Cultured 3T3 Fibroblasts Research Articles

Endogenous lectins from cultured cells: subcellular localization of carbohydrate-binding protein 35 in 3T3 fibroblasts.

In previous studies, a lectin designated as carbohydrate-binding protein 35 (CBP35) has been isolated from cultured 3T3 fibroblasts. In the present study, rabbit antibodies directed against CBP35 were used to analyze the subcellular distribution of CBP35 in 3T3 cells. Several lines of evidence indicate that CBP35 is found externally exposed at the cell surface: immunofluorescent staining of live 3T3 cells; agglutination of suspension of 3T3 fibroblasts by specific antibodies; and isolation, by immunoaffinity chromatography, of a Mr 35,000 component from cells surface-labeled with 125I. In addition to the plasma membrane, CBP35 could also be found intracellularly, as revealed by immunofluorescence studies of fixed and permeabilized 3T3 cells. The staining pattern showed the presence of CBP35 on the nucleus and in the cytoplasm. These results are consistent with the finding that among several subcellular fractions, CBP35 can be found by immunoblotting procedures in the nuclear pellet, the soluble fraction, and the plasma membrane fraction of the postnuclear supernatant.

Mitogenic Factors in Prostatic Tissue and Expressed Prostatic Secretion

Expressed prostatic secretions and extracts of benign prostatic hyperplasia tissue contain a polypeptide growth factor(s) that stimulates the uptake of tritium-labeled thymidine by cultured 3T3 fibroblasts. Mitogenic activity was present in expressed prostatic secretions and extracts of benign prostatic hyperplasia tissue. The apparent molecular weights of the mitogenic fractions were estimated to be 300,000, 150,000 and 60,000 daltons for prostatic tissue extracts, and 30,000 daltons for expressed prostatic secretions. Bioassays yielded a mean of 27 units of mitogenic activity per mg. protein in expressed prostatic secretions obtained from men with normal and enlarged prostate glands. There was no difference in bioassayable mitogenic activity in the expressed prostatic secretions from normal and benign prostatic hyperplasia samples but gel filtration studies revealed a high molecular weight component present only in samples from men with prostatic enlargement. A dialyzable low molecular weight inhibitor of fibroblast growth was found in the prostatic tissues and expressed prostatic secretions. We report the characterization studies and discuss the possible roles of growth factors in the pathogenesis of benign prostatic hyperplasia.

Growth control in cultured 3T3 fibroblasts: neutralization and identification of a growth-inhibitory factor by a monoclonal antibody.

A fibroblast growth regulator, which inhibits the growth and division of proliferating fibroblasts, has been isolated from medium conditioned by exposure to density-inhibited mouse 3T3 cells. This partially purified preparation of the growth-inhibitory activity, termed FGR-s, contained two major polypeptides (Mrs, 10,000 and 13,000). Using FGR-s as the immunogen, we have carried out in vitro immunization of rat splenocytes and have generated hybridoma lines, each secreting an antibody directed against components of the FGR-s preparation. One such monoclonal antibody, designated 2A4, specifically bound the Mr 13,000 polypeptide of FGR-s. Antibody 2A4 also neutralized the growth-inhibitory effect of FGR-s in a concentration-dependent fashion. These results strongly suggest that the Mr 13,000 polypeptide carries growth-inhibitory activity.

Regulation of phosphatidylcholine biosynthesis by mitogenic growth factors

Phosphatidylcholine (PC) biosynthesis in cultured 3T3 fibroblasts was increased in varying degrees by these mitogenic growth factors: fetal bovine serum, insulin, 12- O-tetradecanoylphorbol-13-acetate, epidermal growth factor, vasopressin, fibroblast growth factor and insulin-like growth factors I and II. PC synthesis was increased 2–4-fold by 10% serum, up to 4-fold by growth factors alone, and up to 8-fold by combinations of two or more growth factors. Single growth factors had no effect on the incorporation of [ 3H]choline into the acid-soluble precursors of PC, while serum or combinations of two or more mitogens could increase the incorporation of [ Hcholine into acid-soluble material by up to 2-fold. Serum was shown to increase choline phosphorylation, choline kinase activity and the size of the phosphocholine pool. These data were utilized to calculate the radioactive specific activity of phosphocholine. Serum did not increase phosphocholine specific activity above control values; thus the increased incorporation of labelled choline into PC after serum stimulation resulted from increased PC synthesis and not from a simple change in specific activity of precursor phosphocholine.

Growth regulation of a cellular tumour antigen, p53, in nontransformed cells.

