In Vitro X-Ray Sensitivity in Ataxia Telangiectasia ...cancerres.aacrjournals.org/content/42/10/3950.full.pdf · tion under hypoxia represents a DNA repair defect ... shape of the

[CANCER RESEARCH 42, 3950-3956, October 1982]

In Vitro X-Ray Sensitivity in Ataxia Telangiectasia Homozygote and

HétérozygoteSkin Fibroblasts under Oxic and Hypoxie Conditions

Timothy J. Kinsella,1 James B. Mitchell, Scott McPherson, Angelo Russo, and Frank Tietze

Radiation Oncology Branch, Division of Cancer Treatment, National Cancer Institute [T. J. K., J. B. M., S. M., A. P.] and National Institute of Arthritis and MetabolicDiseases [F. T.] NIH, Bethesda, Maryland 20205

ABSTRACT

Hétérozygotesfor ataxia telangiectasia (AT) are reported tohave an increased risk of cancer especially prior to age 45years. Cultured skin fibroblasts from some AT hétérozygotesare reported to show increased sensitivity to the lethal effectsof X-irradiation under hypoxia which is intermediate between

the response of AT homozygote and normal control fibroblasts.This in vitro colony formation assay has been suggested as aclinical screen for presumed AT hétérozygotes(relatives of AThomozygotes) to identify individuals at high risk for cancer. Ithas also been speculated that this radiosensitivity under hypoxia may represent a previously unrecognized DNA repairdefect.

We have studied the response to X-irradiation of cultured

skin fibroblasts from three AT families and two normal controlsto further explore these observations. In contrast to the previous report, we did not find any difference in radiation survival(D0) of fibroblasts from obligate AT hétérozygotesin thesefamilies compared to normal control fibroblasts. We reportnormal oxygen enhancement ratios comparing radiosensitivity(D0) under hypoxia to air for the fibroblast cultures of AThomozygotes, hétérozygotes,and normal controls. Additionally, we report no difference in glutathione content of log-phase

cells in AT hétérozygotesand normal control fibroblasts.We conclude that the radiation survival in air and hypoxia of

AT hétérozygoteskin fibroblasts is similar to that of normalcontrol cell lines. Low plating efficiency, variable extrapolationnumbers in air and hypoxia, and cell density effects in somecell lines make it difficult to determine a meaningful oxygenenhancement ratio for these cells. We do not find the responseto X-irradiation under hypoxia to be a useful in vitro marker for

AT hétérozygotes.

INTRODUCTION

AT2 is an autosomal recessive human disease with a reported

frequency of approximately 1 of 40,000 live births (16). Thisgenetic disease has a number of interesting phenotypic manifestations in the homozygote including: a progressive degeneration (or cell loss) within the central nervous system, skin,and blood vessels; an immunological defect of both T- and B-

lymphocyte function; an increased risk of cancer, primarilylymphoma and leukemia; and enhanced normal tissue damagefollowing therapeutic irradiation (8, 16). In vitro work on cultured skin fibroblasts and lymphocytes from AT homozygoteshas demonstrated an increased sensitivity to killing by X-rays

1To whom requests for reprints should be addressed.2 The abbreviations used are: AT, ataxia telangiectasia; OER, oxygen en

hancement ratio; D,O%SF.dose which reduced survival to 10%; GSH, glutathione.Received April 20, 1982; accepted June 29, 1982.

and certain chemicals compared to normal control cell lines (7,10, 15, 18, 19), leading some to suggest that AT is the "X-rayanalog" to xeroderma pigmentosum (10, 16).

AT hétérozygotesusually do not have any of the phenotypicmanifestations found in AT homozygotes, as is characteristicof most autosomal recessive disorders. Cultured skin fibroblasts from AT hétérozygotesoften show a normal survivalresponse to X-irradiation in air, although some cells lines have

shown increased sensitivity (1 ). In 1976, Swift eíal. (17) foundan increased risk of various cancers in AT hétérozygotesbasedon an epidemiological study of 27 AT families. This increasedcancer risk included some epithelial cancers as well as leukemia and lymphoma. In AT hétérozygotesunder the age of 45years, the calculated risk of cancer was 5 times greater thanthat of the normal age-adjusted population. The authors esti

mated that AT hétérozygotesrepresent up to 1% of the population and comprise up to 5% of cancer deaths before age 45.

