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Cell cycle dependence of radiation-induced homologous recombination in cultured monkey cells
Mutation Research 374 Ž1997. 233–243
Cell cycle dependence of radiation-induced homologous recombination in cultured monkey cells J. Cao, S.E. DePrimo, J.R. Stringer
)
Department of Molecular Genetics, Biochemistry, and Microbiology, UniÕersity of Cincinnati Medical Center, Cincinnati, OH 45267-0524, USA Received 31 May 1996; revised 5 November 1996; accepted 5 November 1996
Abstract A lacZ transgene recombination system that reports homologous recombination events involving duplicated lacZ segments was used to study recombination in monkey cells exposed to ionizing radiation at different points in the cell cycle. With this system, recombination events can be detected in single cells by histochemical staining soon after exposure of cells to DNA-damaging treatment. Ionizing radiation rapidly induced recombination 5–10-fold in cells that were at the mitosis stage of the cell cycle. Irradiation either of cells at other points in the cell cycle or of nonsynchronized cells had less of an effect on recombination between lacZ segments. Keywords: Recombination; Genomic instability; Radiation; Cell cycle; Mitosis; LacZ gene
1. Introduction Homologous recombination plays a very important role in the repair of DNA damage w1,2x. However, homologous recombination is also potentially dangerous because it can contribute to genomic instability, which can in turn contribute to neoplastic transformation and cancer w3,4x. An association between increased homologous recombination and cancer risk has been long appreciated based on the phenotypes of cells from patients with chromosome instability and cancer predisposition syndromes such as Bloom syndrome ŽBS., ataxia telangiectasia ŽAT., xeroderma pigmentosum ŽXP., and Fanconi anemia ) Corresponding author. Tel.: Ž513. 558-0069; Fax: Ž513. 5588474; E-mail: [email protected]
ŽFA. w5,6x. Homologous recombination is also thought to be the mechanism of mitotic crossing over, which is known to be a pathway by which genes encoding tumor suppressor proteins are lost from somatic cells. It has been shown that ionizing radiation stimulates recombination between homologous chromosomes in yeasts w7,8x, fruit flies w9,10x and mammalian cells in culture w11x. Ionizing radiation also causes sister chromatid exchanges in mammalian cells w12x. Hence it seems reasonable to the expect that ionizing radiation would induce homologous recombination between tandem repeats in mammalian cells. However, strong induction of homologous recombination between tandem repeats has yet to be demonstrated in cultured mammalian cells. Several previous studies have shown that ionizing
0027-5107r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 6 . 0 0 2 3 7 - 0
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radiation had little effect on recombination between tandemly repeated transgenes w13–16x. Similar results were obtained with a mutant allele of the HPRT gene that could revert by homologous recombination between tandem repeats w17x. The slight induction observed in cultured cells contrasts with the induction of recombination observed between two 70 kb regions that are tandemly duplicated in the pink-eyed unstable mutation of mice w18x. Here we studied the possible relationship between cell cycle stage and response to ionizing radiation in cells derived from the monkey cell line CV-1. We found that radiation induced more recombination in cells that were in mitosis than in cells that were in other cell cycle stages.
2. Material and methods 2.1. Synchronization of FSH 1 cells The FSH-1 cell line was used in all experiments. FSH-1 cells were derived from the CV-1 line of monkey cells as previously described w19x. Cells were grown in DMEM ŽGibco. supplemented with 10% fetal calf serum ŽFCS. ŽSigma., L-glutamine ŽGibco., and antibiotic-antimycotic ŽGibco.. Cells were synchronized by the mitotic shake-off method w20,21x. Culture flasks ŽT150. were seeded with 1.5 = 10 6 cells each. After 2 days, the flasks were prepared for harvesting of mitotic cells. To minimize the chance that cells that had already divided would contaminate the mitotic cell population, weakly attached cells were removed and discarded. This was done by adding 15 ml of media to a flask, striking the flask firmly, and submerging the monolayer in the media. Dislodged cells floating in the media were removed. This process was repeated three times, after which media was added and cultures incubated for 30 min at 378C. The shake-off was then repeated 3 times to harvest mitotic cells, which were collected by centrifugation at 250 = g for 10 min at 48C. Cells were resuspended in media at 48C and counted. The mitotic index of such cells was about 85%. Cells obtained by the shake-off procedure were incubated in media containing BrdU following which the cells were held for various time periods, and then assayed
for binding to a fluorescein conjugate of Anti-BrdU ŽBecton-Dickinson.. Labeling was first observed after 9 h, and continued for a 7-h period. This indicated that the G1 period was 9 h and that S-phase lasted approximately 7 h. Mitotic cells were first observed after 20 h. The fraction of cells in G1 , S, and G2rM in unsynchronized cultures was determined by flow cytometry. 2.2. g-Irradiation procedure A portion of the cells obtained by mitotic shake-off was irradiated Žmitotic cells., and the rest were placed in T25 flasks and incubated at 378C. Some of these cells were irradiated after 4 h ŽG1 phase cells., and some were irradiated after 14 h ŽS phase cells.. Gamma-rays were produced by a 60 Co irradiator at dose rate of 0.80 Gyrmin. For asynchronous cells, log phase cell monolayers were seeded at least 1 day before irradiation at a density of 1.0 = 10 6 cellsrT75 flask. 2.3. Analysis of homologous recombination in synchronous FSH 1 cells After irradiation, cells were removed from flasks by trypsin treatment and were replated at a density of 5 = 10 4r60 mm culture dish and incubated at 378C for 2–5 days. For analysis, one culture was trypsinized and counted, and others were fixed and stained with X-gal overnight at 378C. X-gal stain solution was 0.5 mgrml with 44 mM HEPES, 3 mM potassium ferrocyanide, 3 mM potassium ferricyanide, and 0.7 mM MgCl 2 in PBS. Blue cells were counted by microscopic examination.
3. Results 3.1. Recombination in irradiated FSH-1 cells FSH-1 cells contain two nonfunctional lacZ genes arranged as a tandem repeat. The repeated lacZ genes are each 3 kb in size and are separated by about 4 kb. Homologous recombination between the tandem repeats produces a lacZ gene capable of producing b-galactosidase, and cells containing a
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Fig. 1. Recombination following irradiation at different times during the cell cycle. Monolayers of fixed cells were tested for the presence of b-galactosidase activity by staining with X-Gal. Clusters of blue cells Žareas containing one or more blue cells. were scored by microscopy. Lines labeled as controls are data from cultures that had not been irradiated.
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recombined lacZ gene can be detected by staining with X-gal w19x. To study the effect of g-rays on homologous recombination, cultures of FSH-1 cells were exposed to a total dose of 2 Gy. After irradiation, sixteen 60 mm plates were seeded with 5 = 10 4 cells per plate. Plates were incubated at 378C and analyzed after 2, 3, 4 and 5 days. For each time point, the cells from one culture were harvested and counted, and three cultures were fixed and stained to detect cells expressing b-galactosidase. The cell density on the stained plates was sometimes too high to allow discreet colonies of cells to be identified. Therefore, plates were examined under a microscope to visualize individual blue cells. Because each plate contained very few blue cells, clusters of blue cells were assumed to have been derived from the growth of single blue cells. Therefore, the number of discreet
Fig. 2. Dose response of blue cluster induction at various times after irradiation. Blue clusters were scored as described in Fig. 1. ŽA–D. Results obtained when cells were stained after 2 ŽA., 3 ŽB., 4 ŽC. or 5 ŽD. days post irradiation. S phase and G1 phase cells were tested at 2 Gy only. Lines labeled Asy are from cultures growing asynchronously.
