Swiss 3T3 Cells Research Articles

Tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin does not require extracellular signal-regulated kinase activation in Swiss 3T3 cells stimulated by bombesin or platelet-derived growth factor.

The experiments presented here were designed to examine the contribution of the extracellular signal-regulated mitogen-activated protein kinases (ERKs) to the tyrosine phosphorylation of the focal adhesion proteins p125(Fak), p130(Cas), and paxillin induced by G protein-coupled receptors (GPCRs) and tyrosine kinase receptors in Swiss 3T3 cells. Stimulation of these cells with bombesin, lysophosphatidic acid (LPA), endothelin, and platelet-derived growth factor (PDGF) led to a marked increase in the tyrosine phosphorylation of these focal adhesion proteins and in ERK activation. Exposure of the cells to two structurally unrelated mitogen-activated protein kinase or ERK kinase (MEK) inhibitors, PD98059 and U0126, completely abrogated ERK activation but did not prevent tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin. Furthermore, different dose-response relationships were obtained for tyrosine phosphorylation of focal adhesion proteins and for ERK activation in response to PDGF. Putative upstream events in the activation of focal adhesion proteins including actin cytoskeletal reorganization and myosin light chain (MLC) phosphorylation were also not prevented by inhibition of ERK activation. Thus, our results demonstrate that the activation of the ERK pathway is not necessary for the increase of the tyrosine phosphorylation of p125(Fak), p130(Cas), and paxillin induced by either GPCRs or tyrosine kinase receptors in Swiss 3T3 cells.

The GTP-binding protein G iα translocates to kinetochores and regulates the M-G 1 cell cycle transition of Swiss 3T3 cells

The receptor-generated signals that are responsible for driving the cell cycle are incompletely characterised in mammalian cells. It is clear, however, that the cellular messenger systems that stimulate DNA synthesis and mitosis are separable. These are interwoven with biochemical checkpoints that ensure that processes, such as chromosomal replication and microtubule attachment to duplicated chromosomes, are complete before the following phase of the cell cycle is initiated. In some cells, activation of DNA synthesis by factors such as LPA and serum has been shown to require the GTP-binding protein G i. We have found that G i plays an additional role in mitosis activated by both 7-transmembrane receptors and tyrosine kinase receptors, and that this involves the translocation of the α-subunit of G i (G iα) to the nucleus. Here we show by confocal microscopy that G iαmigrates to the nucleus near the onset of mitosis in serum-activated Swiss 3T3 cells and binds to the kinetochore region of replicated chromosomes. Inhibition of G i function with pertussis toxin had no effect on the induction of DNA synthesis by serum, but cell proliferation was inhibited. Flow cytometric analysis showed that this resulted from retardation of the transition through mitosis and into G 1. Additionally, pertussis toxin impaired the activity of p34 cdc2, a cyclin-dependent kinase involved in the transition from M-phase to G 1, but not the S-phase cyclin, cyclin E. These data show that the G-protein G i has a key role in the regulation of mitosis in fibroblasts.

The replication capacity of intact mammalian nuclei in Xenopus egg extracts declines with quiescence, but the residual DNA synthesis is independent of Xenopus MCM proteins

In eukaryotes, the initiation of DNA synthesis requires the assembly of a pre-replicative complex (pre-RC) at origins of replication. This involves the sequential binding of ORC (origin-recognition-complex), Cdc6 and MCM proteins, a process referred to as licensing. After origin firing, the Cdc6 and MCM proteins dissociate from the chromatin, and do not rebind until after the completion of mitosis, thereby restricting replication to a single round in each cell cycle. Although nuclei normally become licensed for replication as they enter G(1), the extent to which the license is retained when cells enter the quiescent state (G(0)) is controversial. Here we show that the replication capacity of nuclei from Swiss 3T3 cells, in Xenopus egg extracts, is not lost abruptly with the onset of quiescence, but instead declines gradually. The decline in replication capacity, which affects both the number of nuclei induced to replicate and their subsequent rate of DNA synthesis, is accompanied by a fall in the level of chromatin-bound MCM2. When quiescent nuclei are incubated in egg extracts, they do not bind further MCMs unless the nuclei are first permeabilized. The residual replication capacity of intact nuclei must therefore be dependent on the remaining endogenous MCMs. Although high levels of Cdk activity are known to block MCM binding, we show that the failure of intact nuclei in egg extracts to increase their bound MCMs is not due to their uptake and accumulation of Cdk complexes. Instead, the failure of binding must be due to exclusion of some other binding factor from the nucleus, or to the presence within nuclei of an inhibitor of binding other than Cdk activity. In contrast to the situation in Xenopus egg extracts, following serum stimulation of intact quiescent cells, the level of bound MCMs does increase before the cells reach S phase, without any disruption of the nuclear envelope.

