Effects of Nitrogen Mustard and Cyclophosphamide Upon the Synthesis of DNA in Vivo and in Cellâ€”Freepreparations'

[CANCER RESEARCH 29, 98â€”109,January 1969]

Effects of Nitrogen Mustard and Cyclophosphamide upon the Synthesis of DNA in Vivo and in Cellâ€”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] 24â€”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

Determination of the Effects of Agents upon the Fixation of In the standard procedure, the reaction mixture minus the â€˜4Cfrom Formate-'4C and from Adenine-8-14C. Hamsters primer was incubated at 37Â°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. Only one of the deoxyribonucleoside mg per kg) or nitrogen mustard (0.5 mg per kg), and, at speci phosphates was radioactive in each reaction mixture. The fol fled times thereafter, groups of three animals were given intra lowing radioactive substrates, having the indicated specific peritoneal injections of thymidine-C3H3 (specific activity, activities in millicuries per millimole, were used: thymidine 75.3 mc per millimole) at a dosage of 2 pc per gram of body 2-'4C monophosphate (8.5, 13.6, or 29.4); deoxycytidine weight. Animals of the control group received injections of 2-'4C monophosphate (29.2); deoxyadenosine-8-'4C mono physiologic saline and thymidine-C3H3. The hamsters were phosphate (7.7); deoxyguanosine-8-'4C monophosphate killed 2 hours after receiving the radioactive compound, and (14.4); deoxyadenosine-8-'4C triphosphate (8.58, 10.0, or the tumors of the three animals were removed, pooled, minced 17.15); thymidine-2-'4C triphosphate (43.7). The enzyme with knives, and passed through a tissue press. A portion of solution was added to the mixture to give a final concentration the tissue was washed two times with 5 percent trichloroacetic of 7.81 mg of protein per ml, and primer DNA was added to acid, two times with water, two times with absolute ethanol, give a concentration of 0.46 mg per ml. Unless otherwise and two times with ether. After the tissues had dried overnight stated, the primer consisted of salmon sperm DNA that had at room conditions, they were ground with a mortar and pestle been denatured by heating an aqueous solution (4 mg of DNA and desiccated over calcium sulfate overnight or longer. Dupli per ml of water) in a boiling water bath for 10 minutes and cate samples (3â€”5mg) of the dried powder were incubated for then rapidly cooling in an ice bath for 15 minutes; it was used 24 hours at 37Â°C with 1 ml of 1 M solution of hydroxide of immediately after preparation. The final volume of the incuba Hyamine [p-(diisobutylcresoxy-ethoxyethyl)dimethylbenzyl tion mixture was 1.25 ml. ammonium hydroxide] in methanol. After the addition of 14

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1969 American Association for Cancer Research. Glynn P. Wheeler and Jo Ann Alexander ml of a toluene solution of scintillators [4 gm of 2,5-diphenyl Io@ooo SENSITIVE RESISTANT oxazole and 50 mg of 2,2'-p-phenylenebis(5-phenyl-oxazole) per liter of toluene] , the samples were assayed for radioactiv ity in a Tri-Carb liquid scintillation spectrometer. a' E The remainder of the wet tissue was used for the preparation of soluble extract by the procedure described above, and the a extract was then assayed for DNA nucleotidyltransferase activ 4,a .a ity. 4, a RESULTS U 0 0 Effects of Single Doses of Agents upon the Growth of

Sensitive and Resistant Plasmacytoma. Chart 1 shows that 0 2 4 6 0 2 4 6 nitrogen mustard at a dosage of 0.5 mg per kg caused tem porary cessation of the growth of the sensitive tumor and that, Days after injection of nitrogen mustard at dosages of 1.0, 2.0, or 3.0 mg per kg, the agent caused regression in the size of the sensitive tumors. The tumors of Chart 1. Effects of nitrogen mustard upon the growth of bilaterally the animals receiving the larger doses continued to grow at growing cyclophosphamide-sensitive and cyclophosphamide-resistant about the normal rate for 1â€”2days following the treatment plasmacytomas. The numbers on the curves indicate the dosages in mg before a decrease in tumor size began. At none of these dos per kg for a single intraperitoneal injection. The calculated weights of ages did nitrogen mustard do more than temporarily slow the the tumors were based upon external caliper measurements, and each point on the curve is the mean value for 3 to 6 tumors. growth of the bilaterally growing resistant tumors. (The anomolous results obtained on Days 5 and 6 for dosages of 0.5 and 1 .0 mg per kg with the resistant tumor are probably not SENSITIVE RESISTANT significant.) 4,000 Chart 2 shows the effects of single doses of cyclophospha mide upon the growth of sensitive and resistant plasmacy I 2@00 tomas. The results are similar to those obtained with nitrogen mustard, but, as would be expected, higher dosages were re quired. I OpO 0 a, Effects of Single Doses of Agents upon the Fixation of â€˜4C E of Formate-'4C and of Adenine-8-'4C. Chart 3 shows the 8,000 effects of two dosages of nitrogen mustard upon the incorpo 0' ration of 14 C from formate-14 C and from adenine-8-14 C into 4, RNA, DNA, and certain compounds of lower molecular 6@00 @. @0 weight. At a dosage of 0.3 mg of nitrogen mustard per kg 4, (which would cause little inhibition of growth of the sensitive 0 tumor), there was inhibition of incorporation of 14 C of for U 4,000 @0I0O 0 mate-14C into soluble purines and related compounds and into 0 the purines of RNA and DNA. Only the data for the nucleic 2,000 acid adenine are shown, but similar effects were observed for guanine. The curves go through maxima during the first 16 0 hours with subsequent inhibition of incorporation. The in 3 5 hibition of incorporation into DNA was greater than that into RNA. The results for the sensitive and resistant tumors were Days after injection of cyclophosphamide similar, but there was some indication of â€œrecoveryâ€•bythe Chart 2. Effects of cyclophosphamide upon the growth of bilaterally resistant tumor at 48 hours. At this dosage of nitrogen mus growing cyclophosphamide-sensitive and cyclophosphamide-resistant tard there was less inhibition of incorporation of 14C of ade plasmacytomas. The numbers on the curves indicate the dosages in mg nine-8-14C into adenosine phosphates and into the adenine of per kg for a single intraperitoneal injection. The calculated weights of RNA and of DNA than occurred for formate-14 C. With ade the tumors were based upon external caliper measurements, and each nine-S-14 C as the substrate, maxima of fixation were again point on the curve is the mean value for 3 to 5 tumors. observed during the first few hours following injection of the nitrogen mustard, and the inhibition of incorporation into of 14C of formate-14C were generally similar to those ob DNA exceeded that into RNA. There was evidence of â€œre tamed at the lower dosages. However, at the higher dosage coveryâ€• of the normal rates of incorporation by 48 hours there was more inhibition of incorporation of 14C of adenine following treatment for both the sensitive and resistant 8-'4C into the adenosine phosphates and the adenine of RNA tumors. and DNA, and there was less evidence of â€œrecoveryâ€•by 48 When the dosage of nitrogen mustard was increased to 2.0 hours. Similar results were obtained for the sensitive and resist mg per kg, the patterns of the effects upon the incorporation ant tumors.

