[CANCER RESEARCH 29, 98—109,January 1969]

and concentrations of the nitrogen mustard, it was possible toinhibit the synthesis of DNA more than that of RNA and toinhibit synthesis in a sensitive tumor without inhibitingsynthesis in a bilaterally growing resistant tumor. Otherstudies (23) showed that multidose treatment of hamstersbearing plasmacytomas with cyclophosphamide [2-[bis(2-chloroethyl)amino]-2H-1,3,2-oxazaphosphorinane 2-oxide]caused decreases in the activity of DNA nucleotidyltransferaseand of thymidylate kinase of soluble cell fractions preparedfrom the plasmacytomas, but that a single dose of cyclophosphamide given 2 hours before killing the animal caused anincrease of the DNA nucleotidyltransferase activity. Theseresults provoked the questions whether the observed decreasedsynthesis of DNA in vivo and the decreased nucleotidykransferase activity were the cumulative effects of multiple doses ofthe agents, whether similar effects would be observed at longerintervals after the administration of single doses of the drugs,and whether decreased nucleotidyltransferase activity is a primary or secondary effect of the drug. In pursuit of answers tothese questions, we have now performed experiments to: (a)determine the relationship between the interval after administering a single dose of nitrogen mustard or cyclophosphamideand the effect upon the synthesis of nucleic acids in vivo, (b)determine the effects of treatment of cell-free preparationsfrom plasmacytomas with nitrogen mustard upon the DNAnucleotidyltransferase activity, (c) determine the effects oftreatment of DNA with nitrogen mustard in vitro upon thepriming activity of the DNA for the DNA nucleotidyltransferase system, and (d) compare the effects of in vivo treatmentwith cyclophosphamide upon the in vivo synthesis of DNA byplasmacytomas and upon the DNA nucleotidyltransferaseactivity of a cell-free preparation from the same plasmacytomas.

MATERIALS AND METhODS

Determination of the Effects of Agents upon the Growth ofTumors. Trocar implants of cyclophosphamide-sensitive (9)and cyclophosphamide-resistant (22) plasmacytomas wereplaced subcutaneously and bilaterally in the axillary regions ofyoung (45- to 55-gram) Syrian hamsters. Fourteen days latersingle intraperitoneal injections of the respective agents wereadministered, and daily measurements of the tumors weremade by means of calipers. Approximations of the weights ofthe tumors were made with the assumption that the tumorswere prolate spheroids with a density of 1.0.

98 CANCER RESEARCH VOL.29

Effects of Nitrogen Mustard and Cyclophosphamide upon the

Synthesis of DNA in Vivo and in Cell—freePreparations'

Glynn P. Wheelerand Jo Ann AlexanderKettering-Meyer Laboratory2, Southern Research Institite, Birmingham, Alabama 35205

SUMMARY

Single doses of nitrogen mustard or cyclophosphamidecaused regression of plasmacytomas in hamsters and decreasesin the rate of synthesis of DNA and RNA by the tumors.Maximum inhibition of synthesis did not occur immediately

following the administration of the agent but was observable24—48 hours later. This inhibition was accompanied by a decrease in DNA nucleotidyltransferase activity of crude cell-freesupernatant fractions prepared from the treated tumors.

The concentrations of nitrogen mustard required for in vitrodeactivation of the crude DNA nucleotidyltransferase and forin vitro deactivation of commercial salmon sperm DNA as aprimer for this system were much greater than those thatwould be present in hamsters following the administration oftherapeutically effective doses. It was concluded that neitherthe direct deactivation of the DNA nucleotidyltransferase norgross interference with the primer activity of DNA is the causeof the observed therapeutic effect upon the tumor or the decrease of synthesis of DNA in vivo.

Transient inhibition of growth and of synthesis of DNA andRNA by drug-resistant tumors was also observed. It is notpresently known whether the resumption of growth andsynthesis of nucleic acids is the result of repair of damage andrecovery by the cells or of killing and elimination of the cellsof the tumor that are most sensitive to the agent. It is evident,however, that inhibition of the synthesis of DNA and RNA bya tumor during the first 48 hours following administration ofnitrogen mustard or cyclophosphamide cannot be considered

to be indicative that a favorable therapeutic effect of the agenthas been accomplished.

INTRODUCTION

Previous studies in this laboratory (27) have shown that

nitrogen mustard (HN2) inhibits the incorporation of radioactive substrates into DNA by Fortner hamster plasmacytomasin vivo and by minces of these tumors. With selected dosages

1ThiS investigation was supported by the Cancer ChemotherapyNational Service Center, National Cancer Institute, USPHS, under contract PH43-66-29 and by grants from the Charles F. Kettering Foundation and the Alfred P. Sloan Foundation, Inc.

2Afflliated with Sloan-Kettering Institute for Cancer Research, NewYork, New York.

Received April 9, 1968 ; accepted September 15, 1968.

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Effects ofNitrogen Mustard and Cyclophosphamide

Determination of the Effects of Agents upon the Fixation of‘4Cfrom Formate-'4C and from Adenine-8-14C. Hamstersbearing bilaterally and subcutaneously growing 13-day-old and1 4-day-old cyclophosphamide-sensitive and cyclophosphamide-resistant plasmacytomas were given single intraperitonealinjections of nitrogen mustard or cyclophosphamide at specifled doses. At intervals thereafter, the animals were given intraperitoneal injections of sodium formate-14 C (specific activity,55, 220, or 298 jic per mg; dosage, 1 pc per gram of bodyweight) or adenine-8-14C (specific activity, 21.7, 83.3, or 129Ilc per mg; dosage, 0.3 @scper gram of body weight). The

animals were killed by carbon dioxide asphyxiation 2 hoursfollowing the injection of the radioactive compounds. Thetumors from 3 or 4 animals were pooled; homogenates, alcoholic extracts, and the purines of RNA and of DNA wereprepared and assayed for radioactivity by procedures that havebeen described (28).

Preparation of Soluble Extracts of Tissues. Hamsters bearing14-day-old subcutaneous plasmacytomas were asphyxiatedwith carbon dioxide, and the tumors were removed and placedin ice-cold containers. After the tumors were minced freehandwith knives and forced through the perforated steel plate of atissue press, 10 grams of the tissue were homogenized bymeans of a Thomas Tissue Grinder with a motor-driven teflonpestle in 30 ml of an ice-cold aqueous solution that was 0.25M with respect to sucrose and 0.02 M with respect to Tris

hydrochloride buffer (pH 8.0). This homogenate was centrifuged at 105,000 X g for 1 hour. The aqueous supernatantlayer was removed by pipet, care being taken to exclude theoverlying lipid layer. This solution was assayed for protein bythe method of Lowry et a!. (16) and was used without furthertreatment as the source of enzymes for the incubations.