Molecular organization of the mouse gastrin-releasing peptide receptor gene and its promoter.

The murine gastrin-releasing peptide receptor (mGRP-R) is a member of the G protein-coupled receptor family and mediates important physiological actions of its specific ligand, the gastrointestinal hormone/neurotransmitter GRP, including mitogenic properties in the mouse Swiss 3T3 fibroblasts. Glucocorticoids and increases in intracellular cAMP are reported to alter GRP-R gene transcription, but the molecular basis for these effects is unknown. To begin to identify possible gene regulatory mechanisms that are responsible for modifying mGRP-R expression, we determined its structure and investigated its basal promoter activity. We isolated and characterized genomic bacteriophage P1 clones encoding the mouse gastrin-releasing peptide receptor (mGRP-R). By DNA sequencing and Southern blot analyses, we determined the protein coding region to be contained in three exons interrupted by two introns 20 and 2kb in length. The open reading frame of the putative GRP-R gene encodes for a 384-amino-acid protein which demonstrates 48% identity with the mouse BRS-3 protein and 53% identity with the mouse NMB-R protein. The mGRP-R gene locus extends over 29kb and was mapped to the X-chromosome (DXMit20) utilizing a minisatellite polymorphism in the 5' UTR and by fluorescent in-situ hybridization (FISH). In Swiss 3T3 cells, which natively express mGRP-R, two gene-specific mRNA species of 3 and 7kb can be detected by Northern blot analysis. With RNase protection assays, and independently with inverse PCR of 5' RACE clones, common mRNA initiation sites were identified clustered between 21 and 61bp downstream of a TTTAAA motif, which is located 450bp upstream of the ATG translation start site. However, different polyadenylation sites are utilized. A 2kb genomic DNA fragment extending from 2147 to 141 bases 5' to the ATG translation start was cloned into a luciferase reporter plasmid and shown to contain promoter activity in Swiss 3T3 and COS-7 cells. Progressive promoter truncations and mutations of a cyclic AMP response element (CRE) located 83bp upstream of the TTTAAA motif demonstrate that transcriptional mGRP-R activation in Swiss 3T3 cells only occurs when both the TTTAAA motif and the intact CRE site are retained. With the availability of the full structure of the mGRP-R gene and the minimal promoter sequences reported in this study, it will be possible in future studies to investigate the molecular basis for transcriptional regulation of the mGRP-R gene by glucocorticoids, cAMP and other factors.

Mammalian homolog of the yeast cyclase associated protein, CAP/Srv2p, regulates actin filament assembly.

Control of cell shape and motility requires rearrangements of the actin cytoskeleton. One cytoskeletal protein that may regulate actin dynamics is CAP (cyclase associated protein; CAP/Srv2p; ASP-56). CAP was first isolated from yeast as an adenylyl cyclase associated protein required for RAS regulation of cAMP signaling. In addition, CAP also regulates the actin cytoskeleton primarily through an actin monomer binding activity. CAP homologs are found in many eukaryotes, including mammals where they also bind actin, but little is known about their biological function. We, therefore, designed experiments to address CAP1 regulation of the actin cytoskeleton. CAP1 localized to membrane ruffles and actin stress fibers in fixed cells of various types. To address localization in living cells, we constructed GFP-CAP1 fusion proteins and found that fusion proteins lacking the actin-binding region localized like the wild type protein. We also performed microinjection studies with affinity-purified anti-CAP1 antibodies in Swiss 3T3 fibroblasts and found that the antibodies attenuated serum stimulation of stress fibers. Finally, CAP1 purified from platelets through a monoclonal antibody affinity purification step stimulated the formation of stress fiber-like filaments when it was microinjected into serum-starved Swiss 3T3 cells. Taken together, these data suggest that CAP1 promotes assembly of the actin cytoskeleton.

