calcimycin and Carcinoma

Downregulation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM1), a cell adhesion molecule with tumor suppressing properties has been observed in a high percentage of carcinomas of the endometrium and other malignancies. The mechanisms for the dysregulation and the role of hormones and cytokines on the expression of CEACAM1 in endometrial carcinomas is unknown. We therefore studied the effect of estradiol, medroxyprogesterone acetate (MPA), RU486, gamma-interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophore A23187 on the expression in the non-expressing endometrial tumor cell lines Hec1B and Skut1B, respectively. No induction of CEACAM1 expression was observed in Hec1B endometrial adenocarcinoma cells in response to hormones and cytokines whereas treatment with TPA and calcium ionophore A23187 resulted in the strong expression of endogenous CEACAM1 on the mRNA and protein levels. In contrast, no induction of CEACAM1 expression was observed in endometrial mixed mesenchymal Skut1B cells. Studies of other members of the CEACAM family revealed that the re-expression in Hec1B carcinoma cells is restricted to CEACAM1 suggesting a cell type-specific and cell type-independent mechanism of CEACAM1 activation via the protein kinase C (PKC) pathway. Induction of CEACAM1 expression was dependent on protein kinase C protein synthesis and luciferase reporter assays with CEACAM1 promoter constructs demonstrated that the re-expression of CEACAM1 is regulated at the transcriptional level. This is the first report demonstrating that activators of PKC are able to specifically induce the expression of CEACAM1 in human carcinoma cells and our findings may provide a basis for the therapeutic inhibition of tumor growth in malignancies in which CEACAM1 is downregulated.

Topics: Anti-Bacterial Agents; Antigens, CD; Calcimycin; Carcinoma; Cell Adhesion Molecules; Endometrial Neoplasms; Estradiol; Female; Hormone Antagonists; Humans; Interferon-gamma; Medroxyprogesterone Acetate; Mifepristone; Phorbol Esters; Protein Kinase C; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha

Prion diseases are transmissible neurodegenerative disorders characterized by extensive neuronal apoptosis and accumulation of misfolded prion protein (PrP(SC)). Recent reports indicate that PrP(SC) induces neuronal apoptosis via activation of the endoplasmic reticulum (ER) stress pathway and activation of the ER resident caspase-12. Here, we investigate the relationship between prion replication and induction of ER stress during different stages of the disease in a murine scrapie model. The first alteration observed consists of the upregulation of the ER chaperone of the glucose-regulated protein Grp58, which was detected during the presymptomatic phase and followed closely the formation of PrP(SC). An increase in Grp58 expression correlated with PrP(SC) accumulation at all stages of the disease in different brain areas, suggesting that this chaperone may play an important role in the cellular response to prion infection. Indeed, in vitro studies using N2a neuroblastoma cells demonstrated that inhibition of Grp58 expression with small interfering RNA led to a significant enhancement of PrP(SC) toxicity. Conversely, overexpression of Grp58 protected cells against PrP(SC) toxicity and decreased the rate of caspase-12 activation. Grp58 and PrP were shown to interact by coimmunoprecipitation, observing a higher interaction in cells infected with scrapie prions. Our data indicate that expression of Grp58 is an early cellular response to prion replication, acting as a neuroprotective factor against prion neurotoxicity. Our findings suggest that targeting Grp58 interaction may have applications for developing novel strategies for treatment and early diagnosis of prion diseases.

Topics: Analysis of Variance; Animals; Anti-Bacterial Agents; Blotting, Western; Brain; Calcimycin; Calcium Signaling; Carcinoma; Cell Line, Tumor; Cell Survival; Disease Models, Animal; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Immunohistochemistry; Immunoprecipitation; Membrane Proteins; Mice; Mice, Inbred C57BL; Phosphopyruvate Hydratase; Prion Diseases; Prions; Protein Disulfide-Isomerases; RNA, Small Interfering; Transfection

The purpose of this study was to determine whether expression of tissue transglutaminase (TG2) and caspase-3 proteins in drug-resistant breast carcinoma MCF-7/DOX cells would render these cells selectively susceptible to apoptotic stimuli. Despite high resistance to multidrug resistance (MDR)-related drug, doxorubicin (> or =150-fold), the MCF-7/DOX cells were extremely sensitive to apoptotic stimuli. Thus, calcium ionophore, A23187 (A23187) and the protein kinase C inhibitor staurosporine (STS) each induced rapid and time-dependent apoptosis in MCF-7/DOX cells. The apoptosis induced by either agent was accompanied by caspase-3 activation and other downstream changes that are typical of cells undergoing apoptosis. The alterations upstream of caspase-3 activation, however, such as loss in mitochondrial membrane potential (DeltaPsi), release of cytochrome c, and activation of caspase-8, and caspase-9, were detected only in STS-treated cells. The A12387 failed to induce any of the caspase-3 upstream changes, implying that A23187-induced apoptosis may utilize one or more novel upstream pathways leading to the activation of caspase 3. In summary, these data demonstrate that MCF-7/DOX cells are much more sensitive to apoptotic stimuli than previously thought and that A23187-induced apoptosis may involve some novel, yet unidentified, upstream pathway that leads to the activation of caspase-3 and other downstream events.

