Inhibition of repair of UV-damaged DNA by caffeine and mutation induction in Chinese hamster cells

Chem.-Biol. Interactions, 6 (1973) 317-332 (C-) Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands

317

INHIBITION OF REPAIR OF U V - D A M A G E D DNA BY C A F F E I N E A N D M U T A T I O N I N D U C T I O N IN CHINESE HAMSTER CELLS

JAMES E. TROSKO AND ERNEST H. Y. CHU

Department of Human Development, Michigan State University, East Lansing, Mich. 48823 and Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Mich. 48104 (U.S.A.) (Received October 20th, 1972) (Revision received, January 15th, 1973) (Accepted February 1st, 1973)

SUMMARY

The effect of caffeine on UV-irradiated Chinese hamster cells in vitro was studied on the cellular and molecular levels. Caffeine (1 raM) was shown to decrease the colony-forming ability and the frequencies of spontaneous and UV-induced mutations in Chinese hamster cells. The effect of caffeine in reducing the frequency of UV-induced mutations was demonstrated only if caffeine was present in the culture medium during the first post-irradiation cell division. Using alkaline sucrose gradient centrifugation, both parental and newly synthesized DNA in UV-irradiated and unirradiated cells were studied in the presence and absence of caffeine. Caffeine affected the sedimentation profile of DNA synthesized in UV-irradiated cells but not in unirradiated cells. Caffeine had no apparent effect on the incorporation of [3H]thymidine into DNA of control or UV-irradiated cells, nor on the small amount of excision of UV-induced pyrimidine dimers. These results may be interpreted by a hypothesis that caffeine inhibits a certain S-phase specific, post-replication, darkrepair mechanism. The hamster and perhaps other rodent ceils exposed to low doses of UV are capable of DNA replication, by-passing the non-excised pyrimidine dimers. This postulated repair process probably involves de novo DNA synthesis to seal the gaps in the nascent strand. This repair may be also responsible for the enzymatic production of mutations.

INTRODUCTION

Biological damage in certain strains of bacterial and mammalian cells, induced by UV or alkylating agents, can be potentiated by post-treatment with caffeine or Abbreviation: TCA, trichloroacetic acid.

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J . E . TROSKO, E. H. Y. CHU

caffeine derivatives. Post-treatment with caffeine of UV-irradiated bacteria l,z or mammalian cells 3-7 decreases the colony-forming ability, although this phenomenon was not observed in HeLa cells 8 or in a different line of Chinese hamster cells 9. It has also been demonstrated in mammalian cells that caffeine enhances the lethal effects of alkylating agents 1° and Mitomycin C 11. Caffeine and its analogues have been shown to increase the frequency of UVinduced mutations in bacteria, ~.12-14 but have an opposite effect in excision-defective (Hcr-) strains, causing a significant diminution o f U V mutagenesis 15. Caffeine reduced recombination in Escherichia coil IS and Schizosaccharomyces pombe 16. Host cell reactivation is also inhibited in TI phage by caffeine 17. Caffeine alone induces chromosome aberrations in mammalian cells t8-21 and potentiates the chromosome damage by various other agents 22, but caffeine by itself appears to be ineffective in the induction of auxotrophic mutants in Chinese hamster cells 2°. The UV-induced mutation frequencies in Chinese hamster cells can be reduced by post-treatment with caffeine 7. On the other hand, ARLETT AND HARCOURT 23 showed that, while caffeine reduces the spontaneous mutation frequency, it increases the frequency of UV-induced mutations in the same line of Chinese hamster cells. Caffeine appears to be non-synergistic with alkylating agents in the production of dominant lethals in mice 2*.2s. Caffeine was shown to be effective in rendering cytoxan-resistant plasmacytomas sensitive to the action of cytoxan, nitrogen mustard and X-rays 26. On the molecular level, caffeine is known to affect membrane permeability 27 and phosphodiesterase activity 28. Caffeine has also been shown to interact with UV-irradiated DNA 29. The molecular mechanism by which caffeine sensitizes bacteria to UV light' has been attributed to the inhibition of the excision of pyrimidine dimers30-a3. However, caffeine does not appear to inhibit the excision of UV-induced dimers in human cells 34 or inhibit unscheduled DNA synthesis in Chinese hamster cells 35. It is clear that in a variety of organisms caffeine has profound biological effects. Especially interesting is its influence on the repair of DNA damage by UV and chemicals. There are, however, considerable differences and even conflicting results obtained from bacteria and mammalian cells and among mammalian cells of different origin. The Chinese hamster cells in tissue culture are particularly suited for UV irradiation, chemical analysis of DNA and quantitative studies of cell inactivation and mutation induction. In addition, the hamster cells appear to be inefficient in removing the UV-induced pyrimidine dimers *a and yet are relatively resistant to UV. It is possible that a repair mechanism other than excision repair might be operating in these rodent cells. The experiments in this report were designed to throw some light on the mechanisms of DNA repair and ultraviolet mutagenesis in these cells. MATERIALS AND METHODS