Lysophosphatidic acid rapidly induces protein kinase D activation through a pertussis toxin-sensitive pathway.

Protein kinase D (PKD) is a serine-threonine protein kinase with distinct structural features and enzymological properties. Herein we demonstrate that lysophosphatidic acid (LPA) induces rapid PKD activation in mouse Swiss 3T3 and Rat-1 cells. LPA induced PKD activation in a concentration-dependent fashion with maximal stimulation (7.6-fold) achieved at 5 microM. Treatment of Swiss 3T3 cells with the protein kinase C (PKC) inhibitors GF-I, Ro-31-8220, and Gö-7874 completely abrogated PKD activation induced by LPA at concentrations that did not inhibit PKD activity when added directly to the in vitro kinase assays. PKD activation induced by LPA was attenuated markedly and selectively by prior exposure of either Swiss 3T3 or Rat-1 cells to pertussis toxin (PTx) in a concentration-dependent manner. In contrast, treatment with the protein tyrosine kinase inhibitor genistein, the MEK inhibitor PD-098059, or the phosphoinositide 3-kinase inhibitor wortmannin did not affect PKD activation in response to LPA. These results provide the first example of PTx-sensitive and PKC-dependent PKD activation and identify a novel G(i)-dependent event in the action of LPA.

Involvement of Rho-kinase in angiotensin II-induced hypertrophy of rat vascular smooth muscle cells.

Angiotensin II (Ang II) is now believed to play a critical role in the pathogenesis of hypertrophy and/or hyperplasia of vascular smooth muscle cells (VSMCs). Several G(i)- and G(q)-coupled receptors, including the Ang II type 1 (AT(1)) receptor, activate Rho and Rho-associated kinase in Swiss 3T3 cells and cardiac myocytes. However, little is known about the role of Rho-kinase in Ang II-induced vascular hypertrophy in VSMCs. In the present study, we explored the role of Rho and Rho-kinase in Ang II-induced protein synthesis in VSMCs. In unstimulated cells, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to Ang II (100 nmol/L). This effect was completely blocked by the AT(1) receptor blocker candesartan but not by the Ang II type 2 (AT(2)) receptor antagonist PD123319. Botulinum C(3) exoenzyme, which inactivated RhoA, attenuated Ang II-induced [(3)H]leucine incorporation. The specific Rho-kinase inhibitor, Y-27632, dose-dependently abolished Ang II-induced protein synthesis and also suppressed Ang II-induced c-fos mRNA expression. On the other hand, Y-27632 had no effect on Ang II-stimulated phosphorylation of p70 S6 kinase and extracellular signal-regulated kinase 1/2, which are reported to be involved in Ang II-induced protein synthesis, nor had it any effect on the Ang II-induced phosphorylation of PHAS-I, a heat- and acid-stable eIF-4E-binding protein. The phosphorylation of PHAS-I is regulating for translation initiation. These observations suggest that the Rho, Rho-kinase, and c-fos pathways may play a role in Ang II-induced hypertrophic changes of VSMCs through a novel pathway.

Intracellular pH modulation of ADF/cofilin proteins.

