Graduation Year

2008

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Chemistry

Major Professor

Mildred Acevedo-Duncan, Ph.D.

Co-Major Professor

Robert Potter, Ph.D.

Keywords

Small interfering RNA, Cell cycle, Apoptosis, Cell survival, Phosphorylation

Abstract

Prostate cancer is one of the leading causes of death among males in the United States. In this study, we hypothesized that an activated PKC-?-dependent anti-apoptotic pathway, drives the cell cycle proliferation and survival of prostate cancer cells. We investigated the role of atypical PKC-iota (PKC-?) in androgen- independent prostate DU-145 carcinoma, androgen-dependent prostate LNCaP carcinoma compared to transformed non-malignant prostate RWPE-1 cells. Western blotting and immunoprecipitations demonstrated that PKC-? is associated with cyclin-dependent activating kinase (CAK/Cdk7) in androgen-dependent, RWPE-1 and LNCaP cells but not in androgen-independent DU-145 cells. Treatment of prostate RWPE-1 cells with PKC-? silencing RNA (siRNA) decreased cell proliferation, cell cycle accumulation at G2/M phase and decreased phosphorylation of Cdk7 and cdk2.

In addition, PKC-? siRNA treatment provoked a decrease in phosphorylation of Bad and increased Bad/Bcl-xL heterodimerization, leading to cell apoptosis. In DU-145 cells, PKC-? is anti-apoptotic and still required for cell survival. Treatment with PKC-? siRNA blocked an increase in cell number, and inhibited G1/S transition. In addition to cell cycle arrest, both RWPE-1 cells and DU-145 cells underwent apoptosis via mitochondria dysfunction and activating apoptosis cascades such as release of cytochrome c, activation of caspase-7, and poly-(ADP-ribose) polymerase (PARP) cleavage. Mechanistic pathways involving aPKCs in the NF-?B survival pathway were established using pro-inflammatory cytokine, tumor necrosis factor alpha (TNFa). Results demonstrated that RWPE-1 cells and DU-145 cells are insensitive to TNFa whereas LNCaP cells are sensitive to TNFa treatment and undergo apoptosis.

In DU-145 cells, TNFa induced PKC-? activation of I?B kinase, IKKa/ß, while in RWPE-1 cells, PKC-? activates IKKa/ß. Both RWPE-1 and DU-145 show degradation of I?Ba allowing NF-?B/p65 translocation to the nucleus. In LNCaP cells, the upstream kinase activation IKKa/ß was not observed, although there have been reports that LNCaP cells weakly activate IKKa and have NF-?B activation. In vivo kinase assay demonstrates that PKC-? is the substrate of IKK?/ß. A putative PKC-? inhibitor (ICA-1) inhibited activation of IKKa/ß in vivo. Hence, PKC-? is an antiapoptotic protein and this suggests that anti-PKC-? therapy may be a viable option for prostate carcinoma cells.

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