Modulation of Akt vs Stat3 activity by the focal adhesion kinase in non-neoplastic mouse fibroblasts.

Adhesion of cells to each other and to the extracellular matrix (ECM) are both required for cellular functions. Cell-to-cell adhesion is mediated by cadherins, and their engagement triggers the activation of Stat3, which offers a potent survival signal. Adhesion to the ECM on the other hand, activates FAK which attracts and activates Src, as well as receptor tyrosine kinases (RTKs), the PI3k/Akt and Ras/Erk pathways. However, the effect of cell density upon FAK and Akt activity has not been examined. We now demonstrate that, interestingly, despite being potent Stat3 activators, Src and RTKs are unable to activate Stat3 in sparsely growing (i.e., without cadherin engagement), non-neoplastic cells attached to the ECM. In contrast, cell aggregation (i.e., cadherin engagement in the absence of adhesion to a solid substratum) was found to activate both Stat3 and Akt. Pharmacologic or genetic reduction of FAK activity abolished Akt activity at low densities, indicating that FAK is an important activator of Akt in this setting. Notably, FAK knockout increased cellular sensitivity to the Stat3 inhibitor CPA7, while FAK reintroduction restored resistance to this drug. These findings suggest a complementary role of integrin/FAK/Akt and cadherin/Stat3-mediated pro-survival pathways, which may be of significance during neoplastic transformation and metastasis.

Modulation of Akt vs Stat3 activity by the focal adhesion kinase in non-neoplastic mouse fibroblasts.

Adhesion of cells to each other and to the extracellular matrix (ECM) are both required for cellular functions. Cell-to-cell adhesion is mediated by cadherins and their engagement triggers the activation of Stat3, which offers a potent survival signal. Adhesion to the ECM on the other hand, activates FAK which attracts and activates Src, as well as receptor tyrosine kinases (RTKs), the PI3k/Akt and Ras/Erk pathways. However, the effect of cell density upon FAK and Akt activity has not been examined. We now demonstrate that, interestingly, despite being potent Stat3 activators, Src and RTKs are unable to activate Stat3 in sparsely growing (i.e., without cadherin engagement), non-neoplastic cells attached to the ECM. In contrast, cell aggregation (i.e., cadherin engagement in the absence of adhesion to a solid substratum) was found to activate both Stat3 and Akt. Pharmacologic or genetic reduction of FAK activity abolished Akt activity at low densities, indicating that FAK is an important activator of Akt in this setting. Notably, FAK knockout increased cellular sensitivity to the Stat3 inhibitor CPA7, while FAK reintroduction restored resistance to this drug. These findings suggest a complementary role of integrin/FAK/Akt and cadherin/Stat3-mediated pro-survival pathways, which may be of significance during neoplastic transformation and metastasis.

Effect of caveolin-1 on Stat3-ptyr705 levels in breast and lung carcinoma cells.

We recently demonstrated that Cav1 (caveolin-1) is a negative regulator of Stat3 (signal transducer and activator of transcription-3) activity in mouse fibroblasts and human lung carcinoma SHP77 cells. We now examined whether the cellular context may affect their levels as well as the relationship between them, by assessing Cav1 and Stat3-ptyr705 amounts in different cell lines. In MDA-MB-231, A549, and HaCat cells, Cav1 levels were high and Stat3-ptyr705 levels were low, consistent with the notion of a negative effect of endogenous Cav1 on Stat3-ptyr705 levels in these lines. In addition, manipulation of Cav1 levels revealed a negative effect in MCF7 and mouse fibroblast cells, while Cav1 upregulation induced apoptosis in MCF7 cells. In contrast, however, line MRC9 had high Cav1 and high Stat3-ptyr705 levels, indicating that high Cav1 is insufficient to reduce Stat3-ptyr705 levels in this line. MCF7 and LuCi6 cells had very low Cav1 and Stat3-ptyr705 levels, indicating that the low Stat3-ptyr705 can be independent from Cav1 levels altogether. Our results reveal a further level of complexity in the relationship between Cav1 and Stat3-ptyr705 than previously thought. In addition, we demonstrate that in a feedback loop, Stat3 inhibition upregulates Cav1 in HeLa cells but not in other lines tested.

