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Epidermal Growth Factor Sensitizes Cells to Ionizing Radiation by Down-regulating Protein Mutated in Ataxia-Telangiectasia

2001; Elsevier BV; Volume: 276; Issue: 12 Linguagem: Inglês

10.1074/jbc.m006190200

1083-351X

Nuri Gueven, Katherine E. Keating, Philip Chen, Toshiyuki Fukao, Kum Kum Khanna, Dianne Watters, P. Rodemann, Martin F. Lavin,

Carcinogens and Genotoxicity Assessment

Epidermal growth factor (EGF) has been reported to either sensitize or protect cells against ionizing radiation. We report here that EGF increases radiosensitivity in both human fibroblasts and lymphoblasts and down-regulates both ATM (mutated in ataxia-telangiectasia (A-T)) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). No further radiosensitization was observed in A-T cells after pretreatment with EGF. The down-regulation of ATM occurs at the transcriptional level. Concomitant with the down-regulation of ATM, the DNA binding activity of the transcription factor Sp1 decreased. A causal relationship was established between these observations by demonstrating that up-regulation of Sp1 DNA binding activity by granulocyte/macrophage colony-stimulating factor rapidly reversed the EGF-induced decrease in ATM protein and restored radiosensitivity to normal levels. Failure to radiosensitize EGF-treated cells to the same extent as observed for A-T cells can be explained by induction of ATM protein and kinase activity with time post-irradiation. Although ionizing radiation damage to DNA rapidly activates ATM kinase and cell cycle checkpoints, we have provided evidence for the first time that alteration in the amount of ATM protein occurs in response to both EGF and radiation exposure. Taken together these data support complex control of ATM function that has important repercussions for targeting ATM to improve radiotherapeutic benefit. Epidermal growth factor (EGF) has been reported to either sensitize or protect cells against ionizing radiation. We report here that EGF increases radiosensitivity in both human fibroblasts and lymphoblasts and down-regulates both ATM (mutated in ataxia-telangiectasia (A-T)) and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). No further radiosensitization was observed in A-T cells after pretreatment with EGF. The down-regulation of ATM occurs at the transcriptional level. Concomitant with the down-regulation of ATM, the DNA binding activity of the transcription factor Sp1 decreased. A causal relationship was established between these observations by demonstrating that up-regulation of Sp1 DNA binding activity by granulocyte/macrophage colony-stimulating factor rapidly reversed the EGF-induced decrease in ATM protein and restored radiosensitivity to normal levels. Failure to radiosensitize EGF-treated cells to the same extent as observed for A-T cells can be explained by induction of ATM protein and kinase activity with time post-irradiation. Although ionizing radiation damage to DNA rapidly activates ATM kinase and cell cycle checkpoints, we have provided evidence for the first time that alteration in the amount of ATM protein occurs in response to both EGF and radiation exposure. Taken together these data support complex control of ATM function that has important repercussions for targeting ATM to improve radiotherapeutic benefit. protein mutated in ataxia-telangiectasia epidermal growth factor, A-T, ataxia-telangiectasia catalytic substrate of DNA-dependent protein kinase granulocyte/macrophage colony-stimulating factor ionizing radiation peripheral blood mononuclear cells gray phosphate-buffered saline dithiothreitol phenylmethylsulfonyl fluoride kilobase pair base pair nuclear protein at the A-T locus cytomegalovirus Functional loss of the ATM1 protein, mutated in the human genetic disorder ataxia-telangiectasia (A-T) (1Savitsky K. Bar-Shira A. Gilad S. Rotman G. Ziv Y. Vanagaite L. Tagle D.A. Smith S. Uziel T. Sfez S. Ashkenazi M. Pecker I. Frydman M. Harnik R. Patanjali S.R. Simmons A. Clines G.A. Sartiel A. Gatti R.A. Chessa L. Sanal O. Lavin M.F. Jaspers N.G.J. Taylor A.M.R. Arlett C.F. Miki T. Weissman S.M. Lovett M. Collins F.S. Shiloh Y. Science. 