The ADF/cofilin (AC) proteins are necessary for the high rates of actin filament turnover seen in vivo. Their regulation is complex enough to underlie the precision in filament dynamics needed by stimulated cells. Disassembly of actin by AC proteins is inhibited in vitro by phosphorylation of ser3 and pH<7.1. This study of Swiss 3T3 cells demonstrates that pH also affects AC behavior in vivo: (1) Wounded cells show pH-dependent AC translocation to alkaline-induced ruffling membrane; (2) The Triton extractable (soluble) ADF from Swiss 3T3 cells decreases from 42+/-4% to 23+/-4% when the intracellular pH (pH(i)) is reduced from 7.4 to 6.6; (3) Covariance and colocalization analyses of immunostained endogenous proteins show that ADF partitions more with monomeric actin and less with polymeric actin when pH(i) increases. However, the distribution of cofilin, a less pH-sensitive AC in vitro, does not change with pH; (4) Only the unphosphorylatable AC mutant (A3), when overexpressed as a GFP chimera, uniquely produces aberrant cellular phenotypes and only if the pH is shifted from 7.1 to 6.6 or 7.4. A mechanism is proposed that explains why AC(A3)-GFP and AC(wt)-GFP chimeras generate different phenotypes in response to pH changes. Phospho-AC levels increase with cell density, and in motile cells, phospho-AC increases with alkalization, suggesting a homeostatic mechanism that compensates for increased AC activity and filament turnover. These results show that the behavior of AC proteins with pH-sensitivity in vitro is affected by pH in vivo.

Human RhoA/RhoGDI complex expressed in yeast: GTP exchange is sufficient for translocation of RhoA to liposomes.

The human small GTPase, RhoA, expressed in Saccharomyces cerevisiae is post-translationally processed and, when co-expressed with its cytosolic inhibitory protein, RhoGDI, spontaneously forms a heterodimer in vivo. The RhoA/RhoGDI complex, purified to greater than 98% at high yield from the yeast cytosolic fraction, could be stoichiometrically ADP-ribosylated by Clostridium botulinum C3 exoenzyme, contained stoichiometric GDP, and could be nucleotide exchanged fully with [3H]GDP or partially with GTP in the presence of submicromolar Mg2+. The GTP-RhoA/RhoGDI complex hydrolyzed GTP with a rate constant of 4.5 X 10(-5) s(-1), considerably slower than free RhoA. Hydrolysis followed pseudo-first-order kinetics indicating that the RhoA hydrolyzing GTP was RhoGDI associated. The constitutively active G14V-RhoA mutant expressed as a complex with RhoGDI and purified without added nucleotide also bound stoichiometric guanine nucleotide: 95% contained GDP and 5% GTP. Microinjection of the GTP-bound G14V-RhoA/RhoGDI complex (but not the GDP form) into serum-starved Swiss 3T3 cells elicited formation of stress fibers and focal adhesions. In vitro, GTP-bound-RhoA spontaneously translocated from its complex with RhoGDI to liposomes, whereas GDP-RhoA did not. These results show that GTP-triggered translocation of RhoA from RhoGDI to a membrane, where it carries out its signaling function, is an intrinsic property of the RhoA/RhoGDI complex that does not require other protein factors or membrane receptors.

INVESTIGATION OF IN VITRO TOXICITY OF JET FUELS JP-8 AND JET A

The in vitro cytotoxicity and electrophysiological toxicity of Jet Propulsion-8 (JP-8 jet fuel) on four cell types: H4IIE liver cell line, NIH Swiss 3T3 cell line, neuroblastoma × glioma NG108-15 cells, and embryonic hippocampal neurons were investigated. H4IIE cells exposed to Jet A (a commercial fuel) and JP-8 demonstrated identical toxicity with an IC50 of 12.6 ± 0.4 μg/ml for the two fuels. Comparison of H4IIE and NIH/3T3 toxicity to JP-8 revealed that NIH/3T3 cells were more sensitive to JP-8 than H4IIE cells, with an IC50 8.5 ± 0.1 μg/ml. JP-8 exposure for the hippocampal neurons proved to be highly toxic (IC50 of <2 μg/ml), while in contrast, the NG108-15 cells were much less sensitive. Electrophysiological examination of NG108-15 cells showed that administration of JP-8 at 1 μg/ml did not alter significantly any of the electrophysiological properties. However, exposure to JP-8 at 10 μg/ml during a current stimulus of +46 pA decreased the amplitude of the action potential to 83 ± 7% (n = 4), the rate of rise, dV/dtMAX to 50 ± 8% (n = 4), and the spiking rate to 25 ± 11% (n = 4) of the corresponding control levels. These results demonstrate JP-8 induced cytotoxic varies among cell types. The possible mechanisms underlying these observations are presented.

