[CANCERRESEARCH38,2709-2718,September1978]0008-5472/78/0038-0000602.00

Postreplication Repair of DNA in Ultraviolet Light-irradiated Normaland Malignantly Transformed Mouse Epidermal Cell Cultures

G. T. Bowden,' B. Giesselbach, and N. E. Fusenig

GermanCancerResearchCenter,Institute for BiochemistryIm NeuenheimerFeld280,6@ Heidelberg1, Germany

ABSTRACT

Alkalinesucrosegradientanalysisof pulse-labeledsingle-strandedDNAwas usedto studythe effect of uitraviolet (UV)irradiationon the sizeof newlysynthesizedDNAin primaryculturesof mouseepidermalcells and in an Invitro malignantly transformed epidermal cell line. it wasfoundin bothcell typesthat the DNAthatwas synthesizedafter UV irradiationwas smaller. This smaller size mayhave been due to gaps in the newly synthesizedDNA orto a slower rate of DNA polymerization.Thus the pulsetime in the case of the treated cellswas increasedsothatequal amountsof DNA synthesisoccurredin controlandtreated cells. Under these conditionsthe pulse-labeledDNA'swere very similar in size, and this suggestedthatthe rate of polymerizationhad been slowed by UV andthat no gapswere present. However,there remainedthepossibility that gaps were present but were being closedrapidly.Caffeinewas thus usedto inhibitgap fillingunderconditions of equalized DNA synthesis in control andtreated cells. If caffeinewas presentduringthe extendedpulse, the newly synthesizedDNA was again smaller inthe UV-treatedcells than in the controlcells. Thus it hasbeen demonstratedthat gaps do occur in newly synthesized DNA of UV-lrradlatedepidermalcells. Pulse-chaseexperiments showed that the gaps in the newly synthesizedDNAfromirradiatedcellswere filled in, andthat theDNA was Joinedand elongated in bothcell types. Theseexperimentsdemonstratethat both normal primary andmalignantlytransformedepidermal cells exhibit a postreplicationgap-fillingmechanismin responseto UV irradiation.

INTRODUCTION

Postreplication repair was first demonstrated in bacteriathrough the study of bacterial mutants that lacked theability to form recombinants (40). These mutants, typifiedby rec A mutants of Escherichia coli, are more sensitivethan are wild-type strains to the effects of UV irradiation,and they lack the ability to link DNA molecules together toform recombinants. Rupp and Howard-Flanders (40) demonstrated that DNA synthesized in UV-irradiated cells had alower molecular weight than did DNA synthesized in nonirradiated cells, which suggested that this newly synthesizedDNA contained gaps. Rupp et al. (24, 25) showed that thesegaps were opposite the UV-induced pynimidine dimers and

I Aided by Grant PF 961 from the American Cancer Society. To whom

requests for reprints should be addressed, at National Cancer Institute, NIH,Building 37, Room 3A19, Bethesda, Md. 20014.

Received December 14, 1977; accepted May 18, 1978.

that on subsequent incubation these gaps were filled in.Evidence obtained later (4, 41) showed that the gaps in thebacterial nascent DNA were filled in by a recombinationalsister strand exchangeof DNA.

Similar effects of UV irradiation on the molecular weightof newly synthesized DNA in mammalian cells have beenobserved, which indicates that postreplication repair mayalso be an important mechanism for by-passing damage inmammalian DNA (7, 9, 11, 12, 16, 18, 26).

The postreplication gaps in the nascent DNA of mammahan cells may be filled in by the same mechanism as inbacteria (recombination) onbyde novo synthesisor by bothmechanisms. Postreplication gap filling in mouse lymphoma cells (26) and human fibroblasts (7) depends largelyupon de novo synthesis, as has been shown by permittingthe incorporation of exogenous 5-bromodeoxyunidineintothe postreplication gaps, followed by photolysis at 313 nmand determination of the number of breaks in the newlysynthesized DNA. Results from these experiments indicatedgaps of about 1000 nucleotides. The experiments performed by these workers did not exclude the possibility thatboth events, recombination and de novo synthesis, may beoccurring. If sister strand exchanges occur in UV-irradiatedmammalian cells, as seems possible, they must be muchshorter than those occurring in bacteria. Published data (5,32, 44) indicate that pynimidine dimers are transferred fromparental to daughter DNA during postirradiation.

Whereas the excision repair mechanism has been postulated to be an “error-free―mechanism with respect tomutation fixation in mammalian cells, it has been postulated that there may be an error-free and an “error-prone―postreplication repair mechanism in mammalian cells.Chang et al. (10) have postulated that in Chinese hamstercells a caffeine-sensitive, “gap-filling―postreplication repair process is an error-free mechanism which is largelyresponsible for cell survival after DNA damage. An induciblecycloheximide-sensitive enhanced rate of DNA strand rejoining has been demonstrated by D'Ambrosio and Setlow(13) in UV or N-acetoxy-2-acetylaminofluorene-treatedChinese hamster cells. This postreplication repair mechanism may be the mammalian cell counterpart for the bactenial “SOS―inducible error-prone repair mechanism (45).The fidelity of the repair in mammalian cells has been testedby the use of caffeine, a specific inhibitor of the postreplication repair mechanism. The literature contains, however,seemingly contradictory observations on the biologicalconsequences of caffeine posttneatment of physical andchemical carcinogen-treated mammalian cells (10, 43). Alsocaffeine has been reported to reduce the recovery of carcinogen-induced tumors in mouse skin (34, 39, 46). Chang eta!(10) have presented data from experiments with Chinesehamster cells that may help to resolve the apparently con

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G. T.Bowdenet

flicting results of experiments with carcinogen-exposedcells posttreated with caffeine. Their results indicated thatcaffeine has multiple but distinct effects on UV-irnadiatedcells: (a) caffeine inhibits an error-free postreplication nepain mechanism; and (b) caffeine can repress induced mutations after they have been “fixedand expressed.―Thusthe timing of caffeine exposure of carcinogen-treated cellsis very important in possible interpretations of results. Thepossible positive on negative role of postreplication repair inmalignant transformation still must be determined.

The establishment of a method to culture mouse epidermal cells allowed us to study the postreplication repairmechanism in a target cell system of defined cellular specificity and in a cell system that retains for the most part themorphology and functions of the in vivo target tissue,mouse skin (19, 22, 23). The establishment of the cell linePDV2 allowed us to compare the postreplication repairmechanism in normal versus transformed cells (20). Thispaper is the first report concerning the postreplication repair mechanism in epidenmal cells and has particular significance with regard to UV-induced cancinogenesis, sinceUV irradiation induces skin tumors in mice (1 , 36).

