Effect of 3T3 plasma membranes on cells exposed to epidermal growth factor

Epidermal growth factor (EGF) induced DNA synthesis in non-confluent, G₀-arrested Swiss 3T3 fibroblasts is partially blocked by plasma membranes isolated from the EGF receptor deficient NR-6 Swiss 3T3 cell line. This inhibition could be due to either a steric block of the receptor by the membranes, a membrane induced down regulation of the EGF receptor, or a signal generated by membrane binding which is antagonistic towards the mitogenic signal generated by EGF. Binding measurements utilizing ¹²⁵I-labeled EGF demonstrated that membranes do not block either the EGF induced down regulation of the receptor or alter the number of receptors on the surface. These results suggest that the membranes exert their inhibitory effect via generation of a signal which is antagonistic to the EGF induced mitogenic signal, with the result expressed as a reduced mitogenic response.     

Identification of a PDGF-like mitoattractant produced by NIH/3T3 cells after transformation with SV40

It has previously been shown that fibroblastic cells transformed by SV40 exhibit a reduced requirement for PDGF for growth. In addition, NIH/3T3 cells lose both their chemotactic response to PDGF and specific cell surface binding of PDGF after transformation with SV40. We have now examined whether the SV40 transformed NIH/3T3 cells are producing a factor which acts similarly to PDGF. Our studies indicate that NIH/3T3 cells transformed with SV 40 produce a factor which shares many biological properties with PDGF. We were unable to detect this activity in conditioned media from nontransformed NIH/3T3 cells. The SV40/NIH/3T3 derived factor appears to possess both chemotactic and mitogenic activity for connective tissue cells but not endothelial or epithelial cells. Furthermore, in preliminary studies, this activity competes with 125I-PDGF for binding to smooth muscle cells. The biochemical properties of the SV40/NIH/3T3 derived factor are different from those of PDGF. The SV40 activity appears to reside in a heat labile acidic protein (pI less than 7.0) of MW less than 30,000 whereas PDGF is a heat stable basic protein (pI9.8) of 30,000 MW. Production of this factor may play a role in the decreased serum requirement for cell replication exhibited by SV40-transformed NIH/3T3 cells by supplying the cells with their own PDGF-like growth factor.     

Actin associated with membranes from 3T3 mouse fibroblast and HeLa cells

A protein component of membranes isolated from 3T3 mouse fibroblasts and HeLa cells has been identified as actin by peptide mapping. Extensive but apparently not total coincidence was found between the peptide maps of these two nonmuscle membrane-associated actins compared to chick skeletal muscle actin. Between 2 and 4 percent of the total membrane protein appears in the actin band on sodium dodecyl sulfate polyacrylamide gels of 3T3 membranes while about 4 percent of the membrane protein appears as the actin band from HeLa membranes. These values represent approximately the same proportion of actin to total protein found in the cell homogenates. Treatment of intact cells with levels of cytochalasin B sufficient to cause pronounced morphological changes did not change the amount of actin associated with the membrane in either 3T3 or HeLa cells. However, incubation of isolated membranes under conditions favoring conversion of actin from filamentous to monomeric form resulted in dissociation of approximately 80 and 60 percent of the actin from 3T3 and HeLa membranes, respectively. Thus, approximately 20 percent of 3T3 membrane actin and 40 percent of HeLa membrane actin remained associated with the membrane even under actin depolymerizing conditions.     

Mitogenic activity elaborated by macrophage-like cell lines acts as competence factor(s) for BALB/c 3T3 cells

The culture medium from several murine macrophage-like cell lines contained a mitogenic activity that functioned synergistically with platelet-poor plasma to induce DNA synthesis in quiescent density-inhibited BALB/c 3T3 fibroblasts. This mitogenic activity was generated by P388D₁ (and other established lines of) macrophage-like cells that were cultured either in medium alone or in medium supplemented with platelet-poor plasma. The amount of mitogenic activity produced was directly related to the length of time the macrophage-like cells were maintained in the medium. Serum-free medium conditioned by macrophage-cells did not stimulate DNA synthesis in density-inhibited 3T3 cells in the absence of plasma; however, a transient (4-hr) exposure to serum-free macrophage-conditioned medium allowed quiescent cells to respond to plasma-derived progression factors. The addition of plasma to 3T3 cells that had been treated with the macrophage-conditioned medium brought about DNA synthesis after a 12-hr lag. The mitogenic activity that was in macrophage-conditioned medium bound to DEAE-Sephadex and eluted in a single peak using a linear NaCl gradient. This macrophage-derived competence factor was not mitogenic for lymphocytes and was clearly separated by DEAE-Sephadex chromatography from the major peak of the previously described mitogenic monokine, Interleukin-I (lymphocyte activating factor).     

