Cyclic AMP increasing agents rapidly stimulate vimentin phosphorylation in quiescent cultures of Swiss 3T3 cells

The results presented here demonstrate that an elevation in the cellular levels of cyclic AMP (cAMP) increases the phosphorylation of an Mr = 58,000 cellular protein in quiescent cultures of Swiss 3T3 cells. The enhancement of 32Pi incorporation into the Mr 58,000 cellular protein was detected as early as 1 min and reached a maximum after 20 min of treatment. The role of cAMP in the phosphorylation of Mr = 58,000 protein is substantiated by the following lines of evidence: a) a variety of agents that cause cAMP accumulation in 3T3 cells, including cholera toxin, 5'-N-ethylcarboxamideadenosine (NECA), PGE1, and 3-isobutyl-1-methyl-xanthine (IBMX) increased the phosphorylation of the same Mr 58,000 cellular protein as demonstrated by peptide mapping; b) inhibitors of cyclic nucleotide phosphodiesterase potentiated the ability of low concentrations of the adenylate cyclase activators NECA, PGE1, and forskolin to increase Mr 58,000 phosphorylation; and c) permeable derivatives of cAMP such as 8BrcAMP were also effective and specific in promoting Mr 58,000 phosphorylation. Detergent extraction, immunoblotting, and immunoprecipitation identified the Mr = 58,000 phosphoprotein as vimentin, the main protein subunit of the intermediate filaments of mesenchymal cells including Swiss 3T3 cells. Studies with intact 3T3 cells revealed that an increase in the intracellular level of cAMP induced a marked redistribution and collapse of the intermediate filaments. These results raise the possibility that an intact intermediate filament network may restrict the reinitiation of DNA synthesis.     

Melittin stimulates Na entry,Na-K pump activity and DNA synthesis in quiescent cultures of mouse cells

Melittin, an amphipatic polypeptide, increases several fold the activity of Na-K pump in quiescent Swiss 3T3 cells. As with other growth factors, melittin increases the activity of the pump by increasing Na entry into the cell. In contrast, other early responses are not elicited by the toxin. At concentrations that promote ion fluxes, melittin stimulates DNA synthesis in quiescent mouse cells acting synergistically with insulin, epidermal growth factor and with the growth factor released by SV40 BHK cells. In contrast, melittin does not interact synergistically with either phorbol esters or vasopressin. The cellular effects of melittin are consistent with the proposal that ion fluxes signal the initiation of mitogenesis in quiescent cells.     

Vasopressin rapidly stimulates NA entry and NA-K pump activity in quiescent cultures of mouse 3T3 cells

Addition of (Arg) vasopressin to quiescent cultures of Swiss 3T3 cells rapidly stimulates an ouabain-sensitive ⁸⁶Rb uptake. In contrast the hormone has no significant effect on the rate of efflux of this cation from preloaded cells. The stimulation of ⁸⁶Rb uptake is cycloheximide-insensitive, occurs within minutes of hormone addition and results from an increase in the Vmax of the uptake system. Vasopressin stimulates ion uptake in a concentration-dependent fashion (1-100 ng/ml); oxytocin also stimulated the Na-K pump but at significantly higher concentrations. The stimulation of the Na-K pump by vasopressin is apparently mediated by an increase in Na entry into the cells, since the hormone (1) strikingly shifts the concentration dependence on Na⁺ of the Na-K pump, (2) increases ²²Na uptake, and (3) increases intracellular Na contents when the efflux of this ion is blocked by ouabain. Since vasopressin is a potent mitogen for Swiss 3T3 cells, the results provide further evidence in support of a possible role of monovalent ion fluxes in signalling the initiation of growth stimulation.     

