247
Mutation Research, 52 (1978) 247--253 © Elsevier/North-Holland Biomedical Press
ABSENCE OF INTERACTION BETWEEN X-RAYS AND UV L I G H T IN INDUCING OUABAIN- AND THIOGUANINE-RESISTANT MUTANTS IN CHINESE H A M S T E R CELLS
JAMES E. CLEAVER
Laboratory of Radiobiology, University of California, San Francisco, Calif. (U.S.A.) (Received 18 January 1978) (Revision received 5 May 1978) (Accepted 15 May 1978)
Summary Chinese hamster ovary cells were irradiated with X-rays at times from 0 to 17 h before being irradiated with ultraviolet (UV) light. No synergism was observed between the t w o radiations for the production of mutants resistant to either ouabain or 6-thioguanine. These experiments were designed to test whether X-rays induced an error-prone repair system that would increase the frequency of mutations produced by UV light, b u t no such system was detected.
Introduction
Most mutations in prokaryotes appear to be produced b y errors in a DNA replication system that is induced by radiations or chemical mutagens [14,16, 18,19,23--25]. Considerable interest has been aroused as to whether mammalian cells contain a similar inducible, error-prone repair system. But because eukaryotic cells do n o t show the characteristic prokaryotic response of DNA degradation after being damaged [2,4], and because the inducible system in prokaryotes appears to be partly involved with this degradation [8], inducible repair, if it exists in eukaryotes, m a y be substantially different from that of prokaryotes. A serious difficulty in designing experiments to detect an inducible errorprone repair system is that the same damage to DNA can be both a potential inducer of the repair system and the damage on which the induced system acts. Therefore, in the experiments described here, I used two different agents: X-irradiation to induce the putative repair system w i t h o u t inducing a large Work p e r f o r m e d
u n d e r t h e auspices o f the U , S . D e p a r t m e n t o f Energy.
248 number of mutants, and ultraviolet (UV) light to cause the damage on which the putative inducible repair system would then act. The effect of X-irradiation at various times before UV irradiation was determined by measuring the frequencies of two kinds of drug-resistant mutants produced by these radiations - - m u t a n t s resistant to ouabain and mutants resistant to 6-thioguanine. It has been suggested that cells can be made resistant to ouabain by point mutations, which can be produced by UV light [1,3,21] but cannot be made resistant to ouabain by deletions or frameshifts, which are the predominant modes of action of X-rays and some chemicals [1,3,7,21]. Cells can be made resistant to 6-thioguanine, however, by many kinds of mutations that result in either loss or changes in function of hypoxanthine-guanine phosphoribosyl transferase. In the experiments reported here, X-rays caused no mutations at one of the loci studied, that of ouabain resistance. Therefore, any synergism between X-rays and UV light in inducing mutations in these experiments could constitute evidence for induction of an error-prone repair system that increases the number of mutations produced by UV light. Materials and methods Chinese hamster ovary (CHO) cells were grown in Eagle's minimum essential medium plus 15% fetal calf serum. Under routine conditions their doubling time was 12 h and the plating efficiency was between 50 and 90%. Stock cultures were maintained by routine subculturing at low density to keep the frequency of spontaneous mutants at very low levels (ouabain-resistant mutants were less than 10 -8 and 6-thioguanine-resistant mutants were less than 2 × 10-6). Cell survival was determined as described elsewhere [10]. For m u t a t i o n experiments with combined X- and UV irradiations, petri dishes containing monolayers of about 106 cells were irradiated with 300 rads of X-rays (100 kVp, Hewlett-Packard Faxitron), allowed to grow for 0--17 h, and then irradiated with 13 J/m 2 of 254-nm UV light (1.3 J/m 2 • sec). The dose rate from this soft X-ray source was estimated by comparing the survival curves for CHO cells irradiated under similar conditions by the Faxitron and by 250kVp from a General Electric Maxitron 300 that had been calibrated with LiF crystals [10]. A dose of 300 rads (100 kVp) caused a growth delay of about 6 h and decreased