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A Chinese hamster ovary cell line hypersensitive to ionizing radiation and deficient in repair replication
109
Mutation Research, 193 (1988) 109-121 DNA Repair Reports Elsevier M T R 06262
A Chinese hamster ovary cell line hypersensitive to ionizing radiation and deficient in repair replication Lloyd F. Fuller and Robert B. Painter Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, CA 94143 (U.S.A.) (Received 6 May 1987) (Revision received 4 September 1987) (Accepted 8 September 1987)
Keywords: Chinese hamster ovary; Radiosensitivity; Excision repair; Hypomutability.
Summary An X-ray-sensitive Chinese hamster ovary cell line was isolated by means of a semi-automated procedure in which mutagenized cells formed colonies on top of agar, were X-irradiated, and were photographed at two later times. We compared the photographs to identify colonies that displayed significant growth arrest. One of the colonies identified in this manner produced a stable line (irslSF) that is hypersensitive to ionizing radiation. The X-ray dose at which 10% of the population survives (D10) is 2.25 Gy for irslSF and 5.45 Gy for the parental line. The new mutant is also moderately sensitive to ethyl methanesulfonate, irslSF performs only half as much X-ray-induced repair replication as the parental line, indicating a defect in excision repair. This defect is believed to be the primary cause of the line's radiosensitivity. Although irslSF repairs DNA double-strand breaks at a normal rate, it repairs single-strand breaks more slowly .than normal, irslSF has an elevated number of spontaneous chromatid aberrations and produces significantly higher numbers of X-ray-induced chromatid aberrations after exposure during the G 1 phase of the cell cycle. The line is hypomutable, with X-ray exposure inducing only one-third as many 6-thioguanine-resistant colonies as the parental line.
There is an increasing interest in the discovery and characterization of cell lines hypersensitive to ionizing radiation. Initially, radiosensitive strains were discovered in the course of other studies. The radiosensitive LY-S strain arose spontaneously in the L5178Y mouse leukemia line (Alexander, 1961). V79/79, a radiosensitive strain derived from the V79 hamster lung fibroblast, was isolated during a study of X-ray-induced small-colony formaCorrespondence: Dr. Robert B. Painter, Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, CA 94143 (U.S.A.).
tion (Sinclair, 1964). The radiosensitivity of ataxia telangleetasia (A-T) cells was discovered during the use of radiotherapy against tumors in patients with this disease (Taylor et al., 1975). More recently, several investigators have isolated radiosensitive lines by screening mutagenized populations for colonies hypersensitive to X-rays. The anchorage-independent L5178Y line was the first line used in these attempts because it could be easily replica plated (Sato and Hieda, 1979; Shiomi et al., 1981; Sat<) et al., 1983). The Chinese hamster ovary (CHO) cell line has been another source of radiosensitive mutants. Thomp-
0167-8817/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
110 son et al. (1980b) used challenging doses of ultraviolet (UV) light, mitomycin C, and ethyl methanesulfonate (EMS) to isolate several lines sensitive to DNA damage, including EM9-1, which is sensitive to EMS and X-rays. Replica-plating techniques were used by Stamato et al. (1983) and Jeggo and Kemp (1983) to isolate the XR-1 line and the 6 xrs lines, respectively. A variety of phenotypes is displayed among the radiosensitive CHO lines. The EM9-1 line has a remarkably high rate of spontaneous sister-chromatid exchange (SCE) and is deficient in DNA single-strand-break rejoining (Thompson et al., 1982). The XR-1 and xrs lines are defective in double-strand-break repair (Giaccia et al., 1985; Kemp et al., 1984). The responses of the xrs lines to UV, methyl methanesulfonate, N-methyl-N'nitro-N-nitrosoguanidine, and EMS range from very sensitive to wild type. Recently, increased radiation-induced chromosome and chromatid damage has been observed in the xrs-6 and xrs-7 lines (Kemp and Jeggo, 1986). The xrs-7 line showed chromatid damage after irradiation in the G 1 phase of the cell cycle, an unusual characteristic shared only by A-T cells (Taylor et al., 1976; Nagasawa and Little, 1983) and the new radiosensitive CHO mutant described here. We report the isolation of an X-ray-sensitive CHO cell line that is competent in double-strandbreak repair but deficient in X-ray-induced repair replication, shows abnormally high rates of spontaneous and X-ray-induced chromatid aberrations, and has a reduced frequency of X-ray-induced mutations. Materials and methods Cell culture
AA8 cells (Thompson et al., 1980a) were used as the parental line. EM9-1, which is sensitive to EMS and X-rays, was used as a positive control (Thompson et al., 1982). All cells were grown in minimal essential medium (Gibco, Santa Clara, CA) with 10% fetal calf serum and 50 /~g/ml gentamycin (Gibco). Mutant isolation
Mutants were isolated by a modification of the technique of Busch et al. (1980) using the colony-
screening system previously described (Glaser and Wattenburg, 1966; Sevastopoulos et al., 1977; Konrad et al., 1977). A population of AA8 cells was mutagenized by a 24-h exposure to 1.5/~g/ml ICR-191, grown for 5 days to express damage, and then sprayed onto 400-600 10-cm petri dishes containing 10 ml of minimal essential medium with 5% fetal calf serum and 0.7% noble agar (Gibco). After 4 more days of growth, the cells had formed approximately 300 small colonies/ dish, and these were irradiated with 1 Gy of X-rays from a Philips RT250 therapeutic unit (250 kVp, 15 mA, half-value layer 1.06 mm Cu). The dishes were photographed 1.5 and 3.5 days after irradiation. The photographic negatives were compared to identify colonies that showed little growth between the time the two photographs were taken, indicating X-ray sensitivity. These colonies were isolated and expanded for further study. Chromosome preparation
Chromosome preparations were made by adding colcemid (final concentration 10-7 M) to the flasks, waiting 30 min, and then gently swirling the flasks to detach mitotic cells. Detached cells were centrifuged and resuspended in 75 mM KC1 for 5 min. The cells were fixed with 3 changes of Carnoy's fixative (3:1, methanol/acetic acid), then dropped onto microscope slides. The slides were stained with 5% Giemsa in water and dried, a coverslip was attached with DePex, and the chromosome preparations were viewed at 1000× magnification. Cell cycle determination
The method of fraction of labeled mitoses was used (Painter and Drew, 1959) to determine cell cycle times. For each cell line, 2 × 103 cells were inoculated into 22 flasks. 10 h before collections began, 11 flasks of each line were incubated for 30 rain with 0.05/~Ci/ml [3H]thymidine ([3H]dThd) (spec. act., 300 mCi/mmole). [3H]dThd was added to the other 11 flasks 30 min before collections began. Mitotic cells were harvested every hour, and chromosome preparations were made as described above. Autoradiographs were made using Ilford IL-4 emulsion, exposed at 4 ° C for 7 days, and developed in Kodak D19 for 5 min. The fraction of labeled mitoses was scored for each time period.
111
X-Ray-induced chromosomal aberrations in GI Cells were synchronized by mitotic detachment (Terasima and Tolmach, 1963) using colcemid. A sample of these cells was used to measure the mitotic index, and only those populations with a mitotic index > 98% were used. The cells were divided into four 75-cm2 flasks and allowed 1 h to attach. Cells in 3 flasks were irradiated with 0, 0.5, or 1 Gy (dose rate, 1.5 Gy/min). To determine the fraction of cells in S phase, cells in the fourth flask were incubated with 0.05/xCi/ml [3H]dThd (300 mCi/mmole) while the other flasks were being irradiated. After irradiation, the medium in all the flasks was replaced with medium containing colcemid. After approximately 18 h the cells were harvested, and chromosome preparations and autoradiographs were made as described above. X-Ray-induced chromosomal aberrations in G2 Flasks containing exponentially growing cells were irradiated with 0.25 or 0.5 Gy (dose rate, 1.5 Gy/min). Mitotic cells were harvested 2 or 3 h after irradiation as described in the cell cycle study, and chromosome preparations were made. Cells from a duplicate set of flasks were pulse labeled with [3H]dThd as described in the cell cycle study and treated in parallel to be sure that the mitotic cells were not in S phase at the time of irradiation. Chromosome preparations and autoradiographs were made as described above. X-Ray dose-response curve Cells were exposed to 1-6 Gy of X-rays (dose rate, 2 Gy/min) as a suspension in medium at 4 ° C under 5% CO2 in air at a dose rate of 2 Gy/min. After irradiation, the cell suspensions were diluted and inoculated into 100-cm polystyrene dishes. After 12 days the colonies were fixed with phosphate-buffered formalin, stained with 5% Giemsa in water, and counted. UV dose-response curoe Either 300 or 3000 cells were inoculated into 10-cm dishes and then incubated for 1 h to allow the cells to attach. The dishes were drained, rinsed with phosphate-buffered saline (PBS), and 200/d of PBS were added to each dish. Two dishes from each cell line were placed in a UV light apparatus (Steier and Cleaver, 1969) and irradiated for 0.5-9
sec at a rate of 1.3 J/mZ/sec. The dishes were then rinsed with PBS and refilled with normal growth medium. After 12 days of growth, the colonies were fixed and counted as described for the X-ray dose-response curve.
