Hyperoxia alters phorbol ester-induced phospholipase D activation in bovine lung microvascular endothelial cells

We investigated the effect of hyperoxia on phospholipase D (PLD) activation in bovine lung microvascular endothelial cells (BLMVECs). Generation of intracellular reactive oxygen species in BLMVECs exposed to hyperoxia for 2 or 24 h was three-fold higher compared with normoxic cells as measured by dichlorodihydrofluorescein di(acetoxymethyl ester) fluorescence. Exposure of BLMVECs to hyperoxia for 2 or 24 h attenuated 12-O-tetradecanoylphorbol 13-acetate (TPA)-mediated PLD activation compared with normoxic cells, however, hyperoxia did not alter basal PLD activity. Antioxidants, such as propyl gallate and pyrrolidine dithiocarbamate, reversed the effect of hyperoxia on TPA-induced PLD activity. Furthermore, the TPA-induced PLD activation was inhibited not only by the protein kinase C inhibitor, Go6976, but also by the tyrosine kinase inhibitor, genistein, and by the Src kinase specific inhibitor, PP-2, suggesting the involvement of protein kinase C and also tyrosine kinases in TPA-induced PLD activation. Western blot analysis of cell lysates from the hyperoxic (2 or 24 h) BLMVECs stimulated with TPA with anti-phosphotyrosine antibody showed an attenuation in overall tyrosine phosphorylation of proteins. In conclusion, we have demonstrated that hyperoxia enhanced the generation of reactive oxygen species in lung microvascular endothelial cells and attenuated TPA-induced protein tyrosine phosphorylation and PLD activation. As protein tyrosine phosphorylation and PLD play important roles in inflammatory responses, this could provide a mechanism for the regulation of endothelial barrier function during hyperoxic lung injury.     

粘着斑激酶活化对平滑肌细胞粘附和迁移的影响

目的:研究粘着斑激酶磷酸化在细胞外基质成份诱导平滑肌细胞粘附和迁移中的作用。方法:通过纤粘连蛋白(FN)诱导培养的平滑肌细胞粘附迁移,以免疫沉淀和Western blolt方法检测粘着斑激酶(FAK)及其磷酸化的表达量。将FAK反义寡核苷酸(ODNs)经脂质体转染细胞,观察对FAK磷酸化、细胞粘附铺展和迁移的影响。结果:FN在显著诱导平滑肌细胞粘附和迁移时,FAK也呈明显表达,20 mg/L FN可使其磷酸化处于较高表达量。脂质体可有效地介导ODNs转染,转染效率为(86.7±4.5)%,FAK磷酸化表达量明显减少,5-60 mg/L不同浓度FN组,细胞铺展率减少17.89%-27.67%,10、20、40和60 mg/L FN组迁移细胞数也分别显著少23.26%、21.63%、19.31%、17.88%(P＜0.05)。结论:活化的FAK是细胞外基质诱导SMCs粘附和迁移的重要信号分子,由其介导的信号转导促进了这一过程,反义FAK ODNs可有效地对此进行抑制。     

Phosphorylation of ribosomal protein S6 and its regulation during differentiation of human leukemic cells.

We attempted to study the role of protein tyrosine kinase (PTK) and protein kinase C (PKC) in the cascade of phosphorylation of ribosomal protein S6 during differentiation of leukemic cells (HL-60, THP-1, and RWLeu-4). Neither activation nor inhibition of colony stimulating factor-1 (CSF-1) receptor''s PTK activity with CSF-1 or genistein respectively affected the phosphorylation of S6. However, vanadate which is a protein tyrosine phosphatase (PTP) inhibitor showed enhancement of S6 phosphorylation. Dimethylsulfoxide which does not affect either PTK or PKC demonstrated no change in S6 phosphorylation. PKC activation by acute 12-0-tetradecanoyl phorbol-13-acetate (TPA) treatment induced monocytic differentiation and S6 phosphorylation. Surprisingly, the more prominent phosphorylation of S6 protein was observed in PKC-depleted cells by prolonged TPA treatment. Our results suggest that PTK/PTP play a lesser role in S6 phosphorylation of HL-60 cells than PKC does. In addition, two different mechanisms seem to be involved in TPA-induced S6 phosphorylation during HL-60 differentiation: PKC activation by acute TPA treatment and PKC depletion which may lead to the synthesis of some endogenous protein responsible for the differentiation by chronic TPA treatment.     

