ErbB3 upregulation by the HNSCC 3D microenvironment modulates cell survival and growth.

Head and neck squamous carcinomas (HNSCC) present as dense epithelioid three-dimensional (3D) tumor nests that can mediate signals via the human epidermal growth factor receptor (ErbB) tyrosine kinase family to promote intratumoral survival and growth. We examined the role of the tumor microenvironment on ErbB receptor family expression and found that the status of intercellular organization altered the receptor profile. We showed that HNSCC cells forced into tumor island-like 3D aggregates strongly upregulated ErbB3 at the level of transcription. Not only was the elevated ErbB3 responsive to HRG-ß1-induced enhanced signaling mechanism, but also analysis by siRNA-knockdown and kinase inhibitor strategies revealed that the ErbB3/AKT signaling pathway was sufficient to enhance tumor cell survival and growth potential. Elevated ErbB3 expression in the high-density 3D culture system was strongly associated with hypoxia-induced HIF-1α. Hypoxia-regulated ErbB3 expression was mediated by the HIF-1α-binding consensus sequence in the ErbB3 proximal promoter. The findings show that the local 3D tumor microenvironment can trigger reprograming and switching of ErbB family members and thereby influence ErbB3-driven tumor growth.

Differential epidermal growth factor receptor signaling regulates anchorage-independent growth by modulation of the PI3K/AKT pathway.

Tumor cells are capable of surviving loss of nutrients and anchorage in hostile microenvironments. Under these conditions, adapting to specific signaling pathways may shift the balance between growth and cellular dormancy. Here, we report a mechanism by which epidermal growth factor receptor (EGFR) differentially modulates the phosphatidylinositol 3'-kinase (PI3K)/AKT pathway in cellular stress conditions. When carcinoma cells were cultured as multicellular aggregates (MCA), cyclin D1 was induced through a serum-dependent EGFR activating pathway, triggering cell proliferation. The expression of cyclin D1 required both EGFR-mediated ERK and AKT activation. In serum-starved MCAs, EGFR activation was associated with active ERK1/2, but not AKT, and failed to induce cyclin D1. Analysis revealed that, under serum-starved conditions, EGFR-Y1086 residue was poorly autophosphorylated and this correlated with failure to phosphorylate Gab1. Accordingly, the EGFR activation failed to induce EGFR/PI3K complex formation or AKT activation, preventing cyclin D1 induction. Furthermore, we show that in serum-starved MCA, expression of constitutively active AKT re-established cyclin D1 expression and induced proliferation in an EGFR-dependent manner. Thus, modulation of the PI3K/AKT pathway by context-dependent EGFR signaling may regulate tumor cell growth and dormancy.

Overexpression of c-met in oral SCC promotes hepatocyte growth factor-induced disruption of cadherin junctions and invasion.

Hepatocyte growth factor (HGF), the ligand for the c-met proto-oncogene product, is a multifunctional protein that enhances tumor cell motility, extracellular matrix invasion, and mitogenic or morphogenic activities of various cell types. In this study we examined the expression of the c-Met receptor in human oral squamous cell carcinoma (SCC) in vivo and in vitro to explore its relationship to tumor progression and invasiveness. Biopsy specimens of human oral SCC were immunohistochemically stained for c-Met. Nearly all primary oral SCC lesions and lymph node metastases consistently showed intense staining for c-Met, whereas normal oral mucosa showed faint to negative staining only on basal cells. In a panel of human oral SCC cell lines, we found a strong correlation between the levels of c-Met expression and the cells' response to HGF in motility and invasion assays. Sensitivity to HGF also correlated with the expression of the c-Met 9-kb mRNA. When the non-invasive HOC-605 cell line, which expresses a low level of c-Met receptor, was transfected with an expression plasmid containing human c-met cDNA, the transfectant cells showed motile and invasive responses to HGF. Immunostaining and immunoprecipitation studies demonstrated that E-cadherin and c-Met were physically associated at SCC cell-cell junctions, suggesting a direct role for c-Met in induction of junctional integrity. Importantly, HGF caused a rapid elevation of unbound beta-catenin, suggesting its availability for nuclear signal transduction and triggering of cell motility and invasiveness. Thus, overexpression of c-Met may facilitate disruption of E-cadherin junctions. Collectively, these results suggest that HGF/c-Met signaling is a common event in oral SCC that may trigger phenotype modulation and enhanced invasion and metastasis.

