[CANCER RESEARCH 41, 1829-1833, May 1981

Photoreactivation of Ultraviolet Radiation-induced Pyrimidine Dimersin Neonatal BALB/c Mouse Skin1

Honnavara N. Ananthaswamy2 and Michael S. Fisher

Cancer Biology Program, National Cancer Institute-Frederick Cancer Research Center, Frederick, Maryland 21701

ABSTRACT

The numbers of ultraviolet light (UV)-induced pyrimidine di-

mers in the DNA of neonatal BALB/c mouse skin were measured by assessing the sensitivity of the DNA to Micrococcusluteus UV endonuclease. Irradiation of neonatal BALB/c micewith FS40 sunlamps caused a dose-dependent induction ofendonuclease-sensitive sites (pyrimidine dimers) in DNA extracted from back skin. Exposure of these UV-irradiated neonatal mice to photoreactivating (PR) light (' 'cool white" fluores

cent lamp and incandescent lamp) caused a reduction in thenumber of pyrimidine dimers in the DNA, as revealed by a shiftin low-molecular-weight DNA to high-molecular-weight DNA. Incontrast, DNA profiles of the skin of either UV-irradiated miceor UV-irradiated mice kept in the dark for the same duration asthose exposed to PR light did not show a loss of UV-inducedendonuclease-sensitive sites. Furthermore, reversing the order

of treatment, i.e., administering PR light first and then UV, didnot produce a reduction in pyrimidine dimers. These resultsdemonstrate that PR of UV-induced pyrimidine dimers occurs

in neonatal BALB/c mouse skin. The optimal wavelength rangefor in vivo PR appears to be in the visible region of the spectrum(>400 nm). Although dimer formation could be detected inboth dermis and epidermis, PR occurred only in the dermis.Furthermore, the PR phenomenon could not be detected in theskin of adult mice from the same inbred strain.

INTRODUCTION

Exposure of mice to UV results in damage to the DNA inmouse skin, mainly by the formation of cyclobutylpyrimidinedimers (pyrimidine dimers) (3, 13, 14), the induction of skincancers in the dermis and epidermis (2, 7, 22), and systemicimmunological alterations that are important in the pathogen-

esis of skin cancer (8, 11, 12).In bacterial cells and in some cells of higher organisms, the

UV-induced pyrimidine dimers can be repaired enzymatically

in situ by subsequent exposure of the cells to longer wavelengths (300 to 600 nm) of light (6, 17-19). This PR3 has been

used to identify pyrimidine dimers as major factors that areresponsible for the lethal, mutagenic, and carcinogenic activities of UV radiation in several organisms (9, 10). Hart andSetlow (10) have demonstrated elegantly the role of pyrimidinedimers in photocarcinogenesis using PR. These workers reported that the injection of UV-irradiated tissue homogenatesof Poecilia formosa into isogenic recipients resulted in thyroidcarcinoma. However, when UV-irradiated tissue homogenates

1Research sponsored by the National Cancer Institute under Contract N01-

C075380 with Litton Bionetics, Inc.2 To whom requests for reprints should be addressed.3 The abbreviations used are: PR. photoreactivation (or photoreactivating);

PRE, photoreactivating enzyme; ESS, endonuclease-sensitive sites.Received June 23, 1980; accepted February 2, 1981.

were exposed to PR light prior to injection, the tumor incidencewas reduced significantly, thus implicating the involvement ofpyrimidine dimers in UV-induced carcinogenesis.

In order to ascertain whether pyrimidine dimers in the DNAof mouse skin are involved in the UV-induced immunological

alterations in BALB/c mice, we first had to determine whetherPR of pyrimidine dimers occurs in vivo in BALB/c mouse skin.Ley ef al. (13) reported that PR of UV-induced pyrimidine

dimers did not occur in the epidermis of hairless mice. Furthermore, PRE activity was thought to be present only inprocaryotes and certain eucaryotes and to be entirely absentin placental mammals until Sutherland (19) demonstrated thepresence of this enzyme in human leukocytes. Sutherland efal. (21 ) also have reported the presence of PRE activity in 3T3Swiss and BALB/c mouse embryonic fibroblasts.