MATERIALSAND METHODS

Chemicalsand Radlochemicals.Solventsandchemicalsused were reagent grade and were used without furtherpurification. Caffeine was obtained from Merck AG, Darmstadt, Germany. [methy!-3H]Thymidine (6.7 Ci/mmol),[methyl-14Cjcholine chloride (10 mCi/mmol), and L-[U14C]leucmne(500 mCi/mmol) were purchased from NewEngland Nuclear, Frankfurt am Main, Germany. [methyl14C]Thymidine(57 mCi/mmol) was purchased from Amersham Buchler, Braunschweig, Germany.

Eagle's minimal essential medium and fetal calf serumwere obtained from Seromed, Munich, Germany.

CellsandTreatmentProtocol.EPDfrompeninatalmouseskin were prepared as described by Fusenig and Worst (21)from newborn C3H mice. The epidermal cells were platedinto plastic Petni dishes (Falcon Plastics, Oxnard, Calif.) ata mean cell density of 4 x 10@cells/sq cm. The cultureswere maintained at 37°in a humidified incubator with anatmosphere of 5% CO2 in air. Sixteen to 18 hr after plating,the culture medium was changed, which removeddead andunattached cells from the cultures. All of the pulse andpulse-chase experiments were carried out with the primarycultures on the second day after plating (40 to 48 hr) whenthe primary cultures were still proliferating to some extentbut were also undergoing a process resembling epidenmaldifferentiation (20).

The PDV cells were derived by Fusenig et a!. (18) by treatment of a primary culture of epidermal cells from B1OLpmice with 10° M dimethylbenz(a)anthracene. Throughoutthese studies a clone of the PDV cells was used. It is designated PDV 2Cb/1 . The PDV cells were plated into plasticPetni dishes with a mean cell density of 8.8 x 1O@cells/sqcm. All of the pulse and pulse-chase experiments were car

a The abbreviations used are: PDv, malignantly transformed epidermal

cell line; EPD, primary epidermal cell cultures; PBS. Dulbecco's phosphatebuffered saline; xl', xeroderma pigmentosum homozygote.

ned out 2 days after plating, when the cells were still in exponential growth.

Both EPD and PDV cells were irradiated by first removingthe medium, washing the dishes with warm PBS, and thencompletely removing all PBS. The dry dishes were thenexposed to UV irradiation of a wavelength predominantly at254 nm, emitted from a mineral light (Ultra-Violet Products,San Gabriel, Calif.) at a dose rate of 1 J/sq rn/sec. Thedishes were kept in semidarkness for the duration of therepair experiments.

At the end of a pulse or pulse-chase labeling period, theEPD cells were harvested by trypsinization. After the medium was removed, the dishes were washed twice with0.2% trypsin (in PBS without magnesium and calcium) andthen trypsinized off the dish with equal volumes of 0.2%trypsin and 0.2% EDTA (in PBS). After incubation of thedishes at 37°for 10 mm, cold medium was added to thedishes, and the cells were collected by centnifugationat 250x g for 10 mm at 4°.The cell pellet was then washed once

with PBS containing 10 mM EDTA. Finally, the cells weresuspended in the proper volume of 10 mM EDTA in PBS.

The PDV cells were harvested by first removing themedium, washing the dishes twice with PBS, and scrapingthe cells with a rubber policeman into the proper volume of10 mM EDTA in PBS.

PDV cellular DNA was prelabeled by adding either[methyl-3H]thymidmne(1 @Ci/mlmedium) or [methyl1@C]thymidine (0.2 @&Ci/mI)for 24 hr before a mediumchange with nonradioactive medium and further incubationfor 2 hr. After the 2-hr growth period in isotope-free medium, the cells were harvested as described above andloaded on the gradients as described below. In someexperiments PDV cultures were given [methyl-3H]thymidine(1 @Ci/ml)together with [methyl-14C]choline chonide (1

@Ci/ml)or L-[U-14C]leucine (0.5 @Ci/ml).In some experiments parental cellular DNA was prela

beled for 24 hr with [methyl-'4C]thymidine (0.2 @Ci/ml),andafter 2 hr of growth in isotope-free medium the cells werepulse labeled with [methyl-3H]thymidine for 30 mm.

In pulse-labeling experiments EPD or PDV cells wereirradiated with UV (1 J/sq m/aec) and I hr later labeled with[methy!-3H]thymidine (40 @Ci/mlfor PDV and 80 @&Ci/mlforEPD cells) for 30 mm. At the end of the pulse period, thecells were harvested and loaded on the gradients. In somepulse experiments caffeine at a moldr concentration of 1.5on 0.5 mM was included in the medium for the time immediately after irradiation through the duration of the 0.5-hrpulse with [methyl-3H)thymidine.

In pulse-chase experiments EPD on PDV cells were UVirradiated at 1 J/sq rn/sec and 1 hr later pulse-labeled with[methyl-3H]thymidmne for 30 mm. At the end of the pulseperiod, the radioactive medium was removed, dishes werewashed twice with warm medium, and nonradioactive medium containing 15 @gunlabeled thymidine and 15 @&gdeoxycytidine in 5 ml was returned to the dishes. Thedishes were then incubated for the designated chase time,and the cells were harvested and placed on the gradients.

Alkaline Sucrose SedimentationAnalysis. The procedure used in this work is essentially that of Peterson et al.(35). A 36-mi gradient of 5 to 25% sucrose, 0.3 M NaOH, 0.7M NaCI, 0.001 M EDTA, and 0.01 M Tnis was formed in a

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DNA Repair in Mouse Epidermal Cell Cultures

where Ci is proportional to the fraction of total radioactivityfor any given fraction and Mi is the M value assigned to thatfraction.

RESULTS

Pafterns of Alkaline Sucrose Sedimentation of DNAfrom Epidermal Cells. Precise measurements by sedimentation techniques of the size and shape of macromoleculesare best achieved by working with preparations that are aspure as possible. However, when whole cells are lysed onsucrose gradients the cellular DNA represents only a smallfraction of the total number of macromolecules present.Thus in order to interpret correctly changesof the sedimentation profile in molecular terms, it is essential to examinethe profiles for evidenceof DNA-DNA,DNA-protein,or DNAlipid aggregates or complexes.

