Department of Medicine, Surgery and Dentistry, Ospedale San Paolo, Medical Genetics, Università degli studi di Milano, Italy.
Anal Cell Pathol (Amst). 2011;34(6):319-38. doi: 10.3233/ACP-2011-0031.
MAP/microtubule affinity-regulating kinase 4 (MARK4) is a serine-threonine kinase expressed in two spliced isoforms, MARK4L and MARK4S, of which MARK4L is a candidate for a role in neoplastic transformation.
We performed mutation analysis to identify sequence alterations possibly affecting MARK4 expression. We then investigated the MARK4L and MARK4S expression profile in 21 glioma cell lines and 36 tissues of different malignancy grades, glioblastoma-derived cancer stem cells (GBM CSCs) and mouse neural stem cells (NSCs) by real-time PCR, immunoblotting and immunohistochemistry. We also analyzed the sub-cellular localisation of MARK4 isoforms in glioma and normal cell lines by immunofluorescence.
Mutation analysis rules out sequence variations as the cause of the altered MARK4 expression in glioma. Expression profiling confirms that MARK4L is the predominant isoform, whereas MARK4S levels are significantly decreased in comparison and show an inverse correlation with tumour grade. A high MARK4L/MARK4S ratio also characterizes undifferentiated cells, such as GBM CSCs and NSCs. Accordingly, only MARK4L is expressed in brain neurogenic regions. Moreover, while both MARK4 isoforms are localised to the centrosome and midbody in glioma and normal cells, the L isoform exhibits an additional nucleolar localisation in tumour cells.
The observed switch towards MARK4L suggests that the balance between the MARK4 isoforms is carefully guarded during neural differentiation but may be subverted in gliomagenesis. Moreover, the MARK4L nucleolar localisation in tumour cells features this MARK4 isoform as a nucleolus-associated tumour marker.
丝氨酸苏氨酸激酶 MAP/微管亲和调节激酶 4（MARK4）以两种剪接异构体 MARK4L 和 MARK4S 的形式表达，其中 MARK4L 是参与肿瘤转化的候选物。
我们进行了突变分析，以确定可能影响 MARK4 表达的序列改变。然后，我们通过实时 PCR、免疫印迹和免疫组织化学分析了 21 种神经胶质瘤细胞系和 36 种不同恶性程度的组织、胶质母细胞瘤衍生的癌症干细胞（GBM CSCs）和小鼠神经干细胞（NSCs）中的 MARK4L 和 MARK4S 表达谱。我们还通过免疫荧光分析了 MARK4 异构体在神经胶质瘤和正常细胞系中的亚细胞定位。
突变分析排除了序列变异作为神经胶质瘤中 MARK4 表达改变的原因。表达谱分析证实 MARK4L 是主要异构体，而 MARK4S 水平显著降低，与肿瘤分级呈负相关。高 MARK4L/MARK4S 比值也表征了未分化细胞，如 GBM CSCs 和 NSCs。因此，仅 MARK4L 在脑神经发生区域表达。此外，虽然两种 MARK4 异构体都定位于神经胶质瘤和正常细胞的中心体和中间体，但 L 异构体在肿瘤细胞中还表现出额外的核仁定位。
观察到向 MARK4L 的转变表明，在神经分化过程中，MARK4 异构体之间的平衡受到严格保护，但在神经胶质瘤发生过程中可能会被颠覆。此外，肿瘤细胞中 MARK4L 的核仁定位使该 MARK4 异构体成为核仁相关的肿瘤标志物。
