Malaghan Institute of Medical Research, Wellington 6012, New Zealand.
Int J Mol Med. 2010 Jun;25(6):883-8. doi: 10.3892/ijmm_00000418.
The transmembrane glycoprotein CD133 is a marker commonly used for isolation and analysis of putative cancer stem-like cells. However, analysis of CD133 expression is potentially confounded by the fact that two of the commonly used anti-CD133 antibodies, AC133 and 293C, only recognize CD133 that has undergone glycosylation. Therefore, our aim was to thoroughly examine antibody recognition and mRNA expression of CD133 in glioblastoma multiforme. Glioblastoma cell lines and primary cultures obtained from resected tumor tissue were analyzed by real-time PCR, Western blot analysis, and flow cytometry for CD133, and immunofluorescence was used to determine cellular localization. The AC133 and 293C antibodies did not detect any CD133 on the surface of the glioblastoma cells despite the fact that a protein was detected using C24B9, an anti-CD133 antibody that recognizes an unglycosylated epitope. This CD133 variant was truncated ( approximately 16 kDa) and, unlike typical expression of full-length CD133 protein, was found throughout the cytoplasm instead of localized to the plasma membrane. Levels of mRNA and protein for the variant increased with stress, indicating potential for it to be a functional molecule. Because AC133 and 293C antibodies do not detect all CD133 variants in glioblastoma cells, alternate detection methods need to be utilized for complete analysis of CD133 expression and for accurately determining the relationship between CD133 and cancer stem-like cells.
跨膜糖蛋白 CD133 是一种常用于分离和分析假定的癌症干细胞样细胞的标志物。然而，分析 CD133 的表达可能会受到以下事实的影响：两种常用的抗 CD133 抗体，AC133 和 293C，仅识别已经发生糖基化的 CD133。因此，我们的目的是彻底检查多形性胶质母细胞瘤中 CD133 的抗体识别和 mRNA 表达。通过实时 PCR、Western blot 分析和流式细胞术分析从切除的肿瘤组织中获得的胶质母细胞瘤细胞系和原代培养物的 CD133，并通过免疫荧光确定细胞定位。尽管使用 C24B9 检测到了一种识别未糖基化表位的抗 CD133 抗体，但 AC133 和 293C 抗体并未在胶质母细胞瘤细胞的表面检测到任何 CD133。这种 CD133 变体是截断的（约 16 kDa），与全长 CD133 蛋白的典型表达不同，它位于细胞质中，而不是位于质膜上。变体的 mRNA 和蛋白水平随着应激的增加而增加，表明它可能是一种功能性分子。由于 AC133 和 293C 抗体不能检测胶质母细胞瘤细胞中的所有 CD133 变体，因此需要使用替代检测方法来进行 CD133 表达的完整分析，并准确确定 CD133 与癌症干细胞样细胞之间的关系。