Modulation of cyclic-nucleotide-gated channels and regulation of vertebrate phototransduction.

Cyclic-nucleotide-gated (CNG) channels are crucial for sensory transduction in the photoreceptors (rods and cones) of the vertebrate retina. Light triggers a decrease in the cytoplasmic concentration of cyclic GMP in the outer segments of these cells, leading to closure of CNG channels and hyperpolarization of the membrane potential. Hence, CNG channels translate a chemical change in cyclic nucleotide concentration into an electrical signal that can spread through the photoreceptor cell and be transmitted to the rest of the visual system. The sensitivity of phototransduction can be altered by exposing the cells to light, through adaptation processes intrinsic to photoreceptors. Intracellular Ca(2+) is a major signal in light adaptation and, in conjunction with Ca(2+)-binding proteins, one of its targets for modulation is the CNG channel itself. However, other intracellular signals may be involved in the fine-tuning of light sensitivity in response to cues internal to organisms. Several intracellular signals are candidates for mediating changes in cyclic GMP sensitivity including transition metals, such as Ni(2+) and Zn(2+), and lipid metabolites, such as diacylglycerol. Moreover, CNG channels are associated with protein kinases and phosphatases that catalyze changes in phosphorylation state and allosterically modulate channel activity. Recent studies suggest that the effects of circadian rhythms and retinal transmitters on CNG channels may be mediated by such changes in phosphorylation. The goal of this paper is to review the molecular mechanisms underlying modulation of CNG channels and to relate these forms of modulation to the regulation of light sensitivity.

Transfection of MCF-7 carcinoma cells with human integrin alpha7 cDNA promotes adhesion to laminin.

The laminin-binding alpha7beta1 integrin receptor is highly expressed by skeletal and cardiac muscles, and has been suggested to be a crucial molecule during myogenic cell migration and differentiation. Absence of integrin alpha7 subunit contributes to a form of muscular dystrophy in integrin alpha7 null mice, whereas specific mutations in the alpha7 gene are associated in humans with congenital myopathy. To examine in more detail the potential role of integrin alpha7 in human-related muscular disorders, we cloned alpha7 cDNA by RT-PCR from human skeletal muscle mRNA and then expressed the full-length human integrin alpha7 cDNA by transfection in several cell lines including MCF-7, COS-7, and NIH3T3 cells. The isolated cDNA corresponds to the human alpha7X2B alternative splice form. Expression of human alpha7 was further confirmed by transfection of chimeric human/mouse alpha7 cDNA constructs. To demonstrate the functionality of expressed human alpha7, adhesion experiments with transfected MCF-7 cells have confirmed the specific binding of human alpha7 to laminin. In addition, mouse polyclonal and monoclonal antibodies were generated against the extracellular domain of human alpha7 and used to analyze by flow cytometry MCF-7 and NIH3T3 cells transfected with the full-length of human alpha7 cDNA. These results show for the first time the exogenous expression of functional full-length human alpha7 cDNA, as well as the development of monoclonal antibodies against the human alpha7 extracellular domain. Antibodies developed will be useful for further analysis of human disorders involving alpha7 dysfunction and facilitate isolation of muscle stem cells (satellite cells) and thereby expand the opportunities for genetically modified transplantation treatment of human disease.

Growth factors regulate phototransduction in retinal rods by modulating cyclic nucleotide-gated channels through dephosphorylation of a specific tyrosine residue.

