Stimulation of plasminogen activator expression and induction of DNA synthesis by microtubule-disruptive drugs

Treatment of confluent Swiss 3T3 cells in serum-free medium with colchicine, a drug known to depolymerize microtubules, results in a dose-dependent increase in both released and cell-associated plasminogen activator levels. Other anti-microtubule drugs (vinblastine and nocodazole) are also active in stimulating plasminogen activator expression. In contrast, cytochalasin B, a microfilament-disruptive drug, has no effect. In addition, treatment with colchicine, vinblastine or nocodazole, but not cytochalasin B, also results in a dose-dependent induction of DNA synthesis in both confluent and quiescent sparse 3T3 cells in the absence of serum. Furthermore, colchicine treatment also mediates a marked morphologic change. Thus, disruption of microtubules may be sufficient to render 3T3 cells in an “activated” state characterized by morphologic alteration, enhanced plasminogen activator expression and induction of DNA synthesis.     

Production of plasminogen activator by established cell lines of mouse origin

The correlation between malignant transformation and increased plasminogen activator synthesis has been studied in a variety of established cell lines. In contrast to the behavior of secondary mouse embryo cultures, which always show increased fibrinolytic activity when transformed, no such correlation was found within the BALB/c 3T3 line and its transformed derivatives. Cell lines were established from tumors initiated in BALB/c mice by several transformed cell lines. These lines were generally found to contain no more plasminogen activator than the cells used for inoculation. A correlation was found between transformation and plasminogen activator synthesis within Swiss 3T3 cell lines. However, the correlation was not maintained by serum revertants of transformed Swiss 3T3 cells.     

Calcium stimulation of plasminogen activator secretion/production by swiss 3T3 cells.

Actively growing Swiss 3T3 cells secret high levels of plasminogen activator which decreases after the cells become confluent. In contrast, simian virus 40-transformed 3T3 cells secrete large amounts of plasminogen activator independent of cell density (Chou, I.-N., O'Donnel, S.P., Black, P.H., and Roblin, R.O. (1977) J. Cell. Physiol. 91, 31-38). These results suggest a correlation between active cell multiplication and plasminogen activator secretion in both 3T3 and simian virus-transformed 3T3 cells. The data reported herein indicate that treatment of both subconfluent and confluent Swiss 3T3 cells with high concentrations of Ca2+ (final 3.0 to 4.9 mM) increases the amounts of both secreted and cell-associated plasminogen activator in a dose-dependent manner. In addition, the ionophore A23187 (19 to 95 nM) in the presence of a normal level of Ca2+ (1.8 mM) stimulates both production and secretion of plasminogen activator from growing 3T3 cells. The Ca2+ stimulation of plasminogen activator production/secretion may be related to the mitogenic effect of Ca2+.     

Hyperthermia can enhance the initiation of DNA synthesis stimulated by growth factors in Swiss mouse 3T3 cells

Confluent quiescent Swiss mouse 3T3 cells can be stimulated to initiate DNA synthesis and to divide by epidermal growth factor (EGF) and prostaglandin F2 alpha (PGF2 alpha), two mitogens of unrelated structure. Heat treatment at 46 degrees C for up to 20 min of confluent quiescent cells, which has no mitogenic effect, can enhance the stimulatory effect of suboptimal concentrations of EGF or PGF2 alpha on the initiation of DNA synthesis. Furthermore, insulin, which is not mitogenic in these cells, enhances the effect of these mitogens, but this effect is not further enhanced by heat treatment. Likewise the combination of EGF and PGF2 alpha is synergistic on DNA synthesis, and this effect is also not enhanced by the heat treatment. Incubation at 46 degrees C for longer than 20 min was inhibitory in all cases. These results suggest that heat treatment induces events which affect the regulation of the initiation of DNA synthesis in a manner depending on the duration of the heat treatment and the stimulation of the cells.     