100 CANCER RESEARCH VOL.29

SENSITIVE RESISTANT SENSITIVE RESISTANT

200

14 â€˜4 â€˜4 â€˜4 160 Formate-. C Formate.- C Formate- C Formate-. C

I 20

C 0 C) 20@ 0 Adenine-8 Adenine- 8@4C Adenine-.8.- Adenine.-8)4C

0 16 32 48 0 16 32 48 0 16 32 48 0 16 32 48 Hours after nitrogen mustard

Chart 3. Effects of single doses (0.3 mg or 2.0 mg per kg) of nitrogen mustard (HN2) upon the in vivo fixation of !4C of formate-14C or of adenine-8-'4C into the adenine of RNA (RNA-Ad), adenine of DNA (DNA-Ad), soluble purines and related compounds (Pu), and adenosine phosphates (AdP) of sensitive and resistant plasmacytomas. Pu: hypoxanthine, xanthine, adenosine, inosine, adenosine monophosphate, adenosine diphosphate, adenosine triphosphate, inosine monophosphate, guanosine monophosphate, uric acid, and allantoin. AdP: the mono-, di-, and triphosphate derivatives of adenosine. The animals were killed 2 hours after the administration of the labeled substrate. The values for RNA-Ad and DNA-Ad are based upon the specific activity of the adenine; the values for Pu and AdP are based upon the sums of the total activities of the respective compounds.

Chart 4 shows the results obtained with cyclophosphamide. When the substrate was adenine-8-1 4C and the dosage of At a dosage of 10 mg per kg (a dose that was sufficient to cyclophosphamide was 10 mg per kg, stimulation of incorpora cause regression of the sensitive tumor), maxima showing tion of the 14C into adenosine phosphates and into RNA and much stimulation of incorporation were observe4 for both DNA occurred during the fkst few hours following treatment. tumors during the first few hours after treatment; delayed This period of stimulation was followed by a period of inhibi inhibition of incorporation of â€˜4Cfrom formate-1 4C into the tion for the sensitive tumor but not the resistant tumor. At a soluble purines and related compounds and into the KNA and dosage of 20 mg of cyclophosphamide per kg there was no DNA occurred in the sensitive tumor, but extensive stimula evidence of stimulation of incorporation of 14C of adenine tion of incorporation occurred in the resistant tumor. At a 8-'4C during the first few hours following treatment, and the dosage of 20 mg per kg, there was no extensive stimulation of extents of inhibition of incorporation of 14C were similar for incorporation during the first four hours, and there was more the sensitive and the resistant tumors during the 48-hour inhibition of incorporation into nucleic acids. periods of observation.

JANUARY 1969 101

Cyclophosphamide , 10 mg/kg Cyclophosphamide ,20 mg/kg

SENSITIVE RESIS TA NT SE@4SITIVE RESISTANT

-Ad

C 0 U

0 Adenine-8J4C Adenine-.8..14C Adenine-8- â€˜4C Adenine-8-14C

6 AdP

2 Ad

0 16 32 48 0 16 32 48 0 16 32 48 0 16 32 48 Hours after Cyclophosphamide

Chart 4. Effects of single doses (10.0 mg or 20.0 mg per kg) of cyclophosphamide upon the in vivo fixation of 14C of formate-14C or of adenine-8-'4C into the adenine of RNA (RNA-Ad), adenine of DNA (DNA-Ad), soluble purines and related compounds (Pu), and adenosine phosphates (AdP) of sensitive and resistant plasmacytomas. See the legend for Chart 3 for definitions of Pu and AdP. The animals were killed 2 hours after the administration of the labeled substrates. The values for RNA-Ad and DNA-Ad are based upon the specific activities of the adenine; the values for Pu and AdP are based upon the sums of the total activities of the respective compounds.

