Phase Exit Research Articles

A comparative study of CrN, ZrN, NbN and TaN layers as cobalt diffusion barriers for CVD diamond deposition on WC–Co tools

Chromium, zirconium, niobium and tantalum nitrides layers have been sputtered onto WC–12 wt.% Co substrates as diffusion barriers and buffer layers for improving performances of diamond surface coating. X-ray diffraction shows under specific reactive sputtering conditions, only MN (M = Cr, Zr, Nb, Ta) type phase exits. Their electric resistivity has been measured on samples deposited onto silica under the same conditions and related to those published. Surface and transverse scanning electron microscopy shows a dense columnar morphology. Thermo chemical computing proves the stability of those nitrides against Co, hydrogen and methane up to 1150 K (877 °C). A computed diagram of nitrogen partial pressure is given for their carburization with methane showing the highest stability for ZrN and TaN. Diamond deposition for 5 h up to 1153 K (880 °C) highlights a different behaviour for each of those materials. Auger Electron Spectroscopy (AES) profiles show a massive diffusion of cobalt through the decomposed CrN layer, a transformation of TaN and NbN into carbide without diffusion of cobalt while ZrN is outstandingly well preserved.

GEF-H1 Modulates Localized RhoA Activation during Cytokinesis under the Control of Mitotic Kinases

Formation of the mitotic cleavage furrow is dependent upon both microtubules and activity of the small GTPase RhoA. GEF-H1 is a microtubule-regulated exchange factor that couples microtubule dynamics to RhoA activation. GEF-H1 localized to the mitotic apparatus in HeLa cells, particularly at the tips of cortical microtubules and the midbody, and perturbation of GEF-H1 function induced mitotic aberrations, including asymmetric furrowing, membrane blebbing, and impaired cytokinesis. The mitotic kinases Aurora A/B and Cdk1/Cyclin B phosphorylate GEF-H1, thereby inhibiting GEF-H1 catalytic activity. Dephosphorylation of GEF-H1 occurs just prior to cytokinesis, accompanied by GEF-H1-dependent GTP loading on RhoA. Using a live cell biosensor, we demonstrate distinct roles for GEF-H1 and Ect2 in regulating Rho activity in the cleavage furrow, with GEF-H1 catalyzing Rho activation in response to Ect2-dependent localization and initiation of cell cleavage. Our results identify a GEF-H1-dependent mechanism to modulate localized RhoA activation during cytokinesis under the control of mitotic kinases.

Aurora B Kinase Maintains Chromatin Organization During the MI to MII Transition in Surf Clam Oocytes

Meiosis represents a specialized cell cycle whereby cells undergo two reductive divisions without an intervening S phase. In oocytes, the transition from meiosis I to II is brief, with paired sister chromatids remaining condensed throughout the interkinesis period. This stands in contrast to mitotic divisions where cytokinesis and the return to interphase is always accompanied by chromatin decondensation and nuclear envelope reformation. Because other aspects of M phase exit are normal, we probed the mechanisms that allow for polar body extrusion while retaining chromatin condensation in Spisula solidissima oocytes. If oocytes were activated in the presence of protein synthesis inhibitors, oocytes progressed normally through MI, but arrested in interkinesis with condensed chromatin, phosphorylated histone H3 and a disorganized MII spindle. Neither inhibition of CDK1- nor MAPK activity in arrested oocytes was sufficient to drive chromatin decondensation or nuclear envelope reformation, suggesting that these kinases were not responsible for the maintenance of chromatin condensation. However, inhibition of Aurora B kinase activity resulted in chromatin decondensation, loss of histone H3 phosphorylation and reformation of the nuclear envelope. Inhibition of Aurora B activity following MI also resulted in chromosome segregation defects during MII and blocked polar body formation, consistent with Aurora B’s well-established role in cytokinesis. Together, these results suggest that extended Aurora B activity between meiotic divisions maintains chromatin condensation, thus allowing for the rapid reassembly of the MII spindle and progression through meiosis.

