Inhibition of DNA synthesis by protein kinase C-activating phorbol esters in NIH/3T3 cells

Fibroblast growth factor (FGF) plus insulin induced DNA synthesis in and proliferation of NIH/3T3 cells. The protein kinase C-activating phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), inhibited both the DNA synthesis and cell proliferation induced by FGF plus insulin. The concentration of TPA required for 50% inhibition of the DNA synthesis was about 5 nM. Phorbol-12,13-dibutyrate, another protein kinase C-activating phorbol ester, also inhibited the DNA synthesis but 4 alpha-phorbol-12,13-didecanoate, known to be inactive for this enzyme, was ineffective. DNA synthesis started at about 12 h after the addition of FGF plus insulin. The inhibitory action of TPA on the DNA synthesis was observed when it was added within 12 h after the addition of FGF plus insulin. These results suggest that phorbol esters exhibit an antiproliferative action through protein kinase C activation in NIH/3T3 cells, and that this action of phorbol esters is due to inhibition of the progression from the late G1 to the S phase of the cell cycle.     

Protein kinase D potentiates DNA synthesis and cell proliferation induced by bombesin, vasopressin, or phorbol esters in Swiss 3T3 cells

We examined whether protein kinase D (PKD) overexpression in Swiss 3T3 cells potentiates the proliferative response to either the G protein-coupled receptor agonists bombesin and vasopressin or the biologically active phorbol ester phorbol 12,13-dibutyrate (PDBu). In order to generate Swiss 3T3 cells stably overexpressing PKD, cultures of these cells were infected with retrovirus encoding murine PKD and green fluorescent protein (GFP) expressed as two separate proteins translated from the same mRNA. GFP was used as a marker for selection of PKD-positive cells. PKD overexpressed in Swiss 3T3 cells was dramatically activated by cell treatment with bombesin or PDBu as judged by in vitro kinase autophosphorylation assays and exogenous substrate phosphorylation. Concomitantly, these stimuli induced PKD phosphorylation at Ser(744), Ser(748), and Ser(916). PKD activation and phosphorylation were prevented by exposure of the cells to protein kinase C-specific inhibitors. Addition of bombesin, vasopressin, or PDBu to cultures of Swiss 3T3 cells overexpressing PKD induced a striking increase in DNA synthesis and cell number compared with cultures of Swiss 3T3-GFP cells. In contrast, stimulation of DNA synthesis in response to epidermal growth factor, which acts via protein kinase C/PKD-independent pathways, was not enhanced. Our results demonstrate that overexpression of PKD selectively potentiates mitogenesis induced by bombesin, vasopressin, or PDBu in Swiss 3T3 cells.     

Endothelin stimulates DNA synthesis in Swiss 3T3 cells. Synergy with polypeptide growth factors.

The vasoactive peptide endothelin is shown to be a potent mitogen for Swiss 3T3 cells. Although endothelin has little effect on DNA synthesis when added alone to cells in serum-free medium, the peptide synergizes very strongly with several other growth factors. A half-maximal response to endothelin is observed at approx. 0.3 nM, with a maximal effect at 3 nM. Over the same concentration range, endothelin stimulates a 2-fold increase in the accumulation of cellular inositol phosphates. Endothelin may prove to be a useful additional agonist for studying the signalling pathways involved in the control of 3T3-cell proliferation.     

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.     

Initiation of DNA synthesis in quiescent Swiss 3T3 and 3T6 cells by lanthanum

La³⁺ stimulated quiescent Swiss 3T3 and 3T6 ceils to enter the DNA-synthesizing S phase of the cell cycle. La³⁺ and insulin interacted synergistically to increase DNA synthesis. A brief exposure of the cells to soluble LaCl₃ optimally stimulated entry into S. La³⁺ was similar to Al³⁺ in its mitogenic properties (J. Cell. Physiol.118 , 298, 1984), but La³⁺ was 10 times more potent than Al³⁺.     

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.     

