P27^Kip1 Is Involved In APL Cell Apoptosis Induced By As₂O₃ And (or) TGF-β1

Following the success of the all-trans retinoic acid treatment of acute promyelocytic leukemia (APL), China scholars found the effect that arsenic trioxide (arsenic trioxide,As₂O₃) treated the disease is equally clear. But the mechanism which As₂O₃ induced apoptosis on APL cell should be in-depth study. Cell cycle is the basic process in life, regulated by a series of regulatory factors, and whether or not did it work played an important role on the multiplication differentiation, tumor occurrence and development. The regulation was mainly decisied with special protein kinase (cyclin-dependent kinase, CDK)in a particular cell cycle point. Research shows that there is a close relationship between disorder cell cycle regulation and tumor occurrence, Now scholars pay more attention on the relationship between the tumor and P27^Kip1.P27^Kip1 is CDK inhibitor, it had a negative role on the cell cycle, involved in the cell differentiation and apoptosis. P27^Kip1 stopped cell multiplication and tumor formation mainly by blocking the cell cycle at G1/S phase, and was regarded as the candidate tumor suppressor gene, and its decreased expression closely related with the occurrence of tumor, so it can be used as independent prognostic indicators judgement in some tumors. P27^Kip1 protein expression was high in the quite cells, started to decline by mitogen stimulation, when DNA synthesis reached its highest point,its expression down to a minimum level. With the completion of the cell cycle, they began to collected, cells re-entered to the static. Currently the research of the mechanism and clinical significance on P27^Kip1 in acute leukemia had become a hot one. The effect of As₂O₃ used for the treatment of APL had been recognized. To observe APL cell apoptosis induced by As₂O₃ and/or TGF-β1, study the role of P27^Kip1,endogenous TGF-β1,Cyclin E and bcl-2 in cell apoptosis, also the level of P27^Kip1, in this study we treated APL cells with As₂O₃ and/or TGF-β1.Materials and Methods1. Drugs and reagentsLymphocyte separation purchased from the China Association medical Institute; As₂O₃ gifted from Harbin Iraq Dalueye Ltd; RPMI 1640 media company purchased from the United States GIBCO; FBS purchased from the China Academy of Medical Sciences Engineering; TGF-β1 purchased from Peprotech EC companies; PI bought from the United States Sigma companies; Babbit Anti- P27^Kip1,Babbit Anti-TGF-β1,Babbit Anti-Cyclin E,Babbit Anti-bcl-2 and SABC purchased from Dr.Germany Biological Engineering Limited; DECP water,HPR-I,DL2000,rTaq,dNTP and MLV purchased from the United States Promega corporation; DAB purchased from Boshide Biological Engineering Limited; PT-PCR primers were made by the Shanghai Public Health Limited Biological Synthesis.2.Study objectThe patients visited to the First Subsidiary Hospital of China Medical University between June 2006 to July 2007. diagnosised by FAB criteria as acute promyelocytic leukemia (APL). All had the patients and / or their families agreements.3.Cell cultivationTake 5 ml bone marrow from APL patients sterile, extracting bone marrow mononuclear cells, adjusted cells to the concentrations of 2×10⁶ cells/ml with RPMI1640(contained 10% fetal calf serum medium), 37℃,5% CO₂,98% humidity conditions sterile culture. The APL cells were divided into four groups according by different treated factors: control group; 5μmol/L As₂O₃ treated group; 5 ng/ml TGF-β1 treated group; 5μmol/L As₂O₃ combined 5 ng/ml TGF-β1 treated group,all were cultured for 24 hours, 48 hours and 72 hours.4.To detect cell apoptosis and cycle with flow cytometryPropidium iodide (PI) was mixed with APL cells for 40 min at 4℃, then cell apoptosis and cycle can be detected with flow cytometry.5.The protein expression of P27^Kip1,endogenous TGF-β1,Cyclin Eand bcl-2APL cells was detected with immunohistochemistry, they were dropped with the SABC work fuild, color-stained, mount, count.6.The mRNA expression of P27^Kip1,endogenous TGF-β1,Cyclin Eand bcl-2(1) To extract the total RNA and semi-quantitative 5-10×10⁶ APL cells were collected. The total RNA was extracted with Trizol, then fully dissolved. 1μl RNA was dissolved by 79μl DEPC, absorbance was measured at 260 nm and 280nm.(2) RT-PCR System for reverse transcription reaction: RNA3μl, 5XBuffer 4μl, dH₂O 8μl, dNTP 2μl, random primers 1.5μl, MLV 1μl, HPR-I 0.5μl.β-actin was used for an internal control.7.Statistical methodDate was analysised with statistical software SPSS11.5.Results1.The morphological changes of APL cells induced by As₂O₃Apoptosis occered in APL cells which was treated with different concentrations of As₂O₃. With the concentration increased, the proportion of apoptotic cells was raised.2.The change of cell apoptosis and cycle on APL cells with different concentrations of As₂O₃The result showed that apoptosis can be induced by As₂O₃ on APL cells ,as the concentration increased,the proportion of apoptotic cells rasied (P<0.05).The same concentration of As₂O₃ on APL cells, as the prolong of time,the proportion of apoptotic cells also rasied (P<0.05).3.The change of cell apoptosis and cycle on APL cells with As₂O₃ and/or TGF-β1Apoptosis can be induced on APL cells in all treated group. Deal with different factors in different time on the APL cells, as the prolong of time,the proportion of apoptotic cells was also raised. 4.The changes of P27^Kip1,endogenous TGF-β1 Cyclin E and bcl-2 inAPL cells which treated by As₂O₃ and/or TGF-β1The statistics analysis showed P27^Kip1 and endogenous TGF-β1 were up regulated,Cyclin E and bcl-2 were down regulated in As₂O₃ -treated group; Cyclin E and bcl-2 were also down regulated in TGF-β1-treated group, the protein of P27^Kip1 was up regulated, but the mRNA of P27^Kip1 did not change; the mRNA of endogenous TGF-β1 was up regulated, but the protein of endogenous TGF-β1 did not change; the effect of TGF-β1 combined with As₂O₃ on improving P27^Kip1 was more strongger than single factor treated group.Conclusion1.Apoptosis was induced by TGF-β1 and/or As₂O₃ on APL cell,and effect of TGF-β1 combined As₂O₃ group was significantly stronger than single treatment groups.2.P27^Kip1,endogenous TGF-β1 were raised on APL cells treated with As₂O₃; when treated with exogenous TGF-β1,the protein of P27^Kip1 was up regulated, but the mRNA of P27^Kip1 did not change. The effect of TGF-β1 combined with As₂O₃ on improving P27^Kip1 was more strongger than single factor treated group.3.Apoptosis was induced by As₂O₃ on APL cells,it was mediated by improving endogenous TGF-β1,then raised the expression of P27^Kip1, so the activity of Cyclin E was inhibited, cell cycle was arreseted at G1 phase,cells went to apoptosis. Exogenous TGF-β1 strengthened the role of As₂O₃.