Metaphase Arrest by Cyclin E-Cdk2 Requires the Spindle-Checkpoint Kinase Mps1

Cytostatic factor (CSF) arrests vertebrate eggs in metaphase of meiosis II through several pathways that inhibit activation of the anaphase-promoting complex/cyclosome (APC/C). In Xenopus, the Mos-MEK1-MAPK-p90(Rsk) cascade utilizes spindle-assembly-checkpoint components to effect metaphase arrest. Another pathway involves cyclin E-Cdk2, and sustained cyclin E-Cdk2 activity in egg extracts causes metaphase arrest in the absence of Mos; this latter finding suggests that an independent pathway contributes to CSF arrest. Here, we demonstrate that metaphase arrest with cyclin E-Cdk2, but not with Mos, requires the spindle-checkpoint kinase monopolar spindles 1 (Mps1), a cyclin E-Cdk2 target that is also implicated in centrosome duplication. xMps1 is synthesized and activated during oocyte maturation and inactivated upon CSF release. In egg extracts, CSF release by calcium was inhibited by constitutively active cyclin E-Cdk2 and delayed by wild-type xMps1. Ablation of cyclin E by antisense oligonucleotides blocked accumulation of xMps1, suggesting that cyclin E-Cdk2 controls Mps1 levels. During meiosis II, activated cyclin E-Cdk2 significantly inhibited the APC/C even in the absence of the Mos-MAPK pathway, but this inhibition was not sufficient to suppress S phase between meiosis I and II. These results uniquely place xMps1 downstream of cyclin E-Cdk2 in mediating a pathway of APC/C inhibition and metaphase arrest.

Docosahexaenoic acid inhibits cancer cell growth via p27Kip1, CDK2, ERK1/ERK2, and retinoblastoma phosphorylation

Docosahexaenoic acid (DHA), a PUFA of the n-3 family, inhibited the growth of FM3A mouse mammary cancer cells by arresting their progression from the late-G(1) to the S phase of the cell cycle. DHA upregulated p27(Kip1) levels by inhibiting phosphorylation of mitogen-activated protein (MAP) kinases, i.e., ERK1/ERK2. Indeed, inhibition of ERK1/ERK2 phosphorylation by DHA, U0126 [chemical MAPK extracellularly signal-regulated kinase kinase (MEK) inhibitor], and MEK(SA) (cells expressing dominant negative constructs of MEK) resulted in the accumulation of p27(Kip1). MAP kinase (MAPK) inhibition by DHA did not increase p27(Kip1) mRNA levels. Rather, this fatty acid stabilized p27(Kip1) contents and inhibited MAPK-dependent proteasomal degradation of this protein. DHA also diminished cyclin E phosphorylation, cyclin-dependent kinase-2 (CDK2) activity, and phosphorylation of retinoblastoma protein in these cells. Our study shows that DHA arrests cell growth by modulating the phosphorylation of cell cycle-related proteins.

Curvaton reheating mechanism in inflation on warped Dvali–Gabadadze–Porrati brane

Abstract An impressed feature of inflation on warped Dvali–Gabadadze–Porrati (DGP) brane is that the inflationary phase exits spontaneously for a scalar inflaton field with exponential potential, which presents a graceful exit mechanism for the inflation. But its reheating mechanism leaves open. We investigate the curvaton reheating in inflation on warped DGP brane model. The reheating may occur in effectively 5-dimensional or 4-dimensional stage. We study the permitted parameter space of the curvaton field in detail. We demonstrate how the inflation model of the warped DGP brane is improved by the curvaton mechanism.

Doublecortin-like Kinase Controls Neurogenesis by Regulating Mitotic Spindles and M Phase Progression

The mechanisms controlling neurogenesis during brain development remain relatively unknown. Through a differential protein screen with developmental versus mature neural tissues, we identified a group of developmentally enriched microtubule-associated proteins (MAPs) including doublecortin-like kinase (DCLK), a protein that shares high homology with doublecortin (DCX). DCLK, but not DCX, is highly expressed in regions of active neurogenesis in the neocortex and cerebellum. Through a dynein-dependent mechanism, DCLK regulates the formation of bipolar mitotic spindles and the proper transition from prometaphase to metaphase during mitosis. In cultured cortical neural progenitors, DCLK RNAi Lentivirus disrupts the structure of mitotic spindles and the progression of M phase, causing an increase of cell-cycle exit index and an ectopic commitment to a neuronal fate. Furthermore, both DCLK gain and loss of function in vivo specifically promote a neuronal identity in neural progenitors. These data provide evidence that DCLK controls mitotic division by regulating spindle formation and also determines the fate of neural progenitors during cortical neurogenesis.

