Deoxyribonucleic Acid Synthesis I. Effect of in Vivo Cyclophosphamide

Home , Deoxyribonucleoside, Thymidine diphosphate

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 expÃ©rimente) 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.

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Downloaded from cancerres.aacrjournals.org on October 1, 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 /Â¿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 37Â°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-ÃŸ\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 mamÃ³les/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

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TABLE I groups of experiments with several other substrates in order to COMPARISONOF DNA SYNTHETASEACTIVITY AS MEASURED gain information on the fate of other substrates. BY VARIOUS RADIOACTIVESUBSTRATES With dAMP-14C as the radioactive substrate, the Chromato

ACTIVITY"SensitiveSYNTHETASE graphie analysis showed that dATP reached a very high peak in a very short time (Table 2). Evidently the tumor is much better EXPEIIICENT SUBSTRATEdTMP-2-'4CfdAMP-8-Â»C\dCMP-3HACTIVITY NO.10A10BIOC20ALABELED (mc/mmole)9.032.42200 supplied with dAMP kinase (and also clGMP and dCMP kinases, control4.33.00.065Â«treated"1.11.40.017'control0.2G0.400.26as will be noted below) than with dTMP kinase. Cyclophospha- mide treatment had no inhibitory action on the dAMP kinase even though this same pair of enzymes exhibited a treated-to- control peak height ratio of 0.33 when tested with dTMP-14C. The dADP (though not completely separated from dATP) was dGMP-8-Â»CfdTMP-2-HC\dCMP-2-14CSPECIFIC28.08.4316.0DNA4.415.35.3Sensitive1.33.11.06Treated/0.290.200.20 obviously reaching its peak concentration well after the dATP peakâ€”again bringing up the possibility that the main pathway of the triphosphate synthesis might be directly from the mono- phosphate rather than via the diphosphate. The only degradation " The data for 14C are in terms of cpm fixed per 0.8 min, cor products present in more than trace amounts were uric acid, rected to a common specific activity of 10 mc/mmole. hypoxanthine, and xanthine; and even these remain at sur b Both sets of enzymes were derived from tumors in 5-day ex prisingly low levels (< 10% each) for 3 hr. In fact, of the 6 sub periments. c These values are not corrected for counting efficiency, and strates used, the dAMP was by far the most resistant to degrada are thus not comparable to the MC data. tive reactions. With AMP-"C as the radioactive substrate and GMP, cytidylic dTTP is derived directly from dTMP rather than from dTDP.7 acid, uridylic acid as the nonradioactive substrates, the con (d) In the end, almost all of the 14C is present as thymidine, version of AMP to ATP was practically quantitative within the 10-min preincubation period (Table 2). Degradative reaction to though this conversion is probably not of importance during the inosinic acid was very rapid (37% in 2 hr), with lesser amounts time when the polymerase reaction is proceeding at its initial, of uric acid, hypoxanthine, and xanthine. No conversion of linear rate, (e) Free thymine concentration also builds up, but AMP to dAMP was detected. this reaction is certainly too slow to be of importance in these With dGMP-"C as the radioactive substrate (Table 2), the studies. conversion of dGMP to dGTP was too rapid to be followed by The curve for dTTP (Chart 2) is thus the only one of direct our assay system. Conversion of radioactivity to uric acid also significance to the present study. We determine the peak of the proceeded at a very rapid rate (to 70% in 40 min for the control curve (heavy dot) and note the corresponding values on the 2 run). In this particular case, poor Chromatographie separations axes (arrows). Within experiments using enzymes prepared in and the extreme speed of the reaction cause the comparison of parallel, the enzymes exhibiting the higher peaks tended to be the ones with the earlier peak times. The peak time is easy to under stand in terms of dTMP kinase activity: the more enzyme activity, the less time required to attain a given concentration of dTTP. The peak height, on the other hand, represents a tran sient equilibrium between the kinase and the several enzymes utilizing dTTP: it represents a crude measure of the kinase activity relative? to all the competing reactions. Of the 2 measures of dTMP kinase activity, we find that the peak time has the greater variability from 1 series of experiments to another; and thus in a quantitative sense, we consider the peak height to be the more dependable. The conclusions of this study are based on experiments with dTMP as the radioactive substrate; but we carried out single