1995; 268: 1749-1753Crossref PubMed Scopus (2364) Google Scholar), leads to a pleiotropic phenotype including neurological abnormalities, immunodeficiency, and a predisposition to develop a number of malignancies, primarily leukemias and lymphomas (2Sedgwick R.P. Boder E. Vianney De Jong J.M.B. Hereditary Neuropathies and Spinocerebellar Atrophies. Elsevier Science Publishers B.V., Amsterdam1991: 347-423Google Scholar). The characteristic most widely studied in A-T is hypersensitivity to ionizing radiation (3Gotoff S.P. Amirmokri E. Liebner E.J. Am. J. Dis. Child. 1967; 114: 617-625Crossref PubMed Scopus (200) Google Scholar, 4Taylor A.M. Harnden D.G. Arlett C.F. Harcourt S.A. Lehmann A.R. Stevens S. Bridges B.A. Nature. 1975; 4: 427-429Crossref Scopus (799) Google Scholar, 5Lavin M.F. Shiloh Y. Annu. Rev. Immunol. 1997; 15: 177-202Crossref PubMed Scopus (538) Google Scholar) that appears to be due to an inability of ATM to recognize and facilitate the repair of a subcategory of double strand breaks or a form of damage that is converted into a double strand break in DNA (6Cornforth M.W. Bedford J.S. Science. 1985; 227: 1589-1591Crossref PubMed Scopus (244) Google Scholar,7Foray N. Priestley A. Alsbeih G. Badie C. Capulas E.P. Arlett C.F. Malaise E.P. Int. J. Radiat. Biol. 1997; 72: 271-283Crossref PubMed Scopus (189) Google Scholar). It is also likely that recognition of a specific form of DNA damage represents the trigger for ATM to activate a number of cell cycle checkpoints (8Beamish H. Lavin M.F. Int. J. Radiat. Biol. 1994; 65: 175-184Crossref PubMed Scopus (186) Google Scholar, 9Nagasawa H. Little J.B. Mutat. Res. 1983; 109: 297-308Crossref PubMed Scopus (92) Google Scholar). A-T cells are defective in the phosphorylation of p53 on serine 15 and serine 20 and dephosphorylation at serine 376 after exposure of cells to ionizing radiation (10Siliciano J.D. Canman C.E. Taya T. Sakaguchi K. Appella E. Kastan M.B. Genes Dev. 1997; 11: 3471-3481Crossref PubMed Scopus (710) Google Scholar, 11Waterman M.J.F. Stavridi E.S. Wateman J.L.F. Halazonetis T.D. Nat. Genet. 1998; 19: 175-178Crossref PubMed Scopus (404) Google Scholar, 12Chehab N.H. Malikzay A. Appel M. Halazonetis T.D. Genes Dev. 2000; 14: 278-288Crossref PubMed Google Scholar). Evidence for a direct involvement of ATM with p53 was provided by the demonstration that these two molecules interact directly (13Khanna K.K. Keating K.E. Kozlov S. Scott S. Gatei M. Lavin M.F. Nat. Genet. 1998; 20: 398-400Crossref PubMed Scopus (403) Google Scholar) and ATM phosphorylates p53 on serine 15 in response to DNA damage (14Banin S. Moyal L. Shieh S.-Y. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reiss Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1697) Google Scholar, 15Canman C.E. Lim D.-S. Cimprich K.A. Taka Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1700) Google Scholar). Pre-existing ATM protein is rapidly activated by ionizing radiation and radiomimetic agents by an undescribed mechanism (13Khanna K.K. Keating K.E. Kozlov S. Scott S. Gatei M. Lavin M.F. Nat. Genet. 1998; 20: 398-400Crossref PubMed Scopus (403) Google Scholar, 14Banin S. Moyal L. Shieh S.-Y. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reiss Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1697) Google Scholar, 15Canman C.E. Lim D.