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© 2001 Oxford University Press Human Molecular Genetics, 2001, Vol. 10, No. 5 519–528 Extra-chromosomal telomeric DNA in cells from Atm–/– mice and patients with ataxia-telangiectasia M. Prakash Hande1,+, Adayabalam S. Balajee2, Andrei Tchirkov1, Anthony Wynshaw-Boris3 and Peter M. Lansdorp1,4,§ 1Terry Fox Laboratory, British Columbia Cancer Agency, 601 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada, for Radiological Research, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, USA, 3School of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0627, USA and 4Department of Medicine, University of British Columbia, Vancouver, BC V6T 2B5, Canada 2Center Received 9 November 2000; Revised and Accepted 12 January 2001 Ataxia-telangiectasia (AT) is an autosomally recessive human genetic disease with pleiotropic defects such as neurological degeneration, immunodeficiency, chromosomal instability, cancer susceptibility and premature aging. Cells derived from AT patients and ataxia-telangiectasia mutated (ATM)-deficient mice show slow growth in culture and premature senescence. ATM, which belongs to the PI3 kinase family along with DNA-PK, plays a major role in signaling the p53 response to DNA strand breaks. Telomere maintenance is perturbed in yeast strains lacking genes homologous to ATM and cells from patients with AT have short telomeres. We examined the length of individual telomeres in cells from Atm–/– mice by fluorescence in situ hybridization. Telomeres were extensively shortened in multiple tissues of Atm–/– mice. More than the expected number of telomere signals was observed in interphase nuclei of Atm–/– mouse fibroblasts. Signals corresponding to 5–25 kb of telomeric DNA that were not associated with chromosomes were also noticed in Atm–/– metaphase spreads. Extrachromosomal telomeric DNA was also detected in fibroblasts from AT patients and may represent fragmented telomeres or by-products of defective replication of telomeric DNA. These results suggest a role of ATM in telomere maintenance and replication, which may contribute to the poor growth of Atm–/– cells and increased tumor incidence in both AT patients and Atm–/– mice. INTRODUCTION Ataxia-telangiectasia (AT) is a pleiotropic inherited disease characterized by neurodegeneration, cancer susceptibility, immunodeficiency, genetic instability, radiation sensitivity and premature aging. ATM (ataxia telangiectasia mutated), the gene responsible for AT, is thought to play a crucial role in a signal transduction network that modulates cell cycle checkpoints, genetic recombination, apoptosis and other cellular responses to DNA damage. Cells derived from AT patients (1) and ATM-deficient mice show slow growth in culture and premature senescence (2–4). Cells from AT individuals display abnormalities in culture such as cytoskeletal defects and hypersensitivity to ionizing radiation (5). They also show chromosome abnormalities in the form of end-to-end associations involving telomeres (6,7). Telomeres, consisting of (TTAGGG)n repeats and associated proteins, protect chromosomes from end fusions, incomplete replication and exonuclease degradation (8–10). Telomere shortening may play an important role in tumorigenesis and aging (11). Peripheral blood lymphocytes from AT patients show accelerated telomere shortening (12) and dysfunctional telomeres in yeast strains lacking ATM homolog genes, tel1+ and rad3, caused self-circularization of chromosomes (13). ATM shows some homology to TEL1 and MEC1 genes of budding yeast and rad3+ and tel1+ genes in fission yeast which are known to be involved in telomere maintenance (13,14) and meiotic and mitotic cell cycle checkpoint control (15). Yeast strains with mutations in genes homologous to ATM such as TEL1 and MEC1 show altered telomere chromatin and redistribution of silencing proteins in response to DNA damaging agents (13,14,16–18). Additionally, the ATM gene has been implicated in recombination of DNA sequences and double-strand break repair. Based on the homology of ATM to TEL1 and MEC1 genes in yeast, it has been suggested that mutations in ATM could also lead to defective telomere maintenance (14,16). In this study, we have evaluated the potential role of the ATM gene in telomere function and chromosome integrity in wild-type and ATM knockout mice. Our results indicate that the functional inactivation of ATM leads to telomere shortening, chromosome instability and the occurrence of extrachromosomal fragments of telomeric DNA suggestive of an important role for the mammalian ATM gene in maintaining telomere integrity. +Present address: Center for Radiological Research, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York, NY 10032, USA §To whom correspondence should be addressed at: Terry Fox Laboratory, British Columbia Cancer Agency, 601 West 10th Avenue, Vancouver, BC V5Z 1L3, Canada. Tel: +1 604 877 6070; Fax: +1 604 877 0712; Email: [email protected] 522 Human Molecular Genetics, 2001, Vol. 10, No. 5 Table 1. Telomere fluorescence and chromosomal abnormalities in metaphase chromosomes from Atm–/– mice; results from Q-FISH (19,20) and cytogenetic analyses from five litters of 129/SvEv mice ATM genotype Metaphases analyzed Telomere fluorescence (mean ± SD) p-arm q-arm all Aneuploid cells End-to-end fusions Deletions Fragments +/+ (five mice) 64 37.7 ± 15.7 52.6 ± 22.9 45.0 ± 20.9 1 (1.4) 0 0 3 (4.1) +/– (five mice) 81 32.0 ± 13.9 39.7 ± 19.2 35.9 ± 17.1 13 (16.0) 8 (9.9) 3 (3.7) 9 (11.1) –/– (six mice) 95 24.0 ± 11.0 31.7 ± 15.5 27.9 ± 13.9 19 (20.0) 22 (23.2) 14 (14.7) 30 (31.6) Data are pooled for comparison. Numbers in parentheses indicate events per 100 cells. End-to-end fusions include Robertsonian-like configurations, telomere associations and dicentric chromosomes; deletions were noticed as chromosomes lacking q-arm telomeres; fragments/breaks include centric fragments, acentric fragments, chromosome breaks and chromatid breaks. RESULTS AND DISCUSSION To study telomeres in mice lacking the ATM gene, the telomere length was measured using quantitative fluorescence in situ hybridization (Q-FISH) (19,20) in metaphase chromosomes from primary cells derived from littermate Atm+/+, Atm+/– and Atm–/– mice (129/SvEv and 129/SvEv/BlSW genetic background). Atm–/– splenocytes from 129/SvEv mice showed extensive telomere shortening (Table 1 and Fig. 1A) corresponding to a 35–40% loss of telomeric DNA (from 44 kb in wild-type to 27 kb in Atm–/– mice). Heterozygous Atm +/– mice also displayed a significant reduction (20–25%) in telomere length compared with their Atm+/+ littermates. A similar degree of telomere shortening was observed in cultured fibroblasts from lung and skin of Atm–/– compared with wild-type Atm+/+ 129/SvEv mice (data not shown). Telomeres in cells from 129/ SvEv/BlSW Atm–/– splenocytes were very heterogeneous in length and also significantly shorter than in Atm+/+ controls (Fig. 1B). Chromosome ends without detectable repeats were frequently observed in Atm–/– but not Atm+/+ cells of mice from both genetic backgrounds (Fig. 1A, B, E and F). Representative FISH images of metaphase spreads from Atm+/+ and Atm–/– mouse splenocytes are shown in Figure 1C and D, respectively. In order to determine the telomere length in non-dividing cells other than splenocytes, the telomere repeat content of interphase cells from thymus and bone marrow of the same mice was measured using FISH of cells in suspension and flow cytometry (flow-FISH) (21). A significant reduction in telomere fluorescence in cells from all the homozygous Atm–/– mice analyzed was observed (Fig. 2), although the telomere length differences were less dramatic compared with the results obtained using Q-FISH. Thymocytes from a single male Atm–/– mouse were an exception and showed slightly elongated telomeres (Fig. 2). Further analysis