[CANCER RESEARCH 29, 1184—1194,June 1969]

1

Department of Chemistry, University ofPennsylvania, and Division ofAnimal Biology, School of Veterinary Medicine, University of Pennsylvania,Philadelphia, Pennsylvania 19104

SUMMARY MATERIALS AND METHODS

Two lines of Ehrlich ascites tumors showing a 10- to 50-folddifference in sensitivity to nitrogen mustard (HN2) have beencompared as to the extent of alkylation and cross-linking oftheir DNA. The resistant line (L2RA) shows approximatelyhalf the alkylation and half the cross-linking of the sensitiveline (LES) after 60 minutes of exposure to HN2 in vivo. Thealkylation and cross-linking functions have been shown to beproportional to the HN2 dosage; a linear relation exists between alkylation and cross-linking so that 7—8%of the DNA iscross-linked per alkylation, in agreement with predictions fromthe frequency of GpC (S' —@3' base sequence in DNA; G,guanine; C, cytosine) sequences in mammalian DNA. Nothreshold effects are observed for alkylation or cross-linking atlow doses of HN2, the reaction being primarily if not entirelygoverned by the reactivity of the guanine in the native, in vivostructure. The data are fully consistent with the original crosslinking hypothesis as the mechanism of action of bifunctionalalkylating agents but are not consistent with lethal mutationhypotheses of cytotoxicity. The resistance of the Ehrlich cellscan be partially explained by the selection by HN2 of cells lesspermeable to the drug, but the extent of resistance acquiredduring selection requires the intervention of a more elaborateset of cellular changes.

INTRODUCTION

The extensive studies of Brookes and Lawley (1, 2, 14, 15)and numerous other investigators (6, 7, 21, 28) have provenbeyond reasonable doubt that the N7-position of guanine isthe major site of attack by nitrogen mustard on DNA and thatthe major products are N7-guaninyl and N7-bis-diguaninylderivatives. These and other studies (4, 8, 13) have also shownthat the formation of N7-bis-guaninyl alkylation products cancross-link DNA in vitro and in vivo so as to prevent its irreversible denaturation. The purpose of this study was to extendthese findings to the low levels of alkylation accompanying thechemotherapeutic use of alkylating agents in order to examinethe quantitative applicability of the findings to the conditionsobtaining in cells showing different sensitivities to HN2.2

1This work was supported by Grant No. CA 05295 from the USPHS.Received July 24, 1968; accepted January 30, 1969.

Ascites Cell lines. The Lettre@-Ehrlich hypotetraploid ascitestumor was used as a source of HN2 sensitive cells. A resistantsubline was obtained by exposing LES cells to single challengesof HN2 (3.0 mgJkg) twenty-four hours after implantation. Surviving cells were harvested after several weeks; selection wasrepeated through seven transfers. The resulting cell line(L2RA) was resistant to HN2 and was stable in the absence of

further challenge. By increasing the dose of HN2, additional,more resistant sublines can be obtained in this manner, including a line currently being studied which will withstand in vivoexposure to 80 mg/kg of HN2.

The growth characteristics of LES and L2RA were established by titrating mean survival time (days) against inoculumsize (cell number) ; the titrations were done in quadruplicateand the data are presented in Chart 1. Skipper et aL (26) haveshown that this procedure permits a reliable estimate of cellsurvival in L1210 leukemia. Chart 1 is a least squares regression plot of the pooled data for L2RA and LES; althoughL2RA is the somewhat slower-growing cell line, the regressionequations for two cell lines do not show a statistically significant difference. The relative resistance of L2R.A and LES toHN2 were then compared in two ways: animals bearing 1-dayold ascites were given 5 daily doses of HN2, and survival wasobserved; alternately, 5 X 10@ cells/mi of pH 7.0, salinediluted ascites fluid were exposed to HN2 at concentrations of106 to 103M, the excess HN2 removed by centrifugation,and 1O@'of the resuspended cells injected into mice for survivalstudies.

Alkylation Studies

Fifteen to twenty Swiss mice bearing 6-day-old growths ofLES and L2RA ascites tumors were given single intraperitoneal

2The following abbreviations are used in the text: LES, LettréEhrlich hypotetraploid ascites tumor; L2RA, a resistant sublime of LES;SSC, Standard saline citrate (0.14 M NaC1, 0.014 M NA3 citrate); HMP,0.01 M potassium phosphate buffer, pH 7.0; EDTA, ethylenediaminetetraacetic acid, sodium salt; HN2, nitrogen mustard. Throughout thispaper, the terms % reversibly renatured DNA and % cross-linked DNAare used interchangeably to connote the fraction of the DNA whichbehaves like native, double helical DNA following thermal denaturationand annealing. GpC and GpG = 5' -+ 3' base sequence in DNA; G,guanine; C, cytosine.

1184 CANCER RESEARCH VOL.29

Differences in the in Vivo Alkylation and Cross-linking of NitrogenMustard-sensitive and -resistant Lines of Lettre'-Ehrlich Ascites Tumors

Edward H. L. Chun, Leon Gonzales,FlorenceS. Lewis,Judith Jones,and Robert J. Rutman

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Table1Moles

ofHN2 bound/b7 daltonsDNAExperiment

1Experiment 2Experiment30.8

1.01.40.8

1.41.40.8

0.91.0Mean

1.07Overall mean ±S.D.1.2 1.06 ±0.080.95

Alkylation and Cross-linkingofEhrlich Tumors

values are expressed as moles HN2 per 30,000 moles P, or30,000 moles nucleotides, or approximately 10 milliondaltons. The reliability of the assay method and the ability toreproducibly estimate low levels of alkylation is demonstratedin Table 1 ; the overall error in these replicate in vitro measurements is <1 0%, which is also the precision of in vivo measurements (see below).

