231

Mutation Research, 5 2 ( 1 9 7 8 ) 2 3 1 - - 2 4 5 © Elsevier/North-Holland

Biomedical Press

MODIFICATION OF UV-INDUCED MUTATION FREQUENCIES IN CHINESE HAMSTER CELLS BY DOSE FRACTIONATION, CYCLOHEXIMIDE AND CAFFEINE TREATMENTS *

C H I A - C H E N G C H A N G 1, S T E V E N M. D ' A M B R O S I O TROSKO I and R.B. SETLOW 2

2,** ROGER

SCHULTZ

1, J A M E S E.

1 Department of Human Development, College of Human Medicine, Michigan State University, E. Lansing, MI 48824 and 2 Department of Biology, Brookhaven National Laboratory, Upton, N Y 11973 (U.S.A.) (Received 13 January 1978) (Revision received 27 June 1978) (Accepted 29 June 1978)

Summary Chinese hamster (V79) cells were irradiated with a fractionated regime of ultraviolet light (UV1 + UV2). The fractionation of a UV dose always increased the colony-forming ability but reduced (or it did not change) the mutation frequencies. Treatment with cycloheximide between the two UV irradiations resulted in two types of effects, depending on the protocols used. Long exposures to cycloheximide (i.e., :>6 h) for the entire period between UVI and UV2 or partial treatment of cycloheximide {i.e., 3 h) long before UV2 always resulted in reduced colony-forming ability and enhanced or unchanged mutation frequencies. Exposure to cycloheximide for the entire period in the short fractionated regime (i.e., 4 h) between UVI and UV2 or partial treatment of cycloheximide just prior to UV2 tended to give the opposite effects. Caffeine treatment before UV2, with or without UV1, significantly increased the mutation frequencies. These results suggest that an error-free postreplication repair system exists in Chinese hamster cells which is inhibitable by particular cycloheximide or caffeine treatments.

* By acceptance of this article, the publisher a n d / o r recipient acknowledges the U.S. G o v e r n m e n t ' s right to retain a nonexclusive, royalty-free l i c e n s e in and t o a n y copyright covering this paper. R e s e a r c h carried o u t u n d e r the auspices of the United States D e p a r t m e n t o f E n e r g y , as well as grants to James E. Trosko from the National Cancer Institute (CA 13048-05 and CA 21104-01) and Chia-cheng Chang from National Institute of Environmental Health Sciences (ES01809-01). ** Present address: D e p a r t m e n t of Radiology, Ohio State University, Columbus, Ohio 4 3 2 1 0 (U.S.A.).

232 Because of a possible role of somatic mutations in the carcinogenic [2,8,20, 37], atherosclerotic [1], teratogenic [29] and ageing [7,34,36] processes, it is useful to characterize the molecular basis for mutagenesis. The role of DNArepair mechanisms in mutagenesis has been firmly established in Escherichia coli (21) and the concepts of "error-free" and "error-prone" DNA repair have been created to differentiate those repair processes which return damaged DNA to its original condition from those which may generate mutations. Radman [25] and Witkin [40] have postulated that an inducible post-replication repair system ("SOS"-repair) is responsible for mutagenesis in microbial systems. Subsequently, genetic [30,32,39,41] and biochemical [18,31] evidence for such an inducible repair system have been reported. In mammalian cells, excision [26] and postreplication repair [6,16,22,27,38, 39] mechanisms have been demonstrated. From the recent report of higher induced mutation frequencies per unit dose but similar frequencies per unit survival in fibroblast cells of the excision-repair deficient xeroderma pigmento~um syndrome (xp) compared to that of normal cells, Maher and McCormick [24] have inferred that the excision-repair mechanism in human cells is an "error-free" process. Fujiwara [15] provided evidence that mouse L cells possess the ability to by-pass photoproducts remaining unexcised in DNA. Higgins et al. [19] have postulated a model for the postreplication repair (replicative by-pass repair) which, on the surface of things, would provide an error-free mechanism to by-pass unexcised lesions in the template DNA of mammalian cells. Sarasin and Hanewalt [28], Lytle et al. [23] and DasGupta and Summers [11] have presented evidence related to ultraviolet light and chemical carcinogen-reactivation of herpes simplex virus and simian virus 40 in m o n k e y kidney cells. They have interpreted their data as being consistent with a hypothesis that an inducible recovery pathway exists in mammalian cells [11,23,28] and that it is mutagenic [11]. D'Ambrosio and Setlow [10], using a split-dose protocol, reported that an e n h a n c e m e n t of postreplication repair (as the result of an initial small dose) was inhibited when cells were incubated with cycloheximide between the two treatments. The experiments in the present study were designed to determine if an inhibition of enhanced postreplication-repair process led to modifications in the production of UV-induced ouabain resistant mutations in Chinese hamster cells. Materials and methods Cell line and tissue culture

