Role of transient receptor potential canonical channels in glioma cell biology.

摘要

Gliomas, primary brain tumors derived from glial cells, constitute the majority of malignant tumors within the central nervous system. The most malignant of these tumors, grade IV Glioblastoma multiforme, are characterized by extensive proliferation, cellular and nuclear atypia, angiogenesis, areas of necrosis, and widespread invasion into the brain parenchyma. Data from our lab and others have implicated ion channels in the invasion and proliferation of glioma cells. Moreover, calcium signaling in gliomas and other cells has been implicated in both migration and proliferation. The aim of this dissertation was to investigate Transient Receptor Potential Canonical (TRPC) channels role in glioma cell biology. TRPC channels are non-selective cation channels whose activation is downstream of the phospholipase C cascade and their role in glioma biology was previously unknown. In this dissertation, I show expression of TRPC channel subunits within glioma cell lines and glioblastoma multiforme patient biopsy lysates. Further, I demonstrate that these subunits form functional channels on the plasma membrane. Whole-cell patch clamp electrophysiology shows that TRPC channel inhibitors block small, linear currents and affect glioma calcium signaling by decreasing store-operated calcium entry. These pharmacological inhibitors additionally chronically impair glioma proliferation. To assess whether TRPC channels specifically impact glioma calcium signaling and proliferation, shRNA plasmids directed against TRPC1 were utilized. These plasmids specifically decrease TRPC1 subunit expression as shown by western blot analysis and I show through whole-cell recordings and calcium imaging that TRPC1 channels are functionally impaired. Furthermore, TRPC1 shRNA plasmids decrease glioma proliferation. With TRPC1 channel inhibition, glioma cells become larger and, often, multinucleated indicating a role for TRPC1 channels in cytokinesis. In this dissertation, I also show that TRPC1 channels localize to lipid raft fractions of the plasma membrane and propose that the localization and timing of TRPC channel activation plays a crucial role in glioma cell biology.