Nucleic Acid Synthesis in the Neoplastic Cell and Impact of Nuclear Changes on the Biochemistry of Tumor Tissue: a Review SAULKIT*

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Nucleic Acid Synthesis in the Neoplastic Cell and Impact of Nuclear Changes on the Biochemistry of Tumor Tissue: A Review SAULKIT*

(Section of N'udeoprotein Metabolism, Department of Biochemistry, University of Texas ^f.D. Anderson Hospital and Tumor Institute, Houston, Texas)

INTRODUCTION assumptions that the viral and somatic mutation The spectacular progress in nucleic acid chem theories of cancer are indeed amenable to syn istry, bacterial genetics, and viral biology has thesis into a larger whole, and that both viral and stimulated renewed efforts at an integration of somatic mutation carcinogenesis are expressed the two major theories of carcinogenesisâ€”the through common mechanisms. The following quo tation from Luria is noteworthy, "The examples somatic mutation theory, and the viral theory. Four converging lines of investigation have pro discussed (in reference 146) illustrate known in vided the basis for such an integration: (a) The stances of mutational changes from genetic ele evidence that nucleic acids constitute the probable ments that behave as nonviral determinants of repositories of genetic information (44); (6) the heredity. These mutational transitions and the discovery that the replication of an entire virus similarity of gene action exerted by viruses in can be initiated following cell infection with the destructive and in persistent types of infection appropriate viral nucleic acid alone (53, 83, 104); indicate that gene control and virus control over (c) the finding that deoxyribonucleic acid (DNA) cellular functions are two aspects of the same is capable of effecting genetic transformations in genetic mechanism. This concept removes any microorganisms (206, 252) ; and (d) the analyses of a priori incompatibility between a viral and a lysogeny and transduction (1). Once the results of genetic theory of cancer etiology. It reduces the these studies were appreciated, it was inevitable interpretation of virus-induced cancer to that of that their implications for oncology should be the control of cellular development and differ pointed out (6, 7, 242). entiation by genetic elements capable, either The integrative theories of cancer are, of intrinsically, or by association with other special ized genetic elements, of assuming an infectively necessity, preliminary in nature. This follows from transmissible form." the fact that many of the central issues of biology are unresolved and the fact that the areas of nu This essay constitutes a summary of current cleic acid chemistry, microbiology, and genetics biochemical thinking on the problems of gene ac are still in a state of rapid development. Evidence tion and the control of metabolism. The plan is as in animal cells for a "lysogenic-like" integration of follows: First, the hypothesis that neoplasia, like tumor-inducing viruses is as yet only circumstan mutation, results from disturbances in the replica tial (189, 235). The chemistry of nucleic acid repli tion and function of nucleic acids will be discussed. cation is by no means settled; and the status of The action of carcinogenic and mutagenic agents ribonucleic acid (RNA) viruses in the over-all at the molecular level will be indicated. Secondly, scheme remains in doubt. attention will be directed to the structure, com Nevertheless, the present paper starts with the position, and conformation of the nucleic acids. This will be followed by an analysis of the experi * Aided in part by grants from the Leukemia Society, Inc.; mental evidence for the proposed Watson-Crick the American Cancer Society (P35-B); and the National Cancer Institute (C-4238 and C-4064). This review is based models of DNA replication. Suggested biochemical upon a paper presented at the Presidential Symposium of the mechanisms for errors in DNA replication will American Association for Cancer Research held in Chicago, then be considered. Such errors represent the April 8, 1960. chemical basis for gene mutation. Received for publication June 6, 1960. The question will then be raised whether bio- 1121

chemical diffÃ©rencesbetween the DXA of normal Although the picture is far too complicated to and tumor tissues can be experimentally demon be resolved by any facile and all-embracing gener strated. Data on the molar base composition, the alization, it is apparent that disturbances of nucleic range of compositional heterogeneity, the chro- acid metabolism may in several instances represent matographic heterogeneity, and the total cellular the common basis for action. The nature of the DNA content of tissues will be reviewed. It will nucleic acid disturbances need not be the same, be shown that estimations of the DNA content and the initial effects may vary, but the end-re support the concept that a remodeling of the sults would be modifications of genetic structure genetic elements has taken place in neoplasia. The and function. The following salient points may be consequences of the genetic reorganization will cited : then be considered. Three aspects of gene action 1. Stanfield Rogers has shown (187, 188) that and metabolic control will be distinguished: (a) disturbances of nucleic acid metabolism are critical control at the gene level; (6) control at the level of ly involved in carcinogenesis by urethan and nitro the enzyme-forming system; and (c) at the enzyme gen mustards. level itself. At the gene level, this will entail a 2. Biesele has demonstrated (27, 28) that discussion of cell architecture, the effect of gene azaserine, 6-diazo-5-oxo-L-norleucine (DON), mutation on protein and enzyme structure and mercaptopurine riboside and thioguanine riboside composition, the effects of polyploidy, hetero- increase the frequency of mitoiic abnormalities in ploidy, and multiple gene dosage, and the conse fibroblast and sarcoma cell cultures. These sub quences of the acquisition by a cell of genetic ele stances are antimetabolites of nucleic acid pre ments from without. The discussion of control at cursors. Azaserine is also a potent bacterial the level of the enzyme-forming system will in mu tagen. volve the phenomena of enzyme repression and 3. Mitotic abnormalities and chromosomal ab induction, a consideration of the sights of syn errations are frequently encountered in primary fu- thesis of RNA, and the properties of the ribo- mors as well as in tumors of long standing (81, 97, somes. An approximate estimate will be presented 100, 140, 215, 230). The chromosomal abnormali of the number of enzyme-forming templates in ties may include aneuploidy, heteroploidy, and bacterial cells and lymphoma cells. Finally, in rela quasidiploidy, alterations in chromosomal mor tion to control at the enzyme level, we will con phology including drastic chromosomal rear sider the suppressor gene phenomenon, negative rangements, and the presence of marker chromo feedback mechanisms, the dissociation of macro- somes. In instances where chromosomal ab molecules which result in apparent enzyme activa normalities have not been detected, there is the tion, and the more familiar processes of the control suspicion that they may, nevertheless, exist and of enzyme kinetics. that their elucidation may be a function of the resolving power of existing cytological tools. DISTURBANCESOF NUCLEIC ACID METABOLISM Moreover, the absence of gross chromosomal ANDCAKCINOGENESIS changes need not rule out more subtle alterations As pointed out by Auerbach (146, 147) and of the genetic elements. Barrati and Tatum (14), many carcinogenic 4. A number of viruses are replicated in the agents are mutagenic in Drosophila and in micro cell nucleus, where they induce manifestations of organisms. Among these are x-rays, ultraviolet nucleolar enlargement and hyperactivity as shown light, nitrogen mustards, urethan, dibenzanthra- by increases in the RNA mass of the nucleolus, in cene, methylcholanthrene, benzpyrene, m'-methyl- some instances at the point of attachment of the p-dimethylaminoazobenzene, aminoazotoluene, nucleolus to the chromosomes (70, 71). Viral syn and feri-butyl peroxide. Some carcinogens which thesis in the nuclei of cells has been demonstrated do not induce gene mutations do, however, in by electron microscopy (11, 67,159,160,217,218), crease the incidence of chromosomal breaks and fluorescent antibody technics (101), biochemical translocations. Among the carcinogenic or muta (163), cytological, and autoradiographic methods genic agents which induce chromosomal transloca tions and fragmentations are nitrogen and sulfur (60, 70, 71, 145). Viruses which proliferate in the mustards, radiation, diepoxides, urethan, amino- nucleus include: polyoma virus (11, 67, 101), the acridines, benzpyrene, methylcholanthrene (28), Shope papilloma virus (159, 218), the virus which and p-dimethylaminoazobenzene (212). In addi induces adenocarcinomas in amphibian renal tion, Lwoff has emphasized (148) that a number tissues (70, 71), RPL-12 lymphomatosis virus (60), of the carcinogen-mutagens induce prophage de herpes simplex virus (163, 217), and the adeno- velopment in lysogenic bacteria. viruses (160).

Disturbances of nucleic acid metabolism could molecules so that they occupy smaller volumes in take one of several forms. For example : solution (2, 3). a) Radiation inhibits the synthesis of nucleic c) Although amino acridine derivatives have acids in microorganisms and a variety of animal not been shown to possess carcinogenic activity, cells (18, 37,119, 148). The effects may be ascribed these substances are strongly mutagenic to Dro- to inhibition of key enzymes or, possibly, to sophila (6), bacteriophage (1, 40), bacteria (241), damage to templates. Irradiation in vitro induces yeast (76), and polio virus (69). The amino partial degradation of DNA which may be de acridines form complex salts with the phosphate tected by viscosity measurements (2, 3, 195) or by groups of nucleic acids (16, 39). When bacteria are Chromatographie procedures (20). Irradiation of infected with T2 bacteriophage in the presence of rats produces an increase in that part of the DNA proflavine, synthesis of protein and DNA occurs, which is eluted from aniÃ³n exchangers by 2 M and the bacteria lyse as usual but do not liberate NaCl/0.1 M NH3 at the expense of the less easily infective particles (1). The bacterial lysates ob eluted components (167). Ultraviolet light causes tained in the presence of proflavine contain the a 1000-fold inactivation of H. influenzae-treLns- same "doughnuts" (empty phage heads) that have forming factor (252). been seen in premature lysates not containing prÃ²- b) Nitrogen and sulfur mustards inhibit DNA flavine. The phage heads contain sulfur and phage- synthesis in growing cultures of E. coli, in tissue specific complement-fixing antigens but no DNA. culture cells, animal cells in vitro, and animal Free DNA containing hydroxymethyl cytosine is tissues in vivo (68, 96). The biological activity of also found, showing that phage-specific DNA has transforming principle is lost upon treatment been formed. Phage production seems to be ar with nitrogen mustard even in concentrations rested at a late stage that may call for the as which do not affect the viscosity (252). In vitro, sembly of these several components. Acriflavine ofnitrogen DNA and mustards RNA esterify (207)." Polyfunctional the phosphate alkylat- groups inhibits cap formation in the alga, Acetabularia (211). Acridine orange is a potent photosensitizer. ing agents can react with two different nucleo- The effects of the dye may in part be mediated philic centers to form a bridge between them. In through its photosensitizing action (107). When dilute nucleic acid solutions, intramolecular cross- animal or bacterial cells are grown in the presence linking can take place with the formation of a of acridine orange in the dark, there is no ap more compact molecular configuration (2, 3, 207). preciable effect on rate of multiplication, replica This is manifested by a decrease in the viscosity tion of DNA, or protein synthesis. When the cul and the radius of gyration of the molecules. Im tures are not shielded from light, these processes mediately after reaction with nitrogen mustards, are inhibited. the molecular weight of DNA is not reduced, but Unlike the aminoacridines, various benzacridine it falls on standing at 37Â°C., because the tri- derivatives (i.e., methyl-1,2 benzacridine) are phosphate esters produced by the alkylating agents strongly carcinogenic (10,136). Benzacridine salts, are unstable and hydrolyze. DNA alterations are like those of acridine itself, have a strong photo- also manifest from the study of the DNA chro- dynamic effect on Paramecia. Lacassagne et al. matographic profiles on ECTEOLA-cellulose ex (136) have emphasized that, in a series of methyl changers (20). At higher DNA concentrations benzacridines, the probability of finding a carcino (sperm), bifunctional agents cross-link different genic compound increases with the increase in DNA molecules together via their phosphate charge of the K region calculated by theoretical groups. It is improbable that the adenine and methods. cytosine amino groups of DNA are the primary d) Although bromodeoxyuridine has not as yet been shown to be carcinogenic, this substance in points of attachment by the nitrogen mustards, duces marked chromosomal abnormalities in tissue since these groups are hydrogen-bonded in the culture cells.1 Bromodeoxyuridine is mutagenic in native structure. Although both nitrogen mus bacteria (253) and bacteriophage (40,41,143). The tards and radiation reduce the viscosity of DNA, mutagenic action of 5-bromouracil is highly specific. the mechanisms may be different. The chemical Comparison of the distribution of m mutations in changes due to x-rays are brought about by the duced by proflavin and by 5-bromouracil shows highly reactive free radicals (such as OH-) which that these two mutagens act on completely dif are formed on irradiating water. Whereas radia ferent groups of sites in the genetic structure (40) tion lowers the viscosity by degrading the poly and also at points differing from those at which mers into smaller molecules, the mustards achieve the same effect by internally cross-linking the 1 L). Billen and T. C. Hsu, personal communication.

