The antiapoptotic protein AAC-11 interacts with and regulates Acinus-mediated DNA fragmentation

Abstract

The nuclear factor Acinus has been suggested to mediate apoptotic chromatin condensation after caspase cleavage. However, this role has been challenged by recent observations suggesting a contribution of Acinus in apoptotic internucleosomal DNA cleavage. We report here that AAC-11, a survival protein whose expression prevents apoptosis that occurs on deprivation of growth factors, physiologically binds to Acinus and prevents Acinus-mediated DNA fragmentation. AAC-11 was able to protect Acinus from caspase-3 cleavage in vivo and in vitro, thus interfering with its biological function. Interestingly, AAC-11 depletion markedly increased cellular sensitivity to anticancer drugs, whereas its expression interfered with drug-induced cell death. AAC-11 possesses a leucine-zipper domain that dictates, upon oligomerization, its interaction with Acinus as well as the antiapoptotic effect of AAC-11 on drug-induced cell death. A cell permeable peptide that mimics the leucine-zipper subdomain of AAC-11, thus preventing its oligomerization, inhibited the AAC-11-Acinus complex formation and potentiated drug-mediated apoptosis in cancer cells. Our results, therefore, show that targeting AAC-11 might be a potent strategy for cancer treatment by sensitization of tumour cells to chemotherapeutic drugs.

Figure 1

Deregulation of AAC-11 increases drug-induced cell death. (A) U2OS cells stably transfected with empty vector (pSingle-tTS) or the AAC-11 shRNA expression constructs (pS AAC-11 #1 and pS AAC-11 #2) were cultured in the presence or absence of doxycycline (1 μg/ml) for 48 h. Cells were lysed and the lysates were immunoblotted with the indicated antibodies. (B) U2OS control or AAC-11 shRNA-stable clones were cultured in the presence or absence of doxycycline for 48 h. Cells were exposed to cisplatin (CDDP, 20 μM), etoposide (ETO, 20 μM), camptothecin (CPT, 10 μM), paclitaxel (PTX, 1 nM) or 5-fluorouracil (5-FU 1 mM) for 16 h or left untreated. The percentage of apoptotic cells was assessed by flow cytometry. Each bar represents the mean±s.d. from three independent experiments. (C) The indicated cell lines were transfected with mock or AAC-11 specific siRNAs. After 48 h, cells were lysed and the lysates immunoblotted with the indicated antibodies. (D) The indicated cell lines were transfected with mock or AAC-11 specific siRNAs. After 48 h, cells were treated as in (B) and the percentages of apoptotic cells were determined as in (B). (E) AAC-11 expression modulates drug-induced cell death. U2OS clones stably expressing T7-tagged AAC-11 or T7-tagged AAC-11 LL/RR were treated as in (B) and the percentages of apoptotic cells were determined as in (B). Expression of T7-tagged constructs was determined by immunoblotting with an anti-T7 antibody. (F) Jurkat or MCF7/Fas/casp3 cells were transfected with either AAC-11 or AAC-11 LL/RR together with a GFP-reporter plasmid. At 36 h after transfection, the cells were exposed to etoposide (ETO, 20 μM) for 16 h or treated with anti-Fas antibody (0.5 μg/ml) plus cycloheximide for 4 h or left untreated and percentages of apoptotic cells were determined as in (B).

