ABSTRACT

1. Untreated cells Adenovirus treated cells Untreated cells + anti-galectin antibody + anti-galectin antibody - anti-galectin antibody J.Proteome Res., 6, 2007, 869 Wild-type p53-transfected Glioblastoma Cells Courtney Bennett1 1Florida State University, Tallahassee Fl. ABSTRACT In recent years proteomics has established itself as an invaluable approach in the investigation of the cellular basis of disease and in the search for disease therapies.1 Proteins are integral to cellular functions because they take part in cell-cycle regulation, cell proliferation, immune response, metabolism, signaling, and cell-to-cell adhesion. P53 is a tumor-suppressor gene that regulates the cell cycle; loss of a functional p53 gene is common in cancer.2 It has been observed that therapeutic treatment with a wild-type p53 gene suppresses tumor growth and significantly affects the protein expression of glioblastoma cells.3,4 P53 has been demonstrated to regulate the secretion of exosomes and it has been hypothosized that exosome secretion may constitute a form of cellular communication.5 Galectin-1 is a carbohydrate-binding protein that is overexpressed in some tumors; it is involved in cell proliferation, tumor metastasis, and apoptosis, and is essential to cancer cell migration and invasivity.3,6 It has been established that galectin-1 is significantly down-regulated by transfection with wild-type p53 and that this decrease in galectin-1 is accompanied by a significant decrease in cell mobility and an increase in cell sensitivity to chemotherapy.3 Here we transfected the U87 glioblastoma cell line with an adenoviral vector carrying a wild-type p53 (wt p53) gene and examined the differential exosome protein content of both empty vector treated and Ad-p53 treated cells. The proteomic analysis of exosomes from Ad-p53 treated glioblastoma cells will increase our understanding of the role of p53 in tumor suppression and aid in the identification of possible new targets for cancer therapy. RESULTS A total of 156 protein identities were assigned; of these, 62 were unique to the Ad-p53 treated cells, 52 were unique to the empty vector (control) cells, and 42 were common to both groups. Figure 3. In vitro staining of cells after Ad-p53 therapy indicates loss of galectin-1. METHODS P53 treatment Cell cultures of glioma cell line U87 MG were treated with adenoviruses (a D1-312 control adenovirus or Ad-p53). Exosome extraction The cells were lysed and the exosomes separated by ultracentrifugation using a Percoll gradient.Preparations were examined by electron microscopy to verify the presence of exosomes. Protein separation 1D polyacrylamide gel electrophoresis was performed using a 1.5 mm thick SDS gel with 10% resolving gel and 2.5% stacking gel composition. The gel was stained with SYPRO Ruby or Bio-Rad stains. Each lane of the final gel was cut into 40 segments before being digested with trypsin. Mass spectrometry The digested protein samples were analyzed using nano LC MS/MS and microelectrospray ionization in an FT-ICR MS equipped with a 14.5 Tesla magnet. Protein identifications Each peptide fragment ion spectrum was compared with protein sequences through database searches using the Mascot program. Functions were assigned on the basis of the Swiss Prot database annotation. Adenoviral-Mediated Gene Therapy Of Human Gliomas 150 100 75 50 CONCLUSIONS We observed distinct protein differences in the exosomes of the control cells and the Ad-p53 treated cells. The exosomes from the Ad-p53 treated cells exhibited a greater diversity of protein functions than did those from the control cells. The list of functions unique to the Ad-p53 exosome proteins include: angiogenesis regulation, cell cycle regulation, cell survival, membrane trafficking, and protease functions. Additionally, galectin-1 and a mannose-binding lectin, basigin, were found only in the Ad-p53 exosomes. Ad-p53 treatment of glioblastoma cells in known to decrease intracellular galectin-1 expression (Fig.3). The mechanism for the decrease in galectin-1 may be exosomal export rather than destruction in the proteasome. The implication of the expression of galectin-1 and other unique proteins in the exosomes from wt-p53 treated cells will require further investigation. 37 Slices 1-38 25 Figure 1. Wild-type p53 was inserted into a human adenoviral construct and used to transfect glioblastoma cells. 20 15 10 Standards U87+P53 Lysozyme U87+DL312 Figure 4. 1 D PAGE of Exosome samples (10% Gel) ACKNOWLEDGEMENTS This project is a collaboration between Dr. Charles A. Conrad at M.D. Anderson Cancer Center and the ICR group at the NHMFL, including Drs. Carol L. Nilsson, Mark. R. Emmett, Huan He, and Alan G. Marshall. Figure 2. Exosomes are secreted vesicles that contain proteins that may be used in cell communication.5 REFERENCES 1Nilsson, C. Mass spectrometry of biomolecules (II): Applications in biological systems, 2000. 2Sherr, C.J. Principles of tumor suppression. Cell, 2004, 116 (2), 235-246. 3Puchades, M. et al. Proteomic Investigation of Glioblastoma Cell Lines Treated with Wild-Type p53 and Cytotoxic Chemotherapy Demonstrates an Association between Galectin-1 and p53 Expression. Journal of Proteome Research, 2007, (6), 869-875. 4Ventura, A. Restoration of p53 function leads to tumour regression in vivo. Nature, 2007, 445 (7128), 661-665. 5Yu, S.The Regulation of exosome secretion: a novel function of the p53 protein. Cancer Research, 2006, 66 (9), 4795-4801. 6Camby, I. et al. Galectin-1: a small protein with major functions. Glycobiology, 2006, 16 (11), 137-157. Figure 5. Protein identification results for gel slice 1. Database search tool MASCOT was used to search the NCBI protein database. Filamin- A