7 However, the evidence of (irav and Smelile (4) for Landschutz ascites cells and of IvÃ©s(6) for NovikotT hcpatoma is much more definitive in establishing the dTDP as an intermediate between dTMP and dTTP. 40 ' 80 8 On the basis of this measurement we have no reason for dis 120 160 200 240 cussing the results in terms of decreased kinase activity rather than increased phosphatase activity, but it would be more effi Reaction time (min ) cient foraceli to control anabolic products by controlling anabolic CHART 2. An example of the time-course of HC distribution rather than eatabolic reactions. Anyhow, we are not concerned among various reaction mixture components. This example cor that the observed dTMP kinase activity may be in some measure responds to the resistant-control curve of Chart 1. Abbreviations relative rather than absolute, since it is only the relative activity used are: dTTP, thymidine-5'-triphosphate; dTDP, thymidino- which is important to the polymerase enzyme. 5'-diphosphate; dTMP, thymidine-5'-phosphate.

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TABLE 2 stimulating effect on the DNA synthetase activity of the cell RATES OF NUCLEOSIDE TRIPHOSPHATE FORMATION FROM extracts (Table 4). This is significant because, in our present VARIOUSNUCLEOSIDEMONOPHOSPHATESUBSTRATES ignorance of the stimulation mechanism in sensitive plasmacytomas, we have no reason to expect a correlation of the short- TRIPHOSPHATEControlMax.OF THE CORRESPONDING term stimulation with long-term inhibition. COMPARISON OF TREATED AND CONTROL SENSITIVE PLASMA- SUBSTRATEEnzymeÂ»C treatedMai. CYTOMAS.Twohr of in vivo treatment with cyclophosphamide (%)Â«9689483*79(min)205040105-Day(%)Â«3809666Â»822419Time(min)400003530result in a cell extract with slightly increased DNA synthetase activity (Table 5); but 5 days of in vivo treatment with cyclo 10dTMP-2-Â»CdAMP-8-Â»CAMP-8-14CdGMP-8-Â»CGMP-8-'4CEnzymeset No. phosphamide result in a cell extract with strongly decreased DNA synthetase activity. From the single 2-hr experiment with 4 times the usual cyclophosphamide dosage (Table 5), it would appear that this stimulating effect of inhibitor can be obscured b3481Time by excessive amounts of inhibitor per se, even apart from a longer reaction time. 20dTMP-2-14CdCMP-2-I4CPEAKset No. For either 2 hr or 3 days of in vivo treatment with cyclophos phamide, there is no inhibition of dTMP kinase activity (Table 3). In fact, there appears to be a small stimulation (null prob ability at 2 hr = <0.25), which we cannot explain, but which 0 This refers to the maximum triphosphate as a % of total does correlate with the slight stimulation observed in the DNA "C, as determined by the procedure of Chart 2. synthetase activity (Table 5) of 2-hr experiments. <â€¢"Instantaneous"values (minus 10 min). No regular peak.

"peak height" values to be undependable; but in any case, this TABLE 3 kinase reaction is far from being a rate-limiting factor in the VARIATIONOF dTMP KINASE ACTIVITYWITH CYCLOPHOS PHAMIDETREATMENTANDWITHTUMORSTRAIN over-all synthesis of DNA. With dCMP-l4C as the radioactive substrate, dCTP concen Av. dTTP MAXIMUM(%)<â€¢ tration increased to about the same ]>eak as the dATP and TREATMENTPERIOD5 dGTP (Table 2). However, unlike the kinases for dAMP and control21.3 control29.7 treated13.1 treated32.4 dGMP, the kinase(s) for dCMP in this single experiment indi (7)11 cated a drop in activity due to cyclophosphamide treatment. days (6) (7) (0)34.7 COMPARISON OF SENSITIVE AND RESISTANT CONTROL PLASMA- 3 days 22.7 (3) 32.3 (3) 34.0 (3) (3) CYTOMAS.Thesensitive subline of the plasmacytoma has about 2hrAll 20.3(4)21.3 32.5(4)31.0 26.0 (3)Resistant39.7 (3) two-thirds as much dTMP kinase activity as the resistant subline (Table 3). After sensitive-to-resistant peak height ratios are experimentsSensitive (14)Resistant(13)Sensitive calculated for the 13 separate experiments, the application of Student's t test defines the 5% confidence interval as 0.67 Â± " This refers to the maximum dTTP as a % of total 14C,as de termined b}' the procedure of Chart 2. 0.11. No other enzymatic difference between the tumor sublines 6Figures in parentheses indicate number of experiments aver was evident from the Chromatographie curves; but the sensitive aged. tumors do grow slower and they have a lesser DNA synthetase activity. The latter point is shown in the combined data for 2-hr, 3-day, and 5-day experiments (data not in any table), TABLE 4 where the average sensitive to resistant ratio, with its 95% THE RELATIVEDNA SYNTHETASEACTIVITYOFCYCLOPHOSPHA- confidence interval, is 0.80 Â±0.11. MIDE-TUEATED AND CONTROL RESISTANT PLASMACYTOMAS

