Purification of a cytotoxic factor from human and rat tissues

Op, Bormer

doi:10.1038/bjc.1976.178

Cited by 3 publications

(1 citation statement)

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“…These factors appear for the most part to be macromolecules (particularly glycoproteins) and very few of small molecular weight have been described. A protease-degradable inhibitor, of molecular weight 1000-5000, has been reported to be produced by a mastocytoma (13) and a cytotoxic amine of molecular weight 700 has been obtained from various animal tissues (14). The autoinhibitors now reported appear to be structurally distinct from any of these substances.…”

Section: Discussionmentioning

confidence: 98%

Production of autoinhibitory factors by mouse lymphoma cells in vitro and its relationship to thiol dependence.

Tanapat¹,

Gaetjens²,

Broome³

1978

Proc. Natl. Acad. Sci. U.S.A.

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Five different kinds of mouse lymphoma cells conditioned culture medium so that it became inhibitory to thiol-dependent cells but not to thiol-independent cells. Conditioning was rapid and appeared to be complete in 1-3 hr. The inhibitory effects could be reversed by concentrations of 2-mercaptoethanol higher than those usually required for growth promotion. Inhibitory factors were concentrated by ultrafiltration and chromatography on Sephadex G-10 which showed their apparent molecular weights to be 300-600. The factors were stable at 1000 and to proteases but were inactivated (after reduction) by iodoacetamide, indicating that they may be thiols or disulfides.Specific thiols and their corresponding disulfides promote the survival and replication in vitro of mouse lymphoid cells, a property which is retained in numerous lymphoma lines (1-4). Such substances, however, are either nonphysiological (particularly 2-mercaptoethanol which has been widely used in experimental systems) or are required at concentrations much greater than occur in vivo. On the other hand, thiol is not required by lymphoma cells when they are cocultivated with macrophages or other cells but, after separation, the supernatant medium does not continue to support growth (5, 6). The mechanism of these effects and their relationship to conditions in vio thus far have not been explained.We here present evidence that thiol-dependent lymphoma cells in vitro are subject to autoinhibition mediated through substances that they liberate into the medium. These have been partially characterized and appear to be thiols or disulfides. Growth promoting thiols (e.g., 2-mercaptoethanol) or cocultivated macrophages interrupt the autoinhibitory system, and these actions may therefore be significant for restoring cell proliferation. The experiments to be described were prompted by observations on the growth promoting effect of macrophages in cocultivation with lymphoma cells (6). Numerous attempts failed to show evidence that macrophages produced a stable conditioning of the medium, and an alternative explanation, that these cells might reverse an autoinhibition by L1210(V) cells, was therefore considered.In most of our experiments we used cells of the thiol-dependent lymphoma L1210(V), whose properties we compared with those of its thiol-independent variant L1210(A) (7). Three other cell lines were examined-EL4, RADA1, and ERLDand in these the same relationships between autoinhibition and thiol dependence were found. MATERIALS AND METHODSTumors. B6D2F1 mice were used to carry the following tumors in ascitic form: L1210(V), L1210(A), and EL4. ERLD (i) Supernatants were concentrated up to 20 times by using a UM05 or other ultrafiltration membrane (Amicon Co., Lexington, MA). Aliquots from these concentrated solutions were tested for their ability to support growth of the various cell lines in tissue culture.(ii) Supernatants were lyophilized to dryness and the residues were dissolved in water and chromatographed on Sephadex G-10.Measurement of Lymphoma Ce...

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Section: Discussionmentioning

confidence: 98%

Production of autoinhibitory factors by mouse lymphoma cells in vitro and its relationship to thiol dependence.

Tanapat¹,

Gaetjens²,

Broome³

1978

Proc. Natl. Acad. Sci. U.S.A.

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Inhibition of lymphocyte mitogenesis by factor(s) released from macrophages isolated from ascitic fluid of advanced ovarian cancer patients

Sheid

Boyce

1984

Cancer Immunol Immunother

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Ascitic fluid from women with advanced ovarian carcinomas was shown to contain factor(s) which inhibit(s) T lymphocyte mitogenesis. The factor(s) was (were) demonstrated to be associated with the infiltrating macrophages. The inhibition was reversible and inhibited mitogenesis at some late event in the cell cycle. The inhibitory substance(s) was (were) noncytotoxic, dialyzable, heat-stable at 70 degrees C for 10 min (but unstable at 100 degrees C for 15 min), and partially resistant to protease treatment (55%-70%). Further experiments demonstrated that macrophages isolated from the ascitic fluid of patients with cirrhosis of the liver also released factor(s) which inhibit(s) T lymphocyte mitogenesis. On the basis of our data and data from other investigators, we propose that in advanced human ovarian cancer of epithelial origin, macrophages which infiltrate the ascitic fluid elaborate nonspecific inhibitors of T lymphocyte blastogenesis within the proximal environment, resulting in localized immunosuppression and the subsequent enhancement of metastasis within the peritoneal cavity, the tumor cells themselves being resistant to the cytocidal action of the macrophages due to genetic selection and/or their inherent biochemical ability to circumvent normal immunosurveillance mechanisms. This may account, at least in part, for the rapid metastasis and poor prognosis of human ovarian adenocarcinomas.

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