Ethyl methanesulfonate dose-response curve The technique used to generate the EMS dose-response curve is modeled on that of Thompson et al. (1980a). Cells in suspension were treated with EMS for 1 h at 37°C in 50-ml polypropylene centrifuge tubes. The cells were then pelleted by centrifugation, resuspended in growth medium, and diluted into dishes. After 12 days of growth, the colonies were fixed and counted as described for the X-ray dose-response curve. Alkaline filter elution Repair of single-strand breaks was measured by alkaline filter elution (Kohn et al., 1981). Cells were labeled by 24-h growth in medium containing 0.01 /~Ci/ml [14C]dThd (spec. act., 50 m C i / mmole), irradiated with 4 Gy of X-rays at 4" C, incubated at 37°C for various times to allow repair, and then immersed in cold PBS. AA8 cells labeled by 24-h growth in 0.1 /~Ci/ml [3H]dThd (50 Ci/mmole) and irradiated with 4 Gy but not allowed to repair were used as internal standard cells. For the elution, 14C-labeled cells were mixed with 3H-labeled internal standard cells, placed on the filters, rinsed with cold PBS, and then lysed and digested for 30 min with a solution containing 0.2% sodium dodecyl sulfate, 25 mM EDTA, pH 9.7, and 0.5 #g/ml proteinase K. This solution was replaced with the pH 12.1 alkaline elution buffer, which was pulled through the filter and collected as 1-ml fractions for 14 h. At pH 12.1 the complementary DNA strands separate and the DNA eluted from the filter is single-stranded. The fractions were neutralized with 1 N HC1 and their radioactivity was determined by liquid scintillation spectrometry. Neutral filter elution Repair of double-strand breaks was measured by neutral filter elution (Bradley and Kohn, 1979). The neutral filter elution technique is the same as the alkaline filter elution technique except for the use of a pH 9.6 elution buffer. At this lower pH,
112 the complementary DNA strands do not separate and the DNA eluting from the filter is doublestranded. Double-strand breaks were induced with 50 or 100 Gy of X-rays (dose rate, 2 Gy/min).
X-Ray-induced repair replication X-Ray-induced repair replication was measured by methods based on those of Painter and Young (1972) and Smith et al. (1981). Cells were labeled by 2 days of growth in medium containing 0.4 /zCi/ml 32p-labeled orthophosphate. Approximately 16 h before irradiation, the 32p was removed, and 2 × 106 cells were placed in each 60-mm dish. 90 min before irradiation, the medium was replaced with medium containing 10 -5 M bromodeoxyuridine (BrdUrd) and 10 -6 M fluorodeoxyuridine (FdUrd). 30 min before irradiation, this medium was replaced with medium containing BrdUrd and FdUrd in the concentrations listed above plus 2 mM hydroxyurea. Just before irradiation, the medium was replaced with medium containing BrdUrd, FdUrd, hydroxyurea, and 100 /~Ci/mi [3H]dThd (50 Ci/mmole). The cells were irradiated with 100-300 Gy (dose rate, 10 Gy/min) at 25 ° C, then incubated at 37 °C for 3 h. The DNA was isolated by treating the cells with a solution containing 50 mM NaC1, 40 mM Tris, pH 8, 20 mM EDTA, 100 /zg/ml proteinase K, and 100 # g / m l RNAase, followed by extraction with phenol and chloroform and dialysis against 3 changes of SSC (0.15 M sodium chloride, 0.015 M sodium citrate). The DNA was sheared using a Virtis homogenizer (1 rain at 20000 rpm). The DNA solution was added to 4.8 g CsC1, 1 g Cs2SO4, 0.5 ml 1 N NaOH, and sufficient SSC to make 10.3 g final weight. The solution was centrifuged at 40000 rpm for 36 h in a 50 Ti rotor (Beckman) and then collected from the bottom of the tube in approximately 22 fractions. Samples of these fractions were counted by scintillation spectrometry to determine the light and heavy DNA peaks. The fractions containing the light peak were pooled, then rebanded by the technique described above. The fractions from the rebanding were collected in test tubes and chilled to 4 ° C. The DNA was precipitated by adding I ml of 0.02% (w/v) salmon sperm DNA in 0.4 N NaOH, then 1 ml of 4% perchloric acid (PCA). The precipitate was collected on glass fiber filters and
rinsed with 4% PCA, followed by a 70% alcohol rinse and a 100% alcohol rinse. The filters were dried and placed in vials for scintillation spectrometry. The specific activity of the DNA from unirradiated cells was determined by isolating DNA by the technique described above, measuring absorbance at 260 nm, and determining 32p radioactivity by scintillation spectrometry.
X-Ray-induced inhibition of DNA synthesis This experiment was performed as described by Painter and Young (1980). Cells (about 5 × 10 4) were inoculated into 20-mm dishes containing medium with 0.05 # C i / m l [14C]dThd (50 m C i / mmole) and allowed to grow for 2 days. The medium was replaced with nonradioactive medium, and 16 h later the cells were irradiated with 5-20 Gy (dose rate, 2 Gy/min) at 25°C, then returned to 37 o C. At various times, duplicate sets of dishes were removed from the incubator and the medium was replaced with medium containing 1 #Ci/ml [3H]dThd (50 Ci/mmole). After 10 min the dishes were quickly drained and immersed in ice-cold SSC. The dishes were drained, 1 ml of 0.02% (w/v) salmon sperm DNA in 0.4 N NaOH was added, and the contents of the dishes were scraped into test tubes. 4% PCA (1 ml) was added to the test tubes, and the precipitate was collected as described above.
Frequency of X-ray-induced mutations Mutant cells were selected by resistance to the drug 6-thioguanine (Hsie et al., 1979; Jostes et al., 1980; Thompson et al., 1980a). Exponentially growing populations of cells were irradiated at X-ray doses chosen to produce survival levels of about 100, 50, 10, and 1% in the two cell lines. Sufficient cells were irradiated so that 100 viable mutant cells were expected in each group, based on wild-type frequencies of mutation induction (1.3 × 10 -5 mutants/Gy) (Cleaver, 1977; Hsie et al., 1978). After irradiation, the cells were grown for 9 days to express the mutant phenotype. Mutant cells were selected by inoculating l0 s cells into 10-cm petri dishes containing medium with 4 /~g/ml 6-thioguanine. Colonies were allowed to form for 12 days and were then fixed with phosphate-buffered formalin and stained with Giemsa.
113
Sister-chromatid exchange
100
The rate of spontaneous SCE was measured by the fluorescence-plus-Giemsa technique described by Perry and Wolff (1974), with the modifications described by Morgan et al. (1983). The cells were grown for two cell cycles in 10 #M BrdUrd, and chromosome preparations were made as described above. Before staining, the slides were immersed in 5 p g / m l of Hoechst 33258 in Sorensen's phosphate buffer (0.067 M Na2HPO 4 and KH2PO4, pH 6.8) for 20 min. The slides were rinsed, the coverslips were mounted with Sorensen's buffer, and the slides were placed on a slide warmer at 55 °C under black light for 4 min. The coverslips were removed and the slides were stained in 55[ Giemsa for 5 min. The slides were then rinsed and dried, and the frequency of SCEs in 100 cells of each line was determined.
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Results 1
Mutant isolation
0
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2
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5
6
7
8
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UV Exposure in seconds (1.3 J/m2-sec)
From approximately 60 000 colonies of mutagenized AA8 cells examined, 66 colonies were cho-
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X-ray Exposure (Gy) Fig. 1. X-Ray survival curves of the AA8 parental line (i), the EMS- and X-ray-sensitive EM9-1 line (A), and irslSF (O). The bars indicate mean + S.D.
0
1~
i 2~
i 4~
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600
900
EMS Concentration (pg/ml) Fig. 2. (A) UV survival curves of AA8 (m) and irslSF (O). (B) EMS survival curves of AA8 (m), irslSF (O), and EM9-1 (A). The bars indicate m e a n + S.D.
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Fraction [3H] DNA Retained Fig. 3. Alkaline filter elution profiles of D N A after irradiation with 4 G y of X-rays. (A) AA8 cells. Minutes of repair: 0 (=a); 10 (i); 20 (12); no X-rays ([]). (B) irslSF. Minutes of repair: 0 ( ~ ) ; 10 (O); 20 (O); no X-ray (~9). 14C-Labeled D N A is from cells that were irradiated and allowed to repair for various times. 3H-Labeled D N A is from internal standard cells that were irradiated but not allowed to repair. Each figure shows 1 set of a pair of duplicate profiles. TABLE 1 X-RAY-INDUCED CHROMOSOME AND CHROMATID ABERRATIONS Treatment
Irradiation in G1 AA8
Clone 22
X-ray dose (Gy)
Labeled mitoses (%)
Total n u m b e r of chromosomes
Chromatid and isochromatid deletions
Chromatid exchanges
0 0.5 1.0
3.6
2182 2133 2318
1 2 6
0 0 2
0 0.5 1.0
2.7
2112 2774 2181
15 a 11 b 21 c
12 a 27 a
0 0 2
2042 2257 2172
0 14 27
Irradiation in G : (Harvested 2 h after irradiation) AA8 0 0.25 0.50 Clone 22
0 0.25 0.50
0 0 0
1970 2251 2051
7c 20 26
AA8
0 0.25 0.50
50 14 13
1995 2044 2034
2 29 32
Clone 22
0 0.25 0.50
36.6 14 9.6
1930 1941 2006
17 a 34 75 a
a Significantly different from AA8 ( p < 0.001, Student's t test). b p < 0.05. c v < 0.01.
