University of Southern California Dissertations and Theses

Effects Of 5-Aminodeoxyuridine And 5-Aminouridine On Metabolism Of Nucleic Acids

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Effects Of 5-Aminodeoxyuridine And 5-Aminouridine On Metabolism Of Nucleic Acids

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Content EFFECTS OF 5-AMINODEOXYURIDINE AND 5-AMINOURIDINE ON METABOLISM OF NUCLEIC ACIDS by Melvyn Friedland A Dissertation Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (Biochemistry and Nutrition) January 19&3 UNIVERSITY O F SOUTHERN CALIFORNIA GRADUATE SCHOOL UNIVERSITY PARK LOS ANGELES 7. CALIFORNIA This dissertation, written by under the direction of }&Jk....Dissertation Com mittee, and approved by all its members, has been presented to and accepted by the Graduate School, in partial fulfillment of requirements for the degree of D O C T O R O F P H I L O S O P H Y MELVYN FRIEDLAND Dean 19.63. DISSERTATION COMMITTEE Chairman Chairman .. ACKNOWLEDGEMENT I wish to express my gratitude to Dr. Donald W. Visser for his encouragement and guidance during this investigation. I wish to thank Dr. Milton R. Heinrich and Dr. Carmel M. Roberts for the time and effort which they generously donated as members of my committee. TABLE OP CONTENTS PAGE INTRODUCTION ........................................ 1 HISTORICAL .......................................... 7 MATERIALS AND METHODS................................ 35 Synthesis of 5-aminodeoxyuridine»HC1.............. 35 Growth of Esoherichia ooli KL2.................... 36 Liquid growth medium............................ 36 Solid cultures.................................. 36 Inoculum........................................ 36 Growth of Escherichia coli K12 for experiments to determine the effect of combinations of 5-aminodeoxyuridine and 5-fluorodeoxyuridine . . 37 Growth of Escherichia coli K12 for isolation of DNA adenine and thymine....................... 37 Ehrlich ascites cells.............................. 38 Growth and passage of tumor. • ................ 38 Incubation of whole cells in vitro ........ 38 Preparation of high speed supernatant fraction of Ehrlich ascites cells ...................... *fl Incubation procedure........................... *+3 Isolation of DNA adenine and thymine from Escherichia coli EL2............................ ^3 Isolation of RNA,DNA, and protein from Ehrlich ascites cells.................................... *+5 Analysis of acid-soluble fractions................ !+5 Acid solubles from whole cells and a large scale cell free system.............................. b5 Acid solubles of the small scale, cell free system k? Paper chromatography......... U-7 Measurement of optical density and turbidity ... *+9 Measurement of radioactivity.................... *+9 iii PAGE RESULTS..................................... 50 iL. Incorporation of label from serine-3“C into DNA adenine and thymine of Escherichia coli £12 .... 50 Growth of Escherichia coli £12 in 5Ee presence of combinations of 5-aminodeoxyuridine and 5-fluorodeoxyuridine 53 Effect of 5-aminouridine on the incorporation of label into nucleic acid and protein of Ehrlich ascites cells......... 57 The conversion of orotic acid to nucleotides by a high speed supernatant fraction from Ehrlich ascites cells...................................... 6*t DISCUSSION.............................................. 67 SUMMARY................................................ 79 BIBLIOGRAPHY............................................ 8*f iv LIST OP TABLES TABLE PAGE I. Krebs Phosphate Buffer Medium III.......... 39 II. Composition of the Amino Acid-Orotic Acid Solution................................. 1+0 III. Composition of the Incubation Mixture for Ehrlich Ascites Cells..................... 1+2 IV. Composition of'the Incubation Solutions for Demonstrating the Effect of 5-Aminouridine on the Conversion of Orotic Acid-6-C1^ to Nucleotides in a Cell Free System ...... ¥+ V. Elution Schedule for Hand-Operated Columns. . . 1+8 VI. Effect of 5-Aminodeoxyuridine on Incorporation of Radioactivity from Serine-3-C1^ into DNA Thymine and Adenine of Escherichia coli K12 . 51+ VII. The Effect of 5"Aminouridine and Inosine on Incorporation of Uracil-2-C1^ into RNA of Ehrlich Ascites Cells in Vitro........... 61 v LIST OF FIGURES FIGURE PAGE 1. Structural Formulae of 5-Aminodeoxyuridine and 5-Aminourldine......... *+ 2. Growth of Escherichia coli K12 in the Presence of 5~Aminodeoxyuridine and 5~Aminodeoxyuridine Plus Thymidine ............ 51 3. Growth of Escherichia coli K12 in the Presence of 5-AminodeoxyuricFine and 5-Aminodeoxyuridine Plus Thymidine......................... 52 if. Graphical Representation of the Effects of Combinations of Two Inhibitors, A and B, in Terms of Their Fractional Inhibitory Concen trations, on Growth. ............ 56 5. Effects of Inhibitor Combinations on the Growth of Escherichia coli K12. ........... 58 6. The Effect of 5-Aminouridine on the Incorpora tion of Label from Orotic Acid-fr-C14, into ENA and DNA and from Valine-1-C1^ into Protein of Ehrlich Ascites Cells In Vitro. • . 60 7. The Effect of 5-Aminouridine on the Conversion of Orotic Acid-6-C14- to Nucleotides by a High Speed Supernatant Fraction from Ehrlich Ascites Cells. ............................ 65 8. The Metabolism of Thymidine Nucleotides. .... 69 9. The Metabolism of the Uridine Nucleotides. ... 7*f vi LIST OP ABBREVIATIONS I angstrom unit AMP adenosine-5*-monophosphate ATP adenosine-5'-triphosphate AU 5-aminouracil AUDR 5-aminodeoxyuridine AUR 5-aminouridine AZC 6-azacytosine AZA 8-azaadenine AZG 8-azaguanine AZH 8-azahypoxanthine AZX 8-azaxanthine AZU 6-azauracil AZUR 6-azauridine AZURMP 6-azauridine-5'-monophosphate BU 5-bromouracil BUDR 5-bromodeoxyuridine BUR 5-bromouridine CP 6-chloropurine CU 5-chlorouracil CUDR 5-chlorodeoxyuridine CUR 5-chlorouridine vii DNA deoxyribonucleic acid dUMP deoxyuridine-5-monophosphate DAP 2,6-diaminopurine FU 5-fluorouracil FGDR 5-fluorodeoxyuridine FUR 5“ f luo rour i d ine FUDRMP 5-fluorodeoxyuridine-5? -monophosphate FIC fractional inhibitory concentration GMP guanosine-5*-monophosphate GTP guanosine-5*-triphosphate IMP inosine-5' -monophosphate IU 5-iodouracil IU3)R 5-iododeoxyuridine MP 6-mercaptopurine MPR 6-mercaptopurosine OMP orotidine-5'-monophosphate PGA 3-phosphoglyceric acid PM puromycin RNA ribonucleic acid sRNA soluble ribonucleic TG 6-thioguanine TU 2-thiouracil TMP thymidine-5*-monophosphate TTP thymidine-5'“triphosphate TMV tobacco mosiac virus UTP uridine-5'-triphosphate viii INTRODUCTION Interest in compounds which we now call antimetabo lites predates any knowledge of the chemical nature of life by hundreds of years. In the sixteenth century Paracelsus introduced the use of mercurials for the treatment of / syphilis. However, it was not until 19^0 that these com pounds were shown to react with sulfhydryl groups of bio logical systems (5). Some knowledge of the chemical transformations which occur in organisms was required before the mechanisms of inhibition could be understood. Since inhibitors usually interfere with the interaction of a substrate and an enzyme, an appreciation of the role of enzymes was also a prerequisite to understanding inhibitory mechanisms. In turn, investigations of compounds which were known to have pharmacological activity often elucidated new aspects of cellular metabolism. Some of the experimental work which led to an understanding of mechanisms involved in inhibi tion is described below. Michaelis and Pechstein in 1913 observed that fructose inhibited the action of invertase, and attributed this effect to the structural similarities of fructose and 1 sucrose (1), The inhibitory action of malonlc acid, a structural analogue of sucoinate, on succinic dehydrogenase activity was demonstrated in 1927 (2, 3). In 1939 p-acetylpyridine and pyridine-3-sulfonic acid were shown to be toxic to nicotinic acid deficient dogs (*f). A year earlier, Pyer while searching for substitutes for methionine found that ethionine was toxic to rats (9). Neither paper explained the toxicity as an interference with the utiliza tion of a normal metabolite. In 1939 McIntosh and Whitby suggested that the inhibitory action of sulfanilamide on growth of pneumococci was due to its interference with the utilization of a nutrient essential to the organism (6), Woods in 19^0 dis covered that the bacteriostatic action of sulfanilamide could be reversed with p-aminobenzoic acid (7). He sug gested that sulfanilamide was a competitive inhibitor of p-aminobenzoic acid utilization due to the structural similarity of the two compounds (7). Pildes and Camb in the same year proposed that other useful chemotherapeutic agents might be produced by altering the structure of vitamins and metabolites to obtain antagonistic ana logues (8). Many compounds similar in structure to intermediates of various phases of cellular metabolism have been synthe sized in an effort to find inhibitors of these biological 3 processes. Many Inhibitory purine and pyrimidine analogues have been synthesized. These have involved placing a nitrogen in a position normally held by carbon as in 6-azathymine (10), 6-azauracil (AZU) (11), and the 8-aza- purines. Hydrogen at the 5 position of pyrimidines has been replaced by an amino group or a halogen to yield 5-amino-uracil (AU) (21), 5-bromouracil (BU) (17), 5-iodouracil (IU) (18, 19) and 5-fluorouracil (MJ) (20). Substitution of sulfur for oxygen produced 6-mercaptopurine (MP) (16), 6-thioguanine (TG) (l*f, 15), 2-thiouracil (TU) (13)» and 5-thiouracil (22). The nucleosides and deoxy- nucleosides of many of these compounds have also been syn thesized (2^-31). The riboside and deoxyriboside of AU were synthe sized as possible inhibitors of pyrimidine nucleotide bio synthesis or utilization (Figure 1) (30, 208). Subsequent studies showed that the inhibitory effect of 5-aminodeoxy uridine (AUDR) on growth of Escherichia coll (E. coli) K12 was reversed by various pyrimidine nucleosides in a manner which suggested that uridine, cytidine, deoxyuridine, and deoxycytidine supplied a precursor to the inhibited reac tion, and that thymidine supplied a product of the reac tion (30). These data led to the conclusion that AUDR inhibited the methylation of deoxyuridine monophosphate (dUMP) to thymidine monophosphate (TMP). o - A 8 V H<u I HCH I H(jOH HG— HCOH H 5-AMINODEOXYURIDINE OH I HCOH HCOH HCOH 5-AMINOURIDINE Rig, 1.— Structural formulae of 5-amino deoxyuridine and 5“aminouridine. 5 The antimetabolic effects of 5-aminouridine were investigated by other workers. An inhibition of the growth of wild type Neuroapora and the pyrimidine-requiring strain 1298 was reported by Roberts and Visser (208), of propaga tion of Thieler's GD VII virus by Visser et al. (Mf) and of growth of various mouse tumors by Visser (209). In rat liver and hepatoma Werkheiser et al. found that AUR inhib ited incorporation of labeled phosphate into RNA and phospholipid (210). Werkheiser and Visser demonstrated an inhibition of incorporation of labeled formate and carbamyl aspartate into RNA purines and pyrimidines of these tissues by the analogue (211). Eidinoff found that when rats bear ing human tumor transplants H.S /L were treated with AUR, incorporation of labeled carbamyl aspartate into pyrimidines of liver nucleic acids was inhibited. AUR also inhibited incorporation of this precursor into RNA cytosine and DNA thymine of tumor tissue. The incorporation of labeled carbamyl aspartate into intestinal nucleic acids and con version of radioactive thymidine to tumor DNA were ndt affected (212). These data indicated that AUR inhibited nucleotide metabolism. The following investigations were designed to add to the knowledge of the mechanisms of action of AUDR and AUR. The effect of AUDR on the incorporation of radio- Tk activity from serine-3“C into DNA adenine and thymine of 6 E. coli EL2, and the effect of combined dosage of AUDR and 5-fluorodeoxyuridine (FUDR) on growth of this organism will be described. A modification of the method of synthesis of AUDR which improved the yield is also reported. The effect of AUR upon incorporation of C labeled precursors into nucleic acid and protein of Ehrlich ascites cells in vitro. as well as its effects on conversion of orotic acid-6-C"1 ’ 1 * into nucleotides by a high speed super natant fraction from this tissue will be described. Data obtained from these studies led to more detailed knowledge of the sites of action of AUDR and AUR, as well as evidence indicating the formation of 5-aminouridine monophosphate. HISTORICAL An intensive search for antagonists of known metab olites began in 19*K) after Woods (7) announced that the inhibitory properties of sulfanilamide were due to competi tion with p-amino-benzoic acid. In 19M* Woolley showed that benzimidazole inhibited the growth of Saccharomyces cerevisiae and that the inhibition could be reversed by adenine or guanine (32). In 19^5 Roblin et al. (12), following the suggestions of Fildes and Camb (8), synthe sized 8-azaguanine (AZG), 8-azaxanthine (AZX), 8-aza- adenine (AZA), and 8-azahypoxanthine (AZH) as potential purine antagonists and showed that each of these anti metabolites inhibited the growth of E. coli and Staphylo coccus aureus (Staph, aureus) • It was also found that the inhibition of each analogue was reversed most effectively by its natural counterpart. From the data obtained through testing the large number of purine and pyrimidine analogues which were syn thesized subsequently, it was found that certain structural changes were most likely to produce effective inhibitors. Substitutions at positions 2 and 5 of the uracil molecule (33) and positions 1 and 6 of purines (3*+, 35, 36) produced 7 8 the most effective inhibitors. Substitution of chlorine, bromine or iodine at the 5 position of uracil produced compounds which were analogues of thymine. The inhibitory nature of these compounds was first demonstrated with Lactobacillus casei (L. easel) (37), Various metabolic abnormalities were found in organ isms inhibited by these drugs. 