The long-term goals of this research project is to understand the factors that confer radio- and chemoresistance to tumor cells. One factor that enhances the survival of mammalian cells, including tumor cells, after exposure to heat, noxious agents, radiation or imbalances in cellular redox is the expression of small stress response proteins (SSRPs). SSRPs are associated with growth, development and apoptosis, and they are likely involved in the gain of cancer therapy resistance through the dysregulation of differentiation, apoptosis, and redox regulation. In an effort to identify differences in gene expression in two mouse B cell lymphoma cell lines that differed in apoptotic propensity, and radiation- and chemoresistance, a previously unidentified gene was cloned that has homology to a family of proteins that include a number of SSRPs. The protein product of this gene (p28dra) is associated with radio- and chemoresistance in the cells from which it was identified and in a battery of mouse tumors that are radio- resistant. In addition, the over-expression of p28dra inhibited anthracycline-induced differentiation of K562 cells. We hypothesize that this protein is involved, through redox regulation, in the gain of resistance seen in some tumor cells and that this gain of resistance is through the loss of treatment-induced apoptosis or through a block in differentiation induction. To test our hypothesis, cells in which p28dra regulation has been altered by inducible expression vectors that either up- or down-regulate p28dra will be challenged by radiation or drug exposure to see if apoptotic or differentiation propensity are altered. Furthermore, redox regulation of both endpoints will be examined by monitoring GSH activity as a result of altered p28dra regulation, and GSH and thiol levels will be manipulated in order to alter p28dra activity. Also, mutations in specific regions of the p28dra gene, the GSH binding motif and a structural domain, will be created to identify critical functional regions within the protein. And finally, because no GST or GPx activity has been described, we will identify other proteins that interact with p28dra by yeast two-hybrid analysis. Understanding how p28dra functions will provide new insights into overcoming resistance to cancer therapy and possibly lead to new clinical strategies in cancer management.
