During the previous grant cycle, we showed expression of voltage-dependent Cl- currents in human glioma cell lines and glioma biopsies. Tumor grade and invasiveness correlated positively with expression of glioma Cl-currents, which was absent in normal glia or neurons. We provide evidence that Cl- currents induce cell shape and cell volume changes in glioma cells as they invade tortuous extracellular brain spaces. Glioma Cl- currents and glioma cell invasion were inhibited by the Cl channel blockers DIDS, tamoxifen and chlorotoxin (ClTx) suggesting that glioma CF channels promote the invasiveness of these tumors, a feature that makes gliomas difficult to treat. ClTx has proven to be a highly specific marker for human gliomas in patient biopsies. The molecular nature of glioma CF channels is unknown, as is their interactions with ClTx. Our preliminary biochemical data suggest that these channels are members of the ClC Cl- channel family, with ClC-2, -3, and -5 being prominently expressed in glioma biopsies. Only in glioma cells, these channels interact with a 72kDa protein, the putative ClTx receptor (ClTx-R), which is absent in normal glia and neurons. We are proposing a series of experiments, which will use antisense strategies to delineate the molecular identity of glioma Cl- channels. Using a combination of cell invasion & migration assays, biophysical and imaging techniques, we will study the specific role of ClC-2, -3, and -5 in cell volume regulation and their respective role in glioma cell migration/invasion. We propose to clone ClTx-R following affinity purification and study its interaction with glioma ClC Cl- channels. The similarities between glioma cell migration and the migration of glial progenitor cells suggest that glioma cells may recapitulate this aspect of early glial development and may therefore serve as a model system to study the migration of CNS cells. This possibility will be explored by studying expression and function of ClC Cl- channels in the migration of glial progenitors.
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