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Endocrinology, doi:10.1210/en.2004-0613
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Endocrinology Vol. 145, No. 12 5679-5687
Copyright © 2004 by The Endocrine Society

Ataxia-Telangiectasia Mutated Gene Controls Insulin-Like Growth Factor I Receptor Gene Expression in a Deoxyribonucleic Acid Damage Response Pathway via Mechanisms Involving Zinc-Finger Transcription Factors Sp1 and WT1

Limor Shahrabani-Gargir, Tej K. Pandita and Haim Werner

Department of Clinical Biochemistry (L.S.-G., H.W.), Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel; and Department of Radiation Oncology (T.K.P.), Washington University School of Medicine, St. Louis, Missouri 63108

Address all correspondence and requests for reprints to: Haim Werner, Ph.D., Department of Clinical Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. E-mail: hwerner{at}post.tau.ac.il.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The IGF-I receptor (IGF-IR) has a central role in cell cycle progression as well as in the establishment of the transformed phenotype. Increased expression of the IGF-IR gene, in addition, is correlated with acquisition of radioresistance for cell killing. The ataxia-telangiectasia mutated (ATM) gene product has a pivotal role in coordinating the cellular response to DNA damage. The present study was aimed at testing the hypothesis that the ability of ATM to coordinate the DNA damage response that will lead to cell survival or, alternatively, to apoptosis depends, to a significant extent, on its capacity to control IGF-IR gene expression. The potential involvement of ATM in regulation of IGF-IR expression and function was investigated in isogenic cells with and without ATM function [AT22IJE-T/pEBS7 (ATM –/–) and ATM-corrected AT22IJE-T/YZ5 (ATM +/+) cells and 293 human embryonic kidney cells transfected with small interfering RNAs targeted to ATM]. In addition, the effect of ATM on IGF-IR expression was assessed in nonisogenic cells with ATM function (HFF + human telomerase reverse transcriptase) and without ATM function (GM5823 + human telomerase reverse transcriptase). Results obtained showed that IGF-IR gene expression and IGF-IR promoter activity were largely reduced in ATM –/– cells. Addition of the radiomimetic agent neocarzinostatin for 4 h, however, induced a significant increase in IGF-IR levels in cells without ATM function. In addition, IGF-I-induced IGF-IR and insulin receptor substrate-1 phosphorylation were greatly impaired in ATM-deficient cells. Furthermore, we identified zinc-finger transcription factors Sp1 and WT1 as potential mediators of the effect of ATM on IGF-IR gene expression. The present data suggests that the IGF-IR gene is a novel downstream target in an ATM-mediated DNA damage response pathway. Deregulated expression of the IGF-IR gene after ionizing radiation may be linked to genomic instability and enhanced transforming capacity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE IGF-I RECEPTOR (IGF-IR) mediates the mitogenic, transforming and differentiating effects of the IGF ligands, IGF-I and IGF-II (1, 2, 3). The receptor contains a tyrosine kinase domain in its cytoplasmic portion that is responsible for the transduction of IGF-mediated signals through the ras-raf-MAPK and phosphoinositide 3-protein kinase B/Akt cascades. The absolute requirement for IGF-IR action in normal development is illustrated by the severe growth retardation and perinatal lethality exhibited by igf-1r knockout mice (4). Most importantly, fibroblasts derived from mice embryos lacking IGF-IR are resistant to transformation by most oncogenes, indicating that the presence of IGF-IR is an important prerequisite for establishment of the malignant phenotype (5, 6). Furthermore, IGF-IR exhibits potent antiapoptotic effects that are consistent with the role of IGFs as cell-survival factors (7, 8).

Evidence accumulated in recent years revealed a strong link between the IGF-IR gene and cell killing after exposure to ionizing radiation (radiosensitivity). Overexpression of wild-type IGF-IR in NIH3T3 fibroblasts conferred radioresistance, and this effect correlated with the transforming capacity of the receptor. Conversely, addition of antisense oligomers against IGF-IR mRNA reversed the radioresistant phenotype. Furthermore, immunohistochemical analysis of primary breast tumors indicated that high levels of IGF-IR correlated with ipsilateral breast tumor recurrence after lumpectomy and radiation therapy (9). In keratinocytes, the only growth factor receptor that provided protection from UV-induced apoptosis was the IGF-IR (10). It seems that the activated IGF-IR may, on one hand, function as a survival factor for irradiated cells and, on the other hand, may induce a postmitotic state that would prevent passage of damaged DNA to daughter cells.

The level of expression of the IGF-IR gene in both physiological and pathological states is primarily determined at the transcriptional level (11). Structural and functional analyses of the IGF-IR gene regulatory region revealed the presence of an initiator motif that directs specific transcription initiation from an internal site (12, 13). Like other TATA-less promoters, the IGF-IR promoter contains multiple GC boxes that are potential binding sites for members of the Sp1 family of zinc-finger transcription factors. In addition, the promoter region includes cis-elements for members of the early growth response family of zinc-finger proteins, including the WT1 Wilms’ tumor suppressor (14, 15). Using electrophoretic mobility shift assays and DNase I footprinting analyses, we have previously shown that the transcriptional activities of these nuclear proteins were correlated with high-affinity binding to specific promoter sequences, demonstrating that the IGF-IR gene constitutes a bona fide downstream target for various families of zinc-finger proteins.