Adhesion to fibronectin's EDbdomain induces tyrosine phosphorylation of focal adhesion proteins in Balb/c 3T3 cells

Balb/c 3T3 cell adhesion on substrata coated with fibronectin's (FN) alternatively-spliced ED_b, implicated in some tumor cell systems, and its neighboring type III repeats (III7 and III8) induced intracellular signaling coincident with morphological responses. These events were analysed using minigene-expressed proteins containing various permutations of type III repeats of FN. Cells adherent to the tri-repeat protein 7-ED_b-8 were compared to those attached to the tri-repeat 8-9-10 which can interact with integrins through RGD and its synergistic sequences. Cell adhesion to 7-ED_b-8 generated rapid tyrosine phosphorylation of several intra-cellular proteins (particularly the complex at 120-130 kD), with the overall phosphorylation level and its sensitivity to tyrosine kinase inhibitors similar to responses on the 8-9-10 tri-repeat. This similarity contrasted with the differential morphological responses of cells mediated by these proteins. Further analysis of the kinetics of pho sphorylation through immunoblotting of two focal adhesion proteins, p125FAK and p130cas, revealed a profile for Balb/c 3T3 adhesion to 7-ED_b-8 distinct from that on 8-9-10. ED_b mono-repeat was also more potent for inducing both limited cell spreading and FAK tyrosine phosphorylation than its neigh-boring repeats III7 or III8. Examination of cellular localization of FAK and vinculin showed that cells spread on the 7-ED_b-8 substratum displayed vinculin-positive focal complex-like structures at the cell periphery, in contrast to the classical focal adhesions seen in 8-9-10-adherent cells. These results suggest that ED_b induces cell signaling events, leading to tyrosine phosphorylation of focal adhesion proteins, through a mechanism different from that mediated by integrins recognizing sequences in III8-9-10. ED_b-dependent signaling may have special significance in some tumor systems. © Rapid Science 1998     

Reactive oxygen species-mediated PKC and integrin signaling promotes tumor progression of human hepatoma HepG2

The poor prognosis and recurrence of HCC are majorly caused by intrahepatic metastasis. Delineating the molecular pathways mediating these processes may benefit developing effective targeting therapies. Using human hepatoma HepG2 as a model, we have found reactive oxygen species (ROS) may cooperate with protein kinase C (PKC) for sustained ERK phosphorylation and migration of HepG2 induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). We further investigated whether integrin signaling is involved. Various antagonists of integrin signaling prevented TPA-induced activation of ERK and PKC, ROS generation and migration of HepG2. On the other hand, TPA-induced phosphorylation of integrin signaling components including focal adhesion kinase (FAK), Src (Tyr416) and paxillin (Tyr31 and Ser178) can be prevented by PKC inhibitor Bisindolylmaleimides (BIS) and antioxidant dithiotheritol (DTT). HepG2 overexpressing PKCα contained elevated phosphorylated paxillin. Also, ROS generator phenazine methosulfate and tert-Butyl hydroperoxide may induce phosphorylation of paxillin and activation of PKC. Taken together, ROS mediated cross talk of PKC and integrin for migration of HepG2 induced by TPA. Furthermore, TPA induced intrahepatic metastasis of HepG2 in SCID mice, which was prevented by BIS or (BIS plus DTT). Elevated phosphorylation of paxillin was observed in tumor of mice treated with TPA as compared with those co-treated with TPA/BIS. In summary, the signal pathways for tumor progression of hepatoma induced by TPA can be established both in vitro and in vivo.     

Combined Experimental and Mathematical Approach for Development of Microfabrication-Based Cancer Migration Assay

