MUTAGENIC PROPER'HES 0F CIS-PLATINUM(II)DIAMMINODICHLORIDE IN ESCHERICHIA COLI Dissertation for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY DORIS JEAN BECK 1974 This'fiis to’eertify that the thesis entitled MUTACENIC PROPERTIES OF- CIS-PLATIWJI)DIAMMINODICHLORIDE IN ESCHERICHIA COLI liresented by 'Doris Jean Beck -‘ has been'aceepted towards fulfillment of the requirements for Phi‘l’D; degree in Microbiology 14/47/4415,, Major professor nasal/w. zj/Mz/e 0-7639 ' amoma 6v ‘5 4mm 3. sons 300K BINDERY INC. L'BRARY BINDERS m 3:35;;F3IT. ““3.“ I. ABSTRACT MUTAGENIC PROPERTIES OF CIS-PLATINUM(II)DIAMMINODICHLORIDE IN ESCHERICHIA COLI By Doris Jean Beck The anti-tumor drug cis-platinum(II)diamminodichloride (PDD) induced extensive filamentation in wild type Escherichia coli and in mutants lacking certain deoxyribonucleic acid (DNA) repair functions (uvrA, recB, recC, and 201A ; viability of mutant filaments in PDD was significantly less than that of wild type cells. PDD was highly toxic to lexl, lexl uvrA6 (where its effect was cummulative), and recAlB mutants, all of which were killed before filamentation occurred. 3H-Thyrnine incorporated into DNA of wild type cells subsequently treated with PDD became trichloroacetic acid soluble at rates similar to those observed after exposure to comparable doses of ultraviolet light (UV) or mitomycin C. PDD, like UV, induced extensive degrada- tion of DNA in £335 organisms. After a 30 min lag, PDD significantly inhibited the synthesis of DNA but not ribonucleic acid (RNA) or protein. Treatment of E. ggli_with PDD was successful in yielding auxotrophic mutants, some of which could be reverted to prototrophy by exposure to 2-aminopurine (Z-AP) and/or N-methyl-N;-nitro-N-nitroso— guanidine (NTG) and/or additional exposure to PDD. None of these OW \J a” o C) Doris Jean Beck PDD-induced mutants were reverted by ICR derivatives suggesting that the p1atinum(II) compound does not cause frameshift mutations. PDD treatment was able to effect reversion of 2-AP-, NTG-, UV-, and ICR191- induced auxotrophic mutants and thus exhibited a wide spectrum of mutagenic activities. In contrast, the isomeric ££2§§_compound was much less effective in inducing revertants of an NTG-induced auxo- troph (N46 try) which reverted to prototrophy at high frequency after treatment with PDD. Studies with N46.£§1 indicated that mutagenesis using PDD was most effective when the bacteria were grown for extended periods in low concentrations of PDD. MUTAGENIC PROPERTIES OF CIS-PLATINUM(II)DIAMMINODICHLORIDE IN ESCHERICHIA COLI By Doris Jean Beck A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1974 ACKNOWLEDGMENTS I would like to sincerely express my gratitude to Dr. Robert R. Brubaker, my major advisor, for his guidance and consideration throughout the period of my doctoral study. Thanks is also extended to Loretta VanCamp for her encouragement and to Loretta and Dr. Barnett Rosenberg for their generous donations of the platinum compounds. The assistance of Prudence Hall is also warmly appreciated. A special thank you is extended to Donna Muirhead for her encouragement and helpful suggestions which sustained me throughout my studies at Michigan State University. 11 ACKNOWLEDGMENTS . . . . . LIST OF TABLES . . . . . LIST OF FIGURES . . . . . INTRODUCTION . . . . . . LITERATURE REVIEW . . . TABLE Biological effects of PDD . . Bacteria . . . . . . Eukaryotic cells. . . Viruses . . . . . . . Antitumor activity. . Deoxyribonucleic acid DNA repair. . . . . . . Excision repair . . . Recombination repair. Mutagenesis . . . . . . 2-Aminopurine (2-AP). Ultraviolet light (UV). N-methyl-N'-nitro-N-nitrosoguanidine (NTG) ICR derivatives . . . Compounds which crosslink nucleic acids . LIST OF REFERENCES . . . ARTICLE 1: ARTICLE 2: OF CONTENTS dichloride in Escherichia coli. APPENDIX: diamminodichloride iii Effect of cis-platinum(II)diamminodichloride on wild type and deoxyribonucleic acid repair deficient mutants of Escherichia coli Colicin induction and plasmid elimination caused by treatment of bacteria with cis-platinum(II)- Page ii iv 27 Mutagenic properties of cis-platinum(II)diammino- 34 61 LIST OF TABLES Table Page ARTICLE 1 1. Characterization of repair-deficient mutants of Escherichia coli strain K-12 . . . . . . . . . . . . . . . 29 2. Comparison of treatments with UV light and cis—platinum- (II)diamminodichloride (PDD) on survival and filamen- tation of DNA mutants of Escherichia coli K-123 . . . . . 30 ARTICLE 2 1. Reversion properties of mutants induced by PDD, ICR, UV, NTG, and z—APa O O O O I O O O O C O O O O O O O O O O O O 49 2. Effectiveness of various platinum compounds in inducing reversion of N46 ££y_and in treatment of Swiss white mice after Sarcoma 180 tumor implantation . . . . . . . . 54 3. Reversion of N46 ££y_when exposed to 150 ug PDD for 1 h in E salts, phosphate buffer, saline, or nutrient broth . 55 4. Reversion of N46 try when exposed to 100 ug PDD per ml TM buffer at pH's 5 to 9a . . . . . . . . . . . . . . . . 56 APPENDIX 1. Elimination of F'lac from Escherichia coli AB785 (lac-lglf lac+) by treatment with cis—platinum(II)diamminodichloride (PDD) a O I C O C I O O O C O C O C C O O O O I O C O I O O 67 2. Induction of colicin E2 production in colicinogenic Salmonella typhimurium D36 (colE2) by treatment with cis-platinum(II)diamminodichloride (PDD)a . . . . . . . . 68 iv Figure l. 2. 3. 4. 5. 6. LIST OF FIGURES ARTICLE 1 Survival (colony-forming ability of filaments of Escherichia coli strain W3350 thy_during incubation in C medium supplemented with thymine (20 ug/ml) at 25 c 0, 30 c (0). 35 c O). and 40 c (g) in the presence of Eégfplatinum(II)diamminodichloride (35 ug/ml) . . . . . . . . . . . . . . . . . . . . . . . . . Loss of trichloroacetic acid-insoluble radioactivity from cells of Escherichia coli strain W3350 thy, previously grown with 3H—thymine, during incubation in C medium containing added unlabeled thymine (40 ug/ml) in the presence of 50 pg (0), 25 pg 0), and no (0) Elg—platinumfll)diamminodichloride per ml, respectively . . . . . . . . . . . . . . . . . . . . . Loss of trichloroacetic acid-insoluble radioactivity from cells of Escherichia coli strain W3350 thy, previously grown with 5H-thymine, during incubation in C medium containing added unlabeled thymine (40 ug/ml) in the absence of treatment «1), after irrad- iation with 4,000 ergs of ultraviolet light per mm2 «l), and upon addition of 50 ug of Sigfplatinum(II)- diamminodichloride (C) or 1 ug of mitomycin C (0) Per m1 of medium . . . . . . . . . . . . . . . . . . . . . Loss of trichloroacetic acid-insoluble radioactivity from cells of Escherichia coli strain mr 2-41 previously grown with 3H-thymine, during incuba- tion in tryptone-E medium containing added unlabeled thymine (40 ug/ml) . . . . . . . . . . . . . . . . . . . Increase in cell mass (A) and rates of deoxyribonucleic acid (B), ribonucleic acid (C), and protein (D) syn- thesis in control cells of Escherichia coli strain W3350 1:111 (O) and cells receiving 15 pg (0) and 35 ug «1), respectively, of gigfplatinum(II)diamminodichlor- ide per m1 of C medium . . . . . . . . . . . . . . . . . Rates of deoxyribonucleic acid synthesis in control cells of Escherichia coli strain W3350 fly (0) and cells treated at the initiation of the experiment with 35 ug of cis-platinum(lI)diamminodichloride per m1 of C mediueriD; A, pulsed for 2 min with 3H-thy- mine and B, pulsed for 5 min with 3H-thymine . . . . . V Page 30 31 31 31 32 32 Figure ARTICLE 2 Photographs of petri plates showing colonies from prototrophic revertants of N46 try induced by expos- ure to 2-AP (A), NTG (B), PDD (C), and ICRl91 (D) . . . Photographs of petri plates showing colonies from prototrophic revertants of N46 ££y_induced by expos- ure to PDD (A) and its trans isomer (B) . . . . . . . Reversion data for N46 £52 grown 18 h in nutrient broth containing various concentrations of PDD Reversion data for N46 try exposed to 0 to 300 ug PDD per ml of nutrient broth for 1 h vi Page . 50 52 O 58 . 6O INTRODUCTION A series of novel inorganic platinum(II) compounds was syn- thesized and purified by Rosenberg et a1. (1965, 1967a, 1967b). These compounds inhibited cell division in Escherichia coli and were later shown to cause successful regression of Sarcoma 180 tumors and Leukaemia L 1210 tumors in mice (Rosenberg et a1., 1969; Rosenberg and VanCamp, 1970). The compound gigfplatinum(II)diamminodichloride (PDD) particulary had a high chemotherapeutic index and was observed to have significant effects against many tumors. In contrast, the corresponding £5222 isomer did not induce filamentation in E, 22$}. and exhibited a poor chemotherapeutic index (Rosenberg et al., 1967b, 1969). The compound dichloroethylenediamineplatinum(II) had similar properties to the gig compound; because of its geometry the latter is capable of binding ligands only in the gi§_position. The biological properties of these compounds may be due to their ability to react with nucleic acids and effect interstrand crosslinking between complementary strands. This study was undertaken to supply evidence for the reactivity of PDD with deoxyribonucleic acid (DNA) in E, £211 and to determine the effects of PDD induced damage on these bacteria. Various DNA repair deficient mutants of E3‘3gli have been characterized and can be used to determine which repair functions are essential in correcting PDD induced damage. On occasion when these repair processes are operative in correcting lesions in DNA, alter- ations of DNA base sequences occur which result in mutation. Accordingly, a second objective of this work was to evaluate the mutagenicity of PDD and to obtain evidence for the types of mutations which might be induced by exposure to the drug. This thesis is organized into four divisions. The first div- ision consists of a literature review which describes research concerned with the biological effects of PDD on various systems and brief reviews of two very extensive areas of research, DNA repair and mutagenesis. The second division is a published manu— script describing effects of PDD on DNA repair deficient and wild type E, 521;, The third division is a manuscript to be submitted for publication on the mutagenic activities of PDD in E, 221$. The final division is an appendix describing effects of PDD on bacteria harboring extrachromosomal DNA. LITERATURE REVIEW Biological effects g£_PDD Bacteria Rosenberg et a1. (1965, 1967a, 1967b) reported that treatment of E. 32;; with PDD caused an inhibition of cell division and resulted in the production of long filamentous cells. Cytokinesis of filamen- tous cells of E, 32;; induced by exposure to this drug was only initi- ated by removal of the compound and not by treatment with pantoyl lact- one, divalent cations or elevation of temperature. Gram negative bacteria were highly sensitive to the drug and formed filaments readily while gram positive bacteria filamented only slightly at near toxic levels of the drug. Howle and Gale (1970a) observed cytological changes in PDD induced filamentous cells which suggested an inhibition of DNA synthesis mediated by the p1atinum(II) compound. PDD was found to be associated with protein, nucleic acid and metabolic intermediates of £3.22li lysates when cells had been treated with 191Pt—labeled PDD (Renshaw and Thomson, 1967). Howle et al. (1972) reported that glgfdichloro—([G-SH]-dipyridine)p1atinum(II) associated strongly with calf thymus DNA, yeast ribonucleic acid (RNA) and transfer ribonucleic acid (tRNA) from bacteria or yeast $2HX$££2_PUP not with bovine serum albumin, dextran or erythrocyte membranes. This binding was inhibited by sodium chloride, possibly because the dissociation of chlorine atoms from the platinum atom was necessary for the reaction. 