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CANCER RESEARCH VOLUME18 JULY 1958 NUMBER6 Cellular Metabolism and Cancer*: A Review SAULKIT ANDA. CLARKGRIFFIN (Departments of Biochemistry, University of Texas M, D. Anderson Hospital and Tumor Institute, and Baylor University Collegeof Medicine, Houston 25, Texas) I. INTRODUCTION CONTENTS I. Introduction II Chromosomes, Deoxyribonucleic Acid, and Heredity 1. Chromosome number and morphology 2. DNA content 3. Heterogeneity of DNA III. 4. DNA function and time of DNA synthesis 5. Chromosome organization 6. Quantitative relationship between cell ploidy and metabolism 7. DNA and cell histones 8. Interaction of nucleus and cytoplasm 9. Relationship between cytoplasmic RNA and protein and enzyme synthesis 10. Extranuclear inheritance 11. Gene content and phenotype Subcellular Metabolic Patterns of Neoplastic Cell Popu lations 1. Distribution of protein and RNA 2. Mitochondria of neoplastic cells 3. Proteins of the supernatant fraction 4. Endogenous metabolites 5. Metabolic dedifferentiation IV. Energy-yielding Mechanisms A. Anaerobic and aerobic glycolysis 1. Glycolytic enzymes 2. Electron transport chain deficiency 3. The citric acid cycle 4. Metabolic restraint as a function of phosphate acceptors B. Carbohydrate metabolism and amino acid biosynthesis 1. Amino acids 2. Pentose formation 3. Thymidine biosynthesis C. Glucose utilization and reductive synthesis D. Sulfhydryl compounds and cell division V. Conclusions VI. References * Based in part on a paper presented at the Symposium on Cancer, Biannual meeting of the American Chemical Society, New York, September 12, 1957. Part of the work described in this paper was carried out during the tenure of grants by the American Cancer Society, the National Cancer Institute, and the Leukemia Society, Inc. Tables and charts reprinted with permission from authors and publishers as indicated. In 1912, Boveri proposed that neoplastic cells are characterized by a definite abnormal chromatin complex (28). This hypothesis was based on the following concepts: (a) Different qualities belong to different chromosomes; (£>) A malignant cell ¡sa cell with an irreparable defect located in the nucleus; (c) This defect goes hand in hand with a changed metabolism of the tumor cell. The tumor cell which lacks certain chromosomes, while it has too large a number of other chromosomes, will produce many substances in too great, others in too small, quantities, or none at all. It is probable that the material of single chromosomes which are otherwise normal act on each other under altered quantitative relations so that the products which result are very different from the normal end-products. Boveri suggested that atypical mitosis was the mechanism by which an unequal distribution of the chromosomes was produced; the essence of the theory was not ab normal mitosis, however, but abnormal chromo some complex. By whatever mechanism this might arise, definite tumor formation may be the endresult. The concept of somatic mutation as a cause of cancer has been an appealing one to many investigators (271). Strong (302, 303) and also Foulds (86) showed that two spontaneous mam mary tumors which had arisen in the same indi vidual gave a different reaction when transplanted to either axilla of related mice. In some mice, both tumors grew progressively; in other mice, neither tumor grew; and in some, there was tem porary growth of both tumors. The conclusion concerning the two tumors was that, despite the fact that they were histologically indistinguishable, they were physiologically different and perhaps 621 This One 7WZC-16F-WAFE Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. 622 Cancer Research genetically different. This idea was reinforced by the knowledge that sudden changes take place during the transplantation of spontaneous adenocarcinomas (86), and the idea that genetic differ ences underlie histocompatibility. Nordling (231, 232) called attention to: (a) the correlation be tween mutagens and carcinogens; (6) the correla tion between cell proliferation and cancer inci dence; (c) the mathematical relation existing be tween cancer frequency and age in man; and (d) the stepwise increase of the malignancy of tumors. Although Boveri was specific in attributing cancer to gene mutations as well as to chromosome alterations, other investigators modified the con cept. To quote Burdette (34) : "With the advent of plasmagenes, a number of investigators incor porated changes in these particles into the hy pothesis. Darlington has emphasized the cyto plasm as the source for the change leading to cancer and Haddow in a review on transformation of cells and viruses has commented on the pos sibility that the mechanism which causes contin ued growth may reside at least in part in the cytoplasm. Darlington believes that the cancer determinants which arise in the cytoplasm are due to mutations in either hereditary plasmagenes, infectious viruses, or pro viruses." In the present paper, the Boveri hypothesis will be used as a point of departure for integrating the biochemical data concerning cancer. We will begin with a description of the genetic determi nants of the cell which are embodied within the chromosomes or, more specifically, within the quantitative and qualitative makeup of the deoxyribonucleoproteins (DNP). Recent biochemical experiments on the replication and function of DNP and nuclear-cytoplasmic interrelations will then be considered. This will be followed by a brief discussion of cytoplasmic inheritance and the relationship of the Boveri hypothesis to cur rent concepts of embryonic differentiation. In the second part of this paper, a general survey will be presented of metabolic activities of malig nant tissues. We will first review chemical and metabolic studies of subcellular particles of cells. Secondly, the respiratory-glycolytic imbalance will be discussed. The high glycolytic response of tu mors will be emphasized in relation to the follow ing: (a) the energy requirements of the cancer cell; (b) essential biosynthetic pathways; and (c) reductive synthesis. Finally, there will be a brief summary of the relation of sulfhydryl groups to cell division. Vol. 18, July, 1958 II. CHROMOSOMES, DEOXYRIBONUCLEIC ACID (DNA) AND HEREDITY 1. Chromosome number and morphology.—In the introduction, we briefly discussed the concept that cancer respresents an "irreversible" alteration in the hereditary determinants of the cell. Since the hereditary determinants are contained in the structure of the chromosomes, it is appropriate to direct attention to chromosome number and variability in normal and malignant tissues. It is probable that the preponderant majority of normal somatic cells contains the diploid chromo some number (2re) characteristic of the species (118). Approximately 1-2 per cent of the cells, however, are polyploid cells. Moreover, not all the cells containing approximately the diploid number of chromosomes have exactly 2re chromo somes (18, 144, 147, 213). The chromosome content of embryonic tissues of mice has been studied by Hsu and Pomerat (144). These investigators found that, exclusive of polyploid or haploid cells, only 87 per cent of the liver cells contained exactly 40 chromo somes, while 91 per cent of heart or lung cells contained this number of chromosomes (147). The remaining aneuploid cells contained from 39 to 46 chromosomes. Although the quantitative sig nificance of chromosome aneuploidy and heteroploidy is still under discussion (140, 147), there would seem to be little doubt of its reality (18, 140). Somatic inconstancy has been observed in at least eleven major organ or tissue systems in man, and it occurs in various other species. In the Chinese hamster, Cricetulus grÃ-seas (2« = 22), there are eleven pairs of chromosomes. Of these, nine are readily recognizable, and the remaining six fall into two distinguishable groups of three. Within the latter groups, the individual chromosomes can be identified with less certainty (Chart 1). Yerganian and associates (210, 309, 340) have shown that, although 73 per cent of the mitotic cells of regenerating liver contained 22 chromosomes, only 80 per cent of these were true diploids (309). In the remaining "quasidiploid cells," some chromosomes were represented three or more times, others only once or not at all. Quasidiploid cells may exist in any tissue of any species, yet may escape the attention of cytologists in those species in which morphological chromo somal distinctions cannot be made. The presence of quasidiploid cells tends to increase the total proportion of aneuploid to diploid cells (140). Although some mammalian tumors contain the diploid chromosome number, the most frequent Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. KIT ANDGRIFFIN—CellularMetabolism and Cancer 623 number in all neoplasms of mammals examined sues and (6) by spectrophotometric measurement appears to be somewhat hyperdiploid or hypo- of individual cells (248, 307, 311). The results tetraploid (118, 119, 205). The Novikoff liver obtained by each of these methods are generally tumor of the rat contains approximately 60 per in agreement. Interpretation of such DNA assays cent diploid class cells and about 40 per cent is, however, subject to the followingIcomplications: tetraploid class cells (140). Heteroploidy and aneu- (a) experimental error; (6) differences in the aver ploidy are particularly common in tumors (143, age DNA content of millions of cells or even 205). In some cases, both diploid and tetraploid of individual cells owing to aneuploidy, polyploidy, or polyteny (6) ; (c) changes in the DNA content cell lines have been obtained from the same tumor (121, 159). During prolonged subcultivation in of a cell owing to the replication of chromosomal tissue culture, tumors and normal tissues undergo material in preparation for a coming mitosis (233, 255); and (e) the possibility that small variations numerical and structural chromosomal alterations (145, 205). of the DNA content of cells occur in relation Many quasidiploid and aneuploid cells are found to physiological function (311). in the methylcholanthrene tumor of the Chinese CHROMOSOMES OF THE CHINESE HAMSTER hamster (340). In some cells, there is an accumula tion of as many as six representations of a given chromosome type (Table 1). In addition to changes (colchicine pretreatment) in chromosome number, cancer cells often manifest bizarre changes in chromosome morphology, includ ing the appearance of unusually large J- or V-shaped chromosomes, or new "minute" chromosomes bor dering on centromere size (118, 121, 139, 140, 142, 274). Mitotic abnormalities such as multipolar mi toses, asymmetrical division, and lagging chromo somes are also encountered. There no longer seems to be any doubt that the progression of tumors toward increased malignancy is connected with and caused by gradual genotypic changes. Is it permissible by homology to extrapolate from this development backward and conclude that the precancerous changes that start off the malignancy are of the same nature? Is it permissible to assume that the original step involves genotypic change CHART1.—Morphology of chromosomes of Chinese Ham (205)? ster (2n = 22) (Tonomura and Yerganian [309]). 2. DNA content.—There is considerable evi dence to suggest that the hereditary determinants Although the DNA content of most cells of of the chromosomes consist chemically of deoxy- a given organism is approximately twice that ribonucleoproteins. This has been amply presented found in the sperm cells (249) (Class I cells), or reviewed by many different investigators: (37, many tissues also contain cells whose contents 125, 191, 192, 198, 226, 248, 263, 298, 299, 311). of DNA is 4 times (Class II) or 8 times (Class Nuclear deoxyribonucleic acid (DNA) does not III) that of the spermatids. This is consistent represent the only substance capable of carrying with the concept that normal tissues contain a genetic information. Plant viruses and certain small proportion of polyploid cells, as evidenced animal viruses do not contain detectable DNA directly by chromosome counts. The DNA content but instead contain ribonucleic acid (RNA) (87). parallels the chromosome content during spermaHowever, the absence of DNA in biological sys togenesis and oogénesis (307, 311). tems is relatively infrequent, and, when DNA The average DNA content of spontaneous rat is present, it is believed to be responsible for liver tumors (63, 214) and many leukemic cells genetic function. The role of RNA as a template corresponds to that of normal rat tissues (204, for protein synthesis and the problem of cyto- 207, 220, 221, 227, 244, 264). Certain other lymplasmic inheritance will be considered later. phomas (184, 246, 288), one of the Ehrlich ascites The DNA content of cells has been determined tumors (88), and various carcinomas, however, in two ways: (a) by chemical measurement of contain approximately twice the DNA content of normal somatic cells. There are found in*the the DNA phosphorus and the deoxyribose of tis Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. Cancer Research 624 venous blood of human patients with leukemia and the lymph nodes of these patients cells with the diploid amount of DNA, cells with the tetraploid, cells with intermediate values, and cells containing somewhat more than twice the amount of normal somatic cells (245). The increased con tent of DNA found in many tumors can, in part, be accounted for on the basis of polyploid cells and in part by the fact that many cells are actively synthesizing DNA in preparation for cell division. However, in view of the known incidence of heteroploidy and aneuploidy in normal tissues and the persistent presence of such cells in tumors, it is not illogical to assume that some of the increase Vol. 18, July, 1958 significance are the findings of Bendich and as sociates (23, 24) with respect to the fractionation patterns of DNA in different tissues of the same organism. On the basis of Chromatographie pro files, one can distinguish the following kinds of DNA: calf thymus, human leukemic leukocyte, various bacteria, T2r, T6r, T6r+ phage, spleen, intestine, kidney, and brain of the rat. DNA patterns of rat brain and kidney are illustrated in Chart 3. In the case of the T6r phage strains, a single genetic change is involved, and a difference in Chromatographie profile is seen. The various DNA molecules in a given cell may differ among themselves not only in composition (32) and se- TABLE1 DISTRIBUTION OFCHROMOSOME TYPESINTUMOR CH-38MC (Chinese Hamster)* TOTALCBBOMOSOHES m CELL 20 21 TYPESI332222535323338n202334123232227mll2232161222433IV0021121011S2221V, CHROMOSOME VI,Vllt786677768894887ViliS2222230121222(?)1IX122122041221223X322211S01226125XI032310 22ÃŽ 22 22 22 22 22 a.'i 24 25 26 27 2S 35 * Data illustrate marked aneuploidy, heteropleudy, and quasidiploidy of tumor cells. Only a few of the many cell types counted by Yerganian are shown. There were also ob served cells containing 28, 44, 45, 46, 48, 50 and 192 chromosomes, with a varying distribu tion of chromosome type. (Livingston and Yerganian, 210.) t Chromosomes V, VI, and VII grouped together. ÃŽ Distribution in normal cell type. is due to the increased chromatin content of the individual cells. Chart 2 shows the chromosome contents and the DNA contents of human HeLa cells grown in tissue culture (143). The distribution of DNA shows remarkable agreement with the chromosome counts. Not only is the DNA content per nucleus frequently increased in tumor cells, but it is also much more variable than in normal cells (219, 311). 3. Heterogeneity of DNA.—If a cell contains 42 chromosomes, there should be at least 21 mo lecular species of DNA within the cell. Probably, each cell contains several hundred different types of DNA. During the last few years, a beginning has been made in the fractionation of the DNA mixtures (32, 56, 212). These studies give promise of direct chemical demonstration of genetic dif ferences between normal tissues and cancer. Of quence but also in shape, size, and metabolic activity (15). 4. DNA function and time of DNA synthesis.— The proposal by Watson and Crick (61, 318) that DNA consists of two complementary helical chains running in opposite directions and winding around the same axis, with the chains being linked together by hydrogen bonds between specifically paired purine and pyrimidine bases, represents a plausible basis for gene specificity, gene replica tion, and for gene mutation. Presumably, DNA controls the synthesis of RNA which, in turn, is responsible for the synthesis of both nuclear and cytoplasmic proteins (211, 265). This, in turn, determines the structural characteristics of the cell including the kinds of antigens, enzyme pro teins, and therefore also the functional activities and metabolic patterns of the cell. Potter has Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. KIT AND GRIFFIN—Cellular Metabolism and Cancer also postulated (251) a cytoplasmic RNA-geneindependent enzyme-forming system which might be lost when cell duplication was faster than the rate of replication of the particular enzymeforming system in question. This would lead to an irreversible change in the cell. On the basis of recombination experiments with bacteriophage, Benzer has calculated that genetic units of re combination may be of the order of magnitude of as few as a dozen nucleotide pairs and that mutations may involve various lengths of "chro mosome" (25). The interesting concept that the sequence of the purine and pyrimidine bases of nucleic acids are in some way a "code" for the sequence of 625 with the occurrence of cell division and that isotopes are retained extensively in the DNA of mitotically inactive and active cells indicate that DNA displays a high biochemical stability (17, 123, 308). Therefore, it is assumed that the extent of the incorporation of natural precursors into DNA can be taken as a measure of the rate of DNA synthesis. In regenerating liver, it is observed that the most active incorporation of glycine-N15 (12, 114), orotic acid-C14 (123), or CHART2.