Noxa/Bcl-2 protein interactions contribute to bortezomib resistance in human lymphoid cells

Abstract

Previous studies have suggested that the BH3 domain of the proapoptotic Bcl-2 family member Noxa only interacts with the anti-apoptotic proteins Mcl-1 and A1 but not Bcl-2. In view of the similarity of the BH3 binding domains of these anti-apoptotic proteins as well as recent evidence that studies of isolated BH3 domains can potentially underestimate the binding between full-length Bcl-2 family members, we examined the interaction of full-length human Noxa with anti-apoptotic human Bcl-2 family members. Surface plasmon resonance using bacterially expressed proteins demonstrated that Noxa binds with mean dissociation constants (K(D)) of 3.4 nm for Mcl-1, 70 nm for Bcl-x(L), and 250 nm for wild type human Bcl-2, demonstrating selectivity but not absolute specificity of Noxa for Mcl-1. Further analysis showed that the Noxa/Bcl-2 interaction reflected binding between the Noxa BH3 domain and the Bcl-2 BH3 binding groove. Analysis of proteins expressed in vivo demonstrated that Noxa and Bcl-2 can be pulled down together from a variety of cells. Moreover, when compared with wild type Bcl-2, certain lymphoma-derived Bcl-2 mutants bound Noxa up to 20-fold more tightly in vitro, pulled down more Noxa from cells, and protected cells against killing by transfected Noxa to a greater extent. When killing by bortezomib (an agent whose cytotoxicity in Jurkat T-cell leukemia cells is dependent on Noxa) was examined, apoptosis was enhanced by the Bcl-2/Bcl-x(L) antagonist ABT-737 or by Bcl-2 down-regulation and diminished by Bcl-2 overexpression. Collectively, these observations not only establish the ability of Noxa and Bcl-2 to interact but also identify Bcl-2 overexpression as a potential mechanism of bortezomib resistance.

FIGURE 1.

Binding of full-length human Noxa to Mcl-1, Bcl-2, and Bcl-x_Lin vitro. A, B, and D, shown is surface plasmon resonance (relative units (RU)) observed when immobilized human Noxa was exposed to increasing concentrations of purified Mcl-1 (A), wt Bcl-2 (B), or Bcl-2 variant 4 (D). C, identity of Bcl-2 sequence variants examined in this study is shown. E, shown are dissociation constants for complexes of various anti-apoptotic proteins with full-length Noxa as determined by surface plasmon resonance. Error bars, ±S.D. of 3 independent experiments using different chips and different protein preparations. TM, transmembrane.

FIGURE 2.

Noxa/Bcl-2 interaction involves Noxa BH3 domain and Bcl-2 hydrophobic pocket. A, shown is surface plasmon resonance (relative units (RU)) observed when immobilized 26-mer Noxa BH3 peptide was exposed to increasing concentrations of purified wild type Bcl-2. B, a surface-filling model of human Bcl-2 (PDB 2021) using Pymol software (DeLano Scientific) shows Arg¹⁴⁶ at base of BH3 binding groove. C, amino acid alignment of human anti-apoptotic Bcl-2 family members shows conservation of Arg in the BH3 binding groove. D, shown is surface plasmon resonance observed when immobilized Noxa was exposed to either 1 μm wild type Bcl-2 (red), 1 μm R146A Bcl-2 (orange), or 1 μm GST-only control (blue).

FIGURE 3.

Coimmunoprecipitation of Noxa and Bcl-2. A, after Jurkat cells were treated for 24 h with 0.1% (v/v) DMSO (lane 1) or bortezomib at 3.75, 7.5, 15, or 30 nm (lanes 2–5, respectively) in the presence of 5 μm Q-VD-OPhe, whole cell lysates were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with the indicated antibodies. B, after Jurkat cells were treated with DMSO or the indicated concentration of bortezomib in the presence of 5 μm Q-VD-OPhe for 24 h, lysates containing protein from 3 × 10⁵ cells/sample as well as known amounts of bacterially expressed and purified human S-peptide/His₆-Noxa were subjected to SDS-PAGE and probed with Noxa antibody. β-Actin served as a loading control for the cells. C and D, 24 h after transient transfection with S peptide-tagged Bcl-2 (C) or S peptide/streptavidin-binding protein-tagged Noxa (D), cells were treated for 24 h with 60 nm bortezomib (Bort, C) or 500 nm MG-132 (D). At the end of the incubation, cell lysates were prepared in isotonic buffer containing 1% CHAPS and incubated with S protein-agarose beads to recover protein complexes. Pulldown assays and inputs were subjected to SDS-PAGE and immunoblotting with S peptide, Noxa, or Bcl-2 antibodies. E, lysates prepared from Jurkat cells transiently transfected with S peptide-tagged Bcl-2 variants and treated with 15 nm bortezomib for 24 h were incubated with S-protein agarose. Pulldown assays and inputs were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. F, lysates prepared from untreated RL cells were immunoprecipitated (IP) with Bcl-2 or isotype control antibody (Ab). Immunoprecipitations and inputs were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. G, HCT116 cells stably expressing S-peptide tagged Mcl-1 or wt Bcl-2 were treated with DMSO or 1 μm camptothecin (CPT) for 48 h. Serial dilutions of lysates and pulldown assays were subjected to SDS-PAGE and immunoblotting with the indicated antibodies. Control represents untransfected, untreated HCT116 cells.

FIGURE 4.

