Hyperthermia has considerable 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. It should be particularly effective in combination with radiation and certain chemotherapeutic 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.
The specific aims of this proposal are to determine if the plasma membrane, mitochondria, nuclei, or the cytosol of cells become more thermostable in thermotolerant cells. Thermostability will be assayed by measuring the temperature at which protein denaturation occurs in the organelles listed above and in whole CHL V79 cells using ultra-high sensitivity differential scanning calorimetry (DSC) and spin labeling. This will give information about whether the state of cellular thermotolerance is due to increased thermostability of cellular organelles and which organelles might be the direct targets of hyperthermia. Each of the isolated organelles investigated will be analyzed for the presence of het shock proteins (HSP's) by SDS-PAGE autoradiography. Thus, it will be determined whether HSP's may be directly responsible for thermotolerance by increasing the thermostability of cellular organelles by binding to them. The direct effects of previously identified hyperthermic sensitizers and protectors on the thermostability of the cellular organelles listed above will also be determined by DSC. A second aspect of this proposal deals with measuring protein denaturation in whole V79 cells at 40-45 degrees C by ultra-high sensitivity DSC and relating the degree of protein denaturation to cell survival determined by colony assay. This will give a rapid (about one hour), relatively simple means of both assaying the hyperthermic sensitivity of cells and the degree of damage induced by a prior heat treatment. This will be extended to the development of a method for assaying the damage produced by a heat treatment in EMT6 tumors previously grown in BALB/c mice. If this is successful, a method will have been developed for determining the effectiveness of a hyperthermic treatment immediately after, or possibly during, treatment.
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