The ataxia-telangiectasia mutated (ATM) gene encodes a 350-kDa protein that includes a phosphoinositide 3 kinase domain close to its C terminus (16, 17, 18). Mutation of the ATM gene in both alleles results in progressive neuronal degeneration, premature aging, immunological abnormalities, and an increased risk of cancer in ataxia-telangiectasia (A-T) patients (19). The central role of the ATM protein in signaling DNA damage is now well established (for a review, see Ref.20). Ionizing radiation (IR), but not UV radiation, enhances ATM kinase activity and phosphorylates a series of target proteins (e.g. p53, BRCA1, c-abl, etc.) (21, 22, 23, 24), which are involved in cell cycle control and repair of DNA damage. The potential role of the IGF-IR gene as a target in an ATM-dependent pathway involved in regulating the radiation response was recently inferred from studies demonstrating that IGF-IR levels were reduced in cells carrying mutations in the ATM gene (A-T cells) (25). Complementation of mutant cells with the ATM cDNA resulted in increased IGF-IR promoter activity and elevated IGF-IR levels. Furthermore, forced expression of the IGF-IR in A-T cells conferred increased radioresistance.

In light of the important role of IGF-IR in radioresistance and in the acquisition of a malignant phenotype, we hypothesized that the ability of ATM to coordinate the DNA damage response that will lead to cell survival or, alternatively, to apoptosis may depend on its capacity to control IGF-IR gene expression. The aim of the present study was to examine the molecular mechanisms responsible for regulation of the IGF-IR gene by ATM. Specifically, we focused on a family of zinc-finger proteins that have been previously shown to participate in basal and, potentially, pathological regulation of IGF-IR gene transcription. The results obtained demonstrate that ATM governs transcription of the IGF-IR promoter via a mechanism or mechanisms that involve transcription factors Sp1 and WT1. Impaired regulation of the IGF-IR gene in A-T patients may be linked to defective cell division, genomic instability, and increased incidence of cancer.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
The immortalized fibroblast cell line AT22IJE-T was originally established from primary A-T fibroblasts (26). The primary cell line exhibits a homozygous frameshift mutation at codon 762 of the ATM gene that results in a truncated protein with reduced stability (27). AT22IJE-T cells were transfected with a full-length ATM open reading frame (clone pFB-YZ5, harboring a FLAG epitope) or with the empty expression vector pEBS7 (28). AT22IJE-T-derived clones were kindly provided by Dr. Yosef Shiloh (Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel). AT22IJE-T/YZ5 (ATM +/+) and AT22IJE-T/pEBS7 (ATM –/–) cells were maintained in DMEM containing 15% fetal bovine serum (FBS), 2 mM glutamine, 100 µg/ml hygromycin B, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 1.25 U/ml nystatin.

GM5823 fibroblasts (Coriell Institute, Camden, NJ), derived from an A-T patient, and HFF normal human primary fibroblasts (29) were immortalized by ectopic expression of the human telomerase reverse transcriptase (hTERT) (30). Cells were maintained in DMEM containing 20% FBS. RNA interference was established in the 293 human embryonic kidney cell line with the pSUPER.retro viral system, using 19 nucleotide primers corresponding to positions 7218 and 1267 of the ATM transcript simultaneously (GenBank accession no. nm 000051). 293 Cells were infected with previously packaged viral particles of both interfering RNAs and selected with puromycin and hygromycin B to achieve a stable cell line (293{Delta}ATM). For control purposes, 293 cells infected with small interfering RNAs against lacZ were used (293lacZ). The 293{Delta}ATM and 293lacZ cell lines were maintained in DMEM supplemented with 10% FBS, 10 µg/ml puromycin, 100 µg/ml hygromycin B, and penicillin/streptomycin/nystatin. The 293{Delta}ATM and 293lacZ cell lines were kindly provided by Drs. Yosef Shiloh and Yaniv Lerenthal (Sackler School of Medicine, Tel Aviv University). DNA damage was induced by treating the cells with the radiomimetic agent neocarzinostatin (NCS; Kayaku Co., Tokyo, Japan) at doses of 200–500 ng/ml.

Drosophila Schneider cells were grown in Schneider’s Drosophila medium containing 10% FBS, 2 mM glutamine, and 20 µg/ml gentamicin sulfate. Schneider cells were grown at room temperature in tightly closed 80-cm2 flasks. Cells were plated at a density of 1.5 x 106 cells/ml in 100-mm dishes 2 h before transfection.

