(CANCER RESEARCH 31, 1573-1579, November 1971]

Cross-linking of DNA by Alkylating Agents and Effects on

DNA Function in the Chick Embryo

Jerome J. McCann, Timothy M. Lo, and D. A. Webster

Department of Biology, Illinois Institute of Technology, Chicago, Illinois 60616

SUMMARY

Drug-induced cross-links are found in the DNA of chickembryos within 6 hr after injection of mitomycin C andmethyl-di-(2-chloroethyl)amine into the egg and 24 hr afterinjection of triethylene thiophosphoramide. Effects on therates of synthesis of DNA, RNA, and protein were studiedwith chemical assays for total content of thesemacromolecules as well as radioactive precursor incorporation.All three drugs inhibited DNA synthesis before RNA andprotein synthesis were affected, but there were discrepanciesbetween the two methods of measuring macromolecularsynthesis; thus, radioactive precursor incorporation is notalways a reliable measurement of macromolecular synthesis inthe chick embryo. When the effects ofmethyl-di-(2-chloroethyl)amine were compared to those ofdimethyl-2-chloroethylamine, a monofunctional analog, theformer was a more effective inhibitor of DNA and RNAsynthesis at equivalent alkylating doses; however, the fact thatthe monofunctional analog had some inhibitory activitysuggests that not all the effects of difunctional alkylatingagents are due to their DNA cross-linking activity.

INTRODUCTION

Alkylating agents are inhibitors of cell division, and for thisreason they have been valuable tools for both basic research incell biology and cancer chemotherapy. It had been speculatedthat the difunctional (and polyfunctional) alkylating agentsexerted their effects because of their ability to cross-link DNA.Although there was evidence for the ability of these agents tocross-link DNA in vitro (4, 8, 12, 15), no such evidence forasimilar activity in vivo was available until Szybalski and Iyer(21) using cesium salt density gradient centrifugation showedthat mitomycin C in the growth medium cross-linked the DNAof bacterial cells and mammalian tissue culture cells. They alsodemonstrated that the amount of cross-linking increased withincreasing amounts of mitomycin C in the medium.Cross-linked DNA, when denatured, will spontaneously"zipper up" to reform double-stranded helical DNA; this

process is concentration independent, and one needs only tobe able to detect a small fraction of double-stranded DNA inthe presence of a large amount of single-stranded DNA. A newassay, which can do this, uses polyethylene glycol and dextranin a 2-phase system in which double-stranded DNA partitionsinto the polyethylene glycol-rich upper phase and

single-stranded DNA partitions into the dextran-rich lowerphase (3). This assay has been used to demonstrate that manypreparations of DNA normally contain some cross-linkedDNA, which was postulated to arise when DNA is shearedduring purification (2, 3). In Escherichia coli, an enzymesystem, consisting of an exonuclease and a ligase, has beenshown to cross-link DNA terminally (25).

When difunctional alkylating agents were injected onto thearea vasculosa of 4-day chick embryos in ovo, within 24 hr aspecific type of macrophage was formed over the entireembryo (24). These macrophages were indistinguishable bylight and electron microscopy from macrophages which areformed during the process of "programmed cell death," a

phenomenon believed important for sculpturing the normalcontours of the limbs (18). The macrophages arise frommesodermal cells adjacent to the dead or dying cells,presumably in response to their moribund state, and engulfand digest the cells and cell debris associated with cell death(18). Besides the difunctional alkylating agents, onlyhydroxyurea and daunomycin could induce the formation ofmacrophages in 4- and 5-day chick embryos; all other testedmetabolic poisons did not have this effect. The latter includedmonofunctional alkylating agents, actinomycin D,cycloheximide, colchicine, cyanide, and many others (24). Onthe basis of these results, it was postulated that programmedcell death may occur by a mechanism similar to that producedby the difunctional alkylating agents, hydroxyurea, anddaunomycin. With the polyethylene glycol-dextran 2-phasesystem, DNA from chick embryos treated with mitomycin C,thioTEPA,1 or Carzinophilin was demonstrated to contain ahigher percentage of cross-linked DNA than untreatedembryos, but DNA from chick embryo tissues containingprospective necrotic cells and necrotic cells contained nohigher percentage of cross-linked DNA than control tissues(24). Although these direct experiments failed to demonstratea role for cross-linked DNA in programmed cell death, they areinconclusive because of the relatively few cells involved, thefact that they comprised only about 1% of the extirpatedtissues, and limitations of the assay, which would haverequired about 700 more cross-links per nucleus to havedetected an increase in cross-linked DNA (24). The positiveeffects of hydroxyurea, a specific inhibitor of DNA synthesis(13), and daunomycin, which also affects nucleic acidmetabolism although the mechanism of its action is not known(11), suggest that an inhibition of DNA synthesis is the

Received April 8, 1971 ¡acceptedJune 15, 1971.

