Induction Of Vimentin Expression Research Articles

CD44v3,8-10 is essential for Slug-dependent vimentin gene expression to acquire TGF-β1-induced tumor cell motility.

CD44 is a widely expressed polymorphic adhesion molecule that has pleiotropic functions in development and tumor progression. Its mRNA undergoes alternative splicing to generate multiple variant (CD44v) isoforms, although the function of each CD44v isoform is not fully elucidated. Here, we show that CD44v plays an important role in the induction of vimentin expression upon transforming growth factor‐β1 (TGF‐β1)‐induced epithelial–mesenchymal transition (EMT). Among multiple CD44v isoforms expressed in NUGC3 gastric cancer cells, CD44v8‐10 and CD44v3,8‐10 are involved in the acquisition of migratory and invasive properties associated with TGF‐β1‐induced EMT, and only CD44v3,8‐10 induces the transcription of vimentin mediated by the EMT transcription factor Slug. In primary tumor specimens obtained from patients with gastric cancer, CD44‐containing variant exon 9 (CD44v9) expression and EMT features [E‐cadherin(−)vimentin(+)] were significantly correlated, and EMT features in the cells expressing CD44v9 were associated with tumor invasion depth, lymph node metastasis, and pStage, which indicate invasive and metastatic properties, and poor prognosis. These results indicate that certain CD44v isoforms promote tumor cell motility and metastasis in gastric cancer in association with EMT features, and CD44v3,8‐10 may contribute to these clinical characteristics.

Efficacy of the CDK7 Inhibitor on EMT-Associated Resistance to 3rd Generation EGFR-TKIs in Non-Small Cell Lung Cancer Cell Lines.

Epithelial to mesenchymal transition (EMT) is associated with resistance during EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy. Here, we investigated whether EMT is associated with acquired resistance to 3rd generation EGFR-TKIs, and we explored the effects of cyclin-dependent kinase 7 (CDK7) inhibitors on EMT-mediated EGFR-TKIs resistance in non-small cell lung cancer (NSCLC). We established 3rd generation EGFR-TKI resistant cell lines (H1975/WR and H1975/OR) via repeated exposure to WZ4002 and osimertinib. The two resistant cell lines showed phenotypic changes to a spindle-cell shape, had a reduction of epithelial marker proteins, an induction of vimentin expression, and enhanced cellular mobility. The EMT-related resistant cells had higher sensitivity to THZ1 than the parental cells, although THZ1 treatment did not inhibit EGFR activity. This phenomenon was also observed in TGF-β1 induced EMT cell lines. THZ1 treatment induced G2/M cell cycle arrest and apoptosis in all of the cell lines. In addition, THZ1 treatment led to drug-tolerant, EMT-related resistant cells, and these THZ1-tolerant cells partially recovered their sensitivity to 3rd generation EGFR-TKIs. Taken together, EMT was associated with acquired resistance to 3rd generation EGFR-TKIs, and CDK7 inhibitors could potentially be used as a therapeutic strategy to overcome EMT associated EGFR-TKI resistance in NSCLC.

Abstract P4-14-02: CSF1/CSF1R axis reprograms epithelial-to-mesenchymal phenotypes in inflammatory breast cancer

