325

Mutation Research, 62 (1979) 325--339

© Elsevier/North-HollandBiomedicalPress

SURVIVAL AND DNA REPAIR IN ULTRAVIOLET-IRRADIATED HAPLOID AND DIPLOID CULTURED FROG CELLS

JEROME HOLMAN

J. FREED, R O N A L D C. M A S S E Y

H. H O E S S *, F R A N K

A. A N G E L O S A N T O

** and

Jr. ***

The Institute for Cancer Research, The Fox Chase Cancer Center, Philadelphia, PA 19111 (U.S.A.)

(Received 8 February 1979) (Revision received 11 April 1979) (Accepted 1 May 1979)

Summary Survival and repair of DNA following ultraviolet (254-nm) radiation have been investigated in ICR 2A, a cultured cell line from haploid embryos of the grassfrog, R a n a pipiens. Survival curves from cells recovering in the dark gave mean lethal dose value (Do) in the range 1.5--1.7 Jm -2 for both haploid and diploid cell stocks. The only significant difference observed between haploids and diploids was in the extent of the shoulder at low fluence (Dq), the value for exponentially multiplying diploid cells (3.0 Jm -2 ) being higher than that found for haploids (1.2 Jm-2). Irradiation of cultures reversibly blocked in the G1 phase of the cell cycle gave survival~urve coefficients indistinguishable between haploids and diploids. Post-irradiation exposure to visible light restored colonyforming capacity and removed chromatographically estimated pyrimidine dimers from DNA at the same rates. After fluences killing 90% of the cells, complete restoration of survival was obtained after 60-min exposure to 500 foot-candles, indicating that in this range lethality is entirely photoreversible and therefore attributable to pyrimidine dimers in DNA. Dimer removal required illumination following ultraviolet exposure, intact cells and physiological temperature, implying that the photoreversal involved DNA photolyase activity. Excision-repair capacity was slight, since no loss of dimers could be detected chromatographically during up to 48 h incubation in the dark and since autoradiographically detected "unscheduled DNA synthesis" was limited to a 2-fold increase saturated at 10 Jm -2. These properties make ICR 2A frog cells useful to explore how DNA-repair pathways influence mutant yield. * Present address: Frederick Cancer R e s e a r c h Center, B o x B, F r e d e r i c k , M D , 2 1 7 0 1 ( U . S . A . ) ** Present address: T h e Institute for Medical Research, C a m d e n , N J , 0 8 1 0 3 ( U . S . A . ) *** Present address: 4 9 1 7 Cedar A v e n u e , P h i l a d e l p h i a , P A , 1 9 1 4 3 ( U . S . A . )

326 Mutation frequency following exposure to a mutagenic agent depends on the mode of DNA repair in microorganisms [53]. Investigations of mutation in vertebrate cells have utilized naturally occurring variations in DNA repair capacities, e.g., those found in human xeroderma pigmentosum cell strains [9]. We have sought to extend this approach to cells capable of photoreactivation, to use this conditional repair function as a tool in studies of mutagenesis in cell culture. We have explored the effects of UV radiation o n I C R 2A, a permanent cultured cell line from tissue of androgenetic haploid grassfrog embryos [15--17]. These cells can be propagated as nearly pure haploid populations for many subculture generations, corresponding diploid derivatives are available, and the isolation of drug-resistant variants can be carried out as in other vertebrate cell cultures [30]. In this investigation, we have compared the survival of haploid and diploid cells to assess the effect of a single chromosome set on UV-induced lethality and we have sought to determine the modes of repair of UV-induced DNA damage in this system. What emerges from our study is that ICR 2A cells express substantial photoreversal capacity but are deficient in excision repair, so that repair can be shifted experimentally from intrinsically error-free photoreversal to post-replication repair pathways thought to be error-prone.