Western immunoblotting
Cells were harvested with ice-cold PBS containing 5 mM EDTA and lysed in a buffer composed of 150 mM NaCl, 20 mM HEPES (pH 7.5), 1% Triton X-100, 10% Nonidet P-40, 2 mM EDTA, 2 mM EGTA, 1 mM phenylmethylsulfonylfluoride, 2 µg/ml aprotinin, 1 mM leupeptin, 1 mM pyrophosphate, 1 mM vanadate, and 1 mM dithiothreitol. In immunoprecipitation experiments, a mixture of two protease inhibitor cocktails was used (catalog no. P2850 and P5726; Sigma, St. Louis, MO). Samples were electrophoresed through 8% SDS-PAGE, followed by transfer of the proteins to nitrocellulose membranes. After blocking with 3% BSA in Tris-buffered saline with Tween 20 (20 mM Tris-HCl, pH 7.5; 135 mM NaCl; and 0.1% Tween 20), blots were incubated with a rabbit polyclonal antihuman IGF-IR ß-subunit antibody (C20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Membranes were then washed extensively with Tris-buffered saline with Tween 20 and incubated with a horseradish peroxidase-conjugated secondary antibody. Proteins were detected using the SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). In addition, blots were probed with antibodies against ATM (MAT3-4G10/8) (31), FLAG epitope (M5; Eastman Kodak Imaging Systems, New Haven, CT), Sp1 (Pep-2; Santa Cruz Biotechnology), WT1 (C19; Santa Cruz Biotechnology), and tubulin or actin as loading controls.

To assess IGF-IR phosphorylation, HFF+hTERT and GM5823+hTERT cells were treated with NCS for the indicated periods of time. Cells were immunoprecipitated with AG-agarose coupled to an IGF-IR antibody. Immunoprecipitates were electrophoresed through 8% SDS-PAGE, followed by transfer of the proteins to nitrocellulose membranes. Membranes were blocked with 3% BSA and then immunoblotted with either antiphosphotyrosine (Ab-1; Oncogene Research Products, Darmstadt, Germany) or anti-IGF-IR antibodies. Proteins were detected as described earlier.

Plasmids and DNA transfections
A genomic DNA fragment extending from nucleotides –476 to +640 (nucleotide 1 corresponds to the transcription initiation site of the rat IGF-IR gene) was subcloned upstream of a promoterless firefly luciferase reporter in the p0LUC vector (15). The promoter activity of the p(–476/+640)LUC construct and the location of the Sp1 and WT1 binding sites have been previously described (14, 15). A WT1 expression plasmid (WT1 –/–, in pcDNA3) lacking the 17-amino acid exon 5-encoded sequence and the three-amino acid exon 9-encoded tripeptide (Lys-Thr-Ser, KTS) was kindly provided by Dr. Charles T. Roberts (Oregon Health and Sciences University, Portland, OR) (32). An ATM expression vector (in pcDNA3) was obtained from Dr. Michael B. Kastan (St. Jude Children’s Research Hospital, Memphis, TN) (33). An Sp1 expression vector driven by the actin 5C promoter (pPacSp1) was a gift of Dr. Robert Tjian (University of California, Berkeley, CA) (14). A WT1 promoter-luciferase reporter plasmid, pGLWTpS-P, including nucleotides –873 to +425 (relative to the major transcription site), was kindly provided by Dr. Mike Eccles (University of Otago, Dunedin, New Zealand) (34).

For transient transfection experiments, AT22IJE-T/pEBS7 and AT22IJE-T/YZ5 cells were seeded in six-well plates the day before transfection and transfected with 1 µg of the p(–476/+640)LUC reporter construct, along with 0.2 µg of a ß-galactosidase expression plasmid (pCMVß; Clontech, Palo Alto, CA) using the Fugene-6 reagent (Roche Molecular Biochemicals, Indianapolis, IN). For cotransfection experiments, 0.5 µg of the WT1 –/– expression vector (or empty pcDNA3) was included in the DNA mix. After transfection, cells were incubated for 48 h at 37 C and exposed to NCS (200 ng/ml) during the last 4–24 h of the incubation period. After NCS treatment, cells were collected, and luciferase and ß-galactosidase activities were measured as previously described (12). Promoter activities were expressed as luciferase values normalized for ß-galactosidase activity. Schneider cells were transfected using a calcium phosphate transfection kit (Invitrogen, Carlsbad, CA) (14). In previous studies, we found that the large amounts of pCMVß DNA that are necessary to elicit detectable ß-galactosidase activity in Schneider cells significantly affected Sp1 transactivation of promoter constructs. Therefore, luciferase values were normalized per total protein in each sample.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The IGF-IR has been identified as a central player in the transformation process (11, 35). To examine whether the ATM gene product is involved in regulation of IGF-IR gene expression, we chose to investigate the A-T-derived fibroblast cell line AT22IJE-T, which was complemented with the full-length ATM open reading frame [AT22IJE-T/YZ5 (ATM +/+)] or transfected with the empty vector [AT22IJE-T/pEBS7 (ATM –/–)]. Western blot analysis using anti-ATM as well as anti-FLAG antibodies confirmed the presence of the approximately 350-kDa ATM protein in extracts of ATM +/+ cells but not ATM –/– cells (Fig. 1AGo).



