Date of Award

11-2-2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biology

First Advisor

Scott Garrett

Abstract

Arsenic and cadmium (Cd+2) are well established environmental pollutants. Human exposure to arsenite (As+3) and Cd +2 have been associated with many types of cancers including skin, bladder, liver, prostate, stomach and lung. Epidemiological studies have associated Cd+2 exposure with breast cancer development while As +3 has not been directly associated with breast cancer development, although both As+3 and Cd+2 have been implicated in estrogen receptor mediated gene transcription and endocrine disruption. In the present study, the ability of both these heavy metals to transform the immortalized human breast epithelial (MCF-10A) cell line was examined. For this purpose, the MCF-10A cells were exposed to 1 μM As+3 or Cd+2 with the end point being transformation, verified by the ability of the transformed MCF-10 cells to form colonies in soft agar. As+3 - and Cd+2 - transformed MCF-10A isolates were subjected to microarray analysis and one of the differentially expressed genes that was identified was neuron specific enolase. Neuron specific enolase (ENO-2, γ-enolase) has been used as a biomarker to help identify neuroendocrine differentiation in breast cancer. The next goal of the present study was to validate the microarray data and determine if ENO-2 expression in the breast epithelial cell is influenced by the environmental pollutants, As+3 or Cd+2, in both acute and chronic exposures. It was shown that both As+3 and Cd+2 exposure caused significant increases in ENO-2 expression under conditions of both acute and chronic exposure. In contrast, ENO-1, the major glycolytic enolase in non-muscle and neuronal cells, was largely unaffected by exposure to either As +3 or Cd+2. Localization studies showed that ENO-2 in the MCF-10A cells transformed by As+3 or Cd+2 had both a cytoplasmic and a nuclear localization. In contrast, ENO-1 was mainly localized to the cytoplasm. The ENO-2 that localized to the cytoplasm was found to co-localize with ENO-1. The results are the first to show that ENO-2 expression in the breast epithelial cells is induced by acute and chronic exposure to As+3 or Cd +2. These observations were extended to the urothelial cell, where environmental exposures are strongly linked to urothelial cancer. The UROtsa, an immortalized nontumorigenic urothelial cell line and its Cd+2 and As +3 transformed counterparts were used as the model. Acute exposure of the parental UROtsa cells to both As+3 and Cd+2 caused significant increases in ENO-2 expression. The expression of ENO-2 was significantly elevated in the Cd+2 and As+3 transformed UROtsa cell lines. The tumors formed from these cell lines showed even higher levels of ENO-2 expression. In contrast, ENO-1, the major glycolytic enolase in epithelial cells was unaffected by exposure to As+3 or Cd+2. The elevated expression of ENO-2 in the transplanted tumors suggests that ENO-2 might participate in tumor initiation at the injection site. These findings extend the evidence suggesting a link between As +3 and Cd+2 exposure and neuroendocrine differentiation in tumors. These results suggest that ENO-2 might be a biomarker of human exposure to Cd+2 and As+3 and this exposure could potentially lead to the development of tumors with neuroendocrine differentiation.

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