Carlet et al. BMC Cancer 2010, 10:638 http://www.biomedcentral.com/1471-2407/10/638 Page 8 of 11 Figure 2 Characterization of T-ALL cell lines with conditional PFKFB2-15A and -15B expression. CEM-PFKFB2-15A #D6 (Figure 2A) and CEM-PFKFB2-15B #66 (Figure 2B) cells expressing PFKFB2-15A and -15B in a doxycycline-dependent manner, respectively, were cultured in the presence of increasing concentration of doxycycline (Dox) for the indicated time and analysed for PFKFB2 expression (see Figures S2 and S3 for the corresponding data for the remaining 4 cell lines used in this study). mRNA expression was assessed by using quantitative real time RT-PCR targeting the exons-3/exon-4 boundary present in both splice variants. The data derive from 3 biological replicates analyzed in triplicate and expressed as -ΔCT (i.e., CT value of PFKFB2-15A minus CT value of TBP). PFKFB2 protein was detected by immunoblotting with antibodies against PFKFB2 and a-TUBULIN as loading control (right panels). Shown are representative examples of cells treated with the indicated amounts of doxycycline for 24 hours. primary malignant lymphoblasts and leukemic cell line models. PFKFB2 was identified by microarray analysis of lymphoblasts isolated from GC-treated children suffer- ing from ALL [5] as one of the most promising candi- date genes, since it was regulated in the majority of patients and its deregulation should entail disturbances in glucose metabolism which, in turn, have been impli- cated in cell death induction in general [12,13]. More specifically, GC resistance was associated with increased glucose consumption [32], and interference with this glucose dependency by simultaneous addition of 2-deoxy-D-glucose (2-DG) increased in vitro sensitivity to GC [32,33]. We therefore hypothesized that the apop- totic effect of GC might be triggered, at least in part, by reducing the glycolytic activity of the cell, and that this GC effect is mediated by upregulation of PFKFB2. To further test this hypothesis, we first performed detailed expression and regulation analysis of the 2 splice variants of this gene and of the 3 other members of the PFKFB gene family in several GC-sensitive and -resistant leukemic T-ALL and preB-ALL cell lines and peripheral blood cells from non-leukemic individuals. This made a possible contribution of the 3 other PFKFB isoenzymes to GC effects in such cells highly unlikely: PFKFB1 and -4 were not, or only very moderately, expressed and were not regulated by GC treatment, PFKFB3 was expressed, but not significantly regulated by GC (Additional file 1, Table S3). Concerning PFKFB2, we observed that both splice variants were expressed at similar levels (with PFKFB2-15A somewhat higher than -15B) and were co-regulated in most sys- tems. Although the number of patients is limited, GC induction was more pronounced (both in terms of fre- quency and extent of regulation) in T-ALLs compared to precursor B-ALLs. Concerning correlation with GC sensitivity, all ALL children were considered GC-sensi- tive because all showed a reduction in blast counts within the first 24 to 48 hours of treatment. Three