Figure 6.
Relationship between hypoxia, HIF-1α expression, mTOR activation, and chemoresistance in T-ALL. (A) Phosphorylation of mTOR (pS2448) and (B) 4EBP1 and S6R. Shown are representative histograms (i) and MFI ratio obtained with leukemic cells harvested from hypoxia compared with normoxia cultures (Aii,Bi,Bii). Every dot is the mean of technical triplicates. In panel A, T-ALL #1, n = 4exp; T-ALL #3, n = 4exp; and T-ALL #5, n = 1exp. In panel B, T-ALL#1, n = 2exp; T-ALL#3, n = 1exp. (C) Effect of rapamycin (Rapa) on T-ALL growth in normoxia. Absolute leukemic cell number recovered from every culture condition. The data are expressed as fold variation between the number of cells recovered after 4 days of coculture in presence or absence of rapamycin compared with cells plated at day 0. Every dot is the mean of technical triplicates. T-ALL #1, n = 5exp; T-ALL #2, n = 3exp; T-ALL #3, n = 3exp; T-ALL #4, n = 1exp; and T-ALL #5, n = 3exp. (D) Rapamycin decreases T-ALL cell cycle progression. Shown are proportions of leukemic cells in G0, G1, S, and G2/M phases, during culture at 21% of O2 in presence or absence of rapamycin. Shown are mean ± SEM of triplicate cultures performed with T-ALL #1, T-ALL #2, and T-ALL #4, n = 6exp. (Ei) Rapamycin protects T-ALL from vincristine in normoxia. Shown are the absolute numbers of cells recovered after vincristine treatment in normoxia in presence or absence of rapamycin. (Eii) Same results are presented as percentage of live cells after vincristine treatment in presence (+Rapa) or in absence (Ø) of rapamycin compared with nontreated cells. Every dot is the mean of technical triplicates from T-ALL #1, n = 2exp; T-ALL #2, n = 2exp; T-ALL #3, n = 3exp; and T-ALL #5, n = 3exp. (F) Leukemic cell number recovered 7 days after replating T-ALL pretreated with vincristine ± rapamycin. Shown are mean ± SEM of technical triplicate cultures. (G) HIF-1α impacts on mTOR phosphorylation in low and high O2 concentration. (i) Phosphorylation of mTOR (pS2448) in a representative experiment with T-ALL #1. (ii) Ratio of MFI from shCTL/T-ALL or shHIF-1α/T-ALL cells cultured in hypoxia compared with those cultured in normoxia. Every dot is the mean of technical triplicates obtained with T-ALL #1, n = 2exp; and T-ALL #3, n = 1exp. Statistics were determined using the Friedman test (A), Wilcoxon test (B-E,G), or Mann-Whitney test (F): *P < .05, **P < .01, or ***P < .001. (H) Percentages of live shCTL/T-ALL and shHIF-1α/T-ALL cells recovered after treatment during 72 hours in hypoxia with vincristine in presence (+Rapa) or in absence (Ø) of rapamycin compared with nontreated cells. (I) Leukemic cell production after 7 days in normoxia without treatment after T-ALL. exp, experiment.

Relationship between hypoxia, HIF-1α expression, mTOR activation, and chemoresistance in T-ALL. (A) Phosphorylation of mTOR (pS2448) and (B) 4EBP1 and S6R. Shown are representative histograms (i) and MFI ratio obtained with leukemic cells harvested from hypoxia compared with normoxia cultures (Aii,Bi,Bii). Every dot is the mean of technical triplicates. In panel A, T-ALL #1, n = 4exp; T-ALL #3, n = 4exp; and T-ALL #5, n = 1exp. In panel B, T-ALL#1, n = 2exp; T-ALL#3, n = 1exp. (C) Effect of rapamycin (Rapa) on T-ALL growth in normoxia. Absolute leukemic cell number recovered from every culture condition. The data are expressed as fold variation between the number of cells recovered after 4 days of coculture in presence or absence of rapamycin compared with cells plated at day 0. Every dot is the mean of technical triplicates. T-ALL #1, n = 5exp; T-ALL #2, n = 3exp; T-ALL #3, n = 3exp; T-ALL #4, n = 1exp; and T-ALL #5, n = 3exp. (D) Rapamycin decreases T-ALL cell cycle progression. Shown are proportions of leukemic cells in G0, G1, S, and G2/M phases, during culture at 21% of O2 in presence or absence of rapamycin. Shown are mean ± SEM of triplicate cultures performed with T-ALL #1, T-ALL #2, and T-ALL #4, n = 6exp. (Ei) Rapamycin protects T-ALL from vincristine in normoxia. Shown are the absolute numbers of cells recovered after vincristine treatment in normoxia in presence or absence of rapamycin. (Eii) Same results are presented as percentage of live cells after vincristine treatment in presence (+Rapa) or in absence (Ø) of rapamycin compared with nontreated cells. Every dot is the mean of technical triplicates from T-ALL #1, n = 2exp; T-ALL #2, n = 2exp; T-ALL #3, n = 3exp; and T-ALL #5, n = 3exp. (F) Leukemic cell number recovered 7 days after replating T-ALL pretreated with vincristine ± rapamycin. Shown are mean ± SEM of technical triplicate cultures. (G) HIF-1α impacts on mTOR phosphorylation in low and high O2 concentration. (i) Phosphorylation of mTOR (pS2448) in a representative experiment with T-ALL #1. (ii) Ratio of MFI from shCTL/T-ALL or shHIF-1α/T-ALL cells cultured in hypoxia compared with those cultured in normoxia. Every dot is the mean of technical triplicates obtained with T-ALL #1, n = 2exp; and T-ALL #3, n = 1exp. Statistics were determined using the Friedman test (A), Wilcoxon test (B-E,G), or Mann-Whitney test (F): *P < .05, **P < .01, or ***P < .001. (H) Percentages of live shCTL/T-ALL and shHIF-1α/T-ALL cells recovered after treatment during 72 hours in hypoxia with vincristine in presence (+Rapa) or in absence (Ø) of rapamycin compared with nontreated cells. (I) Leukemic cell production after 7 days in normoxia without treatment after T-ALL. exp, experiment.

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