In Vitro Study About Apoptosis Of STI571 On Expression Of C-kit In Bone Marrow Cells From Patients With Acute Myelogenous Leukemia

Objective This study was designed to explore the influence about apoptosis of STI571 on Expression of C-kit in Bone Marrow cells from patients with Acute myelogenous Leukemia (AML)and apply with a experience for cure AML in clinical as a new method. Methods: 1.morphological observation: To determine cell morphology by light microscope after treatment with STI571 in different concentration(0,0.25,0.5,1.0,10.0umol/l)at indicated time(24h,48h,72h). 2.MTT assay: Inhibition of proliferation was measured with a colorimetric 3-(4,5- dimethylthiazol-2-yl)-2,5-dipheny tetrazolium bromide (MTT) assay[1.2].To assess their survival after STI571 treatment with different concentration at indicated time. Bone Marrow cells (2×106/ml) were transferred to quadruplicate wells of 96-well microtiter plates in 0.2ml culture mediumafter 24h,48h and 72h incubatiny color intensity was measured with a microliter plate reader (DG-3022A.ELISA.NanJing,P.R china) at a wavelength of 570nm.Absorbance Values for wells containing medium alone was subtracted from the results obtained with the test wells. 3.Flow cytometric analysis Flow cytometric analysis of Caspase-3 expression on bone marrow cells after treatment with STI571, cells were washed twice.The samples were analyzed by FCM.in addition in order to detect the activity of Caspase-3 1×10~6/ml cells were resuspended in 500μl cytofix/cytoperm solution them,the samples were washed and analyzed by FCM. 4.The expression of survivin and smac mRNA were measured on bone marrow cells by semi-quantity reverse transcription polymers chain reaction. Results: 1.morphological observation showed characteristic apoptosis changes including cell shrinkage chromatin condensation DNA fragmentation , membrane blebbing and formation of apoptotic bodies. 2.MTT assay showed that STI at (0.25,0.5,1.0,10.0μmol/l) range for 24h,48h and 72h couled markedly inhibit the proliferation of Acute myelogenous Leukemia cells in a dose-dependent manner as well as a time-dependent manner(P<0.05﹚. 3.FCM showed the apoptosis activity of STI571 is remarkable to bone marrow cells exprssion c-kit patient with AML 4.survivin inhibits cell apoptosis and smac induces cell apoptosis the expression of survivin mRNA decrease and smac mRNA increase. Conclusions: 1. STI571 In Vitro inhibits proliferation Expression of C-kit in Bone Marrow cells from patients with AML. 2. STI571 induces Caspase3, Smac mRNA increase to induce cell apoptosis. 3. STI571 inhibits survivin RNA decreas to inhibit cell antiapoptosis. 4. STI571 induces Apoptosis Expression of C-kit in Bone Marrow cells from patients with AML in a dose-dependent manner as well as a time-dependent manner.