70

60>-

@50

-j 40

>0:30U)

@20Ui

Chart 1. Mean survival time of Swiss mice given i.p. injection ofsensitivity (LES; ®)or resistant (L2RA; •)Ehrlich cells.

injections of ethyl-'4C-labeled HN2 in the dose range 0.5—15

mg/kg of HN2. (HN2 at specific activity of 0.75 to 3.0mc/mmole, obtained either from Merck, Tracerlab, or NuclearResearch Chemicals, was used in the experiments.) The cellswere harvested after 60 minutes, separated from the ascitesfluid, and the DNA extracted and purified by a modificationof the procedure of Marmur (19). Approximately twenty-fivegrams of packed cells were resuspended in 125 ml of 0.14 MNaC1-0.1 M EDTA at 15—20°Ccontaining 2 nil of 25% sodiumlauryl sulfate. After 10 minutes, when the cells appeared transparent, cold 5 M NaC1O4 was added to bring the final concentration of perchlorate to 1 M. The aqueous mixture wasshaken with an equal volume of chloroform:isoamyl alcohol(24:1v/v) mixture.An emulsionwasformedupon vigorousshaking. After 30 minutes of shaking, the emulsion was brokenby centrifugation in a #19 Spinco rotor at 10,000 rpm for 20minutes in a Spinco Model L-2 ultracentrifuge. The supernatant solution was dialyzed in the cold against standard salinecitrate for two days with several changes of external fluid.Most of the detergent and perchlorate were removed. The solution was treated with ribonuclease (Worthington, 50 j.Lg/ml) inthe cold for two days and then shaken with an equal volumeof chloroform:isoamyl alcohol mixture. Centrifugation removed ribonuclease as a protein pellet at the water-chloroforminterface. The supernatant was made 0.3 M in sodium acetateand 0.60 volumes of isopropanol was added. The DNA wasspooled out on a glass rod and dissolved in dilute saline-citrate(SSC/10).

C

U)

C0

U0

I0

0101 102 ,@3 iø@@ 106 io@ 10

InoculumSize(No.of CeIIs)(X)In vitro alkylation ofDNA by nitrogen mustard (HN2). 5 x i0@ M DNA

as DNA-P incubated with 5 x i0@ M HN2 at pH 7.0 for 60 minutes at37°C. DNA dialyzed at 0—4°Cto remove unreacted HN2 and its hydrolysisproducts; assayed for bound ‘4Cas under Materials and Methods.

Renaturation Studies

The estimation of cross-linking at the lower levels of alkylation (approximately 1 alkylation/107 daltons DNA) has beenthe most critical methodologic problem in these studies.Despite the reliability of the basic 7.2 M NaC1O4-0.1 M EDTAthermal denaturation procedure (9), as demonstrated below, itwas nonetheless decided to obtain values for cross-linked reversibly renaturable DNA by four different procedures, so asto provide increased confidence in the quantitative relationship between alkylation and cross-linking. The methods employed are described below:

(1) ThermalDenaturationProcedure: “Reversible―DNAwas estimated according to the procedure of Geiduschek (9).The absorbance-temperature profile of the samples of alkylated DNA in 7.2 M NaC1O4-0.1 M EDTA was obtained in aBeckman DU spectrophotometer equipped with a specially designed low heat loss thermostated cell compartment. Duplicatesamples of the DNA were separately heated to designated tem

peratures along the melting curve, quickly chilled in ice, andreequiibrated at 25°C. The O.D.260 was read against a nativeDNA blank. The forward melting curve (absorbance-temperature profile) was compared with the reversible profileobtained from the duplicate samples and the residual hypochromicity used to calculate the fraction of cross-linked, reversibly renaturable DNA in the alkylated samples (9). Chart 2illustrates a typical experiment of this type, while Table 2

shows that the NaC1O4 procedure provides a highly reproducible estimate of the reversible DNA in the alkylated samples.3

Alkylation Determination

An aliquot of DNA solution, 0.50 to 1.00 ml (0.2 to 0.5 mgof DNA), was mixed with 10 ml of scintillation liquid countingmixture containing 4% Cab-o-sil and assayed for 14C Anotheraliquot was analyzed for phosphorus content. The alkylation

3Although it has been shown (2) that N7-alkylated guanine deoxyribosides are unstable to heating, our own studies indicate that the lossof cross-links is sufficiently slow at temperatures below 70°C in 7.2 MNaC1O4 that no significant change in the extent of reversibility occursunder our test conditions.

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Table2Reversibly

renatured DNA(%)Experiment

IExperiment 2Experiment316,

1516, 1817, 1718,

1818, 1920, 1818,

1818, 1813,17Mean

±S.D. 16.5 ±0.9Overall mean ±S.D.18.5

±0.817.2 ±1.616.7

±1.8

I I I I

20 30 40 50 60 70

E. H. L. Chun, L. Gonzales, F. S. Lewis, J. Jones, and R. J. Rutman

1.4

1.3

A 1.2

I.1

I.0

T(°c)

Chart 2. Curves for the reversible and irreversible melting ofDNA in7.2 M NaClO4 according to Geiduschek (9) with and without prioralkylation by HN2. A/An, ratio offinal to initial OD. at 260 mis. O.D.for irreversible curves for DNA at different levels of alkylation afterquenching from temperature (T°C)to 0°Cand equilibrating at 25°C.(In descending order from the top curve, alkylation levels are: 0.0, 1.5,3.5, and 10 M HN2/107 daltons DNA.)