Regulation of HC11 mouse breast epithelial cell differentiation by the E-cadherin/Rac axis.

It was previously demonstrated that differentiation of some established breast epithelial cell lines requires confluence and stimulation with hydrocortisone, insulin and prolactin inducers. We and others previously demonstrated that E-cadherin engagement, which is favored under conditions of confluence, increases the levels and activity of the Rac small GTPase. To investigate the functional relationship between the transforming ability of Rac and its role as an integral component of the differentiative E-cadherin signaling pathway, we introduced a mutationally activated form of Rac, RacV12, into the mouse breast epithelium-derived cell line, HC11. Our results demonstrate that the strength of the Rac signal is key for the outcome of the differentiation process; cRac1 is critically required for differentiation, and at low levels, mutationally activated RacV12 is able to increase differentiation, presumably reinforcing the E-cadherin/Rac differentiative signal. However, high RacV12 expression blocked differentiation concomitant with E-cadherin downregulation, while inducing neoplastic transformation. Therefore, the intensity of the Rac signal is a central determinant in the balance between cell proliferation vs differentiation, two fundamentally opposed processes, a finding which could also have important therapeutic implications.

STAT5A is regulated by DNA damage via the tumor suppressor p53.

Here we report that the STAT5A transcription factor is a direct p53 transcriptional target gene. STAT5A is well expressed in p53 wild type cells but not in p53-null cells. Inhibition of p53 reduces STAT5A expression. DNA damaging agents such as doxorubicin also induced STAT5A expression in a p53 dependent manner. Two p53 binding sites were mapped in the STAT5A gene and named PBS1 and PBS2; these sites were sufficient to confer p53 responsiveness in a luciferase reporter gene. Chromatin immunoprecipitation experiments revealed that PBS2 has constitutive p53 bound to it, while p53 binding to PBS1 required DNA damage. In normal human breast lobules, weak p53 staining correlated with regions of intense STAT5A staining. Interestingly, in a cohort of triple negative breast tumor tissues there was little correlation between regions of p53 and STAT5A staining, likely reflecting a high frequency of p53 mutations that stabilize the protein in these tumors. We thus reveal an unexpected connection between cytokine signaling and p53.

Differential effects of polyoma virus middle tumor antigen mutants upon gap junctional, intercellular communication.

Gap junctions are channels that connect the cytoplasm of adjacent cells. Oncogenes such as the middle Tumor antigen of polyoma virus (mT) are known to suppress gap junctional, intercellular communication (GJIC). mT associates with and is tyrosine-phosphorylated by cSrc family members. Specific mT phosphotyrosines provide docking sites for the phosphotyrosine binding domain of Shc (mT-tyr250) or the SH2 domain of the regulatory subunit of the phosphatidylinositol-3 kinase (PI3k, mT-tyr315). Binding results in the activation of their downstream signaling cascades, Ras/Raf/Erk and PI3 kinase/Akt, respectively, both of which are needed for full neoplastic transformation. To examine the effect of mT-initiated pathways upon gap junctional communication, GJIC was quantitated in rat liver epithelial T51B cells expressing mT-mutants, using a novel technique of in situ electroporation. The results demonstrate for the first time that, although even low levels of wild-type mT are sufficient to interrupt gap junctional communication, GJIC suppression still requires an intact tyr-250 site, that is activation of the Ras pathway. In sharp contrast, activation of the PI3k pathway is not required for GJIC suppression, indicating that GJIC suppression is independent of full neoplastic conversion and the concomitant morphological changes. Interestingly, expression of a constitutively active, myristylated form of the catalytic subunit of PI3k, p110, or the constitutively active mutants E545K and H1047R increased GJIC, while pharmacological inhibition of PI3k eliminated communication. Therefore, although PI3k is growth promoting and in an activated form it can act as an oncogene, it actually plays a positive role upon gap junctional, intercellular communication.

