inositol-1-4-5-trisphosphate and Carcinoma--Squamous-Cell

Using patch clamp and ion-selective fluorescence dye techniques, we investigated the influence of actin cytoskeleton rearrangements on the activity of calcium entry channels in plasma membrane of human carcinoma A431 cells. It is shown that disruption of actin microfilaments by cytohalasin D has no significant effect on calcium release from the stores and its entry from the extracellular space. It also does not interfere with the activation of inositol 1,4,5-trisphosphate (IP3)-dependent high-selective low-conductance calcium channels Imin. The treatment of cells with calyculin A induces the formation of actin filament layer beneath plasma membrane and also inhibits Imin activation and calcium entry through the plasma membrane, though calcium efflux from the stores was nearly unchanged. Thus, it is concluded that calcium signalling in A431 cells can be modulated by actin cytoskeleton rearrangements, and may be well described in terms of "conformational coupling" model.

Topics: Actins; Calcium; Calcium Channel Agonists; Calcium Channels; Calcium Signaling; Carcinoma, Squamous Cell; Cell Line; Cell Membrane; Cytochalasin D; Cytoskeleton; Electric Conductivity; Extracellular Space; Humans; Inositol 1,4,5-Trisphosphate; Ion Channel Gating; Marine Toxins; Oxazoles; Patch-Clamp Techniques; Thapsigargin; Tumor Cells, Cultured

Accelerated cellular repopulation has been described as a response of tumors to fractionated irradiation in both normal tissue and tumor systems. To identify the mechanisms by which cells enhance their proliferative rate in response to clinically used doses of ionizing radiation (IR) we have studied human mammary and squamous carcinoma cells which are autocrine growth regulated by the epidermal growth factor receptor (EGFR) and its ligands, transforming growth factor-alpha and EGF. Both EGF and IR induced EGFR autophosphorylation, comparable levels of phospholipase C gamma activation as measured by inositol-1,4,5-triphosphate production, and as a consequence oscillations in cytosolic [Ca2+]. Activities of Raf-1 and mitogen-activated protein kinase (MAPK) were also stimulated by EGF and IR by Ca(2+)-dependent mechanisms. All these responses to EGF and IR were dependent upon activation of EGFR as judged by the use of the specific inhibitor of EGFR autophosphorylation, tyrphostin AG1478. Importantly, IR-induced proliferation of A431 cells was also inhibited by AG1478. This is the first report which demonstrates a link between IR-induced activation of proliferative signal transduction pathways and enhanced proliferation. We propose that accelerated repopulation of tumors whose growth is regulated by EGFR is initiated by an IR-induced EGFR activation mechanism that mimics the effects of growth factors.

Topics: Breast Neoplasms; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Carcinoma, Squamous Cell; Cell Division; Enzyme Activation; Enzyme Inhibitors; ErbB Receptors; Humans; Inositol 1,4,5-Trisphosphate; Isoenzymes; Nitriles; Phospholipase C gamma; Phosphotyrosine; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-raf; Quinazolines; Radiation, Ionizing; Receptor Protein-Tyrosine Kinases; Signal Transduction; Tumor Cells, Cultured; Type C Phospholipases; Tyrphostins

Application of 0.2-15.0 mM inositol 1,4,5-trisphosphate (IP3) to excised plasma membrane patches from A 431 cells induced activity of low conductance channels that were highly selective for Ca2+ and Ba2+. With 100 mM Ca2+ or 105 mM Ba2+, the channel conductance was 1.1 pS for the minor conductance substrate. The dose response curve gave an apparent binding constant of 0.18 microM IP3. The channel open probability displayed a strong potential dependence: it decreased markedly upon depolarization, and the half-maximum value was achieved at 73 mV. The dependence of channel activity on the intracellular free Ca2+ concentration was bell shaped, but markedly different from that reported for endoplasmic IP3 receptor. The same channels were detected in intact cells upon application of calcium mobilizing reagents. The activity induced in cell attached mode disappeared after patch excision but could be resumed by application of IP3. The data obtained argue for IP3 acting directly on plasma membrane Ca2+ channels.