-S. Cimprich K.A. Taka Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1700) Google Scholar). Exposure of cells to ionizing radiation fails to change either the subcellular distribution or the total amount of cellular ATM protein (16Watters D. Khanna K.K. Beamish H. Birrell G. Spring K. Kedar P. Gatei M. Stenzel D. Hobson K. Kozlov S. Farrell A. Ramsay J. Gatti R. Lavin M.F. Oncogene. 1997; 14: 1911-1921Crossref PubMed Scopus (169) Google Scholar, 17Brown K.D. Ziv Y. Sadanandan S.N. Chessa L. Collins F.S. Tagle D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1840-1845Crossref PubMed Scopus (149) Google Scholar, 18Lakin N.D. Weber P. Stankovic T. Rottinghaus S.T. Taylor A.M. Jackson S.P. Oncogene. 1996; 13: 2707-2716PubMed Google Scholar). Furthermore, ATM protein levels are relatively constant throughout the cell cycle in human fibroblasts (17Brown K.D. Ziv Y. Sadanandan S.N. Chessa L. Collins F.S. Tagle D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1840-1845Crossref PubMed Scopus (149) Google Scholar). These data point to a post-translational mechanism of activation for ATM. However, there is evidence for alteration in the amount of ATM under certain conditions. Although ATM is not detectable by immunohistochemical staining in quiescent myoepithelial cells lining normal breast ducts, significant expression is observed in the proliferative myoepithelium of sclerosing adenosis (19Clarke R.A. Kairouz R. Watters D. Lavin M.F. Kearsley J.H. Soon-Lee C. Mol. Pathol. 1998; 51: 224-226Crossref PubMed Scopus (27) Google Scholar). In addition the amount of ATM protein changes dramatically in quiescent lymphocytes (PBMCs) in response to mitogenic agents (20Fukao T. Kaneko H. Birrell G. Gatei M. Tashata H. Kasahara K. Cross S. Kedar P. Watters D. Khanna K.K. Misko I. Kondo N. Lavin M.F. Blood. 1999; 94: 1998-2006Crossref PubMed Google Scholar). In the latter study a 6–10-fold increase in ATM in PBMCs was observed in response to PHA, reaching a maximum by 3–4 days. As the amount of ATM protein increased, so too did its protein kinase activity. Clearly this is a slow response compared with the rapid activation of ATM post-irradiation. Alteration in the amount of the ATM protein could occur by transcriptional control at the promoter. The housekeeping geneNPAT/E14/CAND3 lies ∼0.55 kb from the 5′ end of the ATM gene (21Byrd P.J. McConville C.M. Cooper P. Parkhill J. Stankovic T. McGuire G.M. Thick J.A. Taylor A.M.R. Hum. Mol. Genet. 1996; 5: 145-149Crossref PubMed Scopus (95) Google Scholar, 22Imai T. Yamauchi M. Seki N. Sugawara T. Saito T. Matsuda Y. Ito H. Nagase T. Nomura N. Hori T. Genome Res. 1996; 6: 439-447Crossref PubMed Scopus (52) Google Scholar, 23Imai T. Sugawara T. Nishiyama A. Shimada R. Ohki R. Seki N. Sagara M. Ito H. Yamauchi M. Hori T. Genomics. 1997; 42: 388-392Crossref PubMed Scopus (16) Google Scholar). These genes share a bidirectional promoter that contains CCAAT boxes and 4 consensus sites for the Sp1 transcription factor (21Byrd P.J. McConville C.M. Cooper P. Parkhill J. Stankovic T. McGuire G.M. Thick J.A. Taylor A.M.R. Hum. Mol. Genet. 1996; 5: 145-149Crossref PubMed Scopus (95) Google Scholar, 22Imai T. Yamauchi M. Seki N. Sugawara T. Saito T. Matsuda Y. Ito H. Nagase T. Nomura N. Hori T. Genome Res. 1996; 6: 439-447Crossref PubMed Scopus (52) Google Scholar). Further delineation of the ATM promoter is required to assist in understanding the transcriptional regulation of ATM. ATM is located predominantly in the nucleus of proliferating cells, which is in keeping with its role in DNA damage recognition and cell cycle control, but ATM has also been detected in cytoplasmic vesicles (16Watters D. Khanna K.K. Beamish H. Birrell G. Spring K. Kedar P. Gatei M. Stenzel D. Hobson K. Kozlov S. Farrell A. Ramsay J. Gatti R. Lavin M.F. Oncogene. 