Cell cycle-dependent vimentin expression in elutriator-synchronized, TPA-treated MPC-11 mouse plasmacytoma cells.

We correlated cell cycle progression and vimentin expression at the single cell level by multiparameter flow cytometry in populations of MPC-11 cells enriched in different cell cycle phases by centrifugal elutriation and subsequently treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Synchronized, untreated cultures showed a uniform, synchronous progression through the cell cycle during further cultivation. A 6-h TPA treatment of G1-phase-enriched cultures induced both a partial G1-phase arrest in the same cycle and a moderate fraction of cells to become vimentin positive. However, nearly all cells of the cultures enriched in S- or in G2/M-phase cells could be arrested by TPA treatment at the earliest in the G1 phase of the second cell cycle and displayed higher fractions of positive cells as well as higher average levels of vimentin. After 20 h of treatment, the G1-phase arrest was almost complete. In terms of fractions of vimentin-positive cells as well as of average cellular vimentin content, the differences between the cultures resembled, albeit on a higher level, those between the respective cultures treated with TPA for 6 h. These observations might explain the striking bimodal distribution of individual cellular vimentin content detectable in G1-phase fractions of asynchronous, TPA-treated cultures. The pattern of vimentin mRNA accumulation in synchronized cultures after short-term TPA treatment strongly suggests that the cell cycle-dependent pattern of vimentin expression is caused, at least in part, by different levels of vimentin mRNA accumulated in the cells. Since proteinaceous mediator(s) are obviously involved in TPA-induced vimentin expression in MPC-11 cells, cell cycle-dependent vimentin expression in these cells may be dependent on cell cycle-dependent regulation of the activity and/or concentration of such mediator(s).     

High resolution analysis of cell cycle-correlated vimentin expression in asynchronously grown,TPA-treated MPC-11 cells by the novel flow cytometric multiparameter BrdU-hoechst/PI and immunolabeling technique

High resolution, multiparameter analysis using the flow cytometric BrdU/Hoechst quenching technique has been applied to study cell cycle kinetics and vimentin expression in individual cells of asynchronously grown MPC-11 mouse plasmacytoma cell cultures treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce in vitro differentiation. BrdU treatment up to 16 h in the absence or presence of TPA did not affect either cell cycle progression or the kinetics or quantity of vimentin expression. TPA-treated cells became arrested in G1 phase of the second cell cycle; however, this G1 phase arrest was transient only. In addition, G1 phase cells located prior to a putative transition point at the beginning of TPA treatment were completely blocked in cell cycle progression. There is also evidence that cells located in G1 or G2/M phase at the beginning of TPA treatment finally expressed low levels of vimentin. On the contrary, cells located in S phase at TPA exposure showed high vimentin levels after treatment. The results presented here show that, with the flow cytometric BrdU/Hoechst quenching technique, one can correlate time-dependent protein expression at the single cell level in asynchronously grown cultures not only with the actual cell cycle state, but also with the history of cell replication. © 1994 Wiley-Liss, Inc.     

Okadaic acid co-induces vimentin expression and cell cycle arrest in MPC-11 mouse plasmacytoma cells

The effect of the tumor promoter okadaic acid on cell cycle progression and on vimentin expression in MPC-11 mouse plasmacytoma cells was compared with that of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Cell cycle progression of asynchronously grown MPC-11 cells was inhibited by both agents, but, in contrast to the G1 phase arrest caused by TPA, okadaic acid gave rise to G2/M phase and S phase arrest. This effect of okadaic acid was delayed significantly compared to the TPA-caused arrest. Furthermore, okadaic acid was able to induce vimentin expression to an extent comparable to the TPA response. However, vimentin expression was markedly delayed in okadaic acid-treated relative to TPA-treated cells. Another protein phosphatase inhibitor, calyculin A, also induced cell cycle changes and vimentin expression at concentrations at or above 1 × 10^?9M. Based on these observations, we suggest an involvement of protein phosphatase 1 (possibly also phosphatase 2A and/or other phosphatases) in both the G2/M cell cycle block and the induction of vimentin expression in MPC-11 cells by okadaic acid. © 1995 Wiley-Liss, Inc.     

