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L929-derived hybridoma growth factor
nonreceptor-type protein-tyrosine phosphatase 10
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This murine aneuploid fibrosarcoma cell line is used to assay TNF-alpha and TNF-beta (see also: bioassays, cytokine assays), which lyse cells sensitized by pretreatment with actinomycin or cycloheximide. Treatment with TNF initiates apoptosis and subsequent cell death. The level of detection is on the order of 50 pg/mL. Another cell line which is approximately 5-10-fold more sensitive is WEHI-164 (see: WEHI-3B). Some L929 subclones have been developed that allow detection of TNF at concentrations of approximately 30 pmol/L (approximately 500 fg/mL). Sublines that are resistant to TNF have been established also. An alternative and entirely different detection method is RT-PCR quantitation of cytokines. Another cell line with higher sensitivity than L929 is PK15.
L929 cells are greater than 10 times more sensitive to TNF mediated cytotoxicity in the presence of lithium chloride, which induces TNF-alpha secretion. In nude mice (see also: Immunodeficient mice), a combination of TNF and LiCl leads to hemorrhagic necrosis and growth inhibition of L929 tumors, whereas little effect is observed when TNF is administered alone.
TNF-alpha and TNF-beta can be distinguished from each other by using suitable neutralizing antibodies. The L929 cytotoxicity assay can be influenced considerably by heat-inactivated human serum (56 ūC, 30 min) which is also cytotoxic and may lead to a detachment of cells.
Extracellular ATP and adenosine, but not AMP or GTP in concentrations of 0.5 to 2.5 mM, inhibit TNF induced cytolysis of L929 cells in the presence of actinomycin D when present throughout the entire assay period.
Treatment of the cells with TNF, IL1, poly(I)-poly(C), IFN-beta, Calcium ionophore A23187, or phorbol 12-myristate 13-acetate (see also: Phorbol esters), but not with EGF, dibutyryl-cAMP, or the adenylate cyclase activator forskolin induces interferon regulatory factor-1 (see: IRS, interferon response sequence). Treatment of the cells with TNF or IL1 also induces the synthesis of IFN-beta.
TNF-alpha stimulates the release of arachidonic acid from these cells. The addition of hydrocortisone or nordihydroguaiaretic acid (NDGA) decreases the cytotoxic effect of TNF-alpha but exogenously added arachidonate or linoleate, indomethacin and eicosatetraynoic acid (ETYA) are without effect. Suppression of arachidonate metabolism by steroids effectively inhibits TNF mediated cytolysis of L929 cells. An increase in intracellular cAMP levels (obtained by adding cell-permeable dibutyryl cAMP to the culture medium) or culture of L929 cells in the presence of reagents elevating intracellular cAMP concentrations.
Recombinant human TNF-alpha (see also: Recombinant cytokines) alone has no effect on L929 tumor cells at 100 units/mL for 20 hours of continuous exposure. However, under the same conditions, rHTNF markedly enhances the cytotoxicity of Adriamycin, actinomycin D, 4'-(9-acridinylamino)-methanesulfon-m-anisidide, teniposide (VM 26), and etoposide (VP 16), all targeted at DNA topoisomerase II. VM-26 can lower the TNF LD50 to femtomolar levels. The topoisomerase II inhibitors novobiocin and coumermycin, which bind to the enzyme ATPase site, protect L929 cells from TNF cytotoxicity but enhance TNF cytotoxicity in human ME-180 cells.
L929 cells are severely deficient in gap junctional communication and known cell-cell adhesion molecules. L929 cells secrete into the conditioned medium a neurotrophic factor that is related closely to the beta subunit of NGF. Other NGF subunits are not produced. L929 also secrete a factor that promotes the differentiation of porcine peripheral blood mononuclear cells into macrophages and monocytes. This factor is identical with murine M-CSF. Conditioned medium of L929 cells is used frequently, therefore, as a crude source of murine M-CSF. L929 cells also secrete a chemotactic factor specific for monocytes, which is identical with murine JE (see also: Chemotaxis). The cells also secrete an uncharacterized factor (see: THP-1-derived growth-promoting activity) that promotes the growth of a large variety of cell lines.
L929 cells proliferate in response to a variety of growth factors, including IL2, IL3, G-CSF, GM-CSF, and M-CSF.
For an overview of other cell lines used in research on cytokines see: Cell lines in Cytokine Research. For other related/relevant entries see also: Cell types.
Copyright © 2012 by H IBELGAUFTS. All rights reserved.
ENTRY LAST MODIFIED: January 2002
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