The long-term objective of the proposed research is to gain an understanding of the cellular lesions resulting by hyperthermia that kill mammalian cells. Knowledge of the cellular/molecular mechanisms by which heat kills cells is important in developing clinical protocols utilizing hyperthermia alone and in combination with other therapeutic modalities in the treatment of cancer. This proposal focuses on one potential target for critical damage, the subplasmalemmal cytoskeleton. The major aim will be to test the hypothesis that damage to the microfilamentous cytoskeleton represents one of several of the critical lesions induced by heat in mammalian cells, and that this cytoskeletal damage is toxic if not repaired. The role of increased levels of cellular Ca++ on the induction and repair of cytoskeletal damage, induced by drug manipulation and/or heat, will also be assessed. A comprehensive quantitative morphological study of the effects of heat on the cytoskeletal/plasma membrane complex using synchronous CHO cells is proposed. Cells will be examined at varying times beginning immediately after treatment to determine repairable and irrepairable lesions. The subcellular morphological endpoints and Ca++ influx will be compared to cell survival. Local anesthetics, cytochalasin B and D, the calcium ionophore A23187, and the calmodulin antagonist trifluoperazine will be used to reversibly disrupt the cytoskeleton. Two modifiers that protect cells against heat damage--thermotolerance and glycerol--will also be used. The morphological and functional integrity of the microfilamentous cytoskeleton will be studied using light microscopy, timelapse cinematography, fluorescence microscopy, and transmission electron microscopy. Emphasis will be placed on the use of critical point dried cell fragments for quantifying ultrastructural damage to the cortical cytoskeleton. A bioassay for a functional cytoskeleton (the ability of cells to respread following heat) will be used to quantify attachment with cell survival. Ca++ influx will be monitored using 45Ca++. Changes in free Ca++ levels within treated cells will be quantified using the fluorescent quinoline Ca++-indicator """"""""Quin 2"""""""". Results from this project should demonstrate whether or not damage to the microfilamentous cytoskeleton associated with the plasma membrane represents a critical lesion of heat (possibly modulated by increased Ca++ levels), with repair of the damage being a prerequisite for reproductive intregrity.
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