Date of Award

January 2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

First Advisor

Scott Garrett

Abstract

Toxic insult from the heavy metal cadmium is known to induce the expression of metallothioneins (MT) which are cysteine-rich heavy metal binding proteins six to seven kilodaltons in size. Previous research demonstrates that over-expression of MT-3 occurs in the majority of breast cancers and is associated with poor patient outcome. Furthermore, MT-3 has been shown to inhibit the growth of breast cancer and prostate cancer cell lines. Studies have shown that the MT-3 protein contains 7 additional amino acids that are not present in any other member of the MT gene family, a 6 amino acid C-terminal sequence and a Thr in the N-terminal region. The unique N-terminal sequence is responsible for the growth inhibitory activity of MT-3 in the neuronal system, while the function of C-terminal region remains unknown. The unique N- and C-terminal domains of MT-3 may play alternative roles in the differentiation and growth of breast cancer cells.

One goal of this study was to characterize the function of the N- and C-terminal domains of MT-3 in the MCF7 breast cancer cell line. For this purpose six different constructs of MTs were prepared which were as follows: wild type (WT) MT-3, MT-3 N-terminal site directed mutagenesis (MT-3 P7T P9T), MT-3 C-terminal deletion (MT-3 E55_E60del), WT MT-1E, and MT-1E mutated to contain the N -terminal of MT-3 (MT-1E N4_C5insTCPCP), or the C-terminal (MT-1E G52_A53insEAAEAE), or both the N- and the C-terminal of MT-3 (MT-1E N4_C5insTCPCP and G52_A53insEAAEAE). Each of these constructs was transfected into MCF7 cells. Both the growth rate and the

transepithelial resistance (TER) of each cell line were measured. Growth rates were statistically significantly reduced in all cell lines except MCF7 parent, Blank Vector, and MT-1E. Vectorial active transport was increased in WT MT-3 and mutants containing the C-terminal of MT-3 as indicated by the formation of domes and increased TER. Observations of confluent cell cultures of mutant cell lines were also performed to determine the doming phenotype. Doming was observed in cell cultures possessing the C-terminal region of MT-3, and the WT MT-3 cell line. The data obtained suggests that the N-terminal region of MT-3 is involved in growth inhibitory activity even in the absence of the C-terminal domain. The C-terminal region is involved in vectorial active transport which is indicated by the formation of domes in cell culture. Depending on cell culture conditions the N-terminal region of MT-3 may attenuate the C-terminal domain’s ability to confer the doming phenotype.

Microarray analysis of the MCF7 mutants was also performed to determine alterations in gene expression. Overlapping hierarchal clustering demonstrates that the N- and C-terminal mutants have unique expression relationships. Genes that were differentially regulated on the microarray and were further validated included the GAGE family antigens. Several GAGE antigens were investigated to examine differential expression between the N- and C-terminal of MT-3. These included: GAGE12H, GAGE12G, GAGE4, GAGE5, GAGE6, GAGE2E-1, GAGE2E-2, GAGE2E-1E, and GAGE2C. A significant repression of GAGE2C, GAGE2E-1, GAGE2E-2, GAGE5, GAGE6, and GAGE12H antigen expression was seen in MCF7 mutants NT-1E, WT MT-3, and MT-3ΔCT. Up regulation of GAG2C, GAGE2E-2, GAGE5, and GAGE12H antigens occurred in MCF7 mutants MT-1E and CT-1E. The GAGE12G antigen demonstrated increased expression in only MCF7 mutant cell lines MT-1E, CT-1E, and MT-3ΔNT. The GAGE6 antigen was the only antigen to show repression in the presence of the N-terminal of MT-3 and up regulation in the presence of the C-terminal of MT-3 but not in the presence of MT-1E.

In conclusion, this study further characterizes the unique properties of the N- and the C-terminal domain of MT-3 and the potential role that it may play in the differentiation of certain breast cancers. Dendrogram clustering analysis suggests that the N-terminal and C-terminal domains of MT-3 differentially regulate gene expression in MCF7 cells. Increased doming activity and increased doubling times of mutant cell lines containing the C-terminal is suggestive of a differentiated phenotype of the cell line. Differential regulation of GAGE antigens and E-cadherin in the presence of MT-3 could be indicative of MT-3’s role in the epithelial to mesenchymal transition of cancer cells.

COinS