Carlet et al. BMC Cancer 2010, 10:638 http://www.biomedcentral.com/1471-2407/10/638 genome” microarrays (Affymetrix HGU133 plus 2.0) to delineate the GC response in primary lymphoblasts from children suffering from ALL as well as from sev- eral other biological systems of GC-sensitivity and resis- tance [5]. Only a small number of genes were regulated by GC in the majority of patients, which might reflect the heterogeneity of the disease. Among these, PFKFB2 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-2), a key regulator of glycolysis [6], was one of the most frequently regulated genes. It was induced more than 4-fold in all 3 T-ALL cases as well as in the T-ALL cell line CCRF-CEM. More than 2-fold induction was observed in 8/10 children with precursor B-ALL and 1/1 adult with precursor B-ALL, but not in peripheral blood lymphocytes from 2 healthy donors treated with GC (such cells do not undergo apoptosis in response to GC) [5]. Interestingly, the second GC-sensitive ALL cell line tested in this study, 697/EU-3, and mouse thymo- cytes failed to regulate PFKFB2. In conclusion, PFKFB2 was induced in many primary ALL cells (particularly T-ALLs), but not in non-malignant lymphoid lineage cells, raising the possibility that this gene might play a functional role in GC-induced apoptosis in malignant lymphoid cells such as ALL. PFKFB2 is a member of a small gene family encoding 4 PFKFB isoenzymes. PFKFBs regulate formation and degradation of fructose-2,6-biphosphate (F-2,6-P2), a signalling molecule that controls glycolysis by regulating phosphofructokinase-1 (PFK-1) activity [7,8]. PFKFBs function as bifunctional enzymes: either they catalyze the synthesis of F-2,6-P2, a potent allosteric effector of PFK-1, via their 6-phosphofructo-2-kinase activity, or they degrade F-2,6-P2 to fructose 6-phosphate by acting as a fructose-2,6-biphosphatase. The kinase to biphosphatase activity ratio (K:B) is determined by the expression of specific isoforms, post-translational modi- fications of the enzyme as well as by numerous catabolic metabolites such as a-glycerol phosphate, citrate or ATP/GTP levels in the cells [9]. Initially identified in rat hepatocytes [8], 4 mammalian PFKFB isoenzymes have been described in different tissues and developmental stages, i.e., PFKFB1 in liver, skeletal muscles and fetal tissues; PFKFB2 in heart, kidney, pancreas islets and lymphoid tissues; PFKFB3 ubiquitously, particularly in brain; and the testis-specific PFKFB4. All isoenzymes share a highly conserved core structure comprising the kinase and phosphatase activities. The isoenzymes and their isoforms mainly differ in their N- and C-terminal variable regions, however, all of them are active as homodimers where the kinase domains come together in a head-to-head fashion, whereas the phosphatase domains function as monomers [7]. The human PFKFB2 gene codes for 2 isoforms (15A and 15B) generated by alternative splicing of the Page 2 of 11 terminal exon 15, thus differing in their C-terminal amino acid sequence [6]. Interestingly, in the more fre- quently investigated PFKFB2-15A isoform, this 54 amino acid region contains two PKA-sensitive phos- phorylation sites [10], i.e. Ser466 and Ser483, which have been shown to play an important role in the regu- lation of kinase- and phosphatase activity. Thus, phos- phorylation of Ser466 increased kinase affinity to fructose-6-phosphate (Fru-6-P), whereas dephosphoryla- tion of the same residues entailed increased phosphatase activity [11]. Since the PFKFB2-15B isoform lacks the above kinase-activating phosphorylation sites, it might lack kinase activity, raising the possibility that the 2 iso- forms may have opposite functions. Recently, alterations in glucose metabolism have been implicated in cell death and survival decisions, particu- larly in the lymphoid lineage [12] and in transformed cells [13]. Thus, lymphocytes depend upon extracellular signals transmitted via surface receptors and so-called survival kinases, like PKB/Akt, to maintain their viabi- lity. Part of these survival signals impinge on glucose metabolism, e.g., by increasing the expression of glucose transporters [14], with the net outcome of increased gly- colysis and ATP production. Decline in glycolytic activ- ity and ATP/ADP ratios results in integrity loss of mitochondria, the central regulators of metabolism and survival [15], with subsequent Bax-Bak dependent cyto- chrome-C release and cell death. Similarly, tumor cells depend upon glycolytic flux (Warburg effect) [16,17], which provides them with building blocks required for growth and energy. Hence, interference with “tumor metabolism” has become a promising target for cancer therapy [13]. The crucial role of PFKFBs in controlling glycolysis, along with the fact that one member of this small gene family, PFKFB2, was frequently regulated by GC in malignant lymphoblasts from children suffering from childhood ALL [5], makes this enzyme an interesting candidate for the anti-leukemic effects of GC. To further address this issue, we investigated expression and GC regulation of all 4 known PFKFB family mem- bers in the previously published and additional child- hood ALL patients and in additional in vitro leukemic systems. We found that only PFKFB2 showed consis- tent GC-induction, particularly, although not exclu- sively, in T-ALL systems. To assess a possible functional significance of this regulation, we performed conditional over-expression using the 2 known PFKFB2 splice variants in the T-ALL cell model CCRF-CEM in which both variants are dramatically induced by GC. However, neither splice variant replicated the apoptotic GC effects nor entailed over-expression a consistent modulating effect on GC-induced leukemia apoptosis.