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Alteration of poly(adp-ribose) polymerase activity influences the mode of oxidant-induced endothelial cell death

University of British Columbia

Poly(ADP-ribose) polymerase (PARP) is a zinc-finger enzyme activated by DNA strand scissions, as occur during oxidative stress. Extensive PARP activation causes depletion of its substrate, NAD+ , and a consequent decline in cellular energy, which contributes to oncotic cell death. Cell death is categorized either as oncosis, a passive form of death, or apoptosis, an active energy-dependent form of death. This thesis examines the effect of altering PARP activity on the cellular response to oxidative injury and the influence that PARP activity has on the process of endothelial cell (EC) death. Fluorescence microscopy reveals hallmark nuclear features consistent with apoptosis in oxidant-stressed ECs when PARP is inhibited with 1,5- isoquinolinediol and oncosis when exposed to oxidant alone. Caspase-3-like activity, a marker of apoptosis, is negligible in oxidant-treated ECs but increases when PARP activity is inhibited. These results demonstrate that the process of oxidant-induced EC death is switched from oncosis to apoptosis by pharmacological inhibition of PARP activity. In accordance with this switch to an active form of death, PARP inhibition maintains energy-dependent cellular processes, such as retention of low concentrations of intracellular free calcium and lysosomal uptake of acridine orange. Metal replacement in the PARP zinc fingers is a potential mechanism of altering PARP activity that may have important consequences in metal toxicology. Because zinc is essential for both the ability of PARP to bind DNA and the activation of its catalytic activity, the potential for altering the DNA-binding capacity of PARP through metal replacement in the zinc-finger motif is investigated. Purified PARP, derived from a baculovirus expression system, is employed for these studies. Various mass spectrometric methods, including laser ablation-ICPMS, MALDI-TOF MS and ESI-MS, are evaluated for their effectiveness in the detection of metal replacement in purified PARP. Analysis of the native PARP is hindered at neutral pH due to suppression of ionization and protein adsorption to instrument components. However, analysis of the 24 kDa caspase-3-digested PARP fragment containing the 2 zinc fingers is accomplished by ESI-MS using a solvent composed of 20% acetonitrile containing 53 mM 1,1,1,3,3,3- hexafluoroisopropanol pH 5.1. Therefore, the groundwork is laid to examine metal replacement in PARP as a potential mechanism for alteration of PARP activity. In conclusion, alteration of PARP activity, either via its catalytic site or at the zinc-finger site, may have important toxicological consequences in a variety of processes including metal toxicity and oxidant-induced cell death.

Thesis/Dissertation

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2009-07-03

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http://hdl.handle.net/2429/10059

Pharmaceutical Sciences

University of British Columbia

UBCV

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