Cell Contact Inhibition Research Articles

Cell Distribution Profiles in Three‐Dimensional Scaffolds with Inverted‐Colloidal‐Crystal Geometry: Modeling and Experimental Investigations

Limited ingrowth of stromal cells is observed when a three-dimensionally ordered scaffold possessing inverted-colloidal-crystal geometry is used to culture adherent cells. In this work, a computational model explaining, as well as predicting, experimental cell distributions is developed. It incorporates a modified Contois cell-growth model that includes the effects of nutrient saturation, competitive product inhibition, and cell-contact inhibition to describe the scaffold-cell system. Our results agree with the hypothesis that the rapid growth of cells on the surface of the scaffold depletes the nutrient supply to the core, resulting in the preferential growth on the exterior of the scaffold. When the cells are cultured in a scaffold subjected to a uniform velocity field, they penetrate to a greater extent into the scaffold core. Alternative seeding and culture strategies are suggested and evaluated.

Merlin facilitates ubiquitination and degradation of transactivation-responsive RNA-binding protein

The Nf2 tumor suppressor codes for merlin, a protein whose function is largely unknown. We have previously demonstrated a novel interaction between merlin and TRBP, which inhibits the oncogenic activity of TRBP. In spite of the significance of their functional interaction, its molecular mechanism still remains to be elucidated. In this report, we investigated how merlin inhibits the oncogenic activity of TRBP in association with cell growth conditions. In the human embryonic kidney 293 cell line, the level of endogenous merlin increased, whereas that of endogenous TRBP significantly decreased along with the increase in cell confluence. We demonstrated that the carboxyl-terminal region of TRBP was responsible for this phenomenon using stable cell lines expressing deletion mutants of TRBP. The overexpression of merlin decreased the protein level of TRBP, and the ubiquitin-like subdomain of merlin's FERM domain was important for this activity. We also demonstrated that TRBP is ubiquitinylated and the ubiquitinylated forms of TRBP are accumulated by ectopically expressed merlin or cell confluence in the presence of MG132, a proteasome inhibitor. Furthermore, we showed that the regulation of TRBP in response to cell confluence was abolished upon knockdown of merlin expression by specific small interfering RNA. Finally, we showed that ectopically expressed merlin restored cell-cell contact inhibition in cells stably expressing TRBP but not in TRBPDeltac. These results suggest that merlin is involved in the regulation of TRBP protein level by facilitating its ubiquitination in response to such cues as cell-cell contacts.

Review of Growth Inhibitory Peptide as a Biotherapeutic agent for tumor growth, adhesion, and metastasis

This review surveys the biological activities of an alpha-fetoprotein (AFP) derived peptide termed the Growth Inhibitory Peptide (GIP), which is a synthetic 34 amino acid segment produced from the full length 590 amino acid AFP molecule. The GIP has been shown to be growth-suppressive in both fetal and tumor cells but not in adult terminally-differentiated cells. The mechanism of action of this peptide has not been fully elucidated; however, GIP is highly interactive at the plasma membrane surface in cellular events such as endocytosis, cell contact inhibition and cytoskeleton-induced cell shape changes. The GIP was shown to be growth-suppressive in nine human tumor types and to suppress the spread of tumor infiltrates and metastases in human and mouse mammary cancers. The AFP-derived peptide and its subfragments were also shown to inhibit tumor cell adhesion to extracellular matrix (ECM) proteins and to block platelet aggregation; thus it was expected that the GIP would inhibit cell spreading/migration and metastatic infiltration into host tissues such as lung and pancreas. It was further found that the cyclic versus linear configuration of GIP determined its biological and anti-cancer efficacy. Genbank amino acid sequence identities with a variety of integrin alpha/beta chain proteins supported the GIP's linkage to inhibition of tumor cell adhesion and platelet aggregation. The combined properties of tumor growth suppression, prevention of tumor cell-to-ECM adhesion, and inhibition of platelet aggregation indicate that tumor-to-platelet interactions present promising targets for GIP as an anti-metastatic agent. Finally, based on cholinergic studies, it was proposed that GIP could influence the enzymatic activity of membrane acetylcholinesterases during tumor growth and metastasis. It was concluded that the GIP derived from full-length AFP represents a growth inhibitory motif possessing instrinsic properties that allow it to interfere in cell surface events such as adhesion, migration, metastasis, and aggregation of tumor cells.