(2) Thermal Remelting Procedure: DNA heated at 65°Cfor10 minutes in 7.2 M NaC1O4-0.1 M EDTA was quickly

quenched at 0°C and then exhaustively dialyzed against pH7.0, 0.01 M HMP buffer. The absorbance-temperature profileof the dialyzed, alkylated DNA was obtained in 0.01 M HMPbuffer, representing the remelting of the previously renatured,reversible DNA. The hyperchromicity of this remelted samplecompared to the native DNA melted in 0.01 HMP provides adirect measure of the reversible, cross-linked DNA content of

the alkylated sample. Chart 3 shows this comparison for atypical sample.

(3) Counter-CurrentProcedure:Five-nilaliquots of theNaC1O4 denatured, 0.01 M HMP dialyzed samples obtained inb above were added to 3.5 ml of 20% dextran and 1.47 ml of30% ethylene glycol according to the procedure of Tiselius et

T(°C)

C-)0InC@J

I—

0

Chart 3. Remelt curve for alkylated DNA. Alkylated DNA denaturedin 7.2 M NaC1O4 as in Chart 2; quenched to 0°Cand dialyzed against0.01 M phosphate buffer at pH 7.0; remelted in 0.01 M buffer. —original DNA; -0-, alkylated denatured DNA in 0.01 M phosphate;—E3 —, unalkylated denatured DNA in 0.01 M phosphate. T, tempera

ture.

aL (27) and vigorously shaken. The emulsion was broken bylow-speed centrifugation and the two-phase system subjectedto 10 transfers. The absorbancies (O.D.260 ) of the upper andlower phases were read. In the partition diagram (Chart 4), thedenatured DNA is confined to the lower phase of transfer tubes0 and 1. The extent of reversible renaturation can be estimatedby subtraction of the denatured DNA from the total DNApartitioned in the two-phase system.

(4) Density Gradient Procedure: Aliquots of the NaC1O4-denatured and dialyzed DNA in 0.01 M HMP buffer, containing approximately 10 @.zgof DNA, were made up to 5.7 in MCsCl and centrifuged for 20 hr at 44,770 rpm in a Model ESpinco. UV absorption photographs were taken at this timeand density measurements performed on a Joyc-Lobl microdensitometer. The densitometer tracing of a typical in vivoalkylated, NaC1O4-denatured sample is shown in Chart 5 ; therelative absorbancy of the native (p 1.705) and denatured (p= 1.715) peaks provide a direct estimate of the reversibly re

natured DNA.

Estimation of “reversible―DNA following alkylation by nitrogen mustard(HN2). In each experiment, 5 x i03 M DNA as DNA-P incubated for 60minutes with 5 x 106 M HN2. Experiment terminated by chilling anddiluting in 0°C0.02 M NaCl; replicate aliquots taken and brought to 7.2 MNaC1O4 0.1 M ethylenediaminetetraacetic acid and the absorbance-temperature profiles obtained as under Materials and Methods.

1186 CANCER RESEARCH VOL.29

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surviving for >90 days. Essentially the same picture is observed when the in vivo dose is doubled.

The extent of alkylation of the DNA of LES and L2RA cellswas next examined in mice bearing 6 days' growths of theascites tumors, following single intraperitoneal doses ofHN2-'4C. The HN2 was allowed to contact the cells for 60minutes prior to sacrifice of the mice, removal of the cells, andextraction of the DNA. The extent of alkylation, expressed asmoles of HN2 bound/107 daltons of DNA, is shown in Chart8. It is immediately apparent that the resistant cell DNAundergoes significantly less attack than does the DNA of thesensitive cells. The difference is observable at the lowest doses

tested; at this level (0.5 mg/kg of HN2), the frequency ofalkylation of L2RA-DNA is 2.5 per 106 deoxynucleotides.While there is some overlap of the alkylation data, statisticalanalysis, to be discussed below, shows that the dose-responsecurves for the two cell lines are significantly different.

The DNA samples obtained from the alkylation studies wereanalyzed for cross-linked, reversibly renaturable componentsby the four procedures listed under MateriaLs and Methods; the

Alkylation and O'oss-linking ofEhrllch Tumors

E0(0csJwC-)z403

0U)034

.800

0.700

0.600

0.500

0.400

0.300

0.2000 I 2 3 4 5 6 7 8 9 10

Tube No.

I 1 I1690 .700 1.710 1.720 1730

DENSITY gm/mi

Chart 5. CsC1density gradient profiles for Ehrlich ascites DNA; 10 @sgDNA in 5.7 M CsC1 at 44,770 rpm for 20 hr. Density tracings from UVphotographs by microdensitometer. (a), alkylated, 7.2 M NaC1O4 denatured dialyzed DNA; (b), unalkylated, 7.2 M NaClO4 denatured dianalyzed DNA; (c), native DNA; D, denatured; M, Micrococcus lysodeikticus marker; N, native; R, renatured; S, sateffite DNA bands.

Chart 4. Counter-current separation of native and denatured alkylated DNA according to Tiseius et al. (27). Upper phase, native orrenatured DNA; lower phase, denatured DNA. O.D. of Tubes 0 and 1used to estimate denatured fraction.