These possible macromolecular complexes were studiedby the cosedimentation of [methyl-3H]thymidmne-pnelabeledDNA either with [methyl-14C]choline chlonide-prelabeled lipids onwith L-[U-'4Cjleucine-prelabeledprotein in PDVcells.The prelabeled cells were lysed for 2 hr, and the gradientswere centrifuged for 4 hr at 25,000 rpm and 20°.The resultsof these experiments are presented in Chart 1. The bulk ofthe DNA sedimented in a band at the bottom of the gradient,well separated from the band,s of radioactively labeled lipid

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Chart 1. Sedimentation of DNA, protein, and lipid on alkaline sucrosegradients. Log-phase PDV cells were prelabeled with [methyl-3H]thymidine(1 pCI/mI) and either (methyl-―C]choline chloride (1 @Cl/mI)or L-(U

@CJIeucine(0.5 @tCi/mI)for 24 hr. After 2 hr of growth in isotope-freemedium, cells were harvested and lysed in situ on top of an alkaline sucrosegradient for 2 hr before being centrifuged for 4 hr. The data are plotted foreach Isotope as the fraction of total radioactivity versus fraction number.

38.5-mi polyallomer tube. These gradients were centrifugedat 25,000 rpm (81,500 x g) in the SW-27 rotor of a Beckmann Model .L5-65 ultracentrifuge at 20°.For the pulselabeling experiments, the time at full speed was 360 ±5(S.D.) mm, and for the prelabeled parental DNA and theDNA in the pulse-chaseexperiments, the time at full speedwas 240 ±5 mm. The acceleration and deceleration timeswere 8 and 30 mm, respectively.

A layer of 0.2 ml 1 N sodium hydroxide was added to thetop of the gradient prior to addition of cells (1.5 x 10@PDVand 4.0 x 10@EPD cells) in a single-cell suspension in 0.2ml of PBS containing 10 mM EDTA. The lysing layer of 0.4ml was less than 1 mm thick.

The cells were allowed to lysa at 20°for either 2 hr forparentally labeled DNA or 3 hr for the pulse-labeled newlysynthesized DNA. The rationale for these lysing times isgiven in “Results.―

After centnifugation the gradients were fractionated fromthe bottom into approximately 30 fractions of 1.2 ml each.To each fraction were added 100 @gbovine serum albuminand 5 ml 12% tnichloroacetic acid. After precipitation at 4°,the DNA was collected on Whatman GF/c glass fiber filtersand washed with cold 2% tnichloroacetic acid. After thefilters were dried at 80°,they were counted in 10 ml ofscintillator consisting of 5 g PPO and 0.5 g POPOP in 1 litertoluene in a Betazint BF 500 liquid scintillation counter(Berthold, Wildbad, Germany).

Calibrationof the Gradientsby Use of T4 Phage DNA.The alkaline sucrose gradients were calibrated with the useof the results from 12 experiments with T4phage performedby 4 different centrifugations at 25,000 rpm in the SW-27polyallomer tubes. The T4 phage containing 14C-IabeledDNA was a gift from Dr. U. Mueller (Institute for Virology,German Cancer Research Center, Heidelberg, Germany).Approximately 4.5 x 10@plaque-forming units of the labeledphage were added directly to the 0.4-mI lysing solution orwere mixed with a suspension of PDV or EPD cells beforeapplication to the gradients. The values assumed for the520,w and molecular weight of the single-stranded DNA from

T4were 69 S and 60 x 10@daltons, respectively (35).Calculation of Molecular Weights. Data from the gra

dients were stoned in a computer, and a program was usedto do the following calculations. From the S value for eachfraction of the gradient obtained from the calibration, themolecular weight (M) was calculated according to the following equation (14)

520.w BM

in which the constant B for single-stranded DNA in alkalinesolution was assumed to be 0.0528 (42) and the exponent0.4 (31, 42). The molecular weight values were then used tocalculate the number-average (Me) molecular weight andweight-average molecular weight (Me) according to theprocedure described by Ehmann and Lett (14) with the useof the equations

M @Ci@ @(Ci/Mi)

M - @MiCi@ Wi

0 5 10 15 20 25 30Top Fraction Number Bottom

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0 10 15 20 30

Top Fraction Number Bottom

5 25

G. T. Bowdenet al.

and protein which banded towards the top of the gradient.The DNA from bacteriophage [‘4C]T4lysed together with[methyl-3H]thymidmne-labeled PDV cells sedimented normally; none of it was found associatedwith the cellular PDVDNA toward the bottom of the gradient (data not shown).These experiments showed that under the conditions ofIysis and centnifugation there are no apparent DNA-DNA,DNA-protein, or DNA-lipid complexes produced in this procedure, These experiments also showed that DNA molecules of differing molecular weights do not entangle, aggregate, or sediment together in our system.

in pulse-labeling experiments only the growing DNAstrands become radioactively labeled. However, the muchlonger parental strands, although unlabeled, are present,and in alkaline sucrose they can entangle with the growingstrands, rendering interpretation of the profiles meaningless. This effect can be circumvented by degrading the DNAto a small extent so that the long parental strands aresomewhat broken down, but the shorter growing strandsare unaffected. This degradation can be done with X-ray(15, 26) or with extended alkaline (0.5 M NaOH) lysis (30,38). In order to work out the proper alkaline lysis conditions,PDV cells were prelabeled with [methyl-'4C]thymidine for24 hr. Four hr after the ‘4Clabel was removed, the cellswere pulse-labeled for 30 mm with [methyl-3H]thymidmne.The doubly labeled cells were then lysed in situ on top ofthe gradient for various lengths of time at 20°and thencentrifuged at 25,000 rpm for 6 hr. Four different lysis times

5 10 15 20Fraction Number

Chart 2. Effect of 0.5-hr cell lysis and denaturation on the pattern ofprelabeled and pulse-labeled DNA. Log-phase PD'I cells were prelabeled for24 hr with [methyl-'@C]thymidine. After 2 hr of growth in isotope-freemedium, cells were pulsed for 0.5 hr with [methyl-3H]thymidine before beingharvested. The cells were lysed in 0.5 M NaOH for 0.5 hr before centrifugationfor 6 hr.

