Gorbenko Olena, Ovcharenko Galyna, Klymenko Tetyana, Zhyvoloup Olexandr, Gaman Nadia, Volkova Darija, Gout Ivan, Filonenko Valeriy
Department of Cell Signalling, Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv, Ukraine.
Hybridoma (Larchmt). 2009 Aug;28(4):295-300. doi: 10.1089/hyb.2009.0018.
Fibroblast growth factor receptor 3 (FGFR3) is a member of the FGFR family of receptor tyrosine kinases, whose function has been implicated in diverse biological processes, including cell proliferation, differentiation, survival, and tumorigenesis. Deregulation of FGFR3 signaling has been implicated with human pathologies, including cancer. Activating mutations in FGFR3 gene are frequently detected in bladder cancer, multiple myeloma, and noninvasive papillary urothelial cell carcinomas, while the overexpression of the receptor is observed in thyroid lymphoma and bladder cancer. The main aim of this study was to generate hybridoma clones producing antibody that could specifically recognize FGFR3/S249C mutant, but not the wild-type FGFR. To achieve this, we used for immunization bacterially expressed fragment of FGFR3 corresponding to loops II-III of the extracellular domain (GST-His/FGFR3/S249C-LII-III), which possesses oncogenic mutation at Ser249 detected in at least 50% of bladder cancers. Primary ELISA screening allowed us to isolate several hybridoma clones that showed specificity towards FGFR3/S249C, but not FGFR3wt protein. Unfortunately, these clones were not stable during single-cell cloning and expansion and lost the ability to recognize specifically FGFR3/S249C. However, this study allowed us to generate several monoclonal antibodies specific towards both FGFR3wt and FGFR3/S249C recombinant proteins. Produced hybridomas secreted MAbs that were specific in Western blotting towards bacterially expressed FGFR3wt and FGFR3/S249C, as well as the full-length receptors ectopically expressed in Sf21 and HEK293 cells. Moreover, transiently expressed wild-type and oncogenic forms of FGFR were efficiently immunoprecipitated with selected antibodies from the lysates of infected Sf21 and transiently transfected HEK293. In summary, generated antibodies should be useful as tools for examining the expression pattern and biological functions of FGFR3 in normal and pathological cells and tissues.
成纤维细胞生长因子受体3（FGFR3）是受体酪氨酸激酶FGFR家族的成员，其功能涉及多种生物学过程，包括细胞增殖、分化、存活和肿瘤发生。FGFR3信号失调与包括癌症在内的人类疾病有关。FGFR3基因的激活突变在膀胱癌、多发性骨髓瘤和非侵袭性乳头状尿路上皮细胞癌中经常被检测到，而在甲状腺淋巴瘤和膀胱癌中观察到该受体的过表达。本研究的主要目的是产生能特异性识别FGFR3/S249C突变体而非野生型FGFR的抗体的杂交瘤克隆。为实现这一目标，我们使用细菌表达的FGFR3细胞外结构域环II-III对应的片段（GST-His/FGFR3/S249C-LII-III）进行免疫，该片段在至少50%的膀胱癌中检测到Ser249处存在致癌突变。初步的ELISA筛选使我们分离出几个对FGFR3/S249C具有特异性但对FGFR3wt蛋白无特异性的杂交瘤克隆。不幸的是，这些克隆在单细胞克隆和扩增过程中不稳定，失去了特异性识别FGFR3/S249C的能力。然而，这项研究使我们产生了几种对FGFR3wt和FGFR3/S249C重组蛋白均具有特异性的单克隆抗体。产生的杂交瘤分泌的单克隆抗体在蛋白质印迹中对细菌表达的FGFR3wt和FGFR3/S249C以及在Sf21和HEK293细胞中异位表达的全长受体具有特异性。此外，从感染的Sf21裂解物和瞬时转染的HEK293中，用选定的抗体能有效地免疫沉淀瞬时表达的野生型和致癌形式的FGFR。总之，产生的抗体应可作为研究FGFR3在正常和病理细胞及组织中的表达模式和生物学功能的工具。