Illumination of vertebrate rod photoreceptors leads to a decrease in the cytoplasmic cGMP concentration and closure of cyclic nucleotide-gated (CNG) channels. Except for Ca(2+), which plays a negative feedback role in adaptation, and 11-cis-retinal, supplied by the retinal pigment epithelium, all of the biochemical machinery of phototransduction is thought to be contained within rod outer segments without involvement of extrinsic regulatory molecules. Here we show that insulin-like growth factor-I (IGF-I), a paracrine factor released from the retinal pigment epithelium, alters phototransduction by rapidly increasing the cGMP sensitivity of CNG channels. The IGF-I-signaling pathway ultimately involves a protein tyrosine phosphatase that catalyzes dephosphorylation of a specific residue in the alpha-subunit of the rod CNG channel protein. IGF-I conjointly accelerates the kinetics and increases the amplitude of the light response, distinct from events that accompany adaptation. These effects of IGF-I could result from the enhancement of the cGMP sensitivity of CNG channels. Hence, in addition to long-term control of development and survival of rods, growth factors regulate phototransduction in the short term by modulating CNG channels.

Purification of mouse primary myoblasts based on alpha 7 integrin expression.

Fundamental insights have come from the study of myogenesis. Primary myoblasts isolated directly from muscle tissue more closely approximate myogenesis than established cell lines. However, contamination of primary muscle cultures with nonmyogenic cells can complicate the results. To overcome this problem, we previously described a method for myoblast purification based on novel culture conditions (T. A. Rando and H. M. Blau, 1994, J. Cell Biol. 125, 1275--1287). Here we report a refinement of this method that leads directly to an enriched population of mouse primary myoblasts, within significantly fewer population doublings. The method described here avoids using adhesion as a criterion for selection. This advance capitalizes on the ability of the antibody CA5.5 to recognize alpha 7 integrin, a muscle-specific cell surface antigen. Enrichment of myoblasts to greater than 95% of the cell population can be achieved by a single round of flow cytometry or magnetic bead separation. This is the first description of a mouse myoblast purification method based on a cell-type-specific antigen. The ease of this procedure for isolating primary myoblasts should expand the opportunities for (1) using these cells in cell transplantation studies in animal models of human disease, (2) isolating and characterizing mutant myoblasts from transgenic animals, and (3) allowing in vitro studies of molecules that regulate muscle cell growth, differentiation, and neoplasia.

Mechanism of inhibition of cyclic nucleotide-gated channel by protein tyrosine kinase probed with genistein.

Rod cyclic nucleotide-gated (CNG) channels are modulated by changes in tyrosine phosphorylation catalyzed by protein tyrosine kinases (PTKs) and phosphatases (PTPs). We used genistein, a PTK inhibitor, to probe the interaction between the channel and PTKs. Previously, we found that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel alpha-subunit (RETalpha), genistein triggers a noncatalytic inhibitory interaction between the PTK and the channel. These studies suggest that PTKs affects RETalpha channels in two ways: (1) by catalyzing phosphorylation of the channel protein, and (2) by allosterically regulating channel activation. Here, we study the mechanism of noncatalytic inhibition. We find that noncatalytic inhibition follows the same activity dependence pattern as catalytic modulation (phosphorylation): the efficacy and apparent affinity of genistein inhibition are much higher for closed than for fully activated channels. Association rates with the genistein-PTK complex were similar for closed and fully activated channels and independent of genistein concentration. Dissociation rates were 100 times slower for closed channels, which is consistent with a much higher affinity for genistein-PTK. Genistein-PTK affects channel gating, but not single channel conductance or the number of active channels. By analyzing single channel gating during genistein-PTK dissociation, we determined the maximal open probability for normal and genistein-PTK-bound channels. genistein-PTK decreases open probability by increasing the free energy required for opening, making opening dramatically less favorable. Ni(2+), which potentiates RETalpha channel gating, partially relieves genistein inhibition, possibly by disrupting the association between the genistein-PTK and the channel. Studies on chimeric channels containing portions of RETalpha, which exhibits genistein inhibition, and the rat olfactory CNG channel alpha-subunit, which does not, reveals that a domain containing S6 and flanking regions is the crucial for genistein inhibition and may constitute the genistein-PTK binding site. Thus, genistein-PTK stabilizes the closed state of the channel by interacting with portions of the channel that participate in gating.

Integrin alpha3beta1 engagement disrupts intercellular adhesion.

