inositol-1-4-5-trisphosphate and Cell-Transformation--Neoplastic

Phosphoinositide 3-kinases (PI3Ks) control cell growth, proliferation, cell survival, metabolic activity, vesicular trafficking, degranulation, and migration. Through these processes, PI3Ks modulate vital physiology. When over-activated in disease, PI3K promotes tumor growth, angiogenesis, metastasis or excessive immune cell activation in inflammation, allergy and autoimmunity. This chapter will introduce molecular activation and signaling of PI3Ks, and connections to target of rapamycin (TOR) and PI3K-related protein kinases (PIKKs). The focus will be on class I PI3Ks, and extend into current developments to exploit mechanistic knowledge for therapy.

Topics: Autoimmunity; Cell Transformation, Neoplastic; Diglycerides; Enzyme Inhibitors; Eukaryotic Cells; Gene Expression Regulation, Neoplastic; Humans; Hypersensitivity; Inositol 1,4,5-Trisphosphate; Isoenzymes; Neoplasms; Phosphatidylinositol 3-Kinases; Phosphatidylinositol 4,5-Diphosphate; Phosphoinositide-3 Kinase Inhibitors; Second Messenger Systems; TOR Serine-Threonine Kinases

Inositol trisphosphate is a second messenger that controls many cellular processes by generating internal calcium signals. It operates through receptors whose molecular and physiological properties closely resemble the calcium-mobilizing ryanodine receptors of muscle. This family of intracellular calcium channels displays the regenerative process of calcium-induced calcium release responsible for the complex spatiotemporal patterns of calcium waves and oscillations. Such a dynamic signalling pathway controls many cellular processes, including fertilization, cell growth, transformation, secretion, smooth muscle contraction, sensory perception and neuronal signalling.

Topics: Animals; Calcium; Calcium Channels; Cell Cycle; Cell Division; Cell Transformation, Neoplastic; Female; Fertilization; GTP-Binding Proteins; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Male; Models, Biological; Neuronal Plasticity; Protein-Tyrosine Kinases; Receptors, Cell Surface; Receptors, Cholinergic; Receptors, Cytoplasmic and Nuclear; Ryanodine; Ryanodine Receptor Calcium Release Channel; Second Messenger Systems; Signal Transduction; Synapses

Recent discoveries in tumor virology, lipid biochemistry, and ion transport studies promise to revolutionize our understanding of cell proliferation, differentiation, and tumorigenesis. A model is proposed, based on similar schemes presented recently by others, that incorporates these discoveries and provides a focus for future research on the functions of oncogene proteins. The model suggests that the early (competence) events in the initiation of cell proliferation are triggered by activation of phosphatidylinositol (PI) turnover, which releases two second messengers, 1,2-diacylglycerol (1,2-DG) and inositol-1,4,5-trisphosphate (IP3). PI turnover is proposed to be regulated by the oncogene protein kinases (src, ros, abl, fps) either directly (acting as PI kinases) or indirectly (as tyrosine kinases). The IP3 triggers Ca2+ release from internal stores, and the elevation of cytosolic Ca2+ acts synergistically with 1,2-DG to activate the Ca2+- and phospholipid-dependent kinase C. Kinase C copurifies with the receptor for the tumor-promoting phorbol esters. It is suggested that kinase C then activates the Na+-H+ exchange system, resulting in an elevation of cytosolic pH and Na+, and that these ionic signals (including the change in Ca2+), either in concert or individually, induce further events, including expression of the protooncogene c-myc, which together commit the cell to initiate replication. Evidences in support of this model are reviewed, together with complications indicating its present inadequacies, particularly recent data suggesting that 1,2-DG may activate tyrosine kinases independent of kinase C.

Topics: Animals; Cell Differentiation; Cell Division; Cell Transformation, Neoplastic; Cells, Cultured; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Ion Channels; Membrane Lipids; Mice; Mitogens; Neoplasm Proteins; Oncogenes; Phosphatidylinositols; Phospholipids; Protein Kinases; Sodium-Potassium-Exchanging ATPase; Transcription, Genetic

Topics: Animals; Arachidonic Acids; Calcium; Cell Transformation, Neoplastic; Diglycerides; Genes, Viral; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Male; Oncogenic Viruses; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Rabbits; Rats; Receptors, Cell Surface; Receptors, Cytoplasmic and Nuclear; Type C Phospholipases