(0.5, 1.0, 2.0, and 3.0 hn) were studied. The results of the0.5- and 3.0-hr lysis are shown in Charts 2 and 3, respectively. With 0.5 hr lysis (Chart 2), the ‘4C-labeledparentalDNAwas on the bottom of the gradient and the 3H-labelednewly synthesized DNA formed a peak in the middle of thegradient. There was, however,evidence for the presenceofDNA aggregates (parental DNA entangled with newly synthesized DNA) toward the right side of the 3H peak. Underthese centrifugation conditions, T4 phage DNA sedimentedto Fraction 12 to 13. After a 2-hr lysis, the parental DNAsedimented to approximately Fraction 26, and the 3H-Iabeled newly synthesized DNA exhibited some skewing toward the larger molecular weights. The results of a 3.0-hrlysis arepresented in Chart 3. The 14C-labeled parental DNAbanded around Fraction 25, and 3H-Iabeled newly synthesized DNA banded in a symmetrical peak near the center ofthe gradient with little evidence for aggregates.

Thus in all the pulse-label experiments reported in thispaper, the harvestedcells were lysedfor 3 hr in 0.5 MNaOH,and the DNA was centrifuged for 6 hr at 25,000 rpm. Forpulse-chase experiments where larger molecular weightspecies were studied, a 2-hr lysis was followed by centnifugation for 4 hr at 25,000 rpm.

Effectof UVIrradiationonthe Sizeof NewlySynthesizedDNAin EPDand PDVcells. Inorderto testforthe presenceof gaps or discontinuities in the newly synthesized DNA inUV-innadiatedEPDand PDVcells, the cells were irradiatedat different dose levels, and 1 hr later they were pulselabeled with [methyl-3H]thymidmne for 30 mm. The cellswere lysed, and the DNA was centrifuged. The results of thepulseexperimentswith PDVand EPDaredepicted in Charts

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25 30Bottom

Chart 3. Effect of 3.0-hr cell lysis and denaturation on the pattern ofprelabeled and pulse-labeled DNA. Log-phase PDV cells were prelabeled for24 hr with [methyl-'@C]thymidine. After 2 hr of growth in isotope-freemedium, cells were pulsed for 0.5 hr with [methy!-3H]thymidine before beingharvested. The cells were lysed in 0.5 M NaOH for 3.0 hr before centrifugationfor6 hr.

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UVdoselevelCaffeinePulse timeMW.

(daltons)M@M@(J/sq

m)mM(mm)(x 10)(x10')A.003039.255.7503037.363.41003024.544.21503016.733.82003011.727.5B.

002043.871.701.52039.466.01002018.640.41006036.057.6101.56021.741.62002010.529.72009021.245.2201.59014.326.9

DNA Repair in Mouse Epidermal Cell Cultures

Top Fraction Number Bottom

5 10 15 20

G060

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0.050

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I Chart 4. Effect 25 @0 EPD.TWO-day-OId@ irradiation onthe size of newly synth

og-phase PDV. L of UV irradiatiog-phase PDv@ on the size of newly@ P cells wereesized DNA in

of epiderma

. cubatlon, pulse ells were irr

labeledwith atsynthesizedDNAin

irradiatedatre harvested ulse-Iabeled with (methyl

. harvested and lysed for 3 hr(methyl@3Hv]atrt@n@c;L@noesef:terv:I5s

r 6 hr. and lysed for 3 hr in situ in

in situ in 0.5 M NaOH before Table 1Effect of UVirradiation on the sizeof pulse-labelednewly

synthesized DNA in PDV cells4 and 5, respectively. The results obtained with the PDVcells (Chart 4) indicate that at increasing UV dose levels thesize of the pulse-labeled DNA progressively decreases. At10- and 20-J/sq m UV, there were shoulders at highermolecular weights to the right of the main smaller molecularweight peaks. The results obtained with the EPD cells(Chart 5) were similar to those obtained with the PDV cells.However, the profile obtained for nonirradiated PDV cellswas narrower and at lower molecular weights than thatobtained for EPD cells. In fact, all the profiles obtained withthe PDV cells indicated lower-molecular-weight speciesthan those obtained with the EPD cells and might indicateeither a smaller replicon size in the transformed cells or agreater alkaline lability of PDVnewly synthesizedDNA.

Using the results of the gradient analysis with the help ofthe computer program described in “Materialsand Methods,―we calculated number and weight average molecularweights from the profiles presented in Charts 4 and 5. Theresults are presented in Tables 1 and 2. For these calculations those fractions that made up the main part of thepeaks, including the shoulders, were considered. Thosefractions at the top and bottom of the gradients were notconsidered in the calculations. Table 1A shows the resultsfrom experiments with PDV cells. In PDV cells the M@andM@valuesdecreasedprogressivelywith increasing UVdoselevels. Likewise, in EPD cells data presented in Chart 2Aindicated that increasing UV dose levels resulted in progressively decreasing number- and weight-average molecular weights. The molecular weights at any given UV doselevel for the PDV cells were smaller than the molecular

weights for the EPD cells.The differences in the sizes Cf pulse-labeled DNA after

different UV dose levels in either cell type may in part bedue to a slower rate of synthesis in the irradiated cells. Inorder to investigate this possibility, we determined thepulse lengths that would give the sameamounts of synthesis for irradiated and nonirradiated cells. The rate of DNAsynthesis in PDV cells as well as in EPD cells (data notshown) irradiated with UV (10 J/sq m) was approximately 1-third that of nonirradiated cells. With UV (20 J/sq m) it wasapproximately 1-fifth that of nonirradiated cells. Thus nonirradiated PDV on EPD cells were pulsed for 20 mm , UVirradiated (10 J/sq m) cells were pulsed for 60 mm, and UV

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The effectof UV irradiation on the size of pulse-labeled newlysynthesizedDNAin EPDcellsUV

doselevelMW.

(daltons)Caffeine

Pulsetime M@M@(J/sqm)(mM) (mm) (x 10) (x10@)A.

00 30 56.479.15030 50.583.310030 32.264.115030 18.241.520030 15.731.4B.

00 20 50.866.000.520 46.864.210020 22.145.810060 45.763.0100.560 26.949.320020 12.332.220090 25.049.1200.590 19.5 37.1

G. T. Bowdenet a!.

Table 2 level of 0.5 mM had only a small effect on the size of pulselabeled DNA. At higher dose levels caffeine significantlylowered the molecular weight of pulse-labeled DNA incontrol-treated EPD cells. Caffeine (0.5 mM) had the effectof lowering the molecular weight of newly synthesized DNAin UV-irradiated cells (10 J/sq m and 20 J/sq m) pulsed forextended pulse times (so as to give an amount of DNAsynthesis equal to that of control-treated cells).

