Ethanol potentiates the stimulatory effects of phorbol ester, sphingosine and 4-hydroxynonenal on the hydrolysis of phosphatidylethanolamine in NIH 3T3 cells

Ethanol and other alcohols have been shown to specifically stimulate phospholipase-D-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts. Here, we further examined the possible mechanism of this ethanol action. Ethanol (10-300 mM) and the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol 13-acetate (TPA) had synergistic stimulatory effects on the degradation of preformed [14C]PtdEtn when added in combination to [14C]ethanolamine-labelled suspended NIH 3T3 cells 30 min after collection of cells by scraping. Scraping caused a transient increase, lasting for less than 30 min, in the cellular content of 1,2-diacylglycerol, another PKC activator. Initially (0-50 min incubation), the main water-soluble product of [14C]PtdEtn degradation in ethanol plus TPA-treated cells was [14C]ethanolamine, while later (90 min) the main product of [14C]PtdEtn hydrolysis was [14C]ethanolamine phosphate in the presence of these agents. Ethanol also potentiated the specific stimulatory effects of sphingosine (through phospholipase D) and 4-hydroxynonenal (not involving phospholipase D) on PtdEtn hydrolysis. The effects of these latter agents were unrelated to PKC activation. These data indicate that the observed potentiating effects of ethanol on PtdEtn hydrolysis do not involve direct regulation of PKC or phospholipase D activities.     

Phorbol ester stimulates the hydrolysis of phosphatidylethanolamine in leukemic HL-60, NIH 3T3, and baby hamster kidney cells

Treatment of leukemic HL-60, NIH 3T3, and baby hamster kidney (BHK-21) cells, prelabeled with [2-14C]ethanolamine, with 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, resulted in increased degradation of both 14C-labeled phosphatidylethanolamine and its alkenyl (plasmalogen) derivate. A half-maximal and a maximal (approximately 3.4-fold) stimulation of ethanolamine phospholipid degradation required 3 and 10-20 nM TPA, respectively. TPA had a similar concentration-dependent stimulatory effect on the hydrolysis of phosphatidylcholine in cells previously prelabeled with [methyl-14C]choline. Increased phospholipid degradation was not accompanied by the formation of lysophosphatidylethanolamine, indicating that a phospholipase A-type enzyme was not involved. About 80% of total water-soluble degradation products was ethanolamine, suggesting that phospholipid hydrolysis was catalyzed by a phospholipase D-type enzyme. Increased formation of ethanolamine with exposure of cells to TPA was observed only after a 10-min lag period. Mezerein, bryostatin, sn-1-oleoyl-2-acetylglycerol, and polymyxin B, all of which mimic the action of TPA on protein phosphorylation in vivo, also stimulated the hydrolysis of ethanolamine phospholipids in HL-60 cells, suggesting that the TPA effect was mediated by protein kinase C.     

Cooperative effects of ethanol and protein kinase C activators on phospholipase-D-mediated hydrolysis of phosphatidylethanolamine in NIH 3T3 fibroblasts.

In a previous study, ethanol was shown to enhance the stimulatory effect of phorbol 12-myristate 13-acetate (PMA), a prominent activator of protein kinase C (PKC), on phospholipase-D (PLD)-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) in NIH 3T3 fibroblasts (Kiss et al. (1991) Eur. J. Biochem. 197, 785-790). Here, the mechanism and possible significance of ethanol-stimulated PtdEtn hydrolysis was further studied. In [14C]ethanolamine-labeled NIH 3T3 fibroblasts, 10 mM ethanol enhanced PMA-induced hydrolysis of PtdEtn 1.5-2.0-fold during a 2.5-15-min incubation period. Other alcohols, including glycerol, methanol, and 1-propanol, also enhanced PMA-induced PtdEtn hydrolysis. Of the other PLD activators tested, ethanol potentiated the PKC-dependent stimulatory effect of bombesin but failed to alter the apparently PKC-independent stimulatory effect of serum. Pretreatment of [14C]ethanolamine-labeled fibroblasts with 200 mM ethanol for 20 min resulted in increased (approx. 2-fold) hydrolysis of [14C]PtdEtn in isolated membranes. In membranes from ethanol-treated, but not from untreated, cells, PMA further enhanced (approx. 1.5-fold) the production of [14C]ethanolamine. Ethanol exerted none of the above stimulatory effects on phosphatidylcholine hydrolysis. These results suggest that the specific stimulatory action of ethanol on PLD-mediated PtdEtn hydrolysis can occur in vivo and may involve increased binding of a regulatory PKC-isoform to membranes.     

