University of Manchester
1.
Likhatcheva, Maria.
Dissection of the factors involved in chromatin remodelling and dna damage response pathway in hypoxic conditions.
Degree: PhD, 2019, University of Manchester
URL: https://www.research.manchester.ac.uk/portal/en/theses/dissection-of-the-factors-involved-in-chromatin-remodelling-and-dna-damage-response-pathway-in-hypoxic-conditions(1844dcb6-1820-4328-83d8-5f3ce7cee010).html 
;
https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.799315
Hypoxia, a common feature of solid tumours, is associated with poor prognosis, a more aggressive tumour phenotype and therapeutic resistance. Tumour hypoxia induces changes in the chromatin structure, with induction of the heterochromatin mark H3K9me3 frequently observed. The upregulation of H3K9me3 was observed with co-incidental upregulation of the methyltransferase Suv39H1 in three (FTC133, U87 and HCT116) different cancer cell lines exposed to hypoxic conditions (0.1% O₂). Thus, it was hypothesised that Suv39H1 was a candidate for the tri- methylation of H3K9 and the first part of this study was focused on investigating the potential regulatory mechanisms. RT-qPCR analysis showed no changes in the levels of Suv39H1 mRNA in hypoxia suggesting a post transcriptional regulation mechanism. In mild hypoxic conditions (O₂ ≥ 1%) the cells failed to induce H3K9me3 and Suv39H1. However, treatment with proteasome inhibitor MG132 in mild hypoxia induced Suv39H1 upregulation, suggesting the involvement of the proteasomal pathway in the regulation of Suv39H1 stability in hypoxia. Under normal oxygen conditions Suv39H1 is degraded via MDM2 dependent ubiquitination, which is blocked by the protein Sirt1. However, the levels of Sirt1 were highly downregulated and MDM2 protein levels were maintained following hypoxic exposure. It has been previously reported that ATM is activated in response to hypoxia and negatively regulates MDM2 activity in response to DNA damage. To test the involvement of ATM and MDM2 in regulating Suv39H1 levels in hypoxia, the ATM specific inhibitor (Ku55933) was used together with MDM2 knockdown under normal oxygen conditions and hypoxia. As predicted, highest levels of Suv39H1 were observed when ATM is catalytically active (hypoxia) and MDM2 inactive (knockdown). The results of this study indicate an important role that ATM has in regulating chromatin structure in response to stress conditions, such as hypoxia, through regulation of MDM2. As ATM plays a crucial role in the hypoxia induced DNA damage response, the second part of this study was focused on uncovering ATM activation in hypoxia. It has been previously reported that the acetyltransferase Tip60 is involved in ATM activation in response to DNA damage. However, if Tip60 plays a role in the hypoxia induced activation of ATM hasn't been tested to date. In order to do this a Tip60 specific inhibitor, TH1834, was used. FTC133 and HCT116 cells were treated with TH1834 under hypoxic conditions and the levels of the catalytically active form of ATM were assessed by Western blot and IF. The obtained results showed a clear involvement of Tip60 in the hypoxia induced activation of ATM. Finally, as the activation of ATM in hypoxia has been related to S-phase arrest, the cell cycle distribution of FTC133 and HCT116 cells were analysed by FACS. The result showed that ATM activation in FTC133 cells seems independent of S-phase arrest. Heterochromatin is suggested to be important for cancer cell survival. This study proposes that ATM is involved in maintaining…
Subjects/Keywords: Hypoxia ATM Suv39H1
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APA (6th Edition):
Likhatcheva, M. (2019). Dissection of the factors involved in chromatin remodelling and dna damage response pathway in hypoxic conditions. (Doctoral Dissertation). University of Manchester. Retrieved from https://www.research.manchester.ac.uk/portal/en/theses/dissection-of-the-factors-involved-in-chromatin-remodelling-and-dna-damage-response-pathway-in-hypoxic-conditions(1844dcb6-1820-4328-83d8-5f3ce7cee010).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.799315
Chicago Manual of Style (16th Edition):
Likhatcheva, Maria. “Dissection of the factors involved in chromatin remodelling and dna damage response pathway in hypoxic conditions.” 2019. Doctoral Dissertation, University of Manchester. Accessed January 20, 2021.
https://www.research.manchester.ac.uk/portal/en/theses/dissection-of-the-factors-involved-in-chromatin-remodelling-and-dna-damage-response-pathway-in-hypoxic-conditions(1844dcb6-1820-4328-83d8-5f3ce7cee010).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.799315.
MLA Handbook (7th Edition):
Likhatcheva, Maria. “Dissection of the factors involved in chromatin remodelling and dna damage response pathway in hypoxic conditions.” 2019. Web. 20 Jan 2021.
Vancouver:
Likhatcheva M. Dissection of the factors involved in chromatin remodelling and dna damage response pathway in hypoxic conditions. [Internet] [Doctoral dissertation]. University of Manchester; 2019. [cited 2021 Jan 20].
Available from: https://www.research.manchester.ac.uk/portal/en/theses/dissection-of-the-factors-involved-in-chromatin-remodelling-and-dna-damage-response-pathway-in-hypoxic-conditions(1844dcb6-1820-4328-83d8-5f3ce7cee010).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.799315.
Council of Science Editors:
Likhatcheva M. Dissection of the factors involved in chromatin remodelling and dna damage response pathway in hypoxic conditions. [Doctoral Dissertation]. University of Manchester; 2019. Available from: https://www.research.manchester.ac.uk/portal/en/theses/dissection-of-the-factors-involved-in-chromatin-remodelling-and-dna-damage-response-pathway-in-hypoxic-conditions(1844dcb6-1820-4328-83d8-5f3ce7cee010).html ; https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.799315
University of Manchester
2.