During tissue morphogenesis and tumor invasion, epithelial cells must undergo intercellular rearrangement in which cells are repositioned with respect to one another and the surrounding mesenchymal extracellular matrix. Using three-dimensional aggregates of squamous epithelial cells, we show that such intercellular rearrangements can be triggered by activation of beta1 integrins after their ligation with extracellular matrices. On nonadherent substrates, multicellular aggregates (MCAs) formed rapidly via E-cadherin junctional complexes and over time became compacted spheroids exhibiting a more epithelial phenotype. After MCAs were replated on culture substrates, the spheroids collapsed to yield tightly arranged cell monolayers. Cell-cell contact induced rapid elevation in E-cadherin levels, which was due to an increase in the metabolic stability of junctional receptors. During MCA remodeling of cell-cell adhesions, and monolayer formation, their E-cadherin levels fell rapidly. Similar behavior was obtained regardless of which ECM ligand-collagen type I, fibronectin, or laminin 1-MCAs were seeded on. In contrast, when seeded onto a matrix elaborated by squamous epithelial cells, cells in the MCA attached, spread, lost cell-cell junctions, and dispersed. Analysis identified laminin 5 as the active ECM ligand in this matrix, and MCA dispersion required functional beta1 integrin and specifically alpha3beta1. Furthermore, substrate-immobilized anti-integrin antibody effectively reproduced the epithelial-mesenchymal-like transition induced by the laminin 5 matrix. During the early stages of aggregate rearrangement and collapse, cells on laminin 5 substrates, but not those on collagen I substrates, exhibited intense cortical arrays of F-actin, microspikes, and fascin accumulation at their peripheral surfaces. These results suggest that engagement of specific integrin-ligand pairs regulates cadherin junctional adhesions during events common to epithelial morphogenesis and tumor invasion.

Adhesive mechanisms regulating invasion and metastasis in oral cancer.

It is the relentless invasion and growth into surrounding tissue that characterize oral squamous cell carcinoma. Metastasis is perhaps the most challenging and important aspect of cancer progression, in that it generally signifies limited survival and ineffective therapy. Inherent in metastasis is invasion, the process by which cells infiltrate into adjacent tissues, degrading basement membranes and extracellular matrix and disrupting tissue architecture and sometimes organ function. The factors that regulate these processes are complex and likely involve loss of the controls that are normally in place in physiologic tissue modeling. Adhesion receptors and their ligands are important in modulating not only invasion of oral squamous cell carcinoma cells but also their survival and proliferation. Normal oral mucosal epithelial cells use integrins to maintain their anchorage to the basement membrane, whereas the formation of stratifying cell layers depends on the formation of intercellular adhesions mediated by cadherins. The process of squamous cell carcinoma invasion and dissemination requires active cell migration through the extracellular matrix with the simultaneous remodeling of intercellular adhesions. Integrins are clearly important in the invasive process, whereas intercellular adhesion receptors restrain invasion and promote a more differentiated phenotype.

Type I collagen degradation by invasive oral squamous cell carcinoma.

Expression of extracellular matrix-degrading proteases is required for tumor cell invasion. In the present study we examined the production of type I collagen-degrading matrix metalloproteinases (MMPs) in the invasive oral squamous cell carcinoma-derived cell line HSC-3. In the absence of serum or exogenous growth factors, HSC-3 cells displayed no collagen degradation activity. Addition of serum slightly increased collagen proteolysis. However, addition of epidermal growth factor (EGF) resulted in nearly complete degradation of the collagen matrix. Zymography showed that MMP-2 and -9 are secreted by HSC-3 cells. EGF stimulated secretion of an additional gelatinase with a molecular weight similar to that of MMP-1. Immunoblotting of conditioned medium confirmed that EGF and, to a lesser degree type I collagen, increased production of MMP-1. Finally, in situ hybridization revealed intense expression of MMP-1 in oral squamous cell carcinoma specimens. Together, these results indicate that MMP-1 is expressed, induced by EGF, and required for type I collagen degradation in oral squamous cell carcinoma.

Interactions of cyclic nucleotide-gated channel subunits and protein tyrosine kinase probed with genistein.

