Ionized calcium (Ca2+), acting as an intracellular messenger, controls numerous biological processes that are essential for life. However, it is also able to convey signals that result in cell death. The fidelity of Ca2+ as a universal second messenger therefore depends on mechanisms that specifically and dynamically regulate its levels within a cell, as well as maintain resting intracellular Ca2+ concentration ([Ca2+]i) very low. One such mechanism for Ca2+ signaling and homeostasis is the plasma membrane Ca2+-ATPase (PMCA), which is a primary active Ca2+ transporter that translocates Ca2+ from a low intracellular Ca2+ environment to a high extracellular environment. There are four mammalian PMCA isoforms (PMCA1-4), which are differentially expressed depending on tissue or cell type. PMCA isoforms possess different sensitivities to biochemical regulation of Ca2+ efflux activity and are also able to subtly alter the dynamics of Ca2+ signals. These properties suggest that the PMCA is not merely a trivial mechanism for Ca2+ extrusion but is influential in contributing to the Ca2+ signaling requirements and unique physiology of different cells. The indispensable nature of Ca2+ signaling in organs such as the brain, heart and skeletal muscle has been the studied extensively but little is known about the roles and regulation of Ca2+ in the mammary gland. This is despite the fact that the mammary gland is a site of extensive Ca2+ flux during lactation. However, cumulating evidence indicates that upregulation of PMCA2 expression in the mammary gland is a major mechanism for milk Ca2+ enrichment. Therefore, the PMCA is likely to be an important mediator of bulk Ca2+ homeostasis in the mammary gland. Studies in other model systems also suggest that PMCAs may regulate other cellular processes such as cell proliferation, differentiation and apoptosis that are required for normal mammary gland physiology. These basic cellular processes are also disturbed in breast cancer and hence deregulation of PMCA expression in the mammary gland may have pathophysiological consequences. Previous studies show that PMCA1 expression is greater in tumorigenic MCF-7 and MDA-MB-231 human breast cancer cells compared to non-tumorigenic MCF-10A human breast epithelial cells. Furthermore, the expression of PMCA1b and PMCA4b is lower in human skin and lung fibroblasts neoplastically transformed by simian virus 40, compared to non-transformed counterparts. It is therefore hypothesized that regulation of PMCA isoform expression is disrupted in breast cancer and that inhibition of PMCA expression in an in vitro model of breast cancer has important effects in modulating intracellular Ca2+ homeostasis, cell proliferation, differentiation and apoptosis. This thesis describes the use of real time RT-PCR to compare PMCA isoform mRNA expression in tumorigenic and non-tumorigenic mammary gland epithelial cells. It demonstrates that particular breast cancer cell lines overexpress PMCA2, an isoform with restricted tissue distribution and which is present in abundant amounts in the lactating rat mammary gland. Thus, some breast cancers may be characterized by the overexpression of Ca2+ transporters that are normally upregulated during the physiological course of lactation. The pathophysiological significance of PMCA2 overexpression in breast cancer is uncertain and future investigations should look at whether levels of PMCA isoform expression correlate with malignancy, prognosis or survival. To address the second hypothesis of this thesis, a stable MCF-7 Tet-off human breast cancer cell line able to conditionally express PMCA antisense was generated. This strategy was necessary due to the current lack of specific pharmacological inhibitors of the PMCA. This thesis shows that PMCA antisense expression significantly inhibits PMCA protein expression, while subtly affecting PMCA-mediated Ca2+ efflux without causing cell death. However, it also reveals that inhibition of PMCA expression has major effects in mediating cell proliferation and cell cycle progression. Moderate changes in PMCA expression and PMCA-mediated Ca2+ transport result in dramatic consequences in MCF-7 cell proliferation. These studies not only support the supposition that modulation of Ca2+ signaling is a viable therapeutic approach for breast cancer but also suggest that PMCAs are possible drug targets. Alternatively, inhibitors of the PMCA may act as adjuvants to augment the efficacy of other anti-neoplastic agents like tamoxifen that have been shown to modulate Ca2+ signaling. Since the discovery of a new family of primary active Ca2+ transporters, which are related to PMCAs, the opportunities in this field of research are very promising.
| Identifer | oai:union.ndltd.org:ADTP/254319 |
| Creators | Lee, Won Jae |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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