Inhibition of Phosphatidylinositol-3-OH Kinase/Akt Signaling Impairs DNA Repair in Glioblastoma Cells following Ionizing Radiation

Abstract

Radiation therapy is a mainstay in the treatment of glioblastomas, but these tumors are often associated with radioresistance. Activation of the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway, which occurs frequently in glioblastomas due to inactivation of the tumor suppressor phosphatase and tensin homologue (PTEN), correlates with radioresistance. To directly test the link between Akt activation and radioresistance, we utilized PTEN-deficient U251 glioblastoma cells engineered to inducibly restore PTEN upon exposure to doxycycline. These cells showed high basal levels of Akt activation (i.e. high levels of phospho-Akt), but induction of PTEN led to substantially decreased phospho-Akt and was associated with radiosensitization. To investigate whether the PTEN-induced radiosensitization was attributable to impaired sensing versus repair of DNA damage, we assessed levels of γ-H2AX after ionizing radiation in U251 cells induced for PTEN. Initial post-radiation levels of γ-H2AX foci were not decreased in PTEN-induced cells; however, the resolution of these foci was significantly delayed. In contrast to these results, induction of phosphatase-dead PTEN showed no appreciable effect. Finally, exposure of cells to the PI3K inhibitor LY294002 did not decrease the occurrence of γ-H2AX foci after irradiation but did markedly delay their resolution. These results together support a direct link between Akt activation, repair of DNA damage, and radioresistance in glioblastoma. Targeting the PI3K/Akt pathway may modulate DNA repair to improve the efficacy of radiation therapy. Radiation therapy is a mainstay in the treatment of glioblastomas, but these tumors are often associated with radioresistance. Activation of the phosphatidylinositol-3-OH kinase (PI3K)/Akt pathway, which occurs frequently in glioblastomas due to inactivation of the tumor suppressor phosphatase and tensin homologue (PTEN), correlates with radioresistance. To directly test the link between Akt activation and radioresistance, we utilized PTEN-deficient U251 glioblastoma cells engineered to inducibly restore PTEN upon exposure to doxycycline. These cells showed high basal levels of Akt activation (i.e. high levels of phospho-Akt), but induction of PTEN led to substantially decreased phospho-Akt and was associated with radiosensitization. To investigate whether the PTEN-induced radiosensitization was attributable to impaired sensing versus repair of DNA damage, we assessed levels of γ-H2AX after ionizing radiation in U251 cells induced for PTEN. Initial post-radiation levels of γ-H2AX foci were not decreased in PTEN-induced cells; however, the resolution of these foci was significantly delayed. In contrast to these results, induction of phosphatase-dead PTEN showed no appreciable effect. Finally, exposure of cells to the PI3K inhibitor LY294002 did not decrease the occurrence of γ-H2AX foci after irradiation but did markedly delay their resolution. These results together support a direct link between Akt activation, repair of DNA damage, and radioresistance in glioblastoma. Targeting the PI3K/Akt pathway may modulate DNA repair to improve the efficacy of radiation therapy. Glioblastoma multiforme, the most common primary adult brain tumor, has a dismal prognosis. Even with aggressive surgery, radiotherapy, and chemotherapy, the median survival for patients with glioblastomas is under one year (1Hegi M.E. Diserens A.C. Gorlia T. Hamou M.F. de Tribolet N. Weller M. Kros J.M. Hainfellner J.A. Mason W. Mariani L. Bromberg J.E. Hau P. Mirimanoff R.O. Cairncross J.G. Janzer R.C. Stupp R. N. Engl. J. Med. 2005; 352: 997-1003Crossref PubMed Scopus (5361) Google Scholar, 2Stupp R. Mason W.P. van den Bent M.J. Weller