Incubation Mixtures and Enzyme Assays. Each sample forincubation contained the following ingredients at the indicatedconcentrations in micromoles per ml: sucrose, 222; Trishydrochloride buffer (pH 7.5), 8.8; adenosine triphosphate,1.1 ; magnesium chloride, 1 .1 ; potassium chloride, 40; potasslum phosphoenolpyruvate, 5.04; and pyruvate kinase, 0.84microgram per ml. Each sample also contained either themonophosphates or triphosphates of deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine at concentrations of0.046 micromole per ml. Only one of the deoxyribonucleosidephosphates was radioactive in each reaction mixture. The following radioactive substrates, having the indicated specificactivities in millicuries per millimole, were used: thymidine2-'4C monophosphate (8.5, 13.6, or 29.4); deoxycytidine2-'4C monophosphate (29.2); deoxyadenosine-8-'4C monophosphate (7.7); deoxyguanosine-8-'4C monophosphate(14.4); deoxyadenosine-8-'4C triphosphate (8.58, 10.0, or17.15); thymidine-2-'4C triphosphate (43.7). The enzymesolution was added to the mixture to give a final concentrationof 7.81 mg of protein per ml, and primer DNA was added togive a concentration of 0.46 mg per ml. Unless otherwisestated, the primer consisted of salmon sperm DNA that hadbeen denatured by heating an aqueous solution (4 mg of DNAper ml of water) in a boiling water bath for 10 minutes andthen rapidly cooling in an ice bath for 15 minutes; it was usedimmediately after preparation. The final volume of the incubation mixture was 1 .25 ml.

In the standard procedure, the reaction mixture minus theprimer was incubated at 37°Cfor 10 minutes, and the primerwas then added. The time at which the primer was added ishereafter referred to as zero time. In certain experiments theenzyme preparation or a solution of native salmon sperm DNAwas incubated with or without nitrogen mustard and then usedin the standard procedure. At zero time and at designatedintervals (each 10 minutes during the first hour, each 20 minutes during the second hour, and each 30 minutes during thethird and fourth hours) during the ensuing 4 hours, 25-@.tlsamples of the incubation mixtures were transferred to discs offilter paper and allowed to dry at room conditions, whereuponthe discs were dropped into a beaker of cold 5 percent trichloroacetic acid. The beaker contained at least 10 ml of theacid solution for each disc. The discs were allowed to standovernight in the acidic solution and were then washed twotimes by standing for 15 minutes in cold 5 percent solutions oftrichloroacetic acid. The discs were next washed once withcold absolute ethanol and allowed to dry on paper towels atroom conditions. The quantities of 14C present on the discswere determined with a Packard Tri-Carb scintillation spectrometer, and it was assumed that the 14C was present inDNA.

At the same times that samples of the incubation mixtureswere taken for placing upon the paper discs, other samples of10 ;.zl each were spotted on Whatman No. 1 paper for subsequent chromatography using isobutyric acid:water:acetic acid(100:50:1 v/v/v) as the solvent. The positions of the radioactive spots were detected by means of blue-sensitive X-rayfilm, and the radioactive areas were cut from the chromatograms and assayed with the liquid scintillation spectrometer.The identities of the various radioactive components of themixtures were determined by comparison of the RF valueswith those of known compounds that were chromatographedin parallel with them.

Determination of the Effects of a Single Dose of Cyclophosphamide or Nitrogen Mustard upon the in Vivo Fixation of 3Hfrom Thymidine-C3H3 by Plasmacytomas and upon the DNANucleotidyltransferase Activity of the Same Tumors. Hamsters bearing 14-day-old subcutaneously growing plasmacytomas were given single injections of cyclophosphamide (20mg per kg) or nitrogen mustard (0.5 mg per kg), and, at specifled times thereafter, groups of three animals were given intraperitoneal injections of thymidine-C3H3 (specific activity,75.3 mc per millimole) at a dosage of 2 pc per gram of bodyweight. Animals of the control group received injections ofphysiologic saline and thymidine-C3H3. The hamsters werekilled 2 hours after receiving the radioactive compound, andthe tumors of the three animals were removed, pooled, mincedwith knives, and passed through a tissue press. A portion ofthe tissue was washed two times with 5 percent trichloroaceticacid, two times with water, two times with absolute ethanol,and two times with ether. After the tissues had dried overnightat room conditions, they were ground with a mortar and pestleand desiccated over calcium sulfate overnight or longer. Duplicate samples (3—5mg) of the dried powder were incubated for24 hours at 37°C with 1 ml of 1 M solution of hydroxide ofHyamine [p-(diisobutylcresoxy-ethoxyethyl)dimethylbenzylammonium hydroxide] in methanol. After the addition of 14

99JANUARY 1969

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Glynn P. Wheeler and Jo Ann Alexander

ml of a toluene solution of scintillators [4 gm of 2,5-diphenyloxazole and 50 mg of 2,2'-p-phenylenebis(5-phenyl-oxazole)

per liter of toluene] , the samples were assayed for radioactivity in a Tri-Carb liquid scintillation spectrometer.

The remainder of the wet tissue was used for the preparationof soluble extract by the procedure described above, and theextract was then assayed for DNA nucleotidyltransferase activity.

RESULTS

Effects of Single Doses of Agents upon the Growth ofSensitive and Resistant Plasmacytoma. Chart 1 shows thatnitrogen mustard at a dosage of 0.5 mg per kg caused tem

porary cessation of the growth of the sensitive tumor and that,at dosages of 1.0, 2.0, or 3.0 mg per kg, the agent causedregression in the size of the sensitive tumors. The tumors ofthe animals receiving the larger doses continued to grow atabout the normal rate for 1—2days following the treatmentbefore a decrease in tumor size began. At none of these dosages did nitrogen mustard do more than temporarily slow thegrowth of the bilaterally growing resistant tumors. (Theanomolous results obtained on Days 5 and 6 for dosages of 0.5and 1 .0 mg per kg with the resistant tumor are probably notsignificant.)

Chart 2 shows the effects of single doses of cyclophosphamide upon the growth of sensitive and resistant plasmacytomas. The results are similar to those obtained with nitrogen

mustard, but, as would be expected, higher dosages were required.

