       Document 0855
 DOCN  M9620855
 TI    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.
 DT    9602
 AU    Sweet MJ; Hume DA; Centre for Molecular and Cellular Biology, University
       of; Queensland, Brisbane, Australia.
 SO    J Inflamm. 1995;45(2):126-35. Unique Identifier : AIDSLINE MED/96050094
 AB    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.
 DE    Animal  Cell Line  Gene Expression  Genes, Reporter  Human  HIV Long
       Terminal Repeat/*GENETICS  HIV-1/*GENETICS
       Lipopolysaccharides/*PHARMACOLOGY  Luciferase/GENETICS/METABOLISM
       Macrophages/*METABOLISM  Mice  Paclitaxel/*PHARMACOLOGY  Protein Kinase
       C/METABOLISM  Signal Transduction  Tetradecanoylphorbol
       Acetate/PHARMACOLOGY  Transfection  Tumor Necrosis Factor/*PHARMACOLOGY
       JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