In the present study, we have adopted the method of assaydescribed by Achey ef al. (1) for demonstrating the presenceof PR. This method is rapid, is sensitive, and requires noradioactive labeling of DNA.

MATERIALS AND METHODS

Mice. Specific-pathogen-free newborn (24 to 48 hr old) and

inbred young adult (8 week old) BALB/c mice were suppliedby the Frederick Cancer Research Center's Animal Production

Area.UV Irradiation and PR Light Sources. Groups of newborn

mice were immobilized by gently sticking them to an adhesivetape during UV treatment. The adult mice were housed individually for the irradiation, and the dorsal hair was removed withelectric clippers. The light source for initial irradiation was abank of 6 Westinghouse FS40 sunlamps that emitted wavelengths between 255 and 400 nm with a peak at 313 nm. Thedistance between the light source and the target was about 20cm. The average fluence at the surface of the middorsum was9 J/sq m/sec (cosine corrected) as measured by an International Light Spectroradiometer System (IL 700/760/780) containing a PM270D-CM149 detector with a spectral range of

240 to 810 nm.Replicate groups of UV-irradiated mice were kept either in

the dark or exposed to PR light. The light source used for PRtreatment consisted of 2 or 4 incandescent bulbs (25 watts) fornewborn or adult mice, respectively. The lamps delivered anaverage fluence of 4.8 J/sq m/sec (noncosine corrected) at adistance of about 25 cm. PR treatment lasted about 20 hr. Inaddition to an incandescent light source, 6 "black light" (40watts) BLB (300 to 400 nm) and 2 ' 'cool white' '(15 watts) (350

to 700 nm) fluorescent lamps (both from General Electric Co.)were used to determine the optimum wavelength range for PRin neonatal mice. Black light was positioned about 20 cm fromthe mice, and cool white fluorescent light was placed about 12cm from the mice. Light from both these sources was passed

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H. N. Ananthaswamy and M. S. Fisher

through a Mylar filter that cuts off wavelengths below 315 nm.The light intensity for black light was 3.2 J/sq m/sec, and forcool white fluorescent light, it was 4.0 J/sq m/sec.

Isolation of DNA from Mouse Skin and Assay for ESS(Pyrimidine Dimers). Strips of skin (approximately 1x1.5 cm)from 3 to 4 mice/group were excised from the middorsumregion, minced thoroughly, and homogenized with a PyrexModel 7717 tissue grinder (5 uniform up-and-down strokes) in20 ml of ice-cold 0.1 M Tris buffer, pH 8.0, containing 0.1 M

NaCI and 1 HIM EDTA. After 2 to 3 min cooling on ice, thehomogenate was spun in a refrigerated centrifuge at 2500 rpmfor 10 min. The pellet, containing mostly nuclei and some singlecells, was resuspended in 5 ml of the same buffer at roomtemperature and treated with sodium dodecyl sulfate (5 mg/ml), proteinase K (0.1 mg/ml), and heat-treated DNase-free

RNase (0.5 mg/ml). After 16 to 20 hr at room temperature, thelysed preparation was extracted once with H2O-saturated

phenol and once with a 1:1 mixture of chlorofornrphenol. TheDNA was precipitated from the aqueous phase with 2 volumesof cold ethanol and allowed to stand at —20°for 4 hr or longer.

The precipitated DNA was redissolved in 20 ITIMTris buffer, pH8.0, containing 40 mw NaCI and 2 ITIMEDTA. The absorbancewas measured at 260 nm and adjusted to 2.0 in all the groups.

The DNA from adult BALB/c mouse back skin (2- x 3-cmstrips) and ear skin was isolated separately. The dorsal hairwas removed with Surgex hair remover cream before the skinwas excised.

