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. 2012 Sep;22(5):677-88.
doi: 10.1111/j.1750-3639.2012.00566.x. Epub 2012 Feb 21.

Characterization of glioma stem cells through multiple stem cell markers and their specific sensitization to double-strand break-inducing agents by pharmacological inhibition of ataxia telangiectasia mutated protein

Alessandro Raso  1 , Donatella Vecchio, Enrico Cappelli, Monica Ropolo, Alessandro Poggi, Paolo Nozza, Roberto Biassoni, Samantha Mascelli, Valeria Capra, Fotios Kalfas, Paolo Severi, Guido Frosina
Affiliations

Characterization of glioma stem cells through multiple stem cell markers and their specific sensitization to double-strand break-inducing agents by pharmacological inhibition of ataxia telangiectasia mutated protein

Alessandro Raso et al. Brain Pathol. 2012 Sep.

Abstract

Previous studies have shown that tumor-driving glioma stem cells (GSC) may promote radio-resistance by constitutive activation of the DNA damage response started by the ataxia telangiectasia mutated (ATM) protein. We have investigated whether GSC may be specifically sensitized to ionizing radiation by inhibiting the DNA damage response. Two grade IV glioma cell lines (BORRU and DR177) were characterized for a number of immunocytochemical, karyotypic, proliferative and differentiative parameters. In particular, the expression of a panel of nine stem cell markers was quantified by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. Overall, BORRU and DR177 displayed pronounced and poor stem phenotypes, respectively. In order to improve the therapeutic efficacy of radiation on GSC, the cells were preincubated with a nontoxic concentration of the ATM inhibitors KU-55933 and KU-60019 and then irradiated. BORRU cells were sensitized to radiation and radio-mimetic chemicals by ATM inhibitors whereas DR177 were protected under the same conditions. No sensitization was observed after cell differentiation or to drugs unable to induce double-strand breaks (DSB), indicating that ATM inhibitors specifically sensitize glioma cells possessing stem phenotype to DSB-inducing agents. In conclusion, pharmacological inhibition of ATM may specifically sensitize GSC to DSB-inducing agents while sparing nonstem cells.