It may be only a coincidence that the ratio 1.25 (the reciprocal TREATEDTOCONTROLRATIOSOF INITIAL of this 0.80 ratio) represents almost exactly the extent by which RATESOP DNA SYNTHESIS 2-hr cyclophosphamide treatment stimulates the DNA syn -daytreatment1.27 thetase system of sensitive tumor (see below and Table 5). But treatment1.00 treatment0.90 on the other hand, it is possible that the cyclophosphamide "stimulation" after 2 hr is really a case of releasing an inhibition SeparateexperimentsAverage which is present in sensitive tumors but absent from resistant 0.971.001.01 1.240.851.00 1.040.951.020.831.301.07 tumors. COMPARISON OF TREATED AND CONTROL RESISTANT PLASMA- CYTOMAS.Asexpected from the fact that this variant of plasma cytoma is cyclophosphamide-resistant, 5 days of cyclophosphamide treatment had no inhibitory effect on either the DNA ratio synthetase activity (Table 4) or the dTMP kinase activity 90% confidence interval"2-hr 0.93-1.093-day0.64-1.365 0.92-1.22 (Table 3). Neither did the 2-hr cyclophosphamide treatment have any Calculated by Student's t distribution.

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TABLE 5 continue (Table 7). However, this degree of inhibition is without THE RELATIVE DNA SYNTHETASE ACTIVITY OF CYCLO- effect (null probability > 0.50) on the soluble protein content of PHOSPHAMIDE-TREATED AND CONTROL SENSITIVE the cell. PLASM ACYTOMAS With the sensitive tumors, the cyclophosphamide causes an actual regression of tumor size. Although tumors with necrotic TO CONTROLRATIOSOFINITIAL SYNTHESIS2-hrRATESOF DNA areas were not used in these experiments, there is a clear tendency (null probability for 6th day data = <0.10) for regressing treatment1.29 treatment0.73 treatment0.09 tumors to yield somewhat less soluble protein than growing tumors. This trend of protein concentration is not in the direction SeparateexperimentsAv. which would provide an alternate explanation (dilution) to our 1.171.181.21Â«1.09-1.333-day1.031.241.000.57-1.43S-day0.530.260.480.500.180.200.320.19-0.45observed decrease in DNA synthetase activity.