The Micrococcus luteus endonuclease assay, described byAchey ef al. (1) and by Paterson ef al. (16), was used withsome modifications to measure the amount of dimers in theDNA. Ten /il of DNA solution were incubated for 20 min at 37°

with 5 n\ of M. luteus extract (containing damage-specific

endonuclease) prepared according to the method of Carrierand Setlow (4). The reaction was stopped by the addition of 15/il of a stop-reaction mixture containing 0.3 M NaOH, 40%

sucrose, and 0.06% bromocresol green. The samples (30 fil)were analyzed by horizontal slab alkaline gel electrophoresis,according to the procedure of McDonell ef al. (15). A 3-mm-

thick 0.3% agarose gel was run for 20 hr at 20 V. The gel wasthen neutralized with 0.1 M Tris buffer, pH 8.0, stained withethidium bromide, and photographed under UV light.

Molecular Weight Determination. Photographic negativesof the gels were scanned with a densitometer (E-C Apparatus

Corp.), and the brightness contour of each migration band wasrecorded on a strip chart (Omniscribe type EC-146).

The strip chart for each band was graduated into intervals ofequal width on the horizontal axis, beginning at the samerelative location on each chart. The intervals, approximately0.4 inches wide, were numbered consecutively beginning withone. Twenty-five such intervals were required to encompass

the area under the band with the greatest migration. Verticallines were erected at each interval, such that the area underthe curve and above the baseline was divided into severalcolumns. The area in each of these columns was determinedby manually counting the number of grid blocks in each column.The midvalue of each column (;'.e., 0.5, 1.5, etc.) was then

multiplied by the relative area of that column to determine theweighted average depth of band migration.

We produced a standard curve using P1, A, and A DNA cutwith EcoRI restriction enzyme by plotting the weighted averagedepth of band migration versus the logarithm of the average

molecular weight for each of the 6 standards. The curve wasfit by a second-degree polynomial regression to the X-Y coor

dinates for the 6 standards, with mean depth of migration beingtaken as the dependent variable. The log molecular weight foreach of the unknowns was determined from this curve, withthe weighted average depth of migration of each unknowndetermined in the manner described above.

RESULTS

Induction of Pyrimidine Dimers in Neonatal Mouse Skin byFS40 Sunlamps. The M. luteus endonuclease assay usinghorizontal slab gel electrophoresis developed by Achey ef al.(1) provides a convenient alternative to the alkaline sucrosegradient assay for the measurement of UV-induced pyrimidinedimers in the DNA. Using this technique, we have studied theinduction of pyrimidine dimers in vivo in neonatal BALB/cmouse skin by irradiation with FS40 sunlamps. To determinethe dose-response for in vivo induction of pyrimidine dimers,

we exposed groups of neonatal mice to average UV doses of3, 6, or 9 kJ/sq m from the FS40 sunlamps. The DNA wasisolated from the dorsal skin immediately after UV exposureand treated with the M. luteus extract. The induction of ESSthen was analyzed on alkaline agarose gels. The results presented in Fig. 1 show that, in the untreated control, there wasno difference in the DNA profile (mobility) with or withouttreatment with the M. luteus enzyme (Fig. 1, Lanes 1 and 2).This indicates that the M. luteus enzyme preparation has nospecificity for non-UV-irradiated DNA. However, DNA from UV-

irradiated neonatal mouse skin treated with the M. luteusextract exhibited ESS (Fig. 1, Lanes 3 to 5), which reflects thepresence of pyrimidine dimers. The ESS increased with increasing UV exposure, as revealed by the increased mobilityof the DNA after treatment with the M. luteus extract. Incontrast, DNA isolated from UV-irradiated mice, but not treated

with the M. luteus extract, migrated like DNA from unirradiatedmouse skin (Fig. 1, Lane 6).