—Comparison of chromosome counts and DNA measurements on individual nuclei of HeLa strain (Hsu and Moorhead [143]). amino acids of the protein polypeptide chain (318) has stimulated thought as to how four different nucleotides may determine the sequence of twenty different amino acids (93, 285). Gamow and Yeas suggested (93) that the amino acid residues are selected by "overlapping" triplets of nucleotides. If the coding triplets are chosen from four nucleo tides, 64 different triplets are possible. However, on the basis of an analysis of known amino acid sequences in proteins, Brenner1 has demon strated that 64 triplets are insufficient to code the known sequences by overlapping triplets. As an alternative, Crick et al.2 proposed a "nonoverlapping" code of nucleotide triplets, with the restriction that certain overlapping nucleotide triplets should represent "nonsense" coding. The observations that the incorporation of isotopically labeled precursors into DNA is correlated 1S. Brenner, On the Impossibility of All Overlapping Triplet Codes in Information Transfer from Nucleic Acid to Proteins. Proc. Nat. Acad. Sc., 43:687-94, 1957. 2F. H. C. Crick, J. S. Griffith, and L. E. Orgel, Codes With out Commas. Proc. Nat. Acad. Sc., 43:416-21, 1957. O.OtU f>t>a>p>>a'*,pH7 l.OUHoCf O.O/U p CHART3.—Chromatography on ECTEOLA of DNA iso lated from (a) rat kidney and (6) rat brain. In both experi ments, the gradient elution schedule was the same, with the flow rate about 3 ml/hr (Bendich el al. [24]). P32 into DNA takes place at 20-30 hours after partial hepatectomy (17, 233). DNA synthesis in this tissue apparently precedes cell mitosis, since the highest incidence of mitosis is observed at about 3 days after the operation. By radioautography on individual nuclei of bean roots exposed to P32, Howard and Pele have shown that isotope is taken up only during a specific period in interphase lasting for several hours and completed about 2 hours before the beginning of prophase (307). In Tradescantia, the earliest vis ible prophase nuclei contain 2 times the diploid amount of DNA, while early telophase nuclei show the diploid value. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. 626 Cancer Research The RNA of testis becomes maximally labeled by adenine-8-C14 within 1 day after intraperitoneal injection into mice, but DNA labeling increases for 8 days (240). After injection of glycine N16 (12) or orotate-C14 (308) into partially hepatectomized rats, it is observed that cytoplasmic RNA purines are maximally labeled at 14-18 hours and nuclear RNA at 26 hours. The C14 content of the cytoplasmic RNA is sufficiently large so that it could easily account for the maintenance of the acid-soluble pool, which in turn could maintain the nuclear RNA and directly or indirect ly provide the labeled precursors of DNA. DNA doubling need not take place prior to prophase in all tissues. The time of duplication seems to be shortly after division in the micronucleus of a ciliate, while in the Ehrlich ascites tumor cells it is possible that duplication of DNA may take place some time after metaphase (185). A study of DNA content and synthesis in syn chronously dividing tumor cells will undoubtedly prove of value as a means of verifying the time of synthesis of DNA in the mitotic cycle (332). 5. Chromosome organization.—The experiments of Demerec and associates (66-69,116) have clari fied the fine structure of the chromosomes of Salmonella and E. coli. There is apparently a coincidence between the gene sequence in the chromosomes and the sequence of biochemical reactions leading to histidine and tryptophan syn thesis which the aforementioned genes control. Although the findings in Salmonella may not only be confined to bacteria, there is as yet no evidence that they are general (250) ; the analogous histidine and tryptophan gene-loci occur in Neurospora on different chromosomes. However, since the metabolic map is highly branched and the arrange ment of the genes on the chromosomes is presumed to be linear, a complete identity between gene sequence and biochemical sequence is impossible. 6. Quantitative relationship between cell ploidy and metabolism.—The assumption that a given gene is involved in a primary way in the pro duction of but a single enzyme has been strongly supported by the studies of Tatum, Beadle, and associates with Neurospora and other microor ganisms (136). The formation of: (a) an altered enzyme and (6) modifications in various quantita tive aspects of enzyme formation may ensue from a genetic alteration. With respect to (a), Suskind and associates have observed the presence of pro teins antigenically related to tryptophan synthetase in different members of a group of very similar Neurospora mutants which require tryp tophan for growth (304, 305) and which lack the enzyme. The presence of these proteins sug Vol. 18, July, 1958 gests defects in specific and separate phases of the synthesis of tryptophan synthetase. A situa tion analogous to that of tryptophan synthetase has been observed in E. coli (58). There seems to be an altered protein, hemoglobin S, in the human hereditary disease, sickle-cell anemia (239). Of particular interest is the recent finding of Ingram (150) that a small difference in the amino acid sequence in one small part of the polypeptide chain exists between the globin of sickle-cell ane mia and normal globin. Modifications in various quantitative aspects of enzyme formation are implicit in the Boveri hypothesis that cancer cells lack certain chromo somes while containing too large a number of other chromosomes. The limited data available suggest that this concept has considerable validity. Mammalian or plant cells including tumor cells possess attributes that could account for a geo metrical progression in cell and nuclear volumes, namely, an increase occurring as an even integral multiple either in the number of chromosomes or in the number of strands in each chromosome (6, 120, 121, 219, 225, 288, 307). Lymphoma #1 (80 chromosomes, one to seven nucleoli per nucleus) contains about twice the RNA as Lym phoma #2 (44 chromosomes, one to four nucleoli per nucleus) (288). The following parameters are proportional to DNA content in the Lettre-Ehrlich ascites tumor (hyperdiploid) as compared with the Ehrlich tetraploid carcinoma: DNA content, cell size, acid-soluble phosphorus content, phospholipide, and RNA phosphorus content (115). Likewise, the amino peptidase activity and nitro gen content (238) are proportional to DNA content in sublines of the Ehrlich-Lettré tumor. Prelimi nary results carried out in our laboratory indicate that the endogenous cellular respiration, the cytochrome oxidase, succinoxidase, and transaminase activities are proportional to the DNA content per cell of related tumor cells. In haploid, diploid, triploid, and tetraploid strains of yeast cells (236, 237), the DNA, RNA, metaphosphate, respira tion, and aerobic fermentation increase in integral fashion with ploidy. 7. DNA and cell histones.—Found in close as sociation with the DNA of the cell are the protamines and the histones (44). Protamines are associated with DNA in fish sperm; histones in most somatic tissues. There are at least two his tones in calf thymus differing from each other in electrophoretic mobility (65), sedimentation in the ultracentrifuge, and amino acid composition (16). Histones prepared from different tissues of the same animal and similar with respect to Chromatographie behavior have closely similar Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. KIT ANDGRIFFIN—CellularMetabolism and Cancer amino acid compositions (45, 60). During the interphase period prior to cell division, both histone and DNA double simultaneously (27), but during the interphase period of cell function there is apparently a greater degree of binding of DNA by "residual protein." Possibly, a complex of DNA and residual protein exists during the heterosynthetic interphase and is dissociated during the autosynthetic interphase. Cruft et al. (62) have suggested that cell-specific histories may exist. Other investigators (44), how ever, have explained the alleged tissue differences described by Cruft et al. as owing to the presence of differing proportions of two electrophoretically distinguishable histone aggregates or the contam ination of the histone preparations with cytoplasmic proteins. Differences in the concentration of histones in tumor tissues have also not generally been confirmed. 8. Interaction of nucleus and cytoplasm.—Sus tained physiological activity of the cell and the expression of its genetic potentiality are dependent on the transfer of materials between the nucleus and the cytoplasm. Although a complete system for the renewal of the amino acids and the purine and pyrimidine bases of the nucleoproteins is contained in the cytoplasm of enucleated algae or amoebae (29), net protein and RNA synthesis gradually declines. Possibly, some substance (nucleotide coenzyme?) which is required in the cyto plasm is formed in the nucleus. Direct evidence that at least part of the cytoplasmic RNA may originate in the nucleus has been obtained by Goldstein and Flaut (105). Nu clei from amoebae labeled with RNA-P32 were transferred by micromanipulation to unlabeled, enucleated or to normal, unenucleated amoebae. Transmission of labeled material from nucleus to cytoplasm was then traced directly by autoradiography. Evidence of nuclear to cytoplasmic exchange has also been obtained by electron micro graphs of Drosophila, which showed that outpocketings of the nuclear membrane were in timately associated with specific regions of the chromosomes. It was suggested that these "blebs" might become detached and released into the cytosome, where they might contribute to the formation of such cytoplasmic structures as endoplasmic reticulum (96, 97). Nuclear blebbing has also been observed in melanoma cells photo graphed by time-lapse cinematography during cul tivation in tissue culture (141). 9. Relationship between cytoplasmic RNA and protein and enzyme synthesis.—There is consider able evidence for an intimate relationship between cytoplasmic RNA and protein and enzyme syn 627 thesis. This has been adequately reviewed by several investigators (3, 54, 64, 134, 135, 155, 188, 321, 339). Our understanding of the detailed mechanism of intervention of RNA in protein synthesis is as yet limited. It has been proposed that RNA functions as a template for the fixation of amino acids and the determination of amino acid sequences but also that energy is in some manner derived from the RNA molecule to spark protein synthesis. In connection with the latter possibility, Allfrey and Mirsky have suggested that DNA mediates the aerobic synthesis of ATP and other polynucleotides in isolated nuclei (9). At the enzyme level, it has been shown that amino acids may be incorporated into liver, pea seedling, or ascites tumor microsomal ribonucleoprotein as ollow s (322) : 1. Amino acid + ATP ^ Amino acid-AMP + pyrophosphate. 2. Amino acid-AMP + factor ^ Amino acidfactor + AMP. 3. Amino acid-factor + ribonucleoprotein frag ment ?i Ribonucleoprotein fragment containing incorporated amino acid. The activation of the amino acid (reaction 1) can be catalyzed by soluble proteins. The fragment indicated in reaction 3 is probably a polynucleotide. 10. Extranuclear inheritance.—The role of DNA as the substance capable of carrying genetic in formation has been emphasized above. However, the role of RNA as a mediator of genetic informa tion is by no means ruled out. Plant viruses and certain animal viruses contain no detectable DNA. Tobacco mosaic virus (TMV) can be re solved into nucleic acid and protein. The viral RNA is capable of initiating viral infection (albeit at 1-5 per cent the level of the intact tobacco mosaic virus). "Mixed" viruses can be produced from protein and RNA derived from different strains of TMV, and it has been shown that the progeny of such viruses always resemble that strain which has supplied the nucleic acid in regard to both symptomatology and chemical composi tion (87). RNA isolated from Ehrlich ascites tu mor cells infected with West Nile encephalitis virus is also infectious. West Nile virus was iso lated from the brains of mice which died following the intracerebral injection of the RNA prepara tions and was identified by means of specific im mune sera.3 Hence, a brief discussion of several authenticated examples of cytoplasmic inheritance is essential in relation to the Boveri hypothesis. 3J. S. Colter, H. H. Bird, A. W. Moyer, and R. A. Brown, Infectivity of Ribonucleic Acid Isolated from Virus Infected Tissues. Virology, 4:522-32, 1957. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. 628 Cancer Research Examples of cytoplasmic inheritance include Kap pa, the killer factor of Paramecium (293), plastids of plants (336), Sigma, a factor concerned with CO2 sensitivity in Drosophila (208), and a factor in yeast the lack of which results in petite col onies (82). Kappa and plastids are mutable, selfduplicating particles which are formed only when some are already present. However, these particles contain DNA4 (283, 293), while Sigma has some of the properties of a virus or rickettsia (29, 104). Kappa and plastids are adapted to definite intracellular conditions which the genes control, and Kappa disappears in the absence of dominant gene, K. Genes seem to control the functioning and maintenance, but not the initial production, of the plasmagenes. The relevance of Kappa, plastids, and Sigma to animal neoplasia requires substantiation. The formation of petite colonies in yeast is more interesting in relation to the Warburg theory of cancer (317). Yeast strains constantly give rise during their growth to mutants which are stable in vegetative reproduction and are characterized by a reduced colony size on media in which sugar is a limiting factor. They lack cytochrome oxidase. It seems that the mutation is due to the accidental noninclusion in the forming bud of a particulate cytoplasmic autoreproducing factor required for the synthesis of respiratory enzymes. For a critical discussion of cytoplasmic inheritance, see (82, 104). 11. Gene content and phenotype.—When the chromosomal and DNA content of closely related cells differ, the enzymes (10, 151, 156, 175, 315), metabolic patterns, and metabolites (90, 284, and unpublished experiments) of these cells will prob ably also differ. The question arises whether the converse proposition holds. When the enzyme and metabolite patterns of cell populations sub jected to the same environment differ from each other, can these differences be ascribed to genetic differences? This question has implications with respect to the concept of whether the neoplastic transformation constitutes an irreversible change and whether tumor progression involves further hereditary changes in neoplastic cells. An alterna tive concept is that of a reversible change involving differences in controlling factors. (See also dis cussion of phenotype and phenocopy [104].) The latter concept would imply changes in the host whereby the environment for a given cell is somehow altered so that it is free of growth restraint. It is known that the various adult tissues of the same animal have highly characteris4Y. Chiba and K. Sugahara, The Nucleic Acid Content of Chloroplasts Isolated from Spinach and Tobacco Leaves. Arch. Biochem. & Biophys., 71:367-76, 1957. Vol. 18, July, 1958 tic enzyme (111) and metabolite patterns (165). These tissues are differentiated. However, neo plastic cells are frequently referred to as dedifferentiated cells, and reference is made to similarities between tumor cells and the growing, undifferentiated embryonic cells. Moreover, it is commonly believed that all differentiated tissues of a given organism contain the same genome (2n chromo somes), although differentiation is believed to be irreversible. The concept of mutational change as a basis for differentiation has not found favor because of the random and sporadic nature of the latter process. Instead, differentiation has been ascribed to two factors: (a) although all the genes are believed to be present in the tissues of an organism, it is thought that some genes may be functional, whereas others are merely latent; (6) specific environmental differences result in irreversible cytoplasmic changes. Alterations at definite periods during embryogenesis (29) in the morphology and metabolism of arthropod chromosomes may represent an ex ample of gene activation. These alterations include changes in degree of polyteny and structural dif ferences conditioned by this such as coiling and cross-sectional appearance, differences in precision of banding and specific local modifications, such as the development of puffed regions in the chro mosome and the formation of Balbiani rings (22, 84). Other possible examples of variable gene activation are: (a) the reversible transformations to mutually exclusive serological types in Parameciumf (6) shifts from the synthesis of fetal hemoglobin to adult hemoglobin; and (c) the spread of pigmentation after an implantation of autografts of pigmented skin in an area of albino skin in the guinea pig.6 In the latter instance, the authors liken the spread of pigment formation to an infection carried via well developed cyto plasmic connections from melanoblasts to nonpigmented dendritic cells. Since all the pigment cells came from the same animal, presumably having the same genome, the observations were interpreted as the release of the potential ability of the cells to form dopa oxidase. It is not certain, however, whether the above examples reflect dif ferences in gene activity or differences in the stability and function of gene products. Brächet (29) has advanced the idea that infective ribonucleoprotein particles may initiate differentiation by gene ac tivation. The transposition of heterochromatin 5G. H. Beale, The Antigen System of Paramecium aurelia. Int. Rev. Cytol, 6:1-23, 1957. •R.E. Billingham and B. P. Medawar, Pigment Spread and Cell Heredity in Guinea-pig Skin. Heredity, 2:29-48, 1948. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. KIT ANDGRIFFIN—CellularMetabolism and Cancer or other chromosomal elements is a possible mech anism for gene activation or inhibition. As a general phenomenon, gross change in chro mosome number is not accepted as a basis for differentiation (198). In amphibia and sea urchins, normal zygotic number is a prerequisite for orderly ontogeny (28, 118). There are, however, examples of chromatin changes and evidence for nuclear as well as cytoplasmic differentiation in tissues. There is an unequal distribution of chromatin between germ line and somatic cells in inverte brates and plants (22, 104), and hyperpyknotic masses in the retinal rods of some mammals have been noted (22). The transplantation experiments of King and Briggs (162, 163) are indicative of restrictions in potentiality for differentiation 629 absolute amount of any given cellular component. Presumably, it is these cellular parameters which are referable to the altered genetic pattern of neoplastic cells. Attention is directed to the dis tribution of protein and RNA among the cellular organelles of tumors and normal tissues (Table 2). In nine animal and six human tumors, Laird and Barton observed that about 40 per cent of the total protein was attributable to the nuclei and about 40 per cent to the supernatant fraction of the cells. Only about 10 per cent was found in the mitochondria and 10 per cent in the microsomes (194, 195). Thymus and adrenal tissue are also characterized by relatively low propor tions of the cytoplasmic particulate fractions and cytoplasmic protein. Liver and kidney cells con- TABLE 2 PERCENTAGE DISTRIBUTION OFPROTEINSANDRNA AMONGCELLFRACTIONS OFVARIOUS NORMALANDMALIGNANT TISSUES* (From Laird and Barton [195] and Allard et al. [7]) RlBOSE NUCLEIC ACID TISSUE Mt. Micro. Super. Nue.385880¿1161411201122PROTEINMt.8313322688332218NITROGENMicro.1381711173229192220Super.40325187404652314445 Nuc. Mt. Micro. Super. Nuc. Animal tumors 38 8 13 40 32 10 29 30 Normal tissues and thymus, rat 58 3 8 32 15 3 37 61 Adrenal, human 20 13 17 51 16 9 37 37 Liver, mouse 21 32 11 37 11 39 29 21 Kidney, rat 16 26 17 40 14 13 32 40 Pancreas, rat 14 8 32 46 42 51 42 Salivary gland, rat 11 8 29 52 9 3 54 35 Brain cortex, rat Intestinal mucosa, rat Regenerating liver (3 days) * The quantity of protein nitrogen or RNA in each cell fraction is expressed as a percentage of the total protein nitrogen or RNA present in the tissue. Nuc = nucleus; Mt = mitochondria; Micro = microsomes; Super = supernatant fluid. Data on regenerating liver, intestinal mucosa, and brain cortex calculated from (7). on the part of late gastrula nuclei of frogs. The differences in the DNA profiles of the various tissues of the same organism are also of interest in this connection (23). For a fuller discussion the reader is referred to (29, 104). III. SUBCELLULAR METABOLIC PATTERNS OF NEOPLASTIC CELL POPULATIONS 1. Distribution of protein and RNA.—The pre vious discussion has emphasized the heterogeneity of neoplastic cell populations. It is seen that neo plastic cells may differ in chromosome number and DNA content. Yet each of the cells within a population is endowed with the capacity for continued and progressive growth until the death of the host animal. We should now like to em phasize characteristics that neoplastic cells have in common. It is the pattern of cellular organiza tion which will be emphasized rather than the tain relatively high mitochondrial protein nitro gen; two exocrine glands, pancreas and submaxillary gland, are characterized by a very high pro portion of microsomal material but a rather low proportion of mitochondria. About 80 per cent of the RNA of the latter cells is found in associa tion with the submicroscopic particles and the nonsedimentable proteins. The mitochondrial frac tion of liver differs from that of kidney with respect to the RNA to protein-nitrogen ratio. Indeed, the kidney mitochondrial fraction prob ably corresponds to the large mitochondrial subfraction of liver which has a very low concentra tion of RNA. Although the tumor cells were similar to the thymus with respect to protein distribution, they differed from thymus and from all the other cells studied thus far by having a large proportion of the RNA in the nuclear fraction .(102, 204, 244). The RNA per nucleus of spleen cells in spontaneous leukemia was in- Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. Cancer Research creased 1.6-fold, and in transplanted leukemia 4.2-fold, over normal mouse spleen cells (13, 244). The ratio of RNA/DNA is generally higher in many tumors than in normal tissues. The nucleoli of the chromosomes are particularly rich in RNA (184). Turnover of the nuclear RNA of tumors may also be greater than that of normal cells (112). 2. Mitochondria of neoplastia cells.—The de creased mitochondrial protein of tumors might be due to changes in the number per cell, size, or chemical properties. A decrease in the number of mitochondria has been noted in preneoplastic rat livers, in spontaneous rat liver tumors, and in the transplantable Novikoff tumor (8, 282, 301). Electron microscope studies on two rat liver tumors were in agreement with the above re sults (138). Novikoff tumor mitochondria exhib ited very prominent internal membranes and varied in diameter over a greater range than those of normal liver cells. Nucleoli were very prominent in the tumor cells, but organized ergastoplasmic structures were scanty, appearing mainly in the form of vesicles. A notable feature of the Novikoff tumor cells was the large nucleocytoplasmic ratio, due to the decreased cytoplasmic volume. Mitochondrial number is also diminished in regenerating liver cells after partial hepatectomy (7). However, in mouse hepatomas, mitochondrial number per nucleus is about the same as that of normal mouse liver, although the number per unit weight of whole tissue is reduced. Apparently, the mouse hepatoma con tained nuclei that were somewhat more polyploid than normal liver nuclei (282, 301). The mito chondrial protein of tumors is not lower than than of normal spleen or thymus cells. Enzymes of the mitochondria include the en zymes of the citric acid cycle, cytochrome oxidase, cytochrome c, succinoxidase, octanoic acid oxidase, DPN-cytochrome c reductase, TPN-cytochrome c reductase, transhydrogenase, glutamic dehydrogenase, enzymes of oxidative phosphorylation, ATPase, and enzymes for the synthesis of paminohippuric acid (132, 133). Schneider and Hogeboom (280) have reported that mitochondria from mouse hepatoma (98/15) contain less than half as much succinoxidase or cytochrome oxidase activity/mg of mitochondrial nitrogen and less than a fifth the total enzyme content as those from normal mouse liver. The decreased succinoxi dase and cytochrome oxidase activity of hepatoma can be attributed only in part to the decreased amount of mitochondrial material. Hepatoma mi tochondrial ATPase activity was also greatly re duced (281). However, the specific activity of DPN-cytochrome c reductase was greater than Vol. 18, July, 1958 in normal tissues (131). Due caution must be exercised in interpreting data on preneoplastic liver. After the feeding of 3'-methyl-4-dimethylaminoazobenzene, there are extensive proliferation of bile duct epithelium and wide variation in the size of parenchymal cells (301). The sedimentation patterns of soluble proteins obtained from disrupted mitochondria were studied by Hogeboom and Schneider (132). The sedimentation patterns of the mitochondrial pro teins from the regenerating liver were identical with those obtained from normal liver. Three components were observed in the preparations ob tained from hepatoma mitochondria, the most prominent being a reasonably sharp, slowly sedimenting peak corresponding in sedimentation con stant to component No. 1 of normal liver. The two remaining peaks were polydisperse and gave sedimentation constants approximating those of components 3 and 4 of normal liver, but in no instance was a peak corresponding to component No. 2 detected. Another attribute of mitochondria of many tumors is their requirement for exogenous DPN+ for the optimal oxidation of Krebs' cycle me tabolites. Pyruvate oxidation parallels mitochon drial nitrogen content, both parameters being re duced in several tumors and brain as compared with liver, heart, or kidney (329). Addition of DPN+ does not necessarily stimulate oxidative activity of mitochondria from freshly prepared normal tissues but does do so in the case of the tumor and brain mitochondria (329). As a result of these oxidations, ATP is generated (161). Oxidative phosphorylation takes place dur ing the oxidation of glutamate, succinate, or aketoglutarate by mitochondria from Hepatoma 98/15. No fluoride need be added to the incuba tion medium. However, the phosphorylation sys tem of the tumor is more unstable than that of liver. Whereas liver mitochondria suspended in isotonic sucrose retained phosphorylating activity for as long as 24 hours at 0°C. with little loss, tumor mitochondria lost 30-50 per cent of their activity after standing only 2-3 hours at 0°C. Tumor mitochondria aged for 24 hours at 0°C. or for 25 minutes at 28°C. completely lost their ability to phosphorylate. The addition of DPN+ to the aged tumor mitochondria significantly in creased the rate of phosphorylation (Table 3). Oxidative phosphorylation in the presence of fluoride has been demonstrated with various other tumors (193, 209,334). The requirement for DPN+ and fluoride is in part attributable to the intense ATPase and DPN+ase activity of tumor mito chondria (161). The latter enzymes have been Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. KIT AND GRIFFIN—Cellular Metabolism and Cancer investigated by Emmelot and associates (77, 81) by observing the oxidative response to octanoate of mixtures of both tumor and liver mitochondria. Liver but not certain tumor mitochondria oxidize octanoate. The addition of tumor mitochondria inhibited this oxidation. The inhibitory effect could be partially counteracted by adding fluoride, a potent ATPase-inhibitor, or nicotinamide, a DPNase-inhibitor. Fluoride added to the tumor mitochondria alone did not restore fatty acid oxi dation. For a number of tumors, the inhibitory activities were dependent upon the procedure used for isolating the mitochondria. If the latter were isolated in and washed with isotonic sucrose and subsequently suspended in KCl-phosphate buffer at 0°,the mitochondria from all but three of the tumors completely inhibited the fatty acid oxi dation in the combined liver-tumor mitochondrial system. Following the use of isotonic sucrose as the medium for the preliminary suspension, the inhibition shown by the mitochondria from five tumors was markedly less than in the former case. When isotonic sucrose containing versene was used for the isolation of the suspension, the mitochondria from four other tumor strains had lost their inhibitory power. However, four tumor strains remained whose mitochondria did inhibit the fatty acid oxidation of liver mitochondria. It should be emphasized that the ATPase is not tumor-specific. Mitochondria prepared from brain and liver mitochondria preincubated for ^ hour at 37°C. in the absence of substrate show the same properties as described for the tumor mitochon dria, although to a smaller extent. Direct measure ments showed that sucrose-versene-prepared mito chondria from tumors that did not inhibit octano ate oxidation had low DPNase and TPNase ac tivities, and those that did inhibit it had high DPNase activity. Reductive amination of gluta mate from a-ketoglutarate and NHs by the tumor mitochondria exhibiting high DPNase activity was low. Addition of DPN+ promoted glutamate synthesis (78). Transamination between a-keto glutarate and valine was high for the mitochondria of all the tumors studied. Analogous results to those described above were obtained when the tumor-liver mitochondrial system was studied with /3-hydroxybutyrate as substrate. Tumor mitochondria from testes, ovarian tu mors, and the spontaneous mouse hepatomas showed the greatest ability to preserve their bio chemical integrity in vitro against the release of latent ATPase and DPNase; these tumors most resembled fresh liver mitochondria. The sucrose-versene mitochondria from these tumors were themselves able to oxidize octanoate and 631 0-hydroxybutyrate; ATPase and DPNase activity were low, reductive amination and pyruvate oxi dation could be accomplished in the absence of added DPN+, and their TPNase was only moder ately active. However, marked differences in com parison with liver mitochondria still remained. The latter could withstand more drastic handling in vitro than the tumor mitochondria before losing their ability to oxidize fatty acids, and the extent of ATPase activation by dinitrophenol did not follow the same patterns as those of liver (79). Siekevitz and Potter have also emphasized that the balance between ATP breakdown and synthesis is of primary importance and that in tumors where the breakdown of high-energy phosphate TABLE 3 EFFECT OF DPN ONP UPTAKE BYMITOCHONDRIA* (From Kielley [161]) TAKENUP/ F N)PhosphorylatingactivityTumor 10 MIN/MO CONDITION OF ENZTHE Fresh MSOBSTBATE 0.001 DPN0.01 Ma-ketoglutarate +a+0.013 Aged Fresh Aged -Glutamate M +u4.(fiHOLES Lirer10.7 16.118.4 18.83.9 10.217.8 S15.0 11. 23.819.79.6 15.015.7 16.3 * Substrates (a-ketoglutarate, glutamate, and succhiate) oxidized by fresh and aged mitochondria isolated from transplantable mouse hepatoma and mouse liver. Tumor mitochon dria aged 2 hours at 0°C.; liver mitochondria aged 3 hours at 5°followed by 24 hours at 0°C. (161). compounds is rapid, fluoride preserves the respira tory rate (290). Wenner and Weinhouse have suggested (329) that the binding of DPN+ by tumor mitochondria is looser than that of normal tissues. This might be of significance with respect to the glycolytic activity of the tumors, inasmuch as the DPN+ might be available at correspondingly higher con centrations in the soluble portion of the cell where glycolytic enzymes are concentrated. The total pyridine nucleotides of tumors are considerably lower than those of liver, kidney, and muscle and of approximately the same order of magnitude as those of spleen or brain (153, 154). Primary tu mors induced by azo dyes, embryonic liver, and the livers of newborn rats also had reduced amounts of pyridine nucleotides, the values falling in the range of the transplanted tumors. Pyridine nu cleotides were present in all cell fractions of the normal tissues studied but were absent from tumor microsomes. The highest amounts were found Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. 632 Cancer Research in the soluble supernatant fractions. The nuclei and mitochondria of the tumors contained much less pyridine nucleotides than did the correspond ing cell fractions of liver and kidney, and some what less than that of spleen (48-50). Carruthers and associates observed that hepatoma mitochon dria warmed at 38°C. lost nearly all of their pyridine nucleotides; however, liver mitochondria lost either none or only a portion of the nucleotides under similar conditions. The above investigators discounted the idea that the loss was attributable to the diffusion of the DPN+ from the mito chondria to the supernatant; instead, they sug gested that the pyridine nucleotides of the tumors were more easily cleaved by nucleosidases. The content of pantothenic acid and of Coenzyme A is lower in three transplanted tumors and in a spontaneous azo dye tumor than in normal liver. The majority of each factor in the liver was contained in the mitochondria; in the liver tumor, the supernatant contained the largest amount. The remainders of the vitamin and coenzyme were distributed in the nuclear and mitochondrial fraction of the tumor and the nuclear and supernatant of liver (128). The mitochondria of spontaneous liver tumors contained only half as much total riboflavin as did the same fraction of normal livers, but the concentration of the vitamin per gram of protein was increased (256, 257). Differences in the chemical properties of par ticles from closely related tumors have also been observed. A mouse asci tes sarcoma contained high er amounts of mitochondria and microsomes per unit of DNA than the solid tumor from which it was derived (180), and the N/P ratio of the ascites tumor microsomes was lower than that of the solid tumor. 