HIV-1 replication is amplified in H2O2-, O2- or 1O2-treated cells, and this process has been linked to activation of the transcription factor NF-~B, which binds to the long terminal repeat (LTR) promoter/enhancer regions of HIV-1 sequences and initiates HIV-1 gene expression and replication of the virus. NF-B is negatively regulated by an inhibitory binding protein called IB. Reactive oxygen intermediates or UV light activate NF-B by causing IB to dissociate. The goal of this proposal is to investigate the mechanism of photosensitized activation of NF-B, and the possible roles of protein kinases and proteases in this process. For these studies, various photosensitizing visible dyes have been employed to generate reactive oxygen species in cultured cells and phospholipid micellar systems. Visible irradiation of calphostin C (CalC) dissolved in DMSO or solubilized in the non-ionic detergents Tween 20 or Triton X- 100 generated the corresponding semiquinone radical. In the presence of oxygen the O2.- and .OH radicals were detected. However, irradiation of CalC solubilized in phosphatidyl serine micelles generated only small amounts of O2~- and ~OH. Visible irradiation of CalC solubilized in Triton X-100 micelles also generated singlet oxygen (1O2) a highly reactive form of oxygen. However no 1O2 was detected when CalC was dissolved in phosphatidyl serine micelles. Although CalC inhibition was oxygen-dependent inhibition was not reversed by 1O2 quenchers (NaN3,~- carotene), superoxide dismutase or free radical scavengers (dithrothreitol, N-acetylcysteine, BHT). These observations are in agreement with our findings that when bound to phosphatidyl serine CalC does not generate significant amounts of singlet oxygen, superoxide or hydroxyl radical. Similar results were obtained with hypericin (Hyp). These findings show that CalC and Hyp may not be useful for producing the reactive oxygen intermediates needed for this study.
