Radiation exposure results in direct and indirect damage to many different molecules in cells, and radiation-induced DNA damage is believed to be the cause of cell death. Mammalian cells respond to radiation-induced DNA damage through specific, inducible pathways that are regulated through poorly understood mechanisms that result in the activation and expression of specific genes. Some effects of radiation are mediated through signaling pathways composed of sequence-specific DNA binding proteins that are involved in the regulation of responsive genes. In our preliminary studies we have investigated the role of the inducible transcription factor NF-(kappa)B in mediating the expression of a reporter gene directed by a promoter that is induced by radiation damage: the HIV1 LTR. The objective of this proposal is to further define the role of reactive oxygen intermediates (ROIs), DNA damage, and NF-(kappa)B DNA binding in the regulation of radiation-induced gene expression. We have demonstrated that UV irradiation of mouse L cells activated NF-(kappa)B DNA binding and reporter gene expression. In contrast, treatment with ionizing radiation failed to activate reporter gene expression.' Agents such as tumor necrosis factor (TNF) and phorbol ester (PMA), which generate ROIs, activated NF-(kappa)B DNA binding without altering reporter gene expression, suggesting that NF-(kappa)B was required but was not sufficient to activate gene expression in this cell line. A strategy of using cell fusion to construct heterokaryons was devised to functionally test for the presence of additional factors. Heterokaryons formed by fusing UV-irradiated human cells with mouse L cells containing an integrated HIV1 LTR-directed lacZ gene directly demonstrated that UV irradiation activated a trans-acting pathway that can pass from a UV-irradiated nucleus and act on a target gene in a noniiradiated nucleus. DNA damage or distortion in the DNA conformation around the inducible gene is unlikely to be involved, because the reporter gene was never exposed to UV irradiation. This system will be used to directly test the role of UV- and ionizing radiation-damaged DNA in the signaling pathway in the absence of exposing cells to radiation. To further explore the relationship between NF-(kappa)B-mediated gene regulation and the response to ionizing radiation, we have characterized a human cell line that is strongly induced by agents that generate ROIs. Microcell-mediated human chromosome transfer will be used to evaluate the existence of human factors that complement the defect in gene expression in mouse L cells. The information derived from this investigation will increase our understanding of cellular mechanisms involved in radiation-induced gene expression.
