Fig. 3

Nelarabine resistance does not depend on expression of ENT1/2 transporters and is partly due to upregulation of PI3K, MEK, and Bcl2 signaling. a Gene qRT-PCR analysis for ENT1 and ENT2 mRNA expression in T-ALL cell lines, untreated or treated with nelarabine for 48 h. Results are the mean from three different experiments ± SD. b qRT-PCR analysis for Bcl2 and Bcl-xL mRNA expression in T-ALL cell lines, untreated or treated with nelarabine for 48 h. Results are the mean from three different experiments ± SD. c Western blotting documenting an increase of p-AKT (Ser473), as well as p-ERK (Thr202), and Bcl2 in T-ALL resistant cell lines treated with nelarabine. Antibody to β-actin served as a loading control. d Densitometric analysis of western blotting shown in c was performed to quantify Bcl2 protein in resistant cell lines treated with nelarabine at different time points. The amount of protein was normalized to β-actin density and expressed as fold change compared to control (ratio = Bcl2 treated/Bcl2 control). Densitometry scanning of the bands was performed using a Chemidoc 810 Imager with the appropriate software (UVP, Upland, CA, USA). Statistical analyses were performed with the Dunnett’s multiple comparison test. Results showed a significant increase in the Bcl2 protein expression only in PEER cell line, at 48-h treatment, P < 0.05. e Flow cytometric functional efflux activity assay for 170 kDa P-glycoprotein (P-gp) in CEM-R (overexpressing P-gp) cells. The assay is based on extrusion of the fluorescent P-gp substrate, Rhodamine 123. The efflux activity of P-gp is highly temperature sensitive; P-gp functions optimally at 37 °C, but is inactive at 4 °C. When preloaded with Rhodamine 123 and incubated at 4 °C (CTRL), CEM-R cells retained the dye and consequently exhibited high fluorescence. Conversely, cells incubated at 37 °C (CTRL) effluxed the dye. CEM-R cells treated with nelarabine at 37 °C displayed a marked decrease in the pump activity. f Flow cytometric analysis of surface P-gp expression of CEM-R cells treated with nelarabine for 48 h (10 μM). The histograms are representative of two separate experiments