Regulation Effect Of 5-Fu On The Expression Of NKG2D Ligands MICA/B

Comprehensive treatment is critical to cure malignant tumor.The role of immunotherapy is more and more important except conventional chemotherapy, radiotherapy and surgery. Since Rosenberg et al found lymphokine activated killer (LAK) amplified by high-dose IL-2 could cure cancer in the late 80s of 20th century, the conception of NK cells based adoptive immunotherapy has been proposed. NK cells can kill target cells without the need for prior sensitizationa and they have broader anti-tumor spectrum. As the killing activity of NK cells is related to the dose of IL-2, the number of activated NK cells in vivo and the level of tumor surface antigen of MHC-Ⅰexpression, the clinical efficacy of NK cells immunotherapy is different. In clinical, NK cells are used in combination with chemotherapy to treat tumors. The cytotoxic activity of NK cells is regulated by the balance of inhibitory and stimulatory receptors. NKG2D is a very important stimulatory receptor on the surface of NK cells. NKG2D engagement may directly activate NK cells and subsequently attack target cells expressing NKG2D ligands.NKG2D ligands belong to "induced" surface antigen and many factors, such as ionization, heat shock or virus infection, collectively called "cell stress", can regulate their expressions. If tumor cells are treated with drugs (such as 5-Fu) causing DNA damage, the NKG2D ligands expressed on tumor cells' surface can be upregulated and the activation signals of NK cells are enhanced.Therefore, the susceptibility of the tumor cells to NK cells-mediated killing is increased. In 2009, Nice et al demonstrated a new mechanism of NKG2D ligands'expression-posttranslational regulation. His research results showed that the mouse NKG2D ligand-Multl has long intracellular domain with six lysine residues, and its cell surface expression is regulated by ubiquitination and lysosomal degradation. When the target cells are exposed to ultraviolet light, Multl's level of ubiquitination and lysosomal degradation are reduced, leading to the increased expression of Multl on the target cell surface. Nice speculated that because the cell surface transmembrane proteins MICA, MICB and RAET-1G have continuous lysine residues in the cytoplasmic tail, it's possible that they have regulatory mechanism of ubiquitination. Therefore, we studied the regulation effect of 5-Fu on the expression of MICA and MICB in HeLa cells and the cytotoxic susceptibility of HeLa cells to NK cells. We also investigated the regulatory mechanism of surface expression of MICA, focusing on the role of the cytoplasmic tail. Here we constructed the MICA-full-length and the cytoplasmic tail truncated expression vectors and analysed the impact of proteasome inhibitor MG132 on the surface MICA expression.Methods:HeLa cells were treated with different concentrations of 5-Fu and then the changes of RNA and protein of MICA/B were detected by semi-quantitative PCR, fluorescence quantitative PCR or flow cytometry. Cytolysis of HeLa cells were performed in the presence or absence of NKG2D antibody, MICA or MICB antibody. NK cells-mediated killing of HeLa cells was determined by MTT assay. The MICA-full-length and cytoplasmic tail truncated MICA were amplified and inserted into expression vector-pFLAG-CMV-2. HeLa cells were transfected by the constructed vectors and treated with 5-Fu (40μg/ml) for 24 hours or/and MG132 for 8 hours. Then the expression changes of surface MICAexpression were detected by flow cytometry.Results The results of semi-quantitative RT-PCR showed that MICA/B expression was positively related with the concentration of 5-Fu (2.5 to 40μg/ml) and the duration of exposure time (Oh,8h,16h,24h). Quantitative RT-PCR showed that the mRNA expression of MICA and MICB were upregulated 1.8 and 1.4 times respectively. Flow cytometry data demonstrated that MICA expression was upregulated and the upregulation of MICA expression was mainly due to the non-apoptotic cells. However, the MICB expression keeped negative before and after treatment of 5-Fu.The cytolytic effect of NK92 cells to HeLa cells was enhanced by 5-Fu treatment for 24h at different effect/ target ratio. The killing effect was partially inhibited by NKG2D antibody and MICA antibody but not MICB antibody MICA-full-length-pFLAG-CMV-2 and MICA-cytoplasmic tail truncated-pFLAG-CMV-2 were constructed. HeLa cells were transfected by these two vectors and then treated with 5-Fu (40μg/ml) and/or MG132.The results showed that 5-Fu can increase the surface expression of cytoplasmic tail truncated MICA and the surface expression of MICA keeped constant. On the contrary, HeLa cells had oppsite effect on the surface expression of full-length MICA.Conclusion 5-Fu can upregulate the mRNA expression of MICA/B along with the increase of dose and exposure time.5-Fu can also upregulate the surface expression of MICA and enhance the sensitivity of NK92 cells while MICB surface expression was not affected.The difference of MICA/B surface expression may be related to intracellular segment containing ubiquitin site.It suggested that chemotherapy can work in coordination with NK cell-based immunotherapy and enhance the therapeutic effect. It's indicated that the cytoplasmic tail has a role on the regulation of surface expression of MICA in HeLa cells.