Heparin-binding growth factor 1 stimulates tyrosine phosphorylation in NIH 3T3 cells.

Tyrosine phosphorylation of cellular proteins induced by heparin-binding growth factor 1 (HBGF-1) was studied by using the murine fibroblast cell line NIH 3T3 (clone 2.2). HBGF-1 specifically induced the rapid tyrosine phosphorylation of polypeptides of Mr 150,000, 130,000, and 90,000 that were detected with polyclonal and monoclonal antiphosphotyrosine (anti-P-Tyr) antibodies. The concentration of HBGF-1 required for half-maximal induction of tyrosine phosphorylation of the Mr-150,000 Mr-130,000, and Mr-90,000 proteins was approximately 0.2 to 0.5 ng/ml, which was consistent with the half-maximal concentration required for stimulation of DNA synthesis in NIH 3T3 cells. HBGF-1-induced tyrosine phosphorylation of the Mr-150,000 and Mr-130,000 proteins was detected within 30 s, whereas phosphorylation of the Mr-90,000 protein was not detected until 3 min after HBGF-1 stimulation. All three proteins were phosphorylated maximally after 15 to 30 min. Phosphoamino acid analysis of the Mr-150,000 and Mr-90,000 proteins confirmed the phosphorylation of these proteins on tyrosine residues. Phosphorylation of the Mr-150,000 and Mr-90,000 proteins occurred when cells were exposed to HBGF-1 at 37 degrees C but not at 4 degrees C. Exposure of cells to sodium orthovanadate, a potent P-Tyr phosphatase inhibitor, before stimulation with HBGF-1 resulted in enhanced detection of the Mr-150,000, Mr-130,000, and Mr-90,000 proteins by anti-P-Tyr antibodies. Anti-P-Tyr affinity-based chromatography was used to adsorb the HBGF-1 receptor affinity labeled with 125I-HBGF-1. The cross-linked HBGF-1 receptor-ligand complex was eluded with phenyl phosphate as two components: Mr 170,000 and 150,000. P-Tyr, but not phosphoserine or phosphothreonine, inhibited adsorption of the (125)I-HBGF-1-receptor complex to the anti-P-Tyr antibody matrix. Treatment of cells with sodium orthovanadate also enhanced recognition of the cross-linked (125)I-HBGF-1-receptor complex by the anti-P-Tyr matrix. These data suggest that (i) the (125)I-HBGF-1-receptor complex is phosphorylated on tyrosine residues and (ii) HBGF-1-induced signal transduction involves, in part, the tyrosine phosphorylation of at least three polypeptides.     

Ha-ras-transformation alters the metabolism of phosphatidylethanolamine and phosphatidylcholine in NIH 3T3 fibroblasts

Cultured NIH 3T3 fibroblasts were employed to investigate the changes in the phospholipid metabolism induced by Ha-ras transformation. All phospholipid fractions were reduced in ras-transformed fibroblasts except phosphatidylethanolamine (PE). The incorporation of labeled choline and ethanolamine into phosphatidylcholine (PC), PE and their corresponding metabolites were elevated in a similar manner in the transformed cells. The enhanced uptake of choline and ethanolamine correlated with the activation of choline kinase and ethanolamine kinase. Similarly, the uptake of arachidonic, oleic and palmitic acids by PC and PE was higher in ras-cells. Acyl-CoA synthetases, which esterify fatty acid before their incorporation into lysophospholipids, were also activated. However, both CTP:phosphocholine-cytidylyltransferase and CTP:phosphoethanolamine-chytidyltransferase were inhibited in the transformed cells. This fact, taken together with the observed activation of choline- and ethanolamine kinases, led to accumulation of phosphocholine and phosphoethanolamine, which have been presumed to participate in the processes of tumor development. PC biosynthesis seemed to be carried out through the CDP-choline pathway, which was stimulated in the oncogenic cells, whereas PE was more likely, a product of phosphatidylserine decarboxylation rather than the CDP-ethanolamine pathway.     