A Chinese hamster cell line (V79), derived originally from lung tissue, was used for the experiments [14]. Cells were grown in modified Eagle's medium [12] supplemented with non-essential amino acids, 1 mM sodium pyruvate and 5% fetal calf serum for the mutagenesis experiments; or supplemented with 400 pg/ml L-glutamate, 140 units/ml of penicillin, 140 pg/ml of streptomycin and 10% fetal bovine serum in the postreplication-repair experiments. The potassium concentration of the medium is 5.36 mM. The selection medium containing 1 mM ouabain was prepared in batch quantities, stored and frozen until

233 use. Cells were incubated at 37°C in humid air in 9-cm plastic tissue culture dishes (Falcon Plastics).

Mutagenesis experiments Quantitative mutagenesis using ouabain-resistance as a genetic marker has been systematically characterized [5]. Cells were trypsinized and plated for attachment for 3.5 h before UV-irradiation. With the medium removed from the plates, the attached cells were exposed to ultraviolet light from a germicidal lamp {General Electric G25T8-25W) which was positioned to deliver a dose rate of 1 j/m2/sec (10 erg/mm2/sec). Growth medium with and without cycloheximide (or caffeine) was added to plates after the first UV-irradiation (UV~) at various times for various durations described in the protocols accompanying the tables. Medium was decanted prior to the second irradiation (UV2) and growth medium (10 ml) was added to each plate for various expression times until changed for selective medium with 1 mM ouabain. The resistant colonies that developed in selective medium were scored 1 week later. The numbers of cells seeded and plates used for each experiment are indicated in the tables. The survival plates were treated the same way as the mutation plates, except they were n o t exposed to selective medium. The percentage of survivors was determined by dividing the total number of colonies developed by the total number of cells plated, multiplied by 100.

Postreplication-repair experiments Approximately 6 × 10 s cells were plated in 5 ml of Eagle's minimal medium in 60 X 15 mm plastic dishes and incubated at 38°C in a 10% CO2 atmosphere. Parental DNA was labeled by incubation of cells for 15 h with 0.02 pCi of 2-['4C]thymidine (50 Ci/mol) per ml. 30 min before irradiation the m e d i u m was replaced with warm nonradioactive medium. Cells were irradiated after the medium was removed from the plates, with 254-nm radiation at a fluence rate of 0.36 j/m2/sec according to the experimental scheme shown in the figures. Cells were then incubated with or without 3 pg per ml cycloheximide (or 300 pg per ml caffeine) for the periods of time described in the tables and figure legends. The medium was removed just prior to the second UV-irradiation and fresh medium added after irradiation. Irradiated or unirradiated cells were then pulse-labeled with 2.4 and 0.6 pCi/ml of [methyl-3H]-thymidine ([aH]dT) (7 Ci/mmol), respectively for either 30 or 15 min to label approximately equal lengths of DNA. Cells were chased with unlabeled dT (4 pg/ml) for the period of time described in the table and figure legends. After an appropriate chase time, the cells were washed with an EDTA-containing NaC1 solution [33] and exposed to 2000 R (0.52 C/kg air) of X-rays to facilitate the unwinding and separation of DNA strands [13]. The cells were suspended in the same solution, and a b o u t 30 000 cells in 50 pl, containing 2000-cpm of each isotope were lysed in 0.25 ml of 1M NaOH, 0.01 M EDTA solution layered on top of 5.2 ml of a 5--20% (wt./vol.) alkaline sucrose (wt./ vol.) on the b o t t o m . Samples were centrifuged for 130 min at 30 000 rpm at 20 ° in a SW 50.1 rotor of a Beckman model L5-50 ultracentrifuge. Drops were collected onto paper strips and counted for radioactivity as previously described [3].

