Hum Genet (1990) 84:532-534

© Springer-Verlag 1990

Cytogenetic characterization of ataxia telangiectasia (AT) heterozygotes using lymphoblastoid cell lines and chronic T-irradiation

Manjula Waghray 1, Sultan AI-Sedairy 1, Pinar T. Ozand 1' 2, and Mohammed A. Hannan t

t Department of Biological and Medical Research and 2Department of Paediatrics, King Faisal Specialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia

Received August 4~ 1989 / Revised November 17, 1989

Summary. Lymphob las to id cell lines (LCLs) derived f rom two patients identified as ataxia telangiectasia (AT) , two obligate A T he te rozygotes and two controls (heal thy subjects with no k n o w n genetic disease or re- lationship to A T patients) were compared with respect to the induct ion of c h rom osom a l breaks by acute and chronic ?,-irradiation. A l though there was a considerable increase in the f requency of ch romosoma l breaks per cell in the LCLs of A T patients resulting f rom acute irradia- tion, the small increase occurr ing in the LCLs of the A T he te rozygotes m a d e it difficult to distinguish them from the controls. Fol lowing chronic ?,-irradiation, however , the f requency of c h rom osom a l breaks per cell in the LCLs of the A T he te rozygotes occupied a significantly distinct posi t ion f rom that of the controls. These obser- vations suggested that the use of chronic irradiation may be a bet ter choice in the cytogenet ic character izat ion of A T heterozygotes .

Introduction

Ataxia telangiectasia (AT) is an autosomal recessive dis- o rder that is character ized by ch romosoma l instability, increased sensitivity to ionizing radiat ion and predis- posi t ion to malignancies (Taylor 1982; Bode r 1985; L e h m a n n et al. 1987). Howeve r , A T heterozygotes are clinically asymptomat ic but show about a six times in- creased risk of developing cancer compared with the normal popu la t ion (Swift and Chase 1983). Moreover , cells f rom A T he te rozygotes have been repor ted to show mode ra t e sensitivity to radiat ion and chemical carcino- gens (Paterson et al. 1979; Shiloh et al. 1982). As A T he te rozygotes may const i tute up to 7.7% of the popula- t ion (Swift et al. 1986), they m a y represent a large pro- por t ion o f cancer p rone individuals (Swift et al. 1987).

Offprint requests" to: M. Waghray

Therefore , various approaches have been used to detect A T heterozygotes (Kidson et al. 1982; Natara jan et al. 1982; Parshad et al. 1985; Pa terson et al. 1985; Naga- sawa et al. 1987) of ten with ambiguous results. We re- por t here the identification of obligate he terozygotes of A T based on the ch romosomal sensitivity in lymphoblas- toid cell lined (LCLs) to chronic ?,-irradiation.

Materials and methods

Cell lines

The experiments reported here were performed on Epstein-Barr virus (EBV) transformed lymphoblastoid cells lines (LCLs) (Neit- zel 1986) derived from two homozygous AT patients, their hetero- zygous parents and two healthy normal subjects. The established LCLs were stored frozen in liquid nitrogen in lml aliquots with 10% DMSO at a cell density of approximately 107/ml. From the frozen stock, cultures were grown in RPMI 1640 medium supplemented with penicillin (100U/ml), streptomycin (100lag/ ml), glutamine (2mM) and 20% heat-inactivated fetal bovine serum, in 25cm 2 tissue culture flasks. These were incubated at 37°C in a humidified (80%) atmosphere with 5% COz, 95% air.

After 7 days incubation, half of the medium from each flask was replaced with fresh medium containing 10% heat-inactivated fetal bovine serum. The same procedure of renewing medium was repeated twice a week until there were enough cells (1 × 10 6 cells/ ml) for storage and experiments.

Irradiation

For acute irradiation, 10 ml aliquots (each with 10 6 cells/ml) of late log phase LCLs were transferred to 25 cm 2 tissue culture flasks and irradiated with a Cs 137 gamma cell 1000 (Atomic Energy of Canada) at a dose rate of 8 Gy/min. Following irradiation, fresh medium was added to each of the flasks, which were then incu- bated at 37°C. After 72 h, the LCLs were harvested for cytogenetic studies.

For chronic irradiation, the LCLs in 25 c m 2 tissue culture flasks (1 × 10 6 cells/ml) were irradiated inside a CO2 incubator at 37°C, in a humidified (80%) atmosphere using a medical Co 6° gamma source (International Neutronics) at a dose rate of 0.0078 Gy/min for 2 to 8 h. Following irradiation, growth medium was renewed as

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in the case of acute irradiation�9 The LCLs were further incubated for 72 h and then harvested for cytogenetic studies�9

Chromosome preparation

Chromosomes were prepared from the irradiated LCLs using the standard protocols (Moorhead et al. 1960; Yunis 1976). Prior to harvesting, the LCLs were mixed well with a Pasteur pipette to break down any clumps. Colcemid was added at a concentrat ion of 0.05 mg/ml for 45 min. Cells were later exposed to 0.075 M hypo- tonic KC1 solution at 37~ for 20 min and fixed in 1 : 3 acetic acid/ methanol fixative for I h. The cells in suspension were dropped on precleaned chilled slides and the chromosomes were trypsin- Giemsa banded.