Topics: Antibiotics, Antineoplastic; Apoptosis; Blotting, Western; Breast Neoplasms; Calcimycin; Carcinoma; Caspase 3; Caspase 8; Caspases; Cell Line, Tumor; Cytochromes c; Doxorubicin; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme Inhibitors; Female; Humans; Ionophores; Membrane Potentials; Mitochondria; Staurosporine

The aim of this work was to study the action of leukotrienes on the growth of human mammary cancer cells MCF-7.. The growth of the cells was measured by incorporation of 3H-thymidine. The action of leukotriene (LT)B4, LTD4, LTC4, LTE4 or arachidonate (AA) was tested in human mammary cancer cells MCF-7 in vitro.. LTB4 or LTD4 but not LTC4 or LTE4 reduced significant incorporation of 3H-thymidine in MCF-7 cells up to 52% or 56% respectively, when administered in concentrations 0.1-1000 pM. Agents in concentrations of 0.01 pM or 10000 pM did not effect 3H-thymidine incorporation. We have shown, that MCF-7 cells synthesise LTB4 when treated with calcium ionophor A23187 (10 microM). Leukotriene-antagonist LY171883 (10 microM) lifts inhibitory effects of LTB4 or LTD4. Arachidonic acid (10 microM) inhibits 3H-thymidine incorporation up to 72%. 5-lipoxygenase inhibitor MK-886 (100 nM) lifts the inhibitory effect of arachidonate.. LTB4 or LTD4 inhibits MCF-7 breast cancer cell growth. LT-receptors mediate the growth-inhibitory effect of LTB4 or LTD4.

Topics: Acetophenones; Arachidonic Acid; Breast Neoplasms; Calcimycin; Carcinoma; Cell Division; Female; Humans; Indoles; Ionophores; Leukotriene Antagonists; Leukotriene B4; Leukotriene D4; Leukotrienes; Lipoxygenase Inhibitors; Osmolar Concentration; Tetrazoles; Thymidine; Tritium; Tumor Cells, Cultured

The effects of TSH and the activation of the cyclic AMP (cAMP) and Ca(2+)-phosphatidylinositol (Ca(2+)-PI) cascades on the activity and expression of the selenoenzyme thyroidal type-I iodothyronine deiodinase (ID-I) have been studied using human thyrocytes grown in primary culture. Stimulation of ID-I activity and expression was obtained with TSH and an analogue of cAMP, 8-bromo-cAMP. In the presence or absence of TSH, the addition of the phorbol ester, phorbol 12-myristate 13-acetate (PMA) together with the calcium ionophore A23187, caused a decrease in ID-I activity; a decrease in ID-I expression was also observed as assessed by cell labelling with [75Se]selenite. PMA alone had no effect on ID-I activity in the presence or absence of TSH. A23187 alone produced a small but significant reduction in ID-I activity, but only in TSH-stimulated cells. These data provide evidence that the expression of thyroidal ID-I is negatively regulated by the Ca(2+)-PI cascade, and positively regulated by the cAMP cascade.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Calcimycin; Calcium; Carcinoma; Cells, Cultured; Cyclic AMP; Dimethyl Sulfoxide; Enzyme Induction; Graves Disease; Humans; Iodide Peroxidase; Microsomes, Liver; Phosphatidylinositols; Proteins; Selenoproteins; Signal Transduction; Sodium Selenite; Tetradecanoylphorbol Acetate; Thyroid Gland; Thyroid Neoplasms; Thyroid Nodule; Thyrotropin

