During brain development, progenitor cells migrate over long distances through narrow and tortuous extracellular spaces posing significant demands on the cell's ability to alter cell volume. This phenotype is recapitulated in primary brain tumors. We demonstrate here that volume changes occurring spontaneously in these cells are mediated by the flux of Cl- along with obligated water across the cell membrane. To do so, glioma cells accumulate Cl- to approximately 100 mM, a concentration threefold greater than predicted by the Nernst equation. Shunting this gradient through the sustained opening of exogenously expressed GABA-gated Cl- channels caused a 33% decrease in cell volume and impaired the ability of cells to migrate in a spatially constrained environment. Further, dividing cells condense their cytoplasm prior to mitosis, a phenomenon which is associated with the release of intracellular Cl- as indicated by a 40-mM decrease in [Cl-]i. These findings provide a new framework for considering the role of intracellular Cl- in glioma cells. Here, Cl- serves as an important osmotically active regulator of cell volume being the energetic driving force for volume changes required by immature cells in cell migration and proliferation. This mechanism that was studied in CNS malignancies may be shared with other immature cells in the brain as well.
Primary brain tumors (gliomas) often present with peritumoral edema. Their ability to thrive in this osmotically altered environment prompted us to examine volume regulation in human glioma cells, specifically the relative contribution of Cl − channels and transporters to this process. After a hyposmotic challenge, cultured astrocytes, D54-MG glioma cells, and glioma cells from human patient biopsies exhibited a regulatory volume decrease (RVD). Although astrocytes were not able to completely reestablish their original prechallenge volumes, glioma cells exhibited complete volume recovery, sometimes recovering to a volume smaller than their original volumes (V Post-RVD < V baseline ). In glioma cells, RVD was largely inhibited by treatment with a combination of Cl − channel inhibitors, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and Cd 2+ (V Post-RVD > 1.4*V baseline ). Volume regulation was also attenuated to a lesser degree by the addition of R-(+)-[(2- n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1 H-inden-5-yl)oxy]acetic acid (DIOA), a known K + -Cl − cotransporter (KCC) inhibitor. To dissect the relative contribution of channels vs. transporters in RVD, we took advantage of the comparatively high temperature dependence of transport processes vs. channel-mediated diffusion. Cooling D54-MG glioma cells to 15°C resulted in a loss of DIOA-sensitive volume regulation. Moreover, at 15°C, the channel blockers NPPB + Cd 2+ completely inhibited RVD and cells behaved like perfect osmometers. The calculated osmolyte flux during RVD under these experimental conditions suggests that the relative contribution of Cl − channels vs. transporters to this process is ∼60–70% and ∼30–40%, respectively. Finally, we identified several candidate proteins that may be involved in RVD, including the Cl − channels ClC-2, ClC-3, ClC-5, ClC-6, and ClC-7 and the transporters KCC1 and KCC3a.
Programmed cell death (apoptosis) is important in tissue maintenance. Hallmarks of apoptosis include caspase activation, DNA fragmentation and an overall reduction in cell volume. Whether this apoptotic volume decrease (AVD) is a mere response to initiators of apoptosis or whether it is functionally significant is not clear. In this study, we sought to answer this question using human malignant glioma cells as a model system. In vivo, high grade gliomas demonstrate an increased percentage of apoptotic cells as well as upregulation of death ligand receptors. By dynamically monitoring cell volume, we show that the induction of apoptosis, via activation of either the intrinsic or extrinsic pathways with staurosporine or TRAIL, respectively, resulted in a rapid AVD in D54-MG human glioma cells. This decrease in cell volume could be prevented by inhibiting the efflux of Cl(-) through channels. Such suppression of AVD also reduced the activation of caspases 3, 8 and 9 and suppressed DNA fragmentation. Importantly, experimental manipulations that reduce the cell volume to 70% of the original volume for periods of at least 3 hours were sufficient to initiate apoptosis even in the absence of death ligands. Hence, this data suggests that cell condensation is both necessary and sufficient for the induction of apoptosis.