spontaneous mutation most frequently occurs. and Crick constructed DNA models and made the Bromodeoxyuridine is incorporated in place of now classic proposal that DNA consists of two thymine into the DNA of bacteria, bacteriophage, helical polynucleotide chains of opposite polarity and animal cells (41, 92, 143, 253). It has been which are twined round one another. The two shown at the enzyme level that DNA polymerase chains are held together by hydrogen bonds be can incorporate the deoxyribonucleotide triphos- tween the bases, each base being joined to a com phates of several base analogs into DNA. Uracil panion base on the other chain. The pairing of and bromouracil specifically replaced thymine; bases is specific, adenine going with thymine and 5-methyl and 5-bromocytosine replaced cytosine; guanine with cytosine. The phosphate groups are and hypoxanthine replaced guanine (24). accessible to hydrogen or hydroxyl ions and to The mutagenic effect of bromodeoxyuridine on dyes and are therefore on the outside, whereas the phage is found even when the thymine analog is bases occur opposite one another on the inside. taken up by infected bacteria in the presence of Prominent 3.36A spacings along the fiber axis chloramphenicol, i.e., when protein synthesis is were interpreted as due to a succession of flat strongly inhibited. This result implies that DNA nucleotides standing out perpendicular to the formed in the presence of chloramphenicol is fiber axis to form a relatively rigid structure and genetically active and also that protein synthesis consistent with the high density of DNA. is not essential in initiating the series of events The concept of DNA as a stiff-coiled macro- leading to mutation by bromodeoxyuridine. This molecule is also based upon physico-chemical data supports the conclusion that the mutagenic action which suggest particles having a molecular weight of bromodeoxyuridine results from its incorpora of IO6to IO7,an axial ratio of 300:1, a diameter of tion into DNAâ€”the eventual creation of a differ about 500 m//, and a thickness of about 2.5 m/i. ence in natural base sequence being the actual This view gains additional support from direct mutational event. observations with the electron microscope, which Other pyrimidine and purine analogs may be in show the molecules to be threadlike and stiff, with corporated into DNA and RNA. lododeoxyuri- a thickness of 1.5-2.0 mn (for references, see 120). dine may replace thymine in the DNA of human Physico-chemical evidence favoring the Wat leukemic leukocytes following the in vivoadminis son-Crick model of DNA includes: (a) titration tration of the analog. lododeoxyuridine is also curves which suggest that the bases are hydrogen- incorporated into DNA of tissue culture cells (73). bonded; (b) the shape and size of the molecule in Fluorouracil, an inhibitor of RNA synthesis and solution obtained from a combination of light of the methylation of deoxyuridylic acid to thy- scattering, viscosity, and sedimentation measure midylic acid, is itself incorporated in place of uracil ments (175), which show that DNA in solution is into the RNA of bacteria (110), animal cells (47), highly extended but not completely straight, and and tobacco mosaic virus (89, 208). That the that its diameter is compatible with the double presence of fluorouracil in the RNA affects nucleic helix model; (c) kinetic studies on the degradation acid function is suggested by the fact that fluoro of DNA by x-rays, acid, or enzymes, which are uracil almost entirely blocks the induction of consistent with there being two strands in DNA so 0-galactosidase in E. coli. Moreover, the fluoro- that the molecules do not come apart until there pyrimidine prevents any further rise of the ÃŸ- are breaks in both backbones opposite one an galactosidase activity in cells that have previous other ; (d) chemical analyses of the molar ratios of ly been treated with the inducer or in a strain of purine and pyrimidine bases (245) ; and (Ã¨)experi E. coli in which this enzyme is constitutive. ments on the effect of heat on the physico- Thiouracil and 8-azaguanine are other analogs chemical properties, ultraviolet absorption spectra, which are incorporated into the RNA of tobacco chromatography, and biological activity of DNA. mosaic virus (89). The Watson-Crick model has gained additional support from the recent biological and physico- THE STRUCTUREANDCONFORMATION chemical studies by Marmur and co-workers (65, OFDNA ANDRNA 153) on the separation and specific recombination For a proper appreciation of nucleic acid func of the strands of the DNA duplex. tion, a few words are required on the structure There is one well authenticated instance of a and conformation of DNA and RNA. It is now DNA with an exceptional structure. This is the generally accepted that the Watson-Crick model DNA of bacteriophage 0X174, which seems to be (237) adequately describes the properties of most sm

The UNA of animal cells, bacteria, and TMV- the absorption curve after reaction with formalde virus also appear to be single-stranded macro- hyde, whereas native DNA does not, suggests that molecules containing helical regions and intra the amino groups of adenine and cytosine partici molecular hydrogen bonding (125). Evidence for pate in forming the hydrogen-bonded structure the single-stranded coiled structure of RNA has (82). been derived from six experimental approaches: The two RNA components of bacteria, tumor (a) measurement of the changes in optical density cells, and animal tissues have molecular weights of with temperature, formaldehyde, or cation con the order of magnitude of 1 and 2 X IO6, respec centration; (o) experiments on the optical rota tively (125, 144, 227). High molecular weight tion of RNA under various conditions; (c) physico- RNA has also been isolated from liver microsomes chemical studies of the sedimentation rate, (50, 94) and calf thymus nuclei (48). The molecu viscosity, or light scattering properties of RNA; lar weight of tobacco mosaic virus RNA and of (d) investigations of the chromatography of RNA other RNA viruses seems to be about 1.7 X IO6 on aniÃ³n exchangers; (e) x-ray diffraction experi (29,49). ments; and (/) studies of the rate of RNA degra DNA SYNTHESIS dation and the inactivation of biological activity Mechanisms.â€”A plausible mechanism for DNA by heat or enzymes (for references and details, see replication has been suggested by Watson and [125]). In addition, RNA molecules from a num Crick (237). According to this proposal, the twin- ber of sources do not show in their purine and stranded DNA molecule partially unwinds, and pyrimidine molar base composition an equiva each base attracts a complimentary free nucleo- lence of adenine with uracil and of cytosine with tide already available for polymerization within guanine. This is in contradistinction to DNA. the cell. These free nucleotides, whose phosphate Sedimentation studies in the analytical ultra- groups probably already possess the free energy centrifuge of bacterial or animal tissue RNA re necessary for polyesterification, would then link veal the presence of two components having up with one another after being held in place by sedimentation constants of about 80S and 18S. the parental template chain to form a new poly The sedimentation constants of RNA solutions nucleotide molecule of the required nucleotide se decrease as the ionic strength of the medium is quence. Thus, each DNA strand serves as a tem decreased, whereas the viscosities increase. The plate for the synthesis of a complementary strand. viscosity at elevated temperatures is greater than This hypothesis of DNA replication falls in the that at room temperature. A reversible hyper- class of semiconservative mechanisms of replication chromicity is manifested by RNA solutions when in contradistinction to conservative or dispersive the temperature of the solutions is raised to 95Â°C. mechanisms (61). Conservative mechanisms are The optical density-temperature curves for RNA those which do not destroy the integrity of the differ markedly from those of native DNA. In the entire parental DNA duplex in the course of the case of RNA, the optical density of the solutions replication process so that, among the daughter begins to increase at a temperature of about 30Â°- duplexes produced by one or more replications, 40Â°C. and a gradual increase of about 30 per cent there is one which is entirely parental and the rest is observed as the temperature is raised to 95Â°C. entirely new. This could be true, for instance, if The curves may be shifted to higher temperatures DNA were replicated indirectly through the inter by increasing the ionic strength of the solution. mediary of another substance, e.g., protein or Unlike native DNA, RNA reacts rapidly with RNA, which was first synthesized on the surface formaldehyde ; the rate of the reaction is tempera of the intact parental duplex and then served as a ture-dependent. These observations are all con template for the synthesis of the daughter DNA sistent with the hypothetical structure of RNA as duplexes. Semi-conservative mechanisms are those a single-stranded, coiled polynucleotide with which conserve the atomic identity of single chains partial intramolecular hydrogen bonding. The of the parental DNA duplex, although effecting a hyperchromicity with temperature, and depend permanent separation of the two chains from each ence of viscosity, sedimentation, and reaction other in the course of replication. Dispersive with formaldehyde on temperature are all indica tive of alternative structures of the molecules in mechanisms are those which do not conserve the solution so that the molecules are more extended atomic integrity of the chains of the parental and less hydrogen-bonded at low ionic strengths duplex but result in the dispersion of its atoms and higher temperatures (94, 125, 144, 227). The among the replica duplexes. A scheme which in fact that RNA and denatured DNA both manifest volves numerous breaks in the original chains falls hyperchromicity and a shift in the maximum of into this class.

