Telomere dysfunction in normal human epidermal keratinocytes

Minty, Fay (2007) Telomere dysfunction in normal human epidermal keratinocytes. PhD thesis, University of Glasgow.

Abstract

TRF2 is one of the main telomere binding proteins and a key regulator in protecting the telomere, the end of a linear chromosome. The telomere can be artificially "uncapped" by expression of myc-TRF2DeltaBDeltaM, a myc tagged dominant negative version of the protein. Both myc-TRF2 Full Length (FL) and myc-TRF2DeltaBDeltaM were retrovirally-infected into cells as part of an IRES-GFP construct. NHEK expressing GFP (and therefore either myc-TRF2 FL or myc-TRF2DeltaBDeltaM were selected at early time points using flow-sorting and this allowed either low or high expression levels to be isolated. Low or high levels of expression of myc-TRF2DeltaBDeltaM altered colony morphology, reduced clonogenicity and almost completely prevented proliferation of NHEK. Several markers of DNA damage were investigated and a small amount of p53- phosphoS15 was detected 2 days after expressing myc-TRF2DeltaBDeltaM in NHEK. The number of small colonies containing senescent cells was increased in NHEK expressing myc-TRF2DeltaBDeltaM compared with Vector only controls. Induction of DNA damage responses is undoubtedly a contributing factor and senescence at least one cellular outcome of uncapped telomeres in NHEK. Low levels of myc-TRF2 FL expression in NHEK also caused a reduction in cell proliferation, but not as severe as seen by expression of myc-TRF2DeltaBDeltaM. High levels of myc-TRF2 FL demonstrated an even greater reduction in proliferation, equivalent to myc-TRF2DeltaBDeltaM. Despite the reduced cell proliferation, NHEK expressing myc-TRF2 FL did not demonstrate any p53-phosphoS15. Excessive telomere processing may play a role in the dramatic effect seen in NHEK following expression of myc-TRF2 FL. The majority of human cancers are of epithelial derivation. As a major focus of cancer therapy is centered on exploiting the properties of the telomere, a full understanding of it's properties in an epithelial context is warranted. This thesis examines the effects of uncapping telomeres in Normal Human Epidermal Keratinocytes (NHEK), the first time an investigation into telomere status has taken place with a primary epithelial cell system.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Ken Parkinson
• Keywords: Oncology
• Date of Award: 2007
• Depositing User: Enlighten Team
• Unique ID: glathesis:2007-74207
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 23 Sep 2019 15:33
• Last Modified: 23 Sep 2019 15:33
• URI: https://theses.gla.ac.uk/id/eprint/74207