Bcl-2 protects against Noxa-induced apoptosis in Jurkat cells. A, Jurkat cells were transfected with plasmid encoding EGFP-conjugated histone H2B (to mark successfully transfected cells) along with plasmids encoding the indicated Bcl-2 variant and S peptide/streptavidin-binding protein-tagged Noxa or the respective empty vector. After 24 h, cells were stained with APC-conjugated annexin V and subjected to 2-color flow cytometry. Numbers at the right of each dot plot indicate the percentage of EGFP-histone H2B⁺ cells that was also annexin V⁺ (events in upper right quadrant divided by sum of events in two right quadrants). B, shown is a summary of the percentage of EGFP-histone H2B⁺ cells that were annexin V⁺ after each treatment. Error bars, ±S.D. of three independent experiments. *, p < 0.007 by unpaired t test. Inset in B, an immunoblot of cell lysates from cells transfected with plasmids encoding the indicated proteins is shown. GAPDH and the endogenous proteins served as loading controls.

FIGURE 5.

Noxa participates in bortezomib-induced apoptosis in Jurkat cells. A, Jurkat cells were treated for 24 h with the indicated bortezomib concentration in the absence of Q-VD-OPhe, then stained with propidium iodide under conditions that allow extraction of fragmented chromatin as previously described (87, 88). Error bars, ±S.D. of three-five independent experiments. B, the percentage of subdiploid cells obtained when parental Jurkat cells or Jurkat variants, characterized as previously described (44, 89), were treated for 24 h with 7.5 nm bortezomib (Bort) or 25 nm camptothecin or for 5 h with CH.11 agonistic anti-Fas antibody. Error bars, ±S.D. of three or four independent experiments. C and D, 24 h (Noxa) or 48 h (Bak) after transient transfection with the indicated siRNA along with plasmid encoding EGFP-histone H2B, Jurkat cells were treated with bortezomib (Bort) for 24 h, stained with APC-annexin V, and analyzed by flow cytometry. Dot plots from a representative experiment are shown in C. Numbers at right of each plot indicate the percentage of EGFP⁺ cells that are annexin V⁺. Results from this experiment and two additional experiments are summarized in D. Error bars, ±S.D. of three independent experiments. Inset in D, shown is an immunoblot of siRNA transfected cells. E, 24 h after transfection with the indicated siRNA along with plasmid encoding EGFP-histone H2B, Jurkat cells were treated with 15 nm bortezomib for 24 h, stained with APC-annexin V, and analyzed by flow cytometry (left panel) or harvested for RNA, which was analyzed for Bim, Puma, and GAPDH message by quantitative RT-PCR (right panel). Error bars, ±S.D. of three independent experiments. Inset in E, Jurkat cells treated with control or Bim siRNA and harvested for immunoblotting are shown. GAPDH served as a loading control. F and G, 24 h after Jurkat cells were treated with increasing concentrations of bortezomib and/or ABT-737, cells were stained with propidium iodide and analyzed by flow cytometry to determine percentage of cells with <2n DNA content. Panel G shows median effect analysis (90) from the data in panel F. A combination index of <1, seen at all bortezomib concentrations above 2.5 nm, indicates synergy.

FIGURE 6.

Bcl-2 protects against bortezomib-induced death. A and B, 24 h after transfection with pCMS5A vector (which encodes EGFP-histone H2B), pCMS5A/Bcl-2 shRNA, or pCMS5A/Bcl-2 shRNA/Bcl-2* (encodes shRNA-resistant Bcl-2, denoted Bcl-2 shRNA RES), cells were treated with bortezomib (Bort) for 24 h, stained with APC-annexin V, and analyzed by 2-color flow cytometry. Dot plots (A) show representative results after treatment with diluent (top) or 30 nm bortezomib (bottom). B summarizes the percentage of successfully transfected (EGFP⁺) cells that are annexin V-positive at 0, 7.5, or 30 nm bortezomib. Error bars, ±S.D. of three independent experiments. *, different from corresponding empty vector sample at p ≤ 0.04 in paired t tests. Inset in B, 24 h after transient transfection with the indicated plasmids, EGFP-positive cells were collected by fluorescence-activated cell sorting for immunoblotting. C and D, 24 h after Jurkat cells were transiently transfected with plasmids encoding S peptide-tagged Bcl-2 variants or empty vector along with plasmid encoding EGFP-histone H2B, cells were treated with diluent or bortezomib for 24 h, stained with APC-annexin V, and analyzed by flow cytometry. Dot plots (C) show data from one representative experiment after transfection with empty vector, variant 1, or variant 4. D summarizes the mean ±S.D. of three independent experiments. *, different from empty vector sample at p < 0.0001. **, different from variants 1–3 at p < 0.002. Inset in D, shown is an immunoblot of whole cell lysates after transfection with empty vector or the Bcl-2 variants. E, whole cell lysates from untreated lymphoid cell lines were probed with antibodies to Bcl-2, Mcl-1, or as a loading control, GAPDH. F, the cell lines shown in panel E were treated for 24 h with the indicated concentration of bortezomib, stained with propidium iodide, and subjected to flow cytometry as previously described (44, 89). To correct for differences in base-line apoptosis between cell lines, normalized apoptosis was calculated as (observed − base line)/(100 − base line). Results are the mean of four independent experiments. S.D., generally ± 5% or less, have been omitted for clarity. G, whole cell lysates from Jurkat (lanes 1 and 2) or RL cells (lanes 3–7) treated for 24 h with diluent (lanes 1, 3) or bortezomib at 3.75 (lane 4), 7.5 (lane 5), 15 (lane 6), or 30 nm (lanes 2 and 7) were probed with antibodies to Noxa or, as a loading control, c-Raf. Vertical dashed lines in panels E and G indicate removal of intervening, unrelated lanes.