Radiation-induced DNA double-strand breaks and the radiosensitivity of human cells: A closer look

Bi~chimie ( 1997} 79, 507-575 © Soci6t,5 fran(ai~e de biochflnie et bk~It~giem~fl6culaire/ ELsevier.Paris

Review

Radiation-induced DNA double-strand breaks and the radiosensRivity of human cells: A closer look N F o r a y ~, C F A r l e t t t', E P M a l a i s e ~* aLaboratoireb de R, uKobiologie (URA-CNRS 1967) PRl-lnstitut Gu~tave-Rtmssv, 94805 ViHejui/i Fran,'c: MRC (.'ell Mutation Unit, Utliversitv o]'Susse.i, F.Imel; Brighton," BNI 9RR. UK ' (Received 4 June 1997; accepted 13 Novelnber 1997j

Summary ~ A large number of reports suggest that DNA double-strand breaks (DSBI play a m~tjor role in the radiation-induced killing of mammalian cells. However. the arguments supporting the relationship between DSB and radioscnsitiviJy are generally indirect. Furthermore. care nlus! be takef~to allow lot the possible impact of the techniques and of the experimental protocols on the relationship belween DSB and cell dealh. The recent data on DSB induction, repair and misrepair in human cell liues and their correlation with intrinsic radiosen',iti'.'ily aw reviewed. human cells / radioseusitivity / DNA double-strand breaks / DNA repah" Introduction

Ionizing radiation causes several types of DNA damage, alters bases and sugars, induces the formation of DNA-DNA and DNA-protein cross,links and causes single- (SSB) and doublestrand breaks (DSB) 128, 49]. According to several lines of evidence, it is generally accepted that the DSB are the main, if not the only, type of damage that leads to the death of irradiated cells 128.38, 49, 571. Indeed, DSB have long been known to be lethal in bacteria 1301. In yeast, a single residual DSB is linked to a mean lethal dose 1291. In many experimental siluations (studies on hypoxia, thiol groups, linear energy transfer (LET), hyperthermia), cell killing correlates with the number of DSB and not to that of SSB 128, 491. Hydrogen peroxide induces many SSB, but produces few DSB and reduced cell killing [491. A dose-effect relationship has been lbund between the concentration of the restriction enzyme (RE) Pvull, which produces only DSB, and cell death [191. Moreover, there is a direct link between RE and DSB, chromosome aberrations and cell killing [12, 171. There is also good evidence for a direct correlation between unrepaired DSB and residual chromo*Correspondence and reprints Abbreviations: AT, ataxia telangiectasia; bp, base pair; CHO. Chinese hamster ovary; CPT. camptothecin; DSB. DNA doublestrand break; FAR. fr,'tction of activity released: FAP, familial adenomatous polyposis; LET, linear energy transfer: NFE, neutral filter elution; PCC, premature chromosome condensqtion; PFGE, pulsed field gel electrophoresis; RE, restriction endonuclease" SB. strand break; SF2. surviving fraction at 2 Gy; SSB. DNA singlestrand break; TJ12, repair half-time; VRH1; variable repair hall-time; WDR, tryptophan (W)-aspartic acid (DI repeat.

some breaks in human cells 14, 161. Most of the rodent celt lines that are hypersensitive to ionizing radiation ate deftcient in DSB repair and/or display chromosomal instability 11041. The situation in human cell lines appears, ilowcver, to be much less simple, This review examines the mechani:ms including induction level, ;epair rate, irreparable damage and misrepair by which DSI3 could accotmt l~r the ladiation-induced death of htnnan cells.

Methods fiw measuring DS|~ Several methods are available, aft of which h~ve at least two l~alures in common: they newt' measure the number of DSB directly, and they are relatively insensitive. DSB are measured indirectly from the properties, either mech~nfic:~l or electrophoretic, of the fragraents of DNA created by iro radiation, or by the extent of ehromatin relaxation. The techo niques are generally calibrated using DNA labeled will] radioactivity (It-'si]ttridine). As each [~ disintegration creates a single DSB, it is easy ~.o meastn'e the nunaber of DSB II, 40, 71 I. Mathematical models can be used to Ir,'mslatc the results into numbers of DSB 17, 141. Radiation doses of tens or hundreds of Gy are used to measure DSB whereas cell killing is measured in response to a few Gy. A dose of I Gy of low LET radiation generates, in average. 39 DSB in the genome of a diploid human cell [95 I. The total DNA in the human genome is distributed over 46 chromosomes: a situation equivalent to 46 DSB. Therefore, a dose of I Gy leads to DNA fragments whose average size is about 631)0 Mbp/(46 + 40) = 73 Mbp. The resolution of the methods

u ~ is not ~fficient m measure accurately efl~,JtSresulting in ~ m e . t s smaller than 15 Mbp. an effect equivalent to alx)ut 10Gy [7. 81. S~cros¢ gmdie.t sedittte~ltation