Migration of cancer cells is a key determinant of metastasis, which is correlated with poor prognosis in patients. Evidence shows that cancer cell motility is regulated by stromal cell interactions. To quantify the role of homotypic and heterotypic cell–cell interaction in migration, a two-dimensional migration assay has been developed by microfabrication techniques. Two breast cancer cell lines, MDA-MB-231 and MDA-MB-453, were used to develop micropatterns of cancer cells (cell islands) that revealed distinct migration profiles in this assay. Although the individual migration rates of these cells showed only a sevenfold difference, MDA-MB-453 islands migrated significantly lower than MDA-MB-231 islands, indicating differential regulation of migration in isolated cells vs. islands. Island size had the greatest impact on migration, primarily for MDA-MB-231 cells. Migration of MDA-MB-231 islands was decreased by interaction with homotypic cells, and significantly more by heterotypic non-cancer-associated fibroblasts. In addition, a mathematical model of island migration in multi-cellular population has been developed using Stefan–Maxwell’s equation. The model showed qualitative agreement with experimental results and predicted a biphasic relation between cell densities and island sizes. The combined experimental and mathematical model can be used to quantitatively study the impact of cell–cell interactions on migration.     

Ultrastructural study of TPA-induced cell motility: human well-differentiated rectal adenocarcinoma cells move as coherent sheets via localized modulation of cell-cell adhesion

We previously found that 12-O-tetradecanoylphorbol-13-acetate (TPA)-enhanced invasion of Matrigel was associated with augmentation of cell motility but not with metalloproteinase activity in a highly metastatic variant (L-10) of human rectal adenocarcinoma cell line RCM-1. In a two-dimensional cell motility assay, TPA induced active L-10 cell locomotion with characteristic morphology; the cells moved outwards from the cell islands mainly as a localized coherent sheet of cells. The leading cells showed locomotor morphologies with fan-shaped leading lamellae while the following cells had cell contacts on all sides and appeared to lack leading lamellae. In the present ultrastructural study, the following cells frequently showed tapering cytoplasmic protrusions and leading lamella-like processes underlapping a preceding cell, indicating that the locomotion mechanism is almost the same for both the leading and following cells. For this type of locomotion as a coherent sheet we propose that localized modulation of cell-cell adhesion was induced such that wide intercellular gaps occurred at the lower portion of the cells to allow the cells to extend the tapering cytoplasmic processes and leading lamellae while close cell-cell contacts remained at the upper portion of the cells. These TPA-induced changes took place predominantly in the cells at the periphery of the cell islands, while the cells in the middle of the cell islands maintained close cell-cell contacts including complex interdigitation all around the cells, suggesting the modulation of TPA action by cell-cell interaction. Additionally, consistent with the evidence for junctional complexes between the cells moving outwards, the Lucifer-yellow dye transfer studies showed some, limited cell-cell coupling, suggesting the presence of at least some gap junctional intercellular communication in the moving cell sheets.     

Inertial migration of cancer cells in blood flow in microchannels

The circulating tumor cell test is used to evaluate the condition of breast cancer patients by counting the number of cancer cells in peripheral blood samples. Although microfluidic systems to detect or separate cells using the inertial migration effect may be applied to this test, the hydrodynamic forces acting on cancer cells in high hematocrit blood flow are incompletely understood. In the present study, we investigated the inertial migration of cancer cells in high hematocrit blood flow in microchannels. The maximum hematocrit used in this study was about 40%. By measuring the cell migration probability, we examined the effects of cell–cell interactions, cell deformability, and variations in cell size on the inertial migration of cancer cells in blood. The results clearly illustrate that cancer cells can migrate towards equilibrium positions up to a hematocrit level of 10%. We also performed simple scaling analysis to explain the differences in migration length between rigid particles and cancer cells as well as the effect of hematocrit on cancer cell migration. These results will be important for the design of microfluidic devices for separating cells from blood.     

Fibronectin induces phosphorylation of a 120-kDa protein and synergizes with the T cell receptor to activate cytotoxic T cell clones

Fibronectin (FN) has been shown to act as a costimulator in both CD4⁺ and CD8⁺ T cell activation through the T cell receptor (TcR). Consistent with previous studies, we found that FN is able to both enhance the maximal amount of TcR-triggered degranulation and lower the threshold for activation. The density of immobilized anti-CD3 or anti-TcR required to induce degranulation and tyrosine phosphorylation of cellular proteins by several cytotoxic T lymphocyte clones is quantitatively about tenfold lower in the presence of FN. We further demonstrate that FN alone stimulates transient tyrosine phosphorylation of a 120-kDa protein (pp120) in CD8⁺ T cells and when FN is coimmobilized with substimulatory amounts of anti-CD3 or anti-TcR there is a synergistic response, resulting in prolonged and enhanced phosphorylation of pp120. To determine if FN acts as a costimulator in CD8⁺ cells solely through mediating adhesion events or if it also transduces signals in T cells we conducted remote stimulation experiments. Degranulation was induced when FN and sub-stimulatory anti-CD3 were presented on separate surfaces, indicating that FN induces independent transmembrane signals capable of augmenting TcR-induced signals resulting in a functional response. Both FN plus TcR-induced tyrosine phosphorylation of pp120 and degranulation are inhibited by RGD-containing peptides, implying that an RGD-dependent FN receptor is mediating phosphorylation of pp120 and enhancing TcR-mediated degranulation.     