Based on this epidemiological observation in AT hétérozygotes, Paterson ef a/. (9) studied the response of cultured skinfibroblasts from AT hétérozygotesto X-irradiation under air andhypoxia. Of the 4 families screened in their study, the obligatehétérozygotes(parents of an AT homozygote) in 2 familiesshowed an intermediate sensitivity to killing by X-irradiationunder hypoxia between the AT homozygote and normal controlcell lines. There was no difference to X-ray killing in air betweenthe AT hétérozygotesand normal control cell lines. Their OERs,which compare the relative killing under hypoxia to aeratedconditions, for the AT hétérozygotesin these 2 families werereported to be significantly different from the OERs of theirrespective AT homozygotes and the normal controls. The authors concluded that this enhanced X-ray sensitivity underhypoxia might be an in vitro marker for AT hétérozygotesandmay be of clinical significance as a screen for relatives of AThomozygotes who represent a group at high risk for cancer.They also speculate that the increased sensitivity to X-irradiation under hypoxia represents a DNA repair defect related totheir cancer risk.

This in vitro observation of increased sensitivity to X-irradiation under hypoxia in AT hétérozygoteswas intriguing to us forseveral reasons, (a) In calculating the OER, the authors usedthe D,O%SFinstead of the D0, a standard measurement of invitro radiosensitivity (21 ). Since the D,O%SFvalue is often at theinflection area between the shoulder and linear portion of aradiation survival curve, we questioned the technical reproduc-ibility of the OER calculations. (6) Their OER values for X-irradiation of the AT homozygotes and normal control cell lines(1.5 to 1.8) were considerably less than reported previouslyfor most mammalian cell lines (2.5 to 3.5) (5, 21 ). Since oxygenis well known to be a dose-modifying factor for X-ray killing,we questioned whether the lower OERs for AT homozygotesand normal controls reported by Paterson were artifacts of the

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Radiosensitivity of AT Skin Fibroblasta

methods used for hypoxic irradiation. Hypoxia in their experiments was induced by gassing cells in plastic tubes. Plasticculture vessels contain dissolved O2 which can severely compromise hypoxic conditions (2). (c) Based on reports of increased X-ray sensitivity in some GSH-deficient cell lines (3,4), we questioned whether the purported increased radiosen-

sitivity of AT hétérozygotesunder hypoxia might represent anabnormality in GSH metabolism.

In this report, we present our results of X-irradiation in air

and hypoxia for skin fibroblast cell lines from 3 AT families and2 normal controls. We also present measurements of GSHlevels in both the AT and control cell lines.

MATERIALS AND METHODS

Cell Lines. Skin fibroblast cell lines from 3 AT families and 2 normalcontrols were obtained from the Human Genetic Mutation Cell Repository at the Institute for Medical Research in Camden, N. J. All celllines were grown in F12 (6) medium (Grand Island Biological Co.,Grand Island, N. Y.) supplemented with 10% newborn calf serum, 5%fetal calf serum, penicillin, and streptomycin. Exponentially growingcells were used for all experiments. The AT and normal cell lines werestudied between passages 3 and 10.

Irradiation of Cell Cultures. Cells were plated 24 hr prior to eachexperiment in specially designed 60-mm glass Petri dishes and incubated at 37°in a humidified atmosphere of 5% CO2-95% air. The glass

Petri dishes were completely enclosed except for side arms equippedwith glass ground connections which were used to attach the dishes tovarious gassing arrangements.

For irradiations in air, a set of Petri dishes were gassed with 5%CO2-95% air, sealed, and immediately irradiated at room temperature.

Following irradiation, the cells were removed by trypsin, counted, andplated into 100-mm plastic dishes (Falcon Plastics, Oxnard, Calif.).

