Mutation Research, 217 (1989) 219-226 DNA Repair Elsevier

MTR 06332

219

DNA-protein cross[inking in normal and solar UV-sensitive ICR 2A frog cell lines exposed to solar UV-radiation

Bar ry S. Rosens t e in a, L i - W e n La i b, J o n a t h a n M. D u c o r e c a n d Rebecca B. Rosens t e in a

a Department of Radiation Medicine, Box G, Brown University, Providence, RI 02912 and Department of Radiation Medicine and Biology Research, Rhode Island Hospital, Providence, RI 02903, Departments of b Radiology and c Pediatrics, The University of Texas Health

Science Center at Dallas, 5323 Harry Hines Bird, Dallas, TX 75235 and d Department of Community Health, Box G, Brown University, Providence, RI 02912 (U.S.A.)

(Received 28 April 1988) (Revision received 5 Dec, ember 1988)

(Accepted 11 December 1988)

Keywords: Alkaline elution; DNA-protein crosslinks; ICR 2A cells; Solar UV-radiation

Summary

DNA-protein crosslinks (DPC) were measured following exposure to the solar UV wavelengths produced by a fluorescent sunlamp in ICR 2A frog cells and two solar UV-sensitive mutants derived from this cell line. Approx. 5-7 DPC per 101° dalton were induced in these cells by either 150 kJ /m 2 of sunlamp UV > 315 nm plus photoreactivating light (PRL) or 10 kJ /m e of sunlamp UV > 295 nm. The irradiated cells were then incubated for 0-24 h and the level of DPC measured using alkaline elution. It was found for the ICR 2A cells exposed to sunlamp UV > 315 nm that the level of DPC increased about 3-fold during a 2-h postirradiation incubation and then decreased. The mutant cell lines also showed an enhancement in the level of DPC following irradiation, although it was much less pronounced and the levels decreased much more rapidly. In a similar fashion, the level of DPC increased in ICR 2A cells exposed to sunlamp UV > 295 nm with more than a 5-fold enhancement after a 4-h incubation. Once again, the mutant cell lines showed an increase in the level of DPC that was smaller and more transient than the effect in the ICR 2A cells. These results suggests that this enhancement in DPC may be indicative of a process that plays a role in cellular survival following solar UV-irradiation.

Correspondence: Dr. Barry S. Rosenstein, Box G, Brown Uni- versity, Providence, RI 02912 (U.S.A.).

Abbreviations: DPC, DNA-protein crosslinks; PBS, phos- phate-buffered saline; PRL, photoreactivating light; SDS, sodium dodecyl sulphate.

Present address: Department of Pediatrics, University of Cali- fornia at Davis, MS-1A, Room 1134, Davis, CA 95616 (U.S.A.).

In addition to cyclobutane thymine dimers, the 320-400 nm UV wavelengths present at the earth's surface produce a variety of other DNA damages, including pyrimidine (6-4) pyrimidone photo- products, thymine glycols, DNA strand breaks and DNA-protein crosslinks (Haxiharan and Cerutti, 1977; Rosenstein and Ducore, 1983; Peak et al., 1985). In recent years evidence has begun to

0921-8777/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

220

accumulate that these non-dimer DNA damages play a important biological role for this solar UV region (Zelle et al., 1980; Ritter and Williams, 1981; Smith and Paterson, 1981; Suzuki et al., 1981; Keyse et al., 1983; Rosenstein, 1984a, b; Tyrrell, 1984; Rosenstein and Rosenstein, 1985; Rosenstein et al., 1985, 1986).

In order to investigate the repair of these damages in greater detail, mutant cell lines that are hypersensitive to solar UV wavelengths and deficient in the repair of non-dimer DNA damages have been isolated (Rosenstein and Chao, 1985). This has been accomplished using the ICR 2A frog cell line which possess two useful features that have made the isolation of this type of mutant feasible. The first is that it is highly proficient in enzymatic photoreactivation (Freed et al., 1979; Rosenstein and Setlow, 1980). Hence, it is possible to irradiate these cells with solar UV wavelengths, which induce few dimers relative to non-dimer damages, and then eliminate most of the dimers by exposure to photoreactivating light (PRL). This results in the production of a relatively pure popu- lation of non-dimer damages. The second feature these cells possess is that they contain a haploid complement of chromosomes which should in- crease the likelihood of producing a mutant cell line with a particular phenotype (Freed and Mezger-Freed, 1970). The purpose of the experi- ments described in this paper is to examine the induction of DPC in these mutant cell lines and to determine whether this type of DNA damage might play a role in the hypersensitivity to solar UV- radiation exhibited by these cells.

