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.
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.
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), 3.6 and 3.8 Gy(HMGB1/C103F 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.
These results suggest a previous unanticipated role for HDAC1 in modification of HMGB1-mediated radiosensitivity by its direct interaction with HMGB1.
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High mobility group box 1(HMGB1), belonging to the high-mobility group (HMG) protein family, is a cytokine-like 215 amino acid nuclear protein, which is an important mediator of the body′s inflammatory, ischemia and injury[1,2]. It is known that HMGB1 is structured into three domains, two basic HMG boxes (HMG domains A and B) and a highly acidic C-terminal domain, which confer an overall dipolar appearance to this protein. Each of the HMG boxes is formed by two short and one long α-helix that upon folding produce an L- or V-shaped three-dimensional domain structure. The concave surface of the L- or V-shaped HMG box domain contacts the DNA in the minor groove in two slightly different ways introducing important modifications in the structure of DNA, in particular a strong bend[1,2]. Presumably, these features are of relevance for the biological functions in which HMGB1 has been involved (DNA repair, recombination, replication, and transcription)[1,2]. HMGB1 can interact through its HMG box domains with a broad range of proteins ranging from nuclear cell proteins to viral proteins, including the recombination activation gene protein (RAG1), several transcription factor proteins including the tumor suppressor p53, the octamer transcription factors Oct1, Oct2, Oct4 and Oct6, some homeotic (Hox) proteins, the steroid receptors, the general initiation factor human TATA-binding protein (hTBP), and the viral replication proteins Rep78 and Rep68[1,2,3,4,5,6]. HMG box A is important for binding to hTBP and p53, whereas the binding to Oct factors, Hox factors, and hormone receptors can take place through boxes A or B[3,4]. HMGB1 is a critical target for cancer therapy[7].
