Effects of Nitrogen Mustard and Cyclophosphamide Upon the Synthesis of DNA in Vivo and in Cell—Freepreparations'
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[CANCER RESEARCH 29, 98—109,January 1969] Effects of Nitrogen Mustard and Cyclophosphamide upon the Synthesis of DNA in Vivo and in Cell—freePreparations' Glynn P. Wheelerand Jo Ann Alexander Kettering-Meyer Laboratory2, Southern Research Institite, Birmingham, Alabama 35205 SUMMARY and concentrations of the nitrogen mustard, it was possible to inhibit the synthesis of DNA more than that of RNA and to Single doses of nitrogen mustard or cyclophosphamide inhibit synthesis in a sensitive tumor without inhibiting caused regression of plasmacytomas in hamsters and decreases synthesis in a bilaterally growing resistant tumor. Other in the rate of synthesis of DNA and RNA by the tumors. studies (23) showed that multidose treatment of hamsters Maximum inhibition of synthesis did not occur immediately bearing plasmacytomas with cyclophosphamide [2-[bis(2- following the administration of the agent but was observable chloroethyl)amino]-2H-1,3,2-oxazaphosphorinane 2-oxide] 24—48 hours later. This inhibition was accompanied by a de caused decreases in the activity of DNA nucleotidyltransferase crease in DNA nucleotidyltransferase activity of crude cell-free and of thymidylate kinase of soluble cell fractions prepared supernatant fractions prepared from the treated tumors. from the plasmacytomas, but that a single dose of cyclophos The concentrations of nitrogen mustard required for in vitro phamide given 2 hours before killing the animal caused an deactivation of the crude DNA nucleotidyltransferase and for increase of the DNA nucleotidyltransferase activity. These in vitro deactivation of commercial salmon sperm DNA as a results provoked the questions whether the observed decreased primer for this system were much greater than those that synthesis of DNA in vivo and the decreased nucleotidykrans would be present in hamsters following the administration of ferase activity were the cumulative effects of multiple doses of therapeutically effective doses. It was concluded that neither the agents, whether similar effects would be observed at longer the direct deactivation of the DNA nucleotidyltransferase nor intervals after the administration of single doses of the drugs, gross interference with the primer activity of DNA is the cause and whether decreased nucleotidyltransferase activity is a pri of the observed therapeutic effect upon the tumor or the de mary or secondary effect of the drug. In pursuit of answers to crease of synthesis of DNA in vivo. these questions, we have now performed experiments to: (a) Transient inhibition of growth and of synthesis of DNA and determine the relationship between the interval after admin RNA by drug-resistant tumors was also observed. It is not istering a single dose of nitrogen mustard or cyclophosphamide presently known whether the resumption of growth and and the effect upon the synthesis of nucleic acids in vivo, (b) synthesis of nucleic acids is the result of repair of damage and determine the effects of treatment of cell-free preparations recovery by the cells or of killing and elimination of the cells from plasmacytomas with nitrogen mustard upon the DNA of the tumor that are most sensitive to the agent. It is evident, nucleotidyltransferase activity, (c) determine the effects of however, that inhibition of the synthesis of DNA and RNA by treatment of DNA with nitrogen mustard in vitro upon the a tumor during the first 48 hours following administration of priming activity of the DNA for the DNA nucleotidyltrans nitrogen mustard or cyclophosphamide cannot be considered ferase system, and (d) compare the effects of in vivo treatment to be indicative that a favorable therapeutic effect of the agent with cyclophosphamide upon the in vivo synthesis of DNA by has been accomplished. plasmacytomas and upon the DNA nucleotidyltransferase activity of a cell-free preparation from the same plasmacy INTRODUCTION tomas. Previous studies in this laboratory (27) have shown that MATERIALS AND METhODS nitrogen mustard (HN2) inhibits the incorporation of radio active substrates into DNA by Fortner hamster plasmacytomas Determination of the Effects of Agents upon the Growth of in vivo and by minces of these tumors. With selected dosages Tumors. Trocar implants of cyclophosphamide-sensitive (9) and cyclophosphamide-resistant (22) plasmacytomas were placed subcutaneously and bilaterally in the axillary regions of 1ThiS investigation was supported by the Cancer Chemotherapy young (45- to 55-gram) Syrian hamsters. Fourteen days later National Service Center, National Cancer Institute, USPHS, under con tract PH43-66-29 and by grants from the Charles F. Kettering Founda single intraperitoneal injections of the respective agents were tion and the Alfred P. Sloan Foundation, Inc. administered, and daily measurements of the tumors were 2Afflliated with Sloan-Kettering Institute for Cancer Research, New made by means of calipers. Approximations of the weights of York, New York. the tumors were made with the assumption that the tumors Received April 9, 1968 ; accepted September 15, 1968. were prolate spheroids with a density of 1.0. 