Cellular Signal Transduction Mechanisms Research Articles

Chemopreventive actions of polyphenolic compounds in cancer

Oxidative stress and associated mechanisms involving inflammation, aberrant signaling pathways and gap junction intercellular communication is increasingly associated with the pathogenesis of various chronic degenerative disorders such as atherosclerosis, neurodegeneration and cancer. Consumption of fruits, vegetables and beverages like teas continues to be suggested to have the capacity to reduce the incidence of cancer. The bioactive compounds including phenolics may be responsible for the chemopreventive effects. While the free radical scavenging and antioxidant properties of phenolics are well established, emerging literature reports suggest that their chemopreventive effects may also be ascribed to their ability to modulate components of cell signaling pathways. This paper reviews the potential chemoprevention role of phenolics with a focus on cellular signal transduction mechanisms and prevention of gap junction intercellular communication relevant to cancer.

Mechanisms of Cellular Signal Transduction

Mechanisms of Cellular Signal Transduction

Zinc Homeostasis in C6 Glioma Cells: Phospholipase C Activity Regulates Cellular Zinc Export

Zinc homeostasis in mammalian cells is precisely regulated by cellular signal transduction mechanisms. The main result of this study is the finding that modulators of phospholipase C (PLC) activity affect cellular zinc export. Two different PLC inhibitors caused an increase of the total cellular zinc level whereas two different PLC activators caused a decrease. Furthermore, both the inhibition of cyclic nucleotide phosphodiesterases as well as the administration of 8-bromo-cAMP evoked a drop in the intracellular zinc level, indicating the involvement of cAMP in the control of cellular zinc export. It is concluded that the activity of PLC controls cellular zinc transport and that the effect of elevated zinc concentrations on PLC activity might be mediated by cAMP. However, modulation of other major signaling enzymes did not affect the cellular zinc homeostasis. These include activation and inhibition of guanylate cyclase, activation of protein kinase G, activation of protein kinase A, and activation or inhibition of protein kinase C. Furthermore there was no evidence for the existence of a zinc-sensing receptor in C6 glioma cells, which would stimulate PLC activity and evoke a mobilization of intracellular free-calcium levels.

Cellular Mechanisms of Olfactory Signal Transduction

The sense of smell has the exquisite capacity to recognize and discriminate among an immense variety of volatile chemical compounds that are present in the environment. In mammals, olfactory cues are detected by sensory neurons belonging to three different sensory modalities harboured in the nasal cavity. Olfactory and vomeronasal primary sensory neurons (OSNs and VSNs, respectively) detect odorants and pheromones, whereas free nerve endings of the V cranial nerve (N. trigeminus) constitute the common chemical sense and convey pain, touch, temperature and chemosensory information. Our interest is to understand the molecular and cellular mechanisms of chemosensation in the diverse subsystems. A first common step in signal transduction is the specific binding of odorant molecules to receptor proteins located in specialized membrane protrusions: the cilia, microvilli or free nerve endings innervating the nasal mucosa. Olfactory and pheromone receptor proteins of OSNs and VSNs, respectively, are identified and functionally characterized to some extent. The intracellular mechanisms that transduce the initial chemical information into an electrical signal in OSNs involve the adenylate cyclase–cyclic nucleotide-gated channel pathway in addition to a recently identified phosphoinositides pathway. In contrast, a phospholipase C–diacylglycerol– transient receptor potential (TRP) channel pathway seems to play the major role in VSNs. However, the molecular mechanisms used by trigeminal neurons to detect odorant molecules are largely unknown. The beginning characterization of thermosensitive TRP channels among other receptors and ion channels expressed in trigeminal ganglion cells revealed their role in detection of chemical compounds and chemosensory signalling in these cells. The detection and interpretation of chemical cues in an animal’s

Comprehensive and uniform synthesis of all naturally occurring phosphorylated phosphatidylinositols.

