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[CANCER RESEARCH 39, 131-138, January
1979]0008-5472/79/0039@0O00$02.0O
Oncogenesis, Mutagenesis, DNA Damage, and Cytotoxicity in
CulturedMammalian Cells Treated with Alkylating Agents1
A. R. Peterson,2 Hazel Peterson, and Charles Heideiberger
University of Southern California Comprehensive Cancer Center,
Cancer Research Laboratory, Los Angeles, California 90033
esis. One is to trace step by step the process of
oncogenictransformation in cells assembled from components,
DNA,chromatin, nuclei, and cytoplasm, that have been treatedwith
carcinogens under carefully controlled conditions invitro ; the
other approach is to establish uniform associations between the
process of oncogenic transformation andother processes that are
better understood at the molecularlevel. We have chosen the latter
approach for the studieson the effects of monofunctional
methylating and ethylatingagents reported in this series of
papers.
A prerequisite for our approach is that the processes thatwe
study should have end points that can be measuredaccurately and
interpreted with a minimum of ambiguity.For the process of
mutation, colonies of AZG3-resistantcells, and for the process of
alkali-labile DNA damage,changes in the molecular weight of DNA on
alkaline sucrosegradients have been shown to fulfill most of the
criteria foraccuracy (3, 8, 19, 22, 24, 32, 33).4 Some difficulties
remainin the interpretation of the data of mutation and DNAdamage,
but experiments are designed to resolve thosedifficulties in the
course of this work.
In the present study, we report associations betweenmutations to
AZG resistance, cytotoxicity, and alkali-labileDNAdamage. Mutation
to AZG resistance,which could notbe detected in the tetnaploid
C3H/10T1/2 cells, was measured in diploid Chinese hamster V79 cells
to be relatedthrough associations with DNA damage and cytotoxicity
tothe effects of the alkylating agents in the transformablecells.
Preliminary accounts of this work have been published (21).
MATERIALSAND METHODS
Chemicals, Radiochemicals,and Cell Culture Media.MNNGand EMS
were obtained from Sigma Chemical Co.,St. Louis, Mo. ; ENNG and MMS
were obtained from AldrichChemical Co. , Inc. , Milwaukee, Wis. The
purity of thesechemicals was 98 to 99.5% as determined by UV
andinfrared spectroscopy and by gas-liquid chromatography.Radiochem
icals, [2-14C]thymidine (specific activity, 60 mCi/mmoi) and
[6-3H]thymidine (specific activity, 5 Ci/mmol),were supplied by
Amersham/Seanle Corp. , ArlingtonHeights, ill. Media and sera for
cell culture were suppliedby Grand Island Biological Co., Grand
Island, N. V. PlasticPetni dishes for many experiments were
obtained from
3 The abbreviations used are: AZG, 8-azaguanine; MNNG,
N-methyl-N'-
nitro-N-nitrosoguanidine; EMS, ethyl methanesulfonate; ENNG,
N-ethyl-N'-nitro-N-nitrosoguanidine; MMS, methyl methanesulfonate;
LD, dose lethalto percentage of animals indicated by subscript
number; ANNG, N-alkyl-N'nitro-N-nitrosoguanidine.
4 A. A. Peterson and M. Mulkins, Alkali-labile Phosphate Bonds
in the
DNA of Mammalian Cells and Bacteriophages, manuscript in
preparation.
ABSTRACT
Mutation to 8-azaguanine resistance in Chinese hamstercells and
cytotoxicity and production and repair of alkalilabile lesions in
the DNA of Chinese hamster V79 andtransformable mouse embryo
C3H/10T1/2 fibrobiasts weremeasured after the cells had been
treated for 2 hr with themonofunctional alkylating agents,
N-methyl-, and N-ethylN'-nitno-N-nitrosoguanidine and methyl and
ethyl methanesulfonates. in both cell lines, methylating agents
were morecytotoxic than were equimolar concentrations of
ethylatingagents, and N-methyl- and
N-ethyl-N'-nitno-N-nitrosoguanidine were more cytotoxic than were
methyl methanesulfonate, and ethyl methanesuifonate. At equitoxic
concentrations, N-methyl-N'-nitno-N-nitnosoguanidine was 14 timesas
mutagenic as was methyl methansulfonate and produced400 times as
many alkali-labile lesions; while
N-ethyl-N'-nitro-N-nitrosoguanidine and ethyl methanesuifonate
produced similar numbers of alkali-labile lesions and wereequally
mutagenic. A plot of alkali-labile lesions versusmutations was a
straight line, but the frequency of alkalilabile lesions was 7
orders of magnitude greater than wasthe frequency of mutations; the
rate of repair of the alkalilabile lesions varied inversely as the
first power of thenumber of lesions. These findings suggest that
mutagenicand alkali-labile lesions are associatedbut not identical
andthat neither lesion is associated with cytotoxicity. Theresults
are discussed in the light of chemical theories ofalkylation and
mutagenesis and are compared with previousstudies that showed that
oncogenic and alkali-labile lesionsare not uniformly
associated.
