calcimycin and Carcinoma--Squamous-Cell

Tamoxifen is an estrogen receptor (ER) antagonist used as first-line chemotherapy in breast cancer. Recent studies suggest that tamoxifen may be effective not only for ER‑positive but also for ER‑negative cancer cases. The aim of the present study was to investigate the antiproliferative effect of tamoxifen against human non‑melanoma skin cancer cells. Tamoxifen inhibited the proliferation of the skin squamous cell carcinoma (SCC) cell lines A431, DJM‑1 and HSC‑1. A431 cells did not express ER‑α or -β, suggesting that tamoxifen may exert antiproliferative effects on skin SCC cells via a non‑ER‑mediated pathway. Tamoxifen increased the intracellular calcium concentration of skin SCC cells, and this increase in intracellular calcium concentration by calcium ionophore A23187 suppressed the proliferation of skin SCC cells. These data indicate that tamoxifen inhibited the proliferation of human skin SCC cells via increasing intracellular calcium concentration. Voltage-gated calcium channels and non‑selective cation channels are involved in the increase in intracellular calcium concentration induced by tamoxifen. The broad-spectrum protein kinase C (PKC) inhibitor phloretin significantly attenuated the antiproliferative effect of tamoxifen on skin SCC cells. From these data, it may be concluded that tamoxifen inhibits the proliferation of skin SCC cells by induction of extracellular calcium influx via calcium channels in the plasma membrane and by subsequent activation of PKC.

Topics: Antineoplastic Agents, Hormonal; Calcimycin; Calcium; Calcium Channels; Carcinoma, Squamous Cell; Cell Line, Tumor; Cell Membrane; Cell Proliferation; Humans; Phloretin; Protein Kinase C; Protein Kinase Inhibitors; Skin Neoplasms; Tamoxifen

We investigated the mechanisms by which caffeic acid phenethyl ester (CAPE), a phenolic antioxidant, inhibited the stimulation of prostaglandin (PG) synthesis in cultured human oral epithelial cells and in an animal model of acute inflammation. Treatment of cells with CAPE (2.5 microg/ml) suppressed phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA) and calcium ionophore (A23187)-mediated induction of PGE2 synthesis. This relatively low concentration of CAPE did not affect amounts of cyclooxygenase (COX) enzymes. CAPE nonselectively inhibited the activities of baculovirus-expressed hCOX-1 and hCOX-2 enzymes. TPA- and A23187-stimulated release of arachidonic acid from membrane phospholipids was also suppressed by CAPE (4-8 microg/ml). Higher concentrations of CAPE (10-20 microg/ml) suppressed the induction of COX-2 mRNA and protein mediated by TPA. Transient transfections using human COX-2 promoter deletion constructs were performed; the effects of TPA and CAPE were localized to a 124-bp region of the COX-2 promoter. In the rat carrageenan air pouch model of inflammation, CAPE (10-100 mg/kg) caused dose-dependent suppression of PG synthesis. Amounts of COX-2 in the pouch were markedly suppressed by 100 mg/kg CAPE but were unaffected by indomethacin. These data are important for understanding the anticancer and anti-inflammatory properties of CAPE.

Topics: Air; Animals; Anti-Inflammatory Agents, Non-Steroidal; Anticarcinogenic Agents; Arachidonic Acids; Caffeic Acids; Calcimycin; Carcinoma, Squamous Cell; Carrageenan; Cell Membrane; Cyclooxygenase 2; Cyclooxygenase 2 Inhibitors; Cyclooxygenase Inhibitors; Dinoprostone; Enzyme Activation; Enzyme Induction; Epithelial Cells; Genetic Vectors; Humans; Indomethacin; Inflammation; Ionophores; Isoenzymes; Male; Membrane Lipids; Membrane Proteins; Mouth Mucosa; Nucleopolyhedroviruses; Phenylethyl Alcohol; Phospholipids; Promoter Regions, Genetic; Prostaglandin-Endoperoxide Synthases; Rats; Rats, Inbred Lew; Recombinant Fusion Proteins; Tetradecanoylphorbol Acetate; Transfection; Tumor Cells, Cultured

