Bernstein S L, Kutty G, Wiggert B, Albert D M, Nickerson J M
Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892.
Invest Ophthalmol Vis Sci. 1994 Oct;35(11):3931-7.
Two cell lines derived from ocular tumors of a transgenic mouse expressing the SV40 large T antigen have been established as models of human retinoblastoma. One line, TM, originated from a metastasis, and the other, TE, originated from the primary tumor. The authors compared these two lines with the normal adult mouse eye by analysis of the expression of five photoreceptor cell-specific proteins: IRBP, opsin, rod- and cone-specific transducins, and S-antigen. The authors sought to determine which of these proteins was expressed qualitatively and to examine semi-quantitatively for changes in the levels of expression in the cell lines.
Western blot analysis was used to detect photoreceptor-specific intracellular or secreted proteins. Total RNA was prepared from cultured cells or from mouse adult whole eye. Specific messenger levels in total RNA were determined either by northern hybridization analysis or by a semi-quantitative polymerase chain reaction (PCR), coupled to complementary DNA (cDNA) substrates prepared from total RNA.
IRBP was present in the retinoblastoma cell lines and secreted into the medium. Neither S-antigen nor opsin were detectable by immunoblotting. IRBP and cone transducin mRNA were present in both cell lines. In contrast, opsin, rod transducin, and S-Antigen mRNAs were not detectable by PCR. beta-actin was present in the mRNA populations of whole eye and retinoblastoma. SV40 large T antigen mRNA was present only in retinoblastoma cells.
IRBP and cone transducin expression in mouse retinoblastoma cells is independent of signaling provided directly or indirectly through large T antigen or Rb105 regulatory cascades. The pattern of photoreceptor-specific gene expression is similar to that seen in human retinoblastoma cell lines. These murine-derived cell lines may be useful as a tool to study IRBP and cone transducin expression in vitro and to determine early retinoblast expression patterns in the mouse.
已建立两种源自表达SV40大T抗原的转基因小鼠眼部肿瘤的细胞系，作为人类视网膜母细胞瘤的模型。一个细胞系TM源自转移灶，另一个细胞系TE源自原发肿瘤。作者通过分析五种光感受器细胞特异性蛋白（IRBP、视蛋白、视杆和视锥特异性转导蛋白以及S抗原）的表达，将这两种细胞系与正常成年小鼠眼进行了比较。作者试图确定这些蛋白中哪些有定性表达，并半定量检测细胞系中表达水平的变化。
采用蛋白质印迹分析检测光感受器特异性细胞内或分泌蛋白。从培养细胞或成年小鼠全眼中提取总RNA。通过Northern杂交分析或半定量聚合酶链反应（PCR），结合从总RNA制备的互补DNA（cDNA）底物，测定总RNA中的特定信使水平。
IRBP存在于视网膜母细胞瘤细胞系中，并分泌到培养基中。通过免疫印迹未检测到S抗原和视蛋白。两种细胞系中均存在IRBP和视锥转导蛋白mRNA。相比之下，通过PCR未检测到视蛋白、视杆转导蛋白和S抗原mRNA。β-肌动蛋白存在于全眼和视网膜母细胞瘤的mRNA群体中。SV40大T抗原mRNA仅存在于视网膜母细胞瘤细胞中。
小鼠视网膜母细胞瘤细胞中IRBP和视锥转导蛋白的表达独立于通过大T抗原或Rb105调节级联直接或间接提供的信号。光感受器特异性基因表达模式与人类视网膜母细胞瘤细胞系中的相似。这些源自小鼠的细胞系可能作为一种工具，用于体外研究IRBP和视锥转导蛋白的表达，并确定小鼠视网膜母细胞的早期表达模式。