INTRODUCTION
Cells from C3H/10T1/2clone 8 of mouse embryo fibroblasts have
been transformed with a variety of chemical,radiant, and viral
carcinogens to produce foci of cells thatyield malignant
fibrosarcomas in syngeneic, irradiated mice(10, 11). Moreover, the
promoting effects of phorbol estershave been clearly demonstrated
in these cells (16). Therefore, in this cell line under conditions
in which no detectable spontaneous transformation occurs the
molecularevents associated with oncogenesis can be examined
inhomogenous, cloned populations of cells in circumstancesthat can
be controlled more effectively than is possible inexperiments with
whole animals (10, 11).
We envisage 2 kinds of approaches to obtain from C3H/10T1/2
cells information on the molecular basis of oncogen
1 Supported in part by Grant CA-21036 from the National Cancer
Institute,
NIH, and by Grant BC-2H from the American Cancer Society.2 To
whom requests for reprints should be addressed.
Received July 17, 1978; accepted October 10, 1978.
131JANUARY1979
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A. R. Peterson et a!.
T —Total no of AZG1 colonies from treated cultures— Total
no. of dishes after final replating
C —Total AZGrcolonies from acetonetreatedcultures— Total
dishes of replated, acetone-treated cultures
Falcon Plastics, Oxnand, Calif. , until 1977 and were supplied
by Conning Glass Works, Corning, N. V. , thereafter.Density
gradient grade sucrose was obtained fromSchwarz/Mann, Onangeburg,N.
Y.
Culture and Treatment of C3H/10T1/2and V79 Cells.C3H/10T1/2cells
were grown in Eagle's basal mediumsupplemented with 10% fetal calf
serum (26, 27), andChinese hamster V79 cells were grown in
Dulbecco's medium supplemented with 10% dialyzed fetal calf serum
asdescribed previously (3, 24, 25). Exponentially growingcultures
were treated with fresh acetone solutions of alkylating agents for
2 hr at 37°in complete medium. Theconcentration of acetone in the
medium was not toxic anddid not slow the growth of the cells. After
treatment, thecells were washed with complete medium (5 ml) and
coyered with appropriate volumes of fresh medium for measunements
of mutagenesis, cytotoxicity, and DNA repair,which required
posttreatment incubation of the cells.
CytotoxicityAssays. Cytotoxicitywas assayedas
descnibedpreviously (3, 23, 25) by measuring the fraction ofcells
that survived treatment with alkylating agents to produce colonies
of 50 onmore cells within 7 to 10 days aftertreatment. For controls
and treatments with low doses ofalkylating agents (
-
MMS200 400 600 80020
DNA Damage, Mutation, and Cytotoxicity in Cultured Cells
CONCENTRATIONOF ALKYLATINGAGENT(aiM)
0>>
Cl, 120
CI,I—z‘IC100I-,
K80
>-0zLu
CLu
z0
@ 0 2 4 6 8 10 12@ POPULATION DOUBLINGS
Chart 2. The effects of expression time on the frequency of
mutation toAZG resistance in @P9cells occurring spontaneously (@)
or after a 2-hrtreatment with 600 @a.iMMS (0); 1.7 p@ (•),3.4
@iM(A) and 6.8 @.tM(•)MNNG.
MNNG10 15
60
40
20
--AI I I I I0@___
0.10z 0.075
2 o.osoI'-0i 0.025
LD1@,were constructed with at least 5 data points, morebeing
used to delineate shoulder regions, which, particularly in the case
of EMS, were subject to considerablevariability. In some cases,
survival curves were extendedbeyond LD,,@so as to measure D0, the
dose required toreduce the surviving fraction by 1/e in the
exponentialportion of the survival curve (7).
