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Regulation of NF-κB and HIV-1 LTR Activity in Mouse L Cells by Ultraviolet Radiation: LTRtrans-Activation in a Nonirradiated Genome in Heterokaryons

1997; Elsevier BV; Volume: 230; Issue: 1 Linguagem: Inglês

10.1006/excr.1996.3397

1090-2422

Steven C. Miller, Alan C. Taylor, Kenji Watanabe, Kenny Mok, Frank M. Torti,

RNA Research and Splicing

A mouse model system for studying the effect of ultraviolet (uv) radiation on reporter gene expression directed by the human immunodeficiency virus type 1 long-terminal repeat (HIV-LTR) has been developed to address the signals required for LTRtrans-activation in cells with the reporter gene stably integrated into the genome. In a stable mouse L cell clone, L-15, NF-κB DNA binding activity induced by uv-C (254 nm) but not by tumor necrosis factor-α (TNF-α) or 12-O-tetradecanoylphorbol-13-acetate (TPA) correlated with the stimulation of HIV-LTR-directed chloramphenicol acetyltransferase (CAT) activity; uv-C was more effective than uv-B (312 nm), while uv-A (365 nm) had little effect on CAT activity. Inducers of oxidative stress, such as H2O2treatment up to 200 μMor ionizing radiation up to 20 Gy, also had little effect on CAT expression. Pyrrolidine dithiocarbamate (PDTC) inhibited NF-κB DNA binding and stimulation of CAT activity by uv-C in a dose-dependent manner. Unexpectedly, PDTC induced NF-κB DNA binding that was additive with the response with TNF. In an effort to separate uv irradiation and uv-induced DNA damage fromtrans-activation of the HIV-LTR we devised a heterokaryon system. The fusion of uv-irradiated human fibroblasts with a mouse L cell clone containing the HIV-LTR-directedlacZgene resulted in the activation of lacZ activity that was detected in heterokaryons at the single-cell level. These data suggest that uv-induced DNA damage in the chromosomal DNA containing the reporter gene cannot explain activation of the HIV-LTR. This finding demonstrates LTRtrans-activation in a nonirradiated genome.