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A comparative study of halodeoxyuridines

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A comparative study of halodeoxyuridines

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Content A COMPARATIVE STUDY OP HALODEOXYURIDINES by- Bessie Huang A Thesis Presented to the. FACULTY OP THE DEPARTMENT OF BIOCHEMISTRY AND NUTRITION UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree Master of Science January i960 U N IV E R S IT Y O F S O U T H E R N C A L IF O R N IA G R A D U A T E S C H O O L U N IV E R S IT Y P A R K L O S A N G E L E S 7 . C A L IF O R N IA H $74 This thesis, written by Besj.ie..Huang....._..T • . ...,...... _ under the direction of hS£...Thesis Committee, and approved by a ll its members, has been pre- sented to and accepted by the Graduate School, in partial fulfillm ent of requirements fo r the degree of . Master of'Science D a te ................. J a n u a r y j . .. 1 9 . 6 0 THESIS COMMITTEE* a ■ _ Chairman TABLE OP CONTENTS Page LIST OF TABLES ................ .. . .. .. ... . IV LIST OF FIGURES ................... v LIST OF ABBREVIATIONS . ...........■ . ......... vi Chapter I. INTRODUCTION................. 1 II. HISTORICAL BACKGROUND.................... . 7 Discovery, Development and Recent Appli cations Pyrimidine Biosynthesis The Halogen 5-Substituted Derivatives of Pyrimidines I I I . EXPERIMENTAL .......................... 23 Synthesis of 5-Chl'orodeoxyuridine Melting Point Spectrophotometric Data Microbiological Animal Experiment' IV. RESULTS................. •.................. 3^ Microbiological Inhibition Analysis Reversal Studies Combination of Two Halodeoxyuridines Animal Experiment V. DISCUSSION................. 56 VI. SUMMARY ....... 66 BIBLIOGRAPHY.................... 68 iii LIST OF TABLES Table Page I. Growth of E. coli K-12 in the Presence of 5-Fluorodeoxyuridine'or 5-Chlorodeoxy- uridine as a Function of Time........ 36 II. Growth of E. doli K-12 in the Presence of Different Compounds .......... 40 III. Growth of E_. coli K-12. in the Presence of Two Halodeoxyuridines ........... 47 IV. Results of the Isolation of DNA From Ehrlich Ascites Cells . . ... ......... 51 V. Chromatographic Separation of Pyrimidine Bases in DNA - Hydro lysates... ......... 52 VI. . . Spectrophot.ometric and Radioactivity Data of Pyrimidine Bases Isolated From DNA of Ehrlich Ascites Cells ....... 53 iv LIST OF FIGURES FIGURE PAGE 1. The Biosynthesis and Interconversion of Pyrimidine Compounds ................. 12 2. Ultraviolet Absorption Spectrum of 5-Chlorodeoxyuridine' in 0.01 N HC1. ... 25 3. Growth of E. coli K-12 in the Presence of Various 5-Halodeoxyuridines .... 35 4. Reversals by Pyrimidine Nucleosides and Pyrimidine Bases of the Growth,Inhibi tion of E. coli K-12 Produced by 800 pM 5-Chlorodeoxyuridine ........... 38 5. ■ Reversals by Pyrimidine Nucleosides of the Growth Inhibition of E. coli K-12 Produced by 0.5 pM 5-Fluorodeoxy- uridine . .......................... 39 6 . Reversals by Uridine of the Growth In hibition of E. coli K-12 Produced by 5-Chlorodeoxyuridine ................. 42 7. Reversals by Pyrimidine Nucleosides of the Growth Inhibition of E. coli K-12 Produced by 5-Chlorodeoxyuridine . . . 44 8 . Reversals by Pyrimidine Nucleosides of the Growth Inhibition of E. coli K-12 Produced by 5~Fluorodeoxyuridine . . . 45 9. Reversals by 5-BromodeoxyurIdIne of the Growth Inhibition of E. coli K-12 Pro duced by 5-Fluorodeoxyuridine .... 49 v . LIST OF ABBREVIATIONS A - ang s t^orn units ATP- adenosine-5'-triphosphate BrU- 5-bromouracil BrdCy- 5-bromodeoxycytidine BrdUR- 5-bromodeoxyuridine Cy- cytidine CMP- cytidine-51-phosphate CDP- cytidine-5'-diphosphate CTP- cytidine-5r-triphosphate C1U- 5-chlorouracil CldUR- 5-chlorodeoxyuridine CldCy-, 5-chlorodeoxycytidine dCy- deoxycytidine dCMP- deoxycytidine-5*-phosphate dCDP- deoxycytidine-51-diphosphate dCTP- deoxycytidine-51-triphosphate dUR- deoxyuridine dUMP- deoxyuridine-51-phosphate dUDP- .deoxyuridine-51-diphosphate dUTP- deoxyuridine-51-triphosphate DNA- deoxyribonucleic acid FU- 5-fluorouracil vi FUR- 5-fluorouridihe FdUR- 5-fluorodeoxyuridine FdUMP- 5-fluorodeoxyuridine-5’-phosphate IU- 5-iodouracil IdUR- - • 5-iododeoxyuridine RNA- ribonucleic acid TMP- thymidine-5 '-phosphate TDP- thymidine-5 *-diphosphate TTP- thymidine-5 1-triphosphate UR- uridine UMP- uridine- 5 *-phosphate UDP- uridine-5 '-diphosphate UTP- uridine-r5 * -triphosphate ( - molar extinction coefficient X- wave length mp- millimicron pM micromolar or micromoles per liter pm- micromoles CHAPTER I INTRODUCTION Metabolic antagonists are used effectively in many fields of studies today. In biochemistry,, they are help ful in elucidating metabolic pathways, especially in the field of nucleic acid and protein synthesis. In nutrition, proper use of antimetabolites has resulted in a better understanding of vitamin and hormone deficiency diseases. Antimetabolites are being synthesized and' used increasingly for applications in pharmacology and chemotherapy, such as for treatment of infectious diseases, leukemia, tumors, and other illnesses. In biology, they are used in attempts to arrive at a biochemical explanation of the changes which occur in. development and differentiation of organisms, and in the study of mutations (l). It is still not known clearly how antimetabolites exert their inhibitory actions in most instances because of the complexity of many interrelated reactions in the living cell. However, from past studies of the actions of many analogs in vivo and in vitro, several possible sites and mechanisms of action have been postulated (1-3)• Anti metabolites may interfere with a metabolite for formation of an intermediate or product essential for normal 1 metabolic function of the cell. Interference may be by competitive or non-competitive inhibition. Inhibitors may undergo transformations similar to those of the metabolites, thereby producing a series of compounds, each one of which may interfere with normal metabolic trans formations or functions. Antimetabolites may interfere with coenzyme function in.enzyme systems, be incorporated into complex compounds such as nucleic acids or proteins in place of the substrate, inhibit permeability of the cellular membranes, or be catabolized to compounds toxic to the cell.. Another class of inhibitors, the nitrogen mustards (4), change the structures of functional macro molecules such as enzymes or nucleic acids by alkylation or hydrogen bonding to produce biologically inactive macro- molecules. Many analogs of purines, pyrimidines and their precursors have been synthesized. Of the pyrimidine analogs, some have been made by substitutions within the pyrimidine ring. This may be exemplified by 6-azathymine (5), where the number 6 carbon atom in the pyrimidine ring is replaced by a nitrogen atom. Other pyrimidine analogs have been made by substitutions at the 3-position of the ring, like 3-methyluridine ( 6) or 3-methylthymidine ( 7), and still others by substitutions at the 6-position of the ring, like 6-uracil sulfonic acid ( 8), an analog of orotic 3 acid. The 5-substituted analogs are of particular interest as thymine analogs because thymine compounds are specific for DNA synthesis (9). It has been shown that in most instances, the 5-substituted pyrimidine deoxyribosides or ribosides are better inhibitors than the corresponding substituted free bases (10-12). Recently, 5-fluorodeoxy- uridine (FdUR) (48) and 5-bromodeoxyuridine (BrdUR) (7), two very interesting 5-substituted deoxyuridine analogs, were reported. FdUR was found to be not only an excellent inhibitor of nucleic acid metabolism but also an active anti-tumor agent in animals. The drug is currently being tested on human patients suffering from leukemia and various types of cancer. The mechanism of action of FdUR has been studied in various bacterial systems and tumor cells (49-63). BrdUR was also found to inhibit normal nucleic acid metabolism in some bacterial systems and tumor cells (72-74).. However, its major site of inhibition and mechanism of action was shown to be different from that of FdUR. FdUR interferes with deoxyribonucleic acid (DNA) synthesis by inhibiting the methylation of deoxyuridine-5'- phosphate (dUMP) to thymidine-5 1-phosphate (TMP) while BrdUR inhibits the incorporation, of thymine or thymidine into DNA thymine. In addition, 5-bromouracil (BrU), the base analog of BrdUR, is incorporated in place of thymine into the DNA of bacteria ( 6 6,6 7) and bacteriophages 4 (68,71)* "while 5-fluorouracil (FU), the corresponding base analog of FdUR, is incorporated in place of uracil into ribonucleic acid (RNA) of liver, spleen, and Ehrlich ascites cells in mice (5 1,5 8,6 0) and tobacco mosaic virus ( 6l), Furthermore, BrU causes an increase in frequency with which phage mutants of various types arise (6 8) while FU has not been known to cause any kind of mutation. It is of interest that when the two halogens, fluorine and bromine, differing in size ( 1 3,2 6) and electro negativity, were substituted in the 5~position of deoxy uridine (dUR), the resulting halogenated analogs of dUR differ considerably in biological activities. It was therefore of interest to prepare the new compound, 5-chloro deoxyuridine (CldUR.), thus making possible a direct com parison of FdUR, CldUR, and BrdUR as to the mechanisms of action, sites of inhibition, and the relative effective ness as growth inhibitors. It seemed reasonable to pre dict that a substitution at the 5-position of dUR with chlorine, which is intermediate between fluorine and bromine in size ( 1 3,2 6) and electronegativity, would result in a compound possessing some of