Mutation frequencies after MNNG and MMS treatmentwere measured
after various expression times. All the datapoints that did not
differ significantly from the maximummutation frequency were pooled
to produce a mean maximum mutation frequency. Since we (24) and
others (9, 18,32) have repeatedly demonstrated that plots of F
againstdose of alkylating agent are straight lines under the
conditions of these experiments, 3 well-separated data pointswere
generally considered sufficient to delineate the doseresponse for
mutation. Computer-assisted linear regressionanalysis was used to
fit straight lines to the exponentialregion of survival curves and
to the plots of F and alkalilabile lesions against dose. S.E. of
the slopes and interceptswere calculated by equations given in Ref.
5.
RESULTS
Toxicity of Alkylating Agents In C3H/10T1/2 and V79 Cells
The D0 dose levels of MMS, EMS, and ENNG for V79 cellswere lower
than for C3H/10T1/2 cells, but the shoulders ofthe survival curves
of the V79 cells suggest that these cellsincurred about twice as
much sublethal damage as did theC3H/10T1/2 cells (Chart 1).
However, MNNG was not moretoxic to the V79 cells than to the
C3H/10T1/2cells andproduced survival curves without shoulders in
both celllines (Chart 1), suggesting that the cytotoxicity of MNNG
isaffected by factors related to the ploidy and species oforigin of
the cells and also to the mechanism of delivery ofthe MNNGmethyl
group to targets within the cells.
On the other hand, the ANNG were 1 to 3 orders ofmagnitude more
toxic than were equimolar concentrationsof the alkyl
methanesuifonates in both lines of cells (Chart1). Furthermore, the
ethylating agents were consistentlyless toxic than were the
methylating agents.
Mutationin V79cells
Effectsof ExpressionTime. The expressiontime is theinterval
between the removal from the cultures of themutagen-containing
solution and the addition of selectivemedium containing AZG. During
this time, the cultureswere maintained and neplated in fresh medium
to allowthem to undergo several doublings, the number of whichwere
determined from growth curves. In Chart 2 the expression time is
written as the number of doublings.
The spontaneous mutation frequency was unaffected byexpression
time, but the mutation frequency induced bytreatment of the cells
with MNNG and MMS increased withexpression time until 4 population
doublings had occurred,after which further variations in mutation
frequency werenot significantly different (p < 0.05; Chart 2).
This mimimumrequirement of 4 population doublings for
maximumexpression of induced mutants was the same for all
concentrations of MNNG, MMS (Chart 2), ENNG, and EMS (unpub
0.10 ‘ 0.100.075@ \@ 0.075
0.050@ I 1.@Chart I . Dose-response curves of the cytotoxicity
of alkylating agents.
Colony formation from single cells plated immediately after a
2-hr treatmentwith alkylating agent was determined with Chinese
hamster V79 cells (0)and with C3H/1OTV2mouse embryo fibroblasts
(•).
JANUARY 1979 133
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A. R. Peterson et a!.
lished observations). Therefore , the mutation frequencywas
determined either by taking the mean of all measurements made after
4 doublings or by measuring the mutationfrequency after a single
expression time of 6 to 9 doublings.These 2 methods of
determination were compared andfound to yield virtually identical
results.
Dose-Response Curves. Chart 3 shows the dose-response curves for
mutagenesis in V79 cells treated withmonofunctional alkylating
agents. The induced mutationfrequency increased as a linear
function of dose withintercepts —0,the ANNG was —102-foldmore
mutagenicthan were equimolan concentrations of the
correspondingaikyl methanesulfonates and the methylating agents
weremore mutagenic than were the ethylating agents. Thesefindings
show that the mutagenicities of the alkylatingagents used in this
study are influenced both by the structunes of the leaving groups
and of the aikyl groups.
20
16
12
8
4
0
Alkali-labile Lesions in the DNA of V79 and C3HI1OT'/2Cells
Sedimentation of DNA from C3H/10T1/2 and V79 cellsgave broad
symmetrical profiles containing 80% of theradioactivity applied to
the gradient (Chart 4).The peaks ofthese profiles were at 1695 and
1545, respectively, fromwhich M,, values of 7.12 ±0.06 x
10@daltons (18 expeniments with C3H/10T1/2 cells) and 4.62 ±0.41 x
10@daitons(8 experiments with V79 cells) were calculated. After
treatment of the cells with MNNG (6.8 @tM)the DNA sedimentedmore
slowly (—485,0.17 ±0.04 x 10@daltons, 7 expeniments, C3H/10T1/2
cells; —465,0.14 ±0.01 x 10@daltons,3 experiments, V79 cells),
showing that single-strand breakage had occurred. The frequencies
of alkali-labile lesionscalculated from these examples are 5.74
±1.35 lesions/i 08daltons of C3H/1OT'/2 cell DNA and 6.93 ±0.62
Iesions/i08daltons of V79 cell DNA. Profiles representative of
thesesamples are given in Chart 4.
