Chromosomal Aberrations and Mortality of X-Irradiated Mammalian Cells: Emphasis on Repair

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Proceedings of the National Academy of Sciences Vol. 68, No. 3, pp. 667671, March 1971 Chromosomal Aberrations and Mortality of XIrradiated Mammalian Cells: Emphasis on Repair W. C. DEWEY, H.

1 Proceedings of the National Academy of Sciences Vol. 68, No. 3, pp , March 1971 Chromosomal Aberrations and Mortality of XIrradiated Mammalian Cells: Emphasis on Repair W. C. DEWEY, H. H. MILLER, AND D. B. LEEPER Department of Radiation Biology, Colorado State University, Fort Collins 8521 Communicated by Theodore T. Puck, December 3, 197 ABSTRACT Chromosomal aberrations induced by x rays in synchronied Chinese hamster cells correlated well with mortality as the radiosensitivity was made to vary by a factor of by treating the cells with colcemid or 5 bromodeoxyuridine and by irradiating the cells in different parts of the cell cycle. 37% survival was observed whe i the cells sustained about one aberration per cell. Furthermore, the positive correlation was observed during repair of damage. Repair was measured by the reduction in damage observed either as the incubation period between two doses was increased (repair of sublethal lesions), or as the cells were held at 2'C after a single dose of radiation (repair of potentially lethal lesions). Repair of both sublethal and potentially lethal lesions occurred with a halftime of about 12 min and was inhibited at C. These correlations strongly support the hypothesis that xrayinduced cell death results from damage in chromatin structures. Xrayinduced mortality in mammalian cells has been attributed primarily to aberrations or lesions in the chromosomes, and the shoulder on the survival curve was postulated to result from the interaction of independently produced aberrations (1). Interaction of lesions has been demonstrated in terms of both chromosomal damage (2) and mortality (3) by the observation that two doses of radiation produce more than an additive effect if the second dose is given immediately after the first dose. However, if an incubation period of 12 hr is allowed between the two doses, the observed damage is reduced to that expected for an additive effect (26). This reduction in mortality, associated with divided doses, has been attributed to the repair of sublethal lesions (3), and the reduction in chromosomal damage has been attributed to the repair or restitution of lesions in the chromosomes (). To obtain evidence that the two types of lesions are identcal, we have compared the kinetics and magnitude of repair measured by mortality with those measured by chromosomal aberrations. To further test the hypothesis that mortality results from chromosomal aberrations, we have determined the correlation between cell death and chromosomal aberrations as