G protein-coupled receptors (GPCRs) constitute a large protein family of seven transmembrane (7TM) spanning proteins that regulate multiple physiological functions. GPR87 is overexpressed in several cancers and plays a role in tumor cell survival. Here, the basal activity of GPR87 was investigated in transiently transfected HEK293 cells, revealing ligand-independent coupling to Gα

Topics: Animals; Carcinogenesis; Cell Membrane; Cell Transformation, Neoplastic; Chlorocebus aethiops; COS Cells; Cyclic AMP; Female; GTP-Binding Proteins; HEK293 Cells; Humans; Inositol 1,4,5-Trisphosphate; Ligands; Lysophospholipids; Mice; Mice, Inbred BALB C; Mice, Nude; Models, Biological; Mutant Proteins; NIH 3T3 Cells; Receptors, Lysophosphatidic Acid; rho GTP-Binding Proteins; rho-Associated Kinases; Signal Transduction; Transcription Factors; Transfection

Substance P (SP) regulates various physiologic and pathophysiologic responses predominantly by acting through its primary receptor, the neurokinin-1 receptor (NK1R). There are two naturally occurring forms of NK1R: full-length NK1R-FL and truncated NK1R-Tr. SP-coupled NK1R can directly or indirectly regulate the proliferation and metastatic progression of many types of human cancer cells. However, the exact roles played by the two isoforms of NK1R in breast carcinogenesis still remain largely unclear. In the present study, we first examined the expression profile of total NK1Rs, NK1R-FL and NK1R-Tr in multiple breast cancer cell lines as well as in breast tumor samples. We found that total NK1Rs are present in normal, benign and breast tumor tissues; while, NK1R-FL expression are significantly decreased in tumor specimens, particularly in metastatic carcinomas. More interestingly, NK1R-FL is highly expressed in nontumorigenic HBL-100 breast cells, whereas MDA-MB-231, MCF-7 and T47D breast cancer cells express only NK1R-Tr. To further investigate potential implications of NK1R-FL and NK1R-Tr in the malignant phenotypes of breast cancer, we studied the impacts of ectopically overexpressed NK1R-FL and NK1R-Tr in MDA-MB-231 and HBL-100 cells, respectively. Our in vitro and in vivo data showed that NK1R-FL expression was inversely associated with proliferation, invasiveness and metastasis of MDA-MB-231 cells, but overexpression of NK1R-Tr was able to promote malignant transformation of HBL-100 cells and NK1R-Tr may contribute to tumor progression and promote distant metastasis in human breast cancer. A long-term treatment of NK1R antagonist ASN-1377642 exerted antitumor action in breast cancer cells with NK1R-Tr high expression.

Topics: Aniline Compounds; Animals; Breast Neoplasms; Cell Line, Tumor; Cell Transformation, Neoplastic; Female; Gene Expression Regulation, Neoplastic; Humans; Inositol 1,4,5-Trisphosphate; Mammary Glands, Human; Mice, Nude; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurokinin-1 Receptor Antagonists; Phosphorylation; Receptors, Neurokinin-1; Reference Values; Substance P; Triazoles

Non-neuronal expression of components of the glutamatergic system has been increasingly observed, and our laboratory previously had demonstrated the etiological role of ectopically expressed metabotropic glutamate receptor 1 (Grm1/mGluR1) in mouse models of melanoma. We hypothesize that inappropriate glutamatergic signaling in other cell types can dysregulate growth leading to transformation and tumorigenesis. As most cancers are carcinomas, we selected an immortalized primary baby mouse kidney (iBMK) cell model to assess whether Grm1 can transform epithelial cells. These iBMK cells, engineered to be immortal yet nontumorigenic and retaining normal epithelial characteristics, were used as recipients for exogenous Grm1 cDNA. Several stable Grm1-expressing clones were isolated and the Grm1-receptors were shown to be functional, as evidenced by the accumulation of second messengers in response to Grm1 agonist. Additionally activated by agonist were mitogen-activated protein kinase (MAPK) and AKT/protein kinase B signaling cascades, the major intracellular pathways shown by many investigators to be critical in melanomagenesis and other neoplasms. These Grm1-iBMK cells exhibited enhanced cell proliferation in in vitro methylthiazolyldiphenyl-tetrazolium bromide (MTT) assays and significant tumorigenicity in in vivo allografts. Persistent Grm1 expression was required for the maintenance of the in vivo tumorigenic phenotype as demonstrated by an inducible Grm1-silencing RNA. These are the first results that indicate that Grm1 can be an oncogene in epithelial cells. In addition, relevance to human disease in the corresponding tumor type of renal cell carcinoma (RCC) may be suggested by observed expression of GRM1/mGluR1 in a number of RCC tumor biopsy samples and cell lines, and the effects of GRM1 modulation on tumorigenicity therein. Moreover, RCC cell lines exhibited elevated levels of extracellular glutamate, and some lines responded to drugs, which modulate the glutamatergic system. These findings imply a possible role for glutamate signaling apparatus in RCC cell growth, and that the glutamatergic system may be a therapeutic target in RCC.