M. Fisher B. Taphoorn M.J. Belanger K. Brandes A.A. Marosi C. Bogdahn U. Curschmann J. Janzer R.C. Ludwin S.K. Gorlia T. Allgeier A. Lacombe D. Cairncross J.G. Eisenhauer E. Mirimanoff R.O. N. Engl. J. Med. 2005; 352: 987-996Crossref PubMed Scopus (14830) Google Scholar). The phosphatidylinositol-3-OH kinase (PI3K) 3The abbreviations used are: PI3K, phosphatidylinositol-3-OH kinase; PTEN, phosphatase and tensin homologue; DSB, double strand break; Gy, gray; IR, irradiation; DAPI, 4′,6-diamidino-2-phenylindole; ATM, ataxia telangiectasia mutated. signaling pathway is commonly activated in these tumors, often by virtue of PTEN gene mutation but possibly also by epidermal growth factor receptor expression (3Chakravarti A. Zhai G. Suzuki Y. Sarkesh S. Black P.M. Muzikansky A. Loeffler J.S. J. Clin. Oncol. 2004; 22: 1926-1933Crossref PubMed Scopus (326) Google Scholar, 4Choe G. Horvath S. Cloughesy T.F. Crosby K. Seligson D. Palotie A. Inge L. Smith B.L. Sawyers C.L. Mischel P.S. Cancer Res. 2003; 63: 2742-2746PubMed Google Scholar). PTEN encodes a phosphatase that dephosphorylates phosphatidylinositol-3,4,5 triphosphate to convert it to phosphatidylinositol-4,5 bisphosphate. Therefore, inactivation of PTEN leads to increased levels of phosphatidylinositol-3,4,5 triphosphate and increased Akt activation (5Haas-Kogan D. Shalev N. Wong M. Mills G. Yount G. Stokoe D. Curr. Biol. 1998; 8: 1195-1198Abstract Full Text Full Text PDF PubMed Google Scholar). Conversely, restoration of PTEN leads to inhibition of Akt. Chakravarti et al. (3Chakravarti A. Zhai G. Suzuki Y. Sarkesh S. Black P.M. Muzikansky A. Loeffler J.S. J. Clin. Oncol. 2004; 22: 1926-1933Crossref PubMed Scopus (326) Google Scholar) found significantly reduced survival times in patients whose tumors showed PI3K pathway activation. These patients were treated with a combination of surgery with postoperative radiation as the only adjuvant therapy, which suggested that this pathway might play an important role in radiation resistance. One of the factors implicated in radioresistance is activation of the Ras/PI3K/Akt pathway (6Gupta A.K. Bakanauskas V.J. Cerniglia G.J. Cheng Y. Bernhard E.J. Muschel R.J. McKenna W.G. Cancer Res. 2001; 61: 4278-4282PubMed Google Scholar, 7Grana T.M. Rusyn E.V. Zhou H. Sartor C.I. Cox A.D. Cancer Res. 2002; 62: 4142-4150PubMed Google Scholar). Data from numerous investigators show that inhibition of this pathway leads to radiosensitization, not just of glioblastomas but also carcinoma of the colon, bladder, prostate, head and neck, and cervix (6Gupta A.K. Bakanauskas V.J. Cerniglia G.J. Cheng Y. Bernhard E.J. Muschel R.J. McKenna W.G. Cancer Res. 2001; 61: 4278-4282PubMed Google Scholar, 7Grana T.M. Rusyn E.V. Zhou H. Sartor C.I. Cox A.D. Cancer Res. 2002; 62: 4142-4150PubMed Google Scholar, 8Edwards E. Geng L. Tan J. Onishko H. Donnelly E. Hallahan D.E. Cancer Res. 2002; 62: 4671-4677PubMed Google Scholar, 9Gottschalk A.R. Doan A. Nakamura J.L. Stokoe D. Haas-Kogan D.A. Int. J. Radiat. Oncol. Biol. Phys. 2005; 63: 1221-1227Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 10Gupta A.K. Cerniglia G.J. Mick R. Ahmed M.S. Bakanauskas V.J. Muschel R.J. McKenna W.G. Int. J. Radiat. Oncol. Biol. Phys. 2003; 56: 846-853Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 11Gupta A.K. McKenna W.G. Weber C.N. Feldman M.D. Goldsmith J.D. Mick R. Machtay M. Rosenthal D.I. Bakanauskas V.J. Cerniglia G.J. Bernhard E.J. Weber R.S. Muschel R.J. Clin. Cancer Res. 2002; 8: 885-892PubMed Google Scholar, 12Kim I.A. Bae S.S. Fernandes A. Wu J. Muschel R.J. McKenna W.G. Birnbaum M.J. Bernhard E.J. Cancer Res. 2005; 65: 7902-7910Crossref PubMed Scopus (168) Google Scholar, 13Lee C.M. Fuhrman C.B. Planelles V. Peltier M.R. Gaffney D.K. Soisson A.P. Dodson M.K. Tolley H.D. Green C.L. Zempolich K.A. Clin. Cancer Res. 2006; 12: 250-256Crossref PubMed Scopus (115) Google Scholar, 14Li B. Yuan M. Kim I.A. Chang C.M. Bernhard E.J. Shu H.K. Oncogene. 