Study On The Resistance Of CML Cells To IFN-α Mediated By GST Pi

Chronic myelognous leukemia (CML) is a malignant chronic myeloproliferative disorder (MPD) of the hematopoietic stem cell. Interferon-α(IFN-α) is one of the most effective drugs to therapy CML, but the drug resistance ratio is also very high during IFN-αcuring CML. Our laboratory had discovered GSTP1 was over expressed in IFN-αresistance KT-1/A3R CML cell line, but not in IFN-αsensitive strain KT-1/A3, through proteomics, RT-PCR and Western blot analysis. It is indicated that GSTP1 may be play a great role in drug resistance and anti-apoptosis to IFN-αin A3R cells. In the study, we intend to study the effects of GSTP1 expression in apoptosis induced by IFN-αand IFN-αresistance, through inhibition the GSTP1 expression by RNAi technology.Objective: To investigate the effects of GSTP1 in drug resistance and anti-apoptosis of turnout cells, and provide the new tray to study the mechanisms of drug resistance to IFN-αin CML.Methods: 1. Choosing the target sequence to design and synthesize specifically the targeted siRNA to GSTP1 gene, then constructed expression vector pSilencer-GSTP1-A and pSilencer-GSTP1-B. 2. Detecting the expression of GSTP1 in A3R cells by RT-PCR and Western blotting after transfected vector pSilencer-GSTP1-A and pSilencer-GSTP1-B for 48h; 3. Detecting the apoptosis ratio by FCM after transfecting for 48h and 72h, assessed and survival rate by trypan blue and MTT assay after transfecting for 24-96h, respectively. Detecting the expression of bcr/abl in A3R cells by RT-PCR after transfecting for 48h, when A3R cells were transfected pSilencer-GSTP1-A and induced by IFN-α. At the same time, the control group and non-transfection group were set up, and the results were statistically analyzed.Results: 1. After vectors and lipid blends with the rario 1 : 4 (mass/volume) transfecting A3R cells and cultured for 48h, in pSilencer-GSTP1-A and pSilencer-GSTP1-B transfection group, the GSTP1 mRNA expression decreased 78% and 53%, respectively; the protein expression down regulated 71% and 50%, respectively; 2. Comparing with control group, the pSilencer-GSTP 1-A induced apoptosis ratio increasing 4.53% and 5.71% at 48 and 72h, respectively; by using trypan blue staining and MTT assay, the cell growth inhibition at 24-96h were showed 4.56%, 5.94%, 5.83%, 5.16% and 4.48%, 5.48%, 6.06%,4.62%, respectively; At 48h after transfection of pSilencer-GSTP1-A, the bcr/abl mRNA expression decreased 26%.Conclusions: 1.Successfully constructed the siRNA vectors pSilencer-GSTP1-A and pSilencer-GSTP1-B, and the former had the higher inhibition effect than that of the later one. 2. The apoptosis cells induced by IFN-αincreased significantly in A3R cells after tranfected with pSilencer- GSTP1-A (P＜0.05), but the effect on cell growth were little (P＞0.05) . 3. Through inhibition of GSTP 1 expression, the bcr/abl mRNA expression were decreased induced by IFN-α, it is a molecular mechanism which could increase apoptosis finally.