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Telomeres are tandem repeats of the DNA sequence TTAGGG. The 3' end of each telomere consists of a single stranded overhang that is normally concealed in a loop structure at the chromosome ends. Exposure of telomere sequences may induce DNA damage responses that lead to apoptosis and other effects. We have treated five human melanoma cell lines with an 11-base oligonucleotide (5'-GTTAGGGTTAG-3') homologous to the telomere 3' overhang sequence (referred to as T-oligo). T-oligo induces S-phase arrest and apoptosis by 96 hours (29-64%) in four of five melanoma cell lines. We have further investigated the three most responsive cell lines (MU, MM-MC and PM-WK) and found increased levels of E2F1 and p53 transcription factors at 24 and 48 hours after T-oligo treatment. In MU melanoma cells, we also found increased levels of phosphorylated p53 at serine 15 and increased p21. In MU and PM-WK cells, T-oligo treatment resulted in increased levels by Western blot analysis of caspase 9 and caspases 3 and 7, which are downstream effector caspases. A four fold increase in caspase 3 enzyme activity was observed colorimetrically in MU cells by 72 and 96 hours after treatment. We also found down-regulation of survivin, an inhibitor of apoptosis, at 72 hours after T-oligo treatment. In addition to these effects on the apoptosis pathway, the levels of differentiation markers tyrosinase and TRP-1 were increased 96 hours after T-oligo treatment. To investigate whether the apoptosis and other effects seen after T-oligo treatment in p53-responsive MU cells are dependent on p53, we decreased the levels of p53 using siRNA. Apoptosis induced by T-oligo was inhibited by 45% upon reduction of p53 levels with siRNA, indicating that p53 has a substantial but not exclusive role in T-oligo-mediated apoptosis in MU cells. Similarly, the increase in levels of differentiation markers was inhibited after treatment with p53 siRNA. These data demonstrate the therapeutic potential of T-oligo and indicate that p53 can play a major role in T-oligo-mediated apoptosis and differentiation in melanoma cells.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA