347
Mutation Research, 33 (1975) 347--356 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
POTENTIATION BY CAFFEINE OF ULTRAVIOLET-LIGHT DAMAGE IN CULTURED HUMAN CELLS CARTER B. SCHROY and PAUL TODD
Biophysics Department, The Pennsylvania State University, University Park, Pennsylvania 16802 (U.S.A.) (Received June 18th, 1975) (Accepted September 10th, 1975)
Summary Five cultured human cell lines (T-1 kidney, Chang liver, H.Ep. No. 2, HeLa-S3 and HeLa-O) were irradiated with ultraviolet light and immediately exposed to 1.0 and 3.0 mM caffeine for 44 h thereafter. This caffeine treatment reduced the surviving fraction (assayed by colony formation) of the irradiated population, but did not significantly reduce the colony-forming ability of unirradiated control cells. These findings suggest that many cultured human cell lines exhibit post-UV potentiation of potentially lethal damage by caffeine.
Introduction
The effects of caffeine administered after ultraviolet light (UV) irradiation of cultured human cells are unclear. The literature on the subject contains conflicting reports. For example, Buhl and Regan [5] and Lehmann et al. [19] noted that 1 and 1.5 mM caffeine, respectively, did not inhibit post-replication DNA chain elongation (i.e., "gap-filling" and rejoining after "bypass synthesis") in normal diploid human cells. In contrast, Fujiwara and Kondo [9] found that HeLa-S3-9IV cells were inhibited from post-UV gap closure by 2 mM caffeine and stated that "caffeine-sensitive post-replication repair exists in all kinds of cells tested including XP cells". The effect of post-UV caffeine on human cell survival as assayed by colony formation is also questioned. Wilkinson et al. [ 26] found that post-UV exposures of 1--3 mM caffeine for 10--13 days did not suppress the survival of two lines of HeLa cells: the "standard line" and clone M4, which is sensitive to methyl methanesulfonate. Conversely, Arlett [3] presented seldom quoted data demonstrating post-UV suppression of survival by 0.26 and 0.51 mM caffeine and stated that "these HeLa cells do have a repair system sensitive to caffeine". The findings of Wilkinson et al. have occasionally been quoted and extrapolated to mean that human cells in general lack post-UV caffeine sensitivity,
348 e.g. [5], and hence possibly rely less (than rodent cells) on a caffeine-sensitive repair process [17]. As both Arlett and Wilkinson et al. used HeLa cells in their work, we studied various human cell lines from various origins and sources in an a t t e m p t to determine if post-UV suppresson of survival by caffeine were a property of cultured human cells in general or only of particular HeLa cells or clones thereof. Materials and methods Cells used in the experimentation were as follows: (1) HeLa-S3 cells [22], originally taken from a cervical carcinoma of a human Negro female [10]. (2) HeLa-O cells, also referred to as HeLa-rhino, were cloned from HeLa-S3 for their support of the growth of the rhino virus [16], and possess growth and morphological characteristics somewhat different from those of the parental $3 clone. (3) H.Ep No. 2 cells (ATCC CCL No. 23) were originally taken from male human laryngeal carcinoma [20]. (4) Chang liver cells [6] (ATCC CCL No: 13), also referred to as LiCH, are from non-malignant origin but are heteroploid. (5) T-1 kidney cells [4,24], also from non-malignant origin and heteroploid. All cell lines were epithelioid and were maintained in Eagle's Minimal Essential Medium [8] supplemented with 10% fetal calf serum and either penicillinstreptomycin (100 U/ml and 100 pg/ml, respectively) or 50 pg/ml Gentamicin (Schering). All media and sera were purchased from Grand Island Biological Co. or Flow Laboratories. Colony formation experiments were performed in 35-mm tissue culture dishes (Falcon No. 3001) by plating known numbers of cells determined by h e m a c y t o m e t e r count.
Caffeine Caffeine (Calbiochem, lot No. 200200) was prepared by dissolution in doubledistilled water at a concentration of 100 mM (1.94%) and autoclaving (121°C, 15 lbs/in 2) for 15 min. This concentration of caffeine allows it to dissolve completely at 37°C and recrystallize at 4°C during storage. The appropriate a m o u n t of caffeine solution was added directly to the experimental culture medium to give the desired molarity.
Irradiation All irradiations were performed using a General Electric G15T8 15W mercury vapor lamp producing 2.1 + 0.1 J / m 2 sec -1 at a distance of 31 cm from its center. The strongest spectral emmission line was at 254 nm. A UV dosimeter [13] was employed to determine dose-rate. The photocell of the dosimeter was exposed to the source and its current reading noted. A 10-cm plastic Petri dish (Falcon No. 1029) lid was then placed over the photocell (eliminating wavelengths below 295 nm [14]), and t h a t current reading was subtracted from the first to determine the actual dose-rate of far-UV. Irradiations were accomplished by aspirating the medium from the triplicate
349 cultures 3--4 h after plating and placing the three dishes together (without lids) in a sterile 10-cm plastic Petri dish, the lid of which was removed under the UV lamp for the required length of time to deliver the desired dose. Immediately after irradiation, fresh medium with or w i t h o u t caffeine was added to the dishes and all were returned to 37°C. This medium was then removed after 44 h and replaced with medium n o t containing caffeine. The unirradiated control cultures with or w i t h o u t caffeine were prepared in quintuplicate. The dishes were incubated for an average of 12 days with one medium change (in addition to t h a t following the caffeine removal) on the ninth day. The colonies were stained by adding aqueous methylene blue to the medium (final concentration 0.05%) and, after gentle rinsing and drying, were counted under a stereo dissecting microscope. Due to the shadowing of the far-UV by the dishes, colonies at the edges of the dishes were n o t counted on either control or irradiated samples. Results
Comparison of cell lines All cell lines tested exhibited suppression of survival by caffeine with a 1 mM concentration essentially eliminating the survival curve shoulder. Fig. 1 presents survival data in graphical form for 4 of the cell lines. The fifth line, H.Ep. No. 2. had survival curves similar to those of HeLa-S3 but exhibited somewhat more radioresistance as evidenced by its Do (see Table I). Experiments on each cell line were performed three times with various concentrations of caffeine {1--4 mM), and all experiments yielded essentially the same results with small fluctuations in survival curve parameters. None of the cell lines investigated ever failed to exhibit caffeine potentiation of UV damage with a 44 h exposure. Typical survival curve parameters, determined from data such as in Fig. 1, are summarized in Table I.