234 Results

Effect of UV-fractionation and cycloheximide treatment on postreplication repair Fig. I shows the alkaline sucrose gradient profiles of pulse-labeled DNA from cells irradiated with or without 3 J / m 2 of UV (UV~), incubated with or without cycloheximide for 6 h, and then irradiated with 17 J/m 2 of UV (UV2). As shown in this figure, the small dose of UV1 (3 J/m 2) significantly enhanced the amount of postreplication repair compared to that observed after irradiation with 17 J/m 2 alone. If cells were incubated with cycloheximide for the 6 h between the UV, and UV2 irradiations, enhancement of postreplication repair was n o t observed. Some of the pulse-chased DNA from cells incubated with cycloheximide between the two irradiations appeared to be smaller in size than pulse-chased DNA from cells irradiated with UV2 alone. A possible inhibition of the postreplication repair of DNA damaged by UV2 by cycloheximide seems improbable since: (a) there was only a small effect on the repair of UV2damaged DNA in cells incubated with cycloheximide 6 h before UV2 irradiation (Table 1); and, (b) the size of pulse-chased DNA from cells incubated with cycloheximide between UV,and UV2 irradiations was similar in size to pulse-

1

I

UV, CYC

[

I

I

CYCv 2[U

•

(~

SEDIMENT

,R

~t2

d''TPARDENTAL

0~10

I

i"/l

:

5

I0

15 FRACTION

I

1\ \/11

20

25

Fig. 1. S e d i m e n t a t i o n profiles o f D N A f r o m cells g i v e n f r a c t i o n a t e d d o s e s o f U V a n d c y c l o h e x i m i d e . Cells w e r e i r r a d i a t e d (X, e ) o r n o t i r r a d i a t e d (o) w i t h 3 J m -2 U V r a d i a t i o n a n d i n c u b a t e d w i t h (X) or w i t h o u t ( o , e ) 3 #g p e r m l c y c l o h e x i m i d e f o r 6 h. A f t e r r e m o v a l of c y c l o h e x i m i d e , cells w e r e w a s h e d t w i c e w i t h p r e w a r m e d m e d i u m a n d all p l a t e s w e r e i r r a d i a t e d w i t h 17 J m -2 U V r a d i a t i o n . 15 rain l a t e r , i r r a d i a t e d a n d u n l r r a d i a t e d cells w e r e p u l s e d w i t h 2.4 a n d 0.6 #Ci p e r m l [ 3 H ] d T h d f o r 3 0 a n d 15 rain r e s p e c t i v e l y , a n d c h a s e d in u n l a b e l e d m e d i u m f o r 1.7 h b e f o r e s e d i m e n t a t i o n in alkaline s u c r o s e as d e s c r i b e d u n d e r Methods. Cells p r e l a b e l e d w i t h [ 1 4 C ] d T h d (D) w e r e given U V 1 , c y c l o h e x i m i d e a n d U V 2.

235 TABLE 1 EFFECT

OF CYCLOHEXIMIDE

UPON POSTREPLICATION

REPAIR

Percentage o f pulse-labeled

Treatment a UV 1 . -. ---

Cyc 1 .

Cyc 2 .

.

+

UV 2

D N A s e d i m e n t i n g as h i g h molecular weight DNA b

+

40 35 8 6 17

.

+

+

+

-+ +

+

--

--

--

+

+

+

+

+

+

+

+

--

--

+

15

+

--

+

--

+

17

+

--

--

+

+

15

.

+

Cyc 3

.

a The protocol

.

8

for irradiating cells with UV and the time periods for incubation

of cells with 3 #g/ml

c y c l o h e x i m i d e are s h o w n i n F i g . 1. b H i g h - m o l e c u l a r - w e i g h t D N A w a s t a k e n as t h e c u m u l a t i v e f r a c t i o n s f r o m t h e b o t t o m gradients that included 5 0 % o f p a r e n t a l 1 4 C r a d i o a c t i v i t y .

of alkaline sucrose

chased DNA from cells given the sum of the doses, i.e., 20 J/m 2 (unpublished observation).