Phosphorylation and disorganization of vascular-endothelial cadherin in interaction between breast cancer and vascular endothelial cells.

Adherens junctions of the endothelium play a key role in the maintenance of endothelial permeability and are composed of the vascular endothelial (VE)-cadherin/catenin adhesion complex. We report that following tumour cell (MDA MB231 cells) adherence to the HUVECs, there was a rapid (within 5 min) redistribution of VE-cadherin, resulting in its transient loss from regions of endothelial cell-cell contact. The molecule gradually reorganised within the endothelial cell contacts after this time. It was further shown that the overall expression of VE-cadherin did not change, however, the amount of alpha- and beta-catenins coprecipitated with VE-cadherin markedly decreased after 5 min of tumour cell adhesion to the HUVECs. Immunoprobing of these samples with anti-phosphotyrosine antibodies demonstrated that the tyrosine phosphorylation of VE-cadherin was significantly increased following 5 min of tumour cell adhesion. Together, these results suggest that the adhesion of tumour cells to HUVEC promotes the redistribution of VE-cadherin from interendothelial adherens junctions, an effect that may be attributed to the increase in tyrosine phosphorylation of members of the VE-cadherin/catenin adhesion complex. This, in turn, may increase vascular endothelial permeability and facilitate the transendothelial migration of tumour cells during extravasation.     

How Matrix Properties Control the Self-Assembly and Maintenance of Tissues

The mechanism by which cells organize into tissues is fundamental to developmental biology and tissue engineering. Likewise, the disruption of cellular order within tissues is a hallmark of many diseases including cancer and atherosclerosis. Tissue formation is regulated, in part, by a balance between cell–cell cohesion and cell–extracellular matrix (ECM) adhesion. Here, experiments and approaches to alter this balance are discussed, and the nature of this balance in the formation of microvasculature is explored. Using matrices of tailored stiffness and matrix presentation, the role of the mechanical properties and ligand density in angiogenesis has been investigated. Decreasing cell–matrix adhesion by either reducing matrix stiffness or matrix ligand density induces the self-assembly of endothelial cells into network-like structures. These structures are stabilized by the polymerization of the extracellular matrix protein fibronectin. When fibronectin polymerization is inhibited, network formation does not occur. Interestingly, this interplay between substrate mechanics, ECM assembly, and tissue self-assembly is not limited to endothelial cells and has been observed in other cell types as well. These results suggest novel approaches to foster stable cell–cell adhesion and engineer tissues.     

Pervanadate inhibits volume-sensitive chloride current in bovine chromaffin cells

 The role of protein tyrosine phosphorylation in the activation of the volume-sensitive Cl^– current in bovine chromaffin cells was investigated by studying the effects of inhibitors of protein tyrosine kinases (PTKs) and phosphatases (PTPs). The whole-cell current was induced by intracellular guanosine-5’-0-(3-thiotriphosphate) (GTP-[γ-S], 100–250 μM), the nonhydrolysable GTP analogue, or by cell inflation through the patch pipette under voltage-clamp conditions. PTK inhibitors tyrphostin B46 (5–50 μM) and genistein (200 μM) did not inhibit the volume-sensitive Cl^– current nor did they induce it in the absence of other stimuli. In contrast, the PTP inhibitor pervanadate (200 μM) applied intracellularly prevented activation of the current. Voltage-activated Na⁺ and Ca²⁺ currents were unaffected by pervanadate. Neither sodium orthovanadate nor hydrogen peroxide alone mimicked the action of pervanadate. Other PTP inhibitors tested, i.e. ammonium molybdate (10–100 μM), phenylarsine oxide (10 μM), and ZnCl₂ (500 μM), as well as the serine/threonine protein phosphatases inhibitor okadaic acid (200 nM) failed to inhibit the volume-sensitive Cl^– current. It is suggested that the inhibitory action of pervanadate indicates the involvement of protein tyrosine phosphorylation in the regulation of volume-sensitive Cl^– channels in bovine chromaffin cells. The possibility of pervanadate acting via a pathway unrelated to protein phosphorylation is also discussed. Received: 27 June / Received after revision: 15 September / Accepted: 16 September     