revealed that this particular mouse had a thymic lymphoma, which may explain this observation. Since one of the functions of telomeres is to prevent aberrant chromosome end associations (20,22), the severe telomere shortening observed in the Atm–/– mice may result in chromosome abnormalities. Indeed, high levels of chromosome fragmentation (e.g. Fig. 1D–F, asterisks) and end-to-end fusions were observed in Atm–/– mouse splenocytes compared with wild-type cells (Table 1). About 15% of metaphase cells from Atm–/– mice showed deletions characterized by the complete lack of q-arm telomeres (e.g. Fig. 1E and F, asterisks). Most likely, the aneuploidy in the Atm–/– cells resulted from damage to telomeres and/or previous breakage–fusion–bridge cycles (23). Interestingly, cells from heterozygous mice also showed higher levels of chromosome instability compared with the wild-type controls (Table 1). To localize telomeres and to detect the number and distribution of telomere signals in mitotic cells, fibroblasts derived from skin or lung tissues of both Atm+/+ and Atm–/– mice were grown on coverglasses. Cells at a semi-confluent stage were fixed and hybridized with telomeric peptide nucleic acid (PNA) probe (see Materials and Methods). Deconvolution microscopy was used to visualize the telomere signals in the interphase nuclei of such cells (Fig. 3A–C). The reconstructed three-dimensional images were used to count the number of telomere spots in each nucleus. Surprisingly, Atm–/– mouse fibroblasts showed more than the expected number of telomere signals in interphase nuclei (Fig. 3B and C). Cells from Atm +/+ mice displayed an average of 74.5 ± 2.0 telomere signals in their nuclei, close to the expected 80 (40 chromosomes, two ends each) (Fig. 3A and D). In contrast, Atm–/– mouse fibroblasts showed ∼1.3–2 times more telomere spots, depending on the threshold fluorescence intensity used to detect telomere spots (127–181 spots) (Fig. 3D). In view of the short telomeres in cells from Atm–/– mice, the opposite would have been expected from limitations in hybridization efficiency or fluorescence detection. Similarly, clustering of telomeres, as described during meiosis in Atm–/– mice (24), would have yielded opposite results. In view of the large increase in the number of telomere signals, aneuploidy alone in Atm–/– cells seems an unlikely explanation for our observations. Possibly, the surplus telomere signals represent broken telomere fragments. This would also explain why the telomere repeat content in cells from Atm –/– mice measured by flow-FISH (Fig. 2) suggested less telomere shortening relative to Atm+/+ cells than observed at chromosome ends by Q-FISH (Fig. 1). Alternatively, a high proportion of Atm–/– interphase cells could have been in S-phase or telomeres in Atm –/– cells could be replicated earlier than those in wild-type cells. To study these possibilities further, we performed parallel measurements of DNA and TTAGGG repeats by flow cytometry (Fig. 3E). The results of this analysis were compatible with a modest increase in the percentage of Atm–/– fibroblasts in the S- and G2/M-phases of the cell cycle (Fig. 3E and data not shown). A similar trend was also seen in bone marrow cells from the Atm–/– mice (data not shown). The data were furthermore suggestive of early DNA replication of a subset of telomeres in cells from both normal and Atm –/– mice. Human Molecular Genetics, 2001, Vol. 10, No. 5 523 Figure 1. Frequency distributions of telomere fluorescence in 129/svEv (A) and 129/svEv/BlSW (B) mice with and without the ATM gene. Data were collected from Q-FISH studies (19) on metaphase spreads of primary mouse splenocytes of the indicated genotype. The mean and SD for each group of mice is shown. An asterisk indicates P < 0.001 in a Mann–Whitney rank sum test comparing the telomere length distribution in cells from Atm–/– and Atm+/– mice with that in cells from Atm+/+ mice. Individual