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Figures

Figure 1
Figure 1
The DNA damage response pathway, possible target in glioma stem cells (GSC). After DNA damage is sensed by the Mre11‐Rad50‐Nbs1 (MRN)/human single strand DNA binding protein 1 (hSSB1) complex, the checkpoint transducers ataxia telangiectasia mutated (ATM) and ataxia telangiectasia Rad3‐related (ATR) change their conformation and/or localization, resulting in their activation. In turn, ATM and ATR activate a number of downstream molecules, including the checkpoint kinases 1 (Chk1) and 2 (Chk2). Chk1 and Chk2 are responsible for inhibition of cell division cycle (CDC) 25, a phosphatase required for activation of cyclin‐dependent kinases (Cdk) resulting in cell cycle arrest. KU‐55933 and KU‐60019 are specific inhibitors of ATM. Gö 6976 is a specific inhibitor of Chk1. BML‐277 is a specific inhibitor of Chk2. (Adapted and reprinted by permission from the American Association for Cancer Research—ref. (5).)
Figure 2
Figure 2
Characterization of the DR177 cell line. A. Morphological appearance (hematoxylin and eosin, ×100). B. Nestin expression (×100). C. Glial fibrillary acidic protein (GFAP) (×100).
Figure 3
Figure 3
Comparison of BORRU and DR177 glioma cell lines. A,B. Phase‐bright of cell cultures under epidermal growth factor‐fibroblast growth factor (EGF‐FGF)/serum‐free conditions in matrigel‐coated vessels (×20). C,D. Phase‐bright of neurosphere colonies under EGF‐FGF/serum‐free conditions in uncoated vessels (×20). E. Relative quantification of stem cell markers (Prom1, Bmi1, CD15, Msi1, Msi2, Nanog, Nestin, Oct4 and Sox2) in stem (EGF‐FGF) or differentiated [fetal calf serum (FCS)] cells. BORRU under differentiative conditions (FCS) were used as tissue control (ΔCtref). Data are the means ± SD of three experiments. F. Cytofluorimetric analysis of Prom1 on cell lines under EGF‐FGF/serum free conditions; gray profiles indicate the positive cell population, white profiles refer to cells incubated with the second reagent only. G. Proliferative activity; 0.5 × 106 cells were seeded under the indicated conditions and counted after 4 and 8 days. Data are the means ± SEM of three independent experiments. H,I. Phase‐bright of differentiated cells under 10% FCS (×20).
Figure 4
Figure 4
Specific sensitization of BORRU stem cells to ionizing radiation by ataxia telangiectasia mutated (ATM) inhibitors KU‐55933 and KU‐60019. A,B. Cells grown under stem conditions were plated in 96‐well plates in complete medium. The day after, cells were exposed to 1 µM KU‐55933 for 30 minutes at 37°C and then irradiated. Survival was evaluated 192 hours later by the MTT‐based assay. Data are the means ± SEM of 19 independent experiments. C,D. Cell cycle distribution under stem conditions; 90 000 cells were plated in 35 mm dishes, exposed to 1 µM KU‐55933 for 30 minutes, irradiated with 5 Gy and incubated for 192 hours. Cells were then harvested, fixed, permeabilized, stained with propidium iodide (PI) and analyzed by cytofluorimetry. Data are the means ± SEM of five independent experiments. E,F. As in (A,B) but with cells differentiated after exposure to 10% FCS for 2 weeks. Data are the means ± SEM of three independent experiments. G,H. As in (A,B) but with 1 µM KU‐60019. Data are the means ± SEM of 15 independent experiments. EGF‐FGF = epidermal growth factor‐fibroblast growth factor. FCS, fetal calf serum.
Figure 5
Figure 5
Ataxia telangiectasia mutated (ATM) activity and stem cell markers in BORRU cells exposed to KU‐60019 and ionizing radiation. BORRU cells were irradiated with 5 Gy ionizing radiation and extracted at the indicated times. A. The ATM‐dependent Ser15‐p53 phosphorylation was monitored by Western blotting as described under Materials and Methods. Protein loading levels were monitored by probing for actin. B. Relative quantification of stem cell markers (Prom1, Bmi1, CD15, Msi1, Msi2, Nanog, Nestin, Oct4 and Sox2) in control BORRU epidermal growth factor‐fibroblast growth factor (EGF‐FGF) and radiation‐survived BORRU after sensitization with KU‐60019 [(EGF‐FGF) + KU‐60019 + 5 Gy]. BORRU under differentiative conditions [fetal calf serum (FCS)] were used as tissue control (ΔCtref). Data are the means ± SD of three experiments.
Figure 6
Figure 6
Specific sensitization of BORRU stem cells to double‐strand break (DSB)‐inducing drugs by ataxia telangiectasia mutated (ATM) inhibitor KU‐60019. Cells grown under stem conditions were plated in 96‐well plates in complete medium. The day after, cells were exposed to 1 µM KU‐60019 for 30 minutes at 37°C and then treated with (A,B) the DSB‐inducing agent camptothecin (CPT); (C,D) the interstrand crosslinking agent mitomycin C (MMC); (E,F) the nucleotide precursor pool‐depleting agent hydroxyurea (HU). Data are the means ± SEM of at least three independent experiments.
Figure 7
Figure 7
Effects of checkpoint proteins 1 and 2 (Chk1 and Chk2) inhibition in BORRU and DR177 cells. A,B. Cells grown under stem conditions were plated in 96‐well plates in complete medium. The day after, cells were exposed to 0.25 µM Gö 6976 for 30 minutes at 37°C and then irradiated. Survival was evaluated 192 hours later by the MTT‐based assay. Data are the means ± SEM of three independent experiments. C,D. As in (A,B) but with cells exposed to 3 µM BML‐277. Data are the means ± SEM of four independent experiments.
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