Influence of fibric acid derivatives on intermediate filament proteins in myocardiocyte cultures

We analyzed desmin and vimentin accumulation in chick myocardiocyte cultures treated with the fibric acid derivatives bezafibrate, fenofibrate and gemfibrozil. The most noteworthy finding was the 50% decrease in the cytoplasmic desmin fraction in cells treated with gemfibrozil in comparison to control cultures, and the 19% increase in the cytoskeletal fraction in cultures treated with gemfibrozil and with bezafibrate. Vimentin accumulation by cells treated with bezafibrate was similar to that in control cultures, however the cytoskeletal vimentin fraction rose by 26% after treatment with gemfibrozil, and fell 13% after treatment with fenofibrate. No vimentin was found in the cytoplasmic fraction of cell treated with bezafibrate. Given the role of intermediate filaments in heart muscle contraction, fibric acid derivative- induced changes in the cytoplasmic and cytoskeletal concentrations of intermediate filament proteins may be related with the secondary effects of these drugs on heart rate.     

Alteration of cell cycle progression in human leukemia cell line (KOPM-28) induced by 12-o-tetradecanoylphorbol-13-acetate

Terminal cell differentiation usually results in an irreversible arrest in the G1 phase of the cell cycle and loss of cell renewal ability. Human promyelocytic leukemia HL-60 cells induced with 12-o-tetradecanoylphorbol-13-acetate (TPA) differentiate into monocytes/macrophages and accumulate in G1. We determined the effect of TPA on the growth kinetics of a human leukemia cell line (KOPM-28), which developed several of the characteristics of megakaryocytes in response to TPA, such as the surface antigen complex IIb/IIIa, platelet peroxidase and polyploidy. Cell growth was immediately and completely inhibited by TPA. Flow cytometric analysis of cellular DNA content revealed a gradual decrease in cells in G1 and an accumulation of cells in G2. These data suggest that TPA prolonged G1 and rapidly arrested the cells in G2. Synchronized cells were utilized to further analyze the rapid G2 arrest. Cells arrested with aphidicolin at the G1/S interphase were released, and the effects of TPA (added at different intervals) on cell cycle progression were examined 14 h after release. The results showed that TPA added at the end of the S phase, as well as at the G1/S interphase incompletely but distinctly arrested cells in G2. Moreover, G2 arrest was observed when TPA was added to cells released from a colcemid-induced G2/M block, suggesting that cells already in G2 were inhibited by TPA from moving through M to G1. Since some cells became multi-nucleated in the course of incubation with TPA, this G2 accumulation may have resulted at least in part from a prolongation of the phase or a transient G2 block. These changes in cell cycle progression induced by TPA may be characteristic of and/or related to megakaryocytic differentiation of hemopoietic precursor cells.     

TPA induces apoptosis in MPC-11 mouse plasmacytoma cells grown in serum-free medium

Apoptotic-like events could be rapidly induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in cells of the mouse plasmacytoma cell line MPC-11 grown in serum-free medium. Indicators for apoptosis were morphological changes visualized by light and electron microscopy, such as chromatin condensation and the formation of cellular buds and fragments, as well as biochemical indices like the appearance of the so-called ‘DNA ladder’. Additionally, in these cells which are usually devoid of significant amounts of cytoplasmic intermediate filament (cIF) proteins, synthesis and accumulation of the cIF protein vimentin was rapidly induced by TPA treatment and almost all cells became vimentin-positive. Later on, substantial amounts of vimentin and lamin B degradation products appeared, and an increasing fraction of cells displayed low or even undetectable quantities of intact vimentin. This subpopulation was characterized via microscopy to be in the late stages of apoptosis. We suggest that in MPC-11 cells undergoing apoptosis in response to TPA treatment vimentin as well as lamin B are degraded, leading to a rearrangement and eventual loss of their respective filament networks.     