'The abbreviations used are: thioTEPA, triethylene thiophosphoramide; HN2, methyl-di-(2-chloroethyl)amine.

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Jerome J. McCann, Timothy M. Lo, and D. A. Webster

primary mechanism by which all these drugs act and may alsobe a primary mechanism of cell death in programmed celldeath during normal chick embryo development. Thus, itbecame imperative to study the effect of these drugs onmacromolecular synthesis, especially DNA synthesis, duringchick embryo development. First, we undertook a detailedstudy of the cross-linking activity of various drugs with thepolyethylene glycol-dextran 2-phase system but with anabridged procedure relative to that used formerly (24). Thedrugs tested were mitomycin C, HN2, thioTEPA,hydroxyurea, and dimethyl-2-chloroethylamine. The effects ofthese drugs on the incorporation of specific radioactiveprecursors into DNA, RNA, and protein as a function of timeafter injection was determined as well as their effects on totalDNA, RNA, and protein content. The results show thatdifunctional alkylating agents can cross-link the DNA of thechick embryos within 6 hr after their injection and that DNAis the first macromolecule for which synthesis is inhibited bythese drugs.

MATERIALS AND METHODS

The drugs were obtained from the same sources that wereused previously (24). The radioactive compounds werepurchased from International Chemical and Nuclear Corp.,Irvine, Calif., and had the following specific activities:thymidine-methyl-3H (10.5 Ci/mmole), uridine-5-3H (21.7Ci/mmole), L-leucine-4,5-3H (29.8 Ci/mmole), uridine-2-14C(30 mCi/mmole), L-leucine-14C (uniformly labeled, 210

mCi/mmole). The fertile eggs were obtained from C. L. Sharp(Glen Ellyn, 111.). DNase 1 (bovine pancreas) and RNase(bovine pancreas) were purchased from Worthington Biochemical Corp. (Freehold, N.J.). We used Pharmacia T500dextran, Lot 4024, primarily, and General Biochemicals, Inc.(Chagrin Falls, Ohio) polyethylene glycol (M.W.5700 to6700), Lot 88452.

Eggs were incubated for 4 days and candled to determinethe position of the embryo, and the drugs were injected on thearea vasculosa as previously described (24). The radioactiveprecursors were injected into the yolk sac through a holedrilled in the narrow end of the egg. Generally 1 nCi(sometimes 2 fid) of 3H-labeled thymidine and 0.2 nCi ofuridine-'"C or L-leucine-I4C were used at dilutions such that

0.1 ml was injected. After the appropriate incubation, theembryos were removed, stripped of their extraembryonicmembranes, and washed in 0.9% NaCl solution. Only livingembryos were used, generally 5 to 10 per time point. Fordetermination of radioactive incorporation of precursors andtotal content of DNA, RNA, and protein, the embryos werehomogenized for 1 min at top speed in the Sorvall Omni-Mixerwith 5 ml of 0.9% NaCl solution per embryo. Aliquotsof 0.1ml were added to 0.5 ml of cold 1.0 M trichloroacetic acid,and the precipitates were collected on 2.4-cm Whatman GFAglass fiber paper. The discs were washed with 95% ethanol,dried with a heat lamp, and added to vials containing 5 ml of0.4% Omni-Fluor (New England Nuclear, Boston, Mass.) intoluene. Radioactivity was determined on triplicate sampleswith a Beckman Model 1650 liquid scintillation counter. DNAcontent was estimated with the diphenylamine assay (19),

RNA with the orcinol assay (19), and protein with thebiuretassay.