Effect o f a fractionated UV dose and cycloheximide treatment on colonyforming ability and mutagenesis The c y t o t o x i c effects of the concentration of cycloheximide used (5 pg/ml) on colony-forming ability of Chinese hamster cells are small as shown in Fig. 2. Treatment of the cells for 6 h reduced the colony-forming efficiency only to 92% of the control. The plating efficiency and colony sizes gradually reduced with increasing time of treatment. The cycloheximide (or caffeine) treatment also had no effect on the recovery of spontaneous oua r mutants (Table 7, No. 12; Table 8, Nos. 1--3). The results of the experiments designed to determine the effect of fractionating the UV dose and incubating the cells with cycloheximide between UV, and UV2 irradiations on the survival and frequency of UV-induced ouabain-resistant mutants are presented in Tables 2--6 and 8. In the first experim e n t (Table 2), a delay in mutation expression caused by cycloheximide treatm e n t was noted (Table 2, Nos. 7,8 versus Nos. 10,11). Therefore, it was necessary to use longer expression times for the experiments involving cycloheximide treatment. The UV, and UV2 doses were separated by either 4 h (Tables 2, 3 and 6) or 6 h (Tables 4, 5 and 6). In all these experiments, the fractionating of a UV dose always increased the colony-forming ability as compared to the same amount of a single dose of UV radiation. The mutation frequency among the survivors after irradiation with fractionation of the UV radiation was either reduced (Table 2, No. 6 versus No. 9; Table 5, No. 4 versus No. 5) or n o t changed (Table 3, No. 5 versus No. 7; Table 4, 6, No. 4 versus No. 5). Treatment of cycloheximide between two UV irradiations resulted in two different effects on survival and mutations, depending on the protocols used. Longer treatment (i.e., 12 h) for the entire period of time between UV, and UV2 resulted in reduced colony-forming ability b u t higher mutation frequen-

236 ioo, 9o 8o 70 60 6o 4o

~ 30

2o

0

4

8

12

16

20

24

28

Time (hr) Exposed to Cyclohexomide (5/~g/ml) Fig. 2. C o l o n y - f o r m i n g ability o f Chinese h a m s t e r V 7 9 cells e x p o s e d t o c y c l o h e x i m i d e (0.5 m g / m l ) f o r various duxations. TABLE

2

EFFECT OF CYCLOHEXIMIDE TREATMENT UV-INDUCED OUA r MUTATIONS

ON THE

FREQUENCY

OF

Protocol: .{

4 hrs.

UV1

~-48 UV2

hrs.--~.~-12 h r s . - - ~ - 1 2 hrs.--~ ~ 1 wk. ~-,,~

-+ Cycloheximide

UV1 Cycloheximide (3J/m2) (6/~g/ml)

I or

~f

~

Ouabain UV2 [ A = 17J/m21 Survival LB = 20J/m2J (%)

Score for Mutants

Expression Time (hr.)

Mutation Frequency/ 106 Survivors (No. of Mutants)

1

--

--

--

2 3

+ --

---

-+ (A)

87 85 9.8

48 48 48

58 ( 7 6 ) 110 (140) 440 (172)

4 5 6

--

--

+ (B)

4.5

48 60 72

650 (70) 770 (83) 1060 (105)

7 8 9

+

-

+ (A)

8.0

48 60 72

450 (86) 590 (105) 590 (113)

10 11 12

+

+

+ (A)

48 60 72

200 (60) 380 (114) 620 (168)

1-2 3

12

Number of cells per plate (9 cm) used was 1.5 X 106/30 Number of cells per plate (9 cm) used was 4.0 X 106/20

4, 5, 7-9, 11, 12 Number of cells per plate (9 cm) used was 2.4 X 106/12 6, 10, 13 Number of cells per plate (9 cm) used was 2.2 X 106/11

237

TABLE 3

EFFECT OF CYCLOHEXIMIDE TREATMENT ON THE FREQUENCY UV-INDUCED OUA r MUTATIONS Protocol: ,I UVl

4hrs.