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Elcctrophoresis can be u~d on individual cells during lysis. The cells examined optically thus appear as cotttels, and at a twulral pH. the length or m~ment of the tails is proportional to the numberof DSB (comet a~ssay) 123t.

Another m¢lh~l (hl|lo method) uses the radiation~induced DNA unwimling which is mainly due to SSB rather than DSB, The DNA unwinding increases the apparent size of the nttcleus 1731. Alter staining with a dye such as propid i ~ k~id~, the resulting tmlo a ~ its t ~ n , ent may be .~'d ~s ~ ta~asure of the number of breaks I731, Whatever the ~10~; I ~ ~mdiiions of cell lysis prior to the analysis i t . l l considerably i.flae~e the results [59, 101 I. Lysis that pte.~.,.tv~ t ~ ehronuttin t:cganizalion as hx~ps provi~s infor~ t i o n tm the chromafin it,~lf and not simply on the number

ofDSB [ll)}l,

20

30

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PFGE ~parat~.~ DNA fragments in an agam~ gel using their negative charge according to their size 19. 771. Most studies simply me~su~ the amount of DNA that leaves the well and not the migration along the hme it.ll.

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Fill I. Literature data on DSB induction rate in CHO cells in exponential phase of growth. Upper paneh NFE data: ~ 1181;....... 1701: _ _ _ 11I. _ _ _ _ __1601. Lower panel: PFGE data: 1351: _ _ _ wl871; ....... 1971: . . . . 1391: . . . . . . . . . II 1.

Tbe induction it,veil"should always be studi~ alter irradiation at 4"(g to prevent repair. A number of studies perlbrmed o ~ ~ ~ decade using NFE suggested that the dose-effect relationship is curvilit~ar and hence that it is the mirror image el'the ~rvival curve I71 I. This further suggested that the induction of DSB alone can explain the killing effect 171 I. As the~ experiments were carded out in the ab~nce

of the repair of DSB. it was difficult to ascribe the bending of the curves as being due to saturation of repair enzyme systems. Subsequently. it was established that the curvilinear shape of the relationship between radiation dose and DSB was probably a consequence of lysis conditions I59, 691. We have compared the curves published by several

~ B Ind:u~ie~rl

569 groups using NFE and Chinese hamster ovary {CHO} cells in growth phase ~l'ig I. upper panei). The shape of the curve clearly c h a a g e s with the lysis technique used. especially pH and buffer composition. Thus. most dose-effect cur~es appear linear as long as the fraction o f activity released (FAR) does not exceed a certain level (50-70%) [7, 26]. The target theory predicts a linear relationship when in'adiation occurs in the presence o f oxygen and withott| any repair 195], The slope o f this relationship, rather than the shape, can vary for a given cell line according to, for example, the type of electrophoresis used (migration, direction and intensity of electrical fields, etc) (fig 1, lower panel}. Thus, five groups used four types of PFGE and all obtained diflbrent curves with CHO cells in growth phase {fig 1, lower panel}. This demonstrates the importance of

technical parameters ~hen comparing, for example, indue tions of DSB between celt lines, or searching for a correlation between DSB and radiosensitivity. Rehniemahil~ betn'een D S B induction and cell killing

We have identified 26 published studies on the relationship between DSB and intrinsic radiosensitivity of human tumor cells (table !). The results vary without any apparent pattern. The data for all the techniques used (NFE. PFGE and comet assays) indicate that only 7/24 publications found a positive correlation between the DSB induction and the radiosensitivity and this was for a minority of the cell lines studied 126/110). Analysis of the inlbrmation summarized in table 1 (colunm 3) offers no clear explanation as to why the results