Likhatcheva, Maria.
DISSECTION OF THE FACTORS INVOLVED IN CHROMATIN
REMODELLING AND DNA DAMAGE RESPONSE PATHWAY IN HYPOXIC
CONDITIONS.
Degree: 2018, University of Manchester
URL: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:317434
Hypoxia, a common feature of solid tumours, is
associated with poor prognosis, a more aggressive tumour phenotype
and therapeutic resistance. Tumour hypoxia induces changes in the
chromatin structure, with induction of the heterochromatin mark
H3K9me3 frequently observed. The upregulation of H3K9me3 was
observed with co-incidental upregulation of the methyltransferase
Suv39H1 in three (FTC133, U87 and HCT116) different cancer cell
lines exposed to hypoxic conditions (0.1% O2). Thus, it was
hypothesised that Suv39H1 was a candidate for the tri- methylation
of H3K9 and the first part of this study was focused on
investigating the potential regulatory mechanisms. RT-qPCR analysis
showed no changes in the levels of Suv39H1 mRNA in hypoxia
suggesting a post transcriptional regulation mechanism. In mild
hypoxic conditions (O2 ≥1%) the cells failed to induce H3K9me3
and Suv39H1. However, treatment with proteasome inhibitor MG132 in
mild hypoxia induced Suv39H1 upregulation, suggesting the
involvement of the proteasomal pathway in the regulation of Suv39H1
stability in hypoxia. Under normal oxygen conditions Suv39H1 is
degraded via MDM2 dependent ubiquitination, which is blocked by the
protein Sirt1. However, the levels of Sirt1 were highly
downregulated and MDM2 protein levels were maintained following
hypoxic exposure. It has been previously reported that ATM is
activated in response to hypoxia and negatively regulates MDM2
activity in response to DNA damage. To test the involvement of ATM
and MDM2 in regulating Suv39H1 levels in hypoxia, the ATM specific
inhibitor (Ku55933) was used together with MDM2 knockdown under
normal oxygen conditions and hypoxia. As predicted, highest levels
of Suv39H1 were observed when ATM is catalytically active (hypoxia)
and MDM2 inactive (knockdown). The results of this study indicate
an important role that ATM has in regulating chromatin structure in
response to stress conditions, such as hypoxia, through regulation
of MDM2. As ATM plays a crucial role in the hypoxia induced DNA
damage response, the second part of this study was focused on
uncovering ATM activation in hypoxia. It has been previously
reported that the acetyltransferase Tip60 is involved in ATM
activation in response to DNA damage. However, if Tip60 plays a
role in the hypoxia induced activation of ATM hasn’t been tested
to date. In order to do this a Tip60 specific inhibitor, TH1834,
was used. FTC133 and HCT116 cells were treated with TH1834 under
hypoxic conditions and the levels of the catalytically active form
of ATM were assessed by Western blot and IF. The obtained results
showed a clear involvement of Tip60 in the hypoxia induced
activation of ATM. Finally, as the activation of ATM in hypoxia has
been related to S-phase arrest, the cell cycle distribution of
FTC133 and HCT116 cells were analysed by FACS. The result showed
that ATM activation in FTC133 cells seems independent of S-phase
arrest. Heterochromatin is suggested to be important for cancer
cell survival. This study proposes that ATM is involved in…
Advisors/Committee Members: DEMONACOS, CONSTANTINOS C, GIELING, ROBEN R, Williams, Kaye, Demonacos, Constantinos, Gieling, Roben.Subjects/Keywords: Hypoxia ATM Suv39H1
Record Details
Similar Records
Cite
Share »
Record Details
Similar Records
Cite
« Share
❌
APA ·
Chicago ·
MLA ·
Vancouver ·
CSE |
Export
to Zotero / EndNote / Reference
Manager
APA (6th Edition):
Likhatcheva, M. (2018). DISSECTION OF THE FACTORS INVOLVED IN CHROMATIN
REMODELLING AND DNA DAMAGE RESPONSE PATHWAY IN HYPOXIC
CONDITIONS. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:317434
Chicago Manual of Style (16th Edition):
Likhatcheva, Maria. “DISSECTION OF THE FACTORS INVOLVED IN CHROMATIN
REMODELLING AND DNA DAMAGE RESPONSE PATHWAY IN HYPOXIC
CONDITIONS.” 2018. Doctoral Dissertation, University of Manchester. Accessed January 20, 2021.
http://www.manchester.ac.uk/escholar/uk-ac-man-scw:317434.
MLA Handbook (7th Edition):
Likhatcheva, Maria. “DISSECTION OF THE FACTORS INVOLVED IN CHROMATIN
REMODELLING AND DNA DAMAGE RESPONSE PATHWAY IN HYPOXIC
CONDITIONS.” 2018. Web. 20 Jan 2021.
Vancouver:
Likhatcheva M. DISSECTION OF THE FACTORS INVOLVED IN CHROMATIN
REMODELLING AND DNA DAMAGE RESPONSE PATHWAY IN HYPOXIC
CONDITIONS. [Internet] [Doctoral dissertation]. University of Manchester; 2018. [cited 2021 Jan 20].
Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:317434.
Council of Science Editors:
Likhatcheva M. DISSECTION OF THE FACTORS INVOLVED IN CHROMATIN
REMODELLING AND DNA DAMAGE RESPONSE PATHWAY IN HYPOXIC
CONDITIONS. [Doctoral Dissertation]. University of Manchester; 2018. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:317434
Open Access Theses and Dissertations