Ku70 acetylation mediates neuroblastoma cell death induced by histone deacetylase inhibitors

Abstract

Histone deacetylase inhibitors (HDACIs) are therapeutic drugs that inhibit deacetylase activity, thereby increasing acetylation of many proteins, including histones. HDACIs have antineoplastic effects in preclinical and clinical trials and are being considered for cancers with unmet therapeutic need, including neuroblastoma (NB). Uncertainty of how HDACI-induced protein acetylation leads to cell death, however, makes it difficult to determine which tumors are likely to be responsive to these agents. Here, we show that NB cells are sensitive to HDACIs, and that the mechanism by which HDACIs induce apoptosis involves Bax. In these cells, Bax associates with cytoplasmic Ku70, a protein that typically increases chemotherapy resistance. Our data show that in NB cells Ku70 binds to Bax in an acetylation-sensitive manner. Upon HDACI treatment, acetylated Ku70 releases Bax, allowing it to translocate to mitochondria and trigger cytochrome c release, leading to caspase-dependent death. This study shows that Ku70 is an important Bax-binding protein, and that this interaction can be therapeutically regulated in NB cells. Whereas the Bax-binding ability of Ku70 allows it to block apoptosis in response to certain agents, it is also a molecular target for the action of HDACIs, and in this context, a mediator of NB cell death.

Fig. 1.

HDACI induces cell death in N-type NB cells. IMR32 and SH-SY5Y cells were treated with various concentrations of TSA or sodium butyrate as indicated for 24 (▴) or 48 (▪) h. Cell viability was determined by MTT assay. Results are expressed as the percentage of viable cells compared with vehicle-treated controls (mean ± SD, n = 3).

Fig. 2.

TSA induces apoptosis in IMR32 cells. (A) IMR32 cells were either treated with DMSO or 1 μM TSA for 24 h. Interference contrast microscopy was used to determine cell morphology at ×200. (B) DNA content was determined by propidium iodide staining and analyzed by flow cytometry in IMR32 cells treated with TSA (1 μM), Z-VAD (100 μM), or CDDP (10 μg/ml) as indicated for 24 h. (C) IMR32 cells were treated with 1 μM TSA for various times as indicated, and the S100 fraction was immunoblotted by using anti-cytochrome c antibodies. The same blot was reprobed with anti-β-tubulin antibodies as a loading control.

Fig. 3.

Bax mediates TSA-induced apoptosis. (A) Mitochondrial fractions of IMR32 cells isolated after treatment with 1 μM TSA for various times as indicated were immunoblotted by using anti-Bax antibodies. The same blot was probed for cytochrome oxidase subunit IV as a loading control. (B) IMR32 and SH-SY5Y were transfected with either empty vector or HA-tagged Bax. Cell viability was determined by MTT assay 48 h after transfection. Results are expressed as the percentage of control (mean ± SD, n = 3). (C) WT, Bax^-/-, or Bak^-/- MEF cells were treated with various concentrations of TSA as indicated for 24 h. Percent of apoptotic cells was derived quantitatively by measuring the percentage of subG1 population using flow cytometry. Results are expressed as mean ± SD (n = 3). (D) WT and Ku70^-/- MEF cells had the same TSA treatment as described in C. Cell viability was determined by MTT assay.

Fig. 4.

Bax is released from Ku70 in response to TSA. (A) Cytoplasmic and nuclear fractions of IMR32 cells were isolated. Equal amounts of proteins were separated on a 4-20% SDS/PAGE gradient gel and then immunoblotted by using anti-Ku70, anti-Bax, anti-β-tubulin, or anti-histone H1 antibodies as indicated. (B) Endogenous Ku70 before or after 4 h TSA treatment was immunoprecipitated by using goat anti-Ku70 antibodies or normal goat serum (NGS) as a control. The immunoprecipitates were separated by SDS/PAGE and probed with anti-Bax or anti-Ku70 antibodies. Input represents 20% of the samples used in immunoprecipitation. (C) IMR32 cells were transfected with either Flag-Ku70 WT or Flag-Ku70 K539R/K542R mutant expression vectors. Twenty-four hours after transfection, cells were treated with 1 μM TSA for an additional 4 h. The transfected Flag-Ku70s were immunoprecipitated by using anti-Flag antibodies or normal mouse serum (NMS) as a control, separated by SDS/PAGE, and probed with anti-Bax or anti-Flag antibodies. Input represents 20% of the samples used in immunoprecipitation.

Fig. 5.

Lys-539 and Lys-542 of Ku70 are critical for Ku70-dependent, TSA-induced apoptosis. IMR32 cells were transfected with a GFP expression vector plus control vector, Flag-Ku70 WT, or Flag-Ku70 double-lysine mutant expression vector. Twenty-four hours after transfection, cells were treated with vehicle (DMSO) or 1 μM TSA for 24 h. (A) The GFP-positive cells were sorted by flow cytometry. Equal amounts of extracts were separated by 4-20% gradient SDS/PAGE and then the blot was probed with anti-Flag, anti-Ku70, anti-Bax, or anti-β-tubulin antibodies as shown. (B) Apoptotic cells were determined by staining the cells with Hoechst dye and counting cells showing DNA condensation per 100 GFP-positive cells. The results are from three different experiments and expressed as mean ± SD (n = 3).

Fig. 6.

Overexpression of Ku70 WT and the double-lysine mutant has differential effects in blocking TSA, STS, or Dox-induced apoptosis. (A) Expression of Flag-Ku70 WT or the double-lysine mutant in the stable IMR32 cell lines was determined by immunoblotting with anti-Flag, anti-Ku70, anti-Bax, or anti-β-tubulin antibodies as indicated. (B) After 24 or 48 h of 1 μM TSA treatment, the viability of IMR32 cells stably expressing Flag-Ku70 WT or the double-lysine mutant was determined by MTT assay. Results are presented as mean ± SD (n = 3). (C) After 24 h of STS or Dox treatment, the viability of IMR32 cells stably expressing Flag-Ku70 WT or the double-lysine mutant was determined by MTT assay. Results are expressed as the percentage of respective controls (mean ± SD, n = 3).