Enzyme, Nitrogen, and DNA Concentrations Insarcoma 180

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-mercaptopurine 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.â€”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.â€”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.â€”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.â€”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; PÂ¡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.

1 part tissue (wet weight) in 40 volumes of saline. concentration has been expressed as /ig DNA/ml Determination of adenosine-o'-phosphatase,â€” of glass wool-filtered homogenate.6 Adenosine-5'-phosphatase was determined by as Determination of nitrogen.â€”Nitrogen was as say of the PÃŒliberated in a mixture consisting of sayed in aliquots of the glass wool-filtered ho- 20 AmÃ³lesof 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. Determinations were pH 7.5; 0.2 ml. of a 0.0984 M MgCU solution; 0.2 done in duplicate, with a Coleman Junior spectro- ml. of a 0.016 M EDTA solution; and 0.6 ml. of photometer. water. After a 1-minute pre-incubation time, 0.4 Measurement of enzyme activity.â€”All measure ml. of homogenate was added to make a final vol ments of enzyme activities are based on the initial ume of 3.2 ml. The homogenate was prepared from zero order time interval of the reactions. Prelimi 1 part tissue (wet weight) and 30 volumes of saline. nary experiments provided assurance that reaction The temperature of incubation was 37Â°C., and the velocity was directly proportional to the homoge incubation time 30 minutes. The reaction was ter nate concentration. The enzyme activities, with minated by addition of 0.8 ml. of a 50 per cent the exception of cathepsin, were uniformly assayed trichloroacetic acid solution. in the pH range of 7.4-7.5. At this pH glycolysis Determination of inosine-5'-piiosphatase.â€”Ino- proceeds at maximum rate. The role of the above sine-5'-phosphatase was determined by the PÂ¡ enzymes in the over-all glycolysis by Sarcoma 180 liberated in a mixture similar to that described homogenates is at present under investigation. above for adenosine-5'-phosphate except that 9 /umoles of the tetrahydrate of the disodium salt of RESULTS inosine-5'-phosphate (Sigma) served as substrate The histopathologic effect of intraperitoneal ad ministration of 6-MP to the host on Sarcoma 180 and 0.30 ml. of 0.0984 M MgCl2 was used. (5) has been re-examined and confirmed.7 The Determination of adenosine deaminase.â€”Adeno- sine deaminase was determined spectrophoto- principal characteristic cytologie change consisted predominantly of giant, multinuclear cells with metrically by the method of Kaplan, Colowick, both nuclear and cytoplasmic vacuolization. These and Ciotti (19). To 2.95 ml. of phosphate buffer, cytologie changes appear to persist until rÃ©sorp pH 7.5, containing 0.218 AmÃ³les of adenosine tion of the tumor is complete or growth is re (Pabst) 0.05 ml. of tumor homogenate was added sumed. Histologie examination of the tumors from diluted as described for adenosine-S'-phosphatase. mice treated with 6-MP or of control tumors re The decrease in optical density at 265 m/i was ob vealed no necrosis; connective tissue was found served every 30 seconds after the first 30-second only in the peripheral portion of the sarcomas. interval for a total of 4 minutes. The temperature When tumors of nontreated mice were collected in of incubation was 37.3Â°C. the earlier phases of tumor growth (between the Lactic dehydrogenase,â€”Lactic dehydrogenase 4th and 5th day after implantation, Group la) the was estimated by the method of Meister (20). One original transplant was still clearly discernible; this part tissue (wet weight) was homogenized with 40 portion was discarded. Macroscopic examination volumes of saline, filtered through glass wool, and revealed poor vascularization of tumors of 6-MP- then again diluted 30-fold. treated mice (Group 3). About 1 per cent of the Determination of DNA.â€”DNA was extracted tumors of the control groups (Groups 1, la, and 2) were so thoroughly vascularized that a clear-cut from the tumor tissues essentially by the method separation of tumor tissue from macroscopically of Schneider (23). Equal volumes of 10 per cent trichloroacetic acid and glass wool-filtered ho visible blood vessels could not be effected. Such tumors were rejected. mogenate, prepared by homogenizing 1 part tu Since CMC was used as a vehicle for 6-MP and mor tissue with 20 volumes of saline, were mixed the tumors of the 6-MP treated animals (Table 1, and heated for 20 minutes at 92Â°C. DNA stand Group 3)8 were considerably lower in weight than ards in 5 per cent trichloroacetic acid were heated 6The authors are indebted to Dr. L. F. Cavalieri for a at the same time. DNA was determined in the supernatants according to Brady's modification ol generous sample of highly purified DNA which was used as standard. Dische's method (3). Color development at 25Â°C. 7The authors are greatly indebted to Dr. Stephen Sternberg for the histologie examination of the tumor specimen. was allowed to proceed for 30 minutes, after which 8Because of the low weights of the tumors of Group la and it reached a maximum, and the color remained un Group 3, pools of two to three tumors were used for each changed for at least 20 additional minutes. DNA homogenate specimen.