Calpain inhibition stimulates caspase-dependent apoptosis induced by taxol in NIH3T3 cells

Taxol is an anticancer drug that triggers apoptosis in a wide spectrum of cancers such as ovarian, breast, lung, head and neck, and bladder carcinoma by both caspase-dependent and -independent apoptosis mechanisms. However, the exact signaling pathways involved in taxol-induced apoptosis strongly depend on the cellular background and they are not completely established yet. In this study we demonstrate that taxol induces caspase-3-independent apoptosis in NIH3T3 cells by a calpain-mediated mechanism. Taxol treatment produced changes in the mitochondrial membrane potential (Delta Psi m) which could be responsible of Ca(2+) release from the mitochondria and the consequent calpain activation. Interestingly, we show that calpain produced proteolysis of caspase-3 and demonstrate that, accordingly, calpain inhibition increased taxol-induced apoptosis. In addition, we reveal that poly (ADP-ribose) polymerase (PARP) was processed by calpain in taxol-treated cells and by caspase-3 after calpain inhibition. In conclusion, these results demonstrate for the first time that calpain could play an important role modulating taxol-induced apoptosis. Further studies are needed to address the potentiality of inducing apoptosis by a combined use of taxol and calpain inhibitors in cells with increased calpain activity.     

V-fos stimulates expression of the alpha 1(III) collagen gene in NIH 3T3 cells

NIH 3T3 cells that are transformed by the v-fos containing FBR proviral DNA show a selective increase in alpha 1 (III) collagen synthesis, increased levels of alpha 1(III) collagen RNA and an increased synthesis of this RNA.     

Bombesin stimulates the rapid activation of phospholipase A2-catalyzed phosphatidylcholine hydrolysis in Swiss 3T3 cells.

In Swiss 3T3 fibroblasts bombesin stimulated the release of arachidonic acid in a time- and dose-dependent manner. Arachidonate levels were significantly elevated after only a 2-s stimulation with the agonist. Furthermore, by measuring the arachidonate content of cellular phospholipids after cell activation, it was shown that there was selective depletion from phosphatidylcholine over the same time course. The corresponding production of lysophosphatidylcholine suggested the involvement of a phosphatidylcholine-specific phospholipase A2. Initial arachidonic acid release was not dependent on the presence of extracellular calcium, not activated by treatment of the cells with thapsigargin, and was unaffected by down-regulation of protein kinase C activity, or by treatment of the cells with the protein kinase C inhibitor staurosporine. These data strongly suggest that occupation of the bombesin receptor is closely coupled to activation of phospholipase A2 which results in the rapid release of arachidonic acid from phosphatidylcholine.     

Anandamide stimulates phospholipase D activity in PC12 cells but not in NIH 3T3 fibroblasts

The endogenous cannabinoid arachidonoylethanolamide was previously reported to have no effects on the phospholipase D activity in Chinese hamster ovary cells expressing the human brain-specific cannabinoid receptor, while in mouse peritoneal cells, delta9-tetrahydrocannabinol stimulated this enzyme. In this work, arachidonoylethanolamide (0.1-1 microM) was found to stimulate the phospholipase D-mediated phospholipid hydrolysis in rat adrenal pheochromocytoma PC12 cells, but not in mouse NIH 3T3 fibroblasts. The phospholipase D-activating effects of arachidonoylethanolamide were comparable to those elicited by phorbol ester and nerve growth factor, while arachidonic acid (1 microM) had no effects. The results show that, depending on the cell type, arachidonoylethanolamide can be an activator of the phospholipase D system.     

Differential phospholipase D activation by bradykinin and sphingosine 1-phosphate in NIH 3T3 fibroblasts overexpressing gelsolin.

Gelsolin, an actin-binding protein, shows a strong ability to bind to phosphatidylinositol 4,5-bisphosphate (PIP(2)). Here we showed in in vitro experiments that gelsolin inhibited recombinant phospholipase D1 (PLD1) and PLD2 activities but not the oleate-dependent PLD and that this inhibition was not reversed by increasing PIP(2) concentration. To investigate the role of gelsolin in agonist-mediated PLD activation, we used NIH 3T3 fibroblasts stably transfected with the cDNA for human cytosolic gelsolin. Gelsolin overexpression suppressed bradykinin-induced activation of phospholipase C (PLC) and PLD. On the other hand, sphingosine 1-phosphate (S1P)-induced PLD activation could not be modified by gelsolin overexpression, whereas PLC activation was suppressed. PLD activation by phorbol myristate acetate or Ca(2+) ionophore A23187 was not affected by gelsolin overexpression. Stimulation of control cells with either bradykinin or S1P caused translocation of protein kinase C (PKC) to the membranes. Translocation of PKC-alpha and PKC-beta1 but not PKC-epsilon was reduced in gelsolin-overexpressed cells, whereas phosphorylation of mitogen-activated protein kinase was not changed. S1P-induced PLC activation and mitogen-activated protein kinase phosphorylation were sensitive to pertussis toxin, but PLD response was insensitive to such treatment, suggesting that S1P induced PLD activation via certain G protein distinct from G(i) for PLC and mitogen-activated protein kinase pathway. Our results suggest that gelsolin modulates bradykinin-mediated PLD activation via suppression of PLC and PKC activities but did not affect S1P-mediated PLD activation.     

