Defense Date

2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Anatomy & Neurobiology

First Advisor

William Broaddus

Second Advisor

Helen Fillmore

Third Advisor

Timothy Vanmeter

Fourth Advisor

John Bigbee

Fifth Advisor

Lynne Elmore

Sixth Advisor

Martin Graf

Abstract

Previous studies in our laboratory demonstrated the expression of WT1 in a significant number of glioma cells and established its role in promoting tumor cell proliferation. Here, we noted the effect(s) of manipulating WT1 levels on the expression levels of genes that were previously shown to be regulated by WT1. We found no correlation between the expression levels of WT1 and PDGF-A, Snai1 and E-cadherin and a consistent inverse correlation between WT1 and IGF-1R expression in U251-MG cells. To ascertain whether the increased IGF-1R levels resulting from WT1 silencing could account for decreased cellular proliferation, we utilized siRNA mediated knockdown of IGF-1R and found a modest decrease in cellular proliferation. Gene expression profiling in U251-MG cells was then used to identify candidate target genes for WT1. Several genes whose levels directly correlated with WT1 were observed to have putative or established oncogenic role(s) in glioma cells or other malignancies. Among the genes correlated inversely, meanwhile, a tumor-suppressor role was attributed to some. Real time RT-PCR helped to substantiate these microarray findings in U251-MG cells. We also characterized the expression and function of WT1 in U1242-MG and GBM6 cells. Interestingly, in these cells WT1 facilitated cell invasiveness but had no discernible influence on cellular proliferation. The expressions of the candidate WT1 target genes were studied also determined in these 2 cell lines. At least 3 genes were consistently down-regulated with WT1 silencing in the three cell lines- INPP5A, CD97, and TYMS. To determine whether CD97 assisted WT1 in facilitating cellular invasion, we silenced CD97 expression using siRNA and noted a significant decrease in the cells’ ability to invade through Matrigel-coated filters. We propose that WT1 profoundly impacts the glioma cells’ invasive ability, and this function is mediated by CD97 alone or in conjunction with other pro-invasive molecules. Our findings argue for the oncogenic role of WT1 in the specific context of glioma cells. They also point to a novel pro-invasive protein- CD97- in glioma cells. Further studies are necessary to confirm the mechanism by which CD97 promotes invasion as well as to explore its potential as a diagnostic and/or therapeutic target.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

May 2011

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