2004; 23: 4594-4602Crossref PubMed Scopus (143) Google Scholar, 15Nakamura J.L. Karlsson A. Arvold N.D. Gottschalk A.R. Pieper R.O. Stokoe D. Haas-Kogan D.A. J. Neuro-oncol. 2005; 71: 215-222Crossref PubMed Scopus (86) Google Scholar). The precise mechanism by which the PI3K/Akt pathway leads to radioresistance has yet to be determined. Radiation is thought to kill cells by causing DNA damage, specifically double strand breaks (DSBs) (16Willers H. Dahm-Daphi J. Powell S.N. Br. J. Cancer. 2004; 90: 1297-1301Crossref PubMed Scopus (115) Google Scholar). This leads to a DNA damage response to allow repair of the DNA strand breaks prior to undergoing cell division. We investigated whether the PI3K/Akt pathway may influence the DNA damage response. In order to manipulate activity of this pathway we tested a cell line that is normally PTEN-deficient but has been engineered so that wild-type PTEN can be induced by adding doxycycline, U251-PTEN (17Radu A. Neubauer V. Akagi T. Hanafusa H. Georgescu M.M. Mol. Cell. Biol. 2003; 23: 6139-6149Crossref PubMed Scopus (187) Google Scholar). As a control we used another cell line, U251-C124S, that is inducible for a mutant form of PTEN that is devoid of phosphatase activity due to a cysteine 124 alteration to serine. In contrast to the radiosensitization and impaired DNA damage repair noted when wild-type PTEN was induced, doxycycline-mediated induction of U251-C124S showed no effect on clonogenic survival or resolution of H2AX activation. These experiments together link Akt activation with DNA damage repair, a pathway that may be usefully modulated by PTEN. Tissue Culture and Reagents—U251MG, a human malignant glioma cell line that is PTEN-deficient, was obtained from the Brain Tumor Research Center Tissue Bank at the University of California San Francisco. U251MG cells were cultured in Dulbecco’s modified Eagle’s medium (4500 mg/liter glucose; Invitrogen) containing 10% fetal bovine serum (Atlanta Biologicals). U251-PTEN and U251-C124S doxycycline-inducible cells, both derivatives of U251MG cells, were a gift from M. Georgescu (M.D. Anderson Cancer Center, Houston, TX) (17Radu A. Neubauer V. Akagi T. Hanafusa H. Georgescu M.M. Mol. Cell. Biol. 2003; 23: 6139-6149Crossref PubMed Scopus (187) Google Scholar). These cells were cultured in Dulbecco’s modified Eagle’s medium (4500 mg/liter glucose; Invitrogen) containing 10% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA) with G418 (400 μg/ml) and blasticidin (2 μg/ml) added. These cells were cultured in and grown in an incubator containing 5% carbon dioxide and 21% oxygen. Radiation Survival Determination—Cells were seeded into 60-mm dishes at defined densities and irradiated 1 h after addition of drug at a dose rate of 1.6 Gy/min using a Mark I cesium irradiator (J. L. Shepherd, San Fernando, CA). Colonies were stained and counted 10 to 14 days after irradiation. A colony by definition had >50 cells. The surviving fraction was calculated by dividing the number of colonies formed by the number of cells plated × plating efficiency. Each point on the survival curve represents the mean surviving fraction from at least three replicates. Protein Extraction, Western Blot Analysis—Details regarding protein isolation, gel electrophoresis, and Western blotting have been published previously (18Pore N. Jiang Z. Gupta A. Cerniglia G. Kao G.D. Maity A. Cancer Res. 2006; 66: 3197-3204Crossref PubMed Scopus (244) Google Scholar). The following antibodies were used: monoclonal anti-phospho Akt antibody that recognizes P-S473 (Cell Signaling, Danvers, MA), anti-Akt antibody (Cell Signaling) at a dilution of 1:1000, anti-b-actin antibody (Sigma) at a 1:1,000 dilution. The secondary antibody used for these blots was a goat anti-mouse antibody (Bio-Rad). Antibody binding was detected by chemiluminescence using an ECL kit (Amersham Biosciences). Flow Cytometry—Determination of cell cycle status was performed via flow-assisted cytometric analysis as described previously (19Vindelov L.L. Christensen I.J. Cytometry. 