Many transformed cells in culture have been found to express elevated levels of a cellular tumour antigen, termed p53. This protein has also been implicated in the regulation of cellular growth. For these reasons experiments were designed to examine the expression of p53 as quiescent cultures of nontransformed 3T3 fibroblasts were stimulated to reenter the cell cycle. Synchronous populations of cells were obtained by releasing a culture from density-dependent inhibition of growth with the addition of fresh serum. Steady-state levels of p53 protein and mRNA were measured as a function of time after addition of serum to quiescent cultures and the rate of synthesis of p53 protein was analysed at a number of time points. The results, reported here, demonstrate an increase in the synthesis and steady-state levels of p53 protein and mRNA prior to DNA synthesis in late G1, and suggest a role for p53 in the progression of cells from a growth-arrested state to an actively dividing state.

Endogenous lectins from cultured cells. Isolation and characterization of carbohydrate-binding proteins from 3T3 fibroblasts.

Extracts of cultured 3T3 fibroblasts, obtained by homogenization and Triton X-100 solubilization, were fractionated on Sepharose columns covalently derivatized with asialofetuin. Three distinct carbohydrate-binding proteins (CBPs) were purified from the material bound to the affinity column: CBP35 (Mr = 35,000), CBP16 (Mr = 16,000), and CBP13.5 (Mr = 13,500). These CBPs were similar in several key properties. (a) They showed agglutination activity when assayed with rabbit erythrocytes; (b) they all appear to specifically recognize galactose-containing glycoconjugates; (c) they have low isoelectric points, pI 4.5-4.7; (d) their binding activities are rapidly lost in the absence of beta-mercaptoethanol; (e) the CBPs do not interact with each other, and the fractionated proteins can bind to asialofetuin independent of associated polypeptides; and (f) none of the proteins tend to self-associate to form oligomers of identical subunits. Comparisons of these and other properties of the CBPs suggest that CBP16 and CBP13.5 may be the murine counterparts of lactose-specific lectins previously identified in electric eel and in several bovine and avian tissues. In contrast, it appears that CBP35 represents a newly identified protein capable of binding to galactose-containing carbohydrates.

フィブロブラストによるエイコサペンタエン酸由来のプロスタサイクリン様物質の生成

There has been a discrepancy in several investigators whether prostacyclin-like substance (PGI3) might be produced from eicosapentaenoic acid (EPA) in the vessel wall. Production of PGI3 from EPA in 3T3 fibroblasts, which had an ability to produce prostacyclin (PGI2) from arachidonic acid (AA), was studied. After various times of incubation with EPA and 3T3 fibroblasts, aliquots of the culture media were transferred to platelet-rich plasma, to which ADP was added to induce aggregation. Inhibition of platelet aggregation was observed with samples removed after short minutes incubation, but not from longer periods incubation. The addition of tranylcypromine, an inhibitor of prostacyclin synthetase, to the culture media blocked the formation of a labile inhibitor of platelet aggregation. These data demonstrated sufficient synthesis of PGI3 like substance to account for the inhibition of platelet aggregation. But the inhibitory effiect of platelet aggregation by PGI3 like substance from EPA was weaker than by PGI2 like substance from AA. The formations of PGI3 and PGI2 produced from EPA and AA by the culture of 3T3 fibroblasts were measured as their stable metabolites 17Δ-6-keto-PGF1α and 6-keto-PGF1α respectively by gas chromatography-mass spectrometry. Unexpectedly, the amount of 17Δ-6-keto-PGF1α was higher than that of 6-keto-PGF1α. These observations suggest that PGI3 is inferior to PGI2 in the ability to inhibit platelet aggregation.

Retinoids and phorbol esters alter release of fibronectin from enucleated cells.

Addition of 12-tetradecanoylphorbol 13-acetate (TPA) to cultures of intact Swiss mouse 3T3 fibroblasts induced a dose-dependent increase in ornithine decarboxylase (OrnDCase) activity. Over the same concentration range, 10(-9) to 10(-6) M, TPA induced the release of radioactively labeled fibronectin (FN) from the cells into the culture medium. Retinoic acid, a derivative of vitamin A, inhibited in a dose-dependent manner both the increase in OrnDCase activity and the release of FN induced by TPA. To examine the effects of TPA and retinoic acid in enucleated cells, the cells were treated with 7.5 micrograms of cytochalasin B per ml of medium during centrifugation at 10,000 X g for 35 min at 37 degrees C. In a series of five experiments, the treated cells were 94.7 +/- 4.8% (+/- SEM) enucleated as measured by [3H]thymidine incorporation and verified by Giesma staining for nuclei. In the enucleated cells, TPA did not induce increased OrnDCase activity but did induce FN release in a dose-dependent fashion over the same concentration range effective for FN release from intact cells. Moreover, addition of retinoic acid to the enucleated cells inhibited the phorbol ester-induced release of FN in a dose-dependent manner. A series of phorbol ester derivatives showed the same order of activity in causing FN release from the enucleated cells as reported for inducing OrnDcase activity in intact cells or in promoting mouse skin tumors in vivo. Similarly, several retinoids were tested for their ability to inhibit the phorbol ester-induced release of FN from enucleated cells. The efficacy of the retinoids in preventing FN release paralleled their activity in inhibiting phorbol ester-induced OrnDCase activity and skin tumor promotion, as reported in the literature. We conclude that at least one aspect of tumor promotion induced by phorbol esters--the loss of FN--does not require the cell nucleus, and further, that retinoids can inhibit this aspect of tumor promotion without nuclear involvement.

