Understanding the molecular basis of cancer represents a critical step in finding a cure. Unraveling the genetic abnormalities that result in uncontrolled cell proliferation will set the basis for the development of new approaches to halt the progression of cancer. Inactivation of the p53 tumor suppressor gene represents the most common genetic abnormality found in human cancer [1]. Additionally Mdm2, a cellular regulator of p53, has been observed overexpressed in a number of human tumors, many possessing wild-type p53 protein. We recently discovered that a Mdm2 like protein called MdmX is capable of protecting p53 protein from Mdm2 mediated degradation. The mdmX gene was isolated in 1996 and shown to encode a protein with significant homology to Mdm2. MdmX overexpression, like Mdm2, has been shown to block p53 transactivation [2], however the mdmX gene expression is not induced by p53 following DNA damage. Based on the increased stability of p53 in the presence of MdmX the hypothesis of this proposal is that the MdmX protein is capable of maintaining p53 protein levels in undamaged cells and thus represents a novel target to modulate p53 activity.
The specific aims of this proposal are designed to test this hypothesis and explore ways to use MdmX to reactivate p53 protein in tumors harboring wild-type p53 genes and Mdm2 overexpression. (1) To identify the domains of MdmX that affect p53 stability and transactivation. This will involve creation of MdmX and Mdm2 mutant proteins in order to examine how the various regions of MdmX serve to maintain p53 protein levels. Mutants will be overexpressed in cell lines possessing wild-type and mutant p53 genes with either normal or elevated Mdm2 protein in order to assess p53 reactivation. (2) To test the prediction that reactivation of p53 with MdmX mutants will restore tumor suppression. We have discovered an MdmX mutant protein capable of stabilizing p53 and increasing p53 transactivation.
This aim will first use cell lines to characterize the p53 biological effects of cell cycle arrest and apoptosis following MdmX overexpression. Subsequent steps will involve the development of a mdmX gene delivery vector to reactivate p53 in mice harboring tumors with elevated Mdm2 and wild-type p53. (3) To elucidate p53-independent interactions of MdmX. Given that Mdm2 has demonstrated cellular effects that are p53-independent, it is likely that MdmX will also possess p53 independent roles.
This aim will examine the ability of MdmX to associate with specific cellular proteins that interact with Mdm2, assess how mdmX and antisense mdm2 expression affect TGFbeta resistance in tumor cell lines and characterize a novel MdmX transactivation activity.
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