98 CANCER RESEARCH VOL.29 Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1969 American Association for Cancer Research. Effects ofNitrogen Mustard and Cyclophosphamide Determination of the Effects of Agents upon the Fixation of In the standard procedure, the reaction mixture minus the ‘4Cfrom Formate-'4C and from Adenine-8-14C. Hamsters primer was incubated at 37°Cfor 10 minutes, and the primer bearing bilaterally and subcutaneously growing 13-day-old and was then added. The time at which the primer was added is 1 4-day-old cyclophosphamide-sensitive and cyclophospha hereafter referred to as zero time. In certain experiments the mide-resistant plasmacytomas were given single intraperitoneal enzyme preparation or a solution of native salmon sperm DNA injections of nitrogen mustard or cyclophosphamide at speci was incubated with or without nitrogen mustard and then used fled doses. At intervals thereafter, the animals were given intra in the standard procedure. At zero time and at designated peritoneal injections of sodium formate-14 C (specific activity, intervals (each 10 minutes during the first hour, each 20 min 55, 220, or 298 jic per mg; dosage, 1 pc per gram of body utes during the second hour, and each 30 minutes during the weight) or adenine-8-14C (specific activity, 21.7, 83.3, or 129 third and fourth hours) during the ensuing 4 hours, [email protected] Ilc per mg; dosage, 0.3 @scper gram of body weight). The samples of the incubation mixtures were transferred to discs of animals were killed by carbon dioxide asphyxiation 2 hours filter paper and allowed to dry at room conditions, whereupon following the injection of the radioactive compounds. The the discs were dropped into a beaker of cold 5 percent tri tumors from 3 or 4 animals were pooled; homogenates, alco chloroacetic acid. The beaker contained at least 10 ml of the holic extracts, and the purines of RNA and of DNA were acid solution for each disc. The discs were allowed to stand prepared and assayed for radioactivity by procedures that have overnight in the acidic solution and were then washed two been described (28). times by standing for 15 minutes in cold 5 percent solutions of Preparation of Soluble Extracts of Tissues. Hamsters bearing trichloroacetic acid. The discs were next washed once with 14-day-old subcutaneous plasmacytomas were asphyxiated cold absolute ethanol and allowed to dry on paper towels at with carbon dioxide, and the tumors were removed and placed room conditions. The quantities of 14C present on the discs in ice-cold containers. After the tumors were minced freehand were determined with a Packard Tri-Carb scintillation spec with knives and forced through the perforated steel plate of a trometer, and it was assumed that the 14C was present in tissue press, 10 grams of the tissue were homogenized by DNA. means of a Thomas Tissue Grinder with a motor-driven teflon At the same times that samples of the incubation mixtures pestle in 30 ml of an ice-cold aqueous solution that was 0.25 were taken for placing upon the paper discs, other samples of M with respect to sucrose and 0.02 M with respect to Tris 10 ;.zl each were spotted on Whatman No. 1 paper for subse hydrochloride buffer (pH 8.0). This homogenate was centri quent chromatography using isobutyric acid:water:acetic acid fuged at 105,000 X g for 1 hour. The aqueous supernatant (100:50:1 v/v/v) as the solvent. The positions of the radio layer was removed by pipet, care being taken to exclude the active spots were detected by means of blue-sensitive X-ray overlying lipid layer. This solution was assayed for protein by film, and the radioactive areas were cut from the chromato the method of Lowry et a!. (16) and was used without further grams and assayed with the liquid scintillation spectrometer. treatment as the source of enzymes for the incubations. The identities of the various radioactive components of the Incubation Mixtures and Enzyme Assays. Each sample for mixtures were determined by comparison of the RF values incubation contained the following ingredients at the indicated with those of known compounds that were chromatographed concentrations in micromoles per ml: sucrose, 222; Tris in parallel with them. hydrochloride buffer (pH 7.5), 8.8; adenosine triphosphate, Determination of the Effects of a Single Dose of Cyclophos 1.1 ; magnesium chloride, 1.1 ; potassium chloride, 40; potas phamide or Nitrogen Mustard upon the in Vivo Fixation of 3H slum phosphoenolpyruvate, 5.04; and pyruvate kinase, 0.84 from Thymidine-C3H3 by Plasmacytomas and upon the DNA microgram per ml. Each sample also contained either the Nucleotidyltransferase Activity of the Same Tumors. Ham monophosphates or triphosphates of deoxyadenosine, deoxy sters bearing 14-day-old subcutaneously growing plasmacy guanosine, deoxycytidine, and thymidine at concentrations of tomas were given single injections of cyclophosphamide (20 0.046 micromole per ml.