radiation sensitivity of cell strains from families with...

(CANCER RESEARCH 49. 4705-4714. September I. 1989]

Radiation Sensitivity of Cell Strains from Families with Genetic DisordersPredisposing to Radiation-induced Cancer1

John B. Little,2 Warren W. Nichols, Philip Troilo, Hatsumi Nagasawa, and Louise C. Strong

Department of Cancer Biology, Harvard School of Public Health, Boston, Massachusetts 02115 [J. B. L., H. N.]; Merck Research Institute, West Point, Pennsylvania19486 IW. W. N., P. T.]; and The University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030 [L. C. SJ

ABSTRACT

This investigation was designed to test the hypothesis that skin fibro-blasts from patients with genetic disorders characterized by hypersuscep-tibility to X-ray-induced cancer are sensitive to the cytotoxic or clasto-genic effects of X-irradiation in vitro. Cell strains were established from28 specifically ascertained patients from families with nevoid basal cellcarcinoma syndrome, retinoblastoma, or other disorders apparently predisposing to radiation-induced cancer. These included 10 patients with aclear personal or family history of radiation-induced tumors. These cellstrains were examined for the cytotoxic effects of X-irradiation in 3distinct series of separate, blinded experiments, along with a group of 9similarly coded cell bank controls. Cells from 11 of these patients and 6controls were studied for sensitivity to X-ray-induced chromosomal aberrations. Seven of the 37 cell strains were moderately hypersensitive toradiation-induced cell killing; 2 of these were from patients with radiation-induced tumors and 1 was a cell bank control. These results suggest thatsuch isolated cases of hypersensitivity probably do not relate to theunderlying genetic disorder. Overall, the X-ray response of cells fromaffected individuals in this study showed no systematic difference fromthat of cells from nonaffected relatives or cell bank controls for eithercytotoxicity or clastogenicity.

INTRODUCTION

The original observation that skin fibroblasts from individuals with the sun-sensitive disorder xeroderma pigmentosum

were hypersensitive to the mutagenic and cytotoxic effects ofUV light because of a defect in the excision repair pathway forDNA damage gave impetus to the hypothesis that other cancer-prone disorders might also be associated with defective DNArepair (1). Since that time, cells from individuals with mostdisorders associated with an increased susceptibility to cancerhave been surveyed for their sensitivity to the cytotoxic orcytogenetic effects of various DNA-damaging agents. Two au-tosomal dominant conditions are of particular interest, inasmuch as they are both associated with a high frequency ofinduction by X-irradiation of specific types of cancer (2, 3).These are hereditary retinoblastoma and NBCCS.' Patients

with hereditary retinoblastoma are unusually susceptible to theinduction of osteogenic sarcomas by ionizing radiation, whereaspatients with NBCCS may be markedly hypersusceptible to theinduction of basal cell cancers by radiation.

There are several reports in the literature indicating slight tomoderate hypersensitivity to the cytotoxic effects of X-irradiation in skin fibroblast cell strains obtained from certain patientswith these two disorders (e.g., Refs. 4-6). On the other hand,several laboratories have not been able to confirm these findings. Most of these studies have involved the examination of

Received 1/26/89; revised 5/8/89; accepted 5/16/89.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by Grants CA-27925, CA-33624, CA-47542, andES-00002 from the NIH.

2To whom requests for reprints should be addressed.*The abbreviations used are: NBCCS, nevoid basal cell carcinoma syndrome;

EBSS, Earle's balanced salt solutions; MDAH, M. D. Anderson Hospital; D0,

inverse of the slope or dose necessary to reduce survival to 37% on the linearportion of the curve; D|0, initial dose necessary to reduce survival to 10%.

fibroblast cell strains from isolated cases of the disorder, oftenobtained from cell banks.

The present investigation was undertaken to examine thisquestion by studying the cytotoxic and clastogenic response toX-irradiation of cells from specifically ascertained families withthese disorders. Hereditary retinoblastoma and NBCCS werechosen for study because of the known genetic predispositionto specific tumor types and the observed hypersusceptibility toradiation-induced cancers of the same type (2, 3). The casesstudied included those specifically derived from families showing such hypersusceptibility. Four additional patients were studied with other tumors apparently induced by radiation. In allcases, cell strains were received coded and blinded. In mostinstances, the cells were studied at two or three different pointsin time; each time the cells were sent with a new code. The X-ray response of cells from affected individuals with these disorders showed no systematic difference from that of cells fromnonaffected relatives or cell bank controls studied in parallel.

MATERIALS AND METHODS

Patient Ascertainment. Patients were ascertained through the geneticsprogram at the University of Texas M. D. Anderson Cancer Center,with selection for patients with a hereditary predisposition to cancerand the presence of radiation-related tumors. Patients were identifiedfrom systematic surveys (active follow-up of all cases or 3-year survivors) of patients with retinoblastoma (7, 8), NBCCS,4 childhood softtissue sarcoma, and Ewing's sarcoma (9) and from patients referred

specifically for genetic studies with familial and radiation-related cancer. In all instances current medical and family history was obtained bytelephone or in-person interview; reported radiation and/or chemotherapy as well as any history of cancer in patients and their relativeswere confirmed by medical reports.

From the surveys and clinical referrals, all surviving cases withclinical or familial history suggestive of a genetic predisposition tocancer, including those with known chromosomal rearrangements andthose with radiation-related tumors, were identified and invited toparticipate in the study by providing a punch skin biopsy. Multipleaffected and unaffected family members from the same kindred wereincluded, where possible, with the rationale that affected family members should show a common response to irradiation if that responsewere attributable to the common genetic defect and that unaffectedfamily members would serve as ideal controls for other genetic factorsas well as for variation in sample collection or processing. Additionalcases were ascertained from the National Institute of General MedicalSciences Human Genetics Mutant Cell Repository at the Coriell Institute for Medical Research, Camden, NJ, according to the same diagnostic criteria.

The relevant clinical and familial history of the patients and controlsis outlined in Table 1.

Fibroblast Cell Strains. Skin biopsies were obtained in the GeneticsClinic of the University of Texas M. D. Anderson Hospital in Houstonor by local physicians according to the same protocol. The biopsyspecimens were coded (MDAH code numbers) and sent immediatelyto the Coriell Institute for Medical Research, where fibroblast cellstrains were established and the cytogenetic studies were performed.The cell strains were sent to the Harvard School of Public Health where

* Unpublished series.

4705

on July 2, 2018. © 1989 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

http://cancerres.aacrjournals.org/

FAMILIAL CANCER CELL STRAIN RADIATION SENSITIVITY

Table 1 Sources of skin fibroblasl cell strains

CellstrainSourceKindredno.Age

andsexRelationship statusAge

(yr)/diagnosisAge

(yr)/XRT"

(chemotherapy)historyAge

(yr)/XRT-relatedtumorOther

clinical/familialcharacteristicsNevoid

basal cell carcinomasyndrome1'104105106III112113129176MDAHMDAHMDAHMDAHMDAHMDAHMDAHMDAH10410410407307307309110555F24M32M62F65F40F41M4MMotherProbandSiblingProbandSpouseSiblingProbandProbandControl21,BCCface24,

BCCface17,BCCfaceControlControl27,

BCCprimarilyneck,post-auric

ular area;onelesiononface4,

medulloblas-toma,ALL;ribanomalies;??NBCCSNoneNoneNone26,

repeatedlyforBCCReyelid.entire

faceNoneNone6,

repeatedlyforchronicotitis;27.