The evidence for the Watson-Crick semi-con bacteria on a bromouracil-free medium and in servative mechanism of DNA replication is im fected the bacteria with bromouracil-containing pressive but by no means conclusive. Four lines of ("heavy") phage, no "heavy" phage was detected experimental evidence are relevant: (a) equi by CsCl centrifugation, even among the early librium sedimentation studies in a CsCl gradient phage progeny released by prematurely opened of labeled progeny DNA (157,198, 219); (6) radio- cells. Similarly, no UV-resistant bromouracil-free autographic studies of chromosome replication phage could be found among early phage progeny (224) ; (c) the enzymatic replication of DNA (138, released by bromouracil-labeled bacteria infected 139); and (d) various bacteriophage studies of with bromouracil-free phage. The results were in progeny formation (141, 209, 210). terpreted as indicative of a dispersive mode of One method of testing the Watson-Crick hy DNA replication in phage 0X-174. It is possible pothesis of DNA replication is the "transfer ex that the mechanism of DNA replication in phage periment." In such an experiment, the distribu X-174differs from that of bacteria, algae, and tion of labeled parental DNA to progeny mole animal cells. However, the phage experiments are cules is followed. The Watson-Crick hypothesis complicated by the fact that the phage DNA makes some striking predictions concerning its undergoes many more replications prior to study outcome : (a) After just one generation in the non- than does the DNA of the higher organisms. Since labeling medium, each DNA molecule will be only the results are essentially negative in character, half labeled; (o) at any generation after the first, that is, the "heavy" DNA label of the parental there will be found only two classes of molecules, phage was not recovered, the crucial question of half labeled and completely unlabeled. For ex the sensitivity of the method must be raised. ample, at the second generation, there will be There is, however, another reservation to the equal numbers of half labeled and completely un interpretation of the Meselson-Stahl experiment. labeled molecules. This is the question of whether the sub-units of In their transfer experiments, Meselson and DNA represent the complementary DNA strands Stahl permitted N'Mabeled E. coli to multiply in of the Watson-Crick model or monomers arising N14 medium. At intervals during the following from aggregates of DNA (157). On the basis of four generations, samples of the DNA were ex light scattering measurements of the molecular amined with respect to the distribution of N16 weight of DNA and of the kinetics of deoxyribo- label. This was accomplished by determining the nuclease degradation of heated E. coli DNA density distribution of the DNA by the method of preparations, Cavalieri and co-workers have sug equilibrium density-gradient centrifugation. It gested that the unit DNA molecule of E. coli is was found that, when the populations of cells and actually a dimer composed of two double helics, hence of DNA molecules had just doubled, all the laterally bonded together (45). Thus, each double DNA molecules in the culture were exactly half- helix would be conserved intact during cell divi labeled. After just two doublings of the population, sion and the bonds holding the dimer together half-labeled and completely unlabeled molecules would presumably be ruptured during some part were present in equal numbers. Indeed, in all of the replication cycle. On the basis of this inter samples taken after the first generation, the frac pretation, the replication of DNA would be con tion of DNA molecules which were half-labeled servative rather than semi-conservative. was 2'~". These results demonstrate that the DNA An unambiguous answer to this problem cannot molecules of E. coli are composed of two sub-units be derived from the other experiments previously and that, upon duplication, each daughter mole cited. Taylor and co-workers (224) labeled Vicia cule receives one parental sub-unit. The sub-units root tip chromosomes by immersing this actively remain intact through many successive duplica growing region of the plant for a short time in tions. liquid medium containing tritium-labeled thymi- The Meselson-Stahl experiments have been dine. They then observed the distribution of label confirmed by Sueoka (219) who studied mitotic among chromosomes autoradiographically after replication of DNA in the unicellular green alga, one or two duplications in the absence of labeled Chlamydomonas reinhardi, and by Simon (198), thymidine. After one duplication, the two daugh who carried out transfer experiments with HeLa ter chromosomes were seen to be labeled equally. cells in tissue culture. In the latter instance, the Following the second duplication, two of the four DNA was labeled with bromouracil. Transfer ex chromosomes were usually labeled to the same ex periments of parental DNA to the progeny of tent as the first generation chromosomes; the other bacteriophage 0X-174, however, yield a different two were generally completely unlabeled. Objec result. When Kozinski and Szybalski (135) grew tions to the use of colchicine to induce mitotic

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1960 American Association for Cancer Research. KITâ€”NucleicAdd Synthesis in Tumor Tissue: Review 1127 arrest in these experiments (137) are discussed by primers. Interestingly enough, the enzymatically Woods and Schairer (243). One must conclude synthesized DNA manifested the same molar base from the results that a chromosome is composed of composition as the primer which was employed two sub-units which segregate, one into each (138). As in the case of native DNA, the product daughter chromosome, and that each sub-unit is of enzymatic synthesis showed an equivalence of transmitted intact throughout successive genera adenine to thymine and guanine to cytosine. The tions. Here again, it cannot be stated with cer single-stranded <Â¿>X-174DNA also served as tainty that the sub-units represent DNA duplexes. primer. In this instance, a double-stranded From the "star" experiments of Levinthal and product was formed. Heat-denatured DNA is a Thomas (61, 141), there is evidence that a large more effective primer than native DNA (31, 138). piece of the transferred parental DNA of T-even From these findings, it is clear that the equiva bacteriophage is conserved, but the remainder of lence of adenine to thymine and guanine to cyto the transferred parental label is more widely dis sine is an inherent feature of DNA synthesis by tributed. In other experiments, Stent and co- the polymerase enzyme. Furthermore, the data workers (209, 210) estimated the distribution of suggest that the DNA added to the reaction serves the transferred DNA atoms by means of the lethal as a template for the enzymatic replication of effects on the progeny population of the decay of DNA. incorporated radiophosphorus P32 atoms derived If primer DNA is actually single-stranded DNA from the DNA of highly P32-labeled parental and if the synthetic reaction produces double- bacteriophage. In the process of transfer from stranded molecules, then the idealized reaction parent to progeny, one part of the parental DNA should cease when the primer DNA has been appears to be broken up into small framents, doubled in amount. DNA increases of greater whereas another major fraction of the parental than twofold would be possible only with enzyme DNA is not so extensively fragmented but instead systems containing nucleases capable of convert is transferred to a few progeny phage in large ag ing native DNA to denatured DNA. Actually, net gregates. These experiments could mean that the increases in DNA in amounts at least 10 times as mechanism of phage DNA replication is dispersive. great as the primer added have been observed It is not excluded, however, that the hereditary (138). The explanation for this phenomenon re substance of the phage is contained in only a part mains to be elucidated. of the DNA complement of each particle and that To date, it has not been shown that enzy the remainder of the DNA molecules play some matically synthesized DNA is biologically active. nongenetic or physiological role in the initiation of Errors in DNA replication.â€”It is a corollary of intracellular phage growth. In particular, it seems the Watson-Crick hypothesis concerning the possible that the genome of the phage resides in structure and function of DNA that a change in only that portion of the viral DNA which is trans the DNA sequence of one or a few nucleotides will ferred as a large fragment to the progeny and be mutagenic (21, 40, 237). Mechanisms for spon which appears to remain intact in successive trans taneous mutation and for experimentally induced fers. mutations have been suggested on the basis of this The enzymatic synthesis of DNA has been concept. Watson and Crick pointed out (237) that demonstrated with extracts of bacteria (133, 138, the specificity in DNA structure (adenine pairing 139), regenerating rat liver, thymus, intestine with thymine and guanine with cytosine) results (33, 152, 180), and tumor cells (33, 59, 151, 180). from the assumption that each of the bases The enzyme is absent or low in nondividing tissues possesses one tautomerie form which is very much such as normal rat liver, brain, or skeletal muscle, more stable than any of the other possibilities. The but appears at the same time (10 hours after the fact that a compound is tautomerie, however, operation) as DNA synthesis in vivo in regenerat means that the hydrogen atoms can occasionally ing rat liver (34). When partially hepatectomized change their locations. Thus, a spontaneous muta rats were irradiated 6-13.5 hours after the opera tion might be owing to a base occurring very oc tion with x-rays, the radiation produced essential casionally in one of the less likely tautomerie ly complete inhibition of the increase in DNA forms, at the moment when the complementary polymerase (32) that would be expected to occur chain is being formed. For example, whereas between 6 and 24 hours after partial hepa- adenine will normally pair with thymine, if there tectomy. is a tautomerie shift of one of its hydrogen atoms, A primer DNA is required for enzymatic it can pair with cytosine. The next time pairing activity. The E. coli polymerase is able to utilize occurs, the adenine (having resumed its more usual various bacterial DNA's or calf thymus DNA as tautomerie form) will pair with thymine, but the

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1960 American Association for Cancer Research. 1128 Cancer Research Vol. 20, September 1960 cytosine will pair with guanine, and so a change ducible and noninducible by the above two base in the sequence of bases will have occurred. analogs is so drastic that it seems to reveal two Nitrous acid is a mutagenic agent for tobacco kinds of fundamentally different mutagenic ef mosaic virus, 0X-174 phage, T4 phage (226), and fects: (a) a mutagenic effect by which the analog poliovirus (30). Nucleic acids are deaminated by induces forward mutations and their reversions, nitrous acid so that adenine is converted to hypo- and (6) a mutagenic effect which is responsible for xanthine, guanine to xanthine, and cytosine to the formation and reversion of those mutants uracil. The production of mutants by nitrous acid which cannot be induced to revert by the base is a one-hit process, the deamination of a single analogs. Inductions of mutations by bromouracil base presumably being sufficient to produce a and 2-aminopurine presumably arise by mistakes mutant. In the case of RNA, the direct formation in base pairing. Freese has pointed out (84) that, of uracil from cytosine can, obviously, account for whatever the exact mechanism, a purine would be mutations. However, the deaminations of adenine replaced by another purine and a pyrimidine by and guanine in RNA are not so obviously muta another pyrimidine. Because of the purine-pyrim- genic, since one or more additional steps would be idine conservation, the only possible base pair required in the host cell to replace the abnormal changes that can be induced by such base ana bases by normal ones in the new RNA chain. The logs are the "transitions": abnormal bases could provoke a pairing error leading to a new sequence of nucleotides in the progeny RNA. In the case of DNA, none of the deamination products would be normal bases, so (A = adenine, G = guanine, T = thymine, and that all the nitrous acid-induced mutations might C = cytosine, or any derivative which is altered result from some sort of pairing error provoked at the 5 position). Furthermore, each base analog during duplication by the presence of an abnormal should be able to induce "transitions" in both base. The deamination products are essentially directions: in one direction by a pairing mistake base analogs comparable to bromouracil and 2- made by the base analog when it becomes incorpo aminopurine (41, 84, 143). rated into DNA; in the other direction by a pair There is a clear-cut distinction between the ing mistake which the base analog makes in later mutagenic action of nitrous acid on 0X-174 and replications, after it has been incorporated into T4 phage. Mutants of 0X-174 arise in pure clones, DNA. whereas most T4 mutants arise in mixed clones, Proflavine induces mutations of the second suggesting that there is only one copy of genetic kind. Possibly the proflavine-induced mutations information in 0X-174 and at least two copies in involve a molecular transition in which there is a T4. The alteration of one of the T4 copies yields a "transversion" of a nucleotide pair in which a type of hÃ©tÃ©rozygote.Thenumber of copies of purine is replaced by a pyrimidine and vice versa genetic information can be directly related to the (84): apparent number of strands in the DNA molecule. It is inferred that hÃ©tÃ©rozygotesinduced by nitrous acid can consist of double-stranded DNA II II with singular regions in which the base pairs are " not the normal complementary ones. These results are consistent with the picture that the DNA is in the Watson-Crick double-stranded configuration, Rather interesting is a third kind of mutagenic both complementary strands carrying the same effect, that of a lower pH or higher temperature, information. This conclusion is also strongly sup which is very likely due to the separation of single ported by the experiments of Marmur and Doty purines from the sugar phosphate backbone. The (163) in which hybrid DNA duplexes have been removed purine might be replaced by another formed by the exchange of the dissociated double (activated) base in the resting DNA, or during strands of wild type and homologous mutant DNA replication the new DNA chain, using the DNA. altered chain as the complementary template, in Mutants of T4 phage induced by bromodeoxy- corporates a base at the site of the gap which it uridine and 2-aminopurine in the forward reaction would not have incorporated normally. If any of can be induced to revert (84). In contrast, pro- the four bases entered this site more or less at flavin-induced and most spontaneous mutants random, either molecular "transitions" (I) or cannot be induced to revert by bromouracil and "transversions" (II) might result (84). Another 2-aminopurine, although they revert spontaneous possibility is that the deleted base might be ly. The difference between reverting mutants in- skipped during DNA replication so that the