Effect of caffeine alone Table II presents relative efficiencies for the cells in the experiments from which Fig. 1 and parts of Table I were obtained. The 44-h caffeine t r e a t m e n t alone did n o t decrease the plating efficiency of the unirradiated controls. The time dependence of caffeine t o x i t y in H.Ep. No. 2 cells is presented in Table III. A 1 mM concentration of the drug does n o t induce any toxicity in this cell line for up to 7 days, and there is evidence that it actually enhances survival. There is no toxicity observed with a 3 mM concentration of the drug until after the third day.
Effect of method of caffeine preparation An a t t e m p t was made to determine if the caffeine preparation m e t h o d of Wilkinson et al. [26] was responsible for their negative results. A 5% suspension was prepared, sterilized, and used as stock for both T-1 and HeLa-S3 experiments. Both lines exhibited the same suppression of survival as they did when the 1.94% solution was used as stock. Quantitative evaluation of the effectiveness of a 5% suspension was difficult as it was necessary to assume t h a t the caffeine was uniformly dispersed after agitation. Placing the 5% stock bottle
I
I
_-%~
- RA
I
~
I
T-I (EXl~ 91)
O Om M CAFFEINE ,,~ ,, o
L,CH
o\
.\..\
0.01
p-
h, 0.001
--
I
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I
I
I
I
I
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I I
HeLO -0
IC
- ~m~~~.
o~
_ _
-
00m
UV DOSE : J i m 2
Fig. 1, S u r v i v a l c u r v e s f o r 4 h u m a n cell lines. T h e f i f t h cell line, H . E p . N o . 2, h a s s u r v i v a l c u r v e s s i m i l a r to t h e H e L a - S 3 c u r v e s . O p e n c i r c l e s r e p r e s e n t cells r e c e i v i n g n o c a f f e i n e a f t e r i r r a d i a t i o n ; s q u a r e s r e p r e s e n t 1 m M a n d t r i a n g l e s r e p r e s e n t 3 m M o f c a f f e i n e p r e s e n t f o r 44 h a f t e r i r r a d i a t i o n . E r r o r b a r s r e p r e s e n t p r o p o g a t e d s t a n d a r d e r r o r s o f t h e m e a n . S u r v i v a l c u r v e p a r a m e t e r s are p r e s e n t e d in T a b l e I.
TABLE I UV SURVIVAL CURVE PARAMETERS
FOR HUMAN CELLS
S u r v i v a l c u r v e p a r a m e t e r s [ 1 ] ( U V o n l y , n o c a f f e i n e ) f o r h u m a n cells s t a t e d as m e a n ± s t a n d a r d e r r o r . D o s a g e p a r a m e t e r s are in J / m 2. D o = m e a n l e t h a l d o s e , t h e d o s e r e q u i r e d t o r e d u c e s u r v i v a l f r o m 0.1 t o 0 . 0 3 7 l D q = q u a s i - t h r e s h o l d d o s e , t h e d o s e at w h i c h t h e e x p o n e n t i a l p a r t o f t h e c u r v e i n t e r s e c t s t h e 1.0 s u r v i v a l line; D 3 7 = d o s e r e q u i r e d t o r e d u c e s u r v i v a l f r o m l . 0 to 0 . 3 7 ; D I 0 = d o s e r e q u i r e d t o r e d u c e s u r v i v a l to 0 . 1 0 a n d d o s e at w h i c h D I o M F is d e t e r m i n e d ; n = e x t r a p o l a t i o n n u m b e r o f t h e e x p o n e n t i a l p a r t o f t h e c u r v e ; p = t h e " r e p a i r p a r a m e t e r " d e r i v e d f r o m Eq. 3 o f r e f . [ 1 2 ] as p = (f e x p (D/Do)--l)/(1--f) is c a l c u l a t e d a t D = D I 0 f r o m e y e - f i t t e d s u r v i v a l etLrves, a n d is i n c l u d e d f o r p o s s i b l e f u t u r e r e f e r e n c e as i t is i n d e p e n d e n t o f a b s o l u t e d o s i m e t r y ; a n d D 1 0 M F is t h e d o s e m o d i f y i n g f a c t o r at t h e 1 0 % s u r v i v a l level a n d is h e r e d e f i n e d as t h e r a t i o o f t h e D I 0 in t h e p r e s e n c e o f c a f f e i n e t o t h e D 1 0 in its a b s e n c e . Cell line
DO
Dq
D37
D10
n
p
D10MF lmM
3mM
Changliver (LiCH)
3.7±0.4
2.7±0.1
6.2±0.0
10.7±0.3
2.2±0.1
1.0±0.5
0.65
0.26
H.Ep. No. 2
5.2±0.2
2.6±0.4
7.3±0.7
14.0±1.0
1.5±0.1
0.6±0.1
0.76
0.51
HeLa-O
3.2±0.2
1.3±0.3
4.4±0.1
8.6±0.3
1.5±0.1
0.5±0.1
0.77
0.47
HeLa-S3
3.1±0.5
1.4±0.1
4.4±0.1
8.4±0.0
1.5±0.1
0.6±0.1
0.70
0.23
T-l~dney
4.0±0.1
2.2±0.7
5.8±1.1
10.7±1.6
1.8±0.3
0.8±0.4
0.75
0.64
351
TABLE
II
RELATIVE
PLATING
EFFICIENCIES
OF HUMAN
CELL LINES IN 1 AND 3 mM CAFFEINE
A p p r o x i m a t e a b s o l u t e plating e f f i c i e n c i e s ( A A P E ) and relative plating e f f i c i e n c i e s ( R P E ) for t h e c u r v e s in F i g . 1 e x p r e s s e d as m e a n -+ p r o p a g a t e d standard error. N o 4 4 - h c a f f e i n e t r e a t m e n t a l o n e s i g n i f i c a n t l y d e c r e a s e d t h e surviving f r a c t i o n . A A P E w a s e s t i m a t e d via h e m o c y t o m e t e r c o u n t . AAPE (0 mM)
R P E (1 m M )
RPE (3 raM)
Chang liver ( L i C H )
50%
1 0 5 -+ 5 %
1 0 6 +- 6 %
H.Ep. No. 2
80
9 9 -+ 7
9 9 +- 7
HeLa-O
35
9 8 -+ 7
110 + 7
HeLa-S3
20
1 4 1 -+ 1 4
T-1
50
1 1 4 +- 4
TABLE
1 4 8 +- 1 9 9 6 +- 7
III
TIME-DEPENDENT
CAFFEINE
TOXICITY
IN UNIRRADIATED
H.Ep. No. 2 CELLS
T h e e f f e c t s o f c a f f e i n e a l o n e o n the survival o f H . E p . N o . 2 cells. S u r v i v a l s are e x p r e s s e d as p e r c e n t a g e (-+ standard error) o f t h e 0 m M a b s o l u t e c o n t r o l for t h e e x p e r i m e n t . N o t i c e that all I m M p o i n t s are sign i f i c a n t l y a b o v e 1 . 0 s u r v i v a l and that 3 m M c a f f e i n e d o e s n o t b e c o m e t o x i c until after the third d a y . D a y s in c a f f e i n e