The negative from the picture of the DNA gel in Fig. 1 wasscanned in a densitometer in order to quantitate the dose-response for dimer formation. The densitometer scan presented in Chart 1 shows that most of the DNA in the untreated

Decreasing Molecular Weight «(Increasing Number of ESS - )

Chart 1. Microdensitometer scan of the negative from picture of gel in Fig. 1for DNA profiles of no UV (Lane 2) (M.W. = 18.9 x 106). UV (3 kJ/sq m) (Lane3) (M.W. = 15 x 106), UV (6 kJ/sq m) (Lane 4) (M.W. = 7.6x10°), and UV (9kJ/sq m) (Lane 5) (M.W. = 4 x 10e).

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PR in Neonatal Mouse Skin

neonatal mouse skin has a high molecular weight and is relatively homogeneous. As the UV dose increases, the high-molecular-weight DMA peak progressively decreases and appearsas low-molecular-weight DMA. Concomitantly, this low-molecular-weight DMA increases with increasing UV exposure.

The average molecular weights were calculated from thescanning profiles presented in Chart 1 according to the procedure described in "Materials and Methods." These values

were then used to calculate the number of UV-induced ESS in

the DNA of newborn mouse skin according to the followingequation:

No. of ESS/108 daltons = 108 (—1 T77I7-)\M.W.UV M.W.o/

where M.W.UVequals the average molecular weight of the UV-

irradiated DNA after treatment with M. luteus extract and M.W.0equals the average molecular weight of the unirradiated controlor irradiated DNA before treatment with M. luteus extract.

The average molecular weights of unirradiated DNA (Fig. 1,Lane 2) and UV-irradiated (9 kJ/sq m) DNA without treatment

with M. luteus extract (Fig. 2, Lane 1) were calculated to be18.9 x 106 and 17 x 106, respectively. These values appearto be lower than those for the P1 DNA marker (30 x 106)although P1 DNA migrates farther than do the other 2 DNA's

in the gel (Fig. 2). This is because the P1 DNA does not exhibita long "tail," whereas the other 2 DNA's, plus the DNA's in

Fig. 2, Lanes 6 and 7, all exhibit a long tail. Thus, when themolecular weights are averaged, their values become smallerthan the P1 DNA marker.

The data presented in Chart 2 indicate that, with increasingUV exposure, there was an increase in the number of ESS.However, the dose-response curve does not exhibit a linearrelationship between the number of ESS and the dose of UV.This nonlinearity could be due to the errors associated with thedetermination of average molecular weights, the nonrandomdistribution of UV-induced damage in mouse skin DNA, or acombination of these factors.

PR of UV-induced Pyrimidine Dimers in Neonatal Mouse

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Fluence, kJ/m2Chart 2. Dose-response for ESS (pyrimidine dinners) induced by FS40 sun-

lamps in neonatal BALB/c mouse skin DNA.

Decreasing Molecular Weight -(Increasing Number of ESS - )

Chart 3. Microdensitometer scan of the negative from picture of gel in Fig 2for DNA profiles of UV (9 kJ/sq m) - enzyme (ENZ) (Lane i) (M.W. = 17 x106), UV (9 kJ/sq m) -I- 20 hr dark + enzyme (Lane 2) (M.W. = 4.8 x 106), UV(9 kJ/sq m) + 20 hr PR light + enzyme (Lane 4) (M.W. = 7.2 x 106), and 20 hrPR light + UV (9 kJ/sq m) + enzyme (Lane 5) (M.W. = 4.7 x 106).

Skin. After exposure to FS40 sunlamps (9 kJ/sq m), groups of3 or 4 mice were either killed immediately, kept in the dark for20 hr, or exposed to PR light (incandescent lamp) for 20 hr.DNA was isolated from the skin of mice in each group and wassubjected to alkaline agarose gel electrophoresis either beforeor after treatment with M. luteus extract. Typical gel electro-phoretic profiles are shown in Fig. 2. It is apparent from theprofiles that, following treatment with M. luteus extract, theDNA isolated from neonatal mouse skin immediately after UVirradiation exhibits ESS as revealed by the presence of low-molecular-weight DNA (Fig. 2, Lane 2). The DNA profile of