Although Charts 3 and 4 present data for single experiments, these experiments, but, since presentation of all of the data each experimental point on the curves was obtained for pooled would require 24 curves, the results are presented in abbrevi tumors from three to five animals. The similarities of the ated tabular form (Table 1). The data in the columns for the curves of Chart 3 to those of Chart 4 lend credence to the 10- to 30-minute interval reflect the relative rates of fixation general characteristics of the curves of both experiments. The of 14C during the period of most rapid fixation; the data in experimental points are connected by straight lines to indicate the columns for the 10- to 240-minute interval reflect the that â€œbestfitâ€•lines were not determined; therefore, the small relative quantities of 14C fixed when incorporation ceased. irregularities are probably not significant. The data ofTable 1 show that, at concentrations of 106 M, @ Effects of Treating Crude, Cell-free Extracts with Nitrogen 10â€” M, and i0@ M, there was little or no inhibition of @ Mustard upon DNA Nucleotidyltransferase Activity. Data for incorporation of the 4C of deoxyadenosine-8-' 4C triphos complete curves, such as those of Chart 5, were obtained in phate into the acid-insoluble material; in fact, there was some

102 CANCER RESEARCH VOL.29

U00

900

â€¢700 R-T ....----.

_...@ -.@ S-T 500 0. C) 300 0

@0 I00 â€˜C U- 600' dGMP@8- 400W

@ 20 0 S-c

@@@ 0 i i a I i â€¢ i , 0 40 80 120 160 200 240 0 40 80 120 160 200 240

Time of incubation (mm)

Chart 5. Effects of single doses of cyclophosphamide (20 mg per kg) upon the in vitro DNA nudeotidyltransferase activity of plasmacytomas when the animals were killed 48 hours after administration of the cyclophosphamide. Deoxyribonucleoside monophosphates were used as substrates in the assay system, and the labeled substrates in the respective assays are indicated. dAMP-8-14C, deoxyadenosine-8-14C monophosphate; dTMP-2.'4C, thymidine.2-14C monophosphate; dGMP-8-14C, deoxyguanosine-8-14C monophosphate; dCMP-2.1'C, deoxycytidine-2-14C monophosphate; R-C, control resistant tumor; R-T, treated resistant tumor; S-C, control sensitive tumor; S-T, treated sensitive tumor.

1Experiment Table of 14C of 14C during 10â€”30 mm during 10-240 mm @ of control)Fixation (% of control) no.ConcentrationResistant110â€”6 of HN2 (M)Fixation ResistantSensitive Sensitive 77 136 iOâ€”@ i@â€”@177 130 74 108 134 1022iOâ€”4 98 6291 80 220 165 iO@ 57 69 102 112 33iO@ 10292 1 2128 4 62 99 5 x 103 28 59 23 104 10â€”234 148 3 Effects of preincubating the enzyme preparations with nitrogen mustard (HN2) upon the fixation of I 4C from deoxyadenosine-8-' 4C triphosphate into the acid-insoluble material. The nitrogen mustard was added to the enzyme preparation, and the mixture was incubated at 37Â°Cfor 30 minutes prior to addition of this treated enzyme to the assay mixture.