3 Shooter et al. (1972) also suggested that the solvents used to dissolve these compounds may alter their effects possibly due to variations in binding of the different charged species. Shimizu and Rosenberg (1973) reported that exposure to PDD caused a preferential inhibition of DNA synthesis in E, 23;; as they found the amount of DNA in PDD treated cell cultures decreased with increasing concentrations of the drug. Protein synthesis was effected secondarily at higher concentrations followed by subsequent decrease in total RNA at highest doses. Mitomycin C treatment in contrast produced a more immediate and drastic effect as the amount of DNA was greatly reduced at low concentrations with RNA effected secondarily at lower concentrations than protein synthesis. Capabilities for repairing DNA seem to be important for survival of colony forming ability when cells are exposed to PDD. Shimizu and Rosenberg (1973) reported that DNA repair deficient mutants Bs-l and 83-2 of E, ggl1_were more sensitive to PDD than were wild type cells. The g§£_locus was also extremely important for retention of colony form- ing ability when mutants were grown in PDD as presence of the mutation rendered the cells 13 to 23 times more sensitive to the drug than the wild type (Drobnik et al., 1971). Drobnik also found that cells which were Eggf were 2 to 5 times more sensitive while mutants of the ii; locus showed only limited sensitivity to the platinum(II) compound. In contrast, phage exposed to the drug showed essentially identical rates of survival in both hcr+ and her. strains of E, coli. Eukaryotic cells DNA synthesis was shown to be selectively inhibited by PDD in AV3 cells in tissue culture (Harder and Rosenberg, 1970) and Erhlich ascites tumor cells (Howle and Gale, 1970b). At higher concentrations, RNA and then protein synthesis were effected. DNA and RNA synthesis were also in- hibited in lymphocytes stimulated with phytohemagglutinin and then treated with PDD (Howle et al., 1971). Viruses Kutinova et al. (1972a, 1972b) reported that PDD inhibited the rep— lication of SV40 virus in green monkey kidney cells. The treated virus was able to induce the production of T antigen but V antigen production was suppressed. Virus treated with PDD was found to retain its immunogenicity in guinea pigs. In addition, Kara et al. (1971) found that transformation of chick embryo fibroblast cells with Rous sarcoma virus was completely inhibited by pretreatment of cells with PDD. Antitumor activity The compound PDD particularly has a high chemotherapeutic index and was found to have significant effects against Dunning ascitic leukemia and Walker 256 carcinosarcoma in rats (Kociba et al., 1970), Ehrlich ascites tumour (Howle and Gale, 1970b), reticulum cell sarcoma (Talley, 1970), Lewis lung carcinoma (Rosenberg, 1971), leukemia and various neoplasms in man (Leonard et al., 1971; DeConti et al., 1972; Lange et al., 1972, 1973). Welsch (1971) reported that PDD was effective in inhibiting the growth of mammary tumors chemically induced by 7,lZ-dimethylbenz[a]anthracene. PDD was effective against tumors when administered intraperiton— eally but had no effect when taken orally (Carter, 1972). Pre-injection of cysteine or nucleophiles reduced the toxicity of both alkylating agents 6 and PDD but, upon pre-injection, the doses required for tumor inhibition were raised to a point where therapeutic value was lost (Connors, 1971) Moreover Connors (1972) observed that a Walker carcinoma with acquired resistance to the alkylating agent melphalan also exhibited cross resis- tance to other alkylating agents and to PDD. Toxic effects of PDD treat- ment were severe nausea and vomiting, nephrotoxicity, myelosuppression, erythrosuppression, hyperuricemia, and audiologic impairment (Rossof et al., 1972). Zak et al. (1972) reported that ten days after PDD administration to mice, the cell populations in the bone marrow approached normal levels and that lethal consequences due to fluctuation in cellular elements of bone marrow were not observed. The immunosuppressive effects which were observed during therapy with platinum(II) compounds (Berenbaum, 1971; Khan and Hill, 1971, 1972, 1973) might be due to its inhibition of DNA synthesis in the lymphatic cells. Taylor et a1. (1973) treated normal and tumor bearing rats with 195Pt-[lAC]ethy1enediamine platinum[140]ethylenediamine dichloride and dichloride. They found the half life of retention in tissues to range from 2.5 to 7.6 days and suggested that this slow clearing from tissues correlates with the long term effects of the compound on DNA synthesis. They were able to find only small amounts of the labeled compound in the nuclear fraction of cell lysates but possibly this amount might be sufficient to interfere with DNA synthesis. Soon after injection, high levels of the drug were found in the kidneys; this concentration probably caused the renal toxicity observed in patients treated with the drug (Rossof et al., 1972). Hoeschele and VanCamp (1972) found that tumor bearing mice excreted the compound more slowly and thus retained higher levels of the compound. Combination therapy using PDD with other chemotherapeutic agents was found to enhance the successful treatment of neoplasms. Woodman et al. (1973) and VanCamp and Rosenberg (1972) reported that combination therapy of PDD with cytoxan enhanced the therapeutic effect of PDD. Conran and Rosenberg (1972) found that injection of the immunostimulant, zymosan, previously to administration of PDD in tumor bearing mice gave better rates of cure than controls receiving only PDD. Zak and Drobnik (1971) observed that PDD treatment enhanced lethality of x-irradiation in studies of post-irradiation lethality of x—rays in mice. Deoxyribonucleic acid Horacek and Drobnik (1971) reported that treatment of calf thymus DNA with PDD supported renaturation of the DNA and hypothesized that the compound formed crosslinks in the DNA which held both strands in register during heating. Roberts and Pascoe (1972) reported that the drug cross- links complementary strands of DNA in Hela cells although the chemical nature of these crosslinks is yet unknown. Shooter et al. (1972) also obtained evidence for crosslinking in the DNA and RNA of bacteriophage T7 and R17 respectively. Their studies provided evidence for the presence of both inter- and intra- strand crosslinks plus crosslinking of protein to the nucleic acids. These authors concluded that concentrations of the drug which inactivated the bacteriophage probably did not cause lethal inter-strand crosslinking or crosslinking of nucleic acid to protein but hypothesized that intra-strand linking of nucleic acid bases may be the most important reaction for phage inactivation. Studies of the reaction of 1[‘Cv-labeled platinum ethylenediamine dichloride with nucleic acid constituents indicated that the most reactive regions of DNA probably involved guanine moities (Robins, 1973a, 1973b). Horacek and Drobnik (1971) found that PDD caused spectroscopic changes and a decrease in Tm of calf thymus DNA treated 33 yiggg, suggesting that the compound reacted with the bases of the DNA. Only purine bases were observed to undergo a similar spectrosc0pic change in solutions of the compound. Shooter and Merrifield (1972) found that PDD and its £3222 isomer were both capable of crosslinking DNA. The only difference which resulted from the binding of these two compounds to DNA was a conforma- tional change induced in native DNA by PDD but not by the Egggg compound. This conformational change was also observed when DNA was treated with platinum(II)ethylenediaminedichloride. These data suggest that the platinum(II) compounds react with DNA causing damage by formation of crosslinks which, in the case of tumor cells and E. coli, result in inactivation and filamentation, respectively. DNA repair Various tumors have been shown to have differing susceptibilities to the platinum(II) compounds which may be due to their different capa- bilities of DNA repair. It is quite difficult to study this DNA damage and repair in mammalian cells because little is known of the repair mechanisms which are operative in eukaryotic organisms in general. How- ever, various DNA repair mutants of prokaryotes are available and studies of these reactions in E, 33;; may provide additional information regarding the nature of the damage caused by the drug. Reslova (1971/1972) reported that PDD induced production of phage in lysogenic bacteria; this phenomenon was probably dependent on the activities of DNA repair processes. If loss of viability in E. coli is due to damage of DNA, then repair mutants should be more sensitive to the drug than wild type bacteria. Three repair mechanisms function to maintain DNA in E. 33;; (Witkin, 1969). Photoreactivation involves a "photo-reactivating enzyme" which, when activated by visible light, is capable of monomerizing pyrimidine dimers induced by exposure to ultraviolet light (UV). Ex- cision repair is a process which occurs in the dark and involves a phy- sical removal of damaged nucleotides by introduction of single strand breaks on either side of the damaged area followed by release of single stranded oligonucleotides. Local single strand DNA breakdown then occurs (widening the gap) followed by resynthesis of DNA in the resulting gap. Finally, a joining of the strand occurs (Pauling and Hamm, 1968). Post-replication or recombination repair involves the repair of gaps opposite damaged nucleotides which result after replication (Howard- Flanders et al., 1968). This process involves an exchange of genetic material between sister chromosomes so that the missing template is restored and the DNA regains its continuity. Cells that are defective in genetic recombination are more sensitive to radiomimetic agents than are the wild type since the former lack the capabilities for recombina- tion repair (Howard-Flanders et al., 1966, 1968). Photoreactivation is the least likely of these methods to introduce errors in the DNA and thus cause mutations (Witkin, 1969), but since this repair is specific for pyrimidine dimers it functions mainly in the repair of damage induced by UV. From results obtained with excision repair defective strains of E, coli, recombination repair appears to be more prone to error than does 10 excision repair and is thus most likely to result in mutations (Witkin, 1969). Excision repair Three genetic loci, designated as uvrA, uvrB and uvrC appear to function in the recognition and the excision of damaged DNA resulting from UV induced thymine dimers or interstrand crosslinks caused by hi- functional alkylating agents (Boyce and Howard-Flanders, 1965; Howard- Flanders et al., 1966). These mutants are not sensitive to methyl- methanesulfonate induced damage which causes chain breaks independent of enzyme excision, but are sensitive to mitomycin C or UV (Strauss et al., 1968). After exposure to mitomycin C or UV, DNA breakdown occurred to a larger extent in the wild type BEEN strain (Boyce and Howard—Flanders, 1964). Mutation at these loci also prevented the host cell reactivation (Hcr) of ultraviolet irradiated phage (Howard-Flanders et al., 1966). Double 2!