EN2 is a candidate oncogene in human breast cancer

Only a few critical oncogenes have been identified in the more commonly occurring cases of sporadic breast cancer. We provide evidence that EN2 is ectopically expressed in a subset of human breast cancer and may have a causal role in mammary tumorigenesis. Nontumorigenic mammary cell lines engineered to ectopically express En-2 have a marked reduction in their cycling time, lose cell contact inhibition, become sensitive to 17-AAG treatment, fail to differentiate when exposed to lactogenic hormones and induce mammary tumors when transplanted into cleared mammary glands of syngeneic hosts. RNA interference studies suggest that EN2 expression is required for the maintenance of the transformed phenotype of a human breast tumor cell line.

Duration of In Vitro Maturation of Recipient Oocytes Affects Blastocyst Development of Cloned Porcine Embryos

This study investigated the effects of different incubation periods for oocyte maturation and contact inhibition of donor cells as well as different osmolarities for storage of recipient oocytes on fusion rates, cleavage rates, and blastocyst yields of porcine somatic nuclear transfer (SCNT) derived embryos. In addition, the in vivo developmental potential of cloned embryos derived from the most promising SCNT protocol was tested by transfer to recipient gilts. Storage of in vitro-matured oocytes for 7.5 h in calcium-free TL-HEPES medium at 295 or 320 mOsmol prior to activation yielded significantly (p < 0.05) higher parthenogenetic blastocyst rates compared to storage in TL-HEPES with an osmolarity of 270 mOsmol (24.4 +/- 3.0% and 26.2 +/- 4.3% vs. 18.3 +/- 6.4%, respectively, mean +/- SD) and improved the visibility of the polar body. Electrical fusion of fibroblasts to enucleated oocytes matured for 38, 40, or 42 h resulted in similar fusion and cleavage rates (74.8-84.4%). However, nuclear transfer with oocytes matured for 40 h in vitro yielded significantly higher (p < 0.05) development to the blastocyst stage after 7 days of culture (14.7 +/- 1.7%) than with oocytes matured for 38 h (9.5 +/- 2.1%) or 42 h (5.1 +/- 2.1%). Contact inhibition for 24, 48, or 72 h significantly (p < 0.05) increased the proportion of cells at G0/G1 compared with cycling fibroblasts. However, duration of contact inhibition of the donor cells for either 24, 48, or 72 h had no effect on blastocyst rates of SCNT embryos. Four gilts received an average of 150 SCNT embryos (range 138-161) reconstructed with oocytes matured for 40 h; two of these became pregnant; one of them went to term and farrowed four piglets on day 115 of pregnancy. Microsatellite analysis confirmed that the clones were genetically identical with the donor cells. These results show that changes of the in vitro maturation protocol may affect in vitro development of reconstructed porcine embryos, while duration of the contact inhibition period plays a minor role for the success of porcine SCNT. The effects on in vivo development are yet to be determined.

High-density growth arrest in Ras-transformed cells: low Cdk kinase activities in spite of absence of p27 Kip Cdk-complexes

The ras oncogene transforms immortalized, contact-inhibited non-malignant murine fibroblasts into cells that are focus forming, exhibit increased saturation density, and are malignant in suitable hosts. Here, we examined changes in cell cycle control complexes as normal and Ras-transformed cells ceased to grow exponentially, to reveal the molecular basis for Ras-dependent focus formation. As normal cells entered density-dependent arrest, cyclin D1 decreased while cyclin D2 was induced and replaced D1 in Cdk4 complexes. Concomitantly, p27 Kip1 levels rose and the inhibitor accumulated in both Cdk4 and Cdk2 complexes, as these kinases were inactivated. Ras-transformed cells failed to arrest at normal saturation density and showed no significant alterations in cell control complexes at this point. Yet, at an elevated density the Ras-transformed cells ceased to proliferate and entered a quiescent-like state with low Cdk4 and Cdk2 activity. Surprisingly, this delayed arrest was molecularly distinct from contact inhibition of normal cells, as it occurred in the absence of p27 Kip1 induction and cyclin D1 levels remained high. This demonstrates that although oncogenic Ras efficiently disabled the normal response to contact inhibition, a separate back-up mechanism enforced cell cycle arrest at higher cell density.