Mitogenic effect of prostaglandin E1 in Swiss 3T3 cells: role of cyclic AMP

Addition of prostaglandin E1 (PGE1) to quiescent cultures of Swiss 3T3 cells rapidly elevates the intracellular levels of cAMP and increases the activity of adenylate cyclase in particulate fractions of these cells. In the presence of insulin, PGE1 stimulates the reinitiation of DNA synthesis. Both effects (increase in cellular cAMP and stimulation of DNA synthesis) are markedly potentiated by 1-methyl-3-isobutyl xanthine (IBMX) or by 4-(3-butoxy-4 methoxy benzyl)-2-imidazolidine (Ro 20-1724), both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. In the presence of 50 microM IBMX, PGE1 caused a dose-dependent increase in cAMP levels and in [3H]thymidine incorporation into acid-insoluble material at concentrations (5-50 ng/ml) that are orders of magnitude lower than those used in previous studies (50 micrograms/ml) to demonstrate growth-inhibitory effects. Thus, the inhibitory effects produced by adding high concentrations of PGE1 on the initiation of DNA synthesis in Swiss 3T3 cells are not mediated by cAMP and should be regarded as nonspecific. In contrast, the mitogenic activity of PGE1 parallels its ability to increase the intracellular levels of cAMP. The findings support the proposition that a sustained increase in the level of this cyclic nucleotide acts as a mitogenic signal for confluent and quiescent Swiss 3T3 cells.     

Increases in phosphatidic acid levels accompany sphingosine-stimulated proliferation of quiescent Swiss 3T3 cells

Sphingosine, a breakdown product of cellular sphingolipids, has recently been shown to stimulate DNA synthesis and act synergistically with known growth factors to induce proliferation of quiescent Swiss 3T3 fibroblasts (Hong, Z., Buckley, N. E., Gibson, K., and Spiegel, S. (1990) J. Biol. Chem. 265, 76-81). The present study demonstrates that mitogenic concentrations of sphingosine induce early increases in cytosolic phosphatidic acid, which is a potent mitogen for Swiss 3T3 cells. Structurally related analogs of sphingosine, such as N-stearoylsphingosine and other long chain aliphatic amines, did not mimic the mitogenic effect of sphingosine and did not elevate phosphatidic acid levels. Sphingosine not only stimulated [3H]thymidine incorporation with similar efficiency and kinetics as phosphatidic acid, it also induced similar morphological alterations. Both sphingosine and phosphatidic acid acted synergistically with a variety of growth factors, such as, insulin, epidermal growth factor, fibroblast growth factor, and 12-O-tetradecanoylphorbol 13-acetate. In sharp contrast, sphingosine and phosphatidic acid did not have additive or synergistic effects in either the presence or absence of other growth factors. Both sphingosine and phosphatidic acid stimulated DNA synthesis in cells made protein kinase C-deficient by prolonged treatment with phorbol ester and sphingosine still stimulated similar increases in phosphtidic acid in these cells. Furthermore, similar to the actions of phosphatidic acid on signal transduction in Swiss 3T3 cells, mitogenic concentrations of sphingosine also inhibit cAMP accumulation and trigger the hydrolysis of polyphosphoinositides. Our findings indicate that sphingosine and phosphatidic acid control cellular responses in Swiss 3T3 cells through a common pathway. In view of the prominent role of phosphatidic acid in signal transduction and cellular proliferation, our observations that sphingosine, at mitogenic concentrations, increases the level of phosphatidic acid and also mimics the effects of phosphatidic acid on signal transduction, have important implications for the mechanism of action of sphingosine.     

Rete testis fluid contains a growth factor for cultured fibroblasts

A new polypeptide mitogen has been detected at high specific activity in the rete testis fluid of rams (oRTF). The factor, which stimulates DNA synthesis in quiescent Swiss 3T3 cells, has a molecular weight of 45,000 as assessed by gel filtration through Ultrogel AcA 34. The factor is heat stable but is inactivated by proteolytic enzymes and by β-mercaptoethanol. The growth-promoting activity in oRTF does not bind to concanavalin A.     