Materials and methods Cell cultures Monolayer cultures of ICR 2A cells were grown in Falcon plastic flasks at 25°C in diluted Leibovitz L-15 supplemented with 10% fetal bovine serum [16, 17]. Haploid cells were obtained from early subculture generations and diploids from a cell stock that had been propagated through more than 200 subcultures, and in which only diploid derivatives were present (Table 1). Mycoplasma contamination was undetectable by culture methods (Stanford Mycoplasma Laboratory) or by measurements of uridine and uracil incorporation [45]. Identification of ICR 2A as Rana pipiens was confirmed by karyotype analysis and by electrophoretic analysis of several enzymes [19]. TABLE 1 C H A R A C T E R I S T I C S OF T H E S U B S T R A I N S OF ICR 2A E M P L O Y E D Haploids

Diploids

Subculture generations Chromosome counts a Chromosomes:

31--48

238--259

Cells: D N A p e r G1 n u c l e u s ( p g ) b E f f i c i e n c y o f plating c

4 70 1 1 8.0 2 0 . 2 ± 1.1% ( 1 3 )

12

13

25

26

20

23

24

25

26

27

3 1 1 16 16.4 3 4 . 7 ± 2.4% ( 1 7 )

27

2

a R e p r e s e n t a t i v e m e t a p h a s e c h r o m o s o m e c o u n t s at s u b c u l t u r e s 31 ( h a p l o i d s ) a n d 2 3 8 ( d i p l o i d s ) . b D e t e r m i n a t i o n s b y f l o w m i c r o f i u o r i m e t r y at s u b c u l t u r e s 4 5 ( h a p l o i d s ) and 2 5 1 (dtploids), using C H O C h i n e s e h a m s t e r f l b r o b l a s t s as s t a n d a r d a t 7.0 pg per n u c l e u s . c C o l o n y f o r m a t i o n in u n i r r a d i a t e d c o n t r o l c u l t u r e s , ~ o m i n o c u l a t i o n o f 1 0 0 ceils p e r flask. E a c h d e t e r r u i n a t i o n is t h e average o f 6 flasks. T h e m e a n v a l u e ( t o g e t h e r w i t h its s t a n d a z d e r r o r ) is s h o w n for t h e n u m b e r o f d e t e r m i n a t i o n s g i v e n in p a r e n t h e s e s .

327 CHO-K1 Chinese hamster cells and WI-38 human fibroblasts used in some experiments were propagated in monolayer culture in F-12 medium [20] with 10% fetal bovine serum in 5% C02 at 37°C.

irradiation and dosimetry Cells to be irradiated were subcultured to ultraviolet-transparent plastic petri dishes (5213 Permanox 60 × 15 mm Contur Dish, Lux Scientific Corporation, Thousand Oaks, CA 91360) b y adding 5 ml of a cell suspension such that after 24 h, from 4 to 5 × 10 s attached and spread cells were present. Just prior to irradiation a rubber policeman was used to remove the cells from the periphery o f the dish where conditions of exposure differ from the rest of the growth surface. The m e d i u m was replaced during irradiation b y 5 ml o f phosphate-buffered saline (diluted to 60% to be isotonic for frog cells). Pairs of plates were exposed to ultraviolet radiation for the desired time in the irradiation chamber described below. All operations following irradiation were carried o u t in a laboratory from which light o f wavelengths below 500 nm was excluded. The irradiated cultures were rinsed with 5 ml diluted PBS which was replaced with 5 ml growth medium. The cultures were then incubated at 25°C overnight in the dark. Irradiation was carried o u t from below in a cabinet containing an 8-W low pressure mercury arc, a Coming 7-54 filter to reduce visible wavelenghts and a shutter. The fluence rate at the specimen was estimated using a Tektronix J-16 digital p h o t o m e t e r and J 6 5 0 4 radiometric probe exposed through a window made from the b o t t o m of a Permanox petri dish. The response of the photometer was calibrated for the radiation emitted by this source b y potassium ferrioxalate actinometry [23] as described b y Jagger [25]. The fluence rates in the experiments reported here ranged from 9.8 to 15.0 Jm -2 min -1. For photoreactivation experiments, irradiated cultures and appropriate controls were illuminated b y a lamp with t w o 15-W cool white fluorescent tubes. The incident illumination at the level o f the cultures was adjusted to 500 footcandles as measured with a Gossen Luna-Pro photographic exposure meter; the incident energy was measured as 1100 pW/cm 2 with the J-16 p h o t o m e t e r and J 6 5 0 2 calibrated probe. The temperature of the cultures during illumination was measured with a hypodermic thermistor probe, and the conditions described above produced no rise in medium temperature during 60 min o f exposure. In some experiments, the cultures were maintained at 2°C on a surface of crushed ice.