Thus only with the use of caffeine, as an inhibitor ofpostreplication gap filling, could gaps or discontinuities innewly synthesized DNA of UV-inradiated PDV and EPD cellsbe tentatively demonstrated . However, there remained thepossibility that caffeine selectively slowed the overall rateof DNA synthesis in the UV-irradiated cells. This possibilitywas tested by studying the effect of caffeine on continuous[methyl-3H]thymidine incorporation into DNA of controland UV-treated (10 J/sq m) cells. The results obtained withthe PDVcells are depicted in Chart 6. Caffeine had a slightinhibitory effect on [methyl-3H]thymidmne incorporation incontrol-treated cells but a slight stimulatory effect in thecase of UV-irradiated cells. Similar results were obtainedwith EPD cells (data not shown). Thus the shift seen in thepresence of caffeine in UV-irradiated cells cannot be attributed to inhibition by caffeine of the rate of DNA polymenization. Thus there is strong evidence for the presence of

Chart 6. Effect of I .5 mM caffeine on the rate of DNA synthesis innonirradlated and Uv-lrradiated (10 J/sq m) cells which, I hr later, werepulse labeled with (methyl-3H]thymidine continuously. Caffeine (1.5 mM) wasadded immediately after Irradiation and left In the medium for the duration 6fthe pulse. DNA was extracted, and its specific activity was determined incpm 3H per i@gDNA. C, 0.0 mM caffeine; 0, 1.5 mM caffeine.

irradiated (20 J/sq m) cells were pulsed for 90 mm. Theresults of the pulse experiments under conditions of equalized DNA synthesis for PDV cells are given in Table lB. At adose level of 10 J/sq m, there appears to be a very smallshift to lower molecular weights in the case of irradiatedcells, and at 20 J/sq m this shift is more evident. Thesesmall shifts were also observed in the EPD cells (Table 2B)and are also reproducible.

One interpretation of the results obtained in Table 1 and2B is that there are very few if any gaps or discontinuities inthe newly synthesized DNA in irradiated epidermal cells.However, it is possible that the gaps are being formed butare rapidly being filled during the pulse period. This rapidgap filling might be the reasonwhy a langedifference is notseen in the size of pulse-labeled DNA in nonirradiated andirradiated cells when much longer pulse periods are usedto equalize the amount of DNA synthesis.

This hypothesis could be tested if there were some way toblock the gap-filling mechanism. It hasbeenshown in otherrodent cells in culture that caffeine, when applied to irradiated cells, inhibited the gap-filling mechanism (12, 16, 17,28).

Thus in separate experiments with PDV and EPD cells,caffeine at a concentration that did not affect pulse-labeledDNA in control-treated cells was added to the medium ofUV-irradiated cells that were pulsed for extended periods.The results of the experiment with PDV cells are given inTable 1B. Caffeine at 1.5 mp@ihad only a small effect on thesize distribution of pulse-labeled DNA in control-treatedcells. When cells irradiated at 10J/sq m were pulsed for 60mm instead of 20 mm, the molecular weight was close tothat of pulse-labeled DNA in control- treated cells. However,if caffeine (1 .5 mM) was present in the medium during theextended pulse time, the molecular weight was considerably lower than if caffeine was not present. Likewise, at adose level of 20 J/sq m, caffeine caused a decrease in themolecular weight of the pulse-labeled DNA when an extended pulse period was used.

Similar experiments were carried out with EPD cells, andthe results are summarized in Table 2B. Caffeine at a dose

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Theoreticaland experimentalnumber of discontinuities innewlysynthesizedDNAof UV-irradiatedPDVand EPDcellsTheoretical

no.ExperimentalUVdose (discontinui- no. (discontin

level ties/107daltons uities/107dalCelltype (J/sqm) DNA) tonsDNA)PDV

10 1.90.5PDV20 3.80.7EPD10 1.50.4EPD20 3.1 0.5

DNA Repair in Mouse Epidermal Cell Cultures

gaps in the newly synthesized DNA of UV-irradiated PDVand EPDcells.

Some researchers have observed that the d@,stancebetween gaps in the newly synthesized DNA in irradiated cellscorresponds to the interpynimidine dimer distance and havethus proposed that the pynimidine dimens cause the gaps tobe left in the newly synthesized DNA (7, 24). In order to testthis hypothesis in PDV and EPD cells, we calculated thetheoretical number of discontinuities per 10@daltons ofDNA as if every pynimidine dimer present in the DNA causeda gap or discontinuity, and these numbers were comparedto the experimental number of discontinuities per 10@daltons of DNA. The theoretical number of gaps was calculatedfrom the pynimidine dimen data derived by Bowden et al. (2)for PDV and EPD cells. The experimental number of gapswas calculated directly from the number-average molecularweight obtained from an experimental situation in whichUV-irnadiatedcells were pulse-labeled for an extended penod in the presence of caffeine. These number-averagemolecular weights were not corrected for the number-average molecular weight resulting from pulsing nonirradiatedcells. The results of these calculations are presented inTable 3. It is apparent that there are fewer gaps in the newlysynthesized DNA than the number of pynimidine dimers inthe parental DNAfor both PDVand EPDcells.

DNA Gap Filling, Elongation,and Joining in UV-irradiated Epidermal Cells. Pulse-chaseexperiments were carned out to determine whether the EPD and PDV cells werecapable of closing the gaps observed in the pulse expeniments and were capable of elongating and joining thenewly synthesized DNA. Nonirradiated and irradiated cellswere pulsed with [methyl-3H]thymidine for 0.5 hr beforebeing chased for 0.25, 0.5, 1, 2, and 4 hr. In nonirradiatedcells (Chart 7), there was a progressive shift to highermolecular weights of the pulse-chased DNA until 4 hr ofchase when the molecular weight was approximately thatfound for parentally labeled DNAunder the sameconditionsof lysis and centnifugation. In the UV-irradiated cells (Chart8), the rate of chase was markedly slower, but by 4 hr ofchasethe molecular weight of the DNAfrom irradiated cellswas approaching that of the nonirradiated control cells. By24 hr of chase, the molecular weight of the pulse-chasedDNA from irradiated PDV cells was back to that of theparentally labeled DNA (data not shown). Similar experiments were carried out with EPD cells, and a comparison ofthe 2 cell types is presented in Chart 9 where the numberaverage molecular weight (Me) of the pulse-chased DNA isplotted against the time of chase starting at 0 hr chase(immediately after the 0.5-hr pulse). As shown in Chart 9,

Table3

the kinetics of the chase is biphasic in both cell types.There was a rapid rate of elongation and joining in the first0.5 to 1 hr after the pulse, followed by a more gradualincrease in the number-average molecular weight for thenext 3 hr. There was no apparent difference in the kinetics

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Chart 7. DNA elongation and joining in nonirradiated PDV cells as demonstrated by a pulse-chase experiment. Log-phase PDV cells were pulsedwith [methyl-3H]thymidine for 0.5 hr before being incubated in isotope-freemedium (containing thymidine and deoxycytidine) for 0.25, 0.5, 1, 2, and 4hr. At the end of each chase period the cells were harvested, suspended,and lysed in situ on top of the gradient (2 hr) before centrifugation for 4 hr.

