We have recently shown that several inducers of the glucose regulated set of stress proteins (grps) lead to a transient resistance to the anti-cancer drug adriamycin. The focus of this renewal application is to continue our analysis of this drug resistance phenomenon and to determine the mechanism by which it occurs. We will present data in this application which demonstrates that two inducers of grps selectively deplete the nuclear enzyme topoisomerase II. Topoisomerase II, a principal nuclear protein associated with DNA replication and cell division, has been identified as a major target of action for several anti- neoplastic agents including adriamycin. Its disappearance during a grp stress could explain the associated drug resistance. It is the goal of this application to test this hypothesis by determining if other grp inducers also deplete topoisomerase II and if this change in enzyme levels parallels the expression of drug resistance. Secondly, we will also investigate the molecular mechanism by which this major nuclear alteration comes about, specifically by focusing on changes known to be associated with the induction of grps. An in vitro system will be developed to examine the pathway for topoisomerase II loss and to isolate and purify the factor(s) responsible. Since studies have indicated that topoisomerase II levels change in parallel with a cell's proliferative status, we will compare topoisomerase II disappear- ance and cell cycle alterations to distinguish whether the disappearance of the enzyme results from cycle changes or precedes such changes. Finally, since two inducers of this stress protein system are anoxia and glucose starvation, this stressed state is expected to have applications in tumor development in situ. We will therefore continue our in vivo studies by examining topoisomerase II levels in RIF-1 tumors in situ as a method of distinguishing grp induced and drug resistant tumor regions. A variety of techniques from biochemistry, molecular biology and radiation biology will be used to undertake the experiments described in this proposal, including polyacrylamide gel electrophoresis, western blot analysis, northern blot analysis, colony cell survival, use of an in vitro/in vivo tumor system, cell fractionation, and protein purification procedures.
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