Estimation of surviving fraction After overnight incubation in the dark, the cells were detached with trypsin and enumerated with a Coulter electronic cell counter. Cell density was adjusted to 104 cells/ml, and appropriate dilutions made to inoculate groups o f 6 culture flasks (Falcon 3012, 25 cm 2) with 100 or 1000 cells each in 5 ml of growth medium. These cultures were incubated undisturbed in the dark for 10--14 days, then rinsed with PBS, fixed with methanol and stained with Giemsa. The n u m b e r of surviving colonies (groups of more than 30 cells) was determined b y inspection with a stereo microscope.

328

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Fitting and statistical evaluation o f survival curves Pooled data from each series of experiments were fitted by a computer method to the equation S = e-~d[l -- (I -- eXd) n ]

where S is the surviving fraction, /z the initial slope, k the final slope, d the ultraviolet fluence and n the intercept at zero fluence. Values of the coefficients p, k and n were optimized to produce best fit,i.e.,to minimize the sum of squared errors. The procedure is similar to that proposed by Porter [39], and employed an unpublished program developed by Dr. Sam Litwin of this Institute. Examples of curves fitted in this w a y are shown in Figs. 2 and 3. Comparisons between pairs of experimental series were made by assigning the

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separately estimated values o f the coefficients p, k and n in various combinations and testing whether these gave a significantly better fit than if the same values were used for b o t h populations. Significance was tested using a sampled permutation m e t h o d : the data are pooled, assigned at random to t w o populations, new survival curves fitted and new values o f the coefficient estimated. This procedure is repeated 99 times to calculate the differences to be expected from sampling errors, and thus the probability that the differences between the original data sets could arise b y chance. Such curves were used to calculate the best estimates o f Do (increment of fluence reducing S b y 1/e at the final slope) and Dq (fluence corresponding to S = 1 b y extrapolating from the final slope).

Estimation of thymine dimers Cultures for estimation of dimer content were plated in Permanox dishes at 1.0 X 104 cells per dish in medium with 1.0 pCi/ml of [Me-3H]thymidine (Amersham, sp. act. 20 Ci/mmole) and incubated for 48 h prior to irradiation. Following irradiation and further treatment required b y the experiment, the dimer content was determined by one o f t w o methods: (1) The cells were detached with a rubber policeman, washed b y centrifugation in diluted PBS, fixed b y addition of an equal volume of cold 10% trichloroacetic acid, and washed once b y centrifugation in cold 5% trichloroacetic acid. The dried cell pellet was then hydrolysed in formic acid and dimer content determined b y paper chromatography as described by Carrier and Setlow [7]. (2) DNA was precipitated and hydrolysed as described above, b u t dimer content was determined b y one-dimensional thin-layer chromatography as described by Cook and Friedberg [12], using silica-gel plates and ethylacetate--n-propanol--H20 (4 : 1 : 2) as the solvent. In some experiments, dimer analysis was carried o u t on b o t h whole-cell preparations and on isolated nuclei prepared b y the detergent procedure of Penman [37].

Estimation of "unscheduled DNA synthesis" For autoradiography, cells were grown on 2 X 2 cm glass coverslips or in 2 X

330 2 cm wells of Lab-Tek Culture Slide Chambers. Prior to irradiation, the coverslips were rinsed in PBS and inverted in Permanox dishes containing PBS. Subsequent to irradiation, the cultures were returned to complete growth medium and after various periods of incubation exposed for 4 h to 10 gCi/ml of [3H]thymidine. After an additional incubation for 1 h in medium containing 0.1 mM cold thymidine, the cultures were rinsed 3 times in 0.55% saline, fixed in methanol for 5 min, and dried. The preparations were coated with Kodak NTB-2 liquid emulsion, dried and exposed at 4°C for 8--14 days, developed and stained as described b y Perry [38]. The number o f grains per nucleus was counted in samples of 400 nuclei and the frequency distribution of grains per nucleus plotted as a histogram. The occurrence of repair incorporation was inferred from analyses of the distribution of grains per nucleus [ 52]. Results