the biological properties of each of the other two halogen derivatives. In addition, since it has been shown that 5-cfrl°rourac^l (C1U), the base analog of CldUR, is incorporated into the DNA of some strains of Escherichia coli ( 2 6,6 5,6 9), and 5 ' since pyrimidine nucleosides have been shown to be more > active as DNA precursors than, free bases, one might presume that CldUR would be more effectively incorporated into DNA than C1U. Should this occur, the new compound would be of interest for studies in genetics. A method of synthesis of chlorouridine from uridine (UR) had previously been described by Roberts and Visser (6). This procedure seemed applicable for the present synthesis of CldUR from dUR. This background offers the rationale for the synthesis and biological testing of CldUR described in this thesis. Escherichia coli K-12 was chosen as the test organism because preliminary experiments showed that CldUR inhibited the growth of this organism de novo. Since the organism requires no pyrimidine for growth, it is likely that CldUR interfered with some intermediates arising de novo from simple precursors. Inhibition analysis and reversal studies were undertaken in the hope that this approach might provide information concerning possible sites of action of CldUR. The size and nature of the three halogens were then cor related with the biological activity of the three halogen 5-substituted analogs of deoxyuridine. Information concerning the site of action resulting 6 from these studies were extended by tracer studies. It was decided that this work would be done using a mammalian system. From the standpoint of convenience, and because, under the experimental conditions, a rapidly proliferating % system is needed, Ehrlich ascites cells from mice were used for the latter experiment. CHAPTER II HISTORICAL BACKGROUND Discovery, Development, and Recent Applications The concept of antimetabolite action started in .1910-191^- when Michaelis and Pechstein observed that some inhibitors of a carbohydrate-hydrolyzing enzyme were similar1 in structure to the hydrolysis products (14). Among other findings, Quastel. and Wooldridge in 1928 showed that malonic acid, which is structurally similar to succinic acid, competitively inhibits succinic dehydrogenase activ ity (15). Woolley and his co-workers in 1938 found that /3-acetylpyridine and pyridine-3-sulfonic acid, analogs of nicotinic acid, were toxic rather than beneficial to. dogs deficient in'this vitamin (1 6). The idea that structural resemblance is the reason for inhibition slowly gained acceptance and the hypothesis of substrate-inhibitor com petition was gradually established. This concept was popularized when Woods in 19^0 discovered that the sulfonamide drugs were antagonists of p-amino benzoic acid (17). He suggested that the sulfonamide drugs owed their bacteriostatic properties to the fact that they are structural analogues of p-amino benzoic acid, an essential metabolite in these microorganisms, and that the two 7 8 compounds compete for a position on a specific enzyme. This interpretation aroused interest in synthesis of many analogs of metabolites and vitamins which might be useful chemotherapeutic agents against infectious diseases. An outstanding success was the development of analogs of pan tothenic acid., which exert a therapeutic effect in ex perimental malaria, a disease caused by organisms known to require pantothenic acid as growth factor. For example, the' 4-chlorophenyl analog of pantoyl taurine is at least, four times as active as quinine (l8) . Intelligent use of antimetabolites in a number of biological systems has sub sequently resulted in a better understanding of the inter mediary metabolism of amino acids, purines, and pyrimi dines, and of the nature of enzyme action-( 1-3)• Numerous examples of antimetabolite action can be discussed, but this thesis shall be concerned mainly with the 5-substituted pyrimidine analogs. It is of interest to note that substitutions at the pyrimidine nucleus pro duce antimetabolites which differ considerably in their mechanisms of action. Thus, BrU, 5-r.itrouracil, 5-hydroxy- uracil, and 5-aminouracil all inhibit growth of Lacto bacillus casei, but reversal studies have shown that there are differences in their sites of inhibition. BrU proved to be a competitive antagonist of thymine; 5-nitrouracil, an antifolic; 5-hydroxyuracil, a uracil inhibitor; 9 | 5-aminouracil appeared, to act "both as an antifolic and as an inhibitor of thymine utilization (19)• An interesting pyrimidine nucleoside analog is 5-hydroxyuridine. It competitively inhibits the growth stimulation produced by purine in purine-requiring E. Ci Oli_ mutants. This inhibitory effect is completely reversed by uridine or cytidine in a manner indicating that the purines may function in the formation of pyrimidine-like compounds (20) . In 1954, It was reported that 8-azaguanine and 6-mercaptopurine inhibited normal cell function by being incorporated into nucleic acids which resulted in non functional or fraudulent nucleic acids (21-23). This finding caused much excitement because of the possibility of applications of these results for correlating the fields of biochemistry, enzymology, therapeutics and genetics. Subsequently, many antimetabolites have been reported to be incorporated into DNA and RNA and into proteins. Incorporation of analogs into nucleic acids does not necessarily result in inactivation of all func tion of the cell. p-Fluorophenylalanine is incorporated into the proteins of Lactobacillus arabinosus and E. coli where the analog partially replaces phenylalanine and tyrosine. Although several adaptive abilities of the organism are inhibited, incorporation of other amino acids 10 into the proteins is not affected (24.,25) . Although C1U, BrU, 5-iodouracil (IU) (26), and 6-azathymine (27) are in corporated into DNA in place of thymine in bacteria., the mechanism of inhibition of these pyrimidine analogs may not be a result of incorporation into DNA (27-29). 2-ThiouracLl (30) and FU (6l) are incorporated into the RNA of tobacco mosaic virus and these resulted in reduced infectivity of the virus. Ever since the finding that quantitative differ ences exist in the metabolism of tumor and most normal cells (31)5 a popular approach to cancer chemotherapy has involved the use of analogs of pyrimidines, purines, and their precursors as drugs. Some 40,000 compounds are being tested annually for anti-cancer activity. Some have cured cancers in animals and a few are of temporary bene fit in human cancer patients. Thus, 2-thiouracil is used for treatment of thyroid cancer, 6-mercaptopurine and azaguanine, amethopterin and aminopterine, azaserine and many others,. alone or in combination, are used for treat ment of leukemia (3 2). In addition to toxic effects, a relapse eventually develops referred to as drug resistance in almost every case where a favorable response to these drugs is obtained (3 3). With regard to the use of Inhibitors in cancer chemotherapy, it should be mentioned that effective 11 inhibition of bacterial growth does not necessarily ensure activities as anti tumor agents in vivo. Thus., when the effects of uracil antagonists on the inhibition of growth of mammary adenocarcinoma 755 in mice were studied (3^)j it was observed that of the six compounds which behaved as competitive antagonists of uracil in L. easel, only two— 4-thiouracil and 6-azauracil, were active in reducing tumor growth; one-2,4-dithiouracil, had an' effect on the tumor, at near toxic doses; while three-5-fluorouracil, 5-hydroxy- uracil and 5-pnopoxyuracil, were inactive under the con ditions studied. In turn, a number of compounds,vfori. .example, tkrhydroxypyrimidine, 5-carboxyuracil, and urethan, which were inactive microbiologically, proved to be effec tive tumor inhibitors in mice. Pyrimidine Biosynthesis The biosynthesis and interconversion of pyrimidine compounds are shown in Figure 1 (l,35^36). Knowledge of the biosynthesis of deoxyribonucle- otides is still not complete. Using glucose-l-C1^ as * carbon source for growth of E. coli strain B, Cohen and co-worker (37) isolated deoxyribose and ribose of RNA and DNA and found similar labeling. They concluded that in E. coli strain B, the ribose and deoxyribose of nucleic acids are derived from the same common pathway in E. coli. More recently, Spell and Dinning (38) observed that in Oxalacetic Aspartic- -ureido- succmrc dihydro orotic orotic 'UTP- UDP- COenzymes RNA orotidylic acic UMP 'CTP- : cdp CMP" dtJMP- cytosine cytidine- dCy - uracil t >uridine" dUR \k dUMP uracil l . thymine T ^ thymidine dCDP dCTP • TMP TDP TTP Fig. l.-The biosynthesis and interconversion of 3 . pyrimidine compounds. ^Broken lines— doubtful pathways Straight lines--reversible pathways. 13 Lactobacillus leichmannii, vitamin B^2 was required for the conversion of ribose to deoxyribose. While in RNA biosynthesis, the presence of 5-phos- phoribosyl-l-pyrophosphate accounts to a large extent for the formation of ribonucleotides directly from the pyrimi dine bases, there is no evidence for the presence of a deoxyribose analog of PRPP in DNA synthesis. Deoxynucle- oside transglycosylase, first reported.by McNutt (39), is found in a number of bacterial species. The presence.of pyrimidine deoxyribonucleoside phosphorylase, which converts uracil and' thymine but not i cytosine to the corresponding deoxynucleosides, has been observed in liver preparations (40,41), but whether or not this enzyme is the same as that of pyrimidine ribonucle- oside phosphorylase is still not known. In a soluble extract of Salmonella typhimurium LT-2, Grossman (42,43) has shown the net conversion of cytidine and uridine to the corresponding deoxyribonucle oside, the conversion of cytidine-5*-phosphate (CMP) to deoxycytidine-5'-phosphate (dCMP), and the conversion of purine ribosides and ribotides to the corresponding deoxyribosides and deoxyribotides. Experiments with enzyme preparations have revealed the presence of nucleotide kinases and deoxynucleotide kinases but specific kinases have not been isolated. 