1997; 14: 1911-1921Crossref PubMed Scopus (169) Google Scholar, 17Brown K.D. Ziv Y. Sadanandan S.N. Chessa L. Collins F.S. Tagle D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1840-1845Crossref PubMed Scopus (149) Google Scholar, 24Lim D.S. Kirsch D.G. Canman C.E. Ahn J.H. Ziv Y. Newman L.S. Darnell R.B. Shiloh Y. Kastan M.B. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10146-10151Crossref PubMed Scopus (152) Google Scholar). Since ATM has been implicated in more general intracellular signaling and since it responds to mitogenic agents, we studied its possible regulation by epidermal growth factor (EGF) that alters cellular response to radiation. EGF enhances the radiosensitivity of some cell types (25Balaban N. Moni J. Shannon M. Dang L. Murphy E. Goldkorn T. Biochim. Biophys. Acta. 1996; 1314: 147-156Crossref PubMed Scopus (135) Google Scholar, 26Knebel A. Rahmsdorf H.J. Ullrich A. Herrlich P. EMBO J. 1996; 15: 5314-5325Crossref PubMed Scopus (465) Google Scholar, 27Kwok T.T. Sutherland R.M. J. Natl. Cancer Inst. 1991; 81: 1020-1024Crossref Scopus (62) Google Scholar, 28Nelson J. McGivern M. Walker B. Bailie J.R. Murphy R.F. Eur. J. Cancer Clin. Oncol. 1989; 25: 1851-1855Abstract Full Text PDF PubMed Scopus (6) Google Scholar) and increases the radioresistance of other cells (29Wollman R. Yahalom J. Maxy R. Pinto J. Fuks Z. Int. J. Radiat. Oncol. Biol. Phys. 1994; 30: 91-98Abstract Full Text PDF PubMed Scopus (89) Google Scholar). We show here that EGF enhanced the radiosensitivity of both fibroblasts and lymphoblasts, and this was associated with a decrease in ATM. Reduction in ATM protein was accompanied by a decrease in the amount of Sp1 DNA binding activity. We also demonstrate that ATM protein is rapidly restored to constitutive levels by both granulocyte/macrophage colony-stimulating factor (GM-CSF) and ionizing radiation, and this appears to be achieved by increasing Sp1 DNA binding activity. Normal human skin fibroblasts HSF7 and NFF were from healthy donors expressing ATM protein; A-T fibroblasts GM03395 were obtained from Coriell Cell Repositories. Lymphoblastoid cells were established by Epstein-Barr virus transformation from healthy individuals, C3ABR, C28ABR, C35ABR, C2ABR, and C31ABR. All cells were grown at 37 °C in a humidified atmosphere of 5% CO2 and 95% air in RPMI 1640 medium supplemented with 10% fetal calf serum. Irradiation of cells was performed at room temperature using a137Cs source delivering gamma rays at a dose rate of 2.8 Gy/min. EGF (R & D Systems) was added at a concentration of 50 ng/ml to the culture medium of log-phase cultures or serum-reduced fibroblasts for various times as indicated. The antibodies used are as follows: p53 monoclonal antibody (polyclonal antibody 1801) and polyclonal Sp1 antibody (Sp1pEp2) were from Santa Cruz Biotechnology; monoclonal α-actin (A4700) was from Sigma; polyclonal anti-Ku and DNA-PKcs antibodies were obtained from Susan Lees-Miller, University of Calgary, Canada; anti-phosphorylated p53 serine 15 was obtained from New England Biolabs; and monoclonal ATM (CT1) and polyclonal ATM raised in sheep (ATM5BA) antibodies were generated in this laboratory. Logarithmically growing lymphoblastoid cells were incubated with or without EGF (50 ng/ml) for 16 h prior to exposure to ionizing radiation (2.5 Gy/min, 137Cs). Cell viability was determined at daily intervals between 1 and 4 days by adding 0.1 ml of 0.4% trypan blue to a 0.5-ml cell suspension as described previously, and viable cells were counted (30Zhang N. Chen P. Gatei M. Scott S. Khanna K.K. Lavin M.