Poised for Action

The current model for gene expression in response to environmental change involves recruitment of transcription factors and RNA polymerase II (RNA Pol II) to the DNA. Radonjic et al. challenge this view with evidence that, in stationary-phase yeast, RNA Pol II is already bound to intergenic DNA upstream of genes that will be rapidly stimulated in yeast following exit from the quiescent state. Changes in poly(A) mRNA were quantified after stationary cells were introduced into glucose-rich medium, and three peaks in mRNA abundance were detected during the progression back to stationary phase (a 9-day process), as well as a sharp rise in mRNA abundance between 15 and 25 minutes after glucose repletion. Microarray analysis indicated that 769 genes were stimulated between 2- and 35-fold within 6 minutes of exit from stationary phase. There was also a set of genes that were induced just prior to entry of stationary phase and that remained enriched in stationary-phase cells for several days, suggesting active transcription in stationary cells. The abundance of several components of the transcriptional machinery was similar in stationary- and mid-log-phase cells, although the phosphorylation of the C-terminal domain (CTD) of the largest subunit of RNA Pol II was less in stationary-phase cells. Using genome-wide chromatin immunoprecipitation (ChIP on a chip), RNA Pol II was enriched on more than 1300 DNA sites in stationary cells and on ~3200 sites on mid-log-phase cells. One difference was the frequency with which RNA Pol II was found on intergenic regions (higher in stationary-phase cells relative to mid-log-phase cells) and gene-coding regions (higher in mid-log-phase cells). More detailed analysis of the DNA containing a gene that is enriched in stationary phase showed that RNA Pol II was bound along the gene-encoding region for PUT4 , consistent with active transcription. Detailed analysis of a gene that is immediately up-regulated upon exit from stationary phase, SUT1 , showed that RNA Pol II was bound in the intergenic region. The authors emphasize two results from their work. First, transcription is not completely shut down in stationary-phase cells, and they may be capable of responding to environmental changes without leaving stationary phase through the limited active RNA Pol II present. Second, RNA Pol II binding to DNA may not be the rate-limiting step in transcription for the genes that are rapidly stimulated following exit from stationary phase; instead, RNA Pol II may be bound in the intergenic region but the activity may be repressed by the state of phosphorylation, allowing a rapid response at these genes. M. Radonjic, J.-C. Andrau, P. Lijnzaad, P. Kemmeren, T. T. J. P. Kockelkorn, D. van Leenen, N. L. van Berkum, F. C. P. Holstege, Genome-wide analyses reveal RNA polymerase II located upstream of genes poised for rapid response upon S. cerevisiae stationary phase exit. Mol. Cell 18 , 171-183 (2005). [Online Journal]

Novel indication for cancer therapy: Chk1 inhibition sensitizes tumor cells to antimitotics

Paclitaxel (Taxol) is the most-prescribed anti-mitotic agent for a variety of advanced metastatic cancers. It induces mitotic arrest leading to apoptosis through microtubule stabilization. Chk1 is the major cell-cycle checkpoint kinase mediating S- and G2-arrests in response to various DNA-damages. Chk1 inhibitor is anticipated and has been demonstrated to potentiate the cytotoxicity of DNA-damaging agents through abrogation of cell-cycle checkpoints. Paclitaxel does not, however, induce Chk1 activation, and Chk1 has not been shown to function in mitotic checkpoint. Thus, Chk1 inhibitor is not expected to enhance the toxicity of paclitaxel. Here we show that downregulation of Chk1 sensitizes tumor cells to the toxicity of paclitaxel in cell proliferation assay. Fluorescence microscopy showed that Chk1 knockdown augments mitotic catastrophe and apoptosis in paclitaxel-treated cancer cells. Further, we elucidated the mechanism of this sensitization. Chk1 inhibition facilitates paclitaxel-induced M-phase entry by activation of Cdc2 kinase and accumulation of cyclin B1, the required cofactor for Cdc2 kinase activity. Moreover, Chk1 downregulation inhibits M phase exit through induction of the anaphase inhibitor, securin/PDS1. Collectively, Chk1 elimination sustains a more effective mitotic arrest as demonstrated by the more efficient accumulation of M-phase marker phospho-histone H3. We show that Chk1 elimination attenuates the paclitaxel-induced activation of the anti-apoptotic p42/p44 (ERK1/2) MAP kinase pathway, additionally contributing to the sensitization. Our results suggest that in addition to its well-established role as an enforcer of S and G2-checkpoints in response to genotoxic stress, Chk1 also plays a protective role in mitotic checkpoint to lessen mitotic catastrophe and thereby limits cell-death. Therefore Chk1 downregulation can not only potentiate DNA-damaging agents, but also enhance the toxicity of anti-microtubule agents, which significantly broadens its therapeutic applications.