The cGMP sensitivity of cyclic nucleotide-gated (CNG) channels can be modulated by changes in phosphorylation catalyzed by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases. Previously, we used genistein, a PTK inhibitor, to probe the interaction between PTKs and homomeric channels comprised of alpha subunits (RETalpha) of rod photoreceptor CNG channels expressed in Xenopus oocytes. We showed that in addition to inhibiting phosphorylation, genistein triggers a noncatalytic interaction between PTKs and homomeric RETalpha channels that allosterically inhibits channel gating. Here, we show that native CNG channels from rods, cones, and olfactory receptor neurons also exhibit noncatalytic inhibition induced by genistein, suggesting that in each of these sensory cells, CNG channels are part of a regulatory complex that contains PTKs. Native CNG channels are heteromers, containing beta as well as alpha subunits. To determine the contributions of alpha and beta subunits to genistein inhibition, we compared the effect of genistein on native, homomeric (RETalpha and OLFalpha), and heteromeric (RETalpha+beta, OLFalpha+beta, and OLFalpha+RETbeta) CNG channels. We found that genistein only inhibits channels that contain either the RETalpha or the OLFbeta subunits. This finding, along with other observations about the maximal effect of genistein and the Hill coefficient of genistein inhibition, suggests that the RETalpha and OLFbeta subunits contain binding sites for the PTK, whereas RETbeta and OLFalpha subunits do not.

HGF induces FAK activation and integrin-mediated adhesion in MTLn3 breast carcinoma cells.

Expression of hepatocyte growth factor (HGF) and its tyrosine kinase receptor, c-Met, is positively correlated with breast carcinoma progression. We found that in invasive and metastatic MTLn3 breast carcinoma cells, HGF stimulated both initial adhesion to and motility on the extracellular matrix (ECM) ligands laminin 1, type I collagen, and fibronectin. Next, analysis with function-perturbing antibodies showed that adhesion to the different ECM proteins was mediated through specific beta1 integrins. In MTLn3 cells, HGF induced rapid tyrosine phosphorylation and activation of both c-Met and focal adhesion kinase (FAK). Cell anchorage and adhesion to the ECM substrates was required for HGF-induced FAK activation, since HGF failed to trigger tyrosine phosphorylation of FAK in suspended cells. Our results provide evidence that the 2 signaling pathways, integrin/ECM and c-Met/HGF, cooperate synergistically to induce FAK activation in an adhesion-dependent manner, leading to enhanced cell adhesion and motility. Moreover, we found that a FRNK (the FAK-related non-kinase)-like molecule is expressed in MTLn3 cells. Since FRNK acts as a competitive inhibitor of FAK function, our results suggest that a FRNK-like protein could facilitate disassembly of focal adhesions and likely be responsible for the HGF-induced scattering and motility of MTLn3 cells.

Expression of the alpha7beta1 laminin receptor suppresses melanoma growth and metastatic potential.

The alpha7beta1 integrin is a laminin-binding receptor that was originally identified in melanoma. Here, we show that, in clonally derived mouse K1735 melanoma variant cell lines with high (M-2) and low (C-23) metastatic potential, elevated expression of alpha7 correlates with reduced cell motility, metastasis, and tumor growth. Both cell lines showed similar beta1 integrin-dependent adhesion to laminin-1 and the E8 laminin fragment. However, the highly metastatic M-2 cells rapidly migrated on laminin, whereas the nonmetastatic C-23 cells were minimally motile. Laminin-binding integrin profiles showed that the M-2 cells expressed moderate amounts of alpha1 and abundant alpha6 but low or undetectable levels of alpha2 and alpha7. By contrast, C-23 cells expressed low or undetectable levels of alpha1, alpha2, and alpha6 but had up-regulated levels of alpha7. Consistent with the protein data, Northern blot analysis showed that levels of alpha7 mRNA were highest in the poorly metastatic variant cells, whereas alpha6 message was not detected; in contrast, alpha6 mRNA was elevated in the highly metastatic cells, whereas alpha7 message was not detected. Forced expression of alpha7 in the M-2 cells suppressed cell motility, tumor growth, and metastasis. Collectively, these results indicate that, during melanoma progression, acquisition of a highly tumorigenic and metastatic melanoma phenotype is associated with loss of the alpha7beta1 laminin receptor.

Activity-dependent modulation of rod photoreceptor cyclic nucleotide-gated channels mediated by phosphorylation of a specific tyrosine residue.

