Ca~(2+)-NFAT信号通路在骨髓基质细胞介导的费城染色体阳性急性淋巴细胞白血病耐药中的作用
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摘要
目的:探讨Ca~(2+)-活化T细胞核因子(nuclear factors of activated T cells,NFAT)信号通路在骨髓基质细胞介导的费城染色体阳性(Ph~+)急性淋巴细胞白血病(ALL)耐药中的作用。方法:聚合酶链式反应检测Sup-B15细胞及Ph~+ALL原代细胞NFAT mRNA的转录水平;流式细胞术检测Sup-B15细胞P-糖蛋白表达;Western blot检测Sup-B15细胞NFAT蛋白的变化;Annexin V/7-AAD标记细胞,流式细胞术检测细胞凋亡;Fluo 3-AM染料标记细胞,流式细胞术检测共培养后白血病细胞Ca~(2+)浓度变化。结果:Sup-B15细胞及Ph~+ALL原代细胞中均可检测到NFAT表达;流式细胞术未检测到Sup-B15细胞表达P-糖蛋白;临床应用的治疗浓度(2.5和5μmol/L)的环孢素(CAS)可明显抑制NFAT蛋白表达,其中5μmol/L CAS抑制作用更明显;临床治疗浓度CAS(2.5和5μmol/L)对Sup-B15细胞的凋亡无明显影响,而较高浓度CAS(10μmol/L)可诱导Sup-B15细胞的凋亡。骨髓基质细胞OP9可使Sup-B15细胞及Ph~+ALL原代细胞对伊马替尼的敏感性下降;与骨髓基质细胞OP9共培养后Sup-B15细胞Ca~(2+)浓度升高,总NFAT蛋白水平及核蛋白水平均增加;在共培养体系中加入环孢素抑制Ca~(2+)-NFAT信号通路,可降低OP9对Sup-B15细胞的保护作用。结论:Ca~(2+)-NFAT信号通路有助于Ph~+ALL细胞的存活,骨髓基质细胞_+可通过活化Ca~(2+)-NFAT信号通路介导Ph~+ALL细胞对IM的耐药。
Objective: To explore the role of Ca~(2+)-NFAT signaling pathway in Ph~+-ALL drug resistance mediated by bone marrow stromal cells. Methods: The transcription level of NFAT mRNA in Sup-B15 cells and Ph~+ ALL primary cells was detected by polymerase chain reaction. The expression of P-glycoprotein in Sup-B15 cells was detected by flow cytometry. The change of NFAT protein in Sup-B15 cells was detected by Western blot. AnnexinV/7-AAD was used to label cells. Flow cytometry was used to detect cell apoptosis; Fluo 3-AM dye was used to label cells, and flow cytometry used to detect changes of Ca~(2+) concentration in leukemia cells. Results: NFAT expression could be detected in both Sup-B15 and Ph~+ ALL primary cells; P-glycoprotein could not be detected by flow cytometry; CAS could significantly inhibit NFAT protein expression in clinically applied drug concentrations(2.5, 5 μmol/L); Clinically applied concentration of CAS(2.5, 5 μmol/L) has no significant effect on the apoptosis of Sup-B15 cells, while higher concentration of CAS(10 μmol/L) could induce apoptosis of Sup-B15 cells. Bone marrow stromal cells OP9 could,decrease the sensitivity of Sup-B15 cells and Ph~+ ALL primary cells to imatinib(IM); After co-culture with bone were marrow stromal cells, the Ca~(2+) concentration in Sup-B15 cells was enhanced, the levels of NFAT protein and nullear protein in sup-B15 cells also were enhanced. The addition of CAS in co-culture system could inlibit the Ca~(2+)-NFAT signaling pathway, reduce the protective effect of OP9 on Sup-B15 cells. Conclution: The Ca~(2+)-NFAT sigualing pathway, contributes to the survival of Ph~+ ALL cells. Bone marrow stromal cells can mediate the resistance of Ph~+ ALL cells to IM by activating Ca~(2+)-NFAT signaling pathway.
Objective: To explore the role of Ca~(2+)-NFAT signaling pathway in Ph~+-ALL drug resistance mediated by bone marrow stromal cells. Methods: The transcription level of NFAT mRNA in Sup-B15 cells and Ph~+ ALL primary cells was detected by polymerase chain reaction. The expression of P-glycoprotein in Sup-B15 cells was detected by flow cytometry. The change of NFAT protein in Sup-B15 cells was detected by Western blot. AnnexinV/7-AAD was used to label cells. Flow cytometry was used to detect cell apoptosis; Fluo 3-AM dye was used to label cells, and flow cytometry used to detect changes of Ca~(2+) concentration in leukemia cells. Results: NFAT expression could be detected in both Sup-B15 and Ph~+ ALL primary cells; P-glycoprotein could not be detected by flow cytometry; CAS could significantly inhibit NFAT protein expression in clinically applied drug concentrations(2.5, 5 μmol/L); Clinically applied concentration of CAS(2.5, 5 μmol/L) has no significant effect on the apoptosis of Sup-B15 cells, while higher concentration of CAS(10 μmol/L) could induce apoptosis of Sup-B15 cells. Bone marrow stromal cells OP9 could,decrease the sensitivity of Sup-B15 cells and Ph~+ ALL primary cells to imatinib(IM); After co-culture with bone were marrow stromal cells, the Ca~(2+) concentration in Sup-B15 cells was enhanced, the levels of NFAT protein and nullear protein in sup-B15 cells also were enhanced. The addition of CAS in co-culture system could inlibit the Ca~(2+)-NFAT signaling pathway, reduce the protective effect of OP9 on Sup-B15 cells. Conclution: The Ca~(2+)-NFAT sigualing pathway, contributes to the survival of Ph~+ ALL cells. Bone marrow stromal cells can mediate the resistance of Ph~+ ALL cells to IM by activating Ca~(2+)-NFAT signaling pathway.