5”Chlorouracil (CU), BU, and IU were incorporated into the deoxyribose nucleic acid (DNA) of bacteria, virus and mammalian cells (38-^3). These inhibitors replaced thymine, since the DNA thymine content was decreased by an amount equivalent to the quantity of analogue incorporated (^1, *+3). The extent of incorporation into DNA of E. coli I was presumed to be related to the ionic radius of the halogen. The atomic radius of bromine (1.95 X) is most similar to that of the methyl group (2.0 X), and BU was incorporated into DNA to the greatest extent. Since more CU was incorporated than IU, it was suggested that an atomic radius smaller than methyl (Cl = 1,80 X) was more favorable than a larger one (I = 2.15 X) 0f2). Precursors or products of an inhibited reaction, added in sufficient amounts, restore normal growth. Inves tigations of the ability of compounds which supply the sub strate or product of the presumedly inhibited reaction to restore normal growth can often indicate the site of action 9 of an antimetabolite. BU and IU inhibition of the growth of E. coli 15T- was reversed, as expected, with thymine. When CU was the inhibitor, uracil as well as thymine was required to restore normal growth (*fl). These data indi cated that CU inhibited the metabolism of uracil as well as thymine compounds. 5-Bromouracil riboside (BUR) and 5“ chlorouracil riboside (CUR) inhibited growth of Neurosnora 1298, a pyrimidine-requiring mutant (*f7), and propagation of Theiler's GD VII virus (¥+). Inhibition of propagation of Theiler's GD VII virus was reversed by uridine (¥0, and both uridine and cytidine restored control growth to cul tures of Neurspora 1298 (M7). The inhibitory properties of 5-bromouracil deoxy- riboside (BUDR), 5-chlorouracil deoxyriboside (CUDR), and 5-iodouracil deoxyriboside (IUDR) have been demonstrated with bacteria (30, 31, *+9) and those of BUDR and IUDR with various tumor tissues (*+8, 50, 5l). BUDR was a more effec tive inhibitor of bacterial growth than BU or BUR (30, *+5, * + 6) . The incorporation of labeled formate, thymidine, and orotic acid into DNA thymine of various mammalian tissues was inhibited by BUDR and IUDR (50, 51, 52), while incorporation of orotic acid into DNA cytosine was not affected (5l, 52). BUDR was 5 times as effective as IUDR as an inhibitor of thymine incorporation into DNA (52). 10 The primary sites of action of IUDR in mammalian tissues were found to vary in different tissues. In mouse L5178T leukemia cells thymidine kinase and thymidine monophosphate (TMP) kinase were inhibited. TMP kinase activity was also repressed in calf thymus and Ehrlich ascites cells. In some human leukemias the analogue inhibited the incorporation of thymidine triphosphate (TTP) into DNA (53). Inhibitors which affect sequential reactions in a biosynthetic pathway produce inhibitions, when used in com bination, greater than the sum of the degree of inhibition produced by each when used separately. Noninhibitory con centrations of IUDR, GUDR, and BUDR potentiated FUDR inhibi tion of E. coli KL2. As the concentrations of the former compounds were increased growth was stimulated until control growth was obtained (31). Stimulation of growth of L. casei by these compounds was also observed (37). Since FUDR was shown to inhibit thymidylate synthetase (71, 72) these data indicated that IUDR, CUDR, and BUDR inhibited steps after methylation of dUMP, and that substitution of BU, CU or IU for thymine did not necessarily produce non functional DNA. BUDR was also utilized for growth during a limited time by various mammalian cells in culture, replac ing 50 to 80 per cent of the DNA thymine (5 * 0 . Incorporation of BU or CU into E. coli strains 15T-, I, or B/r caused the cells to become distorted in shape 11 (**1, 55). Mutants of T-even phages occurred in the presence of BU, CU and IU, but not MJ (56, 57). Mutants were formed only while DNA was being synthesized (58). BU-containing Bacillus subtilis DNA transformed indol-, methionine-, arginine-, or pyridoxine-requiring strains to cells which did not require these supplements for growth (39, 59). Incorporation of BU into DNA increased sensitivity to UV irradiation. Sensitivity increased with increased amount of incorporation (60). A comparative study has shown that the sensitivity to UV irradiation was greatest for BU-containing cells, intermediate for cells with IU, and least for those with CU. X-irradiation of these cells indicated a decreasing order of sensitivity of cells con taining IU, BU, and CU (6l). IU-containing cells were most sensitive to decay. BU and CU caused about equal sensi tivity. Only when BU and were incorporated into oppo site strands of DNA was an increased sensitivity to the radioactive isotope observed (213). These data indicated that BU-, CU-, or IU-containing DNA could perform some of the functions of DNA, but was not functionally identical with normal DNA. The substitution of fluorine for hydrogen at the 5 position of uracil produced a uracil antimetabolite. Since chlorine, bromine, and iodine, whose atomic radii were similar to that of the methyl group, produced thymine 12 antimetabolites, formation of a uracil antagonist was antic ipated. The dimensions of the atomic radius of fluorine (1.35 X) and hydrogen (1.2 5t) are approximately equal (2*f6). PIT was an effective inhibitor of growth of many microorgan isms (63)» phages (86, 88), and a variety of mouse tumors (63, 6*0. IU was incorporated into RNA of Tobacco Mosaic Virus (TMV) (67)» E. coli (68), and tumor, intestine, bone marrow, and kidney of mice bearing Ehrlich ascites cells (65). Replacement of uracil was demonstrated in E. coli (68) and in TMV (67) where as much as *f7 per cent of the uracil was replaced, while the amounts of the other bases were not affected (67). Incorporation was greater into tumor tissue than into other mouse tissues (65). PU was not found in DNA of Ehrlich ascites cells (65, 66). Ehrlich ascites cells converted FU and 5-fluorouridine (PUR) to ribonucleoside mono-, di-, and triphosphates, and PUDR to 5“fluorodeoxyuridine monophosphate and PUR mono-, di-, and triphosphates (66). It has been shown that RNA is synthesized from ribonucleotide diphosphates (2*+9j 250) or ribonucleoside triphosphates (251, 252), and that DNA is synthesized from deoxynucleoside triphosphates. The incorporation of PU into the RNA and the lack of incorpora tion of this analogue into DNA of Ehrlich ascites cells was a reflection of the ability of this tissue to form PUR 13 diphosphate and triphosphate and its inability to form FUDR triphosphate. In vivo and in vitro studies with Ehrlich ascites cells demonstrated the inhibitory effect of PU on the incorporation of labeled orotic acid or uracil into RNA uracil and DNA thymine. The analogue also inhibited incor poration of formate into DNA thymine (69, 70), but incor poration of thymidine into DNA thymine was not affected (70). The growth of human tumor H.EP/L in tissue culture was inhibited by PU, PUR, or FURR. Thymidine reversed the inhibitory effect of PURR but did not reverse PUR inhibi tion at the same molar ratio of substrate to inhibitor (90). These data suggested that PU inhibited ribonucleotide metabolism and the methylation of dUMP. 5-Fluorodeoxyuri- dine monophosphate (FUDRMP) was shown to inhibit thymidylate synthetase from E. coli B (71) or Ehrlich ascites cells (72). In the presence of adenosine triphosphate (ATP), CUDR and BUDR were also inhibitory, but at much higher con centrations (72). Two mechanisms of resistance to PU have been dis covered. One mechanism involved a decreased sensitivity of thymidylate synthetase to FUDRMP. This was not accompanied by any change in the ability of resistant cells to convert PU to FUDRMP (73 9 7*0. Resistance had also been observed to be due to a decreased ability of cells to form PU Ik nucleosides. Nucleoside phosphorylase activity of acetone powders from Ehrlich ascites cells was inhibited by PU. These preparations converted PU to PUR, and if ATP was added PURMP was formed (75). Acetone powders from Ehrlich ascites cells resistant to PU have a greatly decreased uridine and deoxyuridine phosphorylase activity. Uridine kinase activ ity was also somewhat lower than in sensitive cells (76). Extracts of E, coli resistant to PU, PUR and FUDR were unable to metabolize PU to PURMP or uracil to UMP in the presence of PRPP, although these activities were present in extracts from sensitive cells (77). Development of resistance to PU in four Ehrlich ascites strains occurred without loss of uridine phospho rylase, deoxyuridine phosphorylase, or deoxyuridine kinase activity. All four strains showed decreased uridine kinase activity, but in one strain resistance to PU occurred before any measurable decrease in uridine kinase activity was observed (79). Resistance to PU inhibition was generally due to a loss of enzymes which converted FU to nucleotides either directly or by formation of nucleosides and subse quent phosphorylation. However, it can be seen above that other mechanisms of resistance occurred. E. coli KL2 grown on agar plates after exposure to PU formed fewer colonies than controls. The number of colonies approached control values if the plates were made 15 hyperosmotic with any one of several salts or sugars. Addition of uracil or thymine to the agar plates did not relieve the requirement for a hypertonic medium. When equimolar amounts of PU and uracil were present in the cul ture medium, the number of colonies formed in isotonic agar plates was the same as that of the control. Thymine was only ifO per cent as effective for preventing osmotic sensi tivity. PU treated cells were distorted in shape, but no protoplasts were found (80). Actively growing E. coli K12 cells ruptured in FU-containing isotonic broth (81). E. coli B did not show this osmotic sensitivity (80). coll K12 which had been treated with PU accumu lated cell wall precursors, the molar amounts being related to the degree of osmotic sensitivity (82). Incorporation of glutamate, alanine, glycine, or lycine into cell walls of Staphylococci was inhibited by PU. Hexoseamine-amino acid compounds accumulated similar to those which accumu lated in penicillin-treated Staph, aureus (83, 8^, 253» 25*0. An analogue of a cell wall precursor containing PU, phosphate, muramic acid, alanine, and a compound composed of IU, phosphate, muramic acid, glutamate, lysine, and alanine were found. The two compounds accounted for 70-80 per cent of the intercellular PU (81 *-). These data indicated that PU may inhibit cell wall synthesis of E. coli K12 in a manner somewhat analogous to that of penicillin. It was 16 also possible that the compounds containing FU, muramic acid, and amino acids were the compounds which accounted for the observed inhibition of cell wall synthesis. FU stimulated RNA synthesis in a uracil-requiring strain of E. coli when uracil was present in the medium in sub-optimal amounts. Leucine was not incorporated into