DOI
https://doi.org/10.25772/KF95-B211
Defense Date
2006
Document Type
Thesis
Degree Name
Doctor of Philosophy
Department
Biochemistry
First Advisor
Dr. Kristoffer Valerie
Abstract
Ionizing radiation (IR) triggers many signaling pathways stemming from DNA damage, and, independently, from extra-nuclear events. To generate radio-mimetic DNA double-strand breaks (DSBs) without and minimizing the effects on extra-nuclear radiation targets, human (p53+) glioma and carcinoma cells containing bromodeoxyuridine (BrdU)- substituted DNA were treated with Hoechst 33258 followed by long wave-length UV (UV-A) (BrdU photolysis). BrdU photolysis resulted in well-controlled, dose-dependent generation of DSBs equivalent to 0.2 - 20 Gy of IR, as detected by pulse-field gel electrophoresis, accompanied by dose-dependent H2AX phosphorylation at ser-139 and ATM phosphorylation at ser-1981, indicating ATM activation. Furthermore, BrdU photolysis increased phosphorylation of Chk2 (at thr-68) and p53 (at ser-15). p53 phosphorylation was reduced by the ATM inhibitor caffeine, and H2AX phosphorylation was greatly reduced in AT cells, confirming that phosphorylation was primarily ATM-dependent. We also examined the effects of BrdU photolysis on the major cellular signaling ERK pathways. Interestingly, low-dose (≤ 2 Gy-equivalents) BrdU photolysis stimulated ERK1/2 phosphorylation whereas higher doses (≥ 5 Gy eq.) resulted in Em1/2 dephosphorylation. ERK1/2 phosphorylation was ATM-dependent, whereas dephosphorylation was ATM-independent and DSBs dose-dependent. Thus ERK1/2 appear to be both positively and negatively regulated by ATM depending on the severity of the insult to DNA. In summary, few DSBs trigger ATM-dependent ERK1/2 pro-survival signals whereas more DSBs result in ERK1/2 dephosphorylation consistent with a switch from pro-survival to anti-survival signaling that might affect DSBs repair.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
June 2008