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  		FIG. 1. Effect of NCS treatment on endogenous IGF-IR gene expression in cells with and without ATM function. A, Untreated AT22IJE-T/pEBS7 (ATM –/–) and AT22IJE-T/YZ5 (ATM +/+) cells were lysed as indicated in Materials and Methods, and equal amounts of protein (50 µg) were separated by 6% SDS-PAGE and transferred onto nitrocellulose membranes. ATM expression was assessed using a monoclonal ATM antibody (MAT3-4G10/8, upper panel) and an anti-FLAG epitope antibody (lower panel). The position of the approximately 350-kDa ATM band is indicated by the arrows. IB, Immunoblotting. B, Subconfluent AT22IJE-T/YZ5 (+) and AT22IJE-T/pEBS7 (–) cultures were treated with increasing doses of the radiomimetic drug NCS for 30 min. After incubation, cellular extracts were prepared, electrophoresed through 8% SDS-PAGE, and blotted with an antihuman IGF-IR antibody. The position of the 97-kDa IGF-IR ß-subunit protein is indicated by the arrow. C, AT22IJE-T/YZ5 and AT22IJE-T/pEBS7 cells were incubated with NCS (200 ng/ml) (+) or left untreated (–) for the indicated periods of time. After treatment, cells were lysed, and IGF-IR abundance was assessed by Western immunoblotting, as described in Materials and Methods. Membranes were reprobed with a tubulin antibody as a loading control. The bar graphs represent the densitometric scanning of the IGF-IR bands shown in the autoradiograms normalized to the corresponding tubulin bands. A value of 1 was assigned to the IGF-IR/tubulin levels in untreated ATM +/+ cells. Open bars denote untreated cells, and solid bars represent NCS-treated cells. Bars are the mean ± SEM of two to three experiments. *, P < 0.005 vs. control ATM +/+ cells; **, P < 0.05 vs. untreated ATM –/– cells at 4 h; #, P < 0.005 vs. untreated ATM +/+ cells at 6 h; +, P < 0.001 vs. untreated ATM +/+ cells at 8 h.

 
To determine the optimal dose of the radiomimetic agent NCS that is required to assess the potential regulation of the IGF-IR gene by ATM, ATM +/+ and ATM –/– fibroblasts were treated with 0, 80, 200, and 500 ng/ml of NCS for 30 min, and IGF-IR levels were measured by Western blotting. Basal IGF-IR levels were approximately 5.5-fold higher in ATM +/+ cells than in ATM –/– cells (Fig. 1BGo), which is consistent with previous observations (25). At NCS doses of 200–500 ng/ml, a decrease in IGF-IR levels was observed in control ATM +/+ cells. Therefore, in all subsequent experiments, NCS was used at concentrations of 200–500 ng/ml.

To analyze the time course of the IGF-IR response to IR damage in an ATM-dependent manner, ATM +/+ and ATM –/– fibroblasts were treated with NCS (200 ng/ml) for 0, 4, 6, and 8 h, after which IGF-IR levels were assessed in whole-cell lysates. Results of Western immunoblotting indicated that cells with functional ATM exhibited a significant decrease in IGF-IR levels after 6 and 8 h of treatment (67 and 70% decrease, respectively; Fig. 1CGo, upper panel) in contrast to cells without ATM function, which showed a 2.5-fold increase in IGF-IR levels at 4 h, followed by an attenuated decrease (25 and 67% reduction at 6 and 8 h, respectively; Fig. 1CGo, lower panel). Similar differences in IGF-IR levels were seen between 293{Delta}ATM cells (transfected with small interfering RNAs targeted against ATM) and control 293lacZ cells (data not shown).

To investigate whether ATM modulates early steps in the IGF-I response, we evaluated the tyrosine phosphorylation of IGF-IR in response to IGF-I treatment. For this purpose, HFF+hTERT (ATM +/+) and GM5823+hTERT (ATM –/–) cells were treated with IGF-I (20 ng/ml) for 5 min, after which whole-cell lysates were prepared and immunoprecipitated with an anti-IGF-IR antibody. Precipitates were electrophoresed through 8% SDS-PAGE, transferred to nitrocellulose membranes, and blotted with either antiphosphotyrosine (Fig. 2AGo, upper panel) or anti-IGF-IR (Fig. 2AGo, lower panel) antibodies. IGF-I-induced IGF-IR phosphorylation was significantly impaired in cells without ATM function compared with cells with functional ATM (compare lane 7 vs. lane 3). Thus, IGF-IR phosphorylation was approximately 10-fold lower in ATM –/– cells than in ATM +/+ cells. In addition, IGF-I-induced phosphorylation of the downstream signaling molecule insulin receptor substrate-1 (IRS-1) as well as total IRS-1 levels were reduced in cells without ATM function (Fig. 2BGo).



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  		FIG. 2. IGF-I-induced IGF-IR and IRS-1 phosphorylation in cells with and without ATM function. A, Serum-starved HFF+hTERT (ATM +/+) and GM5823+hTERT (ATM –/–) cells were incubated with IGF-I (20 ng/ml) for 5 min, with or without NCS treatment (200 ng/ml for 10 min). After incubation, cells were lysed as indicated in Materials and Methods, immunoprecipitated with an IGF-IR antibody, electrophoresed through 8% SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with either a phosphotyrosine antibody ({alpha}PY; upper panel) or an IGF-IR antibody (lower panel). IP, Immunoprecipitation; IB, immunoblotting. B, Cell lysates were prepared from IGF-I-treated and untreated HFF+hTERT and GM5823+hTERT cells, and IRS-1 phosphorylation was measured by Western blotting using an antiphospho-IRS-1 antibody. Membranes were reprobed with total IRS-1 and tubulin antibodies.

