Carlet et al. BMC Cancer 2010, 10:638 http://www.biomedcentral.com/1471-2407/10/638 reduced in systems resistant to GC-induced apoptosis (non-leukemic donors, CEM-C7H2-R-lines). Expression and regulation of PFKFB2 isoforms in additional leukemic cell line models To further address whether PFKFB2 regulation was related to GC-sensitivity and/or preB-ALL and T-ALL origin, we treated 9 leukemia cell lines with 10-7M dexa- methasone for 2, 6, 12 and 24 hours in biological tripli- cates and performed real time RT-PCR using primers covering the exon-3/exon-4 boundary present in both splice variants (Figure 1A). To assess the contribution of the individual splice variants, the 24 hour samples were further analyzed using primers specific for PFKFB2-15A and -15B (Figure 1B and 1C). As we previously reported [31], the extent of GC sensitivity in these cell lines was markedly different, i.e., untransfected Jurkat and MOLT4 T-ALL cell lines as well as AT-1 precursor B-ALL and Daudi Burkitt lymphoma cells were resistant to GC-induced apoptosis, while all others were GC-sen- sitive, although with varying kinetics (Additional file 1, Figure S1). Effects of GC on cell cycle progression were also determined [31] (Additional file 1, Figure S1) and showed that GC-induced apoptosis was frequently pre- ceded or accompanied by an increase of cells in the G1 phase of the cell division cycle, whereas the cell lines resistant to GC-induced apoptosis were also resistant to the GC effects on the cell cycle. The notable exception was AT-1, in which G1 cell cycle arrest was observed in the complete absence of apoptosis. As shown in Figure 1A, PFKFB2 induction was a frequent event in these cell lines starting as soon as 2 hours after initial GC expo- sure and increasing up to 24 hours. In those instances where PFKFB2 induction was observed, both splice var- iants appeared to be similarly regulated (Figure 1B). There was no apparent correlation between PFKFB2 induction and GC sensitivity or T/B-lineage origin of the leukemic cell lines. Concerning basal levels, the 2 splice variants showed co-expression across the panel of leukemia cell lines (Figure 1C), with PFKFB2-15A levels slightly exceeding those of -15B, thus resembling the findings on the microarrays. In general, B-lineage cells revealed lower expression levels than T-lineage cells (again resembling the situation in patients), but there was no apparent correlation between basal PFKFB2 levels and GC-sensitivity. For instance, GC-sensitive C7H2 T-ALL cells had similar PFKFB2 levels as GC-resistant MOLT4 T-ALL. The same was true for GC-sensitive JurkatGR and their parental GC-resistant Jurkat cell line. Similar to the situation in patients, GC-dependent induction was more pronounced in T-ALL cells than in B-lineage leukemias, but there was no obvious correla- tion with GC sensitivity across all samples. In the Jurkat Page 6 of 11 T-ALL system, however, induction of PFKFB2 correlated with GC sensitivity, as we have similarly seen in the CCRF-CEM system using microarray technology. Although we were unable to definitively prove regula- tion at the protein level (for reasons explained in Addi- tional file 1), the combined clinical and experimental mRNA data were compatible with the notion that induction of PFKFB2 contributes to GC-induced apop- tosis, particularly in T-ALL cells. Effect of conditional over-expression of PFKFB2 isoforms on cell viability and GC-sensitivity To evaluate a possible contribution of increased PFKFB2 levels to the anti-leukemic effects of GC, we generated cell lines conditionally overexpressing either PFKFB2- 15A or -15B in a doxycycline-dependent manner. Three clones for each isoform (termed CEM-PFKFB2-15A#C3, #D6, #E8 and CEM-PFKFB2-15B#65, #66, #95, respec- tively) were analyzed for regulation of the transgene by quantitative RT-PCR and immunoblotting (Figure 2 and Additional file 1, Figure S2 and S3). In all instances we obtained doxycycline dose- and time-dependent induc- tion of the PFKFB2 splice variants to levels as high as, or even exceeding, those obtained after GC treatment. Nevertheless, no effect was observed on cell viability, suggesting that induction of either PFKFB2 splice var- iant is not sufficient to explain apoptosis seen after GC exposure (Figure 3). In addition, the FACS data of PI-stained nuclei strongly suggested that there was no effect on cell cycle progression or GC-induced cell cycle arrest (data not shown). To investigate whether trans- genic expression of PFKFB2 resulted in altered GC sus- ceptibility, we treated the above T-ALL cell lines with 10-7M dexamethasone in the presence and absence of doxycycline (i.e., with and without transgenic PFKFB2 induction) and analyzed the extent of apoptosis (as measured by FACS analysis of propidium iodide incor- poration) at various time points (Figure 3). Neither PFKFB2-15A nor -15B over-expression significantly changed GC sensitivity in these cells, although both iso- forms were clearly detectable by immunoblotting (Figure 3C). Moreover, in the case of PFKFB2-15A where corre- sponding analyses can be performed, we observed that the activation-specific Ser466 [11] was phosphorylated, suggesting that the transgenic protein was not only sufficiently well expressed, but also active as a kinase. In conclusion, the data show that, at least in the investi- gated model system, neither PFKFB2-15A nor -15B over-expression mimics the anti-leukemic effects of GC, nor does it alter GC sensitivity. Discussion In this study, we addressed the possible functional sig- nificance of GC-dependent PFKFB2 regulation in