Tahle I. Correlation betvceen DSB induction. DSB repair rate and hyperscnsiti',,ity of human tumor cell lines. Rtferences tttltl te~'lmiqlws NFE

Schwartz et al, 1988 1821 Kelland et al. 1988 1421 Schwartz and Vaughau 1989 1781 MacMillan et al, 1989 [471 Peacock et al, 1989 163] Schwartz et al, 1990 1791 MacMillan et al. 1990 1501 Schwartz et al, 1991 181 ] Alaoui-Janlali el al, 1992 121 Zaffann|i el al, 1994 111)31 Olive el al, 1994 1621 Schwarlz et al, 1996 18111 PI,;GI~

Gia¢cia et al, 1992 1321 Smeets el al, 1993 1841 Cedervall et al, 1994 1151 Ruiz de Ahnodovar et al, 1994 1751 Smeets et al. 1994 1851 MacKay et al. 1995 [461 Whitaker el al, 1995 1981 Allalunis-Turner et al, 1995 131 Nunez et al. 1995 [581 Woudstra et al. 1996 [ 101J Woudstra et al, 1996 I I0(ll Contet assay

Miii{er et ij}[-['J94 1551 Olive et al, 1994 [621 Olive and Bannath 1611 Total

Number ~f di[fi'rent cell lines and studie,~'

('.rrclalion belween DSB imh.'lion and rodio.~ensim'il~

Corrt'hllion between I)SB repair rotc .~ld radio,~en.~ili~'it.v

91 lines. 12 stmlics

Obtained with 17/91 lines and with 5/I 1 ~'tudies

Obtained with 73/81 lines wld wilh 6/8' .~tudie.~

12 5 6 2 2 17 9 8 2 20 6 r.]3

No Yes Yes Yes No Yes No Yesa No No No

Yes* at lOOGy + I h Yes Yes at IOOGy + 1 h

33 lim',v 11 ,~lu,tic,~

OI,t, mw,I witll 14132 lin,,,, ttml p,'trlh 2/1{15lHdlr¢'.'~

Oh/aincd with 25/3 ~ /iut'~ and ~ilh o/lO ~t~ttlit,,~

5 2 2 7 4 4 9 2 6 2 3

No No No Yes* No No Yes* No

Yes* at 10Gy + Ih

9 lines. 3 sttldies

Obtaitwd with 0/2 lines. aml with 0/3 similes

3 6 4

No No No

119 lim,s. 26 studies

Obtained n,itlt 26/I I0 lines. and with 7/24 studie,~

*Statistically significant. 'tAt doses lower than I00 Gy.

No No

Yes* a| 101)Gy + l h No Yes '~'at 50 Gy + 2 h No Yc~

No

Ye~ Yes fnr Ihe fast rep~fi," Nn Yes* tin" the slow repair Yes Yes* lor the fast repair No No Obtaim,d with 1/2 lines. and with 1/2 sltulie,~'

Yes No Obtained witll 8W/0/lim~,~. ,rod with 13/20 smdie,~'

