DNA-strand breaks and rejoining following exposure of synchronized Chinese hamster cells to ionizing radiation

Mutation Research

Elsevier Publishing Company, Amsterdam Printed in The Netherlands

459

DNA-STRAND B R E A K S AND R E J O I N I N G F O L L O W I N G E X P O S U R E OF S Y N C H R O N I Z E D C H I N E S E HAMSTER CELLS TO I O N I Z I N G R A D I A T I O N

R. M. HUMPHREY, D. L. STEWARD* AND B. A. SEDITA Department of Physics, The University of Texas, M. D. Anderson Hospital and Tumor Institute at Houston, Texas (U.S.A.)

(Received August i5th, 1968)

SUMMARY Radiation-induced single strand breaks in the DNA of synchronized Chinese hamster cells and the rejoining of broken pieces have been investigated by the use of alkaline sucrose density gradients. The weight average molecular weight of denatured DNA has been determined for cells in mitosis, G, and S phase following irradiation and post-irradiation incubation. The data presented supports the conclusion that the processes which are responsible for the rejoining of broken pieces of DNA are present and functional during all phases of the cell cycle, including mitosis. The molecular weight of den atured DNA from unirradiated control cells consisted of a wide distribution of small and large pieces ranging from about IO. lO 8 to 250" lO 2 with a weight average molecular weight of 50 to 55" IOS. This data is consistent with the hypothesis that mammalian cell chromosomal DNA is arranged in a series of small units held together by linker molecules which are labile to alkaline conditions.

INTRODUCTION Neutral and alkaline sucrose-density gradients have been used at the molecular level to estimate both damage and repair to DNA, a suspected critical target of radiation action3,~,12,13,1~. Exposure to ionizing radiation results in a shift in the molecular weight distribution to a lower value than in the unirradiated control, indicating the induction of breaks in the DNA molecule. Subsequent incubation of the irradiated cells in a normal medium environment showed an increase in the molecular weight of the DNA, thus demonstrating a rejoining of the disrupted units of the DNA strand12,13,17. It has been demonstrated by survival and chromosomal analysis that Chinese hamster cells i n vitro exhibit a cell cycle** dependent response to ionizing radiation 8,23. * Present address : Human Health Research and Development Division, Dow Chemical Company, Zionsville, Ind. (U.S.A.). ** The cell cycle consists of the pre-deoxyribonucleic acid (DNA) synthesis period: G1; the DNA synthesis period: S; the post-DNA synthesis period: G,; the mitotic period: M. Mutation Res., 6 (1968) 459-465

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One explanation for this response may be variations in repair capability during different phases of the cell cycle 5& In this paper we report the use of alkaline sucrose-density gradients to determine the molecular weight distribution of DNA from synchronized Chinese hamster cells, and to demonstrate the capability of cells in different phases of the cell cycle to rejoin breaks in the DNA induced by ionizing radiation. MATERIAL AND METHODS

Cell growth and synchronization Stock cultures of Chinese hamster cells, strain Don C (ref. 9) were grown as a monolayer in McCoy's 5 a medium ~6supplemented with 20 % fetal calf serum (Hyland Labs Inc.). Don C is a diploid fibroblast cell line with a generation time of I o - I 2 h. Rotating glass cylinders (12oo cm 2 surface area) were seeded with 5 ° to 60. lO 6 cells and 15o ml of medium containing 0.5 FC/ml of L3HJTdR (1. 9 C/mM, Schwartz Bio Research, Inc.) to obtain populations of labeled cells. The vessels were rotated at 0.2 rev./min at 37 ° for 16 h. The i3H]TdR labeled cells were arrested in metaphase by the addition of colcemid (0.06/tg/ml) to the medium 2 h prior to selective detachment of the mitotic cells 2~. The resulting cell suspension was centrifuged at 12oo ×g. One aliquot was fixed, stained and scored for mitotic index and a second aliquot was resuspended in o.15 M saline for subsequent irradiation. For experiments with mitotic cells, colcemid was added to the saline (o.o6 Fg/ml) and samples were irradiated. One sample of irradiated cells was lysed immediately and a second was returned to medium plus colcemid for a I 2 h incubation at 37 °. Control cells received identical treatment except for the irradiation. At the end of the incubation period the cells remaining suspended in the medium (unattached mitotic cells) were centrifuged, resuspended in saline and lysed. To obtain Gt and S phase cells, the selected mitotic cells were returned to normal medium and incubated for 1.5 h and 6 h respectively. Cultures were trypsinized, centrifuged, resuspended in saline and irradiated. One sample was lysed immediately and one returned to normal medium for I h post-irradiation incubation with subsequent lysis as described above. Mitotic index and !~C]TdR pulse labeling ~ was conducted as an index of synchrony for the G1 and S cultures.