3. Proteins of the "supernatant" fraction.—The "supernatant" fraction of tissues contains a num ber of component ribonucleoproteins correspond ing to a diameter of about 8-30 m^i (27-107 S). Microsomes, by comparison, are about 100 m/u in diameter (130 S) (241, 243). Of six components obtained by ultracentrifugation, component B (49 S) predominates in normal liver, spleen, and pan creas, and appears to correspond to the 15-m/i granules found on the endoplasmic reticulum by electron microscopy. Components A and B were greatly reduced, while the concentrations of C and E were markedly elevated in regenerating liver, in hepatomas and cholangiomas induced by azo dyes, and in the Ehrlich carcinoma and Jensen sarcoma. The concentration of microsomes in the tumors was reduced to half the normal value. In spontaneous or transplanted leukemic spleen, Vol. 18, July, 1958 the concentration of C is about twice that found in normal spleen. Electrophoretic analysis thus suggests that, in various types of tissues, the macromolecular particles are qualitatively similar but are present in differing amounts (242). The highest rate of amino acid incorporation into cell protein has been localized in the cell fraction which contains the ribonucleoproteins. The slowest sedimenting class of soluble, nonparticulate proteins of rat liver represents one-half of the supernatant proteins or one-fourth of the proteins of rat liver (3.6 S). By electrophoretic analysis, they can be separated into two compo nents; one of these, the "h" component, was found to contain the bulk of the soluble protein-bound azo dye derivatives and to represent approximate ly one-fourth of the proteins. In azo dye-induced hepatomas, the "h" component is reduced in amount. Other tumors contain relatively little "h" (294). Eldredge and Luck (75) have also reported that hepatoma extracts differ from nor mal liver extracts in having less of the more slowly moving components and more of the faster moving ones. Indirect evidence for changes in the profile of soluble proteins have been obtained by immunological methods. Antigens of the H-2 locus are present in all normal tissues of a given animal, but significant quantitative differences between tissues can be shown to exist by absorption tech nics. The greatest absorbing capacity is exhibited by lymphoid tissue and mammary gland, the least by striated muscle and erythrocytes. The erythrocytes of some strains present a curious situa tion: In erythrocytes of C57BL mice (ff-a?6 gene locus, antigens BEF), antigen E is practically absent. Apparently, this antigen depends on nu clear elements for its continued presence because young red cells exhibit some response to antigen E. Antigenic simplification is commonly observed in many tumors (130, 190). 4. Endogenous metabolites.— a) General: The changes in cellular organelles and enzymes inevitably affect the distribution of metabolites within the tumor cell. It has gen erally been noted that tumors contain low levels of glycogen and fat, relatively more water and lactate, and in some cases more pyruvate and aketoglutarate than do most normal tissues (111, 202). The glycogen content of transplanted tumors is particularly low. Tumors possess several-fold higher lactate levels compared with differentiated tissues, reflecting their high rate of aerobic glycolysis (103, 200). Pyruvate is found to be very low in normal tissues and somewhat elevated in venous blood and in neoplastic tissues. Many Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. KIT ANDGRIFFIN—CellularMetabolism and Cancer tumor tissues contain more citrate than almost all normal tissues except skin, bone, hair, and the tissues comprising the seminal vesicle (244, 253). Of the minerals studied, the levels of iron, copper, zinc, and calcium were less in hyperplastic epidermis and in skin carcinoma than in normal epidermis, but it is possible that the drop in calcium and perhaps other constituents may be associated with an altered cell type rather than with a change specific for premalignant epider mis (47). 6) Nucleotides and deoxyribonucleosides: The concentration of deoxyribosidic compounds in mouse tumors is much greater than in most normal tissues (277-279). In rat hepatoma, the concen tration was found to be the same as in normal rat liver. However, it was observed that deoxycytidine accounted for almost all the deoxyribo sidic compounds of liver, whereas in the hepatoma deoxyuridine and thymidine were also present and accounted for only 53-59 per cent of the total acid-soluble deoxyribosidic material. Follow ing partial hepatectomy, the concentration of deoxyribosidic compounds in regenerating liver increased more than 60 per cent. The increase occurred before cell division, in harmony with the thesis that the deoxyribosidic compounds represented precursors of DNA. Unlike the tu mors, the nucleosides of regenerating liver can be accounted for almost entirely as deoxycytidine. The profiles of free nucleotides of acid-soluble extracts of normal tissues and tumors differ from one another with respect to a number of compo nents. Brain and tumor chromatograms resembled each other more than either resembled liver or muscle. Both apparently lacked the ADP-X peak that did appear in liver, and both possessed the UDP derivatives that were not seen in muscle (276). c) Free amino acid pattern: Free amino acids are present in tissues of animals in the post-absorp tive state at 3-9 times their concentration in the blood. The patterns are highly characteristic for each tissue of a given organism and are main tained at relatively constant levels despite phys iological alterations (165, 173). Some of the free amino acids of rat thymus and spleen can be reduced in concentration by prolonged fasting or by the temporary modification of the enzyme pattern of the tissue by the institution of a metabolic block, but the free amino acid content changes are remarkably small following bilateral adrenalectomy, a procedure which profoundly al ters the weight of lymphatic tissue in the body. In some instances, a particular organ may show significant variations from one species to another. 633 Reproducible differences can also be found within the tissues of a given organ. Thus, the gray and white matter of the central nervous system possess different patterns of free amino acids, as do the auricle and ventricle of the heart. The patterns of lymphocytes, polymorphonuclear leukocytes, macrophages, and erythrocytes are distinctly dif ferent. The specific amino acid patterns found in the various tissues of the adult organism must have their origin in time and space during develop ment from the fertilized egg. Changes in the distribution of free amino acids and related com pounds take place at all stages of development from the unovulated egg to a stage at which the final functional and structural patterns are laid down (268). /2-iTAU EA-P* EA-P» e*T SD* SPLEEN CHART4.—Free amino acid patterns of rat spleen, mouse lymphosarcorna, and rat sarcoma cells (induced by methylcholanthrene). The height of each bar represents the mean concentration. Abbreviations are: Tau = taurine; Ala = ala nine; Gly = glycine; Glu = glutamic; Asp = asp; EA-P = ethanolamine phosphate; SD = Sprague Dawley (Kit [165]). The free amino acid patterns of tumors differ greatly from those of related normal tissues (Chart 4). As compared with lymph nodes, thymus, spleen, or appendix cells, lymphatic tumors con tain relatively high levels of alanine, glycine, and proline but reduced amounts of aspartate, ethanolamine phosphate, and very little glutamine. The amino acid patterns of many transplantable and spontaneous tumors are more simi lar to one another than to normal tissues of origin or to embryonic tissues. However, even closely related tumors can be distinguished from one another on the basis of characteristic differences in amino acid levels (172). The free amino acid pattern of leukemia C1498 has been studied during the growth of this tumor in two strains of mice differing from each other by a single histocompatibility gene. In a strain Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. 634 Cancer Research resistant to the tumor, the tumor grows for a time and then regresses. The free amino acid patterns of the tumor grown in the latter mice are the same as the pattern in the susceptible subline for 8 days after transplantation. Subsequently, the tumors of the resistant strain show a relative increase in free glutamic acid, together with the appearance of free glutamine, an amino acid not detected at any time on chromatograms of extracts from tumors grown in the susceptible subline. By the 12th day, the regressing tumors displayed some decrease in serine, an elevation in the amounts of valine and the leucines, significantly greater amounts of free glutamic and aspartic acids, and relatively large amounts of free glutamine (266). Increases in the glutamine content of regressing Yoshida sarcoma cells and in Ehrlich ascites cells damaged by chemotherapeutic agents are also observed (267). Glutamine is required for the synthesis of purines (110), protein, and various other cellular constituents. Sarcomycin-damaged ascites tumor cells take up extracellular glutamine-2-C14 and convert it to glutamic acid. How ever, the tumor cells display a more limited ca pacity to utilize exogenous glutamic acid. These results suggest that the alterations in the gluta mine content of tumors are related to changes in cellular anabolism. 5. Metabolic dedifferentiation.—With time and continued cell division, cancer cell populations become increasingly autonomous. The net result of the genetic, metabolic, and structural altera tions in these populations is the progressive loss of function. Tumor progression and its implica tions have already been reviewed (86, 91, 109). To summarize the essential points: a) Mammary tumors originating in the same mouse differ in transplantability to foreign strains (302). With increasing malignancy, high immunogenetic specificity is replaced by an increasing host range, and heterotransplantability appears (109). There is evidence for correlation between alterations in tumor chromosome number, antigenicitv, and capacity to grow in foreign strains (118, 119, 121, 122). o) Serologie findings give close support to this correlation (11). Diploid tumors are more active than polyploid sublines not only in absorbing antibody from a number of immune sera but also in provoking an immune response. While in keeping with the view that neoplasms have simplified isoantigens (130), new antigens have been found in a number of lymphomas (11, 118, 121). c) There is a progression from hormone de pendency to independence from hormonal stimu lation (86, 91). Vol. 18, July, 1958 d) Slowly growing, highly differentiated neo plasms increase their growth rate and lose most or all visible signs of differentiation. e) Inconvertible solid tumors become convert ible to the ascites form (180-183, 186, 187). /) There is an evolution in tumor cell popula tions from responsiveness to x-rays or chemo therapeutic agents to unresponsiveness (197). Biochemical changes incident to tumor pro gression may be illustrated by the enzymatic alterations of neoplastic liver cells. When a liver becomes neoplastic, many of the specific functional activities markedly decrease or are lost altogether. Normal liver is characterized by a high activity of arginase and cystine desulfhydrase (111). In the hepatoma, these enzymes are either reduced in activity or else have virtually disappeared. Tryptophan peroxidase and the enzymes respon sible for kynurenine disappearance are very low (57), there is a block in the ring opening of histidine, and the mitochondrial enzyme, glutamic dehydrogenase, is reduced to the vanishing point (7). There is a decrease in the ability of hepatoma mitochondrial enzymes to form citrulline, to syn thesize urea from citrulline, or p-amino-hippuric acid from glycine and PABA (310). Riboflavin and the flavin enzymes, D-amino acid oxidase and xanthine dehydrogenase, are decreased in hepatoma and in fetal liver. There are reductions in the oxidative enzymes cytochrome oxidase, cytochrome c, catalase, and uricase (7). Soluble enzymes concerned with the degradation of uracil and dihydrouracil diminish. Interestingly enough, this leads to an enhanced capacity of hepatoma tissue to utilize exogenous uracil for the synthesis of uridylate and the uracil of nucleic acids (46, 124, 272). Hepatomas contain little glycogen, and in the absence of adenylic acid the enzyme, phosphorylase, is relatively inactive (106). There is a decrease in phosphoglucomutase, an increase in the direct oxidation of glucose-6-phosphate and 6-phosphogluconate, a virtual disappearance of the microsomal enzyme, glucose-6-phosphatase, and an in crease in hexose phosphate isomerase (319, 320). Glucose-6-phosphatase is not affected in regenerat ing liver, is decreased in embryonic liver, and great ly increased in diabetes or after fasting. Glucose6-phosphate dehydrogenase, phosphoglucomutase, and phosphohexoseisomerase are essentially nor mal in embryonic or regenerating liver. Trans planted tumors including the hepatoma oxidize fatty acids but produce little or no ketone bodies; they utilize acetoacetate more readily than do liver slices but synthesize relatively little fatty acids from acetate as compared with liver slices (217, 218). Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. KIT ANDGRIFFIN—CellularMetabolism and Cancer Loss of function takes place in stages; there is a persistence in many neoplasms of certain of the biochemical attributes of the differentiated tissue of origin. The transition states in the change from dependency to autonomy have been discussed by Furth (91). Several examples of persisting biochemical function may be listed: (a) the pro duction of melanin by most melanomas, (o) bone formation in osteogenic sarcomas, and (c) hormone secretion in certain glandular tumors (111). Dedifferentiation is not a necessary concomitant of malignancy. In some testicular tumors, enzymes for the production of androgens are lacking; in other tumors, the androgenicity may actually in crease during the course of transplantation (273). Transplantable strains of adrenal tumors which secrete either estrogens, androgens, or corticoids have been isolated by Furth (91). Transplantable Leydig-cell tumors were found to exhibit some features in common with luteomas and cortical adenomas. Transplantable pituitary tumors which secrete thyrotrophic, mammotrophic, or adrenocorticotrophic hormones have also been studied. IV. ENERGY-YIELDING MECHANISMS A. ANAEROBIC ANDAEROBICGLYCOLYSIS The structural alterations of neoplastic cells affect the metabolic processes concerned with the energy economy of these cells (35, 316, 317, 326). The respiration of most tumors is appreciable, though often less than that of the more active normal tissues. The R.Q. is usually moderately low. Nearly all tumors manifest extraordinary rates of anaerobic glycolysis and appreciable rates of aerobic glycolysis, properties shared with few normal tissues (76, 196). Those normal tissues which do exhibit a very high glycolytic rate differ metabolically from tumors in other respects: for example, jejunal mucosa and retina manifest a high absolute Q value for respiration (in the former c ase about equal to the high anaerobic glycolytic Q value, while the Pasteur effect is minimal); kidney medulla manifests a low R.Q. (35). Because of the very active glycolysis, the proportion of ATP generated by the glycolytic system, as com pared with the mitochondrial respiratory system, is potentially very high in most tumors (Table 4). Ascites cancer cells can obtain approximately as much energy in the form of ATP from fermenta tion as from respiration, while liver and kidney may obtain about 100 times as much ATP from respiration as from fermentation. It should be emphasized that a high rate of fermentation is not necessarily characteristic of all growing tissues : although the fermentation of embryonic cells and of animal cells cultivated in the relatively anaero bic environment of tissue culture is appreciable, 635 the fermentation by regenerating liver tissue does not significantly exceed that of normal liver. The rate of anaerobic glycolysis of neoplastic cells is so great that incorporation of glycine-2-C14 into cell protein or nucleic acid purines proceeds as well under anaerobic as under aerobic conditions (203). Aerobically, glucose stimulates the incor poration of amino acids or P32 into the protein or nucleic acids of lymphatic tissues and tumors (83, 174). Anaerobically, with glucose present, P32 uptake by appendix cells into the organic acid-soluble or nucleic acid fractions are markedly inhibited; no such inhibition is observed with lymphosarcoma cells. Warburg regards the fer mentative shift of the neoplastic cells as an ir reversible process and as owing to a mitochondrial change. He states, "The autonomy of the respiring grana, both biochemically and genetically hardly be doubted today . . ." (317). Warburg can has TABLE 4 CONTRAST OFTHEQ VALUES OFSOMENORMALBODY CELLSWITHTHEQ VALUES OFASCITES Calk Liver Kidney Embryo (very young) Cancer emphasized CANCERCELLS Data of Warburg (317) Q»> Q°»„ -15 105 1 1 -15 105 1 1 10« 106 -15 - 7 130 109 25 60 the "damage" 105 49 25 60 to the respiratory ap paratus in the cancer cell and visualizes a selective process following this damage whereby weakly fermenting cells perish while the more strongly fermenting cells stay alive. The selective process continues until the respiratory failure is compen sated for energetically by the increase in fermen tation. Only then has a cancer cell resulted from the normal body cell (317, 326). The views of Warburg thus represent a theory to account for the fermentative activities of tumors and the car cinogenic process per se. The point of view dis cussed in the present paper differs from that of Warburg in that the metabolic imbalance of the neoplastic cells are attributed to changes in the cell nucleus. Damage to respiratory grana is viewed as significant only if nuclear alteration results from primary mitochondrial damage. The point at issue is this: Will the progeny of an animal cell con taining an undamaged nucleus and damaged mito chondria contain normal or damaged mitochon dria? The following hypotheses have been proposed to explain the respiratory-glycolytic imbalance of tumors: (a) an increase in the concentration of glycolytic enzymes, leading to high rates of Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. 