Date of Award

January 2018

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Psychology

First Advisor

Scott H. Garrett

Abstract

Heavy metals Arsenite (As3+) and Cadmium (Cd2+) are known human carcinogens and exposure to them occurs through cigarette smoking, industrialization, and contaminated water sources. Epidemiological studies have shown that prolonged exposure to these heavy metals are associated with several health issues, including bladder cancer. With survival rates directly correlated to detection age and tumor stage, the demand for early detection would effectively cut costs and improve the patient prognosis of human bladder cancer.

Metallothioneins (MT) play an essential role in metal regulation, heavy metal detoxification, and cellular redox chemistry in cellular biological systems and tissues. Previous studies in literature have found elevated MT expression in patients with kidney, breast, prostate and bladder cancers. This increased presence of MT may contribute to the resistance to the chemotherapy drug Cis-diamminediechloroplatinum(II), CDDP (Cisplatin) in human bladder cancer.

Previous studies from our laboratory have shown that As3+ and Cd2+ can malignantly transform the human urothelial cell line UROtsa and these transformed cells can form subcutaneous, as well as, intraperitoneal tumors. The first goal of this study is to determine the expression level of six MT isoforms (MT-1A, MT-1E, MT-1F, MT-1X, MT-2A, and MT-3) in the Parent UROtsa cell line and the 13 independently derived As+3-and Cd+2-transformed cell lines. The second goal of this study is to determine the expression levels of the same six MT isoforms in the Parent UROtsa and in two As+3 (As #1 and As #5) -and two Cd+2 (Cd #1 and Cd #4) -transformed cell lines when they were acutely exposed to various concentrations of the chemotherapy drug, cisplatin.

For this study, Messenger Ribonucleic Acid (mRNA) was isolated from the Parent UROtsa cell line and the 13 independently derived As+3-and Cd+2-transformed cell lines, as well as, two As+3 (As #1 and As #5) -and two Cd+2 (Cd #1 and Cd #4) -transformed cell lines and the Parent UROtsa after the cells were acutely exposed to cisplatin. Real Time Reverse Transcriptase Quantitative Polymerase Chain Reaction (RT-qPCR) was performed to determine the basal expression of the six MT isoforms in the Parent UROtsa cell line and the 13 independently derived As+3-and Cd+2-transformed cell lines. Real Time RT-qPCR was also performed to determine the corresponding expression of the Parent UROtsa and in two As+3 -and two Cd+2 -transformed cell lines after the cells were acutely exposed to cisplatin. The results demonstrated an overall decrease in the expression of the six MT isoforms in the transformed cell lines compared to the Parent UROtsa, as well as, an overall decrease in expression after exposure to cisplatin when compared to the basal expression levels.

In order to further look at the basal expression, Immunohistochemical (IHC) analysis was performed on the subcutaneous transplants generated by the subcutaneous injection of the 13 independently derived As+3-and Cd+2-transformed cell lines into immune compromised mice. The six As3+ and the seven Cd2+ UROtsa mouse subcutaneous tumors were stained for MT isoforms 1/2 and MT isoform 3. MT isoforms 1/2 staining of the 13 UROtsa mouse subcutaneous tumors show moderate or weak-to-moderate staining. MT isoform 3 showed strong and diffuse staining, however the peripheral less differentiated tumor cells showed weaker staining.

Additionally, western blot analysis was performed to determine the levels of MT proteins in the Parent UROtsa and two of the As3+ -and two of the Cd2+ -transformed cell lines. Overall, the results demonstrated a decrease in the levels of total MT in the Parent UROtsa, as well as, the transformed cell lines.

In conclusion, our data shows that exposure to As3+ or Cd2+ decreases the expression of the six MT isoforms. This decrease in MT expression may disrupt the normal cellular redox within the cells, thus promoting the transformation process. In addition, exposure to cisplatin demonstrated a decrease in expression of the six MT isoforms, which may have implications in the treatment of human bladder cancer with this drug.

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