. Changes in mitochondrial structure and in the concentration of mitochondrial cyto chromes correlate with informational content of mitochon drial cytochromes correlate with informational content of mitochondrial DNA, and in vitro abnormal changes in CANCER RESEARCH VOL. 35 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1975 American Association for Cancer Research. Mitochondria mitochondrial DNA are accompanied by changes in the morphology and cytochrome content of mitochondria. In the light of the role of mitochondrial genes in directing the synthesis of key components of the mitochondrion, numerous observations of tumor-associated disturbances of mitochondrial function cannot be ignored. To quote an outstanding authority in the field of cytoplasmic genetics: “Inthe past only nuclear genes were taken into considera tion in planning and evaluation of cancer research studies. With the development of our knowledge about cytoplasmic genetics, it would be useful to reconsider past studies and to design new investigations aimed specifically at examining the possible role of cytoplasmic genes in neoplastic transfor mation― (27). References I. Aisenberg, A. C. The Glycolysis and Respiration of Tumors. New York: Academic Press,Inc., 1961. 2. Attardi, G., Constantino, P., and Ojala, D. Molecular Approaches to the Dissectionof the Mitochondrial Genomein HeLa Cells. In A. M. Kroon and C. Saccone (eds.), The Biogenesis of Mitochondria, pp. 9-29. New York: Academic Press, Inc., 1974. 3. Bernhard, W. Electron Microscopy of Tumor Cells and Tumor Viruses, a Review. Cancer Res., 28: 491-509, 1968. 4. Bernhard, W., and Tournier, P. Modification Persistante des Mito chondries dans des Cellules Tumorales de Hamster Transformée par L'Adenovirus 12. Intern. J. Cancer, 1: 61—80,1966. 5. Beutler, E., and Blaisdell, R. K. Iron Enzymes in Iron Deficiency. J. Lab. Clin. Med., 52: 694-699, 1958. 6. Borst, P. Mitochondrial Nucleic Acids. Ann. Rev. Biochem., 41: DNA During Induction of Petites with Ethidium Bromide. J. Mol. Biol.,52: 323—335, 1970. 16. Koch, J. The Cytoplasmic DNAs ofCultured Human Cells. Effects of Ethidium Bromide on Their Replication and Maintenance. European J. Biochem., 30: 53-59, 1972. 17. Mackler, B., Douglas, H. C., Will, S., Hawthorne, D. C., and Mahler, H. R. BiochemicalCorrelatesof RespiratoryDeficiency.IV. Compo sition and Properties of Respiratory Particles from Mutant Yeasts. Biochemistry,4: 2016-2020, 1965. 18. Mahler, H. R., Mackler, B., Grandchamp, S., and Slonimski, P. P. BiochemicalCorrelatesof Respiratory Deficiency. I. The Isolation of a Respiratory Particle. Biochemistry. 3: 668-677, 1964. 19. Nass, M. M. K. The Circularity of Mitochondrial DNA. Proc. NatI. Acad.Sci.U.S.,56:1215-1222, 1966. 20. Nass, M. M. K. Structure, Synthesis, and Transcription of Mitochon drial DNA in Normal, Malignant. and Drug-treated Cells. in: K. W. McKerns (ed.), Hormones and Cancer, pp. 261-307. New York: Aca demic Press, Inc., 1974. 21. Nass, S., and Nass, M. M. K. Intramitochondrial Fibers with Deoxyribonucleic Acid Characteristics: Observations of Ehrlich As cites Tumor Cells. J. NatI. Cancer Inst., 33: 777-798, 1964. 22. Olsen, R. E., and Kaplan, N. 0. The Effect of Pantothenic Acid Deficiency Upon the Coenzyme A Content and Pyruvate Utilization of Rat and Duck Tissues. J. Biol. Chem., 175: 515-529, 1948. 23. Paoletti, C. A., and Riou, G. Le DNA Mitochondrial des Cellules Malignes. Bull. Cancer,57: 301-308, 1970. 24. Paoletti, C. A., and Riou, G. The Mitochondrial DNA of Malignant Cells. in F. E. Hahn (ed.), Progress in Molecular and Subcellular Biology, pp. 203—248. Berlin: Springer Verlag, 1973. 