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Date of Award

2004

Document Type

Dissertation - Pacific Access Restricted

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

John Livesey

First Committee Member

James Blankenship

Second Committee Member

William Chan

Third Committee Member

Timothy Smith

Fourth Committee Member

Craig Vierra

Abstract

Chemoresistance is a major obstacle in successful chemotherapy. This research explored several genetic and epigenetic factors involved in chemoresistance, angiogenesis and metastasis in the human non-small cell lung cancer cell line A549 and its paclitaxel resistant subclone A549-T24. We characterized various morphological and biochemical differences (with a special focus on the Bcl-2 family of apoptotic regulators) between the two cell lines. Although paclitaxel induced apoptosis in both the cell lines, the subclone was 10 fold more resistant to this drug. Our immunocytochemistry data indicated that VEGF (a potent inducer of angiogenesis) and VEGF receptor-2 mRNA expression levels were higher in A549-T24 cells compared to those in A549 cells. We also observed a higher angiogenic potential in A549-T24 cells as determined by the effect of these cells on endothelial cell growth and cell sprouting using EA.hy926 human umbilical vein endothelial cells and rat aortic ring models, respectively. Our data suggested that tumor cell-induced angiogenesis may involve activation of nitric oxide, calcium and PI3K signaling pathways. Intracellular calcium [Ca 2+ ] i plays a critical role in cellular growth and apoptosis. We characterized alterations in the regulatory pathways of [Ca 2+ ] i handling in our cell lines. While the endoplasmic reticulum calcium store ([Ca 2+ ] er ) was significantly lower, calcium influx pathways were considerably inhibited in A549-T24 cells compared to A549 cells. We investigated the actions of 2-aminoethoxydiphenyl borate to release [Ca 2+ ] er and to block store operated Ca 2+ channels. In addition, we studied the role of Ca 2+ in thapsigargin-induced apoptosis in A549 cells. Integrins, a family of cell adhesion proteins, inhibit apoptosis via activation of survival signals. Integrin (mainly β 3 and α 5 ) gene expression patterns and functions differed between A549 and A549-T24 cell lines, suggesting that adhesion to matrix elements may modulate the response to paclitaxel. Indeed, adhesion to extracellular matrix proteins via integrins resulted in a further decrease in chemosensitivity in A549-T24 cells with simultaneous inactivation of BAD (a pro-apoptotic protein). Cell adhesion mediated drug resistance was successfully reversed using integrin blockers (GRGDS and LM609). In summary, our data suggested that chemoresistance is multifactorial. Understanding the molecular mechanisms of chemoresistance will enable the design of better anticancer agents.

Pages

462

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