D1@The chromatographic procedures used by Brookes and Law

icy (1, 2) for the separation and estimation of di-(guanin-7-ylethyi)methylamine were modified by us so as to secure separations involving the relatively large samples (2—5 mg of DNA)needed to permit radioactivity scanning at very low frequencies of alkylation. A preliminary separation of unalkylated purines and monoalkylated products was accomplishedin Solvent System 1, isobutyric acid:NH4OH:H2O (40: 25:24).This was followed by chromatography in the same direction

using Solvent System 2, methanol:HC1:H2O (7: 2: 1). The suitability of this procedure was confirmed by column chromatography on Dowex 50 H@ using strong HC1 elution. The fraction eluted at 6 N HC1 shows an absorbance at 260 mi/14Cratio of approximately 17, which is approximately twice theratio observed for N7-alkylated guanine and is indicative of 2moles of guanine/mole of HN2-'4 C, as expected for the N7-bis-diguaninyl alkylation product. This fraction chromatographs with a mobility of 0.0—0.05 in Solvent System1—Solvent System 2 unidirectional chromatography. Typicalradioactivity scans of alkylated DNA hydrolysis products areexhibited in Chart 6; the consistency of the results is shown inTable 5.

RESULTS

The differences in sensitivity to HN2 of the Ehrlich asciteslines LES and L2RA are shown in Chart 7 and Table 3. From

Table 3, it appears that the L2RA can withstand at least a10-fold increase in HN2 concentration over that which kills99.99% of the LES cells in vitro. In keeping with this, 5 dailydoses of HN2 at 0.4 mg/kg do not significantly increase themean survival times of mice bearing the L2RA tumor; thesame dose schedule produces a significant extension of longevity in mice bearing the LES tumor, 60% of the treated mice

R‘I

R

(a)

(b)

(C)

1187JUNE 1969

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‘@.‘0.05

DNA105Purinefraction(I.5@MA+G)

“@‘0.1I

Table3Surviva!

times(days)Surviving―LESL2RALESL2RAControl

cells17.815.6[HN2]

for lSminio—@i0—@10—6>90

>3618.3>90

16.916.0<iO@

i0@—‘0.5<i0@

—0.7—-1.0[HN2]

for 60 miniO@iO@106>90

>4617.3>75

17.914.0<i0@

io—@---1.0<iO@

---0.5—-1.0

DNA 101

(2mg DNA, I N Hi

.@RF

E. H. L. Chun, L. Gonzales, F. S. Lewis, J. Jones, and R. J. Rutman

DNA101

I

l00

50

0

I00

50

0

l00

50

0

(9z>>a:

(I,

0

Chart 6. Comparative chromatography of in vivo alkylated Ehrlichcell DNA; tracings of strip scanning assays for ‘4Cfrom HN2-14C.DNA 101 and 105 (see Table 5) purine fractions after 36-hr dialysis vs0.1 N HC1. Total DNA 101 after lO-min hydrolysis in 1 N HC1 at100°C. Bottom and middle panel, one directional chromatography inSolvent Systems 1 and 2; top panel, Solvent System 1 only. A, adenine;G, guanine.

Chart 7. Comparative survival time of Swiss mice inoculated withi07 Ehrlich cells i.p. and given HN2 on Days 1 to 6 following inoculation. Upper panel, control; middle panel, 0.4 mg/kg HN2 per dose;bottom panel, 0.8 mg/kg of HN2 per dose. K, killed, i.e., remaininganimals sacrificed; Sensitive, HN2-sensitive LES cells; Resistant, HN2-resistant L2RA cells.

pooled data for both cell lines are summarized in Table 4. Atlow HN2 doses (<3.0 for L2RA; <1.5 for LES) correspondingto alkylation frequencies of 1.5 X 10@ , the content of reversible DNA is generally <5%, which is at the lower limit ofdetection of all of the methods except Method 2, the thermalremeit procedure. Since this procedure distinguishes betweenhypochromicity due to aggregates of denatured DNA and thatdue to helix formation, its detection limit is closer to 2.5%. AtHN2 doses of 1.5 mg/kg or less, the data do not unambiguously prove the presence of cross-linked DNA nor do theyprovide evidence of differences between the cross-linking ofLES and L2RA. However, at a dose of 3 mg/kg, there is significant cross-linking of LES cell DNA in contrast to the lower,nonsignfficant values for L2RA. As the HN2 dose increases,significant cross-linking is observed in L2RA-DNA, but thevalue remains approximately 50% of that observed for LESDNA. This separation is projected graphically in Chart 9; theregression lines relating dose to cross-linking are significantlydifferent.

The presence of reversible DNA down to very low levels ofalkylation requires that the cross-linked samples contain N7-bis-(alkyl)-guanines (15). A selection of the alkylated DNA's

were subjected to chromatography after hydrolysis and thefractions assayed for radioactivity. From Table 5 it can be seenthat in the frequency range 3 X 106 to 2.5 X 10@ alkyiations per deoxynucleotide, there is a constant proportion of

Comparative sensitivity of LES and L2RA Ehrlich lines to HN2 in vitro.(50 x iO—@cells/ml incubated with HN2 in diluted ascites fluid at 37°C,pH 7.0; excess HN2 removed by centrifugation; cells dispersed in saline andio@injectedinto 10 mice.)

aDetermined by comparison with survival time vs inoculum size ofChart 1.