14 . However, indirect evidence does-, indicate existence of these specific kinases. Canellakis has shown that a high' speed, particle free, supernatant fraction from normal rat liver converts deoxyadenosine-5’-phosphate, dCMP, and deoxy- guanosine-51-phosphate but not TMP to the respective diphosphates and triphosphates (44) . The comparable fraction obtained from regenerating rat liver, in addition, can convert TMP to TDP and TTP (45) . Kornberg and co workers (46) have found in Lactobacillus bulgaricus 09j which requires orotate as a specific growth factor, an enzyme which converts orotate, but not uracil, to UMP. A kinase from E. coli has also been purified by Kornberg (35) which, in the presence of adenosine-51-triphosphate (ATP), converts TMP, dCMP, deoxyadenosine-5!-phosphate, and deoxyguanosine-5 1-phosphate to the respective nucleoside triphosphates. This enzyme, however, does not phosphory- late dUMP. Of interest is the fact that Kornberg and co- workers (47) have also shown that, while dUMP is inactive, deoxyuridine-5 '-triphosphate can substitute for TTP as a precursor of DNA thymine in their cell-free enzyme prepara tion containing DNA pyrophosphorylase. The Halogen 5-Substituted Derivatives of Pyrimidines 5-Fl]uorouracil was first synthesized by Duschinsky et al. (48) in 1957 in an attempt to make a metabolic antagonist of uracil. A uracil antagonist was desired 15 because earlier evidence showed tha-t tumor tissue utilized uracil more effectively than did normal tissue (49,50). Because of the potent inhibitory effect of this uracil derivative on the growth of microorganisms and some animal tumors (51), 5-fluorouridine (FUR), FdUR and other sub stituted F-pyrimidine nucleosides and deoxynucleosides were synthesized (52) . The mechanism of action of the afore mentioned pyrimidine derivatives has been studied ex tensively. Because of the interesting biological activi ties of FdUR and its close relationship to CldUR, the postulated sites of inhibition of the F-pyrimidines are summarized briefly. FU was shown to be 5,000 times more effective than BrU in inhibiting growth of L. leichmannii (51) . Thymidine and thymine reverse growth completely and noncompetitively at the same molar concentration as the inhibitor, while uracil reverses growth competitively at a much higher con centration. Cohen (53) showed in some strains of E_. coli that, of the F-pyrimidines, FdUR was most effective in inhibiting growth. He also demonstrated conversion of FdUR to fluorodeoxyuridine-5'-phosphate (FdUMP) in the cells, and that the latter irreversibly inhibited bacterial thymidylate synthetase. In a human tumor cell strain, Eidinoff and Rich (54) demonstrated that while FdUR inhibited the incorporation of orotic-2-C1^ into DNA 16 thymine, the incorporation of labeled thymidine into DNA thymine was enhanced. Studies on mice bearing Ehrlich ascites carcinoma in vivo (5 5) and in vitro (5 6) showed inhibition by FU of incorporation of uracil-2-C^^, orotic _ ili 14 00 acid-6-C , C -formate and phosphate-Po^ into DNA of liver, spleen, and ascites cells. Incorporation of labeled thymidine, again, was unaffected. On the basis of these data, it was postulated that the primary site of inhibi tion by FU was the methylation of dUMP to TMP and that FdUMP, which had been shown to be produced from FU in enzyme extracts of Ehrlich ascites tumor cells (57) and in studies of whole cells in vitro (5 6,5 8) was the true in hibitor. This conclusion was also supported by the in vestigations of Scheiner et al. In bacteria (59). A second site of inhibition by the fluorinated pyrimidines was postulated to be the interruption of RNA ■ \ synthesis caused by FU or FU ribotides which blocked the 'i utilization of uracil or uracil ribotides. This postulate was supported by the fact that in E. coli K-12, thymine and thymidine did not completely reverse the inhibition caused by FU (51)j that in E. coli strain B In the presence of FdUR, there was a complete return to viability only when uracil and thymine were both added as reversing agents (5 3); that in human tumor cell strain, while thymidine reversed completely the effects caused by FdUR, 17 it did not do so, in the presence of FU and FUR. On the other handj dUR reversed the effects caused by any of the three inhibitors (54). Heidelberger and co-workers.further observed that FU or FUR, but not FdUR, inhibited the in corporation of uracil-2-Cli+ and orotic acid-6-C1^ into RNA of Ehrlich ascites cells in vitro (51)• Skold showed that in acetone powder extracts of Ehrlich ascites cells, FU strongly inhibited the action of pyrimidine nucleoside phosphorylase (57). The postulation that FU blocks the utilization of uracil was further supported by the finding that fluorouracil-2-C1^ was converted to FUR, fluorouridine- 5 '-phosphate, fluorouridine-51-diphosphate, and to FdUMP in Ehrlich ascites cells (55,56). In a later paper, Heidel berger et al. (60), in a newly developed medium containing ascites serum and chick embryo extract, showed the con version of labeled FUR to the corresponding ribonucleotides at all three levels of phosphorylation and the conversion of labeled FdUR to FdUMP as well as its cleavage to FU and consequent conversion to FU ribonucleotides. The presence of fluorodeoxyuridine-5 1-diphosphate and fluorodeoxy- uridine-5'-triphosphate, however, was not detected. In an earlier study, Skold (57) demonstrated the reaction of FU with ribose-1-phosphate in the presence of nucleoside phosphorylase to give FUR, which could further be phos- phorylated by ATP to give FUMP in acetone-powder extracts 18 of Ehrlich ascites tumor cells . These findings suggested a third site of inhibi-. tion, namely the incorporation of the F-pyrimidines in place of uracil into RNA, thereby forming "fraudulent" RNA. Gordon and Stachelin ( 6l) showed incorporation of FU into RNA of tobacco mosaic virus replacing a third of the uracil. At the same time, virus propagation was re duced to 50 per cent. Heidelberger et al. (51,58) found FU-2-C^ in the nucleic acid RNA of liver, spleen and ascites cells in mice bearing Ehrlich ascites tumors. Similar incorporation of FU into RNA of E. coli K-12 was reported by Heidelberger (6 0). As has been mentioned, the fluorinated pyrimidines are being tested for anti-tumor growth activity. In general, when tested on animals, the carcinostatic property and degree of toxicity vary among the series of F-pyrimi- dine derivatives from tumor.to tumor ( 6 2). When FU was tested clinically on cancer patients, tumor regression was noted only in those manifesting severe toxicity (6 0,6 3). FdUR, which produces lesser toxic effects, is being tested clinically. The problem of drug resistance has interested many investigators (33). Skold et al. (64) studied possible enzymatic mechanism for the development of resistance 'against FU in ascites tumors. They found that both the UR and dUR phosphorylase activity were absent. There was a decrease in UR kinase activity, but the dUR kinase activi ty showed little change. In addition., in the resistant cells, labeled uracil was not phosphorylated to uridine- 5'-phosphate (UMP). They concluded that during develop ment of resistance, the cells lose their capacity to con vert uracil to uracil nucleotides. Before the synthesis of FU, other halouracils, C1U, BrU, and"IU had been made and studied as inhibitors of growth of microorganisms. Hitchings and co-workers ( 6 5) in 1945 found that C1U and BrU completely inhibited the growth of L. casei in the presence of thymine, and Weygand and co-workers ( 6 6) observed incorporation of labeled BrU into nucleic acid of Streptococcus faecalls. Since then, C1U, BrU, and IU have been found to be in corporated into DNA, but not RNA, in bacteria and bacterio phages, replacing thymine residues in the nucleic acid molecules ( 6 7-6 9, 28). In 1956, Zamenhof et al.(2 6) observed a direct correlation between the molecular size of the three halouracils and the extent to which they replace thymine. They found that in E. coli strain I, 48 mole per cent BrU was incorporated into DNA in place of thymine, while only 21 mole per cent C1U and 14 mole per cent IU were incorporated. Comparing the atomic radii of the halogens to that of the methyl group, they observed 20 that BrU was closest In size to thymine. This was followed by C1U and then, by IU. As has been mentioned, BrU is also of interest because of its ability to induce specific mutations in bacteriophages ( 7 0). In a T2r+ bacteriophage, it was ob served that addition of C1U or IU to the growth medium also caused several different plague-type mutants to appear ( 7 1) . BrdUR was first synthesized in 1955 (7). This dUR analog was not a very active inhibitor of the groxirth of E. coli K-12. However, in L. arabinosus and L. leichmannii (72), BrdUR proved to be a very effective inhibitor of grox\rth. Thymidine was found competitively to reverse this growth inhibition. In experiments using human tumor trans plants in a medium containing thymidine-C"^ or orotic 14 acid-C in vitro, Eidinoff et al. (73) observed that, upon addition of BrdUR, the specific activity of DNA thymine was depressed by a factor of 100. Using mice bearing Ehrlich ascites tumor cells in vivo (7^0 . > Kit et lli al. observed a reduced Incorporation of formaldehyde-C or formate-C^^ Into DNA thymine of the tumor cells, while incorporation into DNA adenine and guanine x*ras less In hibited. Although DNA synthesis was suppressed, they observed increased labeling of thymine, thymidine and thymidylate in the acid soluble fraction. Kit and co workers concluded that BrdUR was a relatively specific 21 antagonist of the terminal step, of DNA thymine synthesis. 