Mutagenesis, Vol. 16, No. 5, 419-422,
September 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Ataxia telangiectasia: G2 checkpoint and chromosomal damage in proliferating lymphocytes
1 Programa de Genética Humana and Departamento de Pediatría y Cirugía Infantil, Facultad de Medicina, Universidad de Chile, Santiago, Chile, 2 Centro de Investigaciones Biológicas, CSIC, Velázquez, 144 Madrid-28006, Spain and 3 Departamento de Biología, Universidad Autónoma de Madrid, Canto Blanco, Madrid, Spain
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Abstract |
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There is a checkpoint pathway in eukaryotic cells that depends on ATM (ataxia telangiectasia mutated) kinase which activates the processes leading to the repair of DNA damage and also lengthens the G2 stage of the cell cycle. In cells from ataxia telangiectasia patients, due to their lack of active ATM kinase, an increase in chromosomal aberrations and a failure to induce G2 lengthening could be expected. However, the basal G2 timing in ataxia telangiectasia cells was longer than in controls and was further extended after X-ray irradiation (0.4 Gy), although to a lesser extent than in controls. Moreover, in control cells caffeine shortened G2 and increased chromosomal damage 7-fold, while in ataxia telangiectasia cells caffeine only trebled aberration yield without shortening G2. As caffeine is an inhibitor of ATM kinase, these results suggest the existence of some redundant ATM-independent checkpoint in G2 of ataxia telangiectasia cells. The differential response to caffeine of ataxia telangiectasia and control lymphocytes may be explained by the presence of two different subpathways in the G2 checkpoint: one regulating the processing and repair of damaged DNA and the other controlling G2 timing. While in controls both subpathways may be mediated by ATM kinase, in ataxia telangiectasia cells caffeine-sensitive ATR kinase and the caffeine-insensitive DNA-PK kinases might be responsible for DNA repair and the G2 delay subpathways, respectively. Confirmation of this model in ataxia telangiectasia cells with another cell type in which both subpathways are mediated by DNA-PK should define whether a metylxanthine such as caffeine may also have an additional direct inhibitory effect on DNA repair.
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Introduction |
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During G2 the DNA damage checkpoint pathway allows activation of the mitotic cyclin-dependent kinases to permit the cell to enter into mitosis when DNA repair is completed (Elledge, 1996
). However, because checkpoint mechanisms are transient and frail, cells unable to complete DNA repair in G2 will enter into mitosis after a delay. This phenomenon, known as checkpoint override or checkpoint adaptation (Paulovich et al., 1997), is the main source of the chromosomal aberrations observed in mitosis. The relationship between chromosomal breaks in mitosis and the number of double-strand breaks present in G2 (Badie et al., 1995) is modified by the capability of the cell to process DNA damage.
Cells from ataxia telangiectasia (AT) patients are prone to genomic instability because of their lack of functional ATM kinases. These kinases are components of the checkpoint pathway that controls removal of DNA damage before allowing the onset of mitosis (Matsuoka et al., 1998). They have two main functions: one induces DNA repair, while the other lengthens G2 timing (Jeggo et al., 1998; Shackelford et al., 1999). Induction of DNA repair by the ATM-dependent pathway occurs by phosphorylation of nibrin (Nbs-1, the protein mutated in Nijmegen syndrome) and formation and activation of the complex Mre11/Rad50/Nbs1, involved in the repair of double-strand breaks (Wu et al., 2000).
The role of ATM kinase in the control of G2 timing relies on phosphorylation and activation of the Chk1 and Chk2 kinases that, in turn, prevent activation by Cdc25 phosphatase of the mitotic cyclin-dependent kinase (Teyssier et al., 1999). ATM kinase also activates the checkpoint protein p53, which induces synthesis of p21, the physiological inhibitor of most, if not all, cyclin-dependent kinases (Bunz et al., 1998).
As caffeine inhibits ATM kinase (Blasina et al., 1999; Sarkaria et al., 1999) the increase in chromosomal damage that this purine induces might be a secondary effect of the G2 shortening it induces. In the presence of caffeine control cells become phenocopies of AT cells in G2 behaviour, just as they are during S phase (Painter and Young, 1980). This suggestion predicts that caffeine would not modify the number of chromosomal aberrations nor G2 timing in AT cells, as they lack functional ATM kinases (Zhou et al., 2000). However, AT cells could also have a defect in the processing of DNA damage not mediated by G2 shortening (Foray et al., 1997).
The main objectives of the present work were: (i) to detect any ATM-independent DNA damage checkpoint in G2; (ii) to compare the physiological properties of this putative redundant checkpoint with those displayed by the conventional ATM-dependent one.