636 Cancer Research glucose utilization; (6) deficiencies in the mitochondrial electron-transport system; (c) relative deficiencies in the capacity of mitochondrial citric acid cycle enzymes to catabolize the pyruvate produced by fermentation; and (d) glycolytic and respiratory rates as a manifestation of release from regulatory control, whether endocrine or bio chemical. Warburg has emphasized the significance of glycolysis with respect to the energy economy of the cell. We should like also to emphasize the significance of rapid glucose utilization with respect to various anabolic processes and reductive synthesis by the cell. 1. Glycolytic enzymes.—Although the capacity of intact tumor cells to produce lactic acid from glucose is much greater than that of most normal cells, the capacity of tumor homogenates to ferment hexose diphosphate and to esterify inorganic phos phate is not necessarily greater (201). With excess hexose diphosphate, glucose, pyruvate, and fluo ride present, the following Qi^ctate values were observed: heart, 121; muscle, 110; diaphragm, 170; kidney, 96; liver, 82; brain, 80; FlexnerJobling tumor, 73; Walker tumor, 64; Jensen sarcoma, 80. Beck and associates report that gly colysis by homogenates of leukocytes from the blood of patients with chronic lymphatic or myelocytic leukemia was but 16 and 42 per cent, respectively, of that of normal cells (19, 21). The activity of lactic dehydrogenase and 3-phosphoglyceraldehyde dehydrogenase was closely pro portional to the glycolytic rate in the three tissues, while greatly exceeding it in maximal velocity. Conversely, aldolase and triósephosphate isomerase activities were higher than normal in myelocytic leukemia, although the glycolytic rate was lower than normal. Km for substrate and coenzyme at pH optima were essentially identical for the corresponding enzymes of the three tissues, al though differing among different enzymes, sug gesting functional similarity between normal and leukemic enzymes. The lactic dehydrogenase of various neoplastic tissues is of the same order of magnitude as that of normal tissues (111). However, when mouse lymphosarcoma (Q£'= 60) enzymes were com pared with those of appendix (Q?,' = 25), it was observed that lactic dehydrogenase was 8 times as active, phosphoglyceraldehyde dehydrogenase 3 times, hexokinase 2 times, and enzymes of ribose-5-phosphate metabolism 4 times as active in the neoplastic as in the appendix cells (312). A high-malignancy tumor line (1742) grown in tissue culture contained 3 times as much aldolase and 2 times as much a-glycerophosphate dehydro Vol. 18, July, 1958 genase as a low-malignancy line (E2049) (317). As compared with normal liver, hepatoma tissues contain increased concentrations of phosphohexoseisomerase (320). Aldolase content of human adenocarcinoma of the colon or rectum is in creased, as compared with that of adjacent normal mucosa (289). Evidence exists that the hexokinase reaction is rate-limiting in normal and malignant tissues. Normal tissues frequently exhibit low glycolytic rates on glucose owing to the inability to phosphorylate the sugar (201). The "hexokinase" re action is inhibited by some hormonal influence which can be relieved or balanced in vivo but which is nondissociable in vitro. Homogenates of the Flexner-Jobling tumor and rat brain are able to glycolyze and esterify phosphate at maximum rates with very low glucose levels of 25 mg. per cent and show slight increases at very high glucose levels of 1440 mg. per cent, but rat liver, kidney, and diaphragm muscle homogenates are unable to utilize glucose at low levels and are considerably stimulated at very high glucose levels. Addition of insulin permits the maximum effect at much lower glucose levels in these tissues but insulin effects were not obtained with rat brain or tumor homogenates. Hexokinase is also rate-limiting in normal or leukemic human leu kocytes, though in different ways. In normal cells, hexokinase limits by controlling the rate of glucose phosphorylation. An adequate supply of ADP is maintained by hexokinase and phosphofructokinase and ATPase activity which together main tain optimal ATP:ADP ratios. In the human leukemic cells, the chief consequence of the hexo kinase level is a critical lowering of the ADP level since ATPase is abnormally low. Thus ADP formation is doubly impaired and the glycolytic rate correspondingly diminished (20). Although glycolytic enzymes are localized pre dominantly in the soluble supernatant fraction of tissues, they may also be loosely bound by the mitochondria of brain, melanoma, and Ehrlich or Krebs-2 ascites tumor cells (126, 129). Mito chondrial glycolysis is responsive to insulin and anti-insulin hormones, whereas this has not been found with glycolysis by the supernatant fractions prepared from the same tumor. Under anaerobic conditions, glycolyzing homog enates of normal and neoplastic rat tissues are capable of metabolizing pyruvate at a rapid rate to yield compounds other than lactic acid. In the Flexner-Jobling tumor, essentially all the py ruvate not reduced to lactate is converted to propanediol phosphate; in homogenates of normal tissues, the decarboxylation of pyruvate to CÛ2 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. KIT ANDGRIFFIN—CellularMetabolism and Cancer 637 and a two-carbon fragment is an appreciable part to succinate of chorion and embryonic head tissues (basal Qo, = 10-20) was similar to that of the of metabolism (113). 2. Electron transport chain deficiency.—As a re tumors. sult of the rapid utilization of glucose by tumors, The above test situation represents a compari pyruvate and DPNH are generated. The DPNH son of the resting state of the tissue with that of is ordinarily reoxidized via the electron transport added substrate. Greenstein and associates (111) chain of the mitochondria, while the pyruvate compared the state with added substrate to a third state—that in which both substrate and cytois metabolized in the presence of the citric acid cycle enzymes to COa and water. When the ac chrome c were added. Here the tumors rather tivity of the electron transport chain enzymes is than the normal tissues responded with the great limiting, the pyruvate may act as an alternative est percentile increase in respiration. This third response is inversely proportional to the cytohydrogen acceptor for the DPNH, and lactate is formed. There is considerable evidence to sug chrome c content and is independent because gest that relative electron transport chain de of its excess of the cytochrome oxidase. Thus, ficiencies exist in tumors. When tissue slices were the excess of cytochrome oxidase over cytochrome incubated in the presence of Buccinate or p- c was found to be least in the case of tissues such phenylenediamine, the percentile increase in oxi as heart and kidney, greatest in the case of certain dation was generally smaller in the case of neotumors, and intermediate in other tumors and plastic tissues than normal tissues (111). However, in less active normal tissues. Malignant tissues, mouse melanomas show a far greater stimulation in comparison with normal tissues, not only pos of oxygen consumption by p-phenylenediamine sessed low concentrations of cytochrome c, but also than most other tumors (36). Normal tissues fell displayed the greatest disparity between the com ponents of the cytochrome oxidase-cytochrome into two main groups, namely: (a) tissues with high oxidative responses, liver, kidney, brain, and c system. muscle, and (6) tissues with low response to the Sensitive spectrophotometric measurements by Chance and Castor (51) of the cytochrome ctwo test substances, gastrointestinal mucosa, lung, skin, mammary gland, bone marrow, and lymphat cytochrome oxidase balance of ascites tumor cells are not consistent with the observations of Greenic tissues. Benign and malignant tumors showed a behavior closer to that of the second group. stein. Strangely, the amount of cytochrome c a As pointed out by Woods (335), not only is a was unusually great relative to cytochrome and a 3 in Krebs-2, Ehrlich, and thymoma ascites low response to exogenous succinate characteristic of certain non-neoplastic growing tissues as well cells, exceeding that of very highly respiring yeast as tumors, but the percentile response to succinate cells. The respiration of these tumors is also large. is a function of the physiological state and medium The disparity of cytochrome c to a3 was of just bathing the tissue slice. The QO2of S91 melanoma the opposite sense as that discussed by Greenstein. slices and Krebs-2 ascites cells in mouse ascites Further experiments will be required to clarify this discrepancy. On the other hand, Chance and serum were lower than slices of kidney, liver, Castor observed that the cytochrome c to cyto brain, or chorion. Embryonic head tissues were chrome b ratio was over fourfold greater than only slightly higher than the S91 melanoma. In Krebs-Ringer solution, all Q02 values were lower that of mammalian heart muscle. Cytochrome by 15-50 per cent, especially in the case of liver. b was absent from the spectra of the Krebs-2 The per cent response to succinate in Krebs-Ringer and Ehrlich ascites cells and very low in the was in order of increasing magnitude: Krebs-2, thymoma cells. Tissue homogenate studies also generally suggest embryo head, S91 melanoma, brain or kidney, and liver. The percentile succinate effect was that neoplastic tissues contain considerably less less in ascites fluid than in Krebs-Ringer solution. of the enzymes of the electron transport chain relative to liver, kidney, heart, brain, or muscle Moreover, the addition of Coenzyme I to the Krebs-Ringer solution markedly lowered the suc and of the same order of magnitude as the less cinate response but slightly increased the Qo2 active normal tissues such as spleen, thymus, or lung. Tumors in general contain relatively without added succinate. However, even in ascites serum, the Krebs-2 tumor and the S91 melanoma low levels of succinoxidase, cytochrome c, and the electron transport factor which is blocked slices were stimulated only 9 and 19 per cent, by Antimycin A (252). There is an impaired respectively, as compared with 50 and 30 per cent function of the complete "DPNH oxidase" system in Krebs-Ringer, whereas several normal tissues were stimulated 27-64 per cent in ascites serum in hepatoma 98/15 as compared with normal and 91-382 per cent in Krebs-Ringer. The response mouse liver, chiefly owing to a failure of the Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. 638 Cancer Research Vol. 18, July, 1958 cytochrome components of the respiratory chain, though a somewhat diminished diaphorase activity was also noted (199). Cytochrome reducÃ-asewas not lowered in the hepatoma. The very low total oxidative reaction exhibited by transplanted sar comas and adenocarcinomas was likewise due to a deficiency of cytochromes c and oxidase and to diaphorase. Cytochrome c reducÃ-ase activity was superior only to muscle. The activity of cytochrome oxidase in normal epidermis is much lower than in most normal tissues (47). In epidermis, which has received eighteen to 24 treatments with a carcinogen, the activity of this enzyme is increased to near ly twice that of the normal epidermis. The cytochrome oxidase drops slightly in the skin cancer as compared with the hyperplastic skin but, unlike the case of other neoplasms, remains considerably higher than its tissue of origin. If there is a deficiency of the electron transport system in tumors, the addition of an artificial electron transport system should stimulate respira tion and reduce lactate accumulation. Kertesz and Albano have shown that the addition of a terminal respiratory system composed of polyphenol oxidase and of an o-dihydroxyphenol in catalytic amounts can indeed inhibit aerobic lactic acid formation while increasing the respiration of homogenates of the Ehrlich adenocarcinoma (160). It is this simultaneous action which makes it possible to localize with much probability the intervention of the system in the reoxidation of the reduced pyridine nucleotides. The fact that in the presence of the pyocianine dyes glycolysis of tumor slices is inhibited while respiration in creases also suggests that the aerobic lactic acid formation of tumors is due to electron transport deficiency. Likewise, the addition of méthylène blue stimulates the oxygen uptake of intact Ehr TABLE 5 lich tumor cell suspensions (312). At the same time, the accumulation of lactate, ribose, and GLUCOSE METABOLISM OFEHRLICH ASCITES TUMOR CELLS* fructose is reduced (Table 5). At a concentration of 7 X 10-%, méthylène blue doubled the oxida No glucose Glucose Glucose+M.B. tion of glucose-1-C14 to C1402 by the Ehrlich (/imoles)31.80.133.170.04(Amóles)18.029.4532.14.50.62(pmoles)45.730.024.51.40.34 Measurements Û2uptake tumor without affecting the yield from glucoseGlucose utilized 6-C14. At 7 X 10-*M, méthylène blue stimulated Lactic acid accumulated C14O2yield 7 times from glucose-1-C14and twofold Ribose accumulated Fructose accumulated from glucose-6-C14 (335). There was no significant * Cells were washed with Ringer-heparin and the centrifuged cells suspended in Ringer-phosphate. Cell weight, 72.5 pentose accumulation. When added to tumor ho mg.; incubation, 60 min. (after Villavicencio and Barron, 312). mogenates, TPN+ stimulates the oxidation of gluclose-1-C14to CI4O2. Further evidence for a relative deficiency of The oxidation of isotopically labeled glucose the electron transport chain in tumors may be to C14U2by various tumors has been studied imputed from a consideration of the aerobic gly- by Kit (169). In the presence of fluoride and colysis of tumor homogenates and from the effect pyruvate, the formation of C14U2from glucoseof various dyes upon glycolysis. Although the 1-C14, glucose-2-C14, or glucose-6-C14 was stimu oxidative capacity of tumors falls in the range lated, although fluoride and pyruvate prevented of the less active normal tissues, aerobic glycolysis the labeling of alanine, aspartate, and glutamate. persists in tumors but not in the latter tissues. Since the latter observation indicates that the A homogenate system manifesting active aerobic fluoride and pyruvate were preventing the me glycolysis of hexose diphosphate was developed tabolites of radioactive glucose from entering the citric acid cycle, the C14O2was probably formed by Reif and associates (261) in which the per sistence of aerobic lactic acid formation could as a result of the oxidation of glucose via the be interpreted as proportional to DPN-cytochrome hexose monophosphate shunt. For the oxidation c reductase. It was demonstrated that heart, liver, of carbon two of glucose to take place, it is and kidney contained 2-4 times the DPN cyto- necessary for the tissue to recycle the pentose chrome-c reductase activity of spleen, the Flexner- phosphate formed in the phosphogluconate path way to a hexose molecule in which the C-2 of Jobling, Jensen, Walker, or Ehrlich tumors. Bril liant cresol blue (BCB) can act as a direct link the original glucose moiety occupies the C-l posi between