The goal of this proposal is to develop means to selectively regulate the expression of genes associated with the initiation or progression of cancer. The attainment of this goal would constitute a significant new approach to cancer therapeutics. Our strategy is to use peptide combinatorial libraries to identify DNA-binding domains able to specifically recognize promoter regions of individual genes; the binding domains are then coupled to transcriptional regulating domains so as to create novel proteins capable of selective transactivation or repression. Our overall goal is embodied in three Specific Aims.
In Aim I we will build on our recent progress and evaluate the actions of novel repressors targeted to the promoter of the MDR1 multi-drug resistance gene. We will measure the effects of the repressors on P-glycoprotein expression, drug transport, and drug resistance, both in MDR tumor cells in culture and in MDR tumor xenografts in mice.
In Aim II we will exploit our recent progress in the design of novel transcriptional activators that selectively regulate the expression of Bax, a pro-apoptotic gene that is an important target of the p53 tumor suppressor. We will measure the effects of the novel transactivators on Bax expression, Bax-induced apoptosis, and responses to chemotherapeutic agents. This will be done in p53-deficient tumor cells in culture and in tumor xenografts in mice. Our intent is to be able to selectively enhance Bax-driven apoptotic responses to chemotherapy in p53-deficient tumors, but not in normal cells.
In Aim III we will develop approaches for the intra-cellular delivery of the novel selective transcriptional regulator proteins created in Aims I, II. This will be accomplished by linking the novel proteins with 'delivery peptides' designed to promote passage of macromolecules across cell membranes. These protein chimeras will then be tested for their ability to influence expression of MDR1 or of Bax when administered externally to intact cells. The experiments proposed in Aims 1-Ill will provide important general insights into the feasibility of selective modulation of cancer-related gene expression. They will also provide new molecular tools that could prove to be valuable adjuncts for cancer chemotherapy.
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