 
To establish whether the reduced expression of the IGF-IR gene in cells without ATM function was due to decreased transcription of the IGF-IR gene, 293{Delta}ATM cells and control 293lacZ cells were transiently transfected with a luciferase reporter gene under the control of the proximal IGF-IR promoter [p(–476/+640)LUC]. This construct, containing 476 bp of the 5'-flanking region and 640 bp of the 5'-untranslated region of the IGF-IR gene, has been shown to display maximal promoter activity in most cell types (36). As shown in Fig. 3AGo, basal IGF-IR promoter activity in 293{Delta}ATM cells was approximately 4-fold lower than in control 293 cells (column 1). Similarly, differences in basal IGF-IR promoter activity were observed between AT22IJE-T/YZ5 and AT22IJE-T/pEBS7 cells. Thus, IGF-IR promoter activity in ATM-deficient AT22IJE-T/pEBS7 cells was 42.9 ± 7.3% of the activity seen in cells with ATM function (column 2).



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  		FIG. 3. Regulation of IGF-IR promoter activity by ATM. A, 293lacZ (ATM +/+) (column 1, solid bar) and 293{Delta}ATM (ATM –/–) (column 1, open bar) cells were transfected with 1 µg of the p(–476/+640)LUC IGF-IR promoter reporter construct and 0.2 µg of the pCMVß plasmid, using the Metafectene transfection reagent (Biontex Laboratories GmbH, Munich, Germany). AT22IJE-T/YZ5 (ATM +/+) (column 2, solid bar) and AT22IJE-T/pEBS7 (ATM –/–) (column 2, open bar) cells were transfected with 1 µg of the IGF-IR promoter construct along with 0.2 µg of pCMVß, using the Fugene-6 reagent. Transfected cells were incubated for 48 h, after which cells were harvested and the levels of luciferase and ß-galactosidase were measured. Promoter activities are expressed as luciferase values normalized for ß-galactosidase levels. A value of 100% was given to the promoter activity in ATM +/+ cells. Results are the mean ± SEM of three independent experiments performed in duplicate dishes. *, P < 0.001 vs. ATM +/+ cells. B, AT22IJE-T/YZ5 (ATM +/+) (solid bars) and AT22IJE-T/pEBS7 (ATM –/–) (open bars) cells were transfected with 1 µg of the p(–476/+640)LUC IGF-IR promoter reporter construct, along with 0.2 µg of the pCMVß plasmid. Cells were left undisturbed for 48 h (controls) or treated with NCS (200 ng/ml) during the last 4 or 24 h of the incubation period. A value of 100% was given to the promoter activity in untreated cells. Results are the mean ± SEM of three independent experiments performed in duplicate dishes. *, P < 0.05 vs. untreated ATM –/– cells.

 
To examine whether the increase in IGF-IR gene expression in ATM –/– cells after 4 h of NCS treatment was mediated at the level of transcription, AT22IJE-T/YZ5 and AT22IJE-T/pEBS7 cells were transiently transfected with the IGF-IR promoter reporter construct. Transfected cells were left undisturbed for 48 h (controls) or exposed to NCS during the last 4–24 h of the incubation period, after which cells were collected and luciferase and ß-galactosidase activities were measured. Four hours of NCS treatment induced an approximately 37% increase in promoter activity in cells without ATM function (Fig. 3BGo), which is probably responsible for the increase in endogenous IGF-IR levels that is seen in such cells. On the other hand, a negligible change in promoter activity was observed in cells with ATM function at 4 h. After 24 h, IGF-IR promoter activity was reduced by approximately 29% in cells with ATM function and by approximately 28% in cells without ATM function.

The proximal approximately 500 bp of the 5'-flanking region and approximately 700 bp of the 5'-untranslated region of the IGF-IR promoter have been previously shown to contain 12 consensus binding sites for WT1, a zinc-finger transcription factor with tumor suppressor activity (15). The biological relevance of these cis-elements was established by a combination of electrophoretic mobility shift assays, DNase I footprinting analysis, and transfection experiments (37, 38, 39). To examine whether WT1 is differentially expressed in cells with ATM function compared with cells without ATM function, Western blot analysis was performed in whole-cell lysates of AT22IJE-T/YZ5 and AT22IJE-T/pEBS7 fibroblasts, using a WT1 antibody (C19) that is directed against the C terminus of the molecule. The levels of WT1 in cells with ATM function were approximately 3.6-fold higher than in cells without ATM function (Fig. 4AGo). Likewise, WT1 levels were reduced by 23% (n = 3, P < 0.05) in 293{Delta}ATM cells in which ATM was knocked down by the interfering RNA approach compared with control 293lacZ cells (Fig. 4BGo). Next we assessed the ability of transcription factor WT1 to modulate IGF-IR promoter activity in an ATM-dependent manner. For this purpose, AT22IJE-T/YZ5 and AT22IJE-T/pEBS7 fibroblasts were transiently cotransfected with the p(–476/+640)LUC IGF-IR promoter reporter construct together with a WT1 –/– expression vector. The results obtained showed that ectopic WT1 –/– stimulated IGF-IR promoter activity in cells without ATM function by approximately 2.7-fold, whereas in cells with ATM function, WT1 had a reduced effect (Fig. 4CGo).



