Mechanisms of bombesin-induced arachidonate mobilization in Swiss 3T3 fibroblasts

A peptide mitogen bombesin, which activates the phospholipase C-protein kinase C signaling pathway, induces a mepacrine-sensitive, dose-dependent increase in the release of [3H]arachidonic acid and its metabolites ([3H]AA) from prelabeled Swiss 3T3 fibroblasts. The effect is temporally composed of two phases, i.e. an initial transient burst that is essentially independent of extracellular Ca2+, and a following sustained phase that is absolutely dependent on the extracellular Ca2+. The initial transient [3H]AA liberation occurs concomitantly with bombesin-induced 45Ca efflux from prelabeled cells: both responses being substantially attenuated by loading cells with a Ca2+ chelator quin2. However, bombesin-induced intracellular Ca2+ mobilization by itself is not sufficient as a signal for the initial transient [3H]AA liberation, since A23187 potently stimulates 45Ca efflux to an extent comparable to bombesin but fails to induce [3H]AA release in the absence of extracellular Ca2+. The second sustained phase of the bombesin-induced [3H]AA release is abolished by reducing extracellular Ca2+ to 0.03 mM, although bombesin effects on phospholipase C and protein kinase C activation are barely affected by the same procedure. A protein kinase C activator phorbol 12,13-dibutyrate induces an extracellular Ca(2+)-dependent, slowly developing sustained increase in [3H]AA release, and markedly potentiates both phases of bombesin-induced [3H]AA release. Down-regulation of cellular protein kinase C completely abolishes all of the effects of phorbol dibutyrate, and partially inhibits the second but not the first phase of bombesin-induced [3H]AA release. These results indicate that bombesin-induced receptor-mediated activation of phospholipase A2 involves multiple mechanisms, including intracellular Ca2+ mobilization for the first phase, protein kinase C activation plus Ca2+ influx for the second phase, and as yet unknown mechanism(s) independent of intracellular Ca2+ mobilization or protein kinase C for both of the phases.     

Signaling pathways for sphingosylphosphorylcholine-mediated mitogenesis in Swiss 3T3 fibroblasts

Sphingosylphosphorylcholine (SPC), or lysophingomyelin, a wide-spectrum growth promoting agent for a variety of cell types (Desai, N. N., and S. Spiegel. 1991. Biochem. Biophys. Res. Comm. 181: 361-366), stimulates cellular proliferation of quiescent Swiss 3T3 fibroblasts to a greater extent than other known growth factors or than the structurally related molecules, sphingosine and sphingosine-1- phosphate. SPC potentiated the mitogenic effect of an activator of protein kinase C, 12-O-tetradecanoylphorbol 13-acetate, and did not compete with phorbol esters for binding to protein kinase C in intact Swiss 3T3 fibroblasts. However, downregulation of protein kinase C, by prolonged treatment with phorbol ester, reduced, but did not eliminate, the ability of SPC to stimulate DNA synthesis, indicating that SPC may act via both protein kinase C-dependent and -independent signaling pathways. SPC induced a rapid rise in intracellular free calcium ([Ca2+]i) in viable 3T3 fibroblasts determined with a digital imaging system. Although the increases in [Ca2+]i were observed even in the absence of calcium in the external medium, no increase in the levels of inositol phosphates could be detected in response to mitogenic concentrations of SPC. Furthermore, in contrast to sphingosine or sphingosine-1-phosphate, the mitogenic effect of SPC was not accompanied by increases in phosphatidic acid levels or changes in cAMP levels. SPC, but not sphingosine or sphingosine-1-phosphate, stimulates the release of arachidonic acid. Therefore, the ability of SPC to act an extremely potent mitogen may be due to activation of signaling pathway(s) distinct from those used by sphingosine or sphingosine-1- phosphate.     

Diacylglycerol stimulates phospholipase A2 from Swiss 3T3 fibroblasts

We recently demonstrated that diacylglycerol induced arachidonate release and prostaglandin E2 synthesis in 3T3 fibroblasts, and greatly augmented prostaglandin E2 synthesis in response to submaximal and maximal concentrations of bradykinin. We have now partially purified a phospholipase A2 from the cells. When phosphatidyl[3H]choline was used as substrate, several diacylglycerols augmented phospholipase A2 activity. Diacylglycerol was effective at concentrations as low as 30 nM. Protein kinase C inhibition did not affect diacylglycerol's stimulation of phospholipase A2. Diacylglycerol did not alter the calcium requirement for phospholipase A2 or its pH optimum. The present study demonstrates that the effect of diacylglycerol to augment arachidonate metabolism is at the level of phospholipase A2, itself.     

