G2 chromosomal radiosensitivity of ataxia-telangiectasia heterozygotes

Chromosomal Radiosensitivity of Ataxia-Telangiectasia Heterozygotes G2

Ram Parshad, Katherine K. Sanford, Gary M. Jones, and Robert E. Tarone

ABSTRACT: Five lines of skin fibroblasts from individuals heterozygous for ataxia-telangiectasia (AT), compared with six cell lines from age-matched normal controls, show a much higher frequency of chromatid breaks and gaps following x-irradiation during the Gz phase of the cell cycle. The magnitude of this difference suggests that G2 chromatid radiosensitivity could provide the basis for an assay to detect A-T heterozygotes. Though clinically normal, A-T heterozygotes share a high risk of cancer with A-T homozygotes and constitute approximately 1% of the h u m a n population. Farther, we propose that G2 chromosomal radiosensitivity, which appears to result from a DNA repair deficiency, may be associated with a genetic predisposition to cancer.

INTRODUCTION

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder associated with a high frequency of malignant neoplasms and distinct clinical symptoms [1], including cerebellar ataxia, oculomotor apraxia, oculocutaneous telangiectasia, susceptibility to sinopulmonary infections, and immunologic abnormalities [2, 3]. Cells from A-T individuals show chromosome instability [4] and unusual sensitivity to ionizing radiation and to DNA strand-cleaving antitumor agents with respect to cell killing and chromosomal damage [5-9]; furthermore, DNA synthesis is less inhibited by x- or -/-irradiation than in normal cells [10, 11]. A-T heterozygotes, though lacking the major clinical features of the syndrome, share a high risk of cancer with the homozygotes [1]. Because these heterozygotes cannot be diagnosed clinically and constitute approximately 1% of the h u m a n population [1], their detection is important for genetic counseling, preventive medicine, and cancer control. Cells from A-T heterozygotes exhibit spontaneous chromosomal abnormalities [4] and, compared with cells from normal individuals and A-T homozygotes, shown an intermediate radiosensitivity with respect to cell killing and reduced DNA replication [12-15] and an intermediate sensitivity to the DNA breaking agent, neocarzinostatin [16]. However, the degree of hypersensitivity reported previously is not sufficient to provide a reliable test for detecting A-T heterozygotes. We have previously shown a relationship between tumorigenicity of cultured

From the Pathology Department, H o w a r d University College of Medicine. W a s h i n g t o n , D.C. {R.P.), and the Laboratory of Cellular a n d Molecular Biology (K.K.S., G.M.J.] a n d Biometry Branch (R.E.T.), National Cancer Institute, Bethesda, MD.

Address requests for reprints to Dr. K. K. Sanford, National Cancer Institute, Building 37. Room 2Do2, Bethesda, MD 20205. Received November 7, 1983; accepted December 21, 1983.

163 ~; 1985 by Elsevier Science P u b l i s h i n g Co., Inc. 52 Vanderbilt Ave., New York, NY 10017

Cancer Genetics a n d Cytogenetics 14, 163 168 (19851 0165-4608/85/$03.30

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cells w h e n implanted in vivo and e n h a n c e d chromatid damage i n d u c e d by x-irradiation or fluorescent light during the late S or G2 phases of the cell cycle. Both mouse and h u m a n cells transformed to malignant neoplastic cells in culture, as well as cell lines from h u m a n tumors, showed a significantly higher incidence of chromatid breaks and/or gaps than did normal cells when irradiated during late SG2 or G2 [17-20]. When compared to cells from normal donors, skin fibroblasts from i n d i v i d u a l s with genetic disorders predisposing to a high risk of cancer, i n c l u d i n g A-T, Bloom's syndrome, familial polyposis, Fanconi's anemia, Gardner's syndrome, and xeroderma p i g m e n t o s u m (complementation groups C, E, and variant), also exhibited a significantly higher incidence of G2 radiation-induced chromatid damage, but to a lesser extent than in the malignant neoplastic cells [6, 21, 22]. The results of these studies implicate deficiency in DNA repair as a basis for the genetic predisposition to cancer. Because A-T heterozygotes are cancer-prone, our objective in this study was to ascertain whether skin fibroblasts from these i n d i v i d u a l s also show increased chromatid damage following Gz x-irradiation. MATERIALS A N D M E T H O D S

