1. Establishment of IDH1 mutant cell lines We have established lentivirus vector system that encodes pathogenic IDH mutations. By using this virus expression system, we established a series of glioma cell or normal cell lines with the mutant enzyme expression. The IDH mutants in this approach include IDH1 WT, R132C, and R132H; IDH2 WT, R140Q, and R172K. The coding sequences of IDH mutants were further cloned into other vector system, to allow different needs for application, such as fusion expression with EGFP, bi-cistronic expression with EGFP, or doxycyclin inducible expression, etc. The transgenic cell lines provide potent tool for consequent research understanding phenotypic changes in IDH mutant tumors within the group and branch. 2. Observation of cellular locomotion of IDH1 mutant cells We found remarkable enhancement in cellular locomotion in IDH1 mutant cells. By using time-lapse imaging, we observed IDH1 mutant cells adopted amoeba-like locomotion, which shifted from polygonal shaped, slow locomotion nature to fan-shaped, irregular shaped, fast locomotion phenotype. The changes in cellular locomotion was further confirmed by demonstrating increased indicence of lamellipodia formation in 80% IDH1 mutant cells, whereas IDH1 WT cells exhibit 10% lamellipodia formation. Nevertheless, in a LifeAct confocal microscopy assay, we observed that IDH1 mutant cells exhibitted obvious inward movement of plasma membrane in lamellipodia region, which confirmed the enhancement in cellular locomotion and plasma membrane motility. 3. Observation of endocytosis in IDH1 mutant cells The enhancement in locomotion and plasma reorganization suggested enhancement in substance exchange across plasma membrane. Logically with investigated cellular uptake in IDH1 mutant cells. We applied BSA-FITC or Dextran-FITC based cellular uptake assay, and analyzed endocytosis through either confocal microscopy combined with quantitative image analysis, fluorescence measurement, or flowcytometry. We demonstrated 5-10 fold increase in substance uptake in IDH1 mutant cells. Additionally, we confirmed by immunostaining and western blot that enhanced endocytosis involved varied molecular mechanisms, such as caveolin- clathrin-dependent pathway, macropinocytosis and endosomic pathways. Nevertheless, transmission and scanning electron microscopy confirmed the existence of endocytic vesicles and membrane ruffling in mutant cells. 4. Investigation of Rac1 pathway in IDH1 mutant cells We found Ras-related C3 botulinum toxin substrate 1 (Rac1) is essential for the changes in cellular motility and endocytosis in IDH1 mutant cells. Rac1 is a small GTPase that governs lamellipodia formation and endocytosis. The significant changes in cellular shape and substance uptake hinted that Rac1 could be a bridge in IDH1 mutation and phenotypic changes we identified in these cell lines. By immunostaining, we identified Rac1, as well as its active form Rac1-GTP, is enriched in the lamellipodia region of mutant cells. Western blot confirmed an overall increase in Rac1-GTP in mutant cells. To better understand the role of Rac1 in IDH1-associated phenotypic changes, we reduced the Rac1 expression in IDH1 mutant cells through siRNA transfection. We found that Rac1 genetic silencing lead to drastic decrease in both lamellipodia formation (over 85% decrease) and endocytosis (over 80% decrease). The important role of Rac1 in cellular morphology and endocytosis was further demonstrated by constitutive active/inactive mutant Rac1 expression in mutant cells.
| Pang, Ying; Lu, Yanxin; Caisova, Veronika et al. (2018) Targeting NAD+/PARP DNA Repair Pathway as a Novel Therapeutic Approach to SDHB-Mutated Cluster I Pheochromocytoma and Paraganglioma. Clin Cancer Res 24:3423-3432 |
