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Comparative Study
. 2010 Dec;78(6):1096-104.
doi: 10.1124/mol.110.068007. Epub 2010 Sep 21.

Melanoma differentiation associated gene-7/interleukin-24 potently induces apoptosis in human myeloid leukemia cells through a process regulated by endoplasmic reticulum stress

Mohamed Rahmani  1 , Mandy Mayo, Rupesh Dash, Upneet Kaur Sokhi, Igor P Dmitriev, Devanand Sarkar, Paul Dent, David T Curiel, Paul B Fisher, Steven Grant
Affiliations
Comparative Study

Melanoma differentiation associated gene-7/interleukin-24 potently induces apoptosis in human myeloid leukemia cells through a process regulated by endoplasmic reticulum stress

Mohamed Rahmani et al. Mol Pharmacol. 2010 Dec.

Abstract

Melanoma differentiation associated gene-7 (mda-7)/interleukin-24 (IL-24), a member of the IL-10 cytokine gene family, preferentially induces cell death in neoplastic epithelial cells types while sparing their normal counterparts. The effects of mda-7/IL-24 in acute myeloid leukemia (AML) cells have not been extensively characterized. Treatment with recombinant GST-MDA-7/IL-24 potently induced apoptosis in diverse myeloid leukemia cell types including U937, HL60, MV4-11, EOL-1, and MLL/ENL cells. MDA-7/IL-24 also markedly induced apoptosis in and suppressed the colony-forming capacity of primary AML blasts but exerted minimal toxicity toward normal CD34(+) hematopoietic progenitor cells. MDA-7/IL-24 lethality was associated with pronounced endoplasmic reticulum (ER) stress induction in leukemia cell lines and primary AML blasts, manifested by the accumulation of growth arrest and DNA damage-inducible protein 34 (GADD34), 78-kDa glucose-regulated protein (GRP78)/BiP, inositol-requiring enzyme 1α (IRE1α), and eukaryotic initiation factor 2α phosphorylation. It is noteworthy that short hairpin RNA (shRNA) knockdown of IRE1α, GADD34, or GRP78/BiP significantly enhanced MDA-7/IL-24-mediated apoptosis, indicating a protective role for these molecules against MDA-7/IL-24 lethality. MDA-7/IL-24 also down-regulated the antiapoptotic protein Mcl-1 and sharply increased expression of the proapoptotic proteins Bim and Noxa. Ectopic Mcl-1 expression or shRNA knockdown of Bim or Noxa significantly attenuated MDA-7/IL-24-mediated leukemia cell death. Finally, knockdown of Bax or Bak significantly reduced MDA-7/IL-24 lethality. Together, these findings indicate that MDA-7/IL-24 potently induces apoptosis in human myeloid leukemia cells through a process regulated by ER stress induction, Mcl-1 down-regulation, and Bim and Noxa up-regulation. They also suggest that MDA-7/IL-24 warrants further investigation in myeloid leukemia.

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Figures

Fig. 1.
Fig. 1.
GST-MDA-7/IL-24 potently induces apoptosis in various human myeloid leukemia cells. U937 (A), HL60 (B), MV4-11 (C), EOL1 (D), and MLL/ENL (E) cells were exposed for 24 h (□) or 48 h (●) to the designated concentration of GST-MDA-7/IL-24, after which the percentage of apoptotic cells was determined by Annexin V/PI analysis as described under Materials and Methods. F, U937 cells were exposed to GST protein alone for 48 h, after which the extent of cell death was determined as above. Values represent the means for three separate experiments ± S.D.
Fig. 2.
Fig. 2.
GST-MDA-7/IL-24 induces profound mitochondrial injury and caspase activation in human myeloid leukemia cells. A, U937 cells were exposed to the designated concentration of GST-MDA-7/IL-24 for 48 h, after which protein lysates were prepared and subjected to Western blot analysis to monitor caspase-3, caspase-8 cleavage/activation, and PARP degradation. B, HL60, MV4-11, and MLL/ENL cells were exposed to GST-MDA-7/IL-24 (100 nM for HL60 and MV4-11, and 50 nM for MLL/ENL) for the designated intervals, after which Western blot assays were performed as in A. C, U937, HL60, and MV4-11 cells were treated with GST-MDA-7/IL-24 (200 nM for U937 cells and 100 nM for HL60 and MV4-11 cells) for the designated intervals, after which mitochondria-free cytosolic fractions were obtained as described under Materials and Methods and subjected to Western blot analysis to monitor the release of cytochrome c and AIF into the cytosol. For this and all subsequent Western blot assays, lanes were loaded with 20 μg of protein; blots were subsequently reprobed with anti-tubulin (Tub) antibodies to document equivalent loading and transfer. The blots shown are representative of three or more independent experiments.
Fig. 3.
Fig. 3.
GST-MDA-7/IL-24 potently induces apoptosis in primary AML cells. A to E, leukemic blasts were isolated as described under Materials and Methods from the bone marrow of five patients with AML (FAB classification M2) and exposed to the designated concentrations of GST-MDA-7/IL-24 for 24 or 48 h, after which the extent of apoptosis was determined using Annexin V/PI staining assay. On the other hand, protein lysates were prepared from patient 3 and subjected to Western blot analysis to monitor caspase-3 and caspase-8 cleavage/activation and PARP degradation.
Fig. 4.
Fig. 4.
GST-MDA-7/IL-24 induces a marked reduction in the clonogenic potential of primary AML blasts but is relatively sparing toward normal CD34+ progenitor cells. A, leukemic blasts isolated from patient 3 were plated in methylcellulose in the presence of the designated concentrations of GST-MDA-7/IL-24 for 14 days, after which L-CFUs were enumerated and expressed as a percentage relative to untreated cells. B, normal CD34+ cells isolated from three normal subjects (N#1–3) were exposed to GST-MDA-7/IL-24 (50 or 100 nM) for 48 h, after which cell death was determined by flow cytometry using the Annexin V staining assay. C, normal CD34+ cells (N#1–2) were plated in methylcellulose in the presence of the designated concentrations of GST-MDA-7/IL-24 for 8 days, after which CFU-GMs were enumerated and expressed as in A. Each sample was analyzed in triplicate; values represent the means ± S.D.
Fig. 5.
Fig. 5.
GST-MDA-7/IL-24 triggers UPR response in leukemia cells. U937 cells were exposed to the designated concentrations of GST-MDA-7/IL-24 for 48 h (A) or to 200 nM GST-MDA-7/IL-24 for the designated intervals (B and C), after which protein lysates were prepared and subjected to Western blot analysis using the indicated antibodies. D, HL60, MV4-11, and MLL-ENL were exposed to GST-MDA-7/IL-24 (100 nM for HL60 and MV4-11, and 50 nM for MLL/ENL) for the designated intervals, after which Western blot analysis was performed using indicated antibodies.
Fig. 6.
Fig. 6.
GST-MDA-7/IL-24 induces UPR response in primary AML cells. Leukemic blasts isolated from two patients (2 and 5) with AML were exposed to the designated concentrations of GST-MDA-7/IL-24 for 48 h, after which cells were lysed and subjected to Western blot analysis using the indicated antibodies.
Fig. 7.
Fig. 7.
Functional roles of IRE1α, GRP78/BiP, and GADD34 in regulating GST-MDA-7/IL-24-mediated cell death in leukemia cells. A, U937 cells were infected with lentiviruses carrying shRNA constructs against IRE1α, GRP78/BiP, GADD34, or their control GFP counterparts and selected with puromycin for 2 weeks, after which Western blot analysis was performed to monitor the level of knockdown of these proteins. B, these cells were treated with GST-MDA-7/IL-24 (150 nM) for 48 h, after which the extent of apoptosis was monitored using Annexin V/PI analysis assay. Values represent the means for three separate experiments ± S.D. *, significantly higher that values for shGFP-transfected cells (p < 0.05 in each case).
Fig. 8.
Fig. 8.
GST-MDA-7/IL-24 down-regulates Mcl-1 and Bcl-xL and up-regulates Noxa and Bim in human leukemia cells. U937 (A), HL60 (B), MV4-11 (C) cells, or leukemic blasts (D) were exposed to GST-MDA-7/IL-24 for the designated intervals, after which Western blot analysis was performed. The GST-MDA7/IL-24 concentrations used were 200 nM for U937 cells and 100 nM for HL60, MV4-11, and leukemic blasts.
Fig. 9.
Fig. 9.
Knockdown of Bim or Noxa or enforced the expression of Mcl-1 renders cells less susceptible to GST-MDA-7/IL-24 lethality. U937 cells in which Bim (A, inset) or Noxa (B, inset) was stably knocked down with shRNA, and their control counterpart shGFP-transfected cells were lysed, and protein lysates were subjected to Western blot analysis to monitor down-regulation of Bim and Noxa. Cells were then exposed to GST-MDA-7/IL-24 (150 or 200 nM) for 48 h, after which the extent of cell death was determined using the Annexin V/PI staining assay (A and B). C, U937 cells ectopically expressing Mcl-1 or the empty vector (pCEP4) were exposed to GST-MDA-7/IL-24 for 48 h, after which the extent of cell death was monitored as above. Protein lysates were also prepared from cells before treatment and Mcl-1 protein levels were monitored by Western blot analysis (C, inset). *, significantly less than values obtained for pCEP4 cells (P < 0.01). D, U937 cells in which Bak or Bax was stably knocked down with shRNA or their control counterpart cells transfected with GFP-shRNA (shGFP) were exposed to GST-MDA-7/IL-24 for 48 h, after which the extent of cell death was monitored using the Annexin V/PI staining assay. For all studies, values represent the means for three separate experiments ± S.D. For A, B, and D, *, significantly lower than values obtained for shGFP-transfected cells (P < 0.05).
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