Figure 1.
Etoposide as well as doxorubicin downregulates Jak2 and Jak2-V617F when they are inactivated.
(A) After cultured for 9 h in medium without Epo, UT7 cells were left untreated or treated with 5 µM etoposide (VP16) or 0.5 µM doxorubicin (DXR) for 4 hr in the absence or presence of 50 mU/ml Epo, as indicated. Cells were lysed and subjected to immunoblot analysis with anti-Jak2 antibody, followed by reprobing with anti-EpoR and anti-β-actin, as indicated. (B, C) After cultured for 3 h in medium without Epo, 32D/EpoR cells were treated for 5 h in the absence or presence of 100 mU/ml Epo, as indicated, with increasing concentrations of etoposide (C) or doxorubicin (D), as indicated. Cell lysates were analyzed by immunoblotting with antibodies against indicated proteins. (D) 32D/EpoR or parental 32Dcl3 cells, cultured in medium containing 10% WEHI conditioning medium as the source of IL-3, were washed out of cytokine for 1 h. Cells were further cultured with or without 5 µM etoposide (VP16) for 6 h, as indicated, and analyzed. (E, F) After cultured for 9 h in medium without Epo, UT7/Jak2-V617F cells were treated for 1 h with or without 2 µM JakI-1. Cells were subsequently treated with increasing concentrations of etoposide (E; 0, 1, 2, 5 µM) or doxorubicin (D; 0, 0.1, 0.2, 0.5 µM), as indicated, and analyzed. (G) UT7/Jak2-V617F cells starved from Epo were pretreated with indicated concentrations of AG490 for 1 h. Cells were then treated with 5 µM etoposide or 0.5 µM doxorubicin for 6 h, as indicated, and analyzed.
Figure 2.
Involvement of the PI3K/Akt/GSK3β pathway in downregulation of Jak2 in response to DNA damage.
(A) After cultured for 9 h in medium without Epo, UT7 cells were pretreated for 1 h with 50 µM LY294002 (PI3K-I) or 50 µM PD98059 (MEK-I), as indicated, or left untreated. Cells were subsequently treated with or without 10 µM etoposide (VP16) for 4 h, as indicated, in the presence of 20 mU/ml Epo or in its absence (Epo -). Cells were lysed and subjected to immunoblot analysis with anti-Jak2 antibody, followed by sequential reprobing with anti-phospho-GSK3α/β-S9/21 (GSK3β-P), anti-GSK3β, anti-β-actin, as indicated. (B) 32D/Akt-myr (Akt-myr) as well as control 32D/RevTRE (Cont.) cells were cultured for 24 h with 1 µg/ml doxycycline to induced the expression of Akt-myr in 32D/Akt-myr cells and subsequently washed out of WEHI conditioning medium for 12 h. Cells were then pretreated for 1 h with 1 µM JakI-1 or 10 µM LY294002 (PI3K-I), as indicated, or left untreated. Cells were finally treated with or without 10 µM etoposide (VP16), as indicated, for 4 h before analysis with indicated antibodies. (C) After cultured for 9 h in medium without Epo, UT7 cells were pretreated for 1 h with 10 µM SB216763 (SB216), 40 mM LiCl, or okadaic acid at 100 nM (OA100) or 200 nM (OA200), as indicated, or left untreated. Cells were subsequently treated with or without 10 µM etoposide (VP16) for 4 h, as indicated, and analyzed. (D) 32DE/STAT5A1*6 (STAT5A1*6) or control 32DE/pMX (Cont.) cells were pretreated for 1 h with 1 µM JakI-1 or 50 µM LY294002 (PI3K-I), as indicated, or left untreated in the absence of Epo. Cells were further treated with or without 5 µM etoposide (VP16) for 6 h, as indicated, before analysis. (E) 32DE/STAT5A1*6 (STAT5A1*6) or control 32DE/pMX (Cont.) cells were cultured overnight in the absence of Epo. Cells were lysed and subjected to immunoprecipitation of p85. Immunoprecipitates were analyzed by immunoblotting. (F) After cultured for 12 h in medium without Epo, UT7/Jak2-V617F cells were pretreated for 1 h with 50 µM LY294002 (PI3K-I), 2 µM JakI-1, 40 mM LiCl, or 10 µM MG132, as indicated, or left untreated as control (Cont.). Cells were subsequently treated with or without 5 µM etoposide (VP16), as indicated, for 6 h and analyzed.
Figure 3.
Possible involvement of ubiquitin-proteasome pathway and Cbls in downregulation of Jak2.
