Segregation of Newly Replicated DNA in Lethally Irradiated Cultured Mammalian Cells (L5178Y)

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SEGREGATION OF NEWLY REPLICATED DNA IN LETHALLY IRRADIATED CULTURED MAMMALIAN CELLS (L5178Y)

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Recent work on cultured mammalian cells suggests that DNA constitutes at least part of the target molecules responsible for death of mammalian cells as well as for chromosome aberrations (1,2,3). This is in accord with the hypothesis held by radiobiologists for years, namely, that radiation damage to chromosomes (including DNA molecules) must be expressed as chromosome aberrations at M stage in order for the cells to die ( 4 , 5 , 6 , 7 ) . Some evidence which supports this hypothesis in cultured mammalian cells is: (a) The dose required to kill one mammalian cell is of the same order of magnitude as that needed to produce one visible chromosome aberration per cell ( 8 , 9 ) . Moreover, the shape of survival cuwes was said to response curves of chromosome (b) Factors which alter the extent of killing chromosome aberra- . explained from the dose tions to the same extent which ET (12), anoxia (lo), cell stage (11,13) and BUdR However, the appearance of persistent chromosome this hypothesis. For e appearance of chromosome in several systems seems to oppose aberrations in cultured peripheral blood from irradiated persons (e.g., 14) may be explained by assuming that the irradiated stem cells remained undivided for a long time and gradually were released into circulation. The persistent chromosome aberrations in some of the established cell lines'may be explained by assuming that not all chromosome aberrations are lethal, but some are compatible with life and reproduction of mammalian cells. In other words, there is no really conclusive evidence against the chromosome aberration hypothesis.

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cell death. Glucksmann then, is how chromosome aberration leads to and Spear suggested that cells showing chromosome aberrations in theM stage are unable to complete mitosis, and that this results in cell death (4,5,15). From observations of the extreme difference in radio sensitivity of Trades can tia pollen before and after nuclear division, Newcombe, however, stated, " killing of cells resulted from loss of chromosome parts and not from breakage In our previous studies, mouse lymphoma cells (L5178Y), i n suspension culture,irradiated with 1,000 Rof gamma rays, were able t o complete mitosis as i s shown by the rapid rate of decline of the mitotic index (16,17). These cells were also able to divide as can be seen by the increase in cell number a t the time of the first postirradiation mitotic peak (16). Experimental evidence obtained in this study suggested that the cells died at G stage.In another experiment, it has been shown that the first post - irradiation mitotic peak must occur before the cells show the changes associated with cell death (7, 16). Thus, i t seems apparent that lethally injured cells must pass through mitosis in order to die. In the suspension culture of mouse lymphoma, L5178Y, (a neardiploid cell line),..cells completed mitosis there by excluding death during. mitosis as suggested by Glucksmann and Spear, a t least,' for this cell line. The purpose of the present paper is to ' investigate the possibility of death due to loss of chromosomal mater'ial during division as suggested by Newcombe (6). replicated DNA in irradiated cells was segregated among two daughter Therefore,we studied how newly

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Mouse lymphoma cells (L5178Y) were incubated for two hours in the presence of thymidine-C concentration of 4 uCi/ml) to label all cells in the S, G2 and M stages. A l l cells, except those in G stage, were labeled during the incubation period (17,18). The culture was then irradiated with 1,000 R of Co ganma rays and incubated immediately with thymidine-H (specific activity of 67 mCi/mM and concentration of 0.2 uCi/ml). Successive aliquots of the cells were fixed and stained. Radioautographs of the aliquots were made using the double emulsion method (18, 19). In the radioautographs, cells labeled only with tritium were counted; these were the ce,lls which had been in the GI stage at the time of irradiation. Control cultures were treated in the same way except for irradiation. The number of silver grains seen on radioautography in the tritium-containing cells of both irradiated irradiated cell cultures increased gradually as the cells passed through the S period. At the ninth hour post-irradiation, when the cells labeled only with tritium were beginning to divide, the number of silver grains per cell nearly halved. experiment will be reported elsewhere). (Further details of the In slides made nine hours post-irradiation, cells in telophase with a dumbbell shape were selected for study. silver'grains in each half of the dividing nucleus was counted. Since these cells had completed the S period, the number of silver grains The number of was a measure of the newly replicated DNA synthesis in one life cycle. Fifty-eight dumbbell-shaped telophase cells in four experiments were studied frvm non-irradiated cultures and 31 in the irradiated cultures.