Irradiation technique The cell suspensions were irradiated in air at room temperature to doses of 2.5 krad, 5 krad and IO krad with 137Cs 7-rays (0.60 MeV). The dose rate was 7200 rads/nfin determined by ferrous sulfate dosimetry 22.

Lysing technique The cells (1. lO 5 in 0.5 ml) were lysed and the DNA denatured by the addition of 0.5 ml of lysing solution. This solution consisted of 2 % tri-iso-propylnaphthalene sulfonic acid (Eastman Organic Chem.) and 1% p-aminosalieylic acid (Sigma) in a solvent of 6 % secondary butyl alcohol in water which was then adjusted to pH 12. 3 with i.o N NaOH. Elapsed time from lysis to start of centrifugation was about 30-4 ° nfin. In most experiments the total radioactivity in the gradient was 5-6" lO 3 counts/ rain.

Mutalion Res., 6 (i968) 459-465

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Centrifugation technique The cells were lysed in the bottom of polyallomer (Beckman) centrifuge tubes and the gradients were then poured. All gradients were of the constant velocity, exponential type as described b y NOLL19 with a sucrose concentration at the top of 5 %. The sucrose solutions contained 0.06 M p-aminosalicylic acid and were adjusted to p H 12.3 with IO.O N NaOH. Centrifugation was carried out in a Spinco Model L preparative ultracentrifuge at 24000 rev./min, 20 ° for 4 h, using an SW 25.1 rotor. After centrifugation, equal fractions (about I.O ml) of the gradient were collected by bottom puncture on to o.45-/z cellulose nitrate filters, washed twice in cold IO °/, trichloroacetic acid, and assayed (io-min counts) for radioactivity in a liquid scintillation spectrometer (Nuclear Chicago). An IBM 7o94 computer was used for calculations of the sedimentation coefficient (s°20,,,) corresponding to each fraction of the constant velocity sucros:e gradient assuming a particle density of 1.8 g/cm a for alkaline denatured DNA. A value for molecular weight of alkaline denatured DNA [mol.wt. = (s°20,~ 0,c52~)~ was obtained according to STUDIER2~and the number average and ~veight average molecular weight for the distribution were calculated 2. The s°20,~ of 3H-labeled T2 and 14C-labeled 2 bacteriophage DNA (courtesy of Dr. ROGER H E W I T T ) w e r e determined by the above described technique, and used for calibration of the gradient*. RESULTS

The results of a series of experiments performed on asynchronous Chinese hamster cells using the lysing conditions and alkaline sucrose-gradients described TABLE I THE

MOLECULAR

WEIGHT

(~VEIGHT

AVERAGE) ~

OF

DENATURED

CELLS (IO OO0 rads) DURING DIFFERENT

DNA

OBTAINED

FROM

UNIRRA-

DIATED CONTROL CELLS, 1--2 h P O S T - I R R A D I A T I O N

IRRADIATED INCUBATION

AND IRRADIATED CELLS FOLLOWING PHASES OF THE CELL CYCLE

Cell cycle period

Treatment

Mol.wt. x zo 6

Asynchronous

Control IOOOO r a d s IOOOO r a d s plus i h p o s t - i r r a d i a t i o n i n c u b a t i o n

52.7 ~ 6.6 19. 4 ~ 4.0 51.1 ~ 12.8

Mitosis ( m e t a p b a s e )

Control ioooo rads i o o o o r a d s plus I h p o s t - i r r a d i a t i o n i n c u b a t i o n

43.0 ~- 14.o 23.0 ~: 4-7 50-3 :L 11.o

G~

C o n t r o l (M) IOOOO f a d s (M) i o o o o f a d s in M plus i h p o s t - i r r a d i a t i o n i n c u b a t i o n

44.2 ~ 9.0 25. 7 ± 3.6 41.2 ~ 3.2

S

Control ioooo rads i o o o o r a d s plus i h p o s t - i r r a d i a t i o n i n c u b a t i o n

69.5 -~ 17.o 25.8 _+_ 8.0 65-7 ~ 17.4

A

a T h e s t a n d a r d d e v i a t i o n is i n d i c a t e d .

* F r o m t h e s e d a t a we o b s e r v e d t h a t a m i x t u r e of 14C-labeled ~ p h a g e D N A a n d 3H-labeled m a m m a lian cell D N A s e d i m e n t e d i n d e p e n d e n t l y of each other. F u r t h e r m o r e , c e n t r i f u g i n g for di ffe re nt t i m e s did n o t a l t e r t h e c a l c u l a t e d s%0 ' w for ~ D N A i n d i c a t i n g t h e g r a d i e n t s were c o n s t a n t v e l o c i t y for ~ D N A .