Phorbol ester stimulates the synthesis of sphingomyelin in NIH 3T3 cells. A diminished response in cells transformed with human A-raf carrying retrovirus

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated the synthesis of sphingomyelin (CerPCho) from a [¹⁴C]choline-labelled phosphatidylcholine (PtdCho) pool in NIH 3T3 cells. Maximal stimulation (68%) of CerP-Cho synthesis, accompanied by an increase (38%) in its cellular content, required only 2 nM TPA. Higher concentrations of TPA (2–100 nM) had progressively less effect on CerPCho synthesis which correlated with increased hydrolysis of precursor PtdCho. In cells transformed with human or mouse A-raf carrying retroviruses TPA-stimulated PtdCho hydrolysis, but not CerPCho synthesis, suggesting independent regulation of these processes by the TPA-stimulated signal transduction system.     

Characterization of metformin transport system in NIH 3T3 cells.

The biochemical properties of the metformin transport system were studied in NIH 3T3 cells. 14C-metformin uptake appeared to be a sodium dependent process. Iso-osmotical replacement of Na+ by choline chloride in the assay medium resulted in a decrease of metformin uptake. Amiloride (200 microM) inhibited the metformin transport by 35% in these cells. Gramicidin, a channel ionophore, was the most effective in inhibiting the metformin transport as compared to valinomycin, a mobile ion carrier, and Ca2+ ionophore (A 23187). Loading of cells with asparagine, ornithine, or polylysine did not influence the uptake process. However, the addition of lysine or arginine significantly stimulated the metformin uptake by NIH 3T3 cells. Similarly, the addition of metformin stimulated the arginine uptake by these cells, suggesting that metformin shares the y+ transport system. Metformin inhibited competitively the uptake of 14C-spermidine, a molecule of the polyamine family, by NIH 3T3 cells, whereas the latter failed to influence the uptake of the former significantly by these cells. Incubation of NIH 3T3 cells in the presence of difluoromethyl-ornithine (a suicidal inhibitor of polyamine biosynthesis) stimulated the spermidine, but not the metformin, uptake by these cells. Interestingly, a prolonged incubation of these cells in the presence of metformin failed to down-regulate the spermidine transport process. The spermidine- and methylglyoxal-bis(guanylhydrazone), MGBG-transport deficient (3T3MG) cells which do not accumulate exogeneous spermidine or MGBG, took up 14C-metformin. However, 14C-metformin uptake by 3T3MG cells was lower than that by normal NIH 3T3 cells.     

Isolation and characterization of NIH 3T3 cells expressing polyomavirus small T antigen.

The polyomavirus small T-antigen gene, together with the polyomavirus promoter, was inserted into a retrovirus vector pGV16 which contains the Moloney sarcoma virus long terminal repeat and neomycin resistance gene driven by the simian virus 40 promoter. This expression vector, pGVST, was packaged into retrovirus particles by transfection of psi 2 cells which harbor packaging-defective murine retrovirus genome. NIH 3T3 cells were infected by this replication-defective retrovirus containing pGVST. Of the 15 G418-resistant cell clones, 8 express small T antigen at various levels as revealed by immunoprecipitation. A cellular protein with an apparent molecular weight of about 32,000 coprecipitates with small T antigen. Immunofluorescent staining shows that small T antigen is mainly present in the nuclei. Morphologically, cells expressing small T antigen are indistinguishable from parental NIH 3T3 cells and have a microfilament pattern similar to that in parental NIH 3T3 cells. Cells expressing small T antigen form a flat monolayer but continue to grow beyond the saturation density observed for parental NIH 3T3 cells and eventually come off the culture plate as a result of overconfluency. There is some correlation between the level of expression of small T antigen and the growth rate of the cells. Small T-antigen-expressing cells form small colonies in soft agar. However, the proportion of cells which form these small colonies is rather small. A clone of these cells tested did not form tumors in nude mice within 3 months after inoculation of 10(6) cells per animal. Thus, present studies establish that the small T antigen of polyomavirus is a second nucleus-localized transforming gene product of the virus (the first one being large T antigen) and by itself has a function which is to stimulate the growth of NIH 3T3 cells beyond their saturation density in monolayer culture.     