Discussion

SEQUENCE OF CYCLOPHOSPHAMIDEEFFECTSON ENZYME AC TIVITIES. The differences observed in the several types of meas urement are individually significant, but the most interesting ratio90% results of this study are in the correlation of these effects as they confidence interval6TREATED concern the sensitive treated tumors. It is useful to discuss this " The ratio for 1 experiment with 4 times the usual inhibitor in terms of the time sequence of cyclophosphamide effects. But dosage is 0.91. it might be pointed out that what is called a time sequence is 6 Calculated by Student's t distribution. also a sequence of increasing (cumulative) inhibitor dosage. The 1st step in the time-response sequence is the stimulation of TABLE 6 the DNA synthetase system at 2 hr (Table 5). Since we know AVERAGE PROTEIN CONTENT (mg/ml) OF SOLUBLE CELL nothing of the nature of this stimulation, we have no way of FRACTIONS" knowing whether this 1st step is at all related to the later steps. The stimulation of dTMP kinase activity at 2 hr (Table 3) is TYPE AND TREATMENT OF PLASUACYTOMA possibly related. TIME2 The 2nd step in the time-response sequence seems to be the control15.8 control13.9 treated15.6 treated12.2 cessation of growth. In our own experiments we observed com plete cessation of growth on the 4th day following the start of hr (4)* (4) (3) (3)17.4 cyclophosphamide treatment (Table 7). Other work from our 4th day 17.1 (3) 15.5 (3) 15.9 (3) (3) laboratory has shown that these tumors continue to grow for 6th daySensitive15.3 (7)Resistant14.3 (7)Sensitive13.1 (7)Resistant14.9 (7) either 1 or 2 days after the start of treatment (16). " In the "Methods" section, these are the fractions "assayed The 3rd step in the time-response sequence appears to be the for protein." decrease in activity of the DNA synthetase system. The evidence 6 The numbers in parentheses indicate the number of experi for putting this step after the growth inhibition step is not as ments involved in the average. convincing as for the other conclusions of this study, but in all we think it constitutes a reasonable probability. For one thing, It is only in the 5-day experiments that inhibition of dTMP the average DNA synthetase activity in the 3-day experimentsâ€” kinase becomes appreciable. taken at face value despite the poor statistical agreementâ€”shows EFFECTS ON TUMOR SIZE AND PROTEIN CONTENT. From the no decrease of DNA synthetase activity (Table 5). In another data of Table 6 there is some indication that, for all the tumors, line of reasoning, we could stop looking for the time when the the soluble protein content of the tumor passes through a peak effects first become noticeable, and look instead for the time at about the 4th day of the experiment9 (which is about the 17th when the effects approach completion. The same data from the day after tumor implantation). However, this seems to be unim portant with regard to our purposes. TABLE 7 As shown above in comparing sensitive and resistant control TUMOR WEIGHTS AT VARIOUS PERIODS OF TIME AFTER IST tumors, the faster growing, resistant tumor has the greater DNA TREATMENTWITH CYCLOPHOSPHAMIDE synthetase activity, but according to Table 6 it has the lesser content of soluble protein. Relating to the latter conclusion the Av % OF INITIAL WEIGHT mean difference between the sensitive and resistant pairs for all TIME(day)4th5th6thSensitive reaction times is 1.48 mg/ml with a standard deviation of 0.35. control197 control215 treated81 treated161 The growth of the resistant tumors is markedly inhibited by the cyclophosphamide treatment, even though the growth does (9)Â°212 (10)225 (9)60 (10)171 (9)218 (10)231 (9)46 (10)175 (6)Resistant (7)Sensitive (6)Resistant (7) 9Null probabilities comparing 2-hr and 5-day data with 3-day data (sensitive control, resistant control, plus resistant treated " The numbers in parentheses indicate the number of experi data only) were both between P = 0.025 and P = 0.01. ments involved in the average.