Adenosine receptor activation in quiescent Swiss 3T3 cells. Enhancement of cAMP levels, DNA synthesis and cell division

Anti-tubulin immunofluorescence microscopy is used here to demonstrate that eggs of Lytechinus variegatus are induced to assemble cytoplasmic microtubules upon artificial activation. These microtubules progress through three distinct configurations followed by cycles of abortive division. The first of these is a configuration in which microtubules are found in a disordered network near the egg cortex; the progressive thickening of the microtubule-containing layer appears to be responsible for the centripetal movement of the egg nucleus that occurs shortly after activation. These microtubules are replaced at about 40 min by a population of long, radially arrayed microtubules, which are restructured by about 70 min to form the apolar mitotic apparatus. Each of the microtubule configurations characteristic of activated eggs becomes more prominent when eggs are treated at the appropriate times after activation with the microtubule-stabilizing drug taxol. Any microtubule organizing centers within the activated egg must have very limited authority, since aster-like structures are not seen, and microtubules are not observed to be closely associated with the nucleus or egg cortex. Activation of eggs with ammonia in Ca²⁺-free sea water (a treatment that bypasses the cortical reaction and the Ca²⁺ transient) induces the appearance of microtubules as readily and in the same patterns as does treatment with ionophore A23187 or butyric acid, both of which activate by inducing an intracellular calcium release and the cortical reaction.     

Calcium-dependent stimulation of BALB/c 3T3 mouse cell DNA synthesis by a tumor-promoting phorbol ester (PMA).

During the first six passages after their arrival in this laboratory, BALB/c 3T3 mouse cells did not proliferate in serum containing-medium having an ionic calcium concentration of 0.05 mM or less, but by the ninth passage they had become able to multiply in the presence of these lower calcium levels. In low calcium (e.g., 0.02 mM) medium, passage 1-6, cells in sparse cultures were blocked at the Gl/S boundary of their cycle. These blocked cells could be induced to start making DNA within only one hour either by returning the ionic calcium level to a normal range of values (1.25 mM), or by adding 0.05 mug/ml of PMA (12-O-tetradecanoyl-phorbol-13-acetate). PMA probably acted by sensitizing the blocked cells to calcium rather than replacing the ion, because it was ineffective in ionic calcium-free medium. Finally, PMA did not by itself induce proliferation of cells (regardless of the number of passages) which had been proliferatively inactivated by density-dependent factors in confluent cultures. However, PMA did promote DNA synthesis by these cells during their brief transition to the "cycling" state caused by exposure to fresh serum-containing medium.     

The stimulation of DNA synthesis in resting Swiss 3T3 cells by non-histone chromosomal proteins

Nonhistone chromosomal proteins (NHC), isolated from the kidney and liver of intact rats, the liver of rats treated with hepatocarcinogen DEN and the rat hepatoma, stimulate DNA synthesis in Swiss 3T3 cells in resting culture. The maximum stimulating effect was obtained in the presence of narrow NHC fractions eluted with 0.4-0.5 M NaCl from the phosphocellulose column and identified as hetero-organic NHC protein antigens of the kidney origin associated with hepatocellular tumors.     

Growth factor- and dexamethasone-induced proteins in Swiss 3T3 cells. Relationship to DNA synthesis

Dexamethasone synergistically enhances the stimulation of DNA synthesis in quiescent Swiss 3T3 cells by cartilage-derived growth factor (CDGF) while having no consistent effect when added with platelet-derived growth factor (PDGF) or serum. We examined the hypothesis that this difference might be attributed to selective synthesis of individual proteins early in the G1 phase of the cell cycle. Swiss 3T3 cells were treated with CDGF, PDGF, and fetal bovine serum for 3 h, with or without dexamethasone, and [35S]methionine-labeled proteins were separated by two-dimensional electrophoresis on giant gels. Over 3300 proteins could be distinguished; 34 of these were consistently induced more than 3-fold by all three factors, while an additional 30 inductions were variably present. Dexamethasone by itself induced 8 other proteins, and at least 9 growth factor inductions were synergistically enhanced by addition of the hormone. To identify proteins intimately associated with growth control, we looked for inductions that reflected the dexamethasone synergy with CDGF on DNA synthesis and lack of such an effect with PDGF. The induction of only one group of proteins, the Band 1 isoforms (44-46 kDa, pI 6.1-5.9) displayed such selective synergy. The majority of the other growth factor inductions were inhibited by dexamethasone, even in the context of maximal DNA synthesis, implying that their increased synthesis is not required for growth. When 3T3 cells were treated with increasing doses of CDGF with and without dexamethasone, autoradiographic densities of induced proteins varied in a dose-responsive fashion. However, only levels of the Band 1 proteins bore a constant linear relationship to DNA synthesis, suggesting that they play an important role in early control of the cell cycle.     