Appropriate numbers of cells were plated for each dose point to allowfor at least 50 to 75 colonies/dish. The maximum number of cellsplated per 100-mm dish was 105. Each dose point was plated in

triplicate. The dishes were incubated for 14 to 21 days for colonyformation with fresh medium supplementation at 7-day intervals. Fol

lowing the incubation period, the colonies were fixed and stained, andcolonies were scored as survivors that contained at least 50 cells.

For hypoxic irradiations, another set of the glass Petri dishes wereconnected via glass ground connections to a glass manifold for gassing. This glass gassing system was chosen over using plastic dishes ortubes to eliminate the problem of diffusion of oxygen from plasticvessels (2). The dishes were gassed for 1 hr at 37° with a constant

flow (300 ml/min) of humidified 5% CO2-95% nitrogen (Matheson Gas

Co., East Rutherford, N. J.). Oxygen tensions were measured from theeffluent gas flow with a Thermox probe (Ametek, Inc., Pittsburgh, Pa.).After 0.5 hr of gassing, the oxygen tension had equilibrated to 6 ppm±10% (S.D.). After 1 hr of gassing, the individual dishes were sealedby closing off stopcocks in the sidearms, removed from the manifold,and irradiated at room temperature. Following irradiation, the disheswere opened, cells were plated, and colonies were scored as describedabove. The 1-hr gassing period had no effect on the plating efficiency.

Both the air and hypoxic survival curves were determined on the sameday for each cell line.

The effect of X-irradiated feeder cells on the plating efficiency and

shape of the survival curve were determined for some of the cell linesused. In these experiments, fibroblasts of the same line were irradiated(3000 rads) and then plated along with viable fibroblasts to a total cellconcentration of 5 x 10" cells/100-mm dish.

Cell samples were irradiated using a 6-MeV photon beam from a

Mevatron VI linear accelerator. The dose rate was 200 rads/min.Dosimetry was carried out using a Baldwin Farmer ion chamber connected to a Keithly electrometer system having a direct National Bureau

of Standards calibration. Full electron equilibrium was ensured for allirradiations.

Calculation of Radiation Survival Parameters. A least-squares

regression analysis of the individual dose data points on each cell linewas determined to generate radiation survival curves. At least 2 experiments were conducted for each cell line. The curves are presentedwith dose plotted on a linear scale and surviving fraction on a logarithmic scale. The radiation curve for an AT hétérozygoteor normal controlcell line is characterized by a shoulder at low doses merging into anexponential line at higher doses. The AT homozygote cell lines usuallydo not have a shoulder on the semilogarithmic (log-linear) survival

curve. Conventionally, 2 parameters have been used to describe theradiation survival curve: D0, the slope of the exponential portion of thecurve (the dose required to reduce survival to 0.37 on any survival levelon the exponential portion of the curve); and the Ti or extrapolationnumber, which is a measure of the width of the shoulder of the curveand calculated by extrapolating the straight portion of the curve backto the surviving fraction axis (5). Additionally, the D,O%SFwas calculatedfrom the survival curves using the least-squares regression analysis.

The OER has been calculated for both the AT and normal cell linesusing both the D¡>and D,,, ;u ratios. Since oxygen acts as a dose-

modifying agent, the OER is simply a ratio of hypoxic and aerateddoses needed to achieve the same biological effect and assumes thatthe extrapolation number (h) is not significantly different under air andhypoxic conditions. Since the ñvalues under air and hypoxia variedconsiderably in some of the AT and normal control lines, we have alsocalculated the OER by the method of Pike and Alper (12) whichcomputes the D0 values of both the air and hypoxic curves using acommon ("average") ñvalue.

GSH Determination. Cells in exponential growth were used forevaluation of the GSH levels. Cell monolayers in 25-sq cm flasks weretreated with 2 ml of 0.01 N HCI, maintained at 4°for 10 min, and then

dislodged with a rubber policeman. The fluid was then collected, frozenand thawed twice, and maintained at 4°prior to GSH determination.The GSH content was determined by the 5,5'-dithiobis(2-nitrobenzoic

acid)-GSH reducíase procedure (20). Protein determination was done

by the method of Lowry. The GSH content is expressed as ¡igGSH permg protein.