JANUARY 1969 103

indication of stimulation of incorporation at these concentra Table 2 @ tions of nitrogen mustard. At concentrations of 10 M and of 14C of 14C greater, the nitrogen mustard treatment caused decreases in during 10â€”30 mm during 10â€”240mm (% of control) (% ofcontrol)Sensitive @ the rates of 4C fixation by the enzyme preparations from Concentration ResistantiO@100 both the sensitive and resistant tumors, with slightly greater of HN2 (M)Fixation Sensitive ResistantFixation 94iO@56 decreases being observed for the preparations from the sensi 95134 12810243 10983 tive tumor. At the higher concentrations of nitrogen mustard 8651 84 there were also decreases in the capacities (as indicated by the Effects of preincubating native salmon sperm DNA primer with nitrogen data in the last two columns) of the systems, and the decreases mustard (HN2) upon the fixation of â€˜4Cfrom deoxyadenosine-8-' 4C tn were greater for the enzymes from the sensitive tumors. The phosphate into the acid-insoluble material by enzymes from untreated sensi @ decreases in the rates of fixation of 4C during the interval tive and resistant plasmacytomas. The necessary quantity of nitrogen mus 10â€”30 minutes were greater than the decreases in the quanti tard was added to a solution of DNA in water (4 mg per ml), and the mixture was incubated at 37Â°Cfor 30 minutes prior to the addition of this @ ties of 4C fixed during the 10- to 240-minute periods of treated DNA to the assay mixture. incubation. In these experiments the crude enzyme preparations were treated with the nitrogen mustard for 30 minutes, and a por tion of this solution was added to the other components of the assay medium with a resultant dilution of 3- to 4-fold. It is (20mgperkg)tothehamsters,andcell-freepreparationswere recognized that some residual nitrogen mustard might be trans assayed for DNA nucleotidyltransferase with the deoxyribo ferred to the assay mixture and that it might react with some nucleoside monophosphates as substrates. The deoxyribo of these components and hence cause a decrease in the quan nucleoside monophosphates were present in equimolar quan tity of labeled substrate fixed into the acid-insoluble material. tities, but only one was radioactive. Incubation mixtures were The important point, however, is that, whether the observed set up in which each of the deoxyribonucleoside monophos inhibition is caused by reaction of the nitrogen mustard with phates was the labeled substrate. The values for the radioactiv one or more components of the enzyme preparation or with ity for the different labeled substrates have been adjusted to one or more components of the assay mixture, supraphysio bring them to a common basis for comparison; therefore, the logic concentrations of nitrogen mustard are required before curves indicate the relative rates of fixation of the various any inhibition occurs. substrates by the preparation from the sensitive and resistant Similarly, high concentrations of nitrogen mustard were re tumors and the@effects of the treatment thereon. The prepara @ quired to cause decreases in the rate of fixation of 4C into tions from the tumors of the treated animals had lower DNA acid-insoluble material by a DNA nucleotidyltransferase sys nucleotidyltransferase activity regardless of which labeled sub tem derived from leukemia L1210 ascites cells (29). strate was used. These results are consistent with those ob Effects of Treating Salmon Sperm DNA with Nitrogen Mus tamed by Tomisek et a!. (23) in experiments with preparations tani upon the Priming Activity of the DNA for DNA Nucleo from sensitive tumors following multidose treatment of the tidyltransferase. Table 2 contains the data of a single experi tumor-bearing hamsters. Chromatographic analysis of the in ment of this type. Treatment of native DNA with i0@ M cubation mixtures during the course of the assays showed that nitrogen mustard for 30 minutes had little or no effect upon the quantities of the metabolites of the respective labeled sub its priming activity for the DNA nucleotidyltransferase. After strates were similar for the preparations from the treated and treatment with i0@ M or 102 M nitrogen mustard, the untreated animals and that the decreased fixation of 14C by DNA served less efficiently as a primer in the incubation mix the samples corresponding to the treated animals was not due ture containing the enzymes from the sensitive tumor, while to a deficiency of substrate. there was less effect upon its utilization as primer in the mix Effects of in Vivo Treatment with Cyclophosphamide upon ture containing the enzymes from the resistant tumor. Since the Synthesis of DNA in Vivo and upon the DNA Nucleotidyl no steps were taken to remove unreacted nitrogen mustard transferase Activity in Vitro. The experiments described from the incubation mixture with the DNA prior to addition above showed that cyclophosphamide and nitrogen mustard of a portion of this mixture to the total incubation mixture, it interfere with the synthesis of DNA in vivo, that treatment in is possible that a portion of the observed inhibition could be vitro with nitrogen mustard at supraphysiologic concentrations due to the effects of the residual nitrogen mustard upon the can cause decreases in the DNA nucleotidyltransferase activity, enzymes. (There was a dilution of approximately 7-fold upon and that in vivo treatment could cause decreased DNA nucleo addition of the primer solution to the total transferase assay tidyltransferase activity in vitro. This experiment was per mixture.) Nevertheless, it is significant that treatment of the formed to determine whether the decreases of DNA synthesis DNA with i0@ M nitrogen mustard, which is probably a in vivo are accompanied by decreases in DNA nucleotidyltrans supraphysiologic concentration, had little effect upon the ferase activity of the very same tissues. Chart 6 shows that priming activity of the DNA. after 8 hours the decrease in in vivo synthesis of DNA ex Effects of in Vwo Treatment with Cyclophosphamide upon ceeded the decrease in DNA nucleotidyltransferase activity. the DNA Nucleotidyltransferase Activity in Vitro. Chart 5 Assays with monophospates and triphosphates yielded similar shows the results obtained in an experiment in which bilater results. ally growing plasmacytomas were taken from animals 48 hours The curves of Chart 7 show that a single dose of nitrogen following the administration of saline or cyclophosphamide mustard also caused decreases in the in vivo fixation of 3H of

104 CANCER RESEARCH VOL.29

00 100

0 80 C 60 0 0 0 0 40 4-. C 60 0 20 U 0 40 0 4 12 20 28 36 44 52 Hours after cyclophosphamide 20