CANCER RESEARCH VOL.291188

2 4 6 8 10

WEEKS

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Table4Doseof

(mg/kg)No.

ofsamples

S R%

ofreversibleDNA by method―Cross-linking

(mean % ±S.D.)S1

R2 S R3 S R4 S RSR1.0101.53

1(3)b(3)(1)(2)8(3.7 ±4.0)3.0424.5(4.3)8.0(0)9.03.59.0(4.0)7.3 ±3.8 3.0 ±3.56.06

414.56.418.69.317.09.221.313.017.4 ±5.4 9.3 ±4.510.012016282823±515.05

526.512.629.018.731.015.235.021.529.7 ±4.7 16.7 ±4.0

Alkylation and &oss-linking ofEhrlich Tumors

theless, there is sufficient scatter in the data as well as overlapbetween the results to warrant statistical analysis. Accordingly,the various relations were tested for significance using leastsquares determination of regression coefficients. The individual sets of data, conforming to the linear relationship, Ya + bx, are given below:

A. Alkylation (Y = moles HN2/107 daltons DNA) vs HN2dose (x = mg/kg)

1. LES, Y = —0.05±0.15 ±0.34x ±0.03x@ <@2. L2RA, Y = +0.05 ±0.05 ±0.17x ±0.Olx

B. Cross-linking (Y % Reversible DNA) vs HN2 dose (x

mg/kg)1. LES by Method 1, Y 2.0 ±1.0 + 1.7x ±0.2x2. LESbyMethod2,Y4.3±1.2+1.7x±0.2x NS

3. LES by Method 3, Y 3.2 ±1.4 ±1.9x ±0.3x@4. LES by Method 4, Y 6.0 ±1.5 + 2.lx ±O.3x5. LES, pooled data,

Y=4.0±0.7± 1.8x±0.15x6. L2RA, pooled data,

Y= 1.7 ±0.8 ±0.9x± 0.15x(N.S. = not significantly different)

C. Cross-linking (Y % Reversible DNA) vs alkylations (xmoles HN2/107 daltons DNA)

1. LES, Y4.0+5.3x2. L2RA, Y = 1.7 + 5.6x

The regression equations (A-i and A-2) for the in vivo alkylationof DNA are significantly different for the two cell lines (P <0.001), and both regression lines go through the origin. Fromthis it appears justifiable to conclude that L2RA derives itsresistance, at least in part, from an effective dose reductionwhich protects the resistant cell DNA from alkylation. Thedata also strongly suggest that there is no threshold effect withrespect to alkylation or that, if such effect is present, it canonly be observed at doses having negligible cytotoxic action

(<0.1 mg/kg).The regression equations relating HN2 dose to the cross-link

ing of the DNA present a more complex pattern. First, it is to benoted that the regression coefficients b defining the slope ofthe lines relating dose to cross-linking (Equations B-i to B-4)

za

U)C0

0@0

f-0

c'JzIU)4,

0

EU)C0

0>‘

HN2 (mg/kg)

Chart 8. Comparative alkylation of Ehrlich tumor cell DNA by HN2;single i.p. dose administered 6 days after tumor inoculation. q LESsensitive cells; •,L2RA-resistant cells. Right-hand scale, frequency inalkylations/deoxynucleotide ; inset, expanded scale for low doses(points averaged in main diagram).

the N7-bis-guaninyl (Di-G) component in the alkylated sampies. Since this range of alkylation overlaps the data for %

reversible DNA, which is, in turn, proportional to alkylationfrequency, it may be reasonably presumed that the extensionof the regression lines of Chart 9, relating alkylation to crosslinking, to the lowest levels of alkylation, is justified.

DISCUSSION

The relationship between the dosage of HN2, the frequencyof alkylation of the DNA, and the extent of cross-linking inboth the sensitive and resistant tumor cells is clearcut. None

0 5 10 15

Estimates of the cross-linking of DNA following in vivo HN2 treatment. HN2-'4C administered intraperitoneally to Swiss micebearing 6-day growths of LES (S) and L2RA (R) ascites tumors. DNA extracted and assayed for bound alkylating agent and for

cross-linkage.aMethod 1, NaClO4 temperature-absorbance profile; Method 2, counter-current separation of reversible and denatured DNA;

Method 3, 0.01 M phosphate buffer remelting procedure; Method 4, CsCl gradient procedure.bNumbers in parentheses are regarded as unreliable either because of inadequate replication or because of approach to the lower

limit of detection of the method.

JUNE 1969 1189

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Table5DNA

no.Dose

ofHN2

(mg/kg)Moles

HN2bound/107

daltons DNAColumn

chromatography―Paper

chromatography@'(%Di-G)dpm

oncolumndpm

elutedin 6 N HC1%Di@GCLES

103104102

1051010.8

0.81.72.53.00.25

0.25

0.350.750.751400

1635665629772986372

4041645

83449426.6

24.724.728.116.524

2928

20Average24.125.2L2RA

2012022030.5

1.01.00.10

0.150.15810

9901000220

281

19327.128.319.332

28

19Average24.926.3Overall

average ±S. D.25.0 ±4.1

case of CsCi density centrifugation (Equation B-4), that theintercept a departs significantly from 0. The effect of thepositive intercepts, even in the pooled data, is to overestimatethe cross-linking at low levels of alkylation, e.g., Equation B-Spredicts 9.4% cross-links for LES at 3.0 mg/kg HN2, whereasthe pooled data of Table 4 give a value of 7.3%, while thecomparison at 15 mg/kg HN2 is 31% predicted, 29.7% observed. Notwithstanding these limitations, the data support theconclusion that the extent of cross-linking bears a constant,linear relationship to the frequency of alkylation and that thisrelationship is identical for the sensitive and resistant cells usedin this study. Consequently, the resistance of the L2RA cellspresumably also reflects the approximately 50% reduction incovalent cross-linking of the complementary strands of theDNA, which under the conditions of the tests is not accompanied by selective restriction or elimination of this class ofmolecular damage.

