INT. J. RADIAT. BIOL ., 1987, VOL. 51, NO . 2, 209-218

Rejoining of double strand breaks in normal human andataxia-telangiectasia fibroblasts after exposure to "Co y-rays,241Am a-particles or bleomycint

THERESE M. COQUERELLE, KARL F . WEIBEZAHN$and CHRISTINE LUCKE-HUHLEKernforschungszentrum Karlsruhe, Institut fur Genetik and Toxikologie,$ HS/Abteilung Biophysik, Postfach 3640, D-7400 Karlsruhe 1,F.R. Germany(Received 9 July 1986; revision received 28 July 1986 ;accepted 6 August 1986)

The rejoining of DNA double strand breaks (dsb) induced by 60Co y-rays, 24 'Ama-particles or bleomycin was measured by neutral filter elution . In agreementwith their colony-forming ability, ataxia-telangiectasia cells (AT2BE) andnormal fibroblasts exhibited similar dsb rejoining capacity following a-irradiation, but showed marked differences in the rejoining kinetics of dsbinduced by y-rays or bleomycin .Indexing terms : dsb rejoining, ataxia telangiectasia, ionizing radiation, bleomycin .

1 . IntroductionCells from ataxia-telangiectasia (A-T) patients are abnormally sensitive to

ionizing radiation and to bleomycin . This has been shown by colony survival as wellas by cytogenetic methods (Lehman and Stevens 1979, Paterson and Smith 1979,Taylor et al . 1975, 1979) . The high frequency of chromosome aberrations in A-Tlymphocytes after irradiation or bleomycin treatment suggests that unrejoined ormisrepaired breaks are one type of damage responsible for the enhanced cell killingby these agents (Taylor et al . 1976, Taylor 1978, Natarayan and Meyers 1979) .

However, measurements of the breaks by classical sucrose centrifugation(Vincent et al . 1975, Paterson et al . 1976, Lehman and Stevens 1977), by alkalinefilter elution (Fornace and Little 1980, Hariharan et al. 1981) or by fluorometricanalysis of DNA unwinding (Thierry et al. 1985) have not demonstrated an impairedproficiency of A-T cells to rejoin DNA strand breaks . Nevertheless, this does notrule out the possibility that a small subclass of breaks remains unrejoined eventhough the majority of strand breaks are normally rejoined (Taylor 1978, Coquerelleand Weibezahn 1981, van der Schans et al. 1983). This hypothesis is supported bythe work of Cornforth and Bedford (1985) who investigated the breakage andrejoining of prematurely condensed chromosomes in the G1 phase and could showthat after a dose of 6 Gy the fraction of unrejoined breaks is five to six times greater inA-T cells than in normal cells .

In an earlier study (Coquerelle and Weibezahn 1981) we measured the rejoiningof y-induced double strand breaks (dsb) in three different A-T strains by neutralfilter elution and found a difference in the rejoining capacity in the AT2BE strainonly. To analyse this defect in more detail AT2BE fibroblasts have been exposed toagents inducing various kinds of DNA breaks : a-particles, bleomycin and y-rays .

t Dedicated to Professor K. G. Zimmer on his 75th birthday .

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2. Materials and methods2 .1 . Chemicals

[2- 14C]Thymidine with a specific activity of 1 .85-2 . 22 GBq/mmol (50-60 mCi/mM) was purchased from Amersham . Bleomycin was a gift from the Fa .Heinrich Mack, Illertissen, Bavaria .

2.2 . Cell cultures and cell labellingThe normal skin fibroblasts were derived from a normal child and obtained from

Dr R. Eife, University of Munich . The fibroblasts AT2BE (CRL 1343) wereobtained from the American Type Culture Collection Rockville, M .D . Cells weregrown as monolayer cultures in Dulbecco's medium supplemented with 20 per centfoetal bovine serum (Gibco Biocult), 100 units penicillin and 100µg streptomycinper ml. The cell passage number was between 12 and 16 . [2- 14C]Thymidine wasadded to growing cells to a final concentration of 1 .48 kBq/ml for 52 h. One daybefore irradiation the cells were inoculated into Falcon Petri dishes with a diameterof 50 mm at a cell density of 3 x 10 5 cells per dish. For a-irradiation cells were seededinto specially designed dishes consisting of a glass ring and a Melinex foil bottom of6µm thickness .