Chart 6. Effects of single doses of cyclophosphamide (20 mg per kg) upon the in vivo fixation of 3H from thymidine-C3H3 into the acid-insoluble fraction of sensitive plasmacytomas and upon the in vitro IIIIIII nucleotidyltransferase activity of the same tumors when 0 thymidine-2-14C monophosphate (dTMP) and the supplementary 4 12 20 28 deoxyribonucleoside monophsophates or thymidine-2-14C triphosphate (dTTP) and the supplementary deoxyribonucleoside triphosphates were used as substrates for the assay system. The curves for the in vitro Hours after nitrogen mustard assays are based upon the quantities of 14C fixed during the 10- to 40-minute interval of the assay incubation. Chart 7. Effects of single doses of nitrogen mustard (0.5 mg per kg) upon the in viva fixation of 3H from thymidine-C3H3 into the acid-insoluble fraction of sensitive plasmacytomas and upon the in vitro nucleotidyltransferase activity of the same tumors when thymidine-C3H3 by sensitive tumors and in the DNA nucleo thymidine-2-14C monophosphate (dTMP) and the supplementary tidyltransferase activity of these tumors. deoxyribonucleoside monophosphates or thymidine-2-14C triphosphate These results differ with those obtained previously (29) in (dTTP) and the supplementary deoxyribonucleoside triphosphates were experiments with 1,3-bis(2-chloroethyl)-1-.nitrosourea and mice used as substrates for the assay system. The curves for the in vitro bearing leukemia L1210 ascites cells or leukemia L1210 solid assays are based upon the quantities of 14C fixed during the 10- to tumors; in those experiments, decreased in vivo fixation of 3H 40-minute interval of the assay incubation. The vertical lines show the of thymidine-C3H3 occurred in the absence of decreased activ range of values obtained in multiple experiments, and the curves are ity of DNA nucleotidyltransferase. drawn through the mean values. The circles on the vertical lines show the range of values for the experiments with dTMP; the horizontal bars on these same vertical lines show the range of values for the experi DISCUSSION ments with dTTP. The values for the in vivo curve are based upon 4 experiments; the values for the dTMP curve are based upon 2 experiments; the values for the d@VFP curve are based upon 5 The inhibition of synthesis of DNA by alkylating agents has experiments. been recognized as a common effect of these agents for a number of years (25, 26), but the cause of this inhibition has not been established. Although the present study does not The curves of Charts 3 and 4 show that maximum inhibition show the cause, it does yield some additional information rela of the in vivo synthesis of DNA does not occur immediately tive to this inhibition and seems to eliminate certain possible following the treatment, but rather it occurs several hours or causes. days later. Similar delays in attaining maximum inhibition Single-dose treatment of experimental animals permits deter have been observed in this laboratory for 1,3-bis(2-chloro mination of the effects of graded dosages and hence the rela ethyl)-1-nitrosourea (28) and by others with nitrogen mustard tive sensitivities of various metabolic events in the intact (8), with cyclophosphamide (17), and with uracil mustard (2). animal. Such treatment also permits a time-series study, which The maxima of specific activity, occurring during the first few yields information about the sequence of the appearance of hours following treatment, and corresponding to stimulation various effects following treatment and about the change in of synthesis of DNA when low dosages of nitrogen mustard or the magnitude of a single effect as time passes following the cyclophosphamide were used, are of interest. It is obvious that treatment. the observed effects of the agents are not unidirectional and

JANUARY 1969 105

are dependent on the time the observation is made, and this larly nitrogen mustard, it would be expected that the deactiva may be a partial explanation for the discrepancies in results tion of the enzyme would have to occur quickly after admin reported by various investigators. istration of the agent, and hence maximum decrease in syn Tomisek et al. (23) reported an average increase of 21% in thesis of DNA would occur more quickly than has been ob nucleotidyltransferase activity of plasmacytomas 2 hours after served (Charts 3, 4). (b) The concentrations of nitrogen mus a dose (10 mg per kg) of cyclophosphamide, but an average tard required to inactivate the DNA nucleotidyltransferase in 68% decrease in activity after 5 daily treatments (at the same vitro (Table 1) are much greater than would be expected fol dosage) and decreases of 9%, 23%@, and 33%3 after 2 hours lowing a single dose of nitrogen mustard at a level of 2 mg per with a dosage of 40 mg per kg. Some of the data of Table 1 of kg to a hamster. Smellie et al. (21) and Papirmeister (18) also the present report are consistent with stimulation of DNA found that supraphysiologic concentrations of nitrogen mus nucleotidyltransferase activity upon treatment of the enzyme tard and sulfur mustard were required to reduce the activities preparation with low concentrations of nitrogen mustard. One of the polymerizing enzymes involved in the synthesis of might speculate that this stimulation is due to deactivation of DNA. Ruddon and Johnson (19) have recently