Survival curves and the effect o f ploidy In a cell population with a single chromosome set, the greatest sensitivity to inactivation b y radiation would be expected in cells in G1 phase, prior to chromosome replication. To attain this state, ICR 2A cultures were exposed to low serum medium and allowed to become reversibly blocked in G1. As shown in Fig. 1, after 72 h at a serum concentration of 0.25% the fraction of cells in S-phase declined to a minimum. Hypoxanthine (50/~M) was present to meet the purine requirement of ICR 2A cells in L-15 medium [47]; w i t h o u t hypoxanthine the low serum block becomes irreversible (data n o t shown). Following feeding with standard growth medium (10% fetal bovine serum), the cells entered S after a lag o f a b o u t 24 h and reached a peak o f replication at a b o u t 36 h with 80--90% of the cell population in S-phase. Blockade in G1 was confirmed b y estimations of DNA per nucleus b y flow microfluorimetry of samples of formaldehyde-fixed cells, carried o u t at Lawrence Livermore Laboratory. When haploid and diploid cultures were irradiated just prior to releasing the block and allowed to form colonies in the dark, the survival curves were as shown in Fig. 2. Statistical evaluation showed no significant reduction in error from assignment of differences in any constant o f the equation. Therefore, as shown in Table 2, c o m m o n values o f D0 and Dq best represent these data. Other experiments, using ICR 2A cells from cultures in exponential growth, are summarized in Fig. 3. Exponential growth o f these cultures was inferred TABLE 2 S U R V I V A L C U R V E C O E F F I C I E N T S F O R ICR 2A CELLS F O L L O W I N G 254 n m R A D I A T I O N T y p e of culture

DO a

Dq a

Haploid

G1, blocked

Diploid Haploid Diploid

G1, blocked

1.51 1.51 1.69 1.69

2.57 2.57 1.25 3.06 b

Ploidy

Exponential

Exponential

a F l u e n c e in J m - 2 , e s t i m a t e d as d e s c r i b e d in m a t e r i a l s a n d m e t h o d s . b Difference f r o m haploid value significant at P ~ 0.05.

331 from growth curves [16] and from autoradiography of cultures exposed to [aH]thymidine which showed 40--50% o f the cells to be in S-phase, as expected for exponential cultures of ICR 2A [1,52]. As m a y be seen in Fig. 3, the diploid and haploid curves are best fitted b y a c o m m o n final slope, b u t the diploids show a more extended shoulder region than the haploids. As shown in Table 2, this difference is significant at the 5% level. As a test of our irradiation procedure and calculation method, a survival curve for CHO-K1 Chinese hamster cells was determined. This experiment (data n o t shown) yielded Do 6.5 Jm -2 and Dq 3.2 Jm -2. Since these values are comparable to those of other workers [35], we conclude that our procedures are adequate. In summary, our best estimate of the Do value for haploid frog cells is in the range 1.5--1.7 Jm -2, a b o u t one-fourth the value obtained from Chinese hamster cells. Even when irradiated in G1, the cells are able to accumulate sublethal damage, the width o f the shoulder (Dq) being greater than the Do value. Doubling of chromosome number did n o t increase the value of Do, and increased the shoulder value (Dq) in exponential cultures b u t not in G l - b l o c k e d cultures. As is noted below, the absorptions of radiation in haploids and diploids were comparable in these experiments, since the relation between fluence and pyrimidine dimer yield was similar in the two cell strains.