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  		FIG. 4. Effect of ATM on WT1 expression. A, AT22IJE-T/YZ5 (ATM +/+) and AT22IJE-T/pEBS7 (ATM –/–) cells were lysed as indicated in Materials and Methods, electrophoresed through 8% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with a WT1 antibody. The membrane was reprobed with a tubulin antibody as a loading control. B, Cellular extracts were prepared from 293{Delta}ATM (ATM –/–) and 293lacZ (ATM +/+) cells, and proteins were resolved through SDS-PAGE and blotted with WT1 and tubulin antibodies. The bar graph shows the WT1 values normalized per tubulin of three independent experiments (mean ± SEM). A value of 100% was given to the WT1/tubulin levels in ATM +/+ cells. *, P < 0.05 vs. ATM +/+ cells. C, One microgram of the p(–476/+640)LUC reporter construct was cotransfected into AT22IJE-T/YZ5 and AT22IJE-T/pEBS7 cells, together with 0.5 µg of a WT1 expression vector (open bars) or of an empty pcDNA3 vector (solid bars) and 0.2 µg of pCMVß, using the Fugene-6 reagent. Transfected cells were incubated for 48 h, after which cells were lysed and assayed for luciferase and ß-galactosidase levels. One hundred percent represents the promoter activity in both cell lines in the absence of WT1 expression vector. Results are the mean ± SEM of three experiments, each performed in duplicate. *, P < 0.05 vs. pcDNA3-transfected cells. D, HFF+hTERT (ATM +/+) and GM5823+hTERT (ATM –/–) fibroblasts were treated with NCS (200–500 ng/ml) for different periods of time, after which lysates were prepared and WT1 abundance was assessed by Western blotting. The migration positions of the 54-kDa, 52-kDa, and 38-kDa WT1 isoforms are indicated by the arrows. Membranes were reprobed with a tubulin antibody as a loading control. E, 293{Delta}ATM (ATM –/–) and 293lacZ (ATM +/+) cells were transfected with 1 µg of a WT1 promoter luciferase reporter plasmid along with 0.2 µg of a ß-galactosidase control plasmid, using the Metafectene reagent. After 48 h, cells were harvested, and the levels of luciferase and ß-galactosidase were measured. WT1 promoter activities are expressed as luciferase values normalized for ß-galactosidase levels. A value of 100% was given to the promoter activity in ATM-expressing 293 cells. Results are the mean ± SEM of two independent experiments performed in duplicate dishes. *, P < 0.05 vs. ATM +/+ cells.

 
To assess the potential involvement of WT1 in the DNA damage response, cells with and without ATM function were treated with NCS (200–500 ng/ml) for different periods of time, after which WT1 abundance was measured by Western blotting. Interestingly, the basal pattern of expression of WT1 in GM5823+hTERT (ATM –/–) cells differed from that of HFF+hTERT (ATM +/+) cells. Thus, whereas cells with ATM function exhibited a typical approximately 52- to 54-kDa doublet in addition to an approximately 38-kDa isoform, the expression of the 52-kDa band in cells without ATM function was largely reduced. A strong increase in the expression of the 38-kDa WT1 isoform was seen in ATM +/+ cells in response to NCS treatment, whereas no change was seen in ATM –/– cells (Fig. 4DGo). Reduced levels of the WT1 gene product in cells without ATM function can probably result from diminished transcription of the WT1 gene. To establish whether the WT1 promoter is down-regulated in cells without ATM function, 293{Delta}ATM and 293lacZ cells were transfected with a luciferase reporter gene under the control of the WT1 promoter. The WT1 promoter, extending from –873 to +425 relative to the major human transcription start site, has been shown to contain all of the cis-elements required for transcriptional activity in 293 cells (40). Results obtained showed that WT1 promoter activity in cells without ATM function was approximately 32% of the activity seen in cells with ATM function (Fig. 4EGo). NCS treatment of transfected cells had no major effect on WT1 promoter activity (data not shown).

An additional zinc-finger transcription factor, the presence of which is critical for efficient expression of the IGF-IR gene, is Sp1. Previously, it has been shown that Sp1 binds a cluster of GC boxes located at nucleotides –399/–394, –378/–373, –374/–369, and –193/–188 in the IGF-IR 5'-flanking region and enhances promoter activity in Sp1-null Drosophila-derived Schneider cells (12, 14). To evaluate Sp1 expression in ATM-containing or ATM-lacking cells, Western blot analysis was performed in HFF+hTERT (ATM +/+) and GM5823+hTERT (ATM –/–) fibroblasts. Sp1 levels (normalized per tubulin) were about 8.8-fold higher in HFF+hTERT compared with GM5823+hTERT cells under basal conditions (Fig. 5Go). After NCS treatment, Sp1 levels in HFF+hTERT cells decreased by approximately 83% at 10 min and by 95% at 4 h. Of note, an additional Sp1 band, which may correspond to phosphorylated Sp1, was observed in HFF+hTERT cells after short-term NCS treatment.