引文
1 El Fakih R,Jabbour E,Ravandi F,et al.Current paradigms in the management of Philadelphia chromosome positive acute lymphoblastic leukemia in adults.Am J Hematol,2018;93(2):286-295.
2 Boise LH,Petryniak B,Mao X,et al.The NFAT-1 DNA binding complex in activated T cells contains Fra-1 and JunB.Mol Cell Biol,1993;13(3):1911-1919.
3 Pukrop T,Binder C.The complex pathways of Wnt 5a in cancer progression.J Mol Med(Berl),2008;86(3):259-266.
4 Caetano MS,Vieira-de-Abreu A,Teixeira LK,et al.NFATC2transcription factor regulates cell cycle progression during lymphocyte activation:evidence of its involvement in the control of cyclin gene expression.FASEB J,2002;16(14):1940-1942.
5 Horsley V,Allprantis AO,Polak L,et al.NFATc1 balances quiescence and proliferation of skin stem cells.Cell,2008;132(2):299-310.
6 T?tterman TH,Danersund A,Nilsson K,et al.Cyclosporin-A is selectively cytotoxic to human leukemic T cells in vitro.Blood,1982;59(5):1103-1107.
7 Nooter K,Sonneveld P,Oostrum R,et al.Overexpression of the mdr1gene in blast cells from patients with acute myelocytic leukemia is associated with decreased anthracycline accumulation that can be restored by cyclosporin-A.Int J Cancer,1990;45(2):263-268.
8 Meads MB,Gatenby RA,Dalton WS.Environment-mediated drug resistance:a maior contributor to minimal residual disease.Nat Rev cancer,2009;9(9):665-674.
9 Schaniel C,Sirabella D,Qiu J,et al.Wnt-inhibitory factor 1dysregulation of the bone marrow niche exhausts hematopoietic stem cells.Blood,2011;118(9):2420-2429.
10 Sugimura R,He XC,Venkatraman A,et al.Noncanonical Wnt signaling maintains hematopoietic stem cells in the niche.Cell,2012;150(2):351-365.
11张焕新,陈翀,闫志凌,等.Ph阳性急性淋巴细胞白血病细胞株Sup-B15与基质细胞共培养后对伊马替尼敏感性的变化.中华血液学杂志,2015;36(6):460-464.
2 Boise LH,Petryniak B,Mao X,et al.The NFAT-1 DNA binding complex in activated T cells contains Fra-1 and JunB.Mol Cell Biol,1993;13(3):1911-1919.
3 Pukrop T,Binder C.The complex pathways of Wnt 5a in cancer progression.J Mol Med(Berl),2008;86(3):259-266.
4 Caetano MS,Vieira-de-Abreu A,Teixeira LK,et al.NFATC2transcription factor regulates cell cycle progression during lymphocyte activation:evidence of its involvement in the control of cyclin gene expression.FASEB J,2002;16(14):1940-1942.
5 Horsley V,Allprantis AO,Polak L,et al.NFATc1 balances quiescence and proliferation of skin stem cells.Cell,2008;132(2):299-310.
6 T?tterman TH,Danersund A,Nilsson K,et al.Cyclosporin-A is selectively cytotoxic to human leukemic T cells in vitro.Blood,1982;59(5):1103-1107.
7 Nooter K,Sonneveld P,Oostrum R,et al.Overexpression of the mdr1gene in blast cells from patients with acute myelocytic leukemia is associated with decreased anthracycline accumulation that can be restored by cyclosporin-A.Int J Cancer,1990;45(2):263-268.
8 Meads MB,Gatenby RA,Dalton WS.Environment-mediated drug resistance:a maior contributor to minimal residual disease.Nat Rev cancer,2009;9(9):665-674.
9 Schaniel C,Sirabella D,Qiu J,et al.Wnt-inhibitory factor 1dysregulation of the bone marrow niche exhausts hematopoietic stem cells.Blood,2011;118(9):2420-2429.
10 Sugimura R,He XC,Venkatraman A,et al.Noncanonical Wnt signaling maintains hematopoietic stem cells in the niche.Cell,2012;150(2):351-365.
11张焕新,陈翀,闫志凌,等.Ph阳性急性淋巴细胞白血病细胞株Sup-B15与基质细胞共培养后对伊马替尼敏感性的变化.中华血液学杂志,2015;36(6):460-464.