Stimulation by xanthine oxidase of 3T3 Swiss fibroblasts and human lymphocytes

Xanthine oxidase stimulates [3H]thymidine incorporation by 3T3 cells even in the absence of any added xanthine, but in the presence of, and synergistically with, insulin or various other growth-stimulating factors. Optimal stimulation was obtained with 2-3 mU enzyme/ml and higher concentrations were toxic. Xanthine oxidase also stimulated human peripheral blood lymphocytes in the presence of phytohemagglutinin and xanthine.     

Second messengers regulate endosomal acidification in Swiss 3T3 fibroblasts.

Acidification of the endosomal pathway is important for ligand and receptor sorting, toxin activation, and protein degradation by lysosomal acid hydrolases. Fluorescent probes and imaging methods were developed to measure pH to better than 0.2 U accuracy in individual endocytic vesicles in Swiss 3T3 fibroblasts. Endosomes were pulse labeled with transferrin (Tf), alpha 2-macroglobulin (alpha 2M), or dextran, each conjugated with tetramethylrhodamine and carboxyfluorescein (for pH 5-8) or dichlorocarboxyfluorescein (for pH 4-6); pH in individual labeled vesicles was measured by ratio imaging using a cooled CCD camera and novel image analysis software. Tf-labeled endosomes acidified to pH 6.2 +/- 0.1 with a t1/2 of 4 min at 37 degrees C, and remained small and near the cell periphery. Dextran- and alpha 2M-labeled endosomes acidified to pH 4.7 +/- 0.2, becoming larger and moving toward the nucleus over 30 min; approximately 15% of alpha 2M-labeled endosomes were strongly acidic (pH less than 5.5) at only 1 min after labeling. Replacement of external Cl by NO3 or isethionate strongly and reversibly inhibited acidification. Addition of ouabain (1 mM) at the time of labeling strongly enhanced acidification in the first 5 min; Tf-labeled endosomes acidified to pH 5.3 without a change in morphology. Activation of phospholipase C by vasopressin (50 nM) enhanced acidification of early endosomes; activation of protein kinase C by PMA (100 nM) enhanced acidification strongly, whereas elevation of intracellular Ca by A23187 (1 microM) had no effect on acidification. Activation of protein kinase A by CPT-cAMP (0.5 mM) or forskolin (50 microM) inhibited acidification. Lysosomal pH was not affected by ouabain or the protein kinase activators. These results establish a methodology for quantitative measurement of pH in individual endocytic vesicles, and demonstrate that acidification of endosomes labeled with Tf and alpha 2M (receptor-mediated endocytosis) and dextran (fluid-phase endocytosis) is sensitive to intracellular anion composition, Na/K pump inhibition, and multiple intracellular second messengers.     

LacSwitchTM Inducible Mammalian Expression System in mouse Swiss 3T3 fibroblasts

Swiss 3T3 fibroblasts were transfected with the provided plasmids of LacSwitch Inducible Mammalian Expression System (Stratagene). Stable transfectants were selected, expanded and characterised. At first, the production of CAT in these cell lines could be induced by IPTG treatment, but the inducibility was lost after a few months in culture in a reproducible manner. Further analysis revealed that the transfectants did not lose the cat gene nor the lac repressor protein. As a result, we conclude that LacSwitch Inducible Mammalian Expression System needs further modification for use in Swiss 3T3 fibroblasts.     

Protein tyrosine phosphatase gene expression analysis in Swiss 3T3 fibroblasts

The aim of this study was to identify protein tyrosine phosphatases (PTPs) expressed in Swiss 3T3 fibroblasts and to examine their expression levels as well as to characterize quantitative aspects of RT-PCR based on degenerate deoxyoligonucleotides. By using an RT-PCR assay based on degenerate deoxyoligonucleotide primers, expression of mRNAs for two cytoplasmic- and six transmembrane-type PTPs in Swiss 3T3 cells was detected. The sequences of two of them are new. Among nine analyzed PTPs expressed to widely varied extends, only three have mRNA levels high enough to be seen on Northern blots with 10 µg of total RNA per lane. The frequencies with which the examined PTPs are represented among the PCR amplification products, correlate stronger with the primer fidelity, defined as the number of mismatches between the primer- and the cDNA target-sequences, rather than with the PTP expression levels. In conclusion, an RT-PCR assay based on degenerate primers can be successfully used to sample the expressed PTPs and to identify new members of this gene family. However, reliable quantification of their mRNA levels can only be achieved using the classical approaches, like Northern, RNase protection assay or non-degenerate quantitative RT-PCR.     