The pleckstrin homology domains of protein kinase B and GRP1 (general receptor for phosphoinositides-1) are sensitive and selective probes for the cellular detection of phosphatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate in vivo

We have tested the binding specificities of the pleckstrin homology (PH) domains of protein kinase B (PKB) and GRP1 (general receptor for phosphoinositides-1), expressed as green fluorescent protein (GFP) fusion proteins [PH(PKB)GFP and PH(GRP1)GFP respectively] in HEK 293 cells and Swiss 3T3 cells, using confocal microscopy. Stimulation of HEK 293 cells with insulin caused a small, but sustained, increase in PtdIns(3,4,5)P3 levels, detected using a radioligand displacement assay, which was mirrored by the translocation of PH(PKB)GFP and PH(GRP1)GFP from the cytosol to the plasma membrane of live, transfected cells. Similar results were obtained using Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) and expressing either PH(PKB)GFP or PH(GRP1)GFP. Biochemical analyses confirmed the accumulation of both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in response to PDGF, but only the latter was present at increased levels in Swiss 3T3 cells 30 min after an oxidative stress (1 mM H2O2). Concomitantly, only PH(PKB)GFP, and not PH(GRP1)GFP, was localized at plasma membranes after 30 min of treatment with H2O2. The fusion proteins appear accurately to report the spatial and temporal distribution of PtdIns(3,4,5)P3 and PtdIns(3,4)P2 in intact cells. These results establish the lipid selectivity of these PH domains in vivo, and further emphasize the overlapping, but distinct, second messenger roles of PtdIns(3,4,5)P3 and PtdIns(3,4)P2.

The pleckstrin homology domains of protein kinase B and GRP1 (general receptor for phosphoinositides-1) are sensitive and selective probes for the cellular detection of phosphatidylinositol 3,4-bisphosphate and/or phosphatidylinositol 3,4,5-trisphosphate in vivo

We have tested the binding specificities of the pleckstrin homology (PH) domains of protein kinase B (PKB) and GRP1 (general receptor for phosphoinositides-1), expressed as green fluorescent protein (GFP) fusion proteins [PH(PKB)GFP and PH(GRP1)GFP respectively] in HEK 293 cells and Swiss 3T3 cells, using confocal microscopy. Stimulation of HEK 293 cells with insulin caused a small, but sustained, increase in PtdIns(3,4,5)P(3) levels, detected using a radioligand displacement assay, which was mirrored by the translocation of PH(PKB)GFP and PH(GRP1)GFP from the cytosol to the plasma membrane of live, transfected cells. Similar results were obtained using Swiss 3T3 cells stimulated with platelet-derived growth factor (PDGF) and expressing either PH(PKB)GFP or PH(GRP1)GFP. Biochemical analyses confirmed the accumulation of both PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) in response to PDGF, but only the latter was present at increased levels in Swiss 3T3 cells 30 min after an oxidative stress (1 mM H(2)O(2)). Concomitantly, only PH(PKB)GFP, and not PH(GRP1)GFP, was localized at plasma membranes after 30 min of treatment with H(2)O(2). The fusion proteins appear accurately to report the spatial and temporal distribution of PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) in intact cells. These results establish the lipid selectivity of these PH domains in vivo, and further emphasize the overlapping, but distinct, second messenger roles of PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2).

Adrenomedullin suppresses interleukin-1β-induced tumor necrosis factor-α production in Swiss 3T3 cells

We demonstrated that adrenomedullin (AM) inhibited interleukin-1β-induced tumor necrosis factor-α (TNF-α) secretion and gene transcription in Swiss 3T3 fibroblasts maximally to 23% and 18% of control, while the other peptides elevating intracellular cAMP levels elicited much weaker effects. AM rapidly reduced the gene transcript level of TNF-α, inducing a maximal effect within 1 h. The inhibitory effect of AM was restored with an AM receptor antagonist as well as a cAMP-dependent protein kinase inhibitor. These findings indicate that AM is a potent and quick suppressor of TNF-α production in Swiss 3T3 cells acting through the cAMP protein kinase A pathway. As TNF-α is a major inflammatory cytokine and stimulates AM production in fibroblasts, AM is deduced to be an autocrine or paracrine factor suppressing inflammation through the inhibition of TNF-α production.

