calcimycin and Cell-Transformation--Neoplastic

Topics: Amiloride; Animals; Calcimycin; Calcium; Carrier Proteins; Cell Differentiation; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Dimethyl Sulfoxide; Electrolytes; Erythropoiesis; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Leukemia, Experimental; Lipopolysaccharides; Lymphocytes; Lymphoma; Mice; Monensin; Ouabain; Protons; Sodium; Sodium-Hydrogen Exchangers; Sodium-Potassium-Exchanging ATPase

Treatment of serum-deprived fibroblasts with serum or growth factors results in an immediate induction of the c-fos and c-myc proto-oncogenes. Maximal levels of c-fos mRNA are detected 30 minutes after treatment and maximal levels of c-myc mRNA are detected 60 minutes after treatment. The c-fos protein is expressed at high levels for about two hours following induction, yet the cell morphology remains normal. Thus, either an extended period of c-fos expression is required for cellular transformation, or the highly modified form of the fos protein, present in stimulated cells, is biochemically different from the transforming protein and is therefore not capable of inducing transformation. In this system, growth factor treatment results in mitogenesis. However, c-fos and c-myc are also induced in A431 cells, and in subclones derived from A431 cells, treated with epidermal growth factor (EGF). No correlation was found between the effects of EGF on A431 cell proliferation and the induction of c-fos and c-myc. Interestingly, the strongest inducer of c-fos in A431 cells was the calcium ionophore A23187. Induction occurred in almost 100% of the treated cells without prior serum deprivation or growth arrest. Treatment of HL60 cells with 12-0 tetra decanoylphorbol-13-acetate (TPA), which promotes macrophage-like differentiation, also induced c-fos with a time course similar to that observed in mitogen-treated fibroblasts. Thus, in HL60 cells, c-fos induction is associated with differentiation. In normal macrophages c-fos and c-myc can also be induced by CSF-1. However, the kinetics of induction are entirely different from those in growth factor-stimulated fibroblasts. Taken together, the data suggest a more general role for c-fos and c-myc in the transduction of growth factor signals received at the cell membrane, within the nucleus.

Topics: Animals; Blood; Calcimycin; Cell Line; Cell Transformation, Neoplastic; Colony-Stimulating Factors; Gene Expression Regulation; Growth Substances; Humans; Leukemia, Myeloid, Acute; Macrophages; Proto-Oncogenes; RNA, Messenger; Tetradecanoylphorbol Acetate

To Investigate the role of protein kinase C (PKC) signal transduction in the process of HL-60 leukemic cells induced into dendritic cells (DCs) by calcium ionophore A23187.. HL-60 cells were pretreated with protein kinase C (PKC) inhibitor Bis-1 for 24 hours followed by cultured with A23187 for another 36 hours, and the morphology, immunophenotype and function of stimulating proliferation of allogeneic T cells were compared with the cells only treated with A23187.. The morphological changes, surface marker expressions and ability to stimulating the proliferation of allogeneic T cells were inhibited in DCs derived from HL-60 cells treated with PKC inhibitor Bis-1. The percentage of CD83, CD80, CD86 expressing on DCs induced by A23187 significantly decreased to (28.97 +/- 4.05)% vs. (13.23 +/- 2.15)%, (19.10 +/- 5.46)% vs. (9.70 +/- 1.69)%, (41.03 +/- 6.43)% vs. (23.37 +/- 7.50)%, respectively (P < 0.05).. The induction of HL-60 cells induced into DCs by A23187 can be inhibited by PKC inhibitor Bis-1, this suggested that PKC signal transduction pathway might be involved in the process of HL-60 leukemic cells differentiated into DCs by A23187 induction.

Topics: Calcimycin; Calcium; Cell Differentiation; Cell Transformation, Neoplastic; Dendritic Cells; HL-60 Cells; Humans; Ionophores; Protein Kinase C

To explore whether the promyelocytic leukemia cell line HL-60 may differentiate into activated dendritic cells (DCs) by A23187, a calcium ionophore.. The HL-60 cells were cultured in common medium alone or with various concentrations of A23187 (25-1 600 microg/L) and rhGM-CSF (100 microg/L). After culture for 24-96 hours, the cellular morphological change was observed under light microscope and electron microscope. Surface makers on treated HL-60 cells were analyzed by flow cytometry. The proliferation of allogeneic human T cells was detected by MTT colorimetry.. Under the condition of a suitable dose (200 microg/L) of A23187 treatment of HL-60 cells for 24 hours, the expression of CD83 molecule, a characteristic marker on DCs, was highest. The typical dendritic outgrowth appeared at a time when HL-60 cells were treated with A23187 for 72 hours. However, when HL-60 cells were treated with A23187 for 96 hours, the expressions of CD80 (B7.1), CD86 (B7.2), MHC-class II molecule and CD54 on HL-60 cells reached peak, and marked activation of allogeneic T cells occurred.. Calcium ionophore A23187 can induce the HL-60 cells to differentiate into activated DCs-like cells.