Synergistic stimulation of DNA synthesis by bradykinin and vasopressin in swiss 3T3 cells

Vasopressin and bradykinin bind to receptors coupled to GTP-binding proteins and rapidly induce polyphosphoinositide breakdown leading to Ca²⁺ mobilization and activation of protein kinase C. Both peptides are known to induce mitogenesis in the presence of growth factors that act through receptors with intrinsic tyrosine kinase activity. Surprisingly, addition of a combination of vaso-pressin and bradykinin to Swiss 3T3 cells synergistically stimulates DNA synthesis in the absence of any other growth factors. This effect is induced at nanomolar concentrations of the peptides and could be inhibited by addition of specific receptor antagonists or broad spectrum neuropeptide antagonists. Bradykinin, which stimulates transient activation of protein kinase C, induces DNA synthesis in synergy with substances that cause long-term activation of protein kinase C, like vasopression or phorbol 12, 13-dibutyrate. Down-regulation of protein kinase C inhibited the induction of mitogenesis by the combination of vasopressin and bradykinin, thus demonstrating the importance of long-term activation of this enzyme for DNA synthesis. Analysis of tyrosine phosphorylated proteins of M_r = 110,000–130,000 and M_r = 70,000–80,000 revealed a biphasic response after stimulation with bradykinin, whereas the response induced by vasopressin declined after the initial maximum. The combination of bradykinin with vasopressin caused an enhanced and prolonged increase in tyrosine phosphorylation of these proteins as compared with the individual peptides. Inhibition of tyrosine phosphorylation by tyrphostin was paralleled by inhibition of DNA synthesis. Together, these results demonstrate synergistic stimulation of DNA synthesis by bradykinin and vasopressin via prolonged stimulation of multiple signaling pathways and imply that the interactive effects of Ca²⁺ -mobilizing peptides on mitogenesis may be more general than previously thought. © 1994 Wiley-Liss, Inc.     

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+.     

Serum stimulates na entry and the Na-K pump in quiescent cultures of epithelial cells (MDCK)

The growth of an epithelial canine kidney line (MDCK) was reversibly arrested by gradually lowering the serum concentration in the medium over a 3-day period. The cells were demonstrably quiescent by autoradiography after an additional 24 hours in serum-free media. Addition of fresh serum produced DNA synthesis after an 18-hour lag period. The quiescent cells then grew to confluency retaining their transport capacities as seen by the formation of “domes”. This system allows for measurement of monovalent ion fluxes and its relationship to growth regulation. The addition of fresh serum to quiescent MDCK cells increased the uptake of ⁸⁶Rb, a measure of Na-K pump activity. This stimulation was mediated by increased uptake of Na into the cells. Serum-stimulated DNA synthesis was blocked by the addition of ouabain in concentrations that inhibit the Na-K pump. Serum appears to stimulate growth in epithelial cells by increasing the amount of intracellular Na available to the Na-K pump. Monovalent ion transport may play a role in the regulation of epithelial cell proliferation.     

Effects of polyamine depletion on serum stimulation of quiescent 3T3 murine fibroblast cells

Numerous reports have shown that polyamines are required for cell proliferation. A current model for regulating commitment to DNA replication in cultured fibroblasts stimulated from quiescence by serum addition postulates sequential action by specific growth factors. To temporally localize polyamine-dependent steps within this defined sequence, mouse Balb/c-3T3 fibroblasts were partially depleted of polyamines by treatment with DL-alpha-difluoromethylornithine (DFMO), next rendered quiescent by serum deprivation, then stimulated by 10% serum with or without exogenous putrescine (Pu). Depletion of polyamines was verified by HPLC, and entry of cells into S phase was monitored by autoradiography. After 24 h of incubation with [3H]-thymidine, polyamine-depleted cells had labeling indices similar to quiescent cells if they were serum-stimulated without Pu, but progressed to S phase to the same degree as control cultures if polyamines were restored by adding Pu at the time of serum stimulation. These observations suggested that commitment of quiescent cells to DNA replication may require polyamines. To determine if polyamine-dependent steps occur during the pre-commitment period (up to 12 h after serum stimulation) or only in traverse of G1 (12 h to 24 h, post-commitment), polyamine-depleted quiescent cells were serum-stimulated for 12 h without Pu, then returned to low serum with Pu. Labeling indices of these cultures remained nearly as low as those of unstimulated cells. Reducing serum concentration from 10% to 0.5% at 12 h after stimulation did not effect labeling indices of control cells not depleted of polyamines by DFMO. These results supported the postulated requirement for polyamines during pre-commitment events. However, polyamine-deficient quiescent cells serum-stimulated without Pu for periods longer than 24 h had labeling indices at 36 and 48 h significantly greater than at 24 h. This suggested that polyamine depletion may decrease the rate at which quiescent cells commit to DNA replication, rather than producing an absolute blockade during the pre-commitment period.     