diaphorase and oxygen, thereby providing tion. Also, the triósephosphate must be converted a pathway that bypasses the DPN-cytochrome to hexose phosphate by a reversal of the aldolase c reductase enzymes. For four normal tissues, reaction so that C-6 of glucose is converted to BCB had no effect on oxygen uptake, but BCB C-l. Although the oxidation of glucose-1-C14 of strongly stimulated the oxygen uptake in three thymus, spleen, or appendix cells was also in out of four tumors. creased when pyruvate and fluoride were present, Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. KIT ANDGRIFFIN—CellularMetabolism and Cancer the oxidation of glucose-6-C14 or glucose-2-C14 was either less affected or markedly inhibited. Kinoshito recently noted that, in bovine corneal epi thelium, the presence of pyruvate markedly stimu lates the hexose monophosphate shunt (164). The coupled reaction can be demonstrated in dialyzed homogenates with added TPN+ in which there occurs the conversion of pyruvate to lactate, the simultaneous utilization of glucose-6-phosphate, and the production of CC>2.Under anaerobic con ditions, the presence of pryuvate increased the COa radioactivity from glucose-1-C14 8 times but reduced to a third radioactivity in lactate. There was also an increase from 128 counts/min to 2140 counts/min of C14O2from glucose-2-C14 and from 0 to 86 counts/min in the case of glucose6-C14. The corneal epithelium to some extent can use TPN+ in place of DPN+ in the lactic dehydrogenase reaction. The mitochondrial enzyme, transhydrogenase, is absent in this tissue. It ap pears that, in the corneal epithelium, the rate of reoxidation of TPNH is the rate-limiting step in the phosphogluconate oxidation pathway. The Qo, is as great as that of liver, although the citric acid cycle may not play a prominent role. The oxidation of DPNH but not TPNH by liver mitochondria according to Kaplan (158) is as sociated with ATP production. If the same situa tion prevails in the mitochondria of cornea, it is difficult to see how the TPNH formed in the phosphogluconate oxidation pathway could be used for the production of biological energy. In the corneal epithelium, the interaction of dehydrogenases of the shunt with lactic dehydrogenase may provide a means by which ATP can be generated. This could be achieved by the following sequence of events: the TPNH formed via the dehydrogenation reaction of hexose phosphate con verts pyruvate to lactate. The lactate is then reoxidized to pyruvate with the generation of DPNH. The high-energy phosphate bonds are then generated in the oxidation of DPNH by mitochondria. In effect, the coupling of the two dehydrogenase systems would serve as a pyridine nucleotide transhydrogenase in the soluble cyto plasm. Transhydrogenase may function as a regulator of respiration and of energy-releasing reactions (158). This enzyme occurs abundantly in mito chondria of liver, kidney, heart, or muscle; at lower concentrations in brain, spleen, and testes (146); but it has not been detected in the Ehrlich (333) or Novikoff tumors (262) or the cornea. In the mitochondria, the shifting of electrons from TPNH to DPNH would permit oxidative phosphorylation, while the shifting of electrons 639 from DPNH to TPNH would result in a lowering of phosphorylation but an increase in the "reduc ing environment" of the cell. The absence of the transhydrogenase enzyme, together with a very active phosphogluconate pathway, as is ob served in tumors, might lead to the same situation. Although DPN+ is present chiefly in the oxi dized form in tissues, TPN+ is present chiefly and sometimes exclusively in the reduced form. The ratio of DPN+/DPNH ranges from about 1.2 in liver to over 20 in skeletal muscle. Tumors display intermediate values of this ratio of from 2.5 to 4.5. The total DPN+ is in all cases consid erably higher than the total TPN+ (100). In mitochondria, the ratio of reduced to oxidized DPN+ is normally maintained at a low value by the rapid rate of electron transport to molecular oxygen by the electron transfer chain enzymes. It therefore seems unlikely that reductive syn thetic processes could be effectively carried out under these unfavorable conditions. Since the ratio of reduced to oxidized pyridine nucleotides is relatively high in the intact cell, it is probable that reduced nucleotides predominate in the extramitochondrial portion of the cell (152). This is in keeping with the observation that externally added pyridine nucleotides are oxidized very slow ly by intact mitochondria and that other oxidative pathways available to reduced pyridine nucleo tides in the soluble portion of the cytoplasm are slow and rather ineffective when compared with the highly integrated and effective electron trans port chain of the mitochondria. DPN+ is synthesized in the nucleus of the cell. Since many cytoplasmic dehydrogenases are DPN+-dependent, the rate of supply of products derived from an enzyme system localized in the nucleus may markedly influence cytoplasmic re actions; the disturbance of such a system might well have a profound influence on the behavior of the cell, particularly on cell division and dif ferentiation. The rate of synthesis of DPN+ by mammary tumor nuclei is only about one-fifth that of nuclei from lactating mammary gland and one-third that of gland from nonlactating mice. The activities of the enzymes from fetal and very young mouse liver were also very much less than those from the livers of adult mice. These results suggest that a decreased rate of DPN+ synthesis may be one aspect of rapid cell proliferation (30). 3. The citric acid cycle.—The metabolism of acetate, pyruvate, glucose, and other metabolites both in vivo and in vitro have been studied by a number of investigators. The patterns of sub strate utilization differ markedly in many tumors Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. 640 Cancer Research from that of most normal tissues. The tumor utilization pattern is frequently but not always characterized by a diminished tendency to metab olize substrates through the citric acid cycle. a) In vivo experiments: Busch and co-workers administered acetate-1-C14 to tumor-bearing rats. For most normal tissues, the half-time for dis appearance of acetate-1-C14 was 6-15 seconds and for liver it was 48 seconds, but, for the three transplantable tumors, it was 4f minutes. In nor mal tissues, the nonvolatile compound with most activity was glutamate, with smaller amounts in aspartate and succinate. These substances ac counted for 33-75 per cent of the total radioac tivity in nontumor tissues and 4-6 per cent of the isotope in the tumors at 1 minute after the injection of the labeled acetate. In the tumors at the end of 1 minute, 3 per cent of the radio activity was in COj, but in the nontumor tissues 6-43 per cent was in COz (40). The metabolism of acetate through the citric acid cycle can be blocked by injecting malonate prior to acetate1-C14.By 2 hours after the institution of a mal onate block, the Flexner-Jobling tumor accumu lates an average of 4-6 /¿molesof succinate/gm tissue, as compared with a control value of less than 0.1 Mmole. Presumably, the succinate arises from glutamate (43). In heart, lung, spleen, liver, and muscle, the succinate pool accounts for 10-30 per cent of the acetate-1-C14 radioactivity; most of the counts of the Flexner-Jobling tumor remain volatile even 30 minutes after injection. The in vivo findings with the Flexner-Jobling tumor were confirmed with the Jensen sarcoma, Walker 256 tumors, and a lymphosarcoma either in the pres ence or absence of malonate (38). The experiments were repeated with pyruvate-2-C14 as substrate (39). The primary metabolic pathway of pyruvate2-C14leads to alanine, glutamate, aspartate, and lactate in nontumor tissues. In the tumors, the ratio of isotope in lactate to amino acids was 20:1, while the ratio for other tissues was 1:1 or less. When tumor-bearing animals were poi soned with fluoroacetate, citrate accumulated in almost all normal tissues but not in a number of tumors. However, citrate did accumulate in hepatoma 98/15. 6) Slice experiments: Pyruvate metabolism by slices of the Walker or Jensen tumors and a uterine carcinoma was studied after adding glu cose, glutamate, or both to the incubation medium (42). Adding glucose to the tumor slice medium changed the products of pyruvate-2-C14 utiliza tion from 14 per cent in COj, 64 per cent in lactate, and 21 per cent in amino acids to 92 per cent in lactate and 5 per cent in amino acids. Adding Vol. 18, July, 1958 glucose to liver or kidney slices resulted in no significant changes in the metabolite pattern. However, in the presence of glutamate, liver slices transferred 71 per cent of the total isotope to alanine, while kidney slices increased the labeling of glutamate at the expense of CI4C>2and lactate. Brain slices converted 66 per cent of pyruvate2-C14 to amino acids under control conditions, but more isotope to lactate in the presence of glucose and more to alanine in the presence of glutamate. Spleen cells gave a greater per cent of the isotope in amino acids in control media and a greater per cent in COt and in amino acids in the medium to which glucose was added. Tu mors used pyruvate primarily as a hydrogen ac ceptor rather than for amino acid synthesis. After incubation with lactate-2-C14, the ratio of radioactivity in free aspartate :glutamate :ala nine was as follows: 0.2:1.0:2.0 in lymphosarcoma cells and 0.9:1.0:0.6 in spleen cells (178). Both normal and neoplastic lymphatic cell suspensions were capable of oxidizing aspartate-4-C14, glutamate-2-C14, succinate-2-C14, or acetate-2-C14 to C14C>2, of converting these substrates to dicarboxylic amino acids, and of incorporating the label into cell protein (166). Transamination was ex tremely active in all the tissues (170, 172). The failure to metabolize lactate to aspartate was not owing to a complete inability of the tumor cells to effect this conversion. The specific activity of aspartate was as great as that of glutamate with acetate-2-C14 as substrate (166). The addi tion of unlabeled succinate increased the conver sion of acetate-2-C14 to amino acids by the Gardner lymphosarcoma. After incubation with succinate2-C14, the specific activity of aspartate increased to 2-5 times that of glutamate in all the tissues. On the other hand, when both succinate-2-C14 and nonlabeled glucose were present, considerably more glutamate was formed, so that the pattern of radioactivity resembled that found in the ex periments with acetate-2-C14 alone. The failure of citrate to accumulate in tumors in fluoroacetate-treated animals under in vivo con ditions is also not owing to an inherent inability of tumors to form citrate. Tumors contain ample condensing enzyme. Even at room temperature, in the presence of oxalacetate plus acetate, lympho sarcoma cells form citrate, and the addition of fluoroacetate to the medium enhances citrate ac cumulation only slightly (177). However, the net citrate of spleen cells increases little at the end of a 2-hour incubation unless fluoroacetate is added. Possibly, citrate synthesis exceeds utiliza tion in the lymphosarcoma cells. The capacity of transplantable tumors and normal tissues to ac- Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. KIT ANDGRIFFIN-—Cellular Metabolism and Cancer cumulate citrate in the presence of excess sub strate and high atmospheric oxygen tension has been demonstrated by Busch (41). The oxidation of long- and short-chain fatty acids, lactate, and glucose to C14O2 by slices of transplanted mouse or rat tumors at rates com parable to most normal tissues has been adequately demonstrated by Weinhouse and coworkers (323, 325). Differences between liver and tumor slices with respect to oxidation of fatty acids to CO2 are greater with the short-chain fatty acids than with the long-chain fatty acids. Tissue slices of a number of tumors are considerably less effective than most normal tissues in metabolizing acetate to C02 and to dicarboxylic amino acids. The Gardner lymphosarcoma is a partial exception to this rule (166). Lipogenesis from glucose or acetate takes place in several neoplastic tissues; however, the rate is probably too slow to meet the needs of the tumor, so that the tumor is more dependent on blood-borne intermediates than on internal sources (218). Pathways of substrate utilization are in general quite different as between hepatoma and liver slice. Rat hepatoma slices metabolize glucoseTJ-C14 to protein, CO2, and lipide faster than do normal liver slices. With pyruvate-2-C14 as substrate, there is much less gluconeogenesis by hepatoma, more label in protein, and about the same in lipide and CO2 as in normal liver (341). Radioactive butyrate and octanoate are oxidized by mouse hepatoma, C954, about as well as normal liver and kidney (55). Octanoate is used in prefer ence to acetate by this hepatoma. With either octanoate-1-C14 or acetate-1-C14 as substrate, the ratio of label converted to glutamate as compared with glutamine was 6.7 in hepatoma but 0.6 in normal liver. Normal liver converted acetate to glucose, but hepatoma failed to carry out this conversion. Hepatoma was more active than host liver in glucose utilization. When acetate-2-C14 was employed as substrate instead of acetate-1-C14, (33) more isotope was found in hepatoma alanine and lactate. c) Enzymes: Assays of citric acid cycle enzymes do not generally reveal marked deficiencies of tumor enzymes as compared with normal tissues (328). Studies with homogenates show that the limiting enzyme or group of enzymes in the oxidative complex are present in tumors in amounts roughly equivalent to the levels in spleen, thymus, lung, and embryonic tissues and at levels generally lower than that of liver, kidney, or heart. Although aconitase activity may be low in tumors, it is known that aconitase is a very unstable enzyme. Therefore, it is not certain that the low values 641 observed represent a real deficiency or whether its inactivation is more rapid in the tumors than in normal tissues (324). Since experiments with tissue slices show manifestly altered metabolism, it is necessary to examine the restraints of oxidative or glycolytic activity imposed upon both normal and tumor tissues. 4. Metabolic restraint as a function of phosphate acceptors.—The rates of glycolysis and respiration are determined by the availability of phosphate and phosphate acceptors (254). The oxygen con sumption of slices can be increased considerably by the addition of low concentrations of dinitrophenol (330), a substance believed to stimulate "ATPase" activity by effecting hydrolysis of a high-energy phosphate compound formed during oxidative phosphorylation (53). In this way, the Qo: of tumor cells can be increased up to 22 (335). The stimulating effect on respiration of phosphate acceptors can be shown directly on mitochondria. The rate of respiration of heart, muscle, or liver mitochondria is greatly stimulated by adding hexokinase plus glucose, creatine plus creatine kinase, dinitrophenol, or ADP and in organic phosphate. High dinitrophenol concentra tions inhibit respiration, presumably by promot ing excessive ATP breakdown. Tumor homoge nates differ with respect to the balance of ADP/ ATP (254). In homogenates of the Flexner-Jobling tumor, dinitrophenol markedly inhibits respira tion, since ATP breakdown is already high, while fluoride preserves respiration; but mitochondria from hepatoma 98/15 oxidize a-ketoglutarate, succinate, or glutamate in the absence of fluoride with a P/O of 1-2.5. Respiration of tumor slices or cell suspensions can be severely inhibited, im mediately after the addition of glucose, mannose, or fructose (Crabtree effect) (193, 216). In the presence of glucose, méthylèneblue accelerates the oxygen consumption of tumors beyond the control level (Table 5) ; in the absence of glucose, méthylèneblue causes a progressive inhibition. The inhibition is sensitive to the phosphate con tent of the media. Ehrlich tumor respiration is inhibited by glucose concent rations above 30 mg. per cent at a phosphate concentration of 0.02 M; below 30 mg. per cent, there is stimulation. Studies of aerobic glycolysis demonstrate that the glucose had disappeared and that the appear ance of lactate was maximal just before the release of the inhibition. Raising the concentration of orthophosphate to 0.055 M reduced the inhibition markedly (31). At very low glucose concentrations, the oxida tion of glucose-6-C14 to C14U2 is only slightly less than that of glucose-1-C14. When the glucose Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. 