Tissue culture An aneuploid clonai cell line (V79-4), originally derived from the lung tissue

EFFECT OF CAFFEINE ON HAMSTER CELLS

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319

of a male Chinese hamster was used for these experiments. The V79-4 cells were grown in Eagle's minimum essential medium, supplemented with 15~o fetal calf serum. The origin, cytology and growth of this cell line have been previously described 36.

Alkaline sucrose gradients To determine if caffeine might interfere with the synthesis of new D N A (i.e., preventing small fragments from being linked together), the relative molecular weight profiles of D N A molecules, synthesized in the presence or absence of caffeine, were determined by a modified alkaline sucrose gradient technique described by MCGRATH AND WILLIAMS37'38. V79-4 cells were incubated at 37 ° in [3H]thymidine (3/zCi/ml, 20 Ci/mM, New England Nuclear), with or without 1 m M caffeine for 10 h after UV treatment. The 3.6 ml gradients of 5 - 2 0 ~ sucrose contained 0.3 M N a O H , 0.5 M NaCI and 0.01 M EDTA. At the bottom was a cushion of 0.2 ml of 60~o alkaline sucrose. At the top was 0.2 ml of 1 N N a O H into which approximately 3-12 • 103 ceils were gently layered. After remaining at 20 ° for 1 h, the samples were spun for 90 min at 30 000 rev./min in a Beckman SW-56 rotor. The bottom of each centrifuge tube was punctured and 29 _+. I fractions were collected on filter paper discs. The discs were washed with cold 5 ~o TCA and then with ethyl alcohol. After drying, the discs were counted in a scintillation spectrometer. To determine if caffeine interfered with the repair of UV-damaged pre-labelled DNA, cells were labelled with [3H]thymidine for 24 h. The medium was decanted and non-radioactive medium was added several hours before UV-irradiation. After irradiation, non-radioactive medium, with or without 1 m M caffeine, was added to each petri dish and the cells were incubated for another 6 h. Cells were then collected and placed on a gradient as described above. DNA synthesis assay To determine if caffeine treatment affected D N A synthesis (incorporation of [3H]thymidine into TCA-insoluble material), the method described by BOLLUM39 as modified by REGAN AND CHU 4° was used. 8 • 105 cells were inoculated into each 60-mm petri dish and incubated for one day before assay. The old medium was decanted and new media containing [3H]thymidine (3/~Ci/ml, 6.7 Ci/mM), with or without 1 m M caffeine were added. At various times after exposure of cells to [3H]thymidine or [3H]thymidine plus caffeine, duplicate dishes for each treatment were sonicated with a Sonifier (Heat Systems Company, Melville, N.Y.) for 10 sec. Aliquots of 100/d of the sonicate were placed on 3-mm Whatman paper discs for scintillation counting. U V irradiation The UV light irradiation was performed with two 15-W germicidal lamps with output predominantly at 254 nm. The incident dose rate to the cells was adjusted for each experiment. Before irradiation, the medium was decanted from the petri dishes, the edge of the monolayer of cells was scraped with a rubber policeman and the cells

320

J. E. TROSKO, E. H. Y. CHU

were washed twice with Hanks' balanced saline. The cells were irradiated in 0.5 ml of Hanks' saline.