> -~-- 72 hrs.--~ ~- 12 hrs. - ~ UV2 ~or~ ~" L~ ~ Ouabain

+ Cycloheximide

UV 2 f A = 17J/m 2-] Survival LB = 20J/m2/ (%)

UV1 Cycloheximide (3J/m2) (6/zg/ml)

OF

~lwk.

Score for Mutants Mutation Expression Frequency/ Time 106 Survivors (hr.) (No. of Mutants)

1

--

-

120

2 3

+ -

-

120

+ (A)

9.5

4

-

+ (B)

4.7

72 84

1070 950

(90) (8O)

+

+ (A)

8.5

72 84

800 980

(122) (150)

+

+ (A)

72 84

380 600

(106) (168)

5 6

7 8

16

9

72 72 72

5.0 (7) 20 (29) 810 (138)

1-2 Number of cells per plate (9 cm) used was 1.2 X 106/24 3 - 9 Number of cells per plate (9 Cm) used was 1.8 X 106/18

TABLE 4

EFFECT OF THE TIME OF CYCLOHEXIMIDE TREATMENT FREQUENCY OF UV-INDUCED OUA r MUTATIONS Protocol: 6hrs.

>~-65

UVl

hrs.--~,~-- 31 hrs.---~

ON THE

~ 1 wk. Score for Mutants

UV2

.. cyc.

-

3-1

cyc

Ouabain

_ -

3-2 cyc

-

3-3

;;;;;;,',';;;;;;

UV2 UV1 Cycloheximide [ ; = 17J/m2] Survival (3J/m2) (8 pg/ml) = 20J/m2J (%) 2

+

3

-

4

--

5

+

6 7 8

+ + +

-

i

cyc-- 3 - 1 cyc- 3-2 cyc-- 3--3

Expression Time (hr.)

Mutation Frequency/ 106 Survivors (No. of Mutants)

84

65

+ (A)

80 9.8

65 96

130 (111) 370 (76)

+ (B) + (A)

5.0 7.4

96 96

660 570

+ (A) + (A) + (A)

3.8 5.8 6.4

96 96 96

940 (74) 870 (106) 800 (107)

1 Number of cells per plate (9 cm) used was 1.2 X 106/24 2 Number of cells per plate (9 cm) used was 1.05 X 106/21 3--8 Number of cetls per plate (9 cm) used was 2.1 X 106/21

43

(43)

(69) (88)

238 TABLE 5 EFFECT

OF

FREQUENCY

THE T I M E OF C Y C L O H E X I M I D E T R E A T M E N T OF UV-INDUCED O U A r M U T A T I O N S

ON THE

Protocol:

,{

6 hrs.

> ~ - - 6 5 hrs. - - - ~ - - 3 1

UVl

hrs.

b

~ 1 wk,

UV2 cyc -

3-1

Score for Mutants

Ouabain

•c¥c - 3 - 2_ cyc - 3 - 3

,';;;;;/;/,"

.

.

.

.

.

.

.

.

?YC.'T J6

.

.

.

.

.

.

.

.

(hr.)

Mutation Frequency/ 106 Survivors (No. of Mutants)

+ (A)

83 79 10

65 65 96

72 (72) 150 (126) 320 (70)

+ (B) + (A)

5.3 10

96 96

850 (95) 510 (109)

+ + + +

5.1 6.4 10 8.3

96 96 96 96

990 970 760 910

UV 2

UV1 Cycloheximide (3J/m 2) (5/zg/ml

r A = 17J/m2] LB = 20J/m2J

1

-

-

-

2 3

+ -

-

4 5

+

--

6 7 8 9

+ + + +

cyc cyc cyc cyc

--

3-1 3-2 3-3 6

(A) (A) (A) (A)

Survival (%)

Expression Time

(106) (131) (165) (150)