CD44 and its partners in metastasis

The establishment of metastasis requires that tumor cells acquire new adhesion and migration properties to emigrate from primary sites and colonize distant organs. CD44 is a cell membrane protein often overexpressed on tumor cells and, being both a cell–cell and cell–extracellular matrix adhesion protein, is well positioned to contribute to this process. Furthermore the interaction of CD44 with other cellular proteins involved in motogenesis and proteolysis is a determinant factor in cell migration and invasion. This review summarizes current knowledge on the role of CD44 in metastasis, as well as the challenges on understanding how this process operates. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of tumor promoter 12-O-tetradecanoyl-phorbol 13-acetate on CD 7 expression by T lineage cells

Phorbol esters exert diverse effects on cellular activation and differentiation. CD 7, a differentiation antigen appearing early in T cell ontogeny, may be involved in the activation and differentiation processes. CD 7 was found to be rapidly down-regulated by 12-O-tetradecanoylphorbol 13-acetate (TPA) from mature T cell surface. The time course of CD 7 down-regulation was similar to that of other functionally important T cell antigens, CD 3 and CD 4. Within 2 h, TPA at 10 to 30 ng/ml induced a complete down-regulation of CD 7. Twenty-four hours later, the reappearance of CD 7 on TPA-treated cells was observed. This phenomenon was monocyte independent. In contrast, CD 7 expression on thymocytes was resistant to the effect of TPA. In addition, certain leukemic T cells were also resistant to TPA-induced CD 7 down-regulation. The mechanism underlying TPA-induced CD 7 down-regulation was investigated further. Synthetic diacylglycerol, sn-1,2-dioctanoylglycerol, which activates protein kinase C, did not induce down-regulation of CD 7 on mature T cells. Ionomycin, a calcium ionophore, did not down-regulate this antigen either. Thus, it is concluded that the processes of protein kinase C activation and/or cytosolic calcium influx are not sufficient for TPA-induced CD 7 down-regulation; other pathways induced by TPA may be responsible.     

Reduced cholecystokinin receptor phosphorylation and restored signalling in protein kinase C down-regulated rat pancreatic acinar cells

 Receptor phosphorylation in response to agonist stimulation is a key regulatory principle in signal transduction. Previous work has suggested the concerted action of protein kinase C (PKC) and a staurosporine-insensitive receptor kinase in homologous phosphorylation of the cholecystokinin (CCK) receptor in freshly isolated rat pancreatic acinar cells [Gates, Ulrich, Miller (1993) Am J Physiol 264:G840–G847]. The present study shows that down-regulation of PKC by prolonged (2 h) treatment with 0.1 μM 12-O-tetradecanoylphorbol-13-acetate (TPA) markedly reduced basal CCK receptor phosphorylation as well as that induced by TPA (0.1 μM) and cholecystokinin-(26-33)-peptide amide (CCK₈, 0.1 μM). The phosphorylation level reached was the same with both stimulants and equalled basal phosphorylation in untreated control cells. The absence of any CCK₈-stimulated phosphorylation reflecting the activity of a putative staurosporine-insensitive receptor kinase raises the intriguing possibility that a basal level of PKC-mediated receptor phosphorylation is required for the action of such a receptor kinase. Immunoblot analysis revealed that the decrease in receptor phosphorylation coincided with a marked reduction of PKC-α and, to a lesser extent, PKC-ɛ. In addition, TPA-induced inhibition of the increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) evoked by the high-affinity CCK receptor agonist JMV-180 was completely reversed. The time-course of recovery closely matched that of the reduction of PKC-α. Finally, digital imaging microscopy of individual PKC down-regulated cells revealed a marked increase in the duration of JMV-180-evoked oscillatory changes in [Ca²⁺]_i. Taken together, the present findings are in agreement with the idea that PKC-α-mediated receptor phosphorylation leads to a shortening of the duration of the [Ca²⁺]_i oscillations and eventually to inhibition of high-affinity Ca²⁺ signalling through the native CCK receptor in pancreatic acinar cells. Received: 15 September 1997 / Accepted: 2 October 1997     