fluorescence values of p and q arms [expressed in telomere fluorescence units (TFU)] were used to generate the histograms. One TFU corresponds to an estimated 1 kb of telomeric DNA (40). (C–F) FISH analysis of metaphase chromosome spreads from cultured splenocytes of Atm+/+ (C) and Atm–/– mice (D–F). DNA (DAPI) is shown in blue and TTAGGG repeats are shown in red (representing Cy3-labeled telomere PNA probe). Note the overall decrease in telomere fluorescence and heterogeneous telomere signals in the Atm –/– mouse chromosomes. Asterisks indicate fusion events (D) and chromosomes lacking telomeres due to breakage in or outside telomeric DNA (E and F). 524 Human Molecular Genetics, 2001, Vol. 10, No. 5 Figure 2. Results from telomere fluorescence analysis by flow-FISH (21) of primary cells from spleen, bone marrow and thymus. Fluorescence values are expressed as the mean number of molecules of equivalent soluble fluorochromes (MESF). Vertical bars indicate the standard deviation of the mean. The statistical significance using Student’s t-test was: a, P = 0.023; b, P = 0.025; c, P = 0.007; *, male Atm–/– mouse that showed a thymic lymphoma. To further study the excess number of telomere spots seen in interphase cells, we performed telomere FISH on metaphase chromosomes from fibroblasts that were fixed in situ to preserve cytoplasm (Fig. 4). In the cells from Atm–/– mice, we observed that >80% of the mitotic metaphase cells displayed excess telomere signals that were clearly dissociated from the bulk of chromosomal DNA (Fig. 4C and D). Such extrachromosomal spots were rarely observed in Atm +/+ cells (Fig. 4A and B). The presence of extra telomere signals that were not associated with chromosomes but located within the cell was also noticed in 60–75% of the metaphases derived from bone marrow cells of Atm–/– mice. Extra chromosomal telomere signals observed in the metaphase cells of Atm–/– mice correlated with the increased telomere spots observed in the interphase nuclei in these cells. The extra spots could represent broken telomeres or defective replication intermediates such as single-strand G-rich repeats. However, no signals were observed in Q-FISH experiments if heat denaturation was omitted from the protocol, suggesting a double-strand nature of the extra-chromosomal telomere signals. Primary fibroblasts from three normal individuals and four different AT patients were also analyzed to study the presence of extra-chromosomal telomeric DNA in human cells. The fibroblasts were grown on chamber slides and hybridized with Cy3-labeled PNA-telomeric probe. Although the presence of extra-chromosomal telomeric DNA in cells from human AT patients was not as pronounced as in cells from Atm–/– mice, ∼40–50% of the cells analyzed showed a significantly increased number of telomere fluorescence spots not associated with the chromosome ends relative to normal fibroblasts (Table 2). An example of a metaphase spread from a homozygous AT patient is shown in Figure 5. The presence of extra-chromosomal telomeres in cells from both AT patients and Atm–/– mice supports an important role of the ATM gene in maintaining telomere integrity. This notion is supported by control experiments with PNA probes specific for alphoid sequences showing bright staining at centromeres but no extrachromosomal signals in AT-deficient cells from either human or murine origin (data not shown). To find out whether the extra chromosomal telomeres observed in Atm –/– cells are present in the nucleus or in the cytoplasm, we isolated cytoplasmic and nuclear DNA by high salt extraction. In order to verify that the cytoplasmic DNA was free of nuclear DNA contamination and vice versa, dot blot hybridization was performed on equal amounts of cytoplasmic and nuclear DNA blotted onto Hybond-N membrane and hybridized digoxigenin-labeled mitochondrial and ribosomal DNA probes. As expected, the hybridization signal for mitochondrial