II

Chart 8. DNA elongation and joining in UV-lrradiated (10 J/sq m) PD'Icells as demonstrated by a pulse-chase experiment. PDV cells were irradiatedat 10J/sqmand,1hr later,werepulse-labeledfor0.5hrwith(methyl@H]thymidihe.The pulse-labeledDNAwaschasedby incubatingthe cells in

isotope-free medium (containing thymidine and deoxycytidine) for 0.25, 0.5,1, 2, and 4 hr. At the end of each chase period, the cells were harvested,suspended, and lysed in situ on top of the gradient (2 hr) before centrifugationfor4 hr.

0lop

15 20Fraction Number

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G. T. Bowden et al.

synthesized DNA. The results obtained with the primary andmalignantly transformed epidermal cells indicated that thedimers in the parental DNA may not be responsible for thegaps in the newly synthesized DNA because there was only1 gap when there were 4 to 6 dimers present in 10@daltonsof parental DNA. In order to obtain the experimental resultsfor the number of discontinuities, it was necessary to blockthe apparent gap-filling mechanism with the specific inhibitor, caffeine, and then calculate the number of discontinuities. These results with epidermal cells may indicate thatthere is some other type of mechanism whereby the DNAreplication complex of a cell can deal with unexcisedpynimidine dimers in parental DNA that is being replicated.The results could also indicate that the pynimidine dimersare not randomly induced in the DNA in such a way thatthere is a large variation in the interdimer distances andthat the experimental molecular weights obtained in pulseexperiments actually do correspond to the interdimer distances that existed when the pulse experiments were carned out.

The pulse-labeling experiments also indicated that thesize of newly synthesized DNA made in 0.5 hr in nonirradiated and irradiated cells was greater in the primaryepidermal cells than in the malignantly transformed cells. Apossible explanation for the difference was that the actualamount of UV damage to the DNA for a given UV dose levelwas different for the 2 cell types. However, it has beenfound that the percentage of UV-induced dimers for anygiven UV dose level was slightly greater in the PDV cellscompared to the EPDcells (3) (becauseof shielding of UVby multilayered EPD cells) but not different enough toaccount for the difference seen in the sizes of pulse-labeledDNA in the UV-irradiated EPD and PDV cells. It has beenshown similarly that the size of newly synthesized DNA inboth nonirradiated and irradiated HeLa, mouse L-cells, andChinese hamster ovary cells at equal levels of UV-inducedDNA damage was different for the 3 different cell lines (37).Rauth et a!. described this difference as a phenotypiccharacteristic of the cell lines studied, since the differencewas not related to differences either in the rate of DNAsynthesis or in the amount of UV damage in the irradiatedcells.

Caffeine (1.5 mM) was found to have only a small effect onthe size of pulse-labeled DNA in nonirradiated PDV cells.However, caffeine reduced the size of the pulse-labeledDNA in nonirnadiated EPD cells to a size equivalent to thatfound in nonirradiated PDV cells. Similar to the results withthe PDV cells, Fujiwara (16) showed that caffeine (2 mM)had very little effect on the size of pulse-labeled (2-hr pulse)DNA in nonirradiated mouse L-cells. Caffeine is thought toact by binding single-stranded portions of double-strandedDNA, and in the case of UV-irradiated cells it is thought tobind to the gaps left in the newly synthesized DNA and toinhibit the gap-filling mechanism. The sensitivity of thenonirradiated EPD cells and the insensitivity of the PDVcells may indicate differences in the fine structure of theDNA replication complexes in the 2 cell types. Perhapsduring normal DNA replication in EPD cells, single-strandedDNA configurations are more frequent than in PDV cells.

Both normal and malignantly transformed epidermal cellswere capable of gap-filling, joining, and elongating newly

¶

Chart 9. Rate of DNA elongation after uv (10 J/sq m) in EPD and PDVcells. Number average molecular weights for each chase time in both UVirradiated (10 J/sq m) EPD and PDV were calculated, and the data are plottedas the number average molecular weight versus chase time (hr).

of the effective rate of joining and elongation between theirradiated EPD and PDV cells. In both cell types it appearedthat the gaps formed in the newly synthesized DNA in theirradiated cells were filled, and the newly synthesized DNAwas joined and elongated. Thus both EPD and PDV cellsperform postreplication gap filling after UV irradiation.

DISCUSSION

The experiments described in this paper show that inboth normal and malignantly transformed mouse epidermalcells in culture the newly synthesized DNA formed after UVirradiation of the cells contains gaps or discontinuities, andthese gaps are filled and the DNA is elongated and joinedon further incubation. This postreplication repair mechanism after UV irradiation has been described in both human(6, 7, 9) and rodent (11, 12, 16, 26, 43) cell lines but hasnot been described previously in cultures of epidermalorigin or in primary cultures.