protein in this uracil deficient medium. However, if FU was added incorporation was one third that obtained in the medium containing adequate uracil. FU inhibited leucine incorporation 50 per cent when uracil concentration was optimal. The formation of the inducible enzymes /^-galac- tosidase and D-serine dehydrase by uracil-requiring E. coli was inhibited by FU in the absence of uraoil, although the formation of the constitutive enzymes succinic dehydrogenase and catalase was not affected. Induction of/^-galactosidase in wild type E. coli was also inhibited by FU. Under con ditions of the above experiments DNA synthesis was inhibited. Thymine reversed inhibition of DNA synthesis, and uracil, but not thymine, reversed inhibition of/^-galactosidase formation (85). These data demonstrated that FU substituted for uracil to a limited extent. The effect of FU on consti tutive enzymes was variable. There was a selective inhibi tion of /^-galactosidase from E. coli ML 308, and K 12 J-2-1, but not of catalase or succinic dehydrogenase from E. coli 63”86 (85). Phage synthesis in infected E, coli. grown in 17 the presence of FU, required supplements of uracil and thymine. Only DNA was synthesized if thymine alone was added, and only protein synthesis was stimulated by uracil (86). Other investigators, however, found that while uracil stimulated only protein synthesis, both DNA and protein synthesis were stimulated by addition of thymine (87). Cell proteins from E. coli ML 308 and Bacillus megaterium treated with FU and thymidine showed the same elution pattern as control cells when analyzed by chroma tography on DEAE cellulose. However, the incorporation of proline and tyrosine into protein was inhibited, and arginine incorporation was stimulated. -Glucuronidase activity and /-galactosidase induction were inhibited. The activities of various other enzymes were unaffected. Alka line phosphatase synthesized by the cells grown in the presence of FCJ and thymidine was less thermo-stable than that of control cells (89). These data suggested that FU caused the synthesis of enzymes with modified amino acid contents. Sedimentation patterns of ribosomes from E. coli ML 308 or B which have been treated with FU were not the same as those of ribosomes from control cells. Amino acid uptake into protein did not appear to be affected (91). Incorporation of amino acids into protein in cell-free systems from E. coli was stimulated by synthetic polyribo nucleotides. The base composition of these polymers 18 determined which amino acids were incorporated (225, 226, 227). These compounds were assumed to act in a manner similar to that of "messenger" ENA, which had been proposed as the agent which determined the amino acid composition of the proteins synthesized (228). Ribosomal ENA and transfer ENA were also required for protein synthesis (259). FU was shown above to be incorporated into ENA. Its incor poration into "messenger" ENA, transfer RNA, or ribosomal ENA would be expected to produce either nonfunctional RNA or RNA which acted abnormally. Both were shown to occur. Protein synthesis was inhibited, and abnormal proteins were formed in FU treated cells. 2-Thiouracil (TU) was known to be an antithyroid drug and an antimetabolite of nucleic acid metabolism. It was incorporated into the RNA of TMV, Bacillus megatherium, and Cannabus sativa L (92-96). Approximately 20 per cent of the RNA uracil of Bacillus megatherium was replaced by the analogue (95). Growth of L. casei. E. coli. protozoa, experimental tumors (97-101) and the orgamisms mentioned above was inhibited by this compound. The rate of growth of TMV was reduced by TU, but the total number of infec tious particles formed was not altered (93). Horse liver nucleoside phosphorylase converted TU to its riboside or deoxyriboside in the presence of ribose-l-phosphate or deoxyribose-l-phosphate respectively (103). The formation 19 of nucleotide mono-, di-, and triphosphates of the nucleo side analogue may be assumed from the fact that it is incorporated into RNA. To date, only the monophosphate has been isolated, and this through HNA hydrolysis. The inhibitory pyrimidine analogues 5-azauracil, 6-azauracil (AZU), 6-azacytosine, and 6-azathymine have one ring carbon replaced by nitrogen. The growth of some bac teria, tumors, viruses, and parasitic flat worms was inhibi ted by AZU (106-110). 6-Azauridine (AZUR) was a more potent inhibitor of growth of tumors and vaccinia virus than AZU (108, 116, 117). AZUH was formed so efficiently when AZU was present in the culture of E. coli that this was the preferred method of synthesis (111, 112). AZUR was phosphorylated to 6-azauridine monophosphate (AZURMP) by mouse liver, tumor, E. coli and S. faecalis (113, H^» 115). In mouse tissue no higher phosphates were found (113, 116). AZUR mono-, di-, and triphosphates were found in S. faecalis (11^, 115, 218). AZfr was incorporated into RNA of this organism (11*0. The ribonucleoside diphosphate of AZU, which had been chemi cally synthesized, was an inhibitor of E. coli polynucleo tide phosphorylase. AZURMP was not inhibitory (118), An accumulation of orotic acid and orotidine mono phosphate (OMP) or orotidine occurred during AZU and AZUR inhibition of tumor and E. coli. Orotic acid and OMP were 20 found in the urine of mice and humans fed AZU or AZUB (116, 119). The conversion of orotic acid to uridine triphos phate (UTP) by a cell free system from mouse tumor was inhibited by AZUR and AZURMP, but not by AZU. AZUR did not affect the conversion of uracil or uridine to UTP (116). These data indicated that AZUR or AZURMP inhibited the con version of OMP to UMP. Partially purified preparations of yeast orotidylate decarboxylase were inhibited by AZURMP but not AZU or AZUR (116, 168). The effectiveness of vari ous compounds as inhibitors of yeast orotidylate decarboxy lase was compared. AZURMP was a much more effective inhibi tor than azauridine-2'-phosphate, azauridine-3'-phosphate, azauridine-5'-diphosphate, UMP or UDP (168). AZUR competed to some extent with uridine for uridine kinase (113). Mouse leukemia cells resistant to AZUR show a decreased ability to phosphoxylate uridine or AZUR to the monophos phate. OMP decarboxylation in resistant cells was much less sensitive to AZUR inhibition (120). These data also indicated that AZURMP was the inhibitor, and that resistance involved a loss of ability to form this compound. Large amounts of orotic acid appeared in the medium of E. coli incubated in the presence of 6-azacytosine (AZC) (120). AZU was a more effective inhibitor of E. coli growth than AZC (121). The reverse was true of the nucleosides. 6-Azacyto8ine was more effective in suppressing E. coli growth than AZUR (123). E. coll and Paramecium vulgaris deaminated AZC (120). In light of the deamination and the accumulation of orotic acid in the growth medium, inhibi tion of growth of E. coli and Paramecium vulgaris was attributed to the formation of AZU derivatives (120). It was suggested that the greater growth inhibition produced by 6-azacytosine was due to its being incorporated into the cells to a greater extent than AZUR (120), Adenocarcinoma 755 was ten times more sensitive to AZC than AZU (122). Further investigation was needed to determine if this was due to a greater permeability of AZC than AZU, or if AZC- containing compounds were inhibitors. 6-Azathymine and 6-azathymine deoxyriboside inhibi ted growth of various microorganisms, the latter being the more effective inhibitor (12*f, 125, 126). Inhibition by 6-azathymine was reversed by thymine or thymidine (121 *, 125). Mammalian cells converted 6-azathymine to the ribo side and deoxyriboside (127). 5-Azauracil was synthesized (128), and inhibited growth of adenocarcinoma 755 and E. coli B (129, 130). E. coli inhibition was reversed by pyrimidines and their nucleosides or deoxynucleosides (130). A large number of purine analogues have been pre pared. Of these 6-mercaptopurine (MP) and 6-thioguanine (TG) were the most effective inhibitors (16, 31 * - , 35). MP inhibited growth of bacteria and tumor (36, 131, 132). 22 Inhibition of E. coll. L. casei. and S. faecalis was reversed by adenine, hypoxanthine, and xanthine (131) 133) 13*+). Data from various sources suggested that MP inhibi ted conversion of inosine monophosphate (IMP) to adenosine monophosphate (AMP) or guanosine monophosphate (GMP). MP inhibition of Bacillus cereus (B. cereus) was reversed more effectively by hypoxanthine than by adenine or guanine (135). The incorporation of labeled formate into nucleic 11 4 - acid purines of E. coli B (139) and of C glycine into nucleic acid adenine and guanine from four mouse ascites tumors (lMD) was inhibited by MP. Inhibition of various tumors was reversed in a com petitive manner by hypoxanthine or inosine, while reversal by adenine or adenosine indicated a product effect (136). Incorporation of labeled glycine and hypoxanthine into ENA and DNA adenine of L1210 leukemia cells was inhibited by MP. There was no effect on incorporation of label into nucleic acid guanine (137). Adenylosuccinic acid conversion to fumarate and AMP was inhibited by 6-mercapto adenylo succinic acid (138). In these tissues AMP synthesis was the primary site of MP action. S. faecalis strains which had developed resistance to MP or AZG showed decreased ability to utilize guanine or hypoxanthine for nucleic acid synthesis, but showed no impairment of ability to utilize adenine or xanthine (1^1, 23 1^2). MP- and AZG-resistant L1210 leukemia cells demon strated similar properties (1^3). In these organisms the resistance to the purine antimetabolites was probably due to a loss of enzymes which converted guanine and hypoxan thine to GMP and IMP. A loss of ability to convert IMP to adenine was not likely, since these cells utilized zanthine for growth, and the conversion of XMP to AMP would have involved the formation of IMP as an intermediate step (260). The inability to use guanine could have been due to a lack of the enzyme GMP-reductase (261) which converted GMP directly to IMP. The formation of IMP from GMP would not be expected to involve a reversal of GMP formation since the amination of XMP is essentially irreversible (260). MP was converted to the ribotide by bacterial and mammalian tissues (1^3? l1 ^* l*+5). Resistance in L1210 leukemia cells was due to a loss of ability to convert MP to the ribotide. IMP, GMP, or AMP formation from hypoxan thine was also diminished (l*t7) • MP-resistant S. faecalis and adenocarcinoma 755 strains were also resistant to 6-mercaptopurine riboside (MPR) (1^9). These data indicated that either MPR was rapidly cleaved to MP by a nucleosidase or that MP was converted to MPR which was then phospho- rylated. Resting B. cereus cells converted MP to thioxanthine and thiouric acid, neither of which was inhibitory. MP was 2k converted to RNA and DNA adenine and guanine, but no MP was found in NA of these cells (l*f8). The inhibitor was incor porated into RNA and DNA of adenocarcinoma 755 and Harding- Passey melanoma (150). MP inhibited the action of various enzymes. /^-Galactosidase induction of E. coli was inhibited, but no more than total protein synthesis (131). Adenosine deami nase of rabbit intestinal mucosa and adenocarcinoma 755 was inhibited (150). 6-Mercaptopurine riboside triphosphate substituted for GTP to a limited extent in a cell-free sys tem which incorporated amino acids into protein, and showed no inhibitory properties (151). A general inhibition of protein synthesis was to be expected of a nucleic acid antimetabolite. The inhibition was not due to an inter ference with the function of GTP in the transfer of amino acids from "soluble1 1 RNA (sRNA) to ribosome. Inhibition of adenosine deaminase by MP was in keeping with its action as an adenine antimetabolite. TG inhibited growth of many bacteria and tumors (152-155). Inhibition of L, casei was reversed by adenine, guanine, hypoxanthine, or xanthine (133» 153). TG was a more effective inhibitor of various mouse tumors than MP (13^, 15^). TG inhibited the incorporation of labeled guanine into nucleic acid of Ehrlich ascites cells. Incor poration of glycine into nucleic acid adenine was inhibited 25 for a longer period of time than glycine incorporation into nucleic acid guanine. Conversion of IMP, but not adenine or AMP, to nucleic acid adenine was inhibited. TG prevented the accumulation of N-formylglcineamide ribotide in aza- serine-treated cells (156). These data suggested inhibition of two sites of the de novo pathway, one before the forma tion of N-formylglycineamide ribotide and a second between IMP and AMP. The inability to utilize guanine in the presence of TG may have been due to the second inhibitory site. TG was incorporated into RNA and DNA (157). Sensi tivity to this analogue seemed to be correlated with its incorporation into nucleic acids. Resistant cells incor porated less than sensitive cells (158). Decreased incor poration into nucleic acid may have been due to a decreased conversion of TG to nucleotides, as had been found with other inhibitors. 