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Cellular response to stress and DNA damage: the role of Sirtuins in the regulation of autophagy

Garva, Richa

[Thesis]. Manchester, UK: The University of Manchester; 2014.

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Abstract

Autophagy is a regulated and evolutionarily conserved catabolic process that serves to degrade superfluous or damaged organelles and recycle their biochemical components for use in energy production and other biosynthetic reactions. It is a crucial cellular response to various stresses including oxidative stress and starvation. Sirtuins are NAD+ dependent deacetylases that link transcriptional regulation to cellular energy homeostasis, DNA damage, ROS response, cell cycle control, apoptosis and autophagy. The role of autophagy in cancer is complex as autophagy exerts both cell protective and damaging functions depending on the circumstances. The purpose of this study is to investigate the role of individual Sirtuin family members in the regulation of gene expression of the known autophagy markers LC3 and Beclin 1 under DNA damage and oxidative stress. To investigate the regulation of autophagy, we followed the gene expression of autophagy genes LC3 and Beclin 1 under diverse stress conditions in human osteosarcoma cells (U2OS). The protein and mRNA levels of LC3-II and the formation of autophagosomes were increased in etoposide and rotenone treated cells. While LC3-II and LC3-1 protein expressions were increased in Sirt1 overexpressing cells, no significant change was observed in Beclin 1 protein level. However, inhibition of Sirt1 by siRNA did not affect the cellular levels of the autophagy markers suggesting the potential involvement of other Sirtuin family members in the regulation of autophagy. Elevated LC3 mRNA was observed in cells overexpressing any of the Sirtuins family members; however etoposide treatment selectively inhibited Sirt1 and Sirt2 dependent upregulation of LC3 mRNA. Induction of LC3-Luc reporter activity was observed in Sirt5 transfected cells, which was further increased by etoposide treatment. Sirt5 carrying mutation in its catalytic domain is not able to induce autophagy and Sirt5 mediating autophagy is under the control of NF-KB transcription factor. These results support the notion that Sirtuins are important regulators of autophagy and the function of each member is to differentially regulate DNA damage and oxidative stress responses.