Photoreactivation of survival If, instead of being allowed to recover in the dark after ultraviolet irradiation, ICR 2A cells were immediately exposed to visible light, there was a timed e p e n d e n t restoration of survival to control values (Fig. 4). Under the illumination employed, if cells were exposed to ultraviolet doses reducing survival to a b o u t 0.1, there was a linear increase in the "rescued fraction" over the course o f 60 min. However, if survival was reduced only to 0.6, (upper curve) survival was restored completely in 10 min and further illumination had little effect. As will be detailed below, this restoration of survival parallels the apparently enzymatic loss o f pyrimidine dimers from DNA. Capacity for photoreactivation is demonstrable in the haploid cultures from early passages and is fully retained in the late-passage diploid cells.

Equivalence of dimer induction in haploids and diploids In view of the near equivalence of survival in haploids and diploids, the possibility existed that the effective radiation dose (i.e., the energy absorbed b y DNA) at a given fluence might differ in the two cell stocks as a result of differences in cell shape and consequent shielding o f the nucleus b y cytoplasmic nucleoproteins. To investigate this, we determined the yield of dimers produced in haploid and diploid cells exposed to a range of ultraviolet fluences (Fig. 5). Regression of dimer yield on fluence was n o t significantly different between haploids and diploids: both sets of data were pooled to yield a slope of 0.0026% dimers per increment of 1 Jm -2, a value comparable to that reported for Chinese hamster cells [50].

Photoreversal o f dimers If the photoreactivation o f survival in ICR 2A cells is due to the action of

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D N A photolyase, restoration of viability should be coordinate with loss of pyrimidine dimers from D N A . To test this, we investigated loss of thymine dimers from the D N A of prelabeled and irradiated cells exposed to the visible light source used in the survivalexperiments (Fig. 6). Cells exposed to 40 J m -2 and immediately illuminated at 25°C lost dimers from D N A in a linear fashion at the rate of 0.026% dimers per h. In our photoreactivation experiments, the average dose of 6 J m -2 produced killingthat required about 1 h of illumination to reverse (Fig. 4). At this dose, we would expect a dimer yield of about 0.025% (Fig. 5). If the rate of dimer removal after 6 J m -2 is comparable to that measured after 40 J m -2, we would expect I h of illumination to be required for complete dimer removal. Therefore, it would appear that photoreactivation and dimer removal occur at the same rates. Since loss of dimers or inhibition of dimer formation m a y be related to non-

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of time of exposure

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333 enzymatic effects in some biological systems [22], control experiments using 50 Jm -2 were carried o u t to test some o f these possibilities. Exposure to the visible source for 1 h prior to ultraviolet irradiation had no effect on dimer yield, suggesting that photoprotective effects are n o t important in frog cells. When irradiated cultures maintained at 2°C on crushed ice were illuminated for 3 h t h e y retained 80% of the initial dimer content, as would be expected if dimer removal required enzyme activity. When the irradiated cultures were illuminated for 3 h following suspension o f the cells in 5% trichloracetic acid, 95% of the initial dimer content was retained. As discussed below, there was little loss of dimers if intact cells were incubated in darkness. Loss of dimers is thus light~lependent, temperature-dependent and occurs only in DNA illuminated in physiologically intact cells. Therefore the photoreactivation we observe is attributable to enzyme activity and pyrimidine dimers appear to be the predominant lethal lesion in this system. Dimer persistence during incubation in the dark To test for the occurrence of excision repair, the dimer content of prelabeled, irradiated cultures was estimated after incubation in the dark at 25°C for periods up to 48 h. Table 3 summarizes results of several experiments; in no case did we observe the decline in dimer content observed in comparable experiments with excision-competent cells, e.g., human fibroblasts [41]. It has been reported that in mouse cells loss of dimers due to excision repair m a y be masked b y persistence o f dimer-containing TCA-precipitable polynucleotides, b u t is detectable if isolated nuclei are assayed [36]. To test for this effect, expts. 5 and 7 (Table 3) were run with washed nuclei, b u t no loss of dimers could be detected. Since large ultraviolet exposures were used to induce dimer contents high enough for analysis, these experiments might fail to detect a low and readily saturated level of excision activity. However, it can be inferred that the excision p a t h w a y does n o t provide a route o f dimer removal comparable to that induced b y photoreversal.

TABLE 3 DIMER CONTENT DURING INCUBATION IN THE DARK Expt.

