Characterization and Molecular Targeting of the Bcl-2 i-Motif for Modulation of Gene Expression and Induction of Chemosensitivity in Lymphoma

Persistent Link:
http://hdl.handle.net/10150/193638
Title:
Characterization and Molecular Targeting of the Bcl-2 i-Motif for Modulation of Gene Expression and Induction of Chemosensitivity in Lymphoma
Author:
Kendrick, Samantha Lynn
Issue Date:
2010
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
The nature of DNA has captivated scientists for more than fifty years. The discovery of the double-helix model of DNA by Watson and Crick in 1953 not only established the primary structure of DNA, but also provided the mechanism behind DNA function. Since then, the demonstration of DNA secondary structure formation has allowed for the proposal that the dynamics of DNA itself can function to modulate transcription. We demonstrate for the first time the i-motif DNA secondary structure formed from an element within the Bcl-2 promoter region has potential to serve as a cellular molecular target for modulation of gene expression. Unlike typical oncogenes, Bcl-2 acts by promoting cellular survival rather than increasing cellular proliferation. The over-expression of Bcl-2, most notably in lymphomas, has been associated with the development of chemoresistance.Transcriptional regulation of Bcl-2 is highly complex and a guanine- and cytosine-rich (GC-rich) region directly upstream of the P1 site has been shown to be integral to Bcl-2 promoter activity. We have demonstrated that the C-rich strand is capable of forming an intramolecular i-motif DNA secondary structure with a transition pH of 6.6 and a predominant 8:5:7 loop using mutational studies coupled with circular dichroic spectra and thermal stability analyses. In addition, a novel assay involving the sequential incorporation of a fluorescent thymine analog at each thymine position provided evidence of a capping structure within the top loop region of the i-motif. Two different classes of steroids either stabilize or destabilize the i-motif structure and this differential interaction results in the activation or repression of Bcl-2 expression. The i-motif stabilizing steroid significantly up-regulated Bcl-2 gene and protein expression in BJAB Burkitt's lymphoma cells while the destabilizing steroid down-regulated Bcl-2 expression in B95.8 Burkitt's and Granta-519 mantle cell lymphoma cells, as well as in a SCID mouse lymphoma model. More importantly, the down-regulation of Bcl-2 led to chemosensitization of etoposide-resistant lymphoma cells demonstrating that Bcl-2 i-motif interactive small molecules can act as chemosensitizing agents. Conversely, compounds that up-regulate Bcl-2 by stabilization of the i-motif have potential for use as neuroprotective agents.
Type:
text; Electronic Dissertation
Keywords:
Bcl-2; DNA Secondary Structures; i-Motif; Lymphoma
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Cancer Biology; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Hurley, Laurence H
Committee Chair:
Hurley, Laurence H

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleCharacterization and Molecular Targeting of the Bcl-2 i-Motif for Modulation of Gene Expression and Induction of Chemosensitivity in Lymphomaen_US
dc.creatorKendrick, Samantha Lynnen_US
dc.contributor.authorKendrick, Samantha Lynnen_US
dc.date.issued2010en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThe nature of DNA has captivated scientists for more than fifty years. The discovery of the double-helix model of DNA by Watson and Crick in 1953 not only established the primary structure of DNA, but also provided the mechanism behind DNA function. Since then, the demonstration of DNA secondary structure formation has allowed for the proposal that the dynamics of DNA itself can function to modulate transcription. We demonstrate for the first time the i-motif DNA secondary structure formed from an element within the Bcl-2 promoter region has potential to serve as a cellular molecular target for modulation of gene expression. Unlike typical oncogenes, Bcl-2 acts by promoting cellular survival rather than increasing cellular proliferation. The over-expression of Bcl-2, most notably in lymphomas, has been associated with the development of chemoresistance.Transcriptional regulation of Bcl-2 is highly complex and a guanine- and cytosine-rich (GC-rich) region directly upstream of the P1 site has been shown to be integral to Bcl-2 promoter activity. We have demonstrated that the C-rich strand is capable of forming an intramolecular i-motif DNA secondary structure with a transition pH of 6.6 and a predominant 8:5:7 loop using mutational studies coupled with circular dichroic spectra and thermal stability analyses. In addition, a novel assay involving the sequential incorporation of a fluorescent thymine analog at each thymine position provided evidence of a capping structure within the top loop region of the i-motif. Two different classes of steroids either stabilize or destabilize the i-motif structure and this differential interaction results in the activation or repression of Bcl-2 expression. The i-motif stabilizing steroid significantly up-regulated Bcl-2 gene and protein expression in BJAB Burkitt's lymphoma cells while the destabilizing steroid down-regulated Bcl-2 expression in B95.8 Burkitt's and Granta-519 mantle cell lymphoma cells, as well as in a SCID mouse lymphoma model. More importantly, the down-regulation of Bcl-2 led to chemosensitization of etoposide-resistant lymphoma cells demonstrating that Bcl-2 i-motif interactive small molecules can act as chemosensitizing agents. Conversely, compounds that up-regulate Bcl-2 by stabilization of the i-motif have potential for use as neuroprotective agents.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectBcl-2en_US
dc.subjectDNA Secondary Structuresen_US
dc.subjecti-Motifen_US
dc.subjectLymphomaen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineCancer Biologyen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorHurley, Laurence Hen_US
dc.contributor.chairHurley, Laurence Hen_US
dc.contributor.committeememberBowden, Timothy G.en_US
dc.contributor.committeememberCherrington, Nathanen_US
dc.contributor.committeememberDorr, Roberten_US
dc.contributor.committeememberWondrak, Georgen_US
dc.identifier.proquest11344en_US
dc.identifier.oclc752261203en_US
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