Figure 2

Physical interaction between Acinus and the LZ domain of AAC-11. (A) Selection of Drosophila Acinus (CG10473) fragments that interact with Drosophila AAC-11 in the yeast-two-hybrid system. Black lines indicate the fragments of CG10473 that interact with Drosophila AAC-11. Domains of CG10473 from Superfamily (supfam.org) SSF54928 (RBD) and SSF69060 (ARPC3) are indicated. The minimal interacting domain is from aa 298 to aa 390. (^*) indicates a fragment identified two times in the screen. (B) 293T cells were cotransfected with T7-AAC-11 together with Flag-tagged Acinus. Cell lysates were subjected to anti-Flag immunoprecipitation (IP) followed by immunoblotting (IB) with anti-T7. In these and all the following experiments, the expression of proteins under investigation was determined by direct immunoblotting. (C) Endogenous AAC-11 interacts with endogenous Acinus. Cell extracts (500 μg of proteins in 0.3 ml) derived from HeLa cells exposed or not to etoposide (20 μM, 2 h) were subject to immunoprecipitation with a control antibody or anti-Acinus antibody followed by immunoblotting with anti-AAC-11 antibody. (D) AAC-11 interacts with a domain encompassing residues 840 to 918 of Acinus. 293T cells were cotransfected with T7-AAC-11 together with the indicated Acinus Flag-tagged constructs. Immunoprecipitation and western blot analysis was performed as in (B). (E, F) The LZ domain of AAC-11 mediates its interaction with Acinus. (E) 293T cells were cotransfected with T7-Acinus-S together with the indicated AAC-11 Flag-tagged constructs. Immunoprecipitation and western blot analysis was performed as in (B). (F) 293T cells were cotransfected with Flag-Acinus-S together with the indicated AAC-11 LZ GFP-tagged constructs. Cell lysates were immunoprecipitated with anti-GFP and blotted with anti-Flag. (G) Nuclear colocalization of endogenous Acinus and GFP-AAC-11. HeLa cells either nontransfected (control) or transfected with GFP-AAC-11 or GFP-AAC-11 LL/RR were fixed in paraformaldehyde and immunolabelled with anti-Acinus antibody (red) and analysed by confocal microscopy. Nuclei were stained with DAPI. (H) The LZ of AAC-11 can oligomerize. 293T cells were cotransfected with GST-tagged AAC-11 LZ together with the indicated AAC-11 LZ GFP-tagged constructs. Cell lysates were immunoprecipitated with anti-GFP and blotted with anti-GST.

Figure 3

AAC-11 modulates Acinus apoptotic cleavage. (A) U2OS clones stably expressing T7-tagged AAC-11 or T7-tagged AAC-11 LL/RR were exposed to etoposide (20 μM) for 12 h or left untreated. Cell lysates were immunoblotted with the indicated antibodies. (B) Schematic representation of Acinus-S. The putative caspase-3 cleavage sites, as well as the corresponding fragments with theoretical molecular weights are represented. (C) ³⁵S-labelled Acinus-S (left panel) or ICAD (right panel) were pre-incubated with recombinant AAC-11, AAC-11 LL/RR or with BSA for 2 h at 4°C. Active caspase-3 was then added and the reaction mixtures were incubated for 1 h at 37°C. The samples were then analysed by SDS–PAGE and autoradiography.

Figure 4

A role for Acinus and AAC-11 in oligonucleosomal DNA fragmentation. (A) U2OS cells were transfected with siRNA to Acinus or mock siRNA. After 48 h, cells were lysed and the lysates immunoblotted with the indicated antibodies. (B) Deregulation of Acinus does not inhibit apoptotic chromatin condensation. U2OS cells were transfected with siRNA to Acinus or mock siRNA. After 48 h, the cells were exposed to etoposide (ETO, 10 μM, 16 h) followed by 1 μM staurosporine (STS) for another 4 h or left untreated. DAPI stainings of these cells were then visualized under a fluorescence microscope. Results show the mean values±standard deviation from triplicate with 200 nuclei counted per condition. (C) A role for Acinus in oligonucleosomal DNA fragmentation. U2OS cells were transfected with mock siRNA or siRNA to Acinus in the absence or together with Flag-tagged Acinus-L in which two silent mutations preventing interaction between siRNA and the Acinus mRNA were introduced (Acinus mut). After 48 h, the cells were treated as in (B). Subsequently, all DNA samples were extracted and separated on 1.5% agarose gels. (D) AAC-11 expression does not modulate drug-induced chromatin condensation. U2OS clones stably expressing T7-tagged AAC-11 or T7-tagged AAC-11 LL/RR were treated as in (B) and apoptotic chromatin condensation was measured as in (B). (E) AAC-11 expression prevents oligonucleosomal DNA fragmentation. U2OS clones stably expressing T7-tagged AAC-11 or T7-tagged AAC-11 LL/RR the cells were treated as in (B). Subsequently, all DNA samples were extracted and separated on 1.5% agarose gels (top) or sub-G1 fraction was determined by flow cytometry (bottom). Each bar represents the mean±s.d. from three independent experiments. (F) AAC-11 expression does not prevent CAD DNAse activity. U2OS-stable clones were exposed to staurosporine (1 μM) for 2 h or left untreated. CAD was immunoprecipitated from the cell lysates with an anti-CAD antibody and the purified CAD immunocomplexes were incubated with plasmid DNA. The reaction samples were then separated by 1.8% agarose gel electrophoresis to visualize DNA degradation. (G) AAC-11 expression does not prevent H2AX phosphorylation. U2OS-stable clones were treated as in (F). Cell lysates were then analysed by western blotting using the indicated antibodies. (H) Deregulation of AAC-11 increases apoptotic oligonucleosomal DNA fragmentation. U2OS control or AAC-11 shRNA-stable clones were cultured in the presence or absence of doxycycline (1 μg/ml) for 48 h. Cells were treated as in (B). Oligonucleosomal DNA fragmentation was then estimated.