Chromosome analysis, in each cell line, was performed in trip- licate on coded trypsin-Giemsa banded metaphases (a total of 150) for each experimental point. Breaks and rearrangements (i.e., rings, translocations, inversions, dicentrics) were scored and the karyotypic analysis was performed as defined by the ISCN (1985) nomenclature. Rearrangements were assessed as two breaks per cell. The baseline chromosome breakage for each sample was cal- culated from the untreated culture. The statistical analysis was based on the mean number of chromosomal breaks per cell ob- tained in each cell line. Comparisons of groups of cell lines (AT in- dividuals, A T heterozygotes and normal controls) were performed using the standard t test (Snedecor and Cochran 1980). The stan- dard deviations of the mean were calculated; the significance criterion refers to P < 0.05.

Results and discussion

The frequencies of chromosomal aberrations (i.e., breaks per cell) following acute irradiation in the LCLs derived from the AT homozygotes, AT heterozygotes and the two normal controls are illustrated in Fig. 1A. The spontaneous frequency of chromosomal breaks per cell in each cell line was calculated from the respective un-irradiated controls. The AT homozygotes showed a significantly higher frequency of chromosomal breaks per celt with increasing radiation doses than the normal controls, which showed a very low incidence of similar breaks. Although the AT heterozygotes showed a slightly higher frequency of chromosomal breaks, these were not significantly different from the normal controls (P-->0.1).

Figure 1B shows the plotted data of the frequency of chromosomal breaks per cell in the different cell lines following chronic y-irradiation. The frequency of chro- mosomal breaks per cell was significantly higher in the AT homozygotes than in the normal controls. However, in these experiments, the AT heterozygotes were signifi- cantly distinguishable from the normal controls on the basis of the frequency of chromosomal breaks per cell; the values obtained by using the standard t test also proved to be statistically significant (P < 0.01). The com- parative data obtained with acute and chronic y-irradia- tion thus indicated that chronic irradiation enabled a better separation of the AT heterozygotes from the AT homozygotes and the normal controls.

Natarajan et al. (1982) obtained inconsistent results in the cytogenetic characterization of AT heterozygotes following acute irradiation in both skin fibroblasts and lymphocytes. Parshad et al. (1985) carried out cytoge-

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Fig, 1, A Dose response curves of chromosomal breaks per cell fol- lowing acute radiation; values are plot ted as the mean _+ standard deviation. �9 �9 99641 (control), �9 ....... �9 117846 (control), A - - - A 172600 (ataxia telangiectasia heterozygotes; ATH) , �9 ....... �9 244418 (ATH), [] [] 171840 (ataxia telangiectasia homozygotes; AT) , A- - -A 212526 (AT). B dose-response curves of chromosomal breaks per cell following chronic radiation; values are plotted as the m e a n + standard deviation. O ....... �9 99614 (control), �9 �9 117846 (control), A - - - A 172600 (ATH), �9 ....... �9 244418 (ATH), [] [] 171840 (AT), A- - -A 212526 (AT)

netic studies on skin fibroblast cell lines from 5 AT het- erozygotes and compared them with 6 cell lines from normal controls following acute irradiation. Their data showed a slightly increased frequency of chromatid breaks in the AT heterozygotes but this value was not markedly different from that observed in the AT homo- zygotes.

Paterson et al. (1985) recommended the use of chronic irradiation in cell survival analysis to detect AT heterozygotes in the population. Recently, Hannan et al. (1989, 1990) have shown the usefulness of chronic y- irradiation in demonstrating intermediate radiosensitivity in skin fibroblasts from non-Hodgkin's lymphoma pa- tients, and in a patient with neurofibromatosis, which could not be characterized as radiosensitive using acute irradiation. These observations prompted the present study in which the usefulness of chronic 7-irradiation in

534

the cytogenet ic character iza t ion of A T heterozygotes has b e e n explored. A l though fur ther studies are needed , our p re l iminary data on the cytogenet ic analysis of A T heterozygotes appear to suppor t the idea that chronic 7- i r radia t ion may be a be t t e r choice in dist inguishing them from A T homozygotes and no rma l controls .

Acknowledgements. The authors acknowledge the financial and administrative support from the King Faisal Specialist Hospital and Research Centre for this work under Project No. 87-0013. They express their gratitude to Dr. Edward Rabe and Dr. Ge- neroso Gascon for their contribution toward clinical cases. They also thank Mr. Blake Smith and Mr. David Sigut for technical as- sistance and Ms. Leonida Z. Jimenez for secretarial work.

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