Induction of vimentin synthesis in a murine myeloma cell line by TPA is strongly dependent on the composition of the cell culture medium

MPC-11 mouse plasmacytoma cells virtually lacking intermediate filament (IF) proteins can be induced to synthesize and accumulate the IF protein vimentin by treatment with the tumor promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Like MPC-11 cells, X63-Ag8.6.5.3 mouse myeloma cells (Ag8) proved to be vimentin-negative, as assayed by immunoblotting of whole cellular protein using goat antiserum to vimentin and [125I]protein A. Vimentin synthesis could be elicited by a TPA concentration as low as 10(-9) M in cells grown in HB-102 serum-free medium. Transfer of these cells to medium containing 15% fetal calf cerum (FCS) greatly reduced the ability of these cells to synthesize vimentin upon TPA treatment. After 50 generations of culture in the presence of FCS, induction of vimentin synthesis was barely detectable even at a TPA concentration of 10(-6) M. Addition of FCS to cells grown in serum-free medium partially suppressed vimentin induction by TPA. This suppression seems to be due, at least in part, to nondialyzable, heat-sensitive components of FCS, since the dialyzable fraction even enhanced vimentin induction by TPA. When cells grown in the presence of FCS were transferred back to serum-free medium, their ability to synthesize vimentin in response to TPA treatment was readily restored. The individual components of serum-free medium which proved to support vimentin induction by TPA were insulin and the unsaturated fatty acids oleic acid and linoleic acid. An even stronger TPA response could be elicited by a combination of these components.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of extracellular signal-regulated kinase (ERK) in G2 phase delays mitotic entry through p21CIP1

Extracellular signal-regulated kinase activity is essential for mediating cell cycle progression from G(1) phase to S phase (DNA synthesis). In contrast, the role of extracellular signal-regulated kinase during G(2) phase and mitosis (M phase) is largely undefined. Previous studies have suggested that inhibition of basal extracellular signal-regulated kinase activity delays G(2)- and M-phase progression. In the current investigation, we have examined the consequence of activating the extracellular signal-regulated kinase pathway during G(2) phase on subsequent progression through mitosis. Using synchronized HeLa cells, we show that activation of the extracellular signal-regulated kinase pathway with phorbol 12-myristate 13-acetate or epidermal growth factor during G(2) phase causes a rapid cell cycle arrest in G(2) as measured by flow cytometry, mitotic indices and cyclin B1 expression. This G(2)-phase arrest was reversed by pre-treatment with bisindolylmaleimide or U0126, which are selective inhibitors of protein kinase C proteins or the extracellular signal-regulated kinase activators, MEK1/2, respectively. The extracellular signal-regulated kinase-mediated delay in M-phase entry appeared to involve de novo synthesis of the cyclin-dependent kinase inhibitor, p21(CIP1), during G(2) through a p53-independent mechanism. To establish a function for the increased expression of p21(CIP1) and delayed cell cycle progression, we show that extracellular signal-regulated kinase activation in G(2)-phase cells results in an increased number of cells containing chromosome aberrations characteristic of genomic instability. The presence of chromosome aberrations following extracellular signal-regulated kinase activation during G(2)-phase was further augmented in cells lacking p21(CIP1). These findings suggest that p21(CIP1) mediated inhibition of cell cycle progression during G(2)/M phase protects against inappropriate activation of signalling pathways, which may cause excessive chromosome damage and be detrimental to cell survival.     

Effect of the tumor promoter TPA on the distribution of PHA-stimulated human lymphocytes by cell cycle phases

Patterns of the cell cycle distribution in human peripheral blood lymphocytes, stimulated by PHA alone and PHA plus 12-o-tetradecanoylphorbol-13-acetate (TPA), were studied using DNA cytometry in different times after PHA stimulation. In the first period (nearly 3 days after PHA stimulation) TPA induces no significant differences in the characters under consideration, but in the later period, when the proliferation of the cultures stimulated by PHA alone is reducing, in other cultures stimulated by PHA plus TPA the percentage of cells in S-phase does not reduce, whereas the percentage of cells in G2-phase is rising, which may suggest that this phase is blocked. Concurrently the tetraploid cells are appearing. Accumulation of cells in G2-phase can be overcome by the application of chlorpromazine, which is known to inhibit the membrane-associated protein kinase C.     

