Figure 2
Figure 2. ABT-737 inhibits PV erythroblast proliferation and endogenous erythroid colony production. (A) ABT-737 inhibits the proliferation of PV erythroblasts. Erythroblasts at day 4 of liquid culture derived from CD34+ cells of 3 healthy donors (N) or of 3 JAK2V617F-high patients (PV) were plated in medium containing 3 U/mL erythropoietin (for normal HPCs) or 0.3 U/mL erythropoietin (for PV HPCs) in the presence of 20 nM ABT-737 (ABT-737) or an equivalent volume of DMSO (−). Cell growth is expressed as the mean and SEM of samples counted at different days of culture (left panel). Statistical analysis performed by means of 2-way ANOVA with Bonferroni post-tests showed statistical significances (*P < .05 at day 10; **P < .01 at day 13; and **P < .01 at day 15 between ABT-737–treated normal and PV samples). The percentage of growth inhibition exerted by increasing doses of ABT-737 was assessed by cultivating day-4 erythroblasts from 5 JAK2V617F-positive patients (PV) or 5 healthy donors (N) in medium containing 3 U/mL erythropoietin (for normal HPCs) or 0.3 U/mL erythropoietin (for PV HPCs) in the presence of DMSO or of the indicated doses of ABT-737 (right panel). The percentage of growth inhibition is expressed as the mean and SD of values calculated at day 13 of culture as described in “Study design.” Mann-Whitney nonparametric test showed statistical significance (*P = .028 for 5, 10, and 20 nM between normal and PV samples). (B) Number of BFU-E colonies obtained from the semisolid culture of normal (N) and JAK2V617F-high PV CD34+ cells (PV) plated in the presence of 500 nM ABT-737 or of an equivalent volume of DMSO (−). Data were analyzed with Wilcoxon matched pairs test and showed a statistical significance (**P = .007 between untreated and ABT-737–treated PV samples), whereas the difference between untreated and ABT-737–treated normal samples was not statistically significant. (C) Number of endogenous erythroid colonies (EECs) produced by normal (N) and PV peripheral blood mononuclear cells plated in the presence of 500 nM ABT-737 or of an equivalent volume of DMSO (−). **Mann-Whitney nonparametric test showed a statistical significance of P = .008 between untreated and ABT-737–treated PV samples. (D) GATA-1 expression levels in normal (N) and PV erythroblasts cultured with 3 U/mL erythropoietin or 0.3 U/mL erythropoietin, respectively and treated for 4 days with 20 nM ABT-737 or with an equivalent volume of DMSO (−). The right panel shows the quantification (mean ± SD) of bands shown on the left normalized to beta-actin. *Mann-Whitney nonparametric test showed a statistical significance of P < .05 between GATA-1 protein levels of ABT-737–treated normal and PV samples.

ABT-737 inhibits PV erythroblast proliferation and endogenous erythroid colony production. (A) ABT-737 inhibits the proliferation of PV erythroblasts. Erythroblasts at day 4 of liquid culture derived from CD34+ cells of 3 healthy donors (N) or of 3 JAK2V617F-high patients (PV) were plated in medium containing 3 U/mL erythropoietin (for normal HPCs) or 0.3 U/mL erythropoietin (for PV HPCs) in the presence of 20 nM ABT-737 (ABT-737) or an equivalent volume of DMSO (−). Cell growth is expressed as the mean and SEM of samples counted at different days of culture (left panel). Statistical analysis performed by means of 2-way ANOVA with Bonferroni post-tests showed statistical significances (*P < .05 at day 10; **P < .01 at day 13; and **P < .01 at day 15 between ABT-737–treated normal and PV samples). The percentage of growth inhibition exerted by increasing doses of ABT-737 was assessed by cultivating day-4 erythroblasts from 5 JAK2V617F-positive patients (PV) or 5 healthy donors (N) in medium containing 3 U/mL erythropoietin (for normal HPCs) or 0.3 U/mL erythropoietin (for PV HPCs) in the presence of DMSO or of the indicated doses of ABT-737 (right panel). The percentage of growth inhibition is expressed as the mean and SD of values calculated at day 13 of culture as described in “Study design.” Mann-Whitney nonparametric test showed statistical significance (*P = .028 for 5, 10, and 20 nM between normal and PV samples). (B) Number of BFU-E colonies obtained from the semisolid culture of normal (N) and JAK2V617F-high PV CD34+ cells (PV) plated in the presence of 500 nM ABT-737 or of an equivalent volume of DMSO (−). Data were analyzed with Wilcoxon matched pairs test and showed a statistical significance (**P = .007 between untreated and ABT-737–treated PV samples), whereas the difference between untreated and ABT-737–treated normal samples was not statistically significant. (C) Number of endogenous erythroid colonies (EECs) produced by normal (N) and PV peripheral blood mononuclear cells plated in the presence of 500 nM ABT-737 or of an equivalent volume of DMSO (−). **Mann-Whitney nonparametric test showed a statistical significance of P = .008 between untreated and ABT-737–treated PV samples. (D) GATA-1 expression levels in normal (N) and PV erythroblasts cultured with 3 U/mL erythropoietin or 0.3 U/mL erythropoietin, respectively and treated for 4 days with 20 nM ABT-737 or with an equivalent volume of DMSO (−). The right panel shows the quantification (mean ± SD) of bands shown on the left normalized to beta-actin. *Mann-Whitney nonparametric test showed a statistical significance of P < .05 between GATA-1 protein levels of ABT-737–treated normal and PV samples.

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