Chen R, Eshleman J R, Brodsky R A, Medof M E
Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Cancer Res. 2001 Jan 15;61(2):654-8.
Phosphatidylinositol glycan-A (PIGA) is a gene that encodes an element required for the first step in glycosylphosphatidylinositol (GPI) anchor assembly. Because PIGA is X-located, a single mutation is sufficient to abolish cell surface GPI-anchored protein expression. In this study, we investigated whether mutation of the PIGA gene could be exploited to identify mutator (Mut) phenotypes in cancer. We examined eight Mut colon cancer lines and four non-Mut colon cancers as controls. In every case, flow cytometric analyses of cells sorted for low fluorescence after staining for GPI-linked CD59 and CD55 revealed negative peaks in the Mut lines but not in the controls. Single cell cloning of purged and sorted GPI-anchor- HCT116 cells and sequencing of the PIGA gene in each clone uniformly showed mutations. Pretreatment of the Mut lines with anti-CD55 or anti-CD59 antibodies and complement or with the GPI-anchor-reactive bacterial toxin aerolysin enriched for the GPI-anchor- populations. Expansion of purged GPI-anchor+ cells in the Mut lines and analyses using aerolysin in conjunction with flow cytometry yielded PIGA gene mutation frequencies of 10(-5) to 10(-4), values similar to the mutation frequencies of the hprt gene. This novel approach allows for the detection of as yet undescribed repair or replication defects and in addition to its considerably greater ease of use than existing techniques and in principle would not require the production of cell lines.
磷脂酰肌醇聚糖 -A（PIGA）是一种编码糖基磷脂酰肌醇（GPI）锚定组装第一步所需元件的基因。由于PIGA基因位于X染色体上，单个突变就足以消除细胞表面GPI锚定蛋白的表达。在本研究中，我们调查了是否可以利用PIGA基因突变来识别癌症中的突变体（Mut）表型。我们检测了8个Mut结肠癌系和4个非Mut结肠癌作为对照。在每种情况下，对经GPI连接的CD59和CD55染色后分选的低荧光细胞进行流式细胞术分析，结果显示Mut系中出现阴性峰，而对照中未出现。对纯化和分选的GPI锚定缺失的HCT116细胞进行单细胞克隆，并对每个克隆中的PIGA基因进行测序，结果均显示存在突变。用抗CD55或抗CD59抗体及补体或用GPI锚定反应性细菌毒素气溶素对Mut系进行预处理，可富集GPI锚定缺失群体。在Mut系中扩增纯化的GPI锚定阳性细胞，并结合气溶素和流式细胞术进行分析，得出PIGA基因突变频率为10^(-5)至10^(-4)，与hprt基因的突变频率相似。这种新方法能够检测尚未描述的修复或复制缺陷，并且除了比现有技术使用起来更加简便之外，原则上不需要建立细胞系。