Effects of Single Doses of Agents upon the Fixation of ‘4Cof Formate-'4C and of Adenine-8-'4C. Chart 3 shows theeffects of two dosages of nitrogen mustard upon the incorporation of 14 C from formate-14 C and from adenine-8-14 C intoRNA, DNA, and certain compounds of lower molecularweight. At a dosage of 0.3 mg of nitrogen mustard per kg(which would cause little inhibition of growth of the sensitivetumor), there was inhibition of incorporation of 14 C of formate-14C into soluble purines and related compounds and intothe purines of RNA and DNA. Only the data for the nucleicacid adenine are shown, but similar effects were observed forguanine. The curves go through maxima during the first 16hours with subsequent inhibition of incorporation. The inhibition of incorporation into DNA was greater than that intoRNA. The results for the sensitive and resistant tumors weresimilar, but there was some indication of “recovery―by theresistant tumor at 48 hours. At this dosage of nitrogen mustard there was less inhibition of incorporation of 14C of adenine-8-14C into adenosine phosphates and into the adenine ofRNA and of DNA than occurred for formate-14 C. With adenine-S-14 C as the substrate, maxima of fixation were againobserved during the first few hours following injection of thenitrogen mustard, and the inhibition of incorporation intoDNA exceeded that into RNA. There was evidence of “recovery― of the normal rates of incorporation by 48 hoursfollowing treatment for both the sensitive and resistanttumors.

When the dosage of nitrogen mustard was increased to 2.0mg per kg, the patterns of the effects upon the incorporation

Io@ooo SENSITIVE RESISTANT

a'E

a4,a.a4,

a

U

00

0 2 4 6 0 2 4 6

Days after injection of nitrogen mustard

Chart 1. Effects of nitrogen mustard upon the growth of bilaterallygrowing cyclophosphamide-sensitive and cyclophosphamide-resistantplasmacytomas. The numbers on the curves indicate the dosages in mgper kg for a single intraperitoneal injection. The calculated weights ofthe tumors were based upon external caliper measurements, and eachpoint on the curve is the mean value for 3 to 6 tumors.

4,000

I 2@00

I OpO 0

Days after injection of cyclophosphamide

Chart 2. Effects of cyclophosphamide upon the growth of bilaterallygrowing cyclophosphamide-sensitive and cyclophosphamide-resistantplasmacytomas. The numbers on the curves indicate the dosages in mgper kg for a single intraperitoneal injection. The calculated weights ofthe tumors were based upon external caliper measurements, and eachpoint on the curve is the mean value for 3 to 5 tumors.

of 14C of formate-14C were generally similar to those obtamed at the lower dosages. However, at the higher dosagethere was more inhibition of incorporation of 14C of adenine8-'4C into the adenosine phosphates and the adenine of RNAand DNA, and there was less evidence of “recovery―by 48hours. Similar results were obtained for the sensitive and resistant tumors.

a,E

0'

4,

@04,

0

U

00

SENSITIVE RESISTANT

@0I0O

8,000

6@00 @.

4,000

2,000

03 5

CANCER RESEARCH VOL.29100

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14

Formate-. C

Effects ofNitrogen Mustard and Cyclophosphamide

HN2,O.3 mg/kg HN2,2.Omg/kg

SENSITIVE RESISTANT RESISTANT

200

160

I 20

80

40

0 16 32 48

‘4Formate.- C

‘4Formate- C

‘4Formate-. C

0

C0C)

0

20@

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

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 ofadenine-8-'4C into the adenine of RNA (RNA-Ad), adenine of DNA (DNA-Ad), soluble purines and related compounds (Pu), and adenosinephosphates (AdP) of sensitive and resistant plasmacytomas. Pu: hypoxanthine, xanthine, adenosine, inosine, adenosine monophosphate, adenosinediphosphate, adenosine triphosphate, inosine monophosphate, guanosine monophosphate, uric acid, and allantoin. AdP: the mono-, di-, andtriphosphate derivatives of adenosine. The animals were killed 2 hours after the administration of the labeled substrate. The values for RNA-Adand 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 therespective compounds.

Chart 4 shows the results obtained with cyclophosphamide.At a dosage of 10 mg per kg (a dose that was sufficient tocause regression of the sensitive tumor), maxima showingmuch stimulation of incorporation were observe4 for bothtumors during the first few hours after treatment; delayed

inhibition of incorporation of ‘4Cfrom formate-1 4C into thesoluble purines and related compounds and into the KNA andDNA occurred in the sensitive tumor, but extensive stimulation of incorporation occurred in the resistant tumor. At adosage of 20 mg per kg, there was no extensive stimulation ofincorporation during the first four hours, and there was moreinhibition of incorporation into nucleic acids.

When the substrate was adenine-8-1 4C and the dosage ofcyclophosphamide was 10 mg per kg, stimulation of incorporation of the 14C into adenosine phosphates and into RNA andDNA occurred during the fkst few hours following treatment.This period of stimulation was followed by a period of inhibition for the sensitive tumor but not the resistant tumor. At adosage of 20 mg of cyclophosphamide per kg there was noevidence of stimulation of incorporation of 14C of adenine8-'4C during the first few hours following treatment, and theextents of inhibition of incorporation of 14C were similar forthe sensitive and the resistant tumors during the 48-hourperiods of observation.

JANUARY 1969 101

SENSITIVE

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Glynn P. Wheeler and Jo Ann Alexander

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

0

C0U

0

Adenine-8J4C Adenine-.8..14C Adenine-8- ‘4C Adenine-8-14C

6

2

AdP

0 16 32 48 0 16 32 48 0 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 ofadenine-8-'4C into the adenine of RNA (RNA-Ad), adenine of DNA (DNA-Ad), soluble purines and related compounds (Pu), and adenosinephosphates (AdP) of sensitive and resistant plasmacytomas. See the legend for Chart 3 for definitions of Pu and AdP. The animals were killed 2hours 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,each experimental point on the curves was obtained for pooledtumors from three to five animals. The similarities of thecurves of Chart 3 to those of Chart 4 lend credence to thegeneral characteristics of the curves of both experiments. Theexperimental points are connected by straight lines to indicatethat “bestfit―lines were not determined; therefore, the smallirregularities are probably not significant.

Effects of Treating Crude, Cell-free Extracts with NitrogenMustard upon DNA Nucleotidyltransferase Activity. Data forcomplete curves, such as those of Chart 5, were obtained in

these experiments, but, since presentation of all of the datawould require 24 curves, the results are presented in abbreviated tabular form (Table 1). The data in the columns for the10- to 30-minute interval reflect the relative rates of fixationof 14C during the period of most rapid fixation; the data inthe columns for the 10- to 240-minute interval reflect therelative quantities of 14C fixed when incorporation ceased.