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  		FIG. 5. Regulation of Sp1 levels by NCS treatment in cells with and without ATM function. HFF+hTERT and GM5823+hTERT fibroblasts were treated with NCS (200 ng/ml) for 10 min and 4 h. After treatment, cells were lysed, electrophoresed, and immunoblotted with an Sp1 antibody. The position of Sp1 is indicated by the arrow. Membranes were reprobed with a tubulin antibody as a loading control. The figure shows the results of a typical experiment, repeated three times with similar results. C, Control.

 
To assess the potential functional interactions between ATM and Sp1 in transcriptional regulation of the IGF-IR gene, cotransfection experiments were performed in Schneider cells using expression vectors encoding Sp1 and ATM, along with the p(–476/+640)LUC reporter construct. The rationale for performing these studies in Drosophila-derived cells was the fact that Sp1 is ubiquitously expressed by most mammalian cells, making it difficult to assess the effect of exogenously added Sp1. Therefore, the lack of endogenous Sp1 in Schneider cells provides an optimal background for this type of experiment. As previously demonstrated, transfection of an Sp1 expression vector strongly enhanced IGF-IR promoter activity (~56-fold induction) (Fig. 6Go). ATM expression, on the other hand, was unable to stimulate IGF-IR gene transcription in this particular cellular context. Cotransfection of Sp1 and increasing amounts of the ATM expression vector (0.5, 1, and 2.5 µg) resulted in a dose-dependent synergistic transactivation of the IGF-IR promoter (~1.4-fold enhancement with 1 µg of ATM vector compared with cells transfected with Sp1 alone and ~2.1-fold enhancement with 2.5 µg of ATM).



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  		FIG. 6. Functional interactions between ATM and Sp1 in regulation of IGF-IR promoter activity. Drosophila Schneider cells were cotransfected with 5 µg of the IGF-IR promoter construct p(–476/+640)LUC, along with 300 ng of an actin promoter-driven Sp1 expression vector, in the absence or presence of 0.5, 1, and 2.5 µg of an ATM expression vector (or empty pcDNA3), using calcium phosphate. Luciferase activity was measured after 48 h. A value of 100% was given to the Sp1-induced promoter activity in the absence of ATM. Shown are the results of four to six experiments performed in duplicate dishes. *, P < 0.01 vs. cells transfected with Sp1 alone.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The IGFs have been recognized as major cell survival factors in response to several stress states in various cellular systems (41). Activation of the IGF-IR by IGF-I and IGF-II elicits a strong antiapoptotic response that is consistent with the finding that most transformed cells express high levels of IGF-IR mRNA and protein. In addition, IGF-IR expression is correlated with enhanced cell survival after IR exposure. Thus, IGF-IR –/– fibroblasts, which were derived from mice embryos in which the IGF-IR gene was disrupted by homologous recombination, exhibited an increased sensitivity to IR compared with cells that overexpressed the IGF-IR gene (2). Likewise, down-regulation of IGF-IR in mouse melanoma cells was associated with enhanced radiosensitivity (42).

Transcription of the IGF-IR gene involves complex interactions between various families of transcription factors (11). The present study identifies the IGF-IR gene as a downstream target in an ATM-dependent DNA-damage response signaling pathway. Specifically, our data show that IGF-IR levels and IGF-IR promoter activity are tightly regulated by ATM, with significantly reduced values in ATM-deficient cells. IGF-I responsiveness is similarly impaired in ATM –/– cells, as shown by the reduction in IGF-I-induced IGF-IR and IRS-1 phosphorylation. Interestingly, the decreases in IGF-IR and IRS-1 phosphorylation were several-fold larger than the reductions in total IGF-IR and IRS-1 levels. Hence, these results indicate that decreased IGF-IR signaling in cells without ATM function cannot be explained only on the basis of decreased IGF-IR expression and suggest that additional mechanisms may be responsible for the diminished intrinsic sensitivity of IGF-IR to phosphorylation. In addition, DNA damage induces a strong reduction in IGF-IR expression in normal ATM-containing cells, which may lead to cell cycle arrest. This event may allow cells an opportunity to repair damaged DNA. On the other hand, cells derived from A-T individuals exhibit a transient increase in IGF-IR levels and IGF-IR promoter activity after NCS treatment. This abnormal response may be linked to the impaired capacity of ATM-deficient cells to efficiently halt proliferation and repair DNA damage.

Furthermore, we have identified transcription factors Sp1 and WT1 as potential mediators of the effect of ATM on IGF-IR gene expression. Sp1 is a ubiquitous zinc-finger nuclear protein that activates a specific group of promoters transcribed by RNA polymerase II in vertebrates (43). In addition to its role in normal development, Sp1 is aberrantly expressed in human carcinomas, suggesting that it may be also involved in the etiology of malignant processes (44, 45). Analysis of functional and physical interactions of Sp1 at the IGF-IR promoter revealed that Sp1 expression is critical for IGF-IR gene transactivation (12, 14). Reduced Sp1 expression in ATM –/– cells may be responsible, at least in part, for the decrease in basal IGF-IR levels seen in fibroblasts derived from A-T individuals. The molecular mechanism or mechanisms that are responsible for the ATM-dependent expression of Sp1, however, remain to be elucidated. Unfortunately, we were unable to assess the effect of exogenous Sp1 on IGF-IR promoter activity in cells with and without ATM function due to high endogenous Sp1 levels in most mammalian cells. Coexpression studies in Schneider cells, however, suggest that ATM cooperates in a synergistic fashion with Sp1 in stimulation of IGF-IR gene expression. Interestingly, recent studies have shown that epidermal growth factor enhanced the radiosensitivity of fibroblasts and lymphoblasts via down-regulation of ATM levels, and this effect was associated with a decrease in Sp1 DNA-binding activity (46, 47). Together with the present report, these findings suggest that there are reciprocal links between ATM and Sp1, i.e. cellular levels of Sp1 are governed by ATM, the expression of which, in turn, is controlled by Sp1 itself.

