The Hippo pathway regulates density-dependent proliferation of iPSC-derived cardiac myocytes.

Inducing cardiac myocytes to proliferate is considered a potential therapy to target heart disease, however, modulating cardiac myocyte proliferation has proven to be a technical challenge. The Hippo pathway is a kinase signaling cascade that regulates cell proliferation during the growth of the heart. Inhibition of the Hippo pathway increases the activation of the transcription factors YAP/TAZ, which translocate to the nucleus and upregulate transcription of pro-proliferative genes. The Hippo pathway regulates the proliferation of cancer cells, pluripotent stem cells, and epithelial cells through a cell-cell contact-dependent manner, however, it is unclear if cell density-dependent cell proliferation is a consistent feature in cardiac myocytes. Here, we used cultured human iPSC-derived cardiac myocytes (hiCMs) as a model system to investigate this concept. hiCMs have a comparable transcriptome to the immature cardiac myocytes that proliferate during heart development in vivo. Our data indicate that a dense syncytium of hiCMs can regain cell cycle activity and YAP expression and activity when plated sparsely or when density is reduced through wounding. We found that combining two small molecules, XMU-MP-1 and S1P, increased YAP activity and further enhanced proliferation of low-density hiCMs. Importantly, these compounds had no effect on hiCMs within a dense syncytium. These data add to a growing body of literature that link Hippo pathway regulation with cardiac myocyte proliferation and demonstrate that regulation is restricted to cells with reduced contact inhibition.

The differentiation of colorectal cancer is closely relevant to m6A modification.

The occurrence and development of tumors cannot be separated from the influence of differentiation at different stages and levels. Our study found that E-cadherin was significantly increased in cell model induced by sodium butyrate and cell density, while METTL3, METTL16 and WTAP were decreased during the differentiation of cells. In the clinicopathological tissues, E-cadherin was low expressed in poorly differentiated tumor tissues and above three regulators were highly expressed in poorly differentiated tissues. At the levels of clinicopathological differentiation, tissue differentiation and cell differentiation, the result indicated that the poor prognosis of colorectal cancer (CRC) may be closely related to high expression of total m6A level and high expression of METTL3, METTL16 and WTAP.

Single-cell RNA-sequencing reveals transcriptional dynamics of estrogen-induced dysplasia in the ovarian surface epithelium.

Estrogen therapy increases the risk of ovarian cancer and exogenous estradiol accelerates the onset of ovarian cancer in mouse models. Both in vivo and in vitro, ovarian surface epithelial (OSE) cells exposed to estradiol develop a subpopulation that loses cell polarity, contact inhibition, and forms multi-layered foci of dysplastic cells with increased susceptibility to transformation. Here, we use single-cell RNA-sequencing to characterize this dysplastic subpopulation and identify the transcriptional dynamics involved in its emergence. Estradiol-treated cells were characterized by up-regulation of genes associated with proliferation, metabolism, and survival pathways. Pseudotemporal ordering revealed that OSE cells occupy a largely linear phenotypic spectrum that, in estradiol-treated cells, diverges towards cell state consistent with the dysplastic population. This divergence is characterized by the activation of various cancer-associated pathways including an increase in Greb1 which was validated in fallopian tube epithelium and human ovarian cancers. Taken together, this work reveals possible mechanisms by which estradiol increases epithelial cell susceptibility to tumour initiation.

PDGF controls contact inhibition of locomotion by regulating N-cadherin during neural crest migration.

