Szekely L, Uzvolgyi E, Jiang W Q, Durko M, Wiman K G, Klein G, Sumegi J
Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden.
Cell Growth Differ. 1991 Jun;2(6):287-95.
The subcellular localization of the retinoblastoma (RB) protein has been studied in primate cell lines by immunofluorescence staining using different monoclonal and polyclonal antibodies. The protein appeared as granules of heterogeneous size over the interphase nuclei. Computer assisted digital overlap analysis indicated that it was predominantly localized in euchromatic areas with low DNA density. The largest RB positive grains lined up on the heterochromatin/euchromatin boundary. During mitosis, the RB protein dissociated from the condensing chromosomes. It was dispersed throughout the cytoplasm during metaphase and anaphase, and it reassociated with the decondensing chromatin during telophase. A new monoclonal antibody, designated aRB1C1, was raised against a bacterial TrpE/human retinoblastoma protein. It specifically recognized the nonphosphorylated and differentially phosphorylated forms of the RB protein in immunoprecipitation experiments. A collection of RB expressing cell lines gave a positive staining reaction with the antibody, whereas the RB negative Weri-RB-27 retinoblastoma and OHS osteosarcoma cells failed to react. Wild-type RB complementary DNA was introduced into Weri-RB-27 by retrovirus mediated gene transfer. Similar experiments were performed with the DU145 prostatic carcinoma cell line that expresses a mutant RB protein. Reconstituted cells of both lines expressed the normal size RB protein and gave a positive immunofluorescence reaction with the aRB1C1 and other anti-RB antibodies. The new monoclonal antibody, however, showed cell type dependent differences of the staining pattern compared to other anti-RB antibodies, suggesting differentiation dependent accessibility to its epitope.
通过使用不同的单克隆和多克隆抗体进行免疫荧光染色，在灵长类细胞系中研究了视网膜母细胞瘤（RB）蛋白的亚细胞定位。该蛋白在间期细胞核上表现为大小不一的颗粒。计算机辅助数字重叠分析表明，它主要定位于DNA密度低的常染色质区域。最大的RB阳性颗粒排列在异染色质/常染色质边界上。在有丝分裂期间，RB蛋白从凝聚的染色体上解离。在中期和后期，它分散在整个细胞质中，在末期与解聚的染色质重新结合。针对细菌TrpE/人视网膜母细胞瘤蛋白产生了一种新的单克隆抗体，命名为aRB1C1。在免疫沉淀实验中，它特异性识别RB蛋白的非磷酸化和差异磷酸化形式。一组表达RB的细胞系与该抗体产生阳性染色反应，而RB阴性的Weri-RB-27视网膜母细胞瘤和OHS骨肉瘤细胞没有反应。通过逆转录病毒介导的基因转移将野生型RB互补DNA引入Weri-RB-27。对表达突变RB蛋白的DU145前列腺癌细胞系进行了类似的实验。这两个细胞系的重组细胞都表达正常大小的RB蛋白，并与aRB1C1和其他抗RB抗体产生阳性免疫荧光反应。然而，与其他抗RB抗体相比，这种新的单克隆抗体显示出细胞类型依赖性的染色模式差异，表明其表位的可及性与细胞分化有关。
