Studies of microtubule inhibitor combinations on cytoskeleton architecture

by Sonal, Uppal.

Carol A. Heckman, Advisor Previous work from this laboratory employed an assay for cell morphology, which is based on computer-assisted microscopy and sophisticated classification methods. The assay quantifies aspects of three-dimensional shape, based on values of 102 mathematical variables. To evaluate a phenotype in an experiment, variable values for treated and control cells are compared with a database of values for normal and oncogenically transformed cells. In the current work, I evaluated the effect of inhibitor combinations on the cell features defined by the above assay. The studies confirmed the previous finding that treating cells simultaneously with the MT-polymerizing paclitaxel and MT-depolymerizing agent, colchicine, caused a reversal of the cancer cell phenotype. All combinations of paclitaxel with a MTdepolymerizing agent, including podophyllotoxin, nocodazole, or vinblastine, had the same effect. Based on immunolocalization of tubulin, control cells exhibited numerous MTs arranged parallel to cell edge. Paclitaxel, in combination with any depolymerizing agent, caused MTS to be arranged perpendicular to the cell edge. A review of clinical data showed greater reduction of tumor size in patients exposed to simultaneous combination of MT-polymerizing docetaxel and the MT-depolymerizing vinorelbine, than in patients exposed to either agent alone. The effect of a coupled compound, Colchitaxel, on MT organization and MT plus ends, was studied by immunolocalization. By laser confocal scanning microscopy, the EB1 was localized in sheaths around MT ends in control cells. Treated cells exhibited a more diffuse iii localization, which was restricted to the tips of the MTs. Cultures treated for 48 hours with the novel drug showed an elevated percentage of multinuclear cells. The level was even greater in cultures exposed to the paclitaxel and colchicine combination. The conclusions of the studies are: 1) reversal of the morphometric phenotype is not restricted to the combination of paclitaxel with colchicine, 2) the MT arrangement that best correlates with reversal is one where the periphery is cleared of MTs and MTs are arranged perpendicular to the cell edge, and 3) as a cancer therapy, the highest efficacy is observed when the MT-polymerizing and MT-depolymerizing agents are administered together, not interspersed with single agents. iv