1990; 11: 753-770Crossref PubMed Scopus (228) Google Scholar). Cells were flash-frozen via liquid nitrogen immediately after harvesting and resuspension in citrate buffer (40 mm sodium citrate and 5% Me2SO) and stored at -70° until analysis. At analysis, all samples were thawed and stained with propidium iodide simultaneously. Flow analysis was performed on a BD Biosciences FACSCalibur flow cytometer, and the data were analyzed using CellQuest Pro cell cycle analysis program. Assays for γ-H2AX Activation—Following irradiation, cells were assessed via immunofluorescence for unrepaired DNA damage via phosphorylation of H2AX (γ-H2AX), a standard marker of unrepaired double strand DNA damage. For these experiments, cells were grown on coverslips. All groups of cells were fixed in 4% paraformaldehyde with 0.1% Triton-X100 and probed with anti-γ-H2AX antibody (Upstate Biological, Inc., Lake Placid, NY), followed by secondary antibody (anti-mouse Alexa Fluor 594 (Molecular Probes. subsidiary of Invitrogen). After the staining with specific antibody, the coverslips were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) to mark the nuclei. All treatment groups were then assessed for γ-H2AX foci via sequential imaging through each nuclei. A minimum of 300 cells in each treatment group were counted. U251MG glioblastoma cells are PTEN-deficient, resulting in a high level of Akt activation (5Haas-Kogan D. Shalev N. Wong M. Mills G. Yount G. Stokoe D. Curr. Biol. 1998; 8: 1195-1198Abstract Full Text Full Text PDF PubMed Google Scholar). Treatment of these cells with the PI3K inhibitor LY294002 increases their sensitivity to radiation as determined by clonogenic survival assays (Fig. 1A). Because pharmacologic agents may exert nonspecific effects, we wished to employ a genetic approach to verify these findings. To manipulate the level of PTEN and consequently of phospho-Akt in U251MG cells, we used a derivative cell line containing a tetracycline-inducible wild-type PTEN transgene (17Radu A. Neubauer V. Akagi T. Hanafusa H. Georgescu M.M. Mol. Cell. Biol. 2003; 23: 6139-6149Crossref PubMed Scopus (187) Google Scholar). We confirmed that addition of doxycycline to this cell line increased expression of PTEN and decreased the level of phospho-Akt, as expected (Fig. 1B). An isogenic control cell line was also used in which a mutant, phosphatase-dead form of PTEN (124S) was induced with doxycycline, which did not result in a decrease in phospho-Akt expression (Fig. 1B). U251-PTEN cells were treated with doxycycline, irradiated, and then assessed for clonogenic survival. Induction of wild-type PTEN in these cells led to radiosensitization (Fig. 1C), whereas no such effect was seen in U251-C124S, the control counterpart cell line in which mutant PTEN was induced (Fig. 1D). These experiments established that of the PI3K/Akt pathway in these cells led to increased sensitivity to Because with the cell it was important to that induction of wild-type PTEN did not the cell cycle that the addition of doxycycline led to a in the of cells in and in these cells at the of irradiation Therefore, in cell cycle not for the radiosensitization seen with PTEN We investigated whether PTEN induction might modulate unrepaired DNA damage, via of At of γ-H2AX is to as the number of which correlates with the of unrepaired damage J.A. J. P.M. van J. R. 2004; PubMed Scopus Google Scholar). of the U251-PTEN cell line treated with of doxycycline and ionizing radiation These data are in γ-H2AX foci were 1 h of irradiation in both and control These foci had substantially decreased in