2.3 . Irradiation proceduresIrradiation with y-rays was carried out with a 60Co source (Gammacell 220,

Atomic Energy of Canada Ltd) at a dose rate of 1 . 5 Gy/min for survival determin-ation and 35 Gy/min or 1 .5 Gy/min for the dsb estimation . Experiments have provedthat the same dose administered with the two different dose rates induced the samenumber of breaks . Before y-irradiation the cells were kept in a cold room for 75 min at4°C to have experimental conditions comparable to those during a-irradiation . Thecells were irradiated as monolayers in Dulbecco's phosphate buffer (pH 7 . 2) on iceand then incubated at 37°C in conditioned medium for the indicated times . a-irradiations were performed with 4 MeV a-particles as emitted by an 24'Am source(Amersham/Buchler) . Dosimetric details have been described previously in detail(Liicke-Huhle et al. 1982) . The energy of a-particles was reduced to 3 .4 MeV at thebottom cell surface, corresponding to 120 keV/µm . By rotating the Americiumsource, a homogeneous dose distribution was achieved . The dose rate was0 . 35 Gy/min. Irradiation took place in a cold room at 4°C. Maximum irradiationtime was 75 min .

2.4 . Bleomycin treatmentFibroblasts were exposed to bleomycin in their growth medium in plastic dishes

for one hour at 37°C. The final concentration of bleomycin was 400 pg/ml . Aftertreatment, the cells were washed twice with ice-cold phosphate buffer to preventrepair and then incubated at 37°C in prewarmed conditioned medium for differentperiods of time .

2.5 . Survival assaySurvival after irradiation was determined by the ability of single cells to form

colonies. Fibroblasts were seeded along with 5 x 104 feeder layer cells in 25 cm2Falcon plastic flasks in numbers yielding about 30 colonies per flask . Afterincubation for 2-3 weeks, with a medium change at the 9th day, the plating efficiencywas 40 per cent and 19 per cent for normal and A-T fibroblasts, respectively .

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Feeder layer cells were prepared by exposing the human fibroblasts in suspensionto 40 Gy 60Co y-rays, washing and suspending the irradiated cells in new mediumand plating them into the test dishes I day before the actual experiment .

For the survival after bleomycin, 4 x 10 5 cells were incubated overnight in a20 cm' Petri dish, 1 ml of the appropriate concentration of bleomycin was added andthe cells were incubated for 1 h at 37 °C. After washing with buffered saline, the cellswere trypsinized, appropriately diluted and plated onto the feeder layer in a 25 cm 2Falcon flask .

2.6 . Filter elution techniqueThe method used is a modification of that described by Bradley and Kohn (1979) .

After various times of incubation the medium was removed and the cells were treatedwith 0 .25 per cent trypsin in 0 . 5 mm Na 2EDTA. An aliquot containing 3 x 105 cellswas deposited on a polycarbonate filter of 2,um pore size (Nuclepore), lysed at roomtemperature for 1 h with a solution (I) of 4 ml 0 .05 M Tris, 0 .05 M glycine, 0 .025 MNa2EDTA, 2 per cent w/v sodium dodecyl sulphate (SDS) and 0 . 5 mg/ml freshlydissolved proteinase K (Merck, Darmstadt) adjusted to pH 9 . 6 . This lysis solutionwas allowed to flow slowly through the filter without suction and was afterwardsmeasured for radioactivity . Then an eluting solution (I I), similar to the solution (I)except that proteinase K was omitted, was gently added, and the DNA was elutedusing a pump rate of 0 .03 ml/min. Ten fractions were collected for 90 min each andmixed with 10 ml Instagel for scintillation counting . The radioactivity of the lysissolution was not taken into account . Radioactivity remaining on the filter wasdetermined after treating the filters with 0 . 5 ml 1 M HCl at 80°C for 1 h and thenneutralizing by addition of 2 .5 ml 0 .4 M NaOH.