40
a
0 0 5
Chromosome aberrations
Damaged cells (%)
5 16 62
6 17 52
2 35 c 34 h
24 51 56
0 0 2
0 14 27.5
2 4 ~' 5
10 20 31
0 1 4
3 4 1
5 1 31
1 9 b 18 c
7 1 0
21 35 63
4b 15 a 19 c
115
sen for further study. Of the 62 viable colonies, 42 displayed normal or only modest sensitivity on the initial test, and 19 showed initial sensitivity followed by a return to normal sensitivity over a period of approximately 2 months, irslSF was the only clone that showed stable X-ray sensitivity. Similar experiences with instability of X-ray sensitivity have previously been reported during attempts to isolate X-ray-sensitive lines (Sinclair, 1964; Todd, 1968).
Cell cycle times Examination of the curves for fraction of labeled mitoses versus time for the parental and mutant lines showed that AA8 has a cell cycle time of 12 h, with a 1.5-h G 1 phase, 8-h S phase, and 2.5-h G2 + M interval, whereas irslSF has a cell cycle time of 14.5 h, with a 0.5-h G 1 phase, 10.5-h S phase, and 3.5-h G 2 + M interval.
irslSF rejoined 50% of the single-strand breaks in about 11 min, compared to 5 min for AA8 (Fig. 4). Neutral elution profiles and curves of doublestrand-break rejoining that were generated in the same manner show that irslSF rejoins doublestrand breaks as fast as or faster than AA8 (Figs. 5 and 6).
X-Ray-induced repair replication Alkaline CsC1-Cs2SO 4 equilibrium density gradients are shown in Fig. 7 to illustrate the separation of normal-density DNA from BrdUrdsubstituted DNA. Repair replication is plotted in Fig. 8. irslSF performed approximately half as much repair replication in the dose range of 100-300 Gy as did AA8, indicating a significant defect in the excision repair of X-ray-induced base damage in irslSF.
Sensitivity to DNA-damaging agents irslSF is sensitive to X-rays, with a much smaller shoulder on its X-ray survival curve compared to the AA8 parer~tal line (Fig. 1). The D10 value for irslSF is 2.2 Gy, whereas AA8 and EM9-1 have values of 5.4 Gy and 3.15 Gy, respectively, irslSF also displays moderate sensitivity to UV light and EMS (Fig. 2). DNA strand-break repair Profiles of the alkaline elution of DNA from filters, constructed by plotting for each fraction the percentage of 14C-labeled DNA retained on the filter against the percentage of 3H-labeled internal standard DNA retained on the filter, show that irslSF rejoins single-strand breaks more slowly than does AA8 (Fig. 3). Curves showing rate of single-strand-break rejoining are constructed from these elution profiles by measuring the vertical distance between profiles. The vertical distance between the profiles of unrepaired and unirradiated cells is linearly proportional to the total number of breaks originally induced by the radiation exposure, whereas the vertical distance between the profiles of unrepaired and repaired cells is proportional to the number of breaks repaired during the 37 o C incubation period (Kohn et al., 1976). When combined and plotted as a function of time, these measurements indicate that
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20
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Repair Time (min)
Fig. 4. Rate of single-strand-break rejoining determined from the alkaline filter elution profiles shown in Fig. 3. The two points for each cell line and each repair time are from elution profiles from duplicate samples. II, AAS; O, irslSF.
116
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Fig. 5. Neutral filter elution profiles of DNA after irradiation with 50 or 100 Gy of X-rays. (A) AA8 cells after 50 Gy. Hours of repair: 0 (~); 1 (11); 2 (D); no X-rays ([]). (B) irslSF after 50 Gy. Hours of repair: 0 (~); 1 (O); 2 (O); no X-rays (~). (C) AA8 cells after 100 Gy. (D) irslSF after 100 Gy. Symbols are the same as in A and B. 14C-Labeled DNA is from cells that were irradiated and allowed to repair for various times. 3H-Labeled DNA is from internal standard cells that were irradiated with the same dose but not allowed to repair. Each figure shows 1 set of a pair of duplicate profiles.
X-Ray-induced chromosomal aberrations
X-Ray-induced inhibition of DNA synthesis
A very significant ( p < 0 . 0 0 1 ) increase in the n u m b e r of chromatid exchanges was f o u n d in i r s l S F cells after irradiation in G 1 (Table 1). A l t h o u g h normal C H O cells p r o d u c e a few chromatid-type aberrations after irradiation in G a ( D e w e y et al., 1966), i r s l S F produces m a n y times more. i r s l S F also exhibits significantly higher frequencies of chromatid exchange after irradiation in G 2 and has higher rates of spontaneous chromatid deletions.
i r s l S F showed a slightly greater X-ray-induced inhibition of D N A synthesis than did A A 8 ; the inhibition began sooner and lasted longer (Fig. 9).
X-Ray-induced mutations The A A 8 line had a frequency of X-ray-induced mutation to 6-thioguanine resistance of 2.7 × 1 0 - 5 / G y , whereas i r s l S F had a frequency of 0.73 × 1 0 - 5 / G y (Fig. 10A). C o m p a r e d to AAS, therefore, i r s l S F is h y p o m u t a b l e to the induction
117 100,
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Fig. 6. Rate of double-strand-break rejoining determined from the neutral elution profiles shown in Fig. 5. (A) 50 Gy. (B) 100 Gy. B, AA8; O, irslSF.
of mutations either as a function of X-ray exposure (Fig. IOA) or as a function of survival (Fig.
lOB).
Sister-chromatid exchange The frequency of spontaneous SCE in irslSF cells was 0.23+0.09 SCEs/chromosome. The frequency in AA8 cells was 0.23 + 0.08 SCEs/ chromosome, and in EM9 cells was 3.58 + 0.65 SCEs/chromosome. Therefore, irslSF is normal for spontaneous SCE.
Discussion In addition to its X-ray sensitivity, irslSF differs from its parental line in several areas, irslSF is deficient in its ability to rejoin single-strand breaks; it also has a reduced ability to perform repair replication, which indicates a defect in the excision repair of X-ray-induced base damage. irslSF has a higher frequency of spontaneous
chromatid deletions and of X-ray-induced chromatid aberrations, including those formed after irradiation in the G1 phase of the cell cycle. irslSF is hypomutable to the induction of 6-thioguanine-resistant mutants by X-rays. Finally, irslSF shows greater X-ray-induced inhibition of DNA synthesis than does the parental line. The increased frequency of X-ray-induced chromatid aberrations is almost certainly the immediate cause of irslSF's radiosensitivity. It is well known that chromosome aberrations cause cell death by loss of chromosome fragments and the production of anaphase bridges (Marshak and Hudson, 1937; Koller, 1943). The source of the increased number of chromatid aberrations and the ultimate cause of irslSF's radiosensitivity is probably its deficiency in excision repair of Xray-induced base damage. Base damage that is induced in G 1 and still unrepaired in S phase could lead to chromatid aberrations in a manner similar to the induction of chromatid aberrations
118
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3 5 7 9 11 13 15 17 19 21 23 Fraction i
Fig. 7. Alkaline CsCI-Cs2SO4 equilibrium density gradient profiles of DNA from AA8 cells that were prelabeled with 32p, irradiated with (A) 0, (B) 100 Gy, or (C) 200 Gy of X-rays and then incubated with BrdUrd and [3H]dThd. Density increases from right to left. O, 32p; 3H.
O,
Fig. 8. X-Ray-induced repair replication in AA8 and irslSF, determined from alkaline CsC1-Cs2SO4 equilibrium density gradient profiles. The 3H counts/lag of DNA represent the amount of repair replication that took place. All values of repair replication have been normalized so that the average repair replication in the parental cell line (AA8) after a 100-Gy exposure is 13H count//xg of DNA. Each point represents the result from 1 treated population. II, AA8; O, irslSF.
100 90
Fig. 9. X-Ray-induced inhibition of DNA synthesis. (A) AA8. (B) irslSF. Rates of DNA synthesis were measured by exposing cells uniformly labeled with [14C]dThd to a 10-rain pulse of [3H]dThd. Division of the 3H to 14C ratio in DNA from irradiated cells by the 3H to t4C ratio in DNA from unirradiated cells gives the percentage of inhibition of DNA synthesis. Data points are from duplicate samples. ©, 5 Gy; [3, 10 Gy; ,x, 20 Gv.