A fundamental property of neural crest (NC) migration is contact inhibition of locomotion (CIL), a process by which cells change their direction of migration upon cell contact. CIL has been proven to be essential for NC migration in amphibians and zebrafish by controlling cell polarity in a cell contact-dependent manner. Cell contact during CIL requires the participation of the cell adhesion molecule N-cadherin, which starts to be expressed by NC cells as a consequence of the switch between E- and N-cadherins during epithelial-to-mesenchymal transition (EMT). However, the mechanism that controls the upregulation of N-cadherin remains unknown. Here, we show that platelet-derived growth factor receptor alpha (PDGFRα) and its ligand platelet-derived growth factor A (PDGF-A) are co-expressed in migrating cranial NC. Inhibition of PDGF-A/PDGFRα blocks NC migration by inhibiting N-cadherin and, consequently, impairing CIL. Moreover, we identify phosphatidylinositol-3-kinase (PI3K)/AKT as a downstream effector of the PDGFRα cellular response during CIL. Our results lead us to propose PDGF-A/PDGFRα signalling as a tissue-autonomous regulator of CIL by controlling N-cadherin upregulation during EMT. Finally, we show that once NC cells have undergone EMT, the same PDGF-A/PDGFRα works as an NC chemoattractant, guiding their directional migration.

Catenin delta-1 (CTNND1) phosphorylation controls the mesenchymal to epithelial transition in astrocytic tumors.

Inactivating mutations of the TSC1/TSC2 complex (TSC1/2) cause tuberous sclerosis (TSC), a hereditary syndrome with neurological symptoms and benign hamartoma tumours in the brain. Since TSC effectors are largely unknown in the human brain, TSC patient cortical tubers were used to uncover hyperphosphorylation unique to TSC primary astrocytes, the cell type affected in the brain. We found abnormal hyperphosphorylation of catenin delta-1 S268, which was reversible by mTOR-specific inhibitors. In contrast, in three metastatic astrocytoma cell lines, S268 was under phosphorylated, suggesting S268 phosphorylation controls metastasis. TSC astrocytes appeared epithelial (i.e. tightly adherent, less motile, and epithelial (E)-cadherin positive), whereas wild-type astrocytes were mesenchymal (i.e. E-cadherin negative and highly motile). Despite their epithelial phenotype, TSC astrocytes outgrew contact inhibition, and monolayers sporadically generated tuberous foci, a phenotype blocked by the mTOR inhibitor, Torin1. Also, mTOR-regulated phosphokinase C epsilon (PKCe) activity induced phosphorylation of catenin delta-1 S268, which in turn mediated cell-cell adhesion in astrocytes. The mTOR-dependent, epithelial phenotype of TSC astrocytes suggests TSC1/2 and mTOR tune the phosphorylation level of catenin delta-1 by controlling PKCe activity, thereby regulating the mesenchymal-epithelial-transition (MET). Thus, some forms of TSC could be treated with PKCe inhibitors, while metastasis of astrocytomas might be blocked by PKCe stimulators.

High-density lipoprotein contribute to G0-G1/S transition in Swiss NIH/3T3 fibroblasts.

High density lipoproteins (HDLs) play a crucial role in removing excess cholesterol from peripheral tissues. Although their concentration is lower during conditions of high cell growth rate (cancer and infections), their involvement during cell proliferation is not known. To this aim, we investigated the replicative cycles in synchronised Swiss 3T3 fibroblasts in different experimental conditions: i) contact-inhibited fibroblasts re-entering cell cycle after dilution; ii) scratch-wound assay; iii) serum-deprived cells induced to re-enter G1 by FCS, HDL or PDGF. Analyses were performed during each cell cycle up to quiescence. Cholesterol synthesis increased remarkably during the replicative cycles, decreasing only after cells reached confluence. In contrast, cholesteryl ester (CE) synthesis and content were high at 24 h after dilution and then decreased steeply in the successive cycles. Flow cytometry analysis of DiO-HDL, as well as radiolabeled HDL pulse, demonstrated a significant uptake of CE-HDL in 24 h. DiI-HDL uptake, lipid droplets (LDs) and SR-BI immunostaining and expression followed the same trend. Addition of HDL or PDGF partially restore the proliferation rate and significantly increase SR-BI and pAKT expression in serum-deprived cells. In conclusion, cell transition from G0 to G1/S requires CE-HDL uptake, leading to CE-HDL/SR-BI pathway activation and CEs increase into LDs.