Identification of multiple PKC isoforms in Swiss 3T3 cells: differential down-regulation by phorbol ester.

The expression of members of the Ca2+ and phospholipid-dependent protein kinase (PKC) family were studied in murine Swiss 3T3 cells. In addition to PKC-alpha, the presence of immunoreactive PKC-delta, -epsilon, and zeta was detected. Treatment with 500 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) led to the down-regulation of alpha, delta, and epsilon isoforms, but not that of zeta. Higher concentrations of TPA similarly had no effect on the level of PKC-zeta. In contrast to PKC-alpha, the membrane localization of PKC-delta, -epsilon, and -zeta was not enhanced by extraction in Ca(2+)-containing buffers, whereas acute TPA treatment increased membrane association of PKC-alpha, -delta, and -epsilon but not that of PKC-zeta.     

Inhibitory action of tumor-promoting phorbol esters upon beta-adrenergic agonist stimulation of cyclic AMP accumulation in rat prostatic epithelial cells

The pretreatment of rat prostatic epithelial cells with 4 beta-phorbol 12-myristate 13-acetate resulted in an attenuation of beta-adrenergic stimulated cyclic AMP accumulation. The effect was dependent on time and concentration. The maximal extent of isoproterenol stimulation of cyclic AMP production was reduced by 35% after 15-min pretreatment with the phorbol ester at 25 degrees C. Since a similar action was exerted by other protein kinase C stimulators, present results suggest the involvement of this enzyme in a process of desensitization to beta-adrenergic agonists of the adenylate cyclase system in rat prostatic epithelium.     

Different sensitivity to phorbol esters and pertussis toxin of bombesin- and platelet-derived growth factor-induced, phospholipase C-mediated hydrolysis of phosphoinositides in NIH/3T3 cells

Incubation of the serum-deprived cultures of NIH/3T3 cells with bombesin or platelet-derived growth factor (PDGF) induced the phospholipase C-mediated hydrolysis of phosphoinositides. Protein kinase C-activating 12-O-tetradecanoylphorbol 13-acetate (TPA) and pertussis toxin inhibited the bombesin-induced phospholipase C reactions. AlF4-, a direct activator of GTP-binding proteins (G proteins), also induced the phospholipase C reactions and TPA inhibited the AlF4- -induced reactions. These results suggest that a pertussis toxin-sensitive G protein is involved in the coupling of the bombesin receptor to the phospholipase C and that the coupling of the G protein to the phospholipase C is inhibited by protein kinase C. In contrast, neither TPA nor pertussis toxin inhibited the PDGF-induced phospholipase C reactions, indicating that a pertussis toxin-sensitive G protein is not involved in the coupling of the PDGF receptor to the phospholipase C and that this coupling is insensitive to protein kinase C. These results suggest that the regulatory mechanism of the PDGF receptor for the phospholipase C activation is different from that of the bombesin receptor.     

Receptor specificity of growth factor-stimulated synthesis of 3-phosphorylated inositol lipids in Swiss 3T3 cells.