mouse skin exposed to UV and then kept in the dark for 20 hrreveals a slight increase in the low-molecular-weight DNA tohigh-molecular-weight DNA (Fig. 2, Lane 3) as compared to

the profile of DNA isolated from mice immediately after irradiation (Fig. 2, Lane 2). This could be due to either DNA synthesisor some type of dark repair of UV-induced DNA damage thatoccurred in the 20-hr period following UV irradiation. However,the DNA from UV-irradiated neonatal mice that was subjected

to PR light shows a significant shift from low to high molecularweight (Fig. 2, Lane 4). In contrast, reversing the order oftreatment, i.e., administering PR light first and then irradiatingwith UV, did not result in a change in molecular weight of theDNA (Fig. 2, Lane 5).

The number of ESS/108 daltons was calculated for the

relevant groups from the densitometer scan shown in Chart 3.The results indicate that about 15 ESS/108 daltons are present

in the DNA of both the UV-irradiated mice kept in the dark for

20 hr and the mice exposed to PR light first and then irradiatedwith UV. In contrast, the DNA from the skin of mice irradiatedwith UV and then subjected to PR light shows only about 8ESS/108 daltons. Thus, about 47% of UV-induced ESS is

removed in vivo after PR treatment. However, the percentageof ESS removed by in vivo PR varied from experiment toexperiment (30 to 47%). This variation could be caused bysuch factors as the stage of the neonatal mice or errorsassociated with the determination of molecular weight.

Wavelength Range for in Vivo PR. Previous work has shownthat the action spectrum for the human leukocyte PR enzymeextends from 300 to 600 nm, with a peak at 405 nm (9). In

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H. N. Ananthaswamy and M. S. Fisher

order to determine the effective wavelength range for in vivoPR of UV-induced pyrimidine dimers in neonatal mouse skin,we exposed replicate groups of UV-irradiated (9 kJ/sq m) mice

to black light, cool white fluorescent, or incandescent lightsources. The UV-irradiated mice were exposed to equalamounts of energy (~ 350 kJ/sq m) from one of the 3 light

sources, and their cutaneous DMA was isolated and assayedfor pyrimidine dimers after treatment with M. luteus enzyme.The results presented in Chart 4 indicate that incandescentlight was relatively more effective for PR than the cool whitefluorescent light. In contrast, black light (300 to 400 nm) wasineffective and did not show any decrease in the amount ofdimers after PR treatment (data not shown).

Site of PR in Neonatal Mouse Skin. Much attention hasbeen focused recently on the distribution of PRE activity invarious tissues. In addition, Ley et al. (13) have shown that PRwas absent in the epidermis of adult hairless mice. Since wefound that PR does occur in newborn BALB/c mouse skin, wewished to determine whether PR occurs in the epidermis ordermis, or both. Neonatal mice were irradiated with UV (9 kJ/sq m) and then exposed to the PR light source (incandescentlamps) for 20 hr. Following these treatments, the epidermiswas separated from the dermis by the trypsin flotation method,according to the procedure described by Yuspa and Harris(23). DMA was isolated from epidermis and dermis separatelyand assayed for dimers after treatment with M. luteus extract.The gel electrophoretic profile of the DMA revealed that PRoccurred in the dermis but not in the epidermis. Although theseresults were not quantitated, they appeared to tally with theresults of the whole-skin PR experiment in terms of the amount

of dimers photoreactivated on a qualitative basis. The dose ofUV (9 kJ/sq m) used in these studies induced considerablymore pyrimidine dimers in the epidermis than in the dermis. Inorder to test whether excessive dimer formation in the epidermis masked or interfered with PR, we exposed neonatal miceto a lower dose of UV (1.5 kJ/sq m) and then subjected them

Decreasing Molecular Weight-:(Increasing Number oíESS-»