Nitric Oxide Donors Selectively Potentiate Thrombin-Stimulated p70S6k Activity and Morphological Changes in Swiss 3T3 Cells

Thrombin stimulates both DNA synthesis and cell morphological changes in Swiss 3T3 cells, although the mechanism of signal coordination leading to these responses is unknown. We report here that nitric oxide (NO) donors selectively enhance thrombin-stimulated p70S6k activity by 40–60%, an effect that was sustained for 24 h. Potentiation of p70S6k also was observed with cGMP analogues indicating that this effect is mediated by cGMP-activated protein kinase. NO donors also induced morphological changes characterized by spindle-shaped cells in confluent, nondividing cells or by extended protrusions from the trailing edge in subconfluent, polarized cells. NO donors had no significant effects on intracellular Ca2+ mobilization, DNA synthesis, proliferation, or ERKs 1 and 2 and p90RSK activities, indicating that mitogenic responses and cell division are not altered by NO donors. We conclude that NO donors modulate the morphological changes associated with cellular motility in response to thrombin stimulation through selective enhancement of p70S6k activity.

Ocular hypotensive activity of prostaglandin F 2α (PGF 2α) analogs with neutral substituents at position 1 is predicted by the elated cat iris sphincter smooth muscle preparation but not Ca 2+ signaling in Swiss 3T3 cells

Ocular hypotensive activity of prostaglandin F 2α (PGF 2α) analogs with neutral substituents at position 1 is predicted by the elated cat iris sphincter smooth muscle preparation but not Ca 2+ signaling in Swiss 3T3 cells

Membrane transport and in vitro metabolism of the Ras cascade messenger, glycerophosphoinositol 4-phosphate.

The glycerophosphoinositols, phosphoinositide metabolites formed by Ras-dependent activation of phospholipase A2 and a lysophospholipase, have been proposed to be markers of Ras-induced cell transformation. These compounds can have important cellular effects; GroPIns4P is an inhibitor of G protein-stimulated adenylate cyclase and is transiently produced in several cell types after growth factor receptor stimulation of phosphatidylinositol 3-kinase and the small G protein Rac, indicating the importance of defining further its cellular actions and metabolism. We show here that, in postnuclear membranes from Swiss 3T3 cells, there is no high-affinity 'receptor' binding of GroPIns4P. Instead, possibly through the interaction with a transporter, GroPIns4P rapidly equilibrates between medium and cell cytosol, and, at higher concentrations, can concentrate in the cell cytosol. GroPIns4P can be dephosphorylated to GroPIns in vitro by an enzyme that is membrane-associated, Ca2+-dependent, GroPIns4P-selective and has a specific pH profile. Under in vitro phosphorylating conditions, there is production of GroPIns(4,5)P2 and other inositol phosphates. As these in vitro enzyme activities do not fully correlate with the in vivo handling of GroPIns4P, the intracellular GroPIns4P levels may be controlled by its direct physical removal from the cells.

Inhibitory Phosphorylation Site for Rho-associated Kinase on Smooth Muscle Myosin Phosphatase

It is clear from several studies that myosin phosphatase (MP) can be inhibited via a pathway that involves RhoA. However, the mechanism of inhibition is not established. These studies were carried out to test the hypothesis that Rho-kinase (Rho-associated kinase) via phosphorylation of the myosin phosphatase target subunit 1 (MYPT1) inhibited MP activity and to identify relevant sites of phosphorylation. Phosphorylation by Rho-kinase inhibited MP activity and this reflected a decrease in V(max). Activity of MP with different substrates also was inhibited by phosphorylation. Two major sites of phosphorylation on MYPT1 were Thr(695) and Thr(850). Various point mutations were designed for these phosphorylation sites. Following thiophosphorylation by Rho-kinase and assays of phosphatase activity it was determined that Thr(695) was responsible for inhibition. A site- and phosphorylation-specific antibody was developed for the sequence flanking Thr(695) and this recognized only phosphorylated Thr(695) in both native and recombinant MYPT1. Using this antibody it was shown that stimulation of serum-starved Swiss 3T3 cells by lysophosphatidic acid, thought to activate RhoA pathways, induced an increase in Thr(695) phosphorylation on MYPT1 and this effect was blocked by a Rho-kinase inhibitor, Y-27632. In summary, these results offer strong support for a physiological role of Rho-kinase in regulation of MP activity.