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1960 American Association for Cancer Research. KITâ€”NucleicAcid Synthesis in Tumor Tissue: Review 1129 progeny DNA strand would have one less purine agreement with those of Smith and Kaplan (202) at the specific site. The consequences of this event and Prager and Goerner (182) on mouse leukemic would depend on the nature of the genetic code. tissues, and of Kondo and Osawa (132), who studied the DNA of various normal rat or rabbit THE DNA OF TUMOR TISSUES tissues. They are at variance, however, with the One might postulate that neoplasms differ from results of Bendich, Polli, and co-workers (19, 20, normal tissues with respect to DNA structure and 179), who reported Chromatographie and physico- composition. However, definitive proof of such chemical differences between normal human differences has not as yet been obtained. leukocytes and leukemic cells, and also very The purine and pyrimidine molar base ratios of marked differences between the DNA Chromato normal tissues and tumors are similar (19, 122, graphie profiles of rat kidney and brain. 130). In the case of mouse tissues, there is in each Another approach to the problem is the study case an equivalence of adenine to thymine and of the range of compositional heterogeneity. There guanme to cytosine, with. . an adenine : plus ; thymine 7â€”:â€” is fairly clear-cut evidence that, within a given cell, guanine plus cytosme some DNA molecules contain a relative excess of ratio of about 1.25. adenine and thymine and others a relatively DNA IN O.I5MNOCI, IO"3 M PHOSPHATE, IO'3M VERSENATE 150- :sÅ“==HEATINGTIME:ABOUTiÂ°PER10MINUTES

141.7- EHRLICH â€”LETTRE'-EHRLICH6C3HEO-DBA6C3HED. TO C3H

00 85 125-

tÃ¼ g IK? ÃœJ z 1083-

100

60 65 70 75 80 85 90 95 100 TEMPERATURE Â°C CHART1.â€”Effectof temperature on the optical density of tetraploid carcinomas (Lettre-Ehrlich and Ehrlich). All DNA DNA solutions of diploid and tetraploid lymphomas (6C8HED solutions at a concentration of approximately 2.5 X 10~3 per and 6C8HED-DBA to C3H), and of hyperdiploid and hypo- cent. The DNA is, however, heterogeneous. Hetero greater amount of guanine and cytosine. Doty geneity may be demonstrated by fractionating et al. have shown (66) that the optical density- DNA on ECTEOLA-cellulose aniÃ³nexchangers. temperature curves of DNA are characteristic for The DNA Chromatographie profiles of the follow a given organism. A linear relationship exists be ing normal tissues and tumors of mice have been tween the DNA "melting point" (Tm) and the investigated in this laboratory : (a) spleen, thymus, guanine plus cytosme content. Using this method, lung,, kidney, and liver; (6) spontaneous lym we are now engaged in a study of the range of phomas, diploid and tetraploid lymphomas or compositional heterogeneity in mouse tissues and carcinomas; (c) melanotic or amelanotic mela tumors. nomas. In addition, rat kidney, brain, and spleen When dilute solutions of DNA are heated, the have been studied (122, 124). With the exchang optical density remains constant until the ambient ers used, the Chromatographie elution profiles of temperature is raised to about 80Â°C.(Chart 1). At carefully prepared DNA samples from different higher temperatures, the optical density sharply tissues of the same animal or from normal tissues rises to a value of about 40 per cent above the ab and tumors were all similar. This suggests that the sorption at room temperature. A very steep^ molecular size distribution of the DNA molecules optical density-temperature curve is indicative of are in the same range. The above results are in homogeneity of composition, whereas a broad

curve is suggestive of heterogeneity. This follows (212) are especially significant. These investiga from the fact that DNA molecules having a higher tors studied by microspectrophotometric methods proportion of guanine and cytosine "melt" at more the DNA of precancerous and cancerous rat liver elevated temperatures. The exact shape of each cells 2 days after partial hepatectomy. The rats curve and the Tm depend not only upon the com were previously fed p-dimethylaminoazobenzene position and heterogeneity of the DNA sample but (DAB) for 5 months. The DNA of the two also upon the ionic strength of the DNA solution daughter telophase cells or the metaphase cells were and the rate of heating at any temperature. measured. Alterations from genetically homo The curves for four tumor DNA samples are geneous to genetically heterogeneous populations illustrated in Chart 1. The Tm values are all occurred in the precancerous livers before any about 87.8Â°C. One-third of the optical density definite tumors could be recognized. Hepatoma change above and one-third below the Tm take cells were predominantly subtetraploid in DNA place over a range of about 5.4Â°.This is indicative content. Whereas the ratio of DNA in the two of the same order of magnitude of compositional normal telophase cells was very close to 1.00, this heterogeneity as was observed by Doty et al. for ratio was often about 0.80 in cells of animals fed E. coli DNA (66). It is apparent that, although DAB for 5 months, indicating clearly that the these four tumors are known to differ greatly in genetic material was not being equally distributed. chromosome composition, the range of composi Similar results were obtained by Stich and co- tional heterogeneity as well as the average purine workers (213, 214) with human carcinomas. and pyrimidine base composition is about the Aneuploid chromosome and DNA values are same. It would seem that any differences between indicative of a reorganization of the genetic ma them with respect to DNA probably occur at a terial in neoplasia. The full significance of the very subtle level, such as that of the sequence of phenomenon becomes apparent when it is recog bases along the polynucleotide chains. nized that a quantitative relationship exists be There is one way in which the DNA's of these tween the number of replicate genes or chromo tumors definitely do differ; that is, in the amount of somes of a cell and the enzymes or enzyme-form DNA per cell (122). It is now well established ing systems (EFS) of that cell. Changes in the rela that the DNA content per cell is proportional to tive number of certain chromosomes and genes the chromosome number. Tetraploid cells contain compared with others provide a mechanism for about twice as much DNA as diploid cells. This is irreversibly altering the quantitative balance be particularly well illustrated when measurements tween anabolic and catabolic processes in the cell. are made on the chromosomes and the DNA of the This point will be further elaborated below. same cell population (178). The measurements of the total DNA content GENETICLEVELOFCONTROL have actually strengthened the argument that Both qualitative and quantitative control of cancer cells frequently manifest chromosomal ab metabolism may be exerted at the genetic level. normalities and hence genetic differences. Recent This is manifest from the changes in the proteins studies by Ogawa et al. (164) and by Rabotti (184) and enzymes of cells which result from: (a) spon have confirmed and extended earlier findings by taneous or induced gene mutations; (6) gene Leuchtenberger, by Bader, and by Atkins and dosage effects and polyploidy ; or (c) the introduc Richards of DNA values in tumors corresponding tion of new genetic material into a cell following to aneuploid and polyploid chromosome sets. bacterial mating, bacteriophage infection, or trans Rabotti (184) compared the DNA of normal hu formation by DNA solutions. man lymphocytes, primary carcinomas, and their Protein alterations.â€”Bonner (36, 63) Yanofsky own mÃ©tastases. Whereas the DNA values of the (56, 249), Suskind (220, 221) and others have pro primary tumors and the normal lymphocytes fell vided numerous examples of altered proteins re within the range of "diploid" DNA values, the sulting from gene mutations. One such example is mÃ©tastases were generally 4N-8N. This seems con the enzyme tryptophan synthetase. In mutants, sistent with the concept of progression in animal proteins related to this enzyme may be formed; tumors already described by Hauschka, Klein, and these altered proteins manifest changes in affinity others. to coenzymes, sensitivity to temperature or to In many studies of the DNA content of tumors, metal inhibitors. DNA values corresponding to aneuploid chromo Particularly noteworthy are the experiments of some numbers cannot be distinguished from DNA Ingram and associates (112-117) on the hemo increases due to premitotic DNA synthesis. For globins. This work has established that a single this reason, recent studies by Stich and associates gene mutation results in the substitution of one

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1960 American Association for Cancer Research. KITâ€”NucleicAcid Synthesis in Tumor Tissue: Review 1131 picture of infants with hemoglobin "Barts" is like amino acid for another in the protein chain; hence, protein conformation and protein function are that in thalassemia, but this does not persist into changed. Normal adult human hemoglobin is later life, nor do the children develop an abnormal composed of four polypeptide chains of two differ adult hemoglobin. Two interesting hypotheses ent kinds, now referred to as the a and the ÃŸ have been proposed to explain the findings with chains. Each half molecule contains one a and respect to hemoglobin H and "Barts": (a) there one ÃŸchain. The ÃŸchain has an N-terminal valine- may be an overproduction of ÃŸor 7 chains, re histidine sequence, whereas the a chain has a spectively; or (6) alternatively, the production of valine-leucine N-terminal sequence. A number of the a chains may be inhibited. It would follow from abnormal hemoglobins are known, including the latter hypothesis that, although a chains of hemoglobins S, C, E, I, Da, and D0. All these are hemoglobins A and F are chemically identical, they thought to be caused by mutant genes inherited are not controlled by the same gene. in a Mendelian manner. The genes for hemoglobins Species-specific differences in the amino acid se S and C are allelic. Hunt and Ingram have shown quence of proteins and peptides of the pituitary (113) that, in hemoglobins S and C, a glutamic gland hormones, cytochrome c, and albumins have acid residue of the ÃŸchain is replaced by valine and also been studied. For recent reviews, see refer lysine, respectively. In hemoglobin E, a glutamic ences 17, 72, 108. acid residue of the ÃŸchain is also replaced by Gene dosage effects and polyploidy.â€”Quantita lysine, but in this case the amino acid substitution tive control of metabolism is in part effected by occurs in a different part of the protein molecule. the multiplicity of alÃeleswithin a cell. This phe The mutation which gives rise to hemoglobin D0 nomenon may be illustrated by studies of gene also appears to express itself as an alteration in the dosage in homozygotic and heterozygotic diploid ÃŸchain. On the other hand, changes occur in the cells or by comparisons of diploid and tetraploid a chain in hemoglobin I and Da. Peptide 23 of cells. hemoglobin I contains tryptophan, whereas nor A number of enzymes and cellular constituents mal hemoglobin does not. This suggests that vary with chromosome number in diploid and there may be two hemoglobin genes: an a chain tetraploid cells (Table 1). For example, both the gene to control the amino acid sequence of the a DNA and the RNA contents per tetraploid lym- peptide chain and a ÃŸchain gene. Mutations may phoma cells are about twice as great as per diploid affect one or the other of these genes; for example, lymphoma cells. Likewise, the DNA and the RNA the hemoglobin S, C, D0, and E mutations would contents of hyperdiploid and hypotetraploid car be expected to occur on the ÃŸgene, whereas the cinomas vary with the chromosome number (120, hemoglobin Da and I changes would occur as the 122, 124, 125, 178). Stated differently, the ratio result of alterations of the o chain gene. More of RNA to DNA is approximately constant for over, the loci and the two mutations of the a histologically related asynchronously multiplying chain gene would be expected to be very close cells. The ratios for the carcinomas differ, how together, since they effect changes within the ever, from those of the lymphomas. same small peptide. The loci of the 4 ÃŸchain mu A number of parameters have been studied by tations would be found in two pairs, those for ourselves (126, 127), by Shelton (193), Patterson hemoglobins D/j and E close together and those and Podber (124), and Hauschka and co-workers for hemoblogins S and C in the identical location. (97). The following manifest ploidy dependence in It is of considerable interest that doubly abnormal diploid and tetraploid carcinoma cells: cell vol hemoglobin molecules can be formed by the dis ume, nuclear volume, dry weight, DNA, RNA, sociation and recombination in the laboratory of histones, nitrogen content, endogenous respira the chains of hemoglobin I with those of hemo tion, glycolysis, transaminase, glucose-6-phosphate globins S or C. dehydrogenase, glycyl-leucyl peptidase, and the Human fetal hemoglobin differs from adult glutathione, aspartate, alanine, and taurine of the hemoglobin in that it has an a chain (as in adult free amino acid pool. The following manifest hemoglobin) but also a 7 polypetide chain which ploidy dependence in diploid and tetraploid lym differs from the ÃŸpolypeptide chain of adult phoma cells : cell volume, nuclear volume, average hemoglobin. An abnormal fetal hemoglobin (hemo nucleoli per cell, dry weight, DNA, RNA, histones, globin "Barts") has been observed in an infant in endogenous respiration, glycolysis, succinoxidase, which there are 7 chains only (113). It has also and transaminase. been shown that adult hemoglobin H consists These relationships are by no means fortuitous. solely of ÃŸchains. Hemoglobin H is found only in The effects of various modifications of the cellular the presence of the gene for thalassemia. The blood environment upon metabolism and tumor char-