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areas containing one or more blue cells Žblue clusters. was used to estimate the number of induced recombination events. The relationship between radiation treatment and the number of blue clusters is shown in Fig. 1. Irradiation had little effect on the number of blue clusters in asynchronous cells. By contrast, cultures exposed at mitosis contained 7 times as many blue clusters per 10 5 cells as unirradiated synchronized cells 2 days after irradiation. Less induction of blue clusters was observed in cultures containing cells that had been irradiated at either S or G1. In each of the cultures that had elevated numbers of blue clusters at 2 days post irradiation, the number of blue clusters per 10 5 cells declined over the course of the next 3 days. This decline occurred primarily because the cell number increased more rapidly than the did the number of blue clusters. These results suggest that most of the induction occurred soon after irradiation. Recombination was also studied in cells that were not synchronized when irradiated, but were collected by mitotic shake-off at various times after irradiation. In this procedure, cells that were in mitosis when the culture was irradiated shake-off first, fol-
lowed by cells that were in G2 , S and G1. These experiments showed that most of the blue cells were obtained in the first shake-off suggesting that induction was strongest in cells that were in mitosis at the time of irradiation Ždata not shown.. 3.2. Dose response of recombination Because induction of recombination was strongest in mitotic cells, these cells were used to investigate the dose-dependence of recombination. Fig. 2 shows the number of blue clusters observed at four time points ŽA–D. following irradiation with 1 Gy, 2 Gy or 3 Gy. Recombination in cells growing asynchronously was usually weakly affected by exposure to 1 Gy or 2 Gy, with blue cluster counts remaining within a factor of 2 of those seen in unirradiated control cultures. In asynchronous cultures, stronger induction was obtained after exposure to 3 Gy. Fig. 2A shows that asynchronous cultures stained after 2 days contained 4 times as many blue clusters as unirradiated cultures. However, the blue cluster counts declined in asynchronous cultures that were kept longer than 2 days prior to staining, and by day 5, no induction was evident even after exposure to 3
Fig. 3. Size distribution of blue clusters 5 days after irradiation. Blue clusters were scored as described in Fig. 1. Bars labeled as controls are data from cultures that had not been irradiated.
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Gy Žsee Fig. 2D.. By contrast, cultures irradiated while cells were in mitosis generally contained three to five times as many blue clusters as did unirradiated synchronized cultures, and the blue cluster counts remained elevated by at least 4-fold at all time points. 3.3. Viability of cells that had undergone recombination To determine how many of the blue cells present after irradiation were able to undergo cell division, we analyzed plates of cells for the size of blue clusters. Table 1 shows that each culture exhibited blue clusters of various sizes, and that larger clusters were more prevalent at later times after plating. Clusters contained up to 16 blue cells 5 days after plating, indicating that some blue cells had doubled up to four times. This was consistent with results of previous experiments, which showed that a single blue FSH-1 cell present at the time a plate was seeded could produce up to 16 cells after 5 days of incubation w19x. Fig. 3 shows the distribution of blue cluster sizes present in cultures 5 days after irradiation, where the larger clusters of blue cells were most evident. Data on the sizes of blue clusters obtained at other time points Žlisted in Table 1., were
Fig. 4. Growth curves of synchronous and asynchronous populations after irradiation. Relative cell number is the number of cells present at a given time divided by the number of cells plated initially. Lines labeled as controls are data from cultures that had not been irradiated. Synchronous cultures were irradiated when 80% of the cells were in mitosis.
Fig. 5. Total number of blue cells in cultures derived from mitotic cells that had been exposed to irradiation. The line with error bars shows the data. Lines labelled A, B and C are theoretical curves that show what would be expected if all induction had occurred by Day 2 and if 100% ŽA., 55% ŽB., or 25% ŽC. of the blue cells present at Day 2 were unable to divide.
consistent with results obtained at the 5 day time point. The data in Fig. 3 showed that some of the cells that had undergone recombination were able to divide several times, but it was striking that many of the blue cells induced by irradiation of synchronized cells were present as singlets, even 5 days after irradiation. To illustrate, 5 days after a dose of 2 Gy, the fraction of blue cell ‘clusters’ that contained a single blue cell was 65% in cells irradiated during mitosis. By contrast, the fraction of blue cell ‘clusters’ that had a single blue cell was 46% in unirradiated synchronous cultures. The abundance of single blue cells at late times after irradiation could have been due to either of two nonmutually exclusive factors: Ži. division of some blue cells may have been blocked due to radiation damage, in which case single blue cells generated early after irradiation would have failed to produce clusters of blue cells; and Žii. recombination may have remained elevated for days after irradiation Ždelayed recombination.. To examine these factors, we analyzed the growth kinetics of populations of FSH-1 cells that had been irradiated. First, we examined the degree to which radiation induced a general cell-cycle block. Fig. 4 shows that for 3 days following irradiation, the rate at which cell number increased in irradiated cultures
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Table 1 Frequency and size of blue-cell clusters in FSH-1 cell culture Blue cluster size
a
1
2
Synchronous unirradiated Day 2 4.11 " 0.22 Day 3 2.73 " 0.51 Day 4 1.76 " 0.16 Day 5 1.74 " 0.10
b
3 to 4
2.44 " 1.44 1.23 " 0.12 1.24 " 0.30 1.00 " 0.23
1.23 " 0.12 1.14 " 0.21 0.38 " 0.088
Mitotic 1.0 Gy Day 2 Day 3 Day 4 Day 5
27.5 " 2.5 8.64 " 0.74 4.78 " 0.93 6.49 " 0.75
6.28 " 0.72 5.76 " 0.49 2.68 " 0.40 2.64 " 0.22
2.88 " 0.25 2.35 " 0.35 1.46 " 0.34
Mitotic 2.0 Gy Day 2 Day 3 Day 4 Day 5
28.86 " 3.86 11.71 " 0.80 11.73 " 0.06 11.49 " 2.62
10.15 " 3.49 5.82 " 2.18 5.18 " 0.18 3.13 " 0.57
3.52 " 1.02 3.63 " 0.30 1.15 " 0.22
Mitotic 3.0 Gy Day 2 Day 3 Day 4 Day 5
46.43 " 13.57 18.11 " 4.11 17.86 " 2.14 11.93 " 1.61
7.14 " 2.86 5.33 " 1.33 5.83 " 0.83 3.78 " 0.73
2.11 " 0.11 3.05 " 1.62 1.22 " 0.25
G1 2.0 Gy Day 2 Day 3 Day 4 Day 5
13.50 " 1.83 6.97 " 1.37 5.10 " 2.40 2.79 " 0.38
4.33 " 2.33 5.18 " 3.58 1.88 " 0.63 1.26 " 0.27
3.73 " 2.93 1.40 " 0.35 0.70 " 0.21
S 2.0 Gy Day 2 Day 3 Day 4 Day 5
10.63 " 0.63 3.75 " 1.25 3.24 " 0.99 2.94 " 0.16