The aryl hydrocarbon receptor-dependent disruption of contact inhibition in rat liver WB-F344 epithelial cells is linked with induction of survivin, but not with inhibition of apoptosis.

Inhibition of apoptosis by the ligands of the aryl hydrocarbon receptor (AhR) has been proposed to play a role in their tumor promoting effects on liver parenchymal cells. However, little is presently known about the impact of toxic AhR ligands, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on apoptosis in other liver cell types, such as in liver epithelial/progenitor cells. In the present study, we focused on the effects of TCDD on apoptosis regulation in a model of liver progenitor cells, rat WB-F344 cell line, during the TCDD-elicited release from contact inhibition. The stimulation of cell proliferation in this cell line was associated with deregulated expression of a number of genes known to be under transcriptional control of the Hippo signaling pathway, a principal regulatory pathway involved in contact inhibition of cell proliferation. Interestingly, we found that mRNA and protein levels of survivin, a known Hippo target, which plays a role both in cell division and inhibition of apoptosis, were significantly up-regulated in rat liver epithelial cell model, as well as in undifferentiated human liver HepaRG cells. Using the short interfering RNA-mediated knockdown, we confirmed that survivin plays a central role in cell division of WB-F344 cells. When evaluating the effects of TCDD on apoptosis induction by camptothecin, a genotoxic topoisomerase I inhibitor, we observed that the pre-treatment of WB-F344 cells with TCDD increased number of cells with apoptotic nuclear morphology, and it potentiated cleavage of both caspase-3 and poly(ADP-ribose) polymerase I. This indicated that despite the observed up-regulation of survivin, apoptosis induced by the genotoxin was potentiated in the model of rat liver progenitor cells. The present results indicate that, unlike in hepatocytes, AhR agonists may not prevent induction of apoptosis elicited by DNA-damaging agents in a model of rat liver progenitor cells.

LIF-JAK1-STAT3 signaling delays contact inhibition of human corneal endothelial cells.

Human corneal endothelial cells (HCECs) responsible for corneal transparency have limited proliferative capacity in vivo because of "contact-inhibition." This feature has hampered the ability to engineer HCECs for transplantation. Previously we have reported an in vitro model of HCECs in which contact inhibition was re-established at Day 21, even though cell junction and cell matrix interaction were not perturbed during isolation. Herein, we observe that such HCEC monolayers continue to expand and retain a normal phenotype for 2 more weeks if cultured in a leukemia inhibitory factor (LIF)-containing serum-free medium. Such expansion is accompanied initially by upregulation of Cyclin E2 colocalized with nuclear translocation of phosphorylated retinoblastoma tumor suppressor (p-Rb) at Day 21 followed by a delay in contact inhibition through activation of LIF-Janus kinase1 (JAK1)-signal transducer and activator of transcription 3 (STAT3) signaling at Day 35. The LIF-JAK1-STAT3 signaling is coupled with upregulation of E2F2 colocalized with nuclear p-Rb and with concomitant downregulation of p16(INK4a), of which upregulation is linked to senescence. Hence, activation of LIF-JAK1-STAT3 signaling to delay contact inhibition can be used as another strategy to facilitate engineering of HCEC grafts to solve the unmet global shortage of corneal grafts.

Inhibition of tumor cell proliferation and motility by fibroblasts is both contact and soluble factor dependent.