Studies of cellular signal transduction mechanisms involving receptor-mediated generation of inositol phosphates and phosphorylated phosphatidylinositols require easy access to these naturally occurring products. Although numerous synthetic methods have been developed during the past decade, most of these methods suffer from excessive length and lack of generality. In this work we describe the comprehensive and uniform synthesis of all naturally occurring phosphatidylinositols such as phosphatidylinositol, phosphatidylinositol 3-phosphate, 4-phosphate, 5-phosphate, 3,4-bisphosphate, 3,5-bisphosphate, 4,5-bisphosphate, and 3,4,5-trisphosphate, featuring both saturated and unsaturated fatty acid chains.

The release of inflammatory cytokines from human peripheral blood mononuclear cells in vitro following exposure to adenovirus variants and capsid.

Preclinical and clinical studies with adenoviral vectors have clearly illustrated the potential advantages of this gene transfer system. However, many studies have also demonstrated potent immune responses directed at both vector and transduced cells. We examined in vitro responses of human peripheral blood mononuclear cells (PBMC) to virus exposure as a model for this host response. PBMC were isolated from normal donors and incubated with wild-type adenovirus (Ad5), Ad5 variants deleted for segments of E1 and/or E3, and empty viral capsids. Proinflammatory cytokine release was monitored for 96 hr. Induction of TNF-alpha by intact virions was low although stimulation by empty capsid gave a significant and sustained response. Induction of IL-6, GM-CSF, and a panel alpha- and beta-chemokines by intact virions was prominent, often approaching results obtained with 2.5 microg/ml of lipopolysaccharide (LPS). Responses were generally independent of virion genetic composition and were only partially blunted when UV-inactivated virus was used. Dose-response data showed 100-fold increases in virion concentration produced a maximum 3-fold increase in cytokine release, suggesting saturation. Surprisingly, prominent stimulation occurred after addition of empty capsid, which typically provoked responses equivalent to those seen with LPS stimulation. We present arguments that cellular signal transduction mechanisms activated by binding of virions/capsids stimulate transcription of proinflammatory cytokine genes.

Redox Regulatory Mechanisms of Cellular Signal Transduction

Redox Regulatory Mechanisms of Cellular Signal Transduction

Localization of Nitric-oxide Synthase in Plant Peroxisomes

The presence of nitric-oxide synthase (NOS) in peroxisomes from leaves of pea plants (Pisum sativum L.) was studied. Plant organelles were purified by differential and sucrose density gradient centrifugation. In purified intact peroxisomes a Ca(2+)-dependent NOS activity of 5.61 nmol of L-[(3)H]citrulline mg(-1) protein min(-1) was measured while no activity was detected in mitochondria. The peroxisomal NOS activity was clearly inhibited (60-90%) by different well characterized inhibitors of mammalian NO synthases. The immunoblot analysis of peroxisomes with a polyclonal antibody against the C terminus region of murine iNOS revealed an immunoreactive protein of 130 kDa. Electron microscopy immunogold-labeling confirmed the subcellular localization of NOS in the matrix of peroxisomes as well as in chloroplasts. The presence of NOS in peroxisomes suggests that these oxidative organelles are a cellular source of nitric oxide (NO) and implies new roles for peroxisomes in the cellular signal transduction mechanisms.

Activation of the Drosophila C3G leads to cell fate changes and overproliferation during development, mediated by the RAS-MAPK pathway and RAP1.

The cellular signal transduction pathways by which C3G, a RAS family guanine nucleotide exchange factor, mediates v-crk transformation are not well understood. Here we report the identification of Drosophila C3G, which, like its human cognate, specifically binds to CRK but not DRK/GRB2 adaptor molecules. During Drosophila development, constitutive membrane binding of C3G, which also occurs during v-crk transformation, results in cell fate changes and overproliferation, mimicking overactivity of the RAS-MAPK pathway. The effects of C3G overactivity can be suppressed by reducing the gene dose of components of the RAS-MAPK pathway and of RAP1. These findings provide the first in vivo evidence that membrane localization of C3G can trigger activation of RAP1 and RAS resulting in the activation of MAPK, one of the hallmarks of v-crk transformation previously thought to be mediated through activation of SOS.

Heterogeneity of packing: structural approach.

