SFRP1 inhibited canonical Wnt signaling and cell proliferation of t(8;21)–leukemia cells. (A) Expression of Wnt signaling genes in AML cell lines (Kasumi-1, THP-1, NB4, and CMK) and BM samples from 3 patients with t(8;21)–AML was examined by the Human Wnt Signaling Pathway RT² Profile PCR Array, and PCR products were analyzed by 2% agarose gels. (B) TCF/LEF-, AP-1–, and NFAT-responsive luciferase reporters were cotransfected with SFRP1 expression plasmid into U937T-AE-Tet cells (U937T-AE cells cultured in the absence of tetracycline). These reporters contain constitutively expressing Renilla luciferase construct (40:1) for normalization of transfection efficiency. Cotransfection with the same amount of empty pCMV expression plasmid was done in parallel. LiCl was added to the culture medium 6 hours after transfection, and the cells were incubated for an additional of 24 hours before luciferase measurement. Transfection efficiency was normalized according to the cotransfected Renilla luciferase activity. Results are expressed as mean ± SE from at least triplicate assays and are presented as normalized luciferase activity (firefly luciferase [FLuc]/Renilla luciferase [RLuc]). (C) Analysis of nuclear β-catenin, phospho-JNK, and phospho-p38 MAPK proteins in Kasumi-1 cells treated with 500 ng/mL recombinant SFRP1 protein for 24 hours. Treatment with PBS was done as control. (Top) Nuclear β-catenin levels were examined by Western blot analysis. The same blot was probed with a β-actin Ab, which served as loading control. (Bottom) phospho-JNK (pJNK) and phospho-p38 MAPK (pp38 MAPK) proteins were analyzed by flow cytometry. Representative histograms and the median fluorescence intensity (expressed as mean ± SE) from triplicate assays are shown. (D) WST-1 analysis of cell proliferation of Kasumi-1 and THP-1 cells after 3-day treatment with different concentrations of recombinant SFRP1 and SFRP4 proteins. (E top) Real-time RT-PCR analysis of 13 genes related to Wnt signaling and cell growth control in Kasumi-1 cells treated with 500 ng/mL recombinant SFRP1 protein for 48 hours. Expression was normalized to GAPDH. (Bottom) Confirmation of the MYC and CCND1 down-regulation at protein level in the SFRP1-treated Kasumi-1 cells by Western blot analysis of whole-cell lysates. β-actin was used as a loading control. (D-E) Results are presented as percentage of the PBS-treated control group and expressed as mean ± SE from at least triplicate assays. *P < .05.