For the estimation of cross-linked DNA, the embryos werehomogenized in 0.15 M NaCl : 0.1 M EDTA, pH 8.1, 2ml/embryo, with a glass homogenizer fitted with a Teflonpestle. Pronase (Calbiochem, Los Angeles, Calif.), and a 20%solution of sodium dodecyl sulfate were added to give finalconcentrations of 500 ng/ml and 1%, respectively. Afterincubation for 20 to 24 hr at 37°, the solutions were

deproteinized with an equal volume of chloroformibutanol(4:1, v/v) and centrifuged to separate the phases, and thenucleic acids were precipitated from the aqueous phase with95% ethanol. The nucleic acids were collected on a stirring rodand dissolved in cold 0.2 M NaOH:0.02 M EDTA (trisodiumsalt). The solutions were warmed to 45°for 15 min, cooled to0°,and neutralized with 0.3 volume of 1.0 M KH2P04. After

another deproteinization step with chlorofornrbutanol, thesolutions were dialyzed against 1 liter of 0.01 M sodiumphosphate, pH 7.0, for ca. 24 hr at 4°with 1 change of buffer.

Chloroform was added to the dialysis buffer, and the dialysistubing was boiled for 30 min in 0.1 M acetic acid to removeUV-absorbing materials. Cross-linked DNA content of thedialyzed samples was estimated with the polyethyleneglycol:dextran 2-phase system (1) with a stock made up of23% (w/w) polyethylene glycol and 3 volumes 34% (w/w)dextran. One volume of sample was added to 0.8 volume ofstock solution and mixed intermittently with a Vortex mixerfor 1 to 2 hr. Low-speed centrifugation separated the phases,and the upper phase containing the double-stranded DNA wascarefully removed and mixed with an approximately equalvolume of chloroform to precipitate the phase polymers. Aftercentrifugation, the upper aqueous phase was removed andassayed directly for DNA content with the Burtonmodification (6) of the diphenylamine assay except that theprecipitation step was omitted. The DNA content of thedialyzed samples before phase separation was simultaneouslydetermined; all determinations were in duplicate. Appropriatecontrols were run simultaneously in the 2-phase system,including native DNA, heat-denatured DNA, and phosphatebuffer alone.

RESULTS

Cross-linked DNA. The purity of the dialyzed samples, priorto phase separation, was estimated with A2s9:A23o, andsince this ratio was generally 2.20 or greater it was assumedthat the DNA was quite pure. The alkali treatment wasexpected both to denature the DNA and to hydrolyze theRNA. These samples were subsequently assayed for DNA,RNA, and protein with the Burton assay (6), the orcinol assay(19), and the assay of Lowry et al. (16), respectively, and werefound to contain on the average a content ofRNA:DNA:protein of 2.8:1.0:0.27. It therefore becameimperative to know whether these high ratios of RNA andprotein to DNA would interfere with the partition of DNA inthe 2-phase system. To test this, a solution of 1 mg/ml ofcrude yeast RNA and a chick embryo extract, made byblending 6-day embryos 1 min at top speed in the Sorvall

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Effects of Alkylating Agents on Chick Embryo DNA

0.00

-0.50

-1.00

-1.50

-2.00 i

-2.50

-3.00

.il RNA //g ONA

2.0 4.0 6.0 ap 10 .. 20 40T T ï à "̄^^^""T^r*^^^â„¢"

NativeONA

D Protein• RNA

Denatured ONA

0.2 0-8

i/ 'i DNA

1-0" 2.0 4.00.4 0-6

tig Protein/"!

Chart 1. Effect of RNA and protein on partition of DNA inpolyethylene glycol-dextran 2-phase system. Native chick embryo DNA(107 Mg)or heat-denatured chick embryo DNA (358 jug)plus increasingamounts of yeast RNA (1 mg/ml) or embryo extract (2.28 mgprotein/ml) in a final volume of 2 ml were mixed with 1.6 ml of stockphase solution, and the phase separation was performed as described inthe text.

Omni-Mixerin 0.01 M sodium phosphate, pH 7.0, were used assources of RNA and protein "contaminants." Increasing

amounts of the solutions were added to a constant amount ofpurified chick DNA, native and heat denatured, and tested inthe 2-phase system with the results shown in Chart 1. Withinthe limits tested, there was little effect of either RNA orprotein on the partition of DNA in the phase system.

The recovery of purified chick DNA subjected to the entirepurification sequence averaged 71% after dialysis. Therecovery of alkali-denatured DNA after neutralization,deproteinization, and dialysis was 80%.