DNA was confined to the cytoplasmic fraction and that of ribosomal DNA to the nuclear fraction, indicating that there was little contamination of cytoplasmic DNA with nuclear DNA (Fig. 6A). The results of similar hybridizations with 32P-end-labeled telomere probe were in agreement with the FISH data and showed the presence of increased amounts of telomeric DNA in the cytoplasma of primary fibroblasts and thymocytes from Atm–/–compared with Atm+/+ mice (Fig. 6B). Similar results were obtained when undigested nuclear and cytoplasmic DNA isolated from Atm–/– and Atm+/+ cells was run on agarose gel, transferred to nylon membrane and hybridized with a (TTAGGG)3 probe (Fig. 6C). The mechanism responsible for the presence of extrachromosomal telomeric DNA in cells with defective ATM observed in this study is not known. One possibility is that the loss of ATM leads to defective replication of telomeric DNA, which results in the breakage of telomeric DNA. Previous studies in telomerase-negative human cell lines have also described the presence of extra-chromosomal telomeres (25,26). However, Atm–/– mice were shown to have normal levels of telomerase as reported in a recent study (24). Therefore, the telomere dysfunction/regulation noticed in our study could be due to a telomerase-independent process. One intriguing possibility is that telomeric DNA is particularly vulnerable to breakage. It has been reported that single-strand breaks preferentially accumulate at telomeres (27–29) and that DNA repair at telomeres is defective (30). In the absence of ATM, broken single- or double-strand breaks in telomeres may not be detected and/or repaired (31), possibly resulting in the observed fragments and accelerated telomere shortening (32). Human Molecular Genetics, 2001, Vol. 10, No. 5 525 Figure 3. (A) Telomere signal distribution in interphase nuclei of Atm+/+ fibroblasts (see Materials and Methods). Normally ∼70–95 telomere spots are detected in the wild-type cells (D). (B and C) Telomere spots in interphase nuclei of Atm–/– mouse fibroblasts. Note the increased number of telomere signals (B) and occasional telomere clustering (C) in Atm –/– mouse cells. (D) Distribution of telomere signals in interphase nuclei of Atm+/+, Atm+/– and Atm–/– fibroblasts. Telomere FISH was performed on fibroblasts grown on coverslips. Image analysis of images acquired under identical conditions was performed using two different settings (see Materials and Methods). *, P = 0.01; **, P < 0.003. (E) Measurements of DNA and TTAGGG repeat content in single Atm+/+, Atm+/– and Atm–/– mouse fibroblasts using FISH combined with flow-cytometry (see Materials and Methods). Shown are dot plot histograms of PI versus PNA–FITC (telomere fluorescence) as well as fluorescence histograms of total DNA (PI, top) and telomeric DNA (FITC, side). Studies in yeast have suggested that the ATM homologs TEL1 and MEC1 are required to prevent the degradation of telomeric DNA from exonucleases and allow telomerase access to telomeric DNA (33). In mammalian cells, degradation of the telomeres by exonucleases could explain the telomere shortening observed in cells from patients with AT (12) and in the Atm–/– mice reported here. A recent study has shown that presence of double-strand breaks inhibits DNA replication in an ATM-dependent checkpoint pathway (34). Our observations suggest that telomeric DNA fragments accumulate in Atm–/– cells, possibly due to defective replication, recombination or repair of telomeric DNA. ATM is clearly involved in the p53-mediated DNA damage response following telomere fusions and chromosome breakage in 526 Human Molecular Genetics, 2001, Vol. 10, No. 5 Figure 4. Telomere FISH using FITC-labeled (CCCTAA) 3 probe on murine metaphase fibroblasts grown on microscope slides. Mitotic cells were arrested at metaphase by colcemid treatment for 3–4 h. Hypotonic swelling was performed on the slides by incubation for 5 min with 