It was noted above that no threshold could be observed inthe dose-response curve for alkylation of the DNA. However,in the estimation of cross-linking as % reversible DNA, thelower limit of detection occurs at a dose of 1.5 mg/kg; at thispoint the estimate does not differ significantly from “0.―Conceivably the insensitivity of the procedures could conceal athreshold effect, but the chromatographic analysis of alkylation products at lower HN2 dose levels (down to approximately 0.5 mg/kg) argues against such a conclusion. The dataof Table 5 indicate that diguaninyl products are a constantfraction (25 ±4%) of the total alkylations at the lowest doselevels studied. Our observations as to the frequency of diguaninyi formation confirm the conclusions reported by Lawley (15) and the findings ofDoskocil and Sormova (7). In view

20

Chart 9. Comparative cross-linking data for Ebrlich tumor cell DNAcorresponding to alkylation data of Chart 8. Each point is pooled datafor four methods (see text). Percent of cross-linked DNA is % of reversibly renatured DNA present in the in vivo alkylated sample afterdenaturation in 7.2 M NaC1O4, quenching to 0°C,and reequilibration.

do not differ significantly for the four methods of estimatingthe reversible DNA content of the alkylated samples. Secondly,whereas all of the methods used seem to overestimate the %cross-linking at very low levels of alkylation, it is only in the

I0

0

E. H. L. Chun, L. Gonzales, F. S. Lewis, J. Jones, and R. J. Rutman

40

z0300Ui

z

U)U)0

U

0 5 10 15HN2 (mg/kg)

Chromatographic determination of the % of diguaninyl products in in vivo alkylated DNA. Di-G, N7-bis-guaninyl.

“DNAhydrolyzed, pH 1.6, 37°C for 18 hr; dialysate applied to 5.0 x 0.8 cm Dow 50 H@ and elutedin batches with 3.5 N HC1 and 6 N HC1. Fractions concentrated by flash evaporation and assayed for‘4Cand absorbance at 260 mj.z.

bSamples of DNA hydrolyzed at 100°C with 1 N HC1 for 15 min. Chromatographed in Solvent Sys

tem 1 and then rechromatographed in Solvent System 2. Fraction which remains at origin (RF@ 0.05)assayed for ‘4C.

CA5 % of total radioactivity in the HN2-'4C alkylated DNA.

CANCER RESEARCH VOL.291190

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Table6FractionDose

ofHN2ObservedPredicted―surviving(mg/kg)(alkylations/cellcross-link/cell)(events/cell)0.37

(= D0)i040.1 1.02.5

x iO@ 2.5 x i0@2.5 x i0@ 2.5 x io'1.25

x i053 x iO@

Alkylation and Q'oss-linking ofEhrlich Tumors

trasted with the findings of Lawley and Brookes (17) using E.coli, Crathorn and Roberts (5) using cultured lymphoma cells,and Wheeler and Alexander (29) using hamster piasmacytomas. These investigators found no difference in the alkylation of the DNA of sensitive and resistant variants, although itis not entirely clear that the extent of resistance in the varioussystems is comparable. Differences in the manner of selectionof resistant cells (UV for E. coli, in vitro exposure for thelymphoma line, and cytoxan for the piasmacytoma) may alsohave influenced the outcome. In contrast, Reid and Walker(20) and Wolpert and Ruddon (31) have observed decreasednuclear uptakes of HN2 by resistant lines.

Although analysis of the nature of the permeability changes

may well prove to be of interest, it will not suffice to explainthe extent of the resistance acquired by the L2RA cells since a50% dose reduction is not equivalent to the observed increasein resistance. Further evidence that this is so has been obtainedby selecting more resistant lines from L2RA cells. Cell lineswhich become resistant to doses of up to 80 mg/kg of HN2show neither a decrease in permeability or in the extent ofalkylation when compared to L2RA. Several conclusions canbe drawn concerning the resistance exhibited by L2RA cells.First, additional mechanisms must be involved in the recoveryof the alkylated cells, mechanisms which presumably removeand repair critical modifications of the DNA produced byalkylation (5, 13, 17, 22, 24). Second, the hypothesis that celldeath will directly reflect the frequency of inactivation of allcopies of a given gene within the cell and that, consequently,increases in cell ploidy and DNA content will cause increase inresistance, does not appear to be consistent with our data.Following the formulation of Simon (25), it is possible tocalculate, on the basis of random distribution, the alkylationsneeded to produce any given level of survival for a given degreeof ploidy using an assumed target size of 3 X i0@ deoxynucleotide pairs. These calculations are compared with ourexperimental observations in Table 6. Irrespective of whether

total alkylations or cross-linking events are regarded as critical,cell death occurs at 1 to 3 orders of magnitude fewer alkylations than would be required on the basis of random inactivation of all copies of a given gene. The disparity argues for anamplification of the effect of the alkylations by a mechanismmore complex than direct genetic death.

