Recovery of G2 cells in pea root meristems: Survival and mitotic delay following irradiation

Radiation Botany, 1970, Vol. 10, pp. 145 to 154. Pergamon Press. Printed in Great Britain.

R E C O V E R Y OF Gz CELLS IN PEA R O O T M E R I S T E M S : S U R V I V A L AND M I T O T I C DELAY F O L L O W I N G I R R A D I A T I O N * P. L. W E B S T E R a n d

J. V A N ' T H O F

Biology Department, Brookhaven National Laboratory, Upton, New York 11973, U.S.A.

(Received 10 September 1969) effects of y-irradiation on (1) the capacity of G 2 cells to complete one postirradiation mitosis and (2) mitotic delay of these G2 ceils have been investigated. Recovery with respect to both of these parameters of damage can occur in the absence of any progression through the cell cycle; however, the kinetics of recovery suggest that the two parameters, i.e. failure to divide and delay in division, reflect damage to different systems in the cells. Conditions of extreme hypoxia prevent recovery from damage leading to failure to divide, while recovery with respect to mitotic delay is very much less sensitive to such conditions. An inhibitor of DNA synthesis, 5-fluorodeoxyuridine, while preventing passage of cells through S, has no effect on either recovery system. Finally, a comparison of the radiosensitivity of the whole meristem in terms of its subsequent growth with that of a population of its component cells demonstrates that the former may be quite independent of the latter. Abstract--The

R 6 s u m ~ - O n a dtudid les effets des rayons gamma sur ( i ) la capacitd des cellules G2 d'achever leur cycle mitotique apr~s l'irradiation et (2) le retard mitotique de ces cellules G2. La restauration par rapport ~t ces deux param~tres peut se traduire par l'absence de toute progression du cycle mitotique. Cependant, la cin~tique de la restauration sugg~re que les deux param6tres c'est-~t-dire l'absence et le retard de division sont le reflet d'un dammage ~t diffdrents syst+mes dans les cellules. Les conditions d'extrfime hypoxie pr6viennent la restauration du dommage qui conduit/t l'arr~t de la division alors que la restauration par rapport au retard mitotique est beaucoup moins sensible ~ de telles conditions. U n inhibiteur de la synth6se du DNA, la 5-fluorod6soxyuridinepr6vient le passage des cellules en S mais n'a pas d'effe t sur chacun des systems de restauration. Finalement, une comparaison de la radiosensibilit6 de la croissance du mdrist+me entier par rapport ~t une population de ses cellules constituantes ddmontre que ces radiosensibilittSs respectives peuvent 6tre tout ~ fait inddpendantes l'une de l'autre. Z u s a m m e n f a s s u n g - - D i e Wirkung von y-Bestrahlung auf (1) die F~ihigkeit von Gz-Zellen, eine Mitose nach der Bestrahlung zu vollenden und (2) die mitotische Verz6gerung dieser G.-Zellen wurden untersucht. Erholung von diesen beiden Parametern der Sch~idigung karm eintreten ohne ein Fortschreiten des Zellzyklus; die Kinetik der Erholung legt allerdings nahe, dass die beiden Parameter (Verlust der Teilungsf~ihigkeit und Verz6gerung der Teilung) Sch/iden in verschiedenen Systemen in der Zelle widerspiegeln. Bedingungen extremer Hypoxie verhindern die Erholung von Sch/iden, die zum Verlust der Tcilungsf/ihigkeit ftihren, w/ihrend die Erholung von Sch/iden, die zur mitotischen Vcrz6gerung ftihren, solchen Bedingungen gegentiber sehr viel weniger sensibel ist. Eiia Hemmstoff der DNS-Synthese, 5-Fluordeoxyuridin, der die Entwicklung der Zelle w/ihrend der Phase S blockiert, hat auf keinen der beiden Erholungsvorg/inge eine Wirkung. * Research carried out at Brookhaven National Laboratory under the auspices of the U.S. Atomic Energy Commission. 145

146

P. L. WEBSTER and J. VAN'T HOP Schliesslich zeigt ein Vergleich der Radiosensibilit~it des ganzen Meristems beziiglich seines weiteren Wachstums mit der Radiosensibilit~it einer Population aus Zellen dieses Meristems, dass ersteres ziemlieh unabh~ingig sein kann yon letzterem.