Bombesin activates large-conductance chloride channels in Swiss 3T3 fibroblasts.

Using the patch-clamp technique (cell-attached patches), we found that bombesin, a Ca-mobilizing peptide mitogen, activates large-conductance Cl channels in Swiss 3T3 fibroblasts. The channel activation required a lag period of about 50 s and was equally observed whether bombesin was applied to the patch-pipette or to the bath. A23187 (10(-6)M) in the bath induced the similar currents with almost identical current-voltage relationship as bombesin: their slope conductances were 292 +/- 15 (bombesin) and 318 +/- 42 (A23187) pS. In inside-out patches, the induced channels were selective to Cl over gluconate (11:1). These observations strongly suggest that in Swiss 3T3 fibroblasts bombesin activates the Cl channels through a mechanism involving an increase in the intracellular free Ca concentration.     

Spatial organization of centrosome-attached and free microtubules in 3T3 fibroblasts

The spatial organization of microtubules is crucial for different cellular processes. It is traditionally supposed that fibroblasts have radial microtubule arrays consisting of long microtubules that run from the centrosome. However, a detailed analysis of the microtubule array in the internal cytoplasm has never been performed. In the current study, we used laser photobleaching to analyze the spatial organization of microtubules in the internal cytoplasm of cultured 3T3 fibroblasts. Cells were injected with Cy-3-labeled tubulin, after which the growth of microtubules in the centrosome region and peripheral parts of cytoplasm was assayed in the bleached zone. In most cases, microtubule growth in the bleached zone occurred rectilinearly; at distances of up to 5 μm, microtubules seldom bend more than 10°–15°. We considered a growing fragment of the microtubule as a vector with the beginning at the point of occurrence and the end at the point where the growth terminated (or the end point after 30 s if microtubule persistent growth proceeded for longer). We defined the direction of microtubule growth in different parts of the cell using these vectors and measured the angle of their deviation from the vector of comparison. In the area of the centrosome, we directed a comparison vector inside the bleached zone from the centrosome to the beginning of the growing microtubule segment; in the lamella and trailing part of the fibroblast, we used the vector of comparison directed along the long axis of the cell from its geometrical center to periphery. The microtubules growing straight away from the centrosome grew along the cell radius. However, at a distance of 10 μm from the centrosome, radially growing microtubules comprised 40% of the overall number, while at a distance of 20 μm, they made up only 25%. The rest of the microtubules grew in different directions, with the preferred angle between their growth direction and cell radius equaling around 90 °. In the lamella and trailing part of the fibroblast, 80% of all microtubules grew along the long axis of the cell or at an angle of no more than 20 °; 10–15% of microtubules grew along axis of the cell but towards the centrosome. Thus, in 3T3 fibroblasts, the radial system of microtubules is perturbed starting at a distance of several microns from the centrosome. In the internal cytoplasm, the microtubule system is completely disordered and, in the stretched parts of the polarized cell (lamella, trailing edge), the microtubule system again becomes well organized; microtubules are preferentially oriented along the long axis of the cell. From the results obtained, we conclude that the orderliness of microtubules at the periphery of the fibroblast is not a consequence of their growth from the centrosome; rather, their orientation is preset by local factors.     

Expression of a mouse tyrosinase cDNA in 3T3 Swiss mouse fibroblasts

3T3 Swiss mouse fibroblast cell lines expressing tyrosinase, the critical enzyme in melanin synthesis, have been established by co-transfection of a mouse tyrosinase cDNA and a G418-resistance gene. Of sixty-three clones isolated, four are brown in colour, presumably due to synthesis of melanin. Expression of both the tyrosine hydroxylase and dopa oxidase activities of tyrosinase by these pigmented clones has been demonstrated directly by enzyme assays. Electron microscopic studies suggest that the brown pigment is located in membrane-bound cytoplasmic vesicles.     