Abstract Inflammatory breast cancer (IBC) accounts for 2% of breast cancers but 10% of breast cancer-related deaths in the US. Clinical hallmarks of IBC are tumor cell emboli in lymphatic vessels and overexpression of E-cadherin, which promotes cell clustering. Given these hallmarks, IBC is thought to spread via collective invasion and cell clusters. However, we showed that IBC cells underwent epithelial-to-mesenchymal transition (EMT) and metastasized through EMT. Thus, there are two contradictory theories of IBC metastasis. The objectives of this study were 1) to propose a model that reconciles these two models, and 2) to identify target molecules for inhibition of IBC metastasis. Methods: We previously showed that Matrigel culture induced EMT-like changes in SUM149 IBC cells. To test if this transformation from epithelial (E) to mesenchymal (M) in Matrigel culture is unique to IBC cells, a panel of breast cancer cells was cultured in both monolayer and Matrigel-coated plates. The cells were IBC (SUM149, SUM190, KPL4, IBC3), triple-negative breast cancer (TNBC) (MDA-MB-231, MDA-MB-468), and ER+ (MCF7) cells. Phenotypic changes in morphology and expression of EMT markers (E-cadherin, vimentin) were captured with bright field and immunofluorescent (IF) images, respectively. For genome-wide and targeted transcriptional analysis, SUM149 cells cultured in monolayer and Matrigel were processed using DNA microarrays and Taqman qRT-PCR. To correlate the Matrigel gene signature with M features in human breast cancer, a human breast cancer data set was hierarchically clustered with the Matrigel gene signature. Results: SUM149 cells showed a remarkable phenotypic change from E in monolayer culture to M in Matrigel. IF analysis confirmed induction of vimentin expression in Matrigel but stable expression of E-cadherin (thus, we refer to this state as E/M hybrid). This trend was also observed with SUM190 cells. Using qRT-PCR, we confirmed downregulation of E-cadherin and upregulation of M markers (vimentin, Twist1, Snail1, ZEB2) in Matrigel-cultured SUM149 and SUM190 cells compared to monolayer-cultured cells. DNA microarray transcriptional analysis confirmed this trend in SUM149 cells. TNBC has more M-like features than other breast cancer subtypes. Given this evidence, we clustered human breast cancer data using overexpressed genes in Matrigel-cultured SUM149 cells. We identified a cluster of 20 genes in TNBC samples and, assuming that these genes are drivers of E to E/M transition, chose the inflammation-related gene CSF1 as a candidate. The CSF1/CSF1R axis was inhibited by a CSF1R inhibitor, BLZ945; moreover, treatment with BLZ945 reversed the EMT changes in cells in Matrigel culture. Treatment with 5 μM BLZ945 re-induced E-cadherin expression and suppressed Snail1 and Twist1 expression in Matrigel-cultured SUM149 cells. Conclusion: IBC cells are more prone to undergo transition from E to E/M phenotype in Matrigel culture than are cells of other breast cancer subtypes. The CSF1/CSF1R axis plays a role in this E to E/M transition, thus warranting testing its significance using an in vivo IBC model. Phenotypic transition and reversion between E and E/M phenotypes could be a new paradigm that reconciles two contradictory models of IBC metastasis. Citation Format: Kai K, Iwamoto T, Zhang D, Rao AUK, Thompson A, Sen S, Ueno NT. CSF1/CSF1R axis reprograms epithelial-to-mesenchymal phenotypes in inflammatory breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P4-14-02.

Pentabromophenol, a brominated flame retardant derivate, suppresses TGF‐β signaling by sequester TGF‐β receptor from cell surface and further degradation through a caveolae‐mediated endocytosis

Polybormodiphenyl ethers (PBDEs) have been widely used as flame retardants in a variety of consumer products, the possible adverse health effects of PBDEs have focused on their potential endocrine‐disrupting effects and developmental neurotoxicity. In this study, we found that the major biological metabolite of PBDEs, pentabromophenol (PBP) suppressed TGF‐β responsiveness by accelerating the turnover rate of TGF‐β receptor. PBP suppressed TGF‐β1‐mediated cell migration, PAI‐1 reporter gene activation and Smad2/3 activation in various type of cells, abolished TGF‐β1‐mediated repression of E‐cadherin expression, as well as induction of vimentin expression along with Snail and Slug upregulation; thus, blocking the TGF‐β1‐mediated epithelial‐to‐mesenchymal transition (EMT) in A549 calls. TGF‐β superfamily is a key player in the regulation of a wide variety of biological processes from development to pathogenesis including cell proliferation, migration, and the process of cancer development and progression. The in vitro results in this study provide a basis for studies of more detailed relationships between PBP and the modulation of TGF‐β signaling. Given their similarities to PBDEs, additional laboratory and mechanistic study should be considered to assess PBDEs as a potential risk factor for tumorigenesis and other TGF‐β‐related diseases.Support or Funding InformationMinistry of Science and Technology in Taiwan

Syntenin regulates TGF-β1-induced Smad activation and the epithelial-to-mesenchymal transition by inhibiting caveolin-mediated TGF-β type I receptor internalization.

