Ramsamooj P, Notario V, Dritschilo A
Department of Radiation Medicine, Lombardi Cancer Center, Georgetown University, Washington, D.C. 20007, USA.
Radiat Res. 1995 Aug;143(2):158-64.
The responses of cells to ionizing radiation include induction and/or suppression of the expression of genes and proteins. In our investigations of alterations in cellular protein expression in response to ionizing radiation, we have used the techniques of two-dimensional polyacrylamide gel electrophoresis and silver staining. We compared the nuclear protein profiles of control and irradiated (6 Gy, 4 h postirradiation) radioresistant squamous carcinoma cells (SQ-20B) and observed an alteration in the expression of a 40 kDa protein: control nuclei express a protein isoform with pI values between 5.4-5.8, while irradiated nuclei express a more acidic variant with pI values between 5.2-5.5. Using the cyanogen bromide/O-phthalaldehyde method followed by microsequencing analysis, we obtained an internal amino acid sequence and identified the 40 kDa protein as nucleolar protein B23. Western blotting experiments confirmed the internal amino acid sequencing results and showed both species (control, 5.4-5.8, irradiated, 5.2-5.5) to be recognized by an anti-B23 monoclonal antibody. Radiolabeling of control and irradiated samples with [32P]NAD or [32P]orthophosphoric acid revealed the acidic species of B23 to be both ADP-ribosylated and phosphorylated. Therefore, exposure of SQ-20B cells to radiation results in the increase in expression of an ADP-ribosylated and phosphorylated species of B23.
细胞对电离辐射的反应包括基因和蛋白质表达的诱导和/或抑制。在我们对细胞蛋白质表达因电离辐射而发生变化的研究中，我们使用了二维聚丙烯酰胺凝胶电泳和银染技术。我们比较了对照和辐照（6 Gy，辐照后4小时）的耐辐射鳞状癌细胞（SQ - 20B）的核蛋白谱，观察到一种40 kDa蛋白质的表达发生了变化：对照细胞核表达一种等电点值在5.4 - 5.8之间的蛋白质异构体，而辐照后的细胞核表达一种酸性更强的变体，其等电点值在5.2 - 5.5之间。使用溴化氰/邻苯二甲醛法随后进行微测序分析，我们获得了一段内部氨基酸序列，并将这种40 kDa蛋白质鉴定为核仁蛋白B23。蛋白质免疫印迹实验证实了内部氨基酸测序结果，并表明这两种类型（对照，5.4 - 5.8；辐照，5.2 - 5.5）都能被抗B23单克隆抗体识别。用[32P]NAD或[32P]正磷酸对对照和辐照样品进行放射性标记，结果显示酸性形式的B23既发生了ADP - 核糖基化又发生了磷酸化。因此，将SQ - 20B细胞暴露于辐射下会导致ADP - 核糖基化和磷酸化形式的B23表达增加。