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8021-8026Crossref PubMed Scopus (97) Google Scholar). EGF treatment was carried out on log-phase cells for different times as indicated. After washing in PBS, 5 × 106 cells were resuspended in 40 μl of nuclear extraction buffer (25 mm Hepes, pH 8, 0.25 m sucrose, 1 mm EGTA, 5 mm MgSO4, 50 mm NaF, 1 mm DTT, 1 mm PMSF) and disrupted by at least 4 freeze-thaw cycles. Insoluble material was removed by centrifugation at 13,000 × g for 20 min. Protein concentration was determined using a Bio-Rad DC protein assay kit according to the manufacturer's recommendations, and 50–100 μg was used per sample. Protein samples were separated on 5 or 10% denaturing gels and blotted on nitrocellulose membranes. After blocking in 4% milk powder, 0.1% Tween 20, PBS for at least 1 h, the blot was incubated for 1 h at room temperature or overnight at 4 °C, with the relevant primary antibody. The blot was washed three times in 0.1% Tween 20/PBS and incubated with secondary peroxidase-conjugated antibody for 1 h. Following several washing steps, the blot was developed using a chemiluminescence kit (DuPont). Changes in ATM mRNA in lymphoblastoid cells were determined by quantitative PCR as described (31Tashita H. Fukao T. Kaneko H. Teramoto T. Inoue R. Kasahara K. Kondo N. J. Clin. Invest. 1998; 101: 677-681Crossref PubMed Scopus (27) Google Scholar). In short, known amounts of a 402-bp DNA fragment corresponding to an ATM mutation lacking exon 38 (5319 G to A) was used as a competitor during amplification of the corresponding region in ATM cDNA from normal cells, which gives a 544-bp fragment (nucleotides 5122–5665). A series of 2-fold dilutions were employed to find the equivalence point and thus a measure of the amount of ATM mRNA. Expression of mRNA was also determined by Northern blotting. Briefly, total RNA was isolated from log-phase cells with an RNA isolation kit (Qiaquick, Qiagen) according to the manufacturer's instructions, and 20 μg of RNA/lane was separated on a 1% agarose gel. Blotting of RNA on nylon membrane (Hybond-N, Amersham Pharmacia Biotech) was performed overnight by capillary force followed by hybridization with an ATM cDNA probe. The DNA binding activity of transcription factor Sp1 was determined by modification of a method described previously (32Sahijdak W.M. Yang C.R. Zuckerman J.S. Meyers M. Boothman D.A. Radiat. Res. 1994; 138: 47-51Crossref PubMed Scopus (45) Google Scholar). Briefly, 107 cells were washed in ice-cold PBS, resuspended in 400 μl of buffer A (10 mm Hepes, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.1 mm EGTA, 1 mm DTT, 0.2 mm PMSF), and incubated for 15 min on ice. After addition of 25 μl of 10% Nonidet P-40, the cells were subsequently vortexed for 10 s and the nuclei pelleted by centrifugation (1 min, 13,000 × g at 4 °C). The nuclear pellet was resuspended in 40 μl of buffer C (20 mm Hepes, pH 7.9, 400 mm KCl, 1 mmEDTA, 1 mm EGTA, 10% glycerol, 1 mm DTT, 0.2 mm PMSF) and rotated for 30–60 min at 4 °C to elute nuclear proteins. After additional centrifugation for 15 min at 4 °C the supernatant containing the nuclear proteins was aliquoted, frozen in liquid nitrogen, and stored at −70 °C until used in binding reactions. Approximately 35 fmol of 32P-radiolabeled double-strand Sp1 consensus sequence (5′-ATTCGATCGGGGCGGGGCGAGC-3′;Promega) (labeled using T4 polynucleotide kinase) was incubated with 10 μg of nuclear extract in the presence of 1 μg of herring sperm DNA and 20 μg of BSA in binding buffer (10 mm Hepes, pH 7.9, 50 mm KCl, 0.5 mm EDTA, 10% glycerol, 1% Nonidet P-40, 5 mm DTT, 0.2 mm PMSF) in a 20-μl reaction. Binding was for 25 min at room temperature before DNA-protein complexes were separated on a native 5% acrylamide gel. Exposure time to x-ray film