Regulation of the cell cycle of 3T3 cells in culture by a surface membrane-enriched cell fraction

Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: (1) In both cases the cells are arrested in the G₁ protion of the cell cycle; (2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.     

Partial characterization of a growth-inhibiting protein in 3T3 cell plasma membranes

A plasma membrane-enriched fraction from 3T3 cells has been detergent-solubilized, and the supernatant of this solubilization was reconstituted into liposomes using soybean lecithin. When these vesicles were added to actively growing cells, cell growth rates were inhibited to levels that were comparable to those observed with the original plasma membranes (at least 50% of maximum growth rates). Liposomes without proteins, or liposomes containing proteins from SV3T3 plasma membranes did not significantly inhibit growth of 3T3 cells. Treatment of the reconstituted vesicles with urea or high concentrations of salt did not eliminate the growth-inhibiting properties of these reconstituted membranes. These results indicate that the specific growth-inhibiting factor in 3T3 cell plasma membranes is a membrane protein that has significant non-polar interactions with the membrane bilayer.     

Low molecular weight mitogenic factor produced by BRL-3A cultured rat liver cells

A new mitogenic factor has been isolated from medium conditioned by BRL-3A rat liver cells. The factor has been partially purified by a two step procedure involving ion exchange chromatography on Dowex 50 followed by gel filtration chromatography on Sephadex G-75 in 1 M acetic acid. The factor is eluted from the Sephadex G-75 column in the low molecular weight region, behin three peaks of multiplication stimulating activity. The factor is inactivated by treatment with trypsin and dithiothreitol, suggesting that it is a peptide that contains a disulfide linkage. Unlike multiplication stimulating activity, the new factor only weakly stimulates DNA synthesis in quiescent chick fibroblasts, whereas it strongly stimulates DNA synthesis in quiescent NIL8 hamster cells, $\text{BALB}\text{c}$ 3T3 cells, and IMR-90 human fibroblasts.     

Changes in ribosomal binding activity of eIF3 correlate with increased translation rates during activation of T lymphocytes

The rate of protein synthesis in quiescent peripheral blood T lymphocytes increases dramatically following mitogenic activation. The stimulation of translation is due to an increase in the rate of initiation caused by the regulation of initiation factor activities. Here, we focus on eIF3, a large multiprotein complex that plays a central role in the formation of the 40 S initiation complex. Using sucrose density gradient centrifugation to analyze ribosome complexes, we find that most eIF3 is not bound to 40 S ribosomal subunits in unactivated T lymphocytes but becomes ribosome-bound following activation. Immunoblot analyses of sucrose gradient fractions for individual eIF3 subunits show that the small eIF3j subunit is unassociated with the eIF3 complex in quiescent T lymphocytes, but upon activation joins the other eIF3 subunits and binds 40 S ribosomal subunits. Because eIF3j has been shown to be required for eIF3 binding to 40 S ribosomes in vitro, the results suggest that mitogenic stimulation of T lymphocytes leads to an activation of eIF3j, thereby enabling eIF3 to bind to the larger ribosome-free eIF3 subunit complex, and then to the 40 S ribosomes. The association of eIF3j with the other eIF3 subunits appears to be inhibited by rapamycin, suggesting a mechanism that lies downstream from the mammalian target of rapamycin kinase. This association requires ionomycin together with a phorbol ester, which also suggests that calcium signaling is involved. We conclude that the complex formation of eIF3 and its association with the ribosomes might contribute to increased translation rates during T lymphocyte activation.     

Activation of 5-HT1A receptors expressed in NIH-3T3 cells induces focus formation and potentiates EGF effect on DNA synthesis.