£_mutants were not more than 20% more sensitive to UV than were single mutants. A fourth locus, designated as 3352 was mapped near EEEE and differed from the other three 2!; loci phenotypically as EX£E_mutants degrade their DNA "recklessly" after exposure to UV (Ogawa, 1968). A mutation at any of these four loci prevented excision of pyrimidine dimers; Taketo et al. (1972) successfully replaced the defective function of these mutants by pretreating irradiated replicative form DNA with T4 endonuclease 5. This procedure increased survival as judged by assays in spheroplasts of 225 mutants. This endonuclease was purified by Yosuda (1970) and shown to induce single-strand breaks in UV irradiated DNA. The radiation sensitive mutant polA is defective in the production 11 of DNA polymerase l (DeLucia and Cairns, 1969); the latter appeared to function in repair synthesis of DNA by inserting complementary bases into the single strand gaps of damaged DNA (Kato and Kondo, 1970). The mutant was capable of genetic recombination and also recombination re- pair (Gross and Gross, 1969). A series of radiation sensitive mutants designated Egg were isolated by Kato and Kondo (1970) which lack DNA polymerase 1 activity. Both ngE_a d £g§_mutations map near 223E; they promote extensive DNA degradation after UV irradiation suggesting that they may be identical. Coupling of these mutations with g!£_mut- ations did not increase sensitivity to UV irradiation but did result in negligible UV induced DNA breakdown. Therefore, it appears that these genes function after and in parallel with the excision step. There is no direct evidence to indicate that the polymerase 1 enzyme is involved in the process of excision 223:23, since pglé_mutants can excise pyri- midine dimers (Boyle et al., 1970). The final step of excision repair involves the activity of a poly- nucleotide ligase which would rejoin the last 3' to 5'-phosphodiester bond sealing the open DNA molecule. Temperature-sensitive radiation- sensitive mutants which appear to be defective in ligase activity have been isolated. Gellert and Bullock (1970) reported that temperature- sensitive mutants which were radiation-sensitive formed filaments at the nonpermissive temperature. Pauling and Hamm (1968) found that tempera- ture sensitive ligase mutants degraded their DNA extensively after irradiation. 12 Recombination repair Mutants deficient in genetic recombination capabilities were found to be sensitive to radiomimetic agents (Clark et al., 1966). The mutant £££E_is extremely sensitive to UV and totally recombination- deficient (Howard-Flanders et al., 1968; Willets and Clark, 1969). This mutation may alter a regulatory function since an uncoupling of the inhibition of cell division occurs when DNA synthesis is inhibited and thus Eggé_mutants do not form UV-induced filaments (Inouye, 1971). Mutants were able to excise pyrimidine dimers but may be unable to tolerate pyrimidine dimers with opposing gaps which result after rep- lication (Howard-Flanders et al., 1968). Inouye (1971) proposed that septum formation is controlled negatively by the Eggé_gene; thus mutants might continue to divide without repair or replication of DNA therefore resulting in a loss of viability as cells accumulate which lack the full complement of genetic material. Willets and Clark (1969) found that mutants harboring E££E_or Eggg_were limited in their recombination capabilities and were less sensitive to irradiation than were £2251mutants. These mutants are "cautious" in degrading their DNA after UV irradiation; double mutants of Eggé_with the £232 or Eggg_mutations degrade their DNA more slowly after irradiation than do the wild type or Eggé_mutants. These double mutants are still similar to the Eggé_mutant with respect to radiation sensitivity, absence of spontaneous induction of Atphage, and recombin— ation ability. Therefore the reckless degradation of DNA is not respon- sible for the lethality present in recA mutants. 13 _Re_cE_ and 3—939. map close together on the E. 291.; chromosome in the following order; thyAerecC-recB—argA (Willets and Mount, 1969). The 5222 and 523$ loci comprise two groups of mutations which genetically complement each other but not alleles within the same group. The defect thus appears to be in two different recombination genes. These genes appear to code for subunits of an ATP-dependent deoxyribonuclease (DNase) which is absent in both EEEE.andflggg§_mutants (Oishi, 1969; Barbour and Clark, 1970) . Suppressors designated as _s_lg_c_A_ of the £e_c_E_ and LEE phenotype have been shown to lack the ATP-dependent DNase but to possess a high activity of a different ATP-independent DNase (Barbour et al., 1970). Mutations at the EEEé locus do not appear to influence levels of the ATP-dependent DNase (Barbour and Clark, 1970). Other mutations designated EREE suppressed UV sensitivity, mito- mycin C sensitivity and recombination deficiency in £222.3nd.£229.m“tants with a concurrent loss of exonuclease 1 activity (Kushner et al., 1971). The Eggé_mutation suppressed only the two phenotypes, UV sensitivity and mitomycin C sensitivity with simultaneous loss of exonuclease 1 activity (Kushner et al., 1972). Thus it appears that when the ATP-dependent DNase is missing, the 3'0H single stranded ends are needed for resis- tance to mitomycin C and UV but not necessarily for recombination. Mutations at the.§§£.and.$E§ locus are similar in effect and in map location and may be identical (Witkin, 1969). These mutants have an increased sensitivity to radiomimetic agents commensurate with their reduced capabilities to undergo genetic recombination and also degrade their DNA extensively after exposure to radiation. Witkin (1969) found UV mutability was reduced or absent in lex, exr, recA, recB, and recC mutants and hypothesized that UV mutability may require recombination repair. 14 Mutagenesis Orgel (1965) referred to mutations as being heritable altera- tions of nucleic acid sequences in chromosomal DNA or RNA. Freese (1959) characterized and defined the following types of mutations. Transversions are base pair substitutions in which the purine—pyrimi- dine orientation is not maintained but reversed such as the replacement of guanine-cytosine (CO) by CC or thymine-adenine (TA). Transitions are base pair substitutions where a GC base pair is replaced by an AT pair or the reverse, so that the purine-pyrimidine orientation is maintained. Segments of DNA which are reversed and integrated anti— parallel to their normal configuration into the chromosome are inver- sions (Orgel, 1965). A fourth class of mutations were designated as frameshift mutations since these errors consisted of the insertion or deletion of base pairs so that the reading frame of the triplet genetic code was altered and nonsense resulted in the genetic material (Krieg, 1963). Krieg (1963) reported that base analogues which caused transitions were able to revert some transition mutations but not frameshift mutations. Accordingly, frameshift mutations were not reverted by base analogue- induced mutations. Base substitutions caused "missense" mutations where an amino acid substitution resulted in a nonfunctional protein and "nonsense" mutations which resulted in a triplet codon that did not code for an amino acid and caused the termination of protein synthesis. Gorini and Kataja (1965) found that transition mutations were suppressed by streptomycin and related antibiotics in drug sensitive strains as these drugs caused misreading of the genetic code during translation. 15 Suppression of "missense" and "nonsense" mutations has also resulted from additional mutations involving genetically altered tRNA's (Ames and Whitfield, 1966). 2-Amingpurine (2-AP) Z-AP is a base analogue of adenine which can be incorporated into DNA and which can pair correctly with thymine or incorrectly with cyto- sine resulting in an alteration of the DNA nucleotide sequence (Krieg, 1963). Rogan and Bessman (1970) asserted that the mutagenic effect of 2-AP was mainly due to replication errors since they found that it substituted only for adenine in DNA of treated organisms. However, as GMP kinase appeared to be involved in the incorporation of 2-AP the possibility of incorporation errors was not eliminated. Incorporation errors result in CC to AT transitions while replication errors cause AT to CC transitions. Ultraviolet $1555 EEK) Irradiation of DNA with UV resulted in the presence of pyrimidine dimers in the order TT>CT>CC (Setlow and Carrier, 1966). Other photo- products such as single strand breaks and crosslinks were present but not in sufficient number to cause detectable biological effects. Thy- mine dimers, therefore, appeared to be mainly responsible for the muta- genic effect of UV and the inhibition of DNA synthesis which occurred in irradiated cells. Drake (1963) found that approximately half of the UV- induced mutations in T4 phage were GC to AT base transitions. Bockrath and Cheung (1973) suggested that the presence of pyrimidine dimers might direct an error during post-replication repair of gaps in DNA (involving 16 the recA function and resulting in a GC to AT base transition). Witkin (1969) had also hypothesized that UV mutability was dependent on recom— bination repair functions. NametEyl-N'-nitro-N—nitrosggggnidine (NTG) NTG is a powerful mutagen which was shown to mutagenize the replication point region of the chromosome with higher efficiency than other parts of the chromosome (Cerda—Olmedo et al., 1968). NTG is an alkylating agent which was found to methlate guanine, adenine, and cy- tosine in that order of susceptability (Singer and Fraenkel-Conrat, 1969). Base transitions might result from anomalous base pairing due to tautomeric change of methylated nucleotides or base pair deletions might occur following depurination (Singer and Fraenkel-Conrat, 1969). In fact, one class of ICR induced mutations, isolated by Oeschger and Hartman (1970), was found to be revertable with NTG and apparently consisted of base pair additions reverted by the subtraction of a base pair. Yourno and Heath (1969) provided direct evidence for the sub- traction of a GC base pair in an NTG-induced revertant of histidinol dehydrogenase by determining amino acid replacements in enzyme of revertants. ICR derivatives A series of ICR compounds (acridine like substances) were synthe- sized at the Institute for Cancer Research (ICR) by Dr. Hugh J. Greech and co-workers. ICR191 has an alkylating side chain but did not appear to cause base substitutions in mutants isolated by Ames and Whitfield (1966). ICR364-OH lacks an alkylating sidechain and thus is only capable 17 of frameshift mutations; the latter still exhibited potent mutagenic activities (Ames and Whitfield, 1966). Oeschger and Hartman (1970) found that histidine mutants of Salmonella, induced with ICR191 or ICR364-OH, were mainly frameshift mutations. Yourno and Heath (1969) examined amino acid replacements in histidinol dehydrogenase in rever- tants of an ICR191A induced mutant and found that ICR was capable of causing single base pair additions or deletions. Base