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3-day experiments can be considered in this other way. We can 'S LO not be sure the synthetase activity is not decreased by the 4th "5 day, but we are reasonably sure of a decrease between the 4th "o and 6th days.10 By contrast, the growth inhibition is always complete in 3 days, sometimes even in 1 day. Further evidence o 0.8 can be drawn from the analogy with resistant tumor, for which o a marked decrease in growth rate (Table 7) is not accompanied â€¢o by any decreased DNA synthetase activity (Table 4). o As a final consideration, we propose to divide the 3rd step into 0.6 2 on the basis of "normal," uncontrollable differences in degree of inhibitor response from 1 experiment to another. This con sideration is not strictly one of a time series, but, as pointed out above, the whole series could just as well be considered in terms of increasing inhibitor response. The evidence for the 4th step lies in the data of Chart 3, wherein, for each of the seven 5-day experiments, the inhibition of the over-all DNA synthetase system is compared to the inhibition of its dTMP kinase system 0.2 component. One important conclusion from Chart 3 is that some component of the over-all DNA synthetase reaction can be strongly inhibited (to about 50%) while the dTMP kinase system is inhibited hardly at all (to about 10%). The other conclusion 0.2 o.4 0.6 0.8 1.0 is that strong inhibitions in the dTMP kinase system are paral dTMP kinase activity (treated to control ratio) leled by DNA synthetase inhibitions in excess of 50%. A measure of the statistical dependability of these conclusions CHART 3. A comparison of cyclophosphamide effects on the deoxyribonucleic acid synthetase and the thymidylatc kinase is to be noted in the 0.53 Â±0.20 slope of the curve in Chart 3, systems of sensitive Fortner plasmacytomas in 5-day experiments. where the slope is calculated by the least squares method for a curve passing through the origin, and the limits indicated are for 99% probability in Student's t test. This slope is to be com regard to the many experiments in which we observed the pared to the 1.00 slope of the hypothetical curve which would activity of the dTMP kinase system, a 3- to 4-fold difference have been obtained if the 2 enzyme systems had been equally between the zero time and the peak time dTTP concentrations inhibited. Thus, the decrease in dTMP kinase activity is indicated was usual. Perhaps more illustrative were the rare experiments as the 4th step in the time-response series, and the 3rd step in which the inflexion point of the synthetase curve occurred at remains as a complex system requiring further definition in or near (in 1 case even a little before) the time of peak dTTP enzymatic terms. concentration. Redefining the 3rd step in more specific terms, we find that the Having thus concluded that the 4 kinase systems were not critical reaction is seemingly that of the DNA nucleotidyl likely to be responsible for the observed 3rd step in the time- transferase. We do have evidence that the critical reaction is not response sequence, we are left with the DNA nucleotidyl trans one of the 4 kinase systems. One type of evidence provided by ferase as the only other known critical component of the syn our data is applicable to all 4 kinase systems: namely, the over thetase system. We are nonetheless mindful that the synthetase all reaction rate of the synthetase system remains very linear system may eventually be proven to contain a rate-limiting over a period during which the concentrations of the 4 triphos- DNA primer activating factor, such as the DNA phosphatase- phates vary over a wide range. For the specific case of dGMP endonuclease of Escherichia coli (11); and that such a hypo (Table 2, Experiment 10) the linear period of synthetase reaction thetical factor would also be a candidate locus for the 3rd step encompassed a 3-fold range of dGTP concentration: 58% at in the time-response sequence of inhibitions. Liss and Palme (8) zero time to 20% at 45 min. For the specific case of dCMP have studied the short-term effects of the alkylating agents (Table 2, Experiment 20) the linear period of synthetase reaction nitrogen mustard oxide and 2,3,5-triethylenimino-l,4-benzo- encompassed a 7-fold range of dCTP concentration: 81% at the quinone upon Ehrlich ascites cells. Their results were similar to 5-min peak to 12% at 85 min. For the specific case of dAMP our own in that the polymerase appeared to be strongly inhibited (Table 2, Experiment 10) the linear period of synthetase reaction while inhibition of the thymidine kinase system was either includes a 4-fold range of dATP concentration: 68% at the 5-min secondary or absent. peak to 18% at 55 min. In addition, we have noted above that THE REGULATIONOFGROWTH.Of the 4 kinase systems, only the dAMP kinase system was not likely to be rate limiting in that of dTMP is commonly reported to be absent from non- the over-all reaction, since the rate of conversion to dATP and growing tissues (2, 5). This suggests that dTMP kinase might the rate of dAMP degradation were both more favorable than have a special role in controlling DNA synthesis, but Bollum and Potter (2) were unable to determine whether or not the kinase for the other 3 deoxyribonucleotide monophosphates. With preceded the polymerase in time of appearance following partial hepatectomy. Weissman et al. (15) found that, following partial 10Note the lack of overlap for the confidence intervals for the hepatectomy, the kinases for thymidine, dTMP, and dTDP 3- and 5-day experiments (Table 5). appeared sequentially in that order. While our data deal with