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Dose in Gy
50%
20%
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Percent Survival
Fig. 10. Frequency of X-ray-induced 6-thioguanlne-resistant mutants plotted as (A) a function of dose and (B) a function of survival. Spontaneous background frequencies of 6-thioguanine-resistant colonies, 1.89x 10 -5 in AA8 and 3.97 x 10 -5 in irslSF, have been subtracted from the observed frequencies. Data points are from duplicate samples. I , AA8; O, irslSF.
by chemical lesions (Bender et al., 1974). This mechanism has also been suggested to explain the induction of chromatid damage by X-rays in A-T lymphoblastoid cells during G O (Taylor et al., 1976). The deficiency in single-strand-break rejoining observed in the alkaline elution assay may be another component of the defect in excision repair. It is unlikely that this deficiency is a major contributor to irslSF's radiosensitivity, because EM9-1 (Thompson et al., 1982) rejoins X-ray-induced strand breaks more slowly than does irslSF but is less radiosensitive than irslSF. irslSF has some characteristics in common with the other X-ray-sensitive cell lines, but the combination of these characteristics in irslSF is unique; therefore, this line represents a new phenotype. Among the radiosensitive CHO lines, irslSF shares with the xrs-6 and xrs-7 lines the characteristic of increased frequencies of X-ray-induced chromatid damage and with xrs-7 the production of chromatid damage after irradiation in G1 (Kemp and Jeggo, 1986). It differs from the xrs lines and the XR-1 line (Stamato et al., 1983) in its proficiency in double-strand-break repair, irslSF's normal rate of spontaneous SCE distinguishes it from EM9-1, which has an extremely high rate of spontaneous
SCE (Thompson et al., 1982; Dillehay et al., 1983). In addition to radiosensitivity, irslSF shares several characteristics with A-T fibroblasts. Some, but not all, strains of A-T fibroblasts are deficient in X-ray-induced excision repair (Paterson et al., 1977). Irradiation of A-T fibroblasts during G1 produces chromatid damage (Taylor et al., 1976). A-T fibroblasts are also hypomutable to the induction of 6-thioguanine resistance by X-rays (Arlett, 1980; Simons, 1982; Arlett and Harcourt, 1983). The major characteristic of A-T fibroblasts that irslSF cells do not share is the ability to perform radioresistant DNA synthesis, a characteristic found in all A-T fibroblast and lymphoblastoid cells to date. In summary, an X-ray-sensitive CHO cell line has been isolated that is deficient in repair replication, shows an increased rate of spontaneous and X-ray-induced chromatid abnormalities, and has a decreased frequency of X-ray-induced mutations. Further studies of this cell line should contribute to our understanding of radiosensitivity in general and of the relationship between defective excision repair, X-ray-induced chromosome abnormalities, and X-ray-induced mutations.
120
Acknowledgments This work was supported by the Office of Health and Environmental Research, U.S. Department of Energy, contract No. DE-AC03-76SF01012, and by National Cancer Institute grant CA 09272-08 and PHS grant 07232-04.
References Alexander, P. (1961) Mouse lymphoma cells with different radiosensitivities, Nature (London), 192, 572-573. Arlett, C.F. (1980) Mutagenesis in repair-deficient human cell strains, in: M. Alacevic (Ed.), Progress in Environmental Mutagenesis, Elsevier/North-Holland, Amsterdam, pp. 161-174. Arlett, C.F., and S.A. Harcourt (1983) Variation in response to mutagens amongst normal and repair-defective human cells, in: C.W. Lawrence (Ed.), Induced Mutagenesis: Molecular Mechanisms and Their Implications for Environmental Protection, Plenum, New York, pp. 249-266. Bender, M.A, H.G. Griggs and J.S. Bedford (1974) Mechanisms of chromosomal aberration production, lII. Chemicals and ionizing radiation, Mutation Res., 23, 197-212. Bradley, M.O., and K.W. Kohn (1979) X-Ray induced DNA double-strand-hreak production and repair in mammalian cells as measured by neutral filter elution, Nucleic Acids Res., 7, 793-804. Busch, D.B., J.E. Cleaver and D.A. Glaser (1980) Large-scale isolation of UV-sensitive clones of CHO cells, Somat. Cell Genet., 6, 407-418. Cleaver, J.E. (1977) Induction of thioguanine- and ouabain-resistant mutants and single-strand breaks in the DNA of Chinese hamster ovary cells by 3H-thymidine, Genetics, 87, 129-138. Dewey, W.C., R.M. Humphrey and B.A. Sedita (1966) Cell cycle kinetics and radiation-induced chromosomal aberrations studied with C 14 and H 3 labels, Biophys. J., 6, 247-260. Dillehay, L.E., L.H. Thompson, J.L. Minlder and A.V. Carrano (1983) The relationship between sister-chromatid exchange and perturbations in DNA replication in mutant EM9 and normal CHO cells, Mutation Res., 109, 283-296. Giaccia, A., R. Weinstein, J. Hu and T.D. Stamato (1985) Cell cycle-dependent repair o f double-strand DNA breaks in a "y-ray-sensitive Chinese hamster cell, Somat. Cell Mol. Genet., 11, 485-491. Glaser, D.A., and W.H. Wattenburg (1966) An automated system for the growth and analysis of large numbers of bacterial colonies using an environmental chamber and a computer-controlled flying-spot scanner, Ann. N.Y. Acad. Sci., 139, 243-257. Hsie, A.W., J.P. O'Neill, D.B. Couch, J.R. SanSebastian, P.A. Brimer, R. Maclianoff, J.C. Fuscoe, J.C. Riddle, A.P. Li, N.L. Forbes and M.H. Hsie (1978) Quantitative analyses of radiation- and chemical-induced lethality and mutagenesis in Chinese hamster ovary cells, Radiat. Res., 76, 471-492.
Hsie, A.W., J.P. O'Neill, J.R. SanSebastian and P.A. Brimer (1979) The C H O / H G P R T mutation assay: Progress with quantitative mutagenesis and mutagen screening, in: A.W. Hsie, J.P. O'Neill and V.K. McElheny (Eds.), Banbury Report 2, Mammalian Cell Mutagenesis: The Maturation of Test Systems, Cold Spring Harbor Laboratory, New York, pp. 407-420. Jeggo, P.A., and L.M. Kemp (1983) X-Ray-sensitive mutants of Chinese hamster ovary cell line, Isolation and cross-sensitivity to other DNA-damaging agents, Mutation Res., 112, 313-327. Jostes, R.F., K.M. Bushnell and W.C. Dewey (1980) X-Ray induction of 8-azaguanine-resistant mutants in synchronous Chinese hamster ovary cells, Radiat. Res., 83, 146-161. Kemp, L.M., and P.A. Jeggo (1986) Radiation-induced chromosome damage in X-ray-sensitive mutants (xrs) of the Chinese hamster ovary cell line, Mutation Res., 166, 255-263. Kemp, L.M., S.G. Sedgwick and P.A. Jeggo (1984) X-Ray sensitive mutants of Chinese hamster ovary cells defective in double-strand break rejoining, Mutation Res., 132, 189-196. Kohn, K.W., L.C. Erickson, R.A.G. Ewig and C.A. Friedman (1976) Fractionation of DNA from mammahan cells by alkaline elution, Biochemistry, 15, 4629-4637. Kohn, K.W., R.A.G. Ewig, L.C. Erickson and L.A. Zwelling (1981) Measurement of strand breaks and cross-links by alkaline elution, in: E.C. Friedberg and P.C. Hanawalt (Eds.), DNA Repair: A Laboratory Manual of Research Procedures, Vol. l, Part B, Dekker, New York, pp. 379-401. Koller, P.C. (1943) Effects of radiation on pollen grain development, differentiation and germination, Proc. Soc. Edinburgh, 61, 398-429. Konrad, M.W., B. Storrie, D.A. Glaser and L.H. Thompson (1977) Clonal variation in colony morphology and growth of CHO cells cultured on agar, Cell, 10, 305-312. Marshak, A., and J.C. Hudson (1937) Effect of X-rays on chromosomes, Radiology, 29, 669-674. Morgan, W.F., J.L. Schwartz, J.P. Murnane and S. Wolff (1983) Effect of 3-aminobenzamide on sister chromatid exchange frequency in X-irradiated cells, Radiat. Res., 93, 567-571. Nagasawa, H., and J.B. Little (1983) Comparison of kinetics of X-ray-induced cell killing in normal, ataxia telangiectasia and hereditary retinoblastoma fibroblasts, Mutation Res., 109, 297-308. Painter, R.B., and R.M. Drew (1959) Studies on deoxyribonucleic acid metabolism in human cancer cell cultures (HeLa), I. The temporal relationships of deoxyribonucleic acid synthesis to mitosis and turnover time, Lab. Invest., 8, 278-285. Painter, R.B., and B.R. Young (1972) Repair replication in mammalian cells after X-irradiation, Mutation Res., 14, 225-235. Painter, R.B., and B.R. Young (1980) Radiosensitivity in ataxia telanglectasia: A new explanation, Proc. Natl. Acad. Sci. (U.S.A.), 77, 7315-7317. Paterson, M.C., B.P. Smith, P.A. Knight and-A.K. Anderson (1977) Ataxia telangiectasia: An inherited human disease
121 involving radioseusitivity, malignancy and defective DNA repair, in: A. Castellani (Ed.), Research in Photobiology, Plenum, New York, pp. 207-218. Perry, P., and S. Wolff (1974) New Giemsa method for differential staining of sister chromatids, Nature (London), 251, 156-158. Sato, K., and N. Hieda (1979) Isolation and characterization of a mutant mouse lymphoma cell sensitive to methyl methanesulfonate and X rays, Radiat. Res., 78, 167-171. Sato, K., N. Hieda-Shiomi and H. Hama-Inaba (1983) X-Raysensitive mutant mouse cells with various sensitivities to chemical mutagens, Mutation Res., 121, 281-285. Sevastopoulos, C.G., C.T. Wehr and D.A. Glaser (1977) Large-scale automated isolation of Escherichia coil mutants with thermosensitive DNA replication, Proc. Natl. Acad. Sci. (U.S.A.), 74, 3485-3489. Shiomi, T., N. Hieda-Shiomi, K. Sato, H. Tsuji, E. Takahashi and I. Tobari (1981) A mouse-cell mutant sensitive to ionizing radiation is hypermutable by low doses of ~,-radiation, Mutation Res., 83, 107-116. Simons, J.W.I.M. (1982) Studies on survival and mutation in ataxia-telangiectasia cells after X-irradiation under oxic and anoxic conditions, in: B.A. Bridges and D.G. Harnden (Eds.), Ataxia-Telangiectasia: A Cellular and Molecular Link Between Cancer, Neuropathology, and Immune Deficiency, Wiley, Chichester, pp. 155-167. Sinclair, W.K. (1964) X-Ray-induced heritable damage (smallcolony formation) in cultured mammalian cells, Radiat. Res., 21, 584-611. Smith, C.A., P.K. Cooper, and P.C. Hanawalt (1981) Measurement of repair replication by equilibrium sedimentation, in: E.C. Friedberg and P.C. Hanawait (Eds.), DNA Repair: A Laboratory Manual of Research Procedures, Vol. I, Part B, Dekker, New York, pp. 289-305.