analogues such as 2-AP did not elicit reversion of ICR- induced mutants and streptomycin did not cause phenotypic suppression as observed with "missense" mutations (Gorini and Kataja, 1965). ICR— induced mutants were reverted to prototrophy by exposure to ICR deri— vatives. Revertants did not arise by external nonsense suppressors but by intragenic reversion (Oeschger and Hartman, 1970). Since the alkylating ICR derivatives were the most potent mutagens of this series, these compounds may cause mutation during some type of recombination repair of DNA. Ames and Whitfield (1966) theorized that the cause of these mutations may be due to nonreciprocal recombination during DNA repair in regions containing repeating nucleotide sequences. In addition, Riddle and Roth (1972) isolated altered tRNA species in frameshift suppressor strains. They hypothesized that these ICR-induced suppressors might produce a tRNA with a quadruplet anticodon which can read sequences of four bases instead of the usual triplet code. 18 Compounds which crosslink nucleic acids Strauss (1968) reported that bifunctional alkylating agents such as sulfur or nitrogen mustards, nitrous acid, and mitomycin C formed interstrand crosslinks in DNA which was evidenced by the isolation of denaturation-resistant DNA after treatment of organisms with these agents. Cole (1970) has studied the repair of interstrand crosslinks inlE,‘22E1.using psoralen-plus-light treatment to induce crosslinks. He found that both 215 and £225 functions were required for survival after production of approximately 70 crosslinks per genome. Double mutants (EXIT, £325) were killed by treatment causing not more than 1 crosslink per genome. Cole also demonstrated that covalent joining between sister chromosomes occurred during repair of psoralen—plus-light induced crosslinks and postulated that the 235 loci functioned in a partial excision of crosslinks which was followed by strand exchanges between homologous chromosomes under control of the £225 gene. If recombination repair is subject to error, mutation might arise during this process. Alternatively, the presence of crosslinks might promote errors during the recombination process. Kada et al. 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Bacteriol. 100:460-468. Zak, M., and J. Drobnik. 1972. Effect of cis-dichlorodiamineplatinum (II) on the post-irradiation lethality in mice after irradiation with x-rays. Strahlentherapie 142:112-115. Zak, M., J. Drobnik, and Z. Rezny. 1972. The effect of cis-dichloro- diamineplatinum (II) on cytological and morphological changes in bone marrow of mice. Neoplasma lg;305-310. ARTICLE 1 EFFECT OF CIS-PLATINUM(II)DIAMMINODICHLORIDE ON WILD TYPE AND DEOXYRIBONUCLEIC ACID REPAIR-DEFICIENT MUTANTS OF ESCHERICHIA COLI By Doris J. Beck and Robert R. Brubaker Appears In: Journal 2_f_ Bacteriology Volume 116, Number 3, 1973 uRNAL or Bacmmomov. Dec. 197:5, p. 1241-123; ,‘opyright © 1973 American Society for Microbiology Printed in (1..) Effect of cis-P1atinum(II)Diamminodichloride on Wild Type and Deoxyribonucleic Acid Repair-Deficient Mutants of Escherichia coli1 ? DORIS J. BECK AND ROBERT R. BRUBAKER ‘ "omnmnn! nf Mirmhinlnw and Public Health, Michigan State University. East Lansing, Michigan, 48823 JOURNAL or BACTERIOLOGY. Dec. 1973. p. 1247-1252 Copyright © 1973 American Society for Microbiology Vol. 116, No.3 Printed in USA. Effect of cis-Platinum(II)Diamminodichloride on Wild Type and Deoxyribonucleic Acid Repair-Deficient Mutants of Escherichia coli1 DORIS J. BECK AND ROBERT R. BRUBAKER Department of Microbiology and Public Health, Michigan State University. East Lansing. Michigan, 48823 Received for publication 6 August 1973 The anti-tumor drug cis-platinum(II)diamminodichloride (PDD) induced extensive filamentation in wild-type Escherichia coli and in mutants lacking certain deoxyribonucleic acid (DNA) repair functions (uvrA, recB, recC, and polA); viability of repair-deficient mutants treated with PDD was significantly less than that of wild-type cells. PDD was highly toxic to lexl, lexI uvrA6 (where its effect was cummulative), and recAI3 mutants, all of which were killed without formation of filaments. fH-thymine incorporated into DNA of cells subsequently treated with PDD became trichloroacetic acid-soluble at rates similar to those observed after exposure to comparable doses of ultraviolet light (UV) or mitomycin C. PDD, like UV, induced extensive degradation of DNA in recA organisms. After a 30-min lag, PDD inhibited significantly the synthesis of DNA but not of ribonucleic acid or protein in E. coli. However, the relative differences between rates of DNA synthesis observed in PDD-treated and control cells decreased substantially when the duration of pulses (’H-thy- mine) was prolonged from 2 to 5 min. These observations suggest that PDD-induced damage to DNA is reversible, possibly by defined mechanisms of excision and recombination repair. cis-Platinumfll)diamminodichloride (PDD) causes significant regression of a variety of tumors in experimental animals (10, 13, 19, 22, 25, 26) and selectively blocks synthesis of de- oxyribonucleic acid (DNA) in eukaryotic cells (6. 10, 11). PDD forms interstrand cross-links in nucleic acids (7, 16), but the frequency of cross-linkage is insufficient to account for le- thality in bacteriophage (24). Similar to the effects produced by other DNA inhibitors, PDD induces temperate phage in lysogenic bacteria (15), causes filamentation of gram-negative orga- nisms (18, 20, 21), and is exceptionally lethal to certain Exr’, Her‘, and Fil' mutants of Esche- richia coli (5). In this paper, we report studies on PDD- induced filamentation and loss of colony-form- ing ability of a variety of E. coli strains which are blocked in various pathways of DNA repair. Also measured were the inhibition of DNA synthesis and the induction of DNA breakdown in the presence of PDD. The results are in accord with the hypothesis that PDD-induced ‘Article no. 6329 from the Michigan Agricultural Experi- ment Station. lethality in bacteria is caused by the irreversible formation of interstrand cross-links in DNA. MATERIALS AND METHODS Media. Minimal C medium (17), supplemented with glucose (0.01 M) and thymine (20 ug/ml). was used in most experiments. This medium contained unlabeled uracil (20 ug/ml) and cytosine (20 pig/ml) or L-isoleucine (33 ug/ml) and L-val'me (29 lug/ml) when prepared for determination of rates of ribonu- cleic acid (RNA) and protein sythesis. respectively. Complex medium was tryptone-E broth (14). Difco nutrient agar was routinely used as a plating medium; diluent was 0.033 M potassium phosphate buffer, pH 7.0. Bacteria. E. coli strain W 3350 thy was generally employed in experiments concerned with viability of filaments. macromolecular synthesis. and degrada- tion of DNA. The origin of DNA repair mutants has been described (1); their properties are shown in Table l. Macromolecular synthesis. Rates of DNA synthe- sis were measured by pulse labeling. Samples of 1.0 ml of logarithmically growing cells were added to tubes (18 by 150 mm) containing 0.1 ml (10 uCi) of carrier-free ‘H-thymine (63 mCi/mmol). After aera- tion for 2 or 5 min in a model G76 gyratorv water 1347 1248 BECK AND BRUBAKER J. BAcrr-zmor. TABLE 1. Characterization of repair—deficient mutants of Escherichia coli strain K-12 Strain Relevant genotype Remarks Source and reference W3350 rec" L. Snyder ABll57 rect P. Howard Flanders, l AB1886 uvrA6 Mutant from A81 157 P. Howard Flanders, 1 AB2463 recA 13 Mutant from ABll57 P. Howard Flanders, l ABZ494 lexl Mating of AB2474 x AB2383 P. Howard Flanders, l NH4554 lexl Selected from AB2494 P. Howard Flanders, 1 AB2474 uvr/16 lex Mutant from AB1886 P. Howard Flanders. 1 JC5029 rec” thy" prototroph of JC5401 N. S. Willetts, 1 JC5412 recBZI sbc8 P1 transduction to JC5401 N. S. Willetts, 1 JC5426 recC22 P1 transduction to JC5401 N. S. Willetts, 1 JC4583 rec“ S. Barbour, 2, 3 JC4584 recB2I recC22 P1 transduction S. Barbour, 3 JC67 22 recB21 Pl transduction of HF4733 A. J. Clark, 2 mr 2-41 rec+ P1 transduction M. Inouye, 12 an 2-41 recA M. Inouye, 12 W3110 rect P. Delucia, 1 P3470 pol/1 Mutant from W3110 P. Delucia, l bath-shaker (new Brunswick Scientific Co., Inc., New Brunswick, N.J.), incorporation was stopped by addi- tion of 2 ml of cold 5% trichloroacetic acid containing unlabeled thymine (40 ug/ml). After storage for 1 h at DC, the samples were centrifuged and the superna- tant fluids were decanted. The precipitates were suspended in 0.2 ml of 2% NaOH, precipitated with 3 ml of 5% trichloroacetic acid containing unlabeled thymine, and then collected on a OAS-um diameter membrane filter (Millipore Corp., Bedford, Mass). After being rinsed with 20 ml of cold 5% trichloroace- tic acid containing unlabeled thymine, the membranes were dried and immersed in 10 ml of toluene base containing 0.4% 2,5-diphenyloxazole and 0.005% 1.4- di-2-(5-phenyloxazolyl)-benzene; radioactivity was determined in a Packard Tri—Carb scintillation counter. Rates of RNA synthesis were measured similarly by addition of 1.0 ml of culture to tubes containing 0.1 ml (10 pCi) of 'H-uracil (55 mCi/mmol). After pulsing for 5 min, incorporation was stopped by addition of 1 ml of cold 10% trichloroacetic acid containing 40 ug each of unlabeled uracil and cytosine per ml. After storage for 1 h at 0 C, the precipitates were collected on membranes, washed with 20 ml of cold 5% trichloro- acetic acid containing uracil and cytosine, and dried, and radioactivity was determined as described previ- ously. An identical procedure was used to determine rates of protein synthesis by using 0.1 ml (10 uCi) of ’H-L-isoleucine (40 mCi/mmol). In this case the precipitates were washed with 20 ml of cold 10% trichloroacetic acid containing unlabeled L-isoleucine (66 rig/ml) and L-valine (58 rig/ml). Degradation of DNA. Cells of an overnight culture were inoculated into fresh medium containing ’H-thy- mine (2 ug/ml, 1.26 uCi/mmol) and allowed to grow for three to four generations. The cells were collected by centrifugation at 5C (27,000 x g for 10 min), washed twice in cold 0.033 M potassium phosphate (pH 7.0), and then suspended in the same buffer at a concentration of about 5 x 10'/ml. These suspensions were either irradiated with ultraviolet light (UV) or else directly innoculated, at a concentration of 5 x 10‘ cells per ml, into control medium or medium contain- ing PDD or mitomycin C. The concentration of unlabeled thymine in these media was increased to 40 ug/ml. During subsequent incubation at 37 C, duplicate samples of 1.0 ml were removed and precipitated with an equal volume of cold 10% trichloroacetic acid containing unlabeled thymine (40 ug/ml). After stor. age for 1 h at 0 C, each sample received 0.04 ml of a 0.5% solution of bovine serum albumin, in order to facilitate precipitation. After filtration and rinsing. the membranes were dried and radioactivity was determined as described previously. UV irradiation. About 5 ml of tryptone-E broth containing repair mutants or their prototrophs. or phosphate buffer containing cells of strain W3350 thy (approximately 5 x 10" per ml), were placed in a standard glass petri dish. During irradiation, the dish was gentlyishaken beneath a 30-W General Electric germicidal‘lamp. Light intensity, as calibrated by comparisoi of inactivation