1 Number of cells per plate (9 cm) used was 1.2 X 106/24 2

Number of cells per plate (9 cm) used was 1.05 X 106/21

3 - 8 Number of cells per plate (9 cm) used was 2.1 X 106/21 9

Number of cells per plate (9 cm) used was 2.0 X 106/20

cies among the survivors (Table 8, No. 4 versus No. 5). Shorter treatments (i.e., 4 h) for the entire period between UV1 and UV: gave the opposite effect; namely higher survival and lower mutation frequency (Table 3, No. 7 versus No. 9; Table 6, No. 5 versus No. 6). These t w o different effects appear to be separated b y a short period of time, since the 6-h treatment tended to give the opposite results (Table 5, No. 5 versus No. 9; Table 6, No. 7 versus No. 10) to those of the 4-h treatment. These two opposite effects were also found in partial treatments between the fractionated doses of UV. Treatment of cycloheximide for 3 h before the second UV dose always resulted in a reduced colony-forming ability b u t increased mutation frequencies (Table 4, 5, No. 5 versus No. 6; Table 6, No. 7 versus No. 8). This effect was diminished, (Table 4, 5, No. 5 versus Nos. 7 and 8) or even reversed (Table 6, No. 7 versus No. 9) when the cycloheximide treatment shifted closer to the second UV dose. Results from Table 8 (Nos. 7--10) indicate that these t w o different effects are independent of the first dose of UV.

Effect o f caffeine on enhanced repair and mutagenesis Experiments were carried o u t to determine if caffeine can mimic the effect of cycloheximide treatment between the fractionated UV doses. The results

TABLE 6

239

EFFECT OF C Y C L O H E X I M I D E T R E A T M E N T UV-INDUCED OUA r MUTATIONS

ON

THE F R E Q U E N C Y OF

Protocol:
,¢

UVl

6 hrs.

=--~--- 72 h r s . ~

UV 2

UV2

(1)

(2)

ouabain

cyc - 3 - 1

cyc - 3 - 2 caf - 3 - 2 . cyc ,V. 3 - 3 . caf - 3 - 3 cyc -

12

caf - 12 Mutation Frequency/

UV1 (3J/m2)

C y c l o h e x i m i d e (5/~g/ml) or Caffeine(1 m M )

UV2 (17J/m2)

-

Survival (%) 114

-

102 109

37 42 37

1

-

2 3

-

cyccaf-

4 5 6

+ + +

c y c - 12 caf-12

+ (2) +(2) + (2)

8.3 5.0 3.4

630 1190" 900*

(189) (215) (111)

7 8 9 10

-

cyc-3-1 cyc- 3-2 cyc--3-3

+ + + +

(1) (1) (1) (1)

7.2 5.0 5.7 7.6

620 960* 930* 850*

(161) (174) (189) (234)

11 12 13

-

caf - 3 - 1 caf-3-2 caf - 3 - 3

+ (1) + (1) + (1)

6.2 6.9 7.1

790* 790* 930*

(177) (197) (235)

12 12

-

106 Survivors (No. o f Mutants)

1--3

N u m b e r o f cells per plate (9 cm) used was 1.8 X 106/18

4-13

N u m b e r o f cells per plate (9 cm) used was 3.6 X 106/18 *

(76)

(78) (73)

M u t a t i o n frequencies o f c y c l o h e x i m i d e or caffeine treated cells compared to control cells at equivalent doses o f U V were highly significant, p < 1%.

TABLE 9 EFFECT OF CAFFEINE ON THE ENHANCEMENT Treatment a UV I

OF POSTREPLICATION

REPAIR

P e r c e n t a g e o f p u l s e - l a b e l e d D N A s e d i m e n t i n g as h i g h molecular weight DNA b Caffeine

UV 2

(h) --

--

--

31

--

6

--

32

--

0

+

1.6

+

0

+

6.5

+

2

+

3.4

+

4

+

2.0

+

6

+

0.5

a S a m e p r o t o c o l as in Fig. 1 e x c e p t cells w e r e c h a s e d f o r I h . b S e e T a b l e 1.