Evaluation of the role of c-Src and ERK in TCDD-dependent release from contact-inhibition in WB-F344 cells

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is the most potent tumor promoter ever tested in rodents. Although it is known that most of the effects of TCDD are mediated by binding to the aryl hydrocarbon receptor (AhR), the mechanisms leading to tumor promotion remain to be elucidated. Loss of contact-inhibition is one characteristic hallmark in tumorigenesis. In WB-F344 cells, TCDD induces a release from contact-inhibition which is manifested by a twofold increase in DNA-synthesis and cell number when TCDD (1 nmol L-1) is given to confluent cells. Because TCDD leads to phosphorylation of the epidermal growth factor receptor and an increase in c-Src-activation in WB-F344 cells, we investigated the functional relevance of this observation. Pharmacological inhibition of c-Src using PP1 (10 micromol L-1) or genistein (10 micromol L-1) did not prevent TCDD-dependent release from contact-inhibition. In accordance, elevation of cyclin A-a previously identified target of TCDD and marker of S-phase entry-was not reduced in the presence of PP1 or genistein. Western blot analysis revealed that phosphorylation of the EGF-receptor downstream target ERK was not induced in response to TCDD. Furthermore, TCDD-dependent increase in DNA-synthesis was not inhibited by the MEK1/2 inhibitor U0126 (10 micromol L-1). Our data show that neither c-Src-activation, nor ERK-activation are required for TCDD-dependent release from contact-inhibition arguing against a functional role of EGF-receptor activation in response to TCDD in WB-F344 cells.

Notch activation induces endothelial cell cycle arrest and participates in contact inhibition: role of p21Cip1 repression.

Although previous studies demonstrate that appropriate Notch signaling is required during angiogenesis and in vascular homeostasis, the mechanisms by which Notch regulates vascular function remain to be elucidated. Here, we show that activation of the Notch pathway by the ligand Jagged1 reduces the proliferation of endothelial cells. Notch activation inhibits proliferation of endothelial cells in a cell-autonomous manner by inhibiting phosphorylation of the retinoblastoma protein (Rb). During cell cycle entry, p21Cip1 is upregulated in endothelial cells. Activated Notch inhibits mitogen-induced upregulation of p21Cip1 and delays cyclin D-cdk4-mediated Rb phosphorylation. Notch-dependent repression of p21Cip1 prevents nuclear localization of cyclin D and cdk4. The necessity of p21Cip1 for nuclear translocation of cyclin D-cdk4 and S-phase entry in endothelial cells was demonstrated by targeted downregulation of p21Cip1 by using RNA interference. We further demonstrate that when endothelial cells reach confluence, Notch is activated and p21Cip1 is downregulated. Inhibition of the Notch pathway at confluence prevents p21Cip1 downregulation and induces Rb phosphorylation. We suggest that Notch activation contributes to contact inhibition of endothelial cells, in part through repression of p21Cip1 expression.