INTRODUCTION EXISTENCE of post-irradiation

recovery processes has been demonstrated in a wide range of cell types.C8,9,20) Such recovery from ionizing radiation has also been demonstrated in pea root tip meristem ceils; a 24 hr postirradiation period in the absence of sucrose restores the capacity of G1 and G2 cells to initiate DNA synthesis and mitosis respectively at the normal time following provision of sucroseJ 32) A beneficial influence of delayed cell division after irradiation has also been described for survival in yeast(1~) and bacteria(1) and for mutation frequency in Paramecium.(14) It was of interest, therefore, to determine ira stationary phase period between irradiation and the initiation of cell division in root tips by sucrose provision allowed recovery in terms of increased cell survival, and if such a system might be related to the recovery system previously demonstrated in pea root tips. Since true genetic death (i.e. loss of reproductive integrity) is very difficult to measure in as complex a tissue as a root tip meristem, the criterion of survival used in these experiments is the capacity o f G 2 cells to complete their first post-irradiation mitosis. The relationship of this parameter as well as that of mitotic delay of these G2 cells to dose and the effects of a post-irradiation stationary phase period on these aspects of radiation damage are described. The results show that recovery from both types of damage does occur during the starvation period; furthermore, the kinetics of recovery suggest that mitotic delay and failure to divide at all are not simply reflections of quantitative differences in the damage produced in the cell. The system used in the present experiments involved the use of5-fluorodeoxyuridine (FUdR) to prevent G 1 cells from completing S and reaching mitosis. It was important, therefore, in view of the involvement of D N A synthesis in repair of radiation damage in other systems,(ls,~°) to investigate the effects of F U d R on the ability of G~ cells to recover from the two parameters of radiation injury studied in these experiments. THE

The requirements for aerobic metabolism for recovery were also investigated. Since the radiosensitivity of a population of cells within an organized growing tissue was being measured, it was felt important that the radiosensitivity of the whole tissue, i.e. the root meristem, be compared to that of this population. The results indicate that the radiosensitivity of an organized tissue does not necessarily reflect that of individual cells within that tissue.

MATERIAL AND METHODS

Germination and culture Seeds of Pisum sativium (var: Alaska) were sterilized by a 5 rain immersion in Chlorox, then germinated in sterile, moist vermiculite for 4 days at 19°C. Root tips (1 cm) were excised aseptically into White's medium,~ 2s) and cultured with constant shaking at 23°C. All transfers and samplings were carried out aseptically as described elsewhere. (25) The stationary phase distribution(33) was established by culturing the root tips in medium without sucrose. Under such conditions cells accumulate in either G 1 or G2 (Fig. 1), and there is no further progression through the mitotic cycle. (25,33) After 48 hr all roots were transferred to medium containing 2 per cent sucrose to reinitiate progression through the cycle; G a cells enter S (DNA synthesis) and G2 ceils enter mitosis. Fluorodeoxyuridine (FUdR), an inhibitor of D N A synthesis, was added with the sucrose to prevent the G 1 cells from passing through S and to mitosis (see Results). The F U d R was filter-sterilized and added to give a concentration of 10 -6 M. In one experiment tritiated thymidine (3H-TdR) (Schwarz BioResearch, Inc., Spec. Act. 6 Ci/mM, 1 ~Ci/ml) was added instead of FUdR. Root tips to be cultured anaerobically following irradiation were grown under pre-purified nitrogen (99.996 per cent) in the presence of 2 per cent sucrose from 24 to 48 hr. T h e effects of F U d R on the recovery process were investi-

147

RECOVERY OF G2 CELLS

Measurement of mitotic delay

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gated by adding F U d R (10 -6 M) to stationary phase roots for the same 24 hr period.

Mitotic delay was measured directly from the curves of frequency of mitotic figures against time, and was taken to be the time taken to reach one half the m a x i m u m frequency of division figures, minus the time taken to reach the corresponding value in unirradiated roots.