The tachyzoite of Toxoplasma gondii must successfully invade a host cell before it can replicate. Depletion of the Ca2+ in the external medium (EGTA) reduced tachyzoite invasion, suggesting that the initial tachyzoite-host cell interaction is Ca2+ dependent. The interaction of tachyzoites with host cells was also inhibited by Ca2+ channel blockers (verapamil) and calmodulin antagonists (trifluoperazine, calmidazolium). The calmodulin concentrated at the apical end of the tachyzoite could be involved in cytoskeleton movement and conoid extrusion. Invasion also depends on changes in tachyzoite cytosolic calcium. Depletion of Ca2+ with A23187+EGTA and release of Ca2+ from intratachyzoite pools (nuclear and perinuclear areas) inhibited invasion. In contrast, Ca-ionophore and thapsigargin which increase host cell cytosolic Ca2+, significantly decreased tachyzoite invasion. We therefore suggest that the effect of the drug is significantly different from the localized Ca2+ signal that is produced after parasite attachment to its host cell receptors and leads to its internalization into the host cell.

Topics: Animals; Calcimycin; Calcium; Calcium Channel Blockers; Calmodulin; Carcinoma, Squamous Cell; Cytosol; Epithelial Cells; Humans; Imidazoles; Ionophores; Toxoplasma; Trifluoperazine; Tumor Cells, Cultured; Verapamil

Topics: 12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; Adenocarcinoma; Calcimycin; Carcinoma, Small Cell; Carcinoma, Squamous Cell; Cell Communication; Cell Line; Coculture Techniques; Erythrocytes; Humans; Hydroxyeicosatetraenoic Acids; Leukotriene B4; Lung Neoplasms; Models, Biological; Neutrophils; Phospholipases A; Phospholipases A2; Tumor Cells, Cultured

Transforming growth factor alpha (TGF-alpha) is biosynthesized as a membrane-bound precursor protein, pro-TGF-alpha, that undergoes sequential endoproteolytic cleavages to release a soluble form of the factor. In the present study, we have analyzed the biosynthesis and regulation of TGF-alpha production in human tumor-derived cell lines that endogenously express pro-TGF-alpha and the epidermal growth factor (EGF) receptor. These cells biosynthesized membrane-anchored forms of the TGF-alpha that accumulated on the cell surface. Membrane-bound pro-TGF-alpha interacted with the EGF receptor, and complexes of receptor and pro-TGF-alpha contained tyrosine-phosphorylated receptor. Activation of the EGF receptor by soluble EGF or TGF-alpha had a dual effect on TGF-alpha production: an increase in pro-TGF-alpha mRNA levels and an increase in pro-TGF-alpha cleavage. These effects were largely prevented by preincubation with an anti-EGF receptor monoclonal antibody that blocked ligand binding. Growth factor autoinduction of cleavage could be stimulated by several second messenger pathways that are activated by the EGF receptor, including protein kinase C and intracellular calcium, and by other alternative mechanisms. EGF-stimulated cleavage of pro-TGF-alpha could be partially blocked by inhibition of these second messenger pathways. These results suggest that juxtacrine stimulation takes place in human tumor cells that coexpress both the EGF receptor and membrane-anchored TGF-alpha and that TGF-alpha is able to induce its own endoproteolytic cleavage by activating the EGF receptor.

Topics: Animals; Antibodies; Autoradiography; Calcimycin; Carcinoma, Squamous Cell; Cell Membrane; CHO Cells; Cricetinae; Cysteine; ErbB Receptors; Female; Gene Expression Regulation, Neoplastic; Humans; Male; Protein Precursors; Protein Processing, Post-Translational; Rats; Recombinant Proteins; Sulfur Radioisotopes; Tetradecanoylphorbol Acetate; Transfection; Transforming Growth Factor alpha; Tumor Cells, Cultured