Lawley (15) has drawn attention to the correspondence between the in vitro and in vivo patterns of alkylation of DNA;in view of the linear relationships between in vivo HN2 dosage,alkylation, and cross-linking established in this study, it seemsmost probable that the dominant influence in this relationshipis the enhanced reactivity of guanine in the ordered, helical

of the direct proportionality between in vivo [HN2] and DNAalkylation, the constant proportion of diguaninyl alkylationproducts implies that both alkylation and cross-linking areproportional throughout the dose range employed and nothreshold exists for either effect. These findings have an additional implication: Doskocil and Sormova (6, 7) have shownthat di(guanin-7-yl ethyl)methylamine alkylation products canbe formed either as inter- or as intrastrand products, the latterbeing analogous to the thymine dimers formed by ultravioletirradiation, the former supplying complementary strand crosslinks. At low alkylation frequencies (3—6 mg/kg; 1—2alkylations/107 daltons DNA or 3—6/10@ deoxynucleotides), random distribution of diguaninyl products among DNA molecules of M.W. i0'@ daltons, should produce 20 to 40% crosslinking [alkylations/107 gm DNA X fraction guanine alkylalions (0.9) X fraction of Di-G in alkylated guanine (0.25)].The finding that % reversible DNA is no more than a third ofthis value indicates that the majority of the diguaninyl productformation derives from intrastrand events, contrary to theoriginal proposal of Brookes and Lawley (2, 15) but in confirmation of the findings of Doskocil et a!. (7). Our data alsoimply that the ratio of intrastrand/interstrand bifunctionalalkylations remains constant in the chemotherapeutic range,since any fractionation of this effect ought to cause an upwardor downward deflection of the dose-response curve relating %reversible DNA to alkylation.

The manifestations of resistance in the L2RA cells are clearcut. The very small displacement of survival time with multipledoses of HN2 indicates that a major fraction of the L2RA cellssurvive each dose, while the in vitro response suggests thatL2RA cells can withstand at least 10 times and perhaps 50

times as high a concentration of HN2 as can LES cells. Severalchanges are observed in connection with the acquisition ofresistance in this Ehrlich cell line. First, there is the approximately 50% reduction in the alkylation and cross-linking ofthe DNA. This effect can be qualitatively explained by a decrease in the permeability of L2RA cells to HN2; detailedstudies of this, to be published elsewhere, show that at equivalent doses, L2RA cells take up only a fourth to a third asmuch HN2 as do LES cells, but that the partition of the incorporated alkylating agent is identical within both cell lines4.Accompanying the change in permeability, L2RA cells show a25% decrease in cell volume and a comparable decrease inDNA content (i.e., from 15 X 10 12 gm/LES cell to 1 1 X10—12 gm/L2RA cell). Since both of these latter changes arein the direction of greater sensitivity, it would appear thatother rather deep-seated reorganizations of the cell have takenplace of which the permeability change is but one facet.

The findings of Reid and Walker (20) with L-cells and ofGoldenberg and Alexander (1 1) with cultured lymphoma cellsindicate that selective permeability and size changes in response to alkylating agents may have some generality.

The reduced alkylation observed in L2RA cells is to be con

4These findings on permeability are now reported by 0. Klatt et aL,Cancer Res., 29: 286—290, 1969; by M. K. Wolpert and R. W. Ruddon(31), and by R. J. Rutman et a!., Biophys. Biochem. Res. Comm., 32:600—657, 1968.

Comparison of observations with predictions from “lethalmutation―hypothesis.

“Basedon Simon (25).

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E. H. L. Chun, L. Gonzales, F. S. Lewis, J. Jones, and R. J. Rutinan

structure of the native DNA and the limitation this structureplaces on other potential nucleophi.lic sites. C. C. Price et al.(personalcommunication)haveprovidedfurtherquantitativeinsight into this effect by showing that, with diethyl-1.3-chloroethylamine as the reagent, the relative reactivities, comparedto water (competition factors, C.F.), of guanosine, denaturedDNA-guanine, and native DNA-guanine are 3 X i03, 20 X i03,and 150 X i03 respectively.5 The data of Lawley (14),Doskocil and Sormova (6), C. C. Price et al. (personal cornmunication), Walker and Watson (28), and this laboratorypoint to a rigorous adherence to this reactivity, whether invivo or in vitro; so long as the native DNA structure is intact,alkylated guanines exceed 90% of the reaction products. Thisimplies either that the DNA in vivo is in a completely nativestate, i.e., there are no significant denatured regions or that thesingle-stranded regions are shielded from alkylation. This condusion is strengthened by our observation, confirmingBrookes and Lawley (1, 2), that for DNA's of intermediateguanine-cytosine content such as mammalian DNA (21.5%guanine), the ratio of N7-bis-diguaninyi products to totalguanine alkylations is approximately 25% irrespective ofwhether the alkylation takes place in vivo or in vitro. Significant differences between the in vivo structure and the structure of isolated, native DNA would be expected to producedisturbances in this ratio.

Considering the close correspondence of the in vitro and invivo reactions of HN2 with DNA, it seemed of interest tocalculate the extent and pattern of in vivo alkylation from thekinetic parameters of the in vitro reactions using the approxi

mate, expanded form of the second order rate equation, L@ix

K@ [HN2j@ vivo] •[DNA,@ vivo] •L@t . K@ has been experi

mentally determined by us to be 0.07—1.0 moies@ sec @,from studies of the alkylation of DNA by HN2 at pH 7.0 and37°C.6 The respective HN2 and DNA concentrations havebeen approximated as follows for the LES and L2RA cells: 1

ml of packed LES cells contains 8 mg of DNA, equivalent toan apparent concentration of DNA guanine of 5 X 10@ M.One ml of packed LES cells take up approximately 10% of adose of 0.3 @zmoleof HN2, equivalent to an apparent in vivo[HN2J = 3 X 10—@ M; L2RA cells take up approximately2.5% of this dose, producing an apparent in vivo concentrationof 7.5 X 10—6 M HN2. Substituting these terms into theequation and assuming the alkylation to be complete in 10minutes, we have, for

LES: LIx=7 X 10_2 X 5 X i0@ X 2 X i0@ X6X102 6.3 X 106 M alkyl guanine

L2RA: L@x=7 X 102 X 5 X i0@ X 7.5 X 106 X 6 X

102 1.6 X 106 M alkyl guanine

5HN2 has approximately the same competition factor for native DNAguanine as can be seen from the following data: At pH 7.0 Tris buffer(0.01 M) 37°C,60 minutes ofreaction, 5 x 10@ M HN2 hydrolyzes tothe extent of 15% and reacts with 5 x 10@ M DNA (as DNA P) to theextent of 35%. The competition factor is equal to the ratio of reactantconcentrations times the ratio of the extent of reaction; for our datathe value is 1.2 x iO@.