Growth measurements Roots were transferred to medium containing sucrose immediately after irradiation at 48 hr, and the increments in length after a further 6 days expressed as a fraction of the increase in length ofunii'radiated control roots.

Irradiation The roots were irradiated either immediately before transfer to medium containing sucrose or 24 hr before transfer. Irradiation was performed with a 12,000 Ci x3~Cs source at a dose rate of 300 R per minute.

Sampling and cytological analysis Roots were fixed every 3 hr in glacial acetic acid/ethanol (1:3) and the meristems prepared as Feulgen squashes after hydrolysis for I0 min at 60°C in 1 N HC1. Slides of roots exposed to ~H-TdR were coated with K o d a k NTB liquid emulsion and prepared as radioautographs after a 2 day exposure at 4°C. Four meristems from each sample were scored for frequency of mitotic figures, or in the case of 3H-TdR treated roots, frequency of unlabeled mitotic figures. At least 1000 cells were scored from each of these four meristems.

Measurement of survival of G2 cells The number o f G 2 cells passing through mitosis in the presence of F U d R is proportional to the area under the mitotic frequency curve. Hence the fraction of G~ cells surviving to complete a post-irradiation mitosis is expressed as the area under the curve of mitotic frequency as a percentage of the area under the control curve. The validity of such a measurement depends on the ability of the F U d R to prevent any G z ceils reaching mitosis; this was tested (see Results) by comparing the area under the control curve in the presence of F U d R with the area under the curve for frequency of unlabeled mitotic figures in the presence of 8H-TdR.

RESULTS

Inhibition of division of G1 cells by FUdR I n order to ensure that the areas under the mitotic frequency curves represent only G~ cells it was necessary to show that F U d R prevented G z cells from progressing through S and into mitosis. This was done by transferring 48 hr starved stationary phase roots to medium containing sucrose and either F U d R or 8H-TdR. Since G z cells must incorporate 3H-TdR before reaching mitosis, the former G~ ceils are easily distinguishable as unlabeled mitotic figures. I f F U d R allows all of the G 2 cells to divide, but none of the G 1 cells, then the total number of division figures in the FUdR-treated roots should equal the number of unlabeled division figures in the roots exposed to 8H-TdR. It can be seen (Fig. 2) that these two curves are almost identical, indicating that F U d R prevents division of former G a cells without affecting the G~ cells. Hence the area under the mitotic frequency curve in the presence of F U d R is proportional to the number of G~ cells which divide.

Effects of,r-irradiation 1. No post-irradiation stationary phase period. Irradiation of stationary phase root tips immediately prior to the addition of sucrose and F U d R affects division of G2 cells in two distinct ways (Fig. 3). Firstly, all doses used reduce the frequency of G2 cells which divide following addition of sucrose, and secondly, those cells which do survive are delayed in their arrival at mitosis. T h e relative frequencies of Gz cells

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which are able to complete a first post-irradiation mitosis, expressed as log surviving fraction, are plotted against dose (Fig. 5). Although the relative crudity of the measurement of surviving cells makes it impossible to say whether the curve is sigmoidal or exponential, there is a slight shoulder, possibly indicating the existence of a repair process of some sort. T h e extent of mitotic delay is also a function of dose (Fig. 6). I n c l u d e d in Fig. 6 are values taken from a m u c h earlier experiment(~1) in which data on mitotic delay as a function of dose were obtained using intact proliferating pea root tips and X-rays; these points clearly lie close to the curve obtained from the present experiments. T h e extremely low frequency of dividing G 2 cells following the 1200R dose makes it impossible to obtain a meaningful value for mitotic delay at this dose. T w o points should be m a d e about these results: firstly, the survival curve is similar in form to those obtained for m a n y other systems, I

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FIo. 4. Same as Fig. 3 except irradiation 24 hr prior to provision of sucrose. i.e. the n u m b e r of cells which fail to divide increases exponentially with dose, and secondly, the delay exhibited by the surviving cells increased with dose, although the relationship is clearly not linear. These points will be discussed more fully later.