Transforming growth factor-beta1 is not involved in TCDD-dependent release from contact inhibition in WB-F344 cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent tumor promoter ever tested in rodents. Although it is known that most of the effects of TCDD are mediated by binding to the aryl hydrocarbon receptor (AhR), the mechanisms leading to tumor promotion still remain to be elucidated. Loss of contact inhibition is one characteristic hallmark in tumorigenesis. In WB-F344 cells TCDD induces a release from contact inhibition which is manifested by a two- to threefold increase in DNA synthesis when TCDD (1 nM) is applied to confluent cells. Since proliferation of epithelial cells is known to be inhibited by TGF-beta, we investigated whether decreased TGF-beta1 mediates TCDD-dependent release from contact inhibition in WB-F344 cells. Expression of TGF-beta (type II) receptor in WB-F344 cells was analyzed by Western blot analysis. Exposure of 0.1 ng/ml TGF-beta1 to exponentially growing WB-F344 cells resulted in a 40% decrease in DNA synthesis, which was blocked by preincubation with a neutralizing anti-TGF-beta1 antibody, indicating that the TGF-beta receptor in WB-F344 cells is functionally active. Preincubation of confluent, G1-arrested cultures with the neutralizing anti-TGF-beta1-antibody did not lead to an increase in DNA synthesis, ruling out an involvement of TGF-beta1 in mediating contact inhibition in WB-F344 cells. In accord with this, Western blot analysis revealed that protein expression of TGF-beta1 is neither upregulated in confluent cultures nor decreased after TCDD treatment. We conclude that TGF-beta1 is not involved in contact inhibition or in TCDD-dependent release from contact inhibition in WB-F344 cells.

Transforming growth factor beta1 is not involved in 2,3,7,8-tetrachlorodibenzo- p-dioxin-dependent release from contact-inhibition in WB-F344 cells.

TCDD (2,3,7,8-tetrachlorodibenzo- p-dioxin) is the most potent tumor promoter ever tested in rodents. Although it is known that most of the effects of TCDD are mediated by binding to the aryl hydrocarbon receptor (AhR), the mechanisms leading to tumor promotion still remain to be elucidated. Loss of contact-inhibition is one characteristic hallmark in tumorigenesis. In WB-F344 cells, TCDD induces a release from contact-inhibition, which is manifested by a two- to three-fold increase in DNA-synthesis when TCDD (1 nM) is given to confluent cells. Since proliferation of epithelial cells is known to be inhibited by transforming growth factor beta (TGF-beta) we investigated whether decreased TGF-beta expression mediates TCDD-dependent release from contact-inhibition in WB-F344 cells. Expression of TGF-beta (type II) receptor in WB-F344 cells was shown by Western blot analysis. Exposure of exponentially growing WB-F344 cells to 0.1 ng/ml TGF-beta1 resulted in a 40% decrease in DNA synthesis, which could be blocked by pre-incubation with a neutralizing anti-TGF-beta1 antibody indicating that the TGF-beta receptor in WB-F344 cells is functionally active. Pre-incubation of confluent, G1-arrested cultures with the neutralizing anti-TGF-beta1 antibody did not lead to an increase in DNA synthesis, ruling out an involvement of TGF-beta1 in mediating contact-inhibition in WB-F344 cells. In accordance with this, Western blot analysis revealed that protein expression of TGF-beta1 was neither upregulated in confluent cultures nor decreased after TCDD treatment. We therefore conclude that TGF-beta1 is not involved in contact-inhibition nor in TCDD-dependent release from contact-inhibition in WB-F344 cells.

Evidence for ERK1/2 phosphorylation controlling contact inhibition of proliferation in Madin-Darby canine kidney epithelial cells.

Increasing cell density arrests epithelial cell proliferation by a process termed contact inhibition. We investigated mechanisms of contact inhibition using a model of contact-inhibited epithelial cells. Hepatocyte growth factor (HGF) treatment of contact-inhibited Madin-Darby canine kidney (MDCK) cells stimulated cell proliferation and increased levels of phosphorylated ERK1/2 (phospho-ERK1/2) and cyclin D1. MEK inhibitors PD-98059 and U0126 inhibited these HGF-dependent changes, indicating the dependence on phosphorylation of ERK1/2 during HGF-induced loss of contact inhibition. In relation to contact-inhibited high-density cells, low-density MDCK cells proliferated and had higher levels of phospho-ERK1/2 and cyclin D1. PD-98059 and U0126 inhibited low-density MDCK cell proliferation. Trypsinization of high-density MDCK cells immediately increased phospho-ERK1/2 and was followed by a transient increase in cyclin D1 levels. Reformation of cell junctions after trypsinization led to decreases in phospho-ERK1/2 and cyclin D1 levels. High-density MDCK cells express low levels of both cyclin D1 and phospho-ERK1/2, and treatment of these cells with fresh medium containing HGF but not fresh medium alone for 6 h increased phospho-ERK1/2 and cyclin D1 levels compared with cells without medium change. These data provide evidence that HGF abrogates MDCK cell contact inhibition by increasing ERK1/2 phosphorylation and levels of cyclin D1. These results suggest that in MDCK cells, contact inhibition of cell proliferation in the presence of serum occurs by cell density-dependent regulation of ERK1/2 phosphorylation.