kinetics with established values (9) as 13 ergs per mm’ per 8. Miscellaneous. Increase of cell mass was deter- mined spettrophotometrically at 620 nm. Extent of filamentation was monitored by direct observation with a microscope. Reagents. Preparations of PDD were generously provided by Barnett Rosenberg. Mitomycin C and amino acids were purchased from Calbiochern (La Jolla, Calif), and radioisotopes were obtained from New England Nuclear Corp. (Boston, Mass) VOL. 116, 1973 RESULTS Temperature and viability. Viability of PDD-treated cells decreased rapidly as a func- tion of increased temperature of incubation (Fig. 1). For example, the times required to reduce viability of wild-type filaments by 37% in the presence of 35 pg of PDD per ml of medium were about 210 and 60 min at 25 and 40C, respectively. The corresponding value obtained during incubation at 37C was approximately 100 min. DNA repair mutants. A series of mutants blocked in various steps of excision or recombi- nation repair were tested for ability to form filaments and remain viable in the presence of PDD (Table 2). Isolates known to be especially sensitive to UV were rapidly killed by PDD and failed to undergo pronounced filamentation. This response was expressed in particular by recA13 and uvr/16' lexI mutants, whereas single uvrA6 or lead isolates were significantly more resistant to PDD. Also of intermediate sensitiv- ity were polA, recBZI, recC22, and recB21 recC22 isolates which were able to form exten- sive filaments in the presence of PDD. The UV suppressor sbc8 in the recB21 strain JC 5412 (23) was also able to provide protection against PDD. 2i 3 2 o . > \ O: 3 ‘21 m : |-— I Z q 8 -( 0 Ct Lu '0 — -: a E a Z «i I 0 . -I '0 l 1 l A 1 O l 2 3 4 HOURS FIG. 1. Survival (colony-forming ability) of fila- ments of Escherichia coli strain W3350 thy during mcubation in C medium supplemented with thymine (20 [lg/ml) at as c (0), 30 C (c). 35 C (o). and 40 C (C) In the presence of cis-platinamUDdiamminodi- Shllonde (35 pg/ml). The initial optical density was cia-PLATINUM(IDDIAMMINODICHLORIDE 1249 TABLE 2. Comparison of treatments with UV light and cits-platinum (II) diamminodichloride (PPD) on survival and filamentation of DNA mutants of Escherichia coli K-I2‘ Ratio Time to reduce Of For- . . . _ cell . Viability to e ' matron Strain '33:) of UV PDD Ratio ‘ Fila- . to (3) (mm) (8) con- ments‘ trol) A31 157 rect 144 > 90 0.80 ABI886 uvrA6 13 20 0.011 0.81 + AB2463 recA13 2 5 0.007 0.58 0 AB2494 lexI 11 12 0.015 0.67 d: AB4554 lexl 14 12 0.019 0.53 a: AB2474 uvrAB lexl 3 5 0.01 0.55 0 J C5029 rect 155 > 90 0.80 + JC5412 recB2I sbc8 66 > 90 0.86 + JC6722 recB2I 30 20 0.025 0.86 + JC5426 recC22 21 45 0.008 0.89 + J C4583 rec+ 150 > 90 0.91 + JC4584 recB2I recC22 25 10 0.042 1.0 + mr 2-41 rec‘ 90 > 90 1.0 4» mr 2-41 recA l 3 0.006 0.52 0 W31 10 pol A t 204 > 90 l .0 + P3470 polA 24 30 0.013 0.94 + ° Cells growing logarithmically received, at an optical density of 0.1. either saline or PDD in saline to yield a final concentration of 35 pg/ml in tryptone-E medium containing added glucose (0.01 M) and thymine (20 pg/ml). Viability was monitored by plating. and the ratio of optical densities was determined after 3 h of incubation at 37 C. ‘ PPD-induced filamentation: (+), filaments 5 to 10 times longer than control cells: (2;). cells about twice the length of control cells; and (0). cells equal in size or shorter than con- trol cells. Degradation of DNA. Washed wild-type cells which had previously been cultivated with 'H-thymine were inoculated into fresh medium containing PDD; loss of trichloroacetic acid- insoluble radioactivity was monitored during further incubation. About 25% of the total radioactivity became soluble after incubation for 3 h in the presence of 50 pg of PDD per ml of medium (Fig. 2). Release of radioactivity was concentration dependent, as judged by a corre- sponding loss of about 15% after similar incuba- tion with 25 pg PDD per ml. Wild-type cells were treated with approximately equivalent lethal doses of UV, mitomycin C, or PDD; the rate of PDD-induced release of radioactivity was intermediate between that promoted by UV and mitomycin C at the dosages tested (Fig. 3). In view of the known ability of recA mutants to degrade their DNA extensively after irradia- tion with UV, a test of DNA stability was made in the presence of PDD. Addition of 50 pg of PDD per ml failed to induce significant break- down of deoxyribonucleic acid in recA+ cells as 1250 I Y Y t 0 O l: o \ o O ‘3’ 90. mo . _. \o 3 so. a 4 o 2 7o . - g 60. . E P 50» 2 1" JV § not 1' U a O 1 l l o I 2 3 HOURS FIG. 2. Loss of trichloroacetic acid-insoluble radio- activity from cells of Escherichia coli strain W3350 thy, previously grown with 'H-thymine, during incu- bation in C medium containing added unlabeled thymine (40 pg/ml) in the presence of 50 pg (0), 25 pg (0), and no (0) cis-platinum(Indiamminodichloride per ml, respectively. O o IOO 048 o O. ; \B‘O\o . .>. 90 . \0\ . '5 t- 3 so _ o W?“ IE 5 \ .\ g g 70 .. o .x. Ml & ‘9 z 2 6° _ \Nl < 5 5° r :L a: ,1; 4* lot- "i O 1 l 1 l O l 2 3 HOURS FIG. 3. Loss of trichloroacetic acid-insoluble radio— activity from cells of Escherichia coli strain W3350 thy, previously grown with 'H-thymine, during incu- bation in C medium containing added unlabeled thymine (40 pg/ml) in the absence of treatment (0), after irradiation with 4,000 ergs of ultraviolet light per mm’ (0), and upon addition of 50 pg of cis- platinum(IDdiamminodichloride (O) or 1 pg of mi- tomycin C (0) per ml of medium. compared to that in a control culture lacking PDD. Slight breakdown occurred in a control culture of recA cells which was similarly en- hanced by addition of 35 pg of PDD per ml or exposure to 1,000 ergs per mm' of UV light (Fig. 4). Synthesis of DNA. The rate of DNA synthe- sis, as judged by pulse labeling with ’H-thy- mine, became significantly reduced in wild-type cells after 30 min of exposure to PDD. This compound failed to significantly influence the BECK AND BRUBAKER J. BAc'raamL. rate of protein synthesis, although that of RNA may have undergone slight reduction (Fig. 5). The extent of PDD-induced inhibition of DNA synthesis was inversely proportional to the du- ration of pulse with ’H-thymine. For example, an increase of pulse from 2 to 5 min in control cultures yielded an expected 2.5-fold increase in incorporation of isotope (Fig. 6). In contrast, a similar increase in cultures containing PDD resulted in a 35-fold increase in incorporation of ’H-thymine. The observation would be ex- pected if PDD-induced inhibition of DNA syn thesis was reversed with time or if PDD signifi- cantly increased the size of the deoxyribonu- cleotide pool. DISCUSSION Damaged nucleotides are not excised by uvr mutants which must, therefore, rely on proc- esses of recombination to repair irradiated DNA (8, 28). Excision can occur in those repair mutants which are blocked in the process of recombination (8, 23, 28). Cells of both of these 8 O 0 I00 ~ am > P. I:2 _>_ so. .— O < 9 h 9 a: 60. o E i. E < E 40.. U m .— h- E e g 20.. -4 LIJ O. 4 0 L p 1 O l 2 3 HOURS FIG. 4. Loss of trichloroacetic acid-insoluble radio- activity from cells of Escherichia coli strain mr 2—41 previously grown with 'H-thymine, during incubation in tryptone-E medium containing added unlabeled thymine (40 pg/ml). cis-PlatinumUDdiamminodi- chloride (PDD) in saline was added to a culture of recAt cells (0) in this medium. and an equal volume of saline was added to a parallel control culture (0). A culture of recA cells was similarly divided into three subcultures, one of which was treated with 35 pg of PDD per ml (0), another exposed to 1,000 ergs of ultraviolet light per mm’ (0), and the third an untreated control culture (0). VOL. 116, 1973 cis-PLATINUMUDDIAMMINODICHLORIDE OPTICAL DENSITY RADIOACTIVITY (CPM) 19111111l 1 d q l l l l E. ., 0 1 20 40 60 80 IOO I20 ON ’2‘ Q 9 >. t: 2'01 111111" '5 grfiITI_ < :D : Q - c- o - o. s . °~ .. o .. 11 l 1 I 1 l 0 20406080I00|20 1251 MINUTES MINUTES 1'16. 5. Increase in cell mass (A) and rates of deoxyribonucleic acid (B), ribonucleic acid (C), and protein (D) synthesis in control cells of Escherichia coli strain W3350 thy (O) and cells receiving 15 ug (0) and 35 pg (0), respectively, of cis-platinum(IDdiamminodichloride per ml of C medium. Procedures used for pulse labeling are described in the test. I I IIW 1> 1111111: I Him (I) l l lllll] Q 1111111] RADIOACTIVITY (0PM) T 01230] nouns HOURS 4000 E 2000 *- l._l LllLlll.. _L_ 1 2 1 l l l 2 3 u»— 0 HOURS FIG. 6. Rates of deoxyribonucleic acid synthesis in control cells of Escherichia coli strain W3350 thy (O) and cells treated at the initiation of the experiment with 35 ug of cis-platinum(IDdiamminodichloride per ml of C medium (O); A, pulsed for 2 min with ’H-thymine and B, pulsed for 5 min with ‘H-thymine. The difference between the values obtained during 2- and 5-min pulses is shown in C. mutant classes and an isolate lacking DNA polymerase I (polA) were killed by PDD at rates exceeding those of wild-type organisms. PDD was acutely toxic to those recombination mu- tants (uvr/1 and lex) which are known to undergo extensive autodegradation of DNA after treatment with UV. Similar degradation occurred in recA mutants after exposure to PDD. A double mutant blocked in both the excision and recombination pathways of DNA repair (uvrA6 lexl) was more sensitive to PDD than were single-excision (uvrA6) or recombina- tion (lexI) mutants. Both of these pathways may be essential for the repair of interstrand cross-links (4). PDD promoted the release of trichloroacetic acid-soluble fragments of DNA from growing cells. The nature of these fragments was not determined, but they may be analogous to the repair products which arise after treatment of bacteria with UV or certain radiomimetic agents (8, 28). In addition, PDD reversibly inhibited the systhesis of new DNA (Fig. 6), possibly by promoting the degradation of old DNA and thus increasing the size of the non- radioactive deoxyribonucleotide pool. These findings suggest that PDD can directly interact with bacterial DNA and that the resulting damage may be repaired by defined mech- anisms. ACKNOWLEDGMENTS We thank L. Snyder, P. Howard-Flanders. N. S. Willetts, S. Barbour. M. Inouye, A. J. Clark, and P. Delucia for supplying strains. D. J. Beck is a Public Health Service predoctoral trainee supported by grant GM 01911 from the National Institute of General Sciences. LITERATURE CITED 1. Bachmann, B. J. 1972. Pedigrees of some mutant strains of Escherichia coli. Bacteriol. Rev. 36:525—557. 1252 2. Barbour, S. D., and A. J. Clark. 1970. Biochemical and ll. 12. genetic studies of recombination proficiency in Esche- richia coli. 1. Enzymatic activity associated with recB’ and recC‘ genes. Proc. Nat. Acad. Sci. U.S.A. 65:955—961. . Capaldo-Kimball. F.. and S. D. Barbour. 1971. Involve- ment of recombination genes in growth and viability of Escherichia coli K-12. J. Bacteriol. 1062204—‘212. . Cole, R. S. 1973. Repair of DNA containing interstrand crosslinks in Escherichia coli: sequential excision and recombination. Proc. Nat. Acad. Sci. USA. 70:1064-1068. . Drobnik. J., M. Urbankova, and A. Krekulova. 1973. The effect of cis-dichlorodiammineplatinumtll) on Esche- richia coli B. The role of fil, exr, and hcr markers. Mutat. Res. 17:13—21). . Harder. H. C., and B. Rosenberg. 