In both cell lines, alkali-labile lesions increased as a
linearfunction of dose of MNNG, EMS, and ENNG, but in V79
0I I I I
0 10 20 30
0.C)
0
I.
0
>-I—>I—0
0
cr
FRACTION (TOP)Chart 4. Alkaline sucrose gradient sedimentation
profiles of DNA from
C3H/1OTV2cells (A), V79 cells (•),and from V79 cells treated
with 6.8 pMMNNGfor 2 hr, incubatedfor 4 hr, and centrifugedat
13,000rpm for 7 hr(0). A profile of DNA from C3H/1OT'/2 cells
treated with 6.8 pM MNNG for 2hr, incubated for 4 hr, and
centrifuged at 15,000 rpm for 6 hr is also shown
cells the frequency of alkali-labile lesions produced by MMSwas
so low that a significant correlation coefficient couldnot be
obtained for a straight line fit of the data (Chart 5). Inall cases
the intercepts were —0.Chart 5 also shows thatthe ANNG produced
—10k-foldmore lesions than did equimolar concentrations of the
corresponding alkyl methanesulfonates and that the methylating
agents produced about10 times as many lesions as did the ethylating
agents inC3H/10T1/2 cells. However, in V79 cells equimolan
concentrations of EMS and MMS produced similar numbers
ofalkali-labile lesions.
Comparisons of Charts 3 and 5 show that the effectiveness of the
agents in producing mutations parallels theireffectiveness in
producing alkali-labile lesions.
DNA Damageand MutationProducedby EqultoxicDosesof
AlkylatingAgents
Table 1 shows that at equitoxic concentrations MNNGproduced 6
times as many alkali-labile lesions as did MMSin C3H/iOT'/2 cells
and 400 times as many alkali-labilelesions and 14 times as many
mutations as did MMS in V79cells. By contrast, ENNG produced only
1.6 times as manyalkali-labile lesions as did EMS in C3H/10T1/2
cells andnumbers of alkali-labile lesions and mutations in V79
cellsthat were not significantly different from those produced
byEMS. These results show that mutagenesis and cytotoxicity
200 4 8 12 16ENNG
I I I I@
0 2000 4000 6000 8000EMS
OF ALKYLATING AGENT(oM)
0 2 4 6MNNG
I I I I @J
0 200 400 600MMS
CONCENTRATION
0>>
@ 100
Cl)I-.z 80‘4I-
@ 60x
0@ 40
>-0zw
0Ui
U-
z0
‘C
Chart 3. Dose-response curves for mutations to AZG resistance
producedin V79 cells by MNNG R•),correlation coefficient r =
0.983]; MMS [(0), r =0.882];ENNG[(•),r = 0.9931;and EMS[(0), r =
0.860].
I 34 CANCERRESEARCHVOL. 39
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Table1Mutationsand alkali-labile lesions produced by equitoxic
concentrationsofmonofunctional
alkylatingagentsSlopesof dose-response curves of cytotoxicity
(lID0) and alkali-labile lesionsproducedin
mouse C3H/10T1/2 and Chinese hamster V79 cells and of mutations
to AZGresistanceproducedin the V79cells by methylating
(MNNG,MMS)and ethylating (ENNGandEMS)agents.
S.E.'s are calculatedfrom the data points by equations given in
Ref.5.10-@x alkali labile 10@xAZGrD0
lesions x cell x mutationsxCellsAgents (p.@i) D01
ce111xD0'V79MNNG 7.02 ± 0.63 4.74 ±0.74 79.2 ±10V79MMS 164.8
±24 0.011 ±0.04 5.70±1.09V79ENNG 8.13± 1.17 0.714±0.159
19.80±3.3V79EMS 5037 ± 7.00 1.00 ±0.170 17.50
±3.9C3H/1OT'/2MNNG 4.62 ± 0.75 4.79 ±0.24C3H/10T1/2MMS 271
±19.0 0.77 ±0.089C3H/10T1/2ENNG 17.8 ± 3.53 2.21
±0.16C3H/10T1/2EMS 7746 ±19 1 .35 ±0.12
DNA Damage, Mutation, and Cytotoxicity in Cultured Cells
produced by these alkylating agents in V79 cells are
notuniformly associated.