M u t a t i o n Res., 6 (I968) 459-465

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R . M . HUMPHREY, D. L. STEWARD, B. A. SEDITA

91 ~ 6-

_8 "o p "~3-

O 0

O'.5

'

110

Relative distance s e d i m e n t e d ~

Fig. I. T h e s e d i m e n t a t i o n p a t t e r n of d e n a t u r e d D N A o b t a i n e d from cells w h i c h were in t h e S p h a s e of t h e cell cycle. U n i r r a d i a t e d control cells. T h e S%o,w - - 64 a n d t h e w e i g h t a v e r a g e m o l . w t -- 64 • lO 6. T h e arrows indicate t h e m o l e c u l a r w e i g h t of t h e D N A at different positions in t h e g r a d i e n t as described in MATERIALS AND METHODS. 12,

12.

9-

~9 o o o o

o ~6-

o ~6u

rl_

O

o

,

o'.s

,

Relative distGnce sedimented ~

' 1.0

OI 0

'

o'.s

,

~;o

Relative distance sedimented

Fig. 2. T h e s e d i m e n t a t i o n p a t t e r n of d e n a t u r e d D N A o b t a i n e d from cells w h i c h were in t h e S p h a s e of t h e cell cycle. I r r a d i a t e d cells (ioooo rads). T h e s°~0, w = 3c a n d t h e w e i g h t a v e r a g e mol.wt. = 26.8. lO 6. Fig. 3- T h e s e d i m e n t a t i o n p a t t e r n of d e n a t u r e d D N A o b t a i n e d from cells w h i c h were in t h e S p h a s e of t h e cell cycle. I r r a d i a t e d cells w i t h i h p o s t - i r r a d i a t i o n i n c u b a t i o n in n o r m a l m e d i u m . T h e s°20, w ~ 67 a n d t h e w e i g h t a v e r a g e mol.wt. = 87. 4. lO 6. a b o v e a r e g i v e n i n T a b l e I. T h e m o l e c u l a r w e i g h t v a l u e s g i v e n i n T a b l e I a r e t h e weight average molecular weight for the DNA distribution over the gradient excluding t h e r e g i o n o . o - o . I a n d o . 8 5 - 1 . o of t h e r e l a t i v e d i s t a n c e s e d i m e n t e d . I t c a n b e s e e n from an examination of Fig. I that the molecular weight distribution of the denatured DNA from unirradiated c o n t r o l c e l l s w a s r a t h e r l a r g e . I n n e a r l y a l l c a s e s of u n i r r a Mutation

Res., 6 (1968) 459-465

DNA-STRAND BREAKS AND REJOINING

463

diated controls about IO to 15 % of the radioactivity in the gradient was found at the bottom of the centrifuge tube. This would indicate a minimum molecular weight of the fast sedimenting material of 250-300 • lO 6. DNA-strand scission resulting from exposure to ionizing radiation was demonstrated b y a shift of the molecular weight distribution to a lower value. For example immediately following a dose of IOOOOrads the average molecular weight in this series of experiments was reduced to 19. 4 ~ 3" lO6. From a dose range of 2500 to IOOOOrads the number of breaks/grad in asynchronous populations was found to be 4.7 ~: 2. I . lO 12. This value does not differ significantly from that found for mitotic (5.2 ± 1.3- lO12) or S phase cells (4.0 ± 1. 4.1o12). In Fig. 2 it can be seen that the molecular weight distribution of DNA from irradiated cells was rather narrow compared with the controls. Less than 0.3 % of the total radioactivity was found on the bottom of the tube and only lO to 15 % of the radioactivity was at a position in the gradient corresponding to a molecular weight of more than 50" lO 6. When irradiated cells were incubated for I h in normal growth medium at 37 ° the molecular weight distribution returned to approximately that of the unirradiated control value (Fig. 3). In subsequent experiments synchronized populations were utilized. Only populations with a mitotic index of 9 ° % or greater were used. I h after plating the mitotic cells in normal medium, the mitotic index ranged from 2-4 % as the cells progressed into the G1 period. Labeling data indicated that at 5-6 h after plating, 85 % of the cells were in S phase. Figs. 1- 3 show the sedimentation pattern of denatured DNA obtained from cells which were in the S phase at the time of sampling. Similar patterns (not shown) were obtained for cells sampled at mitosis (colcemid arrested metaphase) and the G1 period. For this experiment the weight average molecular weight of each curve was 64- lO s, 26.8. IOL and 87. 4. lO 6 for control, irradiated (IOOOOrads) and irradiated with I h post-irradiation incubation, respectively. The data for the radiation (breakage and rejoining) response during mitosis, G1 and S phase is summarized in Table I. These data illustrate that breakage and rejoining occur to the same extent in all phases of the cell cycle thus far examined. With the present synchrony procedure it was not feasible to examine the response of G2 phase cells due to the extensive decay of synchrony. DISCUSSION All in vitro mammalian cells, thus far analyzed, show a cell cycle dependent response to ionizing radiation 25. Specifically for Chinese hamster cells, the most radiation-responsive phases were G2 and M, whereas the least responsive was the S phase 24. Differences in radiation response and repair capability at the molecular level during the various phases of the cycle is one of the important problems arising from these studies. Several investigators have used the technique of neutral and alkaline sucrosedensity gradients to obtain estimates of the molecular weight of native and denatured DNA from a wide variety of biological material including virus ~, bacteria 1~, slime mold and PPLO (ref. I8), mammalian cells13,14 and mammalian cell chromosomes 3. I t has also been demonstrated that exposure to ionizing radiation resulted in a decrease in the molecular weight of extracted DNA indicating that strand scission occurred in Mutation Res., 6 (1968) 459-465