UV fluence

D i m e r s (%)

(jm-2) 0 h 1 2 3 4 5 6 7

a a a b c b c

26 31 49 40 40 40 40

0.076 0.083 0.163 0.075 0.134 0.075 0.089

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12 h

0.092 0.155

24 h

48 h

0.070 0.092 0.163 0.057 0.110 0.076 0.091

0.076 0.123 0.078 0.086

334

Ultraviolet induction of unscheduled DNA synthesis Cells that carry out excision repair exhibit an ultraviolet-induced incorporation o f thymidine into non-S phase cells. This method of detecting excision has the advantage over dimer measurement that it avoids the problems of estimating small differences between large quantities. Fig. 7 shows histograms of the grain-count distribution in non-S frog cells after sham-irradiation and several levels of ultraviolet exposure. As noted by Viceps-Madore and Mezger-Freed [52] such distributions allow discrimination between S-phase cells with more than 100 grains per nucleus and non-S-phase cells with fewer than 50 grains per nucleus. Ultraviolet exposure induced an approximately 2-fold increase in the mean grain count, significant at P < 0.001. However, the values for 10, 20 and 40 Jm -2 were not significantly different from each other. As illustrated in Fig. 8, the elevation of grain count was not d o s e ~ e p e n d e n t , but appeared to be saturated at 10 Jm -2. In contrast, when excision-competent WI-38 human fibroblasts were evaluated by the same method, the ultraviolet-induced elevation in grain count was greater and was proportional to UV fluence at least up to 20 Jm -2. Unscheduled D N A synthesis in ICR 2A does not vary substantially as a function of time after irradiation. As shown in Fig. 9, ultraviolet induced approximately the same elevation of grain count if isotope was applied immediately following exposure or after a lapse of up to 12 h. The response remained essentially the same to 20 h (see Figs. 7 and 8). These observations leave open several possibilities. There may be a low level of excision activity in frog cells, readily saturated by small numbers of dimers;

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4

8

12

HOURS POST IRRADIATION F i g . 9 . M e a n g r a i n c o u n t s o v e r n o n - S - p h a s e n u c l e i o f h a p l o i d I C R 2 A cells as a f u n c t i o n o f t i m e at w h i c h isotope exposure occurred following irradiation. Open circles, controls; closed circles, following exposure to 20 Jm -2.

thymidine incorporation might reflect repair-replication or there may be repair of lesions other than pyrimidine dimers. Discussion

Survival curves and the effect of ploidy Comparisons of haploid and diploid survival after exposure to ionizing radiation or to UV and other mutagens frequently show a characteristic excess sensitivity of haploids, as in yeasts [29,32,42,54], insect embryos [8] or amphibian embryos [21]. The excess sensitivity of haploids probably cannot be simply equated to susceptibility to recessive lethals, since haploid insect embryos are more resistant that diploids if irradiated during cleavage [8] and scoring of radiation-induced dominant lethals shows the diploids to be twice as sensitive as haploids, as expected [33]. The form of survival curves may be primarily determined by saturation of repair capacity [6,24,42], so that the expectation in a haploid--diploid survival comparison may depend more strongly on physiological properties of the cells than on the number of genetic targets. Ploidy comparisons of survival after ionizing radiation in mammalian cell cultures have necessarily dealt with diploid and higher ploidies, and have produced a rather contradictory literature [5,14]. From the work with yeast cells, one would expect increasing sensitivity with increasing ploidy, reflecting the major influence of dominant lethals. However, in some experiments with mammalian cells tetraploids were found to be more resistant than diploids [2,3, 46] while in others survival was independent of chromosome numer [29,48]. To meet the objection that many of the cell strains compared in the experiments cited above were not isogenic, Burki and Carrano [5] used colchicine to derive a "tetraploid" strain of V-79 Chinese hamster cells and found this to yield a Do value smaller than that from the parental hyperdiploid line, an effect they attributed to a major contribution of chromosome aberrations to lethality. Chromosomal aberrations correlate well with measured survival after ionizing radiation in cell culture [13]. Using UV radiation, Rommelaere and Errera have reported cell-fusion hybrids to have the same UV-survival curve as the parental strains [43].