The A-T homozygous and heterozygous cell lines and one of the normal control lines (GM0500) were obtained from the Institute for Medical Research, Camden, NJ. The remaining age-matched normal control lines {CRL) were obtained from the American Type Culture Collection, Rockville, MD. Line KD was generously provided by Dr. T. Kakunaga [23]. Stock cultures were grown in Dulbecco's modified Eagle's m i n i m u m essential m e d i u m s u p p l e m e n t e d with 10% fetal bovine serum (Flow Laboratories, Inc., McLean, VA), as previously described [24]. Cells and medium were not exposed to light of wavelength <500 nm, as these shorter wavelengths are k n o w n to produce chromatid damage [25]. For chromosome studies, 10 ~ cells in 2 ml m e d i u m were inoculated into Leighton tubes, each containing a 9 x 50 m m coverslip (no. i thickness; Bellco Glass, Inc., Vineland, NJ). After 48 hr of i n c u b a t i o n at 37°C, cultures were irradiated by means of two Philips RT250 opposing therapeutic 250-kV potential x-ray tubes operated at 235 kV, 15 mA, with 0.25 m m Cu and 0.55 m m A1 filters {half-value layer, 0.09 m m Cu), and at a dose rate of 126 R/min at 54-cm target distance. After irradiation, culture fluid was renewed within approximately 10-30 min, 0.1 Fxg colcemid/ml (GIBCO, Grand Island, NY) was added for 1 hr. For chromosome analysis, the experimental and control cells were processed in situ on coverslips by previously described techniques [26]. Chromosome analyses were made on randomized, coded preparations; four cultures were used and 144-200 metaphase cells were examined for each variable. The statistical analyses were based on both the n u m b e r of chromatid breaks or gaps per cell and the percentage of cells showing these abnormalities. Comparisons of groups of cell lines were performed using both the Wilcoxon rank sum test and the standard t test [27]. Comparisons of pairs of cell lines were performed by combining the responses at 25, 50, and 100 R using the Mantel-Haenszel test [27]. Abnormalities scored as breaks showed distinct dislocation and m i s a l i g n m e n t of the chromatid fragment, whereas gaps were achromatic lesions showing no dislocation. By examining metaphase cells 1-1.5 hr after irradiation, we could be assured that the cells were in G2 at the time of x-irradiation. RESULTS

Cells of all lines were p r e d o m i n a n t l y diploid and were of comparable density at the time of x-irradiation. In the first metaphase following irradiation of cells in G2 phase, only two types of chromosome aberrations were o b s e r v e d - - c h r o m a t i d breaks

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and gaps (Fig. 1). Figure 2 presents the frequency of c h r o m a t i d breaks and gaps in skin fibroblasts from normal, A-T homozygote, and A-T heterozygote i n d i v i d u a l s following x-irradiation (0, 25, 50, 100 R) during G2 phase. The c h r o m a t i d damage in both A-T homozygotes and heterozygotes was dose-related. The cells from all five heterozygotes, like those from homozygotes, showed a significantly higher frequency of c h r o m a t i d breaks and gaps at each radiation dose than did normal controis (p < 1 0 4 for each dose level). The incidence of spontaneous c h r o m a t i d gaps was also significantly higher in the A-T and heterozygote cells than in normal cells (p = 0.036 and 0.005, respectively). Cells from the A-T patients exhibited a higher incidence of r a d i a t i o n - i n d u c e d chromatid damage than their heterozygous parents (p <0.01). The heterozygote cell line, GM 3488, had significantly more radiationi n d u c e d chromatid gaps than any of the other heterozygote cell lines (p <0.05), and the A-T offspring (GM3487) of this parent also had a significantly higher incidence of chromatid gaps (p - 0.024) than the other A-T cell line studied. With the exception of cell line GM3488, the remaining four heterozygote lines were homogeneous with respect to r a d i a t i o n - i n d u c e d chromatid breaks and gaps.