was usually 30–45 min. The ATM promoter was amplified from human genomic DNA using primers (forward, 5-TCCCCCGGGGGAGATCAAAACCACAGCAGG, and reverse, 5-CCCAAGCTTGGGCGTTCTCTCGCCTCCTCCCGTG). The 615-bp amplification product was cloned into the pGL3-basic luciferase reporter vector (Promega) using a SmaI/HindIII digest. This construct (pGL3-ATM) was cotransfected with the pRL-CMV vector (Promega) at a ratio of 2:1 into lymphoblastoid cells by electroporation (280 V, 960 microfarads, 1 pulse). Thirty six hours after electroporation the cells were incubated with 50 ng/ml EGF for a further 16 h before cell extracts were prepared. Luciferase activity was measured using the Dual Luciferase Assay (Promega, E1910). Briefly, 100 μl of firefly luciferase substrate (LARII) and 20 μl of cell extract were mixed, and the reaction was immediately measured for 10 s. Then 100 μl of Renilla luciferase substrate including an inhibitor for firefly luciferase (Stop & Glow) was added, and light emission was detected for another 10-s interval. The ratio of both measurements (pGL/pRL) results in the relative luciferase activity, avoiding variabilities in transfection. The absolute values of luminescence measured (relative light unit) was generally in the range of 5·104–5·106. The untreated control was set to 1. ATM kinase activity after EGF treatment was determined using the method described by Canman et al.(15Canman C.E. Lim D.-S. Cimprich K.A. Taka Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1700) Google Scholar). For whole cell lysate isolation, cells were lysed on ice in TGN buffer (50 mm Tris, pH 7.5, 50 mmβ-glycerophosphate, 150 mm NaCl, 10% glycerol, 1% Tween 20, 1 mm NaF, 1 mmNa3VO4, 1 mm PMSF, 2 μg/ml pepstatin, 5 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mmDTT). After centrifugation at 13,000 × g for 1 min, 2 mg of extract was precleared with mouse immunoglobulin G and protein A/G-Sepharose beads. ATM was immunoprecipitated with anti-ATM antibody (ATM5BA) and kinase activity determined as described. Engagement of the EGF receptor by EGF and other ligands initiates signal transduction pathways giving rise to mitogenesis and can also alter the radiosensitivity status of cells (25Balaban N. Moni J. Shannon M. Dang L. Murphy E. Goldkorn T. Biochim. Biophys. Acta. 1996; 1314: 147-156Crossref PubMed Scopus (135) Google Scholar, 26Knebel A. Rahmsdorf H.J. Ullrich A. Herrlich P. EMBO J. 1996; 15: 5314-5325Crossref PubMed Scopus (465) Google Scholar, 27Kwok T.T. Sutherland R.M. J. Natl. Cancer Inst. 1991; 81: 1020-1024Crossref Scopus (62) Google Scholar, 28Nelson J. McGivern M. Walker B. Bailie J.R. Murphy R.F. Eur. J. Cancer Clin. Oncol. 1989; 25: 1851-1855Abstract Full Text PDF PubMed Scopus (6) Google Scholar, 29Wollman R. Yahalom J. Maxy R. Pinto J. Fuks Z. Int. J. Radiat. Oncol. Biol. Phys. 1994; 30: 91-98Abstract Full Text PDF PubMed Scopus (89) Google Scholar). We determined whether EGF might increase the sensitivity of human fibroblasts and lymphoblasts by incubating cells in EGF for 16 h prior to exposure to radiation (1–4 Gy). The results in Fig.1 A demonstrate that EGF-treated lymphoblastoid cells are more sensitive to radiation, and this is observed over the radiation dose range 1–4 Gy (Fig.1 B). The effect of EGF treatment was significant for both dose and time course experiments for C3ABR; p = 0.0023 (α), p = 0.0053 (β) for dose and p= 0.0065 (α) and p = 0.07 (β) for time course. On the other hand treatment of A-T cells with EGF failed to increase the radiosensitivity of these cells (Fig. 1, A andB). A similar sensitization was observed in EGF-treated control fibroblasts, but again no further increase in sensitivity was revealed in EGF-treated A-T fibroblasts (Fig. 1 C). We observed