Adhesion of HT-29 colon carcinoma cells to endothelial cells requires sequential events involving E-selectin and integrin β4

HT-29 colon carcinoma cells attach to TNFα-activated human umbilical vein endothelial cells (HUVECs) by their specific binding to E-selectin. This interaction activates, in the cancer cells, the MAPK SAPK2/p38, which leads to their transendothelial migration (Laferrière et al., J Biol Chem 2001; 276: 33762). In this study, we investigated the role of E-selectin in activating integrins to modulate adhesion and regulate integrin-mediated events. Blocking the integrins from HT-29 cells (α2, α3, α6, αvβ5, β1 and β4) with specific antibodies revealed a role for β4 integrin in their adhesion to TNFα-treated HUVEC. The β4 integrin-dependent adhesion was maximal after 30 min, whereas the-E-selectin-dependent adhesion was maximal after 15 min. Integrin β4 became quickly phosphorylated upon addition of HT-29 cells to endothelial cells and the effect was independent of the expression of E-selectin. Moreover, a recombinant E-selectin/Fc chimera did not induce the phosphorylation of β4. The phosphorylation of β4 is not required for adhesion since adhesion was not affected in HT-29 cells that express a truncated form of β4 that is deleted from its cytoplasmic phosphorylatable domain. However, the expression of the non-phosphorylatable deletant of β4 was associated with decreased transendothelial cell migration underscoring the key role for the cytoplasmic domain of β4 in cell migration. We suggest: 1) that the adhesion of HT-29 cells to activated endothelial cells follows at least two essential sequential steps involving the binding of E-selectin to its receptor on carcinoma cells and then the binding of β4 to its own receptor on endothelial cells; 2) that the phosphorylation of integrin β4 contributes to enhance the motile potential of cancer cells and increase their trans-endothelial migration. Overall, our results indicate that the interaction of metastatic cancer cells with endothelial cells implies a specific sequence of signaling events that ultimately leads to an increase in their efficient transendothelial migration. Abbreviations: ERK – extracellular signal-regulated kinase; GFP – green fluorescent protein; ICAM – intercellular adhesion molecules: JNK – c-Jun NH₂-terminal kinase; MAPK – mitogen-activated protein kinase; MAPKAP K2, MAP kinase-activated protein kinase 2; PI3K – phosphatidyl inositol 3-kinase; SAPK – stress-activated protein kinase; TNFα– tumor necrosis factor-α. This revised version was published online in July 2006 with corrections to the Cover Date.     

Role of Integrin Receptors for Fibronectin,Collagen and Laminin in the Regulation of Ovarian Carcinoma Functions in Response to a Matrix Microenvironment