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Materials and methods |
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Six AT patients (four females, two of them aged 10 and two others aged 9 and 3, and two males, aged 4 and 6 years) were studied. Two of them (AT-5 and AT-6 in Table I
) are siblings. Age- and sex-matched healthy controls were studied in parallel. Diagnostic criteria for AT probands were: (i) the presence of clinical manifestations of the disease (truncal ataxia, elevated 
-fetoprotein, oculomotor apraxia and oculo-cutaneous telangiectasia); (ii) cytogenetical data obtained in phytohaemagglutinin-stimulated lymphocytes (reciprocal translocations involving chromosomes 7 and 14 and chromosomal hypersensitivity to X-rays). The frequency of reciprocal 7/14 translocations varied from 0.9 to 6.9% in the six AT patients, as assessed in G-banded chromosomes. Banding was accomplished by the trypsin–Giemsa method (Seabright, 1971). Data for patient AT-3 have been previously reported (Pincheira and Bravo, 1992).
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Lymphocyte culture and estimation of chromosomal aberrations
Two peripheral blood samples, taken with an interval of 1 month between them, were obtained from each of the six AT patients and from their respective controls.
From 7 to 13 lymphocyte cultures were set up in TC chromosome medium (Difco, USA) for each AT patient and control. Four to six cultures were used to perform the treatments specified in Table I
, while the other three to nine cultures were used to determine G2 duration under the conditions specified in Table II. The cultures were incubated at 37°C for 72 h. Two hours before harvesting, metaphases were blocked with colchicine at a 5x10–7 M final concentration (TC arresting solution; Difco, USA). Cell harvesting and preparation of the slides for scoring chromosomal aberrations were carried out according to standard procedures.
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Caffeine (Merck, Germany) was diluted in TC medium RPMI 1640 (Difco, USA) to obtain fresh solutions for each experiment. Treatments with 5x10–3 M caffeine and 5x10–4 mM colchicine final concentrations were done together in the last 2 h prior to harvesting.
In the X-ray experiments lymphocyte cultures from each AT patient and from the corresponding control were simultaneously irradiated, at room temperature (18–20°C), with Philips radiotherapy equipment operated at 180 kV, 10 mA, 4 mm Al filter, at a dose rate of 0.4 Gy/min. The irradiation time was 1 min. Immediately after irradiation the cultures were relocated to an incubator at 37°C. In experiments designed to validate the effect of X-rays on the induction of chromosomal aberrations the cultures were treated with colchicine 30 min after irradiation and were then harvested 2 h later.
The frequency of chromosomal aberrations was estimated in a minimum of 100 metaphases, from coded slides. The chromosomal aberrations scored included chromatid/isochromatid breaks, chromatid exchanges, chromosome type aberrations (translocations and dicentric chromosomes) and gaps. These last aberrations, however, were excluded from the total aberration yield. Chromatid/isochromatid breaks were classified when the distal segments were dislocated from the chromosome axis or when the unstained segment was larger than the chromatid width. A dicentric chromosome plus an acentric fragment were scored as one single aberration. The ratios between the frequency of chromosomal aberrations in cell cultures treated with each agent and also that under a different condition were estimated (Table III).
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G2 timing
To determine G2 timing under basal conditions three lymphocyte cultures were incubated with [3H]TdR (sp. act. 25 Ci/mmol; Radiochemical Centre, Amersham, UK) at a final concentration of 1 µCi/ml culture medium for the last 3.5, 4.5 and 5.5 h before harvesting. To evaluate the effects of caffeine on G2 timing, similar [3H]TdR treatments plus the addition of caffeine together with colchicine, during the last 2 h before harvesting, were carried out.
The length of G2 in X-ray irradiated lymphocytes was estimated in three cell cultures from each patient and from the corresponding controls. They were irradiated 30 min before incubation with [3H]TdR for either 4, 5 or 6 h. Thus, the cultures were harvested 4.5, 5.5 and 6.5 h after irradiation. Accumulation of cells in metaphase was done by a colchicine treatment 2 h prior to harvesting. Autoradiography of air-dried preparations was performed according to a previously described procedure (Pincheira et al., 1994). The scoring of labelled metaphases from coded slides corresponding to each treatment was independently done by two people with a minimum recording of 300 metaphases per time of fixation. G2 timing was estimated considering the [3H]TdR incubation time at which 50% labelled metaphases were detected.
Statistical analyses
The statistical significance of the differences in G2 timing between control and AT cells under basal conditions as well as that of the mean values for G2 timing in control lymphocytes with X-irradiation or caffeine were estimated by Student's t-test (Table II).
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Results |
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The data in Table I
show that under basal conditions the frequencies of chromosomal aberrations in the lymphocytes from each AT patient, as well as the mean values for the six patients (7.3 ± 3.7%), were higher than in controls (1.2 ± 0.9%). Interindividual heterogeneity was present (P < 0.02, one-tailed Z-test) in the two siblings (AT-5 and AT-6) sharing the same pair of mutant ATM kinases, so they were independent of heterogeneity in the mutant haplotypes of the unrelated probands.