The percentage of rejoined DNA was calculated from the mean values of theentire elution profile using the following normalization :

Percentage DNA dsb rejoined=Log (MV irrad . incub . cells/MV irrad . cells)

Log (MV nonirrad. cells/MV irrad . cells)

MV is the mean value of the 10 fractions of 1 elution .

3 . Results3.1 . Colony forming ability after y- and a-irradiation and after bleomycin treatment

The inactivation curve of normal fibroblasts after y-irradiation shows a slightshoulder. The data points (figure 1, open symbols) can be fitted by a linear-quadraticequation (N/NO =exp-(aD+fD2)) giving the values a=0 .655Gy -1 andf=0.036Gy_

2 . Survival after exposure to a-particles decreases exponentiallyyielding a D37 of 0.27 Gy. Comparing doses at the 10 percent survival level (3 .0-Gy yand 0.6 Gy a) gives an RBE value of 5 . 0 for the 3 .4 MeV a-particles . AT2BE cellsshow exponential inactivation after y- and a-rays (figure 1, closed symbols) . TheD37-values are 0 .57 Gy and 0 . 30 Gy and the D10 values 1 .3 Gy and 0 .7 Gy for y- and

a-rays, respectively . The RBE for a-particles in AT2BE cells is thus 1 .9 . Whilenormal and A-T cells show similar sensitivity to a-irradiation, the A-T cells are by afactor of 2 .3 more sensitive to y-irradiation than normal fibroblasts (table 1) .

Survival curves after bleomycin treatment (figure 1 (C)) show that AT2BE cellsare more sensitive to bleomycin than normal cells as already shown for AT3BI andAT5BI strains (Lehman and Stevens 1979, Zampetti-Bosseler and Scott 1985) . The

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1 .0

0 .1

0.01

0 .0011I111II 11 111111 2

3

4

5

6Dose ( Gy)

7 8

1

2Dose (Gy)

1 2 3 4 5

10Bleomycin (pg/ml)

Figure 1 . Survival curves of fibroblasts from normal human donors (open symbols) andAT2BE fibroblasts (closed symbols) after exposure to 60Co y-rays (a), 24 'Am a-particles (b), or bleomycin (c) . Error bars represent the standard deviation from 3 or 4independent experiments .

Table 1 . Sensitivity of normal and A-T fibroblasts to y-rays, a-particles and bleomycin .

D10 RBE SRF

Normal fibroblasts

y-rays

3 .0Gya-particles

0.6 Gy

5.0Bleomycin

2.5 pg/mlAT2BE (AT CRL 1343) y-rays 1 .3 Gy 2.3

a-particles 0.7 Gy 1.9 0.9Bleomycin

0.7 pg/ml

3.6

SRF, Sensitivity reduction factor at 10 per cent survival (D IO (normal strain)/D IO (ATstrain)) .

survival curves are typically concave . When we compare the relative sensitivities ofAT2BE and normal cells to the lethality of bleomycin at 10 per cent survival, thesensitivity reduction factor (SRF) is 3 .6 (table 1) .

3.2 . Induction of dsb in normal and A-T fibroblasts by y- and a-irradiationThe criterion for the existence of a double-strand break in DNA as detected by

neutral filter elution is the change in DNA length which determines the elution ratethrough polycarbonate filters at non-denaturing pH. The rate of DNA elutionincreases as a function of radiation dose, and the percentage of DNA remaining onthe filter decreases exponentially with the dose . The elution rate per unit dose wasnot significantly different between y- and a-irradiated normal fibroblasts (figure 2) .The results obtained with A-T fibroblasts were the same as with normal fibroblasts(table 2) .

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100

c0 80a.cC=

a)Q 60zQ

00N

40C3

cC3aiX

20

40

60

80

100

Dose (Gy)

Figure 2. Double strand break induction in DNA of normal human fibroblasts as a functionof dose of 60Co y-rays (open symbols) or 241Am a-particles (closed symbols) . Errorbars represent the standard deviation calculated from five independent experiments .

Table 2 . Double strand break induction in DNA and AT2BE fibroblasts as a function ofdose of 60Co y-rays and 241Am a-particles .

