Thiols are generally thought of a antioxidants which protect against oxidative stress imposed by diverse agents and conditions such as ionizing radiation, some chemotherapeutic drugs, neurodegenerative diseases, autoimmune diseases, ischemia/reperfusion, etc. However, in some circumstances, e.g., in the presence of traces of transition metals, some antioxidants become pro-oxidants, causing cell killing, including apoptosis. We have developed a detailed, testable, stepwise model to explain this paradoxical action of thiols in apoptosis. According to the model, under certain cellular conditions, thiols: (i) undergo metal-catalyzed oxidation producing the reactive oxygen species, H2O2, O(2)- and .OH, (ii) disrupt intracellular calcium homeostasis by alteration of critical thiols or disulfides in calcium-regulating proteins, and (iii) activate the transcription factor NF-kB, (iv) resulting in apoptosis. We further hypothesize that bcl-2 can act at one or more steps in this pathway to inhibit thiol-induced apoptosis.
The specific aims are designed to test each step in the model individually as well as to demonstrate the integration of the steps into a pathway from thiols to apoptosis. In addition, we will obtain quantitative data on the relationship between apoptosis and loss of clonogenicity in thiol-treated cells to address whether apoptosis accounts for all the cell death and whether agents that appear to inhibit apoptosis (e.g., calcium chelators or bcl-2) increase long-term cell survival or only delay cell death. Most of these studies will use the model thiol dithiothreitol in human leukemia HL-60 cells. Apoptosis will be shown by morphological and DNA fragmentation criteria and quantitated using DNA fragmentation and TUNEL assays. .OH will be measured using sensitive, specific, fluorometric techniques, calcium will be measured using fluorescent intracellular calcium probes, and NF-kB activity will be assessed using the gel mobility shift assay. Interest in using thiols and other antioxidants to prevent or treat conditions believed to be caused, at least in part, by reactive oxygen species is growing rapidly. Thus, it is important to understand possible detrimental effects of these agents. Furthermore, because of their documented roles in oxidative stress, calcium homeostasis, NF-kB activation and apoptosis, thiols are unique probes for study of chemical, enzymatic and genetic mechanisms involved in each of these processes and in their interactions that cause cell death. Ultimately the knowledge gained in this study may lead to development of novel, specific therapeutics, based on the properties of thiols, that are targeted to particular intracellular domains to """"""""turn on"""""""" protective pathways or """"""""turn off"""""""" undesired cellular processes, e.g., to enhance cell killing in tumor cells or prevent cell killing in AIDS, autoimmune diseases, neurodegenerative diseases, etc., where oxidative stress may play a role.
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