Pyrimidine dimer assay V79-4 cells were labelled for 24 h in [3H]thymidine (10/zCi/ml, 20 Ci/mM). The radioactive medium was decanted 2 h before irradiation and non-radioactive medium was added. To determine if caffeine interfered with the repair of UV-induced pyrimidine dimers, 3 ml of medium, with or without I m M caffeine, were added to appropriate cell cultures immediately after the irradiation. Cells were harvested either immediately after irradiation or after 24 h post-irradiation incubation in the dark. Nuclei were isolated by treating cells with 0.001 M MgCI2-0.01 M Tris buffer, pH 7.5, for 15 min. Cells were spun down and, after decanting the buffer, resuspended and vigorously mixed with 0.1 ~ Nonidet P-40 detergent in the MgCl2-Tris buffer 41. Nuclei were spun down in a clinical centrifuge and washed in cold MgCl2-Tris buffer without the detergent. Nuclei were fixed in cold 5 ~ TCA and the insoluble residue was analyzed for pyrimidine dimers by two-dimensional chromatography after hydrolysis in formic acid 42.

Mutation assay Determination for mutation frequency was done according to the procedure of CHU AND MALLING 43. Forward mutations from azaguanine sensitivity to resistance, reflecting a reduction or loss in the activity of hypoxanthine guanine phosphoribosyl transferase, were studied. Azaguanine resistance behaves like a recessive character in somatic cell hybrids 36. As in man, this locus is likely to be X-linked in the Chinese hamster 44. Immediately after UV exposure, the treated and untreated cells were trypsinized, counted and plated in standard medium in which they were able to divide. Caffeine (1 m M ) was added to the medium for the desired period, as indicated in each experiment. Cytotoxicity was measured by plating 100 cells per 100-mm petri dish, 5 dishes per treatment. After incubation for 6-8 days, the dishes were fixed and stained and the colonies were counted. Cytotoxicity was expressed as the p~'rcentage of the number of colonies in the treated series as compared to the untreated controls. For mutation assay, 1.0-1.5 • l0 s cells per dish were plated. Usually, 10 to 20 dishes were given each treatment. At 42 h after UV irradiation, 8-azaguanine (at a final concentration of 30/~g/ml) was added. The dishes were incubated for 12-16 days and then fixed, stained and the colonies counted. Test of statistical significance of mutation frequencies was done using the method of KASTENBAUMAND BOWMAN45. RESULTS

Caffeine modification of U V-induced mutation frequency The effect of caffeine on cell survival and UV-induced mutations, given as a post-treatment to UV-irradiated V79-4 cells, is illustrated in Table 1. These data are taken from one of the four experiments which were described in a previous report 7,

321

EFFECT OF CAFFEINE ON HAMSTER CELLS in vitro TABLE I

THE EFFECT OF CAFFEINE ON THE FREQUENCY OF U V - I N D U C E D MUTATIONS FROM 8-AZA(;LIANINE SENSITIVITY TO RESISTANCE IN CHINESE HAMSTER CELLS i n v i t r o a

U V dose (erg/mm 2) 0

~ Survival . . No caffeine 100

Mutation freq./105 survivors .

.

. Caffeine 129.1

50 65

126.8 86.2

8.4 4.9

80 100 150

67.4 36.2 7. I

6.7 1.6 0.09

No caffeine

Caffeine

0.17 (5/30" 105) b

0.02 ( I / 3 0 . 105 )

14.77 (382/25.9' 105 )

1.43 ( 2 / I . 5 - 105 )

a Cells in monolayers either were irradiated or were not irradiated with various UV doses, trypsinized and inoculated at different densities to petri dishes. Caffeine (1 raM) was added immediately after inoculation of cells until m u t a n t colonies were scored (approx. 200 h later). The concentration of 8-azaguanine in the medium was 30 ~ug/ml. b N u m b e r of m u t a n t colonies per total n u m b e r of viable cells.