3 .3 . Repair kinetics of radiation-induced dsb in normal human fibroblastsDsb induced by ionizing radiation are rejoined during post-irradiation incub-

ation at 37°C. The rejoining kinetics depend on the type of radiation . After low LETy-irradiation, a fast restoration of the DNA structure occurs (for details of thekinetics see Bradley and Kohn (1979)) . Within 3 min about 60 per cent of the breakshave been rejoined in normal cells . After high LET a-irradiation, the rejoiningkinetics are totally different . The fraction of rejoined dsb increases linearly withinthe first 30 min (figure 3 and table 3) . After 3 h of post-irradiation incubation 95 percent of the y-induced DSB are rejoined, but only 75 per cent of the a-induced dsb(table 3) .

3.4 . Repair kinetics of radiation-induced dsb in A- TfibroblastsAT2BE fibroblasts are impaired in their rejoining capacity for y-induced dsb

(figure 3, open triangles) . Both the fast and the slow part of the rejoining are affected .Within 3 min only 30 per cent instead of 60 per cent of the dsb are rejoined . Thepercentage of nonrejoined breaks in AT2BE cells after longer incubation is alsohigher by 4-6 per cent than in normal cells (table 2) . The significance of these

Mean values of percentage DNA retainedon filter

Dose (Gy) In y-irradiated cells In a-irradiated cells

10 94.1±0.25 92.3±1 . 220 76-0+4-2 81-3+1-337 .5 67.2±1.8 72.3±0.8

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0 60

0 400

20

3 6

10

20 30

Incubation time in minFigure 3 . Rejoining kinetics of double strand breaks in normal human fibroblasts (circles)

and AT2BE fibroblasts (triangles) after 25 Gy of y-rays (open symbols) or 25 Gy of a-particles (closed symbols) . Error bars represent the standard deviation calculated fromfour independent experiments .

Table 3 . Rejoining of dsb in normal and A-T fibroblasts between 30 min and 3 h incubationtime after irradiation .

Dsb rejoined (per cent)

Repair

Normal fibroblasts

AT2BEincubationtime (h)

a-particles

y-rays

a-particles

y-rays

0. 5

45-6+8-6

_ 84-0+7-9

41-1+9-2

81-0+2-01

35 .8+_ 10 . 9

90.0+1.4

44.4+_ 13 . 3

81 .3+5 . 32

60.6 _+10 . 6

88.0+1.0

63. 1+6 .4

82.9+1 . 33

74.2+8 .7

93 .5+0.9

78. 5+4 .2

89.6+3 .6

differences is proven by the pooled t-test (P<0 .02). AT2BE cells rejoin a-induceddsb with the same kinetics as normal fibroblasts . The rejoining is slow and increaseslinearly with time . The maximal rejoining of 75 per cent is reached after 3 h, longerincubations do not increase the amount of rejoined dsb any further .

3 .5 . Repair kinetics of dsb after bleomycin treatmentIn normal fibroblasts the rejoining kinetics of the bleomycin-induced dsb is

biphasic (figure 4) : at 3 min, 30 min and 3 h after treatment 45, 62 and 77 per cent ofthe dsb are rejoined, respectively . In AT2BE cells 55 per cent of the dsb are rejoinedwithin 3 min . During the following 3 h only 10 per cent more breaks are rejoined,leaving about 35 per cent of the dsb unrejoined. Thus the fast repair of thebleomycin-induced dsb occurs normally in AT2BE cells . The difference betweencell types during the first 30 min of incubation time are not statistically significant .The slow repair mechanism occurs with a lower efficiency after bleomycin treat-ment. This is in contrast to our findings for the rejoining of y-induced dsb . After y-

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10 20 30

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Incubation time in min

Rejoining of dsb in normal and A-T fibroblasts

215

100Qzc 80d'o 60

40

20

180

Figure 4 . Rejoining kinetics of double strand breaks in normal human fibroblasts (opensymbols) and AT2BE fibroblasts (closed symbols) treated with 400 µg/ml bleomycinfor 1 h . Error bars represent the standard deviation calculated from four independentexperiments .

irradiation both, the fast and the slow components of rejoining are affected inAT2BE (figure 3) .