NIH-3T3 fibroblasts have been transfected with human serotonin 5-HT1A receptors. Clonal cell lines expressed between 40 and 500 fmol receptor/mg. 5-HT1A agonists strongly inhibited nonstimulated- as well as forskolin- or isoproterenol-stimulated adenylyl cyclase. The effects of 5-HT1A receptor activation on cell growth were investigated. 5-HT1A agonists accelerated cell division, generated foci, and increased DNA synthesis. The stimulation of [3H]thymidine incorporation was much stronger when tyrosine kinase receptors were activated concomitantly. Cyclic AMP (cAMP) elevating agents inhibited DNA synthesis induced by all mitogens tested. The mitogenic activity of 5-HT1A agonists did not seem to be linked to adenylyl cyclase inhibition because 1) we were not able to measure any decrease in intracellular cAMP levels under the conditions of DNA synthesis assay and 2) 2',5'-dideoxyadenosine, which strongly inhibited adenylyl cyclase, was not mitogenic and did not modify the mitogenic effects of 5-HT1A agonists. Pertussis toxin completely blocked potentiation of epidermal growth factor effect induced by 8-hydroxy-di-(n-propyl)aminotetralin, a 5-HT1A agonist, but only partially blocked the one induced by insulin. In conclusion, in transfected NIH-3T3 cells, transforming and mitogenic effects of 5-HT1A agonists involve a pertussis toxin-sensitive G protein but do not seem to be linked to adenylyl cyclase inhibition.     

Bombesin stimulates a high affinity GTPase activity in membranes of Swiss 3T3 fibroblasts

Peptides of the bombesin family are mitogenic for Swiss 3T3 fibroblasts and in these cells stimulate the turnover of polyphosphoinositides. Recent studies have suggested that G protein(s) may be involved in the signal transduction pathway triggered by bombesin. In this study we have found and characterized a high affinity GTPase activity stimulated by bombesin in membranes of Swiss 3T3 fibroblasts. Our results support the involvement of a G protein in the response of Swiss 3T3 cells to bombesin.     

Cell surface-associated growth inhibitory proteins : Evidence for conservation between mouse and human cell lines

The ability of the normal human fibroblast line (IMR91) to exhibit density-dependent regulation of growth has been examined. The line exhibits density-dependent regulation of growth; saturation density in 15% fetal bovine serum is 2 × 10⁵ cells/cm². Membranes prepared from confluent monolayers of these cells contained growth inhibitory factors to both exponentially growing IMR91 and Swiss 3T3 cells. This factor(s) appears to be similar to a previously described factor found on the surface of Swiss 3T3 cells [14]. The inhibition of DNA synthesis in growing IMR91 cultures by membranes was both time- and concentration-dependent. The effect was reversible by high serum. Specificity experiments utilizing membranes prepared from Swiss 3T3 cells indicated some species specificity for inhibition by membranes, but this specificity was no longer exhibited by solubilized membrane preparations. These results are compatible with the suggestion that both the growth inhibitory factors and their receptors are conserved through evolution.     

A low molecular weight factor from dividing cells activates phospholipase D in caveolin-enriched membrane microdomains

Phospholipase D (PLD) activity is elevated in Ras-transformed NIH 3T3 cells. This difference in PLD activity between Ras-transformed and nontransformed parental cells disappeared in isolated membranes from these cells. In reconstitution experiments, heat-denatured cytosolic fractions from Ras-transformed, but not parental, NIH 3T3 cells elevated PLD activity in isolated membranes. This heat-resistant PLD-stimulating activity from the Ras-transformed cells was sensitive to proteases and passed through a 1-kDa MW cutoff membrane, suggesting that the factor is a peptide of less than 10 amino acids. The ability of this PLD-stimulating factor, designated PLD-SF, to elevate PLD activity in isolated membranes was restricted to the caveolin-enriched light membranes, where many signaling molecules are localized. PLD-SF was also elevated in v-Src- and v-Raf-transformed cells and in serum-stimulated NIH 3T3 cells. PLD-SF was detected in a variety of rat tissues but was highest in testes, where a large percentage of cells are dividing. A similar low molecular weight PLD-stimulating activity was found in actively dividing, but not stationary yeast, cells. The data here provide evidence for a highly conserved PLD-stimulating peptide that is elevated in response to mitogenic stimuli.     

Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells.