Activation of protein kinase C relays distinct signaling pathways in the same cell type: differentiation and caspase-mediated apoptosis

Activation of PKC with 5 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 72 h in human U937 myeloid leukemia cells is associated with induction of adherence, followed by monocytic differentiation and G0/G1 cell cycle arrest. In this study, we demonstrate that in addition to these effects about 25% of U937 cells accumulated in an apoptotic subG1 phase after TPA treatment. The appearance of these apoptotic suspension cells was detectable throughout the time course of the culture and was independent of TPA concentrations between 0.5 and 500 nM. Experiments with cells synchronized by centrifugal elutriation revealed dominant susceptibility of G1-phase cells to TPA-mediated apoptosis. While adherent cells expressed differentiation markers including the integrin CD11c, this effect was less pronounced in the TPA-treated suspension fraction. Moreover, previous work has demonstrated cell cycle arrest in differentiating U937 cells. Accordingly, PKC activation by TPA treatment was associated with a significant expression of the cdk/cyclin inhibitor p21WAF/CIP/sdi-1 in the adherent population and subsequent G0/G1 cell cycle arrest. In contrast, suspension cells failed to induce significant levels of p21WAF/CIP/sdi-1 after TPA stimulation. Immunoblotting experiments demonstrated no difference in the expression of the pro-apoptotic factors Bax, Bad, and Bak in either control U937 and TPA-treated adherent or suspension cells, respectively. However, anti-apoptotic factors including Bcl-2, Bcl-xL, and Mcl-1 were significantly induced in the adherent population whereas no induction was detectable in the suspension cells. In this context, incubation with the caspase-3/caspase-7 specific tetrapeptide inhibitor DEVD prior to TPA treatment prevented an accumulation of cells in subG1, respectively, demonstrating an involvement of these caspases. Taken together, these data suggest that PKC activation can relay distinct signaling pathways such as induction of adherence coupled with monocytic differentiation and growth arrest, or induction of caspase-mediated apoptosis coupled with the failure to adhere and to differentiate.     

Loosening of cell cycle controls of human lymphocytes under the action of tumour promoter TPA

The effect of tumour promoter TPA (12-O-tetradecanoylphorbol-13-acetate) on the cell cycle of human peripheral blood lymphocytes stimulated by phytohaemagglutinin (PHA) in vitro was studied and it was found that TPA caused cells to accumulate in all the cell cycle phases. This accumulation took place preferentially at later culture passages, when lymphocytes stimulated by PHA alone stopped mainly in G0/G1 phases. Other effects of TPA were cell induction to enter higher DNA ploidy and to survive and even synthesize DNA under colchicine block of mitosis or under cytochalasin block of cytokinesis. In addition, in experiments in which a transitory block through the G1 phase of cell cycle was applied with use of aminopterin, we could show that a fraction of TPA-treated cells still entered the active phase of DNA synthesis. These findings suggest that TPA causes cell cycle controls to become loose, thereby enhancing adaptability of human lymphocytes to various hindrances in the course of cell cycle and eventually causing them to acquire characteristics known to be common for tumour cells.     

A mitotic GlcNAcylation/phosphorylation signaling complex alters the posttranslational state of the cytoskeletal protein vimentin

O-linked β-N-acetylglucosamine (O-GlcNAc) is a highly dynamic intracellular protein modification responsive to stress, hormones, nutrients, and cell cycle stage. Alterations in O-GlcNAc addition or removal (cycling) impair cell cycle progression and cytokinesis, but the mechanisms are not well understood. Here, we demonstrate that the enzymes responsible for O-GlcNAc cycling, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are in a transient complex at M phase with the mitotic kinase Aurora B and protein phosphatase 1. OGT colocalized to the midbody during telophase with Aurora B. Furthermore, these proteins coprecipitated with each other in a late mitotic extract. The complex was stable under Aurora inhibition; however, the total cellular levels of O-GlcNAc were increased and the localization of OGT was decreased at the midbody after Aurora inhibition. Vimentin, an intermediate filament protein, is an M phase substrate for both Aurora B and OGT. Overexpression of OGT or OGA led to defects in mitotic phosphorylation on multiple sites, whereas OGT overexpression increased mitotic GlcNAcylation of vimentin. OGA inhibition caused a decrease in vimentin late mitotic phosphorylation but increased GlcNAcylation. Together, these data demonstrate that the O-GlcNAc cycling enzymes associate with kinases and phosphatases at M phase to regulate the posttranslational status of vimentin.     