5-Iododeoxyuridine (IdUR) was synthesized very recently by Prusoff et al. (75) . ' This- analog was found to be an effective inhibitor of the growth of S. faecalis ATCC 8043. In studies using Ehrlich ascites cells in a medium supplemented with thymine, thymidine and pteroyl glutamic acid, it was observed that IdUR reversibly in hibited the uptake of orotic acid-C-^, formate-C-^, or thymidine-C1^ into DNA thymine. However, utilization of orotic acid-C1^ for the biosynthesis of DNA cytosine or RNA pyrimidines was unaffected. Prom these data, It was suggested that IdUR inhibited utilization of a thymine- containing precursor of DNA thymine. In a later experiment ( 7 6), IdUR-I1 ^1 was synthesized and Injected into mice bearing Ehrlich.ascites tumor cells. Subsequent Isolation and degradation of the nucleic acids proved the incorpora tion of this unnatural analog into the DNA of the tumor cells . The 5-halodeoxycytidines, 5-bromodeoxycytidine (BrdCy) and 5-chlorodeoxycytidine (CldCy), have been synthesized recently, and their effectiveness, as inhibitors has been tested on Neurospora 1928, E. coli 15 T~ (77)* and E. coli K-12 (78). CldUR, so far as is known, has not been synthesized previously, but the compound was reported by 1/Jacker and 22 co-workers (79) in 1956 and by Dunn and Smith in 1957 ( 6 9)• Both investigators isolated CldUR from DNA after incubation of bacteria in the presence of C1U. The compound has been tested as an inhibitor of L. leichmannii growth by Weygand et al., who reported that both BrdUR and CldUR may serve as growth factors replacing the deoxyriboside requirement or, alternatively, may exert inhibitory effect of their own in the same organism. CHAPTER III EXPERIMENTAL Synthesis of 5-Chlorodeoxyuridine Methods previously described for chlorination'of uridine (6) were used. Powdered deoxyuridine, 5-25 gms. (.023 M), was suspended in 300 ml. of glacial acetic acid at room temperature. Mhen solution occurred, dry chlorine, 10' per cent excess, dissolved in cold anhydrous carbon tetrachloride, was added at room temperature and allowed to stand overnight. The solvents were then removed by lyophilization. The white residue was dissolved in a minimum amount of methanol. To the solution was added 400 ml. of absolute methanol containing 10 to 15 per cent anhydrous ammonia. After standing for three days at room tempera ture, the solvent was removed at reduced pressure until a thick syrup was obtained. Methanol was added, and the solution was again concentrated to a thick syrup at re duced pressure. To the thick syrup was added 30 ml. of absolute methanol. Ethyl acetate was added until the solution became turbid. Air was bubbled through the solu tion until the volume was reduced to half of the original volume. On standing at room temperature, crystals 23 ______________________ 24 appeared. The crystals were dissolved in hot ethanol and evaporated at reduced pressure and room temperature. Mhlte crystals of chlorodeoxyuridine were removed- and the mother liquor was treated with ethanol-ethyl acetate and allowed to Stand, whereupon additional crystalline product was ob tained. This was combined with the first crop. Recrystal lization from absolute ethanol produced white crystals which melted at 1 7 8-I7 9.50 (uncorrected). The total yield was 1.95 gms. (30 per cent). The A of maximum and minimum absorption are 278 mp. and 239 mp, respectively. The s was calculated to be 1 0 .0 6 x 1 0^ and the £ . , 1 .5 5 x 1 0^. i u j l i 1 • Anal. Calcd. for C ^ ^ N g C l : C, 41.15; H, 4.22; N, IO.6 7. Pound: C, 41.05, H, 4.06; N, 10.91. Melting Point The melting point was taken on a Fischer-Johns melting point apparatus. Spectrophotometric Data The ultraviolet absorption spectrum of CldUR (Figure 2) was measured with a Beckman spectrophotometer, model DU. The compound was dissolved in 0.01 N HC1 at a concentration of 20 micrograms per ml. MICROBIOLOGICAL Materials.--Escherichia coli K-12 was obtained 800 700 ^ 600 f 0 o X 500 UJ o z < 400 CD t r O £ 300 CD < UJ 200 100 230 240 250 260 270 280 290 300 WAVE LENGTH MJU Fig. 2.-Ultraviolet absorption spectrum of 5-chlorodeoxyuridine in 0.01 N HC1. through the courtesy of Dr. Richard E. Beltz, College of Medical Evangelists, Los Angeles, California. 5-Flu.oro- deoxyuridine was obtained through the courtesy of Cancer Chemotherapy National Service Center, Washington, D.C. Stock solutions (80).— A double-strength solution of salts (As) was prepared as follows: 0.2 g. of MgSO^.7H?0. • 2.0 g. of (NH4)2S0 4, 8.0 g. of KH2P°4j and 34.4 g.. K2HP0 4. 3HgO per liter. A double-strength glucose solution (Bg) was prepared containing 10 g. glucose per liter of solu tion. Sterile double-strength glucose solution (Bg) was prepared for sterile delivery in the following way: Bs was autoclaved at 121° for 15 minutes in cotton plugged 500 ml. aspirator bottle filled with rubber tubing to inlets on specially prepared 25 ml. burettes. Solid cultures.--0.2 g. of yeast extract, 0.2 g. of casein hydr'olysate, and 1 .5 g. agar were dissolved by heating in 45 ml. of the double strength salt solution (As). The volume was adjusted to 50 ml. with Ag and 5.0 ml. portions of the hot solution were pipetted into test tubes. The tubes were fitted with cotton plugs and auto claved at 121° for 15 minutes. Immediately after auto- claving, 5 ml. of sterile double-strength glucose (Bg) were added aseptically to each tube, and the tubes were allowed to cool in a slanting position. The E. coli culture was 27 transferred to slants and incubated for 18 to 24 hours at 37°. Inoculated slants were stored at 5° and.the cultures were transferred biweekly. Inoculum.--The inoculum was prepared by transfer ring a loopful of the organism to 10 ml. of complete medium made by adding 5 ml. of sterile Bs aseptically to 5 ml. of As which was previously sterilized in a test tube. After incubating 18 hours, three drops (approximaGely .18 ml.) of the cell suspension were transferred to 50 ml. of sterile As, and each assay tube was inoculated with cwo drops (approximately .14 ml.) of the dilute suspension.. Assay.— The test compounds were dissolved at a suitable concentration in double-strength salt solution (As) and stored in the deep freeze. Prior to use, the frozen solutions were allowed to thaw and warm up to room temperature. Otherwise, fresh solutions were prepared. Aliquots of a given-solution were added to 6-inch assay tubes. The volume in each tube was adjusted to 2.5 ml. with AOJ and the tubes were autoclaved at 121° for 15 minutes. 2.5 ml. of sterile glucose (Bs) was added to each tube prior to inoculation, and the tubes were in cubated at 3 5-37° for 18 hours where growth in the control was maximal. Growth was measured as turbidxty xn a Klett-Summerson photoelectric colorimeter (Filter No. 6 6). 28 All determinations were run in duplicates and the control tubes were run in quadruplicates. The per cent maximal growth was calculated by dividing the. Klett reading in the presence of inhibitor or reversing agent by the Klett reading of the control and multiplying the resulting fraction by 1 0 0. ANIMAL EXPERIMENT (55,74,81,82) Animals . --C57 black mice bearing 7-day old trans- plants of Ehrlich ascites carcinoma were used. Transplantation of tumors.--Ehrlich ascites cells were drawn from the abdominal region of.mice bearing 7-hay old transplants of the tumor. 0 .1 cc. of this fluid was injected intraperitoneally Into each experimental mouse. Materials.— Sodium formate-C1^, obtained from Nuclear Chicago, was injected in solution form at a con centration of 400 pc per kilogram weight of the animal. CldUR and BrdUR were dissolved in physiological saline and injected at a concentration of 75 mgm. per kg:. \ weight. All injections were intraperitoneal. The concentration of the solutions to be injected were sufficiently high so that not more than 0 .2 cc. were injected to each mouse at a time. The International Refrigerated Centrifuge, Braun Model PR-1, ran at 38,000 rpm, was used. 29 Isolation of nucleic acids.--The mice were weighed and divided into three groups. CldUR was injected into Group I and BrdUR was injected into Group II. Thirty- minutes later, labeled formate was injected into all three groups of mice. The mice were sacrificed twelve hours 14 after administration of formate-C . Each experimental group consisted of three mice., whose ascitic fluid was pooled. Ascites cells were then separated from the fluid by centrifugation. These cells were washed with five volumes of 0.1 M sodium citrate, pH 7.4, and centrifuged. The process was repeated. Each group of cells was homo genized in an all-glass Potter-Elvejehm homogenizer for twenty minutes. The homogenates were then washed two times in sodium citrate solution and centrifuged. The washings were repeated two times with five volumes of 0.4 N perchloric acid and twice with 95 cent ethanol. All processes performed were done in the cold. The lipids were extracted at 40-50° with three washes of 3:1 ethanol-ether. The precipitates were made up to 8 ml. with 10 per cent NaCl. Phenolphthalein was added, and the pH was adjusted with 1 N NaOH until the indicator turned red. To maintain slight alkalinity, 0.5 ml. satu rated NaHCO^ was added. The samples were extracted for ten minutes with frequent stirring at room temperature and, following centrifugation, were extracted with the same 30 volume of 10 per cent NaCl for one hour at 95°. The hot extraction was repeated for thirty minutes with 5 ml. NaCl. The combined hot and cold extracts were filtered through glass wool into 5 volumes of cold 95 per cent ethanol. To. allow for maximum precipitation of the Na nucleates, the ethanol mixtures were allowed to stand at 0° overnight. After centrifugation, the Na nucleates were washed with ethanol and then with ether. The white fibrous precipi tates were allowed to dry and were weighed. Separation of RNA and further purification.