Author: Jennifer Williams Bergstrom

Publisher:

Published: 2013

Total Pages:

ISBN-13:

Metastatic melanoma and relapsed acute T cell lymphoblastic leukemia remain incurable despite major advancements in our understanding of these cancers. In some ways, the foundation for a cure has already been laid; many molecular targets for drug development are now clearly defined, and recently novel therapeutics have shown promise in the clinical setting. However, this immense progress has not translated into a marked increase in patient survival. This graduate project focused on two aims, which with further research and development may lead to a cure for these cancers. First, the development of a cellular-based high-throughput synergistic assay was looked at, that would allow for rapid screening of multiple drug combinations. Second, cancer-specific alterations in the cell surface high mannose glycoprotein profile were identified in the context of metastatic melanoma and T-ALL cells. The feasibility of these two aims is demonstrated by existing innovations and research. First, protein microarrays have been developed to isolate and characterize a variety of cell types. Second, growth factors and other molecules have been successfully printed onto these arrays. Third, cancer cells and cancer stem cells have been previously shown to have alterations in their cell surface glycome. As part of the first project, micropatterned silane and polyethylene glycol (PEG) coated glass slides were successfully developed, with these modifications acting as a non-fouling surface for cell attachment. On these slides cell-specific antibodies, antiCD3 and anti-GD3, were printed and demonstrated successful capture ofT-ALL and melanoma cell lines, respectively. However, the effectiveness of this cell capture needs further improvement, with regards to uniform cell seeding. Future studies will utilize recently developed epoxide coated slides, which allow for covalent attachment of capture antibodies. Additionally, anti-CD4 antibody will be printed for T-ALL cell capture. With respect to the characterization of the cell surface glycome of malignant melanoma and TALL cell lines versus their non- or less-malignant counterparts, we were able to successfully demonstrate that MHC class I and sodium/potassium A TPase proteins were differentially glycosylated on both of these diverging cancers. MHC class I had significantly higher expression on high metastatic potential B 16F 10 cells compared to B16FO cells (p-value = 0.014), and in Jurkat cells as compared to normal pan T cells (pvalue = 0.011). Additionally, sodium/potassium ATPase had significantly higher expression on B16F10 (p-value = 0.028) and Jurkat cells (p-value = 0.013) verses B16F0 and normal T cells, respectively. These proteins, along with other identified high mannose surface proteins are directly linked to cancer cell proliferation promotion. Additionally, the altered glycosylation patterns identified in this graduate project may have a direct impact on MHC class I and sodium/potassium A TPase' s roles in cancer cell promotion. Based on obtained data, future studies will focus on conforming the presence of identified proteins on the cell surface through antigen immunofluorescence labeling, and foot printing of high mannose glycans. Footprinting will be done to both determine the extent of glycosylation modifications occurring on cancer cells, as well to characterize any changes in protein conformation due to changes in glycosylation, as determined through in silica modeling.