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I n compiling data from the four experiments, the ratios of the number of silver grains in the half of the dumbbell-shaped cell which contains the smaller quantity of radioactivity over the total grain count were calculated and a histogram of the number of cells containing the same ratios were plotted against the ratios(Figure 1). From these data,it is not possible to tell how much ofthe frequency distribution is due to equal segregation and how much t o unequal segregation.Therefore,the theoretical distribution curve for equal segregation was calculated using the following assumptions: (a) the decay of tritium atoms in the cells occurs randomly (Poisson distribution), (b) all betta tracts from tritium decay were derived from DNA-thymidine-H , (c) the number of silver grains is linearly related tothe number of decayed tritium atoms, and (d) newly replicated DNA normally is segregated equally in each half of a dividing cell. The probability (Pi) that a certain ratio of a smaller number o f silver grain counts (N1) of half of a dividing cell over the total grain count (N1 + N2) will fallin a certain range (less than bi and ’ li rger than or equal to a.) is: where N is fhe smaller number of grains in half of a dumbbell-shaped cell,N is the large number, is the probability of having grain number N in the cell population of average grain number, m, and i s equal to (mN1 e-m)/Nl! , and p ’ is the probability of having the grain

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P. (m) was calculated over the range of observed number of grains and the frequencies (sum of P in terms of the expected number of cells with a specified ratio. is shown in Figure 1 as a dotted line. In non-irradiated cells, the frequency distribution of the observed ratio was nearly the same as that expected, indicating that the cells have segregated newly replicated DNA equally among the two grains in two daughter cells of pulse-labeled HeLa cultures deviated Although Kosuka and Moore reported that the number of slightly from that which would have resulted from equal segregation of grain counts to follow Poisson's distribution. These investigations did not consider the Had they used this assumption,as was done in the present paper,.their results would have also indicated equal segregation of newly synthesized DNA. It is interesting to note that equal segregation of DNA contrasts with the unequal segregation of other cellular constituents. For example, Terasima and Tolmach found that the rates of uridine-H uptake in two newly divided HeLa cells differed from one another, suggesting unequal segregation of RNA synthesis mechanisms (21). By estimating the amount of DNA, RNA and dry mass microspectrophotometrically, Killander and Zetterberg also showed that the segregation of DNA among two daughter cells of mouse fibroblasts and leukemia was more nearly equal than that of other cellular constituents (22,23). The rate of formation of two hit type chromosome aberrations in irradiated L5178Y cells was estimated, by Dr. Y. Doida in our laboratory, to be 5 x bridges/cell/(rads)2. This is of the same

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order of magnitude as that in other cell lines (e.g.,Bender, 2 4 ) . After exposure to 500 R, 85% of the total cell population was shown to have abnormal chromosomes. By extrapolating this to the 1,000 R dose level, it is apparent that the number of two hit type chromosome aberrations would be five bridges per cell. If all of these had produced unequal segregation, the histogram of segregation of DNA in the cells irradiated with 1,000 R would have deviated much more strikingly from equal segregation than is evident in Figure 1. Contrary to our original expectation, the exposure to 1,000 R did not prevent most of the cells from segregating newly replicated DNA equally (within variations involved in experimental methods). 18 percent of the cells showed a definite deviation from equal segregation of newly replicated DNA. unequally segregated DNA compares favorably with approximately 20 This fraction of the cells with percent of the cells, which'have been reported to die after the first mitotic peak following the exposure t o 1,000 R (16). This may be ' considered as circumstantial evidence that unequal segregation of the DNA could be an important cause of the post-mitotic G death. Since most of the cells irradiated with 1,000 R would have chromosome aberrations, this means that only a certain type of aberration or that a certain fraction of them produce unbalanced segregation of genetic In L5178Y cells, the previous study (25) shows that the G1 stage requires the highest activation energy. After the division, two daughter cells vith unbalanced genetic materials '(excess and/or , deficient) would fail to meet such an energy requirement, which then would lead to cell death.