Surviving fraction (%)
1
2
3
4
5
6
7
lmM
129 + 8
117 + 10
1 1 7 -+ 1 3
1 1 7 +- 1 1
118 ± 17
117 + 9
1 2 9 -+ 9
3mM
120+
124+
99-+ 9
79-+ 4
10
9
94+
10
66 + 10
7 3 +- 8
in boiling water to effect dissolution of the caffeine was also without consequence as it yielded the same final experimental results. Discussion
Comparison of cell types It is evident that cultured human cell lines exhibit post-UV suppression of survival by non-toxic concentrations of caffeine. Presently, it is n o t possible from the available data to postulate with any certainty why Arlett's HeLa cells exhibited caffeine potentiation at 0.26 and 0.51 mM concentrations and the cells of Wilkinson et al. did not at 1 and 3 mM concentrations. As both used continuous exposures of caffeine after irradiation, there may have been marked clonal differences in caffeine sensitivity or differences in caffeine purity. A similar discrepancy is also indicated in molecular repair experiments. Both Fujiwara and Kondo [9] and Painter [21] used HeLa cells for post-replication repair studies. The former group found that 2 mM caffeine inhibited post-replication gap closure, whereas the latter seemingly did not. Painter [ 2 1 ] , however, did not interpret his data in this manner when presenting his three arguments against the post-replication "gap" concept of Lehmann
352 [18]. One of his arguments was based on the assumption that the effects of caffeine in Chinese hamster cells are the same as in h u m a n cells. He assumed the results of Cleaver and Thomas [7] (i.e., that caffeine inhibits bypasssynthesized DNA strands from rejoining in Chinese hamster cells) to be true for his HeLa cells. Consequently, he used 2 mM caffeine over a 2.5 h period in an a t t e m p t to accumulate gaps while synthesis was occurring in the presence of BUdR. Removal of the caffeine and BUdR and addition of tritiated thymidine would allow the gaps to be closed with the tritiated precursors. In an alkaline equilibrium density gradient, the BUdR-containing strands would sediment apart from the normal-density parental strands and, if gaps were closed with tritiated precursors, the heavier strands would be radioactively labeled as well. However, Painter f o u n d "absolutely no tritium in excess of that expected" and concluded that "gaps of the kind found in bacteria do n o t occur in mammalian cells". One possible reason for his n o t finding tritium label was that the caffeine did n o t prevent gap-filling, and the gaps, if they ever existed per se, were filled during the 2.5-h BUdR incubation. Viewed in this manner, Painter's data would support those of Buhl and Regan [5] and Lehmann et al. [19] (which were obtained with diploid h u m a n fibroblasts) and contradict those of Fujiwara and Kondo [9] (which, as Painter's, were obtained with HeLa cells). It is interesting to note here in the light of Painter's assumption that Roberts and Ward f o u n d that 0.75 mM caffeine inhibited post-replication repair in both sulphur mustard and N-methyl-N-nitrosourea alkylated Chinese hamster cells but not in HeLa cells [23], thus substantiating the possibility of significant differences in the DNA repair mechanisms between hamster and HeLa cells. Caffeine degradation may also be a consideration in the variability of results. A preliminary report by Goth and Cleaver [ 11 ] stated that in I mM tritated caffeine most of the pod in HeLa, normal, Lesch-Nyhan, and XP human cells consisted of caffeine degradation products. They did n o t irradiate their cells, however, and consequently the post-replication condition of the cells was n o t a factor in their findings. Our data (Table III) for 3 mM caffeine (but not for 1 mM) indicates that toxicity occurs to unirradiated H.Ep. No. 2 cells after the third day. Hence, there is an apparent need to clarify the fate of various concentrations of caffeine in unirradiated cells. In irradiated cells one would not expect to observe caffeine potentiation of lethality if the caffeine were essentially all degraded in three hours and the S-phase delay {and hence the repair period) induced by the UV alone were longer than three hours (any synergistic effects notwithstanding). Consequently, it appears that the caffeine responsible for the potentiation of post-UV lethality is n o t degraded in UV-irradiated cells -- at least n o t in 3 h. Also, some of the contradictions may be resolved by determination of the similarities and/or differences between primary cells or diploid cell strains and established lines, cells of normal origin and those of malignant origin, and cells of epithelioid and fibroblastic morphologies. Any one or a combination of the above factors m a y be related to the DNA repair capabilities of any given cell type. The evidence so far available suggests t h a t diploid fibroblasts tend to have a higher resistance to the effects of post-UV caffeine than do established epithelioid cell lines.