570 ~ould differ, Ft~rexample, the probability of positive correl a t ~ does not ~em to be linked to the method used. The difference in the intrinsic radiosensitivity of cell lines may, in some cases, have been too small to detect a statistically significant correlation. The induction of DSB is influenced ~ ph~,e of ~ cell cycle [39, 59[, It is thus possible differences in ~ B induction rates ~ linked to diff e ~ e s in ~ disruption of cells in the cell Cycle [40, 601, Tilts factor ~,amldhowever only have a significant influence if70% o f the cells are in the S pha~, which is nut frequent in human ceil lines [511. One possible explanation for the l i m i ~ positive correlations is sugg~ted by the comparison of ~ results obtained with two tmnor cell lines that were studied using six different methods It(Ill. Three methods gave a positive eorrelation, while the other three showed no differetv:e in the induction of DSB in the two cell lines. An apparently higher DSB induction was found in the hyper~nsitive ~11 line HX 142 using the methods that involved moderate lysis [101l. The authors conclude that the d i f ference between hypersensitive and non-hype~ensitive cell lines lies in the presentability of the lesions | I01 l, Miluer ct at[541 also showed that the DNA-nuclear matrix binding and ~iosensitivity were linked in tumnr~ having the same proportion of DSB [541, More and more workers seem to have taken up the idea that the conformation of the chromatin ~ l d influence both the capacity to detect DNA damage and radio~nsitivity [54, 61, 73, 101 ], But even if this correlation has been demonstrated for a large number of human tumor cell lines, the mechanism hy which the presentability of DSB influences cell killing remains to be explained, The response of hum,~n fibroblasts, whether they are nor° real or hypersensitive cell lines, is difl~rent l'rom that of tumor cells: DSB induction in fibn~hlas! is never correlilled with radiosensitivity llk~r reviews see 14, 271), The exp,~imen~! ¢ottdi|iolts used tot human fibrobla~ts are u~orc for tunlor cells,As eXl~erimenls are carri~:om 6h cells in plateau phase (more than 95% of cells in the ~ 1 phase) it is easy to avoid any possible inf l u e ~ of the cell cycle on DSB measurements: furtherntor¢, all the cells are diploid, The use of unifornt experimental condititms makes it possible to study DSB repair over long periods of time, as irradiated fibmblasts can ~main in plateau phase without detriment for a few days aider irradiation [24, 26, 271, We established that the FAR levd is always about 2% per Gy under these conditions, whether the fibtoblasts are normal or hypersensitive 14, 27|, The results of some studies using the halo assay indicate that ataxia telangicctasia (AT) non-transl~rmed fihtoblasts have mor~ relaxed chromatiu after irrudiation 1911, in agreement with studies on hypersensitive human tumor cells 154, IOII, Parallel experiments using the methud of W~n~atare chromosome condensation (PCC) also suggest thai the induction of chromosome breaks in hyper~usitive and normal control cell lines are not significantly different 141, The number of chromosome breaks detected

per Gy is about 6-7 times smaller than the number of DSB 141. This difference is probably due to tile presentability of the damage, with most of the DSB being masked by tile chromatin condensation l lOll,

DSB repair DSB repair and the shape ¢!f the dose-effect wlationship Tile standard method to study the repair of DSB is Iv irradiate at 4°C and to incubate the cells at 37~'C after irradiation [281: the repair data are expressed as a fi'action of residual damage as a function of time. The technique used has little or no impact on the repair curve, which is in si)arp et~n|rl.~s| with the way tile induction of DSB varies with the technique (fig I). This is because the induction of DSB is obtained by measuring a series of values, all obtained by the same technique and with a specific dose of radiation: thus the technique influences each individual value and hence the ovel~dl doseeffect relationship. However; the repair carve measures the ratio between the value at time t and the value obtained with the ~ m e dose at time 0. Consequently, any bias due to the technique will influence in the same way and to the stone extent both tile numerator and denominator provided that the induction rate is linear. Therelbre, a possible bias will be eliminated. We have summarized in graphical lbnn the resnlts obtained by five groups; they all irradiated CHO cells with a dose of 20 Gy, but u~d four dilTercn! methods t fig 2). A single curve fits all the data points satisfactorily (fig 2, upper panel). While the techniques themselves have little or no influence on the repair curve, the type of carve littiug (by eye or using a model) and the repair time m,ly inlluence the resulls 125 I. The repair curve ulc~tsnrcd Ibr m;iMn|:dia!| cells is generally described as hiphasic 125, 281, The li~.~lcomponent has a very short repair half4hue O'~n}, geuer,'dly l|l~)at 3-.H)mitt, while the =epai.' rate descried by the~secoud component is much slower, with a TI~ of about 411-1r,0 rain 125, 28, 38 I. We have shown that the repair curve cannot be described adequalely as two successive slopes 1251. Fur example, the two Tlt.~ obtained tbr a given ceil line irradiated under the same conditions will differ with the repair time (fig 2). Our description of the repair curve implies that each DSB is repaired ,at its own rate 125, 261. Hence, the slope of the repair curve gradually changes until it reaches a horizontal ba~line corresponding to the irreparable damage (variable repair half-time (VRHT) model) 1251. The VRHT mudel fits the repair curve better than the biphasic model [251. As a result, VRHT curve fitting depends very little on the repair time (fig 2).