Non-small cell lung cancer (NSCLC) is fatal in approximately 90% of all cases due to the failure of systemic therapy, secondary to resistance to chemotherapy. In such malignancies new therapeutic paradigms are needed. One such approach takes advantage of normal physiologic growth regulatory mechanisms, such as terminal cellular differentiation or apoptosis. Suramin, as an antineoplastic drug, has shown efficacy in the treatment of prostate cancer and is capable of promoting differentiation in several human cancer cell lines. Little is known about the differentiating effects of suramin in lung cancer. In the present investigation we evaluated the ability of suramin to induce cross-linked envelope (CLE) formation, as a common marker for squamous differentiation and apoptosis, in three representative human non-small cell lung cancer cell lines: NCI-H226 (squamous), NCI-H358 (bronchoalveolar [adenocarcinoma]), and NCI-H596 (adenosquamous). Among agents that we have tested, suramin demonstrated the unique ability to induce spontaneous CLE formation in the two cell lines with squamous features, NCI-H226 and NCI-H596. Suramin induced CLE formation was accompanied by DNA fragmentation, a marker for apoptosis, in NCI-H596 and NCI-H358, but not in NCI-H226. Stimulation of CLE formation by suramin correlated with the rapid induction of both type II transglutaminase (TG) activity and involucrin expression. These parameters were protein synthesis independent, suggesting posttranslational mechanisms of suramin activity. Induction of differentiation/apoptosis markers by suramin did not correlate with its effect on growth. Modulation of signal transduction is a likely candidate mechanism for suramin activity in lung cancer. The relationship between growth, squamous differentiation, and apoptosis is considered.

Topics: Adenocarcinoma; Antineoplastic Agents; Apoptosis; Calcimycin; Carcinoma, Adenosquamous; Carcinoma, Squamous Cell; Cell Differentiation; Cell Division; DNA Fragmentation; Enzyme Inhibitors; Humans; Ionophores; Lung Neoplasms; Neoplasm Proteins; Protein Kinase C; Protein Precursors; Putrescine; Suramin; Transglutaminases; Tumor Cells, Cultured

The [(3)H]inositol incorporation into the membrane fraction of A-431 human epidermoid carcinoma cells was markedly increased by stimulation of the cells with either epidermal growth factor (EGF), ATP, bradykinin, or a calcium ionophore A23187 in the presence of 1 mM extracellular calcium ions; most incorporated [(3)H]inositol was found to have accumulated as phosphatidylinositol (PI). The EGF- and ATP-stimulated PI synthesis was inhibited by two protein kinase C inhibitors, staurosporine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), and an intracellular calcium chelator, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM), but not by the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7). Pretreatment of cells with pertussis toxin (IAP, islet-activating protein) inhibited the PI synthesis, [Ca(2+)]i elevation, and inositol trisphosphate (IP(3)) production by ATP, suggesting that the phospholipase C(PLC) system coupled with IAP-sensitive G protein is involved in the ATP-stimulated PI synthesis. On the other hand, the ATP stimulation increased the release of [(3)H]choline and [(32)P)phosphatidic acid (PA) from radiolabeled cells, and such release was not inhibited by IAP. In the presence of n-butyl alcohol, which prevents the production of PA by generation of phosphatidylbutanol, the ATP-stimulated PI synthesis was reduced. Because n-butyl alcohol did not inhibit IP(3) production and [Ca(2+)]i elevation, this fact suggests that the lAP-insensitive PLD system is involved in the ATP-stimulated PI synthesis. In A-431 cells, the stimulation of P(2)-purinergic receptors appears to activate the IAP-sensitive PLC system and IAP-insensitive PLD system, both of which are essential for the stimulation of PI synthesis. The present results imply the general prospect that ligand stimulation, which mobilizes second messengers and consumes their precursors, simultaneously provokes the pathway to synthesize and salvage the second messenger precursors as well.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Bradykinin; Calcimycin; Calcium; Carcinoma, Squamous Cell; Chelating Agents; Choline; Diacylglycerol Kinase; Egtazic Acid; Enzyme Activation; Enzyme Inhibitors; Epidermal Growth Factor; ErbB Receptors; Humans; Ionophores; Neoplasm Proteins; Pertussis Toxin; Phosphatidic Acids; Phosphatidylinositol Diacylglycerol-Lyase; Phosphatidylinositols; Phospholipase D; Phosphotransferases (Alcohol Group Acceptor); Pyrimidinones; Receptors, Purinergic P2; Signal Transduction; Staurosporine; Sulfonamides; Thiazoles; Tumor Cells, Cultured; Type C Phospholipases; Virulence Factors, Bordetella

