RAW264 macrophages stably transfected with an HIV-1 LTR reporter gene provide a sensitive bioassay for analysis of signalling pathways in macrophages stimulated with lipopolysaccharide, TNF-alpha or taxol.
Bacterial lipopolysaccharide (LPS) modulates expression of a variety of genes in macrophages, and additionally activates viral promoters including the HIV-1 LTR. The HIV-1 LTR driving the luciferase reporter gene was stably transfected into the murine macrophage cell line, RAW264. In stably transfected cells, luciferase activity was LPS-dependent. As little as 0.01 ng/ml LPS was sufficient to increase luciferase activity over basal levels with maximal stimulation resulting in a 10- to 20-fold response. The cells also responded to human and murine tumour necrosis factor (TNF alpha). Endogenous TNF alpha was not involved in LPS responses, since pretreatment with alpha-TNF alpha antibody did not affect activation. Induction of HIV-1 LTR activity by LPS occurred independently of phorbol myristate acetate (PMA) sensitive protein kinase C (PKC), since depletion of PKC by prolonged exposure to PMA blocked TNF alpha and PMA responses but was not able to abolish LPS action on these cells. Taxol (5-20 micrograms/ml), a chemotherapeutic agent which mimics LPS action on macrophages, was also able to increase expression of the reporter gene driven by the HIV-1 LTR. However, lower doses of taxol that were not sufficient to trans-activate the LTR or to induce TNF alpha expression were cytotoxic to RAW264 cells suggesting that the cytotoxic and LPS-like activities of taxol were not linked. This cell line provides a convenient method for detecting LPS-like activity and is a useful tool for examining LPS and TNF alpha signalling pathways.
Sweet
M J
MJ
Centre for Molecular and Cellular Biology, University of Queensland, Brisbane, Australia.
Hume
D A
DA
D000818:Animals; D002460:Cell Line; D015870:Gene Expression; D017930:Genes, Reporter; D016325:HIV Long Terminal Repeat / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism*; D051379:Mice; D017239:Paclitaxel / Q000494:pharmacology*; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D008070:Lipopolysaccharides; D014409:Tumor Necrosis Factor-alpha; D008156:Luciferases; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D017239:Paclitaxel