5.21 " 1.04 2.42 " 1.92 2.17 " 1.17 1.92 " 0.40
1.58 " 0.08 1.03 " 0.53 0.88 " 0.13
Asynchronous unirradiated Day 2 1.34 " 0.57 Day 3 1.41 " 0.60 Day 4 0.83 " 0.13 Day 5 0.62 " 0.06
2.32 " 0.78 1.18 " 0.37 0.71 " 0.11 0.58 " 0.09
1.15 " 0.07 0.46 " 0.03 0.35 " 0.05
Asynchronous 1.0 Gy Day 2 Day 3 Day 4 Day 5
3.35 " 0.21 3.29 " 0.16 1.16 " 0.16 0.81 " 0.09
4.11 " 0.11 1.69 " 0.19 0.84 " 0.37 0.58 " 0.07
1.47 " 0.09 0.37 " 0.10 0.34 " 0.03
Asynchronous 2.0 Gy Day 2 Day 3
3.92 " 1.20 3.71 " 0.29
2.42 " 0.60 2.50 " 0.50
1.12 " 0.45
5 to 8
0.49 " 0.09 0.42 " 0.10
1.29 " 0.14 0.94 " 0.28
1.52 " 0.81 0.76 " 0.18
0.69 " 0.02 0.75 " 0.28
0.33 " 0.08 0.26 " 0.13
0.68 " 0.43 0.69 " 0.16
0.34 " 0.04 0.28 " 0.08
0.37 " 0.10 0.262 " 0.04
9 to 10
Total
0.270 " 0.03
6.56 " 1.22 5.20 " 0.75 4.63 " 0.76 3.81 " 0.23
1.06 " 0.35
33.78 " 1.78 17.23 " 1.48 11.10 " 0.33 12.59 " 1.60
0.87 " 0.32
39.02 " 7.35 21.05 " 1.96 22.06 " 0.27 17.42 " 3.59
0.54 " 0.10
53.57 " 16.43 25.56 " 5.56 27.43 " 4.57 18.21 " 2.27
0.34 " 0.13
17.83 " 0.50 15.88 " 7.88 8.71 " 3.29 5.34 " 0.79
0.81 " 0.11
15.83 " 1.67 7.75 " 3.25 7.11 " 3.11 7.23 " 0.60
0.23 " 0.03
3.65 " 1.35 3.74 " 1.04 2.33 " 0.21 2.05 " 0.11
0.22 " 0.04
7.46 " 0.32 6.44 " 0.44 2.74 " 0.53 2.20 " 0.18
6.34 " 1.80 7.33 " 0.67
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Table 1 Žcontinued. Blue cluster size
a
1
2
3 to 4
5 to 8
9 to 10
0.68 " 0.03 0.35 " 0.10
0.21 " 0.05
5.39 " 0.59 2.68 " 0.39
0.13 " 0.03
16.35 " 1.15 12.51 " 0.74 6.80 " 1.20 3.15 " 0.15
Asynchronous 2.0 Gy Day 4 Day 5
2.49 " 0.16 1.06 " 0.12
1.17 " 0.33 0.67 " 0.11
1.05 " 0.12 0.40 " 0.06
Asynchronous 3.0 Gy Day 2 Day 3 Day 4 Day 5
10.22 " 0.62 7.39 " 0.39 3.25 " 0.35 1.63 " 0.09
6.13 " 0.53 2.64 " 0.86 1.80 " 0.80 0.77 " 0.07
2.49 " 0.26 0.90 " 0.20 0.31 " 0.07
a b
0.85 " 0.25 0.32 " 0.07
Total
Number of blue cells per cluster. Number of blue clusters per 10 5 cells " SE.
was less than in unirradiated cultures, but by the fourth day, all but one of the irradiated cultures expanded at essentially the same rate as unirradiated cells. The one exception was observed in the cultures irradiated at mitosis, where the rate of increase in cell number paralleled that of unirradiated cells between days 3 and 4, but then appeared to slow slightly between days 4 and 5, such that the number of cells in the plates analyzed at day 5 was 80% as many as would have been expected if the rate at which of cell number increased were the same as between days 3 and 4. This meaningfulness of this break in the curve is unclear given the relatively large variation in cell number per plate. Overall, the growth kinetics data indicated that irradiation did not cause a permanent cell-cycle block in the majority of cells in each culture. We next examined the possibility of a cell-cycle block specific to blue cells. This was done by plotting the number of individual blue cells Žnormalized to 10 5 cells. versus time after irradiation ŽFig. 5.. The normalized number of blue cells declined slightly over the 5-day period studied. These data are inconsistent with what would be expected if all of the single blue cells formed at day 2 had been permanently blocked from going through cell division because if this had been the case, then the normalized number of blue cells would have declined with the kinetics shown by the theoretical curve labeled A in Fig. 5. The growth kinetic data describe a curve that falls between theoretical curves B and C, which describe what would happen if 55% Žcurve B. or 25% Žcurve C. of the single blue cells present 2 days
post-irradiation failed to divide. A cell-cycle block in 50% of the blue cells present at the 2-day time point in irradiated mitotic cells would account for about 30% of the single blue cells present in the culture 5 days post irradiation with 2 Gy. This conclusion stems from the following calculations. At day 2 post exposure, cultures irradiated while in mitosis contained 51 single blue cells. If 50% of these cells failed to divide during the ensuing 3 days, this would account for 26 of the 92 single blue cells present in the mitotic cultures assayed 5 days after irradiation. These data suggest that most of the single blue cells present at 5 days post irradiation were not cells that
Fig. 6. Survival curves of irradiated FSH-1 cells. Data for mitotic cells were obtained with cultures in which 80% of the cells were in mitosis when irradiated. In asynchronous cultures, the population contained 45% G1 cells, 35% S cells and 20% G2 qM cells. The plating efficiency of unirradiated cells was about 20%.
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failed to divide during the 5 day period between plating and staining for b-galactosidase activity. 3.4. Recombination and cell mortality following irradiation The cells in which recombination was most strongly induced by radiation Žmitotic cells. were the cells that were expected to be the most sensitive to the lethal effects of radiation w22,23x. To determine if this were the case in FSH-1 cells, cell survival following irradiation was assayed by determining the number of cells capable of forming colonies. Fig. 6 shows that the survival of FSH-1 cells was reduced in a dose dependent manner after irradiation. As expected w22,23x, survival was influenced by the synchrony of the culture, and by the cell cycle stage at the time of irradiation. Asynchronous cultures were least sensitive to radiation. Mitotic cells were the most sensitive. Cells irradiated during G1 or S phase showed an intermediate degree of sensitivity to radiation. These data established a correlation between the levels of recombination and degree of cytotoxicity induced by ionizing radiation. To assess the possible relationship of recombination and cytotoxicity, we examined induction of blue clusters in cultures that had suffered the same degree of reduction in colony forming capacity following irradiation. As can be seen in Fig. 6, the doses at which colony formation
Fig. 7. The number of blue clusters as a function of time following equitoxic doses of g-radiation. Doses administered and cellcycle stage of cells at the time of irradiation are indicated in inset.
was half that of untreated cells were 1 Gy for mitotic cells, 2 Gy for G1 and S phase cells, and 3 Gy for unsynchronized cells. Fig. 7 shows the number of blue clusters as a function of time following treatments that caused equivalent mortality in cells in M, G1 , S and in unsychronized cells. The curves for cells in G1 and S phases overlapped each other and the curve for unsynchronized cells, supporting the relationship between induction of recombination and mortality. However, the number of blue clusters induced by treatment of mitotic cells with 1 Gy was 2- to 3-fold greater than the number of blue clusters induced by treatment of the other three populations with higher doses. These data suggest that the increased recombination in mitotic cells was not solely due to a greater cytotoxic effect of radiation on mitotic cells.