We used a dominant inhibitory mutation of c-Ha-ras which changes Ser-17 to Asn-17 in the gene product p21 [p21(Asn-17)Ha-ras] to investigate ras function in mitogenic signal transduction. An NIH 3T3 cell line [NIH(M17)] was isolated that displayed inducible expression of the mutant Ha-ras gene (Ha-ras Asn-17) via the mouse mammary tumor virus long terminal repeat and was growth inhibited by dexamethasone. The effect of dexamethasone induction on response of quiescent NIH(M17) cells to mitogens was then analyzed. Stimulation of DNA synthesis by epidermal growth factor (EGF) and 12-O-tetradecanoylphorbol-13-acetate (TPA) was completely blocked by p21(Asn-17) expression, and stimulation by serum, fibroblast growth factor, and platelet-derived growth factor was partially inhibited. However, the induction of fos, jun, and myc by EGF and TPA was not significantly inhibited in this cell line. An effect of p21(Asn-17) on fos induction was, however, demonstrated in transient expression assays in which quiescent NIH 3T3 cells were cotransfected with a fos-cat receptor plasmid plus a Ha-ras Asn-17 expression vector. In this assay, p21(Asn-17) inhibited chloramphenicol acetyltransferase expression induced by EGF and other growth factors. In contrast to its effect on DNA synthesis, however, Ha-ras Asn-17 expression did not inhibit fos-cat expression induced by TPA. Conversely, downregulation of protein kinase C did not inhibit fos-cat induction by activated ras or other oncogenes. These results suggest that ras proteins are involved in at least two parallel mitogenic signal transduction pathways, one of which is independent of protein kinase C. Although either pathway alone appears to be sufficient to induce fos, both appear to be necessary to induce the full mitogenic response.     

Basic streptococcal superantigens (SPEX/SMEZ or SPEC) are responsible for the mitogenic activity of the so-called mitogenic factor (MF)

The mitogenic factor (MF) of group A streptococci has been reported to be a superantigen stimulating human T cells carrying Vbeta2, 4 and 8 and has been designated streptococcal pyrogenic exotoxin F (SPEF). MF was also shown to possess DNase activity. Here we have purified MF from culture supernatants of different Streptococcus pyogenes strains. Surprisingly, the MF preparations from different strains showed different Vbeta specificities depending on the expression of SPEC or SMEZ3 by the producing strain. Their mitogenic activity decreased upon further purification. In addition, the mitogenic activity could be only neutralized by antibodies against the basic streptococcal superantigens SPEC or SPEX (SMEZ3) but not by antibodies against MF itself although the latter were able to neutralize completely the DNase activity of MF. We found that streptodornase type B (SDB) was expressed in two molecular forms (SDBI and SDBII), differing only by one additional N-terminal arginine at SDBI. MF was found identical to the enzyme SDBII but is devoid of superantigenic properties and should no longer be called a superantigen or a pyrogenic exotoxin.     

Purified plasma membranes inhibit polypeptide growth factor-induced DNA synthesis in subconfluent 3T3 cells

Plasma membranes derived from NR-6 cells, a variant line of Swiss mouse 3T3 cells that does not have cell surface receptors for epidermal growth factor (EGF), inhibited EGF-induced stimulation of DNA synthesis by 50% in serum-starved, subconfluent 3T3 cells. Membranes derived from SV3T3 cells were much less effective in inhibiting EGF-induced DNA synthesis. This inhibition on DNA synthesis by NR-6 membranes was not a direct effect of membranes on EGF, nor could it be overcome by high concentrations of EGF. NR-6 membranes were most effective when added 3 h before EGF addition and had little effect when added 2 h or more after EGF. NR-6 membranes also reduced the stimulation of DNA synthesis induced by platelet-derived growth factor or fibroblast growth factor in serum-starved 3T3 cells. These findings indicate that membrane- membrane interactions between nontransformed cells may diminish their ability to proliferate in response to serum polypeptide growth factors.     

A partially purified serum fraction synergistically enhances the mitogenic activity of epidermal growth factor and insulin in quiescent cultures of 3T3 cells.

Epidermal growth factor and insulin need a low concentration of serum to effectively stimulate quiescent 3T3 cells into DNA synthesis. We partially purify a polypeptide component of serum which has no activity by itself but which acts synergistically with epidermal growth factor and insulin to stimulate cultures of 3T3 cells into DNA synthesis as effectively as whole serum. The active fraction is separated from serum by gel chromatography on Sephadex G-100, under acid dissociating conditions, and chromatographs with a molecular weight of 18,000 daltons.We suggest as a general technique, the use of pure growth factors to assay for growth promoting fractions from serum or other sources. Fractions which are not mitogenic by themselves can be detected when assayed together with their complementary pure factors.     