--Q.1 N NaOH (1 ml. per 10 mg. Na nucleate) was added to the Na nucleates and kept at 36° for sixteen hours. The solutions were cooled to 0° and dilute HC1 was added to pH 1. The polymerized DNA which precipitated were quickly separated by centrifugation, washed with .05 N cold HC1, and re dissolved in 0.1 N NaOH. The solutions were neutralized, and again precipitated with 4 volumes of cold ethanol as the Na nucleates. After allowing the mixtures to stand overnight, the Na nucleates were again washed first with ethanol and then with ether. The washed residues were then dried and weighed. Hydrolysis of DNA.— To hydrolyze the DNA, 70 to 72 per cent perchloric acid (5 mg. Na nucleate per 0.1 ml. acid) was added to the Na nucleates in small glass-stoppered j 31 test tubes. The tubes were kept at 100° for one hour with occasional agitation. Upon cooling, the mixtures were diluted with water and neutralized with 5-0 N KOH to pH 2. The precipitates were ground with a glass rod and allowed to stand at 0° for one day to ensure maximum precipitation of potassium perchlorate. After centrifugation, the clear V supernatants were dried by lyophilization and used for chromatographic analysis. Chromatographic analysis.--Each mixture was sepa rated by one-dimensional paper chromatography using Whatman thick paper No. 1, 6 ' x 23*, in a cylindrical glass jar 24' x 12*. The hydrolysates were placed 3 inches from the edge of the paper. Each mixture was run in solvent system (A) isopropanol: concentrated HC1: water (6 9). The posi tions of the spots containing the pyrimidine and purine bases were located with an ultraviolet lamp. Individual bases were then identified by their values. The spots containing thymine and adenine were cut out and eluted with 0.1 N HC1. Each thymine solution from Groups I, II, and III (page 29) was divided into groups X and Y, and was evaporated to near dryness either using a hair drier or a vacuum dessicator. Each adenine solution was similarly treated. Groups X were rechromatographed in solvent . system (B) Butanol; ammonia: water ( 6 9)* (C) butanol: water (84), and again in solvent system (B). Groups Y were rechromatographed in solvent system (B), (C), and (D) butanol: water: formic acid (84). The possibility exists that, under the condition of the in vivo experiment, BrdUR and CldUR might be in corporated in place of thymidine into the DNA of the Ehr lich ascites cells. If this were true, one might reason ably find free BrU or C1U in Groups I and II of the per chloric acid hydrolysates. On chromatographic analysis in system (A), the two halouracils, if present, would be ex pected to move at the same rate as thymine (Table V). On rechromatography on system (B), however, the two analogs should be easily separated from thymine. To detect the presence of BrU and C1U, therefore, paper sections were cut out at positions where the R^ values correspond to that of BrU and C1U. Upon elution with .01 N HC1, these solutions were analyzed for the presence of BrU or C1U by spectro- photometric methods. Spectrophotometric analysis.— The absorbency of the bases was determined at 2 5 0, 260 and 280 mp and at the A. of maximum absorption of the individual base. The identity and purity of the bases were established by the ratios of the readings, A2 5 0 /A260 and A28o/A2 6 0* The pmoles of each base obtained was computed from the read ings at A of maximum absorption. To allow for ultraviolet- absorbing substances in the paper, blanks were cut equal 33 in area to the spots and at equal distances from the start ing line. These were eluted and read at the same A as the corresponding spots. Radioactivity reading.--Each solution containing adenine and thymine was dried and diluted with water. Aliquots were pipetted into duplicate planchets and assayed for radioactivity in a gas flow counter (Nuclear Chicago, Model 183 Scaler, D-47 Gas Flow Head) . The specific acti vities of the bases were then computed and expressed as counts per minute per micromole (cpm). CHAPTER IV RESULTS Microbiological Inhibition Analysis The results of inhibition studies using FdUR, CldUR and BrdUR are summarized in Figure 3, which shows the growth response of E. coli K-12 to different concentra tions of the halodeoxyuridines. The three compounds suppressed the growth of this organism in varying degrees. While .03 pM FdUR produced 50 per cent inhibition of growth of E. coli K-12, 120' pM CldUR and 1,050 pM BrdUR were needed to produce the same inhibitory effect. Table I shows the effectiveness of the inhibitors on the growth of E. coli K-12 with respect to time. At a lower concentra tion of FdUR, temporary inhibition of growth of the organ ism was noted at the end of 18 hours. However, 3 PM FdUR were needed to effect complete suppression of growth' for an indefinite period. Whereas 1,600 pM of CldUR produced complete inhibition of growth for 18 hours, 2,600 pM of the compound did not inhibit growth indefinitely. BrdUR, at a concentration of 2,400 pM, did not completely suppress the growth of the organism at the end of 18 hours . 34 35 5 - F LUORODEOXY URfD IN E O-5-BROMODEOXY- U R ID IN E 100 •-5 -C H L O R O D E O X Y - U R ID IN E 2- 60 4 0 - UJ CC 20 L lI .01 .0 3 .05 .07 JUMOLES PER L IT E R 8 0 0 1600 2 4 0 0 Fig. 3.-Growth of E. coli K-12 in the presence of various 5-halodeoxyuricTines aGrown at 37° in 6-inch test tubes with a total volume of 5 ml. of medium. t>Measured at 18 hours following inocula tion. Points represent average pooled data from one or more duplicate determinations. TABLE I GROWTH OF E. COLI K-12 IN THE PRESENCE OF 5-FLUORODEOXYURIDINE OR 5-CHLORODEOXY- URIDINE AS A FUNCTION OF TIMEa Inhibitors Concentration in pM Time Growth Occurred Following Inoculation FdUR 0 .2 3 days 1 .0 6 days 2 .0 13 days 3,0 no growth CldUR 1600 2 days 2600 2 days aGrown at 37° in 6-inch test tubes with a total volume of 5 ml. of medium. 37 following inoculation. Reversal Studies Several pyrimidine nucleosides and bases, known to participate in the biosynthesis of nucleic acids,, were tested for their ability to reverse the growth inhibition of E. coli K-12 caused by CldUR and FdUR. Because BrdUR is not very inhibitory to the growth of the organism, reversal studies in the presence of■this analog were not attempted. At the concentration of BrdUR appropriate for reversal studies, many complications arise making a direct com parison between BrdUR and CldUR unsuitable. Figures 4 and 5 show the effects of various pyrimidine nucleosides and bases on growth of E. Coli K-12 in the presence of inhibitory concentrations of FdUR or CldUR. Figure 4 shows that uridine or cytidine was a better reversing agent than deoxyuridine or deoxycytidine in the presence of CldUR. UR and Cy produced a reversal to 50 per cent of maximum control growth at concentration of 40 pM and 50 pM, respectively, while 65 pM dUR and 100 pM dCy were needed to obtain similar reversing effects. All these nucleosides reversed the inhibition completely at higher concentrations. Thymine and thymidine did not reverse completely the growth inhibition; 300 pM thymidine reversed the inhibition to only 25 per cent of maximal growth. Orotic acid and thymidylic acid were not effective 38 X h £ o t£ O _l < 2 X < 2 H Z u o cc UJ CL 0 100 2 0 0 3 0 0 4 0 0 JUMOLES PER L IT E R Fig. 4.-Reversals by pyrimidine nucleosides and pyrimidine bases of the-growth inhibition of E. coli K-12 produced by 800 pM'5-chlorodeoxyuridine.^ *8 aGrown at 37° in 6-inch test tubes with a total volume, of 5 nil. of medium. ^Measured at 18 hours following inoculations. Points represent average pooled data from one or more duplicate determinations. 100 8 0 # X-U R ID IN E O -C Y T ID IN E A - DEOXYURIDINE • - DEOXYCYTIDINE 0 -T H Y M ID IN E 1 -T H Y M IN E A - OROTIC ACID □ -TH YM ID YLIC ACID -x© 39 100 tr 8 0 A-D EO XYUR ID INE O -C Y T ID IN E X -U R ID IN E •-T H Y M ID IN E 4 0 20 — • — / / — 1 — 150 200 O 50 100 JLiMOLES PER L IT E R Pig.5.-Reversals by pyrimidine nucleosides of the growth inhibition of E. coli K-12 produced by 0.5 pM 5-f luorodeoxyuridine .a7"b aGrown at 37° in 6-inch test tubes with a total volume of 5 ml. of medium. ^Measured at 18 hours following inoculation. Points represent average pooled data from duplicate determinations. 4o as reversing agents. Figure 5 shows that the growth in hibition of E. coli K-12 produced by .5 pM FdUR was re versed more efficiently by dUR than by UR. A concentration of 10 pM dUR was required as compared to 85 pM UR to attain 50 per cent growth. Thymidine, again, was an ineffective agent. It was of interest to determine whether CldUR, like FdUR ( 6 0), is catabolized to the corresponding free base, which, in turn, could act as an inhibitor. To test this possibility, the effect of C1U on the growth of E. coli K-12 was studied. Table II shows that concentrations up to TABLE II GROWTH OF E. COLI K-12 IN THE 'PRESENCE OF DIFFERENT COMPOUNDS^ Compounds Concentration in pM Per Cent Maximal Growth 5-chlorouracil 200 ‘ 1 0 0 *1 500 1 0 0 *1 1000 . " 1 0 0 *0 uridine 50 95*0 100 9 6. 5 * .5 200 .9 6. 