The analysis of genome stability maintenance mechanisms in human cancer cells and stem cells


Maintaining genome stability is essential for all dividing cells and it promotes longevity. The centromere is one region where stability is critical, as this is where the kinetochore identifies and attaches the spindle microtubules so that each sister chromatid can faithfully segregate. Our knowledge of centromere mechanism has improved vastly over the last decade. A functional role for homologous recombination within centromeric DNA has been proposed whereby covalently closed DNA loops (CCLs) are formed. We examined this model by studying the sites of deposition of the centromere marker, CENP-A, in cells that accumulate Holliday structures throughout there chromosomes. Whilst we found no direct evidence for CENP-A deposition at Holliday junction, tools for further analysis of this important question were developed. Embryonic stem cells (ESCs) are derived from the inner cell mass of the blastocyst. ESCs give rise to lineages leading to all the somatic cells of the developing embryo. Therefore, any epigenetic disturbances or genome defects that occur in the genome at this early stage would have detrimental consequences for the embryo as a whole. Hence, these cells are likely to possess high fidelity genome maintenance mechanisms. It has been shown that human stem cells and embryonal carcinoma cells do not show the same checkpoint-dependent response to DNA replication inhibition. In the present study, we used the SW480 and HCT116 cancer cell lines as non-stem cell controls, with the expectation that these cells would behave like somatic cells. We also used these cells to derive so called colonospheres, which are a proliferating population arising from these cells lines that have some key characteristics of cancer stem cells. We clearly demonstrated that cancer cells appear to respond distinctly to certain chemotherapeutic genome damaging agents when they transition to a more stem cell-like state. A key observation was the dramatic reduction in BLM and GEN1 levels in parallel with the appearance of stem cell markers upon sphere formation. The implications for chromosomal instability following DNA replication stress were also explored and assessed. We also successfully generated induced pluripotent stem cells (iPSCs) from human fibroblasts using modified mRNA. We used these cells and their isogenic parental cells to determine whether these cells differ in responses DNA damaging agent from isogenic fibroblast and/or embryonic stem cells.


Iaith wreiddiolSaesneg
Goruchwylydd / Goruchwylwyr / Cynghorydd
    Dyddiad dyfarnuIon 2014