Telomerase activity and telomere lengths in fibroblast cells treated with ependymin peptide mimetics

by Hirsch, Erica.

Telomerase is an enzyme that helps maintain the telomeric ends of chromosomes during DNA replication. Telomere lengths represent a balance between telomerase activity attempting to elongate their ends, and cell division that causes telomere shortening. As cells age, diminished telomerase activity allows a shortening of telomere lengths until they reach a target length that stimlulates apoptosis. Identifying a drug capable of upregulating telomerase activity may help increase cell (and even organismal) lifespan. The purpose of this thesis was to determine whether treatment of human primary foreskin fibroblast cultures with a 14 amino acid (aa) ependymin peptide mimetic upregulates (or at least maintains) telomerase activity and telomere lengths during cellular ageing. The 14aa peptide was previously shown to significantly increase the murine lifespan by 25%, so its activity was a logical candidate to test in this thesis. In a preliminary set of experiments, the human primary fibroblast cells were shown to respond to the 14aa drug by upregulating the antioxidative enzyme superoxide dismutase (SOD), thus human fibroblast cells likely contain the appropriate receptor for binding this drug. This same dose proved optimal for upregulating telomerase activity in the fibroblast cells an average of 57% relative to untreated cells (p value = 0.003). The upregulation appears to be specific for the sequence of aa in the 14aa drug since a “scrambled” peptide containing the same aa but in a different order showed no upregulation, even at doses 10-fold higher. Treatment of mice once per day or twice per day with the 14aa peptide was also found to upregulate telomerase activity in vivo in brain and heart. The activity was optimal at a 3.3 mg/kg dose for each aged organ, and was generally high in young organs. The activity observed in heart was a total surprise since 2 heart cells are generally thought to be quiescent, and telomerase is usually associated with cell division, so perhaps telomerase has a function other than in cell division. The second part of the hypothesis tested whether treatment of fibroblast cells with the 14aa drug elongated (or prevented from shortening) telomere lengths in aged cells. A telomere length assay (TLA) based on a Southern hybridization approach using a telomere probe appeared to work well, since marker DNAs showed appropriate differences in their “telomere smears”, and aged fibroblast cells showed shorter smears than young cells. However, no difference was observed between drug-treated versus vehicle-treated cells, even at the 10 ng/ml dose previously shown to strongly upregulate telomerase activity. So perhaps the upregulation of telomerase activity was not sufficient to provide a measurable increase in telomere lengths. Telomerase has been shown to extend the lifespan of virus-transformed human cells without showing any visible telomere lengthening (Blackburn et al, 1999), so perhaps telomerase can increase cell lifespan without increasing telomere lengths. To our knowledge, this is the only drug demonstrated to upregulate telomerase activity. Transforming cells with the viral T-antigen can upregulate telomerase, but T-antigen is not a therapeutic drug since it also causes cancer. Telomerase upregulation is known to occur during oncogenesis, but telomerase itself is not an oncogene since oncogenesis also requires the upregulation of oncogenes. Our lab previously showed this peptide does not upregulate the potent oncogene myc. If this proves to be the case for other oncogenes, using this 14aa drug to upregulate telomerase activity without activating oncogenes could prove extremely useful for helping prove telomerase is not an oncogene, and for extending cell lifespan. 3