A plot of alkali-labile lesions versus mutation in V79 cellsgave
a straight line with intercept @0(Chart 6), suggestingan
association between mutagenesis and alkali-labile DNAdamage.
However, Table 1 and Chart 6 show that alkalilabile lesions occur
—i0@times more frequently than domutations, suggesting that the 2
events are not uniformlyassociated, which is supported by further
measurements ofthe kinetics of repair of alkali-labile lesions.
3C,)z0@ 2-“C0
@ 1@ 2 ---@-.@-;-o
0 200 400 600 8001000EMS
CONCENTRATION OF ALKYLATING AGENT (,UM)Chart 5. Dose-response
curves for alkali-labile lesions produced in V79
cells (0) and in C3H/1OT'/a cells (•)by treatment with
alkylating agents for2 hr followed by a 4-hr incubationin fresh
medium.Correlationcoefficient,r = 0.95exceptfor r = 0.02for MMSin
V79cells.
Chart 7 shows that the molecular weight of DNA fromC3H/10T1/2
cells treated with MNNG, MMS, and EMS increased linearly with time
as the cells were incubated infresh medium at 37°,showing that the
alkali-labile lesionswere repaired during the incubation. Repair of
the alkalilabile lesions produced by ENNG could not be detected
inthese experiments. The slopes of the straight lines in Chart7
were plotted against the frequency of alkali-labile lesionsmeasured
4 hr after treatment, and it was found that therates of repair
(slopes) varied as the reciprocal of thenumber of alkali-labile
lesions (Chart 8). Moreover, ethylation lesions were repaired more
slowly than were methylation lesions in the C3H/iOT1/2 cells, and
repair of the lesionsproduced by MMS and MNNG in the V79 cells was
slowerthan in the C3H/10T1/2 cells (Chart 7). Therefore, for
thealkali-labile lesions to be uniformly associated with mutation,
the mutation frequency should vary as the first powerof the
frequency of alkali-labile lesions, and the ethylating
3
0 1 2 3 4 5
Lua-
>-@
00
0a.‘—Co
-
‘ENNG
..@ .
A. A. Peterson et a!.
- MNNG
3.0 -@@‘
2.0
3.0
20
1.0
0 20 40 0 20 40
TIME (HR) AFTER TREATMENTChart7.Theeffectsof
posttreatmentincubationin mediumat37°onthe
molecular weight of DNA from C3H/10T/@ cells treated for 2 hr
with 0.17 pMMNNG, 45.4 pM MMS, 6.21 pM ENNG, and 161 pM EMS
(•);0.34 pM MNNG,90.8 pM MMS, 12.4 pM ENNG, and 322 @iEMS (0);
0.68 @a.iMNNG, 136pM MMS, 24.8 pM ENNG, and 644 pM EMS (A); 1 .36
pM MNNG, and 908 pMMMS (Lx);or 2.72 pM MNNG (s); and 6.80@ MNNG
(0).
agents should be more mutagenic than the methylatingagents.
These conditions are not met (Chart 6). Therefore,our
interpretation of Table 1 and Chart 6 is that the alkalilabile
lesions are not mutagenic, but they represent the setof
O-alkyIation lesions which includes those that are mutagenic.
Furthermore, Table 1 shows that alkylation lesionsthat lead to
alkali-labile DNA damage and mutations to AZGresistance are not
associated with cytotoxicity.
DISCUSSION
We have measured colony formation as a criterion
forcytotoxicity, colony formation in medium containing AZG(40
@.tg/mI)for mutation, and the molecular weight of DNAsedimented
through alkaline sucrose gradients for alkalilabile lesions in DNA.
Before attempting to interpret thesedose-response data, it was
necessary to ensure that theabove criteria do accurately represent
cellular lesions thatlead to cytotoxicity, mutagenesis, and
alkali-labile DNAdamage.
Experiments with the effects of ionizing radiations onmammalian
cells (7) have established that our methods formeasuring
cytotoxicity do represent lesions that preventsingle cells from
undergoing a sufficient number (-‘-6)ofdivisions to form
macroscopic colonies of viable cells.
