HMGB1 increases radiosensitivity by interacting with HDAC1

Abstract

Objective To study the nuclear protein association of high-mobility group box-1(HMGB1) and histone deacetylase 1(HDAC1), and the effect of interaction on radiosensitivity in human breast cancer cells. Methods The protein-protein interaction was determined by immunoprecipitation-Western blot and glutathione-S-transferase capture assays. Cell growth was examined by MTT (methyl thiazolyl tetrazolium)assay and clonogenic assay. Histone deacetylase activity was analyzed by histone deacetylase assay. Results A significant increase of HMGB1 protein and radiosensitivity was observed in MDA-MB-231 and MDA-MB-468 cells transfected with a pCMV-Tag2B expression vector carrying with a full-length of HMGB1 cDNA. HMGB1 binding to HDAC1 was demonstrated as GST(glutathione S-transferase)-pull down and immunoprecipitation Western blot assay, and the association was elevated by irradiation. An LXCXE motif was required for the HMGB1-HADC1 interaction and HMGB1 radiosensitization. A significant difference of IC50 value was observed, for example, 1.8 and 2.2 Gy(wtHMGB1 transfectants, P 0.05), both compared with 3.9 and 4.1 Gy(pCMV-Tag2B transfectants) in MDA-MB-231 and MDA-MB-468 cells, respectively. A specific HDAC1 inhibitor trichostatin A markedly reduced the HMGB1-mediated radiosensitivity, 0.5 Gy in the presence of trichostatin A versus 1.8 Gy in absence of trichostatin A in MDA-MB-231 transfectants, 1.2 Gy (with trichostatin A) versus 2.2 Gy (without trichostatin A) in MDA-MB-468 transfectants, P<0.05. Histone deacetylase activity was also detected in immunoprecipitates prepared from these cells with antibodies to HMGB1, and this activity was abolished by the histone trichostatin A. Conclusions These results suggest a previous unanticipated role for HDAC1 in modification of HMGB1-mediated radiosensitivity by its direct interaction with HMGB1. Key words: High mobility group box; Radiosensitivity; Histone deacetylase 1; Human breast cancer