The amount of cross-linked DNA in chick embryos as afunction of time after injection of mitomycin C, HN2, andthioTEPA is presented in Chart 2, while Chart 3 is adose-response curve for mitomycin C. Even low doses ofmitomycin C (10 to 20 Mg/egg)produce an easily detectableamount of cross-linked DNA within 24 hr. Two other drugsused in this study were hydroxyurea anddimethyl-2-chloroethylamine; they were tested for cross-linking activity (Table 1), and as expected they had none. It wasreported (17) that hydroxyurea caused the appearance of aspontaneously renaturable DNA component in treated E. coli,which could have been either cross-linked DNA or highlyrepetitious DNA, but this component is not present in chickembryos treated with hydroxyurea.

Precursor Studies. The precursors selected for comparingrates of DNA, RNA, and protein synthesis were thymidine,uridine, and leucine, respectively. The specificity of

incorporation of each of these precursors was tested withappropriate enzymes (Table 2). Approximately 25% of theuridine is incorporated into DNA as judged by resistance tohydrolysis by RNase and base. The incorporation of uridine

11.0

10.0

6.0

I 5-°

auK! 4.0

oKu

3.0

2.0

1.0

100,,,, HN2per egg

itomycinper egg

50i/g ThioTEPAper egg

20/<gMitomycinper egg

•Control

12 24 36 48HOURS AFTER INJECTIONS

Chart 2. Cross-linking of DNA by mitomycin C, HN2, and thioTEPAin 4-day chick embryos.

sor

4.0 •

g

3.0

2.0 -

1.0

20 40 60 80 100

lig MITOMYCIN C INJECTED/EGG

Chart 3. Dose-response curve for cross-linking of DNA by mitomycinC in chick embryos after 24 hr, corrected for cross-linking of controls.Average of 2 experiments.

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Jerome J. McCann, Timothy M. Lo, and D. A. Webster

Table 1Assay for cross-linked DNA in 4-day chick embryos treated

with hydroxyurea or dimethyl-2-chloroethylamine

Drug treatment % DNA in top phase

None 0.86Hydroxyurea, 500 /ig/egg, 24 hr 0.71Hydroxyurea, 500 fig/egg, 48 hr 0.75Dimethyl-2-chloroethylamine, 200 ¿ig/egg,24 hi 1.03Dimethyl-2-chloroethylamine, 200 /ig/egg, 48 hr 0.70

Table 2Specificity of incorporation of thymidine, uridine, and

leucine into DNA, RNA, and protein, respectivelyFour-day incubated eggs were given injections of

thymidine-methyl-3 H (5 ¿iCi/egg),uridine-5-3H (0.5 juCi/egg), orL-leucine-4,5-3 H (0.5 /iCi/egg), and after 24-hr incubation homogenates

were prepared from the embryos as described in the text. Aftertreatment with the following enzymes and reagents, the trichloroaceticacid-precipitable cpm were determined on the treated homogenates anduntreated controls with the procedures described in the text. Allenzyme incubations were at 37°.

RadioactiveprecursorThymidineUridineTreatmentDNase

\, 50 Mg/ml, l hrRNase, 42 ¿ig/ml,l hr0.96MNaOH, 50°,1hrDNase

I, 50 fig/ml, 1 hrRNase, 42 Mg/ml, l hr0.96 M NaOH, 50°,1 hi%

of cpmsolubilized67°

6026

7576

Leucine Pronase, 500 /ig/ml, plus 1% 95sodium dodecyl sulfate. 4 hr

0 After 14 hr, 82% of the cpm were solubilized.

60.0001-

50.000 •

40.000 •

30.000

20.000 -

10.000

1 200

/ HU

400

200

10 20 30 40 50 60

HOURS AFTER INJECTIONS

into the DNA of E. coli occurs primarily after its conversion tocytidine (9), and a similar pathway may exist in chickembryos. This should not affect the results reported here aslong as the relative rates of incorporation into RNA and DNAwere the same in control and drug-treated embryos.

