全反式维甲酸诱导经典型霍奇金淋巴瘤B细胞表型
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摘要
目的探讨全反式维甲酸(ATRA)对经典型霍奇金淋巴瘤B细胞表型的诱导作用。方法构建含有G418抗性的B细胞特异性启动子(CD19,CD79a和CD79b)表达载体,转染霍奇金淋巴瘤细胞并筛选稳定克隆。PCR扩增启动子和荧光素酶序列验证其稳定整合,敲除ABF1后检测外源B细胞启动子活性的改变以验证其功能。检测不同浓度的ATRA在不同时间点对外源B细胞启动子活性的诱导作用,通过实时定量PCR进一步证实ATRA对B细胞特异性基因转录水平的影响;检测ATRA与去甲基化药物5-Aza联合处理对B细胞标记物表达的影响;通过流式细胞染色技术仪检测ATRA对霍奇金淋巴瘤细胞表面标记物CD30表达的影响。结果 ATRA能够诱导人经典型霍奇金淋巴瘤B细胞特异性标记物CD19、CD20、CD79a和CD79b的表达,与5-Aza具有协同诱导作用,并下调细胞表面霍奇金特异性抗原CD30的表达。结论 ATRA能够诱导B细胞表型缺失的经典型霍奇金淋巴瘤向B细胞型淋巴瘤转化。
Objective To investigate the induction of B-cell specific phenotype in classical Hodgkin lymphoma( c HL) upon all-trans retinoic acid( ATRA) incubation. Methods To construct B-cell specific promoter( CD19,CD79 a,CD79 b) driven reporter plasmid with NEO cassette to realize stable transfection and selection of c HL reporter cells. To verify the intact integration by amplification of the promoter and luciferase sequences,and to functionally validate the B-cell specific promoter by ABF1 interference and luciferase assay. Repoter cells were incubated with various doses of ATRA and luciferase activity was detected at 24,48 and 72 hours. Reporter cells were treated alone or in combination with5-Aza and ATRA followed by luciferase assay. Endogenous B-cell specific genes( CD19,CD20,CD79 a and CD79 b)transcription and expression levels were detected by real-time PCR and immunoblot,respectively. The expression level of CD30 antigen on Hodgkin lymphoma cell membrane upon ATRA was assessed by flow cytometry. Results ATRA treatment stimulated B-cell specific signature in c HL cells including CD19,CD79 a and CD79 b while down-regulated their CD30 expression. Conclusions ATRA induces B-cell phenotype deficient c HL cells to regain their B-cell transcriptional program while abolishes their Hodgkin-specific machinery.
Objective To investigate the induction of B-cell specific phenotype in classical Hodgkin lymphoma( c HL) upon all-trans retinoic acid( ATRA) incubation. Methods To construct B-cell specific promoter( CD19,CD79 a,CD79 b) driven reporter plasmid with NEO cassette to realize stable transfection and selection of c HL reporter cells. To verify the intact integration by amplification of the promoter and luciferase sequences,and to functionally validate the B-cell specific promoter by ABF1 interference and luciferase assay. Repoter cells were incubated with various doses of ATRA and luciferase activity was detected at 24,48 and 72 hours. Reporter cells were treated alone or in combination with5-Aza and ATRA followed by luciferase assay. Endogenous B-cell specific genes( CD19,CD20,CD79 a and CD79 b)transcription and expression levels were detected by real-time PCR and immunoblot,respectively. The expression level of CD30 antigen on Hodgkin lymphoma cell membrane upon ATRA was assessed by flow cytometry. Results ATRA treatment stimulated B-cell specific signature in c HL cells including CD19,CD79 a and CD79 b while down-regulated their CD30 expression. Conclusions ATRA induces B-cell phenotype deficient c HL cells to regain their B-cell transcriptional program while abolishes their Hodgkin-specific machinery.
引文
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[15]Mathas S.The pathogenesis of classical Hodgkin’s lymphoma:A model for B-cell plasticity[J].Hematol Oncol Clin North Am,2007,21(5):787-804.
[16]李银珍,王芳,邵琼,等.Igh基因单克隆重排检测及其在B细胞性非霍奇金淋巴瘤诊断中的应用[J].中国病理生理杂志,2012,28(11):1994-1998.
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[18]RennéC1,Martin-Subero JI,Eickernjger M,et al.Aberrant expression of Id2,a suppressor of B-cell-specific gene expression,in Hodgkin’s lymphoma[J].Am J Pathol,2006,169(2):655-664.
[19]Losman JA.Cancer therapy:The leukaemia epigenome targeted[J].Nature,2017,543(7647):634-635.
[20]张玲,盛树力,秦川.表观遗传学药物的研究进展[J].中国药理学通报,2013,29(3):297-303.
[21]Ehlers A,Oker E,Bentink S,et al.Histone acetylation and DNA demethylation of B cells result in a Hodgkin-like phenotype[J].Leukemia,2008,22(4):835-841.
[22]Du J,Neuenschwander M,Yu Y,et al.Pharmacological restoration and therapeutic targeting of the B-cell phenotype in classical Hodgkin lymphoma[J].Blood,2017,129(1):71-81.
[23]Viardot A,Goebeler ME,Hess G,et al.Phase 2 study of the bispecific T-cell engager(bite)antibody blinatumomab in relapsed/refractory diffuse large B-cell lymphoma[J].Blood,2016,127(11):1410-1416.
[24]Pérez-Callejo D,González-Rincón J,Sánchez A,et al.Action and resistance of monoclonal CD20 antibodies therapy in B-cell Non-Hodgkin lymphomas[J].Cancer Treat Rev,2015,41(8):680-689.