Topics: Antigens, CD; B7-1 Antigen; B7-2 Antigen; Calcimycin; CD83 Antigen; Cell Proliferation; Cell Transformation, Neoplastic; Dendritic Cells; HL-60 Cells; Humans; Immunoglobulins; Intercellular Adhesion Molecule-1; Ionophores; Membrane Glycoproteins; T-Lymphocytes

Precise control of the level of c-Myc protein is important to normal cellular homeostasis, and this is accomplished in part by degradation through the ubiquitin-proteasome pathway. The calpains are a family of calcium-dependent proteases that play important roles in proteolysis of some proteins, and their possible participation in degradation of intracellular c-Myc was therefore investigated. Activation of calpain with the cell-permeable calcium ionophore A23187 in Rat1a-myc or ts85 cells in culture induced rapid cleavage of c-Myc. This degradation was both calpain- and calcium-dependent since it was inhibited by preincubation with either the calpain-inhibitory peptide calpeptin or the calcium-chelating agent EGTA. A23187-induced c-Myc cleavage occurred in a time-dependent manner comparable to that of FAK, a known calpain substrate, and while calpeptin was able to significantly protect c-Myc from degradation, inhibitors of the proteasome or caspase proteases could not. Exposure of Rat1a-myc or ts85 cells in culture to calpeptin, or to the thiol-protease inhibitor E64d, resulted in the accumulation of c-Myc protein without an impact on ubiquitin-protein conjugates. Using an in vitro assay, calpain-mediated degradation occurred rapidly with wild-type c-Myc as the substrate, but was significantly prolonged in some c-Myc mutants with increased transforming activity derived from lymphoma patients. Those mutants with a prolonged half-life in vitro were also more resistant to A23187-induced cleavage in intact cells. These studies support a role for calpain in the control of c-Myc levels in vivo, and suggest that mutations impacting on sensitivity to calpain may contribute to c-Myc-mediated tumorigenesis.

Topics: Animals; Blotting, Western; Calcimycin; Calcium; Calpain; Cell Line; Cell Transformation, Neoplastic; Chelating Agents; Cysteine Endopeptidases; Dipeptides; Enzyme Activation; Enzyme Inhibitors; Fibroblasts; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Ionophores; Lymphoma; Mammary Neoplasms, Experimental; Mice; Multienzyme Complexes; Mutation; Peptide Hydrolases; Proteasome Endopeptidase Complex; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-myc; Rats

The unique antigen peptide pRL1 on BALB/c radiation-induced leukemia RL(male symbol)1 cells is derived from the normally untranslated 5' region of the mouse c-akt gene. Insertion of an endogenous long terminal repeat into the first coding exon of the gene resulted in the enhanced production of an altered akt protein, RL-akt, and creation of the tumor rejection antigen peptide pRL1. In this study, we constructed an RL-akt-expressing vector to investigate the transforming ability and anti-apoptotic activity of RK-akt in NIH/3T3 cells. RL-akt-expressing clones formed more colonies than did c-akt-expressing clones in soft agar and exhibited increased saturation density, a lower serum requirement for growth, and tumorigenicity on athymic nude mice. Immunoblot analysis of subcellular protein distribution showed that a considerable proportion of RL-akt was distributed in the membrane fraction. Thus, RL-akt expressed in NIH/3T3 cells appeared to behave like the v-akt oncoprotein. Furthermore, the RL-akt gene conferred resistance to the apoptosis induced by the calcium ionophore A23187 and by ultraviolet irradiation of NIH/3T3 cells. These findings indicate that the RL-akt gene is able to transform cells and exerts an anti-apoptotic effect on recipient cells, thereby implicating the gene in leukemogenesis of RL(male symbol)1 cells.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Apoptosis; Calcimycin; Cell Adhesion; Cell Division; Cell Survival; Cell Transformation, Neoplastic; Female; Male; Mice; Mice, Nude; Molecular Sequence Data; Mutagenesis, Insertional; Neoplasms, Experimental; Plasmids; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Proto-Oncogenes; Recombinant Proteins; Repetitive Sequences, Nucleic Acid; Restriction Mapping; Transfection; Ultraviolet Rays

HLA-class II molecules can be induced in low-grade non-Hodgkin B lymphoma cells by either membrane IgM cross-linking or phorbolester stimulation. The ability of phorbolesters to substitute for anti-IgM antibodies in the activation of normal and malignant human B cells has been taken as evidence for the involvement of protein kinase C (PKC) in signals transduced through membrane IgM receptors (mIgR). Here we report on freshly isolated lymphoma B cells from different patients; the cells show a distinct regulation of HLA-class II expression. In certain lymphoma cases phorbol-myristate-acetate (PMA) not only fails to up-regulate HLA-class II molecules but also inhibits anti-IgM or interleukin-4 induced class II expression. This negative signal induced by PMA seems to operate specifically in HLA-class II regulation because PMA can induce other anti-IgM mediated events like blast transformation and induction of IL-4 responsiveness at the same time. Therefore these cells support the concept of functional heterogeneity in low-grade non-Hodgkin lymphoma and may represent a differentiation stage where anti-IgM antibodies and phorbolesters influence the regulation of HLA-class II expression in a contrary direction.

Topics: Antibodies; Calcimycin; Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; Histocompatibility Antigens Class II; Humans; Immunoglobulin M; Interleukin-4; Lymphoma, B-Cell; Protein Kinase C; Receptors, Fc; Receptors, IgE; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Up-Regulation

We have examined the effects on X-ray induced malignant transformation in vitro of a number of activators and inhibitors of protein kinase C (PKC). Several of these substances were found to enhance or inhibit transformation, and the extent of the effects on transformation were found to be consistent with the potencies of the substances in activating or inhibiting PKC. Additionally, the observed transformation enhancement was found to be reversed by the presence of the anticarcinogenic protease inhibitors antipain or the Bowman-Birk inhibitor. These results suggest that activation of protein kinase C may be involved in the mechanism of in vitro X-ray induced malignant transformation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Antipain; Calcimycin; Cell Transformation, Neoplastic; Diglycerides; Enzyme Activation; Isoquinolines; Mice; Mice, Inbred C3H; Neoplasms, Radiation-Induced; Piperazines; Protein Kinase C; Sulfonamides; Trifluoperazine; Trypsin Inhibitor, Bowman-Birk Soybean

