The broad, long-term objective of the project is to understand the relationship between oxidative stress, apoptosis resistance and cancer. Oxidative stress-resistant variants of the murine thymoma-derived WEHI7.2 cell line were established by stable transfection with thioredoxin or catalase cDNA or selection for hydrogen peroxide- resistance. These variants are all resistant to glucocorticoid-induced apoptosis, exhibit delayed cytochrome c release in response to glucocorticoids and have increased NADP(H) pools. Release of cytochrome c from mitochondria marks the commitment point in apoptosis. The hypothesis to be tested is that the NADP(H) pool mediates redox control of apoptosis signaling in response to glucocorticoid treatment of thymocytes; a higher level of NADP(H) may prevent increased levels of reactive oxygen species in response to glucocorticoids or may attenuate subsequent reactive oxygen species-mediated signaling for cytochrome c release. The WEHI7.2 variants provide a unique tool for investigating this hypothesis.
The specific aims of the project are to investigate: 1) the affect of directly modulating NADP(H) pools on sensitivity to glucocorticoid-induced apoptosis by transfecting WEHI7.2 cells and a hydrogen peroxide-resistant variant with genes that respectively raise and lower NADPH synthesis; 2) the relationship between the NADP(H) pool and control of reactive oxygen species by using oxidation-sensitive fluorescein dyes to compare levels of reactive oxygen species following glucocorticoid treatment of WEHI7.2 cells, the oxidative stress-resistant variants and the transfectants from Specific Aim 1; 3) the relationship between the NADP(H) pool and sensitivity of mitochondria to apoptotic signaling by using cell-free assays for cytochrome c release to test whether mitochondria from oxidative stress- resistant WEHI7.2 variants resist releasing cytochrome c in response to apoptotic stimuli; and 4) the relationship between the NADP(H) pool and signaling through critical MAP kinase pathways by using western blots and kinase activity assays to test for differential activation of protein kinase cascades following glucocorticoid treatment of the WEHI7.2 cells vs. oxidative stress-resistant WEHI7.2 variants and transfectants from Specific Aim 1. The knowledge gained from the project may suggest strategies for overcoming the cancer treatment barrier of apoptosis resistance.
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