4. Discussion The data presented above show that radiation can induce recombination in FSH-1 cells, and that the radiation effect is stronger in cells that have completed DNA replication than in cells at other points in the cell cycle. Irradiation of cells growing asynchronously induced less recombination than did irradiation of mitotic cells, even when the dose was administered to the asynchronous cells was three times that given to mitotic cells. Radiation-induced recombination in FSH-1 cells occurred rapidly. The frequency of blue cell clusters reached its peak at the earliest time point tested, 2 days post irradiation. The rapid induction of recombination was most apparent in cells that were irradiated at mitosis because the induction was much stronger in these cells than in cells irradiated during S or G1. Nevertheless, induction of recombination appeared to be equally rapid in FSH-1 cells irradiated at each of the three cell cycle stages. Although recombination events occurred in irradiated cells within 2 days, elevated numbers of single blue cells were present in populations 5 days after radiation. It is not clear what fraction of the single blue cells present at late times after irradiation was due to failure of recombinant cells to divide, and what fraction of single blue cells was due to delayed recombination. However, the growth kinetics of blue
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cells fit a model where some of the blue cells present at late times after irradiation were formed relatively late after exposure to radiation. Prolonged elevation of mutation following exposure to ionizing radiation has been described before in other kinds of cultured mammalian cells w24–26x. In addition, delayed recombination has been reported in irradiated yeast w27,28x. Radiation effects on recombination between mammalian chromosomal repeats have been previously studied by using clonogenic assays to detect cells that have undergone recombination w13–15,17x. In these studies, induction was observed at levels similar to those exhibited by unsynchronized FSH-1 cells in our experiments. This suggests that many if not all of the blue cells observed in our experiments with unsynchronized cells were viable and capable of forming colonies. Clonogenic assays have not been applied to irradiated synchronized cells, so it is not known whether or not the strong induction we observed would be evident by clonogenic assay. About half of the blue cells present in cultures derived from irradiated mitotic cells were able to divide at least once, but the ability of these cells to produce colonies is currently not known. Increased recombination in mitosis may be due to recombination between sister chromatids, as is the case in yeast w29,30x. The mechanism by which radiation induced recombination between lacZ repeats in FSH-1 cells is not known. One possibility is that recombination was used to repair a double-strand break in one of the repeats. It has been shown that a double-strand break in one of a pair of tandem repeats induced homologous recombination between the two repeats w31x. The number of breaks per cell was not measured in our study, but other data have shown that 2 Gy produced an average of one hundred breaks per genome w32,33x. If this were true in FSH-1 cells, and if breaks were randomly located, ten cells in 10 5 would be expected to sustain a break in a lacZ repeat. This fits with the observed frequency of blue cells that were irradiated at mitosis. While radiation-induction of recombination between two chromosomal tandem repeats was not previously evident, radiation-induction of homologous recombination in cultured mammalian cells has been seen before. In a human lymphoblast cell line, X-rays appeared to increase recombination between
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allelic thymidine kinase genes w11x. In addition, experiments utilizing a shuttle vector plasmid with a mutation in an ampicillin-resistance gene showed that the plasmid recombined more frequently with a chromosomal copy of the ampicillin-resistance gene when the plasmid DNA was treated with ionizing radiation prior to transfection into the mammalian cells w34x. Cell cycle dependence of recombination has also been detected in mammalian cells by studying extrachromosomal recombination. In this case, homologous recombination between coinjected plasmids peaked in early to mid-S phase w35x. The difference between these results and those obtained with FSH-1 cells may be a reflection of the different mechanisms thought to be involved in extrachromosomal and intrachromosomal recombination w6,36x. The relatively strong induction of recombination in FSH-1 cells irradiated during mitosis was associated with relatively low survival, which fits with previous data obtained with C3H10T1r2 cells w37,38x. The relatively low resistance of FSH-1 cells in mitosis to the lethal effects of radiation may be related to the fact that cells that survive irradiation at mitosis are more likely to suffer mutation, whether it be via intrachromosomal recombination events or via other mechanisms w39x. The higher rates of mutation sustained by cells irradiated at G2rM may also contribute to the increased number of transformed cells produced by such cultures as compared to cells irradiated at other points in the cell cycle w37,38x. Acknowledgements This work was supported in part by grants PO1ES05652 from the Public Health Service and NIH P30-ES06086 from the Center for Environmental Genetics. J.C. was supported by an NIH Fellowship ŽNIH IT32ES07250. in Environmental Carcinogenesis and Mutagenesis. References w1x Hellgren, D. Ž1992. Mutagen-induced recombination in mammalian cells in vitro, Mutation Res., 284, 37–51. w2x Carr, A.M. and M.F. Hoekstra Ž1995. The cellular responses to DNA damage, Trends Cell Biol., 5, 32–40. w3x Wurgler, F.E. Ž1992. Recombination and gene conversion, Mutation Res., 284, 3–14.
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w20x Terasima, T. and L.J. Tolmach Ž1963. Variation in several responses of HeLa cells to X-irradiation during the division cycle. Biophys. J., 3, 11–33. w21x Miller, R.C., C.R. Geard, M.J. Geard and E.J. Hall Ž1992. Cell-cycle-dependent radiation-induced oncogenic transformation of C3H 10T1r2 cells, Radiation Res., 130, 129–133. w22x Sinclair, W.K. and R.A. Morton Ž1966. X-Ray sensitivity during the cell generation cycle of cultured Chinese Hamster Cells, Radiation Res., 29, 450–474. w23x Sinclair, W.K. Ž1968. Cyclic X-ray responses in mammalian cells in Vitro, Radiation Res., 33, 620–643. w24x Chang, W.P. and J.B. Little Ž1992. Persistently elevated frequency of spontaneous mutations in progeny of CHO clones surviving X-irradiation: associations with delayed reproductive death phenotype, Mutation Res., 270, 191–199. w25x Galloway, S.M. Ž1994. Chromosome aberrations induced in vitro: mechanism, delayed expression, and intriguing questions, Environ. Mol. Mutagen., 23 ŽSuppl. 24., 44–53. w26x Stamato, T.D., R. Weinstein and A. Giaccia Ž1984. Timing of mutation-fixation events in ethyl methane sulfonate-treated Chinese hamster cells, Somat. Cell Mol. Genet., 10Ž4., 429– 434. w27x Fabre, F. and H. Roman Ž1977. Genetic evidence for inducibility of recombination competence in yeast. Proc. Natl. Acad. Sci. USA, 74, 1667–1671. w28x Fabre, F. Ž1983. Mitotic transmission of induced recombination ability in yeast. In: E. Friedberg ŽEd.., Cellular Responses to DNA Damage. New York, Liss, pp. 379–384. w29x Kadyk, L.C. and L.H. Hartwell Ž1992. Sister chromatids are preferred over homologs as substrates for recombinational repair in Saccharomyces cereÕisiae, Genetics, 132, 387–402. w30x Galli, A. and R.H. Schiestl Ž1995. On the mechanism of UV and g-ray-induced intrachromosomal recombination in yeast cells synchronized in different stages of the cell cycle, Mol. Gen. Genet., 248, 301–310. w31x Brenneman, M., F.S. Gimble and J.H. Wilson Ž1996. Stimulation of intrachromosomal homologous recombination in human cells by electroporation with site-specific endonucleases, Proc. Natl. Acad. Sci. USA, 93, 3608–3612. w32x Milligan, J.R., J.A. Aguilera, C.C.L. Wu, J.Y.-Y. Ng and J.F. Ward Ž1996. The difference that linear energy transfer makes to precursors of DNA strand breaks, Radiation Res., 145, 442–448. w33x Ruiz De Almodovar, J.M., C.G. Steel, S.J. Whitaher and T.J. McMillan Ž1994. A comparison of methods for calculating DNA double-strand break induction frequency in mammalian cells by pulsed-field gel electrophoresis, Int. J. Radiat. Biol., 65, 641–649. w34x Mudget, J.S. and W.D. Taylor Ž1990. Recombination between irradiated shuttle vector DNA and chromosomal DNA in African green monkey kidney cells, Mol. Cell. Biol., 10, 37–46. w35x Wong, E.A. and M.R. Capecchi Ž1987. Homologous recombination between coinjected DNA sequences peaks in early to mid-S phase, Mol. Cell. Biol. 7, 2294–2295. w36x Waldman, A.S. and R.M. Liskay Ž1987. Differential effects
J. Cao et al.r Mutation Research 374 (1997) 233–243 of base pair mismatch on intrachromosomal versus extrachromosomal recombination in mouse L cells, Proc. Natl. Acad. Sci. USA, 84, 5340–5344. w37x Cao, J., R.L. Wells and M.M. Elkind Ž1992. Enhanced sensitivity to neoplastic transformation by 137Cs g-rays of cells in the G2 rM-phase age interval, Int. J. Radiat. Biol., 62, 191–199.