66EFFECTS OF CONTACT INHIBITION INTERVALS V. SERUM DEPRIVATION ON DEVELOPMENT OF PORCINE NUCLEAR TRANSFER DERIVED EMBRYOS

Contact inhibition and serum deprivation are commonly used to synchronize donor cells at the G0/G1 state of the cell cycle prior to use in nuclear transfer. Here we compared the effects of serum deprivation (SD) and different intervals of contact inhibition (CI) of the donor cells on the blastocyst rate. One batch from pooled porcine fetal fibroblasts (passage 3) was used in this study. The cells were thawed, seeded to a six-well plate and cultured in Dulbecco‘s Modified Eagles Medium (DMEM) supplemented with 2mM glutamine, 1% non-essential amino acids, 0.1mM mercaptoethanol, 100UmL−1 penicillin, 100mgmL−1 streptomycin containing 10% fetal calf serum (FCS). Serum deprivation was achieved by culturing cells in DMEM containing 0.5% FCS for 48h. Cells of the CI groups were grown to 100% confluency and kept in that state for 24h, 48h and 72h. Immediately after cell cycle synchronization, cells were used in nuclear transfer. Cell cycle state of the cells was evaluated by FACS analysis at 24h after beginning of CI and prior to nuclear transfer. Blastocyst rate was determined 7 days after nuclear transfer. An average of 38-42h in vitro matured oocytes were used in nuclear transfer (NT). NT was performed as described previously (Betthauser J et al., 2000 Nat. Biotechnol. 17, 456–461). There were no differences in the proportion of cells in G0/G1 of the cell cycle in any of the treatment groups (85.0%, 85.8%, 85.5% and 86.3% for SD and CI at either 24h, 48h and 72h, respectively). After nuclear transfer (for each CI group n=336–384 reconstr. embryos; SD n=215) there was a statistically significant difference in the fusion rate between 48h CI and SD cells (74.8% v. 87.5%, t-test P&amp;lt;0.050). Blastocyst rate (blastocysts/fused) differed significantly between SD, 24h CI and 48h CI (17.4%; 9.1%, 9.6%, t-test P&amp;lt;0.050), there was no difference between SD and 72h CI and within the CI groups (72h CI 10.6%). Four transfers of reconstructed embryos (72h CI, n=138–163 embryos/gilt, 1-cell embryos) to prepuberal Landrace gilts led to 2 initial pregnancies determined at Day 25 by ultrasound. One pregnancy was lost at Day 35; the other recipient remained pregnant and farrowed 4 piglets. One piglet was stillborn and one died 7h after birth; the remaining two piglets are healthy and now 4 months old. Four transfers of embryos (n=96–110) reconstructed with SD cells revealed two initial pregnancies determined on Day 25 by ultrasound. Again, one was lost on Day 35, and the other one is now at Day 100. Our results show that, despite similar proportions of cells being in G0/G1 of the cell cycle, cells either contact-inhibited for 72h or serum-deprived both show higher rates of blastocyst development compared to cells contact-inhibited for shorter time periods. Both donor cell preparations can lead to full term development of nuclear transfer-derived embryos. This work was funded by the Deutsche Forschungsgemeinschaft (DFG, SFB 265).

The zinc-finger transcription factor GLI2 antagonizes contact inhibition and differentiation of human epidermal cells.