Biochemical similarities between ras proteins and the GTP-binding proteins and correlation of ras-induced cell transformation with altered transmembrane cation fluxes indicate that ras proteins may act to modulate ion channel activity. To test this idea, whole cell, tight-seal, patch-clamp recording was used to compare macroscopic currents of ras-transformed fibroblasts with currents of their nontransformed counterparts. A prominent calcium-activated, voltage-independent potassium current was observed in 83-100% of cells from three separate fibroblast lines transformed by two different oncogenic ras alleles, whereas the same current was present at much smaller amplitudes in only 0-15% of nontransformed cells. The calcium-activated potassium current is blocked by charybdotoxin and by concentrations of tetraethylammonium above 1 mM, but it is insensitive to apamin. Both normal and ras-transformed cells have another calcium-activated current that is not potassium selective, and, consistent with other studies, normal cells display a voltage-activated calcium conductance. These results suggest that the mechanisms by which ras triggers or maintains cell transformation may involve alterations in the number or activity of certain ion channels, in particular, a type of calcium-activated potassium channel.

Topics: Animals; Apamin; Calcimycin; Calcium; Calcium Channel Blockers; Cell Line; Cell Transformation, Neoplastic; Charybdotoxin; Fibroblasts; Genes, ras; Kinetics; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Potassium Channels; Rats; Scorpion Venoms

Cell hybrids (BIM) were produced between human neuroblastoma cells (IMR-32) and thymidine auxotrophs (B3T) of rat nerve-like cells (B103) in order to obtain cell lines undergoing stable neuronal differentiation. BIM cells exhibited the growth properties of partial transformation: 1) When the cell growth reached a plateau, BIM cells ceased to proliferate and expressed a differentiated phenotype. The shape of the cells changed from flat to round and they extended neurites. 2) When cultured in methylcellulose, BIM cells formed colonies, indicating that BIM cells have the ability of anchorage-independent growth. By Southern blot analysis, BIM cells had both human and rat types of N-myc genes. The human N-myc genes were amplified, but the extent of the amplification was lower in BIM cells than that in the parental cell line IMR-32. The rat N-myc gene was detected at a similar level in BIM, B3T, B103, and rat fibroblastic cells 3Y1. Therefore, the decrease in amplification of human N-myc genes may be involved in the properties of partial reverse-transformation in BIM cells. When treated with various drugs such as db-cAMP, forskolin, and cAMP with isobutyl-methylxanthine, BIM cells expressed a nerve-like phenotype. These findings indicate that cell hybridization yielded partial normalization of transformed nerve-like cells.

Topics: 1-Methyl-3-isobutylxanthine; Animals; Axons; Bucladesine; Calcimycin; Cell Cycle; Cell Transformation, Neoplastic; Colforsin; Cyclic AMP; Fibroblasts; Humans; Hybrid Cells; Methylcellulose; Neuroblastoma; Neurons; Phenotype; Rats; Thymidine; Tretinoin; Tumor Cells, Cultured

Continuous epithelial cell lines from individuals with cystic fibrosis (CF) and normal controls are required to understand the genetic and cellular defects in CF. We used retroviruses to transduce SV40 large T antigen into nasal epithelial cells. Transformed continuous cell lines were isolated that expressed epithelial markers, cytokeratin, and tight junctions. Northern blot analysis shows that all of the cell lines express the putative CF gene mRNA. Studies of transepithelial electrolyte transport show that CF and normal cell lines develop a transepithelial electrical resistance. Normal but not CF cell lines secreted Cl- in response to agonists that increase cellular levels of adenosine 3',5'-cyclic monophosphate (cAMP) (isoproterenol, forskolin, and a membrane-permeant analogue of cAMP) or in response to a tumor-promoting phorbol ester that activates protein kinase C. In contrast, the Ca2(+)-elevating agonist bradykinin and the Ca2+ ionophore A23187 stimulated secretion in both normal and CF cell lines. The continuous cell lines we have produced maintain their proper phenotypes and will serve as useful tools in understanding the pathophysiology of CF.

Topics: Antigens, Polyomavirus Transforming; Base Sequence; Biomarkers; Blood Proteins; Bradykinin; Calcimycin; Calgranulin A; Cell Line; Cell Transformation, Neoplastic; Cells, Cultured; Chloride Channels; Chlorides; Colforsin; Cystic Fibrosis; Epithelial Cells; Epithelium; Genotype; Humans; Ion Channels; Keratins; Membrane Potentials; Membrane Proteins; Molecular Sequence Data; Nasal Mucosa; Oligonucleotide Probes; Phenotype; Polymerase Chain Reaction; Reference Values; Retroviridae; Simian virus 40; Tetradecanoylphorbol Acetate; Theophylline

Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with [35S]methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of [32P]orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation.

