All ETDs from UAB

Advisory Committee Chair

David E Graves

Advisory Committee Members

Tracy Hamilton

Kevin Harris

Aaron Lucius

Donald Muccio

Document Type

Dissertation

Date of Award

2014

Degree Name by School

Doctor of Philosophy (PhD) College of Arts and Sciences

Abstract

Non-helical DNA structures such as hairpins and G-quadruplexes found in the non-coding regions of chromosomes have been implicated in a number of biological processes such as control of genetic expression and in dysregulation causing diseases such as cancer. Recent studies from our laboratory have found that apoptotic DNA released from cancer cells, a synthetic G-quadruplex forming telomeric sequence, and a highly thermal stable DNA hairpin can induce an invasion response from a variety of cancer cell types. This invasion has been demonstrated to be mediated through a toll-like receptor protein found within the endosomes of the cell. We hypothesize that because the telomeres of chromosomes are over extended in cancer cells and can fold into nuclease resistant G-quadruplex structures, these DNA fragments when released from cancer cells undergoing apoptosis are able to survive outside of the cell. Furthermore, the uptake of these structurally stable nuclease-resistant fragments by surviving cancer cells triggers the TLR9-mediated cancer cell invasion response. The research described in this dissertation employs a variety of biophysical techniques to characterize and explore various lengths of telomeric sequence fragments, as well as a highly thermal stable hairpin that our laboratory demonstrated to be both an effective and biologically relevant TLR9 agonist. In order to capture and isolate extracellular telomeric DNA fragments from the apoptotic cancer cells, we also developed a magnetic bead capture assay that utilizes biotin labeled PNA probes. Use of PNAs provides an effective approach to target the G-quadruplex based on the structural motif or on the telomeric sequence. Results from bead capture analyses utilizing complementary PNA (c-PNA) probes indicates that approximately 25% of the total apoptotic DNA released from dying MDA-MD-231 breast cancer cells is telomeric, and that the captured DNA ranges in size from approximately 25 - 200 nucleotides in length. These studies provide a strong foundation for further characterization of the biological roles that telomeric, or other G-quadruplex forming DNA fragments may play.

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