Catechin protection of 3T3 Swiss fibroblasts in culture under oxidative stress

The effects of catechin, a well-known in vitro antioxidant, on 3T3 Swiss fibroblasts are studied under different conditions of oxidative stress leading to cell proliferation or cytotoxicity. Various levels of reactive oxygen species (ROS), generated extracellularly by the xanthine-xanthine oxidase (X-XO) system, are at the origin of the biphasic effect on DNA synthesis by 3T3 Swiss fibroblasts. The addition of 10^?2 U XO/mL, in the absence of exogenous X, catalyzes the production of low levels of $O_2 ^{\dot - } $ and H₂O₂ in the extracellular medium, which stimulate DNA synthesis and cell division. The increase in the level of ROS, by addition of increasing X concentrations, did not enhance this effect proportionally. On the contrary, high levels of ROS inhibit DNA synthesis, the cytotoxicity induced being proportional to the level of H₂O₂ generated by the enzyme system. Catechin does not significantly modify DNA synthesis induced by low levels of ROS, but protects in a dose-dependent manner against the cytoxicity of high levels of ROS.     

Pasteurella multocida toxin stimulates mitogenesis and cytoskeleton reorganization in Swiss 3T3 fibroblasts

Pasteurella multocida toxin (PMT) causes cytoplasmic retraction in epithelial cells, activates osteoclast neoformation, and is a potent mitogen for Swiss 3T3 fibroblasts. In the present study designed to further investigate the effects of PMT on cell shape and proliferation, we report that the mitogenic effect of affinity-purified PMT on quiescent 3T3 cells was even superior at 5 ng/ml to that of fetal bovine serum or bombesin. This positive effect was inhibited by heat denaturation and methylamine treatment (this agent blocks internalization). Preincubation of PMT with gangliosides GM₁, GM₂, or GM₃ counteracted its effect on DNA synthesis, suggesting that the toxin binds to GM-type ceramides on target cells. The distribution of F-actin was analyzed in control/treated cells using FITC-conjugated phalloidin. In comparison with FBS and bombesin, PMT triggered a more rapid and profound reorganization of cortical actin into prominent stress fibers after only 5–10 min. This event lead to the retraction of cells after only 30 min and ultimately to the induction of mitotic figures. Interestingly, methylamine blocked the effects of PMT on stress fiber formation and cell retraction but not the ruffling response, suggesting that some early events may not require toxin internalization. In summary, these findings indicate that PMT concomitantly exerts a strong mitogenic activity and a rapid stimulation of cytoskeletal rearrangements, possibly after binding to membrane gangliosides and subsequent internalization. We propose that this toxin could be used in the future as a defined inducer of transduction signals involved in cellular proliferation and control of cell shape. © 1996 Wiley-Liss, Inc.     

Proliferative and morphological changes induced by vanadium compounds on Swiss 3T3 fibroblasts

Vanadium compounds are shown to have a mitogenic effect on fibroblast cells. The effects of vanadate, vanadyl and pervanadate on the proliferation and morphological changes of Swiss 3T3 cells in culture are compared. Vanadium derivatives induced cell proliferation in a biphasic manner, with a toxic-like effect at doses over 50mM, after 24h of incubation. Vanadyl and vanadate were equally potent at 2.5–10mM. At 50mM vanadate inhibited cell proliferation, whereas slight inhibition was observed at 100mM of vanadyl. At 10mM pervanadate was as potent as vanadate and vanadyl in stimulating fibroblast proliferation, but no effect was observed at lower concentrations. A pronounced cytotoxic-like effect was induced by pervanadate at 50mM. All of these effects were accompanied by morphological changes: transformation of fibroblast shape from polygonal to fusiform; retraction with cytoplasm condensation; and loss of lamellar processes. The magnitude of these transformations correlates with the potency of vanadium derivatives to induce a cytotoxic-like effect: pervanadate>vanadate>vanadyl. These data suggest that the oxidation state and coordination geometry of vanadium determine the degree of the cytotoxicity.     