5* • 5 - aGrown at 37° in 6-inch test tubes with a total volume of 5 ml-.- of medium. ^Measured at 18 hours following inoculation. Values represent average of duplicate determinations and the, aver age deviation. 4l 1,000 pM C1U had no effect on the growth of the organism. To determine whether CldUR is a competitive in hibitor of uridine or the ribotides to which UR is con verted, E. coli K-12 was grown in the presence of two con centrations of CldUR and with varying concentrations of UR. Figure 6 shows that, in the presence of 800 pM and 1,600 pM of CldUR, 40 pM and 90 pM UR were required to reverse the inhibition to 50 per cent of maximal control growth. The inhibition indices (concentration in pM of inhibitor / concentration in pM of reversing agent) alloi'd.ng 50 per cent maximum growth are 20 and 18, respectively. However, in the presence of 800 pM and 1,600 pM CldUR, 60 pM and 200 pM UR were required to reverse the inhibition to 75 per cent of maximal. The inhibition indices allowing 75 per cent maximum growth are' 13 Q-nd 8> respectively. While 150 pM UR reversed the inhibited growth to 94 per cent of the maximal control growth with 800 pM CldUR, 400 pM UR could only reverse this inhibition to 88 per cent of maximal control growth with 1,600 pM CldUR. Hitchings stated that, to be thoroughly convinced of the competitive nature of an inhibition, there must be not only constant effects at constant ratios of inhibitor to metabolite but also complete restoration of growth with an excess of metabolite and complete suppression of growth with an excess of antimetabolite (19)• Since the first two 42 100 < 4 0 • - 800 JUM Cl- DEOX YURIDINE 0-1600 UM CI-DEOXYURIDINE 20 0 4 0 80 120 160 200 3 4 0 0 0 0 JUMOLES PER LITER URIDINE Pig. 6 .-Reversals by uridine of the growth inhibition of E. coli K-12 produced by 5-chlorodeoxyuridine.a*b aGrown at 37° in 6-inch test tubes with a total volume of 5 ml. of medium. ^Measured at 18 hours following inoculation. Points represent average pooled data from duplicate determinations. 43 criteria for competitive inhibition have not been satis fied, it can be concluded that CldUR is not a competitive inhibitor of the utilization of UR. ®ien 5.* C° H K-12 was grown in a medium containing 800 pM CldUR and enough' UR to produce 33 per cent of maximal growth, the addition of increasing amounts of thymidine to "the assay tubes caused a better growth re sponse tfcan when no UR was added (Figure 7)• Experimental results'also showed that when deoxycytidine (dCy).was sub stituted for UR, the same response occurred. However, there was no complete reversal of growth with either re versing agent. Under these conditions, 15 0 pM thymidine reversed growth to 73 per cent of the maximal. E. coli K-12 was grown in the presence of 200 pM CldUR allowing 31 per cent growth and varying amounts of thymidine were added per tube. Figure 7 shows that, thymi dine alone reversed ,the growth inhibition effectively. 50 pM thymidine reversed the growth inhibition from 31 per cent to 80 per cent of maximal control growth. This re versal caused by thymidine in the presence of 200 pM CldUR was more effective than that produced in the presence of 800 pM CldUR and 25 pM UR (Figure 7 ) . Figure 8 shows that FdUR acts in the same manner as CldUR but to a greater degree. Thymidine alone did not reverse the complete inhibition of growth of E. coli K-12 44 100 x l- £ o o c 8 0 0 < 1 6 0 X < 2 I- 4 0 2 Ld O GL 0 Fig. 7.-Reversals by pyrimidine nucleosides of the growth inhibition of E. coli K-12 produced by 5-chlorodeoxyuridine aGrown at 35° in 6-inch test tubes with a total volume of 5 ml. of medium-. ^Measured at 18 hours following inoculation; Points represent average pooled data of one or more duplicate determinations. X -2 0 0 JUMOLE CI-DEOXYURIDINE H 0 - 2 5 JUMOLE URIDINE AND 800 J L I MOLE C I-D EO XYU RID IN E • - 8 0 0 JUMOLE CI-DEOXYURIDINE 0 20 JUMOLES 4 0 6 0 PER L IT E R 8 0 100 TH YM ID IN E 45 100 0 - 5 JUMOLE DEOXYURIDINE AND 0 .5 UM O LE F D EO XYU R ID IN E • - 0 . 5 UM O LE U R ID IN E F - DEOXY- □ - 0 . 0 5 JUMOLE U R ID IN E D EO X Y- O 20 2 0 4 0 60 80 100 JUMOLES PER LITE R THYM IDINE Fig. 8.-Reversals by pyrimidine nucleosides of the growth inhibition of E. coll K-12 produced by 5-fluorodeoxyuridine ' aGrown at 35° in 6-inch test tubes with a total volume of 5 ml.,of medium. ^Measured at 18 hours following inoculation. Points represent average data of one or more dupli cate determinations. 46 caused by 0.5. pM FdUR. However,, if partial reversal (18 per cent of maximal) was achieved by addition of 5 PM dUR, then addition of increasing amounts of thymidine produced a potentiative effect. Only 20 uM thymidine was required to reverse growth to 80 per cent of the control. Also, when the concentration of FdUR was lowered from .5 PM to .05 pM per tube, thymidine alone reversed the growth inhibition as well as that produced in the presence of .5 pM FdUR and 5 pM dUR (Figure 8). Combination of Two Halodeoxyuridines In order to test whether a combination of two halogen-substituted analogs of dUR would produce additive or synergistic effects on growth of E. coli K-12, the bac teria was grown in the presence of .01 pM FdUR causing 95 per. cent maximal control growth and either CldUR or BrdUR 0 at concentrations which permitted approximately 98 per cent of maximal growth, when each inhibitor was added alone. Results in Table III show that no significant ' synergistic effects could be detected by. combining in hibitors under these conditions. It has been shown that FdUR inhibits the methyla- tion of dUMP to TMP (p. 15-16) and that BrU, in low con centrations, is incorporated Into DNA in place of thymine in bacteria and bacteriophages (p. 19-20). It was rationalized that if FdUR were present at a concentration GROWTH OF TABLE III OF E. COLI K-12 IN THE PRESENCE TWO HALODE OXYURIDINESa*b 47 Inhibitor Cone . Addition of Cone. Per Cent Maximal pM Second Inhibitor m Growth FdUR .0 1 CldUR - 95+2 .04 - 2±2 - 50 92+1. .0 1 20 98+3 .01 50 Q5±0 .04 30 0 .04 50 0 - BrdUR 100 93-2 • o H 100 96±2 aGrown at* 37° in 6-inch test tubes with a total volume of 5 ml. of medium. ^Measured at 18 hours following inoculation. Values represent average of duplicate determinations and the average deviation. 48 which primarily blocks the methylation of dUMP to TMP, the organism would not grow because the main pathway for ob taining DNA thymine de novo is blocked. . If BrdUR were present in the medium as a "substitute metabolite" for thymidine, the organism might incorporate this analog into its DNA in place of thymidine for growth. When a small quantity of BrdUR was added to the FdUR inhibited E. coli K-12, there was an increase in turbidity similar to that obtained by addition of thymidine (Figure 9)• At a con centration of FdUR which produced a 33 per cent of maximal growth of E. coli K-12, addition of 60 pM BrdUR to the medium increased the turbidity to 92 per cent of that of the control. Addition of 300 pM BrdUR, however, resulted in a turbidity of 70 per cent of the control. It is questionable whether this increase in turbidity represents an 'increase in growth. The significance of turbidimetric measurement as an indication of growth are discussed on page 57 . When the same test was performed on the combination of FdUR and CldUR, such an effect was not observed (Table III) . Animal Experiment Mice carrying 7-day transplants of Ehrlich ascites carcinoma were individually weighed. The inhibitors and 49 0.04 JUMOLES F - DEOXYURIDINE 0 - 0 . 0 5 M M O LES F DEOXYURIDINE O 2 0 40 60 80 MMOLES PER LITER B-r 100 DEOXYURIDINE Fig. 9.-Reversals by 5-kromodeoxyuridine of the growth inhibition of JE. coli K-12 produced by 5-fluorodeoxyuridlne.a^b aGrown at 35° in 6-inch test tubes with a ^total volume of 5 • of medium. ■ U Measured at 18 hours following inoculation Boints represent average pooled data of one or more duplicate determinations. 50 formate-C1^ were injected as described on pages 28 and 29. A concentration of 75 mgm. BrdUR per kilogram weight of animal was used following Kit's experiment (75). The same concentration of CldUR was used because a preliminary ex periment with, three mice bearing 7-day transplants of ascites cells showed that the animals tolerated a dosage of 75 mgm. CldUR per kilogram weight while a dosage of 100. mgm./ kg. wt. proved toxic. Table IV summarizes the re sults obtained from the- isolation of DNA from Ehrlich ascites cells . Comparing the total weight of the pooled ascitic fluid of each group to the total weight of the mice,‘it was observed that the heavier the mouse, the more ascitic fluid it accumulated. However, the amount of ascitic fluid accumulated increases proportionately faster than the increase in weight of the mice. Oh chromatographic separation of the pyrimidine bases of the ascites DNA, any C1U or BrU present was’ identified by spectrophotometric method. It was observed that there was. no significant absorption when the solutions containing any C1U or BrU were read at wavelengths cor responding to that of maximum absorption of the two analogs, It was concluded that, BrdUR or CldUR was not incorporated into the DNA of Ehrlich ascites cells in detectable quanti ties at the experimental conditions used. Tables V and VI summarize the chromatographic, 51 TABLE IV RESULTS OF THE ISOLATION OF DNA FROM EHRLICH ASCITES CELLSa Inhibitors •Injected Pooled Ascitic Fluid g. Total Solids Following', :LI- pid Extraction ■g. Total Nucleic Acids mg. Total DNA mg. I'BrdUR l6.4' 1.3 45.0 12.4 II CldUR 12.9 1.3 5 0 .0 13.9 III None ^ 8.4 0.7 2 2 .0 6 .0 (Control) Each group consisted of 3 mice with total body weights ranging from 53.0 to 6l.O grams. The inhibitors were