synchronied cells were irradiated in various phases of the cell cycle, which differ in radiosensitivity. Furthermore, this correlation was studied as repair was allowed to occur during a period at 2'C after a single dose of irradiation (7, 8); during this period at 2'C, processes needed to complete the damage (potentially lethal) are apparently inhibited, while repair processes are not. Abbreviations: CHO, Chinese hamster ovary; PE, plating efficiency; N, cellular multiplicity. 667 MATERIALS AND METHODS Lung cells of a male Chinese hamster (Don strain) (9) and hamster ovary cells (CHO) (1, 11), both of which were 95% neardiploid, were carried as monolayers on either glass or Falcon plastic at 37C in an atmosphere of 6% CO2 in Mc Coy's 5a medium (12). The cells were synchronied as described previously (11, 12) by the selective removal of mitotic cells (mitotic indices of 998%) from an asynchronous population, which in some experiments (with Don cells only) had been treated for 2 hr with colcemid (.6 Ag/ml). The synchronied cells were incubated at 37C and were xirradiated (effective energy of 9 kev and dose rate of 3 rad/min (12)) during various phases of the cell cycle. In some experiments, cells were irradiated at room temperature (2327"C) in 6 or 1mm Falcon Petri dishes placed on a rotating wax block. In other experiments, T3 Falcon flasks were irradiated on the rotating wax block placed in a temperaturecontrolled water bath (either, 2, or 37C). To change the temperature rapidly after irradiation (e.g. from or 2 to 37C), we transferred the Tflasks to a 6C water bath for 1 min before they were placed in a 37C water bath. Chromosomal aberrations After xirradiation and plating, the cells were incubated for 1125 hr to allow them to recover from radiationinduced cycle delay. They were then arrested in metaphase by a 23 hr treatment with colcemid (.6.ug/ml). The cell samples were fixed by the hypotonic method when the mitotic ndex reached 13%, and at two or three different times; this ensured an adequate sample of the population of cells as they traversed the first mitosis after irradiation (12, 13). 355 metaphase cells were examined for chromosomal aberrations in each sample. The aberrations were scored as chromatid deletions, isolocus deletions, and exchanges, i.e., chromatid intrachanges and interchanges, and chromosome intrachanges (rings) and interchanges (97% dicentrics) (1). Cell mortality Synchronied cells were plated, treated, and irradiated exactly as those cells studied for chromosomal aberrations. The number of cells plated was adjusted to give 12 colonies per Petri dish or Tflask, and after an incubation time of 69 days, the colonies were fixed and stained (13). Plating efficiencies (PE) were determined from the controls for each experiment. The colony survival values were corrected for the survival of single cells as follows. Cells were

