Defense Date

2008

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Pathology

First Advisor

Shawn Holt

Abstract

Telomerase is a ribonucleoprotein that is reactivated in cancer cells to allow for continuous cellular division and indefinite growth. With telomerase being expressed in more than 85% of all cancer, it is imperative that we understand how to selectively inactivate and degrade this unique DNA polymerase. In doing so, we can specifically target tumor cells to erode their telomeres so that they will undergo apoptosis or senescence. Through this research, we have learned that telomerase can be degraded in the nucleoplasm by Hsp90 chaperone inhibition and in the cytoplasm by the dominant negative mutant, D712A V713I. These findings should guide future drug design to target sites on telomerase that interact with Hsp90 and catalytic divalent metal ions. Previous studies have shown that chaperones function to stabilize the RNP and that their inhibition results in ubiquitin-mediated degradation. However, a detailed understanding of how telomerase is signaled for degradation is not well defined. We showed that Hsp90 inhibition causes telomerase to be degraded by a nuclear ubiquitin/proteasome pathway such that exportation to the cytoplasm is not required. Using confocal fluorescence microscopy and immunoprecipitation /Western analysis, we showed that nucleoplasmic GFP-hTERT is ubiquinated and degraded within 2 hrs of exposure to the Hsp90 inhibitor, Radicicol. Upon combined treatment with the proteasome inhibitor, MG132, degradation is inhibited as shown by Western analysis and fluorescent intensity. Additionally, fluorescent pattern with inhibition of degradation shows telomerase aggregation and co-localization with the nuclear proteasome and not with nucleoli. However, the combined treatment with the exportin inhibitor, Leptomycin B, resulted in complete loss of fluorescence. Taken together, these data suggest that Hsp90 inhibition causes telomerase to immediately undergo nuclear degradation, which may function in the nuclear quality-control of telomerase. The dominant negative expression of telomerase has been shown by many investigators to cause shortening of telomeres. However, the mechanism of how it functions and its fate inside the cell are still unknown. After stably expressing the wild-type and dominant negative mutants GFPhTERT in cells, we show that the D712A V713I mutation causes the ubiquination and degradation of the mutant and wild-type hTERT which eventually leads to the shortening of telomeres. Degradation appears to be cytoplasmic since the additional mutation for the nuclear export signal (nes) and treatment with the exportation inhibitor are able to prevent the reduction in protein levels and fluorescence. Based on this cytoplasmic degradation and the additional co-localization of the GFPDNhTERT to the nucleoli, we propose two new mechanisms of dominant negative hTERT utilizing the theory of interactive dimerization. First, the heterodimer of DNhTERT : wt hTERT may be degraded at a faster rate than the wt hTERT homodimer. Second, the heterodimer may be sequestered in the nucleoli thus diminishing the wild-type hTERT access to the telomere in the nucleoplasm. Overall, we have shown that telomerase can be degraded in the nucleoplasm or cytoplasm depending on the mechanism of inhibition. The significance of this is a better understanding of how Hsp90 inhibition and dominant negative hTERT expression cause the degradation of wild-type hTERT. We have also suggested potential mechanisms of dominant-negative hTERT effect and resistance. With this knowledge, future drug therapies can be designed based on these inhibitors to not only inactivate but also to cause the degradation of an enzyme that is crucially important for the immortalization of cancer cells.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

August 2009

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