2500rad4,

craniospinalXRT;3,chemotherapy(before

biopsy)49,

invasiveBCCRorbitalarearequiring

Rorbitalexen-teration27,

invasiveBCCneck,post-au

ricularareaNoneDeceased,

no follow-uppost-XRT,no family history ofanyNBCCS

characteristicsRetinoblastoma

patients with normalkaryotypes002003004008037015018066CM

1880MDAHMDAHMDAHMDAHMDAHMDAHMDAHMDAHCIMR0860850400900900270041021181IF12M26M23FNewborn,

M17M24

M12M37FProbandProbandProbandProbandSiblingProbandProbandProbandJ/i2,

B-RTB spo

radic'/i

2, B-RTB spo

radic'%2,

U-RTBCfamilial3/i2,

B-RTB famil

ialOffspringVi

2, U-RTBr fa

milialViz,

B-RTB sporadic"/,2,

B-RTB famil

ial2,

U-RTB1

, 4000 radLeye;10,chemotherapy;12,

XRT,chemotherapy'/i

2, 3500 radReye;

2,4000radReye25,

chemotherapy;26,chemotherapyVi2,

4000 radReye,XRT+chemotherapyL

eye;Vi2XRT,chemo-R

eyeControlNoneViz,

3400 radLeye,3000radR

eye2Â°/i2,4000 radReye,

L temporal area,Llateral

neck;2-9,chemother

apyNone

by age 211

2, OS nasopharynx24.

STSperi-pharyngealspace;25,

scalpleio-myosarcomaNoneNone

by age3312,

MFHLmandibleOS,

R distal femur age10Melanoma

chest, age22;Deceasedsibling with B-RTBatage

2/i2;treated with 3500radsReye; developed OS, R zy

goma at age 12.Deceasedsibling had B-RTBatage

'7i2, treated 5000 rad L orbit, developed STS Rnasalfossa

age 14. Father withU-RTBage 2, living and wellat62.Unaffected

by RTB age9.FamilialRTB; no one withSMNby

ages of 30-42. Had sonatage31 with B-RTB,died.Biological

mother reportedwithRTB;patientadopted24,

Paraovarian cyst; 29, multiple uterineleiomyomataRetinoblastoma

patients and relatives with abnormalkaryotypes059060110MCGCGM2718GM5877MDAHMDAHMDAHMDAHMDAHCIMRCIMROSO''08008008008061M37FIM28M31M7F2FUnaffectedcarrierAffectedAffectedUnaffectedUnaffectedProbandProband4,

U-RTBVi2,

U-RTB;l,pi-neoblastomaU-RTB

sporadic3/>2,

B-RTB spo

radicProbable

treatment?Vi2,

photocoagulation46XY,ins(3;13)(pl2;ql3.1;ql4.5)46XX,del(

13)(q 13. 1q; 14.5); moderate mentalretardation.coarse

facialfeatures46XY,deI(13)(ql3.1ql4.5);fail

ure tothrive46XY,dupins(3;13)(pl2;ql3.1ql4.5)

46XY,dupins(3;13)(pl2;ql3.1ql4.5)

46XX,del( 13)(pter q 12.3;q2 1.2qter)46XX,

del( 13)(pter q 14. 1;q2 1.2qter);negative familyhistorySwing's

sarcoma with XRT-relatedosteosarcoma043052MDAHMDAH19010841F25FProbandProband15,

EWSarcRtibia14,

E W Sarc L ilium1

5, 5400 radRleg15,

6500 rad pelvis39,

OS Rfibula21,

OS L ilium

4706




Table 1 â€”¿�Continued

CellstrainSourceKindredno.Age

andsexRelationshipstatusAge

(yr)/diagnosisAge

(yr)/XRT(chemotherapy)

historyAge

(yr)/XRT-relatedtumorOther

clinical/familialcharacteristics

041 MDAH 045 21F

042 MDAH 026 48F

Sarcoma familial cancer syndrome

Proband l.STS; 31, breastcancer

2. 4500 rad R nasal area

14, OS mandible

Medical genetics referral for familial XRT-relaled cancer

Proband 14, extra medullary, blastogenicgranulocyticleukemia 44.AML

14-18, chemotherapy; 18.1200 rad nasopharynx, sinus,adenoids forchronic tonsillitis, sinusitis

Familial sarcoma, breast cancer,brain tumors, "Li-Fraumenisyndrome"'

Familial XRT-related cancer deceased sibling, treated XRTage 6 for tonsillitis, developedmucoid epidermoid carcinomaR tonsil age 17; cousin withlifetime employment in phosphate mine, multiple myelomaage 57; numerous other familial cancers

Cell bank controls

CM275CM

495G

M730CM

1522CM

1650CM

1652CM

2674CM

4390GM

4260CIMRCIMRCIMRCIMRCIMRCIMRCIMRCIMRCIMR42M419

29M343

45F3dayM165

37F165

1IF29F28F60MProbandParentProbandProbandParentProbandProbandProbandProbandApparently

normal.46XYApparently

normal parentofchild

withlowgrowthhor

mone level,46XYApparently

normal.46XXApparently

normalforeskin.46XYApparently

normal, parentofGM1652,46XXApparently

normal,daughterofGM

1650,46XXApparently

normal,46XXApparently

normal.46XXApparently

normal. 46XY

Â°XRT. X-ray therapy; ALL, acute lymphocytic leukemia; B, bilateral; BCC. basal cell carcinoma; EW Sarc. Ewing's Sarcoma; L, left; MFH, malignant fibrous

histiocytoma; OS, osteosarcoma; R, right; RTB. retinoblastoma; AML, acute myelogenous leukemia; SMN, second malignant neoplasm; STS, soft tissue sarcoma;U, unilateral; CIMR. Coriell Institute for Medical Research.

* All individuals were examined for NBCCS by physical examination and chest, skull, and dental X-rays. In addition to the BCC described above, "affected"

individuals (except 176) had evidence of palmar pits, jaw cysts, various skeletal anomalies including fused or bifid ribs and bridging of the sella turcica. and ectopiecalcifications.

' Clinically diagnosed and treated as unilateral retinoblastoma; however, in genetics survey as young adults two retinoblastoma-like lesions were identified in the"unaffected" eye.

d Strong et al. (7).'Little Ã©tal.(11).

the cytotoxicity assays were carried out. In some instances, the strainswere receded at the Coriell Institute and sent to Harvard along withsimilarly coded cell bank controls. In the case of retinoblastoma andNBCCS, two additional series of experiments were carried out. Eachtime, the cell strains were given a new code at the Coriell Institute andsent to Harvard along with additional coded cell bank controls fromthe National Institute of General Medical Sciences repository. All cellstrains were regularly tested for Mycoplasma infection and found to benegative.

Cell Culture and Survival Assay. The cells were maintained at 37Â°C

in a humidified atmosphere of 5% CO2/95% air with regular mediumchanges. They were grown in Eagle's minimal essential medium (Gibco)

with BBSS, supplemented with 15% fetal bovine serum, 900 ng/literD-glucose, 0.6 mg/liter sodium pyruvate, and 50 Â¿ig/mlgentamicin.Reheis serum Lot Y67604 or Hazelton Lot 12103150 were used in allexperiments as indicated.