number by h in the control group and had by In the addition of doxycycline to U251-PTEN cells led to a of cells these foci at h (Fig. In support of the that these foci of unrepaired we the of the foci in surviving cells. At times after irradiation, γ-H2AX foci were which are of that are to unrepaired DNA damage H. Full M. R. G. Res. PubMed Scopus Google Scholar, M.J. J. Cell. PubMed Scopus Google Scholar, M. Res. PubMed Scopus Google Scholar) (Fig. of γ-H2AX foci in in U251 cells induced for wild-type PTEN U251 cells were and treated as described for but fixed for immunofluorescence at h after irradiation. cells were stained for γ-H2AX and then counterstained with to show groups of cells, with the in A or of the group of cells. and show staining and show and show both γ-H2AX and for and and then The of the in by the are at in and B. of γ-H2AX staining with that unrepaired double strand DNA To the that exposure to doxycycline in may the of DNA damage repair, we assessed the isogenic U251-C124S cell line in which PTEN was induced by the addition of doxycycline. (Fig. 1B). In contrast to the seen with the U251-PTEN line (Fig. induction of mutant PTEN showed of resolution of γ-H2AX foci (Fig. that exposure to doxycycline or the induction of an PTEN protein not influence DNA damage that PTEN on through of PI3K activity and of DNA damage As an test of this we assessed the effect of the PI3K inhibitor LY294002 on DNA damage These experiments also tested whether the radiosensitization noted with LY294002 (Fig. was attributable to DNA damage U251 cells were treated with LY294002 and then irradiated in the of the At 1 h after IR, the number of γ-H2AX foci was in the or of the The were seen at in which LY294002 in of γ-H2AX foci (Fig. A and These results that inhibition of PI3K activity in U251 cells not the sensing of DNA damage but leads to a reduced rate of repair of this damage. of a pathway that results in PI3K activity leads to of a marker of unrepaired double strand DNA damage. have that activation of the PI3K/Akt pathway is associated with to radiation in cell (6Gupta A.K. Bakanauskas V.J. Cerniglia G.J. Cheng Y. Bernhard E.J. Muschel R.J. McKenna W.G. Cancer Res. 2001; 61: 4278-4282PubMed Google Scholar, 7Grana T.M. Rusyn E.V. Zhou H. Sartor C.I. Cox A.D. Cancer Res. 2002; 62: 4142-4150PubMed Google Scholar, 8Edwards E. Geng L. Tan J. Onishko H. Donnelly E. Hallahan D.E. Cancer Res. 2002; 62: 4671-4677PubMed Google Scholar, 9Gottschalk A.R. Doan A. Nakamura J.L. Stokoe D. Haas-Kogan D.A. Int. J. Radiat. Oncol. Biol. Phys. 2005; 63: 1221-1227Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 10Gupta A.K. Cerniglia G.J. Mick R. Ahmed M.S. Bakanauskas V.J. Muschel R.J. McKenna W.G. Int. J. Radiat. Oncol. Biol. Phys. 2003; 56: 846-853Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 11Gupta A.K. McKenna W.G. Weber C.N. Feldman M.D. Goldsmith J.D. Mick R. Machtay M. Rosenthal D.I. Bakanauskas V.J. Cerniglia G.J. Bernhard E.J. Weber R.S. Muschel R.J. Clin. Cancer Res. 2002; 8: 885-892PubMed Google Scholar, 12Kim I.A. Bae S.S. Fernandes A. Wu J. Muschel R.J. McKenna W.G. Birnbaum M.J. Bernhard E.J. Cancer Res. 2005; 65: 7902-7910Crossref PubMed Scopus (168) Google Scholar, 13Lee C.M. Fuhrman C.B. Planelles V. Peltier M.R. Gaffney D.K. Soisson A.P. Dodson M.K. Tolley H.D. Green C.L. Zempolich K.A. Clin. Cancer Res. 2006; 12: 250-256Crossref PubMed Scopus (115) Google Scholar, 14Li B. Yuan M. Kim I.A. Chang C.M. Bernhard E.J. Shu H.K. Oncogene. 2004; 23: 4594-4602Crossref PubMed Scopus (143) Google Scholar, 15Nakamura J.L. Karlsson A. Arvold N.D. Gottschalk A.R. Pieper R.O. Stokoe D. Haas-Kogan D.A. J. Neuro-oncol. 