Relatios~ship between DSB relmir and the cellular

h'thulity

We have accumulated 20 published studies on tile relationship between DSB repair and radiosensitivity in human tumor cells. They show an overall positive ton'elation for a

571 majority of the cell lines (St)/10)) (table ilk Thus the situation is clearly different from the induction of DSB. The repair rate ~'md/or the residual damage appe:n" to be important factors influencing lhe radiosensitivity of most human tumor ceil lines, with the more sensitive tumors repairing their DSB more slowly and/or less completely. This defect is seldom as great in human tumor cells as it is in rodent cell lines. There is, however, one exception, in the MO59J cell line, a hypersensitive clone isolated from a tumor line derived from a human glioblastoma [3]. Nevertheless, caution is required, because even though the technique itself has little influence on the repair curve (fig 2), the para-meters (repair time, dose) may have a considerable impact (fig 2), and these parameters vary greatly from one study to another I25, 271. Studies on untransformed human fibroblasts indicate that cells isolated from patients suffering from certain rare disorders are hypersensitive in vitro (nine syndromes are listed in table I1). However, it tnust be emphasized that among these disorders, only AT is always linked to an extreme radiosensitivity [41,901. The cell line 180BR was obtained from a radiosensitive leukemia patient who apparently was suffering from an unknown disease 1641. Cytogenetic studies have often provided the first indication that many of these cell lines had a repair deficiency 14, 5, 161. The cell line 180BR is the only human cell line in which a massive DSB repair deficiency was found I51. The AT cell lines are a striking example of a disease whose link with deficient DSB repair has been the subject of a Io,tg-standing controversy [27,411. We befieve this to be ntainly dne to the irradiation protocol employed. When DSB repair is only studied for the first few hours after irradiation, most studies reveal tit) significant difference between the KF cells aud the controls 1271. In fact, in AT cells vep.'tir may even prove to be faster during the initial 2-3 It 1271, but the residual damage is always about 10% at 24 h, which is significantly higher than in the controls [4, 27]. Low dose rate irradialion at 37°C has now been used by several groups to show that the residual damage at the end of irradiation is always higher in AT cells I10, 24, 27, 102]. Analogous results have been obtained with tumor cell lines [13]. We have shown that DSB induction and repair occur during irradiation at 37°C at rates similar to those found with the other protocols [261. Irradiation at 37°C gradnally selects those DSB which repair at a slower rate, and AT cells have a hu'ge fraction of such DSB [24, 27], as may also have hypersensitive human tumor cells II31. Some degree of DSB repair deficiency is probably an important factm" conlributing to the hypersensitivity of human fibroblasts, or even of tumor cell lines, but the methods (incubation for 24 h after irradiation at 4°C, or low dose rate irradiation at 37°C, and the type of curve fittingl play an important role to detect a repair defect.

DSB misrepair." ~'orretation with celt killing The fideifiy of DSB rejoining ma3~ be studied using p}asraids whose genes are damaged by RE 192]. The fidelity of repair by recombination is studied by codransfection with two piasmids [94] or by transfection with a single plasmid containing two copies of the gene damaged at two different points [521. DSB rejoining is five-fold less accurate in AT cells than in controls [661. Recombination is 80% error-

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Fig 2. Literaturedata tm DSB repairrate after 20 Gy in CHO cells in e×ponentialphase of growth. Upper panel: NFE data: x 1741; comet assaydav~:V 1721;PFGEdata:I'11721:O 181;A [~71. Lower panel: data shown in the upper panel and fitted to two models: biphasi¢ model over 0-2 h (. . . . . model: over 0-.-2 h (..,..). 0-6 h (

), O-O h (.......... ); VRH'I .).

572 T~I~I¢Ii, DSB r e a r deficiency in human genetic disordet~ associated with ionizing radiation hypersensitivily. Syndromes

SF2 ~ (%)

DSB repair de th'iem'y

Minuted gem' prodm't(s)

Ataxia-telangiectasia 18OBR fibroblast cell line H~pogatamagl~line!nia ~ B R tr'tl~rob~lee!l li~) N i j ~ n b t e a ~ e syndrome Ox~inuria U~.er's Cockayne's

3 t~ II

Yes 1271 Yes [51 No"

PI-3 kinase ATM 1761 Ligase LIG I I~]

12 14 16 18

Gard~r's

18 19 20

YesI~ Suggested by SBc studies [221 Suggested by CPTd and cytogenetics studies 168, 861 Suggestt~l by cytogenetics 1891 Suggested by cytogenet~cs 1311

NBS-VI and NBS-V2 [881 Glutathione synthetase GSS 1831 At least five USH genes 1961 WDRc protein CSA, helicase CSB and helicus.eCSC 361 FAP' 1561 Tyrosine kin.use receptor HG 1931 NBCC 1331