353
Synergism and caffeine toxicity Synergism between two agents occurs when the cooperative action of the agents together produces a more effective result than the sum of the results of the agents acting independently. As the survival curves in Fig. 1 are normalized to 1.0 survival for the 1 and 3 mM caffeine concentrations (thus controlling the magnitude of the action of the caffeine agent alone), and the 0 mM curve is present to indicate the magnitude of the action of the UV agent alone, any deviation from the 0 mM survival curve would indicate synergism in the strict meaning of the word. Some workers (e.g. refs. [2,3] ) choose to subscribe to the division probability hypothesis of Whitmore and Till [25] which states that a given agent modifies the probability t h a t a given cell and its progeny will divide and ultimately give rise to a colony. Consequently, for synergism to be expressed within the confines of this hypothesis, the probability for division must differ from the product (not the sum) of the division probabilities taken independently. Therefore, synergism would n o t be readily apparent from survival curves without normal normalization of the data points in accordance with the mathematics of this hypothesis, as the correlation between division probability and plating efficiency is non-linear. If there is no modification of the division probability induced by one of the agents alone, then synergism may be determined with the usual survival curve data without mathematical normalization. Tables II and III indicate that none of the conditions of caffeine treatment used in our UV experiments led to a decrease in plating efficiency and hence division probability. Even within the constraints of the division probability hypothesis [2,25], synergism is observed.
Effect of method of caffeine preparation The results of Wilkinson et al. [26] are difficult to explain. They reported to have used a 5% stock solution of caffeine. It was found in our laboratory that a 5% concentration made an opaque suspension which did n o t dissolve unless autoclaved at 121°C, and, if cooled to 37°C after autoclaving, it recrystallized. It is possible t h a t Wilkinson et al. did n o t have a 5% stock solution as believed but had instead a concentration closer to 2%, similar to ours. This concentration of caffeine renders the drug completely soluble at room temperature with recrystallization occurring at 4°C. If this were the case, then their concentrations of caffeine would have been less than half of that reported, possibly making it too low to yield statistically significant survival depression (judging from our data).
Conclusions and general discussion Contradictions must be resolved before it can be determined what the effects of caffeine on ultraviolet-irradiated h u m a n cells are. Unambiguous and explicit materials and methods sections in papers on the subject are needed, including cell-line origins, primary references to the origin of the clones used, and any obvious peculiarities each cell line is known to possess. A simple designation such as " H e L a cells", for example, is apparently insufficient without further clarification of clonal origin. The manufacturer and lot numbers of any biochemicals used (viz., caffeine) and how they were sterilized should also be stated.
354
Further, extrapolation of data b e y o n d the cell lines used is evidently hazardous. Data obtained with hamster cells alone, for example, are not adequate to make statements concerning "mammalian cells" in general. Perhaps detailed and correlative studies of molecular and survival data of mutants such as those of Klimek et al. [15] would provide useful information. Their subclones of HeLa-S3, particularly clones 46 and 18/2, may provide clues, as the 46 mutant appears that it may be defective for some form of DNA repair. As no correlation between molecular and survival data can be made at this time, it cannot be stated h o w caffeine affects post-replication repair and subsequently survival. Perhaps it can be concluded that cultured human cells in general exhibit post-irradiation suppression of survival by caffeine if the concentration of the drug is high enough and the exposure time is of appropriate duration.
Acknowledgements We wish to thank the following individuals of the Pennsylvania State University Departments of Microbiology and Biology who aided by supplying some of the cell cultures used in this study: Dr. J.J. Docherty, Dr. M.D. Notter, Dr. K.D. Thompson, C.J. Thompson, L. Trusal, L. Richardson and S.C. Thornton.
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355 1 8 L e h m a n n , A . R . , P o s t r e p l i e a t i o n r e p a i r o f D N A in u l t r a v i o l e t - i r r a d i a t e d m a m m a l i a n cells, J. M o l e c . Biol., 6 6 ( 1 9 7 2 ) 4 3 8 - - 4 4 5 . 1 9 L e h m a n n , A . R . , S. K i r k - B e l l , C.F. A r l e t t , M.C. P a t e r s o n , P . H . M . L o h m a n , E . A . d e W e e r d - K a s t e l e i n a n d D. B o o t s m a , X e r o d e r m a p i g m e n t o s u m cells w i t h n o r m a l levels o f e x c i s i o n r e p a i r h a v e a d e f e c t in D N A s y n t h e s i s a f t e r U V - i r r a d i a t i o n , P r o c . N a t l . A c a d . Sci. (U.S.), 7 2 ( 1 9 7 5 ) 2 1 9 - - 2 2 3 . 2 0 M o o r e , A . E . , L. S a b a c h e w s k y a n d H.W. T o o l a n , C u l t u r e c h a r a c t e r i s t i c s o f f o u r p e r m a n e n t lines o f h u m a n c a n c e r cells, C a n c e r R e s . , 1 5 ( 1 9 5 5 ) 5 9 8 - - 6 0 2 . 21 P a i n t e r , R . B . , D N A d a m a g e a n d r e p a i r in e u k a r y o t i c cells, G e n e t i c s 7 8 ( 1 9 7 4 ) 1 3 9 - - 1 4 8 . 2 2 P u c k , T . T . , P.I. M a r c u s a n d S.J. C i e c u r a , C l o n a l g r o w t h o f m a m m a l i a n cells i n v i t r o : g r o w t h c h a r a c t e r istics of H e L a cells w i t h a n d w i t h o u t a ' f e e d e r l a y e r ' , J. E x p t l . M e d . , 1 0 3 ( 1 9 5 6 ) 2 7 3 - - 2 8 4 . 2 3 R o b e r t s , J . J . , a n d K . N . W a r d , I n h i b i t i o n o f p o s t - r e p l i c a t i o n r e p a i r o f a l k y l a t e d D N A b y c a f f e i n e in C h i n e s e h a m s t e r cells b u t n o t H e L a cells, C h e m . - B i o l . I n t e r a c t i o n s , 7 ( 1 9 7 3 ) 2 4 1 - - 2 6 4 . 2 4 v a n d e r V e e n , J . , L. B o t s a n d A. Mes, E s t a b l i s h m e n t o f t w o h u m a n cell s t r a i n s f r o m k i d n e y a n d r e t i c u l e s a r c o m a of l u n g , A r c h . Ges. V i r u s f o r s c h . , 8 ( 1 9 5 8 ) 2 3 0 - - 2 4 5 . 2 5 W h i t m o r e , G . F . , a n d J . E . Till, Q u a n t i t a t i o n o f cellulax r a d i o b i o l o g i c a l r e s p o n s e s , A n n . R e v . N u c l . Sci., 14 (1964) 347--374. 2 6 W i l k i n s o n , R . , J. K i e f f e r a n d A . H . W . Nias, E f f e c t s o f p o s t - t r e a t m e n t w i t h c a f f e i n e o n t h e s e n s i t i v i t y t o u l t r a v i o l e t l i g h t i r r a d i a t i o n o f t w o l i n e s o f H e L a cells, M u t a t i o n R e s . , 1 0 ( 1 9 7 0 ) 6 7 - - 7 4 .