$668 Zoology: Dewey et al. 1.1.1 > IL i.1 I'~~~~G \ '' \G H H IE, DELETIONS AND H EXCHANGES DELETIONe", EXCHANGES 5 c\ G EXCHANGESG 1 2 3 ABERRATIONS PER CELL 5 6 FIG. 1. Colony survival vs.$

2 668 Zoology: Dewey et al > IL i.1 I'~~~~G \ '' \G H H IE, DELETIONS AND H EXCHANGES DELETIONe", EXCHANGES 5 c\ G EXCHANGESG ABERRATIONS PER CELL 5 6 FIG. 1. Colony survival vs. aberrations per cell for synchronied Chinese hamster cells xirradiated at different times in the cell cycle. The letters designate experiments with lung cells (Don): A and B, mitotic and G1 cells, respectively, synchronied without colcemid: C and D, mitotic and G1 cells, respectively, synchronied with colcemid; E, G, and H, Sphase cells synchronied with colcemid. The numbers designate experiments with Chinese hamster ovary cells (CHO) synchronied without colcemid: 1, G1 cells; 3, GI cells treated with 5bromodeoxyuridine; 28, Sphase cells. Data points are shown only for deletions plus exchanges. fixed on control Petri dishes at the time of irradiation and were analyed for cellular multiplicity (N), the average number of cells (1.31.7) per potential colonyforming unit (15). Statistics Each data point for an experiment represents the analysis of either the number of chromosomal aberrations in 751 cells or the number of colonies in 6 Petri dishes. Where indicated in the text or graphs, the confidence intervals represent the standard error of the mean. RESULTS AND DISCUSSION Quantitative relationship between cell killing and chromosomal aberrations The results of several experiments (13, 16), summaried in Fig. 1, indicate the positive correlation between cell killing and chromosomal aberrations for cells irradiated at different times in the cell cycle and after treatment with radiation sensitiers. The change in radiation sensitivity is quantitated as a dosemodifying factor, the ratio of two doses that produce the same amount of damage. For cells synchronied after colcemid treatment, dosemodifying factors (data not shown) were, relative to irradiation during the mitotic phase (which is the most radiosensitive), for irradiation in GI and for irradiation in S phase. The 2hr colcemid treatment sensitied mitotic and G, cells by a factor of 1.52 but had little effect on Sphase cells; and 18hr treatment with 5 bromodeoxyuridine prior to irradiation sensitied GI cells by a factor of A compilation of all the data (Fig. 1) 1 IL i 9 5 U) I N *1.29 PE ' 1 % 7 HR SINGLEa 6 HR SINGLE Proc. Nat. Acad. Sci. USA 68 (1971) J s6 1~ L...^ 2 6 Boo FIG. 2. C) 2 l 5xIO5 CELLS o x l CELLS H T bp A 5 MR SINGLE HOURS AFTER PLATING TOTAL DOSE RADS o 7HR o 6 HR 1 Survival values vs. dose for Don cells irradiated with a single or a divided dose during S phase, i.e., 57 hr after cells synchronied in metaphase with colcemid had been plated. The cells were irradiated at 337C and were incubated at 37C between doses. The arrows indicating the survival fraction corresponding to complete recovery were calculated as follows: A on the assumption that the first dose had the 5hr response and the second dose had the 7hr response (.192 X.151 =.29), and B on the assumption that both doses had the 5hr response (.192 X.192 =.37). The percentage of cells in S phase at the time of irradiation ("per cent labeled") was determined by pulselabeling the cells in replicate Petri dishes for 1 min with [3H]thymidine; the large cell number (5 X 16) represents cell numbers plated for the chromosomal analysis, and the low cell number (1 X 1) represents cell numbers plated for the mortality analy SiS. revealed that the 37% survival level, which specifies an average of one lethal occurrence per cell (17), occurred when the cells sustained an average of about one chromosomal aberration per cell. Furthermore, the logarithm of the survival fraction was linearly related to the aberration frequency, up to about 3 aberrations per cell. Thus, as the radiosensitivity changed by as much as a factor of, the aberration frequency correlated well with colony survival. Magnitude of repair of sublethal lesions After a dose of xrays there are lesions, besides those that result in chromosomal aberrations and cell killing, that do not result in aberrations (2, 6) or killing (3) unless they interact with lesions produced by a second dose of xrays. This phenomenon results in the shoulder or quasithreshold observed when the aberration frequency or the logarithm of survival is plotted as a function of radiation dose (15).