Huntington c h ~ a Nevoid ba~l cell carcinoma

aSurviving tractions at 2 Gy (growing cells] from 12111;bForay et al, unpublished dula; ,~sll'andbreaks; dCanlptolhecin; cWD repeal; q'amili.'d adenomatous polyl~rsi~gene. prone in AT cells 1451. A positive correlation between the accuracy oi DSB rejoining and survival at 2 Gy (SF2) has been found in human tumor cell lines 1651. A high rate of misrepair has been reported for hypersensitive cell lines from bladder minors, in which there was neither an excess of DSB induction nor an excess of residual damage 1671. Thus, repair quality as~ys provide complententary (AT fibroblasts), or new (certain tumor cell lines) information that can be used to fully describe the role of DSB in the radiosensitivity of human cells,

Mechanisms of death of irradiated human cells Basically, Ihree int~ialities of cell death a~"eknown at preso enl; lbese are: mitotic death 1341, permanent arrest in G I 1371 and apoptosis 1211, Now, the question is: what is the r¢lati~nship b~tween DSB and thc~e ihree tuoda!ities of ~ Mitotic ~ath is associated with two grt)ups of chro~ s o ~ a~rrations: exchange iype aberrations (dicentries, rings) and ac~ntfic chromosomes later leading to deletions 1341, Exchange type aberrations are the direct con~quenees of misrepair, invdving at le~u,~ttwo DSB 1341, Deletions may he the con~queuce of unrepaired DSB 1341. While mitotic cell death has been recognized for many years, al~plo~is and permanen| G I are more recent phenomena, Permanenl arrest in GI is directly linked to the presence of unrepaired DSB in the nucleus 1371; and it is particularly important in irr~iated fibroblasts 144, 991, Apoptosis probably also depends on unrepaired DSB, which can trigger a respon~ that is p53 dependent 1531,

Conclusion Two main mes~ges emerge front this review: firstly, it is t~t possible to perform direct measurements of DSB and

their number is derived from a set of biophysical techniques of limited and variable sensitivity. Consequently, the assessmerit of DSB is always likely to be biased by the teclmique employed ~,n :ttty particular study. While this is a major problem when considering the rate of induction it becomes less significant when nteasuring the rate of repair of DSB. Nevertheless, it is likely that the extent of residual damage, after repair, will remain underestimated because of the lack of sensitivity compounded with the limited amount of snclt damage. These limitations may be overcome by the application of improved technology coupled with the use of more efficient models; secondly, it is clear that DSB play a key role in the ionizing radiation lelhality of both normal and tumor derived hum;,m cells. In only a minority (20%) of ttunor cells is the intrinsic radiosensitivity related to the level of induction of DSB° ht contrast, however, lack or !his-repair of DSB appears to he tile inajor mechattism responsible fiw iniliatiug lethality il} human cells.

Acknowledgments This work was supported by the Ligue Nationale Fran~uise Cuntre le Cancer (Comitd des tlauts-de-Seine), the ARCS (Dr Gest) and the Comit~ de Radioprotcctiou d'Elec,,ricit~ de France,

References I Ager DD. DeweyWC ( 19~l}Calihrationof pulsed fieldgel electl~pho~si,~fi~rn~eusurelnentof DNAdouble-strandbreaks. Int .I R~uliat Bio158, 242--259 2 AlaonioJamaliMA, Batist G. Lelmert S (1')91) Radiation-induced damage t~ DNAin drug- and radiatiou-resinlan!sublinc~t;f a haman breast cancercell liue,Radiat lies 129,37-42 3 AIlatunis:ruruerMJ, Zia PKY,BarrunGM. MirzayansR. Day Ill RS ( 1{~5}Radiation-inducedDNAdamage repairin cellsof a radiosensitivehuman malignmugliomacell line.Radial Res 144. 288-293 4 BariteC, IliakisG, Foray N, AIsbeihG, CedervallB. Chawmdra N. Pantelias G. ArleuC. Malaise EP (1995) Inductionand rejoiningof

