Date of Award

1-1-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Mary Ann Sens

Abstract

Arsenic and cadmium (Cd+2) are environmental agents that have been classified as human carcinogens by the International Agency on Research in Cancer (IARC). Numerous epidemiological studies have linked arsenite (As+3) and Cd+2 to the development of bladder cancer. Bladder cancer is the second most common genitourinary malignancy in the United States, and one of the first cancers to be associated with an environmental component. Currently, there are no stand-alone biomarkers for this type of cancer.

Kindlin-2 and Metallothionein (MT) isoform-1X (MT-1X) are two genes that are the focus of this dissertation. Both genes were selected for further experimental analysis based on their common association with bladder cancer. Metallothionein isoforms 1 & 2 (MT I/II) are over-expressed in high-grade muscle-invasive bladder cancers, and in one study, MT-1X showed increased levels of expression compared to the other isoforms. The main goal of the MT-1X study was to investigate the mechanism by which this isoform is over expressed in bladder cancer, and it was predicted that both transcriptional and epigenetic processes were involved in the regulation of the MT-1X promoter. The model system used in this study was the normal human urothelial (UROtsa) cell line and its As+3 or Cd+2 transformed counterparts. The experimental approach was to assess MT-1X mRNA expression, transcription factor binding, as well as, histone modifications in the MT-1X promoter of metal-transformed cells and compare it to that of the non-transformed parent. In addition, the effect of the histone deacetylase inhibitor (HDACi) MS-275 on factor binding and histone modifications was also examined. Results of this study revealed that exposure to MS-275 induced the levels of MT-1X mRNA, and that this induction was further enhanced when the cells were exposed to a combined treatment of zinc (Zn+2), a known inducer of MT transcription, and MS-275. ChIP analysis of the MT-1X promoter showed that there was increased transcription factor binding and histone modifications in the As+3 and Cd+2 transformed cells when compared to the non-transformed parent. Treatment with MS-275 further increased the binding of the transcription factors to the MT-1X promoter. Additionally, the histone modifications that correlated to a transcriptional active state were also increased with MS-275 treatment. The results also highlighted a possible role for the CCAAT-binding transcription factor (NF-I) in the initiation of transcription in the MT-1X promoter.

Kindlin-2 was selected for further investigation in an effort to confirm a microarray analysis that suggested that it might play a role in tumor metastasis. The microarray study revealed that Kindlin-2 was the most consistently repressed gene in the As+3 and Cd+2 transformed UROtsa cell lines that were capable of forming peritoneal tumors. To confirm these results, and to assess whether Kindlin-2 expression correlates with the ability to form tumors in the peritoneum, a common metastatic site in bladder cancer, the current study utilized a combination of RT-PCR, Western, and immunohistochemical analyses to characterize Kindlin-2 expression in As+3 and Cd+2 transformed human cell lines, their tumor transplants in immunocompromised mice, and in archival specimens of normal human bladder and bladder cancer. Results of these experiments did not correlate with the results of the microarray study, but never the less, it was significant since it pointed to a potential role for Kindlin-2 as a prognostic biomarker in a subset of high-grade invasive urothelial cancers that are destined to progression and metastasis.

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