Cyclic nucleotide-gated (CNG) channels are crucial for phototransduction in vertebrate rod photoreceptors. The cGMP sensitivity of these channels is modulated by diffusible intracellular messengers, including Ca2+/calmodulin, contributing to negative feedback during sensory adaptation. Membrane-associated protein tyrosine kinases and phosphatases also modulate rod CNG channels, but whether this results from direct changes in the phosphorylation state of the channel protein has been unclear. Here, we show that bovine rod CNG channel alpha-subunits (bRET) contain a tyrosine phosphorylation site crucial for modulation. bRET channels expressed in Xenopus oocytes exhibit modulation, whereas rat olfactory CNG channels (rOLF) do not. Chimeric channels reveal that differences in the C terminus, containing the cyclic nucleotide-binding domain, account for this difference. One specific tyrosine in bRET (Y498) appears to be crucial; replacement of this tyrosine in bRET curtails modulation, whereas installation into rOLF confers modulability. As the channel becomes dephosphorylated, there is an increase in the rate of spontaneous openings in the absence of ligand, indicating that changes in the phosphorylation state affect the allosteric gating equilibrium. Moreover, we find that dephosphorylation, which favors channel opening, requires open channels, whereas phosphorylation, which promotes channel closing, requires closed channels. Hence, modulation by changes in tyrosine phosphorylation is activity-dependent and may constitute a positive feedback mechanism, contrasting with negative feedback systems underlying adaptation.

Noncatalytic inhibition of cyclic nucleotide-gated channels by tyrosine kinase induced by genistein.

Rod photoreceptor cyclic nucleotide-gated (CNG) channels are modulated by tyrosine phosphorylation. Rod CNG channels expressed in Xenopus oocytes are associated with constitutively active protein tyrosine kinases (PTKs) and protein tyrosine phosphatases that decrease and increase, respectively, the apparent affinity of the channels for cGMP. Here, we examine the effects of genistein, a competitive inhibitor of the ATP binding site, on PTKs. Like other PTK inhibitors (lavendustin A and erbstatin), cytoplasmic application of genistein prevents changes in the cGMP sensitivity that are attributable to tyrosine phosphorylation of the CNG channels. However, unlike these other inhibitors, genistein also slows the activation kinetics and reduces the maximal current through CNG channels at saturating cGMP. These effects occur in the absence of ATP, indicating that they do not involve inhibition of a phosphorylation event, but rather involve an allosteric effect of genistein on CNG channel gating. This could result from direct binding of genistein to the channel; however, the time course of inhibition is surprisingly slow (>30 s), raising the possibility that genistein exerts its effects indirectly. In support of this hypothesis, we find that ligands that selectively bind to PTKs without directly binding to the CNG channel can nonetheless decrease the effect of genistein. Thus, ATP and a nonhydrolyzable ATP derivative competitively inhibit the effect of genistein on the channel. Moreover, erbstatin, an inhibitor of PTKs, can noncompetitively inhibit the effect of genistein. Taken together, these results suggest that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel catalyzed by PTKs, genistein triggers a noncatalytic interaction between the PTK and the channel that allosterically inhibits gating.

Real-time patch-cram detection of intracellular cGMP reveals long-term suppression of responses to NO and muscarinic agonists.

Cyclic GMP (cGMP) is a crucial intracellular messenger in neuronal, muscle, and endocrine cells. The intracellular concentration of cGMP is regulated by various neurotransmitters, including acetylcholine (ACh) and nitric oxide (NO). While much is known about the biochemical steps leading to cGMP synthesis, little is known about cGMP kinetics in intact cells. Here, we use "patch-cramming," in which an excised, inside-out membrane patch containing cyclic nucleotide-gated ion channels is used as a biosensor, to obtain the first real-time measurements of cGMP in intact cells. Patch-cramming experiments on neuroblastoma cells show that both muscarinic agonists and NO rapidly elevate cGMP. NO elicits cGMP responses repeatedly without decrement, whereas responses to muscarinic agonists exhibit a profound and prolonged desensitization. Remarkably, muscarinic agonists also cause long-term (>30 min) suppression (LTS) of cGMP responses elicited by NO. Biochemical measurements reveal that rat sympathetic neurons also exhibit LTS of cGMP, suggesting that LTS is a widespread mechanism that may contribute to synaptic plasticity.