The data ofTable 1 show that, at concentrations of 106 M,10—@ M, and i0@ M, there was little or no inhibition ofincorporation of the@ 4C of deoxyadenosine-8-' 4C triphosphate into the acid-insoluble material; in fact, there was some

102 CANCER RESEARCH VOL.29

RESISTANT

-Ad

Ad

SENSITIVE RESIS TA NT SE@4SITIVE

16 32 48

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Table1Experiment

no.Concentration of HN2 (M)Fixation

of 14Cduring 10—30 mm

@ of control)Fixation

of 14Cduring 10-240 mm

(% of control)

SensitiveResistantSensitiveResistant110—6

iO—@i@—@177

77130 7498 6291

136108 134801022iO—4

iO@10292

22057 691 2128

165102 112433iO@

5 x 10310—234

6228 59

148

9923 104

3

Effects ofNitrogen Mustard and Cyclophosphamide

1,500

U00

900

R-T ....----._...@

-.@ S-T

•700

0.C)

0

@0

‘C

U-

Time of incubation (mm)

Chart 5. Effects of single doses of cyclophosphamide (20 mg per kg) upon the in vitro DNA nudeotidyltransferase activity of plasmacytomaswhen the animals were killed 48 hours after administration of the cyclophosphamide. Deoxyribonucleoside monophosphates were used assubstrates in the assay system, and the labeled substrates in the respective assays are indicated. dAMP-8-14C, deoxyadenosine-8-14Cmonophosphate; 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, treatedsensitive tumor.

500

300

I00

600'

dGMP@8-400W

20 0@ S-c

0 i i a I@@ i@ • i ,

0 40 80 120 160 200 240 200 240

Effects of preincubating the enzyme preparations with nitrogen mustard (HN2) upon thefixation 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 incubatedat 37°Cfor 30 minutes prior to addition of this treated enzyme to the assay mixture.

103JANUARY 1969

dAMP- 8 -.@4C dTMP-.2@4C

0 40 80 120 160

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Glynn P. Wheeler and Jo Ann Alexander

indication of stimulation of incorporation at these concentrations of nitrogen mustard. At concentrations of 10@ M andgreater, the nitrogen mustard treatment caused decreases in

the rates of@ 4C fixation by the enzyme preparations fromboth the sensitive and resistant tumors, with slightly greaterdecreases being observed for the preparations from the sensi

tive tumor. At the higher concentrations of nitrogen mustardthere were also decreases in the capacities (as indicated by thedata in the last two columns) of the systems, and the decreaseswere greater for the enzymes from the sensitive tumors. Thedecreases in the rates of fixation of@ 4C during the interval10—30 minutes were greater than the decreases in the quantities of@ 4C fixed during the 10- to 240-minute periods ofincubation.

In these experiments the crude enzyme preparations weretreated with the nitrogen mustard for 30 minutes, and a portion of this solution was added to the other components of theassay medium with a resultant dilution of 3- to 4-fold. It isrecognized that some residual nitrogen mustard might be trans

ferred to the assay mixture and that it might react with someof these components and hence cause a decrease in the quantity of labeled substrate fixed into the acid-insoluble material.The important point, however, is that, whether the observedinhibition is caused by reaction of the nitrogen mustard withone or more components of the enzyme preparation or withone or more components of the assay mixture, supraphysiologic concentrations of nitrogen mustard are required beforeany inhibition occurs.

Similarly, high concentrations of nitrogen mustard were required to cause decreases in the rate of fixation of@ 4C intoacid-insoluble material by a DNA nucleotidyltransferase system derived from leukemia L1210 ascites cells (29).

Effects of Treating Salmon Sperm DNA with Nitrogen Mustani upon the Priming Activity of the DNA for DNA Nucleotidyltransferase. Table 2 contains the data of a single experiment of this type. Treatment of native DNA with i0@ Mnitrogen mustard for 30 minutes had little or no effect uponits priming activity for the DNA nucleotidyltransferase. Aftertreatment with i0@ M or 102 M nitrogen mustard, theDNA served less efficiently as a primer in the incubation mixture containing the enzymes from the sensitive tumor, whilethere was less effect upon its utilization as primer in the mixture containing the enzymes from the resistant tumor. Sinceno steps were taken to remove unreacted nitrogen mustardfrom the incubation mixture with the DNA prior to additionof a portion of this mixture to the total incubation mixture, itis possible that a portion of the observed inhibition could bedue to the effects of the residual nitrogen mustard upon theenzymes. (There was a dilution of approximately 7-fold uponaddition of the primer solution to the total transferase assaymixture.) Nevertheless, it is significant that treatment of theDNA with i0@ M nitrogen mustard, which is probably asupraphysiologic concentration, had little effect upon thepriming activity of the DNA.

Effects of in Vwo Treatment with Cyclophosphamide uponthe DNA Nucleotidyltransferase Activity in Vitro. Chart 5shows the results obtained in an experiment in which bilaterally growing plasmacytomas were taken from animals 48 hoursfollowing the administration of saline or cyclophosphamide

Table 2

Concentrationof HN2 (M)Fixation

of 14Cduring 10—30 mm

(% of control)

Sensitive ResistantFixation

of 14Cduring 10—240mm

(% ofcontrol)Sensitive

ResistantiO@100

9513494iO@561098312810243

8651 84

Effects of preincubating native salmon sperm DNA primer with nitrogenmustard (HN2) upon the fixation of ‘4C from deoxyadenosine-8-' 4C tnphosphate into the acid-insoluble material by enzymes from untreated sensitive and resistant plasmacytomas. The necessary quantity of nitrogen mustard was added to a solution of DNA in water (4 mg per ml), and themixture was incubated at 37°Cfor 30 minutes prior to the addition of thistreated DNA to the assay mixture.

(20mgperkg)to thehamsters,andcell-freepreparationswereassayed for DNA nucleotidyltransferase with the deoxyribonucleoside monophosphates as substrates. The deoxyribonucleoside monophosphates were present in equimolar quantities, but only one was radioactive. Incubation mixtures wereset up in which each of the deoxyribonucleoside monophosphates was the labeled substrate. The values for the radioactivity for the different labeled substrates have been adjusted tobring them to a common basis for comparison; therefore, thecurves indicate the relative rates of fixation of the varioussubstrates by the preparation from the sensitive and resistanttumors and the@effects of the treatment thereon. The preparations from the tumors of the treated animals had lower DNA

nucleotidyltransferase activity regardless of which labeled substrate was used. These results are consistent with those ob

tamed by Tomisek et a!. (23) in experiments with preparationsfrom sensitive tumors following multidose treatment of thetumor-bearing hamsters. Chromatographic analysis of the in

cubation mixtures during the course of the assays showed thatthe quantities of the metabolites of the respective labeled substrates were similar for the preparations from the treated anduntreated animals and that the decreased fixation of 14C bythe samples corresponding to the treated animals was not dueto a deficiency of substrate.