Normal human and murine fibroblasts can inhibit proliferation of tumor cells when cocultured in vitro. The inhibitory capacity varies depending on the donor and the site of origin of the fibroblast. We showed previously that effective inhibition requires formation of a morphologically intact fibroblast monolayer before seeding of the tumor cells. Here we show that inhibition is extended to motility of tumor cells and we dissect the factors responsible for these inhibitory functions. We find that inhibition is due to two different sets of molecules: (i) the extracellular matrix (ECM) and other surface proteins of the fibroblasts, which are responsible for contact-dependent inhibition of tumor cell proliferation; and (ii) soluble factors secreted by fibroblasts when confronted with tumor cells (confronted conditioned media, CCM) contribute to inhibition of tumor cell proliferation and motility. However, conditioned media (CM) obtained from fibroblasts alone (nonconfronted conditioned media, NCM) did not inhibit tumor cell proliferation and motility. In addition, quantitative PCR (Q-PCR) data show up-regulation of proinflammatory genes. Moreover, comparison of CCM and NCM with an antibody array for 507 different soluble human proteins revealed differential expression of growth differentiation factor 15, dickkopf-related protein 1, endothelial-monocyte-activating polypeptide II, ectodysplasin A2, Galectin-3, chemokine (C-X-C motif) ligand 2, Nidogen1, urokinase, and matrix metalloproteinase 3.

Calreticulin affects cell adhesiveness through differential phosphorylation of insulin receptor substrate-1.

Cellular adhesion to the underlying substratum is regulated through numerous signaling pathways. It has been suggested that insulin receptor substrate 1 (IRS-1) is involved in some of these pathways, via association with and activation of transmembrane integrins. Calreticulin, as an important endoplasmic reticulum-resident, calcium-binding protein with a chaperone function, plays an obvious role in proteomic expression. Our previous work showed that calreticulin mediates cell adhesion not only by affecting protein expression but also by affecting the state of regulatory protein phosphorylation, such as that of c-src. Here, we demonstrate that calreticulin affects the abundance of IRS-1 such that the absence of calreticulin is paralleled by a decrease in IRS-1 levels and the unregulated overexpression of calreticulin is accompanied by an increase in IRS-1 levels. These changes in the abundance of calreticulin and IRS-1 are accompanied by changes in cell-substratum adhesiveness and phosphorylation, such that increases in the expression of calreticulin and IRS-1 are paralleled by an increase in focal contact-based cell-substratum adhesiveness, and a decrease in the expression of these proteins brings about a decrease in cell-substratum adhesiveness. Wild type and calreticulin-null mouse embryonic fibroblasts (MEFs) were cultured and the IRS-1 isoform profile was assessed. Differences in morphology and motility were also quantified. While no substantial differences in the speed of locomotion were found, the directionality of cell movement was greatly promoted by the presence of calreticulin. Calreticulin expression was also found to have a dramatic effect on the phosphorylation state of serine 636 of IRS-1, such that phosphorylation of IRS-1 on serine 636 increased radically in the absence of calreticulin. Most importantly, treatment of cells with the RhoA/ROCK inhibitor, Y-27632, which among its many effects also inhibited serine 636 phosphorylation of IRS-1, had profound effects on cell-substratum adhesion, in that it suppressed focal contacts, induced extensive close contacts, and increased the strength of adhesion. The latter effect, while counterintuitive, can be explained by the close contacts comprising labile bonds but in large numbers. In addition, the lability of bonds in close contacts would permit fast locomotion. An interesting and novel finding is that Y-27632 treatment of MEFs releases them from contact inhibition of locomotion, as evidenced by the invasion of a cell's underside by the thin lamellae and filopodia of a cell in close apposition.

Gerosuppression in confluent cells.

The most physiological type of cell cycle arrest - namely, contact inhibition in dense culture - is the least densely studied. Despite cell cycle arrest, confluent cells do not become senescent. We recently described that mTOR (target of rapamycin) is inactive in contact-inhibited cells. Therefore, conversion from reversible arrest to senescence (geroconversion) is suppressed. I this Perspective, we further extended the gerosuppression model. While causing senescence in regular cell density, etoposide failed to cause senescence in contact-inhibited cells. A transient reactivation of mTOR favored geroconversion in etoposide-treated confluent cells. Like p21, p16 did not cause senescence in high cell density. We discuss that suppression of geroconversion in confluent and contact-inhibited cultures mimics gerosuppression in the organism. We confirmed that levels of p-S6 were low in murine tissues in the organism compared with mouse embryonic fibroblasts in cell culture, whereas p-Akt was reciprocally high in the organism.