RESULTS

The pertinent clinical characteristics of the 3 AT families andthe 2 normal controls are presented in Table 1. Skin fibroblast

Table 1

Description of the cell lines

Cell line is designated according to the repository number from the HumanGenetic Cell Repository, Institute for Medical Research, Camden, N. J.

Cell line Age (yr) Sex

Presumed

genotype Phenotype Relation

Family1GM3395GM3396GM

3397Family

2GM3487GM3488GM

3489Family

3AG3058AG3059AG

3057Normal

controlsAG1522GM344013434284137144847<120MMFMMFFMFMM-/-

Affected—/ +Normal—/+Normal-/-

Affected-/+Normal-/+

Normal-/-

Affected—/+Normal—/ +Normal+

/+Normal+/ + NormalProbandFatherMotherProbandFatherMotherProbandFatherMotherNoneNone

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f. J. Kinsella et al.

cell lines from the obligate hétérozygotes(parents) of each ATfamily were available for study. Two normal control skin fibro-

blast lines, commonly used for radiation survival studies, werealso used in our experiments.

Radiation Survival in Air and Hypoxia. Table 2 lists theplating efficiency and the radiation survival parameters thatwere measured from the survival curves in air and hypoxia ofeach cell line using a least-squares regression analysis. Therewas a considerable variation in the plating efficiency of the ATcell lines as well as a wide range in plating efficiency of thesame cell line from one experiment to another. We did not findany significant increase in plating efficiency by the use of afeeder layer as described. Because of the variation in platingefficiency, the survival data have been normalized to 100%survival in order to facilitate comparison of cell lines. Suchnormalization of cell lines with low variable plating efficienciesmay result in difficulty in interpreting and comparing radiationsurvival data.

We were able to obtain reproducible survival data using thecell lines from AT Families 1 and 2 as well as both normalcontrols as shown in Charts 1 to 3. Assuming the variability inplating efficiency for the cell lines from AT Families 1 and 2,the AT homozygotes are more sensitive to irradiation in bothair and hypoxia than either of the AT hétérozygotes(Table 2)(Charts 1 and 2). The radiation survival curves for the AThétérozygotesdo not differ from the normal controls (Chart 3).The survival curves for the AT hétérozygotesand normal controls under both aerated and hypoxic conditions show smalland variable shoulders with ñvarying from 1.05 to 2.77.

Radiation survival data from AT Family 3 are shown in Chart4. Only one obligate hétérozygotecell line, AG 3057, yieldedadequate survival data. The OER values for this line are withinthe normal range (Table 3). The AT homozygote AG 3058 hada plating efficiency of <1 % and yielded essentially no survivorsto irradiation despite 4 separate experiments with and withoutX-irradiated feeder layers. The other obligate hétérozygotecellline, AG 3059, shows considerable variation in radiation survival as shown in Chart 3. There are 2 data points plotted foreach radiation dose for exposures in air and for hypoxic conditions. These data points represent the maximum-minimum

survival obtained for each dose point when viable cell concentrations per dish were varied by a factor of 4. The 2 dashedand 2 so//d lines therefore represent the maximum-minimumsurvival curves for irradiations in air and hypoxia, respectively.The Do of each curve is indicated in Table 2. When X-irradiatedfeeder cells were utilized using total cell concentrations of 5x 10" cells/dish for all dose points, such dramatic variation in

survival was not observed. Thus, for this AT cell line, the viablecell density per dish was a major variable of radiation responsein air and hypoxia, resulting in OER values from normal tomarkedly abnormal (Table 3).

The OERs using both the D0 and Oi0%sFvalues are presentedin Table 3. Both AT homozygote and hétérozygotecell lineshave OER values within the reported normal range for mammalian cells (OER, 2.5 to 3.5) (5, 21). To account for thedifference in ñunder air and hypoxia in some cell lines (Table2), the OER was also calculated assuming an "average" ñ

according to the method of Pike and Alper (12). Again, theredoes not appear to be any difference in OER comparing the ATcell lines in Families 1 and 2 to the normal controls.

The GSH levels of the AT and normal control cell linesmeasured in exponential growth are given in Table 4. We couldnot distinguish any difference in GSH levels in the AT homozygotes and hétérozygotes.Many GSH values in the AT celllines overlapped our normal control lines (3.5 to 4.0 jug GSHper mg protein).