6-Thioguanosine mono-, di-, and triphos phates were found in Ehrlich ascites cells, liver, and bone marrow (158, 159). These tissues also converted TG to 6-thiouric acid (159). Once incorporated into nucleic acid, TG remained there (158). This stability may have indicated a resistance of TG containing RNA to ribonuclease. 6-Thioguanosine triphosphate, like 6-mercaptopurine tri phosphate, substituted to a limited extent for GTP as a cofactor in protein synthesis, and showed no inhibitory 26 properties (151). A group of purine inhibitors were synthesized which contained a nitrogen in place of the number 8 carbon of the purine ring. 8-Azaadenine (AZA), 8-azaguanine (AZG), 8- azahypoxanthine (AZH), and 8-azaxanthine (AZX) inhibited growth of various microorganisms. AZA inhibited growth of E. coli B/r and TMV, AZX inhibited growth of B. cereus, and AZH inhibited growth E. coli B/r. The RNA of all these microorganisms contained AZG. These data indicated that AZA, AZX, and AZH were converted to AZG. These conversions may have occurred at the nucleotide level due to the actions of enzymes involved in the interconversions of AMP, GMP, XMP, and IMP. The inhibitor in all experiments may have been an AZG containing compound. A small amount of AZG was found in the DNA of B. cereus (62). AZG inhibited growth of protozoa, bacteria, and various tumors (62, 160, 161, 162). Sensitivity to AZG and other azapurines in S. faecalis was found to be correlated with the ability to convert these compounds to nucleotides (l*+5). 9-Ethyl-8-azagu.anine did not inhibit S. faecalis (lMf, l*f5) or 3 human epidermal carcinoma H.Ep /2 strains which were inhibited by AZG (21lf). These data suggested that conversion to the nucleotide was necessary for inhibi tion. RNA synthesis in B. cereus continued in the presence 27 of inhibitory concentrations of AZG. A large amount of the RNA guanine was replaced by AZG (169). Levels of inhibitor which markedly inhibited ribosomal RNA synthesis affected sRNA synthesis only slightly. Guanine of both fractions was replaced by AZG, the replacement being greater in the sRNA. This AZG-containing sRNA combined with amino acids as readily as normal sRNA. Ribosomal and supernatant pro tein synthesis was markedly inhibited (170). In B. cereus. protein synthesis was more sensitive to AZG than DNA or RNA synthesis. RNA synthesis was stimulated at concentrations of the analogue which inhibited protein synthesis. This RNA, which contained AZG, was more acid labile than normal RNA (171, 215). Inhibition of RNA synthesis would not be expected to have been a necessary prerequisite for suppres sion of protein synthesis. In the presence of AZG nonfunc tional RNA might have been synthesized due to replacement of guanine by this analogue, which resulted in a lack of protein synthesis. Although AZG-containing sRNA functioned normally, analogue-containing ribosomal RNA and messenger RNA may have been nonfunctional. The incorporation of sulfur-containing amino acids into protein of B. cereus was inhibited by AZG, although synthesis of total cell protein was not affected (172). Synthesis of protoplasmic protein was inhibited while cell wall syntehsis was stimulated (173)• This difference in 28 response to AZG may have been due to different mechanisms of biosynthesis of cell wall protein and protoplasmic pro tein. Induction of /tf-galactosidase and catalase in Staph. aureus and amylase in B. subtilis was inhibited by AZG (166, 167). Since GTP was required for protein synthesis (259) 8-azaguano sine triphosphate might have been expected to antagonize this function. The inhibition of protein syn thesis shown above could have been due, at least in part, to an interference with this role of GTP. However, 8-azaguanosine triphosphate substituted to some extent for GTP in a cell-free system which incorporated amino acids into protein, and had no inhibitory properties (l5l). 2,6-Diaminopurine (DAP) was an inhibitor of growth of mammalian and bacterial cells (17^, 176, 177, 178). It was also a precursor to nucleic acid adenine and guanine (17^, 175). No unchanged DAP was incorporated (175). DAP was converted to guanosine by a beef liver preparation. DAP and ribose-1-phosphate were converted to 2,6-diamino- purine ribonucleoside. This was then deaminated to guano sine (179). Kinases from yeast phosphorylated DAP riboside to the mono-di- and triphosphate (180). DAP nucleotides were detected in mouse tissue and human erythrocytes (181, 182). 2,6-Diaminopurine riboside triphosphate substituted for ATP as a substrate for yeast hexokinase (182). 29 Inhibition by MP was probably due to interference with the metabolism of adenine compounds in L. easel« since inhibition was reversed with adenine (133). Its conversion to guanosine and incorporation as guanine into nucleic acid of Aerobacter aerogenes (176) indicated action as a guanine antimetabolite in this organism. It had also been sug gested that this compound was a folic acid antimetabolite (177). DNA-resistant strains of Salmonella typhimurium excreted adenine and uracil into the growth medium, as a consequence to development of resistance (183). Resistance was probably due to the loss of ability to convert DNA to nucleotides which was observed with these cells (262). 6-Chioropurine (CP) was a halogen substituted purine whose antimetabolic activities were investigated to some extent. This compound inhibited growth of various mammalian tissues (18*4—187). Incorporation of labeled glycine, and to a lesser extent hypoxanthine, into nucleic acid guanine of Sarcoma 180 was inhibited by CP. Labeling 1* 4 - 6f adenine compounds from glycine-C was not affected. CP increased the conversion of adenine and guanine to nucleic acid guanine (188). Incorporation of *f-amino-5-imidazole- carboxamide, hypoxanthine, and inosine into nucleic acid guanine of Sarcoma 180 was greatly depressed while conver sion to nucleic acid adenine was not diminished. Neither was the incorporation of guanine or guanosine into nucleic 30 acid adenine or guanine affected by the analogue (189). The inhibitory action of CP was potentiated by azaserine (190), an inhibitor of de novo purine biosynthesis (191» 192, 193)• These data indicated that CP inhibited conver sion of IMP to GMP. CP inhibited the degradation of xan thine to C02. It was oxidized to 2,8-dihydroxy-6-chloro- purine which was a more effective inhibitor of xanthine breakdown. CP inhibited xanthine oxidase and 2,8-dihydroxy- 6-chloropurine inhibited uricase (187). Psicofuraine and puromycin were purine antimetabo lites which were produced by two strains of Streptomvces. Psicofuraine was synthesized by Streptomvces hvgroscopious var. deoovinine and inhibited the growth of various micro organisms (19^). Investigations with whole cells and cell- free systems indicated that psicofuraine inhibited the amination of xanthosine monophosphate to GMP (195, 196, 197). Streptomvces alboniger produced the antibiotic puromycin (PM) which inhibited growth of microorganisms (198) and neoplasms (199)* PM suppressed protein synthesis in Pseudomonas fluorescene (200) and rat tissue (201) with out affecting nucleic acid synthesis. RNA which was syn thesized in the presence of PM, unlike that which accumu lated in the presence of chloramphenicol, was stable (200). Polio virus RNA synthesized in HeLa cells whose protein 31 synthesis was blocked by PM was as infective as control viral RNA (201). The appearance of liver tiytophan pyrro- lase activity in new bom guinea pigs was inhibited by PM. It also prevented the increase in activity of this enzyme which normally occurred in liver of adult guinea pigs injected with tryptophan (203). PM was found to inhibit the transfer of leucine from sRNA to microsomal protein of rat liver, but not the earlier steps (202). However, liver of rat and guinea pig treated with PM jLn vivo did not accumulate amino acid-sRNA complexes (203). This was explained by the finding that in cell-free systems from E. coli and rat liver, PM interfered with the transfer of leucine from sRNA to ribosomes but deacylation of sRNA was not inhibited (20*+). The incorporar ■30 tion of label from PJ -containing sRNA to microsomes was not affected by PM although the transfer of labeled leucine from sRNA to microsome was greatly inhibited (205). Studies with Ehrlich ascites cells (206) and erythrocytes (207) suggested that PM caused premature release of protein precursors from the synthetic site on the ribosome. Various nucleic acid antimetabolites were effective as feedback inhibitors. TG, MP, DAP, and to lesser extents AZA, AZG, and AZH inhibited 5-amino-l»—imidazolecarboxamide ribotide synthesis in E. coli B 96 (216). The activities of carbamyl phosphate-aspartate transcarbamylase and 32 dihydroorotase of a cell-free system from Ehrlich ascites cells were inhibited by various pyrimidines, pyrimidine antimetabolites, and their nucleosides and nucleotides (217). Examination of the various phases of biochemistry would show that nucleotides are involved in carbohydrate, lipid, protein and cell wall synthesis as well as in DNA and RNA synthesis. Cytidine diphosphate choline and cyti- dine diphosphate ethanolamine are required for formation of lecithins and cephalins (263). Cytidine diphosphate digly cerides are intermediates in formation of inositol mono- phosphatides (261 *). Uridine diphosphate galactose is an intermediate in the conversion of galactose to glucose (266). Uridine diphosphate glucose is required for the synthesis of sucrose (265), glycogen (267), and cellulose (268). Thymidine diphosphate sugars are involved in the interconversions of glucose, mannose, and rhamnose (269, 270), and the formation of N-acetylglucosamine (271). Activation of amino acids for protein synthesis requires the formation of an AMP-amino acid complex (272, 273). Accumulation of compounds composed of uridine diphosphate, muramic acid, and amino acids was found in penicillin inhibited Staph, aureus (253» 25*0. Muramic acid and the amino acids were also found in the cell wall, and in the same molar ratio as in the uridine nucleotide. 33 From these data, and the observed formation of protoplasts in penicillin treated bacteria it was concluded that the uridine nucleotide-muramic acid-amino acids containing com pounds were intermediates in cell wall synthesis (25*0. Antimetabolites of nucleotide biosynthesis could inhibit the formation of the nucleotide cofactors mentioned above, and thus affect lipid, carbohydrate, protein, or cell wall metabolism. Cells in which CTP formation was inhibited, for example, would also have depressed rates of formation of phospholipids and inosotides. RNA in the form of ribosomes, soluble RNAs, and messenger RNAs are required for protein synthesis (258, 259 > 27*0. DNA was found to be important as part of the genetic determinants of the cell (275). It was shown above that various inhibitors were incorporated into both nucleic acids. DNA which contained antimetabolites could have been inert genetically or could have directed synthesis of frau dulent messenger RNA. Analogue-containing RNA could either have been nonfunctional, or have functioned in abnormal ways. As mentioned above, cells which had incorporated analogues into nucleic acids often grew abnormally. In many cells aberrant proteins were found. Cell growth and protein synthesis were often inhibited. The classic concepts of inhibitor action proposed a compound, similar in structure to a normal metabolite, 3^ which competed with the metabolite for sites on an enzyme. If the rate of utilization of the normal substrate was reduced sufficiently, the rate of growth was depressed. The nucleic acid antimetabolites presented a more complex picture. The classic inhibitor was not a substrate for the enzyme whose action it affected. Many purine and pyrimi dine analogues were metabolized like the normal substrates. They were often exogenously added abnormal substrates which decreased the number of enzyme sites available