plating efficiency of single cells to about 85% of the control value. Cultures were supplied with fresh medium, grown for several days, transferred to roller bottles, and maintained in exponential growth by occasional subculturing as required for at least 6 days after the UV irradiation (about 12 cell divisions). By this time, cells had passed through the period of phenotypic lag (expression time), and mutation frequencies had reached constant levels [ 2 2 ] (Table 1). The minimum expression time for ouabain resistance was 2 days and for 6-thioguanine resistance, 6 days (Table 1). Cultures were then trypsinized, diluted to 5 X 10 s cells/ml, and inoculated into 90-mm petri dishes containing 10 ml of medium supplemented with 3 mM ouabain (final density, 10s--106 cells per dish), 0.06 mM 6-thioguanine (final density, 5 X 104--2 × 10 s cells per dish), or drug-free medium (final density, 5--50 cells per dish). After surviving colonies were allowed to develop for 7--8 days, cultures were fixed, stained, and counted. The m u t a t i o n frequency
249 TABLE 1 FREQUENCIES IRRADIATION
OF DRUG-RESISTANT
Time after UV
CHO CELLS RECOVERED
O u a b a i n - r e s i s t a n t cells (3 m M ) a
AT VARIOUS TIMES AFTER UV
6 - T h i o g u a n i n e - r e s i s t a n t cells b
(XIO $)
(XlO s ) 0days(control) 0days(UV) 2 4 6 7 8 9 10
~0.001 0.10±0.03 2.6 ± 0 . 9 1.9 ± 0 . 3 -2.5 ± 0 . 8 --2.0 ± 0 . 4
~0.2 -10.0±3.3 25.0±5.0 35.8±7.7 31.0±6.1 28.5±5.6 22.8±2.2 32.3±3.0
a O n e e x p e r i m e n t p e r f o r m e d a t 15.6 J / m 2. E r r o r c i t e d is c a l c u l a t e d f r o m P o i s s o n f o r m u l a : i f a t o t a l o f N m u t a n t c o l o n i e s w e r e s c o r e d , e r r o r c i t e d is N1/2. b D a t a p o o l e d f r o m m a n y s e p a r a t e e x p e r i m e n t s at 10 J / m 2. E r r o r c i t e d is s t a n d a r d e r r o r c a l c u l a t e d f o r 4 or m o r e s e p a r a t e d e t e r m i n a t i o n s at e a c h tLme p o i n t .
per dish was calculated from the ratio of plating efficiencies of cells grown with ouabain or 6-thioguanine to plating efficiencies of cells grown with drug-free medium [3]. The mutation frequencies per dish decreased at the higher cell concentrations for 6-thioguanine resistance because of density-dependent cell interaction [1]. Only dishes from the range of cell concentrations for which mutation frequencies were constant were used for calculating average mutation frequencies. Average mutation frequencies (F) were calculated by adding together the m u t a n t colonies from all acceptable dishes according to the formula N +- N 1/2 F =
N0 × P.E.
where N = the total number of clonnies scored, N i n = the random sampling error (assuming Poisson statistics), No = the total number of cells plated, and P.E. = the plating efficiency in drug-free medium. F was calculated after X-irradiation, UV irradiation, and X-irradiation followed by UV (Fx, F u , and Fxu, respectively); the effect of prior X-irradiation on UV-induced mutation frequencies (relative mutation frequency, S) was estimated according to the formula S - Fxu -- Fx Fu Error limits for S were obtained by combining the Poisson errors in Fxu, Fx, and Fu; Fx and Fu were determined for every time interval between X- and UV irradiation. Results
Dose--response relationships for both drug-resistant markers were linear for X-rays and UV light separately [3] ; the yields were 1.3 + 0.4 X 10-7/rad and
250
3.6 -+ 0.2 × 10-s/J/m 2 for 6-thioguanine and 1.0 ± 0.2 × 10-6/J/m 2 for ouabain. X-rays were a less efficient mutagen than UV light at comparable levels of survival for both markers. When cells were irradiated with 300 rads of X-rays (85% survival) followed by 13 J/m 2 of UV light (40% survival} with intervals between the dose of up to 17 h, no synergism was detected at any time interval (Fig. 1). The number of points (2 of 15 for ouabain and 1 of 17 for 6-thioguanine) lying above the level expected for no interaction between X-irradiation and UV irradiation was too few to be significant. These results contrast with those obtained by others for cell survival [9] and transformation [6] in which synergism was detected, although these phenomena were n o t directly measured in the current experiments. Presumably the mechanisms involved in cell killing and transformation are different from the mechanisms of mutagenesis.