Stamato, T.D., R. WeinsteilL A. G-iaccia and L. Mackenzie (1983) Isolation of cell cycle-dependent gamma ray-sensitive Chinese hamster ovary cell, Somat. Cell Genet., 9, 165-173. Steier, H., and J.E. Cleaver (1969) Exposure chamber for quantitative ultraviolet photobiology, Lab. Pract., 18, 1295. Taylor, A.M.R., D.G. Harnden, C.F. Arlett, S.A. Harcourt, A.R. Lehrnann, S. Stevens and B.A. Bridges (1975) Ataxia telangiectasia: a human mutation with abnormal radiation sensitivity, Nature (London), 258, 427-429. Taylor, A.M.R., J.A. Metcalfe, J.M. Oxford and D.G. Harnden (1976) Is chromatid-type damage in ataxia telartgiectasia after irradiation at G O a consequence of defective repair? Nature (London), 260, 441-443. Terasima, T., and L.J. Tolmach (1963) Growth and nucleic acid synthesis in synchronously dividing populations of HeLa cells, Exp. Cell Res., 30, 344-362. Thompson, L.H., S. Fong and K. Brookman (1980a) Validation of conditions for efficient detection of HPRT and APRT mutations in suspension-cultured Chinese hamster ovary cells, Mutation Res., 74, 21-36. Thompson, L.H., J.S. Rubin, J.E. Cleaver, G.F. Whitmore and K. Brookman (1980b) A screening method for isolating DNA repair-deficient mutants of CHO cells, Somat. Cell Genet., 6, 391-405. Thompson, L.H., K.W. Brookman, L.E. Dillehay, A.V. Carrano, J.A. Mazrimas, C.L. Mooney and J.L. Minkler (1982) A CHO-cell strain having hypersensitivity to mutagens, a defect in DNA strand-break repair, and an extraordinary baseline frequency of sister-chromatid exchange, Mutation Res., 95, 427-440. Todd, P. (1968) Defective mammalian cells isolated from Xirradiated cultures, Mutation Res., 5, 173-183.
Mutation Research, 193 (1988) 109-121 DNA Repair Reports Elsevier M T R 06262
A Chinese hamster ovary cell line hypersensitive to ionizing radiation and deficient in repair replication Lloyd F. Fuller and Robert B. Painter Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, CA 94143 (U.S.A.) (Received 6 May 1987) (Revision received 4 September 1987) (Accepted 8 September 1987)
Keywords: Chinese hamster ovary; Radiosensitivity; Excision repair; Hypomutability.
Summary An X-ray-sensitive Chinese hamster ovary cell line was isolated by means of a semi-automated procedure in which mutagenized cells formed colonies on top of agar, were X-irradiated, and were photographed at two later times. We compared the photographs to identify colonies that displayed significant growth arrest. One of the colonies identified in this manner produced a stable line (irslSF) that is hypersensitive to ionizing radiation. The X-ray dose at which 10% of the population survives (D10) is 2.25 Gy for irslSF and 5.45 Gy for the parental line. The new mutant is also moderately sensitive to ethyl methanesulfonate, irslSF performs only half as much X-ray-induced repair replication as the parental line, indicating a defect in excision repair. This defect is believed to be the primary cause of the line's radiosensitivity. Although irslSF repairs DNA double-strand breaks at a normal rate, it repairs single-strand breaks more slowly .than normal, irslSF has an elevated number of spontaneous chromatid aberrations and produces significantly higher numbers of X-ray-induced chromatid aberrations after exposure during the G 1 phase of the cell cycle. The line is hypomutable, with X-ray exposure inducing only one-third as many 6-thioguanine-resistant colonies as the parental line.
There is an increasing interest in the discovery and characterization of cell lines hypersensitive to ionizing radiation. Initially, radiosensitive strains were discovered in the course of other studies. The radiosensitive LY-S strain arose spontaneously in the L5178Y mouse leukemia line (Alexander, 1961). V79/79, a radiosensitive strain derived from the V79 hamster lung fibroblast, was isolated during a study of X-ray-induced small-colony formaCorrespondence: Dr. Robert B. Painter, Laboratory of Radiobiology and Environmental Health, University of California, San Francisco, CA 94143 (U.S.A.).
tion (Sinclair, 1964). The radiosensitivity of ataxia telangleetasia (A-T) cells was discovered during the use of radiotherapy against tumors in patients with this disease (Taylor et al., 1975). More recently, several investigators have isolated radiosensitive lines by screening mutagenized populations for colonies hypersensitive to X-rays. The anchorage-independent L5178Y line was the first line used in these attempts because it could be easily replica plated (Sato and Hieda, 1979; Shiomi et al., 1981; Sat<) et al., 1983). The Chinese hamster ovary (CHO) cell line has been another source of radiosensitive mutants. Thomp-
0167-8817/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)
110 son et al. (1980b) used challenging doses of ultraviolet (UV) light, mitomycin C, and ethyl methanesulfonate (EMS) to isolate several lines sensitive to DNA damage, including EM9-1, which is sensitive to EMS and X-rays. Replica-plating techniques were used by Stamato et al. (1983) and Jeggo and Kemp (1983) to isolate the XR-1 line and the 6 xrs lines, respectively. A variety of phenotypes is displayed among the radiosensitive CHO lines. The EM9-1 line has a remarkably high rate of spontaneous sister-chromatid exchange (SCE) and is deficient in DNA single-strand-break rejoining (Thompson et al., 1982). The XR-1 and xrs lines are defective in double-strand-break repair (Giaccia et al., 1985; Kemp et al., 1984). The responses of the xrs lines to UV, methyl methanesulfonate, N-methyl-N'nitro-N-nitrosoguanidine, and EMS range from very sensitive to wild type. Recently, increased radiation-induced chromosome and chromatid damage has been observed in the xrs-6 and xrs-7 lines (Kemp and Jeggo, 1986). The xrs-7 line showed chromatid damage after irradiation in the G 1 phase of the cell cycle, an unusual characteristic shared only by A-T cells (Taylor et al., 1976; Nagasawa and Little, 1983) and the new radiosensitive CHO mutant described here. We report the isolation of an X-ray-sensitive CHO cell line that is competent in double-strandbreak repair but deficient in X-ray-induced repair replication, shows abnormally high rates of spontaneous and X-ray-induced chromatid aberrations, and has a reduced frequency of X-ray-induced mutations. Materials and methods Cell culture
AA8 cells (Thompson et al., 1980a) were used as the parental line. EM9-1, which is sensitive to EMS and X-rays, was used as a positive control (Thompson et al., 1982). All cells were grown in minimal essential medium (Gibco, Santa Clara, CA) with 10% fetal calf serum and 50 /~g/ml gentamycin (Gibco). Mutant isolation
Mutants were isolated by a modification of the technique of Busch et al. (1980) using the colony-
screening system previously described (Glaser and Wattenburg, 1966; Sevastopoulos et al., 1977; Konrad et al., 1977). A population of AA8 cells was mutagenized by a 24-h exposure to 1.5/~g/ml ICR-191, grown for 5 days to express damage, and then sprayed onto 400-600 10-cm petri dishes containing 10 ml of minimal essential medium with 5% fetal calf serum and 0.7% noble agar (Gibco). After 4 more days of growth, the cells had formed approximately 300 small colonies/ dish, and these were irradiated with 1 Gy of X-rays from a Philips RT250 therapeutic unit (250 kVp, 15 mA, half-value layer 1.06 mm Cu). The dishes were photographed 1.5 and 3.5 days after irradiation. The photographic negatives were compared to identify colonies that showed little growth between the time the two photographs were taken, indicating X-ray sensitivity. These colonies were isolated and expanded for further study. Chromosome preparation
Chromosome preparations were made by adding colcemid (final concentration 10-7 M) to the flasks, waiting 30 min, and then gently swirling the flasks to detach mitotic cells. Detached cells were centrifuged and resuspended in 75 mM KC1 for 5 min. The cells were fixed with 3 changes of Carnoy's fixative (3:1, methanol/acetic acid), then dropped onto microscope slides. The slides were stained with 5% Giemsa in water and dried, a coverslip was attached with DePex, and the chromosome preparations were viewed at 1000× magnification. Cell cycle determination
The method of fraction of labeled mitoses was used (Painter and Drew, 1959) to determine cell cycle times. For each cell line, 2 × 103 cells were inoculated into 22 flasks. 10 h before collections began, 11 flasks of each line were incubated for 30 rain with 0.05/~Ci/ml [3H]thymidine ([3H]dThd) (spec. act., 300 mCi/mmole). [3H]dThd was added to the other 11 flasks 30 min before collections began. Mitotic cells were harvested every hour, and chromosome preparations were made as described above. Autoradiographs were made using Ilford IL-4 emulsion, exposed at 4 ° C for 7 days, and developed in Kodak D19 for 5 min. The fraction of labeled mitoses was scored for each time period.