to the usual substrates. BUDR, CUDR and IUDR were mentioned above to have stimulated growth of E. coli K12 which were deficient in thymidine nucleotides due to the action of FUDR. The analogues substituted for thymine in DNA and thus restored normal growth. They also potentiated FUDR inhibition (31)• These inhibitors functioned both as thymine antagonists and as thymine substitutes. Inhibition of cell metabolism due to synthesis of an abnormal protein, directed by sin analogue-containing messenger RNA would not fit the classic concepts of inhibi tor action. Neither would alterations of cellular metabol ism due to the incorporation of an inhibitor into DNA. These diversified mechanisms of action have made the study of nucleic acid antimetabolites very fascinating. MATERIALS AND METHODS Synthesis of 5-amlnodeoxvurldlne *HC1 Bromine (3.85 g, 0.02^2 mole) was added to deoxy- uridine (5.0 g, 0.0219 mole) dissolved in 10 ml of acetic anhydride and the mixture allowed to stand over night. The solvent was removed at 35° on a Rotovac at reduced pressure and the acetylated 5”bromodeoxyuridine crystallized from ethanol. The yield was 8.31 g (97*1 per cent based on the assumption that 5-*bromodeoxyuridine diacetate was the pro duct ) • Acetylated 5-bromodeoxyuridine was converted directly to AUDR by treatment at 50° for 7 days with 12 ml liquid ammonia in 30 ml absolute ethanol. Ethanol and ammonia were removed on a Rotovac at reduced pressure until a brown syrup was formed. This was dissolved in water and adsorbed on an Amberlite IR 120 resin (H+ form). AUDR was eluted with NH^OH, and the ammonia was removed from the eluent at reduced pressure and room temperature as quickly as possible, since AUDR was unstable in alkaline solution. The pH was adjusted to 3 with concentrated HC1 and the water removed by lyophylization. The residue was ciystal- lized in warm methanol acidified to pH 3 with concentrated 35 36 HC1. The product crystallized as white needles which decom posed at 186°. The yield was 3.0 g (*f9.1 per cent). The AUDR*HC1 migrated at the same rate on paper as AUDR»HC1 pre pared by the original procedure. The paper chromatogram was developed using n-butanol-ethanol-H20 (50:30:20) as the solvent. Growth of Escherichia coli K12 Liquid growth medium.— Double strength salts solu tion was prepared by dissolving 0.2 g MgS0^«7H20, 2.0 g (NHif)2S0if, 26.1 g KgHPO^ 811(1 8.0 g KH2I> 0i+ in one liter of water. Double strength glucose solution was prepared by dissolving 10 g of glucose in one liter of water. The growth medium consisted of equal volumes of each sterile solution (30). All solutions used for growth of E. coli K12 were autoclaved at 15 pounds pressure for 15 minutes. Solid cultures.— E. coli K12 was grown in a medium containing 0.2 g yeast extract, 0.2 g casein hydrolysate, and 1.5 g agar dissolved in 50 ml of double strength salts solution. 5 Ml aliquots were placed in test tubes and autoclaved. Immediately after autoclaving, 5 ml of sterile double strength glucose solution was added to each tube and allowed to cool in a slanting position. Inoculum.— A loopful of E. coli from the solid cul tures was transferred to sterile growth medium and incubated 37 at 37° for 18 hours. For the experiments dealing with the effects of combinations of AUDR and FUDR on growth, a 10 ml culture was prepared. Two drops of the 18-hour culture were transferred to 50 ml of sterile 0.9 per cent saline and one drop of this dilute solution was added to each assay tube. A 200 ml culture was grown as inoculum for experiments involving the isolation of DNA adenine and thymine. Growth of E. coli K12 for experiments to determine the effect of combinations of AUDR and FUDR.— Suitable amounts of inhibitors were dissolved in 2.5 ml of sterile double strength salts solution. 2.5 Ml of sterile double strength glucose was added and the tubes inoculated with one drop of inoculum in sterile saline as described above. The cultures were incubated at 37° for 18 hours. Growth was measured turbidimetrically in a Klett-Summerson photo electric colorimeter (Filter No, 66). Growth of E. coli K 12 for isolation of DNA adenine and thymine.— Cells were grown by adding a 200 ml inoculum to 1800 ml of sterile medium and incubating at 37°. When growth attained 23 per cent of that observed in control cultures at 18 hours, AUDR and thymidine were added. 1^- Serine-3-C was added 65 minutes later. The cells were chilled and harvested by centrifugation 3 hours and k5 38 minutes after addition of AUER and thymidine. Ehrlich ascites cells Growth and passage of the tumor.— The Ehrlich ascites cells were grown in the peritoneal cavity of male Swiss-White mice. After 7 days the ascitic fluid was with drawn and either used as a source of cells for experimental purposes, or 0.1 ml per mouse was used to maintain the cell culture• Incubation of whole cells in vitro.— The cells, after removal from the mice, were centrifuged to determine the packed cell volume. The supernatant fluid was dis carded and the packed tissue washed once with 0.9 per cent saline. The cells were suspended in an equal volume of Krebs phosphate buffer medium III (219) (Table I) modified to contain NaCl in place of the Na salts of the tricarboxy lic acid cycle intermediates. One ml of this suspension (0,5 ml of packed cells) was added to 3 ml of the buffer solution which contained 6.7 mg inosine, 1.0 mg glucose, 0.5 mg glutamine, and inhibitor. These samples were incubated at 37° in a Eubnoff Metabolic Shaker for 10 minutes. One ml of an amino acid solution (Table II) lb 5 (220) containing valine-l-C (8.5 x 10 cpm) and orotic 6 acid-6-C (2.8 x 10 cpm) were added and the incubation continued for 20 minutes. The composition of the incubation TABLE I KHEBS PHOSPHATE BUFFER MEDIUM III 39 I. Stock solutions Grans per 100 ml a. NaCl • 5 b. KC1 5.75 c. CaClg'SHgO 8.66 d. KHgPO^ 10.55 e. MgSOi^THgO 10.29 f. NaHCO^ 6.5 g. Na phosphate buffer 1. 8.9 g Na2HP0if / 100 ml 2. 6.9 g NaHgPO^ / 100 ml 3. Mix 100 ml (1) and 25 ml (2). II. Krebs phosphate buffer medium III Solutions^ (add in order) Ml a. h2o 512 b. NaCl 115 c. KC1 k d. CaCl2 1 e. MgSO^. 1 f. KHgPO^ 1 g* NaHCO^ 3 h. Na phosphate buffer 3 Quantities of stock solutions used. *+0 TABLE II COMPOSITION OP THE AMINO ACID^OROTIC ACID SOLUTION^ Compound MW mg/ml Alanine 89.1 0.19 Arginine *HC1 210 0.12 Aspartic acid 133 0.0025 Cystine 2*4-0 0.075 Glutamic acid 1*4-7 0.19 Glycine 75.1 0.10 Histidine 155 0.075 Isoleucine 131 0.07 Leucine 131 0.10 Lysine *HC1 182 0.156 Methionine 1*4-9 0.0*f5 Phenylalanine 163 0.06 Proline 115 0.15 Serine 105 0.06 Threonine n? 0.125 Tryptophan 20*4- 0.07 Tyrosine 181 0.065 Valine 117 0.20 Orotic acid 156 0.215 -£-Amino acids were used. ■Compounds dissolved in Krebs phosphate buffer m edium III (Table I). *tl mixture is described in Table III. Preparation of high speed supernatant fraction of Ehrlich ascites cells.--The cells were centrifuged to remove ascitic fluid and weighed. They were washed twice with cold 0.9 per cent saline and transferred to a mortar imbedded in ice. Glass powder and 5 to 10 ml of buffer were added, and the cells were ground for 15 to 20 minutes. The buffer was composed of 250 ml 0.25 M sucrose, 15 ml 0.2 M tris HC1 buffer, pH 8.0, and 10 ml of 0.15 M KC1 (221). The ground cells were transferred to centrifuge tubes and spun at *t,000 rpm for 5 minutes in an International Refrigerated Centrifuge Model PR-2 at 0°. The supernatant solution was collected and stored in the cold. The residue was extracted 3 times with equal volumes of buffer, and the supernatant solutions added to that from the first centrifu gation. A total of 10 ml of buffer was used per gram of tissue for grinding, transfer, and extraction. The combined supernatant solutions were centrifuged at 0° and 39,000 rpm in the No. M-0 head of a Spinco Model L preparative centrifuge. The high speed supernatant (HSS) fraction was removed in the cold room using a syringe, and was used as the source of enzymes. Care was taken to avoid contamination with sedimented material or the "fluffy layer" which collected at the top of the solution. k2 TABLE III COMPOSITION OP THE INCUBATION MIXTURE FOR EHRLICH ASCITES CELLSsr Compound MW Amount Inosine 268 6.7 mg Glucose 180 1.0 mg Glutamine 1^6 0.5 mg ADR»HC1 295 variable Amino acid-orotic acid solutions/ 1 ml Ehrlieh ascites _ cell suspension^/ 1 ml -‘ Final volume 5 ml. ^able II. ^J&xrlich ascites cells, packed by centrifugation, were re suspended in an equal volume of Krebs phosphate buf fer (Table I). One ml of cell suspension contained 0.5 ml packed cells. ^3 Incubation procedure.— The incubation mixture con tained ATP, 3_phosphoglyceric acid (PGA), MgClg, ribose-5- ili phosphate, orotic acid-6-C and AUR as well as HSS fraction (Table IV), All solutions except the ATP and HSS fraction were prepared the day before the experiment. ATP and HSS solutions were prepared the day of the experiment. The HSS fraction, ATP, MgC^j ribose-5“ phosphate and inhibitor were incubated at 37° for 10 min utes in a Dubnoff Metabolic Shaker. Orotic acid was added and the incubation continued for 15 minutes. The reactions were terminated by pouring the incubation mixtures into 0.25 volume of *f.O N perchloric acid (PCA). The acid insoluble material was removed by centrifugation at **,000 rpm and 0°. The supernatant solution was decanted and stored in the refrigerator. The residue was extracted with 0.8 volume of 0.6 N PCA. The combined supernatant solu tions were neutralized in the cold to pH 7.0 with 2N KOH using phenol red as indicator. The neutralized solutions were stored in the cold over night to precipitate KClOi,. which was removed by centrifugation at 0°. One large scale incubation was run in the presence of AUR (*+.8 jomoles/ml). All other components were increased 20-fold. Isolation of DNA adenine and thymine from Si. ooli Elf The UNA was isolated by the method of Dunn and TABLE IV COMPOSITION OP THE INCUBATION SOLUTIONsi/POR DEMONSTRATING THE EPPECT OP AUR ON THE CONVERSION OP OROTIC ACID-6-Cllf TO NUCLEOTIDES IN A CELL PREE SYSTEM Compound MW Concentration of Stock Solution?/ (mg/ml) (jjmoles/ml) Volume Added (ml) NagATP^HgO 623 62.3 100 0.1 BaPGA-2/ 32»+ 32 .^f 100 0.25 MgCl2*6H20 203 J+0.7 200 0.25 Ba Ribose-5- phosphate*/ 365 25.0if 68.6 0.1 Orotic acid-6-C^1 * 156 0.98 6.5 0.05 AUR*HC1 295 variable variable 0.2 HSS fraction 2.5 -^otal volume 3*^5 ml. ^11 stock solutions were adjusted to pH 7.*f-7.8 with EOH. Phenol red was used as an internal indicator. barium was removed by passing an acidic solution of these compounds through Bio-Rad AG 50W (sulfonic acid) resin, potassium form. The pH was then adjusted and the solutions made up to volume. ^5 Smith (*+1) and hydrolyzed to puxinea and pyrimidines by the method of Marshak and Vogel (222). The bases were separated by paper chromatography on Whatman No. 1 paper using iso pro panol , 680 ml, HC1, 176 ml, and water to 1 liter as solvent (hi). Adenine was purified by paper chromatography using n-butanol-0.6 N aqueous NH^ (6:1) as the solvent (233)• Adenine was eluted from the paper with 0.01 N HC1, and chromatographed again using n-butanol-water (**3*7) (23*+). , Thymine was purified by paper chromatography using n-butanol- water (*+3s7) as the first solvent, and n-butanol saturated with water, NH^ atmosphere (235) as the second. Isolation of RNA, DNA. and protein from ^hrlich ascites cells The protein and nucleic acids were isolated by the method of Tyner, et al. (236) with the following modifica tions. The 10 per cent NaCl extracts containing the nucleic acids were dialyzed against cold orotic acid, and the pro tein residue was hydrolyzed with 12 N HC1 at 100° for 8 hours • The hydrolysate was centrifuged and the radioactiv ity of the solution was determined. Analysis of acid-soluble fractions Acid solubles from whole cells and a large scale cell free system.— The acid-soluble fractions were analyzed by extended gradient elution as described by Murakami, et al. (237)* Neutralized solutions were adsorbed on 1 z 22 cm columns of Dowex-1 (formate) X10 resin, and the columns washed with water until the absorbancy of the eluate dropped below 0.05. The elution was begun using 1 liter of water in the mixing flask and 2,5 N formic acid in the reservoir. The mixing flask was stirred continually by a magnetic bar encased in Teflon. The reservoir solution was changed to 9 H formic acid after elution of AMP. No ultra violet (UV) absorbing peak was found in the eluate at posi tions where AMP normally appears. The reservoir solution was changed to 9 N formic acid when the eluent concentra tion reached 0.