25. Paoletti, C. A., Riou, G., and Pairault, J. Circular Oligomers in Mitochondrial DNA of Human and Beef Nonmalignant Thyroid Glands. Proc. NatI. Acad. Sci. U.S., 69: 847-850, 1972. 26. Radsack, K., Kato, 333—376, 1972. and Carcinogenesis K., Sato, N., and Kaprowski, H. Effect of F. E., Combs, A. M., and Schutz, B. A. Ethidium Bromide on Mitochondrial and Cytochrome Synthesis in Oxidative Enzymes and Phosphorylation in Hepatic Mitochondria HeLa Cells. Exptl. Cell Res., 66: 410-416, 1971. 27. Sager, R. Cytoplasmic Genes and OrgandIes. New York: Academic Press, Inc., 1972. 28. Sinclair, J. H., and Stevens, D. J. Circular DNA Filaments from Mouse Mitochondria. Proc. NatI. Acad. Sci. U. S., 56: 508-514, 1966. 29. Soslau, G., and Nass, M. M. K. Effects of Ethidium Bromide on the Cytochrome Content and Ultrastructure of L Cell Mitochondria. J. 7. Burch, H. B., Hunter, from Riboflavin-deficient Rats. J. Biol. Chem., 235: 1540-1544, 1960. 8. Clayton, D. A., and Vinograd, J. Complex Mitochondrial DNA in Leukemic and Normal Human Myeloid Cells. Proc. NatI. Acad. Sci. U. S., 62: 1077-1084, 1969. 9. Constantino, P., and Attardi, G. Atypical Pattern of Utilization of Amino Acids for Mitochondrial Protein Synthesis. Proc. NatI. Acad. Sci. U. S., 70: 1490-1494, 1973. 10. DeVries, H., and Kroon, A. M. Euflavin and Ethidium Bromide: Inhibitors of Mitochondriogenesis in Regenerating Rat Liver. Federa tion European Biochem. Soc. Letters, 7: 347-350, 1970. II. DuBuy, H. G., and Woods,M. W. A PossibleCommon Mitochondrial Origin of the Variegational and Virus Diseases in Plants and Cancers in Animals. American Association for the Advancement of Science Conferenceon Cancer, pp. 162—169. Lancaster, Pa.: SciencePress, 1945. 12. Ephrussi, B. The Interplay of Heredity and Environment in the Synthesis of Respiratory Enzymes in Yeast. Harvey Lectures, 46: 45— 76, 1950. 13. Ephrussi, B., Hottinguer, H., and Chimines, A. M. Action de L'Acri flavine sur Levures. I. La Mutation “petite colonie.― Ann. Inst. Pas teur,76: 351—367, 1949. Cell. Biol., 5!:514-524, 1971. 30. Subik, J., Kolarov, J., and Kovac, L. Obligatory Requirement of Intramitochondrial ATP for Normal Functioning of the Eukaryotic Cell. Biochem. Biophys. Res.Commun., 49: 192-198, 1972. 31. Tzagaloff, A., and Akai, A. Assembly of the Mitochondrial Mem brane System. VIII. Properties of the Products of Mitochondrial Protein Synthesis in Yeast. J. Biol. Chem., 247: 6517-6523, 1972. 32. Van Bruggen, E. F. J., Borst, P., Ruttenberg, G. J. C. M., Gruber, M., and Kroon, A. M. Circular Mitochondrial DNA. Biochim. Biophys. Acta, 119:437-439, 1966. 33. Warburg, 0., Posener, K., and Negelein, E. Uber den Stoffwechsel der Carcinomzelle. Biochem. Z., 152: 309-344, 1924. 34. Warburg, 0. On the Origin of Cancer Cells, Science, 123: 309-314, 1956. 35. Warburg, 0. On Respiratory Impairment in Cancer Cells. Science, 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, 1956. 15. Goldring, E. S., Grossman, L. I., Krupnick, D., Cryer, D., and Marmur, J, The Petite Mutation in Yeast: Loss of Mitochondrial 321-390,1967. 37. Woods, M. W., and DuBuy, H. G. Hereditary and Pathogenic Nature of Mutant Mitochondria in Nepeta. J. Nail. Cancer Inst., 11: 11051152,1951. NOVEMBER 1975 Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1975 American Association for Cancer Research. 3335 Is There a Role for Mitochondrial Genes in Carcinogenesis? Henry D. Hoberman Cancer Res 1975;35:3332-3335. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/35/11_Part_2/3332 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1975 American Association for Cancer Research.