Topics: 2,2'-Dipyridyl; Adenosine Triphosphate; Animals; Arsenic; Arsenites; Calcimycin; Carrier Proteins; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Chick Embryo; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum Chaperone BiP; Fibroblasts; Heat-Shock Proteins; Methionine; Mice; Mice, Inbred BALB C; Precipitin Tests; Sepharose; Sulfur Radioisotopes; Tetradecanoylphorbol Acetate; Tunicamycin

Arachidonic acid (C20:4) metabolites were released constitutively from wild-type Rous sarcoma virus-transformed chicken embryo fibroblasts (CEF). 3H-labeled C20:4 and its metabolites were released from unstimulated and uninfected CEF only in response to stimuli such as serum, phorbol ester, or the calcium ionophore A23187. High-pressure liquid chromatography analysis showed that the radioactivity released from [3H]arachidonate-labeled transformed cells was contained in free arachidonate and in the cyclooxygenase products prostaglandin E2 and prostaglandin F2 alpha; no lipoxygenase products were identified. The release of C20:4 and its metabolites from CEF infected with pp60src deletion mutants was correlated with serum-independent DNA synthesis and with the expression of the mRNA for 9E3, a gene expressed in Rous sarcoma virus-transformed cells which has homology with several mitogenic and inflammatory peptides. 3H-labeled C20:4 release was not correlated with p36 phosphorylation, which argues against a role for this protein as a phospholipase A2 inhibitor. CEF infected with other oncogenic viruses encoding a tyrosine kinase also released C20:4, as did CEF infected with viruses that contained mos and ras; however, infection with a crk-containing virus did not result in stimulation of 3H-labeled C20:4 release, suggesting that utilization of this signaling pathway is specific for particular transformation stimuli.

Topics: Animals; Arachidonic Acids; Avian Sarcoma Viruses; Blotting, Northern; Calcimycin; Cell Transformation, Neoplastic; Cells, Cultured; Chick Embryo; DNA Replication; Nucleic Acid Hybridization; RNA, Messenger; Tritium

Injection of molecular probes into unfertilized Xenopus eggs requires suppression of activation. But the unfertilized egg is poised for activity, and pricking, like sperm penetration, triggers the start of the first cell cycle. Methods of suppressing activation generally rely on introduction of drugs into the cell, but some of these techniques are irreversible. I report here that injection without activation can also be accomplished by simply limiting extracellular free Ca2+ to 1-2 microM. The site of injection heals, but the cortex does not contract. Gentle modification of the vitelline envelope, which causes it to become tougher, improves the rate of healing to about 100%. Healed eggs are stable for hours and can be activated when needed. Injection of a plasmid derived from type 1 bovine papilloma virus revealed that replication occurs only after activation, but preloading the DNA markedly increased the efficiency of first-round replication. DNA interaction with the unactivated egg cytoplasm may therefore be required for efficient replication of exogenous DNA. The new procedures described here are likely to be of general utility.

Topics: Animals; Blotting, Southern; Bovine papillomavirus 1; Calcimycin; Calcium; Cell Transformation, Neoplastic; DNA Replication; Female; Kinetics; Microinjections; Oocytes; Papillomaviridae; Plasmids; Suppression, Genetic; Vitelline Membrane

Protein phosphorylation mediated by murine IL3 and other factors has been studied in two different IL3-dependent lines, AC2 and 123. In both lines, responses to rat recombinant IL3 are enhanced or induced by growth in rat spleen lymphocyte conditioned medium. Growth stimulation by murine and rat IL3, by rat lymphokine(s), and by ATP in ATP-responsive cells is closely associated with the rapid (2-4 min) phosphorylation of a 33-kDa protein (p33) in all the cells examined. p33 phosphorylation is not stimulated by another lymphokine, IL4, nor by TPA or calcium ionophore alone, which are unable to stimulate growth by themselves, and is independent of serum. p33 phosphorylation is inhibited by trifluoperazine, an inhibitor of calcium-calmodulin, but is less sensitive to inhibition by H7, an inhibitor of protein kinase c, in AC2 cells. A spontaneous IL3-independent clone of AC2 (AC-) has been isolated. AC- cells are aggressively leukemic, do not produce detectable IL3, but phosphorylate p33 constitutively where it is associated with a particulate cell fraction. It is suggested that p33 is a common intermediate molecule involved in signal transduction by the various ligands which result in growth stimulation and that its constitutive phosphorylation may play a key role in the maintenance of the leukemic state.

Topics: Adenosine Triphosphate; Animals; Calcimycin; Cell Division; Cell Line; Cell Transformation, Neoplastic; Clone Cells; Concanavalin A; Culture Media; Interleukin-3; Interleukin-4; Interleukins; Kinetics; Mice; Molecular Weight; Phosphoproteins; Phosphorylation; Protein Kinase C; Rats; Recombinant Proteins; Spleen; Tetradecanoylphorbol Acetate

Numatrin is a tightly bound nuclear matrix protein (40 kD/pI-5) whose synthesis is markedly and promptly increased in association with cellular commitment for mitogenesis in B lymphocytes. (Feuerstein, N., and J.J. Mond. 1987. J. Biol. Chem. 262:11389-11397). To study whether this event is exclusively associated with proliferation of B lymphocytes, we examined the synthesis of numatrin in T lymphocytes (murine and human) activated by lectins or by anti-T cell antigen receptor monoclonal antibody and in Swiss 3T3 fibroblasts stimulated by growth factors. We showed a close correlation between induction of DNA synthesis and induction of numatrin synthesis in T lymphocytes stimulated by concanavalin A, anti-T cell antigen receptor monoclonal antibody, and IL-2 in murine T cells. Similar results were observed in Swiss 3T3 fibroblasts, thus only combinations of growth factors (insulin/EGF or insulin/B subunit of cholera toxin) or serum, which induced a significant increase in DNA synthesis, were also associated with a significant increase in synthesis of numatrin. Similar to B cells, the increase in numatrin synthesis in fibroblasts was found to occur at early G1 phase. The calcium ionophores, A23187 and ionomycin, previously shown to induce an increase in c-myc and c-fos mRNA levels in fibroblasts, induced a marked increase in the synthesis of a nuclear protein at 80 kD/pI-5 but failed to induce an increase in the synthesis of numatrin indicating that an increase in intracellular Ca++ level is not sufficient for induction of the synthesis of numatrin. This further indicates that the increase in synthesis of numatrin may be more closely correlated with cellular commitment for mitogenesis as compared with other biochemical parameters. Using a polyclonal numatrin antibody we demonstrated that mitogen stimulation is also associated with a marked increase in numatrin abundance, which reached a peak at the onset of S phase and declined at the end of S phase. Evidence is presented to show that numatrin synthesis and abundance is elevated in various lymphoma cell lines. Using indirect immunofluorescence assays we showed that numatrin is abundant in other malignant cells: KB, epidermoid carcinoma, and Hep2 human hepatoma. Immunofluorescence studies further showed that mitogen stimulation of B lymphocytes induced a marked accumulation of numatrin in the nucleoli. This observation is in accord with the recent finding of identity of numatrin with the nucleolar protein B23

