Study On The Mechanism Of HAG Priming To Acute Myelogenous Leukemia Cell Line

Objective: To explore the mechanism of granulocyte colony-stimulating factor(G-CSF) ,cytosine arabinoside(Ara-C) and homoharringtonine(HHT) (HAG) "priming" effect to HL-60 cell in vitro.Methods: HL-60 cell line was used as the experimental model. The cell line was cultured under 8 different conditions: without any drugs, G-CSF (100μg/L) only,HHT(0.05mg/L) only, Ara-C(10mg/L) only, G-CSF with HHT, G-CSF with Ara-C, G-CSF, HHT and Ara-C. The cell counts and the cell morphology were performed at the time of basal state and at 12 hours and 24 hours. The cell growth inhibition rate wre detected by methyl thiazolyl tetrazolium (MTT) assay. The cell cycle, the early apoptotic marker of Annexin V and the cell membrane surface antigen CD11_b and CD33 were determined by flow cytometer at 12 hours and 24 hours. Results:1. After the incubation with G-CSF for 24 hours, the cell number was not increased significantly. After the treatment with HAG for 12 hours, the cell number was the lest than other groups significantly and the morphology indicated the apoptotic body and necrosis cell increased.2. MTT assay found that after incubation for 24 hours the cell growth inhibition rate of HAG group was higher than HA group significantly.(57%±0.09 vs 41 %±0.03,P=0.007).3. G-CSF can not promote the cell into S-phase significantly after the treatment with it for 48 hours. HHT did not change the cell cycle distribution either. HAG can inhibit S-phase cell significantly and increase the SubGi cells, that indicted HAG induce the apoptosis.4. After treatment with HAG for 12 hours the early apoptotic marker of Annexin V elevated significantly more than control and HA group (P= 0.47) .It means G-CSF can enhance Ara-C and HHT inducing the apoptosis.5. After incubation with HAG for 12 hours ,the cell membrane surface antigen CD lib increased higher than HA group (P^ 0.037), which indicated G-CSF induce the differentiation in coordination with HHT and Ara-c. Conclusions:1. G-CSF can not effect HL-60 cell proliferation and cell cycle at early period, and can enhance the cytotoxocity of the drugs such as Ara-c and HHT.2. Low-dose HHT can inhibit proliferation of HL-60 cell effectively without phase specificity. Using Ara-C and HHT together can inhibit the leukemia cells in different cell cycle stage. Priming with G-CSF in HAG does not effect cell cycle significantly.3. In HAG priming therapy ,G-CSF can enhance the apoptosis and the differentiation induced by Ara-C and HHT. This is apart of mechanism of HAG priming therapy regiment.