Date on Master's Thesis/Doctoral Dissertation

5-2006

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Biochemistry and Molecular Biology

Committee Chair

Bates, Paula J.

Author's Keywords

Guanine-rich; Oligonucleotides; Nucleolin; NF-kappaB

Subject

Oligonucleotides

Abstract

Guanine-rich oligonucleotides (GROs) are being developed as a novel anticancer agents. GROs exhibit potent antiproliferative properties against several malignant cell lines and in established in vivo tumor models. In a recent Phase I clinical trial for patients with advanced cancer, one of these GROs was found to have promising signs of clinical activity with no serious side effects. It has been shown that the activity of GROs requires the formation of a G-quartet structure, and their activity correlates with its ability to bind to a specific complex of proteins. One important GRO-binding protein that has been previously identified is nucleolin, a multifunctional protein that is expressed at high levels in cancer cells. In this report, we investigate the novel mechanism of GRO activity by identifying the proteins that bind to an active GRO with use of mass spectrometry. Fourteen nuclear proteins and three cytoplasmic proteins were identified as associating with the active GRO, all of which vary in cellular functions described in the present study. The identified GRO-associated proteins include a protein known as NEMO (NF-êB essential modulator). NF-êB transcription regulates the expression of genes involved in cellular processes such as cell growth, differentiation, and apoptosis. NF-êB signaling is often deregulated in cancer cells, and NEMO is a regulatory component of the cascade. We demonstrate here that GROs bind to NEMO and nucleolin inside the cell, inhibit NF-êB transcription, and prevent phosphorylation of the NF-êB signaling cascade in response to TNFá. To further investigate the mechanism of GROs, we utilized global expression technology to determine the effects of GROs on the entire cell. Microarray gene expression analysis revealed the alteration of 63 genes after 2 hours of treatment with GRO, and 238 genes after 18 hours of treatment with GRO revealing multiple biological systems affected. The data presented in this report reveals important features of the effects of GROs on the cell which gives insight into the antiproliferative mechanism of the novel anticancer agent.

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