When proteins are unable to fold properly in the endoplasmic reticulum (ER), the
resultant formation of misfolded proteins causes stress of the ER. Cells with ER stress
often have a higher abundance of reactive oxygen species (ROS). Previous studies
suggest that ROS could aggravate ER stress by further disrupting the ER protein
folding process. More recent studies suggest that the unfolded protein response
signaling pathways activated by ER stress could lead to the production of ROS. Such
studies lead to the hypothesis that ER stress could be promoted by ROS, and vice
versa. The aim of the present study is to test the above hypothesis by studying how
ROS could be generated in ER-stressed cells. This is followed by investigating if ROS
could increase or decrease the level of ER stress in cells. Finally, the extent of ER
stress induced cell death in the presence and absence of ROS is assessed.
The treatment of HeLa cells with tunicamycin (Tm), a common ER-stress
inducing agent, resulted in the elevation of intracellular ROS that could be detected
with the ROS-reactive probe dichlorodihydrofluorescein (DCF), but not
dihydroethidium which is relatively specific towards superoxide anion. The
Tm-induced elevation of ROS could be prevented by co-incubation of cells with thiol
reductants such as dithiothreitol and N-acetylcysteine but not with the free radical
scavenger ascorbate. The tunicamycin-induced elevation of ROS level could also be
prevented by the over-expression of catalase in HeLa. These data is consistent with
the idea that hydrogen peroxide is a major form of ROS produced in Tm-treated cells.
In addition to elevation of ROS level, HeLa cells treated with tunicamycin also
resulted in the phosphorylation of PERK and eIF2α, and the splicing of XBP-1. In the
presence of cycloheximide to inhibit protein synthesis so as to deplete protein
substrates for folding in the ER, tunicamycin-induced ER stress was greatly
minimized as was evident by the absence of both the phosphorylation of PERK and
splicing of XBP-1. However, the phosphorylation of eIF2α and elevation of
DCF-detectable ROS remained unaffected. The cycloheximde-resistant
phosphorylation of eIF2α could be prevented when cells were co-treated with thiol
reductants, or upon the over-expression of catalase. These data suggest that the
production of ROS in Tm-treated cells does not require the presence of ER stress as a
prerequisite. Furthermore, the ROS so produced could induce phosphorylation of
eIF2α without the need to cause ER stress in the first place.
The quenching of ROS through the use of thiol reductants, or the over-expression
of catalase, had no effect on inhibition of protein synthesis in cells treated with
tunicamycin. However, the extent of cell death was significantly increased. The data
obtained in this study is not consistent with the idea that ROS is a downstream
product of ER stress, capable of inducing more ER-stress by a feedback mechanism.
Therefore, a mutually enhancing effect between ER stress and ROS may not exist.
The ROS found in stressed cells may serve to extend cellular survival under the
condition of continuous stress. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/161520 |
| Date | January 2012 |
| Creators | Lam, Dennis, 林勁行 |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B47869604 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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