3 Proc. Nat. Acad. Sci. USA 68 (1971) XRays and Chromosomal Aberrations 669 * EXP. 17, CHO, PEw76 Na1.3 AA EXP 11 DON, PEu51I N1.5 J J a ) i 3 RADS AT 5HR 9 SINGLE DOSE SECOND DOSE AT: A 5HR 7 HR U *6*HR o 6HR X * 7HR ADDITIVE _> 6 8 l.1] HOURS AFTER PATING.8 F i.6 '3 6 9 DOSE RA DS FIG. 3. Chromosomal exchange frequency vs. dose for Don cells irradiated with a single or a divided dose during S phase (see Fig. 2). Over 95% of the exchanges were of the chromatid type. The lines representing the singledose data were drawn for an exchange frequency proportional to the 1.9th power of the dose. If restitution or repair is complete at the time the second dose is delivered, the response for two 3rad doses will be additive, as indicated. Similar results were obtained for deletions, which, for single doses, were proportional to the 1.5th power of the dose. We studied the extent of repair of these sublethal lesions by comparing the survival fraction and frequency of aberrations produced by divided doses with those produced by a single dose. The cells were irradiated during the S phase, the time in the cyc'e when the cells had the greatest capacity for repair, as evidenced by the largest shoulder on the doseeffect curve (13,18,19). Figs. 2 and 3 illustrate that, as judged either by the survival fraction or number of chromosomal exchanges, repair was essentially complete by 12 hr. The shoulder observed for the first dose of irradiation was also observed for the second dose of irradiation if the cells were allowed to incubate for 12 hr before the second dose was delivered. Thus, sublethal lesions, which do not result in cell killing or chromosomal aberrations unless they interact with similar lesions produced by a second dose of irradiation, were completely repaired during an incubation period of 12 hr. Kinetics of repair of sublethal damage As illustrated in Figs. and 5, the repair both of sublethal lesions and of alterations in chromosomes (which may interact to produce exchanges) had similar repair kinetics. A significant amount of repair occurred during 12 min, and as shown in Figs. 2 and 3, repair was essentially complete by 12 hr. The failure to observe repair of chromosomal damage during S phase in a previous study (5) was probably caused by the fact that the cells scored for aberrations were not synchronied, and thus were not irradiated during a period in which all cells had about the same radiosensitivity. Ideally, repair should be studied when the radiation response does not change during the interval between two portions of a divided dose, but as shown in Figs. and 5, the response did change some. In fact, as shown by the "per cent labeled" curve in Fig., the cells incubated for chromosomal analysis (3 X 15) moved into S phase about 1 hr sooner than the cells plated for survival analysis (1.5 X 1). Thus, during the period between the divided doses, the cells for chromosomal analysis were starting to move into the radiosensitive G2 phase (13, 18) while the cells for survival analysis remained on 2 cr >1 MIN BETWEEN FRACTIONS HOURS AFTER PLATING SYNCHRONIZED CHO CELLS FIG.. Survival values (solid symbols) vs. time after plating for synchronied cells irradiated at 37C during S phase with 6 rads in a single dose; and survival values (open symbols) after two 3rad doses vs. incubation time between the two doses. Since not all erotime doses could be given at the same time, starting times were staggered during the first 2 min. The dotted arrows indicate the time (hours after plating) halfway between any twodose fractions, and the singledose response at this time was used for computing the repair ratios in Fig.!6.'"Per cent labeled" as in Fig. 2. the radioresistant S plateau, which explains why the survival fraction in Fig. and exchanges/cell in Fig. 5. were both increasing as the cells were irradiated at later times after plating. This slight displacement in the cycle between the two populations, however, should have little effect on the comparison of the repair kinetics (19). The repair kinetics from four experiments, in which both survival values and chromosomal aberrations after frac "a U Us w x _. EXP 17.Il MIN BETWEEN FRACTIONS B 1b HOURS AFTER PLATING SYNCHRONIZED CHO CELLS FIG. 5. Chromosomal exchange frequency vs. time after plating or time between two halves of the divided dose, for synchronied CHO cells irradiated at 37C during S phase. See Fig. for further details.