The cells were subcultivated at a 1:4 dilution when they becameconfluent. Cells to be used for experiments were suspended by exposurefor 5 min to 0.25% trypsin in calcium- and magnesium-free Earle's

balanced salt solution 3-4 days after subculture when they were inactive growth. The cells were resuspended in complete medium withserum, counted, and reseeded at low density into 3 replicate 100-mmPetri dishes (Costar) in appropriate numbers such that 30-70 microscopic colonies resulted in each dish when the cloning efficiency andtoxicity of the particular treatment were taken into consideration. Inmost experiments, dishes were seeded at 2 different cell densities foreach dose level; the cell number never exceeded 40,000/dish.

The cells were treated with radiation or drug 18-20 h after seedingand then returned to the incubator for 14-21 days to allow for colonyformation. The colonies were stained and those containing more than50 viable appearing cells were scored as survivors. Surviving fractionswere calculated and the mean survival for each dose level pooled from2-6 separate experiments was fitted to a straight line by linear regression analysis. The D0 and the D,o were derived from the resultantsurvival curve. We found that the D10 was the most consistent andreproducible parameter of survival in multiple experiments with thesame cell strain.

Treatment Protocols. Exposure to radiation or drugs was initiated

4707




18-20 h after seeding at low density as described above. X-rays werederived either from a G.E. Maximar X-ray generator operated at 220kV and 15 mA yielding a dose rate to the cells of 80 rad/min or froma 100-kV Phillips industrial generator operated at 9.6 mA and yieldinga dose rate of 78 rad/min to the cells. UV light irradiation was derivedfrom a bank of five G8T5 G.E. germicida! lamps emitting predominantly 254 nm light. These were housed in a specially constructedradiation chamber. Prior to irradiation with UV light, the medium wasremoved from each culture and the cells were rinsed with EBSS.Immediately following exposure at a dose rate of 0.28 J/m2/s, the

cultures were overlaid with fresh medium and returned to the incubator.Both UV and X-irradiation were carried out at ambient temperature.Mitomycin C and A'-methyl-W-nitro-./V-nitrosoguanidine (both fromSigma) were dissolved in serum-free medium and diluted into eachdish. Following exposure in the dark to each drug for l h at 37Â°C,the

medium containing the drug was removed, and the cells were washedtwice with EBSS, then overlaid with fresh medium containing serum,and returned to the incubator for colony formation.

Plateau Irradiation and GÃ¬Block Measurements. For these experiments, cells were allowed to grow to confluence, after which the culturemedium was changed twice at daily intervals and the experimentsinitiated 48 h after the last medium change. These density-inhibited,confluent cultures were irradiated, and the cells were immediatelysubcultured at low density either to measure the surviving fraction bythe standard colony formation assay or to determine the fractionirreversibly blocked in G,. For the latter measurements, the cells wereseeded at low density into 10 replicate 30-mm plastic Petri dishes(Falcon) and incubated continuously with 1 Â¿iCi/mlof ['H]thymidine(specific activity, 27 Ci/nmol) at 37Â°C.At regular intervals up to 72 h

after subculture, one dish was removed and the cells were fixed on thedishes for autoradiography; the labeling indices were determined byscoring 200 cells on each dish. The results are presented as the fractionor percentage of irradiated cells which entered S phase within 72 h ofsubculture, normalized to the fraction of nonirradiated cells of the samestrain which entered S phase over the same time interval.

Spontaneous and X-ray-induced Chromosomal Aberrations. Chromosomal aberrations were examined following irradiation of confluentfibroblast cultures with X-rays generated by a VarÃanClinac linearaccelerator operated at 4 MeV, yielding a dose rate to the cells of 300rad/min. Cultures were grown and maintained as for the survivalexperiments. Replicate cultures were irradiated with 0, 100, 200, or400 rad, and suspensions of 106 irradiated cells were subcultured intoappropriately labeled 75-cm2 flasks (Lux). Cultures were incubated for

24 h and then were refed and reincubated for 20 h, at which time 0.25MgVelban (Gibco) was added to each flask to arrest cycling cells inmitosis. Metaphases were collected over the next 4 h for a totalincubation period of 48 h. Cultures were then harvested and fixed usinga method previously described by Nichols et al. (10). Slides wereprepared, dried on a 56Â°Chot plate, and stained in 4% Giemsa (Harleco)

for 5-7 min. Nonirradiated control cultures were handled in an identicalfashion.

Metaphases were chosen as appropriate for scoring on the basis ofquality in spreading, staining, and general morphology. Only cells with44-46 chromosomes were scored. Aberrations scored consisted ofchromatid breaks, fragments, triradials, quadriradials, chromosomebreaks, acentric fragments, dicentrics, polycentrics, rings, and abnormalchromosomes resulting from translocations. Chromatid gaps, isochro-matid gaps, prematurely divided medium group chromosomes, andpulverized chromosomes were recorded but not scored as aberrations.

Table 2 X-ray survival parameters for fibroblast cell strains from nevoid basal cell carcinoma syndrome families

RESULTS

Nevoid Basal Cell Carcinoma Syndrome. Cell strains werestudied from four clearly affected and three nonaffected individuals from three different families with NBCCS, as well asone possibly affected individual (176) with multiple primarytumors and rib abnormalities. Two cell bank control strainswere examined in parallel (1650 and 2674). Three separateseries of experiments were carried out over a 2-year period bythree different investigators to determine the cytotoxic effectsof X-rays in these cell strains. In the first series the cells werereceived with the MDAH code numbers (104-176). In thesecond, they were recoded with Coriell Institute numbers(WN25-WN29) but were otherwise grown in the same serumlot (Reheis Y67604) and treated similarly to the first series. Inthe third series of experiments, the cell strains were againrecoded at the Coriell institute with numbers WN45-50 andWN70-71. The codes were not broken nor the relation betweenthem known until all of the experiments in a particular serieswere completed and the results were tabulated. Cell strainsfrom cancer family members were all studied at very similarpassage levels, although passages for the cell bank controls weresomewhat higher.

The results of these experiments are shown in Table 2, inwhich the various code numbers are tabulated as well as theclinical status of the individuals and the mean survival parameters. Data on the cell bank controls examined in this investigation are shown in Table 3. As seen in Table 2, there was nosystematic difference between the cytotoxic response of cellsfrom the affected individuals in the NBCCS families and thosefrom the nonaffected family members and cell bank controls.Only one cell strain (106) showed significantly lower D0 andDIO values than the other cell strains or the usual range ofnormal in our laboratory. Individual survival data for eachexperiment are shown for four of these cell strains in Fig. 1.Although there is a certain amount of unexplained variabilityin some cases, the results were in general remarkably consistentamong experiments carried out by three different individualsover a period of 2 years.