Syntenin, a tandem PDZ domain containing scaffold protein, functions as a positive regulator of cancer cell progression in several human cancers. We report here that syntenin positively regulates transforming growth factor (TGF)-β1-mediated Smad activation and the epithelial-to-mesenchymal transition (EMT) by preventing caveolin-1-mediated internalization of TGF-β type I receptor (TβRI). Knockdown of syntenin suppressed TGF-β1-mediated cell migration, transcriptional responses and Smad2/3 activation in various types of cells; however, overexpression of syntenin facilitated TGF-β1-mediated responses. In particular, syntenin knockdown abolished both the basal and TGF-β1-mediated repression of E-cadherin expression, as well as induction of vimentin expression along with Snail and Slug upregulation; thus, blocking the TGF-β1-induced EMT in A549 cells. In contrast, overexpression of syntenin exhibited the opposite effect. Knockdown of syntenin-induced ubiquitination and degradation of TβRI, but not TGF-β type II receptor, leading to decreased TβRI expression at the plasma membrane. Syntenin associated with TβRI at its C-terminal domain and a syntenin mutant lacking C-terminal domain failed to increase TGF-β1-induced responses. Biochemical analyzes revealed that syntenin inhibited the interaction between caveolin-1 and TβRI and knockdown of syntenin induced a massive internalization of TβRI and caveolin-1 from lipid rafts, indicating that syntenin may increase TGF-β signaling by inhibiting caveolin-1-dependent internalization of TβRI. Moreover, a positive correlation between syntenin expression and phospho-Smad2 levels is observed in human lung tumors. Taken together, these findings demonstrate that syntenin may act as an important positive regulator of TGF-β signaling by regulating caveolin-1-mediated internalization of TβRI; thus, providing a novel function for syntenin that is linked to cancer progression.

46. HIV-induced epithelial–mesenchymal transition in mucosal epithelium facilitates HPV paracellular penetration

Background The incidence of human papillomavirus (HPV)-associated epithelial lesions in HIV-infected individuals is substantially higher than in HIV-negative individuals. HIV may increase the risk of mucosal HPV infection by induction of an epithelial–mesenchymal transition (EMT) phenotype that leads to reduced epithelial barrier functions. Methods: To model the effect of EMT on HPV entry, we used oral epithelial biopsies, and oral and anal keratinocytes from HIV-infected and HIV-uninfected individuals. EMT was defined as loss of adherens and tight junctions, and induction of vimentin expression. To study epithelial HPV entry we used HPV-16 pseudovirions (PsVs) containing a plasmid expressing red fluorescent protein (RFP). PsV penetration was evaluated using confocal microscopy to detect RFP-generated fluorescence. Results: Compared with oral epithelia from HIV-uninfected individuals, oral epithelia from HIV-infected individuals had reduced adherens and tight junctions and expressed vimentin. Keratinocytes isolated from oral and anal tissues of HIV-infected individuals also showed loss of E-cadherin and induction of vimentin expression. Exposure of oral epithelial cells from HIV-uninfected individuals to HIV and HIV tat, gp120, and nef proteins induced EMT with reduced cell junctions and epithelial polarity, and allowed paracellular passage of HPV-16 PsV. Oral mucosal epithelial tissues treated with HIV tat and gp120 also lost tight junctions and allowed PsV paracellular penetration, leading to PsV entry into basal/parabasal cells. Conclusions: HIV-associated EMT in oral epithelia, and oral and anal keratinocytes leads to impaired barrier function. This facilitates entry of HPV/PsV into basal/parabasal cells, and may contribute to the increased incidence of HPV-associated malignancy in HIV-infected individuals.

Epithelial-Stromal Interactions in Human Breast Cancer: Effects on Adhesion, Plasma Membrane Fluidity and Migration Speed and Directness

