Date of Award:

5-2009

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Biology

Committee Chair(s)

Joseph K.-K. Li

Committee

Joseph K.-K. Li

Committee

Gregory J. Podgorski

Committee

Dennis L. Welker

Committee

Scott A. Ensign

Committee

Jeffery R. Broadbent

Abstract

Bluetongue virus (BTV) is a pathogenic member of the Reoviridae family. BTV does not cause disease in humans, but is capable of selectively infecting and killing certain transformed human cell lines. Understanding BTV's oncotrophism may lead to new therapeutics for treating cancer. This study focused on the underlying mechanisms of BTV-induced cell death in carcinoma cell lines. It was our hypothesis that BTV infects human carcinoma transformed cells, produces mRNA and protein, induces a strong inflammatory response, induces mitogen activated protein kinase (MAPK)-based pro-apoptotic signaling, inhibits PKB-based signaling, and eventually kills the cell by inducing apoptosis.

Three carcinoma cell lines (A498, HEP-G2, and A549) were independently infected with BTV. In each cell line we determined: (1) cell viability over the course of infection; (2) BTV induced cytokine expression profile and magnitude of expression; (3) BTV viral RNA expression profile and magnitude of expression; (4) BTV viral protein expression profile and magnitude of expression; (5) changes in BTV induced cell death and cytokine expression in cells with protein kinase B (PKB), p38-MAPK, extracellular receptor kinase (ERK-1/2), stress-activated protein kinase (SAPK-JNK), Src kinase, platelet-derived growth factor receptor (PDGFR) kinase, epidermal growth factor receptor (EDGFR) kinase, or Janus kinase (JAK) activity inhibited; (6) intracellular changes in PKB, p38-MAPK, ERK-1/2, and SAPK-JNK phosphorylation as a result of BTV infection; and (7) BTV-induced changes in tyrosine phosphorylation.

We determined that BTV infects and kills all three cell lines in a cell line dependent manner. Relative cell death between cell lines was proportional to cytokine expression, but inversely proportional to viral protein expression. Only tyrosine kinase inhibitors influenced BTV-induced cell death and cytokine expression. Both A498 and A549 cells constitutively expressed phosphorylated PKB and p38 MAPK, of which both were de-phosphorylated during BTV infection. Tyrosine phosphorylation remained active, with elevated tyrosine phosphorylation exclusively in infected cells.

We conclude that BTV-induced cell death and cytokine expression are a function of the cell's response to infection and are directly related through intracellular signaling. These pathways are only partially poly I:C inducible, but include PKB and tyrosine kinase signaling.

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