NOTCH-1 IN REGULATION OF APOPTOSIS IN TUMOR CELLS

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Notch genes encode transmembrane receptors that control cell differentiation in numerous cell types during development. Humans and mice have 4 homologous notch genes. Mutations causing constitutive activation of notch-1,-2 and 4 are oncogenic in vivo and in vitro. Clinically, constitutive activation of notch-1 is associated with T-cell acute lymphoblastic leukemia (T-ALL), and strikingly increased expression with altered intracellular distribution of notch-1 have been demonstrated in various human malignancies and pre-neoplastic lesions (uterine cervix, colon and lung). We and others have recently discovered that notch-1 has anti-apoptotic activity in vitro in transformed cells in various experimental models. Additionally, we found that downregulating the expression of notch-1 in murine erythroleukemia (MEL) cells in the presence of differentiation- inducing hybrid polar drugs leads to massive apoptosis. Whether notch-1 affects apoptosis in non-transformed cells which do not overexress it remains unknown. Our hypothesis is that notch-1 plays an important role in regulating apoptosis susceptibility in notch-1 expressing tumor cells. This implies that interfering with notch-1 expression or signaling may be used to enhance the efficacy of chemotherapy or radiotherapy in human malignancies expressing notch-1. Our long term objective is the development of therapeutic regimens targeting notch-1 using gene therapy, recombinant notch-1 antagonists or notch-1 monoclonal antibodies for clinical testing in human malignancies expressing notch-1. We have generated experimental agents in each of these catagories. Our Aims our as follows: 1. To determine if notch-1 participates in apoptosis regulation in normal cells, by studying its role in thymocyte apoptosis using notch-1 antagonists and novel transgenic mice we have developed. 2. To elucidate the mechanism(s) through which notch-1 protects MEL cells from apoptosis, by investigating signaling pathways known to be linked to notch-1 which participate in regulating apoptosis. 3. To conduct pre-clinical studies of notch-1 inducible antisense retrovirus vectors, a recombinant notch-1 antagonists and a notch-1 blocking monoclonal antibody in mouse tumor models. The experimental agents will be studied alone and in combination with antineoplastic drugs. Studies will be conducted in both immunodeficient animals with human cervical cancer cells and immunocompetent animals with syngeneic murine tumor cells transformed by human papillomavirus 16 (HPV16). These experiments will elucidate the possible uses of notch-1 as a target for cancer treatment and the mechanisms of notch-1 targeting agents. Additionally, they will provide information on the safety profile of these agents and initial proof of concept for clinical trials of notch-1 targeting agents in notch-1 expressing malignancies.