Repeated H2 O2 exposure drives cell cycle progression in an in vitro model of ulcerative colitis.

The production of hydrogen peroxide (H2 O2 ) drives tumourigenesis in ulcerative colitis (UC). Recently, we showed that H2 O2 activates DNA damage checkpoints in human colonic epithelial cells (HCEC) through c-Jun N-terminal Kinases (JNK) that induces p21(WAF1) . Moreover, caspases circumvented the G1/S and intra-S checkpoints, and cells accumulated in G2/M. The latter observation raised the question of whether repeated H2 O2 exposures alter JNK activation, thereby promoting a direct passage of cells from G2/M arrest to driven cell cycle progression. Here, we report that increased proliferation of repeatedly H2 O2 -exposed HCEC cells (C-cell cultures) was associated with (i) increased phospho-p46 JNK, (ii) decreased total JNK and phospho-p54 JNK and (iii) p21(WAF1) down-regulation. Altered JNK activation and p21(WAF1) down-regulation were accompanied by defects in maintaining G2/M and mitotic spindle checkpoints through adaptation, as well as by apoptosis resistance following H2 O2 exposure. This may cause increased proliferation of C-cell cultures, a defining initiating feature in the inflammation-carcinoma pathway in UC. We further suggest that dysregulated JNK activation is attributed to a non-apoptotic function of caspases, causing checkpoint adaptation in C-cell cultures. Additionally, loss of cell-contact inhibition and the overcoming of senescence, hallmarks of cancer, contributed to increased proliferation. Furthermore, there was evidence that p54 JNK inactivation is responsible for loss of cell-contact inhibition. We present a cellular model of UC and suggest a sinusoidal pattern of proliferation, which is triggered by H2 O2 -induced reactive oxygen species generation, involving an interplay between JNK activation/inactivation, p21(WAF1) , c-Fos, c-Jun/phospho-c-Jun, ATF2/phospho-ATF2, ß-catenin/TCF4-signalling, c-Myc, CDK6 and Cyclin D2, leading to driven cell cycle progression.

Serum can overcome contact inhibition in confluent human pulmonary artery smooth muscle cells.

Pulmonary artery endothelial cells (PAEC) in an intact vessel are continually exposed to serum, but unless injured, do not proliferate, constrained by confluence. In contrast, pulmonary artery smooth muscle cells (PASMC) attain, and maintain, confluence in the presence of minimal serum, protected from serum's stimulatory effects except when the endothelial barrier becomes more permeable. We hypothesized therefore, that confluent PASMC may be less constrained by contact inhibition in the presence of serum than PAEC and tested this idea by exposing confluent non-transformed human PAEC and PASMC to media containing increasing concentrations of fetal bovine serum (FBS) and determining cell growth over 7 days. PAEC that had attained confluence in low serum did not proliferate even when exposed to 5% serum, the highest concentration tested. In contrast, PASMC that attained confluence in low serum did proliferate once serum levels were increased, an effect that was dose dependent. Consistent with this observation, PASMC had more BrdU incorporation and a greater percentage of cells in S phase in 5% compared to 0.2% FBS, whereas no such difference was seen in PAEC. These results suggest that confluent human PAEC are resistant to the stimulatory effects of serum, whereas confluent PASMC can proliferate when serum levels are increased, an effect mediated in part by differences in phosphoinositide 3-kinase activation. This observation may be relevant to understanding the PASMC hyperplasia observed in humans and animals with pulmonary hypertension in which changes in endothelial permeability due to hypoxia or injury expose the underlying smooth muscle to serum.

In situ synthesis and antibacterial activity of copper nanoparticle loaded natural montmorillonite clay based on contact inhibition and ion release.