The results of single experiments in which three cell strainsderived from a single family were exposed to UV light, mito-mycin C, and 7V-methyl-./V'-nitro-./V-nitrosoguanidine are shown

in Table 4. These experiments were carried out in parallel withthe first series of X-ray experiments described above. As can be

Table 3 X-ray survival parameters for cell bank control cell strains

CellstrainCM

495CM

1650GM 1652GM 2674GM 4390GM 4260CIMR

No. of(WN) code experimentsPassage9,55,61,

896,28624, 24, 64581,52632

5248-138-119-109-146-711, 16Cloning

efficiency(%)3-132-66-7

5-2020-23

2-12DÂ»

(rad)86135

144102121121D,o

(rad)188290

285272278257

Cell strain(MDAHcode)104105106111112113129176C1MR"

(WN)

code464948,

7126,4725,5029,7027,4574Clinical

statusNonaffectedAffectedAffectedAffectedNonaffectedNonaffectedAffectedAffected

(?)No.

of experiments33654543Passage4-65-75-83-64-74-63-75-10Cloning

efficiency(%)1-612-325-163-213-127-267-125-17D0(rad)1661149515012510814793D,â€ž(rad)280305183275245281310247

' CIMR, Coriell Institute for Medical Research.

4708




1.0

oK O.I

yÂ£C

0.01

at

O.OOI

(105) (106)

1.0

H 0.1O

iru.

IS

5 o.oi

er

0.001

(112) (129)

200 tOO 60O 800 O ZOO 4OO

DOSE (RADS)

600 800

Fig. 1. X-ray survival curves for cell strains from NBCCS family members.MDAH code numbers: 105, 106, 112 (not affected), and 129. Data from 3 seriesof experiments each identified by a different symbol. These were carried out atdifferent times during a 2-year interval by 3 individuals (see text). Cloningefficiencies and mean survival parameters in Table 2.

Table 4 Survival parameters for cell strains from patients with nevoid basal cellcarcinoma syndrome exposed to three DNA-damaging agents

Cellstrain104

105106176Clinical

statusNonaffected

AffectedAffectedAffected (?)UVWDo1.5

1.41.71.9lightD,â€ž4.9

4.04.65.5MNNG"

(eg/ml)D,,0.9

0.80.50.5D,01.0

2.11.91.3MMC(eg/ml)Do0.12

O.IOO.IO0.17D,00.20

0.220.160.41

Â°MNNG, A'-methyl-A''-nitro-W-nitrosoguanidine; MMC, mitomycin C.

seen Â¡nTable 4, cell strains from the affected family membersshowed no evidence of hypersensitivity to any of the threeDNA-damaging agents in comparison with the response of theunaffected family member or of other cell strains studied undersimilar conditions and at the same time in our laboratory (11).

Chromosomal aberration frequencies for four affected, onenonaffected sibling, and one spouse are shown in Table 5. Oneof the affected individuals had received X-ray therapy and asecond had received X-ray therapy and chemotherapy for amedulloblastoma and acute lymphatic leukemia. In neither ofthese was the baseline aberration frequency markedly elevated.When aberration frequencies from the four affected individuals

were compared to those in the four controls and the twounaffected family members, no significant difference was detected.

Retinoblastoma. The results of X-ray survival experimentsfor 8 individuals with hereditary type retinoblastoma and 5members from one family with D deletion retinoblastoma areshown in Table 6. Five cell bank controls (495, 1650, 1652,2674, 4260) and one nonaffected family member (037) werestudied in parallel. These data result from three series of experiments carried out over the period from early 1984 to mid-1988by three different investigators with two different serum lots.The results shown in Table 6 represent the pooled data fromall of these experiments; results for the control strains studiedin parallel are shown in Table 3.

Six of these 19 control and retinoblastoma cell strains appeared somewhat hypersensitive to the cytotoxic effects of X-irradiation (Dn less than 100 rad and Dâ€ž,less than 250 rad).

Table 5 Percentage of cells with radiation-induced chromosomal aberrations forindividual cell strains

Dose of X-rays(rad)*Cell

strain106111112113129176002003004066CM

1880060GM

2718041GM495GM

1650GM2674GM

4260DesignationNBCCSNBCCSNBCCSNBCCSNBCCSNBCCS

(?)RTB*RTBRTBRTBRTBRTBRTBSTSControlControlControlControlStatus"AffectedAffectedNot

affectedNotaffectedAffectedAffectedAffectedAffectedAffectedAffectedAffectedAffectedAffectedAffected0644101038602002722660100142818181616221824208201396228202006229482938C46.1034593646322824SO3242400786474506875878460927876716060887684

a Refer to Table 1 for complete patient histories.* 50-100 cells scored per data point when possible.c Culture not scored owing to poor growth.d RTB, retinoblastoma; STS. soft tissue sarcoma.

Table 6 X-ray survival parameters for retinoblastoma and other cell strains

CellstrainCIMR"(WN)codeNo.

ofexperimentsPas-

Cloning effi- D0sage ciency (%) (cGy)D,â€ž(cGy)Retinoblastoma

(normalkaryotype)002003004008037015018066GM188079.8775,8477,

86,9376,85,8880,829478,83575884653424-84-104-94-84-105-85-95-76-75-2429-534-191-326-788-1924-557-1119-26106111941291201141109995275316186270335269276221220Retinoblastoma

and/or chromosome 13abnormalities059060110MCGC3,538,

54,735,602,51,727,56533533-143-53-53-53-56-262-632-379-2212-219593129111113236216346270238Other

disorders0420430520419091922022234-64-64-64-610-126-1418-2837-46107120113120290266258320

' CIMR. Coriell Institute for Medical Research.

4709on July 2, 2018. © 1989 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from



Five of these were from patients with retinoblastoma or chromosome 13 rearrangement: MDAH 059 and 060, members ofa D deletion retinoblastoma family (059, an apparent balancedtranslocation carrier; 060, an affected 13q deletion retinoblastoma patient) with D0 values of 84 and 98 and D10 values of190 and 225; GM 1880, a hereditary retinoblastoma patientpreviously studied in this laboratory with D0 of 95 and DK>of220; MDAH 004 and 066, with D0 values of 94 and 99 and D,0values of 184 and 221, respectively. The latter two representtwo of the four retinoblastoma cases with a clear personal orfamily history of retinoblastoma and X-ray-induced second

tumors.Overall, however, cell strains from 8 of 13 patients with

retinoblastoma or chromosome 13 rearrangement showed noevidence of hypersensitivity to X-irradiation including 008, apatient with bilateral retinoblastoma and a radiation-relatedsarcoma and a sibling who also suffered both conditions, and110, another 13q deletion retinoblastoma patient cousin to 060.The lack of a consistent abnormal response to radiation in thetwo 13q deletion retinoblastoma patients from the same family(strains 060 and 110) suggests that the slight hypersensitivityobserved in 060 is not specifically related to the 13q deletion.The last sensitive cell strain, GM 495 with D0 of 89 and D!0 of191, was a cell bank control from an apparently normal individual with a family history of low growth hormone level. Thus,although a small degree of hypersensitivity to X-irradiation wasseen in 4 of 10 retinoblastoma cell strains, and one 3; 13rearrangement cell strain, this is clearly not a systematic findingassociated with retinoblastoma, with retinoblastoma and X-ray-related second tumors, or with retinoblastoma and familial13ql4 deletion. As in the case of NBCCS, there was generallygood agreement between experiments carried out by differentindividuals at different points in time. This is evident in Fig. 2in which data from individual experiments are plotted for 4 ofthe sensitive cell strains including the cell bank control strainGM 495.