but wrongly calculated. The errors in calculation have been corrected. Caffeine alone at the concentration used generally reduces cell survival by about 10% (Table Ili). In this particular experiment, the apparent increase in cell survival in caffeine-treated cells was probably due to experimental error. Caffeine acts together with UV to decrease the colony-forming ability of the UV-irradiated cells. UV significantly increases the frequency of forward mutations from 8-azaguanine sensitivity to resistance (P -<_ 0.01). Incubation of the UV-irradiated cell population in the presence of caffeine for the entire post-irradiation period (about 200 h) significantly reduces the mutation frequency (P _< 0.05). The spontaneous mutation frequency is not significantly lowered by the caffeine treatment. DOMON AND RAUTH 5'6 have shown that caffeine must be present during the first DNA synthesis period of the cells after UV-irradiation in order to potentiate the damage. The experiment in Table II was designed to discover if caffeine is effective only in the same post-irradiation period in reducing the frequency of UV-induced mutations. The average generation time of the V79-4 cells was 12 h. Taking into account the possible mitotic delay due to experimental manipulations, by 18 h after UV-irradiation most cells should have completed one division 43. Accordingly, the same concentration of caffeine was included in the growth medium either during the first cell division after UV-irradiation (0-18 h), during the subsequent two or three divisions (18--42 h), or during the entire post-irradiation incubation period (0-200 h). In order to permit phenotypic expression of induced mutants, 8-azaguanine, at a concentration of 30/~g/ml, was added to all dishes for mutation assay at 42 h after the UV exposure 43. The results in Table II demonstrate that, in order to reduce the frequency of UV-induced mutations, caffeine must be present during

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J. E. TROSKO, E. H. Y. CHU

T A B L E II EIrFECT OF CAFFEINE DURING DIFFERENT PERIODS AFTER U V - I R R A D I A T I O N ON THE INDUCTION OF MU" TATIONS FROM 8-AZAGUANINE SENSITIVITY TO RESISTANCE IN CHINESE HAMSTER CELLS a

UV dose (erg/mm 2)

Caffeine

% Survival

Number of mutant colonies

Total number of viable cells (" 105)

Mutation freq./lO 5 survivors

0 0 0 0

0 0-18 18-42 0 - ~ 200

100 72.3 86.5 46.7 b

25 25 29 23

8 6.5 7.8 3.7

3.13 3.86 3.72 6.17

65 65 65 65

0 0-18 18-42 0 - ~ 200

46.6 1.6 i.7 0

113 0 5 0

3.7 0.1 0.2 ?

30.32 0 29.41 0

a Cells were plated 4 h before the 0 h for attachment. The cells were then rinsed with Hanks' balanced saline and were either untreated or treated with UV and/or caffeine. The UV dose rate was 10 erg/ mm2/sec. Caffeine concentration was 1 raM. The concentration of 8-azaguanine was 30/ag/ml, added at 42 h to all plates for mutation assay. The plating efficiency of the untreated cells in this experiment was 93.1%. b In three other repeat experiments, the cell survival after the prolonged caffeine treatment was 61.9%, 73.5% and 71.5% respectively and did not differ appreciably from those in 0-18-h and 18-42-h series.

TABLE III THE EFFECT OF CAFFEINE (1 m M )

(agg t)

ON COLONY FORMATION OF 8-AZAGUANINE SENSITIVE (azg s) AND RESISTANT

CHINESE HAMSTER CELLS GROWN

in vitro IN

DIFFERENT MEDIA

Part A: Normal medium Number and type of cells plated

Without caffeine

With caffeine

Plating efficiency

% Survival

Plating efficiency

% Survival

100 azg~

112.6

100

100.6

89.3

100 azg r

105.4

100

95.4

90.5

Part B: Normal medium containing 30 #g/1 8-azaguanine Number and type o f cells plated

Without caffeine Mutation freq. per 105 survivors

A verage number of colonies per plate

89.8

--

79.0

4 • 10 4 azg"