(A) 32D/EpoR cells deprived of Epo for 2 h were pretreated with 10 µM MG132 or left untreated as control, as indicated for 1 h in the absence of Epo. Cells were then treated for 6 h with or without 5 µM etoposide, as indicated. Cells were lysed and subjected to immunoblot analysis using indicated antibodies. (B) 293T cells were transfected on 6-well plate with 0.1 µg of pRK5-Ubiquitin-WT and 0.002 µg of pRK5-Jak2-Wt (Wt) or pRK5-Jak2-KE (KE) along with 0.1 µg of pXM-EpoR-Wt or empty plasmid, as indicated. Two days after transfection, cells were lysed, and Jak2 was immunoprecipitated. Immunoprecipitates were analyzed by immunoblotting using antibodies against polyubiquitin (poly-Ubi), Jak2, and Jak2 phosphorylated on Y1007 (Jak2-PY), as indicated. The vertical line indicates the smeary pattern characteristic of ubiquitination. (C) 293T cells were transfected on 6-well plate with 0.3 µg of pRevTRE-Jak2V617F and 0.2 µg of pTet-On along with 0.2 µg of pRK5-Ubiquitin-WT (Ubi), 0.5 µg of pRevTRE-c-Cbl (c-Cbl), or 0.5 µg of pRevTRE-Cbl-b (Cbl-b), as indicated. The amounts of plasmid DNA transfected were equalized by adding pRevTRE. Two days after transfection, cells were lysed, and lysates were analyzed by immunoblotting. The position of unmodified Jak2-V617F and those of ubiquitinated Jak2-V617F are indicated by asterisks and arrows, respectively. (D) Ton.32D/Flt3-Wt (Cont.) and Ton.32D/Flt3-Wt/c-Cbl-RQ/Cbl-b-CA (c-Cbl-RQ/Cbl-b-CA) cells, cultured in doxycycline-containing medium, were cultured for 3 h with or without 5 µM LY294002 (LY), as indicated, in the absence of IL-3. Cells were further treated for 6 h with 10 µM etoposide (VP16) or 1 µM doxorubicin (DXR), as indicated, and lysed. Cell lysates were analyzed by immunoblotting.
Figure 4.
Synergistic induction of apoptosis in UT7/Jak2-V617F by etoposide and JakI-1 and effects of Boc-d-fmk on Jak2 downregulation.
(A) UT7/Jak2-V617F cells were cultured for 12 h with 0.5 µM etoposide (VP16), 0.5 µM JakI-1, 25 µM LY294002, and 1 µM GSK3I-5, as indicated in the absence of Epo, and analyzed for the cellular DNA content by flow cytometry. Percentages of apoptotic cells with sub-G1 DNA content are indicated. (B) UT7/Jak2-V617F cells were cultured for 6 h with 5 µM etoposide (VP16), 1 µM JakI-1, and 100 µM Boc-d-fmk, as indicated, in the absence of Epo and analyzed for the cellular DNA content. (C, D) 32D/EpoR cells deprived of Epo for 2 h were pretreated for 1 h with 100 µM Boc-d-fmk (B-d-f), as indicated, or left untreated. Cells were then treated for 5 h with or without 5 µM etoposide (VP16) or 0.5 µM doxorubicin (DXR), as indicated. Cells were lysed and analyzed by immunoblotting. (E) UT7/Jak2-V617F cells, cultured without Epo for 12 h, were treated with 1 µM JakI-1 and 100 µM Boc-d-fmk (B-d-f), as indicated, or left untreated. Cells were then treated with or without 5 µM etoposide (VP16), as indicated, for 6 h and analyzed.
Figure 5.
Caspase activation is involved in Jak2 degradation in cells under DNA damage stress.
(A) UT7/Jak2-V617F cells, cultured without Epo, were treated for 7 h with or without 1 µM JakI-1, as indicated, with 1 µM etoposide (VP16) added for indicated times before harvest. Cells were then analyzed for activation of Bax or caspase-3 by flow cytometry. Percentages of cells with activated Bax or caspase-3 are plotted. FSC: forward scatter. (B) UT7/Jak2-V617F cells, cultured without Epo, were pretreated for 1 h with 1 µM JakI-1, 100 µM Boc-d-fmk, or 1 µM GSK3I-5, as indicated. Cells were further treated for 6 h with or without 5 µM VP16 and analyzed for loss of mitochondrial membrane potential and activation of Bax or caspase-3, as indicated, by flow cytometry. (C) 32D/EpoR cells were deprived of Epo and pretreated with 100 µM Boc-d-fmk (B-d-f), as indicated, or left untreated. Cells were then treated for 5 h with or without etoposide (VP16), as indicated, before harvest. Immunoprecipitates obtained using anti-Jak2 (06–255) were analyzed by immunoblotting using anti-Jak2 (M-126) and anti-Jak2 (C-20), as indicated. The numbers of cells treated with etoposide were 2.5 times that of cells untreated. An arrow indicates the 100-kDa band. (D) UT7/Jak2-V617F cells, cultured without Epo, were pretreated for 1 h with or without 1 µM JakI-1, as indicated. Cells were then treated for 8 h with or without 5 µM etoposide (VP16), as indicated, before harvest. Immunoprecipitates obtained using anti-Jak2 (06–255) were analyzed by immunoblotting using indicated antibodies. The number of cells treated with etoposide was 2 times that of cells untreated. (E) UT7/Jak2-V617F cells, cultured without Epo, were pretreated for 1 h with 1 µM JakI-1 and 20 µM Boc-d-fmk (B-d-f), as indicated. Cells were then treated for 6 h with or without 5 µM etoposide (VP16), as indicated, before harvest. Immunoprecipitates obtained using anti-Flag was were analyzed by immunoblotting using anti-Jak2 (06–255). The numbers of cells treated with etoposide were 2.5 times that of cells untreated.