The effect of drugs on incorporation of these precursors wasinvestigated in double-labeling experiments with 3H-labeledthymidine and uridine-14 C or leucine-14C. Since an inhibition

of DNA synthesis might cause a secondary inhibition of RNAsynthesis, because there will be less DNA template on whichRNA can be synthesized, we tested hydroxyurea, a knownspecific inhibitor of DNA synthesis (13), to ascertain whethera primary inhibition of DNA synthesis could be distinguishedfrom a secondary inhibition of RNA synthesis. Hydroxyureadoes inhibit DNA synthesis, as measured by both 3H-labeled

thymidine incorporation and total DNA content per embryo,when injected into 4-day chick embryos (Chart 4). The effectsof this drug on 3H-labeled thymidine and uridine-14 Cincorporation in double-labeling experiments were primarilyan inhibition of 3H-labeled thymidine incorporation,

beginning between 10 and 20 hr, and secondarily an inhibitionof uridine-14C incorporation, beginning between 20 and 30 hr

(Chart 5). In this and subsequent charts, the data are expressedas percentage of inhibition relative to controls so that severalexperiments could be averaged because of variations inincorporation of precursors from experiment to experiment.At this stage of development, the embryos are approximatelydoubling in mass every 24 hr. An inhibition of synthesis of amacromolecule of 50% between any 24-hr time point,therefore, represents a complete inhibition of accumulation ofthat macromolecule. For time points differing by 12 and 48hr, the corresponding values are approximately 30 and 75%,respectively. Thus, for hydroxyurea there is littleaccumulation of DNA between 8 and 33 hr, and the inhibitionis approximately 50% at 33 hr (Charts 4 and 5).

Mitomycin C also inhibited DNA synthesis before RNA

60

800 *o

600

50

40

o " 30hiko

20

10

10 20 30 40 50 60

HOURS AFTER INJECTIONSChart 4. Inhibition of DNA synthesis by hydroxyurea (500 jig/egg)

in 4-day chick embryos. Average of 2 experiments, o, control; •, Chart 5. Inhibition of incorporation of precursors into nucleic acidshydroxyurea (HU); , thymidine-3H incorporation per embryo; , in 4-day chick embryos by hydroxyurea (500 Mg/egg).•,thymidine-3H;MgDNA per embryo, o, uridine-1 4C.

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Effects ofAlkylating Agents on Chick Embryo DNA

sor

40 •

E 30eg

i¿v 20

10 •

. thymidine

for each cross-linking drug. Further, DNA from embryostreated with mitomycin C and HN2 was cross-linked within 6hr after treatment (Chart 2), and 3H-labeled thymidine

incorporation was inhibited by 10 to 12 hr (Chart 6), whileDNA from embryos treated with thioTEPA was not cross-linkeduntil 24 hr after treatment (Chart 2), and 3H-labeled

thymidine incorporation was not inhibited until 30 hr.An important question is whether drugs with 2 or more

alkylating sites per molecule (i.e., cross-linking ability) aremore efficacious in inhibiting the synthesis of macromolecules.To answer this question, we compared HN2 and amonofunctional analog, dimethyl-2-chloroethylamine,

10 20 30 40 50

HOURS AFTER INJECTIONS

60 70

Chart 6. Effects of mitomycin C (20 jug/egg) on incorporation ofmacromolecular precursors in 4-day chick embryos.

50p

40 •

zOt: 30mzz

20

10

10 20 30 40 50

HOURS AFTER INJECTIONS

60 70

Chart 7. Effect of mitomycin C (20 jug/egg) on net synthesis ofDNA, RNA, and protein in 4-day chick embryos.

synthesis, but both were inhibited to the same extent afterabout 30 hr (Chart 6). Incorporation of leucine-14C was not

inhibited by mitomycin C, although total protein content perembryo relative to controls decreased, as did total DNA andRNA content (Chart 7). The effects of HN2 and thioTEPA onthese same parameters were also investigated. There was nosignificant difference in the inhibition of incorporation of3H-labeled thymidine and uridine-14C by either of thesedrugs; the incorporation of leucine-14C into acid-precipitable

material was inhibited, but the development of this inhibitiontook longer than the inhibition of incorporation of the nucleicacid precursors. Embryos treated with both drugs showedprimarily a decrease relative to controls of total DNA content,followed by a decrease in RNA and protein content. Thesedata are presented for HN2 (Chart 8).

There is a good correlation between the amount of DNAcross-linked and the percentage of inhibition of DNA synthesis

DNA50r

10 20 30 40

HOURS AFTER INJECTION

60

Chart 8. Effects of HN2 (100 Mg/egg) on net synthesis of DNA,RNA, and protein in 4-day chick embryos.

oi-

40 •

30 •

20

10 •200 ug

•Dimethyl-2-chloroethylaminePer egg

10 20 30 40 50

HOURS AFTER INJECTIONS

Chart 9. Comparison of effects of HN2 anddime thy I-2-chloroe thy lamine on DNA synthesis in 4-day chickembryos. »,inhibition of incorporation of thymidine-3H by HN2; •,inhibition of incorporation of thymidine-3 H by dimethyl-2-chloro-

ethylamine; o, inhibition of net DNA synthesis (total DNA per embryo)by HN2; •,inhibition of net DNA synthesis by dimethyl-2-chloro-

ethylamine.

