The experiments presented and outlined in this proposal are designed to improve the understanding of molecular checkpoint pathways that are altered in chemo-resistant human tumors. Work by the applicant's laboratory, as well as by others, has provided strong evidence which suggests that mdm2 onco-protein overexpression may play a key role in affecting clinical outcome by altering the response of tumor cells to chemotherapeutic agents. The overexpression of mdm2 has been associated with a number of tumors of poor prognosis and has been associated with early treatment failure in childhood acute lymphoblastic leukemia (ALL). It is now accepted that one mechanism by which mdm2 overexpression may provide a selective growth advantage to chemo-treated tumor cells is through its ability to block p53 controlled checkpoint responses. Experimental overexpression of mdm2 has been shown to block the transactivation, cell cycle arrest and apoptotic functions of p53. New data generated by his laboratory suggests that mdm2 overexpression may affect p53 independent pathways mediating chemosensitivity. P53 null cells that have been transfected with an mdm2 expression construct and express elevated levels of mdm2 protein display marked differences in their ability to respond to the chemotherapeutic drug cisplatin when compared to vector alone transfected cells. Furthermore, we have identified that mdm2 can interact with a novel DNA damage checkpoint molecule, DNA polymerase epsilon (DNA pol E). Based on these observations, we hypothesize that mdm2 protein overexpression can alter the response of transformed cells by chemotherapeutic drugs by affecting p53 dependent and independent checkpoint pathways. To test this hypothesis and to identify these p53 independent checkpoint pathways, the following aims are proposed: 1) To determine whether experimental overexpression of mdm2 in p53 null cells alters the response of human tumor cells to chemotherapeutic agents in a drug or tissue specific manner, 2) To determine whether mdm2 protein is overexpressed in primary leukemic cells from relapsed patients harboring p53 gene mutations, 3) To determine whether mdm2 and DNA pol E interact during a DNA damage response and to identify amino acids of both mdm2 and DNA pol E that are required for association and 4) To determine whether mdm2 overexpression can block a DNA pol E mediated checkpoint response to DNA damage. These studies will not only improve the understanding of mdm2 activities in checkpoint pathways, but will also be important for understanding how altered expression of mdm2 affects the response of tumor cells to chemotherapeutic agents. Understanding these processes will hopefully lead to novel therapeutic strategies restoring chemosensitivity to chemo-resistant tumors that overexpress mdm2.
| Asahara, Hitomi; Li, Ying; Fuss, Jill et al. (2003) Stimulation of human DNA polymerase epsilon by MDM2. Nucleic Acids Res 31:2451-9 |