67 Zoology: Dewey et al. Proc. Nat. Acad. Sci. USA 68 (1971) 1.1.8 d6c AtCHM P: 1o CHM Cr.5 g 1..3 2~ I T.a,,12 I. 1 2 3 MIN. 1 2 3 5 6 7 8 9 1 11 12 MIN AT 37C BETWEEN 3RAD DOSES FIG. 6. Repair ratios (R) calculated, as explained in the text, from four different experiments for cells irradiated in S phase with divided doses.

4 67 Zoology: Dewey et al. Proc. Nat. Acad. Sci. USA 68 (1971) d6c AtCHM P: 1o CHM Cr.5 g ~ I T.a,,12 I MIN MIN AT 37C BETWEEN 3RAD DOSES FIG. 6. Repair ratios (R) calculated, as explained in the text, from four different experiments for cells irradiated in S phase with divided doses. The R. ratios calculated from exchanges, or exchanges plus deletions, were much lower (they decreased to about.2 and.38, respectively). CHM, cells treated with 5 jg/ml cycloheximide for a period extending from 15 min before the first dose until 15 min after the second dose. The plot of the damage remaining (I) was obtained from the formula I = 1. (1. Rt)/ (1. R12), where Rt is the repair ratio at any time t, and R12 is the repair ratio at 12 min, i.e., maximum repair. The rate of decline is represented by a constant of.693/12 =.58 min'. RE for chromatid exchanges; ARD for deletions; *Rs for survival; * Rs calculated from deletions. tionated doses were compared with those after single doses, are shown in Fig. 6. The repair ratio for chromosomal aberrations was defined as the number of aberrations for the divided dose divided by the number of aberrations for the single dose. The repair ratio for survival was similarly defined as the survival value for the single dose divided by the survival value for the divided dose. To allow for the small change in radiation response that occurred as the cells progressed in the cycle between the two doses, the response observed after the second dose of irradiation was compared with the singledose response observed at the time halfway between the two doses (dotted lines in Figs. and 5). These results indicated that the repair kinetics for sublethal lesions and alterations in chromo.o I. 2 IA 9 ;.2.1, ~ PE =98 ~~~~~~~ 1.7 L 2 6U 8 1V1u ti 1LI MIN AT 2 C AFTER IRRADIATION (6 RADS) FIG. 7. Repair at 2C of potentially lethal lesions. The synchronied cells were allowed to progress into the S phase during incubation at 37C for 9 hr. They were cooled to 2'C, irradiated with 6 rads, and either immediately returned to 37 C ( min at 2C) or allowed to remain at 2'C for various intervals of time before being returned to 37C. J5.Ot o1IOO MIN AT 2C AFTER IRRADIATION (6 RADS) FIG. 8. Repair at 2C of potential aberrations. See Fig. 7 for details. The chromosomal aberrations scored consisted of the sum of the deletions plus exchanges. The ratio of the exchanges to deletions was not changed significantly by the incubation period (115 min) after irradiation (reduced by a factor of only when compared with min at 2C). somes were similar, and that the halftime is about 12 min. Also, repair did not depend on protein synthesis since it occurred in the presence of cycloheximide, which suppressed the uptake of ['H leucine by 95% (16). Furthermore, repair was seen for both deletions and exchanges, although there appeared to be more repair based on exchanges than on deletions. * This repair observed for deletions supports Revell's hypothesis (1) that chromatid deletions result from an exchange, i.e., the interaction of two lesions. Quantitatively, the amount of repair observed in terms of chromosomal deletions predicted quite well the repair observed in terms of survival; compare in Fig. 6, values for R8 observed for survival with values for R8 calculated by entering deletion data into Fig. 1. Similarity of the repair of sublethal and potentially lethal damage When the cells were irradiated (at 2C) with a single dose and then held at 2C for various periods of time, potentially lethal lesions and potential aberrations were repaired (Figs. 7 and 8). At 2C, metabolism of the cells was greatly reducedincorporation of ['H]leucine was inhibited by 95%which suggests that a process responsible for confirming damage was also inhibited or suppressed, while the repair process was affected very little. The repair kinetics for the lesions with the potential for forming chromosomal aberrations were almost identical to the repair kinetics for the lesionswith the potential for cell death, and very similar to the kinetics for repair of sublethal lesions, either at 37C (Figs. 6) or at 2C (unpublished data). These positive correlations suggest that potential lesions are derived from interactions of sublethal lesions. Furthermore, these potential lesions, if they are not repaired, appear to be responsible for both chromosomal aberrations and cell death. Inhibition of repair of sublethal and potentially lethal damage by holding the cells at WC With an experimental protocol identical with that illustrated in Figs. and 7, no repair in terms of chromosomal aberrations * The ratio of exchanges to deletions (.7 i.8, standard error of mean, for 6 rads) was reduced by a factor of.8 i. when the dose was divided over a period of 112 min.

Proc. Nat. Acad. Sci. USA 68 (1971) or cell mortality was observed when the cells were held at C for as long as 1 hr either between two 3rad doses or after a single dose of 6 rads (data not shown).

