Abstract

Viruses have developed sophisticated strategies to evade host defenses and facilitate the production and spread of progeny. HPV 16, whose immune evasion activities can contribute to cervical cancer, is a prime example of such a virus. In this study, we examined the influence of HPV 16 E6 on TRAIL-induced FADD-dependent and -independent pathways in two cellular models: HCT116 colon carcinoma cells and U2OS osteosarcoma cells. We show that transfection of the HPV 16 E6 oncogene into HCT116 cells provides protection from TRAIL-mediated apoptosis. Additionally, we demonstrate that the protection provided by E6 is dose-dependent because higher levels of E6 provide greater protection. The mechanism underlying this protection involves a rapid reduction in the protein levels of both FADD and procaspase-8, which results in suppression of the activation of caspases 8, 3 and 2. Interestingly, E6 does not interfere with the mitochondrial apoptotic pathway even though HCT116 cells have been classified as type II cells with regard to TRAIL signaling.

To further explore the mechanisms of TRAIL-induced cell death, we used E6 as a molecular tool to probe the TRAIL pathway in U2OS cells. Intriguingly, we found that while E6 protected HCT116 cells from TRAIL, U2OS cells containing E6 remained sensitive to TRAIL. Furthermore, silencing FADD and procaspase-8 expression with siRNA did not prevent TRAlL-induced apoptosis in U2OS cells. However, siBid provided significant protection from TRAIL in these cells, and the cleavage kinetics of Bid and caspase-8 revealed that Bid was cleaved prior to the activation of caspase-8 in this cell death cascade. Although Bid cleavage has been implicated in the JNK and ceramide pathways, neither pathway was found to contribute to TRAlL-induced apoptosis in U2OS cells. However, analysis of cathepsin B activity in these cells revealed that it was significantly activated after exposure to TRAIL. Additionally, the cathepsin B inhibitor, CA074Me, inhibited TRAIL-mediated apoptosis and delayed the cleavage of Bid in U2OS cells exposed to TRAIL. Taken together, these findings demonstrate that TRAIL triggers a FADD-dependent pathway in HCT116 cells and a FADD-independent pathway in U2OS cells, and that the ability of E6 to influence TRAlL-induced apoptosis dependents on which pathway predominates.

LLU Discipline

Microbiology and Molecular Genetics

Department

Microbiology

School

Graduate Studies

First Advisor

Penelope J. Duerksen-Hughes

Second Advisor

Carlos A. Casiano

Third Advisor

Ubaldo Soto

Fourth Advisor

Nathan R. Wall

Fifth Advisor

Carl F. Ware

Degree Name

Doctor of Philosophy (Medical Science)

Degree Level

Ph.D.

Year Degree Awarded

2006

Date (Title Page)

5-2006

Language

English

Library of Congress/MESH Subject Headings

Human papillomavirus 16; Papillomavirus Infections; Apoptosis; Tumor Necrosis Factors

Type

Dissertation

Page Count

xi; 180

Digital Format

PDF

Digital Publisher

Loma Linda University Libraries

Usage Rights

This title appears here courtesy of the author, who has granted Loma Linda University a limited, non-exclusive right to make this publication available to the public. The author retains all other copyrights.

Collection

Loma Linda University Electronic Theses and Dissertations

Collection Website

http://scholarsrepository.llu.edu/etd/

Repository

Loma Linda University. Del E. Webb Memorial Library. University Archives

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