DISCUSSION

The purpose of this study was to test the observation byPaterson ef al. (9) that cultured skin fibroblasts from AT hétérozygotesshowed increased sensitivity to killing by irradiationunder hypoxia. The clinical implication of this observation wasthat this technique might serve as an in vitro marker at AThétérozygotes,a group believed to have a high risk for cancer(17). Additionally, the in vitro radiosensitivity under hypoxiamight reflect a DNA repair defect related to carcinogenesis.

Unfortunately, our results using established skin fibroblastlines in 2 AT families do not confirm the observation by Pater-son ef al. (9). This difference in in vitro results may be ac-

Table 2

Radiation survival parameters of the AT and normal control cell lines

CelllineFamily

1GM3395GM

3396(H)aGM

3397(H)Family

2GM3487GM

3488(H)GM3489(H)Family

3AG3058AG

3059(H)AG3057(H)Normal

controlsAG1522GM3440%

of plating efficiency(range)5.0-173.5-1012-164-61-42-65.4-8.52-3.57-1613-16ñ1.201.631.142.721.971.171.051.551.86IrradiationO«

(rads)44859646111114104-157113118102in

airD,o%sF(rads)110230235150350275240-400265318294Irradiationunderhypoxiañ1.052.771.281.351.901.021.222.722.30D„

(rads)143217322143401313202-P52261265257D,o%SF(rads)330710795370920725470-850650870800*

H, AT hétérozygote.

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Radiosensitivity of AT Skin Fibroblasts

10 c

10200 400 600 800 1000

Dose(rad)

io'

IC8it

I

10

10"

GM 3396(H)

k

B

10'

10-

10-

10'

GM 3397(H)

200 400 800 800 1000 1200 1400

Dose(rad)

200 400 eoo eoo loco

Dose(rad)

1200 1400

Chart 1. Radiation survival curves in air (•,A, •)and under hypoxia (O, A, D) for Family 1 comparing the radiation response of the homozygote (GM 3395) andboth obligate hétérozygotes(GM 3396, GM 3397).

counted for by several reasons. First, AT cell lines often havea low plating efficiency, often less than 10% and at times aslow as 1%. There also is considerable variation in platingefficiency from experiment to experiment using the same cellline. We know of no study which correlates consistent radiationsurvival parameters with variable plating efficiencies, especiallylow variable plating efficiencies. In essence, experiments on

AT cell lines reflect only a minority (1 to 10%) of the total cellpopulation. One may question the usefulness of a cell systemin which 90 to 99% of the cells are dead before experimentaltreatment. Does the survival response of the 1 to 10% ofsurvivors represent the response of the total population? Wefeel that considerations such as these should be seriouslyreckoned with before using the in vitro radiation survival re-

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7. J. Kinsella et al.

I

10

10"

10

10"

10

GM 3487

200 400 600 800 1000

Dose (rad)

- 10

10

10

- 10

- 10-

10

GM 3489(1-1)

200 400 600 800 1000 1200 1400

Dose(rad)

200 400 600 800 1000 1200 1400

Dose(rad)Chart 2. Radiation survival curves in air (•,A, •)and under hypoxia (O, A, G) for Family 2 comparing the radiation response of the homozygote (GM 3487) and

both obligate hétérozygotes(GM 3488, GM 3489).

sponse of AT lines as a screen for presumed hétérozygotes.Second, we do not feel that the DIO%SFis an appropriate

measure of radiosensitivity especially when comparing celllines with variable extrapolation numbers. The DIO%SFoften liesat the inflection area of the radiation survival curve and issubject to experiment fluctuation. In the Paterson study usingDIO%SF,the AT hétérozygotesin 2 of the 4 families screened

showed increased sensitivity to hypoxic irradiation. This increased sensitivity was not evident in their study, however,using the more accurate measure of radiosensitivity, the D0. Inour study, the radiation response of AT hétérozygotesunderhypoxia was similar to our normal controls using either O0orDio%SF.

