Retinoid-dependent up-regulation of TNFR2, TNFAIP2, and ELF4 genes in APL. (A) TNFR2, TNFAIP2, and ELF4 gene expression in APL patient samples by real-time RT-PCR. Leukemia samples from APL patients (with t(15;17)) were examined for retinoid-dependent regulation of TNFR2, TNFAIP2, and ELF4 genes by real-time RT-PCR using TaqMan probes. Leukemic cells obtained at diagnosis were either untreated or treated with ATRA (1 μM) in vitro for (i) 6 hours or (ii) 2 and 5 days (patient no. 3). Retinoid-dependent up-regulation of gene expression is shown as fold induction. ^*Where gene expression (in retinoid untreated) is below detectable level, data point was arbitrarily assigned at the lowest linear expression level in the standard curve to calculate the fold-induction. (B) Retinoid-dependent up-regulation of TNFR2 protein expression. NB4 and NB4-R2 cells were either untreated or treated with ATRA (1 μM) for 24 hours. TNFR2 protein expression was examined by FACS using a PE-labeled monoclonal human TNFR2 antibody. (C) Enhanced granulocytic differentiation with combined treatment of RA and TNFα. (D) Effect of RA and TNFα on apoptosis. NB4 and NB4-R2 cells were cultured in media containing ATRA (0.1 μM) alone, TNFα (10 ng/mL) alone, or ATRA plus TNFα for 7 days. Cells were harvested at 0, 2, 4, and 7 days for the analyses of cell differentiation or apoptosis. Granulocytic differentiation was assessed by measuring CD66b expression using FITC-conjugated CD66b antibody by FACS. Apoptosis was measured by FACS following reactions with FITC-conjugated Annexin V and 7-amino-actinomycin (7-AAD). Annexin V-positive and vital dye-negative cells were counted as apoptotic cells. Means and standard deviations from triplicate experiments are represented.