2. 24-hr post-irradiation stationary phase period. When the roots are irradiated 24 hr prior to the addition of sucrose, i.e. held for 24 hr in the absence of any progression through the mitotic cycle, the response of the G2 cells is considerably modified (Fig. 4). Although the number of G 2 cells surviving through a first post-irradiation mitosis is again reduced with increasing dose (Fig. 5a), the survival curve shows a pronounced shoulder and is displaced to the right, indicating that recovery in terms of the capacity of G 2 cells to divide has occurred. The increase in D O is from 260 R to 355 R, a dose modifying factor of 1.36. A measure of the extent of recovery at any dose can be obtained by expressing the difference

between the fractions killed at that dose as a percentage of the fraction killed when no intervening stationary phase period was allowed. For example, at 300 R 62 per cent of the cells fail to divide if irradiation is carried out immediately before provision of sucrose, while an intervening 24 hr stationary phase period reduces this fraction failing to divide to 28 per cent. Hence, the amount of recovery is (62-28)•62, or 55 per cent. The degree of recovery measured this way falls exponentially with dose (Fig. 5b) indicating that the processes leading to recovery are also sensitive to the irradiation. The 24 hr period in stationary phase between irradiation and sucrose provision also reduces the time taken for the surviving G2 cells to divide (Fig. 6). Again the 1200 R value is omitted because of the very low frequency of division figures seen after this dose. A quantitative estimate of the amount of recovery having occurred can be obtained for each dose. For 150, 300 and 600 R doses, the reductions in mitotic delay are

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69, 72 a n d 67 per cent respectively; i.e. in contrast to the survival parameters, the extent of recovery from mitotic delay remains constant, at least over this dose range. Further evidence that the d a m a g e leading to loss of capacity to complete a first post-irradiation mitosis a n d that leading to mitotic delay affect different systems in the cell can be obtained from the preceding data. I f these different responses reflected only quantitative differences in the a m o u n t of radiation d a m a g e accumulated, then at any one level of survival the survivors should exhibit the same a m o u n t of mitotic delay. This is clearly not the case; for example, 50 per cent of the cells would survive to divide once after a dose of 240 R with no recovery period and after a dose of 440 R with a 24 hr recovery period (Fig. 5). However, the delay in the time taken for the survivors to reach mitosis would be 6.7 hr after the former treatment, yet only 2.6 hr after the latter treatment (Fig. 6). Since at the same level of survival two quite different levels of mitotic delay can result, it is unlikely that mitotic delay is a consequence only of sub-lethal amounts of d a m a g e of the type that prevents ceils dividing at all.

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RECOVERY OF G= CELLS cells from passing through S and G2 to mitosis. If DNA synthesis of the type occurring during normal replication is required for the recovery of G2 cells, then this inhibitory concentration of F U d R should impair the recovery process. The presence of F U d R from the time of irradiation to the time of provision of sucrose, i.e. over the period when recovery normally occurs, prevents neither the increase in survival (Figs. 7a, 5a) nor the reduction in mitotic delay (Figs. 7a, 6) oberved when F U d R is absent during the 24 hr post-irradiation stationary phase period.

Effects of anoxia on recovery Similar preliminary experiments have been carried out to show the effect of complete anoxia on recovery. The results of incubating the roots 40

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under nitrogen for the 24 hr period following irradiation indicate that, while recovery of the capacity to complete a post-irradiation mitosis is considerably reduced by nitrogen (Fig. 5a,b), recovery with respect to mitotic delay is much less affected (Figs. 7b, 6).

Relationship of subsequent root growth to dose In order to compare the sensitivity of the whole meristem to that of the Gz cells within the meristem, stationary phase meristems were irradiated at 48 hr and transferred immediately to medium containing 2 per cent sucrose. The roots were measured after a further 6 days, and the increases in length over this period expressed as a fraction of the corresponding increase in unirradiated roots (Fig. 8). Clearly the sensitivity of the meristem in terms of its subsequent growth (Do=690 R) is much less than that of its component ceils in terms of their ability to divide following irradiation (Do = 260 R).