Mutation Research, 33 (1975) 347--356 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
POTENTIATION BY CAFFEINE OF ULTRAVIOLET-LIGHT DAMAGE IN CULTURED HUMAN CELLS CARTER B. SCHROY and PAUL TODD
Biophysics Department, The Pennsylvania State University, University Park, Pennsylvania 16802 (U.S.A.) (Received June 18th, 1975) (Accepted September 10th, 1975)
Summary Five cultured human cell lines (T-1 kidney, Chang liver, H.Ep. No. 2, HeLa-S3 and HeLa-O) were irradiated with ultraviolet light and immediately exposed to 1.0 and 3.0 mM caffeine for 44 h thereafter. This caffeine treatment reduced the surviving fraction (assayed by colony formation) of the irradiated population, but did not significantly reduce the colony-forming ability of unirradiated control cells. These findings suggest that many cultured human cell lines exhibit post-UV potentiation of potentially lethal damage by caffeine.
Introduction
The effects of caffeine administered after ultraviolet light (UV) irradiation of cultured human cells are unclear. The literature on the subject contains conflicting reports. For example, Buhl and Regan [5] and Lehmann et al. [19] noted that 1 and 1.5 mM caffeine, respectively, did not inhibit post-replication DNA chain elongation (i.e., "gap-filling" and rejoining after "bypass synthesis") in normal diploid human cells. In contrast, Fujiwara and Kondo [9] found that HeLa-S3-9IV cells were inhibited from post-UV gap closure by 2 mM caffeine and stated that "caffeine-sensitive post-replication repair exists in all kinds of cells tested including XP cells". The effect of post-UV caffeine on human cell survival as assayed by colony formation is also questioned. Wilkinson et al. [ 26] found that post-UV exposures of 1--3 mM caffeine for 10--13 days did not suppress the survival of two lines of HeLa cells: the "standard line" and clone M4, which is sensitive to methyl methanesulfonate. Conversely, Arlett [3] presented seldom quoted data demonstrating post-UV suppression of survival by 0.26 and 0.51 mM caffeine and stated that "these HeLa cells do have a repair system sensitive to caffeine". The findings of Wilkinson et al. have occasionally been quoted and extrapolated to mean that human cells in general lack post-UV caffeine sensitivity,
348 e.g. [5], and hence possibly rely less (than rodent cells) on a caffeine-sensitive repair process [17]. As both Arlett and Wilkinson et al. used HeLa cells in their work, we studied various human cell lines from various origins and sources in an a t t e m p t to determine if post-UV suppresson of survival by caffeine were a property of cultured human cells in general or only of particular HeLa cells or clones thereof. Materials and methods Cells used in the experimentation were as follows: (1) HeLa-S3 cells [22], originally taken from a cervical carcinoma of a human Negro female [10]. (2) HeLa-O cells, also referred to as HeLa-rhino, were cloned from HeLa-S3 for their support of the growth of the rhino virus [16], and possess growth and morphological characteristics somewhat different from those of the parental $3 clone. (3) H.Ep No. 2 cells (ATCC CCL No. 23) were originally taken from male human laryngeal carcinoma [20]. (4) Chang liver cells [6] (ATCC CCL No: 13), also referred to as LiCH, are from non-malignant origin but are heteroploid. (5) T-1 kidney cells [4,24], also from non-malignant origin and heteroploid. All cell lines were epithelioid and were maintained in Eagle's Minimal Essential Medium [8] supplemented with 10% fetal calf serum and either penicillinstreptomycin (100 U/ml and 100 pg/ml, respectively) or 50 pg/ml Gentamicin (Schering). All media and sera were purchased from Grand Island Biological Co. or Flow Laboratories. Colony formation experiments were performed in 35-mm tissue culture dishes (Falcon No. 3001) by plating known numbers of cells determined by h e m a c y t o m e t e r count.
Caffeine Caffeine (Calbiochem, lot No. 200200) was prepared by dissolution in doubledistilled water at a concentration of 100 mM (1.94%) and autoclaving (121°C, 15 lbs/in 2) for 15 min. This concentration of caffeine allows it to dissolve completely at 37°C and recrystallize at 4°C during storage. The appropriate a m o u n t of caffeine solution was added directly to the experimental culture medium to give the desired molarity.