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Jerome J. McCann, Timothy M. Lo, and D. A. Webster

injecting twice as much of the latter to have comparablealkylating activities. Dimethyl-2-chloroethylamine should notand does not have cross-linking activity (Table 1), and it is notas effective as its difunctional relative, HN2, in inhibiting DNAsynthesis at comparable alkylating concentrations (Chart 9).The effects of these 2 analogs on total RNA content anduridine-' 4C incorporation were similar to the results for DNA,

i.e., HN2 was a more effective inhibitor as in Chart 9, whereasthey had similar inhibitory effects on protein synthesis.

DISCUSSION

Our results strongly implicate the inhibition of DNAreplication as a primary effect of difunctional alkylating drugson cell division. Assays for drug-induced cross-linked DNAshow its presence within 6 hr after treatment (Chart 2).Although there is evidence for the repair of mitomycinC-induced cross-links in E. coli (22), there is no known repairmechanism for covalent interstrand cross-links in DNA inhigher cells, and since DNA replication requires strandseparation the former will be blocked because the latter isprevented by the covalent cross-links introduced by the drugs.This conclusion is supported by direct measurement of DNAsynthesis with 3H-labeled thymidine incorporation and total

DNA content per embryo. The results with mitomycin C wereparticularly conclusive; both types of measurements showedthat DNA synthesis was inhibited before RNA and proteinsynthesis were inhibited (Charts 6 and 7). Reduction in ratesof RNA and protein synthesis could arise as a consequence ofthe fact that there is less DNA template on which RNA can besynthesized and subsequently less RNA to direct the synthesisof proteins. In E. coli, it was shown early that the primaryaction of mitomycin C was a selective inhibition of DNAformation (20). Although incorporation studies with the other2 cross-linking agents, HN2 and thioTEPA, were not asconclusive, we could demonstrate that net DNA synthesis wasinhibited before net synthesis of RNA and protein wasaffected (Chart 8), suggesting that cross-links affect DNA

synthesis primarily.That precursor incorporation is not always a good

indication of net synthesis is well illustrated in some of theresults reported here. Thus, mitomycin C had no effect onincorporation of leucine-14C into protein for up to 48 hr

(Chart 6), but obviously net protein synthesis was beinginhibited as judged by total protein per embryo (Chart 7). Thesimplest explanation is a mitomycin C-induced breakdown ofproteins so that incorporation of leucine-14C is not inhibited

out net protein content is reduced relative to controls. There isan alternative explanation: the rate of precursor incorporationdepends on pool size and on the accessibility of the precursorto the cell and permeability into the cell, and theseconsiderations must be important here as the precursors areinjected into the yolk sac and there is a difference in the ratesof incorporation of the 3 precursors. After 24 hr, the totalincorporation of uridine-14C, 3H-labeled thymidine, andleucine-14C was about 14, 6, and 3%, respectively. Some early

step on leucine incorporation, such as entrance of leucine intothe circulating blood from the yolk, could be the slow and

rate-limiting step and not affected by mitomycin C treatment.Kay and Handmaker (10) have reached similar conclusionsregarding uridine incorporation and RNA synthesis inlymphocytes. They found that the incorporation of uridinewas probably limited by 1 of the steps prior to the entry ofuridine into the pool of intracellular nucleotides andconcluded that the incorporation of 3H-labeled uridine into

RNA was not a valid measure of the rate of RNA synthesis.Dimethyl-2-chloroethylamine was not as effective an in