111
X-Ray-induced chromosomal aberrations in GI Cells were synchronized by mitotic detachment (Terasima and Tolmach, 1963) using colcemid. A sample of these cells was used to measure the mitotic index, and only those populations with a mitotic index > 98% were used. The cells were divided into four 75-cm2 flasks and allowed 1 h to attach. Cells in 3 flasks were irradiated with 0, 0.5, or 1 Gy (dose rate, 1.5 Gy/min). To determine the fraction of cells in S phase, cells in the fourth flask were incubated with 0.05/xCi/ml [3H]dThd (300 mCi/mmole) while the other flasks were being irradiated. After irradiation, the medium in all the flasks was replaced with medium containing colcemid. After approximately 18 h the cells were harvested, and chromosome preparations and autoradiographs were made as described above. X-Ray-induced chromosomal aberrations in G2 Flasks containing exponentially growing cells were irradiated with 0.25 or 0.5 Gy (dose rate, 1.5 Gy/min). Mitotic cells were harvested 2 or 3 h after irradiation as described in the cell cycle study, and chromosome preparations were made. Cells from a duplicate set of flasks were pulse labeled with [3H]dThd as described in the cell cycle study and treated in parallel to be sure that the mitotic cells were not in S phase at the time of irradiation. Chromosome preparations and autoradiographs were made as described above. X-Ray dose-response curve Cells were exposed to 1-6 Gy of X-rays (dose rate, 2 Gy/min) as a suspension in medium at 4 ° C under 5% CO2 in air at a dose rate of 2 Gy/min. After irradiation, the cell suspensions were diluted and inoculated into 100-cm polystyrene dishes. After 12 days the colonies were fixed with phosphate-buffered formalin, stained with 5% Giemsa in water, and counted. UV dose-response curoe Either 300 or 3000 cells were inoculated into 10-cm dishes and then incubated for 1 h to allow the cells to attach. The dishes were drained, rinsed with phosphate-buffered saline (PBS), and 200/d of PBS were added to each dish. Two dishes from each cell line were placed in a UV light apparatus (Steier and Cleaver, 1969) and irradiated for 0.5-9
sec at a rate of 1.3 J/mZ/sec. The dishes were then rinsed with PBS and refilled with normal growth medium. After 12 days of growth, the colonies were fixed and counted as described for the X-ray dose-response curve.
Ethyl methanesulfonate dose-response curve The technique used to generate the EMS dose-response curve is modeled on that of Thompson et al. (1980a). Cells in suspension were treated with EMS for 1 h at 37°C in 50-ml polypropylene centrifuge tubes. The cells were then pelleted by centrifugation, resuspended in growth medium, and diluted into dishes. After 12 days of growth, the colonies were fixed and counted as described for the X-ray dose-response curve. Alkaline filter elution Repair of single-strand breaks was measured by alkaline filter elution (Kohn et al., 1981). Cells were labeled by 24-h growth in medium containing 0.01 /~Ci/ml [14C]dThd (spec. act., 50 m C i / mmole), irradiated with 4 Gy of X-rays at 4" C, incubated at 37°C for various times to allow repair, and then immersed in cold PBS. AA8 cells labeled by 24-h growth in 0.1 /~Ci/ml [3H]dThd (50 Ci/mmole) and irradiated with 4 Gy but not allowed to repair were used as internal standard cells. For the elution, 14C-labeled cells were mixed with 3H-labeled internal standard cells, placed on the filters, rinsed with cold PBS, and then lysed and digested for 30 min with a solution containing 0.2% sodium dodecyl sulfate, 25 mM EDTA, pH 9.7, and 0.5 #g/ml proteinase K. This solution was replaced with the pH 12.1 alkaline elution buffer, which was pulled through the filter and collected as 1-ml fractions for 14 h. At pH 12.1 the complementary DNA strands separate and the DNA eluted from the filter is single-stranded. The fractions were neutralized with 1 N HC1 and their radioactivity was determined by liquid scintillation spectrometry. Neutral filter elution Repair of double-strand breaks was measured by neutral filter elution (Bradley and Kohn, 1979). The neutral filter elution technique is the same as the alkaline filter elution technique except for the use of a pH 9.6 elution buffer. At this lower pH,
112 the complementary DNA strands do not separate and the DNA eluting from the filter is doublestranded. Double-strand breaks were induced with 50 or 100 Gy of X-rays (dose rate, 2 Gy/min).
X-Ray-induced repair replication X-Ray-induced repair replication was measured by methods based on those of Painter and Young (1972) and Smith et al. (1981). Cells were labeled by 2 days of growth in medium containing 0.4 /zCi/ml 32p-labeled orthophosphate. Approximately 16 h before irradiation, the 32p was removed, and 2 × 106 cells were placed in each 60-mm dish. 90 min before irradiation, the medium was replaced with medium containing 10 -5 M bromodeoxyuridine (BrdUrd) and 10 -6 M fluorodeoxyuridine (FdUrd). 30 min before irradiation, this medium was replaced with medium containing BrdUrd and FdUrd in the concentrations listed above plus 2 mM hydroxyurea. Just before irradiation, the medium was replaced with medium containing BrdUrd, FdUrd, hydroxyurea, and 100 /~Ci/mi [3H]dThd (50 Ci/mmole). The cells were irradiated with 100-300 Gy (dose rate, 10 Gy/min) at 25 ° C, then incubated at 37 °C for 3 h. The DNA was isolated by treating the cells with a solution containing 50 mM NaC1, 40 mM Tris, pH 8, 20 mM EDTA, 100 /zg/ml proteinase K, and 100 # g / m l RNAase, followed by extraction with phenol and chloroform and dialysis against 3 changes of SSC (0.15 M sodium chloride, 0.015 M sodium citrate). The DNA was sheared using a Virtis homogenizer (1 rain at 20000 rpm). The DNA solution was added to 4.8 g CsC1, 1 g Cs2SO4, 0.5 ml 1 N NaOH, and sufficient SSC to make 10.3 g final weight. The solution was centrifuged at 40000 rpm for 36 h in a 50 Ti rotor (Beckman) and then collected from the bottom of the tube in approximately 22 fractions. Samples of these fractions were counted by scintillation spectrometry to determine the light and heavy DNA peaks. The fractions containing the light peak were pooled, then rebanded by the technique described above. The fractions from the rebanding were collected in test tubes and chilled to 4 ° C. The DNA was precipitated by adding I ml of 0.02% (w/v) salmon sperm DNA in 0.4 N NaOH, then 1 ml of 4% perchloric acid (PCA). The precipitate was collected on glass fiber filters and
rinsed with 4% PCA, followed by a 70% alcohol rinse and a 100% alcohol rinse. The filters were dried and placed in vials for scintillation spectrometry. The specific activity of the DNA from unirradiated cells was determined by isolating DNA by the technique described above, measuring absorbance at 260 nm, and determining 32p radioactivity by scintillation spectrometry.
X-Ray-induced inhibition of DNA synthesis This experiment was performed as described by Painter and Young (1980). Cells (about 5 × 10 4) were inoculated into 20-mm dishes containing medium with 0.05 # C i / m l [14C]dThd (50 m C i / mmole) and allowed to grow for 2 days. The medium was replaced with nonradioactive medium, and 16 h later the cells were irradiated with 5-20 Gy (dose rate, 2 Gy/min) at 25°C, then returned to 37 o C. At various times, duplicate sets of dishes were removed from the incubator and the medium was replaced with medium containing 1 #Ci/ml [3H]dThd (50 Ci/mmole). After 10 min the dishes were quickly drained and immersed in ice-cold SSC. The dishes were drained, 1 ml of 0.02% (w/v) salmon sperm DNA in 0.4 N NaOH was added, and the contents of the dishes were scraped into test tubes. 4% PCA (1 ml) was added to the test tubes, and the precipitate was collected as described above.
