Doctoral Dissertations

Tzu-Hao Wang

Date of Award

5-1999

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Jay Wimalasena

Committee Members

Albert T. Ichiki, Joyce Merryman, Donald S. Torry, Wesley D. Wicks

Abstract

The essential cellular functions associated with microtubules have led to a wide use of microtubule-interfering agents in cancer chemotherapy with promising results. Although the most well studied effect of microtubule-interfering agents is an arrest of cells at the G2/M phase of the cell cycle,other effects may also exist. I have observed that paclitaxel (Taxol), docetaxel (Taxotere), vinblastine,vincristine, nocodazole and colchicine activate the c-Jun N-terminal kinase/stress-activated protein kinase(JNK/SAPK) signaling pathway in a variety of human cells. Activation of JNK/SAPK by microtubule interfering agents is dose-dependent and time-dependent and requires interactions with microtubules.Functional activation of the JNKK/SEKl-JNK/SAPK-c Jun cascade was demonstrated by cotransfection with a TPA-response element reporter construct and dominant negative (dn) signal transducers followed by chloramphenicol acetyl-transferase assays. Microtubule-interfering agents also activate both Ras and apoptosis signal-regulating kinase (ASKl), and coexpression of dn Ras and dn ASKl exerted individual and additive inhibition of JNK/SAPK activation by microtubule-interfering agents. These findings suggest that multiple signal transduction pathways are involved with cellular detection of microtubular disarray and subsequent activation of JNK/SAPK.To further examine the role of JNK/SAPK signaling cascades in apoptosis resulting from microtubule dysfunction induced by paclitaxel, I have coexpressed dn signaling proteins of theJNK/SAPK pathway (Ras, ASKl, Rac, JNKK, JNK) in human ovarian cancer cells with a selectable marker to analyze the apoptotic characteristics of cells expressing dn-vectors following exposure to paclitaxel. Expression of these dn signaling proteins had no effect on Bcl-2 phosphorylation, yet inhibited apoptotic changes induced by paclitaxel up to 16 h after treatment. Coexpression of these dna-signaling proteins had no protective effect after 48 h of paclitaxel treatment. These data indicate that: (i) activatedJNK/SAPK acts upstream of membrane changes and caspase-3 activation in paclitaxel-initiated apoptotic pathways, independently of cell cycle stage, (ii) activated JNK/SAPK is not responsible for paclitaxelinducedphosphorylation of Bcl-2, and (iii) apoptosis resulting from microtubule damage may comprise multiple mechanisms, including a JNK/SAPK-dependent early phase and a JNK/SAPK-independent latephase.

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