Epidermal growth factor (EGF) induces rapid actin filament assembly in the membrane skeleton of A431 cells, leading to a approximately 30% rise in cellular filamentous actin levels. EGF-induced actin polymerization depends upon EGF receptor (EGFR) tyrosine kinase activity, since the selective tyrosine kinase inhibitor AG213 abolishes EGF-induced actin polymerization. In accordance, confocal laser scanning microscopy shows that newly assembled actin filaments localize selectively to the tyrosine-phosphorylated EGFR in the plasma membrane, since actin polymerization is not observed at the internalized tyrosine-phosphorylated EGFR. Actin binding proteins (ABP's) are generally believed to regulate actin filament assembly. Ca2+ is known as one of the important regulatory factors for the activity of ABP's in vitro [15]. Therefore, we investigated the importance of the EGF-induced transient rise in [Ca2+]i for the regulation of actin polymerization in vivo. Continuous high [Ca2+]i in the millimolar range induces a prominent rise in cellular filamentous actin levels to approximately 50% over control cells. However, actin polymerization is unimpaired under conditions which effectively block the EGF-induced [Ca2+]i transient. These data demonstrate that EGF-induced actin polymerization localizes to the activated EGFR in the membrane skeleton, independent of the cytosolic free calcium transient.

Topics: Actins; Adenosine Triphosphate; Calcimycin; Calcium; Carcinoma, Squamous Cell; Catechols; Cell Line; Cell Membrane; Cytosol; Egtazic Acid; Epidermal Growth Factor; ErbB Receptors; Fluorescent Antibody Technique; Humans; Kinetics; Macromolecular Substances; Microscopy, Confocal; Nitriles; Phosphorylation; Skin Neoplasms; Time Factors; Tumor Cells, Cultured; Tyrphostins

The regulation of IL-6 mRNA expression was studied in human blood monocytes and in the human epidermoid carcinoma cell line HEp-2. In human monocytes phorbol-12-myristate 13-acetate (PMA) did not induce IL-6 but it increased IL-1 beta and IL-8 mRNA levels. Furthermore, in monocytes, protein kinase C (PKC) activation by PMA even reduced IL-1-induced IL-6 mRNA, and IL-1-induced IL-6 synthesis was increased by the PKC inhibitor staurosporine. IL-6 synthesis in HEp-2 cells was induced by IL-1, PMA, and calcium ionophore A 23187 but not by dibutyryl-cAMP. PMA-, but not IL-1-induced IL-6 synthesis in HEp-2 cells was inhibited by staurosporine. PMA pretreatment of HEp-2 cells abolished PMA-induced IL-6 but the IL-1 effect was not reduced. These data indicate that IL-6 can be induced by a PKC-independent pathway in monocytes and HEp-2 cells. In monocytes PKC activation does not induce IL-6 and PMA interferes with the IL-1 effect. Transcription factors known to be involved with the regulation of IL-6 expression were studied by gel retardation assays. NF-IL-6 and AP-1 activity were constitutively expressed in monocytes and HEp-2 cells under conditions where IL-6 mRNA was not detectable and levels did not change in response to stimulation by IL-1 or PMA. In contrast, NF-kB was increased by both IL-1 and PMA, but only the effect of PMA, and not that of IL-1, was inhibited by staurosporine. In summary, these results show tissue-specific differences in the regulation of IL-6 expression. Induction of IL-6 in monocytes is PKC independent. In the epithelial cell line HEp-2 IL-6 is inducible by PKC as well as by a PKC-independent pathway.