Cyclic AMP specifically blocks proliferation of rat 3T3 cells transformed by polyomavirus.

Elevated exogenous and intracellular levels of cyclic AMP could totally block proliferation of polyomavirus (PyV) transformants derived from rat 3T3 cells without affecting proliferation of normal cells or simian virus 40 (SV40)-induced transformants. Concanavalin A (ConA) had the opposite effect; it could totally block proliferation of both normal cells and SV40 transformants but reduced proliferation of PyV transformants only twofold. Adenylate cyclase was threefold less active in membranes of PyV transformants, and the number of ConA receptors was similar to that of normal cells. Proliferating PyV transformants contained threefold less cyclic AMP than did proliferating SV40 transformants. The sensitivity to cyclic AMP did not correlate with the degree of transformation: cells transformed by Rous sarcoma virus and tumor cells derived from SV40 transformants were not sensitive to cyclic AMP. The differential effect of cyclic AMP and ConA on proliferation was probably due to the activity of an intact middle t protein. The presence of both large T and small t together with middle t was also required for cyclic AMP sensitivity.     

Differentiation between serum stimulation of ouabain-resistant and sensitive Rb influx in quiescent NIH 3T3 cells

Summary The addition of serum to quiescent NIH 3T3 mouse cell cultures resulted in a 10- to 20-fold increase of Rb influx which was resistant to ouabain, and only a three- to fourfold activation of ouabain-sensitive Rb influx. Stimulation of the ouabain-resistant Rb influx following serum addition reached its maximum within 2 min. The stimulation of ouabain-resistant Rb influx was a result ofV _m increase while theK _m for Rb was unchanged. Ouabain-resistant Rb influx, after serum addition, was resistant to amiloride and sensitive to ethacrinic acid. Replacing chloride in the medium by NO₃ ^–, CO₃ ^– and CH₃COO^– resulted in a drastic decrease in the ouabain-resistant Rb influx. It appeared, therefore, that the ouabain-resistant Rb influx in NIH 3T3 cells was Cl^–-dependent.     

Cyclic AMP in plasmacytoma cells

Summary In Balb/c plasmacytoma cells (MOPC 315) the synthesis and secretion of IgA in vitro was suppressed by prior incubation of the tumor cells with anti-Balb/c alloantibody. In tumor cells so treated, the levels of cyclic AMP were found to be increased. The alloantibody, either as alloantiserum or ascitic fluid globulins from allogeneic strains of mice immunized with spleen cells, had been differentially absorbed with Balb/c spleen cells. Such absorption reduces the cytotoxic antibody effect to below detectable levels, as measured by Trypan Blue exclusion, but permits retention of substantial levels of the suppressive antibody effect. The range of dilutions of alloantibody causing the increase in cAMP corresponded with those that caused suppression of IgA secretion. When tumor cells were exposed to agents that increased the level of cAMP either endogenously or exogenously, suppression of secretion of IgA was found, similar to that caused by the alloantibody. When submaximal concentrations of suppressive antibody were combined with submaximal levels of the cAMP-increasing agents greater degrees of suppression were found, suggesting an additive effect. No cytotoxic effect on these tumor cells, as indicated by Trypan Blue exclusion, was caused by these cAMP-increasing agents at the concentrations shown, either alone or in combination with the suppressive antibody.     