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1960 American Association for Cancer Research. 1132 Cancer Research Vol. 20, September 1960 acteristics have been studied. Respiration, glycoly- ethanolamine phosphate content of carcinoma sis, DNA content, and glucose-6-phosphate de- cells were not ploidy-dependent. The glucose-6- hydrogenase activity were not significantly phosphate dehydrogenase activity of tetraploid changed by growing the tumor cells: (a) in male lympkoma cells was about 5-10 times as great as rather than female mice; (6) in heterologous strains in diploid lymphoma cells. However, the fact that of mice; or (c) for from 6 to 11 days in vivo prior so many parameters do manifest ploidy depend to harvesting the cells. Moreover, glycolysis or ence reinforces the notion that metabolism is glucose-6-phosphate dehydrogenase were inde quantitatively related to gene dosage and DNA pendent of temperature-induced stress of the content. tumor-bearing mice. It was observed that, if the The proportionality between chromosome or tumor-bearing mice were kept for 2-4 hours at gene number and enzyme activity is observed in 4Â°C.or overnight at 38Â°C.prior to sacrifice, the a variety of biological systems. Ogur and co- tumor cell DNA content, or the constancy of the workers (165, 166) have shown that, when hap- ratios of glycolysis to DNA or glucose-6-phosphate loid, diploid, triploid, or tetraploid strains of dehydrogenase to DNA, was not changed by the yeast are cultivated under standardized condi temperature treatment. tions, DNA, RNA, dry weight, metaphosphate, TABLE1 METABOLISMOFDIPLOIDANDTKTRAPLOIDLYMPHOMASANDCARCINOMAS

RNA-Riboselig. â€¢6C3HED DNA10Â« Histories,ig. yd.(ig. someno.4076-784678ng. ysis*1.091.021.271.31Succinoxi-dasef0.550.56G-ft-Pdeb cells9.815.311.118.0â€žg.DNA0.330.350.460.47Mg-DNA0.830.982.863.46Glycol DNAt0.311.590

lymphoma6C3HED-DBA-2diploid tetraploidlymphomaLettre-Ehrlich

hyperdiploidcarcinomaEhrlich 440.55 hypotetraploid carci nomaChromo

* ÃŸ\Ca/hour/jug DNA. t M!Oo/hour/Ãtg DNA. Ã®Glucose-6-phosphate dehydrogenase in arbitrary units S. Kit and R. Ragland, unpublished experiments.

The regulation of respiratory processes may be respiration, and glycolysis are proportional to demonstrated by inhibiting respiration through chromosome number. the addition of glucose or by supplying alternative Gene dosage effects have been studied in hydrogen and electron carriers (for example, homozygous and heterozygous plants, yeast, mÃ©thylÃ¨neblue).Inhibition or stimulation from Paramecium, and man. The relative activity of the endogenous level may be elicited with each of invertase in diploid yeast cultures adapted to the tumor cell strains. sucrose is as follows: (a) homozygous recessive, 0; The histologically related tumors did not have (6) heterozygous, 17; (c) homozygous dominant, euphid multiples of the normal chromosome set. 34 (134). In maize, the yellow color of the endo Consequently, an exact quantitative relationship sperm is mainly due to zeaxanthin and propor between chromosome number and all cell enzymes tional to the number of Y genes. The same is true was neither anticipated on theoretical grounds nor of ^-carotene (93). Relative anthocyanine and found experimentally. Cell surface of tetraploid aurone concentrations have been measured in 25 carcinoma cells is only about 1.4 times that of of 27 possible color genotypes of the garden snap diploid cells (97). Many antigens may be localized dragon, Antirrhinum majus (118). At least three on the surface of cells. Tetraploid cells frequently genes, the P, Y, and M genes, control the pigmen manifest antigenic simplification. The free amino tation in these plants. The following generaliza acid content of tissues is related to the genome in tions may be formulated: (a) Increased numbers an extremely complex fashion. It is therefore not of recessive factors result in decreased antho- surprising that most of the free amino acids of cyanin production; (6) genotypes containing lymphoma cells or the glutamic acid, glycine, or pelargonidin glycoside have a significantly lower

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1960 American Association for Cancer Research. KITâ€”NucleicAcid Synthesis in Tumor Tissue: Review 1133 concentration of anthocyanin than genotypes con initially act as constitutive organisms. However, taining cyanidin glycoside; (c) highest concentra after about an hour, the enzyme was no longer tions of aurone are associated with genotypes yy; formed unless an inducer was added. These ex (d) increased numbers of dominant factors result periments will be considered in more detail later. in decreased aurone production; (e) high antho For the present, it is sufficient to note that in the cyanin concentration is related to low aurone heteromerozygote, the z+, i+ genes rapidly func concentration, and vice versa; and (/) the chemical tion in controlling enzyme synthesis. consequence of heterozygosity is an alteration in Pronounced changes in enzyme content also the magnitude of the effect produced by the follow within minutes of infection of E. coli with dominant alÃeles.The complex interrelationships bacteriophage. The following enzymes, which are between genes and gene products in this species not normally present in E. coli, are induced fol will be further elaborated below (see page 1137). lowing infection with T-even phage: (a) deoxy- The galactose-1-phosphate-uridyl transferase cytidylic acid hydroxymethylase; (b) hydroxy- activity per gram hemoglobin in erythrocytes of methyl-deoxycytidylic acid kinase; (c) an enzyme normal and galactosemic individuals is as follows for the transfer of glucose from uridine diphospho- (42): (a) normal, 19.0; (6) carriers, 9.94; (c) gal- glucose directly to the hydroxymethyl cytosine of actosemics, 0. Individuals heterozygous for the DNA; and (d) deoxycytidylic acid phosphatase hemoglobin genes S, C, and E contain roughly 60 (77-80, 131, 133, 203, 204). The T-even phage per cent normal hemoglobin A and 40 per cent of contain in their DNA glucosylated hydroxy the abnormal hemoglobin (239, 240). The expres methyl cytosine in place of cytosine. The deoxy sion of the abnormal gene is under the modifying cytidylic acid-degrading enzyme may have a dual influence of other genetic factors, since lower or function in the promotion of the synthesis of higher proportions of abnormal hemoglobin are a phage DNA: (a) It degrades deoxycytidylic tri- familial characteristic. The presence of the thalas- phosphate to a form which is not utilized as a semia gene exerts a particularly pronounced effect substrate by DNA polymerase; and (6) it provides on the expression of the allelic hemoglobin genes. deoxycytidylic as a substrate for the synthesis of In one case of hemoglobin S-thalassemia disease, hydroxymethyl-deoxycytidylic acid. Further, it 91 per cent hemoglobin S was encountered, while may be a mechanism for the exclusion of phage in a child with hemoglobin C-thalassemia disease other than the T-even variety when E. coli under there was 83 per cent hemoglobin C (156, 199, goes mixed infection with T-even phage and phage 240). which contain cytosine in their DNA. Paramecia homozygous recessive for the kappa The following enzymes are greatly increased in factor (kk) lack the self-replicating kappa particles activity following infection with T-even phage: in their cytoplasm. Homozygous dominant (KK) (a) thymidylate synthetase; (6) DNA poly- individuals of killer stock 51, mating type VII merase; and (c) thymidylic acid and deoxy- contain 851 particles, whereas type VIII contain guanylic acid kinases. Thymidylate synthetase only 371 particles. In the hÃ©tÃ©rozygotes(Kk) activity increases 1000-fold in E. coli Bt- after there are found half as many kappa particles as in phage infection; this strain is normally deficient in the homozygous dominants: 363 particles in mat the enzyme (12, 79, 133, 203). ing type VII hÃ©tÃ©rozygotesand195 in mating Following infection by T5 phage, there is a type VIII heteroxygotes. Moreover, it has been 6-10-fold increase in thymidylate synthetase and shown that killer animals of type VII liberate at in thymidylate, deoxyguanylate, and deoxycyti- least twice as much paramecin in a given time as dylate kinases but a much smaller increase in killer animals of type VIII (15). deoxyadenylate kinase. There are no changes in Enzyme changesfollowing bacterial mating, phage any of the kinases when the bacteria are infected infection, or transformation.â€”E. coli strains con with T3 or T7 phage (23). T3 and T7 seem to taining the i+, z+, y+ genes are inducible for ÃŸ- derive most of their DNA from the host. galactosidase and permease. Strains having the i~ New protein synthesis is necessary for the ap gene are constitutive. An Hfr strain containing the pearance of enzyme activity. Chloramphenicol or z+ i+ genes (i.e., galactosidase-inducible) can be amino acid analogs inhibit the emergence of the conjugated with a receptor F"~ strain that is enzyme activity. Thus, it is unlikely that the genetically z~, i~. Within a few minutes of the enzymes are present in the uninfected cells in a time of entry of the z+, i+ genes, which are closely "masked" form. The attractive hypothesis is that linked, the synthesis of ÃŸ-galactosidasecommences the DNA of the phage replace defective genetic at maximum velocity in the absence of the extra material of the bacteria rather than that they cellular inducer (171, 172). The zygotes, therefore, merely activate dormant genes or eliminate gene