OUABAIN 2.0
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o ii
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I
Mutation Research, 52 (1978) 247--253 © Elsevier/North-Holland Biomedical Press
ABSENCE OF INTERACTION BETWEEN X-RAYS AND UV L I G H T IN INDUCING OUABAIN- AND THIOGUANINE-RESISTANT MUTANTS IN CHINESE H A M S T E R CELLS
JAMES E. CLEAVER
Laboratory of Radiobiology, University of California, San Francisco, Calif. (U.S.A.) (Received 18 January 1978) (Revision received 5 May 1978) (Accepted 15 May 1978)
Summary Chinese hamster ovary cells were irradiated with X-rays at times from 0 to 17 h before being irradiated with ultraviolet (UV) light. No synergism was observed between the t w o radiations for the production of mutants resistant to either ouabain or 6-thioguanine. These experiments were designed to test whether X-rays induced an error-prone repair system that would increase the frequency of mutations produced by UV light, b u t no such system was detected.
Introduction
Most mutations in prokaryotes appear to be produced b y errors in a DNA replication system that is induced by radiations or chemical mutagens [14,16, 18,19,23--25]. Considerable interest has been aroused as to whether mammalian cells contain a similar inducible, error-prone repair system. But because eukaryotic cells do n o t show the characteristic prokaryotic response of DNA degradation after being damaged [2,4], and because the inducible system in prokaryotes appears to be partly involved with this degradation [8], inducible repair, if it exists in eukaryotes, m a y be substantially different from that of prokaryotes. A serious difficulty in designing experiments to detect an inducible errorprone repair system is that the same damage to DNA can be both a potential inducer of the repair system and the damage on which the induced system acts. Therefore, in the experiments described here, I used two different agents: X-irradiation to induce the putative repair system w i t h o u t inducing a large Work p e r f o r m e d
u n d e r t h e auspices o f the U , S . D e p a r t m e n t o f Energy.
248 number of mutants, and ultraviolet (UV) light to cause the damage on which the putative inducible repair system would then act. The effect of X-irradiation at various times before UV irradiation was determined by measuring the frequencies of two kinds of drug-resistant mutants produced by these radiations - - m u t a n t s resistant to ouabain and mutants resistant to 6-thioguanine. It has been suggested that cells can be made resistant to ouabain by point mutations, which can be produced by UV light [1,3,21] but cannot be made resistant to ouabain by deletions or frameshifts, which are the predominant modes of action of X-rays and some chemicals [1,3,7,21]. Cells can be made resistant to 6-thioguanine, however, by many kinds of mutations that result in either loss or changes in function of hypoxanthine-guanine phosphoribosyl transferase. In the experiments reported here, X-rays caused no mutations at one of the loci studied, that of ouabain resistance. Therefore, any synergism between X-rays and UV light in inducing mutations in these experiments could constitute evidence for induction of an error-prone repair system that increases the number of mutations produced by UV light. Materials and methods Chinese hamster ovary (CHO) cells were grown in Eagle's minimum essential medium plus 15% fetal calf serum. Under routine conditions their doubling time was 12 h and the plating efficiency was between 50 and 90%. Stock cultures were maintained by routine subculturing at low density to keep the frequency of spontaneous mutants at very low levels (ouabain-resistant mutants were less than 10 -8 and 6-thioguanine-resistant mutants were less than 2 × 10-6). Cell survival was determined as described elsewhere [10]. For m u t a t i o n experiments with combined X- and UV irradiations, petri dishes containing monolayers of about 106 cells were irradiated with 300 rads of X-rays (100 kVp, Hewlett-Packard Faxitron), allowed to grow for 0--17 h, and then irradiated with 13 J/m 2 of 254-nm UV light (1.3 J/m 2 • sec). The dose rate from this soft X-ray source was estimated by comparing the survival curves for CHO cells irradiated under similar conditions by the Faxitron and by 250kVp from a General Electric Maxitron 300 that had been calibrated with LiF crystals [10]. A dose of 300 rads (100 kVp) caused a