Genome-Wide Analyses Reveal RNA Polymerase II Located Upstream of Genes Poised for Rapid Response upon S. cerevisiae Stationary Phase Exit

The resting state of eukaryotic cells (G0) is relatively uncharacterized. We have applied DNA microarray expression profiling of S. cerevisiae to reveal multiple transitions during a complete 9-day growth cycle between stationary phase (SP) exit and entry. The findings include distinct waves of transcription after the diauxic shift (DS), identification of genes active in SP, and upregulation of over 2500 genes during the first minutes of lag phase. This provides a framework for analyzing large-scale reprogramming of gene expression. Despite global repression, the general transcription machinery is found to be present in quiescent cells but is largely inactive. Genome-wide location analysis by chromatin immunoprecipitation (ChIP on chip) reveals that RNA polymerase II is more predominantly bound at intergenic regions in SP, upstream of hundreds of genes immediately induced upon exit. In contrast to current models of activation-coupled recruitment, the results show that RNA polymerase II is located and maintained upstream of many inactive genes in quiescence.

Zyg-11 and cul-2 regulate progression through meiosis II and polarity establishment in C. elegans.

The mechanisms that ensure coupling between meiotic cell cycle progression and subsequent developmental events, including specification of embryonic axes, are poorly understood. Here, we establish that zyg-11 and the cullin cul-2 promote the metaphase-to-anaphase transition and M phase exit at meiosis II in Caenorhabditis elegans. Our results indicate that ZYG-11 acts with a CUL-2-based E3 ligase that is essential at meiosis II and that functions redundantly with the anaphase-promoting complex/cyclosome at meiosis I. Our data also indicate that delayed M phase exit in zyg-11(RNAi) embryos is due to accumulation of the B type cyclin CYB-3. We demonstrate that PAR proteins and P granules become polarized in an inverted manner during the meiosis II delay resulting from zyg-11 or cul-2 inactivation, and that zyg-11 and cul-2 can regulate polarity establishment independently of a role in cell cycle progression. Furthermore, we find that microtubules appear dispensable for ectopic polarity during the meiosis II delay in zyg-11(RNAi) embryos, as well as for AP polarity during the first mitotic cell cycle in wild-type embryos. Our findings suggest a model in which a CUL-2-based E3 ligase promotes cell cycle progression and prevents polarity establishment during meiosis II, and in which the centrosome acts as a cue to polarize the embryo along the AP axis after exit from the meiotic cell cycle.

Identification of a new APC/C recognition domain, the A box, which is required for the Cdh1-dependent destruction of the kinase Aurora-A during mitotic exit.

The mitotic kinase Aurora A (Aur-A) is required for formation of a bipolar mitotic spindle and accurate chromosome segregation. In somatic cells, Aur-A protein and kinase activity levels peak during mitosis, and Aur-A is degraded during mitotic exit. Here, we investigated how Aur-A protein and kinase activity levels are regulated, taking advantage of the rapid synchronous cell division cycles of Xenopus eggs and cell-free systems derived from them. Aur-A kinase activity oscillates in the early embryonic cell cycles, just as in somatic cells, but Aur-A protein levels are constant, indicating that regulated activation and inactivation, instead of periodic proteolysis, is the dominant mode of Aur-A regulation in these cell cycles. Cdh1, the APC/C activator that targets many mitotic proteins for ubiquitin-dependent proteolysis during late mitosis and G1 in somatic cells, is missing in Xenopus eggs and early embryos. We find that addition of Cdh1 to egg extracts undergoing M phase exit is sufficient to induce rapid degradation of Aur-A. Aur-A contains both of the two known APC/C recognition signals, (1) a C-terminal D box similar to those required for ubiquitin-dependent destruction of cyclin B and several other mitotic proteins, and (2) an N-terminal KEN box similar to that found on cdc20, which is ubiquitinated in response to APC/C(Cdh1). The D box is required for Cdh1-induced destruction of Aur-A but the KEN box is not. Destruction also requires a short region in the N terminus, which contains a newly identified recognition signal, the A box. The A box is conserved in vertebrate Aur-As and contains serine 53, which is phosphorylated during M phase. Mutation of serine 53 to aspartic acid, which can mimic the effect of phosphorylation, completely blocks Cdh1-dependent destruction of Aur-A. These results suggest that dephosphorylation of serine 53 during mitotic exit could control the timing of Aur-A destruction, allowing recognition of both the A box and D box by Cdh1-activated APC/C.