I..cC
¶0
[email protected]
I—-J‘C0
0
‘C
C
0EMS
-@
0.04
0.03
0.02
0.01
0.0I I
2 4J
6 8ALKALI LABILE LESIONS x 10'8 DALTONS
Chart 8. The relationship between the rate of repair (@Mn =
slopes ofcurvesshownin Chart7)andfrequencyat 4 hr aftertreatmentof
alkali-labilelesions produced in C3H/1OT/2 cells by a 2-hr
treatment with MNNG (•),MMS (0), ENNG (A), and EMS
(Es).Resultsobtained from V79 cells treatedwith MNNG (@)and MMS (0)
are also shown.
Those experiments have also been extended to encompassthe
effects of alkylating agents on cultured mammaliancells (3, 9, 18,
23, 28, 32). As for the AZG-resistant phenotype, which is our end
point for mutagenesis, we showedconcurrently with other
laboratories that reproduciblequantitation of mutation at the
hypoxanthine:guanine phosphonibosyltransferase locus requires
stringent selectionconditions that can be satisfied by using 40
@.tgof AZG perml in medium free of hypoxanthine and by replating
mutagen-treated cells at low density prior to selection (8, 18,
19,24, 25, 32). We have now found that expression timeslonger than
we had previously used are necessary foroptimal measurement of the
mutation frequency, in conformity with other reports (8, 18, 19,
32). The developmentand characterization of our alkaline-sucrose
sedimentationprocedure is described in considerable detail4 and
providescomplete corroboration for our interpretation that the
sedimentation measurements used in the present study represent
lesions produced by the alkylating agents in singleDNA strands.
That the lesions become single-strand breakswhen exposed to alkali
has been deduced from our owncalculations (22) and from other
experiments on mammahan cells (33) and bacteriophages (30).
The wide ranges (@-103-fold)in the D0 concentrations ofthe 4
aikylating agents shows that the concentration requined to
penetrate to critical targets for cytotoxicity variesconsiderably
with the structure of the agents. Therefore, itis to be expected
that the concentration required to
penetratetotargetsforotherbiologicaleffectswillalsovaryandthat
comparisons of mutation and DNA damage at equimoIanconcentrations
will reflect differences in the accessibilities of the targets to
the agents, while comparisons atequitoxic doses will reflect
differences in the mechanisms
0
0 h I I I
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DNA Damage, Mutation, and Cytotoxicity in Cultured Cells
of alkylation. These considerations led us to choose
cytotoxicity as a base line for normalizing the results of
mutationand DNA damage, but our decision was also guided by
thestudies of Roberts et a!. (28), who showed in
Chinesehamstercellsthatcytotoxicitywas relatedto the overallextent
of alkylation of the DNA by 3 different methylatingagents.
Furthermore, by normalizing for cytotoxicity weconsider only
lesions that occur in cells that survive treatment with the
alkylating agents.
The dose-response curves for mutation are straight lines.This is
consistent with the theory (12) that relates themutation frequency
to dose according to the formula:
N,,,/N. = Z(G*/G)
where N,,, is the number of mutants, N,, is the number
ofsurvivors, Z is the number of alkylations in the genome,
G*istargetsize,G isgenomesize,and
z = kD
O-alkyiation, some of which should be mutagenic. Howeven,
oncogenic transformation of C3H/10T1/2cells by thesealkylating
agents occurs at too low a frequency (i0@survivors) to be
accurately measured under the conditionscustomarily used for such
experiments (Ref. 26; unpublished observations) but occurs at a
frequency >i0@ survivors in synchronized cells (2) that contain
the same numbens of alkali-labile lesions as do asynchronous cells
(22,23). Therefore, even in cells containing mutagenic
lesions(O-alkylation in DNA) produced by potent carcinogens(MNNG
and ENNG), expression of the transformed phenotype can be diverted
on suppressed.
F We add our findings to the growing weight of evidence( ) from
studies on mutagenesis (1, 24) and carcinogenesis (4,
15, 17, 23) thatthe metabolic processes associated with
theexpression of genetic damage are at least as important asthe
nucleic acid chemistry of the damage itself in detenmining the
biological effects of carcinogens and mutagens.