Spanning binding sites on allosteric proteins with polymer-linked ligand dimers.

One approach to drug design involves determination of the structure of binding sites on target proteins to provide templates for ligand construction. Alternatively, random combinations of chemical groups can be used to generate diverse molecules for screening in the search for effective compounds. Here we report a strategy for developing potent ligands for proteins with multiple binding sites, which combines elements of both approaches: 'polymer-linked ligand dimers', in which two ligands are joined by a polymer chain of variable length. We find that polymer-linked ligand dimers containing two cyclic GMP moieties are up to a thousand times more potent than cyclic GMP in activating cyclic-nucleotide-gated channels and cGMP-dependent protein kinase. Each target protein responds optimally to a polymer-linked ligand dimer with a different average polymer length, even though their cyclic-nucleotide-binding sites are conserved. The tuning of polymer-linked ligand dimers indicates that each protein has a unique spacing of binding sites and provides an estimate of the distance between these sites. As optimal ligands are selected empirically, the polymer-linked ligand dimer strategy enables potent and selective agents to be identified without requiring previous structural information about the target proteins.

E-cadherin regulates anchorage-independent growth and survival in oral squamous cell carcinoma cells.

Integrin-basement membrane interactions provide essential signals that promote survival and growth of epithelial cells, whereas loss of such adhesions triggers programmed cell death. We found that HSC-3 human squamous carcinoma cells survived and grew readily as monolayers, but when they were suspended as single cells, they ceased proliferating and entered into the apoptotic death pathway, characterized by DNA fragmentation. In contrast, if the suspended carcinoma cells were permitted to form E-cadherin-mediated multicellular aggregates, they not only survived but proliferated. However, aggregated normal keratinocytes were unable to survive in suspension culture and rapidly became apoptotic. Anchorage independence and resistance to apoptosis of HSC-3 cell aggregates required high levels of extracellular Ca2+ and was inhibited with function-perturbing anti-E-cadherin antibody. Resistance to suspension-induced apoptosis in cell aggregates paralleled the up-regulation of Bcl-2 but occurred in the absence of focal adhesion kinase activation. Analysis of suspension-induced death in a set of cloned squamous epithelial cell lines with different levels of E-cadherin expression revealed that receptor-positive cell clones evaded apoptosis and proliferated in three-dimensional aggregate culture, whereas cadherin-negative clones failed to survive. Collectively, these observations indicate that cadherin-mediated intercellular adhesions generate a compensatory mechanism that promotes anchorage-independent growth and suppresses apoptosis.

Regulation of cGMP-dependent current in On bipolar cells by calcium/calmodulin-dependent kinase.

The metabotropic receptor mGluR6 is localized to the dendrites of On bipolar cells and mediates synaptic input from photoreceptors. The binding of glutamate to the receptor activates a phosphodiesterase (PDE), which then hydrolyzes cGMP. A nonselective cationic conductance, believed to be gated directly by cGMP, is turned off as a result of the fall in cGMP levels, and the cell hyperpolarizes. Here we present evidence for regulation of the conductance by an additional mechanism that it is independent of cGMP. Whole-cell recordings were obtained from On bipolar cells in slices of tiger salamander retina. Dialysis of cells with 1 microM KN-62 or 10 microM KN-93, two inhibitors of type II calmodulin-dependent protein kinase (CaMKII), depressed cGMP-dependent currents. This depression persisted when hydrolysis of cGMP was prevented with IBMX, a broad-spectrum PDE inhibitor, suggesting that CaMKII acts downstream from the PDE in the cascade. The depression of cGMP-dependent currents was probably not due to a direct interaction of the inhibitors with the channels as neither 1 microM KN-62 or 10 microM KN-93 was found to have any effect on cyclic nucleotide-gated channels when applied directly to excised patches of rod outer segments. We propose that phosphorylation by CaMKII may be an important mechanism for regulating the cGMP-dependent conductance of On bipolar cells.