The stimulation of the initiation of DNA synthesis and cell division in Swiss mouse 3T3 cells by prostaglandin F2 alpha requires specific functional groups in the molecule

Among a number of prostaglandins, PGF2 alpha had the highest specific activity for stimulating the initiation of DNA synthesis in confluent resting Swiss 3T3 cells. At a saturating concentration of 8.5 X 10(-7) M, PGF2 alpha stimulated 21% of the cells to incorporate [ methyl-3H ]thymidine within 28 h. To elicit similar effects, prostaglandins F1 alpha, E1, E2, and D2 were required in 10-fold higher concentrations. Prostaglandins A1, A2, B1, and prostacyclin had no mitogenic activity. Insulin at 10(-8) M enhanced the stimulatory effect of PGF2 alpha and also of prostaglandins F1 alpha, E1, E2, and D2 by increasing the fraction of labeled nuclei. Methyl derivatives of PGF2 alpha were as effective as PGF2 alpha. Epimerization of the hydroxyl group at C-9 abolished the activity of the molecule. In contrast, upon epimerization at C-11 and C-15, some mitogenic activity was retained. In the presence of insulin, the latter molecules were as active as PGF2 alpha. Oxidation of the hydroxyl group at C-15 to a ketone abolished the mitogenic effect, while methyl ether formation led to only a slight loss of activity. Reduction of the delta 13 double bond also led only to a small reduction of activity. Similar differences in the activity of the various prostaglandins and analogues of PGF2 alpha were observed for 2-deoxyglucose uptake and increases in cell number. The relationships between structure and activity of prostaglandins suggest the existence of some specific receptor for PGF2 alpha to confer mitogenic response.     

Protein kinase D potentiates DNA synthesis induced by Gq-coupled receptors by increasing the duration of ERK signaling in swiss 3T3 cells

Protein kinase D (PKD) potentiates cellular DNA synthesis in response to G protein-coupled receptor (GPCR) agonists but the mechanism(s) involved has not been elucidated. Here, we examined whether PKD overexpression in Swiss 3T3 cells regulates the activation/inactivation kinetics of the extracellular-regulated protein kinase (ERK) in response to the mitogenic GPCR agonists bombesin and vasopressin. Addition of bombesin or vasopressin to Swiss 3T3 cells overexpressing PKD induced a striking increase in the duration of MEK/ERK/RSK activation as compared with cultures of either control Swiss 3T3 cells or Swiss 3T3 cells expressing a kinase-inactive PKD mutant. In contrast, the duration of ERK activation in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not increased. Furthermore, bombesin or vasopressin promoted a striking increase in phosphorylation (at Ser-374) and accumulation of c-Fos (the c-fos proto-oncogene product) in Swiss 3T3 cells overexpressing wild-type (but not kinase-inactive) PKD. Inhibition of the sustained phase of ERK/RSK activation abrogated the increase in c-Fos accumulation and DNA synthesis induced by bombesin or vasopressin in PKD-overexpressing cells. Our results demonstrate that PKD selectively potentiates mitogenesis induced by bombesin or vasopressin in Swiss 3T3 cells by increasing the duration of MEK/ERK/RSK signaling.     

Synergistic stimulation of DNA synthesis by cyclic AMP derivatives and growth factors in mouse 3T3 cells

Addition of the cAMP derivatives butcAMP or 8BrcAMP to quiescent cultures of Swiss 3T3 causes synergistic stimulation of DNAk synthesis with insulin, phorbol esters, vasopressin, epidermal growth factor, or fetal bovine serum (2-5%). In the presence of insulin, 8BrcAMP, and butcAMP stimulate [3H]-thymidine incorporation into acid-precipitable material in a dose-dependent manner. The effect of these agents is specific since 8Br5'AMP, 5'AMP, butyrate, or 8BrcGMP fail to stimulate DNA synthesis under identical experimental conditions. Furthermore, the mitogenic effects of the cAMP derivatives were markedly potentiated by 1-methyl-3-isobutyl xanthine and 4-(3-butoxy-4-methoxy benzyl)-2-imidazolidine, both of which are potent inhibitors of cyclic nucleotide phosphodiesterase activity. The growth-promoting effects of the cAMP derivatives were demonstrated by [3H]-thymidine incorporation (either by scintillation counting or by autoradiography), by flow cytofluorometric analysis, and by increase in cell number. When quiescent Swiss 3T3 cells were exposed to butcAMP and insulin, DNA synthesis began after a lag of 17h. The result of sequential additions of cAMP derivatives and insulin to quiescent 3T3 cells suggest that these agents must act simultaneously in G0/G1 to stimulate entry into DNA synthesis in these cells. The findings support the proposition that an increase in cellular levels of cAMP (but not cGMP) act sas a mitogenic stimulus for confluent and quiescent Swiss 3T3 cells.     