Topics: Antibodies, Monoclonal; B-Lymphocytes; Blotting, Western; Calcimycin; Cell Cycle; Cell Division; Cell Line; Cell Transformation, Neoplastic; Concanavalin A; DNA; Electrophoresis, Gel, Two-Dimensional; Fluorescent Antibody Technique; Growth Substances; Humans; Interleukin-2; Kinetics; Nuclear Proteins; Nucleophosmin; Receptors, Antigen, T-Cell; T-Lymphocytes

Topics: 1-Methyl-3-isobutylxanthine; Animals; Avian Sarcoma Viruses; Bucladesine; Calcimycin; Cell Transformation, Neoplastic; Chick Embryo; Cyclic AMP; Fibroblasts; Inositol Phosphates; Kinetics; Phosphorylation; Protein Kinases; Proto-Oncogenes; Tetradecanoylphorbol Acetate

The fluorescent dyes, rhodamine 6G and 123, which specifically stain mitochondria, were used to examine changes in mitochondria that follow malignant transformation. The spatial distribution and shapes of mitochondria differ in untransformed and malignant-transformed cells. In untransformed C3H/10T1/2 clone 8 cells, the mitochondria were distributed radially around the nucleus, and each had a fibrous shape. In chemically transformed MCA clone 16 cells, the mitochondria were distributed randomly in the cytoplasm, and each was shaped like a short rod. Another important mitochondrial change after malignant transformation was the change in the time course of fluorescence emission from the rhodamine present in the mitochondria. A slow increase in fluorescence, which was instantaneous at the time of excitation irradiation occurred in untransformed but not in transformed cells. This slow fluorescence emission, peculiar to untransformed cells was affected by proton ionophore but not by calcium ionophore treatment. The difference in the time courses of fluorescence emission for untransformed and transformed cells may reflect differences in the quenching of the dye fluorescence. The data reported provide evidence that mitochondria are affected by malignant cell transformation.

Topics: Animals; Calcimycin; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Line, Transformed; Cell Transformation, Neoplastic; Clone Cells; Fibroblasts; Fluorescent Dyes; Mice; Microscopy, Fluorescence; Mitochondria; Rhodamine 123; Rhodamines; Staining and Labeling; Xanthenes

We have identified an approximately 85-kD protein in chicken erythrocytes which is immunologically, structurally, and functionally related to the gelsolin found in many muscle and nonmuscle cell types. Cell fractionation reveals a Ca2+-dependent partitioning of gelsolin into the soluble cytoplasm and the membrane-associated cytoskeleton of differentiating or mature erythrocytes. Depending on either the presence of Ca2+ during cell lysis or on the preincubation of the intact cells with the Ca2+-ionophore A23187, up to 40% of the total cellular gelsolin is found associated with the membrane skeleton. Expression of gelsolin shows a strong negative regulation during erythroid differentiation. From quantitations of its steady-state molar ratio to actin, gelsolin is abundant in early progenitor cells as revealed from avian erythroblastosis virus- and S13 virus-transformed cells which are arrested at the colony forming unit erythroid (CFU-e) stage of erythroid development. In these cells, which have a rudimentary and unstable membrane skeleton, gelsolin remains quantitatively cytoplasmic, irrespective of the Ca2+ concentration. During chicken embryo development and maturation, the expression of gelsolin decreases by a factor of approximately 10(3) in erythroid cells. This down regulation is independent from that of actin, which is considerably less, and is observed also when S13-transformed erythroid progenitor cells are induced to differentiate under conditions where the actin content of these cells does not change. In mature erythrocytes of the adult the amount of gelsolin is low, and significantly less than required for potentially capping of all membrane-associated actin filaments. We suggest that the gelsolin in erythroid cells is involved in the assembly of the actin filaments present in the membrane skeleton, and that it may provide for a mechanism, by means of its severing action on actin filaments, to extend the meshwork of the spectrin-actin-based membrane skeleton in erythroid cells during erythropoiesis.

