The long term objective of the proposed research is to gain an understanding of the cellular lesions induced by hyperthermia that kill mammalian cells. Knowledge of the cellular/molecular lesions by which heat kills cells is important in developing clinical protocols using heat alone and in combination with other therapeutic modalities in the treatment of cancer. This proposal focuses on three of the cell's structural systems as potential targets of critical heat damage. These structural systems are: 1) the cytomatrix; 2) the nuclear envelope/lamina complex; and 3) the nuclear matrix. The major aim will be to test the hypothesis that unrepaired heat-induced structural damage to these systems will result in cell death. A comprehensive quantitative morphological study of the effects of heat on these three systems using synchronous CHO cells is proposed. Cells will be examined at varying times beginning immediately after treatment to determine repairable and irreparable lesions. The morphological endpoints will be compared to cell survival. The effects on modifying survival on the morphological endpoints will be assessed using heat sensitizers (local anesthetics, caffeine, low pH) and thermally sensitive CHO mutants with altered morphology will be used in the proposed experiments. The morphological integrity of the various systems will be studied using light microscopy (bright field, phase, and differential interference contrast) fluorescence microscopy, time lapse cinematography, and transmission electron microscopy. Emphasis will be placed on using synchronous cells, sequential extraction of cells that produce well-defined fractions of both morphological and biochemical use, and resinless section electron microscopy for quantifying ultrastructural damage to the three systems. Emphasis will also be placed on the use of the premature chromosome condensation technique as a functional assay of heat-placed on the use of the premature chromosome condensation technique as a functional assay of heat-induced alterations to the nuclear matrix/chromatin complex and for studies of the nuclear envelope/lamina system. Results from this project should demonstrate whether or not heat induces critical lesions to the structural systems under study, with repair of the damage being a pre-requisite for reproductive integrity.
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