Materials and methods

Cell lines and culture conditions ICR 2A, DRP 36 and DRP 153 frog cells were

grown at 25°C in modified Leibowitz medium (Gibeo, Grand Island, NY) supplemented with 10% fetal calf serum (Hyclone, Logan, UT), 100 uni ts /ml penicillin and 100 /~g/ml streptomycin (Gibco). Under these conditions the cells had a doubling time of 48 h.

Labeling and irradiation conditions Cells were plated in either 25-cm 2 or 75-cm 2

flasks (Coming Glass Works, Coming, NY) at a

density of 10 4 ce l l s /cm 2 and either [Me- 3H]thymidine (20 Ci /mmole , New England Nuclear, Boston, MA) or [2-14C]thymidine (59 mCi /mmole) were added to final concentrations of 0.1 /~Ci/ml or 0.02 /~Ci/ml, respectively. Fol- lowing a 72-h incubation the 14C-labeled cells were washed 3 times with phosphate-buffered saline (PBS, Chao and Rosenstein, 1984), covered with 5 ml of PBS and irradiated while being held on ice. The UV was produced by two FS40 West- inghouse fluorescent sunlamps at a fluence rate of 10.4 W / m 2 as measured using a ILl700 radiome- ter. Cultures were exposed to sunlamp UV passed through either the culture flask material alone or the flask plus a sheet of 48A Mylar (DuPont, Wilmington, DE). Under these conditions less than 10% transmittance of wavelengths shorter than either 295 nm or 315 nm respectively took place (Rosenstein, 1984). For the irradiations utilizing 48A Mylar, the cells were also exposed to 30 k J / m 2 of PRL, emitted by two GE F40B blue lamps. The light was filtered through 3 mm lead glass resulting in the elimination of wavelengths shorter than about 350 nm (Rosenstein, 1982). These two irradiation conditions will be referred to as sunlamp UV > 295 nm and sunlamp UV > 315 nm, respectively. A chase period was not empolyed following the incubation with either [14C]thymidine or [3H]thymidine as it was ob- served in previous experiments using a sucrose gradient analysis that essentially no measurable amount of radioactive label was present in nascent DNA chains following a 72-h incubation (unpub- lished data).

Elution conditions and calculations of DPC and DNA strand breaks

Cells were washed twice with PBS and gently scraped off the flasks into ice-cold PBS containing 0.2 m g /m l NazEDTA. 5 × 105 ~4C-labeled cells exposed to a particular treatment were then mixed with 5 x 10 s 3H-labeled cells and irradiated with 10 Gy of X-rays (250 kV Phillips Model RT 250) at a dose rate of approx. 1.1 G y /m in ) while held on ice for the determination of the DPC level (Kohn et al., 1981). In addition, for each treat- ment, 5 × 105 ~4C-labeled cells not exposed to X-rays were mixed with 5 × 10 s 3H-labeled cells that had been irradiated with 3 Gy of X-rays in

order to determine the level of DNA-strand breaks. The following procedure was then used for all elutions. Each cell mixture was loaded on a 25-mm 2-#m pore size polyvinyl chloride filter (Millipore Corp., Bedford, MA), washed twice with cold PBS and lysed with a solution containing 2% sodium dodecyl sulfate (SDS, Gallard-Schlesinger, Carleplace, NY), 0.1 M glycine and 0.02 M EDTA, pH 10. The lysis solution was allowed to flow through the filter by gravity. The cell lysates were then washed with 5 ml of 0.02 M Na2EDTA, pH 10, followed by the addition of an elution solution consisting of 0.1 M tetrapropylammonium hydrox- ide and 0.02 M EDTA (acid form), pH 12.1. The elution solution was pumped through the filters at approx. 0.04 ml/min and 5-ml fractions were col- lected at 3-h intervals. Upon completion of the elution the fractions were made isovolumetric with water and mixed with 10 ml of Budget-Solve (Research Products International Corp., Mount Prospect IL) containing 0.3% acetic acid. Filters were processed as previously described (Kohn et al., 1981). All fractions were counted in a Packard 2000 scintillation counter.

The number of DPC was calculated from the following equation (Peak et al., 1987):

DPCuv = [(1--F)-I /2(PbR + PUV)]

- - [ ( 1 - - r o ) - I / 2 ( P b R ) + P u v ]

where DPCuv is the frequency of sunlamp UV-in- duced DPC, PbR is the frequency of X-ray in- duced single-strand breaks, Pvv is the frequency of sunlamp UV-induced single strand breaks and r and r 0 are the fractions of DNA eluting in the slow component of the elution profiles extrapo- lated to time zero for UV-treated and untreated cells, respectively. The X-ray dose used, 10 Gy, produces approximately 2.7 breaks/10 9 dalton (Kohn et al., 1976). ..

The yield of single-strand breaks induced by the sunlamp UV-irradiations was calculated from the following equation:

Breaks/dalton = 8.1 × 10-10 (By v _ Bunirr )

/ ( B300rad -- Bunir r )

221

where B equals the logarithm of the fraction of DNA retained by the filter after 0 h of elution minus the logarithm of the fraction of DNA re- tained by the filter after 15 h of elution. An X-ray dose of 3 Gy induces approximately 8.1 x 10 -1° single strand breaks per dalton of DNA.

Measurement of cell survival Either 5 X 10 2 cells were plated in 25-cm z flasks

and left untreated, or 5 x l0 s cells were plated and exposedto either 100 k J / m 2 of sunlamp UV > 315 nm or 10 k J /m 2 of sunlamp UV> 295 nm as described above. Following a 2-week incubation the cultures were stained with Giemsa (Harleco, Gibbstown, N J) and the number of colonies (groupings of 32 ore more cells) counted.

Results

DPC induction in cells exposed to sunlamp UV > 315 nm

ICR 2A cells and two solar UV-sensitive cell lines, DRP 36 and DRP 153, were irradiated with 150 kJ /m z of sunlamp UV > 315 nm. As indicated in the Materials and Methods section, these cells were also exposed to PRL, resulting in the elimination of most of the small yield of dimers induced by the sunlamp irradiation. The levels of survival following sunlamp UV > 315 nm-irradia- tion for the 3 cell lines examined are listed in Table 1. The irradiated cells were incubated for either 0, 0.5, 2, 4, 12 or 24 h and then mixed with an equal number of 3H-labeled cells. These mix- tures were next irradiated with 10 Gy of X-rays while being held on ice and then subjected to alkaline elution. Typical results are shown in Fig. 1. Treatment with 10 Gy alone resulted in a very rapid rate of elution. However, sunlamp UV > 315-nm irradiation caused an increase in the amount of DNA retained on the filters indicating DPC were induced. This was confirmed through treatment with proteinase K which eliminated the enhanced retention (data not shown). Upon in- cubation of all 3 cell lines, the retention of the DNA increased, demonstrating a rise in the level of DPC. The level of DPC decreased upon further incubation. This increase in the level of DPC was even greater than suggested by these elution pro- files as the sunlamp U V > 315-nm irradiation

222

TABLE 1

SURVIVAL OF ICR 2A, DRP 36 and DRP 153 CELLS

Cell a Percent survival Percent survival line following exposure following exposure

to 100 kJ /m 2 sunlamp to 10 kJ /m 2 sunlamp UV > 315 nm UV > 295 nm

ICR2A 1.9+0.4x 10 - l b 4.4+1.9 x 10 3

DRP36 7.1 +9.2X 10 -4 6.0+3.3 x 10 3

DRP 153 2.1 +4.2x10 -4 2.7+1.8×10 -3

a Values for percent survival represent the plating efficiency of irradiated cells divided by the plating efficiency of unirradia- ted cells multiplied by 100. The plating efficiencies for unirradiated ICR 2A, DRP 36 and DRP 153 were 0.20, 0.19 and 0.28, respectively.

b Average of 10 values + standard deviation.

c a u s e d a s u b s t a n t i a l i n c r e a s e in t he r a t e o f e l u t i on

b y i t se l f w h i c h is i n d i c a t i v e o f t he i n d u c t i o n o f

D N A - s t r a n d b r e a k s (Fig . 2a). T h e s e s t r a n d b r e a k s

a re t h e r e f o r e i n c l u d e d in the c a l c u l a t i o n o f D P C .

T h e k ine t i c s o f D P C i n d u c t i o n c a n be seen

m o r e c lear ly in Fig . 3 in w h i c h the n u m b e r s o f

D P C were c a l c u l a t e d a n d p l o t t e d . F o r each t r ea t -

m e n t 3 s e p a r a t e i r r a d i a t i o n s w e r e p e r f o r m e d a n d

t h e ave rage level o f D P C a l o n g w i t h t he s t a n d a r d

8O 7O

z o 60 ~- 50 Z

4o rY ~ 30 z kd C.) C~ 20 I . J

o_

10

°~6~

o) SUNLAMP UV> 315 nm | I I i I i Ii

0 6 12

0"-.-,~0~ O.

b) SUNLAMP UV ) 295 nm I I I i I

0 6 12

HOURS OF ELUTION

Fig. 2. DNA strand breakage in sunlamp UV-irradiated cells. Alkaline elution profiles are shown for ICR 2A cells exposed to either (a) 150 kJ /m 2 of sunlamp UV> 315 nm or (b) 10 kJ /m 2 of sunlamp UV > 295 nm and incubated for either 0.5 h (o), 4 h (zx) or 24 (13). Unirradiated cells (e) and cells exposed to 3 Gy of X-rays (A).

e r ro r s s h o w n . F o r e a c h cell l ine, t h e r e w a s a r a p i d

i n c r e a s e in t he level o f D P C w h i c h r e a c h e d a p e a k

w i t h i n a b o u t 2 h f o l l o w i n g i r r a d i a t i o n a n d t h e n

dec l i ned . H o w e v e r , t he i n c r e a s e in t he level o f

D P C w a s m u c h less p r o n o u n c e d in the t w o so la r

U V - s e n s i t i v e cell l ines. Also , t he r a t e a n d e x t e n t o f

z o I - z Ld L rY I-- z Ld 0 12/ i,i 13-

8O 70 60

5O

4O

30

20

10

\A \

I) ICR 2A t - t

0 6 12

•1•0•0•0• ( [] N O~D~

b) 9RP 318 '

0 6 12

D~D-13_D_ c) ?RP I, S3

0 6 12

HOURS OF ELUTION

Fig. l. Alkaline elution profiles for cells exposed to sunlamp UV> 315 nm. (a) ICR 2A, (b) DRP 36 and DRP 153 cells were exposed to 150 kJ /m 2 of sunlamp UV > 315 nm and incubated for either 0 h (o), 2 h (A), or 24 h ([3). These cells were then exposed to 10 Gy of X-rays and subjected to alkaline elution. Unirradiated cells (@) and cells exposed only to 10 Gy of X-rays (A).

25

z 20 o

< o 15

0

n. 10

O _

a_ 5

i i , i , , i

a) ICR 2A

i i i i i i i

0 12 24

i i i , , , i

b) DRP 36

l

i , i i i i J

c) DRP 15..3

f

12 24 0 12 24

HOURS OF INCUBATION

Fig. 3. Kinetics of DPC induction in sunlamp UV > 315-nm irradiated cells. The average numbers of DPC present in either (a) ICR 2A, (b) DRP 36 or (c) DRP 153 cells following exposure to 150 kJ/m: of sunlamp UV > 315 nm are shown. For each incubation period 3 separate irradiations were per- formed. The error bars represent the standard errors.

