Identification of a thymic inhibitor (‘chalone’) of lymphocyte transformation as a spermine complex

Allen, John C.; Smith, Christopher J.; Curry, Merril; Gaugas, J. M.

doi:10.1038/267623a0

Cited by 62 publications

(14 citation statements)

References 10 publications

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“…The system has been implicated in immunoregulation (Byrd et al, 1977;Allen et al, 1977;Gaugas & Curzen, 1978). The phenomena of non-cytotoxicity, G1-phase arrest of cell proliferation and DNA replication, as well as binding of dioxidized spermine to a serum component, provides circumstantial evidence in support of some physiological or pathological role.…”

Section: Discussionmentioning

confidence: 99%

Evidence for serum binding of oxidized spermine and its potent G1-phase inhibition of cell proliferation

Gaugas¹,

Dewey²

1979

Br J Cancer

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Summary.-Serum polyamine oxidase (EC 1.4.3.4) is known to react in vitro with radio-labelled spermine4+ to produce di-oxidized spermine which must incorporate the label. Di-oxidized spermine was compatible with a radio-labelled compound2+ separated from the reaction mixture by ion-exchange chromatography. The compound was measured and had a half-life of about 2-3 h in tissue culture medium. It also rapidly and tightly bound to an unidentified serum component (gel-filtration chromatography indicated a complex of mol. wt 70,000) so that dissociation required treatment with strong acid (ION HCI). Findings suggest that the di-oxidized spermine, in either its free cationic or bound form, potently arrested cell proliferation. This arrest was non-cytotoxic and was confined to the G1 phase of the cell cycle. Products of di-oxidized spermine autodegradation, including trace amounts of stable and cytotoxic acrolein (arrested S phase), were unlikely to have contributed significantly to the arrest.

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Section: Discussionmentioning

confidence: 99%

Evidence for serum binding of oxidized spermine and its potent G1-phase inhibition of cell proliferation

Gaugas¹,

Dewey²

1979

Br J Cancer

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“…Attempts to test whether the inhibitory substances were labile to acid hydrolysis (6 M HC1 at 1100) were not successful because such treatment caused the generation of toxic products in chromatographic preparations even from unconditioned medium. In control experiments, glutathione and ergothioneine were ruled out as possible growth inhibitors, as was spermine, a substance reported to inhibit lymphocyte mitogenesis (9).…”

Section: Resultsmentioning

confidence: 99%

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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“…Our inhibitor is formed and is active when BSC-1 cells are grown in human serum, as well as in calf serum. Human serum is low in plasma amine oxidase activity, and as a result the spermine complex has little inhibitory activity in human serum (13). The BSC-1 inhibitor has very different properties than the inhibitor of in vitro protein synthesis obtained from frozen extracts of Vero M3 cells by Engelhardt (14).…”

Section: Discussionmentioning

confidence: 99%

“…We have excluded the possibility that the substance is a spermine complex, the major inhibitor in a "lymphocyte chalone" preparation (13). Our inhibitor is formed and is active when BSC-1 cells are grown in human serum, as well as in calf serum.…”

Section: Discussionmentioning

confidence: 99%

Density-dependent regulation of growth of BSC-1 cells in cell culture: growth inhibitors formed by the cells.

Holley¹,

Armour²,

Baldwin³

1978

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

103

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Inhibitors formed by a monkey epithelial cell line, BSC-1, play an important role in limiting growth at high cell densities. At least three inhibitors are formed: lactic acid, ammonia, and an unidentified inhibitor that may be an unstable protein. The unidentified inhibitor is destroyed by shaking the conditioned medium, by bubbling gas through the medium, or by heating or storing the medium in the absence of cells. The concentrations of lactic acid and ammonia that accumulate in conditioned medium inhibit growth when added to fresh medium. These results, together with earlier studies, indicate that density-dependent regulation of growth of BSC-1 cells results from the combined effects of (a) inhibitors formed by the cells, (b) decreased availability of receptor sites for serum growth factors as the cells become crowded, and (c) limiting concentrations of low molecular weight nutrients in the medium. In contrast, density-dependent regulation of growth in 3T3 mouse embryo fibroblasts results almost entirely from inactivation of serum factors.Evidence in the literature suggests that there are differences between the growth controls of epithelial cells and fibroblasts in cell culture (1-4). Due to these differences and to the importance of epithelial cells in the origin of tumors, we have made a thorough study of the factors that control the growth of BSC-1 cells, an epithelial cell line of African green monkey kidney origin (5). In previous papers (4, 6) we demonstrated that (a) serum factors and (b) the concentrations of low molecular weight nutrients in the medium contribute to growth regulation of this cell line. The present paper describes the regulation of growth of BSC-1 cells by the formation of inhibitory materials in the culture medium.The initial evidence for the presence of inhibitors was a slight but consistent stimulation of the labeling index in crowded cells whenever conditioned medium with 0.1% serum was replaced by fresh medium without serum. Although the labeling index along the edge of a "wound" in the cell layer often decreased after this treatment, there was a consistent increase of the labeling index in the crowded cell layer. The-increase could not be attributed to fresh serum factors, for serum was not added.Nor could it be due to an increase in the concentrations of nutrients in the fresh medium: the concentrations of nutrients in conditioned medium were found to be at least half those in fresh medium; changing the cultures to fresh serum-free medium with only half the normal concentrations of the nutrients still stimulated DNA synthesis in the cell layer.Tests of known metabolites identified lactate and ammonium ions as significant growth inhibitors in BSC-1 cell cultures. However, the inhibition observed when these ions were added to fresh medium, at the concentrations found in conditioned medium, was not adequate to explain the above results.Evidence that the cells produce an additional, unidentified inhibitor was obtained initially from experiments in which conditioned medium w...

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Identification of a thymic inhibitor (‘chalone’) of lymphocyte transformation as a spermine complex

Cited by 62 publications

References 10 publications

Evidence for serum binding of oxidized spermine and its potent G1-phase inhibition of cell proliferation

Evidence for serum binding of oxidized spermine and its potent G1-phase inhibition of cell proliferation

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

Density-dependent regulation of growth of BSC-1 cells in cell culture: growth inhibitors formed by the cells.

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