Mismatch repair is a vital DNA repair mechanism whose absence leads to a tolerance towards mutations and a predisposition to colon cancer. MLHl is one of the main proteins involved and is highly conserved from E. coli to human. It not only plays a role in repair, but can signal the cell to die if damage levels become too high. The mechanism by which MLHl triggers cell death in response to damage is not entirely clear, and is likely to differ between normal and cancerous cells. Previous work in the Walsh lab had generated MLH1-depleted subclones of a telomerase- immortalised normal human fibroblast cell line. These had been generated by transfecting the parental line hTERT-1604 with an shRNA vector against MLHl and selecting subclones which had reduction in MLHl to various degrees. I further characterised these MLHl knockdown cell lines and revealed that they also exhibit resistance to the methylating agent N-Methyl-N-Nitrosourea. Through the use of various assays we were able to determine that the hTERT immortalised cells did not undergo cell cycle arrest, apoptosis or senescence in response to MNU as colon cancer cell do. Instead, they undergo an MLH1-dependant form of programmed cell death mediated by PARP, but independent of caspase, P53 and ATM/ATR. In 2004, DNMTl deficiency was also implicated in causing MMR defects in mouse embryonic stem cells without a causal mechanism being identified. The effects of DNMTl deficiency are not the same in stem cells and differentiated cells. To determine if depletion of DNMTl can also cause MMR defects in normal human cells, I created DNMT1-depleted hTERT-1604 cells using the same shRNA-mediated strategy as above. Subsequent characterisation of the DNMT1-depleted subclones established that they have a reduction in DNA methylation and the most severely reduced cells are arrested at the G2/M checkpoint. Subclones with significant reductions in DNMT1 also exhibited a decrease in MLH1 expression at the protein, but not the mRNA level. This reduction in MLH1 expression was reversed when a protease inhibitor was employed. The subclone with 31% DNMT1 expression exhibited microsatellite instability, providing further evidence for an interaction between mismatch repair and DNMT1 in human cells and suggesting a mechanism by which this may occur. In conclusion, the work presented here demonstrates a novel role for the mismatch repair protein MLH1 in triggering PARP-dependent cell death in response to damage by MNU. It also shows a link between DNMT1 depletion and MMR deficiency through destabilisation of MLH1.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:551565 |
| Date | January 2009 |
| Creators | Loughery, Jayne Eleanor Patricia |
| Publisher | University of Ulster |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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