In order to further examine the response of retinoblastomafibroblasts, a fourth series of experiments was carried out byH. Nagasawa in confluent, stationary phase cultures with theHazelton serum. Four additional cell strains, numbered WN66-WN69 and including two additional, distinct D deletion retinoblastoma and 2 control cell strains, were also examined atthis time. These data are shown in Table 7 where survival curveparameters are shown for cells irradiated in density-inhibited,confluent growth and subcultured immediately after irradiationto assay for colony-forming ability. The results are generallysimilar to those for proliferating cells presented in Table 6.

When human diploid fibroblasts are irradiated in confluenceand then subcultured at low density, the cells move semisyn-chronously into the S-phase with a delay of 14-18 h followingsubculture. However, a dose-dependent fraction of the cellsappears permanently blocked in the G, phase of the cell cycle;these cells never reach the first S phase after irradiation andsubculture. It has been shown previously that the fraction ofcells blocked in G, under these conditions appears to be largerin retinoblastoma as compared with normal diploid fibroblasts(12). The results of such experiments are also shown in Table7 where the data are presented in terms of the fraction of cellswhich entered S phase within 72 h of subculture followingirradiation with either 200 or 400 rad. These results are shownin Fig. 3 where they have been divided into three arbitraryresponse groups. As can be seen in Fig. 3, 7 of the 8 strainsstudied with chromosome 13 abnormalities or with retinoblastoma fell into Group I, the lowest response group (largest

zo

(Eli

eroto

i.o r

ai

0001

1.0

0.1

:060) ( GM495!

A.\ \

a

\:\

o<ir

0.01

irto

0001

0.0001

(004) (066)

O 200 400 600 800 O 200

DOSE (RADS)400 600 800

Fig. 2. X-ray survival curves for cell strains from retinoblastoma familymembers and one cell bank control: MDAH 060, GM 495 (control), MDAH004, and MDAH 066. Different symbols represent data from each of 3 series ofexperiments as described in text. Cloning efficiencies and mean survival parameters in Table 5.

fraction of cells blocked in G,) consistent with previously reported results (12). However, 2 of the 6 apparently normal cellstrains studied (including GM 495) also fell within this responsegroup, and the two strains with the common 13q deletion andretinoblastoma (060 and 110) are discordant for the responsegroup.

The clastogenic response to X-irradiation was examined infibroblasts from seven patients with retinoblastoma (Table 5).This included four patients with bilateral or familial retinoblastoma and two with the D deletion syndrome. Three of theseseven strains (060, GM 1880, and 004) were those that appearedslightly but significantly hypersensitive to the cytotoxic effectsof X-irradiation. In no case was any statistically significantincrease in radiation-induced chromosomal abnormalities detected when compared to controls. For each subject, a logisticregression of percentage of aberrant cells on radiation wascarried out (13, 14). The regression parameters were thencompared using the Kruskal-Wallis test (15).

Other Disorders. These four strains derived from patientswith other radiation-induced tumors also yielded a normalresponse (Table 6).

4710




Table 7 X-ray response ofrelinoblastoma, chromosome 13 alterations, andcontrol strains irradiated during density inhibition of growth

Results are mean of 2-4 separate experiments for each strain.

Cellstrain059060110MCGCCM

1880CM2718GM5877GM275GM495GM

730AG1522GM4260GM

4390CIMR"(WN)code5353,736051,7256576668595569675258Survival

parameters(cGy)Do

D,o97

3141062759223991

22710523411236090

20013634811035594

283Cloningefficiency45-7323-4316-4117-5012-238-199-174064-8232-73Fraction

cellsentering S-phasepost-subculture200cGy0.570.490.900.680.710.650.580.530.790.520.830.950.610.80400cGy0.340.190.750.390.400.390.350.360.580.240.680.800.350.59Responsegroup*IIinnininin

Â°CIMR, Coriell Institute for Medical Research.* See Fig. 3.

DISCUSSION

There has been considerable interest in the hypothesis thatthe increased susceptibility to spontaneous and induced cancerassociated with certain genetic disorders might be related todefective DNA repair. This hypothesis has been tested in vitroby examining the sensitivity of skin fibroblast cell strains isolated from such individuals to the cytotoxic effects of a varietyof DNA-damaging agents including X-irradiation. The rationale for this approach has been that a significant defect in DNArepair should be manifested by an enhanced sensitivity to thelethal effects of such DNA damaging agents. With the exceptionof xeroderma pigmentosum, ataxia-telangiectasia, and Panconianemia cells which are specifically hypersensitive to ultraviolet

100

100 200 300

DOSE (RADS)

400

Fig. 3. Fraction of cells irreversibly blocked in d following irradiation ofconfluent cultures and subcultivation to low density in the presence of |'H]-

thymidine. Results expressed as percentage of similarly treated, nonirradiatedcells of the same strain which entered the S phase within 72 h of subculture. Datapoints 51 to 68 represent Coriell Institute for Medical Research code numbersfor cell strains as shown in Table 6; 730 = GM 730. Data points represent meanof 2 to 4 separate experiments except for 53 which represents a single experiment.Results are grouped by dashed circles into 3 general response groups, referred toas I, II, and III in Table 6 and text.

light, X-irradiation and crosslinking agents, respectively (1,16),the results of these studies have been at times inconsistent butlargely disappointing. Indeed, only in the case of xerodermapigmentosum has a clear repair defect been identified, althoughthe relationship of this defect to the clinical disorder remainsunclear. Recently, a number of investigators have turned theirattention to other end points such as spontaneous or inducedcytogenetic changes or specific gene mutations. Again, thesestudies have not proved very helpful in elucidating mechanismsfor the majority of cancer-prone disorders.

The two autosomal dominant disorders we have chosen,NBCCS and retinoblastoma, offer particularly good models totest this hypothesis as they both represent genetic predisposition to specific tumors and a high incidence of the same tumortypes in radiation-exposed areas, as well as an available predisposed target tissue, fibroblasts. If a relationship does existbetween in vitro sensitivity to X-irradiation and in vivo hyper-susceptibility to X-ray-induced cancer, this phenomenon shouldbe evident from patients with these syndromes. In both instances fibroblasts would seem to represent a relevant "targettissue," inasmuch as fibromas and fibrosarcomas have been

observed in NBCCS patients, and osteosarcomas and soft tissuesarcomas are observed in excess in hereditary retinoblastomapatients, both spontaneously and following X-ray therapy (2,

3).NBCCS patients treated with radiotherapy may develop mul

tiple basal cell carcinomas within the irradiated areas within 6months to some years after treatment; fibromas and fibrosarcomas of the ovary have also been observed (3, 16). This groupof tumors in NBCCS patients occurs in much larger numbersand with a much shorter latent period than do radiation-inducedbasal cell carcinomas or ovarian fibromas and fibrosarcomasthat occur in survivors of other childhood malignancies. Thelesions in radiation-treated patients appear at an earlier age andthe distribution is unlike that found in the syndrome in generalor in nonirradiated affected members of the same families.Thus, the predisposed susceptible target tissue is specified bythe genetic predisposition, but the distribution and age at onsetare affected by the irradiation.

Patients with hereditary retinoblastoma have an increasedrisk of second malignant neoplasms, especially osteosarcomasand soft tissue sarcomas, both in the irradiated area and elsewhere. Overall, the cumulative risk of a second tumor in ahereditary retinoblastoma patient followed for 20 years hasbeen estimated at 8-14% (2, 17). For radiation-treated hereditary retinoblastoma patients, a dose-dependent risk for secondtumors has been observed (17). While the relative risk associated with radiation was similar to that observed for otherradiation-treated childhood cancer patients, the cumulative riskof a radiation-related second tumor was significantly higher.These findings suggest that retinoblastoma patients are at agreater cumulative risk of a radiation-related second tumor inpredisposed target tissue not because of increased susceptibilityto radiation-induced damage but because of the high spontaneous risk of a second malignant neoplasm.