Frequency of X-ray-induced mutations Mutant cells were selected by resistance to the drug 6-thioguanine (Hsie et al., 1979; Jostes et al., 1980; Thompson et al., 1980a). Exponentially growing populations of cells were irradiated at X-ray doses chosen to produce survival levels of about 100, 50, 10, and 1% in the two cell lines. Sufficient cells were irradiated so that 100 viable mutant cells were expected in each group, based on wild-type frequencies of mutation induction (1.3 × 10 -5 mutants/Gy) (Cleaver, 1977; Hsie et al., 1978). After irradiation, the cells were grown for 9 days to express the mutant phenotype. Mutant cells were selected by inoculating l0 s cells into 10-cm petri dishes containing medium with 4 /~g/ml 6-thioguanine. Colonies were allowed to form for 12 days and were then fixed with phosphate-buffered formalin and stained with Giemsa.
113
Sister-chromatid exchange
100
The rate of spontaneous SCE was measured by the fluorescence-plus-Giemsa technique described by Perry and Wolff (1974), with the modifications described by Morgan et al. (1983). The cells were grown for two cell cycles in 10 #M BrdUrd, and chromosome preparations were made as described above. Before staining, the slides were immersed in 5 p g / m l of Hoechst 33258 in Sorensen's phosphate buffer (0.067 M Na2HPO 4 and KH2PO4, pH 6.8) for 20 min. The slides were rinsed, the coverslips were mounted with Sorensen's buffer, and the slides were placed on a slide warmer at 55 °C under black light for 4 min. The coverslips were removed and the slides were stained in 55[ Giemsa for 5 min. The slides were then rinsed and dried, and the frequency of SCEs in 100 cells of each line was determined.
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0.1
0.05
Fraction [3H] DNA Retained Fig. 3. Alkaline filter elution profiles of D N A after irradiation with 4 G y of X-rays. (A) AA8 cells. Minutes of repair: 0 (=a); 10 (i); 20 (12); no X-rays ([]). (B) irslSF. Minutes of repair: 0 ( ~ ) ; 10 (O); 20 (O); no X-ray (~9). 14C-Labeled D N A is from cells that were irradiated and allowed to repair for various times. 3H-Labeled D N A is from internal standard cells that were irradiated but not allowed to repair. Each figure shows 1 set of a pair of duplicate profiles. TABLE 1 X-RAY-INDUCED CHROMOSOME AND CHROMATID ABERRATIONS Treatment
Irradiation in G1 AA8
Clone 22
X-ray dose (Gy)
Labeled mitoses (%)
Total n u m b e r of chromosomes
Chromatid and isochromatid deletions
Chromatid exchanges
0 0.5 1.0
3.6
2182 2133 2318
1 2 6
0 0 2
0 0.5 1.0
2.7
2112 2774 2181
15 a 11 b 21 c
12 a 27 a
0 0 2
2042 2257 2172
0 14 27
Irradiation in G : (Harvested 2 h after irradiation) AA8 0 0.25 0.50 Clone 22
0 0.25 0.50
0 0 0
1970 2251 2051
7c 20 26
AA8
0 0.25 0.50
50 14 13
1995 2044 2034
2 29 32
Clone 22
0 0.25 0.50
36.6 14 9.6
1930 1941 2006
17 a 34 75 a
a Significantly different from AA8 ( p < 0.001, Student's t test). b p < 0.05. c v < 0.01.
40
a
0 0 5
Chromosome aberrations
Damaged cells (%)
5 16 62
6 17 52
2 35 c 34 h
24 51 56
0 0 2
0 14 27.5
2 4 ~' 5
10 20 31
0 1 4
3 4 1
5 1 31
1 9 b 18 c
7 1 0
21 35 63
4b 15 a 19 c
115
sen for further study. Of the 62 viable colonies, 42 displayed normal or only modest sensitivity on the initial test, and 19 showed initial sensitivity followed by a return to normal sensitivity over a period of approximately 2 months, irslSF was the only clone that showed stable X-ray sensitivity. Similar experiences with instability of X-ray sensitivity have previously been reported during attempts to isolate X-ray-sensitive lines (Sinclair, 1964; Todd, 1968).
Cell cycle times Examination of the curves for fraction of labeled mitoses versus time for the parental and mutant lines showed that AA8 has a cell cycle time of 12 h, with a 1.5-h G 1 phase, 8-h S phase, and 2.5-h G2 + M interval, whereas irslSF has a cell cycle time of 14.5 h, with a 0.5-h G 1 phase, 10.5-h S phase, and 3.5-h G 2 + M interval.
irslSF rejoined 50% of the single-strand breaks in about 11 min, compared to 5 min for AA8 (Fig. 4). Neutral elution profiles and curves of doublestrand-break rejoining that were generated in the same manner show that irslSF rejoins doublestrand breaks as fast as or faster than AA8 (Figs. 5 and 6).
X-Ray-induced repair replication Alkaline CsC1-Cs2SO 4 equilibrium density gradients are shown in Fig. 7 to illustrate the separation of normal-density DNA from BrdUrdsubstituted DNA. Repair replication is plotted in Fig. 8. irslSF performed approximately half as much repair replication in the dose range of 100-300 Gy as did AA8, indicating a significant defect in the excision repair of X-ray-induced base damage in irslSF.
Sensitivity to DNA-damaging agents irslSF is sensitive to X-rays, with a much smaller shoulder on its X-ray survival curve compared to the AA8 parer~tal line (Fig. 1). The D10 value for irslSF is 2.2 Gy, whereas AA8 and EM9-1 have values of 5.4 Gy and 3.15 Gy, respectively, irslSF also displays moderate sensitivity to UV light and EMS (Fig. 2). DNA strand-break repair Profiles of the alkaline elution of DNA from filters, constructed by plotting for each fraction the percentage of 14C-labeled DNA retained on the filter against the percentage of 3H-labeled internal standard DNA retained on the filter, show that irslSF rejoins single-strand breaks more slowly than does AA8 (Fig. 3). Curves showing rate of single-strand-break rejoining are constructed from these elution profiles by measuring the vertical distance between profiles. The vertical distance between the profiles of unrepaired and unirradiated cells is linearly proportional to the total number of breaks originally induced by the radiation exposure, whereas the vertical distance between the profiles of unrepaired and repaired cells is proportional to the number of breaks repaired during the 37 o C incubation period (Kohn et al., 1976). When combined and plotted as a function of time, these measurements indicate that
r
ee
ll;
,~ so
40
20
o
;o
2'o
Repair Time (min)
Fig. 4. Rate of single-strand-break rejoining determined from the alkaline filter elution profiles shown in Fig. 3. The two points for each cell line and each repair time are from elution profiles from duplicate samples. II, AAS; O, irslSF.
116
_
A
0.3 f
_
B
N~liN'~m
rr 7~
~"
1
I
I
I
I
I
I
I
1.0
g if_
o3°I , 1.0
0.5
0.3 110~ 0.5 Fraction[all]DNARetained
0.3
Fig. 5. Neutral filter elution profiles of DNA after irradiation with 50 or 100 Gy of X-rays. (A) AA8 cells after 50 Gy. Hours of repair: 0 (~); 1 (11); 2 (D); no X-rays ([]). (B) irslSF after 50 Gy. Hours of repair: 0 (~); 1 (O); 2 (O); no X-rays (~). (C) AA8 cells after 100 Gy. (D) irslSF after 100 Gy. Symbols are the same as in A and B. 14C-Labeled DNA is from cells that were irradiated and allowed to repair for various times. 3H-Labeled DNA is from internal standard cells that were irradiated with the same dose but not allowed to repair. Each figure shows 1 set of a pair of duplicate profiles.
X-Ray-induced chromosomal aberrations
X-Ray-induced inhibition of DNA synthesis
A very significant ( p < 0 . 0 0 1 ) increase in the n u m b e r of chromatid exchanges was f o u n d in i r s l S F cells after irradiation in G 1 (Table 1). A l t h o u g h normal C H O cells p r o d u c e a few chromatid-type aberrations after irradiation in G a ( D e w e y et al., 1966), i r s l S F produces m a n y times more. i r s l S F also exhibits significantly higher frequencies of chromatid exchange after irradiation in G 2 and has higher rates of spontaneous chromatid deletions.
i r s l S F showed a slightly greater X-ray-induced inhibition of D N A synthesis than did A A 8 ; the inhibition began sooner and lasted longer (Fig. 9).
X-Ray-induced mutations The A A 8 line had a frequency of X-ray-induced mutation to 6-thioguanine resistance of 2.7 × 1 0 - 5 / G y , whereas i r s l S F had a frequency of 0.73 × 1 0 - 5 / G y (Fig. 10A). C o m p a r e d to AAS, therefore, i r s l S F is h y p o m u t a b l e to the induction
117 100,
T
A
B
I
80
=.-
:=¢ n-
60 0O
E Q.
