Induction of RKIP protein and pERK1/2 expression by inhibition of Bcr-Abl in CML cells. A, the effects of Abl kinase inhibitors and Bcr-Abl siRNA on RKIP expression were assessed by Western blotting. After 24 h treatment with Abl kinase inhibitors or 5 days after transfection with control siRNA or Bcr-Abl siRNA, total cell lysates from K562 (upper left) and Meg01 (upper right) cells were prepared and analyzed by immunoblotting. Actin was used as a loading control. Representative blots for two CML cell lines are shown. Relative amounts of mRNAs for Bcr-Abl were measured in K562 (lower left) and Meg01 (lower right) cells. The mRNA expression of Bcr-Abl was assessed by RT-PCR. The expression levels of the target mRNAs were normalized to the relative ratio of the expression of GAPDH mRNA. The results are expressed relative to the untreated control set at 1. Each RT-PCR assay was performed at least three times, and the results are expressed as mean ± S.D. *, p < 0.01 compared with untreated control cells. B, after 24 h treatment with the Abl kinase inhibitors (STI571 (10 μm), AMN107 (10 μm), BMS354825 (10 nm)), ERK1/2 was immunoprecipitated from K562 and Meg01 cells untransfected and 5 days post-transfected with Bcr-Abl siRNA or RKIP siRNA. Immunoprecipitates (IP) were analyzed by Western blot (WB) analysis with ERK1/2-specific antibodies and phosphorylated ERK1/2-specific antibodies. Actin was used as a loading control. Representative blots from two CML cell lines are shown. The upper bar graphs represent the effect of the Abl kinase inhibitors or siRNA knockdown of RKIP and Bcr-Abl on ERK1/2 phosphorylation. Each ERK1/2 phosphorylation normalized to each total ERK1/2 was expressed relative to the untreated control set at 1. Relative amounts of mRNAs for RKIP were measured in K562 and Meg01 cells. The mRNA expression of RKIP was assessed by RT-PCR. The expression levels of the target mRNAs were normalized to the relative ratio of the expression of GAPDH mRNA. The results were expressed relative to the untreated control set at 1. Each RT-PCR assay was performed at least three times, and the results are expressed as mean ± S.D. **, p < 0.05; *, p < 0.01 compared with untreated control cells.

Effects of RKIP overexpression on cell cycle in CML cells. A, K562 and Meg01 cells were transfected with RKIP cDNA. The cells were harvested 5 days post-transfection, and fluorescence-activated cell sorter analysis was performed to determine the fraction in various cell cycle stages. Data are shown as mean ± S.D. in triplicate cultures and are representative of three independent experiments. *, p < 0.01 compared with untreated control cells. B, evaluation of RKIP, FoxM1, KIS, p27^Kip1, and p21^Cip1 protein levels after transfection with RKIP cDNA into the cells. The cells were transfected with RKIP cDNA, harvested after 5 days, and Western blotting was performed by the antibodies for various cell cycle regulator proteins. Actin was used as a loading control. Representative blots for two CML cell lines are shown.

RKIP expression on ALDH^hi hematopoietic progenitor cells derived from CML patients. A, ALDH^hi cells were transfected with RKIP cDNA or RKIP siRNA and then treated with STI571 (10 μm) for 14–17 days. The numbers of CFU-GEMM, CFU-GM, and BFU-E were counted. The rate of the progenitor cells was evaluated as a percentage of the corresponding control. Results are mean ± S.D. from three independent experiments. *, p < 0.01 compared with untreated control cells. B and C, the relative expression levels of RKIP (B) and FoxM1 (C) mRNA of CFU-GEMM, CFU-GM, and BFU-E derived from RKIP cDNA or RKIP siRNA-transfected ALDH^hi cells were assessed after 14–17 days treatment with STI571 (10 μm). For analysis of RKIP and FoxM1 expression, quantitative RT-PCR was performed relative to the GAPDH (G3PDH) gene. Data are shown as mean ± S.D. in triplicate cultures and are representative of three independent experiments (upper panels). **, p < 0.05; *, p < 0.01 compared with untreated control cells. Fourteen days after treatment with STI571 in ALDH^hi cells transfected with RKIP cDNA or RKIP siRNA, the cells from CFU-GEMM, CFU-GM, and BFU-E were harvested. To detect RKIP and FoxM1, RT-PCR was performed. GAPDH is shown as an internal control. The RT-PCR results are representative of three independent experiments (bottom panels).

RKIP mRNA expression in leukemia cells. A, CML (K562, Meg01, and SHG3) and AML (U937, HL60, and YRK2) cell lines were untreated or treated with STI571 (10 μm), ANM107 (10 μm), and BMS354825 (10 nm) for 24 h. CML cells were harvested 5 days after transfection with control siRNA or Bcr-Abl siRNA. AML cell lines were harvested 7 days after transfection with LV-Con or LV-Bcr-Abl. RT-PCR was performed to detect RKIP and Bcr-Abl mRNAs. GAPDH (G3PDH) is shown as an internal control. RT-PCR results are representative of three independent experiments. B, relative amounts of mRNAs for RKIP and Bcr-Abl were measured in K562 (CML), Meg01 (CML), and U937 (AML) cells. CML cells were harvested after 24 h treatment with STI571 (10 μm), ANM107 (10 μm), or BMS354825 (10 nm) or 5 days after transfection with control siRNA or Bcr-Abl siRNA. U937 cells were harvested after 24 h treatment with STI571 (10 μm), ANM107 (10 μm), and BMS354825 (10 nm) or 5 days after transfection with LV-Con and LV-Bcr-Abl. The mRNA expression of RKIP and Bcr-Abl was assessed by RT-PCR. The expression levels of the target mRNAs were normalized to the relative ratio of the expression of GAPDH mRNA. The results were expressed relative to the untreated control set at 1. Each RT-PCR assay was performed at least three times, and the results are expressed as mean ± S.D. *, p < 0.05 compared with untreated control. C, expression of RKIP and Bcr-Abl mRNA in ALDH^hi cells isolated from the bone marrow of a CML patient. Representative flow cytometric analysis of ALDH activity in bone marrow cells from a CML patient and a healthy volunteer. Bone marrow cells from a healthy volunteer and CML patient were incubated with Aldefluor substrate and isolated by using the gated region P1 (0.1 ± 0.08% (upper left panel) and 1.9 ± 0.3% (upper right panel), respectively). The expression of RKIP and Bcr-Abl mRNA in sorted ALDH^hi cells was analyzed after treatment with Abl kinase inhibitors. Representative data from one CML sample are shown (bottom left). The effects of the Abl kinase inhibitors on RKIP expression were assessed by RT-PCR in ALDH^hi cells from CML patients (n = 10). The expression levels of the target mRNAs were normalized to the relative ratio of the expression of GAPDH mRNA. The results are expressed relative to the untreated control set at 1. Each RT-PCR assay was performed at least three times, and the results are expressed as mean ± S.D. *, p < 0.01 compared with untreated control cells.

Anti-proliferative effects of RKIP in K562 cells. A and C, K562 cells were transfected with RKIP cDNA or siRNA. After 48 h, the cells were treated with STI571 (10 μm) for 24, 48, and 72 h. Cell numbers were counted (A) and cell viability (C) was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. The rate of cell survival is expressed as the percentage of the corresponding control. Results are presented as the mean ± S.D. from three independent experiments. B and D, the RKIP cDNA or siRNA-transfected or untransfected K562 cells were collected at the indicated times after the Abl kinase inhibitors. For analysis of RKIP mRNA expression, quantitative RT-PCR was performed relative to the GAPDH gene (B and D). Each RT-PCR assay was performed at least three times, and the results are expressed as mean ± S.D. **, p < 0.05; *, p < 0.01 compared with untreated control cells.

RKIP expression was induced by Abl kinase inhibitor or knock-down of Bcr-Abl in CML progenitor cells. A, the purified ALDH^hi cells from one CML patient were isolated and cultured in semisolid methylcellulose medium (Methocult; H4435). The ALDH^hi cells, which were untransfected or transfected with Bcr-Abl siRNA, were treated with Abl kinase inhibitors for 14–17 days. The numbers of CFU-GEMM, CFU-GM, and BFU-E were then counted. When untreated, the mean number of CFU-GEMM was 42 (range, 37–44). When treated with STI571, AMN107, BMS354825, or Bcr-Abl siRNA transfection, the mean numbers of CFU-GEMM were 4 (3–5), 4.2 (2–5), 1.3 (0–2), and 1.3 (0–2), respectively. When untreated, the numbers of CFU-GM were 224 (189–251). When treated with STI571, AMN107, BMS354825, or Bcr-Abl siRNA transfection, the mean numbers of CFU-GEMM were 31.1 (28–33), 40.8 (37–46), 38.8 (35–49), and 36.1 (31–42), respectively. When untreated, the numbers of BFU-E were 179 (157–191). When treated with STI571, AMN107, BMS354825, or Bcr-Abl siRNA transfection, the mean numbers of CFU-GEMM were 38.9 (33–41), 32.6 (28–36), 25.4 (21–28), and 22.2 (17–26), respectively. CFU-GEMM, CFU-GM, and BFU-E were enumerated after 14–17 days of in vitro culture. These data represent the number of individual colonies produced per 1000 cells plated for doses ranging from 2 × 10² to 2 × 10³ ALDH^hi cells. The rate of the progenitor cells was evaluated as the percentage of the corresponding control (bottom panel). Results are mean ± S.D. of three independent experiments. *, p < 0.01 compared with untreated control cells. B, the relative expression levels of RKIP mRNA of CFU-GEMM, CFU-GM, and BFU-E derived from Bcr-Abl siRNA-transfected ALDH^hi cells were assessed after 14–17 days treatment with the Abl kinase inhibitors. C, the relative expression levels of FoxM1 mRNA of CFU-GEMM, CFU-GM, and BFU-E derived from Bcr-Abl siRNA-transfected ALDH^hi cells were assessed after 14–17 days treatment with the Abl kinase inhibitors. For analysis of RKIP and FoxM1 expression, quantitative RT-PCR was performed relative to the GAPDH gene. Data are shown as the mean ± S.D. in triplicate cultures and are representative of three independent experiments. **, p < 0.05; *, p < 0.01 compared with untreated control cells. Fourteen days after transfection with Bcr-Abl siRNA, mRNA expression of Bcr-Abl was assessed by RT-PCR in CFU-GEMM, CFU-GM, and BFU-E (bottom panels). RT-PCR results are representative of three independent experiments.