Comparing normal fibroblasts after y-irradiation (figure 3 and table 3) orbleomycin treatment (figure 4) it is obvious that the rejoining of the bleomycin-induced dsb is overall less efficient : about 35 and 15 per cent less dsb are rejoinedduring the first 30 min and 180 min, respectively .

4 . DiscussionExposure of normal and A-T fibroblasts to y-rays, a-particles or bleomycin

enables us to study the rejoining of different types of DNA dsb in cells with normalradiosensitivity and those exhibiting a hypersensitivity to low LET radiation(AT2BE) .

Low LET y-radiation produces DNA breaks as well as many other lesions suchas base or sugar damage, subsequently leading to DNA breaks (see review by vonSonntag et al . (1981)). During normal repair processes it is suggested that thecombination of single strand breaks (ssb) and base or sugar damage is enzymaticallyconverted into dsb (Ahnstrum and Bryant 1982) . y-rays also produce dsb by a directhit mechanism but always together with 25 times more ssb and base damage (Elkind1984). Bleomycin, a radiomimetic glycopeptide, induces the release of bases,disruption of phosphodiester bonds and a destruction of the sugar moiety resultingin ssbs and dsbs (Muller and Zahn 1977). On the other hand, high LET radiationgenerates dsb by massive local destruction . Since the damage, however, is verylocalized the induced changes in DNA configuration might be very different fromthose created by y-rays .

In our work the elution rate per unit dose is similar for y- and a-irradiated cells .The level of initial dsb is therefore similar for both radiation types . This is in contrastto the results of Cole et al. (1975), Van der Schans (1983) and Bryant and Blucher(1980) . We do not have any explanation for this discrepancy .

With respect to colony-forming ability normal human fibroblasts show a higher

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RBE value for a-particles (RBE=5 .0) than radiosensitive A-T cells (RBE=1 . 9) .The differences in radiosensitivity between the two cell lines decreases with the highLET radiation . This is in agreement with earlier findings (Cox 1982, Liicke-Huhleet al. 1982, Tobias et al. 1984) and indicates that the repair of damage caused by highLET radiation in A-T cells equals that in normal cells (Van der Schans et al . 1983) .The dsb rejoining data in the present paper support this hypothesis . The similarlinear rejoining kinetics after a-irradiation (figure 3) in normal and A-T cellsindicates that the two strains are similar in their repair capacity for dsb resultingfrom densely ionizing radiation . The interpretation of the different repair efficiencyof y-ray induced dsb is more complicated . From the fact that AT2BE cells rejoin,partly, a-ray induced, y-ray induced and bleomycin-induced dsb, we conclude thatthe repair enzymes involved are present, but that the rate of repair is slower duringthe initial period of the biphasic kinetic of dsb rejoining and less extensive afterbleomycin treatment than for normal fibroblasts .

Cox and coworkers (1984) postulated a disturbed equilibrium between ligationand exonuclease digestion of DNA scissions in A-T cells to explain their resultson repair of defined dsb in plasmid DNA by the action of specific restrictionenzymes. If we assume that y-ray- and bleomycin-induced dsb have overlappingsticky ends, while a-induced dsb are characterized by massive local destruction,differences in a nuclease activity would mainly concern the damage caused by y-raysand bleomycin and thus explain our dsb rejoining data . We cannot, however, atpresent, explain why only in the AT2BE strain dsb rejoining Of 7-induced dsb isimpaired and not in the other A-T complementation groups (Coquerelle andWeibezahn 1981), although all cell lines show hypersensitivity for cell killing .

Mitchel et al . (1984) reported on the failure of heat to sensitize A-T cells againsty-radiation as found for normal fibroblasts . They hypothesized that in normal cellsheat treatment inactivates a process which is already defective in A-T lines . Since itis known that heat has no synergistic effect when combined with high LET radiation(Gernet and Leigh 1977) but inhibits the rejoining of dsb induced by X-rays(Dikomey 1982, Radford 1983) the data demonstrate, like ours, a difference betweenlow LET-induced dsb and high LET-induced dsb with respect to repair in A-Tfibroblasts .