G1 versus G2 cell cycle arrest after adriamycin-induced damage in mouse Swiss3T3 cells

Cell cycle arrest after different types of DNA damage can occur in either G1 phase or G2 phase of the cell cycle, involving the distinct mechanisms of p53/p21 Cip1/Waf1 induction, and phosphorylation of Cdc2, respectively. Treatment of asynchronously growing Swiss3T3 cells with the chemotherapeutic drug adriamycin induced a predominantly G2 cell cycle arrest. Here we investigate why Swiss3T3 cells were arrested in G2 phase and not in G1 phase after adriamycin-induced damage. We show that adriamycin was capable of inducing a G1 cell cycle arrest, both during the G0-G1 transition and during the G1 phase of the normal cell cycle. In G0 cells, adriamycin induced a prolonged cell cycle arrest. However, adriamycin caused only a transient cell cycle delay when added to cells at later time points during G0-G1 transition or at the G1 phase of normal cell cycle. The G1 arrest correlated with the induction of p53 and p21 Cip1/Waf1, and the exit from the arrest correlated with the decline of their expression. In contrast to the G1 arrest, adriamycin-induced G2 arrest was relatively tight and correlated with the Thr-14/Tyr-15 phosphorylation of cyclin B-Cdc2 complexes. The relative stringency of the G1 versus G2 cell cycle arrest may explain the predominance of G2 arrest after adriamycin treatment in mammalian cells.

Role of protein kinase B and the MAP kinase cascade in mediating the EGF-dependent inhibition of glycogen synthase kinase 3 in Swiss 3T3 cells

Epidermal growth factor (EGF), insulin-like growth factor 1 (IGF1) and phorbol myristate acetate (PMA) induce the inhibition of glycogen synthase kinase 3 (GSK3) by stimulating the phosphorylation of an N-terminal serine. Here, we show that protein kinase B (PKB) plays a key role in mediating EGF-induced inhibition of GSK3α and that the classical MAP kinase (MAPK) cascade has two functions in this process. Firstly, it makes a transient contribution to EGF-induced inhibition of GSK3α. Secondly, it shortens the duration of PKB activation and GSK3α inhibition. In contrast, PKB alone mediates the IGF1-induced inhibition of GSK3α, while the MAPK cascade mediates the inhibition of GSK3α by PMA.

Binding of polyomavirus small T antigen to protein phosphatase 2A is required for elimination of p27 and support of S-phase induction in concert with large T antigen.

Although polyomavirus large T antigen readily transactivates S-phase-specific enzymes in serum-starved Swiss 3T3 mouse fibroblasts, it is incapable by itself to efficiently drive such cells into S phase. We describe here that this inability correlates with a weak proficiency of the viral protein to induce the synthesis of cyclin A and cyclin E and to stimulate the respective cyclin/cdk activities. Polyomavirus small T antigen, which together with the large T protein supports S-phase induction, strongly contributes to the synthesis of cyclin A. In addition, small T antigen causes a dramatic induction of cyclin A- and, together with large T antigen, of cyclin E-specific protein kinase activity. This latter function of polyomavirus small T antigen correlates with its competence to provoke the elimination of the kinase inhibitor p27(Kip1). An interaction of the small T antigen with the protein phosphatase 2A is essential for this activity. Hence, the ability to drive quiescent Swiss 3T3 cells into S phase results from the capacity of large T antigen to transactivate DNA synthesis enzymes by its interaction with retinoblastoma-type proteins and from the potential of the large and the small T antigens together to stimulate cyclin A synthesis and cyclin A- and cyclin E-dependent protein kinase activity.