The distribution of aberration types in the sample of AT patients was as follows: 4.3% chromatid/isochromatid breaks, 0.4% chromatid exchanges and 2.6% chromosome-type aberrations, with some interindividual variability. Table I also shows that in AT lymphocytes X-ray irradiation (0.4 Gy) during G2 increases chromosomal aberrations detected under basal conditions. This increment involved chromatid/isochromatid breaks (94.2%), chromatid exchanges (5.3%) and chromosome-type aberrations (0.5%). In control cells the increment in total yield of chromosomal aberrations was higher (13.8 times) than in AT cells (9.6 times) and involved chromatid and isochromatid breaks only.
In relation to the effects of caffeine, Table I also shows that in controls as well as in AT lymphocytes caffeine treatment during G2 (2 h before harvesting) increased chromosomal aberrations, both under basal and X-irradiation conditions. Nevertheless, while in control cells caffeine increased the aberration yield 7-fold in relation to untreated cultures, this yield was only trebled in AT cells. The aberration types involved in the increments recorded in the presence of caffeine were chromatid and isochromatid breaks, both in the controls and AT cells.
With respest to G2 timing, Table II shows that under basal conditions the estimated mean duration of G2 in AT cells was longer (4.4 ± 0.08 h) than in controls (3.9 ± 0.2 h) (P < 0.05, Student's t-test). X-irradiation lengthened by 1.5 h the mean basal G2 time in controls (P < 0.05, Student's t-test), but by only 0.6 h in the two AT patients in which this parameter could be measured. Table II also shows that while in control cells caffeine treatment decreased G2 length from 3.9 to 3.5 h (P < 0.05, Student's t-test), no G2 shortening was detected in cells from the single AT patient where this timing could be evaluated in relation to its control.
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Discussion |
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Irradiation of AT lymphocytes during G2 increased by about an order of magnitude the number of chromosomal aberrations (Table III
, first line), confirming the high sensitivity of these cells to clastogenic agents (Taylor, 1978). This could be a consequence of the lack of ATM kinase in these patients (atm), as this kinase is involved in sensing DNA damage and in transducing its signalling to initiate processing and repair of the DNA lesions in G2 (Pandita et al., 2000). ATM kinase also lengthens G2, so that proliferating cells have time to repair DNA damage before entry into mitosis. Though AT cells lack ATM kinase, the present results indicate that these cells are able to induce both DNA repair and G2 delay by other alternative checkpoint pathways not involving ATM kinase.
Thus, in AT cells G2 length under basal conditions was significantly longer than in control cells (P < 0.05, Student's t-test). Moreover, in the two AT patients in which this parameter could be studied, G2 length was extended after X-ray irradiation, although to a lesser extent than in controls. The present results also show that in AT cells caffeine, an inhibitor of ATM kinases, only trebled the aberration yield (Table III, lines 2 and 3), without shortening G2 in the single AT patient available for study of this last parameter (Table II). In control cells, however, inhibition of ATM kinase by caffeine increased the aberration yield 7-fold and induced a significant delay in G2.
The response of AT cells to caffeine also allows us to discriminate between the two different subpathways of the G2 checkpoint: (i) one subpathway that is depressed by caffeine and activates the processing of DNA damage in order to repair it; (ii) another subpathway which induces G2 lengthening, which is not depressed by caffeine. Depression by caffeine of this second pathway would be restricted to the conventional ATM-dependent checkpoint.
ATM kinase is one member of a family of lipid phosphatidylinositol-3 kinases, also comprising ATR (ATM-related kinase), which acts efficiently in replication checkpoints, and DNA-PK (DNA-dependent protein kinase) (Kim et al., 1999). DNA-PK is the only member of this family insensitive to caffeine (Hall-Jackson et al., 1999; Sarkaria et al., 1999). As caffeine did not modify G2 length in AT cells, its participation in controlling G2 timing in AT cells is feasible (Teyssier et al., 1999). While ATR kinase might control the processing of DNA damage in G2, that the inhibitory effect of caffeine on the process of DNA repair could be a direct one should not be ignored (Kihlman and Natarajan, 1983).
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Acknowledgments |
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The authors wish to thank Drs J.Oroz, C.L.Navarrete and S.Ponce (Hospital Roberto del Río, Santiago, Chile) for their clinical contribution. We wish to thank Mr J.A.Carballo, Ms M.Carrascosa and Mr J.L.Marcilla for their excellent technical help. We thank Mrs B.L.Walker for her revision of the English. This work has been partially supported by a CSIC–University of Chile Agreement (Project 99 CL 0009) and by the Dirección General de Enseñanza Superior del MEC (Spain) (Projects PB96-0909 and PB98-0072). We also thank the Comunidad Autónoma de Madrid for a technician's contract (J.A.Carballo).
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Notes |
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4 To whom correspondence should be addressed. Tel: +34 91 564 45 62; Fax: + 34 91 562 75 18; Email: delatorrec{at}cib.csic.es
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References |
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Received on October 30, 2000; accepted on May 5, 2001.
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