| Summary: | Thesis (M.Sc.)--Memorial University of Newfoundland, 2010. Medicine Includes bibliographical references (leaves 206-226) Mesoderm induction early response 1 (mier1) is a gene encoding Mier1α, a corepressor of ERα (Mercer et al, unpublished data). As breast tumours become invasive, the subcellular localization of MIER1α changes from the nucleus to the cytoplasm, thereby potentially abrogating or changing its function, an action of which may have a role in breast cancer progression (McCarthy et al, 2008). This study analyzed the regulation of mier1 in breast cancer cells by investigating the previously uncharacterized MLP-P1 promoter. The subsequent role of this promoter in the subcellular localization of MIER1α was then studied in order to further elucidate possible mechanisms underlying MIER1α's documented change in subcellular localization. Transient transfection of a MLP-P1 promoter deletion series driving a luciferase reporter gene in multiple cell lines was used to characterize the minimum promoter and revealed that MLP-P1 is the predominately active mier1 promoter. The degree to which its activity was increased over the second mier1 promoter, MAEP-P2, was largest in ER+ breast cancer cell lines. -- Transcriptional activation at MLP-P1 can lead to the incorporation of an alternate exon that generates transcripts harbouring an extra 74bp insert containing either a putative nuclear export sequence (NES) or transmembrane domain. Immunocytochemical analysis showed that MIER1α isoforms resulting from transcripts harbouring this insert (denoted MIER1 3A alpha) localized to the cytoplasm, while MIER1α isoforms lacking this extra amino acid sequence (denoted MIER1 alpha) localized to the nucleus in ER+ MCF7 breast cancer cells. LeptomycinB (LMB), an inhibitor of the nuclear export system, decreased MIER1 3 A alpha cytoplasmic localization while simultaneously increasing its nuclear localization, thereby demonstrating that exon 3A encodes a functional NES. Interestingly, both MIER1α isoforms were equally localized throughout the entire cell in ER- cell lines, a phenomenon which was not affected by LMB treatment. Thus, preferential activation of MLP-P1 in ER+ rather than in ER- breast cancer cells may now be considered a prime candidate mechanism driving the subcellular localization change of MIER1α from the nucleus to the cytoplasm.
|
|---|