PKCepsilon-mediated phosphorylation of vimentin controls integrin recycling and motility

PKCepsilon controls the transport of endocytosed beta1-integrins to the plasma membrane regulating directional cell motility. Vimentin, an intermediate filament protein upregulated upon epithelial cell transformation, is shown here to be a proximal PKCepsilon target within the recycling integrin compartment. On inhibition of PKC and vimentin phosphorylation, integrins become trapped in vesicles and directional cell motility towards matrix is severely attenuated. In vitro reconstitution assays showed that PKCepsilon dissociates from integrin containing endocytic vesicles in a selectively phosphorylated vimentin containing complex. Mutagenesis of PKC (controlled) sites on vimentin and ectopic expression of the variant leads to the accumulation of intracellular PKCepsilon/integrin positive vesicles. Finally, introduction of ectopic wild-type vimentin is shown to promote cell motility in a PKCepsilon-dependent manner; alanine substitutions in PKC (controlled) sites on vimentin abolishes the ability of vimentin to induce cell migration, whereas the substitution of these sites with acidic residues enables vimentin to rescue motility of PKCepsilon null cells. Our results indicate that PKC-mediated phosphorylation of vimentin is a key process in integrin traffic through the cell.     

Additive effects of radiation and docetaxel on murine SCCVII tumors in vivo: special reference to changes in the cell cycle

The purpose of the present study was to investigate the effects of a combination of docetaxel and irradiation in vivo with special reference to docetaxel-arrested G(2)/M-phase cells. At 24 and 48 h after intraperitoneal administration of docetaxel (90 mg/kg), tumor-bearing mice were irradiated with (60)Co gamma rays. Cell cycle distribution was analyzed by a DNA-Ki-67 double staining method using flow cytometry. An accumulation of cells in the G(2)/M phase of up to approximately 40% was observed 24 h after administration of docetaxel. Between 24 and 72 h, the percentage of cells arrested in G(2)/M phase that expressed Ki-67 decreased from 37.2% to 13.8%, in accordance with the increase in the Ki-67-negative G(2)/M-phase fraction. More than half of the cells arrested in G(2)/M phase lost their expression of Ki-67 protein between 24 and 72 h. The G(1)-phase fraction decreased from 28.4% to 8.6% at 24 h after docetaxel treatment; this remained unchanged at 72 h. These flow cytometry data suggested that docetaxel-arrested G(2)/M-phase cells did not enter the next cell cycle and were killed by docetaxel alone. Our data showed that arrest of cells in G(2)/M phase does not contribute to the synergism that has been reported for combinations of docetaxel and radiation in in vivo tumor models.     

A novel matrine derivative WM622 inhibits hepatocellular carcinoma by inhibiting PI3K/AKT signaling pathways

Nasopharyngeal carcinoma (NPC) is a unique subtype of head and neck cancer, with tendency to spread to regional lymph nodes and distant organs at early stage. Vimentin, a major cytoskeletal protein constituent of the intermediate filament, plays a critical role in the epithelial–mesenchymal transition. Overexpression of vimentin is considered to be a critical prerequisite for metastasis in numerous human cancers. Therefore, targeting vimentin for cancer therapy has gained a lot of interest. In the present study, we detected vimentin expression in NPC tissues and found that overexpression of vimentin is associated with poor prognosis of NPC patients. Silencing of vimentin in NPC CNE2 cells by RNAi suppresses cells migration and invasion in vitro. However, blocking vimentin did not affect cell proliferation of CNE2 cells. In addition, the in vivo metastatic potential of CNE2 cells transfected with Vimentin shRNA was suppressed in a nude mouse model of pulmonary metastasis. Silencing of Vimentin in CNE2 cells leads to a decrease of microvessel density and VEGF, CD31, MMP2, and MMP9 expressions in pulmonary metastatic tumors. Importantly, we found that it is easier for the tumor cells from the high vimentin-expressing pulmonary metastatic tumors to reinvade the microvessel and to form stable tumor plaques attached to the endothelial cells, which resemble the resource of circulating tumor cells and are very hard to eliminate. However, depletion of vimentin inhibits the formation of vascular tumor plaques. Our findings suggest that RNAi-based vimentin silencing may be a potential and promising anti-metastatic therapeutic strategy for NPC.     