Interactions occurring between malignant cells and the stromal microenvironment heavily influence tumor progression. We investigated whether this cross-talk affects some molecular and functional aspects specifically correlated with the invasive phenotype of breast tumor cells (i.e. adhesion molecule expression, membrane fluidity, migration) by co-culturing mammary cancer cells exhibiting different degrees of metastatic potential (MDA-MB-231>MCF-7) with fibroblasts isolated from breast healthy skin (normal fibroblasts, NFs) or from breast tumor stroma (cancer-associated fibroblasts, CAFs) in 2D or 3D (nodules) cultures. Confocal immunofluorescence analysis of the epithelial adhesion molecule E-cadherin on frozen nodule sections demonstrated that NFs and CAFs, respectively, induced or inhibited its expression in MCF-7 cells. An increase in the mesenchymal adhesion protein N-cadherin was observed in CAFs, but not in NFs, as a result of the interaction with both kinds of cancer cells. CAFs, in turn, promoted N-cadherin up-regulation in MDA-MB-231 cells and its de novo expression in MCF-7 cells. Beyond promotion of “cadherin switching”, another sign of the CAF-triggered epithelial-mesenchymal transition (EMT) was the induction of vimentin expression in MCF-7 cells. Plasma membrane labeling of monolayer cultures with the fluorescent probe Laurdan showed an enhancement of the membrane fluidity in cancer cells co-cultured with NFs or CAFs. An increase in lipid packing density of fibroblast membranes was promoted by MCF-7 cells. Time-lapsed cell tracking analysis of mammary cancer cells co-cultured with NFs or CAFs revealed an enhancement of tumor cell migration velocity, even with a marked increase in the directness induced by CAFs.Our results demonstrate a reciprocal influence of mammary cancer and fibroblasts on various adhesiveness/invasiveness features. Notably, CAFs' ability to promote EMT, reduction of cell adhesion, increase in membrane fluidity, and migration velocity and directness in mammary cancer cells can be viewed as an overall progression- and invasion-promoting effect.

Abstract 1451: Lipid-based Akt inhibitors preferentially kill lung cancer cells with mesenchymal features

Abstract Most solid tumors are epithelial in origin. A loss of epithelial-cell markers and gain of mesenchymal-cell markers (epithelial mesenchymal transition, EMT) has been observed in patient tumor samples, particularly at the leading edge or invasive front of solid tumors such as non-small cell lung cancer (NSCLC). Pathological EMT is often associated with tumor metastasis and can confer a poor prognosis. EMT is promoted by Akt activation, which contributes to the formation, maintenance, and therapeutic resistance of cancer. In this study, we found that lipid-based Akt inhibitors (LBAI) such as phosphatidylinositol ether lipid analogues (PIAs) and alkylphospholipids (perifosine and miltefosine) preferentially kill tumor cells with mesenchymal features. NSCLC cell lines that exhibited epithelial, intermediate and mesenchymal phenotypes (H322, A549 and H157, respectively) were increasingly sensitive to the cytotoxic effects of LBAI. Prolonged treatment of A549 cells with TGFbeta induced a mesenchymal phenotype and increased sensitivity to LBAI. Because EMT can be controlled by the tumor suppressor RhoB, which we previously showed was transcriptionally activated by LBAI, we investigated the role of RhoB in these cell lines. An inverse relationship between Akt activation and RhoB expression was observed in the parental cell lines as well as A549 cells treated with TGFbeta, which correlated with LBAI cytotoxicity. Stable knockdown of RhoB in A549 cells induced features of EMT including loss of E-cadherin expression, induction of vimentin expression and Akt activation, and increased cellular migration. Stable knockdown of RhoB also increased sensitivity to LBAI. These studies show that lung cancer cells with a mesenchymal phenotype are preferentially sensitivity to LBAI, and that RhoB is a critical mediator of EMT. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1451.

The Clinical Significance of Vimentin-Expressing Gastric Cancer Cells in Bone Marrow

Expression of the mesenchymal marker gene vimentin (VIM) in gastric cancer is associated with a more aggressive form of the disease and poor prognosis. Because epithelial mesenchymal transition (EMT) plays a critical role in the progression of gastric cancer, VIM expression was examined in the bone marrow (BM) of gastric cancer patients. BM samples from 437 gastric cancer patients were collected and analyzed by quantitative RT-PCR. Expression of VIM protein in the primary lesions of resected gastric cancers was evaluated using immunohistochemistry. Furthermore, induction of VIM expression by TGF-beta1 and hypoxia was evaluated in gastric cancer cells. VIM mRNA expression increased concordantly with clinical staging and was significantly associated with tumor invasion and lymph node metastasis (P < .0001). Though cancer cells in the primary lesions did not stain with VIM antibody, some of the cells invading the intratumoral vessels were strongly positive for VIM, but were negative for E-cadherin. Hypoxic conditions and treatment with TGF-beta1 induced VIM expression and repressed E-cadherin in gastric cancer cells, coupled with an alteration of cellular morphology. We found that gastric cancer cells undergo EMT in BM to survive and metastasize. These findings suggest that isolated tumor cells have the potential to undergo EMT, which could increase the malignancy of gastric cancer.

