Date of Award
Doctor of Philosophy (PhD)
Scott, Thomas R
The extracellular matrix (ECM) is a network of proteins for holding tissues and cells together. Among the constituents of the ECM is the large molecular weight glycoprotein laminin. Laminin is predominately found in the basement membrane of tissues. Laminins are composed of α, β, and γ chains with 5 globular (G) subdomains at the carboxyl (C) terminal end of the α chain. Of the 12 different laminin isoforms, laminin-5 has been targeted for cancer research. Specifically, the G3 domain has been studied as the specific binding domain. Cells are able to migrate through cell surface adhesion receptors known as integrins. These heterodimeric receptors are comprised of α and β subunits. Studies have demonstrated integrin α-3 and -6 preferentially bind to laminin-5 alpha3-chain G domains. The G3 domain of the rat laminin-5 alpha3-chain was expressed in a prokaryotic system engineered with chaperone proteins that allow for soluble protein expression in a cold temperature (11.5oC) environment. The expressed recombinant G3 (rG3) has a predicted molecular weight (MW) of 27 kDa, which matches the approximated MW for the sequence including the histidine tag. Following expression and verification through Western Blotting, the protein was purified using chromatography. Upon separation, adhesion and proliferation assays with rG3 were performed on three breast cancer cell lines. The breast cancer cells (MDA-MB-231, MDA-MB-435, and MCF-7) expressed various levels of the α-3, -6, and β-1 integrin, as determined by flow cytometry. For all three breast cancer cell lines, rG3 demonstrated dose-dependent adhesion but an inhibitory effect on proliferation. Each cell line demonstrated morphological changes characteristic of apoptosis at 3, 6, and 12 hours of rG3 treatment. The inhibitory property of rG3 prompted determination of caspase activation. Following a 12-hour treatment with rG3, each cell line was assayed for activation of caspase-3/7, -8, and -9. In each cell line, all three caspases were activated over the levels observed in untreated breast cancer cells. Of particular interest are the elevated caspase-9 levels seen in all cell lines, resulting from altered cell signaling events post-rG3 treatment. Further signaling assays revealed altered levels of phosphorylation of the kinase Akt. Therefore, rG3 binding to cancer cells is specific for the α subunit, but fails to engage the β subunit necessary for induction of cell signaling events for cell survival. This ligand binding specificity leads to reduced cell viability through specific pro-apoptotic signaling events.
Turner, Brittany, "Recombinant G3 Domain Protein of the Rat Laminin-5 α3 Chain Binds To Integrins on Tumorogenic Breast Cancer Cells To Induce Apoptosis" (2008). All Dissertations. 271.