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'It should be pointed out that there may be several different a fraction of the cells die in interphase without mitosis (261, a fraction die in the first post-irradiation mitosis (4,15), a fraction die in the interphase after one or more divisions (16,26,27), a fraction die in mitosis after one or more divisions (16,27,28), and a fraction die after forming giant cells (5,28). leukemic cells, L5178Y, the predominant feature of the early death is the death in the interphase (possibly in G stage) after one or more mitoses, and the death at the mitotic phase after more mitoses (only for a small fraction) (16). P The unequal segregation of genetic materials described in the present paper would apply, at the present time, only to the G1 type death after cell division occurs. Division of Experimental Radiology and Department of Radiation Biology and Biophysics o f Rochester Medical Center

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Dr. Watanabe's present address is the National Institute of Rad' logical Sciences, Chiba, Japan.

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5. Lea, D.E.,in Actions of Radiation on Living Cells, edit. Lea, D.E., (The University Press, Cambridge, 1955). 7. Watanabe, I., and Okada, S., the preceding paper. 8. Puck, T.T., Proc. U.S. Nat. Acad. Sci., 44, 772 (1958). 9. Dewey, W.C., and Humphrey, R.M., Radi at. Res., 16,503 (1962). 12. Schneider, D.O., and Whitmore, G.F., Radiat. Res., l.8, 286 (1963). 14. Bender, M.A., and Gooch, P.C., Radiat. Res. l6, 44 (1962). 15. Shaman, G.B., Intern. J. Rad. Biol., 2, 115 (1959). 16. Watanabe, I., and Okada, S., Radiat. Res. 27, 290 (1966). 17. Whitmore, G.F., and Gulyas, S.,Science 151,691 (1966). 19. Wimber, D.E., and Qua st ler, H., Exp. Cell Res., 30, 8 (1963). 21. Terasima, T., and Tolmach, L.J., Exp. Cell Res., 30, 344 (1963). 22. Kill ander,D., and Zetterberg,A., Exp. Cell Res. 38, 272 (1965). Kosuka, S., and Moore, G.E., J. Nat. Cancer Inst.,, 36, 272 (1965).

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Killander, D., Exp. Cell Res., 40, 2 1 (1965). Bender, M.A., in Mammalian Cytogenetics and Related Problems in Tolmach, L.J., Ann. N.Y. Acad. Sci., 95, 743 (1961).

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THE RATIO OF NUM aER OF SILVER GRAINS OF ONE HALF OF A DIVIDING CELL OVER THAT OF TOTAL GRAINS.

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Legend for Figure 1: Silver Grains of Half of a Dividing Cell Containing the Smaller The Histogram of the Ratio of the Number o f Amount of Radioactivity over the Total Number of Silver Grains in Both Halves of the Dividing Cell. Top Figure: Non-irradiated cells ; Bottom Figure : Irradiated cells. Full lines are the observed histograms and dotted lines are the expected histograms, assuming equal segregation of newly replicated DNA. Since the portions of these histograms where unequal seg rag ation would be most obvious are the tail ends, special attention is paid to these portions.In the non - irradiated cells, the number of cells with a ratio of less than 0.40 was three (3/5&’x 100 = 5percent),while the expected the number of cells with a ratio less than 0.40 was seven (7/31 x 100 = In the irradiated cells, 23percent),while the expected number was 0.81 (0.81/31x 100 = 3 segregation in a range less than 0.40 was 23 - 3 = 20 percent or The fraction of the irradiated cells which showed unequal 23 - 5 = 18 percent. The value at 18 percent of unequally segregated cells as mentioned inthe text was calculated by this method and should be accepted with reservation.