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. 2014 Jan;28(1):203-6.
doi: 10.1038/leu.2013.215. Epub 2013 Jul 15.

mTOR kinase inhibitor sensitizes T-cell lymphoblastic leukemia for chemotherapy-induced DNA damage via suppressing FANCD2 expression

F Guo  1 , J Li  1 , S Zhang  1 , W Du  1 , S Amarachintha  1 , J Sipple  1 , J Phelan  2 , H L Grimes  2 , Y Zheng  1 , Q Pang  1
Affiliations

mTOR kinase inhibitor sensitizes T-cell lymphoblastic leukemia for chemotherapy-induced DNA damage via suppressing FANCD2 expression

F Guo et al. Leukemia. 2014 Jan.
No abstract available

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Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
pp242 sensitizes leukemic cells to chemo-agent-induced apoptosis and DNA damage in vitro through downregulation of FANCD2. (a) Molt-Luc2 cells were pretreated with or without pp242 (2.4μM) or Rapamycin (20 nM) for 3 h, followed by incubation with or without AraC (10μM) or Etoposide (10μM) for additional 24 h. Cell viability (survival) was examined by luminescence and plotted as the percentage of viable cells relative to untreated control. (b) Molt-Luc2 cells were treated as described in (a). The cells were then stained with Annexin V and 7-AAD. Annexin V+ apoptotic cells were determined by flow cytometry. (c) Molt-Luc2 cells were treated as described in (a). γH2AX foci were visualized by immunofluorescence staining. The percentage of cells with ≥ 6 foci was determined. (d) Molt-Luc2 cells were treated as described in (a) and DNA strand breaks were measured using Comet Assay (single-cell electrophoresis). Left, representative images. Right, the length and intensity of DNA tails (DNA strand breaks) relative to heads (nuclei) were determined as olive tail moment. (e) Molt-Luc2 cells were treated with or without pp242 (2.4μM) or Torin 1 (100 nM) for 24 h, and the indicated phosphorylated and total proteins were detected by Western blotting. (f) Molt-Luc2 cells were treated with or without Rapamycin (20 nM) or pp242 (2.4μM) for 24 h or 48 h. FANCD2 expression was examined by Western blotting. Actin or Topo1 was blotted as loading control. (g, h, i) Molt-Luc2 cells were transduced with mock vector or FANCD2 by retroviral infection, and the cells were then pretreated with or without pp242 (2.4μM) for 3 h, followed by incubation with or without AraC (10μM) for additional 24 h. FANCD2 expression was examined by Western blotting. Topo1 was blotted as loading control (g). Apoptotic cells were determined by Annexin V and 7-AAD staining followed by flow cytometry analysis (h). γH2AX content was examined by flow cytometry and mean fluorescence intensity (MFI) was shown (i). *P<0.05; **P<0.01. Error bars represent mean ± SEM.
Figure 2
Figure 2
pp242 enhances anti-leukemia efficacy of AraC in vivo. (a, b) 1×106 Molt-Luc2 T-ALL cells expressing the luciferase maker were injected i.v. into sub-lethally irradiated NSG recipient mice, which after 10 days were injected i.p. with the indicated chemicals daily for 5 days. Survival outcome of the mice was monitored and recorded. Results were analyzed with a log-rank (Mantel-Cox) test and expressed as Kaplan-Meier survival curves. Control: mice without Molt-Luc2 cells and chemical treatment; Vehicle: mice with Molt-Luc2 cells treated with 20% DMSO, 40% PBS, and 40% polyethylene glycol-400; AraC: mice with Molt-Luc2 cells treated with AraC (50mg/Kg); Rapamycin: mice with Molt-Luc2 cells treated with Rapamycin (4 mg/Kg); pp242: mice with Molt-Luc2 cells treated with pp242 (20mg/Kg); Rapamycin + AraC: mice with Molt-Luc2 cells treated with the combination of Rapamycin with AraC; pp242 + AraC: mice with Molt-Luc2 cells treated with the combination of pp242 with AraC. n=5 mice for Control and n=10 mice/group for the rest of experimental groups. **P < 0.01, pp242 + AraC vs Vehicle, pp242 or AraC. n=5 mice for Control and n=10 mice/group for the rest of experimental groups. **P < 0.01, pp242 + AraC vs Vehicle, pp242, or AraC. (c) NSG mice were transplanted with 1×106 Molt-Luc2 cells and treated with chemicals as in (b). Fifteen days after chemical treatment, the distribution and number of Molt-Luc2 cells in the mice were determined by visualizing luciferase using the IVIS system. (d, e, f) NSG mice were transplanted with Molt-Luc2 cells. Fourteen days after transplantation, the mice were treated once with Vehicle, AraC, pp242, or the combination of pp242 with AraC as in (b). The mice were sacrificed at the indicated time points post chemical treatment. Molt-Luc2 cells from the mice were subjected to flow cytometry analysis of human CD45+ cells for apoptosis by Annexin V and 7-AAD staining (d) and DNA damage by γH2AX staining (MFI: mean fluorescence intensity) (e). The cells were sorted and the expression of FANCD2 was quantified by quantitative real-time RT-PCR (f). *P<0.05; **P<0.01. Error bars represent mean ± SEM (n=4 mice/group). (g) pp242 enhances anti-leukemia efficacy of AraC in vivo in primary T-ALL patient sample-xenografted mice. 1×106 primary T-ALL patient cells were injected i.v. into sub-lethally irradiated NSG recipient mice, which after 10 days were injected i.p. with the indicated chemicals daily for 5 days. Survival outcome of the mice was monitored and recorded. Results were analyzed with a log-rank (Mantel-Cox) test and expressed as Kaplan-Meier survival curves. Control: mice without primary T-ALL patient cells and chemical treatment; Vehicle: mice with primary T-ALL patient cells treated with 20% DMSO, 40% PBS, and 40% polyethylene glycol-400; AraC: mice with primary T-ALL patient cells treated with AraC (50mg/Kg); pp242: mice with primary T-ALL patient cells treated with pp242 (20mg/Kg); pp242 + AraC: mice with primary T-ALL patient cells treated with the combination of pp242 with AraC. n=5 mice for Control and n=10 mice/group for the rest of experimental groups. **P < 0.01, pp242 + AraC vs Vehicle, pp242, or AraC.
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