3T3 variants unable to bind epidermal growth factor cannot complement in co-culture

A Swiss-Webster 3T3 variant, called 3T3-ENR7, unable to divide in response to epidermal growth factor was isolated by the mitogen-colchicine selection technique. Like the other EGF non-proliferative variants (3T3-NR6 and 3T3-TNR2), 3T3-ENR7 was unable to bind 125I-EGF. Pairwise co-culture of the three independently isolated EGF non-responsive variants did not restore mitogenic responsiveness to EGF.     

Rapid microassay for protein kinase C translocation in Swiss 3T3 cells

The Ca2+/phosphatidylserine-stimulated protein kinase C (PKC) appears to exist as interconvertible inactive, soluble and active, membrane-bound forms. Changes in the bimodal distribution of PKC induced by diacylglycerol or tumor-promoting phorbol esters have been proposed to regulate the activity of this kinase [Nishizuka, Y. (1984) Nature (London) 308, 693-698]. A rapid microassay for assessment of protein kinase C translocation between cytosol and membranes was developed. This procedure, which relied on the selective digitonin-mediated release of cytoplasmic proteins, eliminated potential homogenization and fractionation artifacts. PKC activity toward histone H1 was determined after limited trypsinolysis, which abolished the Ca2+/phospholipid requirement of the enzyme and prevented interference by inhibitory proteins. Complete translocation of PKC to the membrane fraction and subsequent down-regulation of the kinase in response to 12-O-tetradecanoylphorbol-13-acetate treatment of Swiss 3T3 cells could be demonstrated by this method. Platelet-derived growth factor, insulin-like growth factor 1, vasopressin, and prostaglandin F2 alpha facilitated partial conversions of PKC to the membrane-bound form in quiescent 3T3 cells.     

The trk tyrosine protein kinase mediates the mitogenic properties of nerve growth factor and neurotrophin-3.

The product of the trk proto-oncogene encodes a receptor for nerve growth factor (NGF). Here we show that NGF is a powerful mitogen that can induce resting NIH 3T3 cells to enter S phase, grow in semisolid medium, and become morphologically transformed. These mitogenic effects are absolutely dependent on expression of gp140trk receptors, but do not require the presence of the previously described low affinity NGF receptor. gp140trk also serves as a receptor for the related factor neurotrophin-3 (NT-3), but not for brain-derived neurotrophic factor. Both NGF and NT-3 induce the rapid phosphorylation of gp140trk receptors and the transient expression of c-Fos proteins. However, NT-3 appears to elicit more limited mitogenic responses than NGF. These results indicate that the product of the trk proto-oncogene is sufficient to mediate signal transduction processes induced by NGF and NT-3, at least in proliferating cells.     

Mitogenic and cytotoxic actions of tumor necrosis factor in BALB/c 3T3 cells. Role of phospholipase activation

In addition to its cytotoxic/cytostatic action on many tumor cells in vitro, tumor necrosis factor (TNF) was recently shown to stimulate the growth of some types of cells in culture. We examined the action of TNF in BALB/c 3T3 cells which have been used extensively to study growth regulation. In subconfluent, rapidly dividing 3T3 cultures, murine (Mu) TNF was cytotoxic, while human (Hu) TNF had virtually no antiproliferative action on the cells. In contrast, in density-arrested BALB/c 3T3 cells maintained in a chemically defined, serum-free medium, MuTNF produced a dose-dependent stimulation of DNA synthesis. In stimulating DNA synthesis, MuTNF acted synergistically with both epidermal growth factor or platelet-derived growth factor. While stimulating DNA synthesis in quiescent 3T3 cultures, high doses of MuTNF (100 or 10 ng/ml) were also cytotoxic for a portion of the cells in the same cultures. Cytotoxicity was apparent 2 h after the addition of MuTNF, well before the onset of DNA synthesis. BALB/c 3T3 cell variants selected for their resistance to the cytotoxic action of MuTNF retained the capacity to respond to the mitogenic action of MuTNF, indicating that the stimulation of DNA synthesis by TNF is not a consequence of a TNF "wounding effect." Addition of TNF to density-arrested 3T3 cells resulted in the release of free arachidonic acid and palmitic acid from the cells. Quinacrine, a phospholipase inhibitor, inhibited both cytotoxicity and DNA synthesis in response to TNF, and melittin, a phospholipase activator, mimicked both the cytotoxic and mitogenic actions of TNF in quiescent BALB/c 3T3 cells. These results suggest that phospholipid breakdown is part of the essential early signal transduction events required both for the cytotoxic and mitogenic response to TNF action.