241

indicate that 6- or 12-h caffeine treatments for the entire period between the two UV irradiations reduced the colony-forming ability b u t increased the frequency of mutations (Table 7, No. 7 versus No. 10; Table 8, No. 4 versus No. 6). These results are similar to the cycloheximide treatment. This effect was independent of the first UV dose (Table 7, No. 3 versus No. 6). The results also showed that caffeine pretreatment or partial treatment between two split doses of UV significantly increased the frequency of mutations but it did n o t necessarily reduce the colony-forming ability as was observed when caffeine is given after UV treatment [4]. As shown in Table 9, caffeine, when present for the 6 h between UV, and UV2, completely inhibited the enhancement of postreplication repair by UV fractionation. If caffeine was added immediately after UV, and incubated with the cells for either 2 or 4 h enhanced repair was inhibited 48 and 62% respectively. Discussion Our postreplication repair data show that irradiating cells with an initial small dose of UV, 4--6 h before a larger UV dose, significantly increases the rate by which small DNA, found immediately after UV-irradiation, is converted into high molecular weight DNA. The presence of UV-damaged DNA for at least 1.5 h at any time before UV2 irradiation appears to be sufficient to enhance the rate of postreplication repair and that this enhancement is prevented by cycloheximide treatment. Caffeine present for 6 h between the UV~ and UV2 irradiations inhibits the enhanced rate of repair. Like the effect found with cells incubated for various periods of time with cycloheximide, caffeine must be present the entire period between UV, and UV2. Enhanced repair is inhibited to lesser amounts if cells are incubated with caffeine for only 2 or 4 h before UV2 probably due to an incomplete removal of caffeine from the cells by washing. However, unlike cycloheximide, caffeine does not appear to inhibit protein synthesis i.e., the incorporation of ['4C]-leucine into trichloroacetic acid precipitable material (unpublished observation). Following the observation of D'Ambrosio and Setlow [10] and results presented here that postreplication repair was enhanced by a fractionated dose protocol and that cycloheximide inhibited that enhancement when present between the t w o UV treatments, experiments were designed to determine whether this enhance repair postreplication is correlated with the biological effect b y modifying mutation frequencies. If this enhanced repair postreplication is error-prone in Chinese hamster cells, a fractionated dose protocol similar to that used by D'Ambrosio and Setlow [10] is expected to give higher mutation frequencies which may be abolished by a cycloheximide treatment between the two UV irradiations. The results presented here show that when a small dose of UV precedes a major dose of UV irradiation, the mutagenic effect of UV was reduced or n o t changed while the colony-forming ability was increased compared to a non-fractionated dose. Excluding other possible complications of the protocol which will be discussed later, these results do n o t provide supporting evidence to indicate that enhanced postreplication is error-prone. The phenomenon that we observed could be viewed as a result of dose reduction because of

242 the excision repair of UV, with little or no biological consequence, or of better error-free postreplication repair induced b y UV~, or both. Since Chinese hamster cells excise less than 10% of the dimers produced by UV1 within the 6-h period of our experiments, the contribution due to excision repair is very small. The results from the cycloheximide treatments gave two different effects, depending on the protocols used. The shorter treatment (i.e., 4 h) or treatment immediately before the second b u t major UV irradiation increased the survival b u t reduced the frequency of mutations. The longer treatment (>6 h) or treatment long before UV2, on the other hand, tended to reduce the survival but increase the frequency of mutations. The results from the shorter cycloheximide treatment (i.e., reduced mutation frequency) would be consistent with an inducible error-prone repair system hypothesis; however, the results from the longer cycloheximide treatment (i.e., increased mutation frequency) contradicts this hypothesis. Moreover, the effect of the short cycloheximide treatm e n t seems to promote, rather than inhibit, a repair process, since the reduced mutation frequency was accompanied by an increase in survival. An alternative explanation for the observation that a short treatment of cycloheximide between the UV radiations enhanced the colony-forming ability is readily available, e.g., cycloheximide treatment can delay cell replication and DNA synthesis [17,35], which would provide a longer period of time for errorfree excision repair. Our results from both dose fractionation and cycloheximide treatments, therefore, do n o t provide evidence for the existence of an inducible error-prone repair system in Chinese hamster cells. The two different effects of cycloheximide treatments on cell survival and mutation mentioned previously are of significance. While the mechanisms for the two opposite effects are n o t clear at this stage, the observations point to the dual effects of cycloheximide, one promoting an error-free repair system {i.e., causing the observed reduction in mutation frequency), the other promoting an error-prone repair system (i.e., causing the observed increase in mutation frequency). Without introducing the concept of an inducible error-prone repair system, a simple explanation may be provided as follows. A long cycloheximide treatment may inhibit the synthesis of error-free repair enzymes and cause the decrease in repair and survival, and the increase in mutations by shifting to a constitutive error-prone repair system. The short cycloheximide treatment, as mentioned previously, may cause a delay in cell replication which allows more time for the cell to repair DNA damage b y an error-free excisionrepair mechanism. The two different effects may be attributable to two distinctive effects of cycloheximide, i.e., inhibition of protein and DNA synthesis. It has been shown that the effect of cycloheximide on DNA synthesis was independent of its effect on protein synthesis [35]. Furthermore, Cooney and Bradley [9] have reported that inhibition of DNA synthesis began immediately after addition of 2.5 pg/ml of cycloheximide to cells of Tetrahymena pyriformis, b u t inhibition of protein synthesis was n o t evident until several hours later. The observation is consistent with our explanation that shorter treatment with cycloheximide delays cell replication and may provide more time for error-free repair, whereas longer treatment with cycloheximide affects the synthesis of enzymes involved in error-free repair. Caffeine has been shown to have two different effects on UV-induced

