Figure 1.
Inhibition of Jak2-V617F downregulates etoposide-induced Chk1 activation as well as G2/M arrest and elicits apoptosis in a GSK3-dependent manner.
(A) UT7/Jak2-V617F cells were cultured for 16 h with 0.5 µM etoposide (Etop.), 0.2 µM JakI-1, or 1 µM GSK3-I #5, as indicated, in the absence of Epo. Cells were then analyzed for the cellular DNA content by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content (s-G1) and those of cells in the G2/M phase (G2/M) are indicated. (B) UT7-Jak2-V617F cells were pretreated for 60 min with 1 µM JakI-1 or 40 mM LiCl, as indicated, and subsequently treated with indicated concentrations of etoposide for 6 h. Cells were lysed and subjected to Western blot analysis with antibodies against indicated proteins. Positions of GSK3ß are indicated by arrows, while the caspase-cleaved fragment of PARP by an asterisk.
Figure 2.
Imatinib inhibits the G2/M arrest and prominently induces apoptosis in BCR/ABL-expressing cells treated with etoposide.
(A) 32Dp210 cells were cultured for 24 h with 0.5 µM etoposide (Etop.), 0.6 µM imatinib, or 1 µM SB218078, as indicated, and analyzed for the cellular DNA content by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content and those of cells in the G2/M phase are indicated. (B) K562 cells were cultured for 24 h with 1 µM etoposide, 1 µM imatinib, or 1 µM SB218078, as indicated, and analyzed. (C) 32Dp210 cells were cultured for 16 h with or without 50 ng/ml nocodazole in the presence of 1 µM etoposide and 1 µM imatinib, as indicated, and analyzed for the cellular DNA content by flow cytometry and for the mitotic index, as described under Materials and methods. Each data point represents the mean of three independent experiments, with error bars indicating standard deviations. The asterisk indicates a statistically significant difference determined by Student’s t-test (p<0.01). (D) 32Dp210 cells were cultured for 16 h with 1 µM etoposide and 1 µM imatinib, as indicated, in the presence of 50 ng/ml nocodazole. Cells were analyzed for the DNA content and histone H3 phosphorylated on S10 (H3-S10-P) by flow cytometry. Percentages of cells in G2/M that are positive for H3-S10-P are indicated.
Figure 3.
Imatinib downregulates etoposide- or doxorubicin-induced Chk1 activation and induces apoptosis specifically by inhibiting BCR/ABL.
(A) Ton.B210 cells cultured with DOX to induce BCR/ABL expression (BCR/ABL) in the absence of IL-3 or cultured without DOX in the presence of IL-3 (IL-3) were left untreated as control (Cont.) or treated with 0.6 µM imatinib, 0.5 µM etoposide (Etop.), or 0.1 µM doxorubicin (DXR), as indicated for 12 h, and analyzed for the cellular DNA content. Percentages of apoptotic cells with sub-G1 DNA content and those of cells in the G2/M phase (G2/M) are indicated. (B) Ton.B210 cells expressing BCR/ABL (BCR/ABL) or cultured with IL-3 (IL-3) were left untreated as control (Cont.) or treated with 3 µM imatinib, as indicated, for 1 h. Cells were subsequently treated with 1 µM etoposide or 0.2 µM doxorubicin, as indicated, for indicated times. Cells were lysed and subjected to Western blot analysis with antibodies against indicated proteins. Positions of GSK3α and GSK3ß are indicated. A position of the caspase-cleaved fragment of PARP is indicated by an asterisk.
Figure 4.
Sorafenib inhibits FLT3-ITD to attenuate Chk1 activation and induces apoptosis in cells treated with etoposide.
(A) Ton.32D/FLT3-ITD cells cultured with DOX to induce FLT3-ITD (FLT3-ITD) in the absence of IL-3 or cultured without DOX in the presence of IL-3 (IL-3) were left untreated as control (Cont.) or treated with 0.5 µM sorafenib and indicated concentrations of etoposide (Etop.) for 16 h, and analyzed for the cellular DNA content. Percentages of apoptotic cells with sub-G1 DNA content and those of cells in the G2/M phase (G2/M) are indicated. (B) Ton.32D/FLT3-ITD cells expressing FLT3-ITD (FLT3-ITD) or cultured with IL-3 (IL-3) were left untreated or treated with 0.5 µM sorafenib and subsequently cultured with or without 0.5 µM etoposide, as indicated, for 10 h. Cells were lysed and subjected to Western blot analysis with antibodies against indicated proteins. (C) Ton.32D/Rev-FLT3-ITD cells (FLT3-ITD) or Ton.32D/Rev-FLT3-D835Y cells (FLT3-D835Y) were cultured with 50 nM sorafenib and 1 µM etoposide for 24 h and analyzed. (D) MV4-11 leukemic cells expressing FLT3-ITD werer cultured with 10 µM sorafenib and 0.2 µM etoposide, as indicated, for 24 h and analyzed.
Figure 5.
GSK3ß regulates etoposide-induced Chk1 activation and apoptosis in cytokine- or BCR/ABL-driven hematopoietic cells.
