DNA strand breakage repair in ataxia telangiectasia fibroblast-like cells

357

Mutation Research, 33 ( 1 9 7 5 ) 3 5 7 - - 3 6 6 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in The N e t h e r l a n d s

DNA STRAND B R E A K A G E R E P A I R IN ATAXIA TELANGIECTASIA FIBROBLAST-LIKE CELLS*

R.A. V I N C E N T JR., R.B. S H E R I D A N III and P.C. H U A N G * *

Department of Biochemical and Biophysical Sciences, The Johns Hopkins University, School of Hygiene and Public Health, Baltimore, Maryland 21205 (U.S.A.) (Received A u g u s t 26th, 1 9 7 5 ) (Accepted August 29th, 1975)

Summary Human diploid fibroblast-like cells derived from four patients with the genetic disease ataxia telangiectasia and from two non-mutant donors were examined for the repair of X-ray induced strand breaks in DNA. The ataxia telangiectasia cultures showed no significant differences from the non-mutant cultures in the kinetics and extent of strand repair. This suggests that the increased spontaneous and X-ray induced chromatid aberrations observed in ataxia telangiectasia cells are n o t caused by a defect in the repair of single strand breaks as might be suspected from a general model of aberration production.

Introduction Ionizing radiation causes DNA strand breakage in mammalian cells [13 ]. Failure to repair such damage has been attributed to be a cause of chromosomal aberrations [2,23]. A model has been advanced, based on studies with chromatid exchanges, that cells irradiated in the G2 phase, if n o t repaired, will show achromatic lesions and chromatid aberrations in the next metaphase, and that G2 irradiation mainly induces single strand breaks in DNA [2]. With one possible exception, there are no human cells known to be deficient in the repair of X-ray induced DNA strand breaks. The autosomal recessive disease Progeria, a precocious aging syndrome, may provide the one exception. Fibroblast~like cultures from patients with this disease have been reported to have such a repair defect [7,8]. This observation, however, has been interpreted * A preliminazy r e p o r t o f this work w a s p r e s e n t e d b e f o r e the B i o p h y s i c s S o c i e t y o f A m e r i c a ( B i o p h y s . J., 15 (1975) 16a). ** To whom inquiries should b e a d d r e s s e d .

358 to be biased by the increased sensitivity of DNA in these cells to radiation induced degradation [20] (J. Williams, pers. comm). Karyotypic examinations of these cells have not revealed a higher than usual incidence of X-ray induced chromosomal aberrations [3]. Cultures from patients with ataxia telangiectasia, the Louis-Bar syndrome [ 1 4 ] , another autosomal recessive disease [22], on the other hand, have displayed a higher than normal frequency of spontaneous chromosomal aberrations [9,11,18,21] and a disposition to malignancy [10,17,21]. Furthermore, X-irradiation of ataxia telangiectasia cells at the G2 phase of the cell cycle induces a high incidence of chromatid aberrations in the next metaphase [19]. As predicted by the model of Bender et al. [2] this mutation could be an enzymic defect in the repair of single strand DNA breaks. If this were shown, it would not only offer a explanation for this disease, b u t also yield a useful genetic marker for the study o f DNA repair in eukaryotes. The present study was undertaken to examine the capacity of ataxia telangiectasia cells to repair X-ray induced single strand breaks in DNA. The conventional m e t h o d of analysis for DNA strand breakage, sedimentation in alkaline sucrose gradients, was used. With this method, changes in the molecular weight of single strand DNA may be measured immediately upon irradiation and after designated recovery periods in vivo. Materials and m e t h o d s Four patients with ataxia telangiectasia contributed skin biopsies for this study. Of these, two patients were cousins (RM and VM) who exhibited immunological deficiency [17], and two (AE and KE) were siblings who did not. All of the patients exhibited classical ataxia telangiectasia with cerebellar ataxia, telangiectasia of the bulbar conjunctiva, and elevated s-fetal protein levels [4,12,14,24]. The increased incidence of spontaneous and X-ray induced chromatid aberrations was observed in cultured l y m p h o c y t e s and fibroblast-like cells from each of these patients [19]. Cultures from patients RM and VM were established in this laboratory and those from patients AE and KE were established by Dr. Jack Rary of this institution. The ages and sex of these patients and the two non-mutant donors are given in the legends to Figs. 1--3. Cells used in this study were all diploid (50--200 metaphases examined by Bender and Rary) at the time of assay and were fibroblast-like. They were used at population doublings 5--8. In all experiments, cells were seeded at a density of l 0 s cells/dish on 22 X 22 mm glass cover slips in 35 mm (dia) plastic culture dishes. Incubation was done at 37 ° C in a humidified chamber with 5--8% CO2. Culture medium consisted of reconstituted Eagle's MEM with Earle's salts, fortified with 15% (v/v) fetal calf serum (GIBCO) and 1 X antibiotic--antimycotic mixture (GIBCO). Procedures for culture establishment, subcultivation, maintenance, and mycoplasma assay will be reported elsewhere (manuscript in preparation). Labeling of DNA was done by replacing growth medium with 3 ml of fresh medium containing [3H-methyl]Thd (0.1 pCi/ml, 2 C i / m M ) o r [14C-methyl]Thd (0.0125 pCi/ml, 0.054 Ci/mM). The cultures were incubated in this medium for 46 h, after which, following 2 rinses in Puck's balanced salt solution,