In conclusion our results suggest that AT2BE cells were impaired in the ability torepair y-ray- as well as bleomycin-induced lesions expressed as dsb . Moreover, theresults of both y- and a-rays show that the level of capacity of dsb repair is inaccordance with the D 10 value and suggest that unrepaired dsb might be involved incell lethality .

ReferencesAHNSTROM, G ., and BRYANT, P. E., 1982, DNA double-strand breaks generated by the repair

of X-ray damage in Chinese hamster cells . International Journal of Radiation Biology,41,671-676 .

BRADLEY, M. 0., and KOHN, K. W., 1979, X-ray induced DNA double strand breakproduction and repair in mammalian cells as measured by neutral filter elution . NucleicAcids Research, 7, 793-804.

BRYANT, P. E., and BLOCHER, D., 1980, Measurement of the kinetics of DNA double strandbreak repair in Ehrlich ascites tumour cells using the unwinding method . InternationalJournal of Radiation Biology, 38, 335-347 .

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use

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217

COLE, A., SHONKA, F ., CoRRY, P., and COPPER, W . G ., 1975, CHO cell repair of single-strandand double-strand DNA breaks induced by gamma- and a-radiations. MolecularMechanisms for Repair of DNA, (edited by P. C. Hanawalt and R . B. Setlow (NewYork: Plenum Press), Part B, pp . 665-676 .

COQUERELLE, T. M., and WEIBEZAHN, K . F ., 1981, Rejoining of DNA double-strand breaks inhuman fibroblasts and its impairment in one Ataxia telangiectasia and two Fanconistrains . Journal of Supramolecular Structure and Cellular Biochemistry, 17, 369-376 .

CORNFORTH, M. N., and BEDFORD, J . S., 1985, On the nature of a defect in cells fromindividuals with ataxia-telangiectasia . Science, 227, 1589-1591 .

Cox, R., 1982, A cellular description of the repair defect in ataxia telangiectasia . Ataxia-Telangiectasia, edited by B . A. Bridges and D . G. Harnden (Chichester: Wiley), pp .141-153 .

Cox, R., MASSON, W. K., DEBENHAM, P. G., and WEBB, M. B. T., 1984, The use ofrecombinant DNA plasmids for the determination of DNA-repair and recombinationin cultured mammalian cells . British Journal of Cancer, 49, Suppl. VI, 67-72 .

DIKOMEY, E., 1982, Effect of hyperthermia at 42 and 45 °C on repair of radiation-inducedDNA strand breaks in CHO cells . International Journal of Radiation Biology, 41,603-614.

ELKIND, M. M., 1984, Repair processes in radiation biology . Radiation Research, 100,425-449.

FORNACE, A. J., and LITTLE, J. B., 1980, Normal repair of DNA single-strand breaks inpatients with ataxia telangiectasia. Biochimica Biophysica Acta, 607, 432-437 .

GERNET, E. W., and LEIGH, J . T., 1977, Interaction of hyperthermia with radiations ofdifferent linear energy transfer . International Journal of Radiation Biology, 31,283-288 .

HARIHARAN, P . V ., ELECZKO, S ., SMITH, B. P., and PATERSON, M . C ., 1981, Normal rejoiningof DNA strand breaks in ataxia telangiectasia fibroblast lines after low X-ray exposure .Radiation Research, 86, 589-597 .

LEHMAN, A. R ., and STEVENS, S ., 1977, The production and repair of double-strand breaks incells from normal humans and from patients with ataxia-telangiectasia . BiochimicaBiophysica Acta, 474, 49-60.

LEHMAN, A. R., and STEVENS, S ., 1979, The response of ataxia telangiectasia cells tobleomycin . Nucleic Acids Research, 6, 1953-1960 .

LUCKE-HUHLE, C ., COMPER, W., HIEBER, L., and PECH, M ., 1982, Comparative study of G2delay and survival after 241Am-a and "Co-gamma-irradiation . Radiation and Environ-mental Biophysics, 20,171-185 .