642 Cancer Research Vol. 18, July, 1958 concentration is raised to the level that inhibits mand on the adenine nucleotides by an active endogenous respiration, C14Oifrom ghicose-1-C14 extramitochondrial system of glucose utilization is about twice and from glucose-6-C14 about half as observed in tumor cells may be responsible that obtained at low glucose concentrations (259). for the inhibition of mitochondrial respiration Thus, high glucose concentrations inhibit oxida (Crabtree effect). tion via the Krebs cycle but increase the oxidation The role of phosphate balance in homogenate glycolysis has been convincingly shown by Myerof carbon 1 of glucose by the shunt. This stimula tory effect of high glucose concentrations was hof and Wilson (228). With only catalytic amounts masked in the measurement of oxygen uptake of hexose diphosphate and no fluoride, a QLA from glucose of 50-70 could be obtained in sarcoma because of the pronounced inhibition of endog enous respiration. At high glucose concentrations, homogenates by adding octyl alcohol to inhibit dinitrophenol has only little effect on the oxida a powerful ATPase. The ATPase excess in chick tion of C-l of glucose but has a marked stimulatory embryo over hexokinase is low; therefore, glu cose is glycolyzed well with QLA of 10-25. In effect on the oxidation of C-6. a third situation, it was shown that most of the Glucose greatly accelerates amino acid incor poration into the protein of tumors (83, 177), brain ATPase is particulate-bound. By centrifugeven though respiration may be inhibited owing ing away this ATPase, a phosphate balance was to the Crabtree effect. Glucose can increase the achieved in brain homogenates whereby the QLA transfer of isotope from glutamate-U-C14 to the was 40-50. Insulin and anti-insulin hormones may also protein of Walker tumor slices while decreasing the C14O2production by 57 per cent (234), but, control glycolysis by controlling the entry of gluwhen added to slices of normal tissues, it does close into the cell. Tumors differ in their sensitivity to insulin and anti-insulin hormones. The Krebsnot affect the over-all metabolic pattern. Using sensitive spectroscopic methods, Chance 2 ascites tumor is very insensitive, melanoma S91 relatively sensitive, and amelanotic melano and Hess (52) observed that the endogenous res piration of ascites tumor cells is accelerated by ma, S91A, intermediate in sensitivity (148). The growth of the metabolically sensitive tumor but glucose addition about twofold for approximately a minute, then an inhibition of both respiration not the insensitive tumors can be inhibited by and glucose metabolism occurs to an extent of anti-insulin hormones produced during stress. ten- to 20-fold. The inhibition can be partially Purified enzymes and coenzymes of glycolysis reversed by the uncoupling agent of phosphoryand 5 mg. of rat liver mitochondria protein were lation, dicoumarol. The initial, actively metab combined in a model system (94). The addition olizing state is characterized by a high intracellular of the glycolytic enzymes and glucose inhibited ADP level due to hexokinase activity and the the oxidation of glutamate by the mitochondria, inhibitory phase by a very low level of either but lactate formation was only partly inhibited. phosphate acceptor or phosphate. With 10 mg. of mitochondrial protein, there was The Crabtree effect (inhibition of respiration no inhibition of respiration, while lactic acid for by glycolysis) is the inverse of the Pasteur effect. mation was inhibited 80 per cent. Hexose uptake It has been suggested that the limiting factor was unchanged, but hexose diphosphate accumu in both extramitochondrial glucose utilization and lated. At low orthophosphate concentrations, mi intramitochondrial respiration is the concentration tochondria inhibited glucose uptake and lactate of adenine nucleotides (259). Possibly, adenine formation. The addition of glucose and hexokinase nucleotides are held in the mitochondria during and catalytic amounts of ADP to respiring mito the oxidation of pyruvate, thus diminishing their chondria stimulated respiration. At low ADP con availability for extramitochondrial glucose phos- centrations (3 X 10~4M) adding glucose plus a phorylation. According to this theory, the Pasteur complete glycolytic system inhibited respiration effect can be interpreted as an impairment of (95). There was no inhibition, however, at high the hexokinase reaction owing to the lack of ADP concentrations (2 X 10~3M) or when gly adenine nucleotides at the site of glucose phos- colysis was prevented by leaving out phosphophorylation. Both dinitrophenol and méthylènefructokinase. The inhibition of respiration could blue can uncouple mitochondrial phosphorylation also be relieved by adding dinitrophenol. At the leading to an increase in extramitochondrial ad same time, lactate production increased. When enine nucleotides. The adenine nucleo tides become the two systems compete for both ADP and orthoavailable for the glycolytic process, accelerating phosphate, a pronounced inhibition of hexose up glucose ulitization and thus eliminating the Pas take also takes place. Aisenberg and associates teur effect. On the other hand, an excessive de (4, 5) have studied an analogous system, the Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. KIT ANDGRIFFIN—CellularMetabolism and Cancer coupling of rat liver mitochondria with the super natant enzymes from tumor or rat brain. The model systems described above demonstrate mu tual competition between respiring mitochondria and glycolytic enzymes for limiting amounts of adenine nucleotides. Adenine nucleotides are also required for transphosphorylation reactions. As emphasized by Pot ter (252), the same nucleotides that coordinate fuel consumption with functional load are building blocks of ribonucleic acid. Consequently, a com plex competition may be envisioned, the vectorial resolution of which determines the pattern of metabolism of the cell. To summarize, there is suggestive evidence that certain rate-limiting enzymes of glycolysis may be present in excess, while certain respiratory enzymes may be limiting in many cancer cells. There is considerable evidence for relative defi ciencies of the electron transport system. However, the evidence is not decisive. The concept of War burg that carcinogens specifically damage the re spiratory apparatus of the cell "so that both structure and respiration disappear .. . and that the respiration connected with the grana remains damaged" (317) in particular requires experiment al verification. However, a change in the amounts of key enzymes together with changes in the prop erties of other enzymes, such as substrate affinity or susceptibility to regulation by endocrine fac tors, would presumably result in a marked meta bolic imbalance. Although the detailed mechanism of the cancer imbalance remains unknown, its reality is not questioned. In particular, the im balance between respiration and glycolysis, first emphasized by Warburg, remains the foundation stone of biochemical investigations in cancer (316, 326). B. CARBOHYDRATE METABOLISM AND AMINOACIDBIOSYNTHESIS The structural organization and glycolytic ac tivity of neoplastic cells also have implications with respect to the biosynthesis of: (a) amino acids, (6) pentose compounds, and (c) deoxyribonucleosides. 1. Amino adds.—The capacity of tumor cells and most normal cells to synthesize amino acids is limited. There is a qualitative similarity in the amino acid requirements of a wide variety of human cells in tissue culture deriving from both normal and malignant tissues, including em bryonic and adult cell lines (72, 74). All protein amino acids are required, with the exception of alanine, glycine, serine, aspartate, glutamate, and proline. In the absence of the essential amino 643 acids, cytopathogenic changes develop which cul minate in the death of the cells. The concentration of the individual amino acids necessary for opti mum growth may vary from strain to strain, and minor strain differences with respect to additional components may exist. Thus, rabbit fibroblasts have an additional requirement for serine, while Walker 256 tumor cells require asparagine (215). However, no consistent differences have been noted in this respect between the lines derived from nor mal and from malignant tissues. Glucose, salts, se rum protein, and a number of vitamins are also needed for growth. The vitamins include nicotinamide, pyridoxal, thiamine, riboflavin, pantothenic and folie acids, and choline. Myo-inositol is also a required vitamin for twenty human cell strains, a mouse sarcoma, but not a mouse fibroblast strain (73). Although a requirement for biotin and for vitamin Bi2 has not as yet been proved, these vitamins may also be essential but may be present as trace contaminants in components of the medium. A recent report by Woolley sug gests that the requirement for vitamin BIJ of mice bearing mammary cancer is considerably reduced, as compared with that of normal mice (338). It is not certain whether the tumor-bearing animals can synthesize vitamin BU or are better at trapping and storing the vitamin. The nutritional experiments described above are supported by isotope experiments on amino acid biosynthesis. When normal lymphatic cells or tumors were incubated with isotopically labeled glucose, radioactivity was found only in alanine, glycine, serine, glutamate, aspartate, and in proline (175). Glutamine of brain and diaphragm contained considerable radioactivity, as did brain •y-aminobutyricacid. Tumor proline contained much more radioactivity than did the proline of normal tissues. In the lymphatic tumors and in Ehrlich tumor cells, levels of radioactivity of serine, glycine, and alanine were high relative to glutamate and aspartate. In all the normal tissues, little radioactivity was found in serine and glycine, although biosynthesis was studied under a variety of conditions. Similar results were obtained when amino acid biosynthesis from glycerol-l-C14 was studied in lymphosarcoma or nor mal lymphatic cells (176) (Chart 5). In lympho sarcoma cells, a high percentage of the glycerol was converted to serine and glycine and the prod ucts of their utilization; in normal lymphatic cells, more of the radioactivity was found in aspartate, glutamate, and C14Oi.The relative pat tern of amino acid biosynthesis observed in vitro was also obtained in in vivo experiments. Although both normal and tumor cells con- Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. Cancer Research 644 verted acetate-2-C14 to glutamate and aspartate, lymphosarcoma cells incorporated far more of the acetate-2-C14 to proline, serine, and glycine than did normal cells (167). Serine and glycine are required as building blocks of purines, the methyl group of thymine, porphyrin, glutathione, creatine, ethanolamine, and other cellular com ponents. An enhanced capacity to form these amino acids would have "survival value" for the tumors. Serine and glycine are formed directly from intermediates of glycolysis. Phosphoglycerate, phosphohydroxypyruvate, and phosphoserine (149, 168) are proximal serine precursors. UTILIZATION OF GLYCEIOl-l-CU NEOPIASIIC IY UMPHATIC «ORMAI AND TISSUiS ».0 1.5 1.0 7.5 7.0 4.5 6.0 S.S 5.0 4. 5 4.0 ' 3.5 3.0 - 2.5 - 2.0 I.S 1.0 0.5 0 EXP J 171« I7lb 17«o 179b 110 Chart 5.—Thefollowing abbreviations are used in the chart: T = lymphosarcoma 6C3HED; Sp = mouse spleen cells; Thy = rat thymus cells; App = rabbit appendix cells; Lip = lipides; Pro = protein; Asp = aspartic acid; Glu = glutamic acid; Ala = alanine; Ser = serine; Gly = glycine (Kit and Graham [176]). Also consistent with the nutritional experiments is the fact that no radioactivity is found in tumor glutamine. It is known that exogenous glutamine is very rapidly utilized and decomposed by tumors (266, 267). When labeled glutamine is added to the medium of human carcinoma cells (HeLa) growing in tissue culture, the glutamine is in corporated into the cell protein without prelimi nary degradation (206). Virtually all the glutamine residues of newly synthesized protein arise from the glutamine of the medium. Glutamine and glutamate act independently in protein synthesis, each serving as the direct precursor of its cor responding residue in the protein. 2. Peritose formation.—Pentose compounds are required for the synthesis of nucleic acids and Vol. 18, July, 1958 coenzymes. There are at least two metabolic path ways by which glucose can be converted to ribose: (a) the aerobic phosphogluconate pathway which involves two TPN+-linked dehydrogenases and (b) the anaerobic transketolase-transaldolase path way. Carbon 1 of glucose is lost as COj during the conversion of glucose to ribulose phosphate by the former pathway. Carbon 6 of glucose is not appreciably oxidized to COauntil the glucose is metabolized to pyruvate and the latter passes through the citric acid cycle. By comparing the difference between the yield of C14O2from glucose1-C14 and glucose-6-C14 at short time intervals, an estimate can be made of the capacity of tissues to convert exogenous glucose to pentose via the phosphogluconate pathway. It was estimated that tumor cells formed 2-5 times as much pentose from glucose as normal lymphatic cells (169). The preferential oxidation of glucose-1-C14 to C14Osas compared with glucose-6-C14 is evidence that the phosphogluconate pathway is present in a variety of tumors (2, 80). No preferential oxidation is, however, observed in rat diaphragm, brain, heart, or kidney slices. Enzymes of the phosphogluconate pathway are exceedingly active in adrenal tissue, lymphatic tissues, and mammary tissue and in whole rat embryos in the early stages of development, but these enzymes are low in skeletal muscle or cardiac muscle (11, 312, 331, 333). The levels of the dehydrogenases increase rapidly in lactating mammary tissue from the end of pregnancy to the end of lactation and then fall to very low levels in the involuting mammary gland (98, 101). Consistent with this is the finding that the ratio of radioactivity of C14U2derived from glucose-1-C14as compared with glucose-6-C14 is 1.1 in mammary tissues at the end of pregnancy, 15.7 at 10-18 days of lactation, and 2.1 at the beginning of involution of the mammary tissue. The ratio of CHO2radioactivity is lower for mammary carcinoma than for lactating mammary tissues, though still appreciable (1). The radioactivities of the fatty acids, acetoacetate, acetate, lactate, or alanine have been measured after tissues were incubated with labeled glucose. In this case, more radioactivity was ob served in the alanine after incubation with glucose6-C14than with glucose-1-C14. Radioactivity from glucose-1-C14 can reach the former compounds primarily as a result of the glycolytic pathway, but radioactivity derived from glucose-6-C14 can reach the fatty acids, alanine, or lactate as a result of the metabolism of glucose via the glycolyt ic pathway and also the direct oxidative pathway, provided that pentose phosphate is further de graded to the pyruvate level by the enzymes, Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. KIT ANDGRIFFIN—CellularMetabolism and Cancer transketolase and transaldolase. The excess of radioactivity found in the three carbon compounds or the fatty acids thus provides a means of esti mating the per cent of three carbon compounds derived via the shunt or via the glycolytic path way. It has been estimated that 13-23 per cent of the three carbon compounds of spleen and tumor cells are derived from glucose via the shunt (169). The corresponding values were 25-58 per cent for hepatocarcinomas and lactating mammary tissue and 12-25 per cent for mouse ascites tumors (331). The transketolase-transaldolase pathway has been studied by measuring the conversion of vari ously labeled glucose molecules to the ribose of RNA (179). With normal as well as with malignant lymphatic cells, the incorporation of C14was high est when glucose-2-C14was the substrate, although as much radioactivity was incorporated from glucose-l-C14 as from glucose-6-C14. Approximately 31 and 79 per cent of the radioactivity was due to carbon 5 of ribose when glucose-1-C14 and glucose-6-C14were the respective substrates. With these substrates as well as with glucose-2-C14,only 1-3 per cent of the radioactivity of free serine, alanine, glycine, or lactate was due to the carboxyl carbon atoms. The results suggest that approxi mately half of the RNA purine nucleotide ribose may have been derived by the transketolasetransaldolase pathway in these tissues. Evidence for an active participation of the transketolasetransaldolase pathway in tumors has also been obtained by Hiatt (127), who studied ribose bio synthesis by human HeLa cells grown in tissue culture, and by Wenner et al. (327), who demon strated the active labeling of ribose phosphate by Ehrlich tumor cell suspensions which had been incubated with glucose-1-C14.Enzymes of pentose utilization are 2-5 times as active in lymphosarcoma cells as in normal appendix cells (312). The Flexner-Jobling tumor actively converts glucose-1-C14 to the pentose of RNA and of DNA (275, 276). At 1 hour after the intraperitoneal injection of glucose-1-C14 the RNA of the tumor contained 2,000 counts/min/gm of tissue, while at 5 hours there were 4,380 counts/min/gm tissue. The corresponding values for the livers from the same animals were 0 and 534 counts/min/gm tissue, respectively. The DNA of the tumors con tained 650 and 2,260 counts/min/gm at 1 and 5 hours, respectively, while the DNA of the liver did not incorporate significant levels of C14 at any time. The exact mechanism by which glucose is con verted to deoxyribose is not as yet known. Pos sibly, ribose is an intermediate in the synthesis 645 of deoxyribose from glucose. The evidence suggests that ribose is reduced to deoxyribose while bound in N-glycosidic linkage to purines or pyrimidines (260, 269, 270). Totally labeled cytidine, uridine, deoxycytidine, and thymidine were separately in jected into partially hepatectomized rats, and their incorporation into the pyrimidine part and the sugar moiety of polynucleotide pyrimidines was studied. After injection of labeled pyrimidine ribosides, the ratio between the specific activities of pyrimidine and deoxyribose of the isolated deoxyribosides was very close to the corresponding pyrimidine/ribose ratio of the precursor, indicating a relatively direct conversion of the ribosides to deoxyribosides. fOX. TfffA cerf) H Y L •TNfA r -r 3ftìt"£ | l (V.)