359 3 ml of fresh medium lacking radiolabel was added. After 2 h of incubation, the ctfltures were irradiated with a Keleket Therapy X-ray unit operated at 250 kVp and 15 mA with 1 mm added A1 filtration (0.52 m m Cu h.v.1.). Irradiation was done at 24°C at a dose rate of approx. 2 kR/min for a total dose of 10 kR. Following irradiation the medium was replaced with fresh medium and repair incubation was allowed for designated periods of time. Sucrose gradients were prepared in 38 ml 2.6 × 8.7 cm centrifuge tubes by a modification of a published procedure [1,15]. The b o t t o m of each tube contained 2.5 ml of 60% sucrose in lysis buffer to prevent loss of heavy sedimenting sample. Upon this was layered 33 ml of 5--20% sucrose in lysis buffer. The lysis buffer (pH 13.4) consisted of 0.3 N NaOH, 0.5 M NaC1, and 0.01 M Na2 EDTA. The final pH of the lysis buffer was reduced to 12.8 b y the buffering capacity o f the sucrose. The gradients were stored before use for up to 24 h at 4 ° C. Within 1 h of cell lysis, 2.5 ml of lysis buffer was layered on top of the 5--20% sucrose solution. After irradiation and incubation, each cover slip was rinsed copiously with Puck's balanced salt solution, placed cells down in a nichrome wire basket fabricated around a fiber washer, and lowered onto the surface of the lysis buffer with the washer supported by the rim of the centrifuge tube. Lysis was carried o u t in the dark to eliminate light-induced strand breakage [6] for 4.5 h at 24 ° C. Following lysis the gradients were centrifuged for 15 min at 2 5 0 0 rpm and for 6 h at 26,000 rpm at 5°C in an SW 27 rotor of a Beckman L2-65B preparative centrifuge. After centrifugation, 38 one ml fractions were p u m p e d from the b o t t o m o f the gradient into tubes which, with the exception of every fifth tube, contained 0.5 ml of a 100 pg/ml calf t h y m u s D N A carrier. Every fifth tube was reserved for refractive index determination, and to the remaining tubes 1.5 ml of 14% TCA at 4°C was added. After precipitation on ice for 20 min, each fraction was filtered and rinsed in 5% TCA on 25 mm cellulose acetate filters of pore size 0.45 p, air dried, and counted for 20 min in a toluene-based liquid scintillation solution containing 4.00 g/1 PPO and 50 mg/l POPOP. Results U p o n X-irradiation, allowing time for repair, broken D N A strands rejoined. The shifts toward larger mean DNA subunit sizes for the non-mutant FG and PH cultures are indicated b y the sedimentation patterns of Fig. 1. The sedimentation profiles were found to shift toward larger mean subunit size with all increases in post irradiation repair time, and returned to near the mean subunit size of the unirradiated control after the m a x i m u m incubation period of 60 min. Using 32p-labeled T7 DNA as a standard (mean size 40S), the mean DNA subunit sizes of the unirradiated cultures were c o m p u t e d to be approximately 160S, while those o f the X-irradiated cultures given no post irradiation incubation were 70S. Figs. 2 and 3 show similar shifts in the DNA sedimentation profiles for the ataxia telangieetasia cultures RM, VM, AE and KE. These profiles suggest that, with the exception o f the RM culture, repair is essentially the same as in the