Analysis of the heterogeneity of packing in proteins showed that different groups of the protein preferentially contribute to low- or high-density regions. Statistical distribution reveals the two preferable values for packing density in the form of two peaks. One peak occurs in the range of densities 0.55-0.65, the other occurs in the range 0.75-0.8. The high-density peak is originated primarily by high packing inside the hydrogen bonded backbone and to some extent by side chains. Polar/charged and apolar side chains both contribute to the low-density peak. The average packing density values of individual atomic groups significantly vary for backbone atoms as well as for side chain atoms. The carbonyl oxygen atoms of protein backbone and the end groups of side chains show lower packing density than the rest of the protein. The side-chain atomic groups of a secondary structure element when packed against the neighboring secondary structure element form stronger contacts with the side chains of this element than with its backbone. Analysis of the low-density regions around each buried peptide group was done for the set of proteins with different types of packing, including alpha-alpha, alpha-beta, and beta-beta packing. It was shown that cavities are regularly situated in the groove of secondary structure element packed against neighboring elements for all types of packing. Low density in the regions surrounding the peptide groups and the end groups of side chains can be explained by their positioning next to a cavity formed upon the association of secondary structure elements. The model proposed can be applied to the analysis of protein internal motions, mechanisms of cellular signal transduction, diffusion through protein matrix, and other events.

Reactive Oxygen-Mediated Protein Oxidation in Aging and Disease

Highly reactive oxygen species that are formed during normal metabolism and under conditions of oxidative stress are able to oxidize proteins or convert lipid and carbohydrate derivatives to compounds that react with functional groups on proteins. Among other changes, these ROS-mediated reactions lead to the formation of protein carbonyl derivatives, which serves as a marker of ROS-mediated protein damage. On the basis of this marker, it is established that oxidatively damaged protein is associated with aging and some diseases. The accumulation of oxidatively damaged protein reflects the balance among a myriad of factors that govern the rates of ROS generation and the rate at which damaged protein is degraded. Peroxynitrite, which is formed under normal physiological conditions, is able to oxidize methionine residues in proteins and to nitrate tyrosine residues; however, its ability to do so is dependent on the availability of CO2, which stimulates the nitration of tyrosine residues but inhibits the oxidation of methionine residues. Nitration of tyrosine residues may contribute to peroxynitrite toxicity, as nitration precludes the phosphorylation or nucleotidylation of tyrosine residues and thereby seriously compromises one of the most important mechanisms of cellular regulation and signal transduction.

Analysis of Mechanisms of Cellular Injury on the Micrometer Scale

Non-invasive imaging tools using biosensors and biomarkers for defining the function of living cells and tissues are among rapidly emerging technologies being developed to monitor cellular toxicity mechanisms. Commercially available fluorescence probes and naturally occurring fluorescent molecules can be used to quantitatively monitor a number of functional endpoints in cultured cells. Innovative approaches to exploit the sensitivity, spectroscopy and temporal/spatial resolution properties of fluorescent probes may provide sensitive approaches for analysis of the molecular mechanisms of toxicity in cells exposed to a variety of toxicants. Kinetic analysis of multiple cellular parameters such as intracellular glutathione (GSH) and Ca2+ content, reactive oxygen species (ROS) generation, mitochondrial and plasma membrane potentials, intracellular pH, and gap junction-mediated intercellular communication (GJIC) with cellular component-specific fluorescent probes permits rapid identification of changes in these parameters and the chronology of injury in cells caused by acute toxicant exposure. Cellular signal transduction mechanisms are also likely to be vulnerable to toxic insults.

Reactive oxygen-mediated protein oxidation in aging and disease.