Preclinical characterization of signal transducer and activator of transcription 3 small molecule inhibitors for primary and metastatic brain cancer therapy.

Signal transducer and activator of transcription 3 (STAT3) has been implicated as a hub for multiple oncogenic pathways. The constitutive activation of STAT3 is present in several cancers, including gliomas (GBMs), and is associated with poor therapeutic responses. Phosphorylation of STAT3 triggers its dimerization and nuclear transport, where it promotes the transcription of genes that stimulate tumor growth. In light of this role, inhibitors of the STAT3 pathway are attractive therapeutic targets for cancer. To this end, we evaluated the STAT3-inhibitory activities of three compounds (CPA-7 [trichloronitritodiammineplatinum(IV)], WP1066 [(S,E)-3-(6-bromopyridin-2-yl)-2-cyano-N-(1-phenylethyl)acrylamide, C17H14BrN3O], and ML116 [4-benzyl-1-{thieno[2,3-d]pyrimidin-4-yl}piperidine, C18H19N3S]) in cultured rodent and human glioma cells, including GBM cancer stem cells. Our results demonstrate a potent induction of growth arrest in GBM cells after drug treatment with a concomitant induction of cell death. Although these compounds were effective at inhibiting STAT3 phosphorylation, they also displayed variable dose-dependent inhibition of STAT1, STAT5, and nuclear factor κ light-chain enhancer of activated B cells. The therapeutic efficacy of these compounds was further evaluated in peripheral and intracranial mouse tumor models. Whereas CPA-7 elicited regression of peripheral tumors, both melanoma and GBM, its efficacy was not evident when the tumors were implanted within the brain. Our data suggest poor permeability of this compound to tumors located within the central nervous system. WP1066 and ML116 exhibited poor in vivo efficacy. In summary, CPA-7 constitutes a powerful anticancer agent in models of peripheral solid cancers. Our data strongly support further development of CPA-7-derived compounds with increased permeability to enhance their efficacy in primary and metastatic brain tumors.

Stat3 is a positive regulator of gap junctional intercellular communication in cultured, human lung carcinoma cells.

BACKGROUND: Neoplastic transformation of cultured cells by a number of oncogenes such as src suppresses gap junctional, intercellular communication (GJIC); however, the role of Src and its effector Signal transducer and activator of transcription-3 (Stat3) upon GJIC in non small cell lung cancer (NSCLC) has not been defined. Immunohistochemical analysis revealed high Src activity in NSCLC biopsy samples compared to normal tissues. Here we explored the potential effect of Src and Stat3 upon GJIC, by assessing the levels of tyr418-phosphorylated Src and tyr705-phosphorylated Stat3, respectively, in a panel of NSCLC cell lines. METHODS: Gap junctional communication was examined by electroporating the fluorescent dye Lucifer yellow into cells grown on a transparent electrode, followed by observation of the migration of the dye to the adjacent, non-electroporated cells under fluorescence illumination. RESULTS: An inverse relationship between Src activity levels and GJIC was noted; in five lines with high Src activity GJIC was absent, while two lines with extensive GJIC (QU-DB and SK-LuCi6) had low Src levels, similar to a non-transformed, immortalised lung epithelial cell line. Interestingly, examination of the mechanism indicated that Stat3 inhibition in any of the NSCLC lines expressing high endogenous Src activity levels, or in cells where Src was exogenously transduced, did not restore GJIC. On the contrary, Stat3 downregulation in immortalised lung epithelial cells or in the NSCLC lines displaying extensive GJIC actually suppressed junctional permeability. CONCLUSIONS: Our findings demonstrate that although Stat3 is generally growth promoting and in an activated form it can act as an oncogene, it is actually required for gap junctional communication both in nontransformed lung epithelial cells and in certain lung cancer lines that retain extensive GJIC.

The R(h)oads to Stat3: Stat3 activation by the Rho GTPases.