Effects of in Vivo Treatment with Cyclophosphamide uponthe Synthesis of DNA in Vivo and upon the DNA Nucleotidyltransferase Activity in Vitro. The experiments describedabove showed that cyclophosphamide and nitrogen mustardinterfere with the synthesis of DNA in vivo, that treatment invitro with nitrogen mustard at supraphysiologic concentrationscan cause decreases in the DNA nucleotidyltransferase activity,and that in vivo treatment could cause decreased DNA nucleotidyltransferase activity in vitro. This experiment was per

formed to determine whether the decreases of DNA synthesisin vivo are accompanied by decreases in DNA nucleotidyltransferase activity of the very same tissues. Chart 6 shows thatafter 8 hours the decrease in in vivo synthesis of DNA exceeded the decrease in DNA nucleotidyltransferase activity.Assays with monophospates and triphosphates yielded similar

results.The curves of Chart 7 show that a single dose of nitrogen

mustard also caused decreases in the in vivo fixation of 3H of

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Effects ofNitrogen Mustard and Cyclophosphamide

00

80

60

40

20

0

0

C00

0

100

80

60

40

20

0

04-.

C0U

0

4 12 20 28 36 44 52

Hours after cyclophosphamide

Chart 6. Effects of single doses of cyclophosphamide (20 mg per kg)upon the in vivo fixation of 3H from thymidine-C3H3 into theacid-insoluble fraction of sensitive plasmacytomas and upon the in vitronucleotidyltransferase activity of the same tumors whenthymidine-2-14C monophosphate (dTMP) and the supplementarydeoxyribonucleoside monophsophates or thymidine-2-14C triphosphate(dTTP) and the supplementary deoxyribonucleoside triphosphates wereused as substrates for the assay system. The curves for the in vitroassays are based upon the quantities of 14C fixed during the 10- to40-minute interval of the assay incubation.

thymidine-C3H3 by sensitive tumors and in the DNA nucleotidyltransferase activity of these tumors.

These results differ with those obtained previously (29) inexperiments with 1,3-bis(2-chloroethyl)-1-.nitrosourea and micebearing leukemia L1210 ascites cells or leukemia L1210 solidtumors; in those experiments, decreased in vivo fixation of 3Hof thymidine-C3H3 occurred in the absence of decreased activity of DNA nucleotidyltransferase.

DISCUSSION

The inhibition of synthesis of DNA by alkylating agents hasbeen recognized as a common effect of these agents for anumber of years (25, 26), but the cause of this inhibition hasnot been established. Although the present study does notshow the cause, it does yield some additional information relative to this inhibition and seems to eliminate certain possiblecauses.

Single-dose treatment of experimental animals permits determination of the effects of graded dosages and hence the relative sensitivities of various metabolic events in the intactanimal. Such treatment also permits a time-series study, whichyields information about the sequence of the appearance ofvarious effects following treatment and about the change inthe magnitude of a single effect as time passes following thetreatment.

I I I I I I I

4 12 20 28

Hours after nitrogen mustard

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 theacid-insoluble fraction of sensitive plasmacytomas and upon the in vitronucleotidyltransferase activity of the same tumors whenthymidine-2-14C monophosphate (dTMP) and the supplementarydeoxyribonucleoside monophosphates or thymidine-2-14C triphosphate(dTTP) and the supplementary deoxyribonucleoside triphosphates wereused as substrates for the assay system. The curves for the in vitroassays are based upon the quantities of 14C fixed during the 10- to40-minute interval of the assay incubation. The vertical lines show therange of values obtained in multiple experiments, and the curves aredrawn through the mean values. The circles on the vertical lines showthe range of values for the experiments with dTMP; the horizontal barson these same vertical lines show the range of values for the experiments with dTTP. The values for the in vivo curve are based upon 4experiments; the values for the dTMP curve are based upon 2experiments; the values for the d@VFP curve are based upon 5experiments.

The curves of Charts 3 and 4 show that maximum inhibitionof the in vivo synthesis of DNA does not occur immediatelyfollowing the treatment, but rather it occurs several hours ordays later. Similar delays in attaining maximum inhibitionhave been observed in this laboratory for 1,3-bis(2-chloroethyl)-1-nitrosourea (28) and by others with nitrogen mustard

(8), with cyclophosphamide (17), and with uracil mustard (2).The maxima of specific activity, occurring during the first fewhours following treatment, and corresponding to stimulationof synthesis of DNA when low dosages of nitrogen mustard orcyclophosphamide were used, are of interest. It is obvious thatthe observed effects of the agents are not unidirectional and

JANUARY 1969 105

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Glynn P. Wheeler and Jo Ann Alexander

are dependent on the time the observation is made, and thismay be a partial explanation for the discrepancies in resultsreported by various investigators.

Tomisek et al. (23) reported an average increase of 21% innucleotidyltransferase activity of plasmacytomas 2 hours aftera dose (10 mg per kg) of cyclophosphamide, but an average68% decrease in activity after 5 daily treatments (at the samedosage) and decreases of 9%, 23%@, and 33%3 after 2 hourswith a dosage of 40 mg per kg. Some of the data of Table 1 ofthe present report are consistent with stimulation of DNAnucleotidyltransferase activity upon treatment of the enzymepreparation with low concentrations of nitrogen mustard. Onemight speculate that this stimulation is due to deactivation ofan inhibitor and is observable when low dosages or low concentrations of the agent are used, but is masked to variousextents by other effects when larger quantities of agents areused. It is more likely, however, that the stimulations observedin vivo are due to systemic effects of the agents upon theanimals, because transient stimulation also occurs at the sametime for the de novo synthesis of purines, the conversion ofadenine to nudeotides, the synthesis of RNA, and the synthesis of proteins (G. P. Wheeler and J. A. Alexander, unpublished data). Similar stimulation has been observed in experiments with 1,3-bis(2-chloroethyl)-1-nitrosourea (28).

The similarities of the curves for the soluble compoundsformed from formate-' 4C and from adenine-8-1 4C to thecurves for RNA and DNA (see Charts 3 and 4) are consistentwith the possibility that the decreased synthesis of RNA andDNA might be at least partially due to decreased supplies ofsubstrates. On the other hand, the decreased formation ofthese compounds of low molecular weight might result fromfeedback inhibition or repression of formation of enzymes byaccumulated substrates of nucleic acids. Lack of substrates isevidently not the sole cause of decreased synthesis of DNA,however, because less synthesis of DNA occurred in the cellfree preparations even when the -necessary substrates wereadded to the incubation mixtures. Decreased capacity to phosphorylate deoxyribonucleotides to the corresponding triphosphates is probably not a significant factor in the decrease ofsynthesis of DNA, because similar results were obtained in thein vitro experiments when monophosphates or triphosphateswere used as substrates (Chart 6) and because the quantities ofdeoxyribonucleoside phosphates in the incubation mixturesduring the assays represented by Chart 5 were similar for thesamples corresponding to the treated and the untreated tumors. These results are consistent with the observation ofTomisek et al. (23) that decreased thymidylate kinase wasobserved only after 5 daily treatments of the hamsters withcyclophosphamide (10 mg per kg).