hibitor of DNA and RNA synthesis as HN2 at even slightlymore than the equivalent alkylating dose, but it still inhibitedthe synthesis of both to some extent (Chart 9), and its effectson total protein content were similar to those of HN2. Wheeleret al. (28) have concluded from their studies of the effects ofmonofunctional and difunctional alkylating agents on H.Ep.No. 2 cells that cross-linking of DNA is not a prerequisite fortoxicity by alkylating agents. They did find that higherconcentrations of the monofunctional agents were required toexert effects similar to those of difunctional agents. Thegreater toxicity of the difunctional drugs at low concentrations is presumably due to their cross-linking ability. Brookesand Lawley (5), studying T2 and T4 bacteriophages, suggestedthat inactivation by difunctional alkylating agents is due tointerstrand cross-linking of DNA, while inactivation bymonofunctional alkylating agents is due to degradation ofDNA consequent upon its alkylation. Perhaps this holds truefor procaryotes and eucaryotes also. Dimethyl-2-chJoroethyl-amine, with only 1 alkylating site per molecule, is incapable ofcross-linking DNA (Table 1), but it does cause the same strandscission and depurination reactions that result predominantlyfrom alkylating of guanine at the N-7 position by the syntheticalkylating agents (4, 14); mitomycin C apparently reacts atanother site (23).

Not all of the effects of alkylating drugs can be ascribed tolesions induced in DNA, however. Mitomycin C, for example,is known to react with RNA and ribosomes (26); HN2 is evenmore reactive and should be even less specific as it does notrequire prior reduction as does mitomycin C (21). DeCosseand Gelfant (7) concluded that a major biological result ofnitrogen mustard was independent of DNA replication. It islikely that a wide range of cell functions are affected,depending not only on the specific drug and tissue but also onthe dose, duration of treatment, and the stage of the cell whenthe drug is first used (7, 27).

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2. Alberts, B. M. Characterization of a Naturally Occurring,Crosslinked Fraction of DNA. II. Origin of the Crosslinkage. J.Mol. Biol., 32: 405-421, 1968.

3. Alberts B. M., and Doty, P. M. Characterization of a NaturallyOccurring, Crosslinked Fraction of DNA. I. Nature of theCross-linkage. J. Mol. Biol., 32: 379-403, 1968.

4. Brookes, P., and Lawley, P. D. The Reaction of Mono- andDi-functional Aklylating Agents with Nucleic Acids. Biochem. J.,80: 496-503, 1961.

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6. Burton, K. A Study of the Conditions and Mechanism of theDiphenylamine Reaction for the Colorimetrie Estimation ofDeoxyribonucleic Acid. Biochem. J., 62: 315-323, 1956.

7. DeCosse, J. J., and Gelfant, S. Effects of Nitrogen Mustard duringthe Cell Cyle of the Ehrlich Ascites Tumor. Exptl. Cell. Res., 60:185-190, 1970.

8. Geiduschek, E. P. "Reversible" DNA. Proc. Nati. Acad. Sei. U. S.,

47: 950-955, 1961.9. Karlström, O., and Larsson, A. Signficance of Ribonucleotide

Reduction in the Biosynthesis of Deoxyribonucleotides inEscherichia colt. European J. Biochem., 3: 164-170, 1967.

10. Kay, J. E., and Handmaker, S. D. Uridine Incorporation and RNASynthesis during Stimulation of Lymphocytes by PHA. Exptl. CellRes., 05:411-421, 1970.

11. Kim, J. H., Gelbard, A. S., Djordjevic, B., Kim, S. H., and Perez, A.G. Action of Daunomycin on the Nucleic Acid Metabolism andViability of HeLa Cells. Cancer Res., 28: 2437-2442, 1968.

12. Kohn, K. W., Spears, C. L., and Doty, P. Interstrand Crosslinkingof DNA by Nitrogen Mustard. Appendix: Terminology ofConfigurational Changes of DNA in Solution. J. Mol. Biol., 19:266-288, 1966.

13. Krakoff, I. H., Brown, N. C., and Reichard, P. Inhibition ofRibonucleoside Dephosphate Reductose by Hydroxyurea. CancerRes., 28: 1559-1565,1968.

14. Laurence, D. J. R. Chain Breakage of Deoxyribonucleic Acidfollowing Treatment with Low Doses of Sulfur Mustard. Proc.Roy. Soc. London, Ser. A, 277: 520-530, 1963.

15. Lawley, P. D., and Brookes, P. Interstrand Crosslinking of DNA byDifunctional Alkylating Agents. J. Mol. Biol., 25: 143-160, 1967.

16. Lowry, O. 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. Rosenkranz, H. S. An Anomalous DNA Component inHydroxyurea-treated Escherichia coli. Biochim. Biophys. Acta,729:618-621,1966.

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1971;31:1573-1579. Cancer Res   Jerome J. McCann, Timothy M. Lo and D. A. Webster  Function in the Chick EmbryoCross-linking of DNA by Alkylating Agents and Effects on DNA

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