^5 N formic acid. The normality of formic acid which eluted AMP was calculated using the data of Hurlbert, et al. (238) and the following equation (239). x x = X----------------- antilog --1— 2.3 V x = Concentration of eluent entering the column X = Concentration of reservoir solution v = Volume of eluent passed through the column V a Volume of the solution in the mixing chamber The reservoir containing 9 N formic acid was replaced by a solution containing 9 N formic acid and 1 M ammonium for mate after elution of ADP. This solution was replaced by 2 M ammonium formate, after elution of ATP. k7 UV absorbing peaks were identified by the ratios of their absorbancy at 280 mu to that at 260 mu, which are characteristic for the various nucleotides. These ratios are AMP, 0.25; orotic acid, 1.8; IMP, 0.22; UMP, 0.38; and AUR, 0.6. Acid solubles of the small scale, cell free system.- Hand columns were used in the analysis of the acid-soluble fractions. These columns consisted of glass columns 1 cm in diameter and 15 cm long, which were joined to the base of 125 ml Erlenmeyer flasks. These columns were packed with Dowex-1 (formate) X10 resin to a height of 5 cm. The neutralized acid solubles were adsorbed on the resin and the various fractions eluted by the method of Blair, et al. (Table V) (2*f0). Paper chromatography.— The orotic acid fractions from the ion-exchange chromatography were investigated for purity by paper chromatography. The paper used was Schleicher and Schuell No. 589 Orange Ribbon filter paper. The orotic acid fractions eluted with formic acid (extended gradient elution) were taken to dryness by vacuum desicca tion, and dissolved in k ml of water. The fractions from the hand columns were reduced in volume to 15 to 20 ml by lyophylization. The samples were spotted on paper in small strips about 0.13 inch by 0.75 inch. Nonradioactive orotic **8 TABLE V ELUTION SCHEDULE FOR HAND-OPERATED COLUMNS Fraction No. Eluent Volume (ml) Compounds Eluted I h2o 30 Bases, nucleosides II O.M- N Formic acid 70 Uracil, uridine, AMP III 0.2 M Ammonium formate^/ 210 UMP, orotic acid IV 3.0 N Formic acid 160 ADP V 0.3 M Ammonium formate l»fO UDPX, UDP, OMP VI 0,*t M Ammonium formate 100 OMP VII 0.7 M Ammonium formate 100 UTP, UTPX VIII 1.25 M Ammonium formate 100 ATP -All ammonium formate solutions were adjusted to pH 5.0. b9 acid and orotidine were also spotted on the same strips as references. The chromatogram was developed, and the UV- absorbing spots eluted with water and rechromatographed. The solvents used were n-butanol-water-formic acid (77*13* 10) (2*fl) and NH^HCO^-water (16 g*100 ml) (2^2). UV absorb ing spots were detected with the aid of a Mineralite lamp. Measurement of optical density and turbidity.— Tur bidity of bacterial cultures was measured with a Klett- Summerson photoelectric colorimeter using the No. 66 filter. Optical density of UV-absorbing solutions was measured with a Beckman Model DU spectrophotometer. Measurement of Radioactivity.— Radioactivity of solutions was measured at infinite thinness with a Nuclear- Chicage gas flow counter, Model 13^7, and a Nuclear-Chicago Model C100B actigraph was used for paper strips. A modifi cation of the procedure developed by Chakravarti and Roy for detection of novocaine (2^3) was used to indicate the pres ence of AUR and AURMP on paper. A solution of 1 g of p- dimethylaminobenzaldehyde in 100 ml of glacial acetic acid sprayed on a chromatogram produced a yellow color with AUR and AURMP. A positive result was obtained with 1 x 10 ^ jumoles of AUR. Adenosine, guanosine, uridine, cytosine, inosine, DPN, and DPNH did not produce a color. RESULTS ill Incorporation of label from serine-3-C into DNA adenine and thymine of ~ E. ooli K12 The hydroxy methyl group of serine has been shown to be a good source of the methyl group of thymine (2**5). For 1^. this reason it was felt that serine-3"C would be useful for studying the effect of AUDR on de novo TMP formation. If the inhibitor depressed thymidylate synthetase activity, Ik then incorporation of label from serine-3~C into DNA thymine would be diminished. However, a lack of incorpora tion of label into DNA thymine in the presence of AUDR at concentrations which completely suppressed growth would not indicate a specific effect on de novo TMP formation. It would only show a general lack of DNA synthesis. For this reason thymidine was added as an alternate source of TMP to insure a normal rate of growth and DNA synthesis. Figures 2 and 3 show that the AUDR concentrations employed com pletely inhibited growth of E. coli K12, and that thymidine, in the amounts used, restored control growth. The hydroxy- methyl group of serine has also been shown to be a source of the number 2 and 8 carbons of purines (22*0. If AUDR were a specific inhibitor of thymidylate metabolism then 50 KLETT UNITS 0 130 120 H O lOO 90 80 70 60 50 30 20 lO 225 75 135 195 15 105 30 o TIME IN MINUTES ?ig. 2. Growth of E. coli K 12 in " t l i o presence of AUQS and AUDR thymi dine . 200 mi of an 18 hour oulture were added "t o 1800 ml of sterile gluoose- salts medium and incubated at 37° - -• Control -• 20 Mg/ml AUDH. + * + jig/ml thymidine - * • 20 ja«/ml ATJDR KLETT UNITS o p i w rftt Hi ' Sp,* ►p* to 0 • a h, i 88? 2 0 HMO p i H< H H O D'OOef O O P to tr cr cr|T d d 4 9 0 0 ( S ( f i 0 a<|*H 0 0 0 0 0 0 0 0 0 0 0 0 0 0 o H vn w 0 ■r V* H o vn H V O V fl 2 S labeling of DNA adenine would not be affected. The effect of AUDR on Incorporation of C into DNA adenine and thy mine is shown in Table VI. Only at the higher analogue concentration was the incorporation of label from serine-3“ llf C into DNA thymine inhibited. Specific activity of DNA adenine was not affected. Growth of E. coll VI? in the oresence of comblaa-fclona S? TOR anct"TO)R---------- It was shown above that AUDR inhibited de novo TMP formation. However, at an inhibitor concentration which was in excess of that needed to completely inhibit cell growth thymidylate synthetase activity was only 66 per cent inhibited. A possible explanation of these data is that AUDR inhibits more than one step of thymidine nucleotide biosynthesis. The inhibition of de novo TMP synthesis, shown above, would reduce the amounts of all thymidine nucleotides. The rate of formation of these metabolites would be reduced still further due to AUDR action at the second inhibitory site, producing complete suppression of growth. Elion et al. have developed a method which indi cated whether two inhibitors affected the same sequential steps of a biosynthetic pathway, by the effect of combined dosage of the antimetabolltes on growth (2*40. This method consisted in plotting dose response curves, similar to those of Figures 2 and 3» representing the effect of each TABLE VI EFFECT OF AUDR ON INCORPORATION OF RADIOACTIVITY FROM SERINE-3-Cll+ INTO DNA THYMINE AND ADENINE OF E. COLI K 122/ Addenda^ (jag/ml) Specific Activity (cpm/ml) AUDR Thymidine DNA Thymine DNA Adenine 0 k 35.0 x 103 **3.5 x 103 20 k 33.1 x 103 ^3.5 x 103 0 2 30.7 x 103 50.6 x 103 no 2 10,2 x 103 **7.3 x 103 -200 ml of an 18 hour culture of E. coll K 12 were added to 1800 ml of sterile glucose-salts"~medium and incu bated at 37° for 3 hours and b5 minutes. Thymidine and AUIR were added when turbidity indicated 23 per cent of control growth at 18 hours. Serine-3-C1H- was added 65 minutes later. , ^ach flask contained serine-3”C^lf (30 jag, 13.5 x 10 cpm) • 55 inhibitor on growth when acting alone. Two or three addi tional dose response curves were plotted showing the effects of the inhibitors when acting in combination. Each of these latter assays consisted of a series of tubes in which the concentration of one inhibitor was kept constant, while the concentration of the other was varied. A specific growth response was chosen, which was represented on all the graphs (for example, 60 per cent of maximal growth). The concentrations of analogues in the tubes which showed this growth response was determined. In each assay series the amount of analogue which produced this response, either acting by itself or in combination, was divided by the amount of analogue which produced this response when acting alone. These ratios, termed the "fractional inhibitory concentrations" (PIC), were determined for each inhibitor. The PICs of the inhibitors were then plotted along the ordinate and abscissa of a graph as shown in Pigure *f. If the antimetabolites acted at the same site the plot pro duced the line XY. Points to the left of this line indi cated inhibition of sequential steps of a biosynthetic pathr way. A plot which falls to the right of line XY suggested that one antimetabolite antagonized the inhibitory action of the other. Cohen, et al. (71) and Hartman and Heidelberger (72) have shown that FUBR inhibited thymidylate synthese. FRACTIONAL INHIBITORY CONCENTRATION OF B 5 6 1,0 0.9 0.8 0.7 0.6 0.5 OA 0.3 0.2 0.1 0 0.1 0.2 0.3 OA 0.5 0.6 0.7 0.8 0.9 1.0 FRACTIONAL INHIBITORY CONCENTRATION OF A Fig. *f.— Graphical representation of the effects of combinations of two inhibitors, A and B, in terms of their fractional inhibitory concentrations, on growth. indicates an antagonistic effect. ■^indicates an additive effect, indicates a synergistic effect. 57 The effect of combined dosage of FUDR and AUDR on growth of E. coli K12 was investigated by the method of Elion, et al. (2Mt). FUDR, at the concentrations used, has been shown by Visser, et al. (31) to inhibit only thymidylate synthe tase. If AUDR affected steps subsequent to the methylation of dUMP, then a plot of the FICs of AUDR versus those of FUDR would indicate a potentiation of the inhibition of growth. The growth response to simultaneous dosage of these analogues, shown in Figure 5> indicated a synergism. Effect of AUR on the incorporation of label into nucleic acid and protein of Ehrllch ascites cells Inhibition of incorporation of labeled precursors into RNA and DNA by AUR has been demonstrated by other investigators (210, 211, 212). The results of these experi ments suggested a general Inhibition of nucleotide metabol ism. The effect of AUR dosage on the incorporation of radioactivity from a labeled nucleic acid precursor into RNA and DNA, and from a labeled amino acid into protein was investigated as a possible means of obtaining more specific information about the sites of action of this inhibitor. If incorporation of radioactivity into each of these macro molecules was affected by different concentrations of AUR, then inhibition due to separate sites of action would be indicated. If biosynthesis of RNA, DNA, and protein were P.I.e. OP 5-PLUOROBEOXYURIDISE 1.0 0.9 0.8 L 0.7 0.5 O A 0.3 0.2 0.1 p .i.e. op 5-aminodeoxyhridine Pig. 5.— Effects of inhibitor combinations on the growth of E. coli K12. P. 3.C., fractional inhibitory concentration. The fractional inhibitory concentrations were cal-vj\ culated from dose response curves at turbidities corresponding to 52$ of maximal growth.00 ' 59 equally sensitive to AUR dosage, then the lack of a metabo lite required for synthesis of all three would be inferred. n k , Orotic acid-6-C was used as the nucleic acid pre- iL. cursor and valine-l-C was used as the protein precursor. Ehrlich ascites cells were incubated in vitro in the pres ence of these compounds and increasing concentrations of AUR. It was found that as AUR concentration increased, incorporation of labeled precursors into RNA, DNA and pro tein was inhibited to equivalent degrees (Figure 6). The acid-soluble fraction of the control and the samples which contained the highest AUR concentration were analyzed on Dowex-1 (formate) resin by extended gradient elution (237). The amount of radioactivity in the various fractions was determined. The only significant difference found was that the orotic acid peak from the AUR-containing samples contained more counts (2.60 x 10^ cpm) than this £ peak from the control samples (1.63 x 10' cpm). Paper chromatography of this peak from AUR-containing and control samples showed that orotic acid was the only radioactive material present. Ik During preliminary experiments using uracil-2-C as the labeled nucleic acid precursor, low concentrations of AUR were found to stimulate the incorporation of label into RNA (Table VII). Reichard and Skold (2**7) have shown that acetone powders from Ehrlich ascites cells converted PER CENT OP CONTROL ok o# at* p , W H * lasg p p, « h*Bh , inw ’ 2-etO ? dO p H 4 tty d 0 • t f 0 Hpr o a 0 O' I a ( d <+ (SO dP ctd oitit ddo p p a oo t d dO dP etO 0 p'H H* H P dO>) 0 0 H a P < POOP dHO 4 dP 0 0 9 et# ‘ V B 1 dope 2 *tP et P P et p'c+i # 9 P dd f r * S*fU P 40 9 etP 0 O' cfO J3 Pet d 0 0 P fJLH 0° * 0 ok Sh- h- a. H d O « ) \0v H I nr 0 H do 0 dO' 0 I O' pa* HI p -r i _ SB? f> 0 0 0 ooP a h ? 