103.9

--

89.4

--

1 • 10 5 azg S

19.0

3.1

3.5

100 azg r 1 • lO s azg s

1O0

Average number of colonies per plate

With caffeine Mutation freq. per l0 s survivors

azg r 19.0

EFFECT OF CAFFEINE ON HAMSTER CELLS

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323

the first cell division after irradiation. This result has been confirmed in three further experiments. The question then arises whether 8-azaguanine-resistant mutants in the hamster cells might be selected against in the presence of caffeine. A reconstruction experiment was performed in which the parental V79-4 cells and a previously isolated clone of 8-azaguanine-resistant mutant cells were plated either alone or in mixtures in the presence or absence of caffeine. The results are summarized in Table III. From Part A of the table, it can be seen that both the parental and mutant cells plated at a high efficiency in normal medium. In the presence of caffeine at the concentration used both types of cells lost about 1 0 ~ in colony-forming ability. This reduction in cell survival appears to be independent of the cell's resistance to 8-azaguanine. In Part B of the experiment, the frequency of pre-existing, spontaneous 8-azaguanine-resistant mutants was found to be 19 per l0 s cells at the time of the experiment. This frequency was reduced to 3.5 per 105 survivors when caffeine was added to the medium. When 8-azaguanine-sensitive and -resistant cells were grown in mixed cultures in the presence of 8-azaguanine, the number of colonies that survived would include the pre-existing resistant mutants in the parental cell population as well as the fraction of the 100 8-azaguanine-resistant mutant cells added to the mixture. The results of this experiment clearly demonstrate that under the experimental conditions 8-azaguanineresistant mutant cells were not preferentially selected against in the presence of caffeine. As already shown in Part A, the slight reduction in cell survival in the presence of caffeine is true for both 8-azaguanine-sensitive and -resistant hamster cells. This experiment incidentally also shows the phenomenon of metabolic cooperation. In a more crowded culture (105 vs. 4 • 104 azg s cells), the number of resistant colonies was somewhat reduced due to possible cross-feeding of mutant cells by the parental ceils 46.

Caffeine synergism in the sedimentation profiles of parental and newly synthesized DNA of UV-irradiated 1/79-4 cells To see what effect caffeine might have on the sedimentation profiles of parental D N A of UV-irradiated V79-4 cells, the cells were prelabelled with [3H]thymidine for 24 h, irradiated with UV (75 erg/mm 2) and incubated for an additional 6 h with or without caffeine. The results are shown in Fig. I. Because of the possibility of not detecting a significant shift in sedimentation profile that might be due to caffeine inhibiting the first step of excision repair, other groups of ceils were irradiated with 400 erg/mm z and treated as before. Results are shown in Fig. 2. In both instances, no detectable shift in sedimentation profiles was noted. To determine if caffeine might affect the sedimentation profiles of the newly synthesized D N A in UV-irradiated ceils, cells were irradiated with 80 erg/mm 2 of 254 nm light and then labelled with [aH]thymidine for 10 h in the presence or absence of caffeine. The results are shown in Fig. 3. It can be seen that D N A synthesized in UV-irradiated cells in the presence of caffeine had a broadened sedimentation profile with a peak at a lower molecular weight than the D N A of control or UV-irradiated cells.

324

J. E. TROSKO, E. H. Y. C H U

m~

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10

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Fig. 2

Fig. 1. Sedimentation profiles of D N A from control and UV-irradiated Chinese hamster cells prelabelled with [3H]thymidine. Cells were exposed to 75 erg/mm 2 of 254 nm UV light and then incubated for 6 h in the presence or absence of I m M caffeine. The number on each figure represents the total radioactivity on each gradient. Conditions of centrifugation: 30 000 rev./min for 1.5 h at 20 °.

Fig. 2. Sedimentation profiles of DNA from control and UV-irradiated Chinese hamster cells prelabelled with [aH]thymidine. Cells were exposed to 400 erg/mm 2 of 254 nm UV light and then incubated for 6 h in the presence or absence of 1 mM caffeine. The number on each figure represents the total radioactivity on each gradient. Conditions ofcentrifugation : 30 000 rev./min for 1.5 h at 20°.

The effect of caffeine on the incorporation of [3 H]thymidine in U V-irradiated cells To determine if decreased colony-forming ability, decreased mutation frequency and sedimentation profile shift were related to the incorporation of [3H]thymidine in caffeine-treated UV-irradiated cells, the amount of TCA-precipitable material was measured under the same conditions as in experiments for mutation induction (Table l) and sedimentation profile determinations (Fig. 3). The results are shown in Fig. 4. Caffeine had only a slight inhibitory effect on the incorporation o f [aH]thymidine in control cells after 6 h. There appeared to be no synergistic effect o f caffeine on the incorporation o f [3H]thymidine by UV-irradiated cells.

325

EFFECT OF CAFFEINE ON HAMSTER CELLS ill vitro

9941 ~ lO

10

U V + CAFFEINE

8

i 10

1

1 20

1

i

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10

30

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20

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30

r- 11 OlO

8O48

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CONTROL

10

~

6

5

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m SEDIMENTATION I I I

,~,

10 FRACTION NUMBER

,

,

,

20

,

,

30

Fig. 3. Sedimentation profiles o f D N A from control a n d UV-irradiated Chinese h a m s t e r cells. Cells were exposed to 80 erR/ram 2 o f 254 n m UV light and then incubated for 10 h in [3H]thymidine m e d i u m , with or without I m M caffeine. T h e n u m b e r on each figure represents the total radioactivity on each gradient. Conditions o f centrifugation: 30 000 rev./min for 1.5 h at 20 °.