G1 phase heterogeneity in exponentially growing Swiss 3T3 mouse fibroblasts

The growth rate of normal cultured Swiss 3T3 fibroblasts is function of serum concentration and the fraction of G1 cells, and hence the average residence time in G1, increases with the generation time. Serum contains two sets of factors: competence factors, essentially platelet-derived growth factor (PDGF), which induces competence in quiescent fibroblasts and prevents replicating cells from entering G0, and plasma, which allows progression. The increase in the duplication time and the duration of Gl at low serum concentration could hence be due to the fact that competence factors become limiting. The fraction of non-competent cells, operationally defined as those G1 cells unable to leave G1 in the presence of plasma alone, was determined in populations exponentially growing at serum concentrations between 5 and 20%. To do so exponentially growing cultures were shifted to plasma plus colcemid: one part of the cell population progressed through the cycle and accumulated with a G2 DNA content, whereas non-competent cells remained in G1. Analysis of the DNA distributions performed 24 h after the shift showed that as serum concentration was lowered more cells were found in the non-competent state: they were less than 5% in 20% serum and almost 50% in 5% serum. The non-competent cells constitute a dynamic fraction of the population, since in the presence of serum they can leave Gl and progress in the cycle. These data indicate that one of the steps limiting exponential growth is the acquisition of competence and that this event gives rise to heterogeneity within the G1 population.     

Stimulation of mitogenesis and glucose transport by 1-monooleoylglycerol in Swiss 3T3 fibroblasts

1-Monooleoylglycerol (MOG), a recently reported diacylglycerol kinase inhibitor (Bishop, W. R., Ganong, B. R., and Bell, R. M. (1986) J. Biol. Chem. 261, 6993-7000), exerts potent stimulatory effects on [3H]thymidine incorporation into DNA and glucose transport in Swiss 3T3 fibroblasts. MOG induces a rapid and sustained 2.5-fold increase in the cellular 1,2-diacylglycerol (1,2-DG) content, and phosphorylation of an acidic 80-kDa protein, a putative substrate for the protein kinase C (Ca2+/phospholipid-dependent protein kinase). The effect of MOG is additive to that of bombesin in terms of both an increase in tissue diacylglycerol content and phosphorylation of the 80-kDa proteins. In addition to these effects, MOG potently stimulates release of arachidonic acid from phospholipids. Inhibitors of cyclooxygenase and lipoxygenase have little effect, if any, on MOG-induced stimulation of glucose transport and DNA synthesis, while exogenously applied arachidonic readily stimulates both of these cellular responses. Furthermore, arachidonic acid, at its biologically active concentrations, is found to induce a rapid and sustained increase in cellular 1,2-DG content and stimulate the phosphorylation of the 80-kDa protein, although to a lesser extent than MOG. Prolonged pretreatment of the cells with phorbol 12,13-dibutyrate, which reduces the cellular protein kinase C content, markedly attenuates the effects of both MOG and arachidonic acid on glucose transport and DNA synthesis. These data indicate that MOG increases endogenous 1,2-DG content and thereby acts as a potent activator of protein kinase C, and that activation of protein kinase C is a crucial step in MOG-induced stimulation of mitogenesis and glucose transport.     

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.     

Actinobacillus actinomycetemcomitans adheres to human gingival fibroblasts and modifies cytoskeletal organization

Adherence of Actinobacillus actinomycetemcomitans to human gingival fibroblast cells induces cytoskeletal reorganization. A. actinomycetemcomitans is considered a pathogenic bacteria involved in localized aggressive periodontitis. Studies with epithelial cells have shown an adherent capacity of bacteria that is increased under anaerobic conditions. For adherence to take place, there is a need for interaction between extracellular vesicles and bacterial fimbriae. However, molecular events associated with the adherence process are still unknown. The aim of this study was to investigate whether A. actinomycetemcomitans adherence to human gingival fibroblasts promotes cytoskeletal reorganization. Adherence was determined with light microscopy and scanning electron microscopy. For F-actin visualization, cells were treated with fluorescein-isothiocyanate-phalloidin and samples were examined with epifluorescence optics. Fluorescent was recorded on Kodak T-Max 400 film. We showed that A. actinomycetemcomitans adheres to human gingival fibroblast primary cultures, this property stimulating an increase in the intracellular calcium levels. In human gingival fibroblast primary cultures, we observed that maximal A. actinomycetemcomitans adherence took place 1.5h after culture infection occurred and remained for 6h. The adherence was associated with morphologic alterations and an increased in the intracellular calcium levels. These experiments suggest that A. actinomycetemcomitans adherence cause morphological alterations, induce actin stress fibers and recruitment of intracellular calcium levels.     