Irradiation All irradiations were performed using a General Electric G15T8 15W mercury vapor lamp producing 2.1 + 0.1 J / m 2 sec -1 at a distance of 31 cm from its center. The strongest spectral emmission line was at 254 nm. A UV dosimeter [13] was employed to determine dose-rate. The photocell of the dosimeter was exposed to the source and its current reading noted. A 10-cm plastic Petri dish (Falcon No. 1029) lid was then placed over the photocell (eliminating wavelengths below 295 nm [14]), and t h a t current reading was subtracted from the first to determine the actual dose-rate of far-UV. Irradiations were accomplished by aspirating the medium from the triplicate
349 cultures 3--4 h after plating and placing the three dishes together (without lids) in a sterile 10-cm plastic Petri dish, the lid of which was removed under the UV lamp for the required length of time to deliver the desired dose. Immediately after irradiation, fresh medium with or w i t h o u t caffeine was added to the dishes and all were returned to 37°C. This medium was then removed after 44 h and replaced with medium n o t containing caffeine. The unirradiated control cultures with or w i t h o u t caffeine were prepared in quintuplicate. The dishes were incubated for an average of 12 days with one medium change (in addition to t h a t following the caffeine removal) on the ninth day. The colonies were stained by adding aqueous methylene blue to the medium (final concentration 0.05%) and, after gentle rinsing and drying, were counted under a stereo dissecting microscope. Due to the shadowing of the far-UV by the dishes, colonies at the edges of the dishes were n o t counted on either control or irradiated samples. Results
Comparison of cell lines All cell lines tested exhibited suppression of survival by caffeine with a 1 mM concentration essentially eliminating the survival curve shoulder. Fig. 1 presents survival data in graphical form for 4 of the cell lines. The fifth line, H.Ep. No. 2. had survival curves similar to those of HeLa-S3 but exhibited somewhat more radioresistance as evidenced by its Do (see Table I). Experiments on each cell line were performed three times with various concentrations of caffeine {1--4 mM), and all experiments yielded essentially the same results with small fluctuations in survival curve parameters. None of the cell lines investigated ever failed to exhibit caffeine potentiation of UV damage with a 44 h exposure. Typical survival curve parameters, determined from data such as in Fig. 1, are summarized in Table I.
Effect of caffeine alone Table II presents relative efficiencies for the cells in the experiments from which Fig. 1 and parts of Table I were obtained. The 44-h caffeine t r e a t m e n t alone did n o t decrease the plating efficiency of the unirradiated controls. The time dependence of caffeine t o x i t y in H.Ep. No. 2 cells is presented in Table III. A 1 mM concentration of the drug does n o t induce any toxicity in this cell line for up to 7 days, and there is evidence that it actually enhances survival. There is no toxicity observed with a 3 mM concentration of the drug until after the third day.
Effect of method of caffeine preparation An a t t e m p t was made to determine if the caffeine preparation m e t h o d of Wilkinson et al. [26] was responsible for their negative results. A 5% suspension was prepared, sterilized, and used as stock for both T-1 and HeLa-S3 experiments. Both lines exhibited the same suppression of survival as they did when the 1.94% solution was used as stock. Quantitative evaluation of the effectiveness of a 5% suspension was difficult as it was necessary to assume t h a t the caffeine was uniformly dispersed after agitation. Placing the 5% stock bottle
I
I
_-%~
- RA
I
~
I
T-I (EXl~ 91)
O Om M CAFFEINE ,,~ ,, o
L,CH
o\
.\..\
0.01
p-
h, 0.001
--
I
I
I
I
I
I
I
I
I
I
I I
HeLO -0
IC
- ~m~~~.
o~
_ _
-
00m
UV DOSE : J i m 2
Fig. 1, S u r v i v a l c u r v e s f o r 4 h u m a n cell lines. T h e f i f t h cell line, H . E p . N o . 2, h a s s u r v i v a l c u r v e s s i m i l a r to t h e H e L a - S 3 c u r v e s . O p e n c i r c l e s r e p r e s e n t cells r e c e i v i n g n o c a f f e i n e a f t e r i r r a d i a t i o n ; s q u a r e s r e p r e s e n t 1 m M a n d t r i a n g l e s r e p r e s e n t 3 m M o f c a f f e i n e p r e s e n t f o r 44 h a f t e r i r r a d i a t i o n . E r r o r b a r s r e p r e s e n t p r o p o g a t e d s t a n d a r d e r r o r s o f t h e m e a n . S u r v i v a l c u r v e p a r a m e t e r s are p r e s e n t e d in T a b l e I.