Previous results suggested that there might be a genetic locuson 13ql4 related to increased sensitivity to cell killing in vitroby X-rays that was distinct from but close to the retinoblastomalocus (18). If this is true, discrepancy in reports of X-raysensitivity in retinoblastoma fibroblasts might be explained bygenetic heterogeneity, depending on the region of 13ql4 involved in the retinoblastoma mutation in the specific casesstudied. In the present investigation we examined this possibility by two means: (a) by studying multiple members from the

4711




same family, including one family with a cytogenetic deletion/rearrangement/duplication of 13ql4; and (b) by studying families in which there is clinical evidence for X-ray sensitivityindicated by X-ray-induced tumors in multiple family members.By the careful selection of cases for this study, the findings,which include discordance between affected members of thesame family and lack of consistent findings in patients with X-ray-related tumors, suggest that the isolated instances of in vitroX-ray sensitivity observed are not directly related to the reti-noblastoma or the NBCCS mutation.

There have been scattered reports showing that fibroblastsfrom isolated cases of NBCCS are normal in their cytotoxicresponse to ionizing radiation (5, 19, 20). On the other hand,Chan and Little (6) reported that five NBCCS fibroblast cellstrains were slightly but significantly more sensitive to thecytotoxic effects of radiation than were 6 control strains studiedin parallel. Four of the 6 control strains were derived fromnewborn foreskins. Interestingly, if the mean survival parameters for NBCCS cases reported by Chan and Little (6) arecompared with those derived from the 7 strains studied byFeatherstone et al. (20), the results turn out to be almostidentical. The mean D(lvalues were 98 versus 108 and the meanD,o values were 258 versus 255. The difference between thesetwo studies thus lies in the survival parameters for the normalcontrols; Featherstone et al. (20) found these to be in the samerange as the NBCCS cases whereas Chan and Little (6) foundthe normal survival parameters to be significantly higher. Morerecent results from our laboratory (11), as well as those presented herein in Tables 2 and 3, indicate a broader range forapparently normal strains that would place at least 3 of the 5cases previously reported within the lower limits of normal. Noclear information is available to indicate whether patients inthese previous studies showed clinical hypersensitivity to theinduction of basal cell cancers by irradiation or possessed afamily history of such hypersensitivity.

Cultured fibroblasts from one of the four affected NBCCScases in the present study (MDAH 106) did appear abnormallysensitive to X-irradiation (Table 2), though his affected sibling(MDAH 105) did not manifest the hypersensitivity. Furthermore, a similar degree of hypersensitivity was evident amongone of the controls (GM495; Table 3), whereas fibroblasts fromMDAH patients 111 and 129 with clinical evidence of X-ray-related tumors showed a normal response to X-irradiation.Thus, the significance of the apparent in vitro hypersensitivityof case 106 is not clear. Featherstone et al. (20) reported thatGd-irradiated lymphocytes from patients of NBCCS had slightlybut significantly higher levels of X-ray-induced chromosomalaberrations as compared with normals. Unscheduled DNAsynthesis in lymphocytes from patients with NBCCS has alsobeen reported to be somewhat lower than that found in a groupof healthy controls (21). Generally similar results were reportedin skin fibroblasts by Nagasawa et al. (22). On the other hand,Lehmann et al. (23) found no abnormalities in the response ofthree NBCCS strains to UV light. This latter result is consistentwith the present findings (Table 4).

The results of studies of the in vitro response of cells frompatients with hereditary retinoblastoma have been less consistent. Since the initial reports (4, 18) that fibroblast cell strainsfrom certain patients with retinoblastoma including those withabnormalities of chromosome 13 appeared to be hypersensitiveto the cytotoxic effects of ionizing radiation, considerable interest has developed in studying the response of these cells toDNA-damaging agents in vitro. However, the results have beeninconsistent and at times seemingly conflicting. The findings in

the initial reports were confirmed in several laboratories (5, 24,25). However, several other investigators (26-29) found thatthe radiation sensitivity of hereditary retinoblastoma fibroblastswas not significantly different from that of control strains. Inthe first two of these studies, the negative findings appearrelated in part at least to a low range of normal sensitivitiesfound in their controls.

It is not known which of the patients in these various studiesshowed clinical hypersusceptibility by radiation-induced osteo-sarcoma. In this light, however, the recent study of Woods andByrne (30) is of interest. These authors found no significantdifference in the mean cytotoxic response of 9 hereditary retinoblastoma fibroblast strains to ^-radiation, as compared withthe mean values found in 9 parallel controls. However, whenthe three retinoblastoma cell strains derived from individualswho either developed second cancers or who had a familyhistory of osteosarcoma were analyzed separately, two of thesethree strains were significantly more sensitive than the remainder of the retinoblastoma or normal cell strains and the thirdwas borderline in its sensitivity. Two of five strains from suchpatients were hypersensitive in the present study (Table 5).

No consistent pattern of increased sensitivity has emergedfrom studies of the in vitro sensitivity of retinoblastoma cells tocell killing by other mutagens and carcinogens (31-34). On theother hand, several studies suggest that hereditary retinoblastoma cells may be somewhat hypersensitive to the induction ofcytogenetic damage. These cytogenetic changes include sisterchromatid exchanges (35) and chromosomal damage (36) induced by X-irradiation and sister chromatid exchanges inducedby UV light and mitomycin C (37, 38). In most of these cases,however, the effects were rather small. Indeed, Chaum et al.(39) could find no evidence^for an increase in bleomycin-inducedchromosomal breakage in G2 lymphocytes of hereditary retinoblastoma cases. A similar conclusion was reached by God-dard et al. (40), studying the induction of micronuclei by radiation in fibroblasts, and by Nagasawa and Little (12), whostudied X-ray-induced chromosomal aberrations. Studies of theinduction of gene mutations (25) and transformation to anchorage independence (41) in skin fibroblasts yielded no detectable difference between retinoblastoma and normal cells.Finally, no convincing evidence for a DNA repair defect hasbeen identified in retinoblastoma fibroblasts (42, 43), althoughan unusually large fraction of cells irradiated in confluence andsubcultured to low density appear to be irreversibly blocked inG i (12). Arieti and Priestley (44) reported that fibroblasts fromsome patients with hereditary retinoblastoma had a limitedcapacity for recovery from X-ray-induced potentially lethaldamage in confluent holding experiments. We have not beenable to reproduce this observation convincingly (45, 46).

Taken together, these various results as well as those of thepresent study offer little evidence to implicate cellular hypersensitivity to ionizing radiation or defective DNA repair aspredisposing factors in retinoblastoma. It is possible that arepair defect or other abnormality in DNA metabolism mightoccur spontaneously in isolated cases, unrelated to the centraldisorder itself. The findings in the present report are notinconsistent with this conclusion. Significant hypersensitivityto X-irradiation was evident in 2 of 5 cases from a family withabnormalities of chromosome 13 (MDAH 059 and 060; Table5). However, other affected members of the same family showednormal sensitivity. This finding is similar to that for the nevoidbasal cell carcinoma syndrome shown in Table 2. One of 4affected NBCCS patients studied showed a moderate degree ofradiation hypersensitivity, whereas the remaining affected and

4712




nonaffected individuals from the same family responded normally. The response of the 4 strains from the other patientswith radiation-induced tumors (Table 5) also fell within thenormal range.