—-> HOCHf "KCT/lfe C-/" COr f)-¿t i* HOCH¿ OH C Or ")-ff --V-O *" C?' <""/ \f-MMjl CHART6.—Schematic representation of terminal steps in thymidine biosynthesis. Abbreviations are: C = cytosine;U = uracil; MeC = methyl cytosine; T = thymine; R = ribose; Dr = deoxyribose; P = ortho phosphate; HOCH2-DHUDr = 5-hydroxymethyl-dihydrodeoxyuridine; THFA = tetrahydrofolic acid. 3. Thymidine biosynethesis.-—Terminal steps in the biosynthesis of thymidine (thymidylate) are schematically depicted in Chart 6: An "activated hydroxymethyl" donor reacts with a pyrimidine acceptor to yield the thymidine precursor. For mate, formaldehyde, the beta carbon of serine, or the methyl group of methionine may function as the one-carbon donor compound (171, 258). The activation of the one-carbon donor is me diated by tetrahydrofolic acid, so that NK>hydroxymethyltetrahydrofolic acid is the probable biological reactant with the pyrimidine acceptor compound. Either deoxyuridine or deoxycytidine are potential pyrimidine acceptor compounds. The conversion of uridine or cytidine to DNA-thymine occurs readily, although the reverse of this, the conversion of deoxyribonucleosides to ribonucleosides, does not take place (260). The addition of cytidine, deoxyuridine, or deoxycytidine greatly stimulates the conversion of one-carbon donor compounds to acid-soluble thymine compounds and to DNA-thymine in lymphatic tissues and tumors (171). Thymidine synthesis has been re cently demonstrated in enzyme extracts obtained Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. 646 Vol. 18, July, 1958 Cancer Research from bacteria or from thymus. In each case, tetrahydrofolic acid (THFA), reduced pyridine nucleotide, Mg**, ATP, serine or formaldehyde, and the pyrimidine acceptor compound were re quired (89, 247). According to Blakley (26), deoxyuridine is the pyrimidine acceptor compound; however, other investigators suggest that deoxyuridylic acid is the reactant (85, 89, 247). The product of the reaction may be hydroxymethyluracil deoxyriboside(tide) or a tetrahydrofolic acid derivative of the latter compound (85, 89). In the former case, a reduction followed by a de hydration and rearrangement of the molecule would result in the formation of thymine deoxyriboside(tide). In the latter case, a hypothetical folie acid-pyrimidine intermediate would yield thymidylate and THFA by a process of reductive cleavage similar to that described for the formation of acetic acid from glycine. The hydroxylation step probably precedes the reductive step (85). The thymidylate may next be phosphorylated to thymidine triphosphate and utilized for DNA synthesis (189). The possibility that deoxycytidine may also function as an acceptor pyrimidine in tumor tis sues was recently investigated. Hydroxymethylcytosine is a component of phage DNA, while methylcytosine occurs in the DNA of animal and plant tissues. The conversion of deoxycytidylic acid to hydroxymethyldeoxycytidylic acid in the presence of formaldehyde, THFA, and an enzyme derived from T-6 phage-injected E. coli and the enzymatic deamination of methylcytosine deoxyriboside to thymidine have been demon strated by Cohen and associates (107, 108). Lym phatic cell suspensions or tumors were therefore incubated in the presence of formaldehyde-Cu and a pool of nonlabeled methylcytosine deoxy riboside. The total labeling of the free thymidine, thymine, and thymidylate was not reduced by the presence of the methylcytosine deoxyriboside, thus contra-indicating an obligatory role of the latter compound as an intermediate in thymidine synthesis. Deoxycytidine is probably metabolized to deoxyuridine rather than to methylcytosine deoxyriboside in the tumors. C. GLUCOSEUTILIZATIONAND REDUCTIVESYNTHESIS The reactions shown in Chart 6 are terminal steps in DNA synthesis. It is of interest that four to five of these steps are reductive reactions: (a) the conversion of folie acid to THFA (92), (6) of formyl folie acid to hydroxymethyl tetra hydrofolic acid (108), (c) the hypothetical reduc tion of cytidine to deoxycytidine, and (d) the reductive step in thymidine synthesis. Reduced triphosphopyridine nucleotide is required for the reduction of folie acid by chicken liver enzymes, while dihydrofolic acid was readily reduced by either TPNH or DPNH (92). Reduced triphos phopyridine nucleotide is also required for the reduction of formyl tetrahydrofolic acid to hy droxymethyl tetrahydrofolic acid by a partially purified beef liver enzyme (117) and in the enzy matic synthesis of thymidylate from doexyuridine (26, 89, 247). Since the active utilization of glucose by tumors results in an excess of reducing power, the conditions of tumor metabolism are probably favorable for thymidine synthesis. Indeed, the incorporation of formate or formaldehyde into acid-soluble thymidine compounds proceeds as well or better in tumors anaerobically as aerobically. One wonders whether other key anabolic reac tions are favored by an "excess" of reduced coenzymes? The availability of reduced pyridine nucleotides might in part account for the high en dogenous concentration of proline and the active conversion of radioactive precursors to proline by tumors. There are two reductive steps in the synthesis of proline from glutamic acid: (a) the reduction of glutamate to glutamic semialdehyde and (6) the reduction of pyrroline carboxylic acid to proline (300, 313). DPNH is essential for the latter step (292), and it is quite possible that a pyridine nucleotide is also required for the first step. It may also be significant that glutamic acid can be formed by a pyridine nucloetide-catalyzed reductive amination. Glutamic dehydrogenase ac tivity is several times higher in white blood cells from patients with leukemia and other cancers than in leukocytes of normal individuals or in dividuals with a variety of other diseases (314). D. SULFHYDKYL COMPOUNDS AND CELL DIVISION The idea has long been held that sulfhydryl groups are particularly important in cell division. Support for this idea has come from three lines of evidence: (a) the strong nitroprusside reaction of a number of proliferating tissues, (6) the in hibition of division by thiol poisons, and its re versal by cysteine, glutathione, or thioglycollate; and (c) the fall and rise in concentration of soluble thiols prior to cleavage in the fertilized sea urchin eggs (296). Soluble thiol compounds (largely, though not entirely, due to glutathione) also in crease prior to cell division in plant cells. Gluta thione and ascorbic acid have been demonstrated in cell nuclei (297). Glutathione can be reduced by a reductase utilizing TPNH, suggesting that the excess of reducing power found in tumor Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. KIT ANDGRIFFIN—CellularMetabolism and Cancer tissues may also be optimal for mitotic division. Glutathione reducÃ-ase is very active in Ehrlich asci tes tumor cells (333), but this enzyme is present only to a minor extent in the nucleus. During the phase of accelerative growth of the Murphy lymphosarcoma in rats in which this tumor ulti mately regresses, there is a marked decrease in the sulfhydryl content of the plasma. As the tumors regress, plasma sulfhydryl levels return to normal (286). Plasma sulfhydryl levels have been reported to be low in human cancer patients. In human leukemia, the observation has been made and disputed that leukemic cells have a higher glutathione content than normal leukocytes (287). The growth of lymphocytic leukemic asci tes tumors in mice is accompanied in the later stages by a progressive fall in liver glutathione levels. When administered to mice bearing large tumor growths, A-methopterin inhibited growth and pro duced a marked rise in liver glutathione above normal levels. Nickerson and Falcone (229, 230) have recently found that the reduction of mannan protein disulfide groups of the inner layer of the cell wall is essential for cell division in yeast. These in vestigators call the mitochondrial enzyme which catalyzes this reduction a division enzyme. The disulfide reducÃ-ase is lacking in a yeast mutant which cannot divide. Of interest in connection with cell division is the action of 6-furfurylaminopurine (Kinetin), an adenine derivative first obtained from DNA of herring sperm, which accelerates cell division in plants at a concentration of 100 parts per million provided that indoleacteic acid is also present (222, 223). In the onion root tip, Kinetin promotes mitotic division and induces polyploidy and various forms of pyknosis. Kinetin increases the frequency of mitotic figures in Yoshida sar coma cells (235). The control of cell division has recently been reviewed by Swann (306). V. CONCLUSIONS Populations of neoplastic cells are characterized by profound aberrations with respect to the func tion and morphology of the cell nucleus. Cytological and biochemical evidence supports Boveri's suggestion that the hereditary determinants of tumors differ quantitatively from those of normal cells. There is frequently observed a gross re modeling of the chromatin content of the cancer cells which must entail far-reaching genetic con sequences with respect to cytoplasmic structure and function. The studies of Bendich and others suggest that, as the resolving power of biochemical tools are increased, further subtle differences in 647 the chromatin profiles of tumors and somatic tissues may be observed. The genetic information determining the struc tural and functional characteristics of the cell is probably associated with deoxyribonucleoproteins (DNP). Important strides have been made toward an understanding of the biosynthesis of the building blocks of DNP. A reciprocal relation ship exists between the effects of nuclear function and cytoplasmic function. The cell cytoplasm pro vides the building blocks, accessory factors, and possibly part of the energy required for chromo some and DNP replication. It is not illogical to suppose that the environment prevailing in the cytoplasm may affect the functional activities of the genetic determinants. However, in a subtle but unknown way, the DNP-genes replenish the cytoplasm with essential key components, in the absence of which pathological cytoplasmic changes occur. There have been interesting suggestions that ribonucleoproteins are mediators in nuclear-cytoplasmic relations. The ribonucleic acids of both the nucleus and the cytoplasm are heterogeneous. Experiments with viruses demonstrate that RNA may in some situations transmit genetic informa tion. Some and possibly considerable functional autonomy must be attributed to cytoplasmic organelles. In plant tissues, mitochondria may pos sess extranuclear hereditary factors which through their mutation become the continuing cause of selfperpetuating cellular abnormalities (337). That analogous factors are of significance with respect to cancer in animals has not, however, been proved as yet. Abnormalities can also result from the action of viruses on mitochondrial development and function. In the latter case, the continuing presence of the virus (or provirus) is required, since the mitochondria are not hereditarily changed. Both nuclear and cytoplasmic entities function in space and time. Both are susceptible to the effects of adventitious environmental perturba tions which may alter the availability of raw materials required for replication or function. It is necessary to attribute primacy to nuclear de terminants but total independence of outside in fluence to neither. A hereditary change may be regarded as "ir reversibly" defining cellular metabolic activities over a variable but discrete range. Thus, a quan titative relationship apparently exists between the DNA content of related diploid and tetraploid cells and their enzymatic content or chemical components. However, this integral relationship exists over defined environmental conditions so that the absolute values of the integers may Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1958 American Association for Cancer Research. 648 Cancer Research change as the environment changes. The transduction phenomenon of phage provides us with models whereby the hereditary determinants of a cell may be altered, provided that external DNA is incorporated into the bacterial genome. There is the suspicion that this phenomenon may not be confined to bacteria.7 The possibility must also be considered that external vectors or cellular modifications may persist over many generations so long as drastic environmental changes do not take place, thereby simulating "irreversible" cel lular changes, the elucidation of which may be difficult to analyze experimentally. Biochemical activity has been directed toward an evaluation of the eventual effects of the heredi tary changes on the metabolism of the neoplastic cell; that hereditary chomosomal changes fre quently differentiate neoplasms from their normal counterparts is now almost axiomatic. The bio chemical approach has, however, suffered from the frequent tendency to lump diverse tumors under the generic term, cancer, to assume that bio chemical properties of such cells must be shared, and to neglect the viewpoint that populations consist of individuals which are similar but not necessarily identical in chromosomal content. De spite these limitations, there has been a surprising universality in certain biochemical properties of almost all neoplastic cells. As first pointed out by Warburg, these relate to the imbalance be tween respiration and glycolysis. The apparent controversy in recent years has not been as to the reality of this imbalance but rather as to the causal factors whereby it originates and the mechanisms by which it persists. In sections III and IV, we have explored some of the provocative and interesting ideas which have been put forward in connection with respiratory-glycolytic imbal ance. We believe that there is substantial agree ment about essentials and that the nonbiochemist should not be misled in this regard. 7A recent report describes experiments in which the in cidence of leukemia was increased in a hybrid strain by injec tion into newborn mice of purified nucleic acid prepared from the lymphoid tissues of a high-leukemia donor strain (E. F. Hays, N. S. Simmons, W. S. Beck, Production of Mouse Leu kemia with Purified Nucleic Acid Preparations. Nature, 180 : 1419-20, 1957). The intraperitoneal injection into Pékin ducks of highly polymerized DNA obtained from the erythrocytes or testes of Khaki Campbell ducks also produces various modi fications in the tissues of the former animals (J. Benoit, P. Leroy, C. Vendrely, and R. Vendrely, Des Mutations somatiques dirgéessont-elles possibles chez les oiseau? Comp. Rend. Acad. Se., 224:2320-21, 1957. J. Benoit, P. Leroy, C. Vendrely, and R. Vendrely, Modifications de caractères raciaux observées sur des canetons issus de canes et de canards Pékinpréalablementsoumis à des injections d'acide dèsoxyribonucleique de canard Khaki. Comp. Rend. Acad. Se., 245: 448-51, 1957.) Vol. 18, July, 1958 We have also given consideration to the effects of the respiratory-glycolytic imbalance on the metabolic patterns of the intact cell. The effect on the energy economy of the cell was discussed first. Secondly, we considered the consequences with respect to the biosynthesis of key amino acids, pentose, and other protoplasmic building blocks. Thirdly, we explored the possible relation between respiratory-glycolytic imbalance and re ductive synthesis by the cell. Oxygen competes with other cellular constituents for the hydrogens generated during cell metabolism. In connection with reducing environments, the topic of sulfhydryl groups and cell mitosis was considered. An effort was made to relate biochemical measure ments to the properties which define cancer cells : the requirement for neoprotoplasmic synthesis, and the tendency toward freedom from the regu latory restraints of the intact animal. We agree that an understanding of the biochemical nature of the neoplastic transformation is still remote. It would, however, seem that a persistent focus on alternative metabolic pathways and patterns of metabolism, and on vectorial relationships be tween biochemical properties rather than upon the absolute amounts of the cell contents, will contribute to such an understanding. ACKNOWLEDGMENTS Grateful acknowledgment is due to Dr. T. C. Hsu, of the University of Texas M. D. Anderson Hospital and Tumor In stitute, for many stimulating discussions. REFERENCES 1. ABRAHAM, S.; CADY,P.; and CHAIKOFF,I. L. Pathways of Glucose Metabolism in Tumor and Normal Tissue Slices. Proc. Am. Assoc. Cancer Research, 2:89, 1956. 2. ABRAHAM, S.; HILL, R.; and CHAIKOFF,I. L. 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