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Postgraduate Thesis: A study of biological role of reactive oxygen species in cellular response in stress
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TitleA study of biological role of reactive oxygen species in cellular response in stress
 
AuthorsLam, Dennis
林勁行
 
Issue Date2012
 
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
 
AbstractWhen 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.
 
DegreeDoctor of Philosophy
 
SubjectEndoplasmic reticulum.
Protein folding.
Active oxygen.
Stress (Physiology)
 
Dept/ProgramBiochemistry
 
DC FieldValue
dc.contributor.authorLam, Dennis
 
dc.contributor.author林勁行
 
dc.date.hkucongregation2012
 
dc.date.issued2012
 
dc.description.abstractWhen 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.
 
dc.description.naturepublished_or_final_version
 
dc.description.thesisdisciplineBiochemistry
 
dc.description.thesisleveldoctoral
 
dc.description.thesisnameDoctor of Philosophy
 
dc.identifier.hkulb4786960
 
dc.languageeng
 
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)
 
dc.relation.ispartofHKU Theses Online (HKUTO)
 
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.
 
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License
 
dc.source.urihttp://hub.hku.hk/bib/B47869604
 
dc.subject.lcshEndoplasmic reticulum.
 
dc.subject.lcshProtein folding.
 
dc.subject.lcshActive oxygen.
 
dc.subject.lcshStress (Physiology)
 
dc.titleA study of biological role of reactive oxygen species in cellular response in stress
 
dc.typePG_Thesis
 
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<item><contributor.author>Lam, Dennis</contributor.author>
<contributor.author>&#26519;&#21185;&#34892;</contributor.author>
<date.issued>2012</date.issued>
<description.abstract>&#65279;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 eIF2&#945;, 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 eIF2&#945; and elevation of

DCF-detectable ROS remained unaffected. The cycloheximde-resistant

phosphorylation of eIF2&#945; 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

eIF2&#945; 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.</description.abstract>
<language>eng</language>
<publisher>The University of Hong Kong (Pokfulam, Hong Kong)</publisher>
<relation.ispartof>HKU Theses Online (HKUTO)</relation.ispartof>
<rights>The author retains all proprietary rights, (such as patent rights) and the right to use in future works.</rights>
<rights>Creative Commons: Attribution 3.0 Hong Kong License</rights>
<source.uri>http://hub.hku.hk/bib/B47869604</source.uri>
<subject.lcsh>Endoplasmic reticulum.</subject.lcsh>
<subject.lcsh>Protein folding.</subject.lcsh>
<subject.lcsh>Active oxygen.</subject.lcsh>
<subject.lcsh>Stress (Physiology)</subject.lcsh>
<title>A study of biological role of reactive oxygen species in cellular response in stress</title>
<type>PG_Thesis</type>
<identifier.hkul>b4786960</identifier.hkul>
<description.thesisname>Doctor of Philosophy</description.thesisname>
<description.thesislevel>doctoral</description.thesislevel>
<description.thesisdiscipline>Biochemistry</description.thesisdiscipline>
<description.nature>published_or_final_version</description.nature>
<date.hkucongregation>2012</date.hkucongregation>
<bitstream.url>http://hub.hku.hk/bitstream/10722/161520/1/FullText.pdf</bitstream.url>
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