Copper nanoparticle based clay composite has been synthesized by in situ reduction of a copper ammonium complex ion and characterized by different analytical instruments. The copper nanoparticles were both intercalated and adsorbed on the surface with diameters of <5nm (for intercalated) and 25-30nm (for adsorbed). The composite showed good stability for over 3 months in air. Excellent antimicrobial activity of the composite was observed on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Enterococcus faecalis with mortality rates >90% after 12h. Cellular membrane damage permeated by direct attachment of the composite and indirect damage caused by released copper ion are the primary sources of antibacterial action. Cytotoxicity measurements showed minimal adverse effect on the two human cell lines beyond the M.B.C. value for the microorganisms studied. In the present form the clay composite shows good promise for use in therapeutic applications.

Aryl hydrocarbon receptor-mediated disruption of contact inhibition is associated with connexin43 downregulation and inhibition of gap junctional intercellular communication.

The aryl hydrocarbon receptor (AhR) contributes to the control of cell-to-cell communication, cell adhesion, migration or proliferation. In the present study, we investigated the regulation of connexin43 (Cx43) and Cx43-mediated gap junctional intercellular communication (GJIC) during the AhR-dependent disruption of contact inhibition in non-tumorigenic liver epithelial cells. The contact inhibition of cell proliferation is a process restricting the cell division of confluent non-transformed cells, which is frequently abolished in cancer cells; however, the mechanisms contributing to its disruption are still only partially understood. Disruption of contact inhibition, which was induced by toxic AhR ligands 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or polycyclic aromatic hydrocarbons in epithelial WB-F344 cells, reduced Cx43 protein levels, possibly via enhanced proteasomal degradation, significantly decreased the amount of gap junction plaques and downregulated GJIC, in an AhR-dependent manner. Although both intracellular and membrane Cx43 pools were markedly reduced in cells released from contact inhibition by TCDD, siRNA-mediated Cx43 knock-down was not sufficient to stimulate proliferation in contact-inhibited cells. Our data suggest that downregulation of Cx43/GJIC in non-transformed epithelial cells is an inherent part of disruption of contact inhibition, which occurs at the post-transcriptional level. This process runs in parallel with alterations of other forms of cell-to-cell communication, thus suggesting that toxic AhR agonists may simultaneously abrogate contact inhibition and reduce GJIC, two essential mechanisms linked to deregulation of cell-to-cell communication during tumor promotion and progression.

Structural basis of toxicity and immunity in contact-dependent growth inhibition (CDI) systems.

Contact-dependent growth inhibition (CDI) systems encode polymorphic toxin/immunity proteins that mediate competition between neighboring bacterial cells. We present crystal structures of CDI toxin/immunity complexes from Escherichia coli EC869 and Burkholderia pseudomallei 1026b. Despite sharing little sequence identity, the toxin domains are structurally similar and have homology to endonucleases. The EC869 toxin is a Zn(2+)-dependent DNase capable of completely degrading the genomes of target cells, whereas the Bp1026b toxin cleaves the aminoacyl acceptor stems of tRNA molecules. Each immunity protein binds and inactivates its cognate toxin in a unique manner. The EC869 toxin/immunity complex is stabilized through an unusual ß-augmentation interaction. In contrast, the Bp1026b immunity protein exploits shape and charge complementarity to occlude the toxin active site. These structures represent the initial glimpse into the CDI toxin/immunity network, illustrating how sequence-diverse toxins adopt convergent folds yet retain distinct binding interactions with cognate immunity proteins. Moreover, we present visual demonstration of CDI toxin delivery into a target cell.

Selective activation of p120ctn-Kaiso signaling to unlock contact inhibition of ARPE-19 cells without epithelial-mesenchymal transition.