ACKNOWLEDGMENTS
Part of this work was performed at the McArdle Laboratories for
CancerResearch, University of Wisconsin, Madison, Wis. 53706. We
thank S.Altenbach and D. A. Groom for skilled technical assistance
and Dr. H.Campbell and A. Manaka for some of the computer
programs.
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Cancer Res.,18: 217-366, 1973.
11. Heidelberger, C. Chemical Carcinogenesis. Ann. Rev.
Biochem., 44: 79-121, 1975.
12. Lawley, P. D. Some Chemical Aspects of Dose-Response
Relationshipsin AlkylationMutagenesis.MutationRes.,23:
283-295,1974.
13. Lawley, P. D., and Thatcher, C. J. Methylation of
Deoxyribonucleic Acidin Mammalian Cells by
N-Methyl-N'-nitro-N-nitrosoguanidine. Biochem.J., 116:
693—707,1970.
14. Lehmann, A. A., and Ormerod, M. G. The Replication of DNA in
MurineLymphoma Cells (15178). Biochim. Biophys. Acta, 204: 128-143,
1970.
15. Margison, G. P., Margison, J. M., and Montesanto, A.
Accumulation of0-methyl Guanine in Non-Target-Tissue
Deoxyribonucleic Acid duringchronic Administration of
Dimethylnitrosamine. Biochem J., 165: 463—468, 1977.
16. Mondal, S., Brankow, D. W., and Heidelberger, C. Two-Stage
ChemicalOncogenesis in Cultures of C3H/1OT'/2 Cells. Cancer Res.,
36: 2254-2260, 1976.
17. Nicole, J. W., Swami, P. F., and Pegg, A. E. The
Accumulation of 0'-Methylguanine in the Liver and Kidney DNA of
Rats Treated withDimethylnitrosamine for a Short or a Long Period.
Chem.-BioI. Interac
(G)
where D = dose and k is a constant. The relationshipbetween D
and Z has been established empirically in severallaboratories (13,
28, 33).
Linear dose-response curves for mutation have also beenobtained
by others (9, 19, 32), but the view that the measurement of AZG
resistance in Chinese hamster cells accurately reflects the
mechanism of action of the mutagen (19)is not supported by
experiments that showed no differenceinthe
mutationfrequenciesproducedbyequitoxiclevelsofMNNG and MMS in V79
cells (29). On the other hand, theconditions used in those early
experiments were very different from those that are now considered
necessary formeasuring mutation to AZG resistance.
The dose-response curves for alkali-labile lesions arelinear,
showing that alkylation lesions in DNA increaselinearly with dose
of alkylating agent as demonstrated inprevious studies (13, 28,
33). As to the nature of the lesions,calculations from earlier
experiments where the DNA wassedimenting anomalously had suggested
that the lesionswere depuninations (22). However, Walker and Ewart
(33)and now we have shown that MMS, which would
produceapproximately the same number of depuninations as wouldMNNG
at equitoxic doses (13, 28), produces fewer alkalilabile lesions
than does MNNG. Walker and Ewart (33)provide chromatographic
evidence that their alkali-labilelesions primarily result from
phosphotniesters, and thisagrees with the results of experiments
with bacteriophages(30) and conforms with theory (12, 20) regarding
the mechanisms of aikylation. Therefore, we now consider that
mostof the alkali-labile lesions that we measure result
fromalkylations that give phosphotniesters.
According to chemical theories of the mechanism ofalkylation
(12, 20, 31), an agent that aikylates DNA phosphate oxygens would
also be expected to alkylate otheroxygens in DNA. Alkylation of 06
of guanine would beexpected to result in base substitution
mutations (12, 31).Therefore, mutagenic and alkali-labile lesions
should beassociated but not identical, and in dissociating the
doseresponses for repair of alkali-labile lesions (Chart 8)
andmutagenesis (Chart 6), this is what we have found. The datathat
we present here suggest that C3H/10T1/2 cells treatedwith MNNG,
ENNG, and EMS contain in their DNA extensive
JANUARY1979 137
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A. R. Peterson et a!.
tions,16:301-308,1977.18. O'Neill, J. P., Couch, D. B.,
Machanoff, R., San Sebastian, J. R., Brimer,
P. A., and Hsie, A. W. A Quantitative Assay of Mutation
Induction inChinese Hamster Ovary Cells (CHO/HGPRT System):
Utilization with aVariety of Mutagenic Agents. Mutation Res., 45:
91-101 , 1977.