Topics: Barium; Bradykinin; Calcium; Calcium Channel Agonists; Calcium Channels; Carcinoma, Squamous Cell; Cell Membrane; Dose-Response Relationship, Drug; Electric Conductivity; Heparin; Humans; Inositol 1,4,5-Trisphosphate; Membrane Potentials; Patch-Clamp Techniques; Stimulation, Chemical; Tumor Cells, Cultured; Uridine Triphosphate

A midregion fragment of PTH-related protein (PTHrP), which is intensively conserved across species, has been identified as a secretory product of several different cell types, including keratinocytes and squamous carcinomas. As recent data suggest that a midregion PTHrP fragment may be biologically active, we hypothesized that midregion PTHrPs interact with unique cell surface receptors that mediate autocrine or paracrine action. Dose-dependent transient elevations in intracellular calcium ([Ca2-]i) were observed in fura-2-loaded SqCC/Y1 squamous carcinoma cells exposed to human (h) PTHrP-(67-86)NH2, [Tyr36]hPTHrP-(1-36)NH2, and hPTHrP-(1-141) at concentrations ranging from 1 pM to 1 microM. The effects of maximal stimulatory concentrations of [Tyr36]PTHrP-(1-36)NH2 and PTHrP-(67-86)NH2 on [Ca2+]i were additive. The inhibitory PTH analog, [D-Trp12,Tyr34]bovine PTH-(7-34)NH2, attenuated the [Ca2+]i response to [Tyr36]hPTHrP-(1-36)NH2, but not that to PTHrP-(67-86)NH2. These data suggest that PTHrP-(67-86)NH2 activates a different receptor pathway in SqCC/Y1 cells from the one activated by [Tyr36]hPTHrP-(1-36)NH2. Radiolabeled PTHrP-(67-86)NH2 did not bind to SqCC/Y1 cells, and PTHrP-(67-86)NH2 did not compete for binding of 125I-labeled [Tyr36]PTHrP-(1-36)NH2 to PTH/PTHrP receptors on SaOS-2 osteosarcoma cells. Activation of the phospholipase C pathway by PTHrP-(67-86)NH2 was confirmed by exposing SqCC/Y1 cells to peptide for 1 min and measuring the accumulation of inositol trisphosphates. PTHrP-(67-86)NH2 treatment (100 nM) resulted in maximal stimulation of inositol trisphosphates of 3.1 +/- 0.1-fold over the control value, with an EC50 of 1.5 +/- 1.2 nm. In contrast, PTHrP-(67-86)NH2 (0.1 nM to 1 microM) did not stimulate adenylyl cyclase in SqCC/Y1 cells despite vigorous stimulation of cAMP formation by isoproterenol (1 microM) to 66-fold over the basal value. To determine whether messenger RNA (mRNA) prepared from SqCC/Y1 cells would direct the translation of a receptor protein that mediated a [Ca2+]i response to PTHrP-(67-86)NH2, we performed expression studies in Xenopus oocytes. Fluo-3 fluorescence in Xenopus oocytes expressing SqCC/Y1 mRNA was visualized by confocal video microscopy after exposure to 1 microM PTHrP-(67-86)NH2. Clear increases in [Ca2+]i were detected in mRNA-injected, but not in sham-injected, oocytes. Finally, we examined the effect of PTHrP-(67-86)NH2 treatment on fibronectin secretion from SqCC/YN1 cells. A significant 3.5-fold

Topics: Animals; Biological Transport; Calcium; Carcinoma, Squamous Cell; Cyclic AMP; Cytosol; Fibronectins; Humans; Inositol 1,4,5-Trisphosphate; Oocytes; Parathyroid Hormone-Related Protein; Peptide Fragments; Phosphatidylinositols; Proteins; Receptor, Parathyroid Hormone, Type 1; Receptors, Parathyroid Hormone; Tumor Cells, Cultured; Xenopus

Cholera toxin and pertussis toxin were inhibitory to the incorporation of thymidine into A431 cells in serum-free culture. Cholera toxin enhanced the growth inhibitory effect of epidermal growth factor (EGF) on A431 cells, whereas pertussis toxin attenuated the effect. Cholera toxin increased the concentration of intracellular cyclic AMP (cAMP) to three-times the initial concentration at 120 minutes and it increased the concentration of intracellular inositol trisphosphate (IP3) rapidly but transiently. Pertussis toxin reduced the concentration of IP3 both in EGF treated and untreated A431 cells at 10 minutes. cAMP was not involved in pertussis toxin-mediated effects. In conclusion, the intracellular cAMP and IP3 concentrations in CT-treated A431 cells are compatible with previous reports regarding the growth inhibitory effects on A431 cells. The inhibitory effect of PTX on the EGF-induced increase of intracellular IP3 is thought to be compatible with the finding that PTX attenuated the EGF-induced growth inhibition.

