Date of Award

8-2011

Document Type

Thesis

Degree Name

Master of Science (MS)

Legacy Department

Biological Sciences

Advisor

Scott, Thomas R

Committee Member

McNealy , Tamara

Committee Member

Rice , Charles

Committee Member

Walker , Heather

Abstract

To assess changes in AKT pathway signaling, a recombinant protein of the G3 domain of rat laminin-5 (rG3) that specifically binds the alpha subunit of integrins α6β1 and α6β4 expressed on cancer cells (e.g., MDA-MB-231) was produced. This recombinant protein is believed to interrupt the intracellular signaling events of the AKT pathway, causing a decrease in proliferation and survival of cells after treatment. Viability assays confirmed an apoptotic effect of rG3 on cells in a dose-dependent manner. However, data from gene expression studies of Caspase-9, GRB10, and CDKNIB proved non-conclusive that rG3 is acting upon gene expression, leading to the further investigation of the AKT pathway and proteins involved in this signaling cascade. P53 and phosphorylation of AKT, NFkB/p65, and IKKαβ were evaluated after treatment with rG3 at 0, 3, 6, 9 and 12 hours. Results show significant differences in protein expression for these proteins in cells treated with rG3 compared to untreated cells. Significantly higher levels of AKT and phosphorylated AKT were seen in untreated cells, indicating the inhibitory effect the rG3 protein has on this pathway. Both IKKαβ and the phosphorylated IKKβ catalytic subunit were expressed at a significantly higher level in untreated cells, as were the levels of phosphorylated nuclear NFkB. These results also indicate an inhibition of downstream proteins of the AKT cell survival pathway with rG3 treatment. Cytosolic NFkB, however, was expressed at significantly higher level in cells treated with rG3 when compared with untreated cells because the majority of this protein in actively proliferating untreated cells is in the phosphorylated form. The greatest change was seen in expression of the pro-apoptotic protein, p53. In treated cells, this protein was expressed at greatly higher levels than in the untreated cells, especially at 9 hours after treatment, indicating the large impact rG3 treatment has on the AKT pathway and proves to significantly reduce cell viability through specific signaling events.

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