Growth control in cultured 3T3 fibroblasts: III. Binding interactions of a growth inhibitory activity with target cells

A Fibroblast Growth Regulator (FGR), which inhibits the growth and division of proliferating fibroblasts, has been isolated from medium conditioned by exposure to density-inhibited 3T3 cells. The binding of this FGR to its target cells has been analysed using [ 35S]methionine-labeled inhibitor. The key features of this interaction are ( a) there are approx. 3–4 × 10 5 binding sites/cell; ( b) more ligand is bound at 37 than at 4 °C, possibly due to internalization of the molecule at the higher temperature; ( c) on a per cell basis, approximately the same amount of ligand is bound at high cell density as at low cell density; ( d) there is no evidence of target cell heterogeneity in the binding of the inhibitor; and ( e) the binding can be inhibited partially by calf serum. The results suggest that modulation of cell surface receptor binding by serum and growth inhibitory factors may be an important feature in growth regulation.

Growth control in cultured 3T3 fibroblasts II. Molecular properties of a fraction enriched in growth inhibitory activity.

Treatment of sparse, proliferating cultures of 3T3 cells with medium conditioned by exposure to density-inhibited 3T3 cultures resulted in an inhibition of growth and division in the target cells when compared to similar treatment with unconditioned medium. This growth inhibitory activity was fractionated by ammonium sulfate precipitation and gel filtration, yielding one fraction that was 35-fold enriched in specific activity. Analysis of the chemical and biological properties of this highly active fraction indicated that: (a) it is an endogenous cell product, synthesized by the 3T3 cells and shed into the medium; (b) it is a protein and its activity is sensitive to treatment with pronase; (c) the constituent polypeptide chains have molecular weights of 10,000 and 13,000; and (d) it is not cytotoxic and its effect on target cells are reversible. These results suggest that we have partially purified from conditioned medium an endogenous growth regulatory factor that may play a role in density-dependent inhibition of growth in cultured fibroblasts. We propose the term Fibroblast Growth Regulator to describe this class of molecules.

The effect of bezofibrate and clofibrate on cholesterol accumulation, esterification and removal in cultured 3T3 fibroblasts

3T3 mouse fibroblasts were used to determine the effect of bezafibrate and clofibrate on the cellular metabolism of cholesterol. In cells incubated in normal medium these agents decreased the incorporation of 3H-labelled oleic acid relative to 14C-labelled linoleic acid into the cholesterol ester fraction. When the 3T3 fibroblasts were incubated with cationised low density lipoprotein (LDL) the amount of esterified cholesterol which accumulated in the cells was greatly increased. This accumulation of cholesterol ester was reduced by bezafibrate and clofibrate. These agents decreased the incorporation of both 3H-labelled oleic acid and 14C-labelled linoleic acid into the cholesterol ester fraction of the cells, with a preferential effect on oleic acid as indicated by a reduction in the 3 H 14 C ratio. When cells which had been preincubated with cationised LDL were reincubated in normal medium, the removal of esterified cholesterol from the cells was increased by both bezafibrate and clofibrate. The mechanism of the effects of these agents on the metabolism of cellular cholesterol is discussed.