those of the nontreated control groups (Table 1, with the CMC controls (Table 1, Group 2): tu Groups 1 and 2), it was necessary to evaluate the mor weight, homogenate-N, DNA, and lactic de effect of I.P. injections of CMC on the one hand, hydrogenase (P in all instances <0.01).9 In con of tumor weight on the other, on the parameters trast, highly significant increases were obtained studied. The influence of CMC on the parameters for cathepsin, ATPase, adenosine-5'-phosphatase, of homogenates from tumors harvested between inosine-5'-phosphatase, and adenosine deaminase the 7th and 9th day is presented in Table 1, (P in all instances <0.01). Statistical evaluation Group 2. As compared to the parameters of the showed that the DNA and homogenate-N concen tumor homogenates from tumors of comparable tration and the lactic dehydrogenase activity weight of nontreated mice (Group 1), Group 2 were significantly lower, and all the other enzyme showed no significant changes except in cathepsin activities significantly higher in Group 3 than and adenosine deaminase levels, which were both in Group la, the nontreated group with com increased (P in both instances <0.01). parable tumor weights (P for DNA = 0.02, for all The effect of tumor weight on the various other parameters <0.01). Table 2 shows that the parameters was established as follows: tumors of homogenate-N increased with the weights of the nontreated mice were harvested between the 4th sarcomas. and 5th day after transplantation (Table 1, DISCUSSION Group la) when their weights fell within the range The parameters studied refer solely to that part of the 8-day-old tumors of the 6-MP-treated mice of the tumor tissue extractable by a 1-minute ho- (Table 1, Group 3). The homogenate-N and DNA concentration, as well as the inosine-5'-phospha- mogenization. The connective tissue could not be investigated, owing to its failure to disintegrate, as tase activity of the former tumor homogenates, was significantly lower (P = 0.02 for homogenate- reported above. From the available references con N, <0.01 for DNA and for inosine-5'-phospha- cerned with the composition and metabolism of tase) than in the tumor homogenates from non- connective tissue of various types and origins (2, 24) it appears unlikely that the connective tissue treated mice collected between the 7th and 8th day (Table 1, Group 1). The ATPase, adenosine- portion of the tumor plays a significant part in its 5'-phosphatase, lactic dehydrogenase, adenosine over-all metabolism. The portion of the homoge- 'deaminase, and cathepsin activities showed no sig nizable tissue nitrogen increases in the course of tumor growth; this is shown by the ratio of Â¿ig. nificant change. 9Enzyme activities are based on the nitrogen content of the The following parameters of the tumor homoge glass wool-filtered homogenates rather than on the tissue nates of Group 3, Table 1 (6-MP-treated mice) weights; as shown in Table 2, the homogenizable portion of the showed highly significant decreases as compared tumors is also a function of tumor weight.

TABLE 1 EFFECTOF6-MP ONTUMORWEIGHT,DXA, ANDENZYMEPARAMETERS

THEATEnGroup

1652 la155+ 2710 3200+ Tumor weight (mg) + 179(40)* 68 (113)t + 210 (152)ÃŽ 70 (143 Homogenate-N (jug/ml)# 683 + 54 (14) 566 Â±66 (14)t 630+ 45 (21) 479+ 59 (17 ttÂ§nÂ§ DNA (Mg/ml)# 184+ 30 (14) 137Â±25 (14)t 169+ 23 (21)Ã® 89+ 38 (17 Cathepsin 382+ 80 (17) 290 + 114 (10) 1980 + 598 (27)ft 3540 + 613 (27 nÂ§ ATPase 1860+ 381 (23) 1840+ 172 (10) 20901415 (19) 3810 Â±330 (19 nÂ§ Adenosine -5'-phosphata se 168+ 63 (15) 127+ 37 (15) 176+ 37 (20)Ã® 265+ 50 (18>nÂ§ Inosine-S'-phosphatase 132+ 34 (15) 68+ 7 (ll)t 114Â±32 (19)Ã® 202+ 18 (19 nÂ§ Adenosine deaminase 84+ 12 (15) 94+ 15 (6) 115+ 19 (17)t 145+ 6 (18>nÂ§ Lactic dehydrogenaseNONTREATEDGroup1190Â±127(12)Group 1040+ 90 (12)CMC-TBEATEDGroup1060+ 157 (22)6-MP 671 Â±106 (23tÂ§nÂ§

* Number of tumors or homogenate specimens are given in parentheses. t Denotes statistically significant difference from Group 1. ÃŽDenotesstatistically significant difference from Group la. Â§Denotesstatistically significant difference from Group 2. # Estimated in glass wool-filtered homogenates of 1 part wet tissue in 20 volumes of saline. The enzyme activities are expressed per mg. of homogenate-N as follows: catheptic activity as jug.of tyrosine liberated in 1 hour; ATPase, adenosine-5'-phosphatase, and inosine-5'-phosphatase as jug. of PÂ¡liberated in 1 hour; adenosine deaminase and lactic dehydrogenase, respectively, as jug.of NHs and of lactic acid formed in 1 minute.

