Regulation of NF-kappaB and HIV-1 LTR activity in mouse L cells by ultraviolet radiation: LTR trans-activation in a nonirradiated genome in heterokaryons.
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 LTR trans-activation in cells with the reporter gene stably integrated into the genome. In a stable mouse L cell clone, L-15, NF-kappaB DNA binding activity induced by uv-C (254 nm) but not by tumor necrosis factor-alpha (TNF-alpha) or 12-O-tetra-decanoylphorbol-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 H2O2 treatment up to 200 microM or ionizing radiation up to 20 Gy, also had little effect on CAT expression. Pyrrolidine dithiocarbamate (PDTC) inhibited NF-kappaB DNA binding and stimulation of CAT activity by uv-C in a dose-dependent manner. Unexpectedly, PDTC induced NF-kappaB DNA binding that was additive with the response with TNF. In an effort to separate uv irradiation and uv-induced DNA damage from transactivation 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-directed lacZ gene 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 LTR trans-activation in a nonirradiated genome.
Miller
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SC
Signal Transduction Laboratory, Science and Technology Group, SRI International, Menlo Park, California 94025, USA. smiller@unix.sri.com
Taylor
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A
Watanabe
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Mok
K
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Torti
F M
FM
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002459:Cell Fusion; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage; D004307:Dose-Response Relationship, Radiation; D017930:Genes, Reporter / Q000187:drug effects / Q000528:radiation effects; D016325:HIV Long Terminal Repeat / Q000528:radiation effects*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007739:L Cells; D007763:Lac Operon / Q000528:radiation effects; D051379:Mice; D016328:NF-kappa B / Q000235:genetics*; D018384:Oxidative Stress; D011759:Pyrrolidines / Q000494:pharmacology; D013859:Thiocarbamates / Q000494:pharmacology; D015533:Transcriptional Activation / Q000187:drug effects / Q000528:radiation effects; D014162:Transfection; D014466:Ultraviolet Rays
D000975:Antioxidants; D016328:NF-kappa B; D011759:Pyrrolidines; D013859:Thiocarbamates; C020972:pyrrolidine dithiocarbamic acid; D004247:DNA; D006861:Hydrogen Peroxide; D015500:Chloramphenicol O-Acetyltransferase