0.03 M sodium citrate at 4°C. Cells were fixed in situ by methanol:acetic acid treatment (6–7 drops of formaldehyde were added to 10 ml of fixative to preserve the cytoplasm) for 10 min. Slides were processed for telomere FISH immediately or stored in cold ethanol at –20°C until use. Intact metaphase chromosomes are easily observed in Atm+/+ (A and B) and Atm–/– (C and D) fibroblasts using PI as a counterstain. Images were acquired using a Zeiss Axioplan-2 fluorescence microscope equipped with separate PI and FITC filters and a cooled charge device (CCD) camera (Sensicam). Separately captured PI (A and C) and FITC (B and D) images were superimposed using Northern Eclipse software (Empix Imaging). Images were processed using Adobe Photoshop software and pseudocolors to simultaneously visualize both chromosomes and telomere signals. The magnification from both cell types was similar. Note the abundant signals obtained with the telomere probe that are not associated with chromosomes within the cell boundary. Asterisks point to chromatid breaks. Double asterisks could represent telomeres at chromatid breakage sites. mammalian cells (35). ATM is also a part of p53- and p21mediated cell cycle checkpoint pathways in response to doublestrand DNA breaks (for a review see ref. 1). Recent data has also implicated ATM in DNA damage repair, since ATM directly phosphorylates BRCA1 after ionizing radiation exposure (36). Any of these functions of ATM could participate in the maintenance of telomeres and telomere length in cultured cells. Irrespective of the precise mechanisms involved, the excess telomere signals observed in Atm–/– cells reported here support a major role of ATM in the replication and maintenance of the telomeric DNA in mammalian cells. MATERIALS AND METHODS Mice and cells Atm+/+, Atm+/– and Atm–/– mice of 129/SvEv and 129SvEv/ BlSW (NIH Black Swiss) strains were either purchased from Jackson Laboratories or obtained from the National Institutes of Health (A.W.-B.’s laboratory). A total of 16 mice from five litters (five Atm+/+, five Atm+/– and six Atm–/– mice all derived from Atm+/– crosses) were used in the study. Animals were sacrificed at 8–12 weeks of age and the tissue samples were Human Molecular Genetics, 2001, Vol. 10, No. 5 527 Table 2. Extra-chromosomal telomere fluorescence in human fibroblasts with a heterozygous (AG03059) or homozygous (AG04405, AG04405, GM05823 and GM02052) defect in the ATM gene Normal fibroblasts (BJ) Number of metaphases analyzed Mean SD Standard error Median Maximum Minimum 15 1.7 1.9 0.5 1 6 0 P-value Normal fibroblasts (WI38) 18 1.6 1.7 0.4 1 5 0 Normal fibroblasts (MRC5) 20 1.4 1.6 0.4 1 5 0 A-T +/– fibroblasts (AG03059) 18 2.9 3.1 0.7 2 10 0 Not significant A-T –/– fibroblasts (AG04405) 15 3.7 3.5 0.9 3 14 0 0.05 A-T –/– fibroblasts (AG03058) 15 3.9 2.9 0.7 4 9 0 0.02 A-T –/– fibroblasts (GM05823) 19 4.3 3.2 0.7 4 12 0 0.001 A-T –/– fibroblasts (GM02052) 18 3.9 2.7 0.6 3 8 0 0.001 A Mann–Whitney rank sum test was used to calculate whether the number of extra-chromosomal spots [defined as distinct fluorescence signals with a size and fluorescence intensity >0.5 TFU, comparable to the telomere signals on chromosomes in that metaphase spread (see arrows in Fig. 5)] in the AT cells was significantly increased relative to the normal (BJ, WI38 and MRC5) fibroblasts. tissues, tissues were cut into small pieces and treated with 0.25% trypsin-citrate saline. After 30 min, trypsin was removed and fresh Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum (FBS) was added to the culture dishes. Once sufficient numbers of fibroblasts were released from the tissues and attached to the culture dishes, tissue pieces were removed. The fibroblasts were subcultured and used in the analysis. Human (WI38, MRC5) and AT (GM05823 and GM02052) primary fibroblasts were obtained from Coriell Cell Repository. All the cells were routinely maintained in 2× Eagle’s minimal