A selective inhibitor of cell proliferation from normal serum.

Abstract

A factor in normal serum that selectively and reversibly inhibits proliferation of cells in culture has been enriched 160-fold from calf serum by sequential ammonium sulfate precipitation, gel filtration, and lectin-affinity chromatography. DNA synthesis of normal (but not transformed) rat hepatocytes, human lymphoblast lines, and mitogen-stimulated murine spleen cells is inhibited by >90%, and Vero, murine myeloma, MELC, and a human colon carcinoma cell line to a lesser extent. Growth of other cell lines tested was not affected.Responsive cells are arrested apparently in GI by this inhibitor, the effect of which is maximal by 24 hr and is spontaneously reversible thereafter unless it is renewed. The active fraction is a protein that migrates with the a2-globulins; it is not a lipoprotein, and it is of high apparent molecular weight.Negative signals inhibiting proliferation may, in balance with mitogens, control the growth of at least some cell types (1). Some humoral inhibitors of proliferation have been designated (2-6), but they remain less well characterized than the hormonal growth factors of serum (7)(8)(9)(10)(11)(12). Owing to its content of these mitogens, increasing amounts of serum enhance the rate of proliferation of most cell types in a concentration-dependent manner. In contrast, we found that the proliferation of normal (i.e., nontransformed) hepatocytes in primary culture or in established lines was depressed in proportion as the concentration of serum in the medium was increased above a minimal 2%. This negative effect on growth was found to be due to the presence of an active inhibitory principle in the a2-globulin fraction of normal sera (13) that can also suppress the proliferation of certain other cell types. In this report we describe the occurrence and some characteristics of this serum inhibitor, its partial purification from calf serum, and its effects on growth or mitogenesis of various cell types. The selectivity, reversibility, nontoxicity, and kinetics of growth inhibition by this serum component suggest that it may be of physiologic signifi- Assay of Cell Proliferation and Inhibition of Growth. Assays of inhibitor activity were performed at a concentration of serum (5%) that permitted logarithmic growth without, however, overcoming possible negative effects on growth of putative inhibitory fractions. In the assay system used, a known amount of test fraction in phosphate-buffered saline (pH 7.2) was included in medium containing 5% FCS, to a final concentration of 10% (vol/vol). Control medium was identical but contained 10% phosphate-buffered saline alone. At intervals, as indicated, cells were counted or, more usually, [3Hjthymidine (New England Nuclear, NET 027E, 20 Ci/mol) was added in a final concentration of 1 ,uCi/ml (1 Ci = 3.7 X 1010 becquerels). After 1 or 2 hr, cultures were rinsed with Hanks' balanced salt solution and the label was chased with medium containing excess nonradioactive thymidine. After the cells were harvested and centrifuged, the pellets were disso...

Enhancement of human osteoblast proliferation and phenotypic expression when cultured in human serum

“…Serum contains proteins, polypeptides, hormones, metabolites, nutrients and minerals. In addition, serum may also contain substances inhibiting cell proliferation (Harrington and Godman 1980) and, for this reason, serum is heat-treated, a process that removes complement from the serum and reduces the cytotoxic action of immunoglobulins without damaging polypeptide growth factors. This may explain why heat-treated AHS produced a greater growth response than untreated AHS.…”

Inhibitory Effects of Fetal Calf Serum on Proliferation of Chick Neuroblasts in Culture

“…We have partially purified (around 1,000-fold) an inhibitory factor of neuroblast proliferation from FCS. Other au thors [6] have identified an inhibitory factor from calf serum which inhibits proliferation of several cell types in culture. This factor was only purified by ammonium sulfate pre cipitation and gel filtration: its molecular weight was found to be about 200,000 from its position in the elution profile from Ultrogel AcA 34.…”

“…On the other hand, sera also contain inhibitors of growth [9]. Recently, Harrington and Goldman [6] showed that proliferative activity of normal cells was reduced as the concentration of se rum in the medium was increased. These au thors have identified an inhibitory serum fraction that suppresses the multiplication of certain types of cells, e.g.…”

Identification of Inhibitory Growth Factors from a N-[4-(5-Nitro-2-furyl)-2-thiazolyl]formamide-Induced Murine Bladder Cancer Cell Line

“…McMahon et al [29] have purified an inhibitor of cell proliferation from rat liver, which inhibits only normal rat liver cells but not rat liver tumor cells. Other growth inhibitors have been detected in non-neural cells [30], in calf serum [31], in human plasma-derived serum [32], in rat ascites [33], and in NIH/3T3 cells. Recently in human platelets distinct from the platelet-derived TGF-(3, an EGI was demonstrated, which significantly inhibited the growth of nonmalignant cell lines in monolayer cultures [34].…”