that EGF receptor was activated under these conditions (results not shown). Since radiosensitivity and abnormalities in mitogenesis and other signaling pathways are characteristic of the A-T phenotype, we initially determined whether the radiosensitizing effect of EGF might be due to alteration in the amount of ATM protein in normal control cells. Immunoblotting with anti-ATM antibodies showed that the amount of ATM protein was reduced significantly with time after incubation with EGF in normal fibroblasts (Fig.2 A). No ATM was detected in negative control AT3ABR cells, as expected (16Watters D. Khanna K.K. Beamish H. Birrell G. Spring K. Kedar P. Gatei M. Stenzel D. Hobson K. Kozlov S. Farrell A. Ramsay J. Gatti R. Lavin M.F. Oncogene. 1997; 14: 1911-1921Crossref PubMed Scopus (169) Google Scholar). The same pattern of reduction was observed for the catalytic subunit of DNA-dependent protein kinase, DNA-PKcs, after EGF treatment, but Ku, the DNA damage recognition component of DNA-dependent protein kinase, remained largely unchanged (Fig. 2 A). It is evident that the amount of DNA-PKcs is normal in the A-T cell line, AT3ABR, when compared with C3ABR. EGF treatment of C3ABR lymphoblastoid cells revealed a similar pattern of loss of expression of ATM, down to 30% by 9 h post-treatment (Fig. 2 B). A similar pattern of decrease was also observed for DNA-PKcs (Fig. 1 B). To test the universality of these observations we employed several control lymphoblastoid cell lines. In all four control lines investigated, ATM protein decreased significantly after EGF treatment (Fig. 2 C). It is evident from the positive control (100 μg of extract from the lymphoblastoid cell line, C3ABR) that the amount of ATM protein in HSF7 fibroblasts (at equal loading) is considerably lower than that in lymphoblastoid cells (Fig. 2 A). Since EGF caused a marked decrease in the amount of ATM protein, it was possible that this was due to transcriptional down-regulation. To monitor changes to ATM mRNA in lymphoblastoid cells, we employed quantitative reverse transcriptase-PCR as described previously (31Tashita H. Fukao T. Kaneko H. Teramoto T. Inoue R. Kasahara K. Kondo N. J. Clin. Invest. 1998; 101: 677-681Crossref PubMed Scopus (27) Google Scholar). A DNA fragment (402 bp) corresponding to an ATM mutation (G5319A), which lacks exon 38, was used as a competitor for ATM cDNA amplification (nucleotides 5122–5665, 544 bp in size) in a series of 2-fold dilutions from C3ABR cells. After 8 h of treatment with EGF, an equivalent point was reached at 0.625 attomol/μl of competitor compared with 1.25 attomol/μl for untreated cells representing an ∼2-fold reduction in ATM mRNA in response to EGF (Fig.3 A). After 16 h of treatment with EGF the equivalent point was reached at ∼0.4 attomol representing a 3-fold reduction in ATM mRNA. Northern blot analysis revealed that ATM mRNA was reduced to 60% after incubation of normal fibroblasts (HSF7) with EGF for 8 h and reached a plateau at ∼30% of the untreated value at 12–24 h post-treatment, which parallels the changes shown by reverse transcriptase-PCR in lymphoblastoid cells (Fig. 3 B). A similar genomic organization at the ATM locus exists for the human and mouse genes where ATM and nuclear protein at the A-T locus (NPAT) are arranged ∼0.5 kb apart in a head-to-head configuration (Fig.5 A) (21Byrd P.J. McConville C.M. Cooper P. Parkhill J. Stankovic T. McGuire G.M. Thick J.A. Taylor A.M.R. Hum. Mol. Genet. 1996; 5: 145-149Crossref PubMed Scopus (95) Google Scholar, 22Imai T. Yamauchi M. Seki N. Sugawara T. Saito T. Matsuda Y. Ito H. Nagase T. Nomura N. Hori T. Genome Res. 1996; 6: 439-447Crossref PubMed Scopus (52) Google Scholar). These two genes are transcribed from a central bidirectional promoter that contains several binding sites for the transcriptional factor, Sp1 (33Saito T. Matsuda Y. Ishii H. Watanabe F. Mori M. Hayashi A. Araki R. Fujimori A. Fukumura R. Morimyo M. Tatsumi K. Hori T. Abe M. Mamm. Genome. 