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ouabain-resistant mutations [4]: (a) an enhancement in mutation frequency, when caffeine is given immediately after UV irradiation [inhibiting error-free postreplication repair]; and (b) a reduction in mutation frequency when caffeine is given after the completion of DNA repair [inhibiting mutation expression]. The results from the experiments presented here show that caffeine pretreatment or t r e a t m e n t between the fractionated UV doses always increases the mutation frequency. The results are similar to the enhancing effect of cycloheximide treatment. Since caffeine did not affect excision repair in Chinese hamster cells, the results indicate that caffeine pretreatment may inhibit an error-free postreplication repair system, while allowing error-prone repair to occur.

From the DNA postreplication studies, it was shown that treatment of cycloheximide or caffeine for the whole period of time (6 h) between the fractionated doses significantly inhibited the postreplication repair. The same protocol, on the other hand, significantly increased the mutation frequency. If this observed biological effect is related to the molecular event, we may conclude that this postreplication repair, inhibitable by cycloheximide and caffeine, is an error-free mechanism. This conclusion, however, depends on two assumptions: (a) the postreplication repair measured shortly after the UV2 treatment is the same repair mechanism(s) responsible for the biological effect expressed after a much longer period of time; {b) the rejoining of DNA fragments after UV treatments, that is measured, is related to a repair system, not to the normal DNA synthetic mechanism. Since neither assumption can be substantiated at present, any speculation on the correlation between the biochemical and biological effects is quite tenuous. The biological data, however, are consistent with the existence of an error-free repair mechanism which may be enhanced by dose fractionation and inhibitable by specific type of cycloheximide treatment. The results from radiation or carcinogen reactivation of damaged viruses [11, 23,28] seems to indicate the excistence of an inducible recovery pathway, although other alternative explanations are possible [11]. The discrepancy between the results o f those experiments and the experiments reported here could be due to the use of different cell lines or different protocols. It is noted that m a x i m u m reactivation of damaged viruses occurs when the time interval between host cell UV-irradiation and virus infection is increased by a few days. Since it is technically impossible for us to use a long delay between the UV1 and UV2 doses (i.e., the initial damaged cell will have divided several times before the second UV exposure), our protocol would not have detected such a long delayed induction. It is interesting to note, although it might be quite fortuitous, that mutation enhancement by caffeine treatment also requires a long interval of caffeine treatment following UV-irradiation [4], a p h e n o m e n o n consistent with the notion of delayed induction of error-prone repair enzymes. Phenomenological studies such as these are extremely difficult to interpret because the use of drugs such as caffeine and cycloheximide have multiple effects on the biochemistry of cells, which could interfere with several competing mechanisms related to cell survival. These results clearly point to the necessity of using specific mutants of mammalian cells in order to characterize the nature o f the repair process.

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