Topics: Animals; Carcinoma, Renal Cell; Cell Line, Tumor; Cell Proliferation; Cell Transformation, Neoplastic; Enzyme Activation; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Gene Expression; Glutamic Acid; Humans; Inositol 1,4,5-Trisphosphate; Mice; Mitogen-Activated Protein Kinases; Oncogenes; Phenotype; Proto-Oncogene Proteins c-akt; Receptors, Metabotropic Glutamate; Riluzole

Extracellular acidification accompanies neoplastic transformation of tissues and increases with tumor aggressiveness [1, 2]. The intracellular signaling cascade triggered by this process remains poorly understood and may be linked to recently discovered proton-activated G protein-coupled receptors such as OGR1 and G2A [3, 4]. Here, we report that OGR1 and G2A are expressed in human medulloblastoma tissue and its corresponding neuronal cell line. We show that extracellular acidification activates phospholipase C, IP(3) formation, and subsequent Ca2+ release from thapsigargin-sensitive stores in neurons. The number of responsive cells and the amount of Ca2+ released from stores correlated positively with the extent of extracellular acidification. Ca2+ release recruited the MEK/ERK pathway, providing a mechanistic explanation for how acidification stimulates cell growth. In addition, acidification activated Ca2+-permeable ion channels through a mechanism dependent on phospholipase C but independent of store depletion or a cytoplasmic Ca2+ rise. Hence, extracellular acidification, to levels seen in tumor tissue, activates temporally and spatially distinct pathways that elevate Ca2+ and may be directly relevant for tumor cell biology.

Topics: Calcium Signaling; Cell Cycle Proteins; Cell Line, Tumor; Cell Transformation, Neoplastic; Cerebellar Neoplasms; Extracellular Fluid; Humans; Hydrogen-Ion Concentration; Inositol 1,4,5-Trisphosphate; Medulloblastoma; Receptors, G-Protein-Coupled; Type C Phospholipases

To try to further define the mechanism of action of the putative second messenger inositol 1,3,4,5-tetrakisphosphate (InsP4), we have studied its effects in permeabilized cells expressing different levels of inositol trisphosphate receptor (InsP3R) types I and III and of the GTPase-activating protein GAP1IP4BP. During the growth curve of human HL-60 cells and mouse T15 cells there was an increase in these proteins, which was further increased by differentiation (HL-60) and, marginally, by transformation (T15). T15 cells entering the stationary phase showed much lower concentrations of these proteins and expression was below detection in apoptotic HL-60 cells. Rasp21 showed a different pattern of expression. The ratios of InsP3R subtypes seem to affect the dose-response curve for inositol 2,4,5-trisphosphate Ins(2,4,5)P3. In permeabilized T15 cells the curve was approximately 5-fold to the right of that obtained using HL-60 cells. However, permeabilized untreated and differentiated HL-60 cells and T15 cells all showed a comparable synergistic effect of InsP4 on Ca2+ release stimulated by a concentration of Ins(2,4,5)P3, releasing approximately 20% of the Ins(1,4,5)P3 sensitive Ca2+ pool. The data indicate that under these conditions InsP4 is acting independently of cell type, of the ratio of inositol trisphosphate receptor subtypes, and of the concentration of GAP1IP4BP.

Topics: Animals; Calcium; Calcium Channels; Cell Differentiation; Cell Membrane Permeability; Cell Survival; Cell Transformation, Neoplastic; Fibroblasts; HL-60 Cells; Humans; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Inositol Phosphates; Mice; NADPH Oxidases; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins p21(ras); Receptors, Cytoplasmic and Nuclear; Tretinoin