The role of cytoplasmic microtubules in the regulation of the activity of peptide growth factors

The enhancement of DNA stimulation by peptide growth factors in colchicine-treated cultures of Swiss 3T3 mouse fibroblasts appears to be associated with disassembly of cytoplasmic microtubules. Other antitubulin agents (colcemid, vinblastine, podophyllotoxin, nocodazole, and maytansine) also potentiate the effects of growth promoting factors such as insulin and EGF. The cytoplasmic microtubules are disrupted by antitubulin agents at concentrations very similar to those required for depolymerization of mitotic spindle microtubules. In the presence of highly radioactive [ 3H]thymidine, DNA synthesis can be stimulated in quiescent mouse fibroblasts without completion of the cell cycle. This makes possible studies of the potentiating effects of colchicine during the prereplicative period of the cycle uncomplicated by effects of the antitubulin agent on the mitotic spindle. The enhancing effects of colchicine can be demonstrated with sparse cultures of 3T3 fibroblasts as well as with 3T6 cells. Maytansine and colchicine need not be present during the initial period of interaction of peptides with the cell to produce enhancement of DNA synthesis. The enhancement can be obtained after transient exposure of cells to fetal bovine serum. The potentiating effects of antitubulin agents such as nocodazole, podophyllotoxin, and maytansine can be easily reversed whereas the enhancement with colchicine persists despite washing of cells. The stimulatory effects of insulin and EGF persist in cultures treated with antitubulin agents. The findings support the premise that after internalization the receptor-mitogen complex may remain active within endocytic vesicles when cells are exposed to colchicine.

Growth control in cultured 3T3 fibroblasts. Assays of cell proliferation and demonstration of a growth inhibitory activity.

Treatment of sparse, proliferating cultures of 3T3 cells (target cells) with medium conditioned by exposure to density-inhibited 3T3 cultures resulted in an inhibition of growth and division in the target cells when compared to similar treatment with unconditioned medium (UCM). This differential effect of conditioned medium (CM) and UCM on target cells was demonstrated using three assay systems: (a) assessment of total cell number; (b) measurement of [3H]thymidine incorporated into acid-precipitable DNA; and (c) determination of the percentage of radioactively labeled nuclei in individual cells after incorporation of [3H]thymidine. The difference in the total incorporation of [3H]thymidine in CM-treated and UCM-treated cells was reflected by a difference in the percent of labeled cells. There was no differences in the average number of grains per labeled cell in the two cultures. Moreover, the inhibitory effect of the CM on target cell proliferation was reversible. Finally, this growth inhibitory activity can be collected in serum-free medium, precipitated by ammonium sulfate, and fractionated by gel filtration. In these purification procedures, the inhibitory activity was consistently found to be associated with the protein-containing fractions of the CM. No activity was found upon similar treatment with UCM. These results suggest that a system has been developed for the purification and molecular analysis of growth inhibitory factors that may mediate growth control in culture fibroblasts.

Local anesthetics and wound healing

The effects of local anesthetics, lidocaine and bupivacaine, were tested in tissue cultures of 3T3 and W1-38 fibroblasts, in slices of newborn rat skin and in vivo in granuloma tissue induced by subcutaneous implantation of stainless steel cylinder in rats. The effects on the synthesis or amounts of DNA, collagen, glycosaminoglycans (GAG), noncollagenous proteins, and the activity of prolyl hydroxylase were studied. We found that irrespective of the biological system used, both anesthetics inhibit the synthesis of collagen to a greater extent than noncollagenous proteins. The synthesis of GAG was inhibited as well. We observed no effect on the synthesis or amount of DNA. We conclude that local anesthetics inhibit wound healing by inhibiting the synthesis of major structural macromolecules, collagen and GAG.

Production of macrophage-dependent fibroblast-stimulating activity (M-FSA) by murine macrophages: Effects on BALBc 3T3 fibroblasts

Macrophages cultured in vitro in medium containing platelet-poor plasma serum (PPPS) produce a substance (or substances) that stimulates the proliferation of fibroblasts in vitro. Two alternative assays have been used to study the production of this “macrophage-dependent fibroblast-stimulating activity” (M-FSA) by mouse peritoneal macrophages. The first determined increase in cell numbers induced in cultures of quiescent 3T3 fibroblasts (Growth Curve Assay); the second determined the incorporation of [ 3H]thymidine into the DNA of quiescent 3T3 cells induced by various culture media. Medium containing 5% blood serum (5% BS) resulted in higher final cell numbers (5-fold), and greater incorporation of [ 3H]thymidine into DNA than did medium containing 5% platelet-poor plasma serum (PPPS). Macrophages cultured in PPPS produced two different operationally defined activities. The first, M-FSA, resulted in greater final cell numbers, and was non-dialysable. The second reduced the incorporation of [ 3H]thymidine into DNA, and was found to be dialysable. Maximum levels of M-FSA were produced at a concentration of approx. 5 × 10 6 macrophages/10 ml culture medium containing 5 % PPPS. Successive 18 h cultures of macrophages in fresh changes of medium containing PPPS resulted in equivalent levels of M-FSA in each successive batch of medium. The presence of PPPS in the culture medium appeared to be an absolute requirement for the production of M-FSA. Culture in the absence of serum, in the presence of BSA, FCS, or of RBS did not result in the production of M-FSA. The dialysable factor, on the other hand, was produced non-specifically, in media containing either PPPS, FCS, RBS, BSA, or no serum. The level of production increased with increasing macrophage concentrations. At a concentration of 100 × 10 6 macrophages/10 ml medium, the stimulatory effect of M-FSA in the [ 3H]thymidine incorporation assay was completely masked. The dialysable substance did not affect the proliferation of 3T3 cells. Only the incorporation of [ 3H]thymidine into DNA was affected. It is suggested that M-FSA could be produced by macrophages, indirectly, by activation or de-inhibition of inactive (or inhibitory) substances present specifically in PPPS.