T A B L E IV T H E E F F E C T OF C A F F E I N E O N T H E EXCISION OF U V - I N D U C E D

V79-4

P Y R I M I D I N E D I M E R S IN C H I N E S E H A M S T E R

CELLS .d~

A

Treatment

Counts in X T a c~unts in T

~ XT/T

UV

734/2 237 100

0.032

UV -t- dark incubation

348/1 375 500

0.025

UV + dark incubation + ! m M caffeine

221/1 062 900

0.020

= Cells were irradiated with 100 e r g / m m 2 254 n m U V light at a dose rate o f 20 erg/mm2/sec. X T A

refers to both T T a n d U T dimers, since c h r o m a t o g r a p h y procedures used here do n o t separate t h e m from each other.

326

J. E. TROSKO, E. H. Y. CHU

CONTROL 40

CAFFEINE

30

UV

UV + CAFFEINE

lO 5 I

I

2 4 HOURS IN ~H3 THYMIDINE

I

6

Fig. 4. Effect o f 254 nm U V irradiation and caffeine on the incorporation o f [3H]thymidine into TCA-insoluble material. Bars on the 6 h samples denote the standard errors o f the mean and a 95 ~o confidence interval for four cultures at each treatment. Cells were exposed to UV at 100 e r g / m m 2 and a dose rate of 20 e r g / m m 2 /see.

Effect of caffeine on the excision oJ UV-induced pyrimidine dimers It has been previously reported that Chinese hamster and mouse L-cells do not appear to excise UV-induced pyrimidine dimers as in the bacteria 47- 52. Using a more sensitive technique to measure D N A repair, SETLOW et al. 5a reported that Chinese hamster cells can excise a small fraction of the UV-induced pyrimidine dimers. By isolating the D N A of the UV-irradiated cells and labelling with higher specific activity [3H]thymidine, an attempt was made in the present study to determine if caffeine inhibited the excision of UV-induced pyrimidine dimers. The results in Table IV indicate that caffeine had very little, if any, inhibitory effect on the small amount of excision that was detected. DISCUSSION

We reported 7 that post-treatment of UV-irradiated Chinese hamster cells with l m M caffeine reduces UV-induced mutation frequency, using the quantitative method for the detection of forward mutations to azaguanine resistance. The report contained some errors in calculation, which were soon corrected s4. As pointed out by MORROW5s, in only two out of the four original experiments were the differences statistically significant at the 5 ~ confidence level, although the data all showed the same trend. The situation was further complicated by subsequent work of ARLETT AND HARCOURT 23. They showed that caffeine at 100 Fg/ml (approximately half the concentration used in our experiments) reduces the frequency of UV-induced mutations only when caffeine was present throughout the post-irradiation period, confirming our earlier findings. On the other hand, when caffeine was present during the period allowed for the expression of mutants (0-40 h after irradiation), they found that the mutation frequency was enhanced. The results from the present studies do not resolve the differences between the work of ARLETT AND HARCOURT and ours. We are able now to show in four repeat