inhibitors. In support of this notion is the fact EFS. It is reasonable to assume that the activity that deoxyguanylate kinase of uninfected bacteria of the genetic elements in synthesizing given EFS differs in certain properties from the enzyme may be modified by inhibitors or activators. The found in T2 infected cells. The enzyme from unin concept has long been popular among embryolo- fected bacteria requires the addition of K+ for its gists that, although nuclear activity varies in dif activity, but the enzyme from the coliphage- ferent cells, nuclear potentialities remain un infected cells does not. Although this suggests that changed (158, 176). The accumulation of certain the deoxyguanylic acid kinase from the infected EFS in the cell cytoplasm may perhaps take place cells is a different protein, the results could also be during development and differentiation. explained by an alteration in the K+ binding strength of the enzyme from infected cells (25). CONTROLAT THE LEVEL OF THE ENZYME-FORMINGSYSTEM The acquisition or modification of enzyme activity following infection of animal cells by Enzyme repression and induction.â€”Let us now tumor-inducing viruses has not as yet been ex consider metabolic control at the level of the EFS. tensively studied but remains an attractive possi The control of enzyme-protein content by repres bility. sion and induction are probably examples of con DNA solutions isolated from wild type strains trol exercised at this level. Enzyme repression is a are capable of transforming mutant strains of phenomenon in which the final products of a bio- Bacillus subtilis to nutritional auxotrophy. An synthetic pathway inhibit in some way the syn other type of genetic trait which can be transferred thesis of the enzymes required to catalyze the first by DNA molecules in solution is the capacity to steps of that pathway. A number of examples of form specific enzymes of the inducible type (206). repression are known in bacteria (Table 2). For For example, DNA transformation in Pneu- example, the accumulation of arginine within cer mococci causes the formation of an entirely new tain bacteria represses the further synthesis of biosynthetic pathway for the synthesis of specific acetyl ornithinase and ornithine transcarbamylase, capsular polysaccharides. In the transformation enzymes needed for arginine biosynthesis (170, of the noncapsulated Type III to capsulated Type 186). I, at least three new biochemical systems are The mechanism of enzyme repression is of par formed: (a) oxidation of uridine diphosphoglucose ticular significance. Presumably, there is no effect to uridine diphosphoglucuronic acid, (6) epimeriza- on the formation of the EFS or on its destruction. tion of uridine diphosphoglucuronic acid to uri Rather, the effect is on the action of the EFS dine diphosphogalacturonic acid, (c) polymeriza (149). When the ornithine transcarbamylase sys tion of Type I capsular polysaccharide (9, 206). tem is released from repression in E. coli, there is The following enzymes can be formed in the pres evidence of new protein synthesis, but probably no ence of inducer after transformation by DNA of new RNA is made (186). organisms which were not previously capable of Pardee, Jacob, and Monod (171) have shown forming these enzymes: (a) mannitol phosphate that repression of /3-galactosidase activity depends dehydrogenase ; (6) salicin utilization; (c) ÃŸ- on a dominant gene, i+. Strains of E. coli contain galactosidase; (d) sucrase (206); and (e) alkaline ing the genes i+, z+, are inducible for /3-galactosi- phosphatase (5). It makes no difference whether dase. Those which are genetically i~, z+ are DNA is isolated from an adapted strain or from constitutive for the enzyme. It appears that a re- a nonadapted strain which has the potential for pressor is produced under the influence of the i+ making enzyme. Replication of the genetically gene which prevents the synthesis of new enzyme donated DNA can be demonstrated by the isola molecules. The constitutive state (i~) results from tion of genetically active DNA from transformed loss of the capacity to synthesize active repressor. organisms. It therefore seems likely that the re Possibly, the repressor is a nucleic acid (172). The pair of genetic defects takes place in the DNA of inducer of 0-galactosidase activity counteracts the transformed organisms. However, in this as in the activity of the repressor gene product. other instances discussed in this section, the de Enzyme repression has not yet been fully ex tailed mechanisms of phenotypic expression re plored in animal systems. The repression by crea main to be elucidated. tine of arginine-glycine transamidinase in rats and Turnover of enzyme-forming systems (EFS).â€” chicks may be one such example (236). Another Metabolism may be controlled by the number of apparent example is the depression by glutamine EFS present at any time. Little is known con of the glutamine synthetase levels of HeLa cells cerning the biochemical factors which control the in tissue culture (62). It would be surprising if rate of production and of degradation of particular many more examples are not encountered. Pos-

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1960 American Association for Cancer Research. Kitâ€”Nucleic .leid Synthesis in Tumor Tissue: Review 1135 sibly, enzyme repression may in part account for crease in thymidine kinase and polymerase that the tissue-specific spectrum of enzymes found in would be expected to occur between 6 and 24 the different tissues of an adult animal. According hours after partial hepatectomy (18, 32). Recent to this view, the actual enzyme level in a given ly, Hiatt and Bojarski have shown that, in adult tissue would depend on the competition between liver or kidney, TMP kinase activity can be repressors and inducers at any given time. It markedly increased by thymidine administration. would in part account for the latent enzymatic The rise in TMP kinase ordinarily observed in re potential of a cell. generating liver can be prevented by administer Many examples of enzyme induction are known ing fluorouracil, an inhibitor of thymidylate syn in bacteria, and several have been reported in thesis, thus showing that thymidylate is necessary animal tissues (Table 3). These have been particu for the manifestation of the increased activity of larly studied by Knox and co-workers (128, 129, thymidylate kinase. However, in thymidine- 142). Of special interest is the fact that thymidine treated animals, thymidylate kinase activity in and thymidylate kinase and polymerase may be creases even in the presence of fluorouracil; the inducible. The variation of these enzymes in re increase begins about 1 hour after thymidine ad generating liver has been studied by Potter, Bol- ministration (105). lum, and co-workers (33, 34) and by Mantsavinos Although the experiments of Hiatt and Bojarski and Canellakis (152). These enzymes appear at the are suggestive of enzyme induction, the latter in same time (18 hours after the operation) as DNA vestigators have suggested (107) an alternative synthesis in vivobut continue to increase over the explanation for the experimental results. They period of 18-30 hours while DNA synthesis in vivo have shown that : (a) thymidylate kinase activity is decreasing. When partially hepatectomized disappeared from tissue slices incubated in buffer rats were irradiated 6-13.5 hours after the opera for 3 hours but not from slices incubated with tion with 375-1500 r x-rays, the radiation pro thymidine; (6) a decrease of kinase-specific activ duced essentially complete inhibition of the in ity on dilution of liver extract was prevented by TABLE2 REPRESSIONOFENZYME-FORMINGSYSTEMSINBACTERIA

Reprensor"Arginase""Uracil"ProductssystemAcetyl argininosuc-cinaseCarbamylornithinase, ornithine transearbamylase, 170,186170149, transferaseHistidase,aspartate ofglucosemetabolism"Histidine"3H3P04"Methionine""Tryptophan">Enzyme deliydrogenaseEnzymesurocanase, myoinosital 101,162451,171,17Â«59090 (transaminase,phosphatase,of histidine biosynthesis in Salmonella dehydrase)/3-galactosidase,dehydrogenase, permesseAlkaline phosphataseMethionine synthetaseTryptophan desmolaseTissue differences in animal cells (?)Reference90,

TABLE 3 INDUCEDENZYMESYSTEMSINANIMALCELLSANDBACTERIA

AMMALCELLS BACTEKIA

EnzymeThymidine kinase (?) (liver) phosphate dehydrogenase Thymidylate kinase (?) (liver, kidney) 33, 34, 106, 152 salicin utilization Â¿06 DNA polymerase (?) (liver) 33, 34,152128, sucraseamylomaltase Â¿06171 Transaminases for phenylalanine, tyrosine, tryptophan (liver) 129, 142 Tryptophan(liver)Threonine peroxidase-oxidase 128, 129,142191Enzymemannitolglucuronidase 171 galactokinase 171 dehydrase (liver)Reference33,34,106,152 penicillinaseReference206 171

the presence of boiled extract, thymidine, or sible to assess the validity of these or other thymidylate in the diluent; and (c) thymidine or hypotheses. thymidylate in the homogenizing medium but not in the assay led to increased activity in normal CONTROLATTHEENZYMELEVEL tissues (107). This suggests that the increase after Let us now briefly summarize some of the more thymidine is attributable at least in part to enzyme familiar examples of metabolic control by activa stabilization. Changes in thermal stability of tion or inhibition of enzyme activity. Four general enzymes in the presence of substrate have been categories may be distinguished: (a) control refer encountered by other investigators (91). able to suppressor gene activity; (6) activation Fetal to neonatal life.â€”Drastic variations are related to the dissociation of macromolecules; (c) observed in the enzymatic activity of the livers of negative feedback mechanisms; and (d) various animals during the transition from fetal to post miscellaneous factors which affect enzyme kinetics. natal life. Tyrosine transaminase is absent from the Suppressor genes.â€”Using tryptophan auxo- liver of rats and humans at birth but appears trophs of Neurospora and E. coli, Yanofsky (56, within hours after birth (142, 192). Glucose-6- 248, 249), Bonner (36, 63), and Suskind and co- phosphatase, the enzymes of tyrosine oxidation, workers (220, 221) have greatly contributed to histidase, tryptophan-peroxidase, phenylalanine- the clarification of the action of suppressor genes. pyruvate and phenylalanine-a-ketoglutarate trans The growth dependence of certain mutants on aminase, and phenylalanine hydroxylase are other tryptophan results from the presence of genetical enzymes which are inactive in fetal life but de ly altered enzymes. One such mutant, strain td-24, requires tryptophan for growth at 25Â°C., but it velop postnatally (8, 185). Tyrosine transaminase grows slowly without tryptophan above 30Â°C. can be adaptively increased in the livers of adult rats after parenteral administration of tyrosine. and at this temperature also forms a slight amount Administration of tyrosine to fetal rats in utero of active tryptophan synthetase. In all instances, failed, however, to elicit an appreciable increase in this mutant forms large quantities of a protein, the low activity of the liver enzyme; the large in CRM, which is antigenically related to wild type crease in activity characteristic of adult animals tryptophan synthetase and which possesses activ given these agents is not observed until 7 days ity in reaction (1) but not reactions (2) or (3): after birth. When tryptophan was injected into (1) Indole + triÃ³sephosphate â€”>indole newborn rats during the period when this enzyme glycerol-phosphate was not detected in untreated animals, only small (2) Indole glycerol phosphate + serine â€”Â» amounts of tryptophan peroxidase-oxidase were tryptophan + triÃ³sephosphate synthesized (8). Tryptophan peroxidase-oxidase (3) Indole + serine â€”Â»tryptophan activity was not found in Novikoff hepatoma or in Chang liver cells grown in tissue culture; both It has been possible to obtain highly active of these tissues were originally derived from liver. tryptophan synthetase from this mutant grown at Tryptophan administration failed to elicit enzyme 25Â°by a suitable fractionation of crude inactive activity in these tissues. In the rat, tryptophan extracts. An inorganic inhibitor which completely injection elicits some activity even at an age when inhibits the fractionated mutant enzyme but tryptophan peroxidase-oxidase cannot be demon which has no effect on the wild type enzyme at strated in untreated animals. comparable concentrations can be isolated from Deoxycytidylate deaminase is present in various the inactive td-24 preparations. The inhibitor can tumors, rat thymus, embryonic liver, and rat liver be found in other td mutants and also in wild type which contains proliferating bile duct epithelium, strains. The effect of this inhibitory material can but is absent in normal adult liver and most best be duplicated by Zn++. It appears that with normal tissues (181). The deaminase is also very strain td-24 gene mutation has resulted in the active in chick embryonic tissues (150). Thymidy formation of an altered, metal-sensitive enzyme. late kinase activity is very high in fetal liver and The active enzyme which is obtained by the frac kidney and declines rapidly after birth to the low tionation of crude inactive extracts appears to levels observed in adult liver (105). represent a conversion of CRM to active trypto Two attractive working hypotheses may be in phan synthetase by the dissociation of a metal- voked to explain these findings: (a) increased post protein complex. natal synthesis of EFS for the liver enzymes of A mutation at a second locus, the suppressor amino acid metabolism; and (o) enzyme repression locus, restores the capacity of the mutant, td-24- of thymidylate kinase or deoxycytidylate deami su-24, to grow in the absence of tryptophan. Sus nase. As further data are reported, it will be pos kind and Kurek were able to show (221) that