The data (Table 6) concerning sensitivity to the G, block aremore difficult to interpret. Four of 5 strains from patients withretinoblastoma fell into response group I (greatest G, block)whereas only 2 of 5 normal strains fell in this group; one ofthese, GM 495, was significantly hypersensitive to the cytotoxiceffect of X-rays. On the basis of these findings, it appearsunlikely that the radiosensitive phenotype related to the GÃ¬block is specifically related to retinoblastoma; rather, it may berelated to some other characteristic of certain fibroblast cellstrains including many retinoblastoma strains.

This investigation was undertaken to determine whether cellsfrom specifically ascertained individuals in families with a predisposition to second tumors, particularly radiation-inducedcancers, show significant abnormalities in their response toradiation in vitro when coded samples were studied over a periodof time by several different investigators in one laboratory. Theresults indicate that affected members of such families cannotbe systematically detected by their response to the cytotoxiceffects of X-rays. Apparently conflicting results among laboratories probably relate to the selection of an appropriately broadrange of normal subjects as controls. Occasional cell strains doshow a minor apparent hypersensitivity to radiation-inducedcell killing, as we observed among certain retinoblastoma,NBCCS, and normal cases. It is intriguing that 2 of 4 retinoblastoma patients with histories of radiation-induced secondtumors fell into this group. On the other hand, this was not thecase for the other patients in this study with radiation-inducedtumors (Tables 2 and 6), and none of those tested for X-ray-induced chromosomal breakage showed an abnormal response.

Overall, therefore, our results suggest that such isolated casesof hypersensitivity probably do not relate to the underlyinggenetic disorder. Furthermore, these findings reinforce ouropinion that studies of the response of somatic cells to DNA-damaging agents in vitro are not a very fruitful approach toimprove our understanding of enhanced susceptibility to spontaneous and induced cancer in most genetic disorders or to thedetection of at risk individuals in most families with geneticdisorders predisposing to cancer.

ACKNOWLEDGMENTS

We thank William Dahlberg, John Nove, Annie Schmidt, HelenVetrovs, and Carole Bradt for expert technical assistance. We alsothank Keith Soper for statistical evaluation of the data on radiation-induced chromosomal aberrations.

REFERENCES

1. Little, J. B. Gene-environment interactions in cancer etiology. In: R. S. K.Chaganti and J. L. German (eds.). Genetics in Clinical Oncology, pp. 7-21,New York: Oxford University Press, 1985.

2. Draper, G. J., Saunders, B. M.. and Kingston, J. E. Second primary neoplasms in patients with retinoblastoma. Br. J. Cancer, S3: 661-671, 1986.

3. Strong, L. C. Genetic environmental interactions. In: D. Schottenfeld and J.F. Fraumeni, Jr. (eds.). Cancer Epidemiology and Prevention, pp. 506-516.Philadelphia: W. B. Saunders Co., 1982.

4. Weichselbaum, R. R., Nove, J.. and Little, J. B. X-ray sensitivity of diploidfibroblasts from patients with hereditary or sporadic retinoblastoma. Proc.Nati. Acad. Sci. USA, 75: 3962-3964, 1978.

5. Arlett, C. F., and Harcourt, S. A. Survey of radiosensitivity in a variety ofhuman cell strains. Cancer Res., 40:926-932, 1980.

6. Chan, G. L., and Little, J. B. Cultured diploid fibroblasts from patients withthe nevoid basal cell carcinoma syndrome are hypersensitive to killing byionizing radiation. Am. J. Pathol., ///: 50-55, 1983.

10

12.

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

Strong, L. C., Riccardi, V. M.. Ferrell, R. E., and Sparks. R. S. Familialretinoblastoma and chromosome 13 deletion transmsitted via an insertionaltranslocation. Science (Wash. DC), 213: 1501-1503, 1981.Strong, L. C., Herson, J., Haas, C., Elder. K.. Chakraborty, R., Weiss, K.M., and Majumder. P. Cancer mortality in relatives of retinoblastomapatients. J. Nail. Cancer Inst., 73: 303-311. 1984.Strong, L. C., Herson, J., Osborne, B. M., and Sutow, W. W. Risk ofradiation-related subsequent malignant tumors in survivors of Ewing's sarcoma. J. Nail. Cancer Inst.. 62: 1401-1406. 1979.Nichols. W. W., Miller, R. C.. and Bradt, C. I. In vitro anaphase andmetaphase preparation in mutation testing. In: B. J. Kilbey. M. Legator. W.Nichols, and C. Ramel (eds.). Handbook of Mutagenicity Test Procedures,pp. 225-233. New York: Elsevier/North Holland. 1977.Little, J. B., Nove, J., Dahlberg, W.. Nichols, W. W.. and Strong, L. C.Normal cytotoxic response of skin fibroblasts from patients with Li-Fraumenifamilial cancer syndrome to DNA damaging agents in vitro. Cancer Res., 47:4229-4234, 1987.Nagasawa, H., and Little, J. B. Comparison of kinetics of X-ray-induced cellkilling in normal ataxia telangiectasia and hereditary retinoblastoma fibroblasts. MutÃ¢t.Res., 109: 297-308. 1983.Mosteller, F.. and Tukey, J. W. Data Analysis and Regression, pp. 102-115.Reading. MA: Addison-Wesley, 1977.Fienberg. S. E. The Analysis of Cross-Classified Categorical Data, p. 85.Cambridge. MA: MIT Press, 1977.Hollander. M.. and Wolfe. D. A. Nonparametric Statistical Methods, p. 115.New York: John Wiley & Sons, Inc.. 1973.Harnden, D. G., Edwards, M., Featherstone. T.. Morten, J., Morgan. R..and Taylor, A. M. R. Studies on cells from patients who are cancer proneand who may be radiosensitive. In: H. V. Gelboin et al. (eds.). Genetic andEnvironmental Factors in Experimental and Human Cancer, pp. 231-246.Tokyo: Japan Scientific Societies Press, 1980.Tucker, M. A., D'Angio, G. J., Boice, J. D., Jr., Strong, L. C., Li, F. P.,

Stovall, M., Stone, B. J., Green, D. M., Lombardi, F.. Newton, W., Hoover,R. N., and Fraumeni. J. F. Bone sarcomas linked to radiotherapy andchemotherapy. N. Engl. J. Med., 317: 588-593. 1987.