Whether or not the decreased DNA nucleotidyltransferaseactivity of the treated tumors contributes to the observeddecrease in synthesis of DNA in vivo, it appears to be unlikelythat the decrease in enzyme activity is completely due todirect deactivation of the enzyme by the alkylating agents.This conclusion is derived from two observations: (a) Becauseof the known short biologic half-lives of the agents, particu

kJnpublished results of A. J. Tomisek and B. S. Johnson.

larly nitrogen mustard, it would be expected that the deactivation of the enzyme would have to occur quickly after administration of the agent, and hence maximum decrease in synthesis of DNA would occur more quickly than has been observed (Charts 3, 4). (b) The concentrations of nitrogen mustard required to inactivate the DNA nucleotidyltransferase invitro (Table 1) are much greater than would be expected following a single dose of nitrogen mustard at a level of 2 mg perkg to a hamster. Smellie et al. (21) and Papirmeister (18) alsofound that supraphysiologic concentrations of nitrogen mustard and sulfur mustard were required to reduce the activitiesof the polymerizing enzymes involved in the synthesis ofDNA. Ruddon and Johnson (19) have recently reported thatincubation of a partially purified DNA polymerase fromEscherichia coli with 10@ M nitrogen mustard for 60 minutescaused a 30—35 percent decrease in enzyme activity, whereasincubation with nitrogen mustard at a concentration of iO@M had little effect upon the activity.

At present there is confficting evidence relating to the effectsof alkylation of DNA upon its functioning as a primer for anin vitro DNA-polymerizing system. The small quantity of dataobtained in the present study (Table 2) indicate that it isnecessary to treat commercially obtained salmon sperm DNAwith supraphysiologic concentrations of nitrogen mustard tocause decreased primer activity. Ruddon and Johnson (19)found that the primer activity of native and denatured calfthymus DNA was depressed upon treatment with i0@ M andio—―M nitrogen mustard for 4 hours. On the other hand,Goldstein and Rutman (11) found that in vitro alkylation ofDNA with nitrogen mustard enhanced its primer activity.There is also lack of agreement upon the effect of in vivoalkylation of DNA upon the subsequent priming activity ofthe isolated DNA in in vitro systems. Goldstein and Rutman(11) found that alkylated DNA had decreased primer activity,but Tomisek and Simpson (24) found that their alkylatedDNA had greater primer activity. These discrepancies might bedue partially to differences in the biologic species and to differences in methods and technics of assay.

Although supraphysiologic concentrations of nitrogen mustard are required for the in vitro deactivation of the DNAnucleotidyltransferase (Table 1) and the in vitro alteration ofDNA resulting in decreased primer activity (Table 2), physiologic concentrations of HN2 were sufficient to inhibit theincorporation of 14C from labeled substrates into the nucleicacids of minced tumors (Table 3). The reason for this greatersensitivity of the intact system is not presently known.

The time lag between the administration of nitrogen mustardand the decrease in the rate of synthesis of DNA indicates thatthis apparent inhibition of synthesis of DNA is a secondaryeffect of the agents rather than a primary effect. Studies related to the effects of this agent upon the cell cycle (see Ref.26) indicate that the observed decreases in synthesis of DNAare due to a decreased number of cells carrying out the synthesis of DNA rather than to inhibition of the synthesis per Se.This decrease might result from interference with the progressof cells through the other phases of the cell cycle, and, hence,fewer cells would be present in the S-phase a few hours following treatment. There is some evidence (see Ref. 26) that cellsthat are in the G2-phase at the time of treatment continue

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Table3PrecursorConcentration

of HN2 (M)Speciuic

activity (% ofcontrol)AdenineSensitiveGuanineResistantAdenineGuanineFormate-14C

Hypoxanthine-8-14CiO@

io—@iO@

io—@iO@iO@37

<220

5919

<7<25

<200

4816

<3055

<240

8519

<1244

<250

8913Adenine-8-14Cio—5

io—@iø@69

35264

38090

34280

310

Effects ofNitrogen Mustard and Cyclophosphamide

Effects of nitrogen mustard (1*42) upon the synthesis of DNA by minced tumors. These data are taken froma previous report (27).

tral properties of the isolated DNA. On the other hand, theconcentration of nitrogen mustard required to decrease theprimer activity of DNA for the in vitro RNA-polymerizingsystem is near that which would be expected to exist in ananimal receiving a therapeutic dose of nitrogen mustard.Thus, it is possible that nitrogen mustard, through reactionwith DNA, might interfere with the production of certainmessenger RNA's that are essential to the life of the cells. Ithas been suggested that these messenger RNA's might be involved in the synthesis of DNA-synthesizing enzymes (7). Italso seems possible that the synthesis of messenger RNA'snecessary for the synthesis of proteins required for mitosis(26) might be retarded or prevented. On the other hand,reaction of nitrogen mustard at physiologic concentrationswith messenger RNA's might be great enough to interferewith the coding properties of the RNA's.

Cross-linking of DNA by nitrogen mustard, sufficient toprevent denaturation of the product, might occur at physiologic concentrations of the nitrogen mustard. The primaryeffect of such cross-linking upon the life processes of a cellare not now known. Theoretically, such cross-linking couldprevent semiconservative replication of the DNA and thusprevent mitosis. The effect of cross-linking of DNA upon itsfunctioning as a primer or template for synthesis of RNA isnot known.

It is significant that, at the higher dosages used in thepresent study (nitrogen mustard, 2 mg per kg; cyclophosphamide, 20 mg per kg), there was inhibition of synthesis ofDNA and RNA in the resistant tumors (Charts 3, 4). Atthese dosages there was a transient decrease in the rates ofincrease in the size of the tumors (Charts 1, 2), but then thetumors resumed growth at the normal rate. This might beinterpreted as evidence for repairing of damage and recoveryby the resistant cells or for preferential killing and elirnination of the cells of the tumor that are most sensitive to theagent. The present experiments do not provide data thatwould favor a choice of one of these alternatives over theother. These results, however, do show that inhibition of thesynthesis of DNA and KNA during the first 48 hours following administration of nitrogen mustard or cyclophosphamidecannot be considered as being indicative that a favorabletherapeutic effect of the agents has been accomplished.

