Figure 5.
Figure 5. The calcineurin/NFAT pathway is more active in PPR patients than in PGR patients. (A) PLCγ1 is more phosphorylated in Y783 in PPR (n = 24; mean = 4134 ± 470.7) than in PGR (n = 41; mean = 2992 ± 253.4) T-ALL patients (Mann-Whitney t test, P = .03). Results are presented as means ± SD. (B) Positive correlation (Spearman’s ρ = 0.57, P = .004) between active Src Y416 and PLCγ1 Y783 in PPR patients (left). In PGR patients, Src Y416 and PLCγ1 Y783 are not correlated (right). (C) Expression analysis by quantitative reverse transcription polymerase chain reaction (qRT-PCR) of the NFAT target genes IL-2 and IL-4 after LCK gene silencing in ALL-SIL cells. IL-4 mRNA decreases after LCK silencing (n = 3; paired t test, P = .01). Results are presented as means ± SEM. (D-F) Rescue of LCK activity in GC-sensitive T-ALL cells. (D) P12-ICHIKAWA cell viability was determined by MTT test after treatment with Dex only (48 hr), with anti-CD3 (1 hr) followed by Dex (48 hr), with WH-4-023 (0.01 μM, 1 hr) followed by Dex (48 hr), and with WH-4-023 (0.01 μM, 1 hr) followed by anti-CD3 (1 hr) and by Dex (48 hr). Cell viability of control cells was set to 100%. Results are presented as means ± SEM (n = 3). Anti-CD3 stimulation induced GC resistance in comparison with DMSO only, whereas WH-4-023 pretreatment was able to completely abrogate the anti-CD3 effect (paired t test). (E) WB analysis shows the hyperactivation of Src at Y416, PLC-γ1 at Y783, and NFATc1 in P12-ICHIKAWA cells after anti-CD3 stimulation. (F) qRT-PCR analysis of IL-4 mRNA expression in P12-ICHIKAWA cells after 48 hr of stimulation with anti-CD3 (n = 3, paired t test, P = .04). Unstimulated cells were set at 1. Results are presented as means ± SEM. (G-H) IL-4 affects GC response in T-ALL cells. (G) Increased cell viability measured by MTT assay of P12-ICHIKAWA (right) and KOPT-K1 (left) cells treated or not (CTRL) for 16 hr with IL-4 followed by Dex for 48 hr (n = 3). Cell viability of control cells was set to 100%. Results are presented as means ± SEM. (H) The anti-IL-4-neutralizing antibody increases GC-induced cell death in PPR T-ALL cell lines. Cells were treated with 100 ng/mL of blocking antibody for 16 hr prior to Dex treatment (10 μM for ALL-SIL and 1 μM for TALL-1 and CCRF-CEM, for 48 hr), and cell viability was evaluated by MTT assay, at least in triplicate. Cell viability of control cells was set to 100%. Results are presented as means ± SEM. *P < .05; **P < .01; ***P < .001.

The calcineurin/NFAT pathway is more active in PPR patients than in PGR patients. (A) PLCγ1 is more phosphorylated in Y783 in PPR (n = 24; mean = 4134 ± 470.7) than in PGR (n = 41; mean = 2992 ± 253.4) T-ALL patients (Mann-Whitney t test, P = .03). Results are presented as means ± SD. (B) Positive correlation (Spearman’s ρ = 0.57, P = .004) between active Src Y416 and PLCγ1 Y783 in PPR patients (left). In PGR patients, Src Y416 and PLCγ1 Y783 are not correlated (right). (C) Expression analysis by quantitative reverse transcription polymerase chain reaction (qRT-PCR) of the NFAT target genes IL-2 and IL-4 after LCK gene silencing in ALL-SIL cells. IL-4 mRNA decreases after LCK silencing (n = 3; paired t test, P = .01). Results are presented as means ± SEM. (D-F) Rescue of LCK activity in GC-sensitive T-ALL cells. (D) P12-ICHIKAWA cell viability was determined by MTT test after treatment with Dex only (48 hr), with anti-CD3 (1 hr) followed by Dex (48 hr), with WH-4-023 (0.01 μM, 1 hr) followed by Dex (48 hr), and with WH-4-023 (0.01 μM, 1 hr) followed by anti-CD3 (1 hr) and by Dex (48 hr). Cell viability of control cells was set to 100%. Results are presented as means ± SEM (n = 3). Anti-CD3 stimulation induced GC resistance in comparison with DMSO only, whereas WH-4-023 pretreatment was able to completely abrogate the anti-CD3 effect (paired t test). (E) WB analysis shows the hyperactivation of Src at Y416, PLC-γ1 at Y783, and NFATc1 in P12-ICHIKAWA cells after anti-CD3 stimulation. (F) qRT-PCR analysis of IL-4 mRNA expression in P12-ICHIKAWA cells after 48 hr of stimulation with anti-CD3 (n = 3, paired t test, P = .04). Unstimulated cells were set at 1. Results are presented as means ± SEM. (G-H) IL-4 affects GC response in T-ALL cells. (G) Increased cell viability measured by MTT assay of P12-ICHIKAWA (right) and KOPT-K1 (left) cells treated or not (CTRL) for 16 hr with IL-4 followed by Dex for 48 hr (n = 3). Cell viability of control cells was set to 100%. Results are presented as means ± SEM. (H) The anti-IL-4-neutralizing antibody increases GC-induced cell death in PPR T-ALL cell lines. Cells were treated with 100 ng/mL of blocking antibody for 16 hr prior to Dex treatment (10 μM for ALL-SIL and 1 μM for TALL-1 and CCRF-CEM, for 48 hr), and cell viability was evaluated by MTT assay, at least in triplicate. Cell viability of control cells was set to 100%. Results are presented as means ± SEM. *P < .05; **P < .01; ***P < .001.

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