稳定下调K562细胞株FMI表达对其甲磺酸伊马替尼药物敏感性的影响
|
摘要
目的:探讨稳定下调K562细胞株FMI表达对其甲磺酸伊马替尼药物敏感性的影响及其可能的作用机制。方法:应用Western-blot方法分别检测K562细胞、慢性髓系白血病(CML)病人、慢性髓系急变病人及健康志愿者外周血单个核细胞FMI蛋白表达;设计靶向人FMI的特异性干扰序列,连接入慢病毒载体LV3;用三质粒系统包装慢病毒颗粒,感染K562细胞,筛选稳定下调FMI的K562细胞;应用CCK8法鉴定K562细胞对伊马替尼药物敏感性的变化,AnnexinⅤ/7-AAD双标记的流式细胞术检测细胞凋亡,荧光定量PCR检测白血病细胞FMI、Fz8的转录水平,Western-Blot方法检测白血病细胞FMI、Fz8、NFAT1、BCR-ABL、β-catenin的表达水平。结果:在CML急变病人外周血单个核细胞与K562细胞可检测到FMI蛋白表达,在CML缓解期病人及健康志愿者外周血单个核细胞未检测到FMI表达。成功构建重组慢病毒载体LV3/FMI,并包装慢病毒,筛选出稳定下调FMI蛋白的K562细胞株;稳定下调K562细胞FMI表达后,在相同药物浓度作用下白血病细胞增殖率降低、凋亡率升高,Fz8基因转录水平降低,Fz8蛋白表达水平下降,NFAT1总蛋白水平增高,核转位增加,BCR-ABL融合蛋白表达水平无明显变化,总β-catenin蛋白表达水平下降。结论:稳定下调FMI表达后,K562细胞对伊马替尼药物敏感性增高,凋亡增加,此作用可能是通过抑制FMI-Fz8信号通路而激活Ca2+-NFAT与Wnt/β-catenin信号通路实现的。
Objective: To investigate the effect of stably down-regulating the FMI expression of K562 cells on the sensitivity of K562 cells to Imatinib(IM) and its possible mechanism. Methods: Western-blot was used to detect the expression of FMI protein in K562 cells and peripheral blood mononuclear cells from the patients with chronic myelogenous leukemia, chronic myeloid blast crisis and healthy volunteers. The specific interference sequences targeting at the human FMI gene were designed and ligated into the lentiviral vector LV3; the three plasmid system-packaged lentivirus particles were used to transfect K562 cells to screen K562 cells that stably down-regulated FMI. CCK-8 assay and flow cytometry were used to determine effect of IM on cell proliferation and apoptosis. The transcription level of FMI and Fz8 in leukemia cells was detected by fluorescent quantitative PCR. The protein expression levels of FMI,Fz8, NFAT1, BCR-ABL and β-catenin in leukemia cells were detected by Western-blot. Results: The expression of FMI protein could be detected in peripheral blood mononuclear cells of the patients with CML-BC and K562 cells,the FMI expression could not be detected in all the patients with CML-CP and healthy volunteers. The recombinant lentiviral vector LV3/FMI had been successfully constructed the lentivirus was packaged, and the K562 cells stably down-regulating the FMI protein were screened. After stable down-regulation of FMI expression in K562 cells, the proliferation rate of leukemia cells decreased and the apoptosis rate was increased under the same drug concentration.Both the transcription and protein expression levels of Fz8 decreased. The NFAT1 total protein level increased, as well as the nuclear translocation of protein was enhanced. There was no significant change in the expression level of BCRABL fusion protein. The expression level of β-catenin protein decreased. Conclusion: After the stable down-regulation of FMI expression, the sensitivity of K562 cells to IM and apoptosis of cells increase, which are performed possibly by inhibiting the FMI-Fz8 signaling pathway and activating the Ca~(2+)-NFAT and Wnt/β-catenin signaling pathway.
Objective: To investigate the effect of stably down-regulating the FMI expression of K562 cells on the sensitivity of K562 cells to Imatinib(IM) and its possible mechanism. Methods: Western-blot was used to detect the expression of FMI protein in K562 cells and peripheral blood mononuclear cells from the patients with chronic myelogenous leukemia, chronic myeloid blast crisis and healthy volunteers. The specific interference sequences targeting at the human FMI gene were designed and ligated into the lentiviral vector LV3; the three plasmid system-packaged lentivirus particles were used to transfect K562 cells to screen K562 cells that stably down-regulated FMI. CCK-8 assay and flow cytometry were used to determine effect of IM on cell proliferation and apoptosis. The transcription level of FMI and Fz8 in leukemia cells was detected by fluorescent quantitative PCR. The protein expression levels of FMI,Fz8, NFAT1, BCR-ABL and β-catenin in leukemia cells were detected by Western-blot. Results: The expression of FMI protein could be detected in peripheral blood mononuclear cells of the patients with CML-BC and K562 cells,the FMI expression could not be detected in all the patients with CML-CP and healthy volunteers. The recombinant lentiviral vector LV3/FMI had been successfully constructed the lentivirus was packaged, and the K562 cells stably down-regulating the FMI protein were screened. After stable down-regulation of FMI expression in K562 cells, the proliferation rate of leukemia cells decreased and the apoptosis rate was increased under the same drug concentration.Both the transcription and protein expression levels of Fz8 decreased. The NFAT1 total protein level increased, as well as the nuclear translocation of protein was enhanced. There was no significant change in the expression level of BCRABL fusion protein. The expression level of β-catenin protein decreased. Conclusion: After the stable down-regulation of FMI expression, the sensitivity of K562 cells to IM and apoptosis of cells increase, which are performed possibly by inhibiting the FMI-Fz8 signaling pathway and activating the Ca~(2+)-NFAT and Wnt/β-catenin signaling pathway.