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1960 American Association for Cancer Research. KITâ€”NucleicAcid Synthesis in Tumor Tissue: Review 1137 partially purified preparations of tryptophan particles (64). Ribonuclease appears to be almost synthetase from suppressed mutant strains were exclusively associated with the 80S components of still metal-sensitive. Apparently, the suppressor the 70S particles (74). The ribonuclease activity action of the td-24-su-24 strain may be concerned of the latter becomes active when the structure of with the control of metal availability and the con the nucleoproteins is disrupted. centration of metal-binding enzymes. An acid phosphatase of rat liver is linked to A second mechanism for suppressor gene activ large particles. The bound enzyme has little or no- ity has been suggested by Glass (87) and others. activity toward added glycerophosphate at pH 6. When there are alternative metabolic pathways Aging of the preparations at 0Â°C., short exposure for the utilization of common substrates and limi of the Waring Blendor, or repeated freezing and tations occur in the availability of the common thawing all lead to an irreversible release of the substrates, the suppression of one pathway may enzyme, which then appears to a large extent in a lead to the stimulation of the alternative pathway. soluble form. In contrast to the bound form, the This mechanism may apply to the manifold path free enzyme exhibits a high activity (22). ways of utilization of tryptophan in Drosophila Ascites tumor cells apparently contain a potent (87) and to some extent to pigmentation in plants. ribonuclease inhibitor. The enzyme of these cells The chemical effects of three color factors, P, M, exhibits pH optima at pH's less than 5 and greater and Y, in Antirrhinum majus have been studied than 8, in sharp contrast to that of various normal

Apigen'm

/ .KampferÃ¶l Precursor-! Precursor -2 Pelargonidin .M

"3,4"â€”â€¢* Luteolin Aureucidin k .P

'Quercctin Cyanidin CHART2.â€”Proposed general pathway for flavonoid biosynthesis in A. majus (118)

quantitatively (118). The P factor controls a tissues which had optimal ribonuclease activities special kind of oxidation of the C-3 fragment that within the range, pH 5.6-7.8. In the presence of joints the two 6-carbon rings of the flavonoid pig p-chloromercuribenzoate, a compound which has ments. Anthocyanidins and flavonols are present been shown to reverse the inhibition of ribo only when P is present in the dominant condition nuclease by a naturally occurring ribonuclease in (Chart 2). The M factor controls the oxidation of hibitor of rat liver, the ascites tumor picture the C-6 ring in the flavones, flavonols, and antho- changed completely. The acid and alkaline peaks cyanidins; thus the pigments, quercetin, luteolin, disappeared and were replaced by a broad maxi and cyanidin, are formed in the presence of M; mum between pH 6.5 and 7.2 (54). This suggests kampferol, apigenin, and pelargonidin in its ab that the two optima in the pH activity curves in sence. The Y factor acts in its dominant form to the absence of p-chloromercuribenzoate were a permit the formation of only small amounts of reflection of the dissociation of the enzyme in aurone pigment, and in its recessive form to per hibitor complex at acid and alkaline pH's. mit the production of large amounts of aurone. Negative feedback.â€”Negative feedback repre Suppression effects of one gene upon another may sents still another mechanism for controlling be observed suggesting that genes products may enzymatic activity. In this case, a product of a compete for a limited source of common precursor. metabolic pathway prevents its own further syn (See also page 1133.) thesis, not by inhibiting the EPS as in repression, Dissociation of macromolecules.â€”E. coli ribo- but by inhibiting the enzyme itself. Several ex somes (70S particles) may be reversibly dissoci amples of negative feedback inhibition in bacteria ated by variations in the pH, ionic strength, or bi are shown in Table 4. To illustrate, threonine in valent cation concentrations to 50S and 80S hibits its own biosynthesis by inhibiting homo-

serine kinase activity (244). L-Isoleucine complete molecules are synthesized at chromosomal sites and ly inhibits the action of L-threonine deaminase, then pass to the cytoplasm where they function the initial enzyme in the sequence leading from as components of EFS. There is considerable ex L-threonine to L-isoIeucine. The apparent affinity perimental evidence to support this hypothesis of L-threonine deminase for L-isoleucine is about (38, 121, 125), although several questions remain 100-fold its affinity for L-threonine (234). A nega to be answered: tive feedback mechanism for the control of purine 1. Nucleic acid precursors are rapidly incorpo biosynthesis has been demonstrated in pigeon rated into the RNA of the sailvary gland chromo liver by Wyngaarden and Ashton (246). somes of Chironomus and Drosophila larvae Control of enzyme kinetics.â€”Many factors for (176). In the case of Chironomus, the chromo the control of the kinetics of enzymatic reactions somal puffs and Balbiani rings were intensely are known (170). These include substrate and labeled and the nucleoli became labeled first in the inhibitor concentrations, salts, metal and co- neighborhood of the nucleolar organizers and later enzyme concentrations, pH, temperature, redox in the entire nucleus. potential, oxygen tension, sulfhydryl compounds, 2. Radioautographic studies with tritiated cyt- the ratio of oxidized and reduced DPN and TPN, idine or uridine show that radioactivity is rapidly the products of enzyme reactions, and the levels taken up and concentrated in the RNA of the in inorganic phosphate, ADP, and ATP. High nucleus of plant cells, Ameba, Neurospora, tissue levels of glucose inhibit the respiration of tumor culture cells, or ascites tumors in vivo (for refer TABLE4 ence, see 121,125). After a brief period of cell incu bation, the excess of radioactive precursors may ENZYMESCONTROLLEDBYNEGATIVEFEEDBACK be removed by washing the cells with a medium containing a large "chaser" of nonradioactive uri EnzymeAspartate dine or cytidine. The subsequent localization of carbamylaseHomoserine acidThreonineIsoleueineValineProlineATPGMPAMP,the label may be followed during continued incu kinaseThreonine bation of the cells. Experiments of this kind show deaminaseAcetolactate-forming 244244234170170246149 enzymesGlutamic that in the first 10 minutes the nuclear inter- dehy-hydrogenaseGuanylicsemialdehyde chromatin areas contain the highest number of silver grains (RNA labeling on the radioauto- reducÃaseInosinicacid dehydrogenasePRPP grams), but at later periods the nucleoli are more amidotransferaseEnzyme IMP,GMPHistidineReferences170244234,heavily labeled. Later, the nuclear labeling dimin AMP+ri-boseconverting phos-phoribose-adenylateInhibitorCytidylic-5-phosphate to ishes, and the radioactivity accumulates in the cytoplasm. 3. Goldstein and Flaut have shown that the transplantation of a radioactive Ameba nucleus cells (Crabtree Effect). Permeases may control the to a nonradioactive Ameba was followed by the availability of substrates within a cell. Many transfer of radioactivity to the host cytoplasm hormones probably exert their effects at the enzyme (37, 38). level; for example, epinephrine activates the en 4. Enucleated fragments of Ameba fail to in zyme, phosphorylase. Virus infection may initiate corporate uracil-C14, adenine, or orotic acid into a series of events which lead to enzyme activation ; RNA, although incorporation into the RNA of for example, T-even phage infection seems to fragments containing the nucleus is rapid. The activate deoxyribonuclease. The possibility that earlier indications of some synthesis by anucleated some viruses modify the activity of the "pace fragments can be attributed to the presence of maker" enzymes of metabolic sequences should microorganisms in the food vacuoles of the receive careful consideration. The stability and Amebae (88, 183). turnover of apoenzymes may be altered by the Some adenine turnover in the RNA of enucle concentrations of available substrate. Finally, ated Acetabularia takes place, but there is no net enzyme activity could be indirectly modified by synthesis. Recent experiments indicate that RNA the breakdown of cellular compartments. synthesis can take place in the chloroplasts of this alga, but this is at the expense of other cytoplasmic SYNTHESISANDPROPERTIESOFTHE RNA fractions (microsomes, cell sap) which ENZYME-FORMINGSYSTEM decrease after enucleation (38). The chloroplasts The locus of R\A synthesis.â€”Let us return to must be viewed as unique cytoplasmic elements, the problem of the synthesis of the EFS. It is at since they are mutable and may be endowed with tractive to postulate that single-stranded RNA hereditary characters. Preliminary reports indi-

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1960 American Association for Cancer Research. KITâ€”Nucleic Acid Synthesis in Tumor Tissue: Review 1139 cate that alga chloroplasts can incorporate the 105,000X g (Fraction NP); (6) the nonsedimented DXA precursor, thymidine, into acid-insoluble RNA of this extract (Fraction NSS); and (c) the linkage (216). Moreover, the mere incorporation RNA which is not extracted from the nucleus by of radioactive precursors into RNA in enucleated this procedure (Fraction R) (85, 125, 168). One cells must be regarded with considerable reserve hour after the injection of tritiated uridine into unless it is definitely shown that the incorporation ascites tumor-bearing mice, nuclear fraction NP is into the interior of the polynucleotide chains. has about 6 times the specific activity of the End-group labeling of "transfer-RNA" may be microsomes, whereas nuclear fraction NSS is also mistaken for evidence of RNA synthesis (99, 102, more radioactive than the cytoplasmic super 103). natant fraction. The residual nuclear RNA (Frac 5. Particles morphologically resembling the cy- tion R) is about 20 times as radioactive as the toplasmic ribosomes (8-20 m/Â¿)have been ob microsomes. This residual nuclear fraction is pre served by Swift in the nucleoli and in the inter- sumably associated with the nucleoli and chromo chromatin areas of rat liver nuclei, whereas some somes. what larger particles (30 m^) have been found in Cell nuclei contain amino acid-activating en the interchromatin areas of nuclei of the pancreas zymes (109, 238) and enzyme systems from the of Triturus (222). Particles 10-30 m/i in diameter synthesis of proteins (85). The nuclear amino acid- have also been observed on the lateral loops of activating enzymes from pig liver readily bind lampbrush chromosomes and on the chromosomal alanine to nuclear soluble RNA from pig liver rings of Balbiani (85). Nucleoprotein particles can but are relatively inactive with soluble RNA from be extracted by neutral buffer solutions from iso all other sources including pig liver cytoplasm lated nuclei (85). The particles are revealed by (238). Amino acids are also incorporated into the electron microscopy to consist of granules of 10- RNA of calf thymus nuclei by "pH 5 enzymes" mn diameter. In unextracted nuclei, the presence isolated from the latter source (109). of similar granules may be noted in both the An alternative method for the study of nuclear nucleolus and in the extranucleolar regions of the RNA synthesis has been suggested by the dis nucleus. The possible relationship of such particles covery of Yamana and co-workers (196, 197, 247) to the ribosomes remains to be elucidated. and by Georgiev and associates (86) that the ex 6. The nucleoprotein particles which are ex traction of RNA from tissues by aqueous-phenol tracted from the nucleus by neutral saline may be solutions is incomplete, and that morphologically sedimented by centrifuging the extract at 105,- intact but damaged nuclei persist after such 000 X g for 2 hours in the Spinco preparative phenol extraction. In this fractionation, possible centrifuge (85, 121, 125). The particles contain artifacts arising in the course of the freeze- about 50 per cent RNA by weight and are indis thawing and homogenizing of tissues may be tinguishable from the microsomes with respect to avoided. Moreover, the tissues are exposed to 45 electrophoretic mobility and nucleotide composi per cent phenol within minutes of the time that tion (121, 123, 168). At least one other RNA frac the animals are sacrificed, thereby reducing the tion may be isolated from the nuclei of cells with problem of the control of endogenous nucleases. a molar base composition similar to that of the The results of Yamana and co-workers (197) microsomes (123). High molecular weight RNA is and Georgiev et al. (86) have been confirmed in found in the nucleus as well as the cell ribosomes our laboratory. Table 5 shows the incorporation (48). of titrated uridine into the various kinds of RNA 7. Chemical turnover studies are compatible obtained by the phenol fractionation. Fraction P with the nucleus as a major site of RNA synthesis. is the RNA extracted into the aqueous phase by Shortly after the injection of labeled RNA pre the saline-phenol solution. This fraction is at cursors into rats or mice, the order of specific tributable to cytoplasmic RNA and some nuclear activity is as follows: nucleus > soluble cyto RNA. Approximately 90 per cent of the RNA of plasm > mitochondria-microsomes (13, 57, 58, this fraction may be precipitated with l M NaCl. 154, 155, 201). Of the microsomal subfractions, The soluble 10 per cent has a higher specific activ the ribonucleoprotein granules which are most ity than the RNA precipitable with l M NaCl. active in protein synthesis appear to be least ac Fraction D is the precipitate found at the bottom tive in RNA labeling (26, 194). of the tube below the phenol layer. From the The turnover of three nuclear fractions has been interphase between the saline and phenol, the measured: (a) the ribonucleoprotein particles nuclear fraction is obtained. Fraction E is the which are extracted from the nucleus by neutral RNA which is extracted from the nuclei with saline and sedimented by centrifugation at sodium trichloroacetate, pH 6.9, and phenol. This

Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1960 American Association for Cancer Research. 1140 Cancer Research Vol. 20, September 1960 fraction also contains most of the DNA of the cell. RNA in a ratio of 10:1, there is always the possi Fraction R is the residue. bility that the presence of the DNA distorts the About 80 per cent of the total RNA of the RNA base ratios. Ehrlich tumor cells was found in Fraction P The special case of virus-infected cells requires (Table 5). However, Fraction E had 90 times the additional comment. The question is: Are RNA specific activity of Fraction P, and well over 90 templates synthesized on chromosomal DNA, per cent of the total radioactivity was incorpo or may RNA serve as a template for its own rated into Fraction E plus Fraction R. The RNA replication? Consider an RNA virus. It is known of Fraction E is precipitated from solution em that cell infection by the viral RNA alone is suf bedded in fibrous DNA. It is not as yet known ficient to initiate viral replication (83). Does a whether this represents nascent RNA newly DNA corresponding to the viral RNA preexist in synthesized on the DNA of the chromosomes or the cell? Or, does the viral RNA act as a template for the synthesis of a new DNA intermediate TABLE 5 which might then multiply exponentially to In vivo INCORPORATION(10MINUTES)OFURIDINE-ÃœS finally synthesize new RNA molecules? The two INTO EHRLICHASCITESTUMORRNA FRACTIONS latter mechanisms could preserve the position of DNA as the only macromolecule capable of re cent of producing itself from low molecular weight inter cent of mmÂ«g total Fraction*PSNuclear total mediates. On the other hand, if viral RNA can counts/minin HNA1.17.693.374.02.411.7counts/minâ€žgDNA1.91.3Per R\\80.10.311.86.21.5reproduce itself and transfer its information into a RNA5.40.166.727.50.2Per form usable by the cell, there would be no reason to assume that some species of cellular RNA could ENuclear not do the same. This consideration raises the RDMixed possibility that an independent hereditary system could exist in cells in addition to the one con RNArimili- trolled by DNA. If this were the case, an under standing of the mechanisms by which such RNA * Fraction P extracted from cells with 6 volumes of 0.01 M molecules were replicated could markedly modify NaCl, 10~3M phosphate, 10~3M versenate, and 6 volumes of our concepts of how DNA is replicated. For ex phenol. The RNA is precipitated from the extract by the addi ample, since RNA is a single-stranded molecule, tion of 2 volumes of ethanol and of potassium acetate to a final concentration of 2%. one might conjecture that the chain is duplicated Fraction S was precipitated from the supernatant of the by identical base pairing rather than complemen above step with 5% trichloroacetic acid. Nuclear Fraction E is the RNA extracted from the residue tary base pairing. Or, to preserve the concept of of the NaCl-phenol extraction with 5% trichloroacetic acid, complementary base pairing, one might postulate pH 6.9, and phenol. that the two halves of the chain were comple Nuclear fraction R is the residue from the previous step. Fraction D is a small precipitate formed under the phenol mentary to each other or that chains complemen layer. tary to the viral RNA serve as intermediates in viral RNA synthesis. whether it is merely RNA which has aggregated Although the answers to these questions are of with the DNA. These interesting alternatives are the utmost importance, few definitive experi under investigation. ments are as yet available. Only two statements Sibatani et al. (197, 247) have emphasized that can be made with any assurance: (a) new DNA the molar base composition of Fraction R differs synthesis does not seem to be required for the from that of Fraction P. Differences in the com replication of RNA viruses; and (b) new RNA position of nuclear RNA from cytoplasmic RNA synthesis seems necessary for the replication of have frequently been reported in the past (57, 58, both RNA and DNA viruses (173, 190, 198, 208, 75, 168). Such differences could constitute a 223). Fluorodeoxyuridine or aminopterin, inhibi powerful argument against the role of the nuclear RNA as a precursor of cytoplasmic RNA. It is tors of the methylation of deoxyuridylic acid to our view that gross differences in the composition thymidylic acid and of DNA synthesis, have no of nuclear and cytoplasmic RNA should be re effects on either the rate of formation or the yield garded with reserve unless: (a) the nuclear per cell of the RNA viruses, polio, Newcastle RNA is obtained under conditions where degrada disease virus, and tobacco mosaic virus (190, 198, tion by nucleases is unlikely; and (6) the nuclear 208). Bromodeoxyuridine is also ineffective. These RNA can be shown to be free of any DNA. In antimetabolites do, however, prevent the replica nucleoprotein preparations containing DNA and tion of such DNA viruses as vaccinia and bac-

teriophage. Interference with RNA synthesis The proteins from purified ribonucleoprotein shortly after cell infection inhibits the multiplica particles from rat liver contain a high proportion tion of bacteriophage, vaccinia, adenovirus, herpes, of basic amino acids and only very small amounts and influenza virus (173, 223). of cystine and tryptophan. In this respect and in Until the question of the replication of RNA their low molecular weight, they resemble the viruses is resolved, the problem of the sequence of histones of the nucleus (43, 55,250). As in the case information transfer in normal cells must be con of the histones, the basic proteins of the ribosomes sidered as open. are rich in N-terminal proline and alanine groups Properties of the EPS.â€”By the conventional (43). methods of sucrose-CaCU homogenization and It seems probable that the two high molecular differential centrifugation, the following approxi weight RNA components with sedimentation mate distribution of RNA is found in lymphoma constants of about 30S and 18S, respectively, are cell fractions: microsomes, 54-61 per cent; mito- related to the 50-60S and the 30-40S subunits of chondria-microsomes, 15-20 per cent; cytoplasmic the ribosomal particles (94). For the tumors which "supernatant," 5-7 per cent; nuclear "super we have studied, the molecular weights of these natant," 1-2 per cent; nuclear particles of neutral RNA components are of the order of magnitude of saline extract, 6-7 per cent; and nuclear residue, 2 and 1 X IO6, respectively. Since we know the 10-14 per cent. The cytoplasmic "supernatant" total RNA per cell, the per cent RNA in the fraction is unique in that it has a molecular microsomes, and the approximate molecular weight of about 40,000, contains the unusual base, weight of the RNA of the ribosomes, we can esti 5-ribosyluracil, and functions in the transfer of mate the number of ribosomes and RNA molecules activated amino acids to the microsomes (52, 98, per cell. Let us assume for the sake of argument 99, 111, 169, 251). It is apparent that by either that one ribosomal particle with its two RNA this conventional method of fractionation or by components represents one enzyme-forming tem the phenol fractionation (Table 5), most of the plate and that there are about 2,000 different en cellular RNA is associated with the microsomes. zymes in lymphoma cells. We then estimate that We can arrive at an appreciation of cellular or there are, on the average, about 2,000 templates ganization by certain calculations and comparisons for each diploid lymphoma cell enzyme ! Tissieres of animal cell ribosomes with those of plants, and co-workers (229) have estimated about 90,000 bacteria, and yeast. ribosomes per E. coli in the exponential phase of In exponentially growing bacteria, Tissieres and growth, or, perhaps, 45 templates for each enzyme. co-workers (228, 229) found from 80 to 90 per cent In the stationary phase, or upon incubation in of the cell RNA in ribosomes. Ts'o et al. (231- phosphate buffer, this number decreases by a fac 233) found 50-60 per cent of pea seedling RNA tor of 4-5 to about 20,000 particles per cell. in ribosomes. The composition and molecular It would seem that, compared with E. coli, weight of ribosomes varies with the source and animal lymphoma cells are extremely well buf physiological state of the cell (95, 177). Pea seed fered with respect to enzyme-forming systems. ling, rabbit appendix, and E. coli ribosomes con Thus, changes in the number of EPS under the tain about 60 per cent RNA and about 40 per cent influence of the environment or the complete loss protein (35, 228, 229, 231). Rat liver and yeast of EFS due to genetic alterations must be rela microsomes contain about 40 per cent RNA (95). tively slow processes. The molecular weights of pea seedling, E. coli, The time interval which is required to establish yeast, Neurospora and animal ribosomes are about 4^4.5 X IO6, 2.6 X IO6, 4.1 X IO6, and a new balance of EFS and enzymes may be desig 4.5 X IO6, respectively. These particles manifest nated the phenotypic lag. The factors modifying the phenotypic lag as well as those affecting the sedimentation rates in the ultracentrifuge of about 70S (229) and 75-83S (46, 95, 231). The ribosomal induction and selection of cell lines endowed with particles may be reversibly dissociated in the favorable chromosomal sets are probably impor absence of Mg++ to 30-40S and 40-60S compo tant in tumor progression. A cell which has under nents. These smaller components have approxi gone chromosomal changes and gene mutations mately the same shape, density, and RNA content may still be amenable to control provided that as the larger particles. The molar base ratios of the the enzymatic potential of the cell has not been 30S, 50S, and 70S E. coli particles are also quite expressed. An understanding of the conditions similar (205). Under certain conditions, two 70- which facilitate or retard the expression of the 75S particles may combine to form 100-1 IOS cancer phenotypes would undoubtedly be of particles. significance in the control of neoplasia.

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