Production of plasminogen activator by synchronized normal and rous sarcoma virus-transformed chicken fibroblasts in culture

We studied intracellular activity of the plasminogen activator within the cell cycle of chemically synchronized normal and RSV-transformed chick fibrolasts in culture. Consideration has also been given to the relationship between the plasminogen activator activity and cycles of DNA synthesis or mitosis in cycling fibroblasts after viral infection. The plasminogen activator activity of the cell lysates was assayed on [¹²⁵I]fibrin-coated Petri dishes and was expressed as the radioactivity released from the plates. Normal fibroblasts produced detectable levels of plasminogen activator in the S-phase and late G₂-phase or mitosis of the cell cycle. In contrast, RSV-transformed cells produced high levels of this activator throughout the entire cell cycle although this activity fluctuated and reached a maximum in the G₂-M periods. We also found that the level of plasminogen activator activity in the transformed fibroblasts is influenced by the cycles of DNA synthesis and that cell division is required for the appearance of plasminogen activator activity in the ‘de novo’ virus-infected cultures.     

Diacylglycerols, unlike phorbol esters, do not induce homologous desensitization or down-regulation of protein kinase C in Swiss 3T3 cells

Addition of 1-oleoyl-2-acetyl-glycerol (OAG), 1,2-dioctanoyl-glycerol (diC8) or phorbol-12, 13-dibutyrate (PDBu) to cultures of Swiss 3T3 cells rapidly increases the phosphorylation of the Mr 80,000 protein kinase C (PKC) substrate, inhibits EGF binding and stimulates DNA synthesis. Prolonged incubation (40 h) with PDBu completely blocked these responses to all agents and down-regulated PKC. In contrast, a similar treatment with OAG or diC8, at mitogenic concentrations, neither induced homologous cellular desensitization nor decreased the immunoreactive level or activity of PKC. The results show that PKC down-regulation can be dissociated from PKC-mediated mitogenesis in Swiss 3T3 cells.     

Prostaglandin F(2alpha) (PGF(2alpha)) induces cyclin D1 expression and DNA synthesis via early signaling mechanisms in Swiss mouse 3T3 cells

Prostaglandin F(2alpha) (PGF(2alpha)), a mitogen for Swiss 3T3 cells, triggers cyclin D1 mRNA/protein expression prior to cellular entry into the S phase, but fails to raise cdk4 or cyclin D3 levels, while 1-oleoyl-2-diacylglycerol (OAG), a protein kinase C (PKC) and tyrosine kinase (TK) activator, induces only cyclin D1 expression with no mitogenic response. In contrast, in PKC-depleted or -inhibited cells, PGF(2alpha), but not OAG, increases cyclin D1 expression with no mitogenic response. Finally, OAG, in the presence of orthovanadate (Na(3)VO(4)) or TGF(beta1), induces DNA synthesis. Thus, it appears that PGF(2alpha) triggers cyclin D1 expression via two independent signaling events that complement with TGF(beta1)-triggered events to induce DNA synthesis.     

