Enzyme, Nitrogen, and DNA Concentrations in Sarcoma 180 in Mice Treated with 6-Mercaptopurine* PAULJ. FODOR,DONALDA. CLARKE,ANDOSCARBODANSKY (Division (if Enzymology and Metabolism and Division of Experimental Chemotherapy, Sloan-Kettering Institute for Cancer Research; Department of Biochemistry, Memorial and James Ewing Hospitals; and Sloan-Kettering Divisiion of Cornell University Medical College,New York, N.Y.) SUMMARY The concentrations of homogenate-nitrogen, of DNA, and the activities of various nucleotidases, adenosine deaminase, cathepsin and lactic dehydrogenase have been studied in homogenates of Sarcoma 180 from non treated mice, from mice given injec tions of 6-carboxymethylcellulose in saline, and from mice treated with 6-mercapto- purine suspended in saline containing 6-carboxymethylcellulose. Statistically significant increases in the activities of all the nucleotidases, adenosine deaminase, and cathepsin have been observed in tumor homogenates of mice treated with 6-mercaptopurine. Statistically significant decreases in tumor weight, homo genate-nitrogen, DNA, and lactic dehydrogenase have been observed in the same homogenates. The tumor homogenates of the controls receiving 6-carboxymethylcellulose in saline showed only increases in adenosine deaminase and cathepsin which, however, did not reach the activity levels attained in homogenates of mice treated with 6-mercapto purine. The significance of the findings is discussed. Although the enzymic patterns of tumor growth lism of the regressing tumors (8-10). Within the have been the subject of many investigations (13), past few years the effects of the administration of the processes of regression or arrested growth, alpha peltatin (26), acetylpodophyllotoxin deriva whether spontaneous or induced by chemotherapy, tives (27), chloroethylamine derivatives (11), have received relatively little attention. The study ethyleneimine derivatives (15-18), colchicine (1), of most spontaneously regressing tumors is fraught and of 6-mercaptopurine (21) on glucose utiliza with technical difficulties, since the number of tion, over-all glycolysis, DPN content, and a few such regressions is usually unpredictable, thus other metabolic aspects have been investigated. making unavailable sufficient material for system Among the chemotherapeutic agents studied so atic investigations. Studies on the spontaneously far in their effect on tumor metabolism, 6-mercap regressing Flexner-Jobling carcinoma, which shows topurine occupies an important position because spontaneous regression in about 35 per cent of all of its growth-inhibitory effect on both human and transplants, revealed definite changes in the en- animal tumors (22) with relatively few toxic side zymic pattern and, consequently, in the metabo- effects (4). * This work has been supported in part by the following to This study is the first of a series in which a O.S.: American Cancer Society Grants P-168 and P-164B, systematic effort will be made to elucidate the (irant DRG 332D from the Damon Runyon Memorial Fund metabolic changes induced in tumors by adminis for Cancer Research, and (irant C-4251 (C2S1), from the Na tration of 6-mercaptopurine to the host. Sarcoma tional Cancer Institute, National Institutes of Health, Public Health Service; and to D.A.C. :American Cancer Society Grant 180 has been chosen as the experimental tumor, T-2aB, and Contract SA-43-ph-2445 from The Cancer Chemo since its histology is well known, treatment of the therapy National Service Center, National Cancer Institute, host with 6-mercaptopurine induces no necrosis in National Institutes of Health, U.S. Public Health Service. the tumor, nor do tumors of nontreated hosts con Received for publication August 15, 1960. tain necrotic portions, provided they are collected 232 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1961 American Association for Cancer Research. FoDOR et al.—Sarcoma 180 and 6-Mercaptopurine 233 not later than the 9th day after transplanta tumors that were removed between the 4th tion (5).1 and 5th day after implantation. The average weight of these younger tumors approximated that MATERIALS AND METHODS of the tumors from the 6-MP-treated mice at the Randomly selected female Swiss albino mice end of 7 days of treatment. The younger tumors (HalCR)2 weighing between 18 and 29 gm. served were studied to assess the influence of tumor as experimental animals and also as carriers of the weight on the various parameters. tumor line. The tumor line was maintained by bi Preparation of tumors homogenates.