404

R . M . HUMPHREY, D. L. STEWARD, B. A. SEDITA

the DNA double helix, in bacterial12,1~ cells a rejoining of broken pieces occurred following a post-irradiation incubation period indicating the presence of a repair process. To approach this problem zone-sedimentation analysis of exponential alkaline sucrose-gradients modified for use with mammalian cell DNA were developed. The modifications consisted primarily of a lysing technique which would give complete lysis of the cells at a high pH (Iz.3) in order to denature and deproteinize the DNA. When cells are lysed degradation of the DNA may occur due to mechanical shear or enzymatic action and the present techniques do not completely eliminate this possibility. Initial experiments, in which denatured DNA was allowed to remain in the presence of the lysing material for o.5 3 h indicated that further degradation did not occur. This observation is in agreement with the findings of MCGRATHAND WILLIAMS1~. The data on asynchronous Chinese hamster cells presented in this paper and data for human cells ~5 confirm the original observation of LETT el al. ~a who demonstrated that a DNA rejoining capability exists in X-irradiated mammalian cells. However, the use of synchronized populations has allowed an examination of breakage and rejoining to be made during precise phases of the cell cycle. The data presented in this paper (Table I) and elsewherO 5 support the conclusion that the processes (probably enzymatic in nature) which are responsible for the rejoining of broken pieces of DNA following strand scission induced by ionizing radiation are present and functional during all phases of the cell cycle, including mitosis. Furthermore, the data showed that the amount of initial damage (breaks/grad) produced by a given dose of radiation was independent of the phase of the cell cycle. We have concluded that for DNA-strand scission in mammalian cells, (a) there was no correlation of radiation response and repair capability at the cellular v e r s u s molecular level; (b) that rejoining of the broken pieces of DNA does not require an extensive synthesis of DNA, R N A , or protein since the synthesis of these macromolecules is severely depressed during mitosisI°,2°,2~, 2~. However, the possibility of unscheduled DNA synthesis occurring even during mitosis at these high radiation doses, can not be ruled out2L From the sedimentation pattern shown in Fig. I it appears that the DNA of unirradiated Chinese hamster cells exists as a spectrum of both small and large pieces ranging in size fr(~m about zo- zo" to 25o" zo 6 with a weight average molecular weight of 50 to 55" Ion. Since the total molecular weight of the DNA in each chromosome is about zo 1~ and the average molecular weight per piece was found experimentally to be approx. 5°. Io 6, it follows that the hamster cell chromosome, on the average, may be composed of at least 2ooo (single stranded) pieces of DNA. These data are not in agreement with those of LETT el a l ? 3 who concluded from radiation data that the molecular weight of mammalian cell DNA was greater than 5' zo" and that the DNA probably consisted of only a few units or possibly a single unit. Our present data supports the hypothesis that the mammalian cell chromosomal DNA is arranged in a series of small units~,%~l, TM held together by linker molecules which are labile to high salt or alkaline conditions. ACKNOWLEDGEMENTS

This work was begun in collaboration with Dr. PAUL LOHMAN of the Medical 3/Iuta/ion Res., 6 (1968) 459-465

D N A - s T R A N D BREAKS AND REJOINING

465

Biological Laboratory, TNO, Rijswijk (The Netherlands) while one of the authors (R. M. H.) was a visiting scientist at the Radiobiological Institute TNO, Rijswijk (The Netherlands). This author would like to thank Dr. LOHMAN and the staffs of the RBI and MBL for their help and cooperation. Computations were performed by the Common Research Computer Facility supported by U.S. Public Health Service grant No. FR oo254. The authors would like to thank Mrs. V. WILLINGHAM and Mrs. J. PARKER for expert technical assistance. This work was supported in part by grant CA o4484 from the National Cancer Institute, and by a Public Health Service Special Fellowship (I-F 3 CA-28,42I ) from the National Cancer Institute to R. M. It. (1966 x967). REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16 17 i8 19 20 21 22 23 24 25 26 27 28 29

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Mutation Res., 6 (1968) 459-405