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Fig. 1. DNA s e d i m e n t a t i o n profiles of diploid fibroblast-like cells from n o n - m u t a n t male donors of ages 42 (PH) and 15 (FG) years. Cells were seeded at a density of approx, 104 cells]cm 2, e xpos e d to m e d i u m containing [3 H-rn ethyl]Thd (0,1 #Ci]ml, 2 Ci]mM) for 46 h, irradiated with 10 kR of X-rays, and inCUbated for the times indicated on each panel. Following i n c u b a t i o n for repair, cover slips c o n t a i n i n g approx. 5 X 104 cells were lysed in situ on 38 ml 5--20% alkaline suclcose gradients and centrifuged at 26, 000 zpm for 6 h in a SW-27 rotor. Each c o l u m n of profiles for a given culture c o n s t i t u t e d one centrifug a t i o n run.

n o n - m u t a n t cultures. The sedimentation profile for the RM culture after 60 min post irradiation incubation suggested complete repair of DNA strand breakage since the mean DNA subunit size was the same as that of the unirradiated

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RM culture (Fig. 2). In many cases, however, shoulders appeared at the 160s positions in the other cultures follo~~g 60 min repair incubation. The kinetics of DNA single strand breakage repair for each of the above cultures are presented in Table I. The changes in 5%DNA repair with incubation time suggest that strand breakage repair in ataxia telangiectasia cultures proceeds to no lesser extent than in the cultures of the non-mutant controls.

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The relative broadness of each sedimentation profile is presented in Table II. The values are an estimate of the heterogeneity in DNA subunit sizes for each profile, and show that strand repair yields DNA subunits of increased diversity in size distribution. A decrease in this size diversity, however, was observed for cultures RM and FG following 60 min post-irradiation repair.

363 TABLE I E X T E N T OF D N A R E J O I N I N G I N U N A F F E C T E D A N D A T A X I A T E L A N G I E C T A S I C C E L L S Culture 1

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T h e % r e p a i r w a s c o m p u t e d f r o m t h e s e d i m e n t a t i o n p a t t e r n in Figs. 1--3 at R / T × 100%, w h e r e R is t h e d i s t a n c e b e t w e e n t h e m e a n s u b u r d t size o f g i v e n r e p a i r p r o f i l e a n d t h a t o f t h e 0 r e p a i r t i m e profile, a n d T is t h e d i s t a n c e b e t w e e n the m e a n s u b u n i t size of t h e 0 r e p a i r t i m e p r o f i l e a n d t h a t o f t h e r e s p e c t i v e ~ n i ~ a d i a t e d control profile. T h e F G a n d PH c u l t u r e s w e r e d e r i v e d f r o m n o n - m u t a n t d o n o r s , t h e r e m a i n i n g c u l t u r e s w e r e d e r i v e d from patients with ataxia telangiectasia.

By extrapolation of the repair rates for the post irradiation incubation times given in Table I to 100% repair, it was inferred that complete repair, if attainable, would likely occur within a 90--115 min post irradiation period. In Fig. 4 are shown the cosedimentation profiles of DNA from unirradiated cultures (prelabeled with [14C] Thd) and X-irradiated cultures (pre-labeled with [3H] Thd) given 120 min post.irradiation incubation. Although the X-irradiated cultures demonstrated a larger trailing edge, the mean DNA subunit of the peaks for both the non-mutant and ataxia telangiectasia cultures were within several fractions of each other.

