Hyperthermia has promise as a means of cancer therapy, at least for tumors refractory to other means of treatment but situated such that they can be effectively heated to 43-45 degrees. Most likely it will be used in combination with radiation or certain chemo- therapeutic agents. The molecular effects of hyperthermia are not well understood, in particular the target (if there is a single one) of hyperthermia and the molecular basis of thermotolerance are not known. These two problems are intimately related since thermotolerance is probably due to protection or stabilization of the target. The specific aims of this proposal are to determine if the nucleus and subnuclear components (i.e. nuclear matrix and nucleolus), plasma membrane, and cytoskeleton contain proteins denaturing at the predicted transition temperature of the critical target (46.3 degrees), and if the critical target is stabilized in thermotolerant cells, that is if its denaturation temperature is shifted to higher values (47.5 to 48.7 degrees depending on the degree of thermotolerance). Thermostability will be assayed by measuring the temperature at which protein denaturation occurs in these organelles isolated from control and the thermotolerant cells and in whole CHL V79 cells using high sensitivity differential scanning calorimetry (DSC) and fluorescence spectroscopy in selected cases. These experiments will give information about which cellular organelles can contain the critical target for hyperthermia and whether this target, and thus the organelle containing it, are more thermostable in thermotolerant cells. Whether or not there is an increase in the level of HSP's in any organelle demonstrating increased thermostability will be determined. Thus, the hypothesis that HSP's are responsible for thermotolerance by directly stabilizing cellular proteins and structures will be tested. In addition, the technique of thermal gel analysis will be used to determine the molecular weights of all proteins denaturing during hyperthermia in the isolated organelles. Thus, it should be possible to identify the critical target and determine whether or not it is stabilized by HSP's in thermotolerant cells. The long term goal is to apply the technique of thermal analysis, that is the measurement of cellular protein denaturation by DSC and other physical techniques, to the problem of understanding thermal damage in mammalian cells and tissue. Thermal analysis has potential as a means of analyzing and predicting the thermostability, hence sensitivity to hyperthermia, of normal and tumor tissue.