COSMOLOGICAL EVOLUTION IN A TYPE-0 STRING THEORY

We study the cosmological evolution of a type-0 string theory by employing non-criticality, which may be induced by fluctuations of the D3 brane worlds. We check the consistency of the approach to [Formula: see text] in the corresponding σ-model. The ten-dimensional theory is reduced to an effective four-dimensional model, with only time dependent fields. We show that the four-dimensional universe has an inflationary phase and graceful exit from it, while the other extra dimensions are stabilized to a constant value, with the fifth dimension much larger than the others. We pay particular attention to demonstrating the role of tachyonic matter in inducing these features. The universe asymptotes, for large times, to a nonaccelerating linearly-expanding universe with a time-dependent dilaton and a relaxing to zero vacuum energy a lá quintessence. Our perturbative string solution appears to have initial singularities (Big Bang type), which however, we believe, may be lifted in a nonperturbative way, and they do not represent true singularities of the string theory.

Preparation of high-pressure phase boron nitride films by physical vapor deposition

The high-pressure phases boron nitride films together with cubic, wurtzic, and explosive high-pressure phases, were successfully deposited on the metal alloy substrates by tuned substrate radio frequency magnetron sputtering. The percentage of cubic boron nitride phase in the film was about 50% as calculated by Fourier transform infrared measurements. Infrared peak position of cubic boron nitride at 1006.3 cm−1, which is close to the stressless state, indicates that the film has very low internal stress. Transition electron microscope micrograph shows that pure cubic boron nitride phase exits on the surface of the film. The growth mechanism of the BN films was also discussed.

Coordinated Regulation of M Phase Exit and S Phase Entry by the Cdc2 Activity Level in the Early Embryonic Cell Cycle

In the early embryonic cell cycle, exit from M phase is immediately followed by entry into S phase without an intervening gap phase. To understand the regulatory mechanisms for the cell cycle transition from M to S phase, we examined dependence on Cdc2 inactivation of cell-cycle events occurring during the M-S transition period, using Xenopus egg extracts in which the extent of Cdc2 inactivation at M phase exit was quantitatively controlled. The result demonstrated that MCM binding to and the initiation of DNA replication of nuclear chromatin occurred depending on the decrease of Cdc2 activity to critical levels. Similarly, we found that Cdc2 inhibitory phosphorylation and cyclin B degradation were turned on and off, respectively, depending on the decrease in Cdc2 activity. However, their sensitivity to Cdc2 activity was different, with the turning-on of Cdc2 inhibitory phosphorylation occurring at higher Cdc2 activity levels than the turning-off of cyclin B degradation. This means that, when cyclin B degradation ceases at M phase exit, Cdc2 inhibitory phosphorylation is necessarily activated. In the presence of constitutive synthesis of cyclin B, this condition favors the occurrence of the Cdc2 inactivation period after M phase exit, thereby ensuring progression through S phase. Thus, M phase exit and S phase entry are coordinately regulated by the Cdc2 activity level in the early embryonic cell cycle.

Metaphase to anaphase (mat) transition-defective mutants in Caenorhabditis elegans.

The metaphase to anaphase transition is a critical stage of the eukaryotic cell cycle, and, thus, it is highly regulated. Errors during this transition can lead to chromosome segregation defects and death of the organism. In genetic screens for temperature-sensitive maternal effect embryonic lethal (Mel) mutants, we have identified 32 mutants in the nematode Caenorhabditis elegans in which fertilized embryos arrest as one-cell embryos. In these mutant embryos, the oocyte chromosomes arrest in metaphase of meiosis I without transitioning to anaphase or producing polar bodies. An additional block in M phase exit is evidenced by the failure to form pronuclei and the persistence of phosphohistone H3 and MPM-2 antibody staining. Spermatocyte meiosis is also perturbed; primary spermatocytes arrest in metaphase of meiosis I and fail to produce secondary spermatocytes. Analogous mitotic defects cause M phase delays in mitotic germline proliferation. We have named this class of mutants "mat" for metaphase to anaphase transition defective. These mutants, representing six different complementation groups, all map near genes that encode subunits of the anaphase promoting complex or cyclosome, and, here, we show that one of the genes, emb-27, encodes the C. elegans CDC16 ortholog.