1970. Inhibitory effects of antitumor platinum compounds on DNA, RNA. and protein synthesis in mammalian cells in vitro. Int. J. Cancer 6:207-216. . Horacek, P.. and J. Drobnik. 1971. Interaction of cis- dichlorodiammineplatinum (II) with DNA. Biochem. Biophys. Acta 254:341-347. . Howard-Flanders, P. 1968. DNA repair. Annu. Rev. Biochem. 37:175~200. . Howard-Flanders. P., E. Simson. and L. Theriot. 1964. A locus that controls filament formation and sensitivity to radiation in Escherichia coli K12. Genetics 49:237-246. . Howle. J. A., and G. R. Gale. 1970. Cis-dichlorodiam- mineplatinumfll): persistant and selective inhibition of deoxyribonucleic acid synthesis in vivo. Biochem. Pharmacol. 19:2757—2762. Howle, J. A., H. S. Thompson, A. E. Stone. and G. R. Gale. 1971. Cis-dichlorodiammineplatinum[III; inhibi- tion of nucleic acid synthesis in lymphocytes stimu- lated with phytohemagglutinin. Proc. Soc. Exp. Biol. Med. 137:820—825. Inouye, M. 1971. Pleiotropic effect of the recA gene of Escherichia coli: uncoupling of cell division from de- oxyribonucleic acid replication. J. Bacteriol. 106x339; 542. . Kociba. R. J., S. D. Sleight. and B. Rosenberg. 1970. Inhibition of Dunning ascitic leukemia and Walker 256 carcinosarcoma with cis-diamminedichloroplatinum (NSC-119875). Cancer Chemothe. Rep. Part 1, 54:325-328. . Kohiyama, M. 1968. DNA synthesis in temperature sensitive mutants of Escherichia coli. Cold Spring Harbor Symp. Quant. Biol. 33:317—324. 15. 16. 18. 19. 21. 22. 23. 24. 25. BECK AND BRUBAKER J. BACTERIOL. Reslova. S. 1971/72. The induction of lysogenic strains of Escherichia coli by cis-dichlorodiammineplatinumtII). Chem. Biol. Interact. 4:66-70. Roberts. J. J., and J. M. Pascoe. 1972. Cross-linking of complementary strands of DNA in mammalian cells by antitumor platinum compounds. Nature (London) 235:282—284. . Roberts, R. B.. P. H. Abelson. D. B. Cowie. E. T. Bolton. and R. J. Britten. 1955. Studies of biosynthesis in Escherichia coli. Carnegie Institution of Washington Publication 607, Washington. DC. Rosenberg, B., E. Renshaw, L. VanCamp, J. Hartwick. and J. Drobnik. 1967. Platinum-induced filamentous growth in Escherichia coli. J. Bacteriol. 93:716-721. Rosenberg, B., and L. VanCamp. 1970. The successful regression of large solid sarcoma 180 tumors by plati- num compounds. Cancer Res. 30:1799-1802. . Rosenberg, B., L. VanCamp, E. B. Grimley, and A. J. Thomson. 1967. The inhibition of growth or cell divi- sion in Escherichia coli by different ionic species of platinum (IV) complexes J. Biol. Chem. 242:1347-135‘2. Rosenberg, B., L. VanCamp. and T. Krigas. 1965. Inhibition of cell division in Escherichia coli by elec- trolysis products from a platinum electrode. Nature (London) 205:698—699. Rosenberg, B., L. VanCamp, J. E. Trosko, and V. H. Mansour. 1969. Platinum compounds: a new class of potent antitumor agents. Nature (Londoni 222:385—386. Schlaes. D. M., J. A. Anderson. and S. D. Barbour. 1972. Excision repair properties of isogenic rec‘ mutants of Escherichia coli K-12. J. Bacteriol. 111:723-730. Shooter. K. V., R. Howse, R. K. Merrifeld. and A. B. Robins. 1972. The interaction of platinum 11 com- pounds with bacteriophages T7 and R17. Chem. Biol. Interact. 5:289 307. Talley, R. W. 1970. Chemotherapy of a mouse reticulum cell sarcoma with platinum salts. Proc. Amer. Ass. Cancer Res. 11:78. . Welsch, C. W. 1971. Growth inhibition of rat mammary carcinoma induced by cis-platinumdiamminodichlo- ride-II. J. Nat. Cancer Inst. 47:1071-1078. . Willetts, N. S., and D. W. Mount. 1969. Genetic analy- sis of recombination-deficient mutants of Escherichia coli K-12 carrying rec mutations cotransducible with thyA. J. Bacteriol. 100:923~934. . Witkin. E. M. 1969. Ultraviolet-induced mutation and DNA repair. Annu. Rev. Genet. 3:525—552. ARTICLE 2 MUTAGENIC PROPERTIES OF CIS-PLATINUM(II)DIAMMINODICHLORIDE IN ESCHERICHIA COLI By Doris J. Beck and Robert R. Brubaker Submitted To: Mutation Research SUMMARY The anti-tumor agent cis-platinum(II)diamminodichloride (PDD) induced auxotrophic mutations in Escherichia coli strain K-12 and promoted reversion of certain PDD-, 2-aminopurine (2-AP)-, and N-methyl- N'-nitro-N-nitrosoguanidine (NTG)-induced mutants to prototrophy. PDD- induced mutants were not reverted by ICR derivatives suggesting that PDD does not cause frameshift mutations. Exposure to PDD, however, increased the frequency of prototrophic revertants of some auxotrophic mutants induced by Z-AP, NTG, ultraviolet light and ICR191. Accordingly, PDD can cause base transitions since it reverts base analogue-induced mutations; it may also revert ICRrinduced mutations by causing base subtractions. In contrast, the isomeric £5233 configuration was much less effective in inducing revertants of an NTG-induced auxotroph (N46‘tgy) which reverted to prototrophy at high frequency after treat- ment with PDD. Optimal conditions for obtaining mutants induced by PDD were determined by enumerating revertants of N46 ££y_after exposure to the compound. The highest yield of revertants was obtained when N46 ££y_ was grown for extended periods in nutrient broth containing 10 ug of PDD per m1. Cells suspended in buffer containing PDD for 1 hour yielded few revertants in comparison to actively growing cells exposed to simi- lar concentrations of the compound in nutrient broth for the same period of time. 35 INTRODUCTION .ginglatinum(II)diamminodichloride (PDD) forms interstrand crosslinks in nucleic acids (10, 17) and selectively inhibits synthesis of deoxyribonucleic acid (DNA) in eukaryotic cells (8, 12, 13). Similar to the effects produced by radiomimetic agents, PDD induces phage production in lysogenic bacteria (16), inhibits cell division in gram negative organisms (18, 20, 22) and is exceptionally lethal to DNA repair-deficient mutants of Escherichia coli (3, 6). In this paper, we report studies of PDD—induced mutation in _E. £9}; and the Optimum conditions for PDD-mutagenesis. Various auxotrophic mutations were induced by ultraviolet light (UV), Z-amino- purine (Z-AP), N-methyl-N'-nitro-N—nitrosoguanidine (NTG), ICR191, and PDD. The mutants were then tested for reversion to prototrophy resulting from exposure to each of the chemical mutagens. 36 MATERIALS AND METHODS Bacteria. E, Egli_K-12 strain W3110 (thyA36) was obtained from P. DeLucia. This strain was mutagenized to obtain sets of mutants induced by UV, ICR191, PDD, NTG, and 2-AP. The mutant, N46 551, was obtained after treatment of W3110 with NTG. .Efigié' Difco nutrient broth was routinely used as a complex liquid medium and the medium of Vogel (25) supplemented with 0.01M glucose and 20 ug of thymine per m1 (E medium), was used as a minimal medium. Standard diluent was 0.852 saline. In testing mutants for reversion, the overlayer consisted of E medium containing 0.025% yeast extract plus 1.25% Difco agar and the bottom layer was E medium solidified with 1.5% agar. Difco nutrient agar containing 50 ug streptomycin sulfate per ml in both overlayer and bottom agar was used to identify streptomycin resistant mutants. Unless stated otherwise, solid E medium fortified with 0.12 nutrient broth, was used after mutagenesis to distinguish prototrOphs (large colonies) from potential auxotrophs (small colonies). Solid E medium, fortified with pools of nutrients (9), was also used to identify auxotrophic requirements. TM buffer was described by Adelberg et a1. (1). Chemicals. Dr. Hugh J. Greech (Institute for Cancer Research, Philadelphia, Pa.) generously donated 2—chloro-6dmethoxy-9-[3—(2-chloro- ethyl)aminopropylamino]acridine dihydrochloride (ICR191) and 2-chloro- 6-methoxy-9-[2—(2-hydroxyethyl)aminoethylamino]-l-azaacridine dihydro- chloride (ICR364-OH). PDD and other platinum compounds were kindly 37 38 provided by Dr. Barnett Rosenberg. Penicillin G and 2-AP were obtained from Calbiochem (LaJolla, Calif). NTG was purchased from Aldrich Chem- ical Co., Inc. (Milwaukee, Wis.) and streptomycin sulfate from Sigma (St. Louis, Mo.). Mutagenesis. ICR191 or PDD was dissolved in 0.852 saline immed- iately prior to use and sterilized by filtration using a Nalgene filter unit (0.20 n pore diameter) purchased from Nalge Sybron Corp. (Rochester, N.Y.). The mutagen was added to logarithmically growing cultures, containing about 108 cells per ml, to yield a desired concen- tration ranging from 2.5 to 100 pg per ml. The cells were then incubated overnight at 37 C with aeration in a model G76 gyratory water bath shaker (New Brunswick Scientific Co., Inc. New Brunswick, N. J.). The mutagenized cells were washed with saline, resuspended in nutrient broth, diluted to a density of approximately 108 cells per ml, and incubated at 37 C with aeration for 24 to 48 h, at which time the population approximated 109 organisms per ml. The cells were then washed and suspended in 5 m1 of minimal medium in screwcap tubes (16 x 130 mm) at a density of 2 x 108 per ml and enriched for auxotrophic mutants by selection with penicillin. Cells were mutagenized for 1 h with NTG under Optimal conditions as described by Adelberg et al. (1). Organisms to be irradiated with UV were harvested in the logarithmic growth phase and suspended in saline to a density of~108 cells per m1. Samples of 5 m1 of this sus- pension were irradiated in a glass petri dish for 2 min with gentle shaking under a 30 w General Electric germicidal lamp. Light intensity 39 as calibrated by comparison of inactivation kinetics with established values (11) was 13 ergs per mm2 per sec. The organisms were then grown in nutrient broth prior to selection of auxotrOphs by cultivation in E medium containing penicillin. Cells to be mutagenized with 2-AP were grown in nutrient broth containing 0.25% or 0.5% 2-AP at 37 C without aeration until the pop- ulation approximated 109 organisms per m1, diluted tenfold, and this process was then repeated. Cultures were plated onto nutrient agar and replica plated (14) onto E agar to identify auxotrophs. Penicillin enrichment for auxotrophs. Penicillin G (1000 units per ml) was used to enrich for auxotrophs using two enrichment periods as described by Curtiss et al. (4). Reversion £2 prototrophy. Cells removed from nutrient agar were suspended in saline at a density of approximately 5 x 108 per ml. Sam- ples of 0.1 m1, added to 5 m1 of melted overlayer agar (45C), were layered onto solid E agar in petri dishes. After the overlayer had solidified, crystals of the mutagen to be tested were placed in the center of duplicate plates which were then incubated at 37 C for 3 to 4 days. thimal conditions for PDD mutagenesis. Reversion of N46 try to prototrophy was used to evaluate efficacy of mutagenesis with PDD under various conditions. PDD was added to suspensions of bacteria (108 cells per ml) in 0.852 saline, various buffers, or media. The latter were incubated for periods of l to 18 h whereas the organisms in saline and buffers were removed from PDD by centrifugation after 1 h incubation. The cells were then washed in saline, grown overnight in nutrient broth, 40 and plated on nutrient agar and solid E medium to determine the rever- sion frequency. RESULTS Independent clones of auxotrophic mutants were isolated follow- ing mutagenesis with NTG, UV, 2-AP, ICR191 and PDD. Most of the 41 PDD-induced mutations were obtained by growing g, 521$.W3110 overnight in concentrations of PDD varying from 10 to 25 pg per ml. The resulting mutants were tested for spontaneous reversion to prototrophy and for increased frequency of reversion induced by exposure to chemical mut- agens (Table 1). The PDD-induced mutants did not show increased reversion to prototrophy when exposed to ICR364-OH, a nonalkylating azaacridine which causes frameshift mutations by addition and deletion of nucleotide bases (15). In contrast, reversion of PDD-induced auxo- trophic mutants was greatly enhanced by Z-AP or NTG which cause base pair substitutions (2). PDD exhibited a slightly wider spectrum in its reversion of ICRrinduced mutants than did NTG indicating that both mutagens have similar potential to revert frameshift mutants. PDD was also able to induce reversion of NTG and 2-AP-induced mutants but dis— played a more narrow spectrum of activity than did NTG or 2—AP. Fig. 1 illustrates photographs of an NTG-induced auxotroph, N46.