5 Proc. Nat. Acad. Sci. USA 68 (1971) or cell mortality was observed when the cells were held at C for as long as 1 hr either between two 3rad doses or after a single dose of 6 rads (data not shown). Thus, when the metabolic processes were essentially stopped, as evidenced by more than a 99% decrease in the rate of leucine incorporation, the repair processes that operate at 2'C and 37C were also inhibited. CONCLUSION Xrayinduced cell death results from chromosomal damage The excellent agreement between the amount of cell killing and the number of chromosomal aberrations as the cells were irradiated under different conditions of radiosensitivity (Fig. 1) and the excellent agreement between the repair of lesions responsible for cell death and those responsible for chromosomal aberrations (Figs. 28) strongly support the hypothesis that xrayinduced cell death results from damage in chromatin structures. The observation that about 1 aberration per cell on the average corresponded to a survival level of 37% suggests that the lethal lesion is a chromosomal aberration. [As shown by Poisson statistics applicable to target theory (15), lethal events occurring at random will reduce the survival to 37% when there is an average of one lethal event per cell. ] However, the situation is probably not this simple because there may be lesions in chromosomes undetectable at the cytological level which do cause death, while there are probably detectable aberrations (for instance, many chromatid exchanges) which do not. For example, repair measured by survival correlated best with repair measured in terms of deletions (Fig. 6), probably because most deletions, according to the exchange hypothesis (6, 1), should affect both sister chromatids near the same locus and should thus affect both daughter cells. A chromatid exchange, however, could leave one sister chromatid, and thus one daughter cell, unaffected; this one unaffected daughter cell could then result in a colony of cells. Thus, the particular lesion or lesions in the chromosomes responsible for lethality is not clearly defined. XRays and Chromosomal Aberrations 671 This research was supported in part by the Public Health Service Research Grant CA8618 and Atomic Energy Commission Postdoctoral Fellowship Grant AT(111)186. We thank Dr. L. E. Stone for his assistance in the cycloheximide experiments. 1. Puck, T. T., Rev. Mod. Phys., 31, 33 (1959). 2. Sax, K., Proc. Nat. Acad. Sci. USA, 25, 225 (1939). 3. Elkind, M. M., and H. Sutton, Radiat. Res., 13, 556 (196).. Wolff, S., in Radiation Protection and Recovery, ed. A. Hollaender (Pergamon Press, New York, 196), Vol. 7, p Dewey, W. C., and R. M. Humphrey, Exp. Cell Res., 35, 262 (196). 6. Evans, H. J., in Radiation Res., ed. G. Silini (NorthHolland Publishing Co., Amsterdam, 1967), p Phillips, R. A., and L. J. Tolmach, Radiat. Res., 29, 13 (1966). 8. Whitmore, G. F., S. Gulyas, and J. Kotalik, in Time and Dose Relationships in Radiation Biology as Applied to Radiotherapy, Carmel Conference, Sept. 1969, p. 1, Brookhaven Natl. Lab. 9. Hsu, T. C., and M. T. Zenes, J. Nat. Cancer Inst., 32, 857 (196). 1. Tjio, J. H., and T. T. Puck, J. Exp. Med., 18, 259 (1958). 11. Tobey, R. A., E. C. Anderson, and D. F. Petersen, J. Cell Physiol., 7, 63 (1967). 12. Dewey, W. C., and H. H. Miller, Exp. Cell Res., 57, 63 (1969). 13. Dewey, W. C., S. Furman, and H. H. Miller, Radiat. Res., 3, 561 (197). 1. Revel, S. H., Proc. Roy. Soc. Edinburgh Sert. B., 15, 563 (1959). 15. Elkind, M. M., and G. F. Whitmore, The Radiobiology of Cultured Mammalian Cells (Gordon and Breach, New York, 1967), pp. 2, 1, 237, Dewey, W. C., in Time and Dose Relationships in Radiation Biology as Applied to Radiotherapy, Carmel Conference Sept. 1969, p. 51, 11, Brookhaven Natl. Lab. 17. Lea, D. E., Actions of Radiations on Living Cells (University Press, Cambridge, England, 1956). 18. Sinclair, W. K., and R. A. Morton, Radiat. Res., 29, 5 (1966). 19. Sinclair, W. K., Suppl. Japan J. Genet.,, 11 (1965).

Early Repair Processes in Marrow Cells Irradiated and Proliferating in Vivo1

RADIATION RESEARCH 18, 96-105 (1963) Early Repair Processes in Marrow Cells Irradiated and Proliferating in Vivo1 J. E. TILL AND E. A. McCULLOCH Department of Medical Biophysics, University of Toronto,