Highly reactive oxygen species that are formed during normal metabolism and under conditions of oxidative stress are able to oxidize proteins or convert lipid and carbohydrate derivatives to compounds that react with functional groups on proteins. Among other changes, these ROS-mediated reactions lead to the formation of protein carbonyl derivatives, which serves as a marker of ROS-mediated protein damage. On the basis of this marker, it is established that oxidatively damaged protein is associated with aging and some diseases. The accumulation of oxidatively damaged protein reflects the balance among a myriad of factors that govern the rates of ROS generation and the rate at which damaged protein is degraded. Peroxynitrite, which is formed under normal physiological conditions, is able to oxidize methionine residues in proteins and to nitrate tyrosine residues; however, its ability to do so is dependent on the availability of CO2, which stimulates the nitration of tyrosine residues but inhibits the oxidation of methionine residues. Nitration of tyrosine residues may contribute to peroxynitrite toxicity, as nitration precludes the phosphorylation or nucleotidylation of tyrosine residues and thereby seriously compromises one of the most important mechanisms of cellular regulation and signal transduction.

From ion currents to genomic analysis: recent advances in GABAA receptor research.

The gamma-aminobutyric acid type A (GABAA) receptor represents an elementary switching mechanism integral to the functioning of the central nervous system and a locus for the action of many mood- and emotion-altering agents such as benzodiazepines, barbiturates, steroids, and alcohol. Anxiety, sleep disorders, and convulsive disorders have been effectively treated with therapeutic agents that enhance the action of GABA at the GABAA receptor or increase the concentration of GABA in nervous tissue. The GABAA receptor is a multimeric membrane-spanning ligand-gated ion channel that admits chloride upon binding of the neurotransmitter GABA and is modulated by many endogenous and therapeutically important agents. Since GABA is the major inhibitory neurotransmitter in the CNS, modulation of its response has profound implications for brain functioning. The GABAA receptor is virtually the only site of action for the centrally acting benzodiazepines, the most widely prescribed of the anti-anxiety medications. Increasing evidence points to an important role for GABA in epilepsy and various neuropsychiatric disorders. Recent advances in molecular biology and complementary information derived from pharmacology, biochemistry, electrophysiology, anatomy and cell biology, and behavior have led to a phenomenal growth in our understanding of the structure, function, regulation, and evolution of the GABAA receptor. Benzodiazepines, barbiturates, steroids, polyvalent cations, and ethanol act as positive or negative modulators of receptor function. The description of a receptor gene superfamily comprising the subunits of the GABAA, nicotinic acetylcholine, and glycine receptors has led to a new way of thinking about gene expression and receptor assembly in the nervous system. Seventeen genetically distinct subunit subtypes (alpha 1-alpha 6, beta 1-beta 4, gamma 1-gamma 4, delta, p1-p2) and alternatively spliced variants contribute to the molecular architecture of the GABAA receptor. Mysteriously, certain preferred combinations of subunits, most notably the alpha 1 beta 2 gamma 2 arrangement, are widely codistributed, while the expression of other subunits, such as beta 1 or alpha 6, is severely restricted to specific neurons in the hippocampal formation or cerebellar cortex. Nervous tissue has the capacity to exert control over receptor number, allosteric uncoupling, subunit mRNA levels, and posttranslational modifications through cellular signal transduction mechanisms under active investigation. The genomic organization of the GABAA receptor genes suggests that the present abundance of subtypes arose during evolution through the duplication and translocations of a primordial alpha-beta-gamma gene cluster. This review describes these varied aspects of GABAA receptor research with special emphasis on contemporary cellular and molecular discoveries.

Cellular mechanisms of signal transduction for neurotrophins

The molecular cloning of new neuroactive growth factors and their receptors has greatly enhanced our understanding of important interactions among receptors and signaling molecules. These studies have begun to illuminate some of the mechanisms that allow for specificity in neuronal signaling. Model cell systems, such as the PC-12 pheochromocytoma cell line, express receptors for these different neurotrophic factors, leading to comparisons of signaling pathways for these factors. Upon binding their ligands, these receptors undergo phosphorylation on tyrosine residues, which directs their interaction with signaling proteins containing src homology (SH2) domains, sequences that mediate associations with tyrosine-phosphorylated proteins. These SH2 proteins translate the tyrosine kinase activity of receptors into downstream events that result in the specific cellular response. Investigations such as these have revealed that molecular specificity in signaling pathways may arise from combinatorial diversity in interactions between receptors and key regulatory proteins.