T A B L E II BROADNESS

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OF DNA

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1.29 1.47 1.70 1.60 1.73 1.70

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1.35 0.89 0.94 0.53 0.51 0.77

T h e p r o f i l e w i d t h w a s m e a s u r e d a t 1 / 2 t h e m a x i m u m p e a k h e i g h t a n d n o r m a l i z e d to t h e u n i r r a d i a t e d ~ontrol of each culture. The FG and PH cultures were derived from n o n - m u t a n t donors, the remaining cultures were derived from patients with ataxia telangiectasia. 3 Unirr adiated controls.

364 9

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Fig. 4. D N A c o s e d i m e n t a t i o n p r o f i l e s f o r d i p l o i d f i b r o b l a s t - l i k e cells f r o m n o n - m u t a n t d o n o r s a n d p a t i e n t s affected with ataxia telangiectasia, (: -c~) u n i r r a d i a t e d c u l t u r e s e x p o s e d f o r 46 h to m e d i u m c o n taining [14C-methyl]Thd (0.0125 pCi/ml, 0.054 Ci/mM)..-. , c u l t u r e s e x p o s e d to [ 3 H - m e t h y l ] T h d , i r r a d i a t e d , a n d i n c u b a t e d f o r 1 2 0 m i n as d e s c r i b e d in t h e l e g e n d to Fig. 1. T h e c o v e r slip c u l t u r e s f r o m t h e u n i x r a d i a t e d a n d i r r a d i a t e d c u l t u r e s w e r e l y s e d s i m u l t a n e o u s l y b a c k to b a c k in t h e s a m e t u b e . All of t h e p r o f i l e s in this F i g u r e w e r e d e r i v e d f r o m o n e c e n t r i f u g a t i o n run.

Discussion

We have shown in this study that fibroblast-like cells from ataxia telangiectasia patients do undergo repair of X-ray induced single strand breaks in DNA as assayed by alkaline sucrose gradients. It is further shown that immunological involvement in this disease apparently does n o t contribute to differences at this level of DNA repair. The results are comparable for all four ataxia telangiectasia patients, with or w i t h o u t immuno-deficiencies, as Well as the n o n - m u t a n t donors in both the rate (Table I) and extent (Fig. 4) of repair. The apparent faster rate

365 of repair with the RM cells (Table I) and slight differences in the final extent of repair (Fig. 4) may n o t be significant due to the innate variation in these measurements. Also, the slower rate of repair (i.e. 90--115 min for complete repair) as compared to that observed in other studies [16] can only be attributed to difference in quality of radiation and culture conditions. The possibility t h a t DNA of ataxia telangiectasia cells may yield higher numbers of single strand breaks than DNA of non-mutant cells in unirradiated or X-irradiated cultures has been considered. Using the mean sedimentation values of DNA in alkaline sucrose gradients as a measurement, the native size of single stranded DNA in the cells studied here has been calculated to be 160S. This same mean molecular size was observed for all the cultures which received no X-irradiation (Figs. 1--3). In results not shown, cosedimentation of DNA from ataxia telangiectasia and n o n - m u t a n t cells which were radiolabeled differently, i.e., one by [,4 C] Thd and the other by [3 H] Thd, gave also identical profiles for X-irradiated cultures in which no recovery period was allowed. This suggests that there was no difference in the initial number of single strand DNA breaks induced by X-rays in these cells. The initial X-ray induced reduction in the size of the single strand DNA was similar in all cells studied here. All showed a reduction in DNA size from the native 160S to 70S upon irradiation (Figs 1--3). This result further indicates that DNA of ataxia telangiectasia cells is n o t particularly sensitive to X-irradiation, nor is it specially susceptible to manipulative handling during assay. We have observed an increased size distribution of DNA upon repair incubation (Table II). It is possible that DNA may regain its size by ligation of smaller pieces asynchronously, with 160S being the ultimate size recoverable. There was indeed an accumulation of DNA at the leading portion (160S) of the sedimentation profile as repair incubation proceeded, concomitantly with the reduction of DNA at the trailing edge (70S). It should be pointed out that although sedimentation in alkaline sucrose gradients is by far the most c o m m o n m e t h o d of estimating the size distribution of single strand eukaryotic DNA, it is insensitive to the detection of misrepair and is vulnerable to experimental artifacts [5]. In view of this, extra precautions were carried out in this study, such as lysing the cells in situ on the gradient to avoid artifactual shearing of the DNA during handling, lysing in the dark to avoid light-stimulated strand breakage, and using minimal amounts of radiolabel of low specific activity. Because of this, the present result is of special significance. It shows that defective repair of single strand DNA breaks is n o t the major contributing cause for the observed chromatid aberrations in ataxia telangiectasia cells. The observation that these cells have the ability to undergo a significant degree of DNA repair under the high dosage (10 kR) of radiation used in this study allows one to predict that exposure of the same cells to nonlethal doses of X-rays such as those used to induce chromatid aberrations are insufficient to cause a measurable, damaging degree of single strand DNA breaks. The present study does n o t differentiate double strand breakage from single strand breakage; therefore, double strand breakage cannot be ruled o u t as a cause of chromatid aberrations. The evidence from this study suggests that single strand breakage does not play a role in the formation of X-ray induced chromatid aberrations in cells from patients with the disease ataxia telangiectasia.