Nove, J., Little, J. B.. Weichselbaum. R. R.. Nichols, W. W.. and Hoffman.E. Retinoblastoma. chromosome 13, and in vitro cellular radiosensitivity.Cytogenet. Cell Genet., 24: 176-184, 1979.Weichselbaum. R. R., Nove, J., and Little, J. B. X-ray sensitivity of fifty-three human diploid fibroblast cell strains from patients with characterizedgenetic disorders. Cancer Res., 40: 920-926, 1980.Featherstone, T., Taylor, A. M. R., and Harnden. D. G. Studies on theradiosensitivity of cells from patients with basal cell naevus syndrome. Am.J. Hum. Genet., 35: 58-66, 1983.Ringborg, U., Lambert, B., Landegren. J.. and Lewensohn, R. DecreasedUV-induced DNA repair synthesis in peripheral leukocytes from patientswith the nevoid basal cell carcinoma syndrome. J. Invest. Derm., 76: 268-270. 1981.Nagasawa. H.. Little, F. F., Burke. M. J., McCone, E. F., Targovnik. H. S.,Chan. G. L.. and Little. J. B. Study of basal cell nevus syndrome fibroblastsafter treatment with DNA-damaging agents. In: R. R. Tice and A. Hollaender(eds.). Sister Chromatid Exchanges, pp. 775-785. New York: Plenum Publishing Corp., 1984.Lehmann. A. R., Kirk-Bell. S., Arlett, C. F., Harcourt, S. A., de Weerd-Kastelein, E. A.. Keijzer, W., and Hall-Smith. P. Repair of ultraviolet lightdamage in a variety of human fibroblast cell strains. Cancer Res.. 37: 904-910. 1977.Paterson. M. C., Gentner, N. E., Middlestadt. M. V., and Weinfeld, M.Cancer predisposition, carcinogen hypersensitivity and aberrant DNA metabolism. J. Cell. Physiol. Suppl.. 3: 45-61, 1984.Wang, Y., Parks, W. C., Wigle, J. C., MÃ¤her,V. M., and McCormick, J. J.Fibroblasts from patients with inherited predisposition to retinoblastomaexhibit normal sensitivity to the mutagenic effects of ionizing radiation.MutÃ¢t.Res. 175: 107-114, 1986.Ejima. Y.. Sasaki. M. S.. Utsumi. H.. Kaneko. A., and Tanooka. H. Radi-osensitivily of fibroblasts from patients with retinoblastoma and chromosome-13 anomalies. MutÃ¢t.Res., 103: 177-184. 1982.Kossakowska. A. E.. Gallie. B. L., and Phillips. R. A. Fibroblasts fromretinoblastoma patients: enhanced growth in fetal calf serum and a normalresponse to ionizing radiation. J. Cell. Physiol., ///: 15-20. 1982.Zampetti-Bosseler. F., and Scott, D. Cell death, chromosome damage andmitotic delay in normal human, ataxia telangiectasia and retinoblastomafibroblasts after X-irradiation. Int. J. Radial. Biol.. 39: 547-558. 1981.Morten, J. E. N. Cellular studies on retinoblastoma. Int. J. Radial. Biol., 49:485-494, 1986.Woods, W. G., and Byrne, T. D. In vitro sensitivity of normal and hereditaryretinoblastoma fibroblasts to DNA-damaging agents. Cancer Res., 46:6305-6310. 1986.Fujiwara, Y'., Miyazaki, N.. Kano. Y., Takahashi. T.. and Kaneko. A. X-ray.

UV and chemical mutagen sensitivities of skin fibroblasts from patients withfamilial and chromosome 13q-retinoblastomas. J. Radial. Res.. 22:472-476,1981.Barfknecht. T. R., and Little, J. B. Survival of hereditary retinoblastomahuman skin fibroblasts after treatment with DNA-damaging chemicals. MutÃ¢t.Res., 105: 189-194, 1982.Fabricant, J. D., Au, W., Fabricant, R. N., and Morgan, K. S. SCE and UDSstudies in a family with retinoblastoma: a case study. Teratogen. Carcinogen.Mutagen.. 2: 85-90, 1982.

4713




34. St. Gainer, H. C., and Kinsella. A. R. Analysis of spontaneous, carcinogen- by micronucleus induction in vitro. MutÃ¢t.Res., 752: 31-38, 1985.induced and promoter-induced chromosomal instability in patients with 41. Wang, Y., Kateley-Kohler, S., MÃ¤her,V. M., and McCormick, J. J. MCohereditary retinoblastoma. Int. J. Cancer, 32:449-453, 1983. radiation-induced transformation to anchorage independence of fibroblasts

35. Abramovsky-Kaplan, I., and Jones, I. S. Sister chromatid exchange induced from normal persons and patients with inherited predisposition to retino-by X-irradiation of retinoblastoma lymphocytes. Invest. Ophthalmol. Vis. blastoma. Carcinogenesis (Lond.), 7: 1927-1929, 1986.Sci., 25: 698-702. 1984. 42. Woods, W. G., Lopez, M., and Kalvonjian, S. L. Normal repair of y

36. Morten, J. E. N., Harnden, D. G., and Taylor, A. M. R. Chromosome radiation-induced single-strand and double-strand DNA breaks in retinoblas-damage in G0 X-irradiated lymphocytes from patients with hereditary reti- toma fibroblasts. Biochim. Biophys. Acta, 695:40-48, 1982.noblastoma. Cancer Res., 41: 3635-3638, 1981. 43. Cleaver, J. E., Char, D., Charles, W. C., and Rand, N. Repair and replication

37. Der-Sarkissian, H., Bonaiti-Pellic, C., Briard-Guillemot, M-I.., and /ucker, of DNA in hereditary (bilateral) rctinoblastoma cells after X-irradiation.J-M. Sister chromatid exchanges in patients with retinoblastoma. Cancer Cancer Res., 42: 1343-1347, 1982.Genet. Cytogcnet., 7:73-77, 1982. 44. Arieti, C. F., and Priestley, A. Defective recovery from potentially lethal

38. Takabayashi, T., Lin, M. S., and Wilson. M. G. Ultraviolet light and damage in some human fibroblast cell strains. Int. J. Radial. Biol., 43: 157-mitomycinC induced sisler-chromatid exchanges in fibroblasts from patients 167, 1983.with retinoblastoma. Mutai. Res., /â€¢//.101-104, 1984. 45. Litllc, J. B. Biological consequences of X-ray induced DNA damage and

39. ( h.muÃ.E., Doucette, L. A., Ellsworth, R. M., Abramson, D. H., llaik, B. repair processes in relation to cell killing and carcinogencsis. In: E. C.G., Kit. hm F. D., and C'haganti, R. S. K. Bleomycin-induced chromosome Friedberg, P. C'. llanawalt. and C. F. Fox (eds.), DNA Repair Mechanisms,breakage in G2 lymphocytes of retinoblastoma patients. Cytogcnet. Cell pp. 701-711. New York: Academic Press, 1978.Genet., ÃŒ8:152-154, 1984. 46. Wcichselbaum, R. R.. Tomkinson, K., and Little, J. B. Repair of potentially

40. Goddard, A. D., Hcddlc, J. A., Gallic, B. L., and Phillips, R. A. Radiation lethal X-ray damage in fibroblasts derived from patients with hereditary andsensitivity of fibroblasts of bilateral retinoblastoma patients as determined D-delction rclinoblastoma. Int. J. RadiÃ¢t.Biol.. 47:445-456, 1985.

4714



1989;49:4705-4714. Cancer Res John B. Little, Warren W. Nichols, Philip Troilo, et al. Disorders Predisposing to Radiation-induced CancerRadiation Sensitivity of Cell Strains from Families with Genetic

Updated version

http://cancerres.aacrjournals.org/content/49/17/4705

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/49/17/4705To request permission to re-use all or part of this article, use this link



http://cancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



radiation sensitivity of cell strains from families with...

Documents