In stratified epidermis, activation of the Hh/Gli signal transduction pathway has been implicated in the control of cell proliferation and tumorigenesis. The zinc-finger transcription factor Gli2 has been identified as critical mediator of the Hh signal at the distal end of the pathway, but the molecular mechanisms by which Gli2 regulates cell proliferation or induces epidermal malignancies such as basal cell carcinoma are still unclear. Here, we provide evidence for a role of human GLI2 in antagonizing contact inhibition and epidermal differentiation. We show by gene expression profiling that activation of the GLI2 oncogene in human keratinocytes activates the transcription of a number of genes involved in cell cycle progression such as E2F1, CCND1, CDC2 and CDC45L, while it represses genes associated with epidermal differentiation. Analysis of the proliferative effect of GLI2 revealed that GLI2 is able to induce G1-S phase progression in contact-inhibited keratinocytes. Detailed time-course experiments identified E2F1 as early transcriptional target of GLI2. Further, we show that GLI2 expression in human keratinocytes results in a marked downregulation of epidermal differentiation markers. The data suggest a role for GLI2 in Hh-induced epidermal neoplasia by opposing epithelial cell cycle arrest signals and epidermal differentiation.

RhoA activation stimulates IEC-6 cell proliferation by decreasing p21Wafl1/Cip1 and increasing Cdk2 expression in a polyamine dependent manner

Background & Aims: Although RhoA plays an important role m ceil proliferation and in Ras transformation in fibroblasts and mammary epithelial cells; its role in intestinal epithelial cells is still unknown. In our previous study, we showed that polyamine depletion (DFMO treatment) strongly inhibits proliferation of intestinal epithelial cells (IEC-6). In this report, we examined the effects of active RhoA and the mechanism underlying 1EC-6 cell proliferation, contact inhibition, and focus formation. We also examined whether polyamine depletion inhibits cell proliferation m the presence of constitutively active RhoA. Methods: Constitutively active RhoA and vector transfected 1EC-6 cell lines were used. Cells were grown in the presence or absence of DFMO, an irreversible inhibitor of omithine decarboxylase (ODC). Growth characteristics were assessed by microscopic observation, cell counting, and ceil cycle analysis by flow cytometry. Cell cycle regulators were evaluated by western blot, functional assay, and real-time PCR. Results: RhoA transfection significantly increased the rate of cell proliferation. These cells also demonstrated attenuated contact inhibition and conspicuous loci when they were fully confluent, increased proliferation was accompanied by decreased p21Wafl/Cipl, increased Cdk2 expression and activity. The inhibition of p21Wafl/Cipl was independent of p53. There was no activation of the Ras-Raf-Mek-Erk pathway in the RhoA transfected cell line. Polyamine depletion totally prevented the effect of activated RhoA on 1EC-6 cell proliferation and focus formation, decreased Cdk2 expression and activity independent of p21Wafl/Cipl. Conclusions: First, RhoA activation decreases p21, increase~ Cdk2 protein and Cdk2 activity, leading to the stimulation of intestinal epithelial cell proliferation and transformation; second, polyamine depletion totally prevents stimulating effect of RhoA on proliferation by decreasing Cdk2 expression and activity.

Cell cycle independent infection and gene transfer by recombinant Sendai viruses

A common problem for viral vectors in the field of somatic gene therapy is the dependence of an efficient cellular transduction on the cell cycle phase of target cells. An optimized viral vector system should therefore transduce cells in different cell cycle phases equally to improve transduction efficiencies. Recent observations that recombinant Sendai viruses (SeV) can infect a broad range of different tissues suggested SeV to be a good candidate for future gene therapeutic strategies in which dividing and non-dividing cells have to be reached. However, detailed data on the influence of distinct cell cycle phases on the infection of SeV or related viruses are missing. We report that synchronization of NIH 3T3 cells as well as contact inhibition of human fibroblast cells did not exhibit any negative influence on SeV infection rates. Furthermore, different attractive target tissues like human umbilical cord derived cells or primary human hepatocytes can be reached by SeV efficiently. As an important information for further cell cycle studies of paramyxoviruses we discovered surprisingly that the DNA polymerase inhibitor aphidicolin (induces a G 1/M arrest) functions as an inhibitor of SeV but not of an adenoviral expression vector. In conclusion, the results demonstrate SeV based vector particles to be an ideal tool to reach equally cells coexisting in different cell cycle phases.