366

Acknowledgment We thank Mrs. Patricia Bohdan for her able technical assistance, Drs. Michael Bender and Timothy Merz for their thoughtful discussions, and Dr. John Leavitt for the preparation of 32p-labeled T7 DNA. This research was supported by N.I.H. postdoctoral fellowship GM 57140 (R.A.V.), a Kunitz--Worthington Scholarship (R.B.S.) and the National Foundation/March of Dimes (Grant CRBS 300). References 1 A y a d , S . R . , W.R. BonsaU a n d S. H u n t , A s i m p l e m e t h o d f o r t h e p r o d u c t i o n o f a c c u r a t e l i n e a r gradients using a constant speed peristaltic pump, Analyt. Biochem., 22 (1968) 533--535. 2 B e n d e r , M.A., H . G , Griggs a n d J.S. B e d f o r d , M e c h a n i s m s of c h r o m o s o m a l a b e r r a t i o n . III. C h e m i c a l s and ionizing radiation, Mutat, Res., 23 (1974) 197--212. 3 B e n d e r , M,A. a n d J.M. R a r y , S p o n t a n e o u s a n d X - r a y i n d u c e d c h r o m o s o m a l a b e r r a t i o n s in p r o g e r i a , R a d i a t . Res., 59 ( 1 9 7 4 ) 181a~ 4 B o d e r , E. a n d R.P. S e d g w i c k , A t a x i a - t e l a n g i e c t a s i a a familial s y n d r o m e o f p r o g r e s s i v e c e r e b e l l a r a t a x i a , o c u l o - c u t a n e o u s t e l a n g i e c t a s i a a n d f r e q u e n t p u l m o n a r y i n f e c t i o n , Univ. S o u t h e r n Calif. Med. Bull., 9 ( 1 9 5 7 ) 1 5 - - 2 7 , 51. 5 Cleaver, J . E . , C o n f o r m a t i o n o f D N A in a l k a l i n e s u c r o s e : t h e s u b u n i t h y p o t h e s i s in m a m m a l i a n cells, B i o c h e m . B i o p h y s . Res. C o m m . , 5 9 ( 1 9 7 4 ) 9 2 - - 9 9 . 6 E l k i n d , M.M., S e d i m e n t a t i o n o f D N A r e l e a s e d f r o m C h i n e s e h a m s t e r cells, B i o p h y s . J., 11 ( 1 9 7 1 ) 504--520. 7 E p s t e i n , J., J . R . Williams, a n d J.B. 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