2005; 71: 215-222Crossref PubMed Scopus (86) Google Scholar). We show in this using a PTEN-deficient cell line, that treatment with the PI3K inhibitor LY294002 or PTEN expression using a doxycycline-inducible leads to radiosensitization. this One is that might be of cells into of the cell flow showed that induction of wild-type PTEN in the cell cycle at the of irradiation this is to be the for the radiosensitization. H2AX is a that is on J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) by ataxia telangiectasia and kinase C. D. E. K. W. Curr. 2002; 12: PubMed Scopus Google Scholar, S. M. A. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, T. M. N. K. M. Cancer Res. 2004; PubMed Scopus Google Scholar) in response to DNA This occurs with a and by 10 J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of γ-H2AX is a and specific for unrepaired γ-H2AX the of repair factors such as and to foci at V. M. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, C. A. A. Biol. 2003; PubMed Scopus Google Scholar). is that the of these foci increases the of repair and repair of Therefore, γ-H2AX is not a marker for DNA damage repair but an role in the repair with this for both of the gene are but and are to ionizing radiation J. H. C. K. R. U. S. A. 2002; PubMed Scopus Google Scholar, A. S. B. V. E. M.J. C. A. M. L. K. A. 2002; PubMed Scopus Google Scholar). In the described we genetic and pharmacologic to show that a mechanism for the radiosensitization by PTEN is of the DNA damage repair that PTEN modulated the repair of DNA damage have investigated the of PI3K on γ-H2AX At the kinase activity of PI3K G. R. M.M. A. R. C. L. J. G. Cancer Res. Google Scholar). et al. S. M. A. J. Biol. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) found that 1 of the drug did not the induction of γ-H2AX after DNA damage. at γ-H2AX activation was These results are with the inhibition of PI3K, protein at high levels of Smith Cancer Res. Google Scholar, Clin. Cancer Res. Google Scholar, A.A. DNA 2004; PubMed Scopus Google Scholar, G.J. J. C. G. J. PubMed Scopus Google Scholar). et al. T. M. N. K. M. Cancer Res. 2004; PubMed Scopus Google Scholar) found that LY294002 did not decrease γ-H2AX foci in cells were for ATM, as the of the et al. V. M. Curr. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar) found that of cells with a high of with activation of γ-H2AX addition of the of after irradiation did not γ-H2AX foci the of foci resolution were not A genetic approach can nonspecific that pharmacologic An to using a cell line is that this results in specific of the PI3K/Akt pathway inhibition of kinase such as kinase or results are with of et al. M. U. S. J. K. M. E. Clin. Cancer Res. 2006; 12: PubMed Scopus Google γ-H2AX foci in carcinoma cells h after irradiation and found that the number of was in cells treated with LY294002 and radiation in cells treated with radiation this result is to with their that LY294002 γ-H2AX induction following radiation as determined by Western blotting in these cells M. U. S. J. K. M. E. Clin. Cancer Res. 2006; 12: PubMed Scopus Google Scholar). we found that inhibition by PTEN induction or by LY294002 did not expression of γ-H2AX the the number of 1 h after radiation was the in control cells as in the PTEN-induced or cells. the γ-H2AX foci in the PTEN-induced or cells. all the foci had from control cells but were in the treated cells. The that of the foci were seen in cells with (Fig. is with the that unrepaired DNA damage in these cells leads to cell radiation that then to the of both and Cells containing a at the after irradiation or form colonies N. Radiat. Res. PubMed Scopus Google Scholar). In we found that of PI3K/Akt signaling by PTEN induction or by treatment with the PI3K inhibitor LY294002 led to of unrepaired double strand DNA damage. These also that the PI3K/Akt pathway can modulate DNA damage repair in response to radiation and may have to the efficacy of radiation therapy in of human in which this pathway has been We for in flow and for the U251-PTEN and U251-C124S cells.