Transforming Growth Factor-β Promotes Invasion in Tumorigenic but not in Nontumorigenic Human Prostatic Epithelial Cells

Transforming growth factor-beta (TGF-beta) is a pleiotropic growth factor with actions that are dependent on circumstances, including dose, target cell type, and context. TGF-beta can elicit both growth-promoting and growth-suppressive activities. In normal tissues, TGF-beta generally acts to restrict growth and maintain differentiation. However, during tumorigenesis, changes in TGF-beta expression and cellular responses can promote tumorigenesis. The present study examines the effects of TGF-beta on the nontumorigenic human prostatic epithelial cell line BPH1 and on three derivative tumorigenic sublines BPH1(CAFTD)1, BPH1(CAFTD)3, and BPH1(CAFTD)5. The data show that TGF-beta has different effects on the nontumorigenic and tumorigenic cells. The nontumorigenic cells are growth inhibited by TGF-beta. In contrast, the tumorigenic sublines are not growth inhibited but instead undergo an epithelial to mesenchymal transformation (EMT) in response to TGF-beta. The tumorigenic lines show constitutively elevated levels of phosphorylated Akt, which modulates their response to TGF-beta by blocking Smad3 and p21 nuclear translocation. On TGF-beta stimulation of the tumorigenic sublines, the activated Akt allows the cell to escape cell cycle arrest. The phosphatidylinositol 3-kinase/Akt pathway is also involved in TGF-beta-induced EMT, defined here by induction of vimentin expression and enhanced cellular motility. In vivo, tumorigenic cells with constitutively active TGF-beta signaling show increased invasion with EMT, which express vimentin, located specifically at the invasive front of the tumor. These data indicate that following malignant transformation TGF-beta can play a direct role in promoting prostatic cancer and further that these responses are context specific in vivo.

Reduced progesterone metabolites protect rat hippocampal neurones from kainic acid excitotoxicity in vivo.

The ovarian hormone progesterone is neuroprotective in some animal models of neurodegeneration. Progesterone actions in the brain may partly be mediated by the locally produced metabolites 5alpha-dihydroprogesterone and 3alpha,5alpha-tetrahydroprogesterone. The neuroprotective effects of these two metabolites of progesterone were assessed in this study. Ovariectomized Wistar rats were injected with kainic acid, to induce excitotoxic neuronal death in the hippocampus, and with different doses of 5alpha-dihydroprogesterone and 3alpha,5alpha-tetrahydroprogesterone. The number of surviving neurones in the hilus of the dentate gyrus of the hippocampus was assessed with the optical disector method. The administration of kainic acid resulted in a significant decrease in the number of hilar neurones and in the induction of vimentin expression in reactive astrocytes, a sign of neural damage. Low doses of 5alpha-dihydroprogesterone (0.25 and 0.5 mg/kg body weight, b.w.) prevented the loss of hilar neurones and the appearance of vimentin immunoreactivity in astrocytes. Higher doses (1-2 mg/kg b.w.) were not neuroprotective. By contrast, low doses of 3alpha,5alpha-tetrahydroprogesterone (0.25-1 mg/kg b.w.) were unable to protect the hilus from kainic acid while higher doses (2-4 mg/kg b.w.) were protective. The different optimal neuroprotective doses of 5alpha-dihydroprogesterone and 3alpha,5alpha-tetrahydroprogesterone suggest that these two steroids may protect neurones using different mechanisms. The neuroprotective effects of 3alpha,5alpha-tetrahydroprogesterone may be exerted by the inhibition of neuronal activity via the GABAA receptor. This latter possibility is supported by the observation that 3beta,5alpha-tetrahydroprogesterone, an isomer of 3alpha,5alpha-tetrahydroprogesterone that does not bind to GABAA receptor, was not neuroprotective. In summary, our findings suggest that progesterone neuroprotective effects may be, at least in part, mediated by its reduced metabolites 5alpha-dihydroprogesterone and 3alpha,5alpha-tetrahydroprogesterone.

The calpastatin-derived calpain inhibitor CP1B reduces mRNA expression of matrix metalloproteinase-2 and -9 and invasion by leukemic THP-1 cells.