TABLE I UV SURVIVAL CURVE PARAMETERS
FOR HUMAN CELLS
S u r v i v a l c u r v e p a r a m e t e r s [ 1 ] ( U V o n l y , n o c a f f e i n e ) f o r h u m a n cells s t a t e d as m e a n ± s t a n d a r d e r r o r . D o s a g e p a r a m e t e r s are in J / m 2. D o = m e a n l e t h a l d o s e , t h e d o s e r e q u i r e d t o r e d u c e s u r v i v a l f r o m 0.1 t o 0 . 0 3 7 l D q = q u a s i - t h r e s h o l d d o s e , t h e d o s e at w h i c h t h e e x p o n e n t i a l p a r t o f t h e c u r v e i n t e r s e c t s t h e 1.0 s u r v i v a l line; D 3 7 = d o s e r e q u i r e d t o r e d u c e s u r v i v a l f r o m l . 0 to 0 . 3 7 ; D I 0 = d o s e r e q u i r e d t o r e d u c e s u r v i v a l to 0 . 1 0 a n d d o s e at w h i c h D I o M F is d e t e r m i n e d ; n = e x t r a p o l a t i o n n u m b e r o f t h e e x p o n e n t i a l p a r t o f t h e c u r v e ; p = t h e " r e p a i r p a r a m e t e r " d e r i v e d f r o m Eq. 3 o f r e f . [ 1 2 ] as p = (f e x p (D/Do)--l)/(1--f) is c a l c u l a t e d a t D = D I 0 f r o m e y e - f i t t e d s u r v i v a l etLrves, a n d is i n c l u d e d f o r p o s s i b l e f u t u r e r e f e r e n c e as i t is i n d e p e n d e n t o f a b s o l u t e d o s i m e t r y ; a n d D 1 0 M F is t h e d o s e m o d i f y i n g f a c t o r at t h e 1 0 % s u r v i v a l level a n d is h e r e d e f i n e d as t h e r a t i o o f t h e D I 0 in t h e p r e s e n c e o f c a f f e i n e t o t h e D 1 0 in its a b s e n c e . Cell line
DO
Dq
D37
D10
n
p
D10MF lmM
3mM
Changliver (LiCH)
3.7±0.4
2.7±0.1
6.2±0.0
10.7±0.3
2.2±0.1
1.0±0.5
0.65
0.26
H.Ep. No. 2
5.2±0.2
2.6±0.4
7.3±0.7
14.0±1.0
1.5±0.1
0.6±0.1
0.76
0.51
HeLa-O
3.2±0.2
1.3±0.3
4.4±0.1
8.6±0.3
1.5±0.1
0.5±0.1
0.77
0.47
HeLa-S3
3.1±0.5
1.4±0.1
4.4±0.1
8.4±0.0
1.5±0.1
0.6±0.1
0.70
0.23
T-l~dney
4.0±0.1
2.2±0.7
5.8±1.1
10.7±1.6
1.8±0.3
0.8±0.4
0.75
0.64
351
TABLE
II
RELATIVE
PLATING
EFFICIENCIES
OF HUMAN
CELL LINES IN 1 AND 3 mM CAFFEINE
A p p r o x i m a t e a b s o l u t e plating e f f i c i e n c i e s ( A A P E ) and relative plating e f f i c i e n c i e s ( R P E ) for t h e c u r v e s in F i g . 1 e x p r e s s e d as m e a n -+ p r o p a g a t e d standard error. N o 4 4 - h c a f f e i n e t r e a t m e n t a l o n e s i g n i f i c a n t l y d e c r e a s e d t h e surviving f r a c t i o n . A A P E w a s e s t i m a t e d via h e m o c y t o m e t e r c o u n t . AAPE (0 mM)
R P E (1 m M )
RPE (3 raM)
Chang liver ( L i C H )
50%
1 0 5 -+ 5 %
1 0 6 +- 6 %
H.Ep. No. 2
80
9 9 -+ 7
9 9 +- 7
HeLa-O
35
9 8 -+ 7
110 + 7
HeLa-S3
20
1 4 1 -+ 1 4
T-1
50
1 1 4 +- 4
TABLE
1 4 8 +- 1 9 9 6 +- 7
III
TIME-DEPENDENT
CAFFEINE
TOXICITY
IN UNIRRADIATED
H.Ep. No. 2 CELLS
T h e e f f e c t s o f c a f f e i n e a l o n e o n the survival o f H . E p . N o . 2 cells. S u r v i v a l s are e x p r e s s e d as p e r c e n t a g e (-+ standard error) o f t h e 0 m M a b s o l u t e c o n t r o l for t h e e x p e r i m e n t . N o t i c e that all I m M p o i n t s are sign i f i c a n t l y a b o v e 1 . 0 s u r v i v a l and that 3 m M c a f f e i n e d o e s n o t b e c o m e t o x i c until after the third d a y . D a y s in c a f f e i n e
Surviving fraction (%)
1
2
3
4
5
6
7
lmM
129 + 8
117 + 10
1 1 7 -+ 1 3
1 1 7 +- 1 1
118 ± 17
117 + 9
1 2 9 -+ 9
3mM
120+
124+
99-+ 9
79-+ 4
10
9
94+
10
66 + 10
7 3 +- 8
in boiling water to effect dissolution of the caffeine was also without consequence as it yielded the same final experimental results. Discussion
Comparison of cell types It is evident that cultured human cell lines exhibit post-UV suppression of survival by non-toxic concentrations of caffeine. Presently, it is n o t possible from the available data to postulate with any certainty why Arlett's HeLa cells exhibited caffeine potentiation at 0.26 and 0.51 mM concentrations and the cells of Wilkinson et al. did not at 1 and 3 mM concentrations. As both used continuous exposures of caffeine after irradiation, there may have been marked clonal differences in caffeine sensitivity or differences in caffeine purity. A similar discrepancy is also indicated in molecular repair experiments. Both Fujiwara and Kondo [9] and Painter [21] used HeLa cells for post-replication repair studies. The former group found that 2 mM caffeine inhibited post-replication gap closure, whereas the latter seemingly did not. Painter [ 2 1 ] , however, did not interpret his data in this manner when presenting his three arguments against the post-replication "gap" concept of Lehmann
352 [18]. One of his arguments was based on the assumption that the effects of caffeine in Chinese hamster cells are the same as in h u m a n cells. He assumed the results of Cleaver and Thomas [7] (i.e., that caffeine inhibits bypasssynthesized DNA strands from rejoining in Chinese hamster cells) to be true for his HeLa cells. Consequently, he used 2 mM caffeine over a 2.5 h period in an a t t e m p t to accumulate gaps while synthesis was occurring in the presence of BUdR. Removal of the caffeine and BUdR and addition of tritiated thymidine would allow the gaps to be closed with the tritiated precursors. In an alkaline equilibrium density gradient, the BUdR-containing strands would sediment apart from the normal-density parental strands and, if gaps were closed with tritiated precursors, the heavier strands would be radioactively labeled as well. However, Painter f o u n d "absolutely no tritium in excess of that expected" and concluded that "gaps of the kind found in bacteria do n o t occur in mammalian cells". One possible reason for his n o t finding tritium label was that the caffeine did n o t prevent gap-filling, and the gaps, if they ever existed per se, were filled during the 2.5-h BUdR incubation. Viewed in this manner, Painter's data would support those of Buhl and Regan [5] and Lehmann et al. [19] (which were obtained with diploid h u m a n fibroblasts) and contradict those of Fujiwara and Kondo [9] (which, as Painter's, were obtained with HeLa cells). It is interesting to note here in the light of Painter's assumption that Roberts and Ward f o u n d that 0.75 mM caffeine inhibited post-replication repair in both sulphur mustard and N-methyl-N-nitrosourea alkylated Chinese hamster cells but not in HeLa cells [23], thus substantiating the possibility of significant differences in the DNA repair