The signal transducer and activator of transcription-3 (Stat3) is a member of the STAT family of cytoplasmic transcription factors. Overactivation of Stat3 is detected with high frequency in human cancer and is considered a molecular abnormality that supports the tumor phenotype. Despite concerted investigative efforts, the molecular mechanisms leading to the aberrant Stat3 activation and Stat3-mediated transformation and tumorigenesis are still not clearly defined. Recent evidence reveals a crosstalk close relationship between Stat3 signaling and members of the Rho family of small GTPases, including Rac1, Cdc42 and RhoA. Specifically, Rac1, acting in a complex with the MgcRacGAP (male germ cell RacGAP), promotes tyrosine phosphorylation of Stat3 by the IL6-receptor family/Jak kinase complex, as well as its translocation to the nucleus. Studies have further revealed that the mutational activation of Rac1 and Cdc42 results in Stat3 activation, which occurs in part through the upregulation of IL6 family cytokines that in turn stimulates Stat3 through the Jak kinases. Interestingly, evidence also shows that the engagement of cadherins, cell to cell adhesion molecules, specifically induces a striking increase in Rac1 and Cdc42 protein levels and activity, which in turn results in Stat3 activation. In this review we integrate recent findings clarifying the role of the Rho family GTPases in Stat3 activation in the context of malignant progression.

Roads to Stat3 Paved with Cadherins.

The engagement of cadherins, cell-to-cell adhesion proteins, triggers a dramatic increase in the levels and activity of the Rac/Cdc42 GTPases, through the inhibition of proteasomal degradation. This leads to an increase in transcription and secretion of IL6 family cytokines, activation of their common receptor, gp130, in an autocrine manner and phosphorylation of the signal transducer and activator of transcription-3 (Stat3) on tyrosine-705 by the Jak kinases. Stat3 subsequently dimerizes, migrates to the nucleus and activates the transcription of genes involved in cell division and survival. The Src oncogene also increases Rac levels, leading to secretion of IL6 family cytokines and gp130 activation, which triggers a Stat3-ptyr705 increase. Interestingly, at the same time, Src downregulates cadherins in a quantitative manner, while cadherins are required to preserve gp130 levels for IL6 family signalling. Therefore, a fine balance between Src527F/Rac/IL6 and Src527F/cadherin/gp130 levels is in existence, which is required for Stat3 activation. This further demonstrates the important role of cadherins in the activation of Stat3, through preservation of gp130 function. Conversely, the absence of cadherin engagement correlates with low Stat3 activity: In sparsely growing cells, both gp130 and Stat3-ptyr705 levels are very low, despite the fact that cSrc is active in the FAK (focal adhesion kinase)/cSrc complex, which further indicates that the engagement of cadherins is important for Stat3 activation, not just their presence. Furthermore, the caveolin-1 protein downregulates Stat3 through binding and sequestration of cadherins to the scaffolding domain of caveolin-1. We hypothesize that the cadherins/Rac/gp130 axis may be a conserved pathway to Stat3 activation in a number of systems. This fact could have significant implications in Stat3 biology, as well as in drug testing and development.

Activated Src requires Cadherin-11, Rac, and gp130 for Stat3 activation and survival of mouse Balb/c3T3 fibroblasts.

We previously demonstrated that engagement of cadherins, cell to cell adhesion molecules, triggers a dramatic increase in levels and activity of the Rac/Cdc42 small GTPases, which is followed by secretion of IL6 family cytokines and activation of their common receptor, gp130, in an autocrine manner. This results in phosphorylation of the Signal Transducer and Activator of Transcription-3 (Stat3) on tyrosine-705, which then dimerizes, migrates to the nucleus, and activates transcription of genes involved in cell division and survival. In the present report we demonstrate that, in mouse Balb/c3T3 fibroblasts, mutationally activated Src527F also increases Rac levels, leading to secretion of IL6 family cytokines and gp130 activation, which triggers the Stat3-ptyr705 increase. Interestingly, our results also demonstrate that cadherin-11 is required to preserve gp130 levels for IL6 family signaling. At the same time, however, activated Src527F downregulates cadherin-11, in a quantitative manner. As a result, Src527F expression to intermediate levels allows sufficient cadherin-11, hence gp130 levels for Stat3 activation, as expected. However, expressed to high levels, Src527F eliminates cadherin-11, hence gp130 signaling, thus abolishing Stat3-ptyr705 stimulation. Taken together, these data establish for the first time a loop between Src, cadherin-11, gp130, and Stat3 activation. This fine balance between Src527F and cadherin-11 levels which is required for Stat3 activation and cellular survival could have significant therapeutic implications.