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w38x Cao, J., R.L. Wells and M.M. Elkind Ž1993. Neoplastic transformation of C3H Mouse Embryo Cells, 10T1r2: Cellcycle dependence for 50 kV X-rays and UV-B light. Int. J. Radiat. Biol., 64, 83–92. w39x Little, J.B. Ž1994. Changing views of cellular radiosensitivity, Radiation Res., 140, 299–311.
Cell cycle dependence of radiation-induced homologous recombination in cultured monkey cells J. Cao, S.E. DePrimo, J.R. Stringer
)
Department of Molecular Genetics, Biochemistry, and Microbiology, UniÕersity of Cincinnati Medical Center, Cincinnati, OH 45267-0524, USA Received 31 May 1996; revised 5 November 1996; accepted 5 November 1996
Abstract A lacZ transgene recombination system that reports homologous recombination events involving duplicated lacZ segments was used to study recombination in monkey cells exposed to ionizing radiation at different points in the cell cycle. With this system, recombination events can be detected in single cells by histochemical staining soon after exposure of cells to DNA-damaging treatment. Ionizing radiation rapidly induced recombination 5–10-fold in cells that were at the mitosis stage of the cell cycle. Irradiation either of cells at other points in the cell cycle or of nonsynchronized cells had less of an effect on recombination between lacZ segments. Keywords: Recombination; Genomic instability; Radiation; Cell cycle; Mitosis; LacZ gene
1. Introduction Homologous recombination plays a very important role in the repair of DNA damage w1,2x. However, homologous recombination is also potentially dangerous because it can contribute to genomic instability, which can in turn contribute to neoplastic transformation and cancer w3,4x. An association between increased homologous recombination and cancer risk has been long appreciated based on the phenotypes of cells from patients with chromosome instability and cancer predisposition syndromes such as Bloom syndrome ŽBS., ataxia telangiectasia ŽAT., xeroderma pigmentosum ŽXP., and Fanconi anemia ) Corresponding author. Tel.: Ž513. 558-0069; Fax: Ž513. 5588474; E-mail: [email protected]
ŽFA. w5,6x. Homologous recombination is also thought to be the mechanism of mitotic crossing over, which is known to be a pathway by which genes encoding tumor suppressor proteins are lost from somatic cells. It has been shown that ionizing radiation stimulates recombination between homologous chromosomes in yeasts w7,8x, fruit flies w9,10x and mammalian cells in culture w11x. Ionizing radiation also causes sister chromatid exchanges in mammalian cells w12x. Hence it seems reasonable to the expect that ionizing radiation would induce homologous recombination between tandem repeats in mammalian cells. However, strong induction of homologous recombination between tandem repeats has yet to be demonstrated in cultured mammalian cells. Several previous studies have shown that ionizing
0027-5107r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved. PII S 0 0 2 7 - 5 1 0 7 Ž 9 6 . 0 0 2 3 7 - 0
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radiation had little effect on recombination between tandemly repeated transgenes w13–16x. Similar results were obtained with a mutant allele of the HPRT gene that could revert by homologous recombination between tandem repeats w17x. The slight induction observed in cultured cells contrasts with the induction of recombination observed between two 70 kb regions that are tandemly duplicated in the pink-eyed unstable mutation of mice w18x. Here we studied the possible relationship between cell cycle stage and response to ionizing radiation in cells derived from the monkey cell line CV-1. We found that radiation induced more recombination in cells that were in mitosis than in cells that were in other cell cycle stages.
2. Material and methods 2.1. Synchronization of FSH 1 cells The FSH-1 cell line was used in all experiments. FSH-1 cells were derived from the CV-1 line of monkey cells as previously described w19x. Cells were grown in DMEM ŽGibco. supplemented with 10% fetal calf serum ŽFCS. ŽSigma., L-glutamine ŽGibco., and antibiotic-antimycotic ŽGibco.. Cells were synchronized by the mitotic shake-off method w20,21x. Culture flasks ŽT150. were seeded with 1.5 = 10 6 cells each. After 2 days, the flasks were prepared for harvesting of mitotic cells. To minimize the chance that cells that had already divided would contaminate the mitotic cell population, weakly attached cells were removed and discarded. This was done by adding 15 ml of media to a flask, striking the flask firmly, and submerging the monolayer in the media. Dislodged cells floating in the media were removed. This process was repeated three times, after which media was added and cultures incubated for 30 min at 378C. The shake-off was then repeated 3 times to harvest mitotic cells, which were collected by centrifugation at 250 = g for 10 min at 48C. Cells were resuspended in media at 48C and counted. The mitotic index of such cells was about 85%. Cells obtained by the shake-off procedure were incubated in media containing BrdU following which the cells were held for various time periods, and then assayed
for binding to a fluorescein conjugate of Anti-BrdU ŽBecton-Dickinson.. Labeling was first observed after 9 h, and continued for a 7-h period. This indicated that the G1 period was 9 h and that S-phase lasted approximately 7 h. Mitotic cells were first observed after 20 h. The fraction of cells in G1 , S, and G2rM in unsynchronized cultures was determined by flow cytometry. 2.2. g-Irradiation procedure A portion of the cells obtained by mitotic shake-off was irradiated Žmitotic cells., and the rest were placed in T25 flasks and incubated at 378C. Some of these cells were irradiated after 4 h ŽG1 phase cells., and some were irradiated after 14 h ŽS phase cells.. Gamma-rays were produced by a 60 Co irradiator at dose rate of 0.80 Gyrmin. For asynchronous cells, log phase cell monolayers were seeded at least 1 day before irradiation at a density of 1.0 = 10 6 cellsrT75 flask. 2.3. Analysis of homologous recombination in synchronous FSH 1 cells After irradiation, cells were removed from flasks by trypsin treatment and were replated at a density of 5 = 10 4r60 mm culture dish and incubated at 378C for 2–5 days. For analysis, one culture was trypsinized and counted, and others were fixed and stained with X-gal overnight at 378C. X-gal stain solution was 0.5 mgrml with 44 mM HEPES, 3 mM potassium ferrocyanide, 3 mM potassium ferricyanide, and 0.7 mM MgCl 2 in PBS. Blue cells were counted by microscopic examination.