We have investigated synthesis of 3-phosphorylated inositol lipids in growth factor-stimulated Swiss 3T3 cells. Those growth factors tested which act via tyrosine kinase-containing receptors (platelet-derived growth factor (PDGF), insulin growth factor I (IGF-I), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF)) caused the rapid synthesis of [32P]PtdIns(3,4)P2 and [32P]PtdIns(3,4,5)P3 (PtdIns is phosphatidylinositol) in [32P]P(i)-prelabeled cells and the appearance of an inositol lipid 3-OH kinase in antiphosphotyrosine immunoprecipates. In contrast, those growth factors tested which act via G-protein-coupled receptors (bombesin, vasopressin, prostaglandin E1) were unable to stimulate either of the above responses. Furthermore, while PDGF was able to increase the formation of PtdIns(3,4)P2 and PtdIns(3,4,5)P3 in streptolysin-permeabilized cells, guanosine 5'-3-(thio)triphosphate and guanyl-5'-yl imidodiphosphate were not. These results suggest that Swiss 3T3 cells possess the machinery for tyrosine kinase but not G-protein-mediated activation of PtdIns(4,5)P2 3-OH kinase; a situation which is the inverse to that recently described for human neutrophils. The tyrosine kinase-containing receptors differed markedly in their relative abilities to elevate the levels of [32P] PtdIns(3,4,5)P3 (ranked in the order PDGF greater than or equal to IGF-I greater than EGF greater than bFGF), [32P]Ptd-OH (PDGF greater than EGF greater than bFGF; undetectable for IGF-I), and [32P]PtdIns4P (EGF greater than bFGF greater than PDGF; undetectable for IGF-I) in [32P]P(i)-prelabeled cells. These differences are epitomized by IGF-I, which was the joint most powerful stimulus for [32P] PtdIns(3,4,5)P3 formation, but was unable to stimulate a measurable accumulation of [32P]Ptd-OH (and hence, by deduction, was unable to stimulate phospholipase C). These results indicate that there is a differential ability among the tyrosine kinase-containing receptors present in a single cell to recruit phospholipase C and PtdIns(4,5)P2 3-OH kinase into their signalling complexes and further emphasizes the notion that the rapid synthesis of PtdIns(3,4,5)P3 may be a signalling event.     

Sites of in vivo phosphorylation of the T cell tyrosine protein kinase in LSTRA cells and their alteration by tumor-promoting phorbol esters

The LSTRA cell line contains an elevated level of a tyrosine protein kinase of apparent molecular weight of 56,000 (pp56Tcell). Analysis of the tryptic fragments of this protein labeled in vivo with 32P shows that it contains four sites of tyrosine phosphorylation and one site of serine phosphorylation. Two of the sites of in vivo tyrosine phosphorylation are also labeled in vitro when membranes are incubated with [gamma-32P]ATP. One of the sites that is labeled in vivo and in vitro (site 1) is identical in sequence with the major site of tyrosine phosphorylation in the transforming protein of the Rous sarcoma virus. Analysis of the sites of in vivo phosphorylation in pp56Tcell from LSTRA cells treated with 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) reveals that this agent induces at least four new sites of serine phosphorylation. Treatment with PMA also causes a selective reduction in the level of tyrosine phosphorylation in site 1. Thus PMA causes new sites of serine phosphorylation in pp56Tcell and reduces the amount of phosphate in one of the sites of tyrosine phosphorylation.     

Aluminum ions stimulate DNA synthesis in quiescent cultures of Swiss 3T3 and 3T6 cells

Micromolar concentrations of AI3+ are shown to be strongly mitogenic for quiescent cultures of Swiss 3T3 and 3T6 cells. AI3+ caused a striking shift in the dose-response curve for the effect of fetal bovine serum on 3H-thymidine incorporation. In the absence of serum the mitogenic effect of aluminum was greatly potentiated by insulin or cholera toxin, but not epidermal growth factor or 12-0-tetradecanoyl-phorbol-13-acetate. The stimulation of DNA synthesis was maximal by 15-20 microM AI3+ X AI3+ at 100 microM had no inhibitory effect on DNA synthesis. AI3+ had no significant effect on cellular cyclic adenosine monophosphate in the presence or absence of insulin or an inhibitor of cyclic nucleotide phosphodiesterases.     

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.     

Effect of dihydrocytochalasin B on the induction of DNA synthesis by epidermal growth factor, insulin and serum in Swiss 3T3 cells

The addition of a microfilament-disorganizing agent--dihydrocytochalasin B B (5-10 micrograms/ml)--to to quiescent confluent or sparse (in 0.5% serum) Swiss 3T3 cells, 1-2 hours prior to stimulation, inhibited the initiation of DNA synthesis induced by an epidermal growth factor (7.5-10 ng/ml) and insulin (0.5-1.0 micrograms/ml), but exerted a low effect on serum stimulation. DNA synthesis was measured 21-23 hours after the growth factor administration by 14C-thymidine incorporation in acid-insoluble material and the ratio of this fraction to exogenous thymidine uptake. Moreover, the polar solvent dimethylsulfoxide, present in culture medium at low concentration (0.1-0.5%), also caused a decrease in the basal level of 14C-thymidine incorporation in resting cells, and a less decrease in the induced incorporation.     

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.