Activated Rac1 requires gp130 for Stat3 activation, cell proliferation and migration.

Rac1 (Rac) is a member of the Rho family of small GTPases which controls cell migration by regulating the organization of actin filaments. Previous results suggested that mutationally activated forms of the Rho GTPases can activate the Signal Transducer and Activator of Transcription-3 (Stat3), but the exact mechanism is a matter of controversy. We recently demonstrated that Stat3 activity of cultured cells increases dramatically following E-cadherin engagement. To better understand this pathway, we now compared Stat3 activity levels in mouse HC11 cells before and after expression of the mutationally activated Rac1 (Rac(V12)), at different cell densities. The results revealed for the first time a dramatic increase in protein levels and activity of both the endogenous Rac and Rac(V12) with cell density, which was due to inhibition of proteasomal degradation. In addition, Rac(V12)-expressing cells had higher Stat3, tyrosine-705 phosphorylation and activity levels at all densities, indicating that Rac(V12) is able to activate Stat3. Further examination of the mechanism of Stat3 activation showed that Rac(V12) expression caused a surge in mRNA of Interleukin-6 (IL6) family cytokines, known potent Stat3 activators. Knockdown of gp130, the common subunit of this family reduced Stat3 activity, indicating that these cytokines may be responsible for the Stat3 activation by Rac(V12). The upregulation of IL6 family cytokines was required for cell migration and proliferation induced by Rac(V12), as shown by gp130 knockdown experiments, thus demonstrating that the gp130/Stat3 axis represents an essential effector of activated Rac for the regulation of key cellular functions.

Differentiation of Mouse Breast Epithelial HC11 and EpH4 Cells.

Cadherins play an important role in the regulation of cell differentiation as well as neoplasia. Here we describe the origins and methods of the induction of differentiation of two mouse breast epithelial cell lines, HC11 and EpH4, and their use to study complementary stages of mammary gland development and neoplastic transformation. The HC11 mouse breast epithelial cell line originated from the mammary gland of a pregnant Balb/c mouse. It differentiates when grown to confluence attached to a plastic Petri dish surface in medium containing fetal calf serum and Hydrocortisone, Insulin and Prolactin (HIP medium). Under these conditions, HC11 cells produce the milk proteins ß-casein and whey acidic protein (WAP), similar to lactating mammary epithelial cells, and form rudimentary mammary gland-like structures termed "domes". The EpH4 cell line was derived from spontaneously immortalized mouse mammary gland epithelial cells isolated from a pregnant Balb/c mouse. Unlike HC11, EpH4 cells can fully differentiate into spheroids (also called mammospheres) when cultured under three-dimensional (3D) growth conditions in HIP medium. Cells are trypsinized, suspended in a 20% matrix consisting of a mixture of extracellular matrix proteins produced by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells, plated on top of a layer of concentrated matrix coating a plastic Petri dish or multiwell plate, and covered with a layer of 10% matrix-containing HIP medium. Under these conditions, EpH4 cells form hollow spheroids that exhibit apical-basal polarity, a hollow lumen, and produce ß-casein and WAP. Using these techniques, our results demonstrated that the intensity of the cadherin/Rac signal is critical for the differentiation of HC11 cells. While Rac1 is necessary for differentiation and low levels of activated RacV12 increase differentiation, high RacV12 levels block differentiation while inducing neoplasia. In contrast, EpH4 cells represent an earlier stage in mammary epithelial differentiation, which is inhibited by even low levels of RacV12.