Integrins play an important role in cellular matrix interactions requisite for cancer cell adhesion, growth, migration and invasion. In this study, we have investigated the expression of integrin subunits α3, α6, αv and β1 in normal ovaries, benign ovarian tumors and ovarian carcinomas of different pathological grades. The expression of these integrins in ovarian cancer cell lines was also investigated, and their role in sustaining proliferation, adhesion, migration and invasion in cohort with the activation of signaling pathways in response to extracellular matrices (ECM) was evaluated. We demonstrate a differential expression pattern of α3, α6, αv and β1 integrin subunits in ovarian carcinomas compared to normal ovaries and benign ovarian tumors. Ovarian cancer cell lines (Hey, Ovcar3 and Peo.36) demonstrated significantly high expression of α3, α6, αv and β1 integrin subunits. A significant increase in proliferation and adhesion (P<0.05) in response to collagen 1 (Coll) and laminin (LM), ligands for integrin receptor α3β1 and α6β1 was observed in ovarian cancer cell lines. On the other hand, fibronectin (FN), a receptor for αvβ1 integrin, increased proliferation in all ovarian cancer cell lines studied but only enhanced adhesion in Hey cell line (P<0.05). Neutralizing antibodies against α3, α6, αv and β1 integrin subunits inhibited ECM-induced proliferation, but increased adhesion to ECM was inhibited by β1 integrin subunit antibody. No suppression of Coll, LM and FN-induced (Hey cells only) adhesion was observed in the presence of α3 or αv subunit antibodies but LM-induced adhesion was inhibited by blocking α6 subunit functions. LM, FN and Coll enhanced chemotactic migration in Hey cells, but direct invasion across ECM was observed only in the presence of LM and Coll. Blocking antibodies against α3, α6 and β1 integrin subunits inhibited both chemotactic migration and invasion of Hey cells in response to respective ECM. Adhesion of ovarian cancer cells to FN, Coll and LM activated Ras, Erk and Akt pathways. Neutralizing αv and β1 functions did not inhibit FN-induced activation of Ras and Erk pathways but inhibited the Akt pathway. On the other hand, antibodies against α6 and β1 subunits, but not α3 subunit, inhibited LM-induced activation of Ras but did not inhibit the downstream Akt pathway. Neutralizing β1 subunit function however, inhibited LM-induced Erk activation. Coll-induced activation of Ras, Erk and Akt pathways was inhibited by α3 and β1 integrin subunit antibodies. These results indicate that α3β1, αvβ1 and α6β1 integrin mediate proliferation, adhesion, migration and invasion of ovarian cancer cells in response to ECM and targeting these integrins to modulate integrin–ECM interactions in tumor cells may be a promising tool to reduce the dissemination of ovarian carcinoma in vivo.     

Defining the function of beta-catenin tyrosine phosphorylation in cadherin-mediated cell-cell adhesion

β-catenin is a key protein in cadherin–catenin cell adhesion complex and its tyrosine phosphorylation is believed to cause destruction of junctional apparatus. The broad spectrum of substrates for kinases and phosphatases, however, does not rule out tyrosine phosphorylation of other junctional proteins as the main culprit in reduction of cell adhesion activity. Further, the endogenous β-catenin perturbs detailed functional analysis of phosphorylated mutant β-catenin in living cells. To directly evaluate the effect of β-catenin tyrosine phosphorylation in cell adhesion, we utilized F9 cells in which expression of endogenous β-catenin and its closely related protein plakoglobin were completely shut down. We also used α-catenin-deficient (αD) cells to evaluate the role of α-catenin on β-catenin tyrosine phosphorylation. We show that β-catenin with phosphorylation mutation at 654th tyrosine forms functional cadherin–catenin complex to mediate strong cadherin-mediated cell adhesion. Moreover, we show that 64th and 86th tyrosines are mainly phosphorylated in F9 cells, especially in the absence of α-catenin. Phosphorylation of these tyrosine residues, however, does not affect cadherin-mediated cell adhesion activity. Our data identified a novel site phosphorylated by endogenous tyrosine kinases in β-catenin. We also demonstrate that tyrosine phosphorylation of β-catenin might regulate cadherin-mediated cell adhesion in a more complicated way than previously expected.     

Paracrine communication between malignant and non-malignant prostate epithelial cells in culture alters growth rate,matrix protease secretion and in vitro invasion

Epithelial cancer cell invasion is facilitated by stromal cells, immune cells, endothelial cells and other epithelial cells. We have used two human papilloma immortalized prostate cell lines, CA-HPV-10 from a carcinoma and PZ-HPV-7 cells from normal prostatic epithelium to study cell–cell influences on growth, gelatinase secretion, invasion and responses to TGFβ1. We found that co-culture with CA-10 carcinoma cells stimulates proliferation of the PZ-7 epithelial line. TGFβ1 inhibited growth of both lines, but while inhibitory effects on the CA-10 cells diminished after removal of the peptide, inhibition of PZ-7 was lasting. Interestingly, the TGFβ-induced growth inhibition in PZ-7 cells could be partially reversed by co-culture with CA-10 cells. Co-culture with CA cells in a 3-chamber invasion assay also promoted invasion of PZ cells. CA-10 invasion was enhanced by co-culture with TGFβ1-treated-PZ-7 cultures and this enhancement was associated with TGFβ1-induced secretion of matrix metalloproteinase-9. Our observations suggest that interaction between prostate cancer cells and prostate epithelial cells may promote proliferation of the epithelial cell population and produce a paracrine source of MMP-9 which may facilitate early cancer cell invasion. This revised version was published online in July 2006 with corrections to the Cover Date.