mechanisms between hamster and HeLa cells. Caffeine degradation may also be a consideration in the variability of results. A preliminary report by Goth and Cleaver [ 11 ] stated that in I mM tritated caffeine most of the pod in HeLa, normal, Lesch-Nyhan, and XP human cells consisted of caffeine degradation products. They did n o t irradiate their cells, however, and consequently the post-replication condition of the cells was n o t a factor in their findings. Our data (Table III) for 3 mM caffeine (but not for 1 mM) indicates that toxicity occurs to unirradiated H.Ep. No. 2 cells after the third day. Hence, there is an apparent need to clarify the fate of various concentrations of caffeine in unirradiated cells. In irradiated cells one would not expect to observe caffeine potentiation of lethality if the caffeine were essentially all degraded in three hours and the S-phase delay {and hence the repair period) induced by the UV alone were longer than three hours (any synergistic effects notwithstanding). Consequently, it appears that the caffeine responsible for the potentiation of post-UV lethality is n o t degraded in UV-irradiated cells -- at least n o t in 3 h. Also, some of the contradictions may be resolved by determination of the similarities and/or differences between primary cells or diploid cell strains and established lines, cells of normal origin and those of malignant origin, and cells of epithelioid and fibroblastic morphologies. Any one or a combination of the above factors m a y be related to the DNA repair capabilities of any given cell type. The evidence so far available suggests t h a t diploid fibroblasts tend to have a higher resistance to the effects of post-UV caffeine than do established epithelioid cell lines.
353
Synergism and caffeine toxicity Synergism between two agents occurs when the cooperative action of the agents together produces a more effective result than the sum of the results of the agents acting independently. As the survival curves in Fig. 1 are normalized to 1.0 survival for the 1 and 3 mM caffeine concentrations (thus controlling the magnitude of the action of the caffeine agent alone), and the 0 mM curve is present to indicate the magnitude of the action of the UV agent alone, any deviation from the 0 mM survival curve would indicate synergism in the strict meaning of the word. Some workers (e.g. refs. [2,3] ) choose to subscribe to the division probability hypothesis of Whitmore and Till [25] which states that a given agent modifies the probability t h a t a given cell and its progeny will divide and ultimately give rise to a colony. Consequently, for synergism to be expressed within the confines of this hypothesis, the probability for division must differ from the product (not the sum) of the division probabilities taken independently. Therefore, synergism would n o t be readily apparent from survival curves without normal normalization of the data points in accordance with the mathematics of this hypothesis, as the correlation between division probability and plating efficiency is non-linear. If there is no modification of the division probability induced by one of the agents alone, then synergism may be determined with the usual survival curve data without mathematical normalization. Tables II and III indicate that none of the conditions of caffeine treatment used in our UV experiments led to a decrease in plating efficiency and hence division probability. Even within the constraints of the division probability hypothesis [2,25], synergism is observed.
Effect of method of caffeine preparation The results of Wilkinson et al. [26] are difficult to explain. They reported to have used a 5% stock solution of caffeine. It was found in our laboratory that a 5% concentration made an opaque suspension which did n o t dissolve unless autoclaved at 121°C, and, if cooled to 37°C after autoclaving, it recrystallized. It is possible t h a t Wilkinson et al. did n o t have a 5% stock solution as believed but had instead a concentration closer to 2%, similar to ours. This concentration of caffeine renders the drug completely soluble at room temperature with recrystallization occurring at 4°C. If this were the case, then their concentrations of caffeine would have been less than half of that reported, possibly making it too low to yield statistically significant survival depression (judging from our data).
Conclusions and general discussion Contradictions must be resolved before it can be determined what the effects of caffeine on ultraviolet-irradiated h u m a n cells are. Unambiguous and explicit materials and methods sections in papers on the subject are needed, including cell-line origins, primary references to the origin of the clones used, and any obvious peculiarities each cell line is known to possess. A simple designation such as " H e L a cells", for example, is apparently insufficient without further clarification of clonal origin. The manufacturer and lot numbers of any biochemicals used (viz., caffeine) and how they were sterilized should also be stated.
354
Further, extrapolation of data b e y o n d the cell lines used is evidently hazardous. Data obtained with hamster cells alone, for example, are not adequate to make statements concerning "mammalian cells" in general. Perhaps detailed and correlative studies of molecular and survival data of mutants such as those of Klimek et al. [15] would provide useful information. Their subclones of HeLa-S3, particularly clones 46 and 18/2, may provide clues, as the 46 mutant appears that it may be defective for some form of DNA repair. As no correlation between molecular and survival data can be made at this time, it cannot be stated h o w caffeine affects post-replication repair and subsequently survival. Perhaps it can be concluded that cultured human cells in general exhibit post-irradiation suppression of survival by caffeine if the concentration of the drug is high enough and the exposure time is of appropriate duration.
Acknowledgements We wish to thank the following individuals of the Pennsylvania State University Departments of Microbiology and Biology who aided by supplying some of the cell cultures used in this study: Dr. J.J. Docherty, Dr. M.D. Notter, Dr. K.D. Thompson, C.J. Thompson, L. Trusal, L. Richardson and S.C. Thornton.
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