TABLE 2

DPC INDUCTION IN ICR 2A, DRP 36 AND DRP 153 CELLS

Cell Hours of DPC per 1010 dalton line incubation following exposure

to 150 kJ /m 2 sunlamp UV > 315 nm

DPC per 101° dalton following exposure to 10 kJ /m 2 sunlamp UV > 295 nm

223

ICR2A 0.0 7.2±1.8 a 5.3±1.0 0.5 11.7±3.6 22.2±4.4 2.0 17.3±2.8 23.8±9.3 4.0 16.9±2.5 24.3±7.4

12.0 5.7±1.4 14.2±2.3 24.0 2.3±0.5 7.0±0.5

DRP36 0.0 7.3±0.9 6.1±1.0 0.5 8.5±1.5 9.8±1.6 2.0 12.8±3.5 13.7±1.8 4.0 7.7±1.3 *b 15.7±1.2

12.0 2.5±0.9* 9.8±3.1 24.0 0.1±0.1" 2.2±1.0

DRP153 0.0 6.9±0.9 6.3±2.4 0.5 6 .8±1.4" 10.4±3.0 2.0 10.3±3.3" 13.5±2.7 4.0 4 .5±1.7" 13.3±1.9

12.0 2 .1±0.6" 3.5±1.2 24.0 0.4±0.1" 3.1±1.3

a Average of 3 values + standard deviation. b Analysis of variance was used to test for significant differences in DPC induction between the 3 cell lines for each incubation time

and for each fluence. When the F-statistic was significant, contrasts comparing the DRP 36 and DRP 153 cell lines with the ICR 2A cell line were then evaluated. Significant differences (p ~ 0.05) are marked with an asterick.

decrease in the level of DPC was much greater for I ~8 D R P 36 and D R P 153 than that exhibited by the 80

70 ICR 2A parental cell line (Table 2). z 60 ICR 2A cells were also exposed to 300 k J / m 2 o

F- 50 of sunlamp UV > 315 nm which resulted in a z

t~ 40 percent survival comparable to that observed with L the mutant cell lines irradiated with 150 k J / m 2. tw 30 F-- The yields of DPC per 10 a° dalton at 0, 0.5, 2, 4, z 12 and 24 h were 12.8, 18.7, 26.2, 22.7, 19.3 and o 20 10.6, respectively. Hence, the enhancement in DPC " ' o_ upon incubation was similar to that observed for these cells exposed to the lower fluence.

DPC induction in cells exposed to sunlamp UV > 295 nm

ICR 2A, DRP 36 and DRP 153 cells were also treated with 10 k J / m 2 of sunlamp UV > 295 nm which results in the production of a high level of pyrimidine dimers in addition to DPC. These cells were incubated for 0 -24 h and the level of DPC

10

•'•O-[Z]-r- 0-0~0~ C

\

~ , i i i

u, xO-O-o_(

O'-D-B-u -o-oL-ol x

D~n..B._

,CR 2A , b) DRP 3,6, , £) DRP 1,5

0 6 12 0 6 12 0 6 12

HOURS OF ELUTION

Fig. 4. Alkaline elution profiles for cells exposed to sunlamp UV > 295 nm. (a) ICR 2A, (b) DRP 36 and DRP 153 cells were exposed to 10 kJ /m 2 of sunlamp UV > 295 and incubated for either 0 h (o), 2 h (A), or 24 h (12]). These cells were then exposed to 10 Gy of X-rays and subjected to alkaline elution. Unirradiated cells (0) and cells exposed only to 10 Gy of X-rays (A).

224

(/3 Z o

.< 0

0

r Y

i , i Q _

o F I

1 2 3

2 5 i i i 2ol 15

0

5 o

e) ICR 2A

I l t l l l l l l ~ ' l ~ l l

6

o

o b) DRP ,:36 c) DRP 153 0 t i t i , i i I t i i i i i i , i i i , ,

0 12 24- 0 12 24 0 12 24

HOURS OF INCUBATION Fig. 5. Kinetics of DPC induction in sunlamp UV > 295-nm irradiated cells. The average numbers of DPC present in either (a) ICR 2A, (b) DRP 36 or (c) DRP 153 cells following exposure to 10 kJ/m 2 of sunlamp UV > 295 nm are shown. For each incubation period three separate irradiations were performed. The error bars represent the standard errors.

measured using alkaline elution. Once again, an enhancement in DNA retention was observed in sunlamp UV > 295-nm irradiated cells that in- creased upon incubation (Fig. 4). For this ex- posure, very few DNA-strand breaks were also induced (Fig. 2b). However, for these treatments an even greater increase in the level of DPC was observed which reached a maximum following about a 4-h incubation (Fig. 5). The level then declined, although the number of DPC did not reach the baseline level exhibited in unirradiated cells even following a 24-h incubation. In a similar fashion to that observed for the longer wavelength irradiations, the increase in DPC was much smaller for DRP 36 and DRP 153 than exhibited by the ICR 2A cells.

