ICANCER RESEARCH 26 Part 1, 1466-1472,July 1966] Deoxyribonucleic Acid Synthesis I. Effect of in Vivo Cyclophosphamide Treatment on the in Vitro Activity of the Deoxyribonucleic Acid Synthetase System of Sensitive and Resistant Plasmacytomas1 ARTHUR J. TOMISEK, MARTHA BRUCE IRICK, AND PAULA WEDELES ALLAN Kettering'-Meyer Laboratory,2 Southern Research Institute, Birmingham, Alabama Summary term DNA-synthetase. In addition to this measurement of over-all DNA synthetase activity, we used the same experiments We have shown that the in vivo treatment of Fortner plasma- to determine the time course of radioactivity distribution among cytomas with Cyclophosphamide can lead to strong inhibitions of both deoxyribonucleic acid nucleotidyl transferase and thymi- the soluble components of the synthetase reaction mixtures. Our data show that the observed decreases in enzyme activity dylatc kinase activities in the soluble cell fractions. However, in are among the possible consequences of growth inhibition in the allowing only 2 hr for the inhibitor to act, the effect observed on tumor. the transferase was an unexplained stimulation rather than an inhibition. We have also provided some evidence that the inhibition of Materials and Methods growth precedes the inhibition of deoxyribonucleic acid nucleo ENZYME PREPARATION.Fortner hamster plasmacytoma tidyl transferase activity. ("sensitive") (3) and ite cyclophosphamide-resistant subline (12) were used for bilateral s.c. implantation into groups of 6-12 Introduction Golden Syrian hamsters, the animals in each experiment being uniform with respect to commercial subline, sex, and approxi Previous studies in this laboratory have shown that several mate age. On the 12th-14th postimplant day the animals were alkylating agents inhibited the in vivo synthesis of DNA by divided into 2 subgroups, to receive daily i.p. injections of Fortner hamster plasmacytomas and the in vitro synthesis of either saline or 10 mg/kg of Cyclophosphamide4 for either 1, 3, DNA by minces of these tumors (16). These studies have dealt or 5 injections. Hereafter these are referred to as "2-hr experi with the intact tumor-host system and with whole tumor cells. ments," "3-day experiments," and "5-day experiments." During As extensions of these studies, this and a related paper (17) deal the experimental period, animals were weighed daily and tumor with subcellular fractions from tumors which have been treated weights were estimated daily from caliper measurements. One in vivo with Cyclophosphamide. day after the final injection (except 2 hr for the 2-hr expérimente) For the present study the experiments of Mantsavinos and animals were stunned by carbon dioxide and killed by decapita Canellakis (10) and of Bollum (1) served as models for assaying tion. Pooled tumors were cooled, weighed, minced, passed for that portion of the DNA synthesis pathway which extends from the 4 substrates dTMP, dCMP, dAMP, and dGMP3 to through an extruder, and ground with 3 volumes of cold, buf fered (0.02 M Tris, pH 8) 0.25 M sucrose in a Potter-Elvejhem DNA. It is this multi-enzyme system to which we apply the homogenizer. After centrifugation at 100,000 X g for 1 hr, the clear aqueous supernatant was assayed for protein (9) and 1This investigation was supported by grants from the Alfred stored in a freezer till needed. In practice, the 4 enzymes of a set P. Sloan Foundation, the Charles F. Kettering Foundation, The were prepared in parallel and simultaneously assayed for syn John A. Hartford Foundation, Inc., and by the Cancer Chemo thetase activity within 1-7 days. Subsequent freezing-and- therapy National Service Center, National Cancer Institute, under the National Institutes of Health Contract No. SA-43-ph- thawings of these enzymes (for purposes other than the present 2433. A preliminary report was presented before the American report) showed that, within this age limit, the synthetase activity Association for Cancer Research, Chicago, Illinois, April 1964. 2Affiliated with the Sloan-Kettering Institute, New York, guauosine-5-triphosphate; GMP, guanosine-5'-phosphate; AMP, New York. adenosine-S'-phosphate; ATP, adenosine-S'-triphosphate; Tris, 3The abbreviations used are: dTMP, thymidine-5'-phosphate; tris(hydroxymethyl)aminomethane. (1TDP, thymidine-5'-diphosphate; dTTP, thymidine-5'-triphos- 42 - [Bis(/3-chloroethyl)amino] - 2H - 1,3,2 - oxazaphosphori- phate; dCMP, deoxycytidiiie-5'-phosphate; dCTP, deoxycytidine- nane-2-oxide. CCNSC 26271. Supplied by Cancer Chemotherapy 5-triphosphate; dAMP, deoxyadenosine-S'-phosphate; dADP, National Service Center. deoxyadenosine-5'-diphosphate; dATP, deoxyadenosine-5'-tri- Received for publication June 21, 1965; revised January 10, phosphate; dGMP, deo.\yguanosine-5'-phosphate; dGTP, deoxy- I960. 