内质网滞留型抗TfR胞内抗体的构建及其对肿瘤细胞增殖与凋亡的影响研究
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摘要
铁是细胞生长增殖、代谢的必需元素,转铁蛋白受体(TfR)介导的胞吞作用是细胞摄取铁的主要途径。肿瘤细胞由于增殖生长旺盛,对铁的需求高,所以肿瘤细胞有比正常细胞更多的与铁代谢相关受体的表达和对铁的摄取率,使得肿瘤细胞对铁剥夺治疗更敏感。研究证明铁剥夺可在体内外抑制肿瘤细胞生长,诱导其凋亡。
胞内抗体技术是指应用基因重组技术在非淋巴细胞内表达具有生物活性的抗体,并通过对抗体分子进行适当修饰,使之定向分布于细胞核、细胞浆或某些细胞器中,从而特异性干扰或阻断分布于该部位的某些生物大分子的活性或加工、分泌过程,引起细胞的一系列生物过程发生改变。在肿瘤基因治疗领域,胞内抗体已应用于阻断、下调与其增殖密切相关的高表达受体或其他癌相关蛋白,如erbB-2、EGFR、VEGFR、叶酸受体等。胞内抗体的发展开辟了一条通过抗原中和进行表型或功能敲除的新途径。
【目的】本研究拟在前期研究基础上,构建表达内质网滞留型抗TfR的胞内抗体,通过抗体与胞内TfR相互作用抑制肿瘤细胞膜TfR的高表达,减少肿瘤细胞铁的转运和吸收,从而抑制肿瘤细胞生长,诱导其凋亡。探索通过抗TfR胞内抗体达到肿瘤细胞铁剥夺抗肿瘤的可行性。
【方法】利用两步法加端PCR技术在抗TfR单链抗体cDNA两端引入信号肽序列、HA-tag序列及KDEL滞留信号,构建内质网滞留型抗TfR单链抗体真核表达载体pIRES2-scFv-HAK-EGFP ,同时设无滞留信号的表达载体pIRES2-scFv-HA-EGFP作为对照;采用脂质体将重组体转染乳腺癌细胞株MCF-7;通过RT-PCR、western blot、激光共聚焦显微术以及免疫电镜检测scFv基因表达及亚细胞定位;间接免疫荧光染色流式细胞术(FCM)检测肿瘤细胞表面TfR表达,real-time PCR检测铁蛋白、转铁蛋白受体mRNA相对水平,了解转染细胞铁代谢状况;PI染色FCM分析细胞周期,MTT法检测细胞增殖与活力,荧光染色观察细胞凋亡。
【结果】①表达载体通过酶切电泳鉴定可见目的基因条带,测序结果证明含有信号肽序列、HA-tag序列、KDEL滞留信号以及TfR-scFv目的基因,表明内质网定位表达的抗TfR胞内抗体真核表达载体构建成功;②RT-PCR和western blot检测可见有scFv相应的基因和蛋白质条带;③激光共聚焦显微术以及免疫电镜观察可见细胞内质网有scFv的定位表达;④间接免疫荧光FCM结果显示,转染pIRES2-scFv-HAK-EGFP的肿瘤细胞表面TfR的表达显著降低,提示胞内表达的TfR-scFv能与胞内转铁蛋白受体结合,抑制其向细胞表面转运;而real time PCR检测结果表明转铁蛋白受体mRNA水平显著升高,提示有肿瘤细胞铁吸收减少;⑤PI染色FCM检测细胞周期显示转染胞内抗体基因scFv-HAK的肿瘤细胞有明显G0/G1期阻滞和细胞凋亡,细胞增殖和活力检测有明显的肿瘤细胞生长抑制。
【结论】首次提出并证明通过内质网滞留型抗转铁蛋白受体胞内抗体可以成功下调肿瘤细胞表面转铁蛋白受体的高表达,减少肿瘤细胞铁的吸收并抑制肿瘤细胞生长,诱导肿瘤细胞凋亡,本研究探索了一条以TfR高表达为靶点的肿瘤胞内抗体生物治疗研究新途径。
Iron is an essential element for cell proliferation and metabolism. Transferring receptor (TfR)-mediated endocytosis is a major pathway for cellular iron uptake, and the TfR is highly overexpressed in malignant cells. This characteristic renders tumor cells more sensitive to iron depletion which is well known to cause cell cycle arrest and apoptosis. Intrabodies are defined as recombinant antibodies which are expressed intracellularly and addressed in specific subcellular compartments where they can bind, and consequently neutralize or modify the activity of their target antigens. In the field of cancer, intrabodies have been used to modulate the expression of proteins upregulated in tumors, such as erbB-2, IL-2 receptor, epidermal growth-factor receptor (EGFR), vascular endothelial growth factor receptor-2 (KDR), folate receptor, et al. The development of intracellularly expressed antibodies introduced a novel way of antigen neutralization causing a phenotypic or functional knockout.