Mastoparan, a novel mitogen for Swiss 3T3 cells, stimulates pertussis toxin-sensitive arachidonic acid release without inositol phosphate accumulation

Mastoparan, a basic tetradecapeptide isolated from wasp venom, is a novel mitogen for Swiss 3T3 cells. This peptide induced DNA synthesis in synergy with insulin in a concentration-dependent manner; half-maximum and maximum responses were achieved at 14 and 17 microM, respectively. Mastoparan also stimulated DNA synthesis in the presence of other growth promoting factors including bombesin, insulin-like growth factor-1, and platelet-derived growth factor. The synergistic mitogenic stimulation by mastoparan can be dissociated from activation of phospholipase C. Mastoparan did not stimulate phosphoinositide breakdown, Ca2+ mobilization or protein kinase C-mediated phosphorylation of a major cellular substrate or transmodulation of the epidermal growth factor receptor. In contrast, mastoparan stimulated arachidonic acid release, prostaglandin E2 production, and enhanced cAMP accumulation in the presence of forskolin. These responses were inhibited by prior treatment with pertussis toxin. Hence, mastoparan stimulates arachidonic acid release via a pertussis toxin-sensitive G protein in Swiss 3T3 cells. Arachidonic acid, like mastoparan, stimulated DNA synthesis in the presence of insulin. The ability of mastoparan to stimulate mitogenesis was reduced by pertussis toxin treatment. These results demonstrate, for the first time, that mastoparan stimulates reinitiation of DNA synthesis in Swiss 3T3 cells and indicate that this peptide may be a useful probe to elucidate signal transduction mechanisms in mitogenesis.     

Protein kinase D2 potentiates MEK/ERK/RSK signaling, c-Fos accumulation and DNA synthesis induced by bombesin in Swiss 3T3 cells

Protein kinase D (PKD) plays an important role in mediating cellular DNA synthesis in response to G protein-coupled receptor (GPCR) agonists but the function of other isoforms of the PKD family has been much less explored. Here, we examined whether PKD2 overexpression in Swiss 3T3 cells facilitates DNA synthesis and the activation of the extracellular regulated protein kinase (ERK) pathway in response to the mitogenic GPCR agonist bombesin. We show that PKD2 overexpression markedly potentiated the ability of this agonist to induce DNA synthesis. Addition of bombesin to Swiss 3T3 cells overexpressing PKD2 also induced a striking increase in the duration of MEK/ERK/RSK activation as compared with cultures of control cells. In contrast, neither DNA synthesis nor the duration of ERK activation in response to epidermal growth factor, which acts via protein kinase C/PKD2-independent pathways, was increased. Furthermore, bombesin promoted a striking accumulation of c-Fos protein in cells overexpressing PKD2. Our study demonstrates that PKD2, like PKD, facilitates mitogenesis and supports the hypothesis that an increase in the duration of the ERK signaling leading to accumulation of immediate gene products is one of the mechanisms by which isoforms of the PKD family enhance re-initiation of DNA synthesis by Gq-coupled receptor activation.     

Cyclic AMP does not mediate inhibition of DNA synthesis by interferon in mouse Swiss 3T3 cells

The purpose of this study was to determine whether cyclic AMP (cAMP) plays any direct or indirect role in the antiproliferative effect of mouse L-cell interferon in Swiss 3T3 cells. Firstly, we found that interferon did not affect intracellular levels of cAMP in these cells in the absence or the presence of cAMP-elevating agents. Secondly, we examined the effect of interferon on the stimulation of DNA synthesis of quiescent 3T3 cells by a range of cyclic AMP-elevating agents, including cholera toxin, cAMP derivatives, and prostaglandin E, added in the presence of insulin or vasopressin. Interferon inhibited cyclic AMP-stimulated DNA synthesis as measured by incorporation of radioactive thymidine into acid-insoluble material and autoradiographic analysis of the fraction of labelled cells. Dose-response curves and kinetics of inhibition were identical to those obtained in cultures stimulated by combinations of growth factors that do not increase the intracellular level of cAMP. The inhibition by interferon of cAMP-stimulated DNA synthesis was also observed in secondary cultures of mouse embryo fibroblasts, where cAMP-elevating agents provide a mitogenic signal in the absence of other added growth factors. These results show that the inhibitory effect of interferon on DNA synthesis in Swiss 3T3 cells is not mediated by cyclic AMP.     