—The ani lateral implantation of tumor fragments of equal mals were killed by cervical dislocation: the tu size; the site of implantation was the subcutaneous mors were excised, weighed, washed briefly in ice- ventro-lateral region of the host mice. It was cus cold saline, and dissected to remove macroscopi- tomary to harvest these tumors on the 7th day cally visible blood vessels. The tumor slices were after implantation. Twenty-six fragments, each again briefly washed in saline, freed quickly from approximately 5 mg. wet weight, were regularly any adhering moisture by being pressed between prepared from one tumor and transplanted into filter paper, weighed on a torsion balance to with the right ventro-lateral region of as many animals. in 1 mg., and immediately transferred to glass Thirteen of the tumor-bearing mice received homogenizing tubes immersed in crushed ice. daily treatment with 6-MP for 7 successive days, Homogenization was effected in saline in a poly- beginning at 24 hours after tumor implantation tetrafluoroethylene (Teflon) homogenizer. The (Group 3). The daily dose was 75 mg/kg of body time required for homogenization did not exceed weight, suspended in 1.0 ml. of saline containing 1 minute. The homogenates were then filtered 0.5 per cent of CMC.3 This dose was divided into through a thin layer of glass wool to remove 2 parts of 0.5 ml.; one was injected regularly in macroscopicaJly visible pieces of nonhomogeniz- the morning of each day and the other 7-8 hours able material, mostly connective tissue.4 The ho later. A second group of thirteen mice (Group 2), mogeneity of the filtrates was ascertained by the serving as controls to the 6-MP-treated group, re fact that aliquots were found to contain equal ceived comparable injections of CMC in saline amounts of nitrogen estimated as described below. only. The tumors from all mice were harvested The time lapse between homogenization and incu regularly between the 7th and 9th day following bation with the substrate was not allowed to ex implantation. Three tumors from each group were ceed 15 minutes. selected at random for histologie examination; the Assay of enzyme activity.—Catheptic activity remainder were employed for the biochemical as was estimated by determination of tyrosine as sessments of the present study. Another control sayed in the NPN fraction after incubation of the group consisted of mice given implants of tumor. homogenates with a preparation of purified horse These mice received no injections of any kind blood hemoglobin. The method of assay and the (Group 1). These animals were also sacrificed preparation of the hemoglobin solution have been between the 7th and 9th day after implanta described (8). Two ml. of hemoglobin were mixed tion and their tumors removed to ascertain with 2 ml. of acetate buffer, pH 3.5, and 1 ml. of that the various parameters under study did homogenate was added. The incubation time was not vary significantly from normal values es 15 minutes for homogenates of Groups 2 and 3 and tablished for Sarcoma 180 as a preliminary was extended to 30 minutes for those of Group 1 study to the present one. A final control group and la. Homogenates were prepared of 1 part tis (Group la) consisted of nontreated mice with sue (wet weight) and 30 volumes of saline. 'The following abbreviations will be used: 6-MP for 6- Determination of A TPase.—ATPase was de mercaptopurine; CMC for 6-carboxymethylcellulose; ATP termined as previously described (9), but EDTA and ATPase, respectively, for adenosine triphosphate and was added to a final concentration of 0.001 M,and adenosine triphosphatase; DNA for deoxyribonucleic acid; the MgCl2 concentration increased to 0.2 ml. NPX for nonprotein nitrogen; homogenate-N for homogenate nitrogen; P¡for orthophosphate; EDTA for ethylendiamino- of a 0.0984 M solution in a total of 3.2 ml. of tetraacetic acid. The term "CMC-treated" implies I.P. injec reaction mixture.6 Homogenates were prepared of tions of 6-carboxyinethylcellulose in saline; the term "6-MP- 1It was not possible to prepare stable dispersions of the treated" indicates that the mice received I.P. injections of tumor connective tissue; treatment of the tissue in an ultra 6-mercaptopurine in saline containing 6-carboxymethylcellu sonic disintegrator at maximum number of oscillations for 45 lose. minutes, (M.S.E. Ultrasonic Disintegrator, Measuring & 2Millerton Research Farm, Inc. Scientific Equipment, Ltd.) resulted only in coarse, unstable 36-MP in the dose administered is insoluble at, or near, suspensions. neutrality. CMC has been used to stabilize the finely dispersed *In all instances where EDTA was added the optimum suspensions of the compound. "concentration was established. Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1961 American Association for Cancer Research. 234 Cancer Research Vol. 21, February 1961 1 part tissue (wet weight) in 40 volumes of saline. concentration has been expressed as /ig DNA/ml Determination of adenosine-o'-phosphatase,— of glass wool-filtered homogenate.6 Adenosine-5'-phosphatase was determined by as Determination of nitrogen.—Nitrogen was as say of the PÃŒliberated in a mixture consisting of sayed in aliquots of the glass wool-filtered ho- 20 Amólesof adenosine-5'-phosphoric acid (Sigma) mogenates used for enzyme assays by micro- disolved in 0.6 ml. of water and adjusted to pH digestion with sulfuric acid and HjOs followed by 7.5; 1.20 ml. of 0.038 M diethyl barbiturate buffer, nesslerization of aliquots.