Three intracellular signal transduction pathways have been found to be utilized by gamma-interferon (IFN-gamma) in the induction of HLA-DR in several cell types, mainly monocytes/macrophages and B-cells: the protein kinase A (PKA); Ca(2+)-calmodulin; and protein kinase C (PKC) pathways. In this study, we investigated the role of these pathways in IFN-gamma-induced HLA-DR expression in normal and neoplastic human thyroid cells. The PKA pathway seemed to inhibit both neoplastic and normal IFN-gamma-induced HLA-DR expression; addition of thyroid-stimulating hormone to normal thyroid cells, as well as 8-bromo cyclic AMP and forskolin to normal and neoplastic cells, reduced the amount of IFN-gamma-induced HLA-DR. Moreover, H-8, a PKA inhibitor, enhanced such IFN-gamma-induced HLA-DR expression. The calcium-calmodulin pathway does not seem to play a role in IFN-gamma-induced HLA-DR expression in normal and neoplastic thyrocytes, since the Ca-ionophore A23187, EGTA, and the calmodulin antagonist, W-7, neither induced HLA-DR nor showed any effect on HLA-DR expression induced by IFN-gamma. Alone, phorbol 12-myristate 13-acetate, a PKC activator, did not induce HLA-DR on thyroid cells. However, its addition to neoplastic cells together with IFN-gamma caused a synergistic elevation of the expressed HLA-DR, whereas it significantly inhibited IFN-gamma-induced HLA-DR in normal thyrocytes. TPA had to be added before or together with IFN-gamma for optimal function. If added more than 6 h after IFN-gamma, TPA was not effective. An inactive TPA analogue did not affect HLA-DR induction, while an active analogue mimicked TPA. Staurosporine, a PKC inhibitor, reduced the TPA enhancing effect in neoplastic thyrocytes and cancelled TPA inhibition in normal cells. Moreover, when added to IFN-gamma without TPA in normal thyroid cells, staurosporine increased 3- to 4-fold the amount of HLA-DR. Thus, in normal thyroid cells the PKC pathway is activated by IFN-gamma and inhibits HLA-DR expression. In neoplastic thyrocytes, although IFN-gamma does not induce HLA-DR via PKC, this pathway augments HLA-DR expression.

Topics: Alkaloids; Calcimycin; Carcinoma; Cells, Cultured; Dose-Response Relationship, Drug; Drug Administration Schedule; Goiter; HLA-DR Antigens; Humans; Interferon-gamma; Isoquinolines; Signal Transduction; Staurosporine; Tetradecanoylphorbol Acetate; Thyroid Neoplasms; Tumor Cells, Cultured

NCI-H295 is a recently described human adrenocortical carcinoma cell line that makes a variety of steroid hormones. We sought to determine if steroidogenesis in these cells employs the same enzymes as those used in normal adrenal steroidogenesis, and if the genes encoding those enzymes exhibit characteristic responsiveness to activators of the protein kinase-A and -C pathways of intracellular second messengers. Northern blots show that NCI-H295 cells contain abundant mRNAs for three key steroidogenic enzymes, cytochrome P450scc, cytochrome P450c17, and cytochrome P450c21. These mRNAs accumulated in a time- and dose-dependent fashion in response to 8-bromo-cAMP (8Br-cAMP), forskolin, cholera toxin, and 3-isobutyl-1-methylxanthine, all activators of the protein kinase-A pathway. Nuclear run-on assays and actinomycin-D transcriptional inhibition experiments show that cAMP regulates the expression of all three genes primarily at the transcriptional level. Inhibition of protein synthesis with cycloheximide did not prevent the cAMP-induced accumulation of P450scc or P450c17 mRNAs, but did inhibit accumulation of P450c21 mRNA, suggesting that cAMP is acting through a mechanism dependent on protein synthesis to promote accumulation of P450c21 mRNA. Stimulation of the protein kinase-C pathway with phorbol ester decreased P450scc and P450c17 mRNAs, but stimulated the accumulation of P450c21 mRNA. RNase protection experiments, Northern blot hybridizations, and reverse transcription-polymerase chain reaction show that NCI-H295 cells express both the 11 beta-hydroxylase (P450c11 beta) encoded by the P450c11B1 gene and the aldosterone synthetase (P450c11AS) encoded by the P450c11B2 gene. 8Br-cAMP increased the abundance of both of these mRNAs with similar kinetics, with maximal accumulation of both after about 24 h. NCI-H295 cells also contain the mRNAs for aromatase and insulin-like growth factor-II. 8Br-cAMP increased the abundance of aromatase mRNA and decreased the abundance of IGF-II mRNA. These studies show that NCI-H295 cells express most of the enzymes needed for human adrenal steroidogenesis, and that the genes encoding these enzymes respond to stimulation of second messenger pathways in a manner similar to that of human adrenals. NCI-H295 cells appear to be a good model for studying the molecular regulation of human adrenal steroidogenesis.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adrenal Cortex; Adrenal Cortex Hormones; Adrenal Cortex Neoplasms; Adrenocorticotropic Hormone; Angiotensin II; Aromatase; Base Sequence; Calcimycin; Carcinoma; Cycloheximide; Cytochromes; Enzymes; Gene Expression Regulation, Enzymologic; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Insulin-Like Growth Factor II; Molecular Sequence Data; RNA, Messenger; Second Messenger Systems; Tetradecanoylphorbol Acetate; Transcription, Genetic; Tumor Cells, Cultured; Zona Glomerulosa