EFFECT OF CAFFEINE O N HAMSTER CELLS

in ritro

327

experiments (the result of one being presented in Table I1) that the caffeine effect in reducing the frequency of UV-induced mutations is only observable when caffeine is present during the first cell division after UV irradiation. Since caffeine was added in the absence of azaguanine, the result may eliminate the problem of possible interaction between the two purine analogues. The result of a reconstruction experiment (Table III) shows that 8-azaguanineresistant mutants added to the mixed cell population were not selected against when caffeine was present. The possibilit), exists that caffeine may alter the processes by which cell to cell contact leads to the phenotypic conversion of resistance to sensitivity under conditions of overcrowding 46. This may explain the reduction in the frequency of pre-existing mutants as observed both by us and by ARLETT AND HARCOURT23. The reconstruction experiment, however, does not test the survival of the newly formed mutants. A difference in technique between the experiments of ARLErr AND HARCOURT and ours is the concentration of caffeine. It has been found in bacteria that its effect is dose-dependent and that at high concentrations caffeine may inhibit the errorprone component of the repair process and thus result in fewer induced mutations. Unfortunately, the combined treatment of hamster cells with UV and I m M caffeine reduces the cell survival (Tables I and 1I) to such a low level that a meaningful distinction cannot be made when caffeine was present either at 0-18 h or or at 18-42 h. When caffeine at 100 #g/ml was added to UV-irradiated hamster cells, survival was nearly 50 ~ and the caffeine effect on survival disappeared by 20 h after irradiation 23. Since the concentrations of caffeine used in the two studies were different and since in bacteria, at least, the caffeine effect on mutation induction is dose-dependent, clearly more experimental data are needed to clarify the apparent differences between the two sets of results. In the present studies with the Chinese hamster cell cultures, caffeine was found to interact with UV-irradiation to decrease both the colonyforming ability and mutation frequency as well as to lead to the production of a relatively low molecular weight D N A synthesized in the first S period after irradiation. Several explanations for the molecular basis for the combined effect of caffeine and UV light on Chinese hamster cells might be suggested from previously reported experiments. In some bacterial strains, caffeine appears to enhance UV damage by inhibiting the excision of UV-induced pyrimidine dimers 3°- 33. If excision repair plays a major role in the hamster ceils, our observations that the molecular weight of D N A synthesized in the UV-irradiated and caffeine-treated hamster cells was lower than that synthesized in control cells and that there was no shift in sedimentation profiles of pre-existing UV-irradiated D N A incubated in caffeine might be interpreted as evidence for caffeine inhibition of excision repair. CHIU AND RAUTH47 have also shown that in mouse L-cells UV alone did not affect the sedimentation profile of prelabelled parental DNA. Nevertheless, rodent cells appear either not to recognize all pyrimidine dimers as lesions or can repair most of the UV-induced damage in D N A by some mechanism other than excision 4s- 52. Recent evidence indicates that some pyrimidine dimers can be removed by rodent cells 53.s6, but within the sensitivity of the pyrimidine dimer assay used in this study caffeine appears not to inhibit excision

328

J. E. TROSKO, E. H. Y. CHU

in the Chinese hamster cells. This lack of an inhibitory effect of caffeine in excising pyrimidine dimers was also observed in human cells 34 and Drosophila tissue culture cells (TROSKO, unpublished results). In addition, caffeine fails to inhibit unscheduled DNA synthesis in Chinese hamster cells35; caffeine (2 mM) does not inhibit the activity of purified mammalian DNAase III, DNAase IV, or terminal deoxynucleotidyltransferase, nor does it impair the ability to degrade an UV-irradiated substrate in vitro 57.

If pyrimidine dimers are the significant lesions induced by UV in Chinese hamster cells and are responsible for inhibiting the synthesis of DNA (incorporation of [3H]thymidine into TCA-insoluble material) and if caffeine inhibits the excision of some of the pyrimidine dimers, one would predict that caffeine would decrease the incorporation of [3H]thymidine into DNA. This was not observed in the experiment described in Fig. 4. On the other hand, we did observe that in the UV-irradiated hamster cells caffeine increases cell killing, reduces mutation frequency and interferes with the ligation of the newly synthesized DNA and these effects appear to be S-phase specific. Therefore, an interpretation other than the inhibition of excision repair seems necessary to account for the inhibitory effect of caffeine during the repair of UVdamage in DNA, leading to the observed biological consequences. RuPp AND HOWARD-FLANDERS 5a showed that some strains of E. coil, which were unable to excise pyrimidine dimers, survived via a recombinational repair mechanism. The DNA synthesized from a non-excision repaired UV-irradiated DNA strand has a lower molecular weight than that from unirradiated cells, indicating that the nascent DNA contained gaps opposite pyrimidine dimers in the parental strands sg. The observations that size of the nascent DNA increased with further incubation and end-to-end association of daughter and parental DNA occurs after UV irradiation 6° support the hypothesis that recombination repair is responsible for filling in the gaps in E. coli. The possible existence of this type of recombination repair mechanism in mammalian cells has been inferred 7'3s'6t-63. However, LEHMANN6a has provided evidence that many dimers are not excised before DNA synthesis in UV-irradiated mouse lymphoma L5178Y cells and that the gaps formed in the nascent DNA are filled in, not by the postulated bacterial recombinational mechanism, but by de novo DNA synthesis. Furthermore, KLiMEK AND VANICEK 65 AND CHIU AND RAUTrt47 have suggested that at low UV exposures, there might be a continuous by-pass mechanism whereby, when a growing fork reaches a dimer, instead of leaving a gap, it continues around the the block. Only at high UV-doses would gaps be formed. In normal human cells, HeLa cells and those from individuals with Xeroderma pigmentosum, the repair of DNA after UV-irradiation involves the elongation and joining of the newly synthesized DNA segments of low molecular weight, despite the presence of some non-excised pyrimidine dimers 66'6~. CLEAVER AND THOMAS 61 demonstrated that the molecular weight of DNA synthesized in UV-irradiated hamster cells increased with time, except in the presence of 2 m M caffeine. They also showed that caffeine did not interfere with DNA synthesized in unirradiated cells. They interpreted their data as indicating that caffeine interferes with semi-conservative DNA replication. Although the possibility is not

