Tumor Suppressor Gene Induced Apoptosis Of Chronic Myeloid Leukemia Cells In Vitro

Objective：To study tumor suppressor gene induced apoptosis of chronicmyeloid leukemia cells in vitro and interacted with imatinib in a highlysynergistic way.Methods：1、Preparation and culture of CML cells.2、Production lentiviralvectors，use lentiviral vector transfer system to transduced tumor suppressorgene p14^ARFinto leukemia cell lines and leukemia cells sources of patients，gene transduction efficiency was detected under fluorescence microscopy.3、Analysis of p14^ARF-induced proliferation and apoptosis in CML leukemiacells by Leukemic colony forming assay and flow cytometry with annexin VFITC/PI double staining.4、To set up p14group, vector group and controlgroup，virus-infected leukemia cells were cultured in vitro for48h，co-culturedwith different concentrations of imatinib，cell proliferation was detected byWST-8， and draw the concentration-response curves, analysis cellproliferation curve.Results:1、 VSV-G pseudotyped lentiviral vectors infected CMLleukemia cell，transduction efficiency was almost100%for K562and blastcells from CML-BC1，transduction efficiency of blast cells from the3othersrespectively were CML-BC275±2.65%，CML-BC392±1.63%，CML-BC485±1.41%，which confirming quite high potential of lentiviral vectors in bloodsystem cells.2、After HIV-p14infection，the ratio of apoptotic cells ofCML-BC1、CML-BC2、CML-BC3、CML-BC4respectively were80±3.37%，95±4.08%，36±0.82%，69±2.58%， compared with the control group，there was a significant difference（p＜0.05）.3、Leukemia cells infected by the virusvector. CML-BC1, CML-BC2, CML-BC3, CML-BC4colony numberscompared with the control group reduced significantly, the difference wasstatistically significant （p＜0.05）.4、Added different concentrations of imatinibto the medium，and co-cultured with the chronic myeloid leukemia cell lines bythe ARF gene infected.Cell proliferation was detected after72hours, thegrowth was inhibited obviously compared with the control group，thedifference was statistically significant （p＜0.05）.Although ARF cooperatedwith imatinib inhibited k562cell proliferation with dose dependently, butcouldn’t make dose effect curve to left shift, Showed that synergistic effects areadditive rather than multiplicat.These results indicated that exogenous forceof the ARF gene expression was enhanced imatinib inhibited the growth ofk562cells.Conclusion:The VSV-G pseudotyped lentiviral vectors expressing p14^ARFcould infect chronic myeloid leukemia cells at high efficiency，inhibitedleukemic cell proliferation apparently, and leaded to apoptosis.In addition，theexpression of exogenous ARF protein enhanced the killing effect of imatinib inPh+chronic myeloid leukemia cells.These results suggest that the combinationof ARF protein and imatinib can be used for the treatment of Ph-positiveleukemias)，and provides experimental data for molecular targeted agents incombination with gene therapy.