Topics: Actins; Aging; Alpharetrovirus; Animals; Calcimycin; Calcium-Binding Proteins; Cell Transformation, Neoplastic; Chick Embryo; Chickens; Cytoplasm; Cytoskeleton; Embryonic and Fetal Development; Erythroblasts; Erythrocytes; Erythropoiesis; Gelsolin; Homeostasis; Humans; Microfilament Proteins

Tumor promotion in mouse skin depends upon establishment of hyperproliferation as well as inflammation and involves disturbance of normal communication between dermis and epidermis. As the collagenous matrix of the dermis is known to play an important role in the maintenance of normal dermal-epidermal interactions, alterations of the dermal collagen types during tumor promotion with 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were investigated. In TPA-treated samples no difference between the relative content of type I and III collagen was observed, whereas the content of type V was significantly increased. When TPA treatment was discontinued before tumor development no increase of type V collagen was observed. Furthermore, treatment with the non-promoting mitogens 4-O-methyl-TPA and Ca-ionophore A 23187 did not result in any alterations of the matrix composition. These data indicate that the increase of type V collagen content is part of the disturbed tissue interactions between dermis and epidermis that facilitate tumor development.

Topics: Animals; Calcimycin; Cell Transformation, Neoplastic; Collagen; Epidermis; Female; Mice; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate

Our previous work demonstrated that NIH-3T3 cells expressing high levels of the mutated cellular ras oncogene (EJ-ras gene) exhibited reduced hormone-sensitive adenylate cyclase and platelet-derived growth factor-stimulated (PDGF) phospholipase A2/C activities. We now report that although the ras-transformed cells display markedly reduced phospholipase C activity, as measured by the levels of inositol 1,4,5-trisphosphate synthesized after PDGF-stimulation, normal levels of phospholipase A2 activity can be uncovered; thus, similar levels of prostaglandin E2 were synthesized in EJ-ras transformed and control cells after stimulation with phorbol myristate acetate (PMA) and/or the calcium ionophore A-23187, agents which stimulate protein kinase C and intracellular Ca2+ levels, respectively. These data suggest that the EJ-ras gene product uncouples the PDGF receptor from the phospholipase C, resulting in reduced PDGF-stimulated Ca2+ mobilization, protein kinase C stimulation and an apparent decrease in Ca2+-dependent phospholipase A2.

Topics: Animals; Calcimycin; Cell Transformation, Neoplastic; Cells, Cultured; Dinoprostone; Kinetics; Mice; Mice, Inbred Strains; Oncogenes; Phospholipases; Phospholipases A; Phospholipases A2; Platelet-Derived Growth Factor; Prostaglandins E; Tetradecanoylphorbol Acetate; Type C Phospholipases

Extracellular calcium is required in the induction of neoplastic transformation of preneoplastic mouse JB6 epidermal cells by 12-O-tetradecanoylphorbol-13-acetate (TPA). Depleting extra-cellular calcium by chelation or by use of commercial calcium-depleted medium inhibited TPA-promoted transformation of promotion-sensitive JB6 cells with a half-maximal inhibition at 1.2 mM calcium. Inhibition was reversible by the addition of calcium. The calcium channel blockers lanthanum and nifedipine inhibited promotion of anchorage-independent transformation by TPA maximally at 10.0 microM and 1.0 nM, respectively, suggesting that calcium entry occurs partially via cell channels. None of the above treatments altered expression of transformation in anchorage-independent tumorigenic JB6 cell lines, indicating that the extracellular calcium requirement was at the level of induction, not expression of transformation. The calcium requirement was not merely a requirement for proliferation; calcium concentrations from 0.2 to 1.8 mM had no effect on JB6 cell monolayer growth. Extracellular calcium was required for 7 days for maximal colony induction. The calcium ionophore A23187 was not a promoter and moderately inhibited TPA-promoted transformation, indicating that increases in free cytosolic calcium concentrations are not sufficient for promotion of transformation and may even activate calcium-dependent antipromoting events. The results suggest that a cellular calcium pool supplied by extracellular calcium, but not distinguishable by ionophoretic increases in free cytosolic calcium, is essential in TPA-promoted neoplastic transformation.

Topics: Animals; Calcimycin; Calcium; Cell Line; Cell Transformation, Neoplastic; Egtazic Acid; Extracellular Space; Lanthanum; Mice; Nifedipine; Phorbols; Tetradecanoylphorbol Acetate

The rare earth elements lanthanum and terbium (0.1-1.0 mM), pharmacological analogs of calcium, induced neoplastic transformation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-sensitive (P+) and to a lesser extent TPA-resistant (P-) preneoplastic mouse JB6 epidermal cells. A maximum of 2500 anchorage-independent colonies per 10(4) cells were induced in P+ lines, a response comparable to that induced by phorbol esters (1.6-16 nM). The maximum lanthanide-induced colony yield in P- lines was 20% of that in P+ lines (approximately 550 colonies), and was observed under conditions where TPA induced less than 30 colonies per 10(4) cells. Lanthanides and TPA produced a synergistic effect on colony size in P+ cells. Lanthanides are not promoting transformation merely by mimicking high calcium: adding exogenous extracellular calcium (up to 50.0 mM) or using calcium ionophore (up to toxic concentrations) to increase intracellular calcium does not promote transformation. Lanthanum will substitute for calcium in activating partially purified protein kinase C (PKC), the calcium-dependent phorbol ester receptor. However, lanthanides must be promoting transformation by a mechanism other than PKC activation because lanthanides failed to activate PKC in intact JB6 cells. Three independent experiments showed a lack of lanthanum effect on PKC-dependent events in intact cells. First, in contrast to TPA, lanthanum pretreatment of JB6 cells did not produce elevated phosphorylation of an 80-kd substrate. Second lanthanum pretreatment did not cause decreased PKC activity after prolonged exposure. Third, lanthanum and TPA affected epidermal growth factor binding with a different magnitude, time course and calcium dependency. We found, however, a PKC substrate in P+, P- and tumorigenic cell lines that is sensitive to lanthanum and increases its migration in sodium dodecylsulfate-polyacrylamide gels from 23 to 21 kd. The above data suggest that: (i) alterations in cation binding may be sufficient for inducing the transformed phenotype; and (ii) lanthanide promotion of neoplastic transformation may be linked to a lanthanide-sensitive PKC substrate, but is not due to a direct PKC activation.

