We hypothesized that if normal cells carry a regulator which is inactivated in cancer cells, the transfer of this regulator from a normal cell to a cancerous one should inactivate the telomerase. Available in the laboratory was a panel of mouse/human microcell hybrids, each containing a tagged normal human chromosome fragment integrated into a mouse melanoma (telomerase-positive) background. We screened these hybrids for telomerase activity using both the polymerase chain reaction (PCR)-based telomeric repeat amplification protocol (TRAP) assay and mTERT mRNA amplification. / We were able to find a telomerase negative microcell hybrid, B78MC27. Following multiple passages in culture most of the cells undergo senescence; however a small population reactivates telomerase to ensure their survival. The revertant B78MC27-R4, where the human tagged chromosome has been removed, retains telomerase inhibition and also behaves like B78MC27 at later passages. In spite of our efforts, PCR mapping and fluorescence in situ hybridization (FISH) analysis on these hybrids failed to identify the exact region containing the putative repressor. Transfection of a reporter gene (luciferase) under the control of the hTERT promoter into these hybrids confirmed that the telomerase regulation is affecting the hTERT transcription level. / Finally, upon chromosome transfer containing the telomerase repressor into the PC-3M-Pro4GP1 human prostate cancer cell line, we observed hTERT gene transcriptional down-regulation and inhibition of telomerase activity. Thus, telomerase regulation is subject to a dominant negative inhibition likely involving the direct binding of a repressor on the hTERT promoter. The discovery of the natural telomerase regulator could eventually lead to potential cancer therapy, particularly for tumors having short telomeres, as it is seen in prostate cancer.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.84503 |
| Date | January 2003 |
| Creators | Deault-Bonin, Marie |
| Contributors | Chevrette, Mario (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 002032076, proquestno: AAINQ98236, Theses scanned by UMI/ProQuest. |
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