We are using the bacterial cat gene under control of the human immunodeficiency virus 1 (HIV-1) promoter region stably integrated in the genome to study radiation-induced gene expression in human cells. This system provides a sensitive, responsive, and convenient assay for radiation-induced gene expression. Using this assay our previous studies demonstrated that ultraviolet light (UV) activates HIV-1 gene expression within hours, possibly through a chromatin decondensation step, while gamma-irradiation does not. Herein, we would like to test the hypothesis that UV-activated HIV-1 gene expression consists of two phases; (i) an early, fast phase, and (ii) a late, slow phase. The early phase is primarily modulated by chromatin structure, via a transient decondensation step, allowing pre-existing transcription factors better access to the HIV-1 promoter. This step does not occur after gamma-irradiation. The late phase is primarily modulated by transcription factors, themselves activated by radiation, such as those encoded by certain proto-oncogenes. The late phase occurs after both types of radiation. The following experiments will be carried out to test this hypothesis: (1) After radiation or treatment with various genotoxic agents, monitor changes in chromatin structure by microscopic examination of mitotic HIVcat/HeLa cell undergoing chromatin decondensation, expression of c-fos and c-jun (AP-1), and NF-kappaB, and the level of HIV-1 cat gene expression by CAT assay and RNA protection. (2) Determine the immediate (minutes to hours) and late (days to a week) effects on HIVcat gene expression after radiation. (3) Determine the importance of topological and structural changes in chromatin important for UV-activated gene expression by using inhibitors of different types of topoisomerases, histone acetylation and poly ADP-ribosylation, and investigate a possible involvement of protein kinase C in this process. (4) Determine what influence the cell cycle has on radiation-induced gene expression. (5) Determine if human cells with deficiencies in DNA repair/processing are impaired at the level and/or rate of radiation-induced gene expression at transcriptionally active and/or-inactive chromosomal loci.
