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Isolation and initial characterization of tumoricidal monokine(s) from the human monocytic leukemia cell line thp-11, 2

  • Author(s): Armstrong, CA
  • Klostergaard, J
  • Granger, GA
  • et al.
Abstract

A cloned subline of the human monocytic leukemia cell line, THP-1, was induced to produce high levels of cytotoxic activity following an 18-hour phorbol myristate acetate (CAS: 16561-29-8) stimulation in vitro. This activity, termed monocyte cell line cytotoxin(s) (MCCT), was tested in vitro on different human continuous cell lines (Chang, ESH-7, GM3104A, HeLa, HT-1080, K562, Mel, T-24) and on primary human fibroblasts (GM3468, Manz). The continuous cell lines exhibited a spectrum of sensitivity to MCCT-containing supernatants whereas the primary fibroblasts were resistant to lysis. Enzymatic degradation and heat denaturation studies indicate that MCCT is a protein. Its bioactivity can be resolved into three lytic peaks after molecular sieving on Ultrogel AcA 44. The major peak, designated ±MCCT, eluted with a molecular weight of 100, 000140, 000 daltons. A minor peak, MCCT, was seen at 60, 00080, 000 daltons, and a third, unstable minor peak, MCCT, eluted at less than 10, 000 daltons. The peak was examined further and was found to migrate as a single peak in 7% native polyacrylamide gel electrolysis tube gels with an rfof 0.250.30. None of the MCCT forms were immunologically cross-reactive with human. Various protease inhibitors known to inhibit monokine- and macrophage-mediated direct cell lysis in vitro were tested for their inhibitory effects on ±MCCT activity. The irreversible binding inhibitor-L-lysyl chloromethyl ketone inhibited the biologic activity of ±MCCT. The reversible binding inhibitors N-L-arginine methyl ester and soybean trypsin inhibitor were able to block in vitro lytic activity when added to ±MCCT in the presence of the target cell. In contrast, the inhibitors phenylmethylsulfonyl fluoride,L-1-tosylamide, 2-phenylethyl chloromethyl ketone, andL-lysine methyl ester were not effective in blocking cytolysis. Finally, the hydrogen peroxide scavenger catalase inhibited MCCT lytic activity in vitro; however, the hypochlorous acid scavengers alanine, serine, and valine were without effect. © 1985 Oxfor University Press. All Rights Reserved.

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