Objective: Aim to deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, in this study we attempt to intracellularly block surface expression of TfR by intracellular antibody technology, and consequently induce deprivation of iron uptake and affect the proliferation of tumor cells.
Methods and results: we constructed two expression plasmid vectors coding for intracellular single-chain antibody against TfR extracellular domain with (scFv-HAK) or without (scFv-HA) endoplasmic reticulum (ER) retention signal, and then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Realtime-PCR assay indirectly indicates that iron uptake of scFv-HAK transfected cells was significantly decreased. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups.
Conclusions: Our results presented here demonstrate that the intracellular expression of an anti-TfR single-chain antibody with ER retention signal dramatically inhibited the surface expression of TfR, by trapping the TfR and targeting it to the ER. Further, we show that retention of the receptor in the ER caused its function impaired and induced cell cycle G1 arrest and apoptosis, and consequently inhibits tumor cells proliferation. For the first time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors.
胞内抗体技术是指应用基因重组技术在非淋巴细胞内表达具有生物活性的抗体,并通过对抗体分子进行适当修饰,使之定向分布于细胞核、细胞浆或某些细胞器中,从而特异性干扰或阻断分布于该部位的某些生物大分子的活性或加工、分泌过程,引起细胞的一系列生物过程发生改变。在肿瘤基因治疗领域,胞内抗体已应用于阻断、下调与其增殖密切相关的高表达受体或其他癌相关蛋白,如erbB-2、EGFR、VEGFR、叶酸受体等。胞内抗体的发展开辟了一条通过抗原中和进行表型或功能敲除的新途径。
【目的】本研究拟在前期研究基础上,构建表达内质网滞留型抗TfR的胞内抗体,通过抗体与胞内TfR相互作用抑制肿瘤细胞膜TfR的高表达,减少肿瘤细胞铁的转运和吸收,从而抑制肿瘤细胞生长,诱导其凋亡。探索通过抗TfR胞内抗体达到肿瘤细胞铁剥夺抗肿瘤的可行性。
【方法】利用两步法加端PCR技术在抗TfR单链抗体cDNA两端引入信号肽序列、HA-tag序列及KDEL滞留信号,构建内质网滞留型抗TfR单链抗体真核表达载体pIRES2-scFv-HAK-EGFP ,同时设无滞留信号的表达载体pIRES2-scFv-HA-EGFP作为对照;采用脂质体将重组体转染乳腺癌细胞株MCF-7;通过RT-PCR、western blot、激光共聚焦显微术以及免疫电镜检测scFv基因表达及亚细胞定位;间接免疫荧光染色流式细胞术(FCM)检测肿瘤细胞表面TfR表达,real-time PCR检测铁蛋白、转铁蛋白受体mRNA相对水平,了解转染细胞铁代谢状况;PI染色FCM分析细胞周期,MTT法检测细胞增殖与活力,荧光染色观察细胞凋亡。
【结果】①表达载体通过酶切电泳鉴定可见目的基因条带,测序结果证明含有信号肽序列、HA-tag序列、KDEL滞留信号以及TfR-scFv目的基因,表明内质网定位表达的抗TfR胞内抗体真核表达载体构建成功;②RT-PCR和western blot检测可见有scFv相应的基因和蛋白质条带;③激光共聚焦显微术以及免疫电镜观察可见细胞内质网有scFv的定位表达;④间接免疫荧光FCM结果显示,转染pIRES2-scFv-HAK-EGFP的肿瘤细胞表面TfR的表达显著降低,提示胞内表达的TfR-scFv能与胞内转铁蛋白受体结合,抑制其向细胞表面转运;而real time PCR检测结果表明转铁蛋白受体mRNA水平显著升高,提示有肿瘤细胞铁吸收减少;⑤PI染色FCM检测细胞周期显示转染胞内抗体基因scFv-HAK的肿瘤细胞有明显G0/G1期阻滞和细胞凋亡,细胞增殖和活力检测有明显的肿瘤细胞生长抑制。
【结论】首次提出并证明通过内质网滞留型抗转铁蛋白受体胞内抗体可以成功下调肿瘤细胞表面转铁蛋白受体的高表达,减少肿瘤细胞铁的吸收并抑制肿瘤细胞生长,诱导肿瘤细胞凋亡,本研究探索了一条以TfR高表达为靶点的肿瘤胞内抗体生物治疗研究新途径。
Iron is an essential element for cell proliferation and metabolism. Transferring receptor (TfR)-mediated endocytosis is a major pathway for cellular iron uptake, and the TfR is highly overexpressed in malignant cells. This characteristic renders tumor cells more sensitive to iron depletion which is well known to cause cell cycle arrest and apoptosis. Intrabodies are defined as recombinant antibodies which are expressed intracellularly and addressed in specific subcellular compartments where they can bind, and consequently neutralize or modify the activity of their target antigens. In the field of cancer, intrabodies have been used to modulate the expression of proteins upregulated in tumors, such as erbB-2, IL-2 receptor, epidermal growth-factor receptor (EGFR), vascular endothelial growth factor receptor-2 (KDR), folate receptor, et al. The development of intracellularly expressed antibodies introduced a novel way of antigen neutralization causing a phenotypic or functional knockout.