The ubiquitous proteases mu- and m-calpain are Ca(2+)-dependent cysteine endopeptidases. Besides involvement in a variety of physio(patho)logical processes, recent studies suggest a pivotal role of calpains in differentiation of hematopoietic cells and tumor cell invasion. However, the precise actions of calpains and their endogenous inhibitor, calpastatin, in these processes are only partially understood. Here we have studied the role of the calpain/calpastatin system in the invasion of leukemic cells under basal and differentiation-stimulating conditions. To further differentiate the human leukaemic cell line THP-1 (monocytic), the cells were treated for 24 hours with the differentiation-stimulating reagents phorbol 12-myristate 13-acetate (PMA) and dimethyl sulfoxide (DMSO). Macrophage- and granulocyte-like differentiation was confirmed by induction of vimentin expression as well as by microscopic and fluorescence-assisted cytometric analysis. Extracellular matrix (ECM) invasion of both the basal and differentiation-stimulated cells in a Matrigel assay was inhibited by pre-incubation of the cells with the specific calpain inhibitor CP1B for 24 hours. Inhibition of invasiveness correlated with decreased mRNA expression and secretion of the matrix metalloproteinases MMP-2 and MMP-9. In contrast, addition of CP1B only during the invasion process did neither influence transmigration nor MMP release. This is the first report showing that the calpain/calpastatin system mediates MMP-mRNA expression of the leukemic THP-1 cells and as a consequence their invasiveness.

Tetracycline regulated expression of vimentin in fibroblasts derived from vimentin null mice.

Fibroblast cell lines were derived from vim-/- mice that express a mouse vimentin transgene in a tetracycline regulatable manner. Vimentin null mouse primary embryo fibroblasts were transformed with SV-40 early genes and vim- cell lines were isolated. A vim- cell line was then serially transfected with an expression plasmid encoding the tetracycline regulatable transactivator (tTA) and a mouse vimentin cDNA expression plasmid under the regulation of Escherichia coli tet operator and minimal CMV promoter sequences. Two stable cell lines were obtained that contained little or no vimentin in the presence of low concentrations of tetracycline but rapidly expressed abundant vimentin filaments after removal of tetracycline. The vimentin content of one cell line was similar to that of control vim+/+ fibroblasts. The level of transgene expression could be regulated by the concentration of tetracycline in a dose dependent fashion. Induction of vimentin expression in these cells did not observably affect cell growth, the distribution of microfilaments or microtubules, or the shape of the nucleus. Enucleation studies indicated that while disassembly of microfilaments significantly increased the sensitivity of the cells to enucleation, the presence or absence of vimentin had no detectable effect on the degree of enucleation with increasing sedimentation force. Monolayer wounding experiments demonstrated that vimentin expression did not alter the mobility of polarized cells at the edge of the wound. Experiments to more directly test the effect of vimentin expression on the capacity of these fibroblasts to survive mechanical trauma indicated that vimentin expression had no obvious effect on the survival of suspension cells subjected to nitrogen cavitation or the fraction of cells that survived the mechanical scraping of monolayer culture. These studies indicate that vimentin expression in a single population of cells does not have an obvious effect on cytoplasmic organization and provides a useful system to study the effects of IFs on the capacity of individual cells to resist mechanical injury.

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 x 10(-9) M. 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.

Growth-regulated expression of vimentin in hamster fibroblasts is a result of increased transcription.

We have previously shown that vimentin is a growth-regulated gene whose mRNA levels increase after serum stimulation of quiescent hamster fibroblasts. In this study, the control of the growth-regulated expression of vimentin was determined in ts13 cells induced to proliferate by serum. Both transcriptional and post-transcriptional mechanisms of regulation were examined by determining transcriptional rates, cytoplasmic transcript abundance, transcript stability, and protein abundance. We observed a fourfold increase in vimentin transcripts in the cytoplasm of serum-stimulated ts13 cells. Since transcripts are stable in both quiescent and stimulated cells, this induction of vimentin expression is a result of a fivefold increase in vimentin-specific transcriptional activity. As a result of this increased transcript availability, the abundance of polymerized vimentin protein increased following serum stimulation of quiescent fibroblasts. Overall, the induction of vimentin expression in fibroblasts by serum is a consequence of increased vimentin-specific transcriptional activity. The significance of this with regard to cytoskeletal organization and cell division is discussed.