Chart 4. Microdensitometer scan of the negative from a picture of gel showingmigration patterns of DMA from mouse skin given UV (9 kJ/sq m) + dark -enzyme (ENZ). UV (9 kJ/sq m) + dark + enzyme, UV (9 kJ/sq m) + cool-whitefluorescent light (~350 kJ/sq m) + enzyme, and UV (9 kJ/sq m) + incandescentlight (~350 kJ/sq m) -I- enzyme. DMA samples were handled as described in

Fig. 1. However, electrophoresis was carried out for approximately 22 hr insteadof the usual 20 hr. A newly prepared batch of M. luteus extract was used in thisexperiment.

to PR light. The epidermal DMA profiles indicated that PR didnot occur even at this low dimer-inducing dose of UV.

PR in Adult Mouse Skin. Ley ef al. (13) have reported thatPR is absent in the epidermis of adult hairless mice. However,it is not known whether PR occurs in the dermis of adult mice.In addition, our observation that PR of UV-induced pyrimidinedimers occurs in the dermis, but not in the epidermis, ofnewborn mouse skin prompted us to look for PR in the adultmouse skin. Groups of adult (8 week old) BALB/c mice wereirradiated with various fluences (9, 18, 22, and 44 kJ/sq m) ofUV from the FS40 sunlamps and were either placed in the darkor exposed to PR light (incandescent lamps) for 20 hr. DMAwas isolated separately from the dorsal skin and ear skin andassayed for pyrimidine dimers before and after treatment withM. luteus extract. The gel electrophoretic profile revealed nodifferences in the amount of pyrimidine dimers between DMAfrom UV-irradiated mice kept in the dark and DMA from UV-

irradiated mice exposed to PR light in both the dorsal skin andthe ear skin. Thus, these results indicate that PR of UV-inducedpyrimidine dimers does not occur in adult BALB/c mouse skinunder conditions that permit optimal PR in neonatal skin.

DISCUSSION

There has been much debate concerning the presence orabsence of PR in mouse tissues (13, 21). The data presentedhere indicate that PR of UV-induced pyrimidine dimers does

occur in the skin of newborn BALB/c mice. Previous studiesby Sutherland ef al. (21) have shown that PRE activity ispresent in mouse embryo fibroblasts in culture. In contrast, Leyef al. (13) have reported that PR of UV-induced pyrimidine

dimers does not occur in the epidermis of hairless adult mice.Our results with adult mice are consistent with this observation.In addition, they show that PR is also absent in the dermis ofadult mice.

The results reported here apparently resolve the discrepancybetween the findings of Sutherland ef a/. (21) and Ley ef a/.(13). It is possible that Sutherland ef al. (21) observed PREactivity in mouse fibroblasts because they used embryoniccells, whereas Ley ef al. (13) were unable to detect PR inhairless mice because they used epidermis from adult mice intheir experiments. In this regard, our results indicate furtherthat PR does not occur in the epidermis of newborn mice either.

The observation that PR occurs in the dermis of newbornBALB/c mice but not in adult mice implies that, sometimebetween birth and 8 weeks of age, PR disappears in the dermisfor reasons that are unclear. Since it is known that certaingenes are turned either "on" or "off" during development, it

is plausible to theorize that the gene(s) that control PR areturned off during this period of development. Alternatively, thedisappearance of PR in adult mice could be attributed tochanges in the cellular content of dermis.

The wavelength range for optimal in vivo PR in newbornBALB/c mice apparently is in the visible region of the spectrum(>400 nm). Recently, Sutherland ef al. (20) have demonstratedthat pyrimidine dimers induced in human skin by an FS20sunlamp could be photoreactivated in situ with visible light froman incandescent light bulb. Chiang and Rupert (5) have shownthat longer wavelengths can photoreactivate dimers in culturedmarsupial cells. Studies by Sutherland ef al. (21) have shown

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PR in Neonatal Mouse Skin

that the human leukocyte PRE exhibited optimal activity at 405nm and extended to 600 nm; an action spectrum for PR by themouse fibroblast enzyme has not yet been determined. However, it remains to be shown that the in vivo PR that weobserved in neonatal BALB/c mouse skin is really an enzyme-

catalyzed event rather than a nonenzymatic photochemicalevent.