growth delay of about 6 h and decreased plating efficiency of single cells to about 85% of the control value. Cultures were supplied with fresh medium, grown for several days, transferred to roller bottles, and maintained in exponential growth by occasional subculturing as required for at least 6 days after the UV irradiation (about 12 cell divisions). By this time, cells had passed through the period of phenotypic lag (expression time), and mutation frequencies had reached constant levels [ 2 2 ] (Table 1). The minimum expression time for ouabain resistance was 2 days and for 6-thioguanine resistance, 6 days (Table 1). Cultures were then trypsinized, diluted to 5 X 10 s cells/ml, and inoculated into 90-mm petri dishes containing 10 ml of medium supplemented with 3 mM ouabain (final density, 10s--106 cells per dish), 0.06 mM 6-thioguanine (final density, 5 X 104--2 × 10 s cells per dish), or drug-free medium (final density, 5--50 cells per dish). After surviving colonies were allowed to develop for 7--8 days, cultures were fixed, stained, and counted. The m u t a t i o n frequency
249 TABLE 1 FREQUENCIES IRRADIATION
OF DRUG-RESISTANT
Time after UV
CHO CELLS RECOVERED
O u a b a i n - r e s i s t a n t cells (3 m M ) a
AT VARIOUS TIMES AFTER UV
6 - T h i o g u a n i n e - r e s i s t a n t cells b
(XIO $)
(XlO s ) 0days(control) 0days(UV) 2 4 6 7 8 9 10
~0.001 0.10±0.03 2.6 ± 0 . 9 1.9 ± 0 . 3 -2.5 ± 0 . 8 --2.0 ± 0 . 4
~0.2 -10.0±3.3 25.0±5.0 35.8±7.7 31.0±6.1 28.5±5.6 22.8±2.2 32.3±3.0
a O n e e x p e r i m e n t p e r f o r m e d a t 15.6 J / m 2. E r r o r c i t e d is c a l c u l a t e d f r o m P o i s s o n f o r m u l a : i f a t o t a l o f N m u t a n t c o l o n i e s w e r e s c o r e d , e r r o r c i t e d is N1/2. b D a t a p o o l e d f r o m m a n y s e p a r a t e e x p e r i m e n t s at 10 J / m 2. E r r o r c i t e d is s t a n d a r d e r r o r c a l c u l a t e d f o r 4 or m o r e s e p a r a t e d e t e r m i n a t i o n s at e a c h tLme p o i n t .
per dish was calculated from the ratio of plating efficiencies of cells grown with ouabain or 6-thioguanine to plating efficiencies of cells grown with drug-free medium [3]. The mutation frequencies per dish decreased at the higher cell concentrations for 6-thioguanine resistance because of density-dependent cell interaction [1]. Only dishes from the range of cell concentrations for which mutation frequencies were constant were used for calculating average mutation frequencies. Average mutation frequencies (F) were calculated by adding together the m u t a n t colonies from all acceptable dishes according to the formula N +- N 1/2 F =
N0 × P.E.
where N = the total number of clonnies scored, N i n = the random sampling error (assuming Poisson statistics), No = the total number of cells plated, and P.E. = the plating efficiency in drug-free medium. F was calculated after X-irradiation, UV irradiation, and X-irradiation followed by UV (Fx, F u , and Fxu, respectively); the effect of prior X-irradiation on UV-induced mutation frequencies (relative mutation frequency, S) was estimated according to the formula S - Fxu -- Fx Fu Error limits for S were obtained by combining the Poisson errors in Fxu, Fx, and Fu; Fx and Fu were determined for every time interval between X- and UV irradiation. Results
Dose--response relationships for both drug-resistant markers were linear for X-rays and UV light separately [3] ; the yields were 1.3 + 0.4 X 10-7/rad and
250
3.6 -+ 0.2 × 10-s/J/m 2 for 6-thioguanine and 1.0 ± 0.2 × 10-6/J/m 2 for ouabain. X-rays were a less efficient mutagen than UV light at comparable levels of survival for both markers. When cells were irradiated with 300 rads of X-rays (85% survival) followed by 13 J/m 2 of UV light (40% survival} with intervals between the dose of up to 17 h, no synergism was detected at any time interval (Fig. 1). The number of points (2 of 15 for ouabain and 1 of 17 for 6-thioguanine) lying above the level expected for no interaction between X-irradiation and UV irradiation was too few to be significant. These results contrast with those obtained by others for cell survival [9] and transformation [6] in which synergism was detected, although these phenomena were n o t directly measured in the current experiments. Presumably the mechanisms involved in cell killing and transformation are different from the mechanisms of mutagenesis.
OUABAIN 2.0
>~J Z 1.0 14J
o ii
Z
_o
I
I--
i
J
I
