Dale Haines - US grants

DESCRIPTION: (adapted from the investigator's abstract) 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.

DESCRIPTION (provided by applicant): Spt23p and Mga2p of Saccharomyces cerevisiae are members of the highly conserved family of NF-kappaB like transcriptional regulators. Similar to their mammalian orthologues, proteins encoded by SPT23 and MGA2 undergo a unique limited proteasome-dependent event that generates the transcriptional competent polypeptides. In addition, the generated transcriptionally active polypeptides are held in a latent state outside of the nucleus and their nuclear mobilization is dependent on ubiquitination and the proteasome. A major difference between the yeast and NF-kappaB proteins is that Spt23p and Mga2p contain a carboxy-terminal transmembrane domain and are expressed as ER anchored proteins. Genetic studies have indicated that in addition to the proteasome, the highly conserved ubiquitin ligase RspSp and the ubiquitin binding Cdc48p- Npl4p-Ufd1 p segregase complex are required for the OLE1 -inducing function of Spt23p and Mga2p. In the case of Spt23p, RspSp is required for proteasome-dependent processing and recent studies indicate that this is mediated by a single RspSp interacting LPKY motif that is present with the carboxy-terminus of Spt23p. Curiously, RspSp and the LPKY motif is dispensable for Mga2p processing and recent published data suggests that RspSp facilitates Mga2p release by a mechanism that is distinct from Spt23p. The goal of experiments proposed here is to further define the various and poorly understood roles of the ubiquitin proteasome pathway in Spt23p and Mga2p expression and activation. The Specific Aims of this proposal are 1) To perform a detailed structure-function based analysis of RspSp, Mga2p and Spt23p, 2) To determine the role of RspSp in release of transcriptionally competent Mga2p and Spt23p from the ER, and 3) To elucidate the mechanism of Mga2p90 and Spt23p90 generation by the proteasome. These studies will provide important new details relating to how the ubiquitin-proteasome pathway impinges on the activity and regulation of NF-kappaB like and membrane-localized proteins in eukaryotic cells.