Experiments measuring the size of pulse-labeled DNA inUV-irradiated human HeLa cells (9), human fibroblasts fromXP patients (29), and mouse L5178Y (26) cells indicate thatapproximately 1 gap is left in the newly synthesized DNA forevery pynimidine dimer. These data support the idea thatdimers interrupt normal DNA replication and that synthesisresumes beyond the dimens. However, although UV-irradiated Chinese hamster ovary and B14 cells (33, 37) alsosynthesize DNA in shorter units, the units are longer thanthe distance between dimers. It has also been shown thatmouse L-cells (11) synthesized DNA in normal size segments after low UV dose levels. Hamster, human, andmouse DNA's synthesized at long times after irradiation areapproximately the same size as that synthesized by nonirradiated cells although a large percentage of the dimersremain in the parental DNA (8, 27). These results indicatedthat dimers are not responsible for the gaps in the newly

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DNA Repair in Mouse Epidermal Cell Cultures

synthesized DNA after UV irradiation. In both cell types thegap-filling mechanism operated rapidly. Similarly, Lehmann(26) found that mouse L5178Y closed the gaps rapidly (after1 hr of chase). Both EPD and PDV cells displayed biphasickinetics, and a chase period of 4 hr was required to obtainDNA species with molecular weights over 150 x 10@daltons.Similar experiments were carried out by Lehmann et a!.(29), using normal human fibroblasts and fibroblasts fromXP patients. It was found that in normal fibnoblasts irradiated with UV(12.5J/sq m) the time taken to synthesizeDNAwith a molecular weight in excess of 150 x 10@daltons was1.5 to 2.5 hr. In classical XP cells the time was 2.5 to 5 hr,whereas in the XP variants (these cells exhibit normal DNAexcision repair after irradiation) the time was much longer(between 4 and 8 hr). In comparison, the epidermal cellsrequired the same amount of time as did the classical XPcells, which do not excise pyrimidine dimers. These variousobservations indicate that, in these 2 repair mechanisms,excision and postreplication repair operate completely independently of each other. It does appear as if cells that donot excise pynimidmnedimers depend upon postreplicationrepair mechanism(s) to deal with unexcised pynimidinedimers when the cells replicate their DNA. Those cells thatlack excision repair and that are defective in postreplicationrepair should be very sensitive to the toxic effects of UVirradiation.

The epidenmal cells of any mammal are constantly beingexposed to the deleterious effects of UV light. Damage(UVinduced) to the DNAof these cells can be repaired throughthe process of excision repair whereby the lesions arespecifically removed from the DNA; however, this processis relatively slow (measurable out to 24 hr after irradiation)and incomplete. The second repair mechanism, postreplication repair, must operate relatively efficiently to deal withthe unexcised pynimidine dimers in the basal cells of theepidermis. For the basal cells this second repair mechanismmust operate efficiently because these cells, being stemcells, are constantly going through rounds of replication.We have presented evidence indicating that postreplicationgap-filling mechanism in both normal primary and malignantly transformed epidermal cells is in fact a rapid processthat deals with unexcised UV-induced DNA damage duringDNA synthesis.

REFERENCES

1. Blum, H. F. Carcinogenesis by Ultraviolet Light. Princeton, N. J.:Princeton University Press, 1959.

2. Bowden,G.T., Hohneck,G., and Fusenig,N. E.DNAExcisionRepairinUltraviolet-irradiated Normal and Maglignantly Transformed Mouse Epidermal Cell Cultures. Cancer Res., 37: 1611-1617, 1977.

3. Bowden, G. T., Trosko, J. E., Shapas, B. G., and Boutwell, R. K.Excision of Pyrimidine Dimers from Epidermal DNA and Nonsemiconservative Epidermal DNA Synthesis following Ultraviolet Irradiation ofMouse Skin. Cancer Res., 35: 3599-3607, 1975.

4. Bridges, B. A. Mechanism of Radiation Mutagenesis in Cellular andSubcellular Systems. Ann. Rev. NucI. Sci., 19: 139-178, 1969.

5. BuhI, S. N., and Regan, J. D. Repair Endonuclease-sensitive Sites inDaughter DNA of Ultraviolet-irradiated Human Cells. Nature, 246: 484-485, 1973.

6. BuhI, S. N., and Regan, J. D. Effect of Caffeine on Postreplication Repairin Human Cells. Biophys. J., 14: 519-526, 1974.

7. Buhl, S. N., Setlow, R. B., and Regan, J. D. Steps in DNA ChainElongation and Joining after Ultraviolet Irradiation. Intern. J. RadiationBiol., 22: 417-424, 1972.

8. Buhl, S. N., Setlow, R. B., and Regan, J. D. Recovery of the Ability to

Synthesize DNA in Segments of Normal Size at Long Times afterUltraviolet Irradiation of Human Cells. Biophys. J.. 13: 1265-1275, 1973.

9. BuhI, S. N., Stillman, R. M., Setlow, R. B., and Regan, J. D. DNA ChainElongation and Joining in Normal Human and Xeroderma PigmentosumCells after Ultraviolet Irradiation. Biophys. J., 12: 1183-1190, 1972.

10. Chang, C. C.. Philippe, C., Trosko, J. E., and Hart, R. Mutagenic an@iEpigenetic Influence of Caffeine on the Frequencies of UV-InducedOuabain-ResistantChineseHamsterCells. MutationRes.,45: 125-136,1977.

11. Chiu, S. F. H., and Rauth, A. M. Nascent DNA Synthesis in UltravioletLight Irradiated Mouse L-Cells. Biochim. Biophys. Acta, 259: 164-174,1972.

12. Cleaver, J. E., and Thomas, G. H. Single Strand Interruptions in DNAand the Effects in Chinese Hamster Cells Irradiated with UltravioletLight. Biochem. Biophys. Res. Commun., 36: 203-208, 1969.

13. D'Ambrosio, S., and Setlow, R. B. Enhancement of PostreplicationRepair in Chinese Hamster Cells. Proc. NatI. Aced. Sci. U. S., 73: 2396-2400, 1976.

14. Ehmann, U. K., and Left, J. K. Review and Evaluation of MolecularWeight Calculations from Sedimentation Profiles of Irradiated DNA.Radiation Res., 54: 152-162, 1973.

15. ElkInd, M. M., and Kamper, C. Two Forms of Repair of DNA in Mammahan Cells following Irradiation. Biophys. J., 10: 237-245, 1970.

16. Fujiwara, Y. Characteristics of DNA Synthesis following UltravioletIrradiation in Mouse L-Cells: Postreplication Repair. Exptl. Cell Res., 75:483-489, 1972.

17. Fujiwara, V., and Kondo, T. Caffeine-sensitive Repair of Ultraviolet LightDamage to DNA of Mouse L-Cells. Biochem. Biophys. Res. Commun.,47: 557-564, 1972.

18. Fujiwara,Y., andKondo,T. PostreplicatlonRepairof UltravioletDamageto DNAof XerodermaPigmentosum,OtherHumanand MouseCells inCulture. Radiation Res., 15: 81—89,1974.

19. Fusenig,N. E. Isolationand Cultivationof EpidermalCellsfrom Embryonic Mouse Skin. Die Naturwissenschaften, 8: 421-422, 1971.