Topics: Alkaloids; Base Sequence; Calcimycin; Carcinoma, Squamous Cell; DNA-Binding Proteins; Gene Expression Regulation; Interleukin-1; Interleukin-6; Laryngeal Neoplasms; Lipopolysaccharides; Molecular Sequence Data; Monocytes; NF-kappa B; Organ Specificity; Polymerase Chain Reaction; Protein Kinase C; Signal Transduction; Staurosporine; Tetradecanoylphorbol Acetate; Transcription Factors; Tumor Cells, Cultured

Epidermal growth factor (EGF) activates a well characterized signal transduction cascade in human A431 epidermoid carcinoma cells. The influence of gravity on EGF-induced EGF-receptor clustering and early gene expression as well as on actin polymerization and actin organization have been investigated. Different signalling pathways induced by the agents TPA, forskolin and A23187 that activate gene expression were tested for sensitivity to gravity. EGF-induced c-fos and c-jun expression were decreased in microgravity. However, constitutive beta-2 microglobulin expression remained unaltered. Under simulated weightlessness conditions EGF- and TPA-induced c-fos expression was decreased, while forskolin- and A23187-induced c-fos expression was independent of the gravity conditions. These results suggest that gravity affects specific signalling pathways. Preliminary results indicate the EGF-induced EGF-receptor clustering remained unaltered irrespective of the gravity conditions. Furthermore, the relative filamentous actin content of steady state A431 cells was enhanced under microgravity conditions and actin filament organization was altered. Under simulated weightlessness actin filament organization in steady state cells as well as in EGF-treated cells was altered as compared to the 1 G reference experiment. Interestingly the microtubule and keratin organization in untreated cells showed no difference with the normal gravity samples. This indicates that gravity may affect specific components of the signal transduction circuitry.

Topics: Calcimycin; Carcinogens; Carcinoma, Squamous Cell; Colforsin; Epidermal Growth Factor; Gene Expression Regulation, Neoplastic; Gravitation; Humans; Ionophores; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Rotation; Signal Transduction; Space Flight; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Weightlessness; Weightlessness Simulation

Keratinocyte intercellular adhesion molecule (ICAM)-I expression is induced by interferon (IFN)-gamma. It has been previously reported that IFN-beta suppresses IFN-gamma-induced ICAM-I expression in A431 cells, a human squamous cell carcinoma cell line. In this study, the suppression mechanisms were investigated at the post second messenger level. Both 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophore (A23187) induce ICAM-I expression in A431 cells. ICAM-I expression induced by either was not suppressed with cotreatment with IFN-beta. Furthermore, IFN-beta did not inhibit the translocation of protein kinase C (PKC) by TPA. It appears that the pathways involved in ICAM-I expression induced by activation of PKC or increased in intracellular Ca++ are not affected by IFN-beta.

Topics: Calcimycin; Carcinoma, Squamous Cell; Cell Adhesion Molecules; Cell Line; Enzyme Activation; Humans; In Vitro Techniques; Intercellular Adhesion Molecule-1; Interferon-beta; Keratinocytes; Protein Kinase C; Second Messenger Systems; Skin Neoplasms; Tetradecanoylphorbol Acetate

The CaSki cell line derived from an epidermoid carcinoma of the uterine cervix produces and releases a tumor associated-antigen, TA-4. The authors have already reported that EGF stimulated the production and secretion of TA-4 by the CaSki cells. EGF receptor is known to be one of the proteins phosphorylated by C-kinase. In order to elucidate a possible role of signal transduction systems (cAMP-A-kinase, diacyglycerol-C-kinase and Ca(2+)-calmodulin) in the regulation of TA-4 production and secretion by human cervical epidermoid carcinoma cells, the effects of cholera toxin (CT), an activator of adenylate cyclase, phorbol 12-myristate 13-acetate (PMA), a protein kinase C activator, and Ca2+ ionophore A23187, an activator of Ca2+ modulation on TA-4 production and secretion by CaSki cells were evaluated. TA-4 in the cultured cells and media were measured with a SCC RIA-Kit. The addition of PMA or Ca2+ ionophore to the medium caused increases in the cellular levels of TA-4 and TA-4 levels in the medium in a dose-dependent manner shortly after the addition. Combined treatment with PMA and Ca2+ ionophore did not cause additive increases in TA-4 levels in the cells and medium compared to the treatment with PMA alone or Ca2+ ionophore alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antigens, Neoplasm; Calcimycin; Carcinoma, Squamous Cell; Cholera Toxin; Female; Humans; Serpins; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Uterine Cervical Neoplasms

