The effects of physical and chemical agents on the cytoarchitecture of pheochromocytoma, PC-12 cells

grantor: University of TorontoThe purpose of this investigation was to determine the relationship of hydrostatic pressure induced changes in the cytoarchitecture to regulation of gene expression in PC-12 cells. Cell viability in undifferentiated and 4 day differentiating cells at 10,000 psi for 20 min was 96% and 95%, respectively; these values were not significantly different from cells at atmospheric pressure. In contrast cell proliferation (plating efficiency) in undifferentiated and 4 day differentiating cells at 10,000 psi for 20 min was reduced to 86% and 80%, respectively, relative to cells at atmospheric pressure. Actin mRNA levels at 6,000 and 10,000 psi for 20 min were 78% and 64%, respectively, in undifferentiated cells and 81% and 72% in 4 day differentiating cells, respectively, relative to untreated controls. Tubulin mRNA levels at 6,000 and 10,000 psi for 20 min were 75% and 67%, respectively, in undifferentiated cells and 84% and 74%, respectively, in 4 day differentiating cells. Confocal microscopy in association with three dimensional reconstruction showed that microtubules and microfilaments were disrupted in a dose dependent manner following pressure treatment. Microtubules and microfilaments were disrupted at 4,000 psi for 20 min. of compression. Neurite extensions were retracted at 4,000 psi and at 8,000 psi. The apparent volume of microtubules and microfilaments out of a total of 100 arbitrary units was 22 and 11 respectively, in cells at atmospheric pressure. At 4,000 psi and 8,000 psi the apparent microtubule volume was reduced to 16 and 12 arbitrary units, respectively, and the apparent microfilament volume was reduced to 8 and 5 arbitrary units, respectively. Hydrostatic pressure induced disruption of the cytoskeleton was accompanied by decreases in the level, and changes in the localization of tubulin and actin mRNA at the highest pressures and durations. The cytoskeletal/soluble fraction index (CSFI50), which is a measure of mRNA cytoskeletal association, indicated that actin mRNA localization was more sensitive to the effects of hydrostatic pressure compared to the CSFI50 of tubulin mRNA. In the presence of a destabilizing concentration of cytochalasin D, the CSFI50 of tubulin and actin mRNA localization was shifted to a lower pressure index compared to untreated controls. The reduction of microtubule and microfilament volumes, and mRNA levels and localization is attributed to the pressure induced disruption of the cytoarchitecture.Ph.D