Contact-inhibition ubiquitously exists in non-transformed cells and explains the poor regenerative capacity of in vivo human retinal pigment epithelial cells (RPE) during aging, injury and diseases. RPE injury or degeneration may unlock mitotic block mediated by contact inhibition but may also promote epithelial-mesenchymal transition (EMT) contributing to retinal blindness. Herein, we confirmed that EMT ensued in post-confluent ARPE-19 cells when contact inhibition was disrupted with EGTA followed by addition of EGF and FGF-2 because of activation of canonical Wnt and Smad/ZEB signaling. In contrast, knockdown of p120-catenin (p120) unlocked such mitotic block by activating p120/Kaiso, but not activating canonical Wnt and Smad/ZEB signaling, thus avoiding EMT. Nuclear BrdU labeling was correlated with nuclear release of Kaiso through p120 nuclear translocation, which was associated with activation of RhoA-ROCK signaling, destabilization of microtubules. Prolonged p120 siRNA knockdown followed by withdrawal further expanded RPE into more compact monolayers with a normal phenotype and a higher density. This new strategy based on selective activation of p120/Kaiso but not Wnt/ß-catenin signaling obviates the need of using single cells and the risk of EMT, and may be deployed to engineer surgical grafts containing RPE and other tissues.

Nuclear p120 catenin unlocks mitotic block of contact-inhibited human corneal endothelial monolayers without disrupting adherent junctions.

Contact inhibition ubiquitously exists in non-transformed cells that are in contact with neighboring cells. This phenomenon explains the poor regenerative capacity of in vivo human corneal endothelial cells during aging, injury and surgery. This study demonstrated that the conventional approach of expanding human corneal endothelial cells by disrupting contact inhibition with EDTA followed by bFGF activated canonical Wnt signaling and lost the normal phenotype to endothelial-mesenchymal transition, especially if TGFß1 was added. By contrast, siRNA against p120 catenin (CTNND1) also uniquely promoted proliferation of the endothelial cells by activating trafficking of p120 catenin to the nucleus, thus relieving repression by nuclear Kaiso. This nuclear p120-catenin-Kaiso signaling is associated with activation of RhoA-ROCK signaling, destabilization of microtubules and inhibition of Hippo signaling, but not with activation of Wnt-ß-catenin signaling. Consequently, proliferating human corneal endothelial cells maintained a hexagonal shape, with junctional expression of N-cadherin, ZO-1 and Na(+)/K(+)-ATPase. Further expansion of human corneal endothelial monolayers with a normal phenotype and a higher density was possible by prolonging treatment with p120 catenin siRNA followed by its withdrawal. This new strategy of perturbing contact inhibition by selective activation of p120-catenin-Kaiso signaling without disrupting adherent junction could be used to engineer surgical grafts containing normal human corneal endothelial cells to meet a global corneal shortage and for endothelial keratoplasties.

Restoration of contact inhibition in human glioblastoma cell lines after MIF knockdown.

BACKGROUND: Studies of the role of the cytokine macrophage-migration-inhibitory-factor (MIF) in malignant tumors have revealed its stimulating influence on cell-cycle progression, angiogenesis and anti-apoptosis. RESULTS: Here we show that in vitro targeting MIF in cultures of human malignant glioblastoma cells by either antisense plasmid introduction or anti-MIF antibody treatment reduced the growth rates of tumor cells. Of note is the marked decrease of proliferation under confluent and over-confluent conditions, implying a role of MIF in overcoming contact inhibition. Several proteins involved in contact inhibition including p27, p21, p53 and CEBPalpha are upregulated in the MIF antisense clones indicating a restoration of contact inhibition in the tumor cells. Correspondingly, we observed a marked increase in MIF mRNA and protein content under higher cell densities in LN18 cells. Furthermore, we showed the relevance of the enzymatic active site of MIF for the proliferation of glioblastoma cells by using the MIF-tautomerase inhibitor ISO-1. CONCLUSION: Our study adds another puzzle stone to the role of MIF in tumor growth and progression by showing the importance of MIF for overcoming contact inhibition.