Inorganic phosphate is necessary for the stimulation of DNA synthesis in swiss 3T3 cells by pure mitogenic agents

We compared the ability of dialysed fetal bovine serum and of combinations of purified growth-promoting factors such as insulin, epidermal growth factor (EGF), vasopressin, fibroblast-derived growth factor and antitubulin agents to stimulate DNA synthesis in 3T3 cells maintained in the absence or presence of inorganic phosphate (P_i). When DNA synthesis was stimulated by serum in the absence of P_i the level induced was 70% of that observed in P_i-containing medium. In contrast, combinations of growth-promoting factors in the absence of P_i stimulated less than 8% of the DNA synthesis which they induced in complete medium. Addition of as little as 50 μM P_i fully restored the ability of the factors to stimulate DNA synthesis. Cells stimulated by purified mitogens in the absence of P_i became blocked in early G1, and for up to 48 h the block was reversible by readdition of p_i. The effectiveness of dialysed serum to stimulate DNA in the absence of P_i suggest that dialysed serum might contain a component capable of supplying P_i to support DNA synthesis. Indeed, delipidization of serum by solvent extraction resulted in loss of ability to stimulate DNA synthesis in the absence of p_i, but delipidized serum stimulated DNA synthesis virtually, as well as dialysed serum in the presence of P_i. Previous conclusions suggesting that P_i is not essential for DNA synthesis appear to require re-evaluation.     

Cyclic AMP phosphodiesterase activity of murine T and B lymphocytes.

Recent evidence suggests that adenosine 3′,5′-monophosphate (cyclic AMP) may play a role in lymphocyte responses. The nature of this role is yet unclear since it has been reported that increases in intracellular cyclic AMP can both inhibit and enhance immune functions. In an attempt to determine how lymphocyte cyclic AMP metabolism is regulated we have measured cyclic nucleotide phosphodiesterase in populations of murine T and B lymphocytes. Results indicate that the enzyme activity in B cells was over fourfold higher than that found in thymocytes. Further analyses by preparative polyacrylamide gel electrophoresis revealed that these cell populations contained different multiple forms of the enzyme.     

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.     

The ultrastructure of primary cilia in quiescent 3T3 cells

Electron microscopy was used to investigate primary cilia in quiescent 3T3 cells. As in the case of primary cilia of other cell types, their basal centriole was found to be a focal point of numerous cytoplasmic microtubules which terminate at the basal feet. There are also intermediate filaments which appear to converge at the basal centriole. Cross-striated fibers of microtubular diameter, reminiscent of striated rootlets of ordinary cilia, appear associated with the proximal end of the basal centriole. Usually less than nine cross-banded basal feet surround the basal centriole in a well-defined plane perpendicular to the centriolar axis. The ciliary shaft was found to be entirely enclosed in the cytoplasm of fully flattened cells. In rounded cells, it could be found extending to the outside of the cell. Periodic striations along the entire shaft were observed after preparing the cells in a special way. The tip of the shaft showed an electron-dense specialization. Several unusual forms of primary cilia were observed which were reminiscent of olfactory flagella or retinal rods.Using tubulin antibody for indirect immunofluorescence, a fluorescent rod is visible in the cells [18] which we demonstrate is identical with the primary cilium.     

Stabilization of mRNA following serum-induction of quiescent 3T3 cells

The stability of mRNA has been measured in 3T3 cells in the resting and the growing states, and also during the transition from the resting to the growing state. Pulse labeled poly(A)⁺ mRNA chased with uridine and cytidine supplemented growth medium decayed with a half-life of 6.5 hr in the resting state, 26 hr during the transition from the resting to the growing condition, and 18 hr during serum-stimulated growth. The half-life of poly(A)⁺ mRNA determined by steady state labeling yielded similar results in resting and serum-stimulated 3T3 cells. Thus during the transition from resting to serum-stimulated growth in 3T3 cells poly(A)⁺ mRNA becomes more stable.     

Stimulation of quiescent 3T3 cells by phosphatidic acid-containing liposomes

Liposomes containing phosphatidic acid were capable of stimulating DNA synthesis in quiescent Swiss 3T3 cells while liposomes composed of other phospholipids were not. These results show that liposomes, which are usually employed to deliver non-lipid molecules into cells, can themselves have profound effects on cell growth. The possible mechanism of phosphatidic acid-mediated cell stimulation and its relation to other growth factors are discussed.