Date of Award

Spring 2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program/Concentration

Biomedical Sciences

Committee Director

Patricia Hentosh

Committee Director

David Gauthier

Committee Member

Dennis Peffley

Committee Member

Emilia Oleszak

Abstract

This is the first evidence that a plant-derived compound–perillyl alcohol regulates telomerase activity via the mammalian target of rapamycin (mTOR) pathway in prostate cancer cells. Telomerase–the enzyme responsible for immortalizing cells through telomeric repeats addition–is de-repressed early in an aspiring cancer cell. We hypothesized that perillyl alcohol regulates hTERT (human telomerase reverse transcriptase) at the translational and post-translational levels via its effects on the mTOR pathway. A rapid suppression of telomerase activity was detected in prostate cancer cell lines (PC-3 and DU145) in response to biologically-relevant concentrations and short incubations of perillyl alcohol or the mTOR inhibitor—rapamycin.

Western blot analysis revealed a decrease in hTERT protein levels in response to either agent that did not coincide wholly, with loss of telomerase activity suggesting a further level of regulation. Using immunoprecipitation we established the presence of a hTERT-mTOR-S6K (p70 S6 kinase)-Hsp90 (Heat shock protein 90)-Akt complex previously detected in activated NK cells in DU145 prostate cancer cells. Further, western blot analysis demonstrated that perillyl alcohol or rapamycin disrupted the binding interactions between RAPTOR and hTERT, mTOR, S6K, and Hsp90, establishing an additional mechanism by which these agents decrease telomerase activity.

Prostate cancer cells overexpress eIF4E (eukaryotic initiation factor 4E) the rate-limiting protein that mediates cap-dependent translation by way of mTOR signaling. Immortalized Chinese hamster ovary (CHO) control cells (pMV7) and CHO cells with forced eIF4E-overexpression (rb4E) were used to elucidate the role of eIF4E in telomerase regulation by perillyl alcohol and rapamycin. Telomerase activity and TERT protein levels were dramatically attenuated in rb4E cells by perillyl alcohol or rapamycin, but the pMV7 cells were unresponsive to either agent. Through western blot analysis we determined eIF4E-overexpression activates Akt–an upstream regulator of mTOR–through a positive-feedback loop thereby increasing the phosphorylation of downstream targets of Akt. These findings demonstrate that eIF4E-overexpression in CHO cells alters protein synthetic processes and gene regulation, thus enabling the inhibitory effects of perillyl alcohol and rapamycin on telomerase activity and TERT protein levels. This study provides evidence for a unique link between perillyl alcohol- and rapamycin-mediated regulation of mTOR and hTERT.

DOI

10.25777/nnr5-bx62

ISBN

9781267395887

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