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The system used in the present experiments, a modification of that described previously,(Z2,sn) has several obvious advantages for studying the

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P. L. WEBSTER andJ. VAN'T HOF

effects of radiation on cell division. Firstly, progression through the mitotic cycle can be rigorously controlled, allowing cells to be retained in G 1 and G 2 both before and after irradiation. Secondly, cells in G2 only are considered, since the G 1 cells can be prevented from reaching mitosis. Thirdly, direct measurements of mitotic delay can be obtained, as well as an unambiguous parameter of survival, the capacity of G 2 cells to complete at least one post-irradiation mitosis. Finally, the radiosensitivity of the meristem can be compared to that of one population of its component cells measured within the tissue. It should be stressed, however, that this criterion of survival is not equivalent to that customarily used. For example, there is no evidence that the G 2 cells which fail to divide in the 36 hr period following irradiation do not divide after this time; however, the mitotic index does reach almost zero before the sampling period is over, suggesting that all of the cells which are able to divide do so in this period. Conversely, the high frequency of aberrations observed in these cells suggests that m a n y of the "survivors" might not divide a second time. Furthermore, this parameter cannot be equated to survival of the root in terms of its subsequent growth. Firstly, no account is taken of the survival o f G 1cells--these would of course contribute to growth. Secondly, the ability of a root to grow following irradiation appears to be at least as much a function of its reorganisational capacity as of the number of cells whose reproductive integrity is left unimpaired. The existence of a quiescent center in a root meristem could make irrelevant the number of cells killed by irradiation.(5,e) Such recovery systems at the organisational level could account for the difference in sensitivity between the meristem and its component cells. It is clear that, at least in root tip meristems, patterns of injury at the cellular level need not be reflected at the tissue level. Mitotic delay has been extensively studied in m a n y different cell types,(3,19,29) including root meristem cells, ~1e,31) although the mechanism is poorly understood. Duration of delay is almost always dose-dependent, but the exact nature of t his relationship differs in different cells. A linear relationship between the delay period and dose

has been well established for cultured m a m malian cells.~ 7,23) Such a relationship is not found in the present experiments; the delay per R clearly falls with increasing dose. This has been found in m a n y other systems, including grasshopper neuroblasts, newt cornea and rat retina cells.(15, pp.299-30°) WHITMORE et (l/.(29) who obtained a similar dose response curve for cultured mouse L cells, suggest that heterogeneity of the cell population is responsible. This is unlikely to be responsible for the nonlinear relationship obtained in the present experiments, since only G 2 cells are considered. The reasons for the discrepancies are not clear; t h e hypothesis that a recovery system is responsible, as suggested by LEA, (15) is attractive in view of the demonstration in these experiments that G 2 cells can recover in the absence of progression through the cycle. A second, not necessarily unrelated, possibility is that cellular interactions might modify the response. For example, FROESE(10) has shown that irradiated cells are delayed less in their division if they are mixed with unirradiated cells, and vice versa. Such interactions might be expected to play a more important role in an organized root meristem than in a single cell suspension. WALTERS a n d PETERSON(23) have demonstrated that radiation-induced delay in Chinese hamster cells is independent of the position of the cell in the mitotic cycle, and suggested 124) that some translational process in protein synthesis, common to all stages of the cycle, was being affected. Since entry of G 1 cells into S and G 2 cells into mitosis are delayed to the same extent following 300 R in pea root meristems,(32,33) and since protein synthesis but not RNA synthesis is required for initiation of S and mitosis by these G 1 and G 2 cells,C26) it is not unreasonable to conclude that inhibition of protein synthesis at the translational level might also be responsible for the delay in the root meristem cells. It should be pointed out, however, that in both H e L a cells(22) and mouse L cells( TM the extent of mitotic delay does appear to be a function of the position of the cell in the cycle. The relationship between mitotic delay and survival has also been considered previously, c21,22) Although FROESE and CORMACK(n) have shown a correlation between division delay and colony-

RECOVERY O F Go CELLS forming ability, ELKINDet al.~ ~) found no difference in delay between surviving and nonsurviving cells. Furthermore, the variation in response throughout the life cycle of mammalian cells is different for these two parameters.~ 2~) The present results suggest that mitotic delay and survival do in fact reflect damage to different systems in the cell. Finally, let us consider the recovery which occurs in pea root tip meristem cells. It has previously been shownl 3~) that over the period during which recovery does occur, the rate of oxygen consumption is low and decreasing. The present results show, however, that the amount of recovery in terms of survival of G 2 cells is considerably reduced under nitrogen. WOLFF and LmPPOLDI3°) first demonstrated an oxygen requirement for rejoining of chromosomes following irradiation, while actual recovery of cells of both OedogoniumIl3) and Vicia(12) has also been shown to require oxygen. The present results are also compatible with the hypothesis that recovery is a metabolic process requiring an energy source. However, the results also suggest that the recovery processes involved in the reduction of mitotic delay are less sensitive to hypoxia. In this respect it is of interest that the recovery of the capacity of Gx cells to enter S without delay is also unaffected by Hypoxia.(34) It is conceivable, therefore, that repair of damage resulting in delays in progression of cells through the mitotic cycle differs from repair of damage leading to reduction of survival in that the former can derive sufficient energy from anaerobic metabolism while the latter cannot. It was previously suggestedl~2) that recovery from delay in progression through the cell cycle might involve a DNA repair system similar to that operating in bacterial cells. Inhibitors of DNA synthesis have also been shown to enhance killing of UV-irradiated mammalian cellsOS,~) although CL~.AVER(4) has demonstrated that concentrations of F U d R which reduce survival do not inhibit DNA repair replication. In the present experiments, irradiated G 2 cells do divide in the presence of a concentration of F U d R which prevents G1 cells from passing through S; furthermore, the presence of F U d R during the 24 hr post-irradiation period, in

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which recovery of G2 cells can occur, does not prevent this recovery. It is possible, however, that any DNA synthesis required for recovery might be different in nature from that occurring during S, as has been suggested by CLEAVER.I4) It is also of interest that, in agreement with the observations of BELL and WOLFF,(2) there was no evidence in the present experiments of any inhibition of chromosome rejoining in those cells exposed to F U d R following irradiation. REFERENCES

1. ALPERT. and GILLIESN. E. (1958) Restoration of Escherichia coli strain B after irradiation: Its dependence on sub-optimal growth conditions. 07. Gen. Microbiol. 18, 461-472. 2. BELL S. and WOLFF S. (1964) Studies on the mechanism of the effect of fluorodeoxyuridine on chromosomes. Proc. Wall Acad. Sci. U.S. 51, 195202. 3. CARLSONJ. G. (1941) Effects of X-radiation on grasshopper chromosomes. Cold Sprbzg Harbor Symp. Quant. Biol. 9, 104-I 12. 4. CLEAVER J. E. (1969) Repair replication of mammalian cell DNA: Effects of compounds that inhibit DNA synthesis or dark repair. Radiation Res. 37, 334-348. 5. CLOWNSF. A. L. (1959) Reorganization of root apices after irradiation. Ann. Botany (London) N.S. 23, 205-210. 6. DAVIDSOND. (i 960) Protection and recovery from ionizing radiation: Mechanisms in seeds and roots, pp. 175-211. In A, HOLLANDER(ed.), Radiation protection and recovery. Pergamon Press, London. 7. ELKINDM. M., H_ANA. and VOLZ K. W. (1963) Radiation response of mammalian cells grown in culture. IV. Dose dependence of division delay and post-irradiation growth of surviving and nonsurviving Chinese hamster cells. 07. Natl Cancer last. 30~ 705-72 i. 8. ELKINDM. M., KAMFER C., MOSES W. B. and SUTTON-GILBERTH. (1967) Sub-lethal radiation damage and repair in mammalian cells. Brookhaven Symp. Biol. 20, 134-157. 9. EVANS H. J. (1967) Repair and recovery at chromosomal and cellular levels: Similarities and differences. Brookhaven Symp. Biol. 20, 111-131. 10. FROESEG. (1967) An interaction between neighboring cells. Exptl Cell Res. 47, 285-301. 11. FROESEG. and CORMACKD. V. (1969) A correlation between division delay and loss of colonyforming ability in Chinese hamster cells irradiated in vitro. Intern. 07. Radiation Biol. 14, 589-592.

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