Beyond structure, to survival: activation of Stat3 by cadherin engagement.

Cells in normal tissues or in tumors have extensive opportunities for adhesion to their neighbors and the importance of cell to cell contact in the study of fundamental cellular processes is beginning to emerge. In this review, we discuss recent evidence of dramatic changes in the activity of an important signal transducer found to be profoundly affected by cell to cell adhesion, the signal transducer and activator of transcription-3 (Stat3). Direct cadherin engagement, growth of cells to postconfluence, or formation of multicellular aggregates were found to induce a striking increase in the levels of Stat3 activity, Rac1/Cdc42, and members of the IL6 receptor family in different settings. This activation was specific to Stat3, in that the levels of the extracellular signal regulated kinase (Erk1/2), a signal transducer often coordinately activated with Stat3 by a number of growth factors or oncogenes, remained unaffected by cell density. Density-dependent Stat3 activation may play a key role in survival, and could contribute to the establishment of cell polarity. It is clear that at any given time the total Stat3 activity levels in a cell are the sum of the effects of cell to cell adhesion plus the conventional Stat3 activating factors present.

PI3k and Stat3: Oncogenes that are Required for Gap Junctional, Intercellular Communication.

Gap junctional, intercellular communication (GJIC) is interrupted in cells transformed by oncogenes such as activated Src. The Src effector, Ras, is required for this effect, so that Ras inhibition restores GJIC in Src-transformed cells. Interestingly, the inhibition of the Src effector phosphatidyl-inositol-3 kinase (PI3k) or Signal Transducer and Activator of Transcription-3 (Stat3) pathways does not restore GJIC. In the contrary, inhibition of PI3k or Stat3 in non-transformed rodent fibroblasts or epithelial cells or certain human lung carcinoma lines with extensive GJIC inhibits communication, while mutational activation of PI3k or Stat3 increases GJIC. Therefore, it appears that oncogenes such as activated Src have a dual role upon GJIC; acting as inhibitors of communication through the Ras pathway, and as activators through activation of PI3k or Stat3. In the presence of high Src activity the inhibitory functions prevail so that the net effect is gap junction closure. PI3k and Stat3 constitute potent survival signals, so that their inhibition in non-transformed cells triggers apoptosis which, in turn, has been independently demonstrated to suppress GJIC. The interruption of gap junctional communication would confine the apoptotic event to single cells and this might be essential for the maintenance of tissue integrity. We hypothesize that the GJIC activation by PI3k or Stat3 may be linked to their survival function.

Regulation of Differentiation of HC11 Mouse Breast Epithelial Cells by the Signal Transducer and Activator of Transcription-3.

BACKGROUND/AIM: The differentiation of the mouse breast epithelial cell line HC11 is known to require confluence as well as the addition of hydrocortisone, insulin and prolactin. MATERIALS AND METHODS: Since confluence, which triggers the engagement of the cell-to-cell adhesion molecule E-cadherin, induces a dramatic increase in the activity of signal transducer and activator of transcription-3 (Stat3), we examined the role of Stat3 in HC11 cell differentiation. RESULTS: Stat3 inhibition abolished differentiation, indicating that Stat3 activity is critically required. However, expression of the mutationally activated form of Stat3 (Stat3C), rather than promoting, it was found to block cell differentiation, even when expressed in low levels, and in the absence of full neoplastic conversion. CONCLUSION: The strength of the E-cadherin/Stat3 signal is key for the outcome of the differentiation process.

Electrode assemblies used for electroporation of cultured cells.