Topics: Calcimycin; Calcium Channel Blockers; Cell Line; Cell Transformation, Neoplastic; Enzyme Activation; ErbB Receptors; Metals, Rare Earth; Phosphorylation; Protein Kinase C; Tetradecanoylphorbol Acetate

Antigen binding to its specific receptor on T cells initiates a series of intracellular events that result in cell differentiation, activation, and clonal expansion. However, the mechanism by which these antigen-occupied receptors induce the transmembrane signal transduction needs clarification. Because this mechanism appears to involve an increase in intracellular free Ca2+ concentration and activation of protein kinase C (PKC), we tested the effect of Ca2+ ionophores and PKC activators on alloantigen-specific primary mixed leukocyte culture cells. Both calcium ionophores, A23187 and ionomycin, in conjunction with 12-O-tetradecanoylphorbol 13-acetate (TPA) mimicked the effect of antigen or interleukin 2 (IL 2) by inducing strong proliferative and alloantigen-specific cytotoxic responses. In addition, Ca2+ ionophore and TPA induced IL 2 receptor expression and IL 2 secretion. The capacity of other phorbol esters or a non-phorbol ester tumor promoter (teleocidin) to replace TPA in induction of cell activation correlated with their ability to bind to and to activate PKC. In addition, the synergistic effect of Ca2+ ionophore and TPA was blocked by either a Ca2+ chelator (EGTA) or cAMP, which is thought to inhibit phosphatidylinositol metabolism. To determine whether the induction of this cytotoxic activity was mediated by a direct effect of Ca2+ ionophore and TPA on cytotoxic T (Tc) cells or was secondary to IL 2 secretion by activated helper T (Th) cells, we tested the effect of Ca2+ ionophore and TPA on isolated populations of cloned, alloantigen-specific Th and Tc cells. Both agents induced cell proliferation and IL 2 production by Th cells, but not by Tc cells. Activation of mixed clones of Th and Tc cells, but not of Tc cells alone, resulted in cytotoxic activity, an effect that could be blocked by anti-IL 2 receptor antibodies. The results thus demonstrate that an increased concentration of intracellular Ca2+ in conjunction with PKC activation can bypass the signal provided by antigen-receptor interaction on Th cells, but does not substitute for IL 2 in activating cytotoxicity by isolated Tc cells.

Topics: Animals; Calcimycin; Carcinogens; Cell Transformation, Neoplastic; Clone Cells; Cyclic AMP; Cyclosporins; Cytotoxicity, Immunologic; Drug Synergism; Egtazic Acid; Female; Immunosuppressive Agents; Interleukin-2; Isoantigens; Lymphocyte Activation; Lymphocyte Culture Test, Mixed; Male; Mice; Mice, Inbred C57BL; Mice, Inbred DBA; Protein Kinase C; Rats; Rats, Inbred Lew; Receptors, Immunologic; Receptors, Interleukin-2; T-Lymphocytes; Tetradecanoylphorbol Acetate

The alterations of stimulus-induced membrane potential changes, superoxide (O2-)-producing capacity and phagocytic activity during differentiation of human granulocytes were investigated in the human leukemia cell lines HL-60 and KG-1 differentiating in vitro and in human leukemic granulocytes obtained from chronic myelogenous leukemia patients. HL-60 cells incubated with dimethyl sulfoxide or with retinoic acid showed progressively increasing O2- production as well as membrane potential changes (depolarization) on contact with phorbol myristate acetate or the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine, with a concomitant increase in the proportion of mature cells of the granulocytic type. Phagocytosis of latex particles, yeast, and oil droplets appeared 24 h after incubation with dimethyl sulfoxide and anteceded the increment of O2- production and membrane potential changes, both of which appeared concomitantly 3 d after incubation with dimethyl sulfoxide. Similar findings were observed when immature and mature granulocytes obtained from chronic myelogenous leukemia patients were stimulated by phorbol ester, the chemotactic peptide, or calcium ionophore A23187, and the amount of O2- production was parallel to the magnitude of membrane potential changes. HL-60 and KG-1 cells incubated for 1-6 d with phorbol myristate acetate showed neither O2- production nor membrane potential changes on contact with phorbol ester, chemotactic peptide, or A23187, although such cells resembled macrophages morphologically, and their phagocytic activity was significantly increased. O2- production and membrane potential changes in normal granulocytes induced by phorbol ester, chemotactic peptide and A23187 were inhibited by 2-deoxyglucose. These findings indicate that the O2--producing system and the system provoking membrane potential changes may develop concomitantly as human granulocytes mature and differentiate, and that the development of these systems and of phagocytic activity may be independently regulated.

