Publication Date
2010
Document Type
Thesis
Committee Members
Scott Baird (Committee Member), Mill Miller (Advisor), Larry Ream (Advisor)
Degree Name
Master of Science (MS)
Abstract
The HIV protein Rev is a nucleolar protein that regulates late gene expression in infected cells by promoting the export of under-spliced viral RNAs (Pollard and Malim, 1998). Its over-expression can also inhibit progression through mitosis (Miyazaki et al., 1995), possibly through its ability to depolymerize microtubules (Watts et al., 2000). Consequently, Rev may activate the spindle assembly checkpoint in mitotic cells and increase the frequency of apoptosis. Rev also binds the nucleolar protein B23 involved in ribosome maturation and centrosome duplication. Because loss of B23 function stimulates apoptosis (Ahn et al., 2005), Rev expression may promote apoptosis by inhibiting B23.
Regardless of its mechanism, it is plausible to hypothesize that Rev expression stimulates apoptosis. To test this hypothesis, Rev and three mutants defective in multimerization, nuclear import, and nuclear export (M4, M6, and M10, respectively) and are known to cause defects in mitosis were transiently and stably over-expressed in HeLa cells. Three separate assays were then used to assay for apoptotic cell death. Since apoptosis is characterized by cell shrinkage, cell rounding, chromatin condensation and fragmentation (Kerr et al., 1993; Lawen, 2003), Rev expressing cells were fixed with formaldehyde, stained with DAPI and examined for overt signs of apoptosis. In cells transiently expressing Rev-GFP, there was a two-fold increase in apoptosis compared to YFP expressing controls. Transient expression of Rev mutants M4, M6, and M10 resulted in comparable increased frequencies of apoptosis. This visual assay is limited by the apparent lack of sensitivity as demonstrated by the inability to detect apoptosis induced by treatment with actinomycin D.
To clarify these results, two additional biochemical assays were used. The first, an ELISA assay that quantifies chromatin fragmentation by measuring the release of mono- and oligo-nucleosomes, showed there was no statistical difference in apoptotic frequencies between Rev-YFP and YFP expressing cells (p=0.43). These results were confirmed by an immunoblot assay that was unable to detect the presence of activated Caspase 3 in Rev expressing cells. Thus it appears that the cell cycle defects induced by Rev expression are either corrected before mitosis is completed or not detected by the spindle assembly checkpoint.
Page Count
48
Department or Program
Department of Neuroscience, Cell Biology & Physiology
Year Degree Awarded
2010
Copyright
Copyright 2010, all rights reserved. This open access ETD is published by Wright State University and OhioLINK.