In cystic fibrosis (CF), epithelial cells are unable to normally up-regulate apical membrane Cl- secretion in response to agents which increase cyclic AMP, but they do increase Cl- secretion in response to increases in intracellular Ca2+. Since intracellular divalent cations regulate the expression of many genes, we hypothesized that mobilization of intracellular Ca2+ and/or other divalent cations might modulate not only Ca(2+)-dependent Cl- channels but also cystic fibrosis transmembrane conductance regulator (CFTR) gene expression. To evaluate this concept, HT-29 human colon carcinoma cells were cultured under various conditions designed to manipulate intracellular divalent cation concentrations and CFTR gene expression was quantified at the levels of transcription, mRNA accumulation, mRNA half-life, and protein. Exposure to the divalent cation ionophores A23187 and ionomycin (agents which increase intracellular divalent cation concentrations) caused dose- and time-dependent reductions of CFTR mRNA levels, which could be blocked by the use of Ca(2+)- and Mg(2+)-free media. Ionophore-induced CFTR gene modulation was also observed with T84 human colon carcinoma cells and freshly isolated normal human bronchial epithelial cells. Incubation of HT-29 cells with thapsigargin, an agent that releases Ca2+ from intracellular stores, or in medium containing increased extracellular concentrations of Ca2+ or Mg2+ also caused down-regulation of CFTR mRNA levels. Transcription run-on analysis showed that, parallel with the decrease in CFTR mRNA levels, A23187 reduced the rate of transcription of the CFTR gene, while CFTR mRNA transcript half-life was unaffected. Consistent with the down-regulation of CFTR gene expression, CFTR protein levels also decreased after exposure to A23187. Thus, despite the independence of Ca(2+)-dependent Cl- channels and cyclic AMP-dependent CFTR-related Cl- channels in epithelial cells, increases in intracellular divalent cation concentrations down-regulate the expression of the CFTR gene at the transcriptional level, with consequent decreases in CFTR mRNA and protein.

Topics: Calcimycin; Calcium; Carcinoma; Cations, Divalent; Colonic Neoplasms; Cystic Fibrosis; Cystic Fibrosis Transmembrane Conductance Regulator; Down-Regulation; Half-Life; Humans; Membrane Proteins; RNA, Messenger; Terpenes; Thapsigargin; Transcription, Genetic; Tumor Cells, Cultured

Based on our in vivo observation that growth of VX2 carcinoma transplanted in rabbits paralleled development of hypercalcemia, we studied the regulation of VX2 tumor growth using a clonal cell line isolated from VX2 tumor (VX2-L). VX2-L cell growth was dependent on prostaglandins released by the cultured cells into the medium, since indomethacin suppressed VX2-L growth, and prostaglandins A2, E1, E2, F1 alpha, and F2 alpha stimulated VX2-L proliferation. In contrast, prostaglandins D2 and I2 inhibited VX2-L proliferation. In contrast to previous reports, increases in extracellular calcium concentration promoted VX2-L growth not only directly but indirectly through augmentation of prostaglandin E synthesis. Antagonists of the intracellular calcium binding protein calmodulin inhibited cell replication. Increases in extracellular calcium also stimulated production of a nonprostaglandin macromolecular bone-resorbing factor. This factor may account for the hypercalcemia which we were unable to block by indomethacin. These results suggest a close relationship between VX2-L growth, prostaglandin production, and hypercalcemia. It is proposed that calcium blockers and anticalmodulin drugs might be powerful anticancer and/or antihypercalcemic agents for malignant cells such as VX2-L.

Topics: Animals; Bone Resorption; Calcimycin; Calcium; Calmodulin; Carcinoma; Cell Division; Cell Line; DNA Replication; Fibroblasts; Indomethacin; Kinetics; Nifedipine; Prostaglandins; Rabbits; Skin; Sulfonamides

The secretion of protein, like cell proliferation, is an integrated response that reflects structural organization of the cell periphery. Stimulation of protein secretion was thus utilized for comparison of integrated responses of the cell periphery in pancreatic acinar carcinoma of the rat and integrated responses in normal rat pancreas. Results of this comparison include: a) The stimulation of protein secretion in acinar carcinoma fragments by carbamylcholine chloride and cholecystokinin octapeptide, pancreatic secretagogues that interact with specific plasma membrane receptors, was only a fraction (one-fifth to one-half) of that observed in normal pancreatic minilobules. b) The Ca2+ ionophore A23187 and the cyclic nucleotide N6,O2'-dibutyryl cyclic AMP, secretagogues that act independently of specific membrane receptors, did not stimulate secretion in the acinar carcinoma. The observed quantitative and qualitative differences in protein secretion indicate fundamental differences in cell periphery organization between the normal and transformed acinar cells of pancreas.

Topics: Animals; Bucladesine; Calcimycin; Carbachol; Carcinoma; Cell Membrane; Cholecystokinin; Dose-Response Relationship, Drug; In Vitro Techniques; Pancreas; Pancreatic Neoplasms; Peptide Fragments; Proteins; Rats; Secretory Rate; Sincalide; Temperature