336

Haploid--diploid comparisons in ICR 2A permit a sensitive test for recessive lethality in cell culture, since we can control factors potentially confusing in ploidy studies with mammalian cells. First, the photoreversibility of UV-inactivation confirms that we are dealing primarily with effects on the genome. Second, the strains compared are likely to be isogenic, since diploid ICR 2A derivatives appear to arise only by defective cytokinesis [51] and b o t h haploid and diploid strains used here are stable and euploid by cytogenetic criteria [30]. Thus, differences in ultraviolet lethality in the two cell stocks should reflect the addition of one complete and redundant chromosome set. Our results suggest that recessive lethality is n o t a major determinant of UV inactivation over the dose range we have studied. In cells blocked in G1, differences in the survival-curve coefficients of haploids and diploids were less than the error in our methods, while in exponentially growing cells haploid and diploid mean lethal doses were indistinguishable, the only significant difference due to doubling of chromosome number being an increased shoulder at low fiuence (Table 2). In consequence of this broader shoulder on the diploid survival curve, one would expect somewhat lower survival among haploids than among diploids exposed to arbitrary doses of UV in mutagenesis experiments, a result we have consistently observed. Recessive lethality also appears to be a secondary factor in response of ICR 2A cells to other mutagens, e.g., acridine mustards and X-rays [30]; in the case of the acridine mustards non-genetic lethality appears to be important [52]. The mean lethal dose of ultraviolet for frog cells appears to be significantly smaller than that of mammalian cells [35]. Since our measurements of dimer induction show a relation between exposure and damage comparable to that in similarly exposed mammalian cells [50], internal screening effects can apparently be considered equal. Frog cells are thus probably more sensitive to inactivation b y a given extent of DNA damage than are mammalian cells. This might reflect the larger size of frog chromosomes: in haploid Rana pipiens cells, 8 pg of DNA is distributed in 13 chromosomes, of which the five largest may account for more than 60% of the total genome on the basis of length at metaphase [31].

Repair functions in ICR 2A The retention o f photoreactivation capacity in ICR 2A is expected since DNA photolyase activity has been demonstrated in many tissues o f amphibians [11] and the enzyme is of widespread occurrence in lower vertebrates [ 10]. A cultured cell line from Xenopus laevis was reported to have repair properties much like those we have found in ICR 2A [ 10,40]. While we have n o t assayed our cells directly for the DNA-photolyase enzyme, our control experiments make it unlikely that we are dealing with the interfering nomenzymatic effects that have been reported in other systems [22]. A notable feature of photoreversal in ICR 2A is its efficiency in the intact cell. Relatively brief exposures to visible light remove dimers completely and fully restore viability. This makes it possible to avoid deleterious effects of visible wavelengths on growth medium [34] or on the cells themselves [4]. Since the time required for photoreactivation is short, we can hold the cells in a non-nutrient salt solution, and thus avoid exposure to toxic p h o t o p r o d u c t s

337 formed in nutrient mixtures. Photoreactivation of marsupial cells, in contrast, appears to require damaging levels of exposure to visible light [27], or exposure to large fluences of long-wave ultraviolet [49]. Other mammalian cells are apparently incapable of photoreactivation of survival [22]. Excision repair does not seem to be a major pathway in ICR 2A. The results of our autoradiographic experiments and our observations on the stability of dimer content in irradiated cells suggest that frog cells resemble rodent cells, rather than human fibroblasts, in their capacity to remove dimers by this route. The Xenopus cell line investigated by Regan et al. [40] appears to have been similarly deficient in excision, since Cook could detect no loss of dimers during incubation in the dark [10]. The question that then arises is the nature of the repair implied by the shoulder on the survival curve for haploid cells irradiated in G1 (Fig. 2, Table 2). This could reflect the slow but long~ontinued excision repair suggested by autoradiography (Fig. 9) or could be the result of postreplication-repair pathways. Further experiments will be required to explore this; these may be aided by use of recently obtained derivatives of ICR 2A deficient in repair replication [44].

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