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17 18 19 20 21

22 23 24

DNA double-straud breaks and interphase chrolil~F-.~lnlc Im'aks after exposure to X-rays in one Ilorula~ and tX~Ohypersensitive huutan libroblast cell lines Rru~a~ Res 144.2i~-35 Badie C, lliakis G. |qmty N. Alsbeih G. Pan,elias GE. Okayasn R. Cheong N. Rus',e] NS. Begg AC. Arletl CI~ Malaise lip i 1995~ De~ective repair of DNA double-strand breaks lind chronlosoule damage in fibl~ablasts fixnl'~ a radiosensiti'~e leukemia patient. ('amer Res 55 1232-1234 Barnes DE. Tomkinson AE, Lebmann AR. Webster ADB. Liudahl T 0992) Mutations in the DNA ligase I gent of an individual with inummodefieiencies and cellular h~,persensitivity to DNA-damaging ngems. Cell 69. 495-503 BRicher D (199t)) |o CHEF eleetmphoresis a linear induction of DSB corresponds to a nonlinear fraction of extracted DNA with dose. hzt ,! Radial Bio157, 7-12 BI6eher D, Einspenner M, Zajackowski J (19891 CHEF electrophoresis: a sensitive teehoique for the determination of DNA doublestrand breaks. Im ,I R.diat Bh*156, 437~td8 Bl(icher D, Kunhi M (1990) DNA dotible-strand break aualysis by CiIEF electrophoresis. Int ,I R.dh~t Rio/58, 23.34 Bl(icher D. Sisal D. Hanuan MA( 1991 ) Fibroblasls (toni ataxia tclaugiectasia (AT) and AT iletel'ozygotes show aud enhanced level (ll" residual DNA double-strand breaks tilter low dose-rate 7-irradialion as assayed by pulsed field gel eleelmphoresis, hit ,I Radial BiM 6t), 791-802 Bradley MO, Kohn KW t l979) X-ray induced DNA double-slrand break production and oapair ill mannllalian tells us measured hy ucntral filter elution. Nuc&h' Acid Res 7. 793-804 Bryant PE (1988| Use of restriction endonucleases to study t~lationships between DNA double-strand breaks, chromosomal aberrations lind oilier cud-points in mamnlalian cells, hit ,I R, tdhtt Biol 54, 86989O Cassoui AM, MacMillan TJ, Peacoek .IH. Steel GG (1992) D i f ferenees in the level of DNA double-strand hreaks in human cell lines following low dose-rate imtdiation. EnrJ Cancer 28A, 161(|-1614 Cedervall B. Kalhnan P ( 19941 Randomly distributed DNA doublestrand breaks as measured by pulsed field gel electrophnresis - A series of expJaaalory calculations. Radial En t,/r,m Bi~q~hys 33. q-21 Cedervall B. Sirzea I'~ Bmdm O, Lewensohn R t 19941 Less iaitial rt~joiniag of X-rily-iltditced of DNA double.strand breaks in cells of a small cell UI285 etnnpared It) a hu'ge cell tll81(I laug carcinoma cell lille. Rtuliat Res 139, 34-39 Conlftwth N. Bedlord JS (1987) A tlaantitali~,e comparisou tit' pulcntially lelhal danlage repair and the rejoining of intcrphasc chl'omo some breaks ill low passage nolulal htunan fibrol~lasts. Badiat Re~ 11 I, 385.405 Costa ND. Brym~t PE (19911} The intlttcliolt o1' DNA douhle-strand breaks in CHO cells by Pvttll: kinetics using neultatl elation (pll 9.6), hit J Radial Bio157, 933o.938 Cosht ND, Bryatlt PE (1991 } Elevated levels of DNA dottble-slrand breaks (dsb) in tvstriction endunucleaseobvated xrs5 cells correlate with Ihe reduced capacity to repair dsb. Mutat Res 255.2t9-.226 Costa ND, Thacker J (19931 Response of radialion-sensitive Imman cells to defined DNA breaks, hit J Radial Bio164, 523-529 Dcsehavtmue PL Fertil B ( 19961 A review of human cell radiosensitivity in vitro, hit .I Radiat Oncol Bhd Phys 34. 251-266 Duchaud E. Ridet A. Stoppe-Lyonnet D, Janin N, Moustacchi E, Rosstill F ( 19961 Deregulaled apoptosis ia Ataxia telangieclasia: association wilh clinical stigmata and radiosensitivily, Cttncer Res 5b, 14011-1404 Edgren M, Revesz L, Larsson A ( 1981 ) Induction and repair of siuglcstrand DNA breaks after X-irradiation of Imman fibroblasts deficient in gluthalioac h~t ,I Radial Biol 4(I, 355-363 Fairbairn DW. Olive PL, O'Neill KL ( 19951 Tile cornel assay: a con> prehensivc review. M.tat Res 339, 37-59 Foray N. Arlett C E Malaise El) (1995} Dose-rate effect nu indactkm and repair rate of radiation-induced DNA douhle-strand breaks ia a nornlal and aa alaxia lel:lllgieclasia Ittlman fibrohlasl cell lille. Biochimie 77. 900-9|)5