Cytotoxicity of the isoflavone genistein in NIH 3T3 cells

Genistein is one of the naturally occurring isoflavones present in plants such as soybeans and is commonly found in a variety of human foods. A number of studies indicated that this class of compounds exerts anticancerogenic and antimutagenic effects in various in vitro systems and in vivo animal models. We studied the effects of genistein on NIH 3T3 cells in in vitro models. The isoflavone genistein has been identified as having antiproliferative and apoptotic effects on various malignant cell types derived from solid tumors. Therefore, the cytotoxic and apoptotic properties of this compound were studied by MTT assay and Hoechst 33258/propidium iodide staining technique. The morphological changes of cells were examined in inverted fluorescent microscope. The oxidation of protein thiol groups and thiobarbituric-acid-reactive species (TBARS) was also determined. The cells were exposed to different concentrations of genistein (0-90 microM) after 24 h of incubation. The results revealed that genistein in concentrations higher than 20 microM significantly reduced cell viability, caused cell morphological changes and induced apoptotic and necrotic cell death. Oxidative modification of protein increased in the cells exposed to genistein in a dose- and time-dependent manner. In conclusion, our preliminary in vitro studies demonstrate the damaging effects of genistein on the mouse embryonic fibroblast cell line.     

Cholera toxin stimulates division of 3T3 cells.

Cholera toxin was used in an attempt to inhibit epidermal growth factor stimulated 3T3 cell division. Instead, cholera toxin alone at low concentrations (10(-10) M), was able to stimulate cell division and could augment EGF stimulated cell division. The mitogenic effect of cholera toxin can occur despite a dramatic increase in the intracellular levels of cAMP in 3T3 cells. Cholera toxin stimulated mitogenesis could not be mimicked by choleragenoid, the binding but inactive subunit of cholera toxin, or by other agents which elevate cAMP levels in 3T3 cells.     

Epidermal growth factor-induced phosphoinositide hydrolysis in permeabilized 3T3 cells: lack of guanosine triphosphate dependence and inhibition by tyrosine-containing peptides.

The possible involvement of a stimulatory guanosine triphosphate (GTP)-binding (G) protein in epidermal growth factor (EGF)-induced phosphoinositide hydrolysis has been investigated in permeabilized NIH-3T3 cells expressing the human EGF receptor. The mitogenic phospholipid lysophosphatidate (LPA), a potent inducer of phosphoinositide hydrolysis, was used as a control stimulus. In intact cells, pertussis toxin partially inhibits the LPA-induced formation of inositol phosphates, but has no effect on the response to EGF. In cells permeabilized with streptolysin-O, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) dramatically increases the initial rate of inositol phosphate formation induced by LPA. In contrast, activation of phospholipase C (PLC) by EGF occurs in a GTP-independent manner. Guanine 5'-O-(2-thiodiphosphate) (GDP beta S) which keeps G proteins in their inactive state, blocks the stimulation by LPA and GTP gamma S, but fails to affect the EGF-induced response. Tyrosine-containing substrate peptides, when added to permeabilized cells, inhibit EGF-induced phosphoinositide hydrolysis without interfering with the response to LPA and GTP gamma S. These data suggest that the EGF receptor does not utilize an intermediary G protein to activate PLC and that receptor-mediated activation of effector systems can be inhibited by exogenous substrate peptides.     

A new lectin in red kidney beans called PvFRIL stimulates proliferation of NIH 3T3 cells expressing the Flt3 receptor

A new legume lectin has been identified by its ability to specifically stimulate proliferation of NIH 3T3 fibroblasts expressing the Flt3 tyrosine kinase receptor. The lectin was isolated from conditioned medium harvested from human peripheral blood mononuclear cells activated to secrete cytokines by a crude red kidney bean extract containing phytohemagglutinin (PHA). Untransfected 3T3 cells and 3T3 cells transfected with the related Fms tyrosine kinase receptor do not respond to this lectin, which we called PvFRIL (Phaseolus vulgaris Flt3 receptor-interacting lectin). When tested on cord blood mononuclear cells enriched for Flt3-expressing progenitors, purified PvFRIL fractions maintained a small population of cells that continued to express CD34 after 2 weeks in suspension cultures containing IL3. These cultures did not show the effects of IL3's strong induction of proliferation and differentiation (high cell number and exhausted medium); instead, low cell number at the end of the culture period resulted in persistence of cells in the context of cell death. These observations led to the hypothesis that PvFRIL acts in a dominant manner to preserve progenitor viability and prevent proliferation and differentiation.     