injected at a concentration of 75 mgm. per kilogram weight of mice. 400 uc sodium formate-Cl^ per kilogram weight of mice were injected thirty minutes later. The mice were sacrificed 12 hours following administration of formate-C-^. TABLE V CHROMATOGRAPHIC SEPARATION OF PYRIMIDINE BASES IN DNA HYDROLYSATESa Solvent, System. A Solventc System B Solvent, System C Solvent System De Gua- Ade- Cyto- Thy- BrU C1U nine nine sine mine Ade- Thy- BrU C1U nine mine Ade- Thy- nine mine Ade- Thy- nine mine Observed Rjn ' .22 .32 .44 .80 .44 .30 .34 .50 many many spots spots Reported Rf .25 .36 .47 .78 .75 .71 .42 .38 .21 .18 .38 .52 .33 .56 aAfter separation on system (A), each compound was divided into two groups: Group X was subjected to system (B), (C), and (B), and Group Y to system (B), [C), and (D) • ^Ran in isopropanol: cone. HC1: water ( 65/1 6.4/18.6 v/v) for 24 hours at room temperature ( 6 9). cRan in n-butanol: water (86/14 v/v) with 5 per cent by volume of ammonia in the vapor phase for 13 hours at room temperature ( 6 9). ^Ran in n-butanol: water (86/14 v/v) for 16 hours at room temperature (84). eRan in n-butanol: water: formic acid (77/13/10 v/v) (84). ^ ro TABLE VI SPECTROPHOTOMETRIC AND RADIOACTIVITY DATA OP PYRIMIDINE BASES ISOLATED FROM DNA OP EHRLICH ASCITES CELLS Groups Compounds Inhibitors Isolated3" ' Injected0 Spectrophotometric Data^ Radioactivity Data a2 8 0 /a 260 a2 5 0 /a 260 tota^ cpm/pm cprn observed reported ooserVed reported I Thymine BrdUR II Thymine CldUR III Thymine none .57 .71 .56 .53 .71 .67 .55 -73 71± 4.6 2350 71± 4.6 860 ' 67+4.2 4800 I Adenine BrdUR' II Adenine CldUR III Adenine none • 3b .75 .38 .37 .80 .76 .32 .73 115+ 7.5 3080 4l5±27 5610 1 5 5 -1 0 10800 Compounds Isolated^ - I Thymine BrdUR II Thymine CldUR III Thymine none • .53 .59 .55 ' .53 .71 .67 -(lost) ■ 50+ 1 .6 ■ 2990 48.51 1.5 986 I Adenine BrdUR II Adenine CldUR III Adenine none .35 - 7b • 37 -37 .77 .76 -(lost) 80± 2 .b 7750 238+ 7 .8 6280 aAfter chromatography in solvent system (A), (B), and (C), (p. 31). t>After chromatography in solvent system (A), (b)a and (C) . cSee p.29 for method of injection & concentration of both inhibitors & radioisotopes. ^Observed values were corrected for individual blank cuvette absorption.Reported values were taken from a publication of the California Corporation for Biochemical Research ( 8 5). ecounts' per minute and average deviation for a 90 per cent confidence interval ( 8 6). fSpecific activity.Concentration was calculated using molar extinction coefficients of 7.89 x 103 for., thymine at 264 mp, pH 2 and 13.1 x 102 for adenine at 263 mp, pH 2. u> spectrophotometric and radioactivity data. Table V shows that* after separation of thymine and adenine by solvent system (A)* when one group (Group Y) was purified by re chromatography in solvent system (B) and subsequently (C)* one single spot was obtained., confirming the purity of the compound. However* when these were subjected to further rechromatography in system (D)* many spots appeared in the paper. Controls containing pure thymine and adenine solu tion spotted on the same paper* however* traveled only as single spots. Wien the other group (Group X) was run in solvent system (B)* (C)* and (B)* one single spot was ob tained in every run having R^. values similar to the re ported values. The compounds in Group X were subjected to spectrophotometric analysis and radioactivity readings. Further purification by chromatography was not possible because insufficient material was available. Table VI shows the effects of BrdUR and CldUR on incorporation of formate-C^ into DNA of Ehrlich ascites cells . After chromatography of the perchloric hydrolysates in solvent system (A)* (B)* and (C)* the incorporation of the labeled precursor into DNA adenine in the presence of BrdUR and CldUR was one-third and one-half of the control* respec tively* while incorporation into DNA thymine was one-half and. one-fifth of the control* respectively. Although the control thymine and adenine were lost in the process* further rechromatography of the thymine and adenine from Groups I and II in solvent system (b) showed'significant increase in specific activity of the adenine isolated from Group I in the presence of BrdUR. This significant in crease in specific activity suggests a need for further rechromatography to establish the purity of the compounds. CHAPTER V DISCUSSION At concentrations of the three halodeoxyuridines which inhibited E. coli K-12 to 50 per cent of control growth, FdUR is 4,000 times as active as Cld'UR which, in turn, is nine times as active as BrdUR. This is consistent with the finding that CldCy is more active than BrdCy in inhibiting the growth of E. coli K-12 (78). It is diffi cult to explain why a particular 5-halodeoxyuridine should be more active than another. One explanation may be based on known differences in electronegativity of the halogens. A second explanation is obvious from consideration of the large differences or similarities of the atomic dimension of the halogen which displaces the usual substituent in the 5-position of the pyrimidine. ‘Fluorine is similar in size to hydrogen but considerably smaller than the methyl group (atomic radii - 1.35^ 1.2, 2.0 A, respectively) (13,26). If fluorine is substituted in place of hydrogen at the 5-position of dUR, the compound, FdUR, behaves more like an antagonist of dUR utilization than an inhibitor of thymidine utilization. As an example, FdUMP competitively inhibits the methylation of dUMP but neither inhibits thymidine utilization nor is incorporated into DNA (p. 15- 16). Furthermore, FU is incorporated in place of uracil into RNA (p. 18). Bromine has an atomic radius of 1.95 A {26), and is therefore very similar in size to a methyl group. Wien Br is substituted at the 5-position of dUR, the resulting compound., BrdUR, inhibits DNA synthesis, from thymidine. In addition, BrU is incorporated in place of thymine into DNA (p. 19). Chlorine (atomic radius - 1.8 A) ( 2 6) is intermediate between fluorine and bromine in size and electronegativity of the atom. In the same way, the compound, CldUR, appears to have biological properties intermediate between FdUR and CldUR. As an example, in the growth of E. coli K-12, the inhibitory activity of CldUR is intermediate between that of FdUR and BrdUR (Figure 3)• Preliminary experiments indicate that the same is true of the activity of CldUR in inhibiting the incor poration of formate-C^^ into DNA of Ehrlich ascites cells (Table VI). Thus, these data support the suggestion ( 2 6) that the size of the substituent at the 5-pc>sition is an important factor in determining specificity. It should be mentioned that the growth of E. coli K-12 was determined experimentally by turbidimetric meas urement. This method, however, does not distinguish be tween growth resulting from normal cell division and that resulting from abnormal increase in cell size. The fact 58 that the organism does increase in size when grown with inhibitors concerned in these studies is evidenced by the finding of Beltz ( 8 3), Frisch and Visser (77). Microscopic examination of E. coli K-12 cells grown in the presence of BrdUR, BrdCy, or CldCy revealed that the cells were ab normally elongated and swollen. It is likely that CldUR may affect the cells in a similar manner. Measurement of growth by turbidity is also not a direct measurement of the viability of the cells. It is therefore possible that the cells taken from a medium containing CldUR or BrdUR may possess very low viability as compared to that taken from a FdUR-containing medium. More extensive’ studies are needed to clarify these questions. Studies on reversals by the pyrimidine nucleosides of the growth inhibition caused by FdUR and CldUR indicate a difference in the sites of inhibition of these analogs. Since Beltz and Visser showed that dCy, dUR or thymidine, when added alone to the growth medium neither inhibited nor stimulated the growth of E. coli K-12 (7) and experi- 1 ments with UR indicate similar results (Table II), it is concluded that a response produced by these pyrimidine nucleosides is due to elimination of the inhibitory effects caused by the presence of the analogs. It is of interest that dUR is a better reversing agent than UR in a FdUR-inhibited system (Figure 5)j while the reverse is true in a CldUR-inhibited system (Figure 4). These effects may be explained in several ways. FdUR is converted to FdUMP and this compound inhibits strongly the methylation of dUMP to TMP (pp. 15-16). Since dUR is a more immediate precursor of dUMP than UR (Figure 1)* it is a better re versing agent in a FdUR-inhibited system. CldUR, on the other hand, might not be converted as readily to chloro- deoxyuridine-5'-phosphate by the kinases as is FdUR because of the difference in size between chlorine and hydrogen. As a result, the unconverted CldUR might inhibit the converr- sion of dUR to dUMP. Thus, UR would be a better precursor than dUR in a CldUR-inhibited system. A second explanation may be due to the possibility that FdUMP blocks the con version of UMP to dUMP while chlorodeoxyuridine-51-phosphate* does not block this step. A third possibility is that the enzyme which converts dUR to uracil in E. coli K-12 is more active than that converting UR to uracil. It is known that FdUR is degraded readily to FU which, in turn, inhibits uracil utilization (6o). If dUR provides more uracil, it will be a better reversing agent than UR in a FdUR-inhibi ted system. In a CldUR-inhibited system, there would be no effect since Table II shows that, even if CldUR were con verted to C1U, the latter is not inhibitory to the growth of the organism at concentrations up to 