Investigating the mode of action of histone deacetylase inhibiting agents

Warrener, Robyn (2012). Investigating the mode of action of histone deacetylase inhibiting agents PhD Thesis, UQ Diamantina Institute, The University of Queensland.

       
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Author Warrener, Robyn
Thesis Title Investigating the mode of action of histone deacetylase inhibiting agents
School, Centre or Institute UQ Diamantina Institute
Institution The University of Queensland
Publication date 2012
Thesis type PhD Thesis
Supervisor Brian Gabrielli
Nicholas Saunders
Total pages 158
Total colour pages 31
Total black and white pages 127
Language eng
Subjects 0601 Biochemistry and Cell Biology
Formatted abstract
Histone deacetylase inhibitors (HDACi) are a class of drug that has gained interest due to their selective cytotoxic properties. HDACi impose a range of effects in treated cells, including up and down regulation of transcription, cell cycle arrests, increased histone and non-histone protein lysine acetylation, aberrant mitotic phenotypes and apoptosis. In the treatment of cancer, traditional therapies are non-selective, typically targeting all actively cycling cells and thus targeting both healthy and cancerous tissue. HDACi have been identified to selectively impose checkpoint arrests in normal cells that confers a resistance to HDACi induced cell death, thus giving these agents an advantage over conventional chemotherapeutic agents, imposing a resistance conferring G2 arrest in primary cells, which is not functional in almost all tumour cells. These HDACi sensitive cells progress into mitosis, where chromosome alignment is disrupted coupled with the failure of the protective spindle assembly checkpoint. Cells undergo an aberrant mitotic division and apoptosis is induced in the subsequent G1. Due to their chemotherapeutic properties, HDACi have been trialled and approved for clinical use. However many actions of HDACi are still being elucidated. The nature of the G2 checkpoint arrest which confers resistance has not been identified. Cells which do not impose this protective checkpoint arrest enter mitosis. The application of HDACi during S phase causes cells to undergo an aberrant mitosis, where chromosomes fail to line up at the metaphase plate and the spindle assembly checkpoint is overridden.

This thesis investigates the changes to histone H3 lysine 9 (Lys9) methylation induced by increased acetylation and whether this is involved in the mode of action of HDACi on sensitive cells. HDACi cause rapid increases to the level of histone acetylation, with this increase anticipated to have dramatic effects on other histone modifications, in particular the methylation of histone H3 lys9. Analysis of the acetylation and methylation patterns of histone H3 lys9 during the cell cycle after being treated with HDACi in S phase revealed that whilst increases to histone acetylation were rapid, reductions to the level of methylation on this site were minor and less dramatic than anticipated. The complete depletion of histone H3 lys9 trimethylation modifications by the overexpression of specific histone demethylases failed to induce aberrant mitosis in all mitotic cells identified. HDACi treated cells were progress through the G2/M phase of the cell cycle slower than untreated cells as determined by FACS analysis of individual cells. Creation of a cell line stably expressing a histone protein fused with the green fluorescent protein (H2B-GFP) allowed live cells to be visualised as they progressed through mitosis, and analysis of time lapse microscopy images allowed us to determine the cell cycle delay was specifically occurring in mitosis.

Cell cycle variation of histone H3 lys9 methylation patterns in response to HDACi treatment was less affected than anticipated, based on the dramatically increased level of histone acetylation. The complete removal of trimethylation from this residue failed to induce aberrant mitosis. The mechanism of action of HDACi in inducing aberrant mitosis therefore seems to be independent of changes to histone H3 lys9 methylation and subsequent HP1 binding to this modified residue. Recent developments in this field have identified that changes to the level of other histone modifications on other sites may still be involved in the activation of the G2 checkpoint in resistant cells, the occurrence of an aberrant mitosis and the subsequent overriding of the spindle assembly checkpoint, all of which are hallmarks of HDACi treatment on cells.
Keyword Histone deacetylase (HDAC) inhibitors
Aberrant mitosis
Histone methylation
Cell cycle

 
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Created: Wed, 15 Aug 2012, 16:03:27 EST by Robyn Warrener on behalf of Scholarly Communication and Digitisation Service