CONSTRUCTION AND CHARACTERIZATION OF A NOVEL FUSION PROTEIN FROM THE EXTRACELLULAR DOMAIN OF MULT1 AND TRANSMEMBRANE AND INTRACELLULAR DOMAINS OF FAS AND ITS THERAPEUTIC EVALUATION FOR CANCER TREATMENT USING AN ADENOVIRAL DELIVERY SYSTEM
Date of Award
Doctor of Philosophy (PhD)
Wagner , Thomas E
Chen , Wen Y
Yu , Xianzhong
Scott , Thomas R
One of the strategies that tumor cells adopt to evade immunosurveillance mounted by elements of the innate immune system, such as NK cells, is to down-regulate certain cell surface molecules through a process also called shedding. Mouse UL16-binding protein-like transcript 1 (MULT1), which can activate NK cells through NK cell receptor NKG2D, is one of such molecules. Tumor cells can also avoid Fas mediated apoptosis by down-regulating its expression, secreting antagonistic `decoy' receptors, or expressing anti-apoptotic molecules. In this study, we report the design and evaluation of the antitumor activity of a novel fusion protein MULT1E/FasTI, consisting of the extracellular domain of MULT1 and transmembrane and intracellular domains of Fas. We hypothesized that this protein, when expressed on a cell, would not only activate NK cells and other NKG2D expressing killer cells through its MULT1E region but also send death signals to induce apoptosis of the cell through the FasTI region. We cloned cDNA encoding the extracellular domain of MULT1 gene from thymus of new born mice and ligated it to the transmembrane and intracellular domains of mouse fas cDNA. The resulting fusion cDNA was inserted into a mammalian cell expressing vector under the control of CMV promoter. The vector was then transfected into mouse TC-1 lung epithelial cancer cells; and stable cell lines expressing the fusion protein were established. In vitro cell culture studies demonstrated that the binding of NKG2D/Fc, a recombinant protein of mouse NK cell receptor, to MULT1E/FasTI expressed on tumor cells was able to elicit apoptosis as assayed by Annexin V-FITC staining and caspase-3 ELISA and also activated NK cells as indicated by enhanced interferon-gamma; expression. In vivo subcutaneous tumor studies demonstrated that tumor cells expressing MULT1E/FasTI grew significantly slower than tumors without the protein. Pulmonary metastasis studies showed that most of the mice completely rejected tumor cells expressing MULT1E/FasTI. We also examined the use of a replication-defective adenovirus as a gene therapy vector to deliver the fusion protein into tumor cells. In vitro and in vivo studies not only demonstrated that the novel fusion protein can be successfully delivered by adenoviral vectors but also confirmed antitumor activity of the fusion protein. Therefore, the reported fusion protein strategy represents a novel and hopeful new anticancer agent for cancer patients.
Kotturi rajeshwar, Hari shankar, "CONSTRUCTION AND CHARACTERIZATION OF A NOVEL FUSION PROTEIN FROM THE EXTRACELLULAR DOMAIN OF MULT1 AND TRANSMEMBRANE AND INTRACELLULAR DOMAINS OF FAS AND ITS THERAPEUTIC EVALUATION FOR CANCER TREATMENT USING AN ADENOVIRAL DELIVERY SYSTEM" (2009). All Dissertations. 370.