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Date of Award

Fall 2012

Degree Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

Supervisor

André Bédard

Language

English

Abstract

Cell transformation by the Src tyrosine kinase is characterized by extensive changes in gene expression. Previous studies have indicated that many of these changes are dependent on the activity of transcription factors belonging to the AP-1, STAT and Ets families. This study took advantage of transformation-deficient and temperature sensitive mutants of the Rous sarcoma virus to characterize transformation-induced patterns of gene expression of two primary cell types, chicken embryo fibroblasts (CEF) and chicken neuroretina (CNR). In these cells, v-Src alters the expression of up to 6% of the protein coding genes. Comparison of this program with independent breast carcinoma data sets identified a group of 42 v-Src inducible genes associated with reduced disease-free survival. Pathway and ontological analyses of the genes differentially regulated by transformation in CEF and CNR indicated a generalized program of de-differentiation induced by Src.

To investigate the role of AP-1 inSrc-mediated transformation, a gene profiling study was conducted to characterize the transcriptomes of v-Src-transformed CEF expressing the Jun dominant-negative allele or the JunD short-hairpin RNA (shRNA). Microarray data analysis indicated a cluster of 18 co-regulated probe-sets activated in v-Src-transformed CEF with repressed AP-1 activity but not activated in normal CEF or CEF transformed only by v-Src. One gene, death-associated protein kinase 1 (DAPK1), is a C/EBPβ-regulated mediator of apoptosis in IFN-γ-induced cell death. Inhibition of DAPK1 abrogated cell-death in v-Src-transformed CEF expressing the JunD shRNA and expression of DAPK1 was dependent on C/EBPβ but antagonized by AP-1. Chromatin immunoprecipitation indicated that C/EBPβ, but not JunD, is recruited to the DAPK1 promoter. In conclusion, JunD promotes survival by indirectly antagonizing C/EBPβ-dependent expression of DAPK1.

McMaster University Library

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