One possibility is that the reduced responsiveness of HCT116 cells with defective p53 to ATMi is due to that ATM signaling is already partially impaired in these cells

One possibility is that the reduced responsiveness of HCT116 cells with defective p53 to ATMi is due to that ATM signaling is already partially impaired in these cells. to ATMi radiosensitization. The mechanism for this p53 effect involves a propensity to undergo mitotic catastrophe relative to cells with wild-type p53. and significantly reduced migration, invasion, and growth, and, when infused directly into mouse orthotopic gliomas prior to repeated doses of radiation, significantly increased the survival of mice compared to radiation or drug alone treatments (21). However, except for the broad spectrum PI3K/mTOR inhibitor (NVP-BEZ235) that also inhibits ATM and DNA-PKcs (22), there are no reports, to our knowledge, of small-molecule ATM inhibitors that significantly cross the blood-brain barrier (BBB) during systemic treatment and demonstrate effective radiosensitization of gliomas studies, AZ31 was prepared in 10% DMSO + 90% Captisol (30%w/v) to 10 mg/ml, and AZ32 in hydroxypropyl-methyl cellulose (0.5%w/v)/0.1%w/v polysorbate-80 to 20 mg/ml. Both compounds were administered by oral gavage 1 hr prior to radiation. Plasmids and viruses Lentiviruses were generated in HEK293T cells (23). Lentivirus expressing H2B-mCherry has been described (23). The computer virus expressing the fusion EGFP-Centrin2 was constructed from pEGFP-Centrin 2 (provided by E. Nigg; Addgene plasmid #41147) and pWPXLd (23). Lentivirus shp53 pLKO.1 puro (provided by R. Weinberg; Addgene plasmid #19119) was used to knock down p53 with pBabe-puro (provided by H. Land, J. Morgenstern, and R. Weinberg; Addgene plasmid #1764) ACVR1B as vacant vector control. Cell culture Certified malignant glioma LN18, T98G, Hs683, SW1088 (anaplastic astrocytoma), SW1783 (anaplastic astrocytoma), U118MG, U138MG, M059J, A172, U87MG, H4, CF5STTG1 cells were obtained from the American Type Culture Collection (ATCC). AstraZeneca purchases cell lines from reputable providers such as ATCC and the European Collection of Authenticated Cell Cultures (ECACC) to ensure cell line authenticity. The AstraZeneca Global Cell Lender performs comprehensive quality inspections on all cell lines including species-specific Short Tandem Repeat (STR) profiling against a minimum of 9 published markers per species. Human glioma U1242, U87/luc-DsRed-p53(281G), and cell GSK-3787 derivatives expressing reporter genes were previously described (21). Mouse glioma GL261 cells were infected with Fluc-DsRed2 lentivirus (21) and sorted prior to cell injections. Similarly, certified NCI-H2228 non-small lung cancer cells were obtained from the ATCC. These cells were also modified to express luciferase (NCI-H2228-Luc) suitable for BLI. Cells were acquired and altered between 2009 and 2016. Cells were grown in complete Dulbeccos Modified Eagles Medium (Gibco) supplemented with 10% FBS and penicillin-streptomycin at 37C and 5% CO2. Cultures were maintained for no longer than 2 month and routinely tested unfavorable for mycoplasma. Radiosurvival (CFA) experiments were carried out as described (20, 21). Antibodies Primary antibodies include p-ATM Ser1981 (Epitomics), ATM (Genetex), p-KAP-1 Ser824 (Bethyl), KAP-1 (CalBiochem), p-p53 Ser15 (Cell Signaling), anti-p53 (Calbiochem or Santa Cruz DO-1), anti-p(T68)-CHK2 (Cell Signaling), anti-CHK2 (Cell Signaling), cleaved caspase 3 rabbit mAb (5A1E), anti–tubulin (Cell Signaling), and anti-GAPDH (EMD Millipore). Secondary antibodies for westerns were anti-rabbit Dylight-800 (Cell Signaling) and anti-mouse Alexa Fluor-680 (Invitrogen). Secondary antibodies for immunofluorescence were AlexaFluor-488, AlexaFluor-568, and AlexaFluor-647 anti-IgG (Invitrogen). Western blotting Western blotting of cell extracts were done as described (20). Mouse surgery and irradiation All procedures were carried out in accordance with protocol AM10197 approved by the VCU (Richmond, VA) Institutional Animal Care and Use GSK-3787 Committee (IACUC). Partial (head only) irradiation was performed using an MDS Nordion Gammacell 40 irradiator with a Cs-137 source at a dose rate of 1 1.05 Gy/min. For conformal micro-irradiation of mice a SARRP (Gulmay) was utilized. Mouse brain tumors were irradiated with 55-mm field either laterally or from the top of the head on the side of the tumor. Microscopy Cells were produced on chamber slides and processed for immunocytochemistry as described (24, 25). Brain tissues were processed as GSK-3787 described (21). Cell nuclei were counterstained with DAPI and mounted in VECTASHIELD mounting medium (Vector Laboratories). Imaging was performed on a Zeiss LSM 710 laser scanning confocal microscope and images were analyzed using Zeiss Zen software. Live cell imaging U87/H2B-mCherry/Centrin2-EGFP and U87/shp53/H2B-mCherry/Centrin2-EGFP cells were seeded on 4-chamber, glass-bottom CELLview tissue culture dishes (Grenier Bio-One) and allowed to GSK-3787 grow for 48C72 hrs. Time-laps videos were captured using a Zeiss Cell Observer SD spinning disk confocal microscope as described (26). AZ32.

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