107

through mitosis but that cells that are in the S-phase at thetime of treatment are subsequently blocked in the G2-phaseand do not reach mitosis. Unpublished data obtained bySimpson-Herren and coworkers in this laboratory show thelengths of the phases of the cell cycle for 10-day-old nitrogenmustard-sensitive plasmacytoma cells to be :@@ 4.9 hr; Ts,5.9 hr; TG, 3.5 hr; TM@ 0.5 hr. If the series of events suggestedin the preceding sentences occurred, the number of cells in theS-phase should be approximately constant for the first 9.0 hrfollowing treatment with nitrogen mustard, and there shouldbe a progressive decrease in the number of cells in the S-phaseduring the next 6 hours or longer (perhaps much longer, if thedispersion in the rates at which the individual cells progressthrough the cycle is large). Such timing would be in fair agreement with the curves of Chart 3. (It is also likely that othereffects contribute to the determination of the shapes of thecurves ofChart 3.)

In order to determine which of the multiple effects of alkylating agents are pertinent to the effectiveness of these agentsin chemotherapy of cancer, it is necessary to correlate biochemical events with responses of tumors. By determining thedosages or concentrations of agents required to cause observable biochemical effects and those required to cause observable effects upon the viability of the neoplastic cells, itmight be possible to eliminate superfluous effects and to distinguish between primary and secondary effects. Table 4 contains data showing the concentrations of nitrogen mustard thatcause a variety of physicochemical, biologic, and biochemicaleffects. A number of the effects are caused only at concentrations of the agent that exceed the concentration of the drugthat would be present in a hamster following a dose sufficientto cause regression of the tumor. Therefore, one might logically question the contribution of those particular effects tocausation of regression of the tumor. Thus, it appears thatgross deactivation of the DNA nucleotidyltransferase activitythat is measured by the assay procedure used in the presentstudy and gross deactivation of DNA (as accomplished by invitro treatment of DNA with nitrogen mustard) as a primer forthe synthesis of DNA are not primary modes of action ofnitrogen mustard. It also appears that the extent of reaction ofnitrogen mustard with DNA in vivo, at dosages used in chemotherapy, would be too low to alter the TM@ viscosity, or spec

JANUARY 1969

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Table 4Required

concentrationofPropertynitrogenmustardReferenceCause

of regression of plasmacytoma1.0mg/kg (5.2 x 106 moles per kg)This paper

Glynn P. Wheeler and Jo Ann Alexander

Inhibition of synthesis of DNAIn vivo by plasmacytomaIn vitro by minced plasmacytomaIn vitro by cultured L cellsIn vitro by Ehrlich ascites cellsIn vitro by cell-free supernatantIn vitro by cell-free supernatantIn vitro by a partially purified bacterial enzyme

Inhibition of synthesis of RNAIn vivo by plasmacytomaIn vitro by minced plasmacytomaIn vitro by Ehrlich ascites cellsIn vitro by a partially purified bacterial enzyme

Inhibition of priming and/or template action of DNANative

For synthesis of DNA

For synthesis of RNA

DenaturedFor synthesis of DNAFor synthesis of RNA

Inhibition of protein synthesis by cell-free systemInterference with coding capacity of

polyuridylic acidInterference with coding capacity of

polycytidylic acidInterference with coding capacity of

polyadenylic acidInterference with functioning of ribosomesDecreased binding of treated polyuridylic acid

to ribosomesDecreased binding of phenylalanyl-tRNA to complex

of treated polyuridylic acid with ribosomesDecreased binding of phenylalanyl-tRNA to complex

of polyuridylic acid with treated ribosomess-100 enzymes for amino acid polymerizations-100 enzymes for aminoacyl-tRNA synthesis

Altered TM of DNA

Hyperchronic effect on DNA

Altered viscosity of DNA

Prevention of denaturation of DNA

Inhibition of cloning of cultured H. Ep. -2 cells

Antimitotic effect on cultured cells

Giant cell formation

Decreased cell survival after exposing Ehrlich cellsin vitro and reimplanting into mice

Decreased cell survival after exposing culturedL1210 cells in vitro and implanting into mice

0.3 mg/kg (1.6 x 10—6moles per kg)iO@ M106 Miø@ MiO@ M

>2 x iO@ Mi@—@M

0.3 mg/kg (1.6 x 106 moles per kg)io—@MiO@ MiO@ M

io—sM

iO@ M

i0—@Mio@ M

>5 x iO-@ M

>5 x i0@ M

5 x iO—@M1 )@10—6M5 )( iO@ M

i0—@M1.5 x iO@ M3 )< iO@ M

102 M

4 x iO@ —2 x iO@ M1.5 x i0—@M

>102 M

5 >( 10' M

106 M

106 M10_6 M

5 x 106 M5 )( 106 M

106 M106 M

5 x 106 M

This paperThis paper

88

This paper2119

This paper

27819

This paper19719

1919

12

12

1212

12

12

121919

3073

7

5710

13

a

31

156

13,4

614

20

iO—@—102 MiO@ —iO@ M

io—@M5 x i07 M

iO@ Mio—@—io—@M

iO@ M

5 x i0—@M

5x107M 31

Concentrations of nitrogen mustard that cause certain physicochemical, biologic, and biochemical effects. tRNA, transicr RNA; TM@ midpoint of the absorbance-temperature transition profile of DNA.

aUnpublished data from this laboratory.

108 CANCER RESEARCH VOL.29

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Effects ofNitrogen Mustard and Cyclophosphamide

of a Nitrogen Mustard on Survival, Growth, Protein and NucleicAcid Synthesis of Mammalian Cells in Vitro. Exptl. Cell Res., 31:19—30,1963.

16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J.Protein Measurement with the Folin Phenol Reagent. J. Biol.Chem., 193: 265—275, 1951.

17. Palme, G., Lisa, E., Oeff, K., and Platis, A. Der Einfluss vonCyclophosphamid auf die Desoxyribonucleinsäure-Synthese vonnormalen proliferierenden Zellen sowie Ascites-Tumorzellen.Arzneimittel-Forsch., 13: 1034—1039, 1963.

18. Papirmeister, B. On the Mechanism of Inhibition of T2 Bacteriophage by Mustard Gas. CRDL Special Publication 2-45, ArmedServices Tech. Information Agency, Arlington, 1961. (Via Reference11above.)

19. Ruddon, R. W., and Johnson, J. M. The Effects of Nitrogen Mustard on DNA Template Activity in Purified DNA and RNA Polymerase Systems. MoL Pharmacol., 4: 258—273, 1968.

20. Rutman, R. J., Steele, W. J., and Price, C. C. ExperimentalChemotherapy Studies II. The Reactions of Chloroquine Mustard(CQM) and Nitrogen Mustard (HN2) with Ehrlich Cells. CancerRes.,21:1134—1140,1961.

21. Smellie, R. M. S., McArdle, A. H., Keir, H. M. and Davidson,J. N.The Incorporation of (4-3H) Thymidine and (8-14C) Adenine intoDNA by Particle-free Extracts of Mammalian Cells. Biochem. J.,69: 37P, 1958.