The phospholipase C (PLC)-mediated hydrolysis of membrane phosphoinositides is an important signal transduction pathway coupled to the cell-surface receptors for several hormones and growth factors. In addition, PLC activity can be modulated by changes in intracellular calcium and activation of GTP binding proteins. In this report, differential activation of PLC in the human keratinocyte cell line SCC-12F was studied as judged by specific patterns of inositol phosphate formation. Several hormones and growth factors previously shown to stimulate PLC in a variety of cell types were screened for activity in SCC-12F cells. Only bradykinin was active, stimulating the PLC-dependent generation of inositol (1,4,5) triphosphate (Ins(1,4,5)P3). Ins(1,4,5)P3 was rapidly metabolized to inositol(1,4)biphosphate (Ins(1,4)P2) and inositol(1,3,4,5)tetrakisphosphate (Ins(1,3,4,5)P4), and subsequently degraded to inositol monophosphates. The response elicited by bradykinin was concentration dependent (EC50 value of 50 nM), suggesting involvement of a specific bradykinin receptor. Treatment of these cells with the calcium ionophore A23187 appeared to result in the direct formation of Ins(1,4)P2 without Ins(1,4,5)P3 as precursor. Treatment of the cells with AIF4-, a putative activator of GTP binding proteins, resulted in the generation of inositol monophosphates as the major metabolites in the absence of detectable Ins(1,4,5)P3 formation. Taken together, these observations suggest that the PLC complex present in SCC-12F cells can be differentially activated to yield either Ins(1,4,5)P3, Ins(1,4)P2, or InsP. The observed effects may be due to a direct PLC-dependent hydrolysis of the appropriate membrane phosphoinositide.

Topics: Bradykinin; Calcimycin; Calcium; Carcinoma, Squamous Cell; Chromatography, High Pressure Liquid; Growth Substances; Humans; Inositol; Inositol Phosphates; Kinetics; Skin Neoplasms; Sodium Fluoride; Tritium

The intracellular signal transduction mechanism leading to desmosome formation in low-calcium-grown keratinocytes after addition of calcium to the medium was studied by immunofluorescence using antibodies to desmoplakins I and II (cytoplasmic desmosomal proteins) and by electron microscopy before and after addition of calcium; protein kinase C (PKC) activators 12-O-tetradecanoylphorbol-13-acetate (TPA), phorbol-12,13-dibutyrate (PDBu), and 1,2-dioctanoylglycerol (DOG); calcium ionophore A23187; selective PKC inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine; and a Ca2+/calmodulin-dependent kinase inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). In previous studies using a low-calcium-grown human epidermal squamous cell carcinoma, we have shown that an increase in extracellular Ca2+ caused a four-fold increase in PKC activity and addition of TPA (10 ng/ml) induced a transient increase in membrane-bound PKC activity in association with cell-cell contact formation. The present study showed that TPA (10 ng/ml). PDBu (10 ng/ml), and DOG (1 mg/ml) induced a rapid cell-cell contact and redistribution of desmoplakins from cytoplasm to the plasma membrane with desmosome formation within 60-120 min, which was similar, although less marked, to the effect of increased Ca2+. The TPA-induced desmosome formation was inhibited by selective PKC inhibitors, H-7 (20 microM) or staurosporine (100 nM). On the other hand, calcium ionophore A23187 induced only a temporary increase in the number of desmoplakin-containing fluorescent spots in the cytoplasm and a temporary cell-cell attachment without desmosome formation. The calcium-induced desmosome formation was partially inhibited by 20-100 microM H-7 or 100 nM staurosporine; however, it was not inhibited by W-7 at a concentration of 25 microM, at which this agent selectively inhibits calmodulin-dependent protein kinase. These results suggest that PKC activation plays an important role in desmoplakin translocation from the cytoplasm to the plasma membrane as one of the processes of calcium-induced desmosome formation.