Homologous and heterologous mitogenic desensitization of Swiss 3T3 cells to phorbol esters and vasopressin: role of receptor and postreceptor steps

Prolonged treatment of Swiss 3T3 cells with phorbol 12,13 dibutyrate (PDB) rendered the cells refractory to subsequent mitogenic stimulation by both PDB and vasopressin. In contrast, the cells retained full responsiveness to a wide variety of other mitogens. An early response to vasopressin and phorbol esters, inhibition of (125I)-labeled epidermal growth factor [(125I)-EGF] binding, was also substantially decreased in PDB pretreated cells. The cross desensitization was not produced by vasopressin; this ligand induced homologous but not heterologous desensitization. Exposure of Swiss 3T3 cells to PDB caused a down regulation of (3H)-PDB receptors but did not reduce the binding of vasopressin to refractory cells. The time-course (t1/2 = 7 h) and dependence on PDB concentration (half maximal at 20 nM) for this phorbol ester receptor loss paralleled the induction of the mitogenic desensitizations to both PDB and vasopressin. However, the time-course of recovery revealed an important dissociation between receptor presence and mitogenic response. When Swiss 3T3 cultures, which had been pretreated with PDB, were washed to remove this ligand and incubated in its absence for 24 h, both (3H)-PDB receptors and PDB or vasopressin inhibition of (125I)-EGF binding were almost completely restored to control levels. However the homologous and heterologous mitogenic desensitizations showed a very different reversal time. After a 24-h recovery period PDB-treated refractory cells were still unable to synthesize DNA in response to PDB or vasopressin. The mitogenic desensitizations were however completely reversible; after a 48-h incubation in the absence of PDB the cells responded fully to the mitogenic actions of PDB or vasopressin. This finding suggests that a further postreceptor step was also desensitized by prolonged PDB treatment. The presence of a low level of cycloheximide during the PDB pretreatment blocked induction of this postreceptor refractoriness. We propose that this refractory postreceptor step selectively blocks both PDB and vasopressin stimulation of DNA synthesis and may represent the point at which the mitogenic pathways of phorbol esters and vasopressin converge.     

Amyloid beta protein precursor is a mitogen

The form of the secreted amyloid beta-protein precursor which contains the protease inhibitor sequence is mitogenic for Swiss 3T3 cells, while the precursor molecule lacking the protease inhibitor domain is not. A ten-fold stimulation of DNA synthesis occurs at 8 x 10(-9) M protein.     

Sphingomyelinase and cell-permeable ceramide analogs stimulate cellular proliferation in quiescent Swiss 3T3 fibroblasts.

Sphingomyelin or the products derived from its metabolism may constitute a signaling system involved in a variety of cellular processes. The activation of a plasma membrane neutral sphingomyelinase, which catalyzes the first step in sphingomyelin turnover, has been suggested to play an important role in cellular differentiation. We have studied the effect of exogenous staphylococcal sphingomyelinase on DNA synthesis and on the composition of membrane sphingolipids in quiescent Swiss 3T3 fibroblasts. Sphingomyelinase stimulated proliferation of Swiss 3T3 cells and potentiated the mitogenic action of other growth factors, such as insulin, epidermal growth factor, and bombesin. Treatment with sphingomyelinase produced a significant decrease in sphingomyelin accompanied by a corresponding increase in ceramide levels. No significant increases were detected in the levels of products derived from ceramide, i.e. ceramide 1-phosphate, sphingosine, or sphingosine 1-phosphate. To further investigate the role of ceramide in cellular proliferation, we studied the effect of cell-permeable analogs of ceramide on DNA synthesis in quiescent Swiss 3T3 cells. Both N-hexanoylsphingosine and N-acetylsphingosine at low concentrations stimulated [3H]thymidine incorporation and acted synergistically with a wide variety of growth factors known to induce proliferation of quiescent Swiss 3T3 fibroblasts. Similar effects were observed with bovine brain ceramides. These results suggest that ceramide may be involved in the regulation of cellular proliferation.