Figure 5

(A) Sequences of the wild-type and mutant peptides corresponding to the LZ subdomain of AAC-11. The Antennapedia sequence is in bold. In the mutant peptide, mutations (leucines to arginines) are underlined. (B) Fluorimetry quantification of the cellular uptake of fluorescein-labelled peptides after 1 h in U2OS cells. Each bar represents the mean±s.d. from three independent experiments. (C) The wild-type peptide prevents LZ–LZ interaction. 293T cells were cotransfected with GFP-AAC-11 (361–400) together with GST-AAC-11 (361–400). At 24 h after transfection, cells were exposed for 3 h to the indicated peptides (10 μM), lysed and cell lysates were immunoprecipitated with anti-GFP and blotted with anti-GST. (D) The wild-type peptide disrupts endogenous AAC-11–Acinus interaction. U2OS cells were exposed for 3 h to the indicated peptides (10 μM), lysed and cell lysates were immunoprecipitated with an anti-Acinus antibody and blotted with an AAC-11-1 antibody. (E, F) The wild-type peptide enhances Acinus apoptotic degradation and oligonucleosomal DNA fragmentation. (E) U2OS cells were exposed for 3 h to the indicated peptides (10 μM). The cells were then exposed to etoposide (20 μM) for 12 h, lysed and the lysates were immunoblotted with an anti-Acinus antibody. (F) U2OS cells were exposed for 3 h to the indicated peptides (10 μM). The cells were then exposed to staurosporine (1 μM) for 4 h or left untreated. Oligonucleosomal DNA fragmentation was then estimated.

Figure 6

The wild-type peptide increases drug-induced cell death. (A) U2OS, HeLa, A549 or Molt-4 cells were exposed for 3 h to the indicated peptides (10 μM). The cells were then exposed to etoposide (ETO, 20 μM) or camptothecin (CPT, 10 μM) for 16 h or cultivated under low-serum conditions or left untreated. The percentages of apoptotic cells were then determined as in Figure 1(B). (B) The wild-type peptide increases drug-induced effector caspases activity. U2OS cells were exposed for 3 h to the indicated peptides (10 μM) and exposed to etoposide (ETO, 20 μM) or camptothecin (CPT, 10 μM) for 5 h and caspase-3/7 activity was measured. (C) Induction of E2F1 in U2OS-E2F1 cells after removal of tetracycline. U2OS-E2F1 cells cultured in normal serum conditions were plated and the induction of E2F1 was blocked with 1 μg/ml tetracycline. After 24 h, the tetracycline was removed by washing the cells with fresh complete media and the cells were collected 8 and 16 h later. The cells lysates were immunoblotted using the indicated antibodies. (D) The wild-type peptide enhances E2F1- and camptothecin-induced apoptosis. U2OS cells cultured in normal serum conditions were exposed for 3 h to the indicated peptides (10 μM). E2F1 was then induced or not, and the cells exposed to camptothecin (CPT, 10 μM) for 16 h or left untreated. The percentages of apoptotic cells were then determined as in Figure 1(B).