TCDD‐dependent downregulation of γ‐catenin in rat liver epithelial cells (WB‐F344)

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is the most potent tumor promoter ever tested in rodents. Although it is known that most of the effects of TCDD are mediated by binding to the aryl hydrocarbon receptor (AHR), the mechanisms leading to tumor promotion still remain to be elucidated. Loss of contact-inhibition is a characteristic hallmark in tumorigenesis. In WB-F344 cells, TCDD induces a release from contact-inhibition manifested by a 2- to 3-fold increase in DNA-synthesis and the emergence of foci when TCDD (1 nM) is given to confluent cells. We focussed our interest on potential cell membrane proteins mediating contact-inhibition in WB-F344 cells, namely E-cadherin, alpha,- beta,- and gamma-catenin (plakoglobin). Using indirect immunofluorescence, E-cadherin, alpha-, beta- and gamma-catenin were detected at cell adhesion sites in untreated, confluent cells. After TCDD-exposure, gamma-catenin was exclusively localized in the cytoplasm whereas localization of E-cadherin, alpha- and beta-catenin remained unaffected. Cytoplasmic gamma-catenin could be extracted by Triton X-100 treatment, demonstrating that gamma-catenin was no longer bound to the actin cytoskeleton. Western blot analysis showed downregulation of gamma-catenin protein levels. This effect was not blocked by pre-incubation with the selective proteasome inhibitor MG-132, indicating that proteolytical degradation of gamma-catenin by the proteasome system was not increased by TCDD. Because mRNA-levels of gamma-catenin were markedly diminished after TCDD-exposure, we conclude that transcriptional downregulation or destabilization of the mRNA contributes to the decrease in gamma-catenin protein levels in response to TCDD. Because gamma-catenin is considered to be a tumor suppressor, our findings might give more insight into the tumor promoting actions of TCDD.

2,3,7,8-Tetrachlorodibenzo- p-dioxin-Dependent Release from Contact Inhibition in WB-F344 Cells: Involvement of Cyclin A

2,3,7,8-Tetrachlorodibenzo- p-dioxin (TCDD) is the most potent tumor promoter ever tested in rodents. Although it is known that most of TCDD actions are mediated by binding to the aryl hydrocarbon receptor (AhR), the mechanisms leading to tumor promotion still remain to be elucidated. Loss of contact inhibition is one characteristic hallmark in tumorigenesis. In rat liver epithelial WB-F344 cells, TCDD induces a release from contact inhibition, which is manifested by a twofold increase in cell number when TCDD (1 nM for 48 h) is added to confluent cells in the presence of serum, but not when given to exponentially growing or subconfluent, serum-deprived WB-F344 cells. Loss of G1 arrest was also shown by flow cytometric analysis. We demonstrate that TCDD treatment significantly increases cyclin D2 and cyclin A protein levels and show by immunofluorescence that these proteins accumulate in the nucleus. Although TCDD treatment leads to a strong increase in cyclin D2/cdk4 and cyclin A/cdk2 complex formation, we could only detect an elevation of cyclin A/cdk2 activity. In accordance with a lack of elevated cdk4 activity, no decrease in the amount of hypophosphorylated retinoblastoma protein could be shown after TCDD treatment. The importance of increased cyclin A/cdk2 activity for TCDD-dependent release from contact inhibition was shown by the fact that the cdk2/cdc2-specific inhibitor olomoucine (25 μM) abolished TCDD response. These data indicate cyclin A-dependent loss of G1 arrest after TCDD treatment mainly downstream of the retinoblastoma protein.

