| IntroductionAcute promyelocytic leukemia (APL) is a malignant diseases of the blood system, the cloning of leukemia cell proliferation control, differentiation obstacles blocked apoptosis, and stagnation in the different stages of cell development. NB4 is with the morphological and biological characteristics of APL.P27Kip1 as a tumor suppressor gene-CDKI (cyclin dependent kinase inhibitor), with cyclinE-CDK2 complexes combination inhibits its activity to prevent the cells to enter S phase cells in the G1 phase of stagnation.CyclinE,as a oncogene,has been confirmed to enter S phase cells is the key regulatory gene.This study investigated cell cycle distribution,apoptosis,the level of P27Kip1,cyclin -E,TGF-β1 changes in NB4 cell after treated with As2O3 and /or TGF-β1,hoping to clarify the pathogenesis of leukemia and to find better treatment strategies and reveals clinical prognosis provided some clues.Methods1. MTT assaysCells, plated in 96-well plates (8×104/ml for NB4 cells), were treated with different concentrations of As2O3.At 24 hours, 48 hours, 72 hours, 20 mL 5mg/mL MTT was added. After incubation for 4 hours, the MTT medium was removed and 150mL DMSO was added. The number of viable cells was assessed by the percentage of absorbance of treated cells relative to that of solvent controls, using 570-nm wave length on a spectrophotometer.2. As2O3 and /or TGF-β1 on NB4 cellsCells were cultured in RPMI-1640 medium supplemented with 10% FBS,at 37℃ in a humidified atmosphere containing 5%CO2. Adjusted cell concentration of 5×105/ml to 5μmol/L As2O3 and/or 5ng/ml TGF-β1, collect cells at different times.3. Trypan blue dye exclusion assay cell growth inhibition rateCollect cells treated with 5μmol/L As2O3 and/or 5ng/ml TGF-β1 at 24,48,72 hours.Take a drop of cell suspension,add two drops of 2% trypan blue, and after two minutes under the microscope records the percentage of living cells.4. Wright-Giemsa staining of apoptotic morphological changes5μmol/L As2O3 and /or 5ng /ml TGF-β1 on NB4 cells for 48 hours, after centrifu-gation, Wright staining one minute, coupled with Giemsa staining liquid water rinse after 15 minutes natural dried, light microscopic observation cell apoptosis.5. Flow cytometry to detect cell cycle and apoptosisCells were collected in centrifuge tubes, 1000r/min centrifugal five minutes, wash twice, add 1ml 80% ethanol,4? refrigerator overnight.1000r/min centrifugal five minutes, add PI dye, 4? dark staining after 30 minutes by flow cytometry test.6. Semi-quantitative RT-PCR was used to detect P27Kip1, cyclinE and endogenous TGF-β1 in mRNA levels(1) total cellular RNA extraction and semi-quantitative Cells were collected, RNA was extracted with Trizol.Then dissolved by 6-15ul DEPC water. Measuring 260, 280 OD value.UV spectrophotometer quantitative, from 2.0≥OD260/OD280≥1.8 of the RNA, will be diluted to a concentration of 3μg/μl.(2) RT-PCR RT reaction system for the RNA 3μl,5×Buffer 4μl, dH20 8μl, 2μl dNTP, random primers 1.5μl,MLV 1μl,HPR-I 0.5μl. RT-PCR system includes reaction product cDNA 3μl, 10×PCR buffer 2.5μl,1.5μl dNTP, 0.2μl TaqDNA polymerase, dH20 15.8μl, the upper and lower reaches of the purpose of primers 1μl.B-actin as an internal control. 13ul of PCR products by adding 0.5mg/L EB 2% agarose gel electrophoresis in the running. UV transmission analyzer (UVP) to observe and analyze the results.7. statistical analysisAll calculations were performed by SPSS for Windows, Versionl 1.5.Results1. The growth inhibition of As2O3 and/or TGF-β1 on NB4 cells As2O3 and/or TGF-β1 can significantly restrain the growth of NB4 cells and promote the apoptosis of NB4 cells. The growth inhibition and apoptosis of NB4 cells treated with As2O3 are in a dose-time dependent.IC50 is about 12μmol/L for 24h, about 5μmol/L for 48h and about 3μmol/L for 72h, respectively.2. NB4 cells apoptosis and cell cycle distribution changes.With increasing concentration of As2O3,NB4 increased in the number of apoptosis. TGF-β1 alone (5 ng / ml) also cause NB4 cells apoptosis, but no more change in cell morphology than As2O3 (5μ, mol / L). The apoptosis treated with combination group was significantly higher than As2O3, TGF-β1 treated group alone. As2O3 and/or TGF-β1 may cause NB4 cells arrest, The blockage of NB4 cells treated by 5μmol/L As2O3 was in G2/M phase, and 5ng/ml TGF-β1 in G1 phase.however,the blockage of NB4 cells by combination of As2O3 and TGF-pi was in S phase.3. P27Kip1, cyclinE, endogenous TGF-β1 and bcl-2 mRNA expression changes after NB4 cells treated with As2O3 and/or TGF-β1.With the increasd concentration of As2O3, P27Kip1 and endogenous TGF-β1 up-regulate mRNA expression and cyclinE, bcl-2 mRNA expression reducted.TGF-β1 dealt with separately NB4 cells, P27Kip1, cyclinE, bcl-2 mRNA expression are the same with As2O3. Combination group showed that TGF-β1 can enhance the effect of As2O3.Exogenous TGF-β1 concentration<5ng/ml, endogenous TGF-β1 increased mRNA expression, exogenous TGF-β1 concentration of 10ng/ml, endogenous TGF-β1 down-regulate mRNA expression.DiscussionAs2O3 is found to be effective in the treatment of APL. Chen found As2O3 induced apoptosis in leukemia cells of endogenous TGF-β1 and accompanied by increased expression of bcl-2 expression reduced. TGF-β1 is known as the strongest factor inhibiting cell proliferation in the blood. Mainly through its cell cycle arrest and apoptosis.The results show that, when As2O3≥5μmol / L, NB4 cells showed significant G2/M phase cell cycle arrest, its causes may be As2O3 work in NB4 cells, G2/M phase reactive oxygen species within the cells can be more than the level of tolerance of the cells, the cells are unable to carry out normal mitosis and arrest in G2 /Mand trigger apoptosis. TGF-pl also promote NB4 cells apoptosis. And the combination of As2O3 and TGF-β1-induced apoptosis in NB4-in As2O3 single drug application, and a S-phase cell cycle arrest, we can see that exogenous TGF-β1 can strengthen NB4 cell apoptosis induced by As2O3. Experimental results indicate that cross-channel may exist in the induction of apoptosis mechanism.The results showed that TGF-β1 and As2O3 regulate P27Kip1, cyclinE, bcl-2 gene expression is the same.As2O3 may increase TGF-β1 promote NB4 cells apoptosis. TGF-β1 is P27Kip1 signal transduction pathway downstream molecules, cell proliferation is a negative regulator. CyclinE is cell cycle regulation factor, and cyclinE,P27Kip1 to maintain interaction between G1/S restriction point of the positive and negative control steady. TGF-β1 may directly inhibit the cyclinE expression, or through the increased expression of P27Kip1 inhibit cyclinE activity, resulting in cell cycle arrest.The results of this study also suggest that as for NB4 cells, the concentration of endogenous TGF-β1<5ng / ml, endogenous TGF-β1 increased mRNA expression, while exogenous TGF-β1 concentration of 10ng/ml, endogenous TGF-β1 reduced mRNA expression. Visible role in NB4 cells, the concentration of endogenous TGF-β1 may be too strong antagonism of endogenous TGF-β1 expression, or the saturation of the target receptor. Therefore the future of TGF-β1 in clinical treatment of leukemia may have guiding significance.Conclusion1.As2O3 and TGF-β1 can induce apoptosis and cell cycle distribution anomaly.2.As2O3 and exogenous TGF-β1 may up-regulate endogenous TGF-β1, so that NB4 cells was induced to apoptosis through high expression of P27Kip1.TGF-β1 may lead to cell cycle arrest by inhibiting the expression of cyclinE directly, or through the increased expression of P27Kip1 feedback inhibiting the activity of cyclinE.3.High concentration of endogenous TGF-β1 could antagonize endogenous expression of TGF-β1, there maybe a target saturation.4.While P27Kip1 gene expression increased, the antagonistic cyclinE gene expression sharply decreased, further prove P27Kip1 and cyclinE may be key intermediate effects of As2O3 and TGF-β1 that cause NB4 cell cycle arrest. |
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