波形蛋白在细胞中的潜在功能

波形蛋白(vimentin)是主要存在于间充质细胞中的一种中间纤维蛋白,近些年研究显示其作为细胞骨架成分,除具有支撑和维持细胞形态的功能外,还参与调节细胞的多种生命活动如:细胞粘附和迁移、信号转导、基因表达和细胞凋亡等。本文就近年来发现的vimentin在细胞中具有的潜在功能进行综述。     

Membrane Ig-cytoskeletal interactions. III. Receptor cross-linking results in the formation of extensive filamentous arrays of vimentin

The proposed function of intermediate filaments is to provide a cell type-specific structural framework that maintains cell shape and organelle distribution and mediates signal transduction through its connections with the plasma membrane and the nucleus. Vimentin is the intermediate filament protein expressed in B lymphocytes. Immunocytochemical analysis of the high salt-stable cytoskeletons from B cells stimulated with anti-Ig revealed an increased accumulation of vimentin in the cytoskeleton compared to nontreated controls. This increased accumulation of vimentin in the cytoskeleton was manifested by the organization of vimentin into extensive filamentous arrays (EFA) as viewed in the fluorescent microscope. In contrast to the effects of anti-Ig, activation of B cells with LPS did not induce the organization of vimentin into EFA. This suggested that signals unique to anti-Ig directed EFA formation. Immunocytochemical results were verified by biochemical analysis showing that vimentin was more abundant in isolated cytoskeletons from anti-Ig activated B cells, than cytoskeletons isolated from LPS-activated B cells. These observations established a relationship between increased content of vimentin in the cytoskeleton and the formation of EFA. By testing a wide variety of activating agents, we were able to correlate increased vimentin expression in the cytoskeleton to activating agents that cross-link membrane Ig. It appeared that treatment of B cells with LPS prohibited the induction of EFA by anti-Ig because cotreatment with both anti-Ig and LPS resulted in decreased vimentin accumulation in the cytoskeleton to a level less than that in resting cells. The significance of these results with regard to B cell biology is discussed.     

Vimentin expression influences flow dependent VASP phosphorylation and regulates cell migration and proliferation

The cytoskeleton plays a central role for the integration of biochemical and biomechanical signals across the cell required for complex cellular functions. Recent studies indicate that the intermediate filament vimentin is necessary for endothelial cell morphogenesis e.g. in the context of leukocyte transmigration. Here, we present evidence, that the scaffold provided by vimentin is essential for VASP localization and PKG mediated VASP phosphorylation and thus controls endothelial cell migration and proliferation. Vimentin suppression using siRNA technique significantly decreased migration velocity by 50% (videomicroscopy), diminished transmigration activity by 42.5% (Boyden chamber) and reduced proliferation by 43% (BrdU-incorporation). In confocal microscopy Vimentin colocalized with VASP and PKG in endothelial cells. Vimentin suppression was accompanied with a translocation of VASP from focal contacts to the perinuclear region. VASP/Vimentin and PKG/Vimentin colocalization appeared to be essential for proper PKG mediated VASP phosphorylation because we detected a diminished expression of PKG and p^Ser239-VASP in vimentin-suppressed cells, Furthermore, the induction of VASP phosphorylation in perfused arteries was markedly decreased in vimentin knockout mice compared to wildtypes. A link is proposed between vimentin, VASP phosphorylation and actin dynamics that delivers an explanation for the important role of vimentin in controlling endothelial cell morphogenesis.