The coupling of multiple signal transduction pathways with steroid response mechanisms

In a human breast carcinoma-derived cell line engineered to contain a hormone-responsive luciferase reporter gene, manipulation of cell growth conditions or cellular signal transduction in a variety of ways can enhance or impair glucocorticoid-mediated induction of a target gene. Induction may be enhanced as much as 10-fold or inhibited 90% by different treatments. For example, two different inhibitors of protein phosphatase-1 and -2A potentiated the hormone-dependent induction of luciferase. Activation of protein kinase-A via addition of 8-bromo-cAMP or forskolin also potentiated the hormonal induction, whereas 8-bromo-cGMP was ineffective. In contrast, activating protein kinase-A by inhibiting cAMP turnover with the phosphodiesterase inhibitors isobutylmethylxanthine or Ro20-1724 inhibited the hormone response rather than potentiated it. The inhibitory activity of isobutylmethylxanthine was evident even when activators of protein kinase-A are administered simultaneously. Isobutylmethylxanthine must, therefore, activate a signal transduction pathway in addition to the protein kinase-A pathway. Activation of protein kinase-C potentiated the hormone response in a cell-specific manner. Treatment with epidermal growth factor and imposition of cell stress by heat shock or inhibition of protein synthesis also enhanced the glucocorticoid response. Thus, our results suggest an elaborate coupling of the steroid response pathway with other cellular signal transduction mechanisms that permits an additional layer of control to be imposed on hormone-mediated transcriptional responses. It is proposed that cell-specific phosphorylation events influence steroid receptor interaction with the basal transcription apparatus, thereby altering receptor-mediated induction mechanisms.

The coupling of multiple signal transduction pathways with steroid response mechanisms.

In a human breast carcinoma-derived cell line engineered to contain a hormone-responsive luciferase reporter gene, manipulation of cell growth conditions or cellular signal transduction in a variety of ways can enhance or impair glucocorticoid-mediated induction of a target gene. Induction may be enhanced as much as 10-fold or inhibited 90% by different treatments. For example, two different inhibitors of protein phosphatase-1 and -2A potentiated the hormone-dependent induction of luciferase. Activation of protein kinase-A via addition of 8-bromo-cAMP or forskolin also potentiated the hormonal induction, whereas 8-bromo-cGMP was ineffective. In contrast, activating protein kinase-A by inhibiting cAMP turnover with the phosphodiesterase inhibitors isobutylmethylxanthine or Ro20-1724 inhibited the hormone response rather than potentiated it. The inhibitory activity of isobutylmethylxanthine was evident even when activators of protein kinase-A are administered simultaneously. Isobutylmethylxanthine must, therefore, activate a signal transduction pathway in addition to the protein kinase-A pathway. Activation of protein kinase-C potentiated the hormone response in a cell-specific manner. Treatment with epidermal growth factor and imposition of cell stress by heat shock or inhibition of protein synthesis also enhanced the glucocorticoid response. Thus, our results suggest an elaborate coupling of the steroid response pathway with other cellular signal transduction mechanisms that permits an additional layer of control to be imposed on hormone-mediated transcriptional responses. It is proposed that cell-specific phosphorylation events influence steroid receptor interaction with the basal transcription apparatus, thereby altering receptor-mediated induction mechanisms.

IgE and adenosine 5′ triphosphate receptors on immature murine mast cells are functionally linked to signal transduction mechanisms

Calcium mobilization in response to IgE-receptor cross-linking by antigen was assessed in immature murine mast cells cultured from bone marrow to determine whether the early-expression of IgE receptors on such cells may be of functional significance. IgE receptors were expressed by approximately 30% of cells after 1 week in culture and by an increased proportion at 2 and 3 weeks. The ability of a non-IgE-dependent stimulus, adenosine 5′ triphosphate (ATP), to increase intracellular calcium in these cells was also tested. Calcium mobilization in large numbers of individual cells was monitored with use of a fluorimetric reagent and flow cytometry. Both antigen and ATP had significant effects on intracellular calcium in cells cultured for as little as 1 week with interleukin-3, when few cells exhibited morphologic or functional chracteristics of mast cells. Longer times in culture were associated with an increase in the proportion of cells responding to these stimuli with calcium mobilization, but not with a change in the magnitude of the response. We conclude that the early expression of IgE receptors during mast cell development may be functionally significant, since these receptors appear to be linked to cellular signal transduction mechanisms. The data additionally imply a possible role for ATP in mast cell development.

