Date of Defense

5-5-2004

Department

Chemistry

First Advisor

David Reinhold, Chemistry

Second Advisor

Susan Stapleton, Chemistry

Third Advisor

Bruce Bejceck, Biology

Abstract

Tumor cells exhibit abnormal growth properties when compared to normal cells. Previous research with inhibitors has suggested that phosphatidylinositol 3-kinase (PI3K) plays a role in the abnormal growth of fibrosarcoma cells. In this study, we further examined the potential involvement ofthe PI3K/Akt pathway in the abnormal growth rate of fibrosarcoma cells using several approaches. First, we studied tumor cells transfected with a plasmid coding for a dominant-negative PI3K. We attempted to determine if these cells were expressing the dominant-negative protein. If expression ofthe dominant-negative protein was found, we could then determine the effect ofthis on the growth of fibrosarcoma cells in serum-free media. The results ofWestern blotting, however, showed no indication that the dominant-negative protein was expressed. This may have resulted from the selection process ofthe cells. We also used tumor and normal fibroblast cell lines and compared the difference of their Akt activity. Akt is an enzyme involved in the PI3K signal transduction pathway, which has been hypothesized to be involved in cell growth. Using a commercial assay for Akt activity we found significantly higher enzyme activity in fibrosarcoma cells when compared to normal human fibroblast cells. This data indicated that the Akt activity induced in the fibrosarcoma cell lines may be involved in their abnormal growth. In order to further the link ofthe Akt activity to abnormal cell growth, three experiments were performed. The first ofthese was an assay to test the activity ofthe enzyme when the PI3K pathway was inhibited. Our hypothesis was that Akt activity would be decreased when the pathway that leads to cell growth is inhibited. We found that the activity of the Akt enzyme was not affected by the PI3K inhibitor LY294002, which has been shown to inhibit fibrosarcoma cell growth. This implies that Akt is not involved in the abnormal growth of thefibrosarcoma cells. Wethen performed experiments to determine whether the cause of the greater Akt enzyme activity in the fibrosarcoma cells is due to increased activation ofthe Akt enzyme via phosphorylation orthat there is simply a greater amount ofenzyme. We found equal amounts of Akt enzyme in all cell lines, but higher levels of phosphorylation in the fibrosarcoma cell lines when compared to the normal fibroblasts. This indicates that increased Akt activity is due to increased phosphorylation and not higher levels of the enzyme in the cells.

Access Setting

Honors Thesis-Campus Only

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