3. Results 3.1. Recombination in irradiated FSH-1 cells FSH-1 cells contain two nonfunctional lacZ genes arranged as a tandem repeat. The repeated lacZ genes are each 3 kb in size and are separated by about 4 kb. Homologous recombination between the tandem repeats produces a lacZ gene capable of producing b-galactosidase, and cells containing a
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Fig. 1. Recombination following irradiation at different times during the cell cycle. Monolayers of fixed cells were tested for the presence of b-galactosidase activity by staining with X-Gal. Clusters of blue cells Žareas containing one or more blue cells. were scored by microscopy. Lines labeled as controls are data from cultures that had not been irradiated.
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recombined lacZ gene can be detected by staining with X-gal w19x. To study the effect of g-rays on homologous recombination, cultures of FSH-1 cells were exposed to a total dose of 2 Gy. After irradiation, sixteen 60 mm plates were seeded with 5 = 10 4 cells per plate. Plates were incubated at 378C and analyzed after 2, 3, 4 and 5 days. For each time point, the cells from one culture were harvested and counted, and three cultures were fixed and stained to detect cells expressing b-galactosidase. The cell density on the stained plates was sometimes too high to allow discreet colonies of cells to be identified. Therefore, plates were examined under a microscope to visualize individual blue cells. Because each plate contained very few blue cells, clusters of blue cells were assumed to have been derived from the growth of single blue cells. Therefore, the number of discreet
Fig. 2. Dose response of blue cluster induction at various times after irradiation. Blue clusters were scored as described in Fig. 1. ŽA–D. Results obtained when cells were stained after 2 ŽA., 3 ŽB., 4 ŽC. or 5 ŽD. days post irradiation. S phase and G1 phase cells were tested at 2 Gy only. Lines labeled Asy are from cultures growing asynchronously.
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areas containing one or more blue cells Žblue clusters. was used to estimate the number of induced recombination events. The relationship between radiation treatment and the number of blue clusters is shown in Fig. 1. Irradiation had little effect on the number of blue clusters in asynchronous cells. By contrast, cultures exposed at mitosis contained 7 times as many blue clusters per 10 5 cells as unirradiated synchronized cells 2 days after irradiation. Less induction of blue clusters was observed in cultures containing cells that had been irradiated at either S or G1. In each of the cultures that had elevated numbers of blue clusters at 2 days post irradiation, the number of blue clusters per 10 5 cells declined over the course of the next 3 days. This decline occurred primarily because the cell number increased more rapidly than the did the number of blue clusters. These results suggest that most of the induction occurred soon after irradiation. Recombination was also studied in cells that were not synchronized when irradiated, but were collected by mitotic shake-off at various times after irradiation. In this procedure, cells that were in mitosis when the culture was irradiated shake-off first, fol-
lowed by cells that were in G2 , S and G1. These experiments showed that most of the blue cells were obtained in the first shake-off suggesting that induction was strongest in cells that were in mitosis at the time of irradiation Ždata not shown.. 3.2. Dose response of recombination Because induction of recombination was strongest in mitotic cells, these cells were used to investigate the dose-dependence of recombination. Fig. 2 shows the number of blue clusters observed at four time points ŽA–D. following irradiation with 1 Gy, 2 Gy or 3 Gy. Recombination in cells growing asynchronously was usually weakly affected by exposure to 1 Gy or 2 Gy, with blue cluster counts remaining within a factor of 2 of those seen in unirradiated control cultures. In asynchronous cultures, stronger induction was obtained after exposure to 3 Gy. Fig. 2A shows that asynchronous cultures stained after 2 days contained 4 times as many blue clusters as unirradiated cultures. However, the blue cluster counts declined in asynchronous cultures that were kept longer than 2 days prior to staining, and by day 5, no induction was evident even after exposure to 3
Fig. 3. Size distribution of blue clusters 5 days after irradiation. Blue clusters were scored as described in Fig. 1. Bars labeled as controls are data from cultures that had not been irradiated.
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Gy Žsee Fig. 2D.. By contrast, cultures irradiated while cells were in mitosis generally contained three to five times as many blue clusters as did unirradiated synchronized cultures, and the blue cluster counts remained elevated by at least 4-fold at all time points. 3.3. Viability of cells that had undergone recombination To determine how many of the blue cells present after irradiation were able to undergo cell division, we analyzed plates of cells for the size of blue clusters. Table 1 shows that each culture exhibited blue clusters of various sizes, and that larger clusters were more prevalent at later times after plating. Clusters contained up to 16 blue cells 5 days after plating, indicating that some blue cells had doubled up to four times. This was consistent with results of previous experiments, which showed that a single blue FSH-1 cell present at the time a plate was seeded could produce up to 16 cells after 5 days of incubation w19x. Fig. 3 shows the distribution of blue cluster sizes present in cultures 5 days after irradiation, where the larger clusters of blue cells were most evident. Data on the sizes of blue clusters obtained at other time points Žlisted in Table 1., were
Fig. 4. Growth curves of synchronous and asynchronous populations after irradiation. Relative cell number is the number of cells present at a given time divided by the number of cells plated initially. Lines labeled as controls are data from cultures that had not been irradiated. Synchronous cultures were irradiated when 80% of the cells were in mitosis.
Fig. 5. Total number of blue cells in cultures derived from mitotic cells that had been exposed to irradiation. The line with error bars shows the data. Lines labelled A, B and C are theoretical curves that show what would be expected if all induction had occurred by Day 2 and if 100% ŽA., 55% ŽB., or 25% ŽC. of the blue cells present at Day 2 were unable to divide.