[25]Bander NH,Czuczman MS,Younes A.Antibody-drug conjugate technology development for hematologic disorders[J].Clin Adv Hematol Oncol,2012,10(8 Suppl 10):1-16.
[2]Townsend W,Linch D.Hodgkin’s lymphoma in adults[J].Lancet,2012,380(9844):836-847.
[3]Küppers R,Rajewsky K,Zhao M,et al.Hodgkin disease:Hodgkin and Reed-Sternberg cells picked from histological sections show clonal immunoglobulin gene rearrangements and appear to be derived from B cells at various stages of development[J].Proc Natl Acad Sci U S A,1994,91(23):10962-10966.
[4]Marafioti T,Hummel M,Foss HD,et al.Hodgkin and ReedSternberg cells represent an expansion of a single cloneoriginating from a germinal center B-cell with functional immunoglobulin gene rearrangements but defective immunoglobulin transcription[J].Blood,2000,95(4):1443-1450.
[5]Slovak ML,Bedell V,Hsu YH,et al.Molecular karyotypes of Hodgkin and Reed-Sternberg cells at disease onset reveal distinct copy number alterations in chemosensitive versus refractory Hodgkin lymphoma[J].Clin Cancer Res,2011,17(10):3443-3454.
[6]刘晓健,龙志国,乔忠杰.经典型霍奇金淋巴瘤表观遗传学异常及其对免疫逃逸的影响[J].实用肿瘤学杂志,2017,31(1):73-77.
[7]Steidl C,Connors JM,Gascoyne RD.Molecular pathogenesis of Hodgkin’s lymphoma:Increasing evidence of the importance of the microenvironment[J].J Clin Oncol,2011,29(14):1812-1826.
[8]Chen X,Esplin BL,Garrett KP,et al.Retinoids accelerate B lineage lymphoid differentiation[J].J Immunol,2008,180(1):138-145.
[9]Sirven A,Ravet E,Charneau P,et al.Enhanced transgene expression in cord blood cd34(+)-derived hematopoietic cells,including developing T cells and NOD/SCID mouse repopulating cells,following transduction with modified trip lentiviral vectors[J].Mol Ther,2001,3(4):438-448.
[10]Sigvardsson M,Clark DR,Fitzsimmons D,et al.Early B-cell factor,e2a,and pax-5 cooperate to activate the early B cell-specific mb-1 promoter[J].Mol Cell Biol,2002,22(24):8539
[11]Akerblad P,Rosberg M,Leanderson T,et al.The b29(immunoglobulin beta-chain)gene is a genetic target for early B-cell factor[J].Mol Cell Biol,1999,19(1):392-401.
[12]Mathas S,Janz M,Hummel F,et al.Intrinsic inhibition of transcription factor E2A by HLH proteins ABF-1 and Id2 mediates reprogramming of neoplastic B cells in Hodgkin lymphoma[J].Nat Immunol,2006,7(2):207-215.
[13]Ushmorov A,Leithuser F,Sakk O,et al.Epigenetic processes play a major role in B-cell-specific gene silencing in classical Hodgkin lymphoma[J].Blood,2006,107(6):2493-2500.
[14]Ushmorov A,Ritz O,Hummel M,et al.Epigenetic silencing of the immunoglobulin heavy-chain gene in classical Hodgkin lymphoma-derived cell lines contributes to the loss of immunoglobulin expression[J].Blood,2004,104(10):3326-3334.
[15]Mathas S.The pathogenesis of classical Hodgkin’s lymphoma:A model for B-cell plasticity[J].Hematol Oncol Clin North Am,2007,21(5):787-804.
[16]李银珍,王芳,邵琼,等.Igh基因单克隆重排检测及其在B细胞性非霍奇金淋巴瘤诊断中的应用[J].中国病理生理杂志,2012,28(11):1994-1998.
[17]Jundt F,Acikg9z O,Kwon SH,et al.Aberrant expression of notch1 interferes with the B-lymphoid phenotype of neoplastic B cells in classical Hodgkin lymphoma[J].Leukemia,2008,22(8):1587-1594.
[18]RennéC1,Martin-Subero JI,Eickernjger M,et al.Aberrant expression of Id2,a suppressor of B-cell-specific gene expression,in Hodgkin’s lymphoma[J].Am J Pathol,2006,169(2):655-664.
[19]Losman JA.Cancer therapy:The leukaemia epigenome targeted[J].Nature,2017,543(7647):634-635.
[20]张玲,盛树力,秦川.表观遗传学药物的研究进展[J].中国药理学通报,2013,29(3):297-303.
[21]Ehlers A,Oker E,Bentink S,et al.Histone acetylation and DNA demethylation of B cells result in a Hodgkin-like phenotype[J].Leukemia,2008,22(4):835-841.
[22]Du J,Neuenschwander M,Yu Y,et al.Pharmacological restoration and therapeutic targeting of the B-cell phenotype in classical Hodgkin lymphoma[J].Blood,2017,129(1):71-81.
[23]Viardot A,Goebeler ME,Hess G,et al.Phase 2 study of the bispecific T-cell engager(bite)antibody blinatumomab in relapsed/refractory diffuse large B-cell lymphoma[J].Blood,2016,127(11):1410-1416.
[24]Pérez-Callejo D,González-Rincón J,Sánchez A,et al.Action and resistance of monoclonal CD20 antibodies therapy in B-cell Non-Hodgkin lymphomas[J].Cancer Treat Rev,2015,41(8):680-689.
[25]Bander NH,Czuczman MS,Younes A.Antibody-drug conjugate technology development for hematologic disorders[J].Clin Adv Hematol Oncol,2012,10(8 Suppl 10):1-16.