honiogenate-N/weight of tumor (mg.) (Table 2), results in tumors with a much lower weight aver and this indicates the relative decrease in connec age but is also associated with a lower DNA con tive tissue. Similar observations have been made centration in the tumor homogenates as compared with two rat sarcomas, where the amount of with the control groups. However, the DNA as connective tissue was determined by histologi- sayed derives solely from the saline-extractable cal technics (12). The increase in homogenate-N in material of the tumor tissue, representing the the course of the growth of Sarcoma 180 in non- nuclei and the cytoplasm, but not the connective treated mice parallels the increase in DNA fairly tissue. It thus represents the major but not neces well. Of the enzyme parameters only inosine-5'- sarily the total amount of DNA. phosphatase was a function of tumor weight, be Increases in the activities of a variety of en ing significantly decreased in the younger tumors. zymes, notably of nucleotidases and of cathepsin, Comparison of tumor homogenates of 6-MP- have been observed in homogenates of spontane treated mice with those of either nontreated or ously regressing Flexner-Jobling carcinomas (8- CMC-injected mice showed highly significant dif 10), and of cathepsin in extracts of dystrophic ferences in all parameters. As has already been muscle tissues (28, 29). The significance of in stated, the CMC control tumor homogenates had creased levels of nucleotidases, especially of significantly higher adenosine deaminase and ATPase or apyrase, has been studied in spontane cathepsin levels than the nontreated controls but ously regressing tumors and found to be associated showed no changes in the other parameters, e.g., with a shift from ADP rephosphorylation to ADP breakdown, terminating in the formation of ino- TABLE 2 sine (10). The ensuing uncoupling of phosphory- Tr.woRWEIGHTANDTOTALSOLUBLENITROGENRECOV lation was evident in a decreased phosphate up ERIESOFTUMORSOFNONTREATEDMICE take and lactic acid production (9). It is of in terest that a 50 per cent decrease in both oxygen total uptake and glycolysis was observed in previous homogenate-N/tumor homogenate-N Tumor weight nate-N/ml* studies with tissue slices of Sarcoma 180 collected (mg.)155Â± Gig)/tumor vvt. (Mg.)566 (fg-11,754 (mg.)11.3 from mice treated with 6-MP (21). The possibility that a mechanism similar to the one reported 68 Â±66 + 204 + 3.00 above is operative in this instance is at present un-' 680 + 250 683 Â±54 9,888+ 778 13 5 + 3.11 1,220 + 280Homoge-870 Â±61Total21,230 Â±1,488Ratio17.4 + 5.31 der investigation. Lactic acid dehydrogenase has been so far the * In glass wool-filtered homogenates of 1 part wet tissue in only enzyme studied in tumor homogenates of 20 volumes of saline. 6-MP-treated mice which showed decreased in homogenate-N, DNA, ATPase, adenosine-5'- stead of increased activity. The significance of this phosphatase, inosine-5'-phosphatase, and lactic observation on the over-all glycolysis by these homogenates will be assessed within the frame dehydrogenase. work of a general study of the glycolysis and respi As the data that have been presented show, the ration of all four groups of homogenates. significantly different values for all the parameters of the homogenates of the 6-MP-treated group ACKNOWLEDGMENTS cannot be explained on the basis that these tu The authors wish to express their appreciation to Misses mors weigh less than those of the two control Tina Marmorale, Angeline Tosti, and Elinor Williams for groups. Comparison with homogenates of tumors their excellent technical assistance. within the same weight range implanted into mice which received no subsequent treatment (Table 1, REFERENCES Group la) showed the same pattern of differences 1. BLOCH-FRANKENTHAL,L.,and BACK, A. Effect of Col- chicine on Tumor Growth and Tumor Pyrophosphatase. as compared with homogenates of tumors of great Proc. Soc. Exper. Biol. & Med., 76:105-9, 1951. er weights (Table 1, Group 1). 2. BOYD,E. S., and NEUMAN,W. F. Chondroitin Sulfate Administration of 6-MP has been shown to in Synthesis and Respiration in Chick Embryonic Cartilage. hibit the growth of the Flexner-Jobling carcino Arch. Biochem. & Biophys., 51:475-86, 1954. ma (25). Heidelberger and Keller (14) found that 3. BHODY,F. S. A Spectrophotometric Study on the De- administration of 6-MP to rats carrying this tumor soxypentose Nucleic Acid-Cysteine Reaction. Acta Chem. is associated with a diminished uptake of C14- Seandinav., 7:502-Â«,1953. 4. BURCHENAL,J. H.; MURPHY,M. L.; ELLISON,R. R.; labeled guanine and adenine into the DNA frac SYKES,M. P.; TAN, T. C.; LEONE,L. A.; KARNOFSKY, tion of the tumor. Our data show that treatment of D. A.; GRAVER,L. F.; DARGEON,H.W.; and RHOADS,C. the host with 6-MP (Table 1, Group 3) not only P. Clinical Evaluation of a New Antimetabolite, 6-Mcr-

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Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1961 American Association for Cancer Research. Enzyme, Nitrogen, and DNA Concentrations in Sarcoma 180 in Mice Treated with 6-Mercaptopurine

Paul J. Fodor, Donald A. Clarke and Oscar Bodansky

Cancer Res 1961;21:232-237.

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