19. O'Neill, J. P., and Hsie, A. W. Chemical Mutagenesis of
Mammalian Cellscan be Quantified. Nature, 269: 815—817,1977.
20. Osterman-Golkar, S., Ehrenberg, L., and Wachtmeister, C. A.
ReactionKinetics and Biological Action in Barley of Mono-Functional
Methanesulfonic Esters. Radiation Botany, 10: 303-327, 1970.
21. Peterson, A. R. A Stratagem for Experiments on Oncogenesis
in vitro.In: U.Saffioti andH.Autrup(eds.),In
VitroCarcinogenesis,Guideto theLiterature, Recent Advances, and
Laboratory Procedures, National Cancer Institute Carcinogenesis
Technical Report Series No. 44, pp. 205-211, Washington, D. C.: U.
S. Dept. of Health, Education and Welfare,1978.
22. Peterson,A. R., Bertram,J. S., and Heidelberger,C.
DNADamageandIts Repair in Transformable Mouse Fibroblasts Treated
with N-methylN'-nitro-N-nitrosoguanidine. Cancer Res., 34:
1592-1599, 1974.
23. Peterson, A. R., Bertram, J. S., and Heidelberger, C. Cell
Cycle Dependency of DNA Damage and Repair in Transformable Mouse
FibroblastsTreated with N-Methyl-N'-nitro-N-nitrosoguanidine.
Cancer Res., 34:1600-1607, 1974.
24. Peterson, A. R., Krahn, D. F., Peterson, H., Heidelberger,
C., Bhuyan, B.K., and Li, L. H., The Influenceof SerumComponentson
the Growthand Mutation of Chinese Hamster Cells in Medium
Containing 8-Azaguanine. Mutation Res., 36: 345-356, 1976.
25. Peterson, A. R., Peterson, H., and Heidelberger, C. The
Influence ofSerumComponentson the Growth and Mutationof
ChineseHamster
Cells in Medium Containing Aminopterin. Mutation Res., 24:
25—33,1974.
26. Reznikoff, C. A., Bertram, J. S., Brankow, D. W., and
Heidelberger, C.Quantitative and Qualitative Studies of Chemical
Transformation ofCloned C3H Mouse Embryo Cells Sensitive to Post
Confluence Inhibitionof Cell Division. Cancer Res., 33: 3239-3249,
1973.
27. Reznikoff, C. A., Brankow, D. W., and Heidelberger, C.
Establishmentand Characterization of a Cloned Line of C3H Mouse
Embryo CellsSensitive to Post Confluence Inhibition of Division.
Cancer Res., 33:3231-3238, 1973.
28. Roberts, J. J., Pascoe, J. M., Plant, J. E., Sturrock, J.
E., and Crathom,A. R. Quantitative Aspects of the Repair of
Alkylated DNA in CulturedMammalian Cells. I. The Effect on HeLa and
Chinese Hamster CellSurvival of Alkylation of Cellular
Macromolecules. Chem.-BioI. Interactions, 3: 29—47,1971.
29. Roberts, J. J., Sturrock, J. E., and Ward, K. N. DNA Repair
andAlkylation-Induced Toxic, Mutagenic and Cytological Effects in
Mammalian cells. In: P. 0. P. T'so and J. A. DiPaolo (eds.),
ChemicalCarcinogenesis, Part A. pp. 401-423. New York: Dekker,
1974.
30. Shooter, K. V. The Kinetics of the Alkaline Hydrolysis of
Phosphotriestersin
DNA.Chem.-BioI.Interactions,13:151—163,1976.
31. Singer, B. All Oxygens in Nucleic Acids React with
Carcinogenic Ethylating Agents. Nature, 264: 333-339, 1976.
32. Van zeeland, A. A., and Simons, J. W. I. M. Linear
Dose-ResponseRelationships after Prolonged Expression Times in V-79
Chinese Hamster Cells. Mutation Res., 35: 129—138,1976.
33. Walker, I. G., and Ewart, D. F., The Nature of
Single-Stranded Breaks inDNA following Treatment of L-CelIs with
Methylating Agents. MutationRes.,19:331-341,1973.
CANCERRESEARCHVOL. 39I 38
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1979;39:131-138. Cancer Res A. R. Peterson, Hazel Peterson and
Charles Heidelberger Cultured Mammalian Cells Treated with
Alkylating AgentsOncogenesis, Mutagenesis, DNA Damage, and
Cytotoxicity in
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