Topics: Carcinoma, Squamous Cell; Cell Division; Cholera Toxin; Cyclic AMP; DNA; Epidermal Growth Factor; Humans; Inositol 1,4,5-Trisphosphate; Kinetics; Mitosis; Pertussis Toxin; Thymidine; Tumor Cells, Cultured; Virulence Factors, Bordetella

The precise mechanism for acantholysis after pemphigus IgG binds to the cell surface is as yet unknown, although involvement of proteinases such as plasminogen activator (PA) has been suggested. We previously reported that pemphigus IgG, but not normal nor bullous pemphigoid IgGs, caused a transient increase in intracellular calcium ([Ca++]i) and inositol 1,4,5-trisphosphate (IP3) concentration in cultured DJM-1 cells (a squamous cell carcinoma line). To clarify whether phospholipase C is involved in this process after the antibody binds to the cell surface, we examined the effects of a specific phospholipase C inhibitor (U73122) on the pemphigus IgG-induced increase in [Ca++]i, IP3, PA secretion, and cell-cell detachment in DJM-1 cells. [Ca+2]i and IP3 contents were determined with or without 30-min pre-incubation with U73122 or an inactive analogue (U73343) with fura-2 acetoxymethylester and a specific IP3 binding protein, respectively. PA activity in the culture medium was measured after various incubation periods with pemphigus IgG by two-step amidolytic assay. The detachment of cell-cell contacts was examined by detecting the retraction of keratin filament bundle from cell-cell contact points to the perinuclear region by immunofluorescence microscopy using anti-keratin antibody. Pemphigus IgG immediately increased [Ca++]i and IP3 content. PA activity in the culture medium has also been increased at 24 h after pemphigus IgG was added in association with cell-cell detachment. However, pre-incubation with U73122 (1-10 microM), but not with U73343 (10 microM), dramatically reduced the pemphigus IgG-induced increases in [Ca++]i, IP3, and PA activity and inhibited the pemphigus IgG-induced cell-cell detachment. Both U73122 and U73343 caused no effects on cell viability and IgG binding to the cell surface. These results suggest that phospholipase C plays an important role in transmembrane signaling leading to cell-cell detachment exerted by pemphigus IgG binding to the cell surface.

Topics: Calcium; Calcium Channel Blockers; Carcinoma, Squamous Cell; Cell Communication; Cell Membrane; Cell Survival; Estrenes; Humans; Immunoglobulin G; Inositol 1,4,5-Trisphosphate; Intracellular Membranes; Pemphigus; Plasminogen Activators; Pyrrolidinones; Skin Neoplasms; Tumor Cells, Cultured; Type C Phospholipases

It is still unclear what kinds of mechanisms are involved in blister formation after antibodies bind to the antigens in pemphigus and bullous pemphigoid. The effects of IgGs from pemphigus vulgaris, pemphigus foliaceus, and bullous pemphigoid sera on intracellular calcium concentration ([Ca++]i) and inositol 1,4,5-trisphosphate were examined in a human squamous cell carcinoma cell line (DJM-1 cells) and in cultured human keratinocytes to clarify whether signal transduction via calcium is involved. IgGs were purified with protein A affinity column from the sera of five pemphigus vulgaris patients, three pemphigus foliaceus patients, eight bullous pemphigoid patients, and 14 normal volunteers. Keratinocytes were cultured in Eagle's minimum essential medium containing 1.8 mM Ca++ and loaded with fura-2/AM, followed by addition of the IgGs. Subsequently, [Ca++]i was determined by measuring the fluorescence ratio (F340/F360) with videomicroscopy. Pemphigus IgGs (seven of eight cases) induced a rapid and transient increase in [Ca++]i in both the cells, whereas a [Ca++]i increase was caused by very few IgGs from bullous pemphigoid (one of eight cases) and normal sera (two of 14 cases). The pemphigus IgG-induced transient [Ca++]i increase was not affected by chelating extracellular Ca++ with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetracetic acid. In addition, monoclonal antibodies acid. In addition, monoclonal antibodies against 180-kD and 230-kD antigens did not exert this change. Pemphigus IgGs that caused a [Ca++]i increase induced rapid and transient production of inositol 1,4,5-trisphosphate, peaking at 20 seconds. These findings suggest that IgG from pemphigus induces Ca++ mobilization by inositol 1,4,5-trisphosphate from internal stores, and that mechanisms of antibody-transmitted signaling in pemphigus may differ from those in bullous pemphigoid.

