The Selective Regulation Of NKG2D Ligands By 5-FU And MG132

NK cells are major effector cells of innate immune system, playing an important role in tumor immunity, antiviral infection and removal of'non-self'cells [1-4]. The difference between T cells and NK cells is that toxic effect of the latter is not restrained by MHC- I molecules of tumor cells, and the cell activity is the result of the interaction of various receptor molecules with corresponding ligands.NKG2D (NK group 2, member D) is receptor of activation and belongs to the type of c-lectin superfamily. NKG2D is widely expressed on many immune cells [5,6], such as all NK cell surface and some NKT cells, part of the T cells[7]. NKG2D ligands are MHC-I related molecules (MHC class I-related molecules MICs), including MICA, MICB, and ULBP1-5 [8] (Human cytomegalo virus UL16 protein binding proteins 1-5). MICs are induced cell surface antigen [10,11]. In recent years it has beeen found in studies that many factors, such as cell stress, heat shock, infection, DNA damage and transformation can up-regulate their expression [13-17].Research shows that due to the lost or variation of MHC- I molecules on tumor cells, the specific MHC restrictive cytotoxic T cells can not play a role. In this case, the engagement of NKG2D to non MHC-I molecules (MICs) and subsequent dissolution of tumor cells in the immune response plays an important role [18,19]. Following the interaction of NKG2D and corresponding ligands induced on surface of target cells, NKG2D binds to adaptor protein and transmits activated signal to NK cells. In this way NK cells obtain the ability to attack the target cells [20]. In addition, CD8 + T-cells are provided with the necessary synergy stimulating signals, thus the activation of NKG2D pathway largely determines the intensity of cells immunity against cancer.At present, NK cells based immuno-therapy combined with chemotherapy is clinically widely applied, but it is still unclear that how chemotherapy regulates the antitumor activity of NK cells, especially how NKG2D-mediated killing mechanism works. Why there are so many different ligands of the same NKG2D receptor exist on the cells? What are the different roles between them? What are the differences between their expression regulation? Different chemotherapy drugs cause different damages, then what is the difference of the regulation of NKG2D ligands expression between the different pathways? Although NKG2D ligands'coding regions are conservative, the homology of their 5 ' non-translation region is very low, which hints that the expression regulation of different NKG2D ligands may be specific by different stimuli or lesions. Therefore, we use lung adenocarcinoma cell line A549 as the cell model to investigate the regulation effect of chemotherapeutic drug 5-FU and proteinase inhibitor MG132 on the expression of NKG2D ligands and the susceptibility of A549 cells to NK lysis. We also studied the possible regulatory mechanisms involed, focusing on the role of DNA damage response pathway. Objective: The research is to study the regulation effect of 5-FU and MG132 on the expression of NKG2D ligands on A549 cell lines and the mechanisms involved.Methods: Genome DNA of A549 cells was isolated and the full-length and five truncated MICA/B promoter were amplified using polymerase chain reaction (PCR) method. The promoter genes were then cloned into the pGL3-Basic reporter vector and transfected into A549 cell. The promoter activity was analysed with Dual-Luciferase Reporter Assay System. The inhibition rates of A549 cells by different concentrations of 5-FU and MG132 were determined by MTT assay. A549 cells were treated with 300μM 5-FU and 10μM MG132 and the changes of MICA/B promoter activity, the mRNA expression of NKG2D ligands as well as the cell-surface expression levels of NKG2D ligands were measured by Dual-Luciferase Reporter Assay, fluorescence quantitative polymerase chain reaction (PCR) and Flow cytometry respectively. In order to investigate whether DNA damage was occurred after the treatment with 5-FU and MG132, We also performed comet assay to observe the DNA alteration. As for the mechanism research, the phosphorylation of Chk1 and Chk2, the molecules involved in the DNA damage response pathway, were tested by Western blot. In order to study whether blocking of ATM and Chk1 could interfere with the induction of NKG2D ligands, specific inhibitors were used to treat A549 cells and the expression of NKG2D ligands in present or absent of 5-FU and MG132 were investigated.Results: We cloned MICA, MICB promoter and their five truncated genes. The activities of MICA/B promoter were 1.48 and 1.87 fold up-regulated respectively treated by 5-FU. The activity of MICB promoter was 1.77 fold up-regulated treated by MG132. After the treatment with 5-FU, the mRNA expression of MICB and ULBP2 were up-regulated 1.76 and 3.28 fold respectively, and the surface ULBP2 was increased by 16.05%, whereas the surface expressions of MICA/B did not change. After 8 hours'treatment with MG132, the mRNA expression of MICB, ULBP1 and ULBP3 were up-regulated 10.62, 11.09 and 7.25 fold respectively, and the surface MICB and ULBP1 were increased by 68.18% and 23.65%. 5-FU or MG132 treatment improved the cytotoxicity of NK cells and the A549 cell lysis were partly blocked by NKG2D antibody, ULBP2 antibody and MICB, ULBP1 antibody. KU55933(ATM Kinase Inhibitor), SB218078(Chk1 Inhibitor)and Wortmannin(the PI3 Kinase Inhibitor)could inhibit the surface expression of ULBP2 and MICB, ULBP1 induced by 5-FU and MG132. We observed a notable trailing phenomenon in A549 cells after treatment with 5-FU or MG132. Phosphorylation of Chk1, an event associated with DNA damage, was found after treatment of 5-FU. Phosphorylation of Chk2, an event associated with DNA damage, was found after treatment of MG132.Conclusion: The results indicate that 5-FU and MG132 selectively up-regulate surface expression of ULBP2 and MICB on A549 cells, and increase the NKG2D mediated cytotoxicity of NK cells. The regulation effect of 5-FU and MG132 is related to the activation of Chk1 and Chk2, an event associated with DNA damage.