consistent with results obtained at the 5 day time point. The data in Fig. 3 showed that some of the cells that had undergone recombination were able to divide several times, but it was striking that many of the blue cells induced by irradiation of synchronized cells were present as singlets, even 5 days after irradiation. To illustrate, 5 days after a dose of 2 Gy, the fraction of blue cell ‘clusters’ that contained a single blue cell was 65% in cells irradiated during mitosis. By contrast, the fraction of blue cell ‘clusters’ that had a single blue cell was 46% in unirradiated synchronous cultures. The abundance of single blue cells at late times after irradiation could have been due to either of two nonmutually exclusive factors: Ži. division of some blue cells may have been blocked due to radiation damage, in which case single blue cells generated early after irradiation would have failed to produce clusters of blue cells; and Žii. recombination may have remained elevated for days after irradiation Ždelayed recombination.. To examine these factors, we analyzed the growth kinetics of populations of FSH-1 cells that had been irradiated. First, we examined the degree to which radiation induced a general cell-cycle block. Fig. 4 shows that for 3 days following irradiation, the rate at which cell number increased in irradiated cultures
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Table 1 Frequency and size of blue-cell clusters in FSH-1 cell culture Blue cluster size
a
1
2
Synchronous unirradiated Day 2 4.11 " 0.22 Day 3 2.73 " 0.51 Day 4 1.76 " 0.16 Day 5 1.74 " 0.10
b
3 to 4
2.44 " 1.44 1.23 " 0.12 1.24 " 0.30 1.00 " 0.23
1.23 " 0.12 1.14 " 0.21 0.38 " 0.088
Mitotic 1.0 Gy Day 2 Day 3 Day 4 Day 5
27.5 " 2.5 8.64 " 0.74 4.78 " 0.93 6.49 " 0.75
6.28 " 0.72 5.76 " 0.49 2.68 " 0.40 2.64 " 0.22
2.88 " 0.25 2.35 " 0.35 1.46 " 0.34
Mitotic 2.0 Gy Day 2 Day 3 Day 4 Day 5
28.86 " 3.86 11.71 " 0.80 11.73 " 0.06 11.49 " 2.62
10.15 " 3.49 5.82 " 2.18 5.18 " 0.18 3.13 " 0.57
3.52 " 1.02 3.63 " 0.30 1.15 " 0.22
Mitotic 3.0 Gy Day 2 Day 3 Day 4 Day 5
46.43 " 13.57 18.11 " 4.11 17.86 " 2.14 11.93 " 1.61
7.14 " 2.86 5.33 " 1.33 5.83 " 0.83 3.78 " 0.73
2.11 " 0.11 3.05 " 1.62 1.22 " 0.25
G1 2.0 Gy Day 2 Day 3 Day 4 Day 5
13.50 " 1.83 6.97 " 1.37 5.10 " 2.40 2.79 " 0.38
4.33 " 2.33 5.18 " 3.58 1.88 " 0.63 1.26 " 0.27
3.73 " 2.93 1.40 " 0.35 0.70 " 0.21
S 2.0 Gy Day 2 Day 3 Day 4 Day 5
10.63 " 0.63 3.75 " 1.25 3.24 " 0.99 2.94 " 0.16
5.21 " 1.04 2.42 " 1.92 2.17 " 1.17 1.92 " 0.40
1.58 " 0.08 1.03 " 0.53 0.88 " 0.13
Asynchronous unirradiated Day 2 1.34 " 0.57 Day 3 1.41 " 0.60 Day 4 0.83 " 0.13 Day 5 0.62 " 0.06
2.32 " 0.78 1.18 " 0.37 0.71 " 0.11 0.58 " 0.09
1.15 " 0.07 0.46 " 0.03 0.35 " 0.05
Asynchronous 1.0 Gy Day 2 Day 3 Day 4 Day 5
3.35 " 0.21 3.29 " 0.16 1.16 " 0.16 0.81 " 0.09
4.11 " 0.11 1.69 " 0.19 0.84 " 0.37 0.58 " 0.07
1.47 " 0.09 0.37 " 0.10 0.34 " 0.03
Asynchronous 2.0 Gy Day 2 Day 3
3.92 " 1.20 3.71 " 0.29
2.42 " 0.60 2.50 " 0.50
1.12 " 0.45
5 to 8
0.49 " 0.09 0.42 " 0.10
1.29 " 0.14 0.94 " 0.28
1.52 " 0.81 0.76 " 0.18
0.69 " 0.02 0.75 " 0.28
0.33 " 0.08 0.26 " 0.13
0.68 " 0.43 0.69 " 0.16
0.34 " 0.04 0.28 " 0.08
0.37 " 0.10 0.262 " 0.04
9 to 10
Total
0.270 " 0.03
6.56 " 1.22 5.20 " 0.75 4.63 " 0.76 3.81 " 0.23
1.06 " 0.35
33.78 " 1.78 17.23 " 1.48 11.10 " 0.33 12.59 " 1.60
0.87 " 0.32
39.02 " 7.35 21.05 " 1.96 22.06 " 0.27 17.42 " 3.59
0.54 " 0.10
53.57 " 16.43 25.56 " 5.56 27.43 " 4.57 18.21 " 2.27
0.34 " 0.13
17.83 " 0.50 15.88 " 7.88 8.71 " 3.29 5.34 " 0.79
0.81 " 0.11
15.83 " 1.67 7.75 " 3.25 7.11 " 3.11 7.23 " 0.60
0.23 " 0.03
3.65 " 1.35 3.74 " 1.04 2.33 " 0.21 2.05 " 0.11
0.22 " 0.04
7.46 " 0.32 6.44 " 0.44 2.74 " 0.53 2.20 " 0.18
6.34 " 1.80 7.33 " 0.67
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Table 1 Žcontinued. Blue cluster size
a
1
2
3 to 4
5 to 8
9 to 10
0.68 " 0.03 0.35 " 0.10
0.21 " 0.05
5.39 " 0.59 2.68 " 0.39
0.13 " 0.03
16.35 " 1.15 12.51 " 0.74 6.80 " 1.20 3.15 " 0.15
Asynchronous 2.0 Gy Day 4 Day 5
2.49 " 0.16 1.06 " 0.12
1.17 " 0.33 0.67 " 0.11
1.05 " 0.12 0.40 " 0.06
Asynchronous 3.0 Gy Day 2 Day 3 Day 4 Day 5
10.22 " 0.62 7.39 " 0.39 3.25 " 0.35 1.63 " 0.09
6.13 " 0.53 2.64 " 0.86 1.80 " 0.80 0.77 " 0.07
2.49 " 0.26 0.90 " 0.20 0.31 " 0.07
a b
0.85 " 0.25 0.32 " 0.07
Total
Number of blue cells per cluster. Number of blue clusters per 10 5 cells " SE.
was less than in unirradiated cultures, but by the fourth day, all but one of the irradiated cultures expanded at essentially the same rate as unirradiated cells. The one exception was observed in the cultures irradiated at mitosis, where the rate of increase in cell number paralleled that of unirradiated cells between days 3 and 4, but then appeared to slow slightly between days 4 and 5, such that the number of cells in the plates analyzed at day 5 was 80% as many as would have been expected if the rate at which of cell number increased were the same as between days 3 and 4. This meaningfulness of this break in the curve is unclear given the relatively large variation in cell number per plate. Overall, the growth kinetics data indicated that irradiation did not cause a permanent cell-cycle block in the majority of cells in each culture. We next examined the possibility of a cell-cycle block specific to blue cells. This was done by plotting the number of individual blue cells Žnormalized to 10 5 cells. versus time after irradiation ŽFig. 5.. The normalized number of blue cells declined slightly over the 5-day period studied. These data are inconsistent with what would be expected if all of the single blue cells formed at day 2 had been permanently blocked from going through cell division because if this had been the case, then the normalized number of blue cells would have declined with the kinetics shown by the theoretical curve labeled A in Fig. 5. The growth kinetic data describe a curve that falls between theoretical curves B and C, which describe what would happen if 55% Žcurve B. or 25% Žcurve C. of the single blue cells present 2 days
post-irradiation failed to divide. A cell-cycle block in 50% of the blue cells present at the 2-day time point in irradiated mitotic cells would account for about 30% of the single blue cells present in the culture 5 days post irradiation with 2 Gy. This conclusion stems from the following calculations. At day 2 post exposure, cultures irradiated while in mitosis contained 51 single blue cells. If 50% of these cells failed to divide during the ensuing 3 days, this would account for 26 of the 92 single blue cells present in the mitotic cultures assayed 5 days after irradiation. These data suggest that most of the single blue cells present at 5 days post irradiation were not cells that
Fig. 6. Survival curves of irradiated FSH-1 cells. Data for mitotic cells were obtained with cultures in which 80% of the cells were in mitosis when irradiated. In asynchronous cultures, the population contained 45% G1 cells, 35% S cells and 20% G2 qM cells. The plating efficiency of unirradiated cells was about 20%.