20. Fusenig, N. E., Samsel, W., Thon, W., and Worst, P. K. M. MalignantTransformation of Epidermal Cells in Culture by DMBA. Inst. de Ia Santeet de Ia Rech. Med., 19: 219-228, 1973.

21. Fusenig, N. E., Thon, W., and Amer. S. M. Growth and Differentiation inEpidermal Cell Cultures from Embryonic Mouse Skin. Federation EuropeanBiochem.Soc. Proc.,24: 159-163,1972.

22. Fusenig, N. E., and Worst, P. K. M. Mouse Epidermal Cell Cultures I.Isolation, Characterization and Cultivation of Epidermal Cells from AdultMouse Skin. J. Invest. Dermatol., 63: 187-193, 1974.

23. Fusenig, N. E., and Worst, P. K. M. Mouse Epidermal Cell Cultures II.Isolation, Characterization and Cultivation of Epidermal Cells fromPerinatal Mouse Skin. Exptl. Cell Res., 93: 443-457, 1975.

24. Howard-Flanders, P., Rupp, W. D. Wilkins, B. M., and Cole, R. S. DNAReplicationand Recombinationafter U―.'Irradiation.ColdSpringHarborSymp. Quant. Biol. 33: 195-207, 1968.

25. Iyer,v. N., and Rupp,W. D. Usefulnessof BenzoylatedNaphthoylatedDEAE-Celluloseto Distinguishand FractionateDouble-StrandedDNABearing Different Extents of Single-Stranded Regions. Biochim. Biophys.Acta,228: 117-226, 1971.

26. Lehmann, A. R. Postreplication Repair of DNA in Ultraviolet IrradiatedMammalian Cells. J. Mol. Biol., 66: 319-337, 1972.

27. Lehmann, A. R. Post-Replication Repair in Ultraviolet-Irradiated Mammalian Cells: No Gaps in DNA Synthesized Late after Ultraviolet Irradiation. European J. Biochem., 31: 438—445,1972.

28. Lehmann,A. R.,andKirk-Bell,S. Effectof CaffeineandTheophyllineonDNA Synthesis in Unirradiated and UV-lrradiated Mammalian Cells.Mutation Res., 26: 73-82, 1974.

29. Lehmann, A. R., Kirk-Bell, S., Arlett, C. F., Paterson, M. C., Lohman, P.H. M., de Weerd-Kastelein, E. D., and Bootsma, P. Xeroderma Pigmentosum Cells with Normal Levels of Excision Repair Have a Defect in DNASynthesis after UV-lrradiation. Proc. NatI. Aced. Sci. U. S., 72: 219-223,1975.

30. Left, J. T., Kluas, E. S., and Sun, C. On the Size of the DNA in theMammalian Chromosome Structural Subunits. Biophys. J., 10: 277-292,1970.

31. Levin, D., and Hutchinson, F. Relation between Single-Strand DNA Massand Sedimentation Distance in Alkaline Sucrose Gradients. J. Mol. BioI.,75:495-502,1973.

32. Meneghini, R., and Hanawalt, P. T-4 Endonuclease V-sensitive Sites inDNA from Ultraviolet-Irradiated Human Cells. Biochim. Biophys. Acta,425: 428-437, 1976.

33. Meyn, R. E., and Humphrey, R. M. Deoxyribonucleic Acid Synthesis inUltraviolet-Light-Irradiated Chinese Hamster Cells. Biophys. J., 11: 295-301, 1971.

34. Nomura, T. Diminution of Tumorigenesis Initiated by 4-Nitroquinoline-1-oxide by Post-treatment with Caffeine in Mice. Nature, 260: 547-549,1976.

35. Peterson, A. R., Bertram, J. S., and Heidelberger, C. DNA Damage andIts Repair in TransformableMouseFibroblastsTreatedwith N-Methyl

SEPTEMBER1978 2717

on June 5, 2018. © 1978 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

G. T. Bowdenet a!.

N'-nitro-N-nitrosoguanidine. Cancer Res., 34: 1592-1599, 1974.36. Pound, A. W. !@iducedCell Proliferation and the Initiation of Skin Tumor

Formation in Mice by Ultraviolet Light. Pathology, 2: 269-275, 1970.37. Rauth, A. M., Tammemagi, M., and Hunter, G. Nascent DNA Synthesis

in Ultraviolet Light-Irradiated Mouse, Human, and Chinese HamsterCells. Biochim. Biophys. Acta, 14: 209-220, 1973.

38. Regan, J. D., Setlow, R. B., and Ley, R. P. Normal and Defective Repairof Damaged DNA in Human Cells: A Sensitive Assay Utilizing thePhotolysis of Bromodeoxyuridine. Proc. NatI. Acad. Sci., U. S., 68: 708-712. 1971.

39. Rothwell, K. Dose Related Inhibition of Chemical Carcinogenesis inMouse Skin by Caffeine. Nature, 252: 69-70, 1974.

40. Rupp, W. D., and Howard-Flanders, P. Discontinuities in the DNASynthesized in an Excision-defective Strain. J. Mol. Biol., 31: 291-304,1968.

41. Rupp, W. D., Wilde, C. E., Reno, D. L., and Howard-Flanders. P.

Exchanges between DNA Strands in Ultraviolet-Irradiated Escherichiacoli. J. Mol. Biol., 61: 25-44, 1971.

42. Studier, F. W. Sedimentation Studies of the Size and Shape of DNA. J.Mol. Biol., 11: 373-390, 1965.

43. Trosko, J. E., and Chu, E. H. Y. Inhibition of Repair of UV-Damaged DNAby Caffeine during S-Phase of the Cell Cycle in Chinese Hamster Cells.Chem.-Biol. Interactions, 6: 317-332, 1973.

44. Waters, R., and Regan, J. D. Recombination of UV-lnduced PyrimidineDimers in Human Fibroblasts. Biochem. Biophys. Res. Commun., 72:803-807,1976.

45. Witkin, E. M. UV-lnduced Mutations and DNA Repair. Ann. Rev. Genet.,3:.525-552, 1969.

46. Zajdela, F., and Latarjet, R. Effet lnhiblteur de Ia Cafeine Sar I;Induction de Cancers Cutanéspar lea Rayons Uftraviolets. C. R. Acad.Sd.,277:1073-1076,1973.

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1978;38:2709-2718. Cancer Res   G. T. Bowden, B. Giesselbach and N. E. Fusenig  CulturesNormal and Malignantly Transformed Mouse Epidermal Cell Postreplication Repair of DNA in Ultraviolet Light-irradiated

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