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1966 American Association for Cancer Research. Arthur J. Tomisek, Martha B. Irick, and Paula W. Allan the order of disappearance of enzymes in a regressing tissue Landschutz Ascites-Tumor Cells. Biochem. J., 89: 486-91, rather than the order of appearance in a tissue about to begin 1963. growing, it is nonetheless pertinent that we find evidence for the 5. Hiatt, H. H., and Bojarski, T. B. Studies of Thymidylate disappearance sequence: growth, polymerase, and dTMP kinase Kinase Activity in Mammalian Tissues. Federation Proc., 19: 309, 1980. system. The latter .sequence is not consistent with either the 6. IvÃ©s,D.H. Evidence for Thymidine Diphosphate as the Pre dTMP kinase system or the polymerase playing a regulatory cursor of Thymidine Triphosphate in Tumor. J. Biol. Chem., role in growth. Indeed, this would seem to imply that the primary 240: 819-24, 1965. site of cyclophosphamide action might better be sought among 7. Kielly, R. K. Patterns of Synthesis of Thymidine Triphos- the control mechanisms of growth, as distinct from the enzymatic phale and Other Deoxyribonucleoside Triphosphates in Mouse capacity for the various DNA synthesis steps. Liver and in Mouse Ascites Hepatoma. Cancer Res., 23: 801- EFFECTOF CYCLOPHOSPHAMIDEONTHE dCMP KINASESYS 10, 1963. TEM.Kielly (7) has shown that, in extracts of mouse hepatoma 8. Liss, E., and Palme, G. Der Einflusz von alkylierenden Cyto- statica auf den Nncleinsaurestoffwechsel von Ehrlich-Ascites- cells, the activity of the dCMP kinase system was almost as Tumorzellen. Z. Krebsforsch., 66: 196-206, 1964. low as that of the dTMP kinase system. This does bring up the 9. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, possibility that in certain cases the dCMP kinase system might R. J. Protein Measurement with the Folin Phenol Reagent. act as a control on DNA synthesis; but we were nonetheless J. Biol. Chem., 193: 265-75, 1951. surprised that the single experiment we carried out with dCMP- 10. Mantsavinos, R., and Canellakis, E. S. Studies on the Biosyn 14Csubstrate (Table 2) showed a rather strong decrease of dCMP thesis of DNA by Cell-free Extracts of Mouse Leukemic kinase activity in plasmacytoma due to treatment for 5 days Cells. Cancer Res., 19: 1239-43, 1959. with cyclophosphamide. The exact place of dCMP kinase ac 11. Richardson, C. C., and Kornberg, A. A. Deoxyribonucleic tivity loss in the time-response series is hardly to be inferred Acid Phosphatase-Exonuclease from Escherichia coli. J. Biol. from the data we have thus far. Chem., 239: 242-50, 1964. 12. Skipper, H. E., and Schabet, F. M., Jr. Experimental Evalua tion of Potential Anticancer Agents. VII. Cross Resistance of Acknowledgments Alkylating Agent-Resistant Neoplasms. Cancer Chemother The authors wish to thank Mr. Charles A. Kelley and Miss apy Rept., gg: 1-22, 1962. Tommie L. Barker for the animal work, Miss Mary Kate Tinkle- 13. Tomisek, A. J. and Allan, P. W. Water Content of Paper as a paugh and Mr. Thomas C. Herren for the radioactivity measure Variable in Paper Chromatography. J. Chromatog., 14: 232- ments, Miss Elizabeth A. Dulmadge for protein assays, and Dr. 37, 1964. William S. Wilcox for advice on statistical procedures. 14. Turba, F., and Turba, M. Trennung von AbkÃ¶mmlingender adenosintriphosphorsÃ¤ure (ATP) durch Verteilungschroma tographie in Filterpapier. Naturwissenschaften, 38: 188-89, References 1951. 1. Bollum, F. J. Thermal Conversion of Nonpriming Deoxyribo- 15. Weissman, S. M., Smellie, R. M. S., and Paul, J. Studies of nucleic Acid to Primer. J. Biol. Chem., 234: 2733-34, 1959. the Biosynthesis of Deoxyribonucleic Acid by Extracts of 2. Bollum, F. J., and Potter, Y. R. Nucleic Acid Metabolism in Mammalian Cells. IV. The Phosphorylation of Thymidine. Regenerating Hat Liver. VI. Soluble Enzymes which Convert Biochem. Biophys. Acta, 45: 101-10, 1960. Thymidine to Thymidine Phosphates and DNA. Cancer Res., 16. Wheeler, G. P., and Alexander, J. A. Studies with Mustards. 19: 561-65, 1959. VI. Effects of Alkylating Agents upon Nucleic Acid Synthesis 3. Fortner, J. G., Mahy, A. G., and Cotran, R. S. Transplant able in Bilaterally Grown Sensitive and Resistant Tumors. Cancer Tumors of the Syrian (Golden) Hamster. Part II. Tumors of Res., 24: 1338^6, 1964. the Hematopoietic Tissues, Genitourinary Organs, Mammary 17. Wheeler, G. P., and Stephens, Z. Studies with Mustards. VII. Glands, and Sarcomas. Ibid., Â£/(Supp.): 199-234, 1961. Effects of Alkylating Agents in Vitro and in Vivo upon the 4. Grav, H. J., and Smellie, R. M. S. The Mechanism of For Thermal Properties of DNA's from Sensitive and Resistant mation of Thymidine 5'-Triphosphate by Enzymes from Plasmacytomas. Ibid., 25: 410-16, 1965.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1966 American Association for Cancer Research. 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 Plasmacytomas

Arthur J. Tomisek, Martha Bruce Irick and Paula Wedeles Allan

Cancer Res 1966;26:1466-1472.

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