In summary, we have shown that (a) FS40 sunlamps causea dose-dependent induction of ESS (pyrimidine dimers) in theDNA of neonatal BALB/c mouse skin; (b) exposure of UV-

irradiated neonatal BALB/c mice to PR light causes a reductionin the amount of pyrimidine dimers in the DNA; (c) the PRactivity appears to be present in the dermis and not in theepidermis of newborn BALB/c mouse skin; and (d) PR isabsent in adult BALB/c mouse skin.

ACKNOWLEDGMENTS

We are indebted to Charles Riggs for developing a method for determining theaverage molecular weight from the alkaline agarose gels. We thank Dr. M. L.Kripke for her suggestions and critical comments and Tom Farris for his excellenttechnical assistance.

REFERENCES

1. Achey. P. M., Woodhead, A. D.. and Setlow. R. B. Photoreactivation ofpyrimidine dimers in DNA from thyroid cells of the teleost, Poecitia formosa.Photochem. Photobiol., 29. 305-310, 1979.

2. Blum, H. F. Carcinogenesis by Ultraviolet Light. Princeton, N. J.: PrincetonUniversity Press, 1959.

3. Bowden, G. T., Trosko, J. E.. Shapas, B. G., and Boutwell, R. Excision ofpyrimidine dimers from epidermal DNA and nonsemiconservative epidermalDNA synthesis following ultraviolet irradiation of mouse skin. Cancer Res..35. 3599-3607, 1975.

4. Carrier, W. L., and Setlow, R. B. Endonuclease from Micrococcus luteuswhich has activity toward ultraviolet-irradiated deoxyribonucleic acid: purification and properties. J. Bacteriol., »02.178-186, 1970.

5. Chiang, T., and Rupert, C. S. Action spectrum for photoreactivation ofultraviolet-irradiated marsupial cells in tissue culture. Photochem. Photobiol..

30: 525-528, 1979.6. Cook. J. S. Photoreactivation in animal cells. In: A. C. Griese (ed.). Photo-

physiology, Vol. 5, pp. 191-233. New York: Academic Press. Inc., 1970.7. Epstein, J. Y. Ultraviolet carcinogenesis. In: A. C. Griese (ed.), Photophy-

siology. Vol. 5. pp. 235-273. New York: Academic Press, Inc., 1970.8. Fisher, M. S., and Kripke, M. L. Systemic alteration induced in mice by

ultraviolet light irradiation and its relationship to ultraviolet carcinogenesis.Proc. Nati. Acad. Sei. U. S. A., 74: 1688-1692. 1977.

9. Harm, H. Repair of UV-irradiated biological systems: photoreactivation. In:S. R. Wang (ed.). Photochemistry and Photobiology of Nucleic Acids, Vol. 2.pp. 219-263. New York: Academic Press. Inc.. 1976.

10. Hart, R. W.. and Setlow, R. B. Direct evidence that pyrimidine dimers in DNAresult in neoplastic transformation. In: D. C. Hanawalt and R. B. Setlow(eds.). Molecular Mechanisms for Repair in DNA, Part B, pp. 719-728. NewYork: Plenum Publishing Corp., 1975.

11. Kripke, M. L. Antigenicity of murine skin tumors induced by ultraviolet light.J. Nati. Cancer Inst., 53. 1333-1336, 1974.

12. Kripke. M. L., and Fisher, M. S. Immunologie parameters of ultravioletcarcinogenesis. J. Nati. Cancer Inst., 57: 211-215. 1976.

13. Ley, R. D., Sedita, B. A., and Grube, D. D. Absence of photoreactivation ofpyrimidine dimers in the epidermis of hairless mice following exposures toultraviolet light. Photochem. Photobiol., 27. 483-485, 1978.