引文
1 Soverini S,Hochhaus A,Nicolini FE,et al.BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors:recommendations from an expert panel on behalf of European LeukemiaNet.Blood,2011;118(5):1208-1215.
2 Giambra V,CR Jenkins,Stem Cells.Leukemia.Springer Netherlands,2012;267-306.
3 Kirstetter P,Anderson K,Porse BT,et al.Activation of the canonical Wnt pathway leads to loss of hematopoietic stem cell repopulation and multilineage differentiation block.Nat Immunol,2006;7(10):1048-1056.
4 Shanavas M,Messner HA,Kamel-Reid S,et al.A comparison of long-term outcomes of donor lymphocyte infusions and tyrosine kinase inhibitors in patients with relapsed CML after allogeneic hematopoietic cell transplantation.Clin Lymphoma Myeloma Leuk,2014;14(1):87-92.
5 Bogner C,Schneller F,Hipp S,et al.Cycling B-CLL cells are highly susceptible to inhibition of the proteasome:involvement of p27,early D-type cyclins,Bax,and caspase-dependent and-independent pathways.Exp Hematol,2003;31(3):218-225.
6 Yang Y,Mallampati S,Sun B,et al.Wnt pathway contributes to the protection by bone marrow stromal cells of acute lymphoblastic leukemia cells and is a potential therapeutic target.Cancer Lett,2013;333(1):9-17.
7 Chen WS,Antic D,Matis M,et al.Asymmetric homotypic interactions of the atypical cadherin flamingo mediate intercellular polarity signaling.Cell,2008;133(6):1093-1105.
8 Heidel FH,Bullinger L,Feng Z,et al.Genetic and pharmacologic inhibition ofβ-catenin targets imatinib-resistant leukemia stem cells in CML.Cell Stem Cell,2012;10(4):412-424.
9 Clevers H,Nusse R,Wnt/β-catenin signaling and disease.Cell,2012;149(6):1192-1205.
10 Nusse R,Clevers H,Wnt/β-catenin signaling,disease,and emerging therapeutic modalities.Cell,2017;169(6):985-999.
11 Voronkov A,Krauss,S,Wnt/beta-catenin signaling and small molecule inhibitors.Curr Pharm Des,2013;19(4):634-664.
12 Robbs BK,Cruz AL,Werneck MB,et al.Dual roles for NFATtranscription factor genes as oncogenes and tumor suppressors.Mol Cell Biol,2008;28(23):7168-7181.
13 Ashihara E,Takada T,Maekawa T,Targeting the canonical Wnt/β-catenin pathway in hematological malignancies.Cancer Sci,2015;106(6):665-671.
2 Giambra V,CR Jenkins,Stem Cells.Leukemia.Springer Netherlands,2012;267-306.
3 Kirstetter P,Anderson K,Porse BT,et al.Activation of the canonical Wnt pathway leads to loss of hematopoietic stem cell repopulation and multilineage differentiation block.Nat Immunol,2006;7(10):1048-1056.
4 Shanavas M,Messner HA,Kamel-Reid S,et al.A comparison of long-term outcomes of donor lymphocyte infusions and tyrosine kinase inhibitors in patients with relapsed CML after allogeneic hematopoietic cell transplantation.Clin Lymphoma Myeloma Leuk,2014;14(1):87-92.
5 Bogner C,Schneller F,Hipp S,et al.Cycling B-CLL cells are highly susceptible to inhibition of the proteasome:involvement of p27,early D-type cyclins,Bax,and caspase-dependent and-independent pathways.Exp Hematol,2003;31(3):218-225.
6 Yang Y,Mallampati S,Sun B,et al.Wnt pathway contributes to the protection by bone marrow stromal cells of acute lymphoblastic leukemia cells and is a potential therapeutic target.Cancer Lett,2013;333(1):9-17.
7 Chen WS,Antic D,Matis M,et al.Asymmetric homotypic interactions of the atypical cadherin flamingo mediate intercellular polarity signaling.Cell,2008;133(6):1093-1105.
8 Heidel FH,Bullinger L,Feng Z,et al.Genetic and pharmacologic inhibition ofβ-catenin targets imatinib-resistant leukemia stem cells in CML.Cell Stem Cell,2012;10(4):412-424.
9 Clevers H,Nusse R,Wnt/β-catenin signaling and disease.Cell,2012;149(6):1192-1205.
10 Nusse R,Clevers H,Wnt/β-catenin signaling,disease,and emerging therapeutic modalities.Cell,2017;169(6):985-999.
11 Voronkov A,Krauss,S,Wnt/beta-catenin signaling and small molecule inhibitors.Curr Pharm Des,2013;19(4):634-664.
12 Robbs BK,Cruz AL,Werneck MB,et al.Dual roles for NFATtranscription factor genes as oncogenes and tumor suppressors.Mol Cell Biol,2008;28(23):7168-7181.
13 Ashihara E,Takada T,Maekawa T,Targeting the canonical Wnt/β-catenin pathway in hematological malignancies.Cancer Sci,2015;106(6):665-671.