££Z) showing its pattern of reversion when exposed to the 4 mutagens. PDD was the most effective agent tested in reverting N46 532, Photographs of N46 £51 show few revertant colonies induced by trans-p1atinum(II)diamminodichloride 41 42 (Fig. ZB) in contrast to the high level of reversion obtained with the gig compound (Fig. 2A). This reversion data correlates with the effect- iveness of these compounds as antitumor agents (4, 24). Table 2 compares these and other platinum compounds which were tested for their effect- iveness in reversion of N46 ££y_to prototrOphy with results of their use in treatment of Sarcoma 180 tumors implanted in mice (4). Near-optimal conditions for mutagenesis with PDD were determined with resting and growing cells of mutant N46 try, The spontaneous frequency of reversion of this mutant was approximately 10-7 whereas values ranging from less than 10-5 to 10“6 were obtained following exposure of resting cells to PDD for l h in 0.852 saline or various buffered solutions (Tables 3, 4). A neutral or slightly acidic reaction appeared most favorable for mutagenesis. The greatest frequency of reversion (10-2) was detected in cul- tures following overnight incubation with 10 to 20 ug of PDD per ml of nutrient broth (Fig. 3). This value was significantly greater than that determined following exposure for 1 h to higher levels of the compound (Fig. 4). Similar results were obtained in experiments designed to determine the mutation frequency to streptomycin resistance (not illustrated). DISCUSSION The results of this study revealed that PDD was mutagenic, an observation which was expected since this compound had been shown to form crosslinks in nucleic acids (10, 17, 24). The efficacy of muta- genesis with PDD in various solutions and for long and short exposure periods was evaluated by determining the reversion frequency of N46.££X (an NTG induced auxotrOph) under specified sets of conditions. PDD was most effective in causing reversion of N46 try when logarithmic cultures were suspended in low concentrations (10 ug PDD per ml) of the drug and allowed to grow until entry into the stationary phase. The reversion frequency was also significantly raised when cells were exposed for 1 h to high concentrations of the drug in nutrient broth but results were variable. The highest reversion frequency for a 1 h exposure period was attained when cells were suspended in nutrient broth containing 100 ug PDD per ml; if the organisms were allowed to remain in this solution for l or 2 additional h the reversion frequency was not increased. These results may be due to a requirement for act- ive metabolism during the period of mutagenesis, possibly related to DNA replication or repair since DNA synthesis is selectively inhibited by PDD (3). The fact that metabolism is required for effective muta- genesis is also emphasized by the low reversion level of N46 Erz" treated with PDD for 1 h in various buffered solutions. The low levels 43 44 of increase in the reversion frequency which were obtained in these experiments might possibly be due to some retention of the compound within cells after the period of mutagenesis. These experiments did not define the mechanism whereby PDD induces mutations, however, many PDD—induced mutants were reverted by Z-AP and NTG which would be expected if base transitions had been induced. The reversion of 2-AP— and NTG-induced mutations by PDD strengthens this suggestion. None of the PDD-induced auxotrOphs reverted to prototrophy when exposed to ICR derivatives; in contrast some ICRsinduced mutants were reverted by PDD suggesting that PDD may rarely cause base subtractions compared to its ability to induce base substitution. These same mutants were generally also revertable by NTG, which has been shown to revert that class of ICR-induced mutants which result from base additions (15). Other platinum compounds were tested for their efficiency in inducing the reversion of N46 552, Dichloroethylenediamineplatinum(II) was similar to PDD in inducing reversion, while Eggggrplatinum(II)- diamminodichloride was relatively ineffective, an observation which appears to correlate with the anti-tumor activity characteristic of these compounds. These 2 effective anti—tumor compounds have been shown to crosslink nucleic acids (23, 24); thus the mutations which they induce might arise from errors resulting from.repair processes utilized in removal of such crosslinks (3). REFERENCES Adelberg, E. A., M. Mandel, and G. Chein Ching Chen, Optimal conditions for mutagenesis by N—methyl—N'-nitro—N-nitrosoguani- dine in Escherichia coli K12, Biochem. Biophys. Res. Commun. l§. (1965) 788-795. Ames, B. N., and H. J. Whitfield, Frameshift mutagenesis in Salmonella, Cold Springs Harbor Symp. Quant. Biol. _3_l (1966) 221-225. Beck, D. J., and R. R. Brubaker, Effect of gigrplatinum(II)- diamminodichloride on wild type and deoxyribonucleic acid repair deficient mutants of Escherichia coli, J. Bacteriol. (In Press). Cleare, M., and J. Hoeschele, Studies on the antitumor activity of group VIII transition metal complexes; I P1atinum(II) complexes, Bioinorganic Chem. '2 (1973) 187-210. Curtiss, R., L. J. Charamella, C. M. Berg, and P. E. Harris, Kinetic and genetic analyses of d-cycloserine inhibition and resistance in Escherichia coli, J. Bacteriol. 22_ (1965) 1238-1250. Drobnik, J., M. Urbankova, and A. Krekulova, The effect of cis-dichlorodiammineplatinum(II) on Escherichia coli B. The role of fil, exr, and hcr markers, Mutat. Res. .11 (1973) 13-20 a Fischer, R., and H. Peresie, Personal communication (1974). 45 10. 11. 12. 13. 14. 15. 16. 46 Harder, H. C., and B. Rosenberg, Inhibitory effects of antitumor platinum compounds on DNA, RNA, and protein synthesis in mammalian cells Ell-£3.99 Int. J. Cancer _6_ (1970) 207-216. Holliday, R., A new method for the identification of biochemical mutants of micro-organisms, Nature .118 (1965) 987. Horacek, P., and J. Drobnik, Interaction of gig-dichlorodiammine- platinum(II) with DNA, Biochem. BiOphys. Acta .224 (1971) 341-347. Howard-Flanders, P., E. Simson, and L. Theriot, A locus that controls filament formation and sensitivity to radiation in Escherichia coli K12, Genetics 42_ (1964) 237-246. Howle, J. A., and G. R. Gale, gig-dichlorodiammineplatinum(II)z persistant and selective inhibition of deoxyribonucleic acid syn- thesis in 3333, Biochem. Pharmacol. ‘12 (1970) 2757-2762. Howle, J. A., H. S. Thompson, A. E. Stone, and G. R. Gale, Gig-dichlorodiammineplatinum(II): inhibition of nucleic acid synthesis in lymphocytes stimulated with phytohemagglutinin, Proc. Soc. Exptl. Biol. Med. .131 (1971) 820-825. Lederberg, J., and E. M. Lederberg, Replica plating and indirect selection of bacterial mutants, J. Bacteriol. '63 (1952) 399-406. Oeschger, N. S., and P. E. Hartman, ICR-induced frameshift mutations in the histidine operon of Salmonella, J. Bacteriol. 191_ (1970) 490-504. Reslova, 8., The induction of lysogenic strains of Escherichia coli by cis-dichlorodiammineplatinum(II), Chem. Biol. Interactions _t_._ (1971/72) 66-70. 17. 18. 19. 20. 21. 22. 23. 24. 47 Roberts, J. J., and J. M. Pascoe, Cross-linking of complementary strands of DNA in mamalian cells by antitumor platinum compounds, Nature 322_ (1972) 282-284. ' Rosenberg, B., E. Renshaw, L. VanCamp, J. Hartwick, and J. Drobnik, Platinum-induced filamentous growth in Escherichia coli, J. Bacteriol. 22_ (1967) 716-721. Rosenberg, B., L. VanCamp, J. E. Trosko, and V. H. Mansour, Platinum compounds, a new class of potent antitumor agents, Nature .222 (1966) 385-386. Rosenberg, B., L. VanCamp, and T. Krigas, Inhibition of cell division in Escherichia coli by electrolysis products from a platinum electrode, Nature .295 (1965) 698-699. Rosenberg, B., and L. VanCamp, The successful regression of large solid Sarcoma 180 tumors by platinum compounds, Cancer Res. ‘29 (1970) 1799-1802. Rosenberg, B., L. VanCamp, E. B. Grimley, and A. J. Thomson, The inhibition of growth or cell division in Escherichia coli by different ionic species of platinum (IV) complexes, J. Biol. Chem. 242’ (1967) 1347-1352. Shooter, K. V., R. Howse, R. K. Merrifield, and A. B. Robins, The interaction of platinum 11 compounds with bacteriophages T7 and R17, Chem.-Biol. Interactions‘ 5 (1972) 289-307. Shooter, K. V., and R. K. Merrifield, Changes in the hydrodynamic properties of DNA induced by interaction with platinum (II) compounds, Biochim. Biophys. Acta 187 (1972) 16-27. 25. 48 Vogel, H. J., and D. M. Bonner, Acetylornithinase of Escherichia coli: partial purification and some properties, J. Biol. Chem. 218 (1956) 97-106. .mucmufia USU HO mwumh COHmH0>0H mDOQCNUCOQm 03H mumafiuww Ou UGwD mum»? mcmwmufia uSO Inufia mmumHa Houuoou m>fiufimom d wounam mum3 vmuwmu on on commune onu mo maoummuo .mHHoo cannouuoxam .nuwmusa may wcsoum waaaouw moHaoHoo unmuum>mu mo wows o no vmumwmcoo umou .mmmv e co m Mom 0 um um woumnsocw mmumao «so was mwumam oumofiamsv mo mumuaoo map ca .mmumHn Huumm aw umwm m vfiaom coco vmum%ma mma OH x m Saws powwow can uumuuxo awash nufiz voamuuuom .uoxmauo>o wows m woodman n ma 0 o c on NH n ma ma 0 o m mm m5 mm c mm m o . o o o o o o a NN MUH w on o o O 0H 3 N on m N n o .3 mam uuo> MUH lop MUH mmu mucousa unmoumm mo uoaabz consuaz mm<|~ was .692 .>D .moH .Qnm up voonvafi mucousa mo moguuomoun soamum>mm .H manna 50 Fig. 1. Photographs of petri plates showing colonies from prototrophic revertants of N46 ££y_induced by exposure to 2-AP (A), NTG (B), PDD (C), and ICR191 (D). Melted E agar overlayer, forti- fied with 0.025% yeast extract and seeded with 5 x 107 auxotrophic cells of N46 £32, was layered onto solid E agar in petri plates: Crystals of the mutagens were placed in the centers of triplicate plates and the plates incubated at 37 C or 42 C for 3 days. The best reversion data is displayed and was achieved here by incuba- tion at 42 C for 2-AP and NTG and 37 C for PDD and ICR. 51.4w -. ,- 52 Fig. 2. Photographs of petri plates showing colonies from prototrophic revertants of N46 ££y_induced by exposure to PDD (A) and its trans isomer (B). The method is described in the legend to Fig. l. Plates were incubated at 37 C for 3 days. 54 Table 2. Effectiveness of various platinum compounds in inducing reversion of N46 try and in treatment of Swiss white mice after Sarcoma 180 tumor implantation Compound Reversionb Sarcoma 180 inhibition T/cc '2is-Platinum(II)diamminodichloride '+++ l trans-P1atinum(II)diamminodichloride 4- 83 Oxalatodiammine platinum(II) ++ 9 Malonatodiammine p1atinum(II) ++ 7 Uracildiammine platinum bluea - 6-Methyluracildiammine platinum bluea + P1atinum(II)ethylenediaminedichloride ++ 27 8These are well defined compounds but their structure is unknown (7). bThe method is described in Table l. The symbols used indicate the following: no significant increase in reversion (-), a slight increase in reversion noted as a few revertant colonies encircling mutagen (+), an intermediate effect in reversion producing a ring of colonies around mutagen (++), an intense effect in reversion producing a wide (1cm) ring of colonies encircling mutagen (+++). cSwiss white mice were treated with 1 shot intraperitoneally on day 1 after tumor implantation. On day 10 T/C ratios were determined as the percent treated to the control tumor weight. Data from paper published by Cleare and Hoeschele (4). 