Electroporation was initially developed for the introduction of DNA into cells which grow in suspension and was performed in a cuvette with two flat electrodes on opposite sides. Different configurations were subsequently developed for the electroporation of adherent cells in situ, while the cells were growing on nonconductive surfaces or a gold-coated, conductive support. We developed an assembly where the cells grow and are electroporated on optically transparent, electrically conductive indium-tin oxide (ITO). This material promotes excellent cell adhesion and growth, is inert and durable, and does not display spontaneous fluorescence, making the examination of the electroporated cells by fluorescence microscopy possible. The molecules to be electroporated are added to the cells and introduced through an electrical pulse delivered by an electrode placed on top of the cells. We describe several electrode and slide configurations which allow the electroporation of large numbers of cells for large-scale biochemical experiments or for the detection of changes in cell morphology and biochemical properties in situ, with control, nonelectroporated cells growing on the same type of ITO-coated surface, side by side with the electroporated ones. In a modified version, this technique can be adapted for the study of intercellular, junctional communication; the pulse is applied in the presence of a fluorescent dye, such as lucifer yellow, causing its penetration into the cells growing on the conductive half of the slide, and the migration of the dye to the nonelectroporated cells growing on the nonconductive area is microscopically observed under fluorescence illumination. An assembly is also described for the electroporation of sensitive cells without the use of an upper electrode.

Electroporation of adherent cells in situ for the study of signal transduction and gap junctional communication.

Cultured adherent cells can be electroporated in situ, as they grow on a glass slide coated with electrically conductive, optically transparent indium-tin oxide (ITO). Although the introduction of DNA is a common use, the technique of electroporation in situ is valuable for studying many aspects of signal transduction. This is because, under the appropriate conditions, in situ electroporation can be remarkably nontraumatic, while a large variety of molecules, such as peptides, oligonucleotides, or drugs, are introduced instantly and into essentially 100% of the cells, making this technique especially suitable for kinetic studies of effector activation. Following the introduction of the material, the cells can be either extracted or biochemically analyzed, or their morphology and gene expression can be examined by immunocytochemistry. In this chapter, we describe the introduction of a peptide blocking the Src-homology 2 domain of the adaptor Grb2 to inhibit the activation of the downstream effector Erk1/2 by EGF. The setup includes nonelectroporated, control cells growing side by side with the electroporated ones on the same type of ITO-coated surface. In a modified version, this assembly can be used very effectively for studying intercellular, junctional communication: cells are grown on a glass slide half of which is ITO-coated. An electric pulse is applied in the presence of the fluorescent dye lucifer yellow, causing its penetration into the cells growing on the conductive part of the slide, and the migration of the dye to the nonelectroporated cells growing on the nonconductive area is microscopically observed under fluorescence illumination.

Stat3 is required for full neoplastic transformation by the Simian Virus 40 large tumor antigen.

To investigate the role of Stat3 (signal transducer and activator of transcription-3) in neoplastic transformation by the Large Tumor antigen of Simian Virus 40 (TAg), murine fibroblasts were rendered deficient in Stat3 activity through expression of a Stat3-specific siRNA or a Cre-loxP recombination system. The results demonstrate that growth rate, formation of foci overgrowing a monolayer of normal cells and colony formation in soft agar were dramatically reduced in Stat3-deficient cells. In addition, TAg expression led to increased Stat3 tyrosine phosphorylation, DNA binding, and transcriptional activity, suggesting that Stat3 is required for TAg-mediated neoplasia. Stat3 activation was prevented by blocking the binding of TAg to pRb (retinoblastoma-susceptibility gene product), whereas genetic ablation of pRb increased Stat3 activity, suggesting that pRb inactivation by TAg might be responsible for the observed Stat3 activation. Stat3 activation by TAg was suppressed after inhibition of c-Src, JAKs or the insulin-like growth factor receptor. On the other hand, targeted disruption of the Fer kinase or pharmacological inhibition of Abl had no effect. Inhibition of Src activity led to Stat3 down-regulation as well as apoptosis of sparsely growing, TAg-transformed cells. However, Src inhibition was relatively ineffective in confluent cells, consistent with previous results indicating that cell to cell adhesion activates Stat3 by a Src-independent mechanism. Direct Stat3 inhibition on the other hand induced apoptosis very effectively in confluent cells, which could have significant therapeutic implications. Taken together, our results suggest that Stat3 is an important component of a pathway emanating from TAg and leading to neoplastic conversion.