(A) 32D/EpoR/pMXs-IG cells (Cont.) or the cells overexpressing the kinase inactive (KI) or S9A (SA) mutant as well as the wild-type (WT) GSK3ß, as indicated, were cultured for 16 h with 0.5 µM etoposide in the presence of 0.1 U/ml Epo and analyzed for the cellular DNA content. Percentages of apoptotic cells with sub-G1 DNA content and those of cells in the G2/M phase are plotted. Each data point represents the mean of three independent experiments, with error bars indicating standard deviations. The asterisks indicate statistically significant differences determined by Student’s t-test (*p<0.05, **p<0.01). (B) The cells indicated as in A were treated with indicated concentrations of etoposide for 7 h in the presence of 0.1 U/ml Epo and analyzed by Western blot analysis using indicated antibodies. Relative levels of Chk1-S345P, determined by densitometric analysis, are shown. (C) 32Dp210 cells were cultured for 16 h with 1 µM etoposide alone (Cont.) or also with 1 µM imatinib and 1 µM GSK3-I #5 (GSK3-I), as indicated, and analyzed for the cellular DNA content. (D) 32Dp210 cells were pretreated for 1 h with 1 µM imatinib, 1 µM GSK3-I #5, or 5 µM MG132, as indicated, or left untreated (Cont.). Cells were then treated with 1 µM etoposide for indicated times and analyzed by Western blot analysis.
Figure 6.
PI3K/Akt upstream of GSK3ß regulates etoposide-induced Chk1 activation and apoptosis in cytokine- or BCR/ABL-driven cells.
(A) 32Dp210/Rev (Cont.) or 32Dp210/Rev-Akt1-myr (Akt-myr) cells were cultured with 0.5 µM etoposide (Etop.) or 2 µM imatinib, as indicated, for 24 h and analyzed for the cellular DNA content. (B) Ton.B210 cells cultured with DOX to induce BCR/ABL expression (BCR/ABL) in the absence of IL-3 or cultured without DOX in the presence of IL-3 (IL-3) were pretreated for 1 h with 1 µM GDC-0941 (GDC) or 5 µM MK-2206 (MK) or left untreated as control (Cont.). Cells were subsequently treated with 0.5 µM etoposide for 12 h or left untreated as control (Cont.), as indicated, and analyzed for the cellular DNA content. (C) Ton.B210 cells expressing BCR/ABL (BCR/ABL) or cultured with IL-3 (IL-3) were pretreated for 30 min with 2 µM GDC-0941, 3 µM MK-2206, or 20 µM SB216763, as indicated, or left untreated as control. Cells were subsequently treated with 1 µM etoposide for 8 h or left untreated, as indicated. Cells were lysed and subjected to Western blot analysis with antibodies against indicated proteins. A position of the caspase-cleaved fragment of PARP is indicated by an asterisk. (D) 32Dp210 cells were precultured for 1 h with 20 µM SB216763 or left untreated for control, as indicated. Cells were then treated for 24 h with 0.5 µM etoposide and 1 µM GDC-0941 or 5 µM MK-2206, as indicated, and analyzed.
Figure 7.
Sorafenib or GDC-0941 inhibits etoposide-induced Chk1 activation and enhances apoptosis in cells expressing T315I-mutated BCR/ABL.
(A) Ton.B210/T315I cells cultured with DOX to induce BCR/ABL with T315I (BCR/ABL-T315I) in the absence of IL-3 or cultured without DOX in the presence of IL-3 (IL-3) were left untreated as control (Cont.) or treated with 5 µM sorafenib and 1 µM etoposide (Etop.), as indicated, for 16 h, and analyzed for the cellular DNA content. (B) Ton.B210/T315I cells expressing BCR/ABL with T315I (T315I) or cultured with IL-3 (IL-3) were left untreated or treated with 10 µM sorafenib, as indicated, for 1 h. Cells were subsequently cultured with or without 1 µM etoposide for 6 h and subjected to Western blot analysis. A position of the caspase-cleaved fragment of PARP is indicated by an asterisk. (C) Ton.B210/T315I cells cultured with DOX were left untreated as control (Cont.) or treated with 5 µM imatinib, 50 nM dasatinib, 1 µM GDC-0941 (GDC) in the presence or absence of 0.5 µM etoposide, as indicated, for 16 h, and analyzed for the cellular DNA content. (D) Ton.B210/T315I cells cultured with DOX were left untreated or treated with 5 µM imatinib, 50 nM dasatinib, 1 µM GDC-0941, as indicated, for 1 h. Cells were subsequently cultured with or without 1 µM etoposide for 6 h and subjected to Western blot analysis.
Figure 8.
A schematic model for enhancement of apoptosis by inhibition of the aberrant tyrosine kinases in hematopoietic cells treated with chemotherapeutics.
Chemotherapeutics induces Chk1-mediated G2/M cell cycle arrest to downregulate induction of apoptosis. Inhibition of BCR/ABL, FLT3-ITD, or Jak2-V617F by imatinib, sorafenib, of JakI-1, respectively, as well as inhibition of PI3K or Akt by GDC-0941 or MK-2206, respectively, drastically enhances apoptosis in hematopoietic cells treated with chemotherapeutics. The enhancing effect, prevented by inhibition of GSK3 by SB216763, may be at least partly due to the inhibition of PI3K/Akt pathway leading to activation of GSK3, which may prevent Chk1-mediated G2/M cell cycle arrest by chemotherapeutics. Inhibition of Chk1 by its inhibitor SB218078 also prominently enhances apoptosis induced by chemotherapeutics.