14. Ley, R. D., Sedita. B. A., Grube. D. D., and Fry. R. J. M. Induction andpersistence of pyrimidine dimers in the epidermal DNA of two strains ofhairless mice. Cancer Res.. 37. 3243-3248. 1977.

15. McDonell, M., Simon, M. N., and Studier, F. W. Analysis of restrictionfragments of T7 DNA and determination of molecular weights by electropho-resis in neutral and alkaline gels. J. Mol. Biol., ) W: 119-146, 1977.

16. Paterson, M. C.. Lohman, P. H. M., and Sluyther, M. L. Use of a UVendonuclease from Micrococcus luteus to monitor the progress of DNArepair in UV-irradiated human cells. Mutât.Res., 79. 245-256. 1970.

17. Rupert, C. S. Enzymatic photoreactivation: overview. In: P. C. Hanawalt andR. B. Setlow (eds.). Molecular Mechanisms for Repair of DNA. Part A. pp.73-87. New York: Plenum Publishing Corp., 1975.

18. Setlow, J. K., and Setlow. R. B. Nature of the photoreactivable ultravioletlesion in deoxyribonucleic acid. Nature (Lond.). 197 560-562, 1963.

19. Sutherland. B. Photoreactivating enzyme from human leukocytes. Nature(Lond.), 248. 109-112, 1974.

20. Sutherland, B. M.. Harber. L. C.. and Kochevar, I. E. Pyrimidine dimerformation and repair in human skin. Cancer Res.. 40: 3181-3185. 1980.

21. Sutherland, B. M., Runge, P., and Sutherland, J. C. DNA Photoreactivatingenzyme from placental mammals. Origin and characteristics Biochemistry,Õ3:4710-4715, 1974.

22. Urbach, F. The Biologic Effects of Ultraviolet Radiation. New York: PergamonPress, 1969.

23. Yuspa, S. H.. and Harris, C. C. Altered differentiation of mouse epidermalcells treated with retinyl acetate in vitro. Exp. Cell. Res., 86: 95-105, 1974.

bed .

NO UV

Decreasing Molecular Weight -*•(Increasing Number of ESS *- )

Fig. 1. Alkaline agarose gel electrophoresis of DNA from the skin of neonatalBALB/c mice exposed to FS40 sunlamps. All DNA samples except A DNA wereadjusted to A260 = 2.0 and treated with ( + ENZ) or without (-E/VZ) M. luteusextract before electrophoresis. \ DNA (500 ng) and \ DNA cut with EcoRIrestriction enzyme were used as molecular weight markers. Electrophoresis wascarried out for 20 hr at 20 V. The molecular weight of single-stranded DNA forthe various markers is as follows: A = 15.4 x 106; A cut with EcoRI = 7.1 x 106(a), 2.4 x 106 (b), 1.9 x 106 (c). 1.6 x 10s (d), and 1.1 x 106 (e).

A DNA

P1 DNA

NO UV + PR

9

B

+ ENZ 7

[-ENZ 6

PR+ UV +ENZ 5

UV+ PR +ENZ 4

UV+DARK +ENZ 3

Õ+ENZ 2

-ENZ 1UV<

-I

Decreasing Molecular Weight »-(Increasing Number of ESS >- )

Fig. 2. Alkaline agarose gel electrophoresis of DNA from the skin of UV-irradiated neonatal BALB/c mice exposed to PR light DNA samples werehandled as mentioned in Fig. 1. (Lanes 7 and 2), UV (9 kj/sq m); (Lane 3). UV(9 kj/sq m) + 20 hr dark; (Lane 4), UV (9 kJ/sq m) + 20 hr PR light(incandescent): (Lane 5). UV (9 kj/sq m) + 20 hr PR light: and (Lanes 6 and 7),no UV + 20 hr PR light. P1 DNA (Lane 8) and \ DNA (Lane 9) were used asmolecular weight markers. The molecular weight of single-stranded P1 DNA is30 x 106. Electrophoresis was carried out for 20 hr at 20 V.

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