Effects of signal transduction pathways in TCDD-induced neoplastic transformation of human cells were assessed with respect to PLC-coupled signaling pathways, adenylyl cyclase-mediated responses and PKC isozyme expressions. A lower stimulation of the intracellular free calcium levels with exposure to extracellular ATP or histamine was observed in the transformed cells, as compared to the parental cells. While the steady-state level of IP3 was higher in the transformed cells, the magnitude of stimulation of IP3 generation by ATP or histamine was significantly lower in the transformed cells than the parental cells. These results indicate that a downregulation PLC-coupled signaling pathways may be involved in the TCDD-induced transformation of human cells. While the steady-state levels of cAMP accumulation were similar between the two cell lines, treatment of PGE2, a potent differentiation inducer, stimulated a higher accumulation of cAMP in the parental cells but isoproterenol, a typical beta-adrenergic agonist, did not induce a significant difference between the two cell lines. These results suggest that desensitization of cAMP-mediated response to extracellular signals including differentiation signals may be associated with a possible mechanism of the carcinogenesis. Elevated expression of PKC-alpha, -gamma, -zeta, -epsilon, -lambda, and -tau were observed in TCDD-transformed cells, indicating a possible association of altered expression of PKC isozymes with TCDD-induced transformation of human cells. The present study demonstrates that alterations of signal transduction pathways are involved in the TCDD-induced transformation of human cells and provides a valuable basis to investigate effects of signaling pathway as a possible mechanism of TCDD-induced carcinogenesis in human cells.

Topics: Blotting, Western; Calcium; Carcinogens; Cell Transformation, Neoplastic; Cyclic AMP; Humans; Inositol 1,4,5-Trisphosphate; Isoenzymes; Polychlorinated Dibenzodioxins; Protein Kinase C; Signal Transduction; Tumor Cells, Cultured

Morphological transformation of Chinese hamster ovary (CHO) cells can be induced by exogenous addition of cyclic AMP (cAMP) or through the stimulation of G protein-coupled receptors ectopically expressed in these cells. The morphological transformation has been shown to represent a phenotypic suppression of CHO cell tumorigenic potential. Studies were undertaken to determine which receptor-activated signal transduction pathway initiates the progression from a tumorigenic to a non-tumorigenic phenotype. Stimulation of CHO cells expressing the dopamine D1 receptor (CHOD1) with a D1 selective agonist, SKF38393, resulted in an increase in cAMP accumulation which correlated with morphologic transformation. SKF38393 had no effect on intracellular calcium levels, arguing against a requirement for phospholipase C or calcium mobilization in the D1-stimulated morphology change. In contrast, stimulation of muscarinic m5 (CHOm5) or vasopressin V1a (CHOV1a) receptors expressed in CHO cells with carbachol or arginine vasopressin (AVP), respectively, did not result in an increase in intracellular calcium and a morphology change. The time course of carbachol-stimulated calcium influx correlated with the time course of morphological transformation, but not with carbachol-stimulated cAMP or inositol, 1,4,5-trisphosphate (IP3) accumulation. Furthermore, no increase in cAMP accumulation was observed in AVP-stimulated CHOV1a cells, suggesting a cAMP-independent stimulation of the transformation process. Carbachol-stimulated CHO cells expressing the m2 muscarinic receptor (CHOm2) failed to undergo a morphological transformation, yet released IP3. Therefore, phospholipase C-mediated signal transduction is not sufficient for the morphological transformation of CHO cells. It appears that receptor-stimulated morphologic transformation of CHO cells can be induced via two independent signaling pathways, mediated by adenylate cyclase or receptor-operated calcium channels.

Topics: 2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-benzazepine; Animals; Arginine Vasopressin; Calcium; Carbachol; Cell Line, Transformed; Cell Transformation, Neoplastic; CHO Cells; Cricetinae; Cyclic AMP; Dinoprostone; Dopamine Agonists; GTP-Binding Proteins; Inositol 1,4,5-Trisphosphate; Kinetics; Receptors, Cell Surface; Receptors, Dopamine D1; Receptors, Muscarinic; Receptors, Vasopressin; Signal Transduction; Time Factors

Alkaline stress transforms Madin-Darby canine kidney (MDCK) cells as indicated by loss of epithelial structure, multilayer cell growth and formation of foci. In the present study we report that transformed MDCK cells (MDCK-F cells) exhibit spontaneous and lasting oscillations of intracellular Ca2+ concentration ([Ca2+]i), which are absent in non-transformed cells. Oscillations, as revealed by Fura-2 video imaging, were due to the activity of an inositol 1,4,5-trisphosphate-(InsP3)-sensitive Ca2+ store since their frequency was dependent on bradykinin concentration and they were abolished by the phosphoinositidase C inhibitor U73122. Moreover, blockers of the cytoplasmic Ca(2+)-ATPase, thapsigargin and 2,5-di-(tetr-butyl)-1,4-benzohydroquinone inhibited oscillatory activity. In contrast, neither injection of ruthenium red, ryanodine nor caffeine had any effect on oscillations. Analysis of the spatial distribution of [Ca2+]i showed that Ca2+ transients originated from an initiation site constant for a given cell and spread through the cell as an advancing Ca2+ wave. Oscillations started in a random manner from single cells and spread over neighbouring cells, suggesting a kind of intercellular communication. We conclude that MDCK-F cells have acquired the ability for endogenous Ca2+ release through transformation. Oscillations are primarily due to the activity of an InsP3-sensitive cytosolic Ca2+ oscillator.