Third, the OER values in the Paterson report were consis-

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Radiosensitivity of AT Skin Fibroblasts

10

10'

10'

GM 3440 10

10"

10"

10

10'

AG 1522

B200 400 600 BOO 1000 1200 1400 1600 200 400 600 8OO 10OO 120O 1400 1600

Dose (rad) Dose (rad)Chart 3. Radiation survival curves in air (•,A ••) and under hypoxia (O, A) for 2 normal control skin fibroblast lines.

10'

•I"'

£1 io-

10'

AG 3057(H)10

10'

10"

10

10

AG 3059(H)

BZOO 400 6OO 800 1000 1200

Dose(rad)

200 400 600 800 1000 1200

Dose (rad)Chart 4. Radiation survival curves in air (•.A. •»)and under hypoxia (O, A, D, O) for the obligate hétérozygotesof Family 3. For AG 3059, the data points in

air (•)and hypoxia (A) represent the maximum-minimum survival at each dose when the viable cell concentrations per dish were varied by a factor of 4 on 2 separateexperiments. and , the maximum-minimum survival curves for irradiations in air and hypoxia, respectively.

tently below 1.75 which is considerably lower than previousreports using mammalian cell lines. We have already noted thedifficulty in using plastic dishes or tubes to perform hypoxicirradiation (2). On the basis of the low OER values of Patersonfor the AT homozygotes and controls (1.5 to 1.75), we can onlyconclude that irradiation was performed under technically inadequate hypoxic conditions. Indeed, our values for the OERusing the D0, Di0%sF and r7au(Table 3) show a range of 2.5 to

3.5 for both AT homozygotes and hétérozygoteswhich isconsidered to be normal (14). Additionally, we found that viablecell density per dish may be an important variable in theradiation response in some AT cell lines.

Based on our findings of a normal response of most AThétérozygotecell lines to hypoxic irradiation, it is not surprisingthat the GSH content in exponentially growing cells does notdiffer from AT homozygote and normal control cell lines. Al-

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Table 3

OEfls of the AT and normal control cell lines using D,AD,o% SFand n„

CelllinesFamily

1GM 3395GM 3396 (H)°

GM 3397(H)Family

2GM 3487GM 3488 (H)GM 3489(H)Family

3AG 3058AG 3059 (H)AG 3057(H)Normal

controlsAG 1522GM 3440DER

Do3.26

2.563.353.12

3.602.751.29-2.42

2.312.25

2.51OERD,o%3F3.00

3.093.382.47

2.632.641.18-3.54

2.453.28

2.72OER

n.v3.07

2.853.192.64

2.552.692.422.63

2.65*H, AT hétérozygote.

Table 4

GSH content of AT and normal control cell lines measured during exponentialgrowth

CelllineGM

3395GM 3396GM 3488GM 3489GM 3057GM 3440GenotypeHomozygote

HétérozygoteHétérozygoteHétérozygoteHétérozygoteNormalGSH

(¿ig/mgprotein)2.72±0.17a

2.96 ±0.293.43 ±0.073.16 ±0.093.76 ±0.313.59 ±0.20

8 Mean ±S.D.

though there is some evidence that the increased radiosensi-

tivity of AT homozygote cell lines reflects an inability to repairdirect DNA damage (11,13), further study of indirect effects ofionizing radiation mediated via free radical formation is underinvestigation in our laboratory.

We conclude that the radiation survival of AT hétérozygotecell lines in air and hypoxia is similar to normal control celllines. Furthermore, due to the variability in the extrapolationnumbers (ñ)of the AT hétérozygotesurvival curves in air andhypoxia, it is difficult to determine a meaningful OER. Possiblefactors contributing to this difficulty include a low plating efficiency of the cell lines, cell density effects, the technique ofhypoxic irradiation, and a poorly understood DNA repair defect.We do not find the OER to be a useful in vitro marker for AThétérozygotes.

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1982;42:3950-3956. Cancer Res   Timothy J. Kinsella, James B. Mitchell, Scott McPherson, et al.   Conditionsand Heterozygote Skin Fibroblasts under Oxic and Hypoxic

X-Ray Sensitivity in Ataxia Telangiectasia HomozygoteIn Vitro

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