Role of human cytomegalovirus immediate-early proteins in cell growth control.

Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus that has been implicated in several disorders, including an association between HCMV reactivation and the overproliferation of arterial smooth muscle cells observed in restenosis. Although HCMV can mediate a growth-arrest phenotype in infected cells, the virus can also promote an environment conducive to proliferation. Here, we present evidence that the HCMV immediate-early (IE) proteins, IE1-72 and IE2-86, may be responsible for inducing this proliferative environment by altering cell cycle control. We find that expression of either of these IE proteins can alter the cell cycle distribution of randomly cycling cells towards S and G(2)/M phases. Additionally, we find that expression of IE2-86, but not IE1-72, induces quiescent cells into S phase and delays cell cycle exit. In the absence of p53, IE1-72 expression can induce S phase and delay cell cycle exit. We also demonstrate that p53 protein levels increase in fibroblasts following the expression of IE1-72. The observed accumulation of p53 protein in IE1-72-expressing cells may account for the inability of IE1-72 to induce S phase and delay cell cycle exit. Our data suggest that expression of HCMV IE1-72 and IE2-86 is sufficient to alter the cell cycle to generate an environment conducive to proliferation.

A destructive role for the Polo-like kinases

DESCOMBES, P. and NIGG, E. A. (1998) The polo-like kinase Plx1 is required for M phase exit and destruction of mitotic regulators in Xenopus egg extracts EMBO J. 17, 1328–1335 KOTANI, S. et al. (1998) PKA and MPF-activated Polo-like kinase regulate Anaphase-Promoting Complex activity and mitosis progression Mol. Cell 1, 371–380 SHIRAYAMA, M., ZACHARIAE, W., CIOSK, R. and NASMYTH, K. (1998) The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae EMBO J. 17, 1336–1349

The polo-like kinase Plx1 is required for M phase exit and destruction of mitotic regulators in Xenopus egg extracts.

Polo-like kinases (Plks), named after the Drosophila gene product polo, have been implicated in the regulation of multiple aspects of mitotic progression, including the activation of the Cdc25 phosphatase, bipolar spindle formation and cytokinesis. Genetic analyses performed in yeast and Drosophila suggest a function for Plks at late stages of mitosis, but biochemical data to support such a function in vertebrate organisms are lacking. Here we have taken advantage of Xenopus egg extracts for exploring the function of Plx1, a Xenopus Plk, during the cell cycle transition from M phase to interphase (I phase). We found that the addition of a catalytically inactive Plx1 mutant to M phase-arrested egg extracts blocked their Ca2+-induced release into interphase. Concomitantly, the proteolytic destruction of several targets of the anaphase-promoting complex and the inactivation of the Cdc2 protein kinase (Cdk1) were prevented. Moreover, the M to I phase transition could be abolished by immunodepletion of Plx1, but was restored upon the addition of recombinant Plx1. These results demonstrate that the exit of egg extracts from M phase arrest requires active Plx1, and they strongly suggest an important role for Plx1 in the activation of the proteolytic machinery that controls the exit from mitosis.

High glucose stimulates expression of p27Kip1 in cultured mouse mesangial cells: relationship to hypertrophy.

Hypertrophy of mesangial cells is an early hallmark of diabetic nephropathy. We have previously shown that murine mesangial cells (MMC), cultured in high-glucose medium, are arrested in the G1 phase of the cell cycle and undergo hypertrophy. This study was undertaken to test whether high glucose-containing medium influences the expression of p27Kip1, an inhibitor of G1 phase active cyclin-dependent kinases (CDK). Incubation of MMC, in the absence of other factors for 48-96 h, in medium containing high D-glucose (450 mg/dl), stimulated p27Kip1 protein expression but failed to influence mRNA abundance. These effects were independent of the osmolarity of the medium. High glucose-stimulated expression of p27Kip1 involved activation of protein kinase C and was partly dependent on induction of transforming growth factor-beta (TGF-beta). Immunoprecipitation experiments revealed that only small amounts of p27Kip1 protein from MMC grown in high-glucose medium preferentially associates with CDK2 but not with CDK4. The p27Kip1 antisense, but not missense, oligonucleotides inhibited high glucose-stimulated total protein synthesis and facilitated G1 phase exit. Our data showed for the first time that expression of p27Kip1 protein is pivotal in mesangial cell hypertrophy induced by high ambient glucose. These findings may be important in the deciphering of molecular processes causing diabetic glomerular hypertrophy.