Date of Award

January 2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Jane R. Dunlevy

Abstract

The development of bladder cancer is known to have a strong association with environmental toxins. This laboratory employs the human UROtsa cell line model to explore the relationship between As+3 and Cd+2 exposure and the development of urothelial cancer.

The parental UROtsa cells and their As+3 and Cd+2 transformed counterparts have been used to define the mechanism of cell death (apoptosis and/or necrosis). A third mechanism of cell death, autophagy, has not yet been investigated. The hypothesis for the current study is that the autophagy pathway involving beclin-1plays a role in UROtsa cell death mechanisms. A combination of real time RT-PCR, western analysis, and immunohistochemistry showed that beclin-1 is expressed in the urothelium of normal human bladder, but large alterations in beclin-1 and its associated autophagy genes are not found in heavy metal induced bladder cancer cells.

SPARC, a glycoprotein with counter adhesive properties, has the ability to modulate cell-cell and cell-matrix interactions. Microarray analysis indicated that SPARC gene expression was greatly decreased between parental and all transformed UROtsa cell lines. The hypothesis for this study is that a reduction in SPARC expression is necessary for a malignant phenotype to develop. SPARC expression was determined in human parental UROtsa cells, their Cd+2 and As+3 transformed counterparts, and in archival specimens of human bladder cancer using a combination of RT-PCR, western

analysis, immunofluorescence localization, and immunohistochemical staining. This study showed that exposure to As+3 or Cd+2 greatly reduced SPARC expression in UROtsa cells.

To further analyze SPARC expression, SPARC was stably transfected into select transformed UROtsa cell lines which were characterized based on growth rates, morphology, wound closure, migration, invasion, and tumorigenicity. Tumors generated by injection of the SPARC-transfected cell lines into nude mice, showed an absence of SPARC expression within the epithelial tumor component, but were positive for the transfected vector. This study suggests post-transcriptional down-regulation of SPARC expression in urothelial carcinoma cells within the mouse tumor environment. Overall, results from this study show that autophagy does not play a large cell death role within the UROtsa system however, down-regulation of SPARC expression does strongly correlate with the malignant phenotype.

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