Topics: Animals; Biological Clocks; Bradykinin; Calcium; Calcium-Transporting ATPases; Cell Communication; Cell Line; Cell Transformation, Neoplastic; Cytoplasm; Dogs; Estrenes; Hydrogen-Ion Concentration; Inositol 1,4,5-Trisphosphate; Ionomycin; Pyrrolidinones; Terpenes; Thapsigargin; Type C Phospholipases

Transformed Mardin-Darby canine kidney-focus (MDCK-F) cells exhibit spontaneous Ca2+ oscillations from an inositol 1,4,5-triphosphate-sensitive cytoplasmic Ca2+ store. In this study, Ca2+ entry from the extracellular space and its role in generation of oscillations were investigated by means of Ca2+ video imaging and the Fura-2/Mn2+ quenching technique. Oscillations were dependent on extracellular Ca2+ concentration and were inhibited by extracellularly applied La3+, Co2+ and Ni2+. Depolarization of the cell membrane with high K+ concentrations and the L-type Ca2+ channel blocker nifedipine had no effect on oscillations, indicating the lack of involvement of voltage-gated Ca2+ channels. Mn2+ quenching experiments disclosed significant Ca2+ influx into MDCK-F cells. The rate of this influx was constant between Ca2+ spikes, but markedly increased during the spontaneous Ca2+ spikes. Similar transient increases in Ca2+ entry could be mimicked by agents triggering intracellular Ca2+ release such as bradykinin and thapsigargin. We conclude that the plasma membrane of MDCK-F cells exhibits a marked voltage-independent Ca2+ permeability permitting Ca2+ entry into the cytoplasm. The rate of Ca2+ entry which determines the frequency of oscillations is most likely to be regulated by the cytoplasmic Ca2+ concentration.

Topics: Animals; Biological Clocks; Bradykinin; Calcium; Calcium-Transporting ATPases; Cell Line; Cell Membrane Permeability; Cell Transformation, Neoplastic; Cytoplasm; Dogs; Extracellular Space; Inositol 1,4,5-Trisphosphate; Ion Transport; Manganese; Terpenes; Thapsigargin

Microspectrofluorometry (fura-2) was combined with the whole-cell patch-clamp technique to study bradykinin-activated calcium (Ca2+) influx in single control or v-Ki-ras-transformed NIH/3T3 (DT) fibroblasts. Application of bradykinin on DT cells, but not on control NIH/3T3 cells, evoked cytosolic Ca2+ oscillations in the presence of extracellular Ca2+, but not in the absence of external Ca2+. This effect of zero external Ca2+ concentration could be mimicked by holding at depolarized membrane potentials. Cytosolic Ca2+ oscillations observed at holding potentials of -20 to -80 mV were terminated by holding at -10 mV or more depolarized potentials. The frequency of Ca2+ oscillations increased with membrane hyperpolarization. Bradykinin significantly enhanced the hyperpolarization-induced increases in the intracellular free Ca2+ concentration ([Ca2+]i) upon membrane hyperpolarization only in DT cells but not in control cells. No [Ca2+]i increase upon hyperpolarization was observed in bradykinin-stimulated DT cells in the absence of external Ca2+, suggesting that bradykinin activates Ca2+ influx. [Ca2+]i increased upon application of inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) into control and DT cells in an extracellular Ca(2+)-dependent manner, indicating that NIH/3T3 fibroblasts have an Ins(1,3,4,5)P4-gated Ca2+ influx pathway. Ins-(1,3,4,5)P4, however, produced the sustained [Ca2+]i increase in DT cells, but not in control NIH/3T3 cells, suggesting that ras may lock the Ca2+ influx pathway at the activated state. Cytosolic Ca2+ oscillations, bradykinin-enhanced Ca2+ influx, and Ins(1,3,4,5)P4-induced Ca2+ influx were all similar in that activity was increased by membrane hyperpolarization. The results suggest that bradykinin-induced cytosolic Ca2+ oscillations in ras-transformed NIH/3T3 cells are maintained by bradykinin-activated continuous Ca2+ influx which may use Ins(1,3,4,5)P4 as an intracellular messenger.