reported that an inhibitor and is observable when low dosages or low con incubation of a partially purified DNA polymerase from @ centrations of the agent are used, but is masked to various Escherichia coli with 10 M nitrogen mustard for 60 minutes extents by other effects when larger quantities of agents are caused a 30â€”35 percent decrease in enzyme activity, whereas used. It is more likely, however, that the stimulations observed incubation with nitrogen mustard at a concentration of iO@ in vivo are due to systemic effects of the agents upon the M had little effect upon the activity. animals, because transient stimulation also occurs at the same At present there is confficting evidence relating to the effects time for the de novo synthesis of purines, the conversion of of alkylation of DNA upon its functioning as a primer for an adenine to nudeotides, the synthesis of RNA, and the syn in vitro DNA-polymerizing system. The small quantity of data thesis of proteins (G. P. Wheeler and J. A. Alexander, unpub obtained in the present study (Table 2) indicate that it is lished data). Similar stimulation has been observed in experi necessary to treat commercially obtained salmon sperm DNA ments with 1,3-bis(2-chloroethyl)-1-nitrosourea (28). with supraphysiologic concentrations of nitrogen mustard to The similarities of the curves for the soluble compounds cause decreased primer activity. Ruddon and Johnson (19) formed from formate-' 4C and from adenine-8-1 4C to the found that the primer activity of native and denatured calf curves for RNA and DNA (see Charts 3 and 4) are consistent thymus DNA was depressed upon treatment with i0@ M and with the possibility that the decreased synthesis of RNA and ioâ€”â€•M nitrogen mustard for 4 hours. On the other hand, DNA might be at least partially due to decreased supplies of Goldstein and Rutman (11) found that in vitro alkylation of substrates. On the other hand, the decreased formation of DNA with nitrogen mustard enhanced its primer activity. these compounds of low molecular weight might result from There is also lack of agreement upon the effect of in vivo feedback inhibition or repression of formation of enzymes by alkylation of DNA upon the subsequent priming activity of accumulated substrates of nucleic acids. Lack of substrates is the isolated DNA in in vitro systems. Goldstein and Rutman evidently not the sole cause of decreased synthesis of DNA, (11) found that alkylated DNA had decreased primer activity, however, because less synthesis of DNA occurred in the cell but Tomisek and Simpson (24) found that their alkylated free preparations even when the -necessary substrates were DNA had greater primer activity. These discrepancies might be added to the incubation mixtures. Decreased capacity to phos due partially to differences in the biologic species and to dif phorylate deoxyribonucleotides to the corresponding triphos ferences in methods and technics of assay. phates is probably not a significant factor in the decrease of Although supraphysiologic concentrations of nitrogen mus synthesis of DNA, because similar results were obtained in the tard are required for the in vitro deactivation of the DNA in vitro experiments when monophosphates or triphosphates nucleotidyltransferase (Table 1) and the in vitro alteration of were used as substrates (Chart 6) and because the quantities of DNA resulting in decreased primer activity (Table 2), physio deoxyribonucleoside phosphates in the incubation mixtures logic concentrations of HN2 were sufficient to inhibit the during the assays represented by Chart 5 were similar for the incorporation of 14C from labeled substrates into the nucleic samples corresponding to the treated and the untreated tu acids of minced tumors (Table 3). The reason for this greater mors. These results are consistent with the observation of sensitivity of the intact system is not presently known. Tomisek et al. (23) that decreased thymidylate kinase was The time lag between the administration of nitrogen mustard observed only after 5 daily treatments of the hamsters with and the decrease in the rate of synthesis of DNA indicates that cyclophosphamide (10 mg per kg). this apparent inhibition of synthesis of DNA is a secondary Whether or not the decreased DNA nucleotidyltransferase effect of the agents rather than a primary effect. Studies re activity of the treated tumors contributes to the observed lated to the effects of this agent upon the cell cycle (see Ref. decrease in synthesis of DNA in vivo, it appears to be unlikely 26) indicate that the observed decreases in synthesis of DNA that the decrease in enzyme activity is completely due to are due to a decreased number of cells carrying out the syn direct deactivation of the enzyme by the alkylating agents. thesis of DNA rather than to inhibition of the synthesis per Se. This conclusion is derived from two observations: (a) Because This decrease might result from interference with the progress of the known short biologic half-lives of the agents, particu of cells through the other phases of the cell cycle, and, hence, fewer cells would be present in the S-phase a few hours follow ing treatment. There is some evidence (see Ref. 26) that cells kJnpublished results of A. J. Tomisek and B. S. Johnson. that are in the G2-phase at the time of treatment continue