Topics: Adult; Aged; Calcium; Carcinoma, Squamous Cell; Female; Humans; Immunoblotting; Immunoglobulin G; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Male; Middle Aged; Pemphigoid, Bullous; Pemphigus; Tumor Cells, Cultured

Somatostatin (SS) and its analogue inhibit the growth of some human tumors in vivo. Surprisingly, we found that SS-14 and its analogue (SMS 201-995) exhibited unexpected proliferative effects on two lines of cells, A431 and KB human epidermoid carcinoma cells, in vitro. The level of intracellular cyclic AMP, an important second messenger that controls the growth of A431 cells, was unaffected in A431 cells by either SS-14 or SMS 201-995. In contrast, SS-14 (20 nM) and SMS 201-995 (10 nM) reduced the level of intracellular inositol 1,4,5-trisphosphate rapidly but transiently.

Topics: Binding, Competitive; Carcinoma, Squamous Cell; Cell Division; Cyclic AMP; Humans; Inositol 1,4,5-Trisphosphate; Kinetics; Octreotide; Receptors, Somatostatin; Second Messenger Systems; Somatostatin; Tumor Cells, Cultured

The basal levels of inositol monophosphate, inositol bisphosphate, and inositol trisphosphate (InsP3) in A-431 cells incubated in Na(+)-Hanks' solution were, respectively, 1.23 +/- 0.18, 0.17 +/- 0.03, and 0.69 +/- 0.07% of the total radioactivity in the cell. When cells were heated, InsP3 increased in a temperature-dependent manner related to the duration of heating. The active form of InsP3, inositol 1,4,5-trisphosphate, increased 237 +/- 17% after heating (45 degrees C, 20 min) then returned to baseline within 15 min after the return to 37 degrees C. The heat-induced increase in InsP3 was not observed in the absence of extracellular Ca2+ or with amiloride treatment. Treatment with the nonhydrolyzable GTP analogue 5'-guanylylimidodiphosphate stimulated that component of the InsP3 increase due to G proteins. U-73122, an inhibitor of phospholipase C-mediated processes, blocked the increase in InsP3 resulting from heat exposure. Both pertussis toxin (30 ng/ml, 24 h), an inhibitor of G inhibitory protein, and cholera toxin (1 microgram/ml, 1 h), a stimulator of G stimulatory protein, increased InsP3 in unheated cells, and heating failed to induce a further increase, suggesting that heat activates G proteins. Likewise, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), 3-isobutyl-1-methylxanthine, Ro 20-1724, or forskolin increased InsP3 in unheated cells, and heat did not cause an additional increase. The InsP3 increase induced by 8-BrcAMP was inhibited by removal of extracellular Ca2+ or treatment with verapamil, suggesting that an influx of extracellular Ca2+ stimulates InsP3 production.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acids; Calcium; Carcinoma, Squamous Cell; Cyclic AMP; GTP-Binding Proteins; Hot Temperature; Humans; Inositol 1,4,5-Trisphosphate; Intracellular Membranes; Osmolar Concentration; Shock; Tumor Cells, Cultured; Type C Phospholipases