1400 CANCER RESEARCH VOL. 26 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1966 American Association for Cancer Research. Deoxyribonucleic Acid Synthesis. I decrease was an average of 0.20 for each freezing and thawing cycle. However, the variability in this decrease was so great that we were unable to estimate the effect of enzyme storage per se. In this report, each pairing of treated and control enzymes and of sensitive and resistant enzymes is with other enzymes of the same set. ENZYMKASSAY.The enzyme assay reaction mixture (usually 1.25 ml total volume) contained the following ingredients (all given in /¿moles/mlof reaction mixture): sucrose, 222; Tris (pH 7.5) buffer, 8.8; ATP, 1.1; magnesium chloride, 1.1; potassium 3-phosphoglycerate, 5.0; potassium chloride, 40; dAMP, dGMP, and dCMP, 0.23 each; dTMP-14C, 0.046. The only other addi tions were enzyme fraction, to 4.62 mg protein/ml, and DXA primer, to 0.45 mg/ml. The primer used was high molecular weight calf thymus DNA (Worthington), heat-denatured shortly before the assay. When other radioactive substrates were substituted for dTMP-"C, the above protocol was preserved in always using the radioactive substrate at 0.20 the concentration of the other 3 substrates. In tests for possible RNA synthesis, the appropriate 40 80 120 160 200 240 4 ribonucleoside monophosphates were substituted. Reaction mixture minus the primer was preincubated for 10 Reaction time (min.) min at 37°Ctopermit conversion of the deoxyribonucleotidephos- CHART1. A set of curves for determination of deoxyribonucleic phates to their triphosphate forms. At the start of the incubation acid synthetase activities. The curves of this example are for and each 10 min thereafter, carefully measured 25-ß\aliquots control and 5-day cyclophosphamide-treated plasmacytomas of were removed and placed on paper discs for assay of DXA both the sensitive and cyclophosphamide-resistant sublincs. synthesis by the procedure of Bollum (1). Simultaneously with the removal of these aliquots, other aliquots (10 jul) were placed directly5 on paper for 1-dimensional chromatography. The which we can attach a dependable physical interpretation. In paper used was Whatman No. 1 except as noted. An isobutyric terms of absolute activity, the average incorporation of dTMP- 14Cinto insoluble DNA by sensitive control enzymes was 1.05 acid solvent (14) was found very effective when the radioactive substrate was dTMP. The common butanol-propionic acid mamóles/hr/ml of reaction mixture. But since these data are too solvent (13) was satisfactory when the radioactive substrate variable, we consider only enzyme activity ratios within each was AMP or dAMP, except that the respective di- and triphos- set of simultaneous assays. phates were not separated. An ammonium isobutyrate solvent6 For 2 sets of enzymes the reproducibility of the assay was gave passable separations when the radioactve substrate wasi checked by the use of alternative radioactive substrates (Table GMP, but it gave poor reparations when the radioactive sub 1). strate was dGMP. Phenol-water on Schleicher and Schnell No. The possibility of deoxyribonucleotide conversion to ribonu- 589 orange-label paper (13) was effective when the radioactive cleotides and subsequent synthesis of RNA was checked by using substrate was dCMP, except for the unimportant separation of the appropriate 4 ribonucleotides in place of the deoxyribonucleo- deoxycytidine from deoxyuridine and lesser degradation products. tides. When AMP-8-14C was the labeled substrate, "RNA" The separated reaction mixture components were cut from the synthesis did not begin till after 2 hr; and when GMP-8-14C was papergrams and assayed for 14Ccontent by liquid scintillation the labeled substrate, there appeared to be no RNA synthesis technics. at all. INTERPRETATIONOF THE CHROMATOGRAPHICANALYSES.Typi Results cal RF values obtained with the isobutyric acid solvent system were: 72, thymine; 64, thymidine; 54, unknown (maximum 5% NATURE OF THE SYNTHETASE ASSAY. Chart 1 shoWS a SCt of of total 14C after 2 hr); 32, dTMP; 27 unknown (maximum 1% curves obtained in a typical DNA-synthetase assay experiment. of total 14Cafter 2 hr) ; 17, dTDP; 10 and 5, dTTP. The initial, linear slopes of the curves are almost the only data to Chart 2 is an example of the data obtainable by the Chromato graphie analysis. From this and similar charts, the following 6 In a few experiments, the values for dTTP by this procedure conclusions are drawn: (a) At first, the kinase and phosphatase were found to average ^ less than for a procedure of placing the of dTMP compete in reducing the dTMP concentration to a aliquots in 80% ethanol before applying to the paper. But the low level. (6) After the dTMP level gets low enough, a thymidine direct application procedure resulted in much smoother time curves. The treated to control ratios were essentially the same kinase action can often be seen to cause an actual downturn in by both procedures. the thymidine curve, (c) The dTDP peak occurs well after the "Pabst Laboratories, Circular OR-18, April, 1961. Solvent IV. dTTP peak, which might possibly be taken to indicate that JULY I960 1407 Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1966 American Association for Cancer Research. Arthur J. Tomisek, Martha B.