essential medium supplemented with 15% FBS (Gibco BRL), vitamins, essential amino acids, non-essential amino acids and antibiotics. The cultures were maintained at 37°C in a humidified 5% CO2 atmosphere. FISH on interphase nuclei and telomere spot analysis Figure 5. Example of extrachromosomal telomere signals (arrows) in a metaphase spread obtained from fibroblasts of a patient with AT (AG 03058) visualized using standard Q-FISH procedures (40). Although spots that were apparently not attached to metaphase chromosomes were occasionally also observed in normal fibroblasts (Table 2), the frequency of such extrachromosomal events in standard metaphase preparation was significantly higher in cells from patients with AT. collected aseptically. Isolation and culturing of splenocytes were described previously (37). For metaphase preparations, splenocytes were grown for 40–42 h in the presence of mitogens and treated with colcemid for 2 h before harvesting (37). Bone marrow cells and thymocytes were isolated by standard techniques. To release fibroblasts from lung and skin Fibroblasts derived from skin/lung were grown on coverglasses until the cells were semiconfluent. Cells were then fixed with 4% paraformaldehyde and dehydrated using cold ethanol series. Coverglasses were used either immediately before telomere FISH or after storage at –20°C. The method used for FISH was as described (20) except that 90% glycerol/ DABCO was used as mounting medium for DAPI. The cells were analyzed using a Delta Vision deconvolution optical sectioning microscope. A stack of images (0.2 µm intervals) was acquired using Cy-3 for telomeres and DAPI for DNA for both the Atm+/+ and Atm–/– fibroblasts under similar exposure conditions. Approximately 20 images were acquired for each genotype studied. The three-dimensional reconstruction of the acquired images was performed using Delta Vision deconvolution software. Telomere spots were counted using two different threshold settings to segment telomere spots from the background. DNA analysis combined with flow-FISH For simultaneous analysis of DNA as well as telomere fluorescence, DNA staining and flow-FISH methods were combined. To obtain clear DNA histograms, cells were fixed in absolute ethanol (–20°C) for 12–16 h before processing for flow-FISH. 528 Human Molecular Genetics, 2001, Vol. 10, No. 5 Figure 6. Dot blot analysis of Atm +/+ and Atm –/– murine cells. (A) Cytoplasmic and nuclear DNA was isolated from Atm+/+ and Atm–/– fibroblasts and hybridized with digoxigenin-labeled mitochondrial and ribosomal DNA probes. The hybridization signals indicate a good separation of cytoplasmic and nuclear DNA. (B) Dot blot analysis of telomeric DNA in cytoplasmic and nuclear DNA isolated from Atm+/+ and Atm–/– fibroblast and thymocytes. The filters were analyzed after hybridization with 32P-labeled (TTAGGG) 3 telomere probe. Note that telomeric DNA is detected in the cytoplasma of Atm–/– but not Atm+/+ cells. (C) Southern blot analysis of cytoplasmic and nuclear DNA isolated from Atm+/+ and Atm–/– cells. Samples were run on a 1% agarose gel and subjected to Southern blot analysis with 32P-labeled (TTAGGG) probe (see Materials and Methods). The blots of cytoplasmic DNA were overexposed to show visible signals. Gels from nuclear and cyto3 plasmic DNA were subjected to autoradiography films for different exposure time. The differences in the amount of DNA that was loaded on the gels and the differences in exposure time make these blots/gels unsuitable for quantitative analysis. Human Molecular Genetics, 2001, Vol. 10, No. 5 Then the cells were pelleted and resuspended in 1 ml phosphate-buffered saline. The subsequent steps were as described for flow-FISH with fluorescein isothiocyanate (FITC)-labeled telomeric PNA probe (21) and finally DNA staining was done using propidium iodide (PI) at a concentration of 0.2 µg/ml. Isolation of cytoplasmic and nuclear DNA DNA was isolated