Topics: 3T3 Cells; Animals; Bradykinin; Calcium; Cell Line, Transformed; Cell Membrane; Cell Transformation, Neoplastic; Cytosol; Electrophysiology; Fibroblasts; Genes, ras; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Membrane Potentials; Mice; Time Factors

1. Inositol phospholipid metabolites were measured from virally and chemically transformed cells. 2. Increased levels of PIP and PIP2 were observed from both transformed cell lines as compared with controls. 3. Intracellular levels of IP3 were also increased approximately three folds in BPV-1 infected ID 13 cells and in 3-MC transformed NIH 3T3 cells. 4. The results suggest that phosphorylation of phosphatidylinositols and enhanced generation of IP3 second messenger molecules are the common signal transducing process leading to the cell transformation.

Topics: Animals; Bovine papillomavirus 1; Cell Division; Cell Line, Transformed; Cell Transformation, Neoplastic; Cell Transformation, Viral; Hydrolysis; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Methylcholanthrene; Mice; Phosphatidylinositols; Phosphorylation; Signal Transduction

The effects of the expression of the protein tyrosine kinase pp60v-src on endothelin- and thrombin-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) production and calcium responses were investigated in Rat-1 fibroblasts. The ability of endothelin-1 to induce the accumulation of these second messengers was dramatically amplified by v-src transformation, with 6- and 3-fold enhancements of the peak Ins(1,4,5)P3 and peak calcium responses, respectively. In contrast, thrombin-dependent responses were slightly reduced following v-src transformation, demonstrating that the augmentation of endothelin-stimulated signal transduction is a selective effect. The magnitude of the stimulated accumulation of Ins(1,4,5)P3 presumably depends upon both the functional activation of phospholipase C to produce Ins(1,4,5)P3, and the activity of the enzymes that metabolize Ins(1,4,5)P3. Although the metabolism of Ins(1,4,5)P3 was strikingly altered by expression of pp60v-src, with a bias towards the production of higher inositol polyphosphates that is consistent with an activated Ins(1,4,5)P3 3-kinase, this change could not account for the marked increase in endothelin-stimulated signaling induced by v-src transformation. This suggests that an effect of pp60v-src is expressed at the level of the plasma membrane, through an interaction with one or more components in the receptor/guanine nucleotide binding protein (G protein)/phospholipase C system that transduces the endothelin signal into Ins(1,4,5)P3 production. Preparation of membranes from normal and v-src-transformed cells showed that, while there was no change in the number of high-affinity endothelin binding sites, the release of Ins(1,4,5)P3 in response to guanine nucleotides and endothelin-1 was significantly increased following v-src transformation. In contrast, the Ins(1,4,5)P3 responses to thrombin and high Ca2+ concentrations were unaffected by transformation. Thus the selective interactions within the G protein system that couples the endothelin receptor to phospholipase C are potential sites at which the v-src transformation process may act to amplify endothelin-dependent Ins(1,4,5)P3 production.

Topics: Animals; Calcium; Cations, Divalent; Cell Transformation, Neoplastic; Endothelins; Fibroblasts; Genes, src; Inositol 1,4,5-Trisphosphate; Iodine Radioisotopes; Kinetics; Rats; Second Messenger Systems; Signal Transduction; Substrate Specificity; Thrombin

Bradykinin (BK) induced a biphasic increase in 1,2-diacylglycerol (DAG) in both K-ras-transformed fibroblasts (DT) and the parent NIH-3T3 cells. The first phase was coincident with the increase in Ins(1,4,5)P3 resulting from PtdIns(4,5)P2 hydrolysis, and the second, sustained, phase was derived from phosphatidylcholine (PtdCho) hydrolysis. In NIH-3T3 cells, stimulation by BK induced greater production of choline than phosphocholine in [3H]choline-labelled cells and appreciable phosphatidylethanol (PtdEtOH) formation in [3H]myristic acid-labelled cells, suggesting that PtdCho was hydrolysed mainly by a phospholipase D (PLD) activity. Pretreatment with propranolol, an inhibitor of phosphatidate phosphohydrolase, markedly diminished the second DAG accumulation, supporting the above notion. In DT cells, BK induced predominantly phosphocholine generation and little PtdEtOH formation, indicating that the PtdCho hydrolysis was due to a phospholipase C (PLC) activity. The BK-induced oscillations in intracellular Ca2+ concentration ([Ca2+]i) observed in single DT cells [Fu, Sugimoto, Oki, Murakami, Okano & Nozawa (1991) FEBS Lett. 281, 263-266] were detected as a sustained [Ca2+]i elevation when assayed in a cell suspension. A receptor-operated Ca2+ channel blocker, SK&F 96365, suppressed both the BK-induced phosphocholine generation and the sustained [Ca2+]i elevation in a similar dose-dependent manner. These results thus suggested that oscillations in [Ca2+]i are involved in the activation of PtdCho-specific PLC in DT cells.