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~

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~,5 ~i~=d Jl: ( 19S8 ~| ) N , \ damage pmduued h~ mmging +adzatb,~i !u mare iit;~l};mll kCH~: }detlIHit.,~. Illethatt|i'qils Oi [ll[tl}all~ll amd tcpa!aNli{. P r . w Am h'i~ h Zd R, ~ Mol HiM 35. ~ 5 125 '~l~ \R:td~tc S. 1)el Kah+u*lma \ M. b;ctlh,xs M P~!k mcu~ [{ S.,,II I~ \ I:ieldillg lletj*llarlcik 11'. ShcHiekt \ ( " Smilh RJH 4 I~,%~r.l.~,~ah/ati,,n el lilt k:sh¢l n'.ndromc t'.pe II) gene ~l..htl)~ to dmmlusumc I{I I I . m M . / (h'm'~ 5. ]|,8tl ~ I¢~t.a2 97 Whitaker SJ. MacMilkm TJ {1992i Pub, cd-fietd ~el electr(/ph~resP, in the nleastlrelnent of DNA double-qrand break repair in xrs-b and CHO cell litles: DNA degradation under some conditiom, intertklcs with Ihe assessment of double-strand break rejoining. R . d i . l R~x 1311. 389-392 98 Whitaker S J, Ung YC. MacMillan TJ (191)51 DNA duuNe-.,Irand break iudtlclion and rejoining as d¢l¢llllillalltn tll hulllall lUll/Of cell radiosensiti~ily. A pulsed tick! gel electrophurcsis stud}' In| .I R . d i . I Bi,d 6% 7- I 8 99 Williams K.I. Bu) l¢ JM. Birch, .IM. Norton ID, Sc~tt I) ( 1~4971 (Teli cycle ;tl'res| del+.'cl ill I.i+Fraumelli +~ylld[tllllC; a !llCChIHIIMll(11 CatlCCl lUedinptb~,ition ? ~21~ ~' ' ~L~'~:~ ~ ' 1 4 , 277-282 IIII) ~'otzdslra I:.C. I"kullsting Jl:. I:loeniug JM. Koaingn A'~T. Kanlpmga ftt| I19961 Radiation-induced DNA damage and dantage lepail in Ihree human I1.1111111cell liues, a$~l~llll .,~'~ 302, 51 59 II)l ~'oudstra I~C, Rc+esink JM, Rtlscnlauu M. Bl-uusling JF. Dlien,,cll ( ' Orta T. Kon ing,+ AWT. Peacuc k .IH. K ampi nga H It ( I qg01 ('Ira mlalln Sll'llCtl.U+e ;llld celhdaf I'adioseusitixit}: a cOlllpatinou O| 19, O Jlkllllalll Ihi,,or cell lilieS, lilt .1 R,rli,a Bi,,! 71), 6q3 7111 1112 Vvurnl R, I:luruet Nli, Duggal N. Yarnold .JR. Peacock lit ( 19~14~( e l lular radiosensitivity and DNA damage in prinlary hunlan 1ibrublasts I.t .I Radmt O.,',d Bi, d I'hv~ 311.~25-¢,33 103 Zaffaroni N. Orlandi L. Villa R. Bear]alto A. Rob, lad |-K. Sd,.estrini R I lqgdl DNA double-strand break repair attd radiatiuu resptmsc ia htullan lUlIIIR+prilnary ,2ultur¢,, Im .I R~rliat Bi.I h(+, 27q 285 1114 Zdzieuieka MZ 119q5) Mammaliall inulalllS detccti,.e ia tile ru.,l~tmne Io ionizin.~ radiatiou-induced DNA damage ,~.l.mt Re,. 3~e< 203.213