from fibroblast and thymocytes from Atm–/– and Atm+/+ mice. The cells were suspended in ice-cold buffer (0.15 M NaCl, 10 mM Tris–HCl pH 7.4, 2 mM MgCl2 and 1 mM dithiothreitol) and homogenized by repeated pipetting. Nonidet P-40 was added to the homogenate. After incubation for 30 min on ice, the homogenate was spun at 15 000 g for 5 min. The supernatant was used as a cytoplasmic DNA extract. The nuclear pellet was resuspended in NET buffer (150 mM NaCl, 10 mM EDTA and 50 mM Tris–HCl pH 7.5) containing 100 µg/ml proteinase K and 0.5% SDS. The DNA was isolated using high salt as described (38). Dot blot hybridization and Southern blot hybridization of cytoplasmic and nuclear DNA The efficiency of fractionation of cytoplasmic and nuclear DNA was determined by dot blot hybridization using digoxigenin-labeled mitochondrial and ribosomal DNA probes. Human mitochondrial DNA probe spanning the region from 5905–7433 bp was generated by PCR. The ribosomal DNA pABB probe (kindly provided by J.E. Sylvester, University of Pennsylvania, PA) is a 1.4 kb human 28S rDNA which is homologous to mouse sequence. Mitochondrial and ribosomal DNA probes (500 ng of each) were labeled with digoxigenin using the high prime labeling kit of Roche Diagnostics. Cytoplasmic and nuclear DNA samples (∼250 ng) derived from Atm+/+ and Atm–/– mice were dot blotted onto Hybond-N membrane (Amersham Pharmacia Biotech). The conditions for pre-hybridization, hybridization, washing and immunological detection of the hybridization signal were essentially according to the instructions of the manufacturer. The hybridization signal was detected by enhanced chemiluminescence followed by exposure to X-ray film. To study the presence of telomeric DNA in cytoplasmic and nuclear DNA, ∼1–2 µg of fractionated DNA isolated from Atm–/– and Atm+/+ mice was dot blotted on to Hybond-N membranes. The oligonucleotides (TTAGGG)3 and (CCCTAA)3 were obtained from Gibco BRL. DNA probes (250 ng) were 5′ end-labeled by the exchange reaction using T4 polynucleotide kinase (Roche) following the manufacturer’s instructions. The unincorporated nucleotides were removed by Sephadex G-50 column chromatography. The filters were hybridized overnight at 37°C in the hybridization solution (Gibco BRL) containing 0.5% SDS and 10 mM EDTA. After hybridization, the filters were washed for 30 min, once in 2× SSC/0.1% SDS and twice in 0.5× SSC/0.1% SDS. The filters were then exposed to autoradiography films at –80°C for a few hours. For Southern hybridization, ∼1–2 µg of cytoplasmic and nuclear DNA isolated from Atm–/– and Atm+/+ mice were digested with HinfI and RsaI (Gibco BRL) according to the instructions of the manufacturer. The DNA was fractionated on a 1.2% agarose gel and transferred to Hybond-N membrane using capillary blotting in a standard protocol (39). The oligonucleotides 529 (TTAGGG)3 and (CCCTAA)3 were obtained from Gibco BRL. DNA probes (250 ng) were 5′ end-labeled using T4 polynucleotide kinase (Roche) following the instructions from the manufacturer. The unincorporated nucleotides were removed by Sephadex G-50 column chromatography. The filters were hybridized overnight at 37°C in hybridization solution (Gibco BRL) containing 0.5% SDS and 10 mM EDTA. After hybridization, the filters were washed for 30 min, once in 2× SSC/0.1% SDS and twice in 0.5× SSC/0.1% SDS. The filters were then exposed to autoradiography films at –80°C for a few hours. ACKNOWLEDGEMENTS Denise Larson, Cam Smith, Viktoriya Dobrovinska and Elizabeth Chavez are thanked for assistance. We thank Drs Howard Lieberman and Tej Pandita for reagents. Drs David J Brenner and Charles R. Geard are acknowledged for their support. This study was supported by grants from the Canadian Institute of Health Research (MOP 38075), the NIH (R01A129524) and by a grant from the National Cancer Institute of Canada with funds from The Terry Fox Run. 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