106 CANCER RESEARCH VOL.29

3PrecursorConcentration Table control)AdenineSensitiveGuanineResistantAdenineGuanineFormate-14C activity (% of

of HN2 (M)Speciuic

ioâ€”@ <22 <20 <24 <25 iO@ 0 0 0 0 ioâ€”@ 59 48 85 89 13Adenine-8-14Cioâ€”5Hypoxanthine-8-14CiO@ iO@ 19 16 19 iO@37 <7<25 <3055 <1244

ioâ€”@ 35 38 34 31 iÃ¸@69 264 090 280 0 Effects of nitrogen mustard (1*42) upon the synthesis of DNA by minced tumors. These data are taken from a previous report (27).

through mitosis but that cells that are in the S-phase at the tral properties of the isolated DNA. On the other hand, the time of treatment are subsequently blocked in the G2-phase concentration of nitrogen mustard required to decrease the and do not reach mitosis. Unpublished data obtained by primer activity of DNA for the in vitro RNA-polymerizing Simpson-Herren and coworkers in this laboratory show the system is near that which would be expected to exist in an lengths of the phases of the cell cycle for 10-day-old nitrogen animal receiving a therapeutic dose of nitrogen mustard. @@ mustard-sensitive plasmacytoma cells to be : 4.9 hr; Ts, Thus, it is possible that nitrogen mustard, through reaction @ 5.9 hr; TG, 3.5 hr; TM 0.5 hr. If the series of events suggested with DNA, might interfere with the production of certain in the preceding sentences occurred, the number of cells in the messenger RNA's that are essential to the life of the cells. It S-phase should be approximately constant for the first 9.0 hr has been suggested that these messenger RNA's might be in following treatment with nitrogen mustard, and there should volved in the synthesis of DNA-synthesizing enzymes (7). It be a progressive decrease in the number of cells in the S-phase also seems possible that the synthesis of messenger RNA's during the next 6 hours or longer (perhaps much longer, if the necessary for the synthesis of proteins required for mitosis dispersion in the rates at which the individual cells progress (26) might be retarded or prevented. On the other hand, through the cycle is large). Such timing would be in fair agree reaction of nitrogen mustard at physiologic concentrations ment with the curves of Chart 3. (It is also likely that other with messenger RNA's might be great enough to interfere effects contribute to the determination of the shapes of the with the coding properties of the RNA's. curves ofChart 3.) Cross-linking of DNA by nitrogen mustard, sufficient to In order to determine which of the multiple effects of alky prevent denaturation of the product, might occur at physio lating agents are pertinent to the effectiveness of these agents logic concentrations of the nitrogen mustard. The primary in chemotherapy of cancer, it is necessary to correlate bio effect of such cross-linking upon the life processes of a cell chemical events with responses of tumors. By determining the are not now known. Theoretically, such cross-linking could dosages or concentrations of agents required to cause ob prevent semiconservative replication of the DNA and thus servable biochemical effects and those required to cause ob prevent mitosis. The effect of cross-linking of DNA upon its servable effects upon the viability of the neoplastic cells, it functioning as a primer or template for synthesis of RNA is might be possible to eliminate superfluous effects and to dis not known. tinguish between primary and secondary effects. Table 4 con It is significant that, at the higher dosages used in the tains data showing the concentrations of nitrogen mustard that present study (nitrogen mustard, 2 mg per kg; cyclophospha cause a variety of physicochemical, biologic, and biochemical mide, 20 mg per kg), there was inhibition of synthesis of effects. A number of the effects are caused only at concentra DNA and RNA in the resistant tumors (Charts 3, 4). At tions of the agent that exceed the concentration of the drug these dosages there was a transient decrease in the rates of that would be present in a hamster following a dose sufficient increase in the size of the tumors (Charts 1, 2), but then the to cause regression of the tumor. Therefore, one might logi tumors resumed growth at the normal rate. This might be cally question the contribution of those particular effects to interpreted as evidence for repairing of damage and recovery causation of regression of the tumor. Thus, it appears that by the resistant cells or for preferential killing and elirnina gross deactivation of the DNA nucleotidyltransferase activity tion of the cells of the tumor that are most sensitive to the that is measured by the assay procedure used in the present agent. The present experiments do not provide data that study and gross deactivation of DNA (as accomplished by in would favor a choice of one of these alternatives over the vitro treatment of DNA with nitrogen mustard) as a primer for other. These results, however, do show that inhibition of the the synthesis of DNA are not primary modes of action of synthesis of DNA and KNA during the first 48 hours follow nitrogen mustard. It also appears that the extent of reaction of ing administration of nitrogen mustard or cyclophosphamide nitrogen mustard with DNA in vivo, at dosages used in chemo cannot be considered as being indicative that a favorable @ therapy, would be too low to alter the TM viscosity, or spec therapeutic effect of the agents has been accomplished.

JANUARY 1969 107

Table 4Required ofPropertynitrogen concentration mustardReferenceCause of regression of plasmacytoma1.0mg/kg (5.2 x 106 moles per kg)This paper Inhibition of synthesis of DNA In vivo by plasmacytoma 0.3 mg/kg (1.6 x 10â€”6moles per kg) This paper In vitro by minced plasmacytoma iO@ M This paper In vitro by cultured L cells 106 M 8 In vitro by Ehrlich ascites cells iÃ¸@ M 8 In vitro by cell-free supernatant iO@ M This paper In vitro by cell-free supernatant >2 x iO@ M 21 i@â€”@M In vitro by a partially purified bacterial enzyme 19 Inhibition of synthesis of RNA In vivo by plasmacytoma 0.3 mg/kg (1.6 x 106 moles per kg) This paper In vitro by minced plasmacytoma ioâ€”@M 27 In vitro by Ehrlich ascites cells iO@ M 8 In vitro by a partially purified bacterial enzyme iO@ M 19 Inhibition of priming and/or template action of DNA Native For synthesis of DNA iOâ€”@â€”102 M This paper iO@ â€”iO@ M 19 For synthesis of RNA ioâ€”@M 7 5 x i07 M 19 Denatured For synthesis of DNA iO@ M 19 For synthesis of RNA ioâ€”@â€”ioâ€”@M 19 Inhibition of protein synthesis by cell-free system Interference with coding capacity of polyuridylic acid ioâ€”sM 12 Interference with coding capacity of polycytidylic acid iO@ M 12 Interference with coding capacity of polyadenylic acid i0â€”@M 12 Interference with functioning of ribosomes io@ M 12 Decreased binding of treated polyuridylic acid to ribosomes >5 x iO-@ M 12 Decreased binding of phenylalanyl-tRNA to complex of treated polyuridylic acid with ribosomes >5 x i0@ M 12 Decreased binding of phenylalanyl-tRNA to complex of polyuridylic acid with treated ribosomes 5 x iOâ€”@M 12 s-100 enzymes for amino acid polymerization 1 )@10â€”6M 19 s-100 enzymes for aminoacyl-tRNA synthesis 5 )( iO@ M 19 Altered TM of DNA i0â€”@M 30 1.5 x iO@ M 7 3 )< iO@ M 3 Hyperchronic effect on DNA 102 M 7 Altered viscosity of DNA 4 x iO@ â€”2 x iO@ M 5 1.5 x i0â€”@M 7 >102 M 10 Prevention of denaturation of DNA 5 >( 10' M 13 Inhibition of cloning of cultured H. Ep. -2 cells 106 M a Antimitotic effect on cultured cells 106 M 3 10_6 M 1 5 x 106 M 15 5 )( 106 M 6 Giant cell formation 106 M 1 106 M 3,4 5 x 106 M 6 iO@ M 14 Decreased cell survival after exposing Ehrlich cells in vitro and reimplanting into mice 5 x i0â€”@M 20 Decreased cell survival after exposing cultured L1210 cells in vitro and implanting into mice 5x107M 31 Concentrations of nitrogen mustard that cause certain physicochemical, biologic, and biochemical effects. tRNA, trans @ icr RNA; TM midpoint of the absorbance-temperature transition profile of DNA. aUnpublished data from this laboratory.