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failed to divide during the 5 day period between plating and staining for b-galactosidase activity. 3.4. Recombination and cell mortality following irradiation The cells in which recombination was most strongly induced by radiation Žmitotic cells. were the cells that were expected to be the most sensitive to the lethal effects of radiation w22,23x. To determine if this were the case in FSH-1 cells, cell survival following irradiation was assayed by determining the number of cells capable of forming colonies. Fig. 6 shows that the survival of FSH-1 cells was reduced in a dose dependent manner after irradiation. As expected w22,23x, survival was influenced by the synchrony of the culture, and by the cell cycle stage at the time of irradiation. Asynchronous cultures were least sensitive to radiation. Mitotic cells were the most sensitive. Cells irradiated during G1 or S phase showed an intermediate degree of sensitivity to radiation. These data established a correlation between the levels of recombination and degree of cytotoxicity induced by ionizing radiation. To assess the possible relationship of recombination and cytotoxicity, we examined induction of blue clusters in cultures that had suffered the same degree of reduction in colony forming capacity following irradiation. As can be seen in Fig. 6, the doses at which colony formation
Fig. 7. The number of blue clusters as a function of time following equitoxic doses of g-radiation. Doses administered and cellcycle stage of cells at the time of irradiation are indicated in inset.
was half that of untreated cells were 1 Gy for mitotic cells, 2 Gy for G1 and S phase cells, and 3 Gy for unsynchronized cells. Fig. 7 shows the number of blue clusters as a function of time following treatments that caused equivalent mortality in cells in M, G1 , S and in unsychronized cells. The curves for cells in G1 and S phases overlapped each other and the curve for unsynchronized cells, supporting the relationship between induction of recombination and mortality. However, the number of blue clusters induced by treatment of mitotic cells with 1 Gy was 2- to 3-fold greater than the number of blue clusters induced by treatment of the other three populations with higher doses. These data suggest that the increased recombination in mitotic cells was not solely due to a greater cytotoxic effect of radiation on mitotic cells.
4. Discussion The data presented above show that radiation can induce recombination in FSH-1 cells, and that the radiation effect is stronger in cells that have completed DNA replication than in cells at other points in the cell cycle. Irradiation of cells growing asynchronously induced less recombination than did irradiation of mitotic cells, even when the dose was administered to the asynchronous cells was three times that given to mitotic cells. Radiation-induced recombination in FSH-1 cells occurred rapidly. The frequency of blue cell clusters reached its peak at the earliest time point tested, 2 days post irradiation. The rapid induction of recombination was most apparent in cells that were irradiated at mitosis because the induction was much stronger in these cells than in cells irradiated during S or G1. Nevertheless, induction of recombination appeared to be equally rapid in FSH-1 cells irradiated at each of the three cell cycle stages. Although recombination events occurred in irradiated cells within 2 days, elevated numbers of single blue cells were present in populations 5 days after radiation. It is not clear what fraction of the single blue cells present at late times after irradiation was due to failure of recombinant cells to divide, and what fraction of single blue cells was due to delayed recombination. However, the growth kinetics of blue
J. Cao et al.r Mutation Research 374 (1997) 233–243
cells fit a model where some of the blue cells present at late times after irradiation were formed relatively late after exposure to radiation. Prolonged elevation of mutation following exposure to ionizing radiation has been described before in other kinds of cultured mammalian cells w24–26x. In addition, delayed recombination has been reported in irradiated yeast w27,28x. Radiation effects on recombination between mammalian chromosomal repeats have been previously studied by using clonogenic assays to detect cells that have undergone recombination w13–15,17x. In these studies, induction was observed at levels similar to those exhibited by unsynchronized FSH-1 cells in our experiments. This suggests that many if not all of the blue cells observed in our experiments with unsynchronized cells were viable and capable of forming colonies. Clonogenic assays have not been applied to irradiated synchronized cells, so it is not known whether or not the strong induction we observed would be evident by clonogenic assay. About half of the blue cells present in cultures derived from irradiated mitotic cells were able to divide at least once, but the ability of these cells to produce colonies is currently not known. Increased recombination in mitosis may be due to recombination between sister chromatids, as is the case in yeast w29,30x. The mechanism by which radiation induced recombination between lacZ repeats in FSH-1 cells is not known. One possibility is that recombination was used to repair a double-strand break in one of the repeats. It has been shown that a double-strand break in one of a pair of tandem repeats induced homologous recombination between the two repeats w31x. The number of breaks per cell was not measured in our study, but other data have shown that 2 Gy produced an average of one hundred breaks per genome w32,33x. If this were true in FSH-1 cells, and if breaks were randomly located, ten cells in 10 5 would be expected to sustain a break in a lacZ repeat. This fits with the observed frequency of blue cells that were irradiated at mitosis. While radiation-induction of recombination between two chromosomal tandem repeats was not previously evident, radiation-induction of homologous recombination in cultured mammalian cells has been seen before. In a human lymphoblast cell line, X-rays appeared to increase recombination between
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allelic thymidine kinase genes w11x. In addition, experiments utilizing a shuttle vector plasmid with a mutation in an ampicillin-resistance gene showed that the plasmid recombined more frequently with a chromosomal copy of the ampicillin-resistance gene when the plasmid DNA was treated with ionizing radiation prior to transfection into the mammalian cells w34x. Cell cycle dependence of recombination has also been detected in mammalian cells by studying extrachromosomal recombination. In this case, homologous recombination between coinjected plasmids peaked in early to mid-S phase w35x. The difference between these results and those obtained with FSH-1 cells may be a reflection of the different mechanisms thought to be involved in extrachromosomal and intrachromosomal recombination w6,36x. The relatively strong induction of recombination in FSH-1 cells irradiated during mitosis was associated with relatively low survival, which fits with previous data obtained with C3H10T1r2 cells w37,38x. The relatively low resistance of FSH-1 cells in mitosis to the lethal effects of radiation may be related to the fact that cells that survive irradiation at mitosis are more likely to suffer mutation, whether it be via intrachromosomal recombination events or via other mechanisms w39x. The higher rates of mutation sustained by cells irradiated at G2rM may also contribute to the increased number of transformed cells produced by such cultures as compared to cells irradiated at other points in the cell cycle w37,38x. Acknowledgements This work was supported in part by grants PO1ES05652 from the Public Health Service and NIH P30-ES06086 from the Center for Environmental Genetics. J.C. was supported by an NIH Fellowship ŽNIH IT32ES07250. in Environmental Carcinogenesis and Mutagenesis. References w1x Hellgren, D. Ž1992. Mutagen-induced recombination in mammalian cells in vitro, Mutation Res., 284, 37–51. w2x Carr, A.M. and M.F. Hoekstra Ž1995. The cellular responses to DNA damage, Trends Cell Biol., 5, 32–40. w3x Wurgler, F.E. Ž1992. Recombination and gene conversion, Mutation Res., 284, 3–14.
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