Topics: 3T3 Cells; Animals; Bradykinin; Calcium; Cell Line, Transformed; Cell Transformation, Neoplastic; Choline; Diglycerides; Genes, ras; Inositol 1,4,5-Trisphosphate; Kinetics; Mice; Myristic Acid; Myristic Acids; Phosphatidylcholines; Phospholipase D; Time Factors; Type C Phospholipases

Topics: 1-Phosphatidylinositol 4-Kinase; Amino Acid Sequence; Animals; Cell Division; Cell Transformation, Neoplastic; Cell Transformation, Viral; Chick Embryo; Diglycerides; Fibroblasts; Inositol 1,4,5-Trisphosphate; Mitogens; Molecular Sequence Data; Oncogene Protein pp60(v-src); Oncogene Proteins v-erbB; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Phosphotransferases; Rats; Retroviridae Proteins, Oncogenic; Second Messenger Systems; Sequence Homology, Nucleic Acid

We used Ha-ras-transformed Madin-Darby canine kidney (MDCK) cells as a model to study possible signal transduction mechanisms underlying the induction of glucagon responsiveness by the differentiation inducers prostaglandin E2 (PGE2) and 8-bromo-cyclic (8-Br-cAMP) AMP and the inhibition of induction by phorbol ester or a serum factor. The steady-state level of inositol 1,4,5-trisphosphate (IP3) was higher in Ha-ras-transformed MDCK cells than in parental MDCK cells. In contrast, the steady-state level of intracellular cAMP of transformed cells was similar to that of normal cells. PGE2 and 8-Br-cAMP increased cAMP content but decreased IP3 levels in a concentration-dependent fashion after 5 days of treatment. We examined the time course for effects of PGE2 and 8-Br-cAMP and found that there was a lag period of 8 to 16 h between elevation of cAMP after the addition of 8-Br-cAMP or PGE2 and the decrease of IP3 levels. Another lag period of 2 days existed before the induction of differentiation. Both the reduction of IP3 levels and the induction of glucagon responsiveness were blocked by phorbol-12-myristate-13-acetate or serum, suggesting that a decrease in the IP3 level might be causally involved in induction of differentiation in transformed MDCK cells. However, induction of differentiation was not due to changes in the expression or guanine nucleotide-binding properties of p21 protein. It is likely that cAMP has a direct regulatory effect on the phospholipid signaling pathway. We conclude that perturbation of the inositol phosphate signaling pathway may be responsible for the induction of differentiation by PGE2 and 8-Br-cAMP in transformed MDCK cells.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Cyclic AMP; Diglycerides; Dinoprostone; Dogs; Genes, ras; GTP-Binding Proteins; Guanine Nucleotides; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Isoproterenol; Oncogene Protein p21(ras); Phospholipids; Tetradecanoylphorbol Acetate; Time Factors; Virulence Factors, Bordetella

It is proposed that a phosphatidylinositol-specific phospholipase C (PLC) enzyme may be present in abnormally high concentrations in certain cancer cells, and that the elevated activity may explain many, if not all, of the neoplastic characteristics of the cancer cells. There have thus far, been two reports in which PLC activity has been found to be elevated several fold in neoplastic cells. The products of the action of PLC on the phosphoinositides, including diglycerides and inositol phosphates, have been shown to activate the process of cell division by elevating the intracellular concentration of calcium ions and by stimulating the activity of protein kinase C. An elevated content of PLC in at least certain neoplastic cells could thus explain uncontrolled proliferative processes in those cells.