ROCK and mDia1 antagonize in Rho-dependent Rac activation in Swiss 3T3 fibroblasts

The small GTPase Rho acts on two effectors, ROCK and mDia1, and induces stress fibers and focal adhesions. However, how ROCK and mDia1 individually regulate signals and dynamics of these structures remains unknown. We stimulated serum-starved Swiss 3T3 fibroblasts with LPA and compared the effects of C3 exoenzyme, a Rho inhibitor, with those of Y-27632, a ROCK inhibitor. Y-27632 treatment suppressed LPA-induced formation of stress fibers and focal adhesions as did C3 exoenzyme but induced membrane ruffles and focal complexes, which were absent in the C3 exoenzyme-treated cells. This phenotype was suppressed by expression of N17Rac. Consistently, the amount of GTP-Rac increased significantly by Y-27632 in LPA-stimulated cells. Biochemically, Y-27632 suppressed tyrosine phosphorylation of paxillin and focal adhesion kinase and not that of Cas. Inhibition of Cas phosphorylation with PP1 or expression of a dominant negative Cas mutant inhibited Y-27632-induced membrane ruffle formation. Moreover, Crk-II mutants lacking in binding to either phosphorylated Cas or DOCK180 suppressed the Y-27632-induced membrane ruffle formation. Finally, expression of a dominant negative mDia1 mutant also inhibited the membrane ruffle formation by Y-27632. Thus, these results have revealed the Rho-dependent Rac activation signaling that is mediated by mDia1 through Cas phosphorylation and antagonized by the action of ROCK.     

Active involvement of Ca2+ in mitotic progression of Swiss 3T3 fibroblasts

Global Ca2+ transients have been observed to precede nuclear envelope breakdown and the onset of anaphase in Swiss 3T3 fibroblasts in 8% (vol/vol) FBS. The occurrence of these Ca2+ transients was dependent on intracellular stores. These Ca2+ transients could be (a) abolished by serum removal without halting mitosis, and (b) eliminated by increasing intracellular Ca2+ buffering capacity through loading the cells with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) buffer, via the tetra(acetoxymethyl) ester, without hindering the transition into anaphase. Microinjection of sufficient concentrations of BAPTA buffer could block nuclear envelope breakdown. Pulses of Ca2+ generated by flash photolysis of intracellularly trapped nitr-5, a "caged" Ca2+, could precipitate precocious nuclear envelope breakdown in prophase cells. In metaphase cells, photochemically generated Ca2+ pulses could cause changes in the appearance of the chromosomes, but the length of time required for cells to make the transition from metaphase to anaphase remained essentially unchanged regardless of whether a Ca2+ pulse was photoreleased during metaphase. The results from these photorelease experiments were not dependent on the presence of serum in the medium. Discharging intracellular Ca2+ stores with ionomycin in the presence of 1.8 mM extracellular Ca2+ doubled the time for cells to pass from late metaphase into anaphase, whereas severe Ca2+ deprivation by treatment with ionomycin in EGTA-containing medium halted mitosis. Our results collectively indicate that Ca2+ is actively involved in nuclear envelope breakdown, but Ca2+ signals are likely unnecessary for the metaphase-anaphase transition in Swiss 3T3 fibroblasts. Additional studies of intracellular Ca2+ concentrations in mitotic REF52 and PtK1 cells revealed that Ca2+ transients are not observed at all mitotic stages in all cells. The absence of observable global Ca2+ transients, where calcium buffers can block and pulses of Ca2+ can advance mitotic stages, may imply that the relevant Ca2+ movements are too local to be detected.     

GTP gamma S activation of proto-oncogene expression in transiently permeabilised Swiss 3T3 fibroblasts

A technique of transient permeabilisation has been used to show that the introduction of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a non-hydrolysable analogue of GTP, into intact Swiss 3T3 fibroblasts stimulates phosphoinositide hydrolysis, cyclic AMP accumulation and the activation of c-fos and c-myc proto-oncogenes. Of a number of nucleotide triphosphates introduced into the cells, only GTP and its non-hydrolysable analogues activated inositol phosphate release, suggesting that this response is mediated by guanine nucleotide regulatory (G) protein(s). The data demonstrate that transient permeabilisation provides a method of examining the involvement of G-proteins in nuclear activation.