Discussion

The levels of DPC present in ICR 2A cells were measured following exposure to the solar UV wavelengths produced by a fluorescent sunlamp. In addition, DRP 36 and DRP 153 which repre- sent two cell lines derived from ICR 2A were examined. These cells were isolated on the basis of hypersensitivity to irradiation with sunlamp UV > 315 nm and are presumably deficient in the repair

of a non-dimer D N A damage as very few dimers result from this treatment (Rosenstein and Chao, 1985). In contrast, these cells exhibit a similar level of sensitivity to that shown by the parental ICR 2A to sunlamp UV > 295 nm which results in the production of a substantial dimer yield. It is likely that the cause for cell death following irradi- ation with these shorter UV wavelengths is due to the presence of dimers in DNA and the non-dimer DNA damages are of little consequence. This is consistent with the high photoreactivable sector exhibited by ICR 2A cells exposed to sunlamp UV > 295 nm (Rosenstein, 1984).

In the work described in this paper it was found that the level of DPC increased upon incubation of UV-irradiated cells. This is con- sistent with previous results obtained with UV- irradiated V79 and normal human skin fibroblasts (Chiu et al., 1984; Lai et al., 1987) and recently in 405 nm-irradiated human epithelial teratocar- cinoma P3 cells (M. Peak, personal communica- tion). However, in the normal human cell work the increase in DPC was not observed in sunlamp UV > 315-nm irradiated cells as only a 0 and 24 h incubation were performed and the increase at shorter times was missed. In the experiments de- scribed in this paper, the increase in the level of DPC was lower and the decline more rapid for cells exposed to sunlamp UV > 315 nm than for cells treated with sunlamp UV > 295 nm. It is possible that this difference may be a reflection of the total yield of D N A damage in that 10 k J / m 2 of sunlamp UV > 295 nm induces about 2400 dimers per 10 9 dalton whereas 150 k J / m 2 of sunlamp UV > 315 nm results in the presence of only about 24 dimers per 10 9 dalton at the end of the treatment (Rosenstein and Mitchell, 1987). It is therefore possible that there may be a protein which becomes covalently associated with DNA following the induction of DNA damage. This protein may be involved in a DNA repair process or some other cellular response to UV irradiation.

One possible explanation that could be pro- posed for the decrease in crosslinking observed following a 12- or 24-h incubation, is that cellular lysis may have occurred resulting in the detach- ment of the most heavily damaged cells. However, an indication of cell degredation or measureable loss of cells was not observed in the cultures of

either the parental or mutant cell lines 24 h after irradiation. In addition, there was n o significant increase in the amount of radioactive counts eluted from the filters during the lysis procedure for any of the samples indicating that there was no sub- stantial D N A breakdown in these cells.

It was found that the DRP 36 and DRP 153 cells exhibited a smaller enhancement in DPC and the levels fell more rapidly than in ICR 2A cells exposed to sunlamp UV > 315 nm. As these cells are also hypersensitive to this treatment, the pro- cess by which DPC form may be of importance for cellular survival. These experiments also showed that this differential enhancement in DPC in the mutant and parental cell lines takes place following sunlamp UV > 295-nm irradiation, al- though a hypersensitivity to this treatment is not exhibited by the mutant cell lines. This is not surprising, because, as already indicated, dimers are the cause for cellular lethality in this shorter wavelength region and it is unlikely that a de- ficiency in a different repair or recovery process would have a significant effect upon cell survival.

Acknowledgements

Susan Baird is thanked for her expert technical assisstance. This work was supported by DHHS grant CA45078 from the National Cancer In- stitute.

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