Journal: Cancer Science

Article Title: Glutathione S‐transferase M1 inhibits dexamethasone‐induced apoptosis in association with the suppression of Bim through dual mechanisms in a lymphoblastic leukemia cell line

doi: 10.1111/j.1349-7006.2009.01432.x

Figure Lengend Snippet: Viral transduction of glutathione S‐transferase M1 (GSTM1) can influence dexamethasone sensitivity in an intracellular glutathione‐independent manner. (a) Genomic PCR analysis of the GSTM1 gene in the indicated cell lines. β‐actin was evaluated as an internal control. (b) Two CCRF‐CEM (CEM) clones expressing GSTM1 (CEM/M1‐4 and CEM/M1‐9) were selected by western blot analysis. Similar results were obtained in repeat blotting experiments. (c) GST activity of GSTM1‐expressing clones and control cells assayed by a colorimetric assay. Data are expressed as the mean ± SEM of three experiments. *P < 0.01; **P < 0.05 compared with CEM/mock. (d) Effect of the glutathione synthesis inhibitor (BSO) on cellular sensitivity to dexamethasone. The cells were incubated for 24 h with or without 50 μm BSO, exposed for a further 72 h to various concentrations of dexamethasone and then assayed in the XTT cell viability assay. Data are the mean ± SEM of at least three separate experiments. Treatment with BSO alone had no effect on cell viability. (e) The cells were incubated for 24 h with or without 50 μm BSO, exposed for a further 72 h to various concentrations of the dexamethasone, 4‐HC, carmustine, or chlorambucil and then assayed in the XTT cell viability assay. The data represent the ratio of the IC50 in the absence of BSO to the IC50 in the presence of BSO.

Article Snippet: Cell viability was evaluated using the XTT assay kit (Roche, Basel, Switzerland) as previously described. ( 23 ) Apoptotic cells were detected by the Annexin V–FITC Apoptosis Detection kit (Roche) according to the manufacturer’s instructions.

Techniques: Transduction, Clone Assay, Expressing, Western Blot, Activity Assay, Colorimetric Assay, Incubation, Viability Assay

Journal: Cancer Science

Article Title: Glutathione S‐transferase M1 inhibits dexamethasone‐induced apoptosis in association with the suppression of Bim through dual mechanisms in a lymphoblastic leukemia cell line

doi: 10.1111/j.1349-7006.2009.01432.x

Figure Lengend Snippet: NF‐κB p50 is responsible for glutathione S‐transferase M1 (GSTM1)‐induced resistance to dexamethasone. (a) The DNA binding activity of NF‐κB p50 and p65 subunits in the indicated CCRF‐CEM (CEM) cells was measured by ELISA. Relative activities are shown as the mean ± SEM of three separate experiments. **P < 0.05, and N.S., not significant, compared with CEM/mock. (b) Bcl‐3 mRNA expression was measured by quantitative RT‐PCR. Data are the mean ± SEM of relative expression levels in four independent experiments. *P < 0.01; **P < 0.05 compared with CEM/mock. (c) Cells were treated with or without 3 μm BAY11‐7082 (BAY) for 3 h, and the activity of p50 and p65 was measured by ELISA. Relative activities are shown as the mean ± SEM of three separate experiments. (d) Cells were treated for 12 h with or without BAY (3 μm), exposed for a further 36 h to 1 μm dexamethasone or medium and the amount of Bim protein was then determined by western blot analysis. Similar results were obtained in repeat experiments. (e) Cell viability was measured by the XTT assay and the mean IC50 was calculated from at least three separate experiments. *P < 0.01 compared with respective controls.

Article Snippet: Cell viability was evaluated using the XTT assay kit (Roche, Basel, Switzerland) as previously described. ( 23 ) Apoptotic cells were detected by the Annexin V–FITC Apoptosis Detection kit (Roche) according to the manufacturer’s instructions.

Techniques: Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot, XTT Assay