1998; 9: 769-772Crossref PubMed Scopus (11) Google Scholar). Since ATM mRNA and protein were reduced after EGF treatment, we predicted that this might be due to interference with or reduced Sp1 DNA binding activity. Use of gel-shift analysis with an oligonucleotide-binding consensus sequence for Sp1 revealed the presence of a single, well defined, retarded band (Fig.4 A). The amount of Sp1 binding in extracts from C3ABR cells decreased with time after EGF treatment (Fig. 4 A). A decrease of ∼50% by 12 h post-treatment, leveling off at later times, paralleled the decrease in the amount of ATM mRNA and protein seen previously. To establish the specificity of this binding, we added anti-Sp1 antibody to the incubation mixture and observed a supershift of the retarded band (Fig.4 A, lower panel). A control antibody against p53 did not alter migration of the band. In addition excess cold Sp1 binding consensus oligonucleotide successfully competed for binding, whereas cold AP-1 binding oligonucleotide failed to do so (results not shown). Under these conditions the amount of Sp1 protein did not change (Fig.4 B). When Sp1 binding was determined in four additional cell lines a similar decrease was observed in all cases (Fig.4 C).Figure 4Effect of EGF on nuclear protein binding to an Sp1-binding consensus sequence in lymphoblastoid cells. A, nuclear extracts (20 μg), prepared at different times after EGF addition from C3ABR, were incubated with32P-labeled double-stranded oligonucleotide (22-mer) containing a single Sp1-binding site. Free and retarded fragments were separated on native 5% acrylamide gels. 1st lane is free fragment only. An Sp1 supershift assay was employed to establish the specificity of binding. Incubation of nuclear extract and labeled fragment was carried out followed by addition of 1 μg of anti-Sp1 antibody (Upstate Biotechnology) for 30 min on ice prior to gel electrophoresis. Anti-p53 antibody (Oncogene Science) was used as a control. Quantitation of binding activity was determined by densitometry.B, Sp1 levels were determined by immunoblotting 20 μg of nuclear extract from each sample in A. Actin was used as a loading control. C, several different lymphoblastoid cells (C3ABR, C28ABR, C35ABR, and C31ABR) were treated with EGF for 16 h prior to preparation of extracts for binding to the Sp1 consensus sequence as described above.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To confirm the effect of EGF on the transcriptional regulation of ATM, we cloned the common promoter region between ATM and NPAT into a luciferase reporter construct (Fig.5) and carried out transient transfections in control lymphoblastoid cells. Exposure of transfected C3ABR cells to EGF for 16 h led to a significant down-regulation of luciferase activity in cells transfected with the ATM promoter construct in agreement with loss of Sp1 binding activity (Fig. 5). The parallel decrease of ATM and Sp1 binding activity together with the down-regulation of ATM promoter activity after EGF treatment suggested that the decrease in Sp1 binding led to reduced ATM. To test this we employed GM-CSF that has been shown to increase markedly the DNA binding activity of Sp1 (34Borellini F. Glazer R.I. J. Biol. Chem. 1993; 268: 7923-7928Abstract Full Text PDF PubMed Google Scholar). As observed above, treatment of cells with EGF for 16 h down-regulated the amount of Sp1 binding activity (Fig. 6 A, 3rd lane) and subsequent addition of GM-CSF to these cells caused a rapid (within 1 h) re