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ACKNOWLEDGMENTS of a Nitrogen Mustard on Survival, Growth, Protein and Nucleic Acid Synthesis of Mammalian Cells in Vitro. Exptl. Cell Res., 31: The authors express their appreciation to Mrs. Janet D. Boothe and 19â€”30,1963. Mr. Anthony J. Moore for technical assistance in performing the ex 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. periments, to the Chemotherapy Department of Southern Research Protein Measurement with the Folin Phenol Reagent. J. Biol. Institute for supplying the tumor-bearing animals, to Miss Tommie Chem., 193: 265â€”275, 1951. Lou Barker for caring for and dissecting the animals, and to Mr. T. C. 17. Palme, G., Lisa, E., Oeff, K., and Platis, A. Der Einfluss von Herren for performing the radioassays. Cyclophosphamid auf die DesoxyribonucleinsÃ¤ure-Synthese von normalen proliferierenden Zellen sowie Ascites-Tumorzellen. REFERENCES Arzneimittel-Forsch., 13: 1034â€”1039, 1963. 18. Papirmeister, B. On the Mechanism of Inhibition of T2 Bacterio 1. Alexander, P., and Mikulski, Z. B. Differences in the Response of phage by Mustard Gas. CRDL Special Publication 2-45, Armed Leukemia Cells in Tissue Culture to Nitrogen Mustard and to Services Tech. Information Agency, Arlington, 1961. (Via Refer Dimethyl Myleran. Biochem. Pharmacol., 5: 275â€”282, 1961. ence11above.) 2. Booth, B. A., Creasey, W. A., and Sartorelli, A. C. Alterations in 19. Ruddon, R. W., and Johnson, J. M. The Effects of Nitrogen Mus Cellular Metabolism Associated with Cell Death Induced by Ura tard on DNA Template Activity in Purified DNA and RNA Poly cil Mustard and 6-Thioguanine. Proc. Natl. Acad. Sci. U. S., 52: merase Systems. MoL Pharmacol., 4: 258â€”273, 1968. 1396â€”1402,1964. 20. Rutman, R. J., Steele, W. J., and Price, C. C. Experimental 3. Brewer, H. B., and Aronow, L. Effects of Nitrogen Mustard on Chemotherapy Studies II. The Reactions of Chloroquine Mustard the Physicochemical Properties of Mouse Fibroblast Deoxyribo (CQM) and Nitrogen Mustard (HN2) with Ehrlich Cells. Cancer nucleic Acid. Cancer Res., 23: 285â€”290, 1963. Res.,21:1134â€”1140,1961. 4. Brewer, H. B., Jr., Comstock, J. P., and Aronow, L. Effects of 21. Smellie, R. M. S., McArdle, A. H., Keir, H. M. and Davidson,J. N. Nitrogen Mustard on Protein and Nucleic Acid Synthesis in The Incorporation of (4-3H) Thymidine and (8-14C) Adenine into Mouse Fibroblasts Growing in Vitro. Biochem. Pharmacol., 8: DNA by Particle-free Extracts of Mammalian Cells. Biochem. J., 281â€”287, 1961. 69: 37P, 1958. 5. Butler, J. A. V., and Smith, K. A. The Action of Ionizing Radix 22. Skipper, H. E., and Schabel, F. M., Jr. Experimental Evaluation tions and of Radiomimetic Substances on Deoxyribonucleic Acid. of Potential Anticancer Agents. VII. Cross Resistance of Alky Part I. The Action of Some Compounds of the â€œMustardâ€•Type. lating Agent-resistant Neoplasms. Cancer Chemotherapy Rep., 22: J. Chem. Soc., 1950: 3411â€”3418,1950. 1â€”22, 1962. 6. Caspersson, T., Farber, S., Foley, G. E., and Killander, D. Cyto 23. Tomisek, A. J., Irick, M. B., and Allan, P. W. Deoxyribonucleic chemical Observations on the Nucleolus-ribosome System. Effects Acid Synthesis I. Effect of in Vivo Cyclophosphamide Treatment of Antinomycin D and Nitrogen Mustard. Exptl. Cell Res., 32: on the in Vitro Activity of the Deoxyribonucleic Acid Synthe 529â€”552, 1963. tase System of Sensitive and Resistant Plasmacytomas. Cancer 7. Chmielewicz,Z. F., Fiel, R. J., Bardos,T. J., and Ambrus,J. L. Res.,26: 1466â€”1472,1966. Alterations of Some Macromolecular and Biochemical Properties 24. Tomisek, A. J., and Simpson, B. T. Effect of in Vivo Cyclophos of Calf Thymus DNA Caused by â€œDualAntagonistsâ€• and Nitro phamide Treatment on the DNA-Priming Ability of DNA from gen Mustard. 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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1969 American Association for Cancer Research. Effects of Nitrogen Mustard and Cyclophosphamide upon the Synthesis of DNA in Vivo and in Cell-free Preparations

Glynn P. Wheeler and Jo Ann Alexander

Cancer Res 1969;29:98-109.

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