55 Table 3. Reversion of N46 ££y_when exposed to 150 ug PDD for l h in E salts, phosphate buffer, saline, or nutrient broth Mutagenizing solution Revertants per 106 cells Minimal E salts 5 0.033 M Potassium phosphate buffer, pH 7.0 2 0.852 Saline 4 Nutrient broth 76 aDuplicate logarithmic cultures of N46 try were washed, suspended in mutagenizing solutions containing PDD, incubated l h at 37 C with aeration, washed and resuspended in nutrient broth. The cells were then incubated at 37 C with aeration until the pOpulations approx- imated 109 organisms per m1, diluted and plated on nutrient agar to determine numbers of viable cells and solid E agar to determine prototrophic revertants. 56 Table 4. Reversion of N46 ££z_when exposed to 100 ug PDD per ml TM buffer at pH's 5 to 98 TM buffer pH Revertants per 106 cells 5.0 14 6.0 88 7.0 59 8.0 20 9.0 43 8Method is described in legend of Table 3. l-l‘I-Hl'l‘. .':a .‘J' 57 Fig. 3. Reversion data for N46 try grown 18 h in nutrient broth containing various concentrations of PDD. Duplicate logarithmic cultures at a density of 108 cells per ml were grown in nutrient broth containing 0 to 200 ug PDD per ml at 37 C with aeration. After mutagenesis the cells were washed, diluted, suspended in nutrient broth, and incubated at 37 C with aeration until the population approx- imated 109 organisms per ml. The cultures were then plated on nutrient agar to determine viable cells and solid E agar to determine prototromfic revertants. Concentrations of 30 to 200 ug PDD per ml nutrient broth gave no increase in reversion level over untreated controls. 58 snag) 80' led slums/veg 601 50 Concentration PDD (ug/ml) 59 Fig. 4. Reversion data for N46 ££y_exposed to 0 to 300 pg PDD per ml of nutrient broth for l h. Duplicate logarithmic cultures of N46 ££y_at a density of 108 cells per ml were exposed to PDD for 1 h, washed with 0.85% saline, resuspended in nutrient broth, and incubated at 37 C with aeration until the population approximated 109 organisms per ml. The cultures were then diluted and plated on nutrient agar to determine the numbers of viable cells and solid E agar to determine prototrophic revertants. —**fl_—_ 6O £6 I I I l I 7.3 0 -- -I (I) g 334 O. U) E U §2 6‘3 8‘ “'0 . O ICC 200 300 Concentration PDD (pg/ml) APPENDIX COLICIN INDUCTION AND PLASMID ELIMHNATION CAUSED BY TREATMENT OF BACTERIA WITH CIS-PLATINUM(II)DIAMMINODICHLORIDE 62 Colicin production was induced in colicinogenic Salmonella typhimurium D36 colE2 and also F'lac plasmids were eliminated from Escherichia coli AB785 (lac-/F'lac+) by treatment of these bacteria with cis-platinum(II)diamminodichloride (PDD). 63 The antitumor agent gig-platinum(II)diamminodichloride (PDD) causes crosslinking of nucleic acids (5, 12, l6, l7) and inhibits synthesis of deoxyribonucleic acid (DNA) in eukaryotic cells (4, 6, 7). PDD also induces phage production in lysogenic bacteria (11), inhibits cytokinesis in gram negative bacteria (13, 14, 15), and is exceptionally lethal to DNA repair deficient mutants of Escherichia coli (1). These properties are characteristic of effects produced by radiomimetic agents and suggest that PDD might also increase the frequency of elimination of plasmid DNA in bacteria and induce production of colicin in colicinogenic bacteria as observed with other DNA damaging drugs (8, 9). Salmonella typhimurium strain D36 (colE2) and E, coli strain AB785 (met, lac-IF'lac+) were obtained from our stocks. E. coli strain W3350 thy_was supplied by Dr. Loren Snyder. Organisms were routinely cultivated on blood agar base slants. To enhance elimination of plasmid DNA a logarithmically growing culture of E, 221$.AB785.l2274§LlESf at a cell density of 108 per ml was treated with O to 40 ug PDD per ml in tryptone broth (10) during incubation at 37 C for 4 h with aeration. The cultures were then suit- ably diluted and plated on Levine EMB agar (Baron, Spilman and Carey from Baltimore Biological Laboratory, Baltimore, Maryland) supplemented with 0.5% lactose. Cells which had lost the ability to ferment lactose appeared as uncolored colonies in contrast to dark colonies of organisms which were able to ferment lactose. Treatment of organisms with increas— ing concentrations of PDD resulted in increasing frequency of plasmid 64 elimination as determined by the increase in number of colonies unable to ferment lactose (Table 2). A concentration of 40 ug PDD per ml elimina- ted Eilflfi in 65% of treated cells in contrast to 23% of cells which spontaneously lost Ellgg_in untreated control cultures. Logarithmically growing cells of E, typhimurium D36 colE2 were suspended in brain heart infusion (BHI) broth and incubated at 37 C with aeration. When the cells numbered about 108 per ml, PDD dissolved in 0.85% saline was added to cultures to yield the desired concentration (0 to 60 pg per m1) and the cells incubated at 37 C for 4.5 h. Cultures were then sterilized with chloroform and the cell debris eliminated by centrifugation. The supernatant fluid was dialyzed against cold distilled water and the average bacteriocin content of duplicate cultures determined as described by Hu et a1. (8). The units of colicin per ml of supernat- ant fluid increased with exposure of cultures to increasing concentrations of PDD (Table l). The reciprocal of the highest dilution showing anti- bacterial activity against E, ggl$_strain W3350 EEy_was 2200 units/ml at the highest concentration of PDD tested (60 ug/ml); this was a signifi- cant increase over untreated cultures which yielded 30 units per ml of the bacteriocin. Colicinogenic cells which were irradiated with 400 ergs per mm2 ultraviolet light as described by Hu et a1. (8) yielded 6600 units per m1 colicin. These data supply additional evidence for the reactivity of PDD with DNA since PDD induces production of colicin and elimination of plasmid DNA in appropriate strains of bacteria as do radiomimetic agents. REFERENCES Beck, D. J., and R. R. Brubaker. 1973. Effect of cis-platinum(II)- diamminodichloride on wild type and deoxyribonucleic acid repair deficient mutants of Escherichia coli. J. Bacteriol. 116:1247-1253. Beck, D. J., and R. R. Brubaker. 1974. Mutagenic properties of cis-platinum(II)diamminodichloride in Escherichia coli. To be submitted for publication. Drobnik, J., M. Urbankova, and A. Krekulova. 1973. The effect of cis-dichlorodiammineplatinum(II) on Escherichia coli B. The role of ELL, 939;, and flmarkers. Mutat. Res. gzl3-20. Harder, H. C., and B. Rosenberg. 1970. Inhibitory effects of antitumor platinum compounds on DNA, RNA, and protein synthesis in mammalian cells $E_y$££g, Int. J. Cancer E3207-216. Horacek, P., and J. Drobnik. 1971. Interaction of gig-dichloro- diammineplatinum(II) with DNA. Biochem. Biophys. Acta 224,341-347. Howle, J. A., and G. R. Gale. 1970. Egg-Dichlorodiammineplatinum(II): persistant and selective inhibition of deoxyribonucleic acid syn- thesis }g_z$!g, Biochem. Pharmacol. E232757-2762. Howle, J. A., H. S. Thompson, A. E. Stone, and G. R. Gale. 1971. gig-Dichlorodiammineplatinum(II): inhibition of nucleic acid syn- thesis in lymphocytes stimulated with phytohemagglutinin. Proc. Soc. Exptl. Biol. Med. E21:820—825. Hu, P. C., G. C. H. Yang, and R. R. Brubaker. 1972. Specificity, induction, and absorption of pesticin. J. Bacteriol. 112:212—219. 65 10. ll. 12. 13. 14. 15. l6. 17. 66 Ikeda, Y., T. Iijima, and K. Tajima. 1967. Elimination of F-episome from a male strain of Escherichia coli by treatment with sarkomycin and a related antibiotic. J. Gen. Appl. Microbiol. 123247-254. Kaiser, A. D. 1955. A genetic study of the temperate coliphage A. Virology.£:424-443. Reslova, S. 1971/72. The induction of lysogenic strains of Escherichia .2211 by gig-dichlorodiammineplatinum(II). Chem.-Biol. Interactions 3366-70. Roberts, J. J., and J. M. Pascoe. 1972. Cross-linking of complement- ary strands of DNA in mammalian cells by antitumor platinum compounds. Nature'E2§:282-284. Rosenberg, B., E. Renshaw, L. VanCamp, J. Hartwick, and J. Drobnik. 1967. Platinum-induced filamentous growth in Escherichia coli. J. Bacteriol. 22,716-721. Rosenberg, B., L. VanCamp, and T. Krigas. 1965. Inhibition of cell division in Escherichia coli by electrolysis products from a plat- inum electrode. Nature 205:698—699. Rosenberg, B., L. VanCamp, E. B. Grimley, and A. J. Thomson, 1967. The inhibition of growth or cell division in Escherichia coli by different ionic species of platinum (IV) complexes. J. Biol. Chem. 23331347-1352. Shooter, K. V., R. Howse, R. K. Merrifield, and A. B. Robins. 1972. The interaction of platinum II compounds with bacteriophages T7 and R17. Chem.-Biol. Interactions E3289-307. Shooter, K. V., and R. K. Merrifield. 1972. Changes in the hydrodynamic properties of DNA induced by interaction with platinum (II) compounds. Biochim. Biophys. Acta 187:16-27. 67 Table l. Elimination of F'lac+ from Escherichia coli AB785 (lac-lF'lac+) by treatment with cis-platinum(II)- diamminodichloride (PDD)a Concentration of PDD . Elimination % 0 23 10 27 20 33 40 65 3Cells of E, 22;; AB785 (lac-IF'lac+) were grown 4 h in tryptone broth containing 0 to 40 ug PDD at 37 C with aeration. The organisms were then diluted tenfold in tryptone broth, incubated overnight, and plated on EMB agar with lactose. Cured cells produced white colonies on this medium since they were unable to ferment lactose in contrast to dark colonies produced by organisms which were able to ferment this sugar. 68 Table 2. Induction of colicin E2 production in Salmonella typhimurium D36 (colE2) by treatment with cis-platinum(II)diammino- dichloride (PDD)a Concentration of PDD Colicin E2b O 30 10 700 20 700 40 700 60 2200 aCells 0f.§° typhimurium D36 (colE2) were grown 4.5 h in BHI broth containing 0 to 60 pg PDD per ml at 37 C with aeration. Chloroform (l/lO vol.) was added to cultures which were shaken vigorously and then incubated at 4 C for 0.5 h. Subsequently the cultures were centrifuged at 48,000 x g for 10 min at 5 C and the resultant super- natant fluid dialyzed overnight against cold distilled H20 ( 4 C). Dilutions of the supernatant were spotted on BAB agar plates, which were dryed for l h at 25 C, sterilized with chloroform vapor, and over- layered with 4 ml of CaEDTA agar containing 105 cells of E._ggli‘K-12 strain W3350 as indicator strain for bacteriocin. bReciprocal of highest dilution of supernatant which exhibited anti- bacterial activity against E, coli W3350. "'Iflifitflfltflln‘ljju{IIIIITLIIJIIMQTIIIIWEs