Topics: Calcimycin; Calcium; Carcinoma, Squamous Cell; Cell Membrane; Cytoskeletal Proteins; Cytosol; Desmoplakins; Desmosomes; Diglycerides; Dose-Response Relationship, Drug; Humans; Phorbol 12,13-Dibutyrate; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Phorbol esters specifically reduce the binding of epidermal growth factor to surface receptors in intact cells, but not when added directly to isolated membranes. We show that after treatment of intact cells with phorbol myristate acetate, 125I-EGF binding is reduced in membranes prepared subsequently. High-affinity binding of 125I-EGF is modulated by an intracellular calcium-dependent regulatory process. Preventing calcium entry with EGTA or enhancing intracellular calcium with A23187 in intact cells modulates EGF receptor affinity in membranes isolated subsequently. Also, EGTA attenuates the usual inhibition of EGF binding caused by phorbol esters. Membrane preparations do not respond to phorbol ester treatment because the calcium- and phospholipid-dependent protein kinase C is removed or inactivated during membrane isolation. Reconstitution of unresponsive membranes with purified C kinase alters phosphorylation of the EGF receptor and restores the inhibitory effect of phorbol esters on 125I-EGF binding previously observed only in intact cells. Thus, activation of the Ca++-dependent enzyme, C kinase, modulates EGF receptor affinity, possibly via altered receptor phosphorylation.

Topics: Animals; Calcimycin; Calcium; Carcinoma, Squamous Cell; Cell Line; Egtazic Acid; Epidermal Growth Factor; ErbB Receptors; Phosphorylation; Protein Kinase C; Protein Kinases; Rats; Receptors, Cell Surface; Tetradecanoylphorbol Acetate

We have studied the effects of the potent tumour promoter phorbol, 12-myristate, 13-acetate (PMA) and two non-promoting hyperplasiogenic compounds ethyl phenylpropriolate (EPP) and the divalent cation ionophore A23187 on the terminal differentiation of normal and transformed human keratinocytes using the loss of cloning efficiency and the formation of cornified envelopes as markers of the differentiated state. PMA induced terminal differentiation in a far greater proportion of normal keratinocytes than it did in the squamous cell carcinoma line SCC-27 but EPP and the calcium ionophores A23187 and Br-X537A had no such differential effect, possibly explaining the poor promoting ability of the last three compounds.

Topics: Alkynes; Calcimycin; Carcinoma, Squamous Cell; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Clone Cells; Humans; Hyperplasia; Keratins; Phorbols; Skin; Tetradecanoylphorbol Acetate

Epidermal growth factor (EGF) stimulates the incorporation of 32Pi and [3H]inositol into phosphatidylinositol (5-10-fold) in A-431 cells. EGF also stimulates the incorporation of 32Pi into phosphatidic acid (up to 10-fold). These effects are attributed to an acceleration of the turnover of phosphatidylinositol as a consequence of the binding of EGF to its membrane receptor. The extent of the phosphatidylinositol response to EGF parallels the extent of hormone binding. The phosphatidylinositol response to EGF appears to be dependent on an influx of calcium since (a) external calcium is required for the enhancement of phosphatidylinositol turnover, (2) the accumulation of 45Ca by A-431 cells is stimulated by EGF, (3) blockage of calcium influx with LaCl3 inhibits stimulation of phosphatidylinositol turnover, and (4) calcium influx via ionophore A23187 is sufficient to stimulate phosphatidylinositol turnover. Since the binding, internalization, and degradation of 125I-labeled EGF in A-431 cells are unaffected by the omission of calcium from the medium, external calcium and phosphatidylinositol turnover are not necessary for the internalization and degradation of the EGF-receptor complex.

Topics: Biological Transport, Active; Calcimycin; Calcium; Carcinoma, Squamous Cell; Cell Line; Epidermal Growth Factor; ErbB Receptors; Humans; Kinetics; Lanthanum; Magnesium; Phosphates; Phosphatidylinositols; Receptors, Cell Surface