Figure 1 Metaphase spread of A-T cells showing chromatid breaks (arrows) and gaps (arrowheads) after G2 x-irradiation (X2318).

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CHROMATID BREAKS

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Figure 2 Frequency of chromatid breaks and gaps in skin fibroblasts from normal, A-T, and A-T heterozygote individuals following x-irradiation during G2 phase. Cell line GM3395 is derived from an A-T patient whose parents were donors of lines GM3396 and GM3397. Similarly, line GM3487 is from another A-T, and GM3488 and GM3489 are from his parents. Each bar represents one standard error of the mean.

DISCUSSION Skin fibroblasts from parents (obligate heterozygotes) of A-T i n d i v i d u a l s compared with fibroblasts from age-matched normal controls show a m u c h higher frequency of chromatid breaks and gaps following x-irradiation during G2 phase. The magnitude of this difference suggests that G2 radiosensitivity could provide the basis for an assay to detect A-T heterozygotes. In three recent attempts to detect A-T heterozygotes by cytogenetic techniques following ionizing radiation [13, 28] or bleomycin treatment [29], only one succeeded in finding a difference between normal and A-T heterozygote cells [13]. This difference in results could be due to the different times of irradiation or bleomycin treatment relative to the cell cycle, as we have shown that the extent of chromatid damage seen at the first posttreatment metaphase is d e p e n d e n t on the stage of the cell cycle at the time of the insult [19]. In recent studies, increased chromatid damage following x-irradiation of cells d u r i n g G 2 appears to be associated with a genetic predisposition to cancer [6, 21, 22]. The present study further supports this concept, in that A-T heterozygotes, which, like homozygotes, are cancer-prone, also show a high incidence of chromatid breaks and gaps following x-irradiation of cells during G2. This e n h a n c e d radiosensitivity of G2 cells probably results from deficient DNA repair during G2-prophase, as suggested previously for cells from cancer-prone i n d i v i d u a l s as well as malignant neoplastic cells [6, 17-22].

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U n l i k e cells f r o m o t h e r genetic m u t a n t s p r e d i s p o s i n g to a h i g h risk of c a n c e r [22], A-T and A-T h e t e r o z y g o t e cells are u n i q u e in s h o w i n g a d o s e - r e l a t e d i n c r e a s e in c h r o m a t i d gaps f o l l o w i n g G 2 x - i r r a d i a t i o n w i t h 25, 50, and 100 R. A c c o r d i n g to the m o n o n e m e t h e o r y of c h r o m a t i d structure, e a c h c h r o m a t i d c o n t a i n s a single cont i n u o u s D N A d o u b l e strand. T h e r e f o r e , r a d i a t i o n - i n d u c e d c h r o m a t i d breaks result from u n r e p a i r e d D N A d o u b l e strand breaks, w h e r e a s gaps m a y result f r o m D N A single strand breaks. T h e D N A strand breaks c o u l d arise d i r e c t l y or i n d i r e c t l y f r o m i n c o m p l e t e e x c i s i o n repair of base d a m a g e [for r e v i e w , see r e f e r e n c e 30]. T h e doserelated i n c r e a s e in r a d i a t i o n - i n d u c e d c h r o m a t i d gaps in A-T and A-T h e t e r o z y g o t e cells suggest that t h e s e result f r o m direct r a d i a t i o n - i n d u c e d D N A strand breaks. P r e v i o u s c y t o g e n e t i c o b s e r v a t i o n s s u p p o r t this c o n c e p t , in that A-T cells s h o w m o r e e x t e n s i v e c h r o m a t i d d a m a g e t h a n do n o r m a l cells f o l l o w i n g t r e a t m e n t w i t h D N A s t r a n d - b r e a k i n g agents g i v e n d u r i n g G2 [8]. A l t h o u g h the c l i n i c a l m a n i f e s t a t i o n of A-T is i n h e r i t e d as a recessive, the epid e m i o l o g i c data on i n c i d e n c e of c a n c e r in A-T f a m i l i e s and the p r e s e n t data suggest that c a n c e r p r o n e n e s s and G2 c h r o m a t i d r a d i o s e n s i t i v i t y are m a n i f e s t w i t h a single A-T gene dose.

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