MITCHEL, R. E . J ., CHAN, A., SMITH, B. P., CHILD, S. D., and PATERSON, M . C., 1984, Theeffects of hyperthermia and ionizing radiation in normal and ataxia telangiectasiahuman fibroblasts lines . Radiation Research, 99, 627-635 .

MULLER, W . E. G., and ZAHN, R. K., 1977, Bleomycin, an antibiotic that removes thyminefrom double stranded DNA . Progress in Nucleic Acid Research and Molecular Biology,20,21-57 .

NATARAJAN, A. T., and MEYERS, M ., 1979, Chromosomal radiosensitivity of ataxia telan-giectasia cells at different cell cycle stages . Human Genetics, 52, 127-132 .

PATERSON, M. C ., and SMITH, P . J ., 1979, Ataxia telangiectasia: An inherited human disorderinvolving hypersensitivity to ionizing radiation and related damaging chemical .Annual Review of Genetics, 13, 291-318 .

PATERSON, M . C., SMITH, B . P., LOHMAN, P. H . M., ANDERSON, A. K., and FISHMAN, L .,1976, Defective excision repair of gamma-ray damaged DNA in human (ataxiatelangiectasia) fibroblasts . Nature, 260, 444447 .

RADFORD, I . R., 1983, Effects of hyperthermia on the repair of X-ray-induced DNA doublestrand breaks in mouse L cells . International Journal of Radiation Biology, 43, 551-557 .

TAYLOR, A . M. R., 1978, Unrepaired DNA strand breaks in irradiated ataxia telangiectasialymphocytes suggested from cytogenetic observations . Mutation Research, 50, 40-418 .

TAYLOR, A . M. R., HARNDEN, D . G., ARLETT, C . F., HARCOURT, S . A., LEHMAN, A . R .,STEVENS, S., and BRIDGES, B . A ., 1975, Ataxia telangiectasia : A human mutation withabnormal radiation sensitivity . Nature, 258, 427-429 .

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TAYLOR, A. M. R ., METCALFE, J . A., OXFORD, J .M., and HARNDEN, D . G., 1976, Is chromatidtype damage in ataxia telangiectasia after radiation at Go a consequence of defectiverepair? Nature, 260, 441-443 .

TAYLOR, A. M. R ., ROSNEY, C . M., and CAMPBELL, J . B ., 1979, Unusual sensitivity of ataxiatelangiectasia cells to bleomycin . Cancer Research, 39, 1046-1050 .

THIERRY, D ., RIGAUD, 0., DURANTON, I ., MOUSTACCHI, E ., and MAGDELENAT, H ., 1985,Quantitative measurement of DNA strand breaks and repair in gamma-irradiatedhuman leukocytes from normal and ataxia telangiectasia donors . Radiation Research,102,347-358 .

TOBIAS, C. A ., BLAKELY, E . A., CHANG, P. Y., LOMMEL, L., and ROOTS, R., 1984, Response ofsensitive human ataxia and resistant T-1 cell lines to accelerated heavy ions . BritishJournal of Cancer, 49, Suppl . VI, 175-185 .

VAN DER SCHANS, G. P ., PATERSON, M. C., and CROSS, W. G., 1983, DNA strand break andrejoining in cultured human fibroblasts exposed to fast neutrons or gamma rays .International Journal of Radiation Biology, 44, 75-85 .

VINCENT, R. A ., SHERIDAN, R. B., and HUANG, P. C., 1975, DNA strand breakage repair inataxia telangiectasia fibroblast-like cells . Mutation Research, 33, 357-366 .

VON SONNTAG, C., HAGEN, U., SCHON-BOPP, A., and SCHULTE-FROHLINDE, D., 1981,Radiation-induced strand breaks in DNA : chemical and enzymatic analysis of endgroups and mechanistic aspects . Advances in Radiation Biology, Vol . 9, edited by J. T .Lett and H. Adler (New York : Academic Press), pp . 109-142 .

ZAMPETTI-BOSSELER, F., and SCOTT, D., 1985, The response of normal and ataxia-telangiectasia cells to bleomycin: Relationship between chromosome damage, cell cycledelay and cell killing . Mutation Research, 151, 89-94 .

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