The Wilms’ tumor protein, WT1, is a member of the early growth response family of transcriptional activators. WT1 was originally identified as a tumor suppressor, and the inactivation of WT1 was implicated in the etiology of Wilms’ tumor, a pediatric kidney cancer (48). WT1 includes, at its C terminus, a DNA-binding domain that comprises four zinc fingers of the C2-H2 class (49). Consistent with its tumor suppressor role, we have shown in earlier studies that particular isoforms of WT1 lacking an alternatively spliced KTS tripeptide between zinc fingers 3 and 4 strongly suppressed the activity of cotransfected IGF-IR promoter constructs (37). However, an important body of work demonstrated that, in certain cellular contexts, WT1 is required to inhibit apoptosis both in vivo and in vitro. This oncogenic role of WT1 was found to be associated with its capacity to up-regulate antiapoptotic genes such as bcl-2 (50). Furthermore, we have recently shown that the capacity of WT1 to elicit its biological actions depends, to a large extent, on the cellular status of p53 (39). Taken together, our results indicate that ATM is able to regulate WT1 gene expression at the level of transcription, as shown by the reduced WT1 promoter activity in ATM-depleted cells. Interestingly, the approximately 38-kDa WT1 isoform, the expression of which was strongly stimulated by NCS treatment in HFF+hTERT, has been shown to be generated through translation initiation from a second in-frame AUG of the WT1 mRNA (51). Thus, these results suggest that ATM can regulate WT1 expression at additional control levels. Paradoxically, augmented levels and/or activity of WT1 were shown to positively modulate IGF-IR gene expression. In response to DNA damage, however, there is an increase in WT1 levels in ATM +/+ cells but not ATM –/– cells. Impaired activation of WT1 in ATM –/– cells may contribute to the transient increase in IGF-IR gene expression that is seen at 4 h. Potential differential effects of the various WT1 isoforms in cells with and without ATM function cannot be discounted. Likewise, the possible phosphorylation of WT1 in an ATM-dependent manner needs to be addressed in a systematic manner.

In addition to transcription factors bearing zinc-finger motifs, we have previously identified other nuclear proteins that are directly involved in IGF-IR gene regulation. Specifically, tumor suppressors p53 and BRCA1 were shown to inhibit transcription of the IGF-IR gene via a mechanism(s) that involved protein-protein interactions with DNA-binding proteins, including Sp1 and TBP, the TATA box-binding component of TFIID (36, 52, 53, 54). The fact that p53 and BRCA1 were characterized as downstream targets for ATM action suggests that ATM can modulate IGF-IR gene expression through multiple pathways (21, 22, 23). Suppression of the IGF-IR gene is usually associated with a reduction in cell-surface IGF-I binding sites, thus allowing the terminally differentiated cell to remain out of the cell cycle. Loss-of-function mutation of tumor suppressors in cancer may result in transcriptional derepression of the IGF-IR gene, with ensuing increases in the levels of IGF-IR and increased proliferative capacity.

In conclusion, we have presented evidence showing a significant reduction in basal IGF-IR values, together with an impaired IGF-IR response after DNA damage, in cells deficient in ATM function. Furthermore, we have identified zinc-finger transcription factors Sp1 and WT1 as potential mediators of the effect of ATM on IGF-IR gene expression. Diminished receptor concentrations in cells derived from A-T individuals are compatible with most of the growth, metabolic, and neurological abnormalities seen in these patients. Deregulated expression of the IGF-IR after IR may be linked to genomic instability, defective cell division, impaired checkpoint arrest, and enhanced transforming capacity.


   Acknowledgments
 
This work was performed in partial fulfillment of the requirements for a Ph.D. degree by Limor Shahrabani-Gargir in the Sackler Faculty of Medicine, Tel Aviv University. We thank Drs. Yosef Shiloh, Yaniv Lerenthal, Charles T. Roberts, Jr., Michael B. Kastan, Robert Tjian, and Mike Eccles for providing cell lines and reagents.


   Footnotes
 
This work was supported by National Institutes of Health Grant NS 34746 (to T.K.P.).

Abbreviations: A-T, Ataxia-telangiectasia; ATM, ataxia-telangiectasia mutated; FBS, fetal bovine serum; hTERT, human telomerase reverse transcriptase; IGF-IR, IGF-I receptor; IR, ionizing radiation; IRS-1, insulin receptor substrate-1; NCS, neocarzinostatin.

Received May 27, 2004.

Accepted for publication August 27, 2004.


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 Introduction
 Materials and Methods
 Results
 Discussion
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