Temporal sequence of hormonal interactions during the prereplicative phase of quiescent cultured 3T3 fibroblasts.

After addition of prostaglandin F2alpha (PGF2alpha) and insulin to quiescent cultured 3T3 fibroblasts a constant lag phase of 15 hr occurs before an increased rate of cellular exit from G1 is observed. The latter process follows first order kinetics, which can be quantified by a rate constant k. The temporal relationship of the interactions of PGF2 alpha, insulin, and hydrocortisone to produce alterations in the rate of exit from G1 was investigated. PGF2alpha establishes a constant lag phase and produces alterations in the rate constant k. These two effects can be partially separated by adding two concentrations of PGF2alpha at different times. Insulin fails to establish the lag phase but can stimulate the effect of PGF2alpha when added at any time after PGF2alpha. Hydrocortisone reduces the value of k when added between 0 and 3 hr after PGF2alpha. These results show that the lag phase can beseparated into temporal regions during which hormones can interact to produce changes in the rate of cellular exit from G1.

Stimulation of the activity of prolyl hydroxylase in 3T3 fibroblasts by 1,10-phenanthroline

In confluent cultures of 3T3 fibroblasts, incubated for 24 h with 1,10-phenanthroline at 10 −5–10 −9 M, the activity of prolyl hydroxylase was significantly increased. 1,10-Phenanthroline was inhibitory at concentrations greater than 10 −4 M. The stimulatory effect of 1,10-phenanthroline manifets itself after 6 h inhubation and increased with time up to 48 h. 2,2′-dipyridyl and 5,6-dimethyl-1-1,10-phemamthroline were also stimulatory; a nonchelating analog, 1,7-phenanthroline had no effect. Cycloheximide did not modify the 1,10-phenanthroline effect. The stimulatory effect does not seem to depend on the shift of an inactive precursor of prolyl hydroxylase to an active form because 1,10-phenanthroline was shown to be ineffective in logarithmically growing cells. While dialysis of washed and homogenized cells significantly increased prolyl hydroxylase activity in cell extracts, undialyzed 1,10-phenanthroline treated samples exhibited higher prolyl hydroxylase activity than dialyxed controls. These data suggested to us that 1,10-phenanthroline and other chelating agents may be forming complexes with certain metal ions or protein-metal ions which are inhibitory towards prolyl hydroxylase.

Requirement for cellular protein synthesis in reversal of ethidium-bormide-induced inhibition of cell transformation by murine sarcoma virus.

Cultures of mouse Balb 3T3 fibroblasts exposed to a noncytotoxic dose of ethidium bromide for 16-18 hr are unable to produce foci after infection with murine sarcoma virus. Such cultures regain susceptibility to infection when incubated for 6-8 hr in drug-free growth medium. Pretreated but not untreated cultures exhibit sensitivity toward brief (6 hr) exposure to cycloheximide, chloramphenicol, and actinomycin D before infection. Pretreatment with cordy-cepin inhibits the ability of cultures to produce foci after infection. The recovery of ethidium-bromide-treated cultures requires the synthesis of cellular proteins which may have some important role in the establishment of RNA tumor virus infection.

Cross-link patterns of collagens synthesised by cultures of 3T6 and 3T3 fibroblasts and by fibroblasts of various granulation tissues

A study has been made of the pattern of reducible cross-links in collagens synthesised by 3T6 and 3T3 fibroblasts in culture and by fibroblasts in three different types of granulation tissue in guinea pigs: scar, silica granuloma and carrageenin granuloma. All the granulation tissue collagens have hydroxylysinohydroxynorleucine as the major cross-link after reduction. This may be due to excess hydroxylation of lysine residues some of which are involved in the cross-linking process. In contrast, the 3T6 and 3T3 fibroblasts produce collagens which have a cross-linking pattern quite different from and more complex than the granulation collagen pattern.