Recent studies indicate that viruses may influence polyphosphoinositide levels. This study has examined the effects of vaccinia virus infection on phospholipase C activity. Infection of BS-C-1 cells, an African Green Monkey kidney cell line, or A431 cells, a human carcinoma cell line, with vaccinia virus inhibits receptor-mediated phospholipase C activation. As a consequence, agonist-mediated Ca2+ mobilization in BS-C-1 cells also was inhibited by vaccinia virus infection. Alleviation of the inhibition of phospholipase C activation was observed in vaccinia virus-infected cells treated with cycloheximide without influencing uninfected cells. Treatment of infected cells with alpha-amanitin, an inhibitor of host mRNA synthesis but not virus mRNA synthesis, failed to alleviate the inhibition of phospholipase C activation. Together these results suggest that a virus-encoded gene product mediates the inhibition of phospholipase C activation without the need of a virus-induced host factor. Analysis of the processes involved in the formation of inositol (1,4,5)-trisphosphate and mobilization of intracellular Ca2+ indicate that the vaccinia virus gene product exerts its inhibitory effects at the level of phospholipase C activity. This may occur by either directly reducing the amount of phospholipase C, reducing the specific activity of phospholipase C, or by inhibiting the association of phospholipase C with its substrate, phosphatidylinositol 4,5-bisphosphate.

Topics: Adenosine Triphosphate; Amanitins; Animals; Calcium; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Viral; Chlorocebus aethiops; Cycloheximide; Enzyme Activation; Humans; Inositol; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kidney; Kinetics; RNA, Messenger; Tumor Cells, Cultured; Type C Phospholipases; Vaccinia virus

Monoclonal antibody 425 binds to a protein epitope of the human EGF receptor and blocks EGF dependent functions such as EGF receptor phosphorylation and mitogenesis (1). We now show that MAb 425 blocks TGF alpha-induced second messenger signals, namely inositol 1,4,5 triphosphate and Ca2+ in two carcinoma cell lines, A 431 and SW-948. In this study we have further characterized the specificity of this antibody for inhibiting TGF alpha induced mitogenesis in MRC-5, a EGF-receptor expressing fibroblast cell line.

Topics: Antibodies, Monoclonal; Calcium; Carcinoma, Squamous Cell; Cell Line; DNA Replication; ErbB Receptors; Humans; Inositol 1,4,5-Trisphosphate; Kinetics; Second Messenger Systems; Transforming Growth Factor alpha

Exposure of A431 human epidermoid carcinoma cells to epidermal growth factor (EGF), bradykinin, and histamine resulted in a time- and concentration-dependent accumulation of the inositol phosphates (InsP) inositol monophosphate, inositol bisphosphate, and inositol trisphosphate (InsP3). Maximal concentrations of EGF (316 ng/ml; approximately 50 nM), bradykinin (1 microM), and histamine (1 mM) resulted in 3-, 6-, and 3-fold increases, respectively, in the amounts of inositol phosphates formed over a 10-min period. The K0.5 values for stimulation were approximately 10 nM, 3 nM, and 10 microM for EGF, bradykinin, and histamine, respectively. EGF and bradykinin stimulated the rapid accumulation of the two isomers of InsP3, Ins(1,3,4)P3, and Ins(1,4,5)P3 as determined by high performance liquid chromatography analysis; maximal accumulation of Ins(1,4,5)P3 occurred within 15 s. EGF and bradykinin also stimulated a rapid (maximal levels attained within 30 s after addition of hormone) and a sustained 4- and 6-fold rise, respectively, in cytosolic free Ca2+ levels as measured by Fura-2 fluorescence. EGF and bradykinin also produced a rapid, although transient, 3- and 5-fold increase, respectively, in cytosolic free Ca2+ after chelation of extracellular Ca2+ with 3 mM EGTA. These data are consistent with the idea that EGF elevates intracellular Ca2+ levels in A431 cells, at least in part, as a result of the rapid formation of Ins(1,4,5)P3 and the consequential release of Ca2+ from intracellular stores.

Topics: Bradykinin; Calcium; Carcinoma, Squamous Cell; Cell Line; Chromatography, High Pressure Liquid; Cytosol; Egtazic Acid; Epidermal Growth Factor; Histamine; Humans; Inositol 1,4,5-Trisphosphate; Inositol Phosphates; Kinetics; Sugar Phosphates