Abrogation of the effects of aphidicolin on NIH3T3 and V79 cells by caffeine

In this communication I show that caffeine (1,3,7-trimethylxanthine) stimulates [³H]thymidine incorporation in aphidicolin-treated V79 and NIH3T3 cells. Flow microfluorometric analysis showed that caffeine, partially or fully, abrogates the cell cycle progression block produced by aphidicolin. Increased cell growth is also observed in cultures treated with both aphidicolin and caffeine compared to cultures treated with aphidicolin only. Microscopic examination of V79 cultures treated with aphidicolin for 8 h showed a marked reduction in the freqeuncy of round mitotic cells, as is expected from a drug which inhibits progression through the cell cycle by inhibiting DNA replication; this effect of aphidicolin was also reduced by caffeine. Biochemical analysis showed that caffeine did not directly interfere with the inhibition of DNA polymerase-α by aphidicolin. Analysis of dNTP pools indicated that caffeine increased the level of dCTP in V79 cells. In aphidicolin-treated V79 cells, the increase in the dCTP level due to exogenous cytidine was almost completely blocked; caffeine also substantially overcame this effect of aphidicolin. These results indicate that caffeine produces its effects on aphidicolin-treated cells by altering the dCTP metabolism.     

ADAM15 overexpression in NIH3T3 cells enhances cell-cell interactions.

ADAM15 is a member of the family of metalloprotease-disintegrins that have been shown to interact with integrins in an RGD- and non-RGD-dependent manner. In the present study, we examined the effects of ADAM15 overexpression on cell-matrix and cell-cell interactions in NIH3T3 cells. Tetracycline-regulated ADAM15 overexpression in NIH3T3 cells leads to an inhibition of migration on a fibronectin-coated filter in a Boyden chamber assay and in a scratch wound model. The effects of ADAM15 overexpression on cell migration are not due to changes in matrix attachment or to the lack of extracellular signal-regulated kinase signaling response to PDGF or fibronectin. However, a decrease in monolayer permeability with ADAM15 overexpression and altered cell morphology suggest a possible increase in cell-cell interaction. Analysis of adhesion of NIH3T3 cells to a polyclonal population of cells retrovirally transduced to overexpress ADAM15 demonstrates a 45% increase in cell adhesion, compared with enhanced green fluorescent protein-expressing control cells. In addition, we demonstrate localization of HA-epitope-tagged ADAM15 to cell-cell contacts in an epithelial cell line that forms extensive cell-cell contact structures. Thus, overexpression of ADAM15 in NIH3T3 cells appears to enhance cell-cell interactions, as suggested by decreased cell migration, altered cell morphology at the wound edge, decreased monolayer permeability, and increased cell adhesion to monolayers of cells expressing ADAM15 by retroviral transduction.     

Human c-fgr induces a monocyte-specific enzyme in NIH 3T3 cells.

The mutant c-fgr protein (p58c-fgr/F523) containing Phe-523 instead of Tyr-523 exhibited transforming activity in NIH 3T3 cells like other protein-tyrosine kinases of the src family, but normal p58c-fgr (p58c-fgr/wt) did not. The mutant protein showed tyrosine kinase activity threefold higher than that of the normal protein in vitro. Surprisingly, transfection of the normal c-fgr gene into NIH 3T3 cells resulted in induction of sodium fluoride (NaF)-sensitive alpha-naphthyl butyrate esterase (alpha-NBE), a marker enzyme of cells of monocytic origin, which was not induced in v-src-, v-fgr-, or lyn-transfected NIH 3T3 cells. The NaF-sensitive alpha-NBE induced in c-fgr transfectants was shown by isoelectric focusing to have a pI of 5.2 to 5.4, a range which was the same as those for thioglycolate-induced murine peritoneal macrophages and 1 alpha,25-dihydroxyvitamin D3-treated WEHI-3B cells. Immunoblotting studies with antiphosphotyrosine antibodies revealed that 58-, 62-, 75-, 120-, 200-, and 230-kDa proteins were commonly phosphorylated at tyrosine residues in NIH 3T3 cells transfected with normal and mutated c-fgr, while 95-kDa protein was significantly phosphorylated at tyrosine residues in cells transfected with the mutated c-fgr. These findings suggest that tyrosine phosphorylation of specific cellular substrate proteins is important in induction of NaF-sensitive alpha-NBE and cell transformation by p58c-fgr.