Modulation of meiotic arrest in mouse oocytes by guanyl nucleotides and modifiers of G‐proteins

Guanyl nucleotide binding-proteins, or G-proteins, are ubiquitous molecules that are involved in cellular signal transduction mechanisms. Because a role has been established for cAMP in meiosis and G-proteins participate in cAMP-generating systems by stimulating or inhibiting adenylate cyclase, the present study was conducted to examine the possible involvement of G-proteins in the resumption of meiotic maturation. Cumulus cell-free mouse oocytes (denuded oocytes) were maintained in meiotic arrest in a transient and dose-dependent manner when microinjected with the nonhydrolyzable GTP analog, GTP gamma S. This effect was specific for GTP gamma S, because GppNHp, GTP, and ATP gamma S were without effect. Three compounds, known to interact with G-proteins, were tested for their ability to modulate meiotic maturation: pertussis toxin, cholera toxin, and aluminum fluoride (AlF4-). Pertussis toxin had little effect on maturation in either cumulus cell-enclosed oocytes or denuded oocytes when meiotic arrest was maintained with dibutyryl cAMP (dbcAMP) or hypoxanthine. Cholera toxin stimulated germinal vesicle breakdown (GVB) in cumulus cell-enclosed oocytes during long-term culture, but its action was inhibitory in denuded oocytes. AlF4- stimulated GVB in both cumulus cell-enclosed oocytes and denuded oocytes when meiotic arrest was maintained with hypoxanthine but was much less effective in dbcAMP-arrested oocytes. In addition, AlF4- abrogated the inhibitory action of cholera toxin in denuded oocytes and also that of follicle-stimulating hormone (FSH) in cumulus cell-enclosed oocytes. Cholera toxin or FSH alone each stimulated the synthesis of cAMP in oocyte-cumulus cell complexes, whereas pertussis toxin or AlF4- alone were without effect. Both cholera toxin and AlF4- augmented the stimulatory action of FSH on cAMP. These data suggest the involvement of guanyl nucleotides and G-proteins in the regulation of GVB, although different G-proteins and mediators may be involved at the oocyte and cumulus cell levels. Cholera toxin most likely acts by ADP ribosylation of the alpha subunit of Gs and increased generation of cAMP, whereas AlF4- appears to act by antagonizing a cAMP-dependent step.

Adrenergic receptors as models for G protein-coupled receptors.

Approximately 80% of known hormones and neurotransmitters activate cellular signal transduction mechanisms by activating G protein-coupled receptors (Birnbaumer et al 1990). Studying the structure and function of these receptors has been challenging because they are not naturally abun­ dant proteins and they require a lipid environment to be fully active. The eDNA or genomic clones for many members of this class of receptors have recently been obtained. Applying the techniques of molecular biology to the study of G protein-coupled receptors has helped circumvent many of the technical difficulties that had complicated previous efforts to charac­ terize these proteins biochemically. The adrenergic receptors have been one of the most extensively studied classes of G protein-coupled receptors. Genomic and/or cDNA clones for nine types of adrenergic receptors have been obtained: two types of (XI receptors (Cotecchia et al 1988; Schwinn et al 1990); three types of C(2 receptors (Kobilka et al 1987; Lomasney et al 1990; Regan et al 1988; Weinshank et aI 1990); the /31 receptor (Frielle et aI 19 87); the /32 receptor (Dixon et al 1986); the /3 3 receptor (Emorine et al 1989) ; and the avian /3 receptor (Yarden et al 1986). Significant progress has been made in eluci­ dating the structural domains involved in ligand binding, G protein acti­ vation, and desensitization, and much of what is learned about the mech­ anism of action of adrenergic receptors will likely apply to the other