T-oligo and its Associated Proteins in Apoptosis and Senescence
thesisposted on 13.12.2012, 00:00 by Richard E. Mulnix
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. An 11-base oligonucleotide homologous to the 3’ end of the telomere sequence known as T-oligo has been shown to induce DNA damage responses such as apoptosis and senescence, and has also been shown to reduce tumorigenicity. This study presents T-oligo’s ability to induce anti-cancer responses in melanoma and lung cancer cell lines, and investigates the effect T-oligo has on senescence and apoptosis inducing proteins along with proteins that are known to bind to T-oligo. Published literature shows that when telomeres become critically short or have damaged DNA, the telomere overhang is exposed inducing apoptosis or senescence in cancer cells. T-oligo treatment which mimics the exposure of the telomere overhang sequence is able to induce senescence and apoptosis in cancer cells since they have altered cell cycle checkpoints. T-oligo may also induce DNA damage responses through binding to telomere-associated proteins or other T-oligo binding proteins. We hypothesize that T-oligo will not induce DNA damage responses in normal bronchial epithelial cells (NBEC), however it will induce DNA damage responses in lung cancer and melanoma cell lines. This study demonstrates that NBEC treated with T-oligo did not show apoptosis, senescence, or inhibition of proliferation, nor was there upregulation of p53 and p21. However, T-oligo did induce several DNA damage responses in cancer cells. T-oligo induced apoptosis in lung cancer cells. T-oligo reduced clonogenic capacity in both lung cancer and melanoma cell lines, and induced senescence in MU melanoma cells and H358 and SW1573 lung cancer cells. T-oligo induced proteins mediating senescence such as p21, p27, and p33 in vitro and induced proteins such as p53 and p73 which induce apoptosis. It was found that T-oligo associated proteins hnRNP C, Pur-β, and Msi-1 were modulated by T-oligo. T-oligo upregulated hnRNP C and Pur-β, and downregulated Msi-1. T-oligo was further able to reduce lung cancer tumors in a mouse model, and found to upregulate senescence associated proteins p21, p27, and p33 in vivo. These results verify our hypothesis that T-oligo is able to induce anti-cancer responses, and demonstrate the proteins and pathways that might be involved in mediating T-oligo induced DNA damage responses.