0® EH 0 >>0 g* H et 0|0 HP H 10 0 P 0 0. , dH i\ Ifgg <gg d m 0 d w 0 g d Hct I d 92 V s! OP 0 * •P 0 4 H 0 I OJ 10 'Jl I H 0 0 I 0 H O 10 0 bi 0 ■r o c > O' 0 3 00 vfi 0 0 H 8 H H O H r o 10 ■F r 00 H O' ♦ 0 *0 t 1 0 d b 1 , 0 9 61 TABLE VII THE EFFECT OF AUR AND INOSINE ON INCORPORATION OF URACIL-2-Cllf INTO RNA OF EHRLICH ASCITES CELLS IN VITROi/ I. Effect of AUR CPM/Absorbancy Per cent of Control Control 37.1 AUR *+.88 nmoles/ml 337 908 AUR 2.M+ ;umoles/ml 296 798 AUR 1.22 pmoles/ml 206 555 II. Effect of inosine CPM/Absorbancy Per cent of Control Control 118 Inosine 5 jumoles/ml 1310 1110 Inosine 10 ;xmoles/ml 1370 1160 Inosine 5 nmoles/ml + AUR 2.M* jzmoles/ml 2130 1810 -Ehrlich ascites cells were incubated in Krebs phosphate buffer medium III supplemented with glucose, glu tamine, inosine, and AUR at 37° as described under "Mate rials and Methods" with the following modification. Uracil-2-C1^ was used as the radioactive precursor in place of orotic acid-6-C1^. 62 uracil to UMP by two pathways. One involved a one step formation of UMP by reaction of uracil and phosphoribosyl pyrophosphate. The second entailed reaction of uracil with ribose-l-phosphate to form uridine, and subsequent phospho rylation of uridine to UMP. The majority of the UMP was formed by this latter pathway. These investigators also found that UMP was formed in the presence of inosine, uraci], and ATP (2^7). Inosine acted as a source of ribose-l- phosphate for uridine synthesis. The stimulation observed in the presence of low AUR concentrations was attributed to similar mechanisms. Pre sumably AUR was acted upon by a nucleoside phosphorylase and cleaved to AU and ribose-l-phosphate, thus increasing the amount of ribose-l-phosphate within the cell. Due to this increased ribose-l-phosphate pool size larger amounts Ik of UMP were formed from uracil-2-C . This stimulation made an evaluation of the inhibitory effects of AUR impossi ble. The increased amount of label incorporated into RNA Ik from uracil-2-C might only represent an enhanced conver sion of uracil to UMP and not reflect any changes in the rate of RNA synthesis. It has been shown that UMP acted as a feedback inhibitor of its own de novo synthesis by depres sing the rates of formation of carbamyl aspartate, dihydro- orotic acid (217), and UMP (276). The conversion of uracil to UMP would depress de novo nucleotide biosynthesis, making 63 an evaluation of the effect of low concentrations of AUB even more difficult. Inosine was added to the incubation mixtures as a possible means of overcoming these difficulties. It had been shown that this nucleoside was an effective source of the sugar moiety of UMP (2^7). If inosine was a better source of ribose-l-phosphate than AUR, than AUR cleavage might not markedly affect the pool size of this pentose. Under these conditions AUR stimulation of uracil incorpora tion would be negligible. Inosine did stimulate labeling of RNA, and AUR, in the presence of inosine, was less effec tive in increasing the amount of label incorporated into this macromolecule (Table VII). However, the stimulatory effect of the inhibitor was still great enough to obscure its inhibitory effects. AUR, at higher concentrations, iL. inhibited uracil-2-C incorporation into RNA and DNA about 1*+ as effectively as orotic acid - 6-C incorporation. (At an inhibitor concentration of 7.2 mg AUR*HCl/5 ml the incor- lL poration of orotic acid-6-C into RNA was inhibited 77 per lu cent, and uracil-2-C incorporation was inhibited 90 per cent.) Due to these complications orotic acid-6-C^1 * was used as the labeled nucleic acid precursor. Orotic acid is on the de novo pathway of nucleotide biosynthesis. It reacts with PRPP rather than ribose-l-phosphate. Since l^f orotic acid-6-C incorporation into RNA was only slightly (h stimulated by AUR (Figure 6), it can be inferred that breakdown of the inhibitor did not markedly affect the PRPP pool size. The conversion of orotic acid to nucleo tides by a high speed supernatant frac tion from khrliohascites cells The effect of AUR on the conversion of orotic 1^. acid-6-C to nucleotides was investigated using a high speed supernatant fraction from Ehrlich ascites cells (Figure 7)• The major product was UTP which accounted for 88 per cent of the nucleotides formed. AUR inhibited the formation of UTP, and this inhibition was reflected in a lack of utilization of orotic acid. There was no accumula tion of OMP, UMP, UDP or UDP-sugar compounds. Low inhibitor concentrations stimulated the incorporation of label into the UTP fraction (38.2 per cent at 1.2 jamoles/ml AUR). Higher analogue concentrations were required for inhibition in the high speed supernatant fraction than in whole cells (Figures 6 and 7). In whole cells H-.88 ^moles/ml AUR 1*+ inhibited incorporation of label from orotic acid-6-C into RNA 77 per cent. The conversion of this precursor to UTP by the high speed supernatant fraction was depressed 9^.7 per cent at 8.^ ^unoles/ml AUR and 26.0 per cent at 7.2 jamoles/ml AUR. When the mechanism of action of a nucleoside anti metabolite has been studied it has usually been found that 65 100 UTP OROTIC ACID UDP OMP o o r. zz 3.6 M-.8 6.0 2.k 1.2 7.2 0 AUR CONCENTRATION (umolcs per ml) Pig. 7.— This effect of AUR on the conversion of orotic acid-6-C1^ to nucleotides by a high speed supernatant fraction from Ehrlich ascites cells.±/ high speed supernatant fraction was incubated with ATP^.MgClp, PGA, ribose-l-phosphate, AUR, and orotic acid-6-C14" as described under "Materials and Methods." 66 conversion to the nucleotide was a prerequisite for inhibi tion. Examples of this are AZUR (116, 168) and PUDR (71, 72)• By analogy it seemed likely that AUR was converted to AURMP by Ehrlich ascites cells. The ability of the high speed supernatant fraction from Ehrlich ascites cells to form AUR nucleotides was investigated. The incubation con ditions were the same as those described above for the con- version of orotic acid-6-C to nucleotides, except that the amounts of all components were increased 20-fold. The AUR concentration was if .8 umoles/ml. The acid soluble fraction was analyzed by extended gradient elution on Uowex-1 (formate). AUV-absorbing peak was eluted having a ratio of absorbancy at 280 nju to that at 260 character istic of AUR. When solutions containing this peak were spotted on paper and tested the p-dimethylaminobenzaldehyde reagent a yellow color was produced. These data indicate that this was an AU-containing compound. It was eluted just prior to the position in the elution sequence where AMP is normally found. Prom its position in the elution sequence it was inferred that this was AURMP. The concen tration of AURMP in the eluate was calculated using the extinction coefficient of AUR. Prom this datum and the volume of the eluate it was determined that 10.9 ^unoles (**.25 mg) of AURMP were formed, which represented 2.2 per cent of the AUR added (**95 jamoles). There was no indica tion of the formation of AUR diphosphate or triphosphate. DISCUSSION The sites of action of AUDR and AUR were investi gated. The data obtained from these investigations were interpreted as indicating a specific site of AUDR inhibi tion and a specific site of AUR inhibition. Additional possible sites of action were also suggested for each analogue. Since one analogue is a deoxyribonucleoside and the other is a ribonucleoside they affect different path ways of nucleotide metabolism. Therefore each will be dis cussed separately. The effect of AUDR on the incorporation of label Ilf from serine-3”C into DNA thymine was studied using two inhibitor concentrations, which were 2 and 11 times the minimum concentration required to inhibit growth completely. Since the hyroxymethyl group of serine is a source of the methyl group of thymine and the number 2 and 8 carbons of purines, an inhibition of the incorporation of isotope into DNA thymine without an effect on the labeling of DNA purines would indicate that AUDR specifically inhibited thymidylate synthetase. Thymidine was added to the growth media as a potential source of THP. Under these conditions the requirement of thymidine nucleotides for DNA synthesis was 67 68 supplied either by de novo synthesis or by utilization of exogenously added thymidine (Figure 8). Only at the higher AUDR concentration was an Tk inhibition of the incorporation of label from serine-3”C into DNA thymine observed. The incorporation of isotope from the same source into DNA adenine was not affected by either concentration of AUDR. These data indicated that thymidylate synthetase was inhibited at the higher AUDR concentration• No inhibition of labeling of DNA thymine was observed at the lower AUDR concentration. Since AUDR com pletely inhibited growth of E. coli K12 at this concentra tion in the absence of thymidine, it was inferred that the lack of effect on thymidylate synthetase activity was due to the presence of thymidine in the culture media. The following two explanations are proposed to account for this effect of thymidine on AUDR inhibition. The first proposes that the inhibition of thymidylate synthetase was due to the action of an AUDR nucleotide, and that phosphorylation of AUDR is a prerequisite to inhibition. Cohen, et al. (71) and Hartman and Heidelberger (72) have shown that phosphorylation of FUDR to the monophosphate is required for inhibition of thymidylate synthetase. Ry analogy it seemed likely that the inhibition of thymidylate synthetase observed above was due to the action of AUDR monophosphate. Thymidine Thymidine nucleotide cofactors Pig. 8.— The metabolism of thymidine nucleotides. Site of AUDR inhibition. Possible additional sites of AUDR inhibition. 70 If AUDR and thymidine were phosphorylated by the same kinase, then when both deoxynucleosides were present they would compete for sites on the same enzyme. If there were a limited amount of enzyme, then the amount of nucleotide formed from either nucleoside would be a function of the ratio of the AUDR concentration to the thymidine concentra tion. By this line of reasoning the lack of inhibition at the lower AUDR concentration (20 jog/ml) in the presence of thymidine (k jig/ml) would indicate that noninhibitory amounts of AUDR monophosphate were formed at this analogue to thymidine ratio. When the AUDR to thymidine ratio was increased 11 fold, adequate AUDR monophosphate could have been formed to inhibit thymidylate synthetase. The second explanation was mentioned under "Results.” It was proposed that AUDR also inhibited some step of thymidine nucleotide metabolism subsequent to TMP formation. At the concentrations of substrates normally found in the cells, AUDR would not completely suppress enzyme activity at either site. However, AUDR inhibition of thymidylate synthetase would decrease the amounts of all thymidine nucleotides formed subsequent to this step (Figure 8). Due to the decreased amount of substrate, AUDR would be a more effective inhibitor at the second site, causing complete suppression of growth. Addition of thymidine to the cul ture medium would restore normal levels of thymidine 71 nucleotides and thus prevent AUDR action at the second site of inhibition. As a means of determining which of the two explana tions is correct the effect of combined dosage of AUDR and FUDR on the growth of E. coli KL2 was investigated. If AUDR inhibited the metabolism of thymidine nucleotides sub sequent to TMP formation then combined dosage of AUDR and a compound known to affect thymidylate synthetase would potentiate inhibition of growth. Visser, et al. (31) have shown that control growth is restored when thymidine is added to a medium containing sufficient FUDR (0.05 MM) to suppress growth completely of E. coli K12. At higher con centrations of FUDR thymidine alone was ineffective as a reversing agent of FUDR inhibition. These data showed that FUDR, at the lower concentration, only inhibited thymidy late synthetase. A synergistic effect was observed when E. coli K12 were grown in the presence of both AUDR and FUDR (0.01-0.05 pM) (Figure 5)» indicating that AUDR did inhibit thymidylate metabolism subsequent to TMP formation. Although the potentiation of this inhibition by the combined use of FUDR and AUDR showed that the latter analogue suppressed utilization of thymidine nucleotides, it did not indicate which steps of thymidylate metabolism were affected. Thymidine nucleotides are required for DNA synthesis and cofactor functions (Figure 8). It is 72 conceivable that AUDR could inhibit any of these processes. These possible sites of action were not investigated fur ther. The above data indicate that AUDR inhibits sequen tial steps of thymidylate metabolism, namely TMP formation and utilization of thymidine nucleotides for DNA or cofac tor synthesis. These data were also interpreted as evidence that AUDR phosphorylation is a prerequisite for inhibition, and that AUDR and thymidine are substrates for the same kinase. In an effort to determine the sites of action of AUR the effect of this analogue on the incorporation of label from orotic acid-6-C^ into RNA and DNA and from Ik valine-l-C into protein was investigated. It is reason able to assume that if the syntheses of all three macro molecules were equally sensitive to inhibition by AUR then a diminished rate of formation of a metabolite required for biosynthesis of all three is indicated. It seems unlikely that if AUR inhibited various enzymes each of which was specifically involved in biosynthesis of one macromolecule that all of the enzymes would be equally affected at vari ous AUR concentrations. It was found that RNA, DNA, and protein biosynthe sis were equally sensitive to inhibition by AUR. It was therefore inferred that the formation of a metabolite 73 required for the biosynthesis of all three macromolecules was inhibited* The following discussion will show that inhibition of UDF synthesis would suppress SNA, DNA, and protein formation in a manner which is consistent with the results* It has been shown that cellular RNA is formed from nucleoside diphosphates by the enzyme polynucleotide phosphorylase (2*t9» 250) and from nucleoside triphosphates by RNA polymerase (251, 252). In cell-free systems from E. coli it has been shown that the first step of de novo formation of pyrimidine deoxynucleotides involved the reduc tion of uridine diphosphate or cytidine diphosphate to the corresponding deoxynucleoside diphosphate (231, 232, 233). If the reasonable assumption is made that Ehrlich ascites cells also synthesize RNA from ribonucleoslde diphosphates or triphosphates, and if deoxynucleotide formation also occurs at the diphosphate level, then a depressed rate of UDF formation would be expected to inhibit RNA and DNA synthesis (Figure 9). Frotein synthesis requires the participation of three types of RNA, namely soluble RNA, ribosomal RNA, and what has been termed "messenger" RNA. It has been shown that soluble RNA of E. coli (20*+) and Ehrlich ascites cells (225) participates in protein synthesis without intermediate breakdown. Synthesis of ribosomal RNA in E. coli has been shown to stop within two minutes after infection with T^. Orotic acid PRPP ►UTP n uridine Ribo se-l-pho sphate Fig. 9.— The metabolism of the uridine nucleotides Site of AUR inhibition. Possible additional sites of AUR inhibition 7? phage (227), Therefore it is necessary that ribosomes present before phage infection be utilized for the rapid and continued synthesis of protein required for phage pro duction after infection. This continued synthesis of pro tein after cessation of ribosomal RNA synthesis suggests that ribosomal RNA participates in protein synthesis with out intermediate breakdown. Messenger RNA, however, has been characterized by a high turnover rate (226-229). Con tinual synthesis of protein would therefore require a con stant formation of messenger RNA. Thus it can be inferred that the inhibition of protein synthesis observed in the presence of AUR was due to a lack of messenger RNA, since ribosomes and soluble RNA synthesized prior to addition of AUR were still available for protein synthesis. AUR inhibited orotic acid-6-C^ incorporation into nucleic acids of Ehrlich ascites cells, and also depressed lM- the conversion of orotic acid-6-C to nucleotides by a high speed supernatant fraction from this tissue. In cell- free incubations there was no accumulation of the compounds which are intermediates in the conversion of orotic acid to UTP. Prom these data it was inferred that a site of action of AUR was the conversion of orotic acid to OMP. An inhibi tion of OMP decarboxylation would also be consistent with these data if the following reactions occurred. OMP, which would be expected to accumulate if its decarboxylation were 76 inhibited, could be dephosphorylated to orotidine. The orotidine could then be cleaved by a nucleoside phospho- rylase to orotic acid and ribose-l-phosphate. The net result would be an apparent lack of orotic acid utilization. It has been shown that OMP accumulates in cell “free systems from mouse L-5178-Y lymphoma cells in which OMP decarboxy lase is inhibited by addition of AZUB or AZURMP (113). These cells contained enzymes which degraded OMP to oroti dine, but showed no orotidine phosphorylase activity (113). If similar enzyme activities are assumed for Ehrlich ascites cells then either OMP or orotidine would accumulate when OMP decarboxylation is inhibited. The absence of OMP or orotidine accumulation, and the lack of utilization of orotic acid in the presence of AUR is therefore interpreted as indicating an inhibition of OMP formation. AUR also inhibited the incorporation of isotope iL. from uracil-2-C into nucleic acids. Uracil has been shown to be converted to UMP by way of two pathways by ace tone powders from Ehrlich ascites cells (Figure 9). One pathway involves the direct formation of UMP from uracil and PRPP. The other pathway involves synthesis of uridine from uracil and ribose-l-phosphate, and subsequent phospho- lylation of uridine to UMP (2^-7). Thus, incorporation of Ilf radioactivity from uracil-2-C into nucleic acids does not involve intermediate formation of orotic acid or OMP. 77 Therefore inhibition of the incorporation of label from this precursor into RNA and DNA by AUR indicates a site of action other than OMP formation. A possible explanation for the inhibition of the conversion of uracil to nucleic acids, and of the inhibition of OMP formation, is that AUR inhibits enzymes which utilize PRPP. By this line of rea soning AUR could be expected to inhibit the conversion of uracil and PRPP to UMP. Another possible site of inhibi tion is uridine kinase. AUR is phosphorylated to AURMP. Therefore it is suggested that AUR could inhibit uridine phosphorylation due to competition for sites on uridine kinase. No further investigations were conducted on these possible sites of inhibition. When the high speed supernatant fraction from Ehrlich ascites cells was incubated with AUR, an AU- containing compound was formed which was eluted from Dowex-1 (formate) just prior to the eluate fraction which normally contains AMP. The position of the AU-containing compound in the elution sequence was interpreted as indicating that this compound was AURMP. About 2.2 per cent of the AUR added was converted to this compound. There was no indica tion of the formation of AUR diphosphate or triphosphate. Studies of the mechanism of action of nucleoside analogues have generally indicated that these compounds were active after conversion to nucleotides. A few examples 78 of this are AZUR which inhibits OMP decarboxylase after conversion to AZURMP (116), PURR which inhibits thymidylate synthetase after phosphorylation to FURRMP (71, 72), and BURR, CURR, and IURR which are incorporated into RNA (*+2). In context with the requirement for phosphorylation demon strated for other nucleic acid antimetabolites the fact that AURMP is formed from AUR strongly suggests that AURMP may be the compound which inhibits nucleotide biosynthesis. Naturally occurring pyrimidines, various pyrimidine analogues, and their nucleosides and nucleotides have been shown to be feedback inhibitors of the formation of carbamyl aspartate and dihydroorotic acid (217). UMP has been shown to be a feedback inhibitor of OMP decarboxylation (276). The inhibitory effects of AUR or AURMP may be due to their action as feedback inhibitors of de novo pyrimidine biosyn thesis. Investigations may show that AUR inhibits de novo pyrimidine nucleotide biosynthesis at steps prior to orotic acid formation. SUMMARY The results of investigations designed to locate the sites of action of AUDR and AUR were described. E. coli K12 was grown in liquid medium supplemented with tl AUDR and serine-3”C . Thymine was added as a potential source of unlabeled TMP to insure a normal rate of DNA synthesis and growth of E, coli in the presence of AUDR. Incorporation of radioactivity into DNA was not inhibited in cultures which contained 20 jjg/ml AUDR and ^ pg/ml thymidine. When the inhibitor concentration was increased to 110 jig/ml and the thymidine concentration lowered to 2 jig/ml the incorporation of radioactivity into DNA thymine was inhibited 66 per cent. Both concentrations completely inhibited growth of E. coli K12 in the absence of thymidine. l l i . Incorporation of isotope from serine-3-C into DNA adenine was not affected. These data were interpreted as indicating the following relationships concerning the effects of AUDR on tlie metabolism of E. coli K12. (1) De novo TMP synthesis was inhibited by AUDR. (2) Formation of an AUDR nucleotide was a prerequisite for inhibition. (3) Thymidine and AUDR were phosphorylated by the same kinase, and therefore these 79 80 nucleosides competed for sites on the enzyme, (b) Only at the higher ratio of AUDR to thymidine was enough AUDR nucleotide formed to inhibit thymidylate synthetase. Inhibition of growth of E. coll EL2 was potentiated by a combined dosage of AUDR and FUDR. Since FUDR has been shown to inhibit only thymidylate synthetase (31) at the concentrations used, the synergism indicated that AUDR inhibited thymidylate metabolism subsequent to TMP forma tion. It was therefore concluded that AUDR inhibited sequential steps of thymidylate metabolism, namely TMP formation and utilization of thymidine nucleotides for DNA or cofactor synthesis. A modification of the method of synthesis of AUDR was also described which increased the yield seven fold over the previously reported yield. AUR was equally effective as an inhibitor of the l*t incorporation of orotic acid-6-C into RNA and DNA, and of l*t valine-l-C into protein of Ehrlich ascites cells at vari ous inhibitor concentrations. It was inferred that this uniform effect was due to an inhibition of the formation of a metabolite which was required for biosynthesis of all 3 macromolecules. UDP is an intermediate in the formation of RNA and DNA, and a lack of "messenger" RNA synthesis would be expected to produce an immediate inhibitory effect on protein synthesis. It was therefore concluded that AUR inhibited some step leading to UDP biosynthesis. 81 , l1 * - l^f Conversion of orotic acid-6-C to UTP-C by a high speed supernatant fraction from Ehrlich ascites cells was inhibited by AUR. The intermediates, OMP, UMP, or UDP did not accumulate. These data were interpreted as indi cating that the conversion of orotic acid to OMP was affected• Incorporation of uracil-2-C into the RNA of Ehrlich ascites cells was stimulated by low concentrations of AUR. Stimulation was attributed to an increase in the ribose-l-phosphate pool due to cleavage of AUR to AU and ribose-l-phosphate by a nucleoside phosphoiylase. This stimulation obscured the inhibitory effects at low concen trations of AUR. Addition of inosine, which is also a source of ribose-l-phosphate, to control and AUR-containing samples decreased the stimulatory effect of AUR, but not sufficiently to permit accurate evaluation of the degree of inhibition produced by low AUR dosage. At higher AUR con- 1*+ centrations, incorporation of uracil-2-C into nucleic acid 1 h was inhibited as effectively as when orotic acid-6-C was used as the labeled precursor. 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Creator Friedland, Melvyn (author)

Core Title Effects Of 5-Aminodeoxyuridine And 5-Aminouridine On Metabolism Of Nucleic Acids

Degree Doctor of Philosophy

Degree Program Biochemistry and Nutrition

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

Tag chemistry, biochemistry,OAI-PMH Harvest

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Advisor Visser, Donald W. (committee chair), Heinrich, Milton R. (committee member), Roberts, Carmel M. (committee member)

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