6A constant of approximately 0.05 liter mole1 sec@ can be estimated from the published data ofDoskocil and Sormova (7).

These figures give predicted frequencies of LES and L2RAalkylation, respectively, of 2.5 X i04 and 6 X i0@ perdeoxynucleotide, as compared to the observed frequencies of3 X 10@ and 1.5 X 10@ respectively. The 5- to 10-folddiscrepancy probably relates to an overestimation of the effective in vivo [HN2] because of the failure to account forextensive competitive reactions with proteins and RNA.

Prediction of the partition of HN2 between N7-bis-diguaninyl and N7-monoguaninyi products depends upon knowledge of the frequency of GpC and GpG sequences in the totalDNA guanine as given by nearest neighbor analysis (12) orcalculated from isostich frequency data (23). Neither of thesefrequency types of sequence data are available for Ehrlich cellDNA; however, both types are available for calf-thymus DNAwhich has an identical guanine-cytosine content; since the frequency of GpC's and GpG's in DNA's of similar content showslittle variation (12), we have used calf thymus DNA as a modelfor this calculation. The appropriate data are gathered in Table7. As can be seen, both the observed diguaninyl content of invivo alkylated DNA and the extent of cross-linking per alkylation are in excellent agreement with the values predicted fromthe sequence data. Obviously, this correspondence can only beaccepted as suggestive until sequence data for Ehrlich cellDNA become available.

The theoretical and experimental data indicate that a constant fraction (approximately 7%) of the alkylations of tumorcell DNA produce stable, covalent cross-links.7 \When applied tothe low frequency of 1 alkylation/i06 deoxynucleotides foundby Wheeler and Alexander (29) in the DNA of solid tumorsunder chemotherapeutic conditions, this factor leads to a totalof approximately 1500 cross-links per diploid cell and anaverage of approximately 30 per chromosome, a figure not farremoved from the current estimate of replication points perchromosome (3). These data are consistent with the view thatcross-linking is the critical alkylation event (10, 18), unless itcan be assumed that covalent linkages of the complementarystrands within a DNA molecule (or within a chromosome) donot interfere with semiconservative replication. It may, however, be argued that at very low alkylation frequencies, socalled sensitive cells have sufficient corrective capacity torepair limited damage to their DNA, which presumably indudes the ability to excise at least one of the bases of thecross-linked base pair, and, therefore, other alkylation damagemust be responsible for the death of such cells (30). Sinceresistance in bacterial and mammalian cells is known to beaccompanied by increased ability to excise and remove alkylation products from DNA (5, 13, 17, 22, 24), previous argument leads to the anomaly that at low aikylation frequenciescell death is due to events other than the cross-linking of DNA,but at high frequencies survival results from the ability toremove and repair alkylation lesions. From the foregoing itfollows that either cross-links are irrelevant to the mechanismof action of bifunctional alkylating agents and to the development of resistance or that the original cross-linking hypothesis

7Studies of the alkylation of L1210 cells in vivo, to be publishedelsewhere, show entirely analogous relations between alkylation andcross-linking.

1192 CANCER RESEARCH VOL.29

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Table 7Nearest

neighborfrequency―Isostichfrequencyb%

of DNA bases in GpC's% of DNA G's in GpCExpected % cross@link/alkylationcObserved % cross-link/alkylation―% of DNA bases in GpG's%ofGinGpG% ofG in GpG + GpCExpected % Di-G formationObserved % Di-G formationRatio intrastrand/interstrand Di.G formation5.0

11.510.0

4.822.333.87.0—8.0

3025 ±5

2.54.0

10.09.0

5.023.333.3

Alkylation and &oss-linking ofEhrlich Thinors

Predicted diguaninyl formation on the basis of nearest neighbor and isostich frequency analysis. A,adenine; C, cytosine; G, guanine; GpC and GpG S -@3base sequence in DNA;Di-G,N7-bis-guaninyl.

“J.Josse(12).bShapfro and Chargaff (23). To calculate GpC content, it is assumed that each purine isostich class

(Pun)containingn-xG'sandx A's,hasarandomdistributionofallsequencepermutations.Accordmgly, the GpC frequency is the sum of the frequencies of sequences terminating in G multiplied bythe random probability that the adjacent pyriinidine will be C.

C(% DNA C's in GpC) x fraction of alkylations/G.

dCalcuiated from Equation C-i in range 1 to 5 alkylations.

of Goldacre et a!. (10) remains the most plausible proposalconcerning the mechanism of action of chemotherapeuticalkylating agents.

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1969;29:1184-1194. Cancer Res   Edward H. L. Chun, Leon Gonzales, Florence S. Lewis, et al.   Ascites Tumors

Lettré-EhrlichNitrogen Mustard-sensitive and -resistant Lines of Alkylation and Cross-linking ofin VivoDifferences in the

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