Objective: Aim to deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, in this study we attempt to intracellularly block surface expression of TfR by intracellular antibody technology, and consequently induce deprivation of iron uptake and affect the proliferation of tumor cells.
Methods and results: we constructed two expression plasmid vectors coding for intracellular single-chain antibody against TfR extracellular domain with (scFv-HAK) or without (scFv-HA) endoplasmic reticulum (ER) retention signal, and then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Realtime-PCR assay indirectly indicates that iron uptake of scFv-HAK transfected cells was significantly decreased. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups.
Conclusions: Our results presented here demonstrate that the intracellular expression of an anti-TfR single-chain antibody with ER retention signal dramatically inhibited the surface expression of TfR, by trapping the TfR and targeting it to the ER. Further, we show that retention of the receptor in the ER caused its function impaired and induced cell cycle G1 arrest and apoptosis, and consequently inhibits tumor cells proliferation. For the first time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors.
引文
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25. Paz K, Brennan LA, Iacolina M,et al. Human single-domain neutralizing intrabodies directed against Etk kinase: a novel approach to impair cellular transformation. Mol Cancer Ther. 2005;4(11):1801-9.
26. Heike Y, Kasono K, Kunisaki C,et al.Overcoming multi-drug resistance using an intracellular anti-MDR1 sFv. Int J Cancer. 2001;92(1):115-22.
27. Eric Tse and Terence H. Rabbitts.Intracellular antibody-caspase-mediated cell killing: An approach for application in cancer therapy. PNAS, 2000,97(22):12266–12271.
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30. Marasco WA.Intrabodies as antiviral agents. Curr Top Microbiol Immunol. 2001;260:247-70.
31. Rondon IJ, Marasco WA.Intracellular antibodies (intrabodies) for gene therapy of infectious diseases. Annu Rev Microbiol. 1997;51:257-83.
32. Marasco WA, Chen S, Richardson JH,et al.Intracellular antibodies against HIV-1 envelope protein for AIDS gene therapy. Hum Gene Ther. 1998;9(11):1627-42.
33. Wayne A. Marasco, Joyce LaVecchio, Aaron Winkler.Human anti-HIV-1 tat sFv intrabodies for gene therapy of advanced HIV-1-infection and AIDS. Journal of Immunological Methods .1999,231: 223–238.
34. BouHamdan M, Strayer DS,Wei D,et al. Inhibition of HIV-1 infection by down-regulation of the CXCR4 co-receptor using an intracellular single chain variable fragment against CXCR4. Gene Ther. 2001;8(5):408-18
35. Robert C. Murphy, Anne Messer.A single-chain Fv intrabody provides functional protection against the effects of mutant protein in an organotypic slice culture model of Huntington’s disease. Molecular Brain Research 121 (2004) 141–145.
36. Paolo Paganetti,Verena Calanca,Carmela Galli,et al.β-site specific intrabodies to decrease and prevent generation of Alzheimer’s AβPeptide. JCB; 2005.168 (6).
37. David W. Colby, YiJia Chu, John P. Cassady,et al. Potent inhibition of huntingtin aggregation and cytotoxicity by a disulfide bond-free single-domain intracellular antibody. PNAS ; 2004,101(51): 17616–17621
38. Annette Busch,Wayne A. Marasco,Cornelia Doebis,et al. MHC class I manipulation on cell surfaces by gene transfer of anti-MHC class I intrabodies—a tool for decreased immunogenicity of allogeneic tissue and cell transplants. Methods 34 (2004) 240–249
39. Mhashilkar AM, Doebis C, Seifert M, et al. Intrabody-mediated penotypic knockout of major histocompatibility complex class I expression in humanan and monkey cell lines and in primary human keratinocytes.Gene Ther,2002,9(5):307-319.
40. Caron de Fromentel C, Gruel N, Venot C, Debussche L, et al. Restoration of transcriptional activity of p53 mutants in human tumour cells by intracellularexpression of anti-p53 single chain Fv fragments. Oncogene. 1999;18(2):551-7.
41. Shaki-Loewenstein S, Zfania R, Hyland S, et al.A universal strategy for stable intracellular antibodies. J Immunol Methods. 2005;303(1-2):19-39. Lobato MN, Rabbitts TH.Intracellular antibodies as specific reagents for functional ablation: future therapeutic molecules. Curr Mol Med. 2004;4(5):519-28.
42. M. Natividad Lobato and Terence H. Rabbitts.Intracellular antibodies and challenges facing their use as therapeutic agents. TRENDS in Molecular Medicine Vol.9 No.9 September 2003,9(9).