2,3,7,8-Tetrachlorodibenzo-p-dioxin-Dependent Release from Contact Inhibition in WB-F344 Cells: Involvement of Cyclin A

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most potent tumor promoter ever tested in rodents. Although it is known that most of TCDD actions are mediated by binding to the aryl hydrocarbon receptor (AhR), the mechanisms leading to tumor promotion still remain to be elucidated. Loss of contact inhibition is one characteristic hallmark in tumorigenesis. In rat liver epithelial WB-F344 cells, TCDD induces a release from contact inhibition, which is manifested by a twofold increase in cell number when TCDD (1 nM for 48 h) is added to confluent cells in the presence of serum, but not when given to exponentially growing or subconfluent, serum-deprived WB-F344 cells. Loss of G1 arrest was also shown by flow cytometric analysis. We demonstrate that TCDD treatment significantly increases cyclin D2 and cyclin A protein levels and show by immunofluorescence that these proteins accumulate in the nucleus. Although TCDD treatment leads to a strong increase in cyclin D2/cdk4 and cyclin A/cdk2 complex formation, we could only detect an elevation of cyclin A/cdk2 activity. In accordance with a lack of elevated cdk4 activity, no decrease in the amount of hypophosphorylated retinoblastoma protein could be shown after TCDD treatment. The importance of increased cyclin A/cdk2 activity for TCDD-dependent release from contact inhibition was shown by the fact that the cdk2/cdc2-specific inhibitor olomoucine (25 μM) abolished TCDD response. These data indicate cyclin A-dependent loss of G1 arrest after TCDD treatment mainly downstream of the retinoblastoma protein.

Protocadherin LKC, a new candidate for a tumor suppressor of colon and liver cancers, its association with contact inhibition of cell proliferation.

Protocadherins are a major subfamily of the cadherin superfamily, but little is known about their functions and intracellular signal transduction. We cloned a novel human protocadherin gene, containing seven EC domains, and identified functional aspects of this gene. The gene was predominantly expressed in liver, kidney and colon tissues, and was thus designated Protocadherin LKC. The expression of Protocadherin LKC is markedly reduced in cancers arising from these tissues at both transcriptional and protein levels. To investigate the effects of Protocadherin LKC expression in colon cancer, we introduced the gene into colon cancer cell line HCT116, which does not express this gene. Significantly, Protocadherin LKC expression induced contact inhibition of cell proliferation although it did not affect growth rate. When grown to post-confluence in monolayer cells cultures, Protocadherin LKC-expressing HCT116 no longer formed multiple cell layers and showed the typical paving stone morphology of normal epithelial cells. Furthermore, expression of Protocadherin LKC suppressed tumor formation of HCT116 cells in a nude mouse model. In addition, we identified a protein, hMAST205 (microtubule-associated serine/threonine kinase-205 kDa), which interacted with Protocadherin LKC; the interaction occurring between the PDZ domain of hMAST205 and C-terminal tail of Protocadherin LKC. Our results suggest that Protocadherin LKC, which directly binds PDZ protein, is a molecular switch for contact inhibition of epithelial cells in the liver, kidney and colon tissues.

Participation of small GTPases Rac1 and Cdc42Hs in myoblast transformation.

We have previously shown that expression of active Rac1 and Cdc4Hs inhibits skeletal muscle cell differentiation. We show here, by bromodeoxyuridine incorporation and cyclin D1 expression, that the expression of active Rac1 and Cdc42Hs but not RhoA impairs cell cycle exit of L6 myoblasts cultured in differentiation medium. Furthermore, expression of activated forms of Rac1 and Cdc42Hs elicits the loss of cell contact inhibition and anchorage-dependent growth as measured by focus forming activity and growth in soft agar. RhoA was once again not found to have this effect. We found a constitutive Rac1 and Cdc42Hs activation in three human rhabdomyosarcoma-derived cell lines, one of the most common causes of solid tumours arising from muscle precursors during childhood. Finally, dominant negative forms of Rac1 and Cdc42Hs inhibit cell proliferation of the RD rhabdomyosarcoma cell line. These data suggest an important role for the small GTPases Rac1 and Cdc42Hs in the generation of skeletal muscle tumours.