Carlet et al. BMC Cancer 2010, 10:638 http://www.biomedcentral.com/1471-2407/10/638 donors and GC-sensitive and -resistant CCRF-CEM derivates. Data for individual patients and cell lines are depicted in Additional file 1, Tables S1 and S2. PFKFB1 and PFKFB4 isoenzymes were neither detectably expressed in any of the investigated systems nor regu- lated by GC. The brain/placenta- and tumor-specific PFKFB3 isoenzyme showed expression levels comparable to those of PFKFB2 (for comparisons see Table 1), how- ever, in contrast to PFKFB2, its expression was not upregulated by GC. Interestingly, expression of PFKFB3 was also observed in peripheral blood lymphocytes from non-leukemic donors, indicating that this isoenzyme is not restricted to placenta and brain or malignant tissues. In conclusion, the PFKFB1 and 4 isoenzymes were neither expressed nor regulated by GC and, hence, might not play a role in the anti-leukemic GC effects. PFKFB3 is expressed, but shows no significant GC-regu- lation, excluding it from the list of likely candidate genes. Expression and regulation of PFKFB2-15A and -15B in lymphocytes and malignant lymphoblasts Next, we analyzed expression and GC regulation of the 2 splice variants of PFKFB2 in the above biological sys- tems. As summarized in Table 1, basal expression of both PFKFB2 isoforms was variable: almost all T-ALLs (known to show particularly strong response to GC) and ETV6/RUNX1 positive ALLs (which have a good prog- nosis) showed intermediate, all other subgroups, no or very low expression. In all groups with detectable expression of PFKFB2, mRNA levels (as measured by signal intensity) of PFKFB2-15A were ~2-fold higher than those of PFKFB2-15B. Concerning regulation, T-ALL patients showed upregulation of both splice var- iants at both time points, with mean M values between 1.7 and 2.7. This was also true for precursor B-ALL Page 5 of 11 patients, although the extent of regulation was consider- ably less (mean M values between 0.6 and 1.1). On an individual basis (Additional file 1, Table S1), the T-ALL group appeared quite homogeneous, with 5/6 T-ALL children showing induction (as defined by an M-value >1, i.e., >2-fold) after 6 and/or 24 hours of GC- treatment. In hyperdiploid precursor B-ALL children, isoform PFKFB2-15A was upregulated in 5/7 cases, with one child presenting a tendency for downregulation (M- value = -0.6), while PFKFB2-15B induction was present only in 1 of the 7 children after 6 hours. The subgroup of children with the ETV6/RUNX1 translocation showed induction of PFKFB2-15A in 4/10 children, 2 showed a slight downregulation, and 4 lacked PFKFB2-15A regula- tion. A more homogeneous situation was observed for the PFKFB2-15B isoform, where induction was detected in 8/10 children, with the remaining 2 showing a ten- dency to downregulate PFKFB2-15B after the first time point. The last subgroup [low hyperdiploid, E2A/PBX1 translocation, t(8;4) and no chromosomal abnormalities] showed upregulated PFKFB2-15A in 7/8 cases and PFKFB2-15B in 4/8, and no regulation in the others. In contrast to the ALL samples where PFKFB2 induction was a frequent event, this was seen only once and only for PFKFB2-15A in the non-leukemic donors. Concern- ing the in vitro cell lines systems, all 6 GC-sensitive CEM-C7H2 subclones showed clear induction of PFKFB2-15A and -15B, with M values ranging from 1.6 to 4.0 (15A) and 1.1 to 2.3 (15B). The 6 R-lines resulted in a remarkably heterogeneous picture, with M-values ranging from 0.0 to 5.1 (15A) and 0.5 to 3.5 (15B). Thus, despite the complexity of the expression profiling data, PFKFB2 induction appeared to be a frequent fea- ture in the GC response of T-lineage ALL and also occurred in precursor B-ALL, albeit with lower fre- quency. Moreover, PFKFB2 induction was absent or Table 1 Expression and regulation of PFKFB2 in different lymphoid systems1 samples T-ALL (6) Precursor B-ALL (25) - hyperdiploid (7) - EVT6/RUNX1 (10) - others (8) Non-leukemic donors (6*) CEM-C7H2-S-lines (6) CEM-C7H2-R-lines (6) Expression (mE-value) Regulation (mM-value) Regulation (mM-value) 15A 6.3 ± 1.5 4.6 ± 2.7 2.1 ± 0.7 6.7 ± 1.7 4.2 ± 2.7 3.4 ± 0.2 2.3 ± 0.1 2.7 ± 0.3 15B 4.0 ± 1.9 3.0 ± 1.7 1.5 ± 0.2 4.4 ± 1.5 2.4 ± 1.3 2.0 ± 0.4 1.8 ± 0.1 1.8 ± 0.1 15A 6/8 h 1.7 ± 1.5 0.7 ± 0.8 0.7 ± 0.6 0.5 ± 1.1 0.9 ± 0.8 0.5 ± 0.7 3.1 ± 0.9 2.0 ± 2.0 15A 24 h 2.4 ± 1.4 1.1 ± 1.2 0.9 ± 1.1 0.8 ± 1.5 1.5 ± 0.8 ND ND ND 15B 6/8 h 1.9 ± 2.3 0.6 ± 0.9 0.3 ± 0.4 0.8 ± 1.1 0.6 ± 0.8 0.2 ± 0.3 1.8 ± 0.4 1.6 ± 1.3 15B 24 h 2.7 ± 2.3 0.9 ± 1.1 0.3 ± 0.3 1.1 ± 1.2 1.3 ± 1.2 ND ND ND 1Expression and regulation of PFKFB2 in ChALL patients, non-leukemic controls (adult healthy donors and epileptic children) and sensitive (S) and resistant (R) CEM-C7H2 cell lines. Basal mean expression, before GC-treatment, is reported as mE-value ± SD; mean regulation was assessed after 6 and 24 hours and is expressed as mM-value ± SD. The total number of cases is reported in brackets next to the subgroup name. Data were assessed by analysis of Affymetrix HGU133 plus 2.0 microarrays. Abbreviation: ND, not determined.