Molecular Targets of Glucocorticoids in Context of Apoptosis Induction in Childhood Precursor T-Cell ALL (T-ALL).

Glucocorticoids (GCs) are principal therapeutic agents in ALL. In spite of their prominent clinical significance, molecular targets of GCs and, consequently, the mechanisms of GC-resistance are still unclear. In the present study, we focussed on molecular mechanisms of GC cytotoxicity in T-ALL, and investigated GC-specific response in leukemic cells at transcriptional and cellular levels. To this end, T-ALL samples (n=14) were investigated for GC-specific gene expression changes before onset of apoptosis (10 μM dexamethasone, 6 hr of incubation). Gene expression data obtained using Affymetrix HG Focus microarrays were normalized by variance stabilization on probe level, and genes consistently changed in GC-treated vs -untreated cells were identified using a SAM-like analysis of log ratios in an unbalanced permutation test. By these procedures, a group of 195 genes (false discovery rate = 0.005) was identified. Furthermore, the T-ALL samples were investigated for GC-specific apoptosis (10 μM dexamethasone, 20 hr). Apoptotic response to dexamethasone was highly heterogeneous with an apoptosis rate varying from 0% to 56% and a mean value of 23% (assessed by annexin V/propidium iodide staining). To identify genes with the highest impact on GC-specific apoptosis, we investigated the correlation between GC-specific gene expression changes and apoptosis rate in T-ALL samples. Expression changes of 25 genes were found to correlate significantly with the rate of apoptosis (Spearman regression analysis; correlation coefficients rs=0.54–0.81, p<0.05). First, we found that the GC-specific apoptosis was associated with a downregulation of genes encoding for cell cycle-promoting proteins (PTTG1, CDC7) and an upregulation of antiproliferative factors (ID2, BTG1), thus indicating that antiproliferative and cytotoxic activities of GCs are coupled in T-ALL. Furthermore, the rate of apoptosis correlated with a suppression of the commonly known survival signaling pathways, as indicated by the upregulation of the NFkappaB inhibitor IkappaB-alpha and downregulation of c-myc. In addition, we observed a downregulation of Bcl11A, a survival factor in the T-cell development. Finally, GC-specific apoptosis correlated with an upregulation of genes encoding for an actin depolymerizing factor DSTN and catabolic enzymes ISG20 (exonuclease) and CLPP (protease). Taken together, investigation of gene expression changes in the context of apoptotic cellular response identified common and T-cell specific survival pathways as GC-targets in T-ALL. In addition, our data suggest a novel GC-specific mechanism of cytotoxicity via direct transcriptional regulation of genes encoding for cellular catabolic proteins.