The Effects Of IFN-α Reactivity With The CML Cell Surface Polypeptide Marker

Background and Objective:Chronic myeloid leukemia (CML) is characterized by bone marrow and extramedullary myeloid cell production. Interferon alpha (IFN-α) is one of the most effective pharmaceuticals for CML treatment. However, there are many CML patients who are resistant to IFN-α treatment, and restrict the widespread usage of IFN-α in clinical treatment. In a previous study, by employing phage display, our research group found a heptapeptide KLWVIPQ, which can bind to IFN-α-sensitive KT-1/A3cells. In this study, we have used the heptapeptide KLWVIPQ to investigate its function on IFN-α recognition and their combined effect on target cells. It might be a clue to analyze the mechanism of IFN-α efficacy and resistance in the treatment of CML.Methods:In this study, IFN-α was used to treat different sensitive CML cell lines KT-1/A3and KT-1/A3R cells. We constructed the pEGFP-KLWVIPQ recombinant expression vector and synthesized chemical peptide. Real Time-PCR was used to test the differences of P53mRNA expression after transfected the pEGFP-KLWVIPQ vector into KT-1/A3and KT-1/A3R cells. The proliferation and growth status of KT-1/A3and KT-1/A3R cells were analyzed by using WST-1cell proliferation and cytotoxicity assay kit. Caspase3activity was measured in different groups by using commercial assay kit. Cell apoptosis rate were examined by flow cytometry at48h after transfection of the pEGFP-KLWVIPQ recombinant expression vector. After chemosynthetic peptide induction of KT-1/A3and KT-1/A3R cells, we have again used cell counting assay and WST-1assay to test the proliferation and growth status. And Western blotting was used to analyze the difference of P53and P210protein expression in the conditions with or without the induction by chemosynthetic peptide.Results:Real Time-PCR results showed that P53expression in KT-1/A3cell was significantly increased after the transfection of the pEGFP-KLWVTPQ recombinant expression vector, but P53expression was reduced by IFN-a treatment. WST-1and flow cytometry assay showed the similar trend of results, i.e., the proliferation and cell viability status were reduced in the group of transfected the pEGFP-KLWVIPQ recombinant expression vector, and the result was opposite in IFN-a added group. Compared with the control group, caspase3activity was not significantly increased in the KT-1/A3cell transfected the pEGFP-KLWVTPQ recombinant expression vector, but KT-1/A3cell with transfected the pEGFP-KLWVIPQ recombinant expression vector made the effect of IFN-a weaken. Cell counting assay and WST-1assay showed that0.01μg/mL chemosynthetic peptide reduced the KT-1/A3cell number, and the inhibiting effect of peptide and IFN-α were weakened when they were working together. Western blotting results showed that IFN-a and peptide induced P53protein expression and inhibited the P210protein expression. When they worked together, the inhibiting effect to KT-1/A3cell was reduced.Conclusions:IFN-a and KLWVIPQ can effectively induce apoptotic response of KT-1/A3cell, and they have no effect on KT-1/A3R cell.