40
20
•
i
0
1
!
~
0
1
2
Tima (hours)
Fig. 6. Rate of double-strand-break rejoining determined from the neutral elution profiles shown in Fig. 5. (A) 50 Gy. (B) 100 Gy. B, AA8; O, irslSF.
of mutations either as a function of X-ray exposure (Fig. IOA) or as a function of survival (Fig.
lOB).
Sister-chromatid exchange The frequency of spontaneous SCE in irslSF cells was 0.23+0.09 SCEs/chromosome. The frequency in AA8 cells was 0.23 + 0.08 SCEs/ chromosome, and in EM9 cells was 3.58 + 0.65 SCEs/chromosome. Therefore, irslSF is normal for spontaneous SCE.
Discussion In addition to its X-ray sensitivity, irslSF differs from its parental line in several areas, irslSF is deficient in its ability to rejoin single-strand breaks; it also has a reduced ability to perform repair replication, which indicates a defect in the excision repair of X-ray-induced base damage. irslSF has a higher frequency of spontaneous
chromatid deletions and of X-ray-induced chromatid aberrations, including those formed after irradiation in the G1 phase of the cell cycle. irslSF is hypomutable to the induction of 6-thioguanine-resistant mutants by X-rays. Finally, irslSF shows greater X-ray-induced inhibition of DNA synthesis than does the parental line. The increased frequency of X-ray-induced chromatid aberrations is almost certainly the immediate cause of irslSF's radiosensitivity. It is well known that chromosome aberrations cause cell death by loss of chromosome fragments and the production of anaphase bridges (Marshak and Hudson, 1937; Koller, 1943). The source of the increased number of chromatid aberrations and the ultimate cause of irslSF's radiosensitivity is probably its deficiency in excision repair of Xray-induced base damage. Base damage that is induced in G 1 and still unrepaired in S phase could lead to chromatid aberrations in a manner similar to the induction of chromatid aberrations
118
2.5
1800
1500 1200
A •
2.0
/
A
~. s
600
z a~E 1.5
1800 1500
B
1200
eo
I /
T
E
9OO
~ 1.0 300e.e~.e,,..Qo~.e.e...e,m,,l_O~+ i
i
i
I
i
I
I
I
i"
~
_
I
I
1800
0.5 /
1500o, 1200.~ 9oo 6o0
C~ oI 0
. O
).Q.O
100
200
3 0
X-ray Exposure (Gy)
"
3 5 7 9 11 13 15 17 19 21 23 Fraction i
Fig. 7. Alkaline CsCI-Cs2SO4 equilibrium density gradient profiles of DNA from AA8 cells that were prelabeled with 32p, irradiated with (A) 0, (B) 100 Gy, or (C) 200 Gy of X-rays and then incubated with BrdUrd and [3H]dThd. Density increases from right to left. O, 32p; 3H.
O,
Fig. 8. X-Ray-induced repair replication in AA8 and irslSF, determined from alkaline CsC1-Cs2SO4 equilibrium density gradient profiles. The 3H counts/lag of DNA represent the amount of repair replication that took place. All values of repair replication have been normalized so that the average repair replication in the parental cell line (AA8) after a 100-Gy exposure is 13H count//xg of DNA. Each point represents the result from 1 treated population. II, AA8; O, irslSF.
100 90
Fig. 9. X-Ray-induced inhibition of DNA synthesis. (A) AA8. (B) irslSF. Rates of DNA synthesis were measured by exposing cells uniformly labeled with [14C]dThd to a 10-rain pulse of [3H]dThd. Division of the 3H to 14C ratio in DNA from irradiated cells by the 3H to t4C ratio in DNA from unirradiated cells gives the percentage of inhibition of DNA synthesis. Data points are from duplicate samples. ©, 5 Gy; [3, 10 Gy; ,x, 20 Gv.
¢0
80
u~ =_
70
g
100
"6
90 80 70
60
Hours alter Irradiation
119
2O ~._
0
18 16
B
N
jI
~0
i'
6
J
4 2 0
w 1
2
3
4
5
6
7
Dose in Gy
50%
20%
5%
Percent Survival
Fig. 10. Frequency of X-ray-induced 6-thioguanlne-resistant mutants plotted as (A) a function of dose and (B) a function of survival. Spontaneous background frequencies of 6-thioguanine-resistant colonies, 1.89x 10 -5 in AA8 and 3.97 x 10 -5 in irslSF, have been subtracted from the observed frequencies. Data points are from duplicate samples. I , AA8; O, irslSF.
by chemical lesions (Bender et al., 1974). This mechanism has also been suggested to explain the induction of chromatid damage by X-rays in A-T lymphoblastoid cells during G O (Taylor et al., 1976). The deficiency in single-strand-break rejoining observed in the alkaline elution assay may be another component of the defect in excision repair. It is unlikely that this deficiency is a major contributor to irslSF's radiosensitivity, because EM9-1 (Thompson et al., 1982) rejoins X-ray-induced strand breaks more slowly than does irslSF but is less radiosensitive than irslSF. irslSF has some characteristics in common with the other X-ray-sensitive cell lines, but the combination of these characteristics in irslSF is unique; therefore, this line represents a new phenotype. Among the radiosensitive CHO lines, irslSF shares with the xrs-6 and xrs-7 lines the characteristic of increased frequencies of X-ray-induced chromatid damage and with xrs-7 the production of chromatid damage after irradiation in G1 (Kemp and Jeggo, 1986). It differs from the xrs lines and the XR-1 line (Stamato et al., 1983) in its proficiency in double-strand-break repair, irslSF's normal rate of spontaneous SCE distinguishes it from EM9-1, which has an extremely high rate of spontaneous
SCE (Thompson et al., 1982; Dillehay et al., 1983). In addition to radiosensitivity, irslSF shares several characteristics with A-T fibroblasts. Some, but not all, strains of A-T fibroblasts are deficient in X-ray-induced excision repair (Paterson et al., 1977). Irradiation of A-T fibroblasts during G1 produces chromatid damage (Taylor et al., 1976). A-T fibroblasts are also hypomutable to the induction of 6-thioguanine resistance by X-rays (Arlett, 1980; Simons, 1982; Arlett and Harcourt, 1983). The major characteristic of A-T fibroblasts that irslSF cells do not share is the ability to perform radioresistant DNA synthesis, a characteristic found in all A-T fibroblast and lymphoblastoid cells to date. In summary, an X-ray-sensitive CHO cell line has been isolated that is deficient in repair replication, shows an increased rate of spontaneous and X-ray-induced chromatid abnormalities, and has a decreased frequency of X-ray-induced mutations. Further studies of this cell line should contribute to our understanding of radiosensitivity in general and of the relationship between defective excision repair, X-ray-induced chromosome abnormalities, and X-ray-induced mutations.
120
Acknowledgments This work was supported by the Office of Health and Environmental Research, U.S. Department of Energy, contract No. DE-AC03-76SF01012, and by National Cancer Institute grant CA 09272-08 and PHS grant 07232-04.
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Stamato, T.D., R. WeinsteilL A. G-iaccia and L. Mackenzie (1983) Isolation of cell cycle-dependent gamma ray-sensitive Chinese hamster ovary cell, Somat. Cell Genet., 9, 165-173. Steier, H., and J.E. Cleaver (1969) Exposure chamber for quantitative ultraviolet photobiology, Lab. Pract., 18, 1295. Taylor, A.M.R., D.G. Harnden, C.F. Arlett, S.A. Harcourt, A.R. Lehrnann, S. Stevens and B.A. Bridges (1975) Ataxia telangiectasia: a human mutation with abnormal radiation sensitivity, Nature (London), 258, 427-429. Taylor, A.M.R., J.A. Metcalfe, J.M. Oxford and D.G. Harnden (1976) Is chromatid-type damage in ataxia telartgiectasia after irradiation at G O a consequence of defective repair? Nature (London), 260, 441-443. Terasima, T., and L.J. Tolmach (1963) Growth and nucleic acid synthesis in synchronously dividing populations of HeLa cells, Exp. Cell Res., 30, 344-362. Thompson, L.H., S. Fong and K. Brookman (1980a) Validation of conditions for efficient detection of HPRT and APRT mutations in suspension-cultured Chinese hamster ovary cells, Mutation Res., 74, 21-36. Thompson, L.H., J.S. Rubin, J.E. Cleaver, G.F. Whitmore and K. Brookman (1980b) A screening method for isolating DNA repair-deficient mutants of CHO cells, Somat. Cell Genet., 6, 391-405. Thompson, L.H., K.W. Brookman, L.E. Dillehay, A.V. Carrano, J.A. Mazrimas, C.L. Mooney and J.L. Minkler (1982) A CHO-cell strain having hypersensitivity to mutagens, a defect in DNA strand-break repair, and an extraordinary baseline frequency of sister-chromatid exchange, Mutation Res., 95, 427-440. Todd, P. (1968) Defective mammalian cells isolated from Xirradiated cultures, Mutation Res., 5, 173-183.