1,000 uM. The fact that CldUR is not a competitive inhibitor 6o of UH (Figure 6) may be interpreted to mean that CldUR inhibits more than one site in DNA synthesis. However, non-competitive inhibition might also result from incor poration of CldUR or CldUR phosphates in place of thymidine or thymine nucleotides into DNA, thus affecting the rate of cell division or producing mutants with variable growth rates. The latter reasoning is supported by the fact that at higher concentration of CldUR, 400 pM UR does not re verse growth inhibition completely. As noted in page 13, Heidelberger and co-workers demonstrated the ineffectiveness of thymine or thymidine as reversing agent of FU-inhibited E. coli K-12, while Cohen reported similar results with E. coli strain B. Re sults shown in Figure 5 are consistent with these data. At concentrations at which the other deoxynucleosides were good reversing agents, thymidine could not reverse the growth inhibition caused by FdUR. Figure 5 shows that, in the presence of .5 jiM FdUR, 200 pM thymidine reversed the inhibited growth of E. coli K-12 by only 10 per cent, while 100 uM dUR, Cy, or 200 uM UR reversed the growth inhibition completely. It is of interest that similar but less pro nounced effects were observed with CldUR (Figure 4). With 800 juM CldUR, 300 pM thymidine reversed the inhibited growth of E. coli K-12 to 25 per cent of maximal while 150 pM UR, 150 pM Cy,.300 pM dUR, or 300 pM dCy reversed the • 6i inhibited growth almost completely. The difference between the action of thymidine and other deoxynucleosides cannot be due to a difference in cell permeability since thymidine is known to reverse,growth completely in aminodeoxyuridine- inhibited _E_. coli K-12 ( 8 7). It is of interest that,, when the concentration of FdUR or CldUR were lowered in the growth media, thymidine was able to reverse growth. Figures 7 and 8 show that thymidine is a good reversing agent in the presence of .05 PM FdUR and a fair reversing agent in the presence of 200 pM CldUR. This may be interpreted to mean that both inhibitors affect two or more different metabolic pathways. At low concentration, the inhibitors primarily affect one metabolic sequence. However, at high concentra tions of the inhibitors, other additional metabolic path ways are affected. Since thymidine reverses growth at low concentrations of FdUR or CldUR, the inhibitors may primari ly affect either thymidine synthesis or thymidine utiliza tion. However, at high concentrations, the inhibitors affect growth of the organism at some additional.sites and this effect cannot be alleviated by thymidine alone, but can be by the other nucleosides. It is known that dUR or UR. can serve as precursors of both RNA uracil synthesis and DNA thymine synthesis while thymidine is a precursor only to DNA thymine synthesis. Figure 8 shows that, in the presence of .5 PM FdUR and sufficient dUR to provide 17 per 62 cent growth, thymidine is a good reversing agent. Similar results were reported by Cohen et al. (53)} who found that in FdUR--inhibited E. coli strain B, complete reversal of growth was observed in the presence of both uracil and thymine. The theory that,, in addition to primarily affect ing TMP synthesis, FdUR secondarily affects uracil utiliza tion explains why thymidine alone cannot reverse complete ly the growth inhibition caused by high concentrations of FdUR, where both TMP synthesis and uracil utilization are affected (pp.. 15,-17). With high concentrations of CldUR, however, the secondary site of inhibition cannot be in the utilization of uracil (see p. 4Q). , :However, CldUR or its metabolic products, CldUR phosphates, may secondarily block the utilization of dCy or cytosine deoxyribotides for DNA synthesis or it may block the normal functioning of coenzymes. These inhibitions may be reversed by UR, dUR, Cy, or dCy but not by thymidine. A more logical explana tion is based on the possibility that in addition to affecting DNA thymine synthesis de novo, high concentra- i tions of CldUR also inhibit the conversion of thymidine to TMP effectively. Thus thymidine is not a good reversing agent. This explanation is supported by the reasoning that, because the size of the chlorine atom is similar to that of the methyl group, CldUR might be predicted to be a good inhibitor of the conversion of thymidine to IMP. This rationale is also supported by the results shown in Figure 63 7. In the presence of 800 pM CldUR and enough UR to reverse the inhibited growth of E. coli K-12 to 33 per cent., a secondary block occurs. Thus, although thymidine reverses growth, this reversal is not as effective as that produced by thymidine in the presence of 200 pM CldUR, which caused 31 per cent maximal growth in the absence of thymidine. On the other hand, these effects may, again, be explained by the increased incorporation of CldUR into DNA, thus causing increased cell death. These results, therefore, suggest that, at high concentrations of FdUR and CldUR, the analogs inhibit the growth of E. coli K-12 at a secon dary site other than.DNA thymine synthesis. This secondary site in a FdUR-inhibited system is concerned with the utilization of uracil, but in the CldUR-inhibited system, this secondary site may be the inhibition of the conversion of thymidine to TMP. Since CldUR inhibits the growth of E. coli de novo, it must interfere with some metabolite arising de novo from simple precursors. From reversal studies and the known behavior of FdUR and BrdUR, one might postulate that CldUR either affects TMP synthesis or TMP utilization. Pre liminary experiments'using tracer studies support this view (p. 64). One might, further, postulate that, because the size of the chlorine atom is more similar to that of bromine than of fluorine, CldUR will act more like BrdUR, 64 therefore, will inhibit primarily the terminal steps of DNA thymine synthesis. At the same time, CldUR might be incor porated like BrdUR in place of thymidine into DNA. Figure 9. shows that BrdUR acts as a "substitute metabolit,e" of thymidine resulting in incorporation of the analog into DNA in a FdUR-inhibited' system. This is similar to the results obtained by Bardos et al. (72), who reported that in L. leichmannii, BrU and BhdUR reverse the inhibitory action caused by the presence of 5-nitrouracil. There is no evidence that CldUR acts as a "substitute metabolite" of. thymidine in a FdUR-inhibited system (Table III). The fact that CldUR does not reverse growth in a FdUR-inhibited system even at low concentrations can be interpreted in two ways: either CldUR is not incorporated at all into DNA, or a small amount which is incorporated into DNA is toxic to the organism. The animal experiment was designed to prove con clusively that one site of inhibition of CldUR is in DNA thymine synthesis and to compare the inhibitory effect of CldUR, FdUR and BrdUR. Formate-C^ was chosen as a labeled precursor; so that an analysis of certain metabolic events from the methylation of dUMP to DNA synthesis as affected by the presence of the inhibitors may be obtained. In addition, an analysis of the amount of incorporation of formate-C^ into DNA thymine in the inhibited and control 65 samples makes possible conclusions concerning thymidine - synthesis. Although the results obtained.(Tables V and VI) are not thoroughly conclusive., preliminary data indicate that DNA synthesis is depressed in the presence of CldUR and BrdUR at the experimental conditions and that CldUR is 14 a better inhibitor of the incorporation of formate-C into DNA thymine in Ehrlich ascites cells than is BrdUR. Comparing this preliminary experiment to the results re ported by Heidelberger et al. on the incorporation of formate-C"^ into Ehrlich ascites cells in vivo using FdUR as inhibitor (53)* it; can also be said that FdUR is a con siderably better inhibitor than is- CldUR. Thus., it again appears that CldUR has activity intermediate between BrdUR and FdUR as an inhibitor of the incorporation of formate- C1^ into DNA thymine of Ehrlich ascites cells . CHAPTER VI SUMMARY The 5-substituted derivative of deoxyuridine, 5-chlorodeoxyurIdine, .was synthesized for the first time. The compound was shown to Inhibit the growth of Escherichia coli K-12. The relative effects on the growth of E. coli K-12 were compared to those of FdUR and BrdUR. It was con cluded that CldUR has inhibitory activity which is inter mediate between that shown by FdUR and BrdUR. Similarities and differences in the mechanisms of inhibition of these three halogen compounds were compared by inhibition analy- r sis and reversal studies using E. coli .K-12 as the test organism. It was observed that UR was a better reversing agent than dUR where CldUR was present in the medium, ' while the reverse was true for FdUR-inhibited E. coll K-12. It was also found that, while thymidine did not reverse growth with high concentrations of FdUR or CldUR, the metabolite reversed growth with low concentrations of either one of the inhibitors. 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Creator Huang, Bessie Yu (author)

Core Title A comparative study of halodeoxyuridines

School Department of Biochemistry and Nutrition

Degree Master of Science

Degree Program Biochemistry and Nutrition

Degree Conferral Date 1960-01

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Visser, Donald H. (committee chair), [illegible] (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-778842

Unique identifier UC11346513

Identifier EP41343.pdf (filename),usctheses-c17-778842 (legacy record id)

Legacy Identifier EP41343.pdf

Dmrecord 778842

Document Type Thesis

Rights Huang, Bessie

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA