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. 2010 Aug 2;30(19):4575–4594. doi: 10.1128/MCB.01595-09

FIG. 4.

FIG. 4.

Bcr/abl influences the expression of ICSBP, Gas2, and β-catenin in myeloid progenitor cells. (A) Bcr/abl increases GAS2 transcription in an ICSBP-dependent manner in undifferentiated myeloid cells. U937 cells were cotransfected with a GAS2 promoter/reporter vector (1.0 kb of the 5′ flank), a vector to express Bcr/abl or the vector control, and a vector to overexpress ICSBP or the vector control. Transfectants were assayed for reporter activity with or without IFN-γ-induced differentiation. *, statistically significant difference in reporter activity with Bcr/abl expression (P < 0.01, n = 4); **, statistically significant difference in reporter expression in Bcr/abl+ transfectants with versus without ICSBP (P < 0.01, n = 4). The lack of a statistically significant difference in reporter activity in IFN-γ-differentiated transfectants is indicated by the bracket. (B) Bcr/abl decreases ICSBP mRNA and increases Gas2 mRNA in U937 myeloid leukemia cells. Stable U937 transfectants were generated with a vector to express p210 Bcr/abl or with empty vector control. RNA was analyzed by real-time PCR for ICSBP and Gas2 expression. Since Gas2 is a calpain inhibitor, the level of expression of calpain was determined. The results were normalized to those for 18S RNA. *, statistically significant decrease in ICSBP expression in Bcr/abl+ cells; **, statistically significant increase in Gas2 mRNA (P < 0.0001, n = 3). The lack of a statistically significant change in calpain mRNA expression with or without Bcr/abl is indicated by the bracket (P = 0.2, n = 3). (C) Bcr/abl decreases ICSBP protein and increases Gas2 and β-catenin protein in U937 myeloid leukemia cells. Cell lysates from the U937 transfectants described above were analyzed by Western blot assay for the expression of ICSBP, Gas2, and tubulin (as a loading control). Since Gas2 inhibits calpain and β-catenin is a calpain substrate, the blots were also probed with antibodies to these proteins. Since calpastatin inhibits calpain in some cells, the blots were probed with an antibody to this protein. Anti-Bcr antibody was used as a control for Bcr/abl expression in the stable transfectants. (D) Increased Gas2 and β-catenin expression in Bcr/abl+ U937 cells is reversed by ICSBP overexpression. U937 stable transfectants were generated with a vector to express Bcr/abl or the empty vector control or with a vector to overexpress ICSBP or the empty vector control. Cell lysates were analyzed by Western blot assay for ICSBP, Gas2, β-catenin, or GAPDH (to control for loading). Blots were also probed with an antibody to Bcr as a control for Bcr/abl expression. (E) Kinase-inactive Bcr/abl does not alter the expression of ICSBP, Gas2, or β-catenin protein in U937 cells. Stable U937 transfectants were generated with a vector to express a kinase-inactive mutant of p210 Bcr/abl (KI-Bcr/abl) or the empty vector control. Cell lysate proteins were analyzed by Western blot assay for the expression of ICSBP, Gas2, β-catenin, Bcr, and GAPDH (loading control) as described for the p210 Bcr/abl-expressing cells. (F) Bcr/abl increases Gas2 mRNA in WT myeloid progenitors, but not in myeloid progenitors from ICSBP−/− mice. Myeloid progenitor cells were isolated from the bone marrow of WT or ICSBP−/− mice and cultured in GM-CSF, IL-3 and SCF. Cells were transduced with a retroviral vector to express p210 Bcr/abl or empty vector control. Some cells were treated with the imatinib (IM). RNA from the transduced cells was analyzed for ICSBP and Gas2 expression by real-time PCR. Results were normalized to those for 18S RNA. *, statistically significant decrease in ICSBP expression in Bcr/abl+ WT progenitor cells (P < 0.0001, n = 3); **, statistically significant increase in ICSBP expression in Bcr/abl+ WT progenitor cells treated with IM (P < 0.0001, n = 3); # or ##, statistically significant increase in the expression of Gas2 in Bcr/abl+ WT cells or decrease in the expression of Gas2 in Bcr/abl+ cells treated with IM, respectively (P < 0.001, n = 3). The lack of a statistically significant difference in Gas2 expression in ICSBP−/− progenitor cells with or without Bcr/abl is indicated by the bracket (P = 0.1, n = 3). Error bars indicate standard errors.