Topics: Calcium; Cell Division; Cell Transformation, Neoplastic; Diglycerides; Fatty Acids, Unsaturated; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Protein Kinase C; Type C Phospholipases

The role of ras proteins in signal transduction was assessed by studying inositol phospholipid metabolism and inositol phospholipid-mediated cellular responsiveness to agonists in cells transformed by ras and other oncogenes. Specific alterations were observed in the inositol phospholipid cycle of ras-transformed fibroblasts, but similar changes were also produced by spontaneous transformation or transformation mediated by either membrane-associated oncogenes, such as src, met, or trk, or cytoplasmic oncogenes, mos and raf; the nuclear oncogenes fos and myc did not produce these changes. The alterations included (i) stimulation of phospholipase A2 activity as indicated by elevated levels of glycerophosphoinositol and nonesterified arachidonic acid and (ii) specific uncoupling between surface receptor-mediated stimulation by platelet-derived growth factor, bombesin, or serum and activation of intracellular phospholipase C. These findings suggest the existence of common biochemical pathways for transformation by cytoplasmic and membrane-associated oncogenes and are not consistent with the hypothesis that 21-kDa ras proteins (p21) are direct or distinct regulatory elements of phospholipase C or phospholipase A2 in inositol phospholipid signal transduction pathways.

Topics: Animals; Cell Line; Cell Line, Transformed; Cell Transformation, Neoplastic; Cells, Cultured; Genes, ras; Inositol; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Mice; Oncogenes; Rats; Sugar Phosphates; Tritium

Expression of a transforming Harvey or Kirsten ras gene caused opposing effects in the ability of platelet-derived growth factor (PDGF) and bradykinin to activate phospholipase C-mediated phosphoinositide hydrolysis. In [3H]inositol-labeled rat-1 fibroblasts, PDGF (5 ng/ml) resulted in a 2-fold increase in the level of [3H]inositol trisphosphate (InsP3) after 2 min and, in the presence of LiCl, a 3- to 8-fold increase in the level of [3H]inositol monophosphate (InsP1) after 30 min. However, in EJ-ras-transfected rat-1 cells, which exhibit near normal levels of PDGF receptors, PDGF resulted in little or no accumulation of either [3H]InsP3 or [3H]InsP1. Similarly, marked stimulations by PDGF were observed in NIH 3T3 cells, as well as in v-src-transformed 3T3 cells, but not in 3T3 cells transformed by Kirsten sarcoma virus or by transfection with v-Ha-ras DNA. This diminished phosphoinositide response in ras-transformed cells was associated with a markedly attenuated mitogenic response to PDGF. On the other hand, both phosphoinositide metabolism and DNA synthesis in ras-transformed fibroblasts were stimulated several-fold by serum. In NIH 3T3 cells carrying a glucocorticoid-inducible v-Ha-ras gene, a close correlation was found between the expression of p21ras and the loss of PDGF-stimulated [3H]InsP1 accumulation. In contrast to this ras-induced desensitization to PDGF, ras-transformed NIH 3T3 cells exhibited an enhanced sensitivity to bradykinin; this effect was associated with an elevated level of high-affinity [3H]bradykinin binding. We propose that a ras gene product (p21) can, directly or indirectly, influence growth factor-stimulated phosphoinositide hydrolysis, as well as DNA synthesis, via alterations in the properties of specific growth factor receptors.

Topics: Animals; Bradykinin; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; DNA Replication; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Mice; Oncogenes; Phosphatidylinositols; Platelet-Derived Growth Factor; Rats; Receptors, Bradykinin; Receptors, Cell Surface; Receptors, Neurotransmitter; Receptors, Platelet-Derived Growth Factor

Steady-state cellular levels of phosphatidylinositol-4,5-bisphosphate (PIP2), 1,2-diacylglycerol (DAG), and inositol phosphates have been measured in two different fibroblast cell lines (NIH 3T3 and NRK cells) before and after transformation with three different ras genes. At high cell density the ratio of DAG to PIP2 was 2.5- to 3-fold higher in the ras-transformed cells than in their untransformed counterparts. The sum of the water-soluble breakdown products of the polyphosphoinositides, inositol-1,4-bisphosphate and inositol-1,4,5-trisphosphate, was also elevated in ras-transformed NRK cells compared with nontransformed NRK cells. These findings suggest that the ras (p21) protein may act by affecting these levels, possibly as a regulatory element in the PIP2 breakdown pathway.

Topics: Animals; Cell Line; Cell Transformation, Neoplastic; Diglycerides; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Oncogenes; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Rats

Topics: 1-Phosphatidylinositol 4-Kinase; Avian Sarcoma Viruses; Cell Transformation, Neoplastic; Diglycerides; Genes, Viral; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Oncogenes; Phosphatidylinositol Phosphates; Phosphatidylinositols; Phosphorylation; Phosphotransferases