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excluded that caffeine stimulates DNAase activity in UV-irradiated cells, our data are consistent with the hypothesis that caffeine interferes with an S-phase specific, nonexcision type of repair mechanism. A considerable body of literature has been accumulated in recent years to document the close but complicated relationship between UV mutagenesis and repair processes in bacteria (see refs.~8'69). Current working hypotheses are as follows: Pyrimidine dimers, which are the major kinds of UV-photoproducts, can be repaired by excision, which is a largely 'error-free' repair mechanism. Unrepaired dimers cause mutations by inducing the formation of daughter strand gaps. These gaps, which are lethal if not repaired, cannot be mended by repair replication because each is located opposite a non-coding lesion. It has been postulated that they are reparable by genetic recombination and aberrant repair produces mutations. Caffeine decreases mutation frequencies in Chinese hamster cells which do not excise all their UV-induced pyrimidine dimers. It may be inferred that the hamster cells can tolerate the unexcised dimers by some S-phase specific, post-replicational dark-repair mechanism. On the basis of LEHMANN'S 64 observation, this process is not a recombinational event but involves de novo DNA synthesis to fill the gaps in the nascent DNA strand. It is conceivable that infidelity in the nucleotide sequence and/or composition could occur during this gap-filling process, leading to the production of mutations. Thus, repair of UV-induced damage in mammalian DNA by the postulated mechanism will increase cell survival but give rise to mutation in a fraction of the surviving cells. Caffeine inhibits this repair (and misrepair), resulting in a reduction in cell survival and mutation yield. These phenomena are evident in rodent cells which are essentially 'excision-deficient' but may exist in other mammalian or eukaryotic cells as well. Our observation that caffeine reduces UV mutagenesis in the hamster cells is similar to that of WITKIN AND FARQUHARSON15 in the excision-defective Hcrstrains in E. coli. According to these authors, caffeine could diminish UV mutagenesis in such bacterial strains by modifying the recombinational repair in a way that reduces its inaccuracy. We are proposing a similar repair mechanism for the hamster cells, the difference being that in the hamster cells caffeine affects the post-replicationai repair synthesis but not a recombinational type of repair. Furthermore, we infer that repair in this manner is error-prone because there is a high frequency of UV-induced mutations over the spontaneous frequency in Chinese hamster cells (Table II, and refs.7'23). A test of this proposed repair mechanism and an elucidation of the molecular mechanisms involved in the UV mutagenesis in mammalian cells would be facilitated if mutants could be found in mammalian cells, as in microorganisms, that modify the processes of mutation and recombination. It would be particularly interesting to study these phenomena in human cells derived from patients with Xeroderma pigmentosum in which UV-endonuclease for excision repair is deficient. ACKNOWLEDGMENTS

Research was supported by Contract AT (11-1)-1704 from the Atomic Energy Commission, grant CA 13048-01 from the National Cancer Institute. J.E.T. is a

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C a r e e r D e v e l o p m e n t A w a r d e e o f the P u b l i c H e a l t h Service ( 1 K 4 C A 2 4 , 085-01). A p o r t i o n o f t h i s w o r k w a s p e r f o r m e d in t h e B i o l o g y D i v i s i o n , O a k R i d g e N a t i o n a l L a b o r a t o r y a n d w a s j o i n t l y s p o n s o r e d b y the N a t i o n a l C a n c e r I n s t i t u t e a n d A t o m i c Energy Commission under contract with Union Carbide Corporation.

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