22. Skipper, H. E., and Schabel, F. M., Jr. Experimental Evaluationof Potential Anticancer Agents. VII. Cross Resistance of Alkylating Agent-resistant Neoplasms. Cancer Chemotherapy Rep., 22:1—22, 1962.

23. Tomisek, A. J., Irick, M. B., and Allan, P. W. DeoxyribonucleicAcid Synthesis I. Effect of in Vivo Cyclophosphamide Treatmenton the in Vitro Activity of the Deoxyribonucleic Acid Synthetase System of Sensitive and Resistant Plasmacytomas. CancerRes.,26: 1466—1472,1966.

24. Tomisek, A. J., and Simpson, B. T. Effect of in Vivo Cyclophosphamide Treatment on the DNA-Priming Ability of DNA fromFortner Plasmacytoma. Proc. Am. Assoc. Cancer Res., 7: 71,1966.

25 . Wheeler, G. P. Studies Related to the Mechanisms of Action ofCytotoxic Alkylating Agents: A Review. Cancer Res., 22:651—688, 1962.

26. Wheeler, G. P. Some Biochemical Effects of Alkylating Agents.Federation Proc., 26: 885—892, 1967.

27. Wheeler, G. P., and Alexander, J. A. Studies with Mustards. VI.Effects of Alkylating Agents upon Nucleic Acid Synthesis in Bilaterally Grown Sensitive and Resistant Tumors. Cancer Res., 24:1338—1346, 1964.

28. Wheeler, G. P., and Bowdon, B. J. Some Effects of 1,3-bis(2-chloroethyl)-1-nitrosourea upon the Synthesis of Protein andNucleic Acids in Vivo and in Vitro. Cancer Res., 25:1770—1778, 1965.

29. Wheeler, G. P., and Bowdon, B. J. Effects of 1,3-bis(2-chloroethyl)-1-nitrosourea and Related Compounds upon the Synthesis ofDNA by Cell-free Systems. Cancer Res., 28: 52—59,1968.

30. Wheeler, G. P., and Stephens, Z. H. Studies with Mustards. VII.Effects of Alkylating Agents in Vitro and in Vivo upon ThermalProperties of Deoxyribonucleic Acids from Sensitive and Resistant Plasmacytomas. Cancer Res., 25: 410—416, 1965.

31. Wilfkoff, L. J., Dixon, G. J., Dulmadge, E. A., and Schabel, F. M.,Jr. Effect of 1,3-Bis(2-chloroethyl)-1-nitrosourea (NSC-409962)and Nitrogen Mustard (NSC-762) on Kinetic Behavior of CulturedL1210 Leukemic Cells. Cancer Chemotherapy Rept., 51: 7—18,1967.

109JANUARY 1969

ACKNOWLEDGMENTS

The authors express their appreciation to Mrs. Janet D. Boothe andMr. Anthony J. Moore for technical assistance in performing the experiments, to the Chemotherapy Department of Southern ResearchInstitute for supplying the tumor-bearing animals, to Miss TommieLou Barker for caring for and dissecting the animals, and to Mr. T. C.Herren for performing the radioassays.

REFERENCES

1. Alexander, P., and Mikulski, Z. B. Differences in the Response ofLeukemia Cells in Tissue Culture to Nitrogen Mustard and toDimethyl Myleran. Biochem. Pharmacol., 5: 275—282, 1961.

2. Booth, B. A., Creasey, W. A., and Sartorelli, A. C. Alterations inCellular Metabolism Associated with Cell Death Induced by Uracil Mustard and 6-Thioguanine. Proc. Natl. Acad. Sci. U. S., 52:1396—1402,1964.

3. Brewer, H. B., and Aronow, L. Effects of Nitrogen Mustard onthe Physicochemical Properties of Mouse Fibroblast Deoxyribonucleic Acid. Cancer Res., 23: 285—290, 1963.

4. Brewer, H. B., Jr., Comstock, J. P., and Aronow, L. Effects ofNitrogen Mustard on Protein and Nucleic Acid Synthesis inMouse Fibroblasts Growing in Vitro. Biochem. Pharmacol., 8:281—287, 1961.

5. Butler, J. A. V., and Smith, K. A. The Action of Ionizing Radixtions and of Radiomimetic Substances on Deoxyribonucleic Acid.Part I. The Action of Some Compounds of the “Mustard―Type.J. Chem. Soc., 1950: 3411—3418,1950.

6. Caspersson, T., Farber, S., Foley, G. E., and Killander, D. Cytochemical Observations on the Nucleolus-ribosome System. Effectsof Antinomycin D and Nitrogen Mustard. Exptl. Cell Res., 32:529—552, 1963.

7. Chmielewicz,Z. F., Fiel, R. J., Bardos,T. J., and Ambrus,J. L.Alterations of Some Macromolecular and Biochemical Propertiesof Calf Thymus DNA Caused by “DualAntagonists― and Nitrogen Mustard. Cancer Res., 27: 1248—1257, 1967.

8. Drysdale, R. B., Hopkins, A., Thomson, R. Y., Smellie, R. M. S.,and Davidson, J. N. Some Effects of Nitrogen and Sulfur Mustards on the Metabolism of Nucleic Acids in Mammalian Cells.Brit. J. Cancer, 12: 137—148, 1958.

9. Fortner, J. G., Mahy, A. G., and Cotran, R. S. TransplantableTumors of the Syrian (Golden) Hamster. Part II: Tumors of theHematopoietic Tissues, Genitourinary Organs, Mammary Glandsand Sarcomas. Cancer Res. (Suppl.), 21: 199—234,1961.

10. Gjessing, E. C., and Chanutin, A. The Effect of Nitrogen Mustards on the Viscosity of Thymonucleate. Cancer Res., 6:593—598,1946.

11. Goldstein, N. 0., and Rutman, R. J. Experimental ChemotherapyStudies. VII. The Effect of Alkylation on the in Vitro Thymidine-Incorporating System of Lettr@-Ehrlich Cells. Cancer Res.,24: 1363—1367, 1964.

12. Johnson, J. M., and Ruddon, R. W. Interaction of Nitrogen Mustard with Polyribonucleotides, Ribosomes and Enzymes Involvedin Protein Synthesis in a Cell-free System. Mol. Pharmacol., 3:195—203, 1967.

13. Kohn, K. W., Spears, C. L., and Doty, P. Inter-strand Crosslinking of DNA by Nitrogen Mustard. J. Mol. Biol., 19:266—288, 1966.

14. Levis, A. G., and Dc Nadai, A. Nucleic Acid and Protein Synthesis in Nitrogen Mustard Induced Giant Cells in Vitro. Exptl. CellRes., 33: 207—215, 1964.

15. Levis, A. G., Spanio, L., and Dc Nadai, A. Radiomimetic Effects

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