Topics: Adult; Calcimycin; Cell Differentiation; Cell Transformation, Neoplastic; Deoxyglucose; Dimethyl Sulfoxide; Gramicidin; Granulocytes; Humans; Leukemia, Myeloid; Membrane Potentials; Phagocytosis; Superoxides; Tetradecanoylphorbol Acetate; Tretinoin

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

The purpose of this investigation was to determine whether cells transformed by herpes simplex virus type 2 (HSV-2) can be stimulated to synthesize prostaglandins (PG). Stimulation was determined by measuring the release of PG into overlay fluids from cell monolayers prelabeled with [3H]arachidonic acid. Results showed that Ca2+ ionophore A-23187 markedly stimulated arachidonic acid release starting 30 min after treatment of HSV-2-transformed and nontransformed rat embryo fibroblast cells. However, only HSV-2-transformed cells were stimulated in production of PG. HSV-2-transformed, nontumorigenic, rat embryo fibroblast, line G, clone 2.0 cells synthesize nearly equal amounts of prostaglandin E2 (PGE2) and prostaglandin F2 alpha, while tumor (rat fibrosarcoma) cells synthesize primarily PGE2. Stimulation of PGE2 synthesis by Ca2+ ionophore A-23187 or 12-O-tetradecanoyl-phorbol-13-acetate decreased as rat fibrosarcoma cells were serially passaged in tissue culture. At low passage of parental rat fibrosarcoma cells, four distinct morphological clonal cell lines were isolated, which varied markedly in their capacity to be stimulated in PG synthesis by 12-O-tetradecanoyl-phorbol-13-acetate. There was correlation between the capacity of clone 1 cells to be stimulated in PGE2 synthesis by serum alone and capacity of the tumors produced by the clone 1 cells to metastasize to the lungs of syngeneic tumor-bearing rats. In summary, cell transformation by HSV-2 appears to be essential for stimulation of PG synthesis in cells. The capacity to be stimulated in arachidonic acid metabolism and PG synthesis may be important in the process of carcinogenesis by a putative human cancer virus.

Topics: Animals; Calcimycin; Cell Division; Cell Transformation, Neoplastic; Clone Cells; Dinoprost; Dinoprostone; Embryo, Mammalian; Fibrosarcoma; Kinetics; Neoplasm Metastasis; Phorbols; Prostaglandins; Prostaglandins E; Prostaglandins F; Rats; Simplexvirus; Tetradecanoylphorbol Acetate

Isolated dog mastocytoma cells sensitized with dog anti-ragweed IgE and challenged with ragweed antigen or incubated with ionophore A23187 or the carboxy-terminal dodecapeptide of platelet factor 4, PF4(59-70), release histamine and concurrently generate leukotrienes B4, C4, and D4. In contrast, the exposure of mastocytoma cells to 0.1-3 micrograms/ml of 15-hydroxyeicosatetraenoic acid (15-HETE) stimulates selectively the generation of leukotrienes, in the absence of histamine release, while 0.1-1 micrograms/ml of compound 48/80 releases histamine without enhancing the generation of leukotrienes. That natural stimuli are capable of selectively activating one synthetic or secretory compartment of mast cells suggests that separate subsets of receptors as well as different biochemical events may serve to mobilize each class of mediators.

Topics: Allergens; Animals; Arachidonic Acids; Calcimycin; Cell Transformation, Neoplastic; Dogs; Histamine Release; Hydroxyeicosatetraenoic Acids; Kinetics; Leukotriene B4; Mast-Cell Sarcoma; p-Methoxy-N-methylphenethylamine; Platelet Factor 4; SRS-A

F-actin aggregates have been found near the substrate attachments in a variety of transformed cells (Carley et al., 1981). Interference reflection microscopy shows that these aggregates are present in central close adhesion areas in Rous sarcoma virus (RSV)-transformed rat kidney cells. If these transformed cells are incubated with N6, O2-dibutyryl 3':5'-cyclic monophosphoric acid (db-cAMP), adenosine 5'-monophosphoric acid (5'-AMP) or adenosine, the F-actin aggregates and their associated close adhesion areas disappear, and the cells flatten out. Treatment of untransformed cells with db-cAMP spreads their focal adhesion plaques and thickens microfilament bundles. Furthermore, F-actin aggregates are substantially more resistant to cytochalasin B and the Ca2+ ionophore A23187 than microfilament bundles in untransformed cells. These differences between F-actin complexes in untransformed and in RSV-transformed cells, with respect to morphology and sensitivities to db-cAMP and cytoskeleton-disrupting drugs, define properties of the change in F-actin regulation and association with the plasma membrane due to transformation.

Topics: Actins; Adenosine; Animals; Avian Sarcoma Viruses; Bucladesine; Calcimycin; Cell Adhesion; Cell Aggregation; Cell Line; Cell Transformation, Neoplastic; Intercellular Junctions; Kidney; Kinetics; Rats

To investigate the type of phospholipase activated by agents that stimulate prostaglandin synthesis, we used transformed mouse cells whose phospholipids were doubly labeled with [14C]inositol and [3H]arachidonic acid. [14C]Inositol was incorporated mostly into the phosphatidylinositol and [3H]arachidonic acid was distributed into the various phospholipids. When these cells were incubated with bradykinin, a stimulator of prostaglandin synthesis, the release of 3H radioactivity from cellular phospholipids and the synthesis of prostaglandin were initiated within seconds and reached a maximum in 40 to 70 s. Analysis of the intracellular lipids revealed a concomitant increase of radioactivity associated with lysophosphatidylinositol, which was detectable within 5 s of incubation with bradykinin and reached a maximum between 40 and 70 s. Lysophosphatidylinositol which could be formed either from a phospholipase A1 or phospholipase A2 reaction, was identified by its chromatographic properties and conversion to glycerophosphorylinositol. We found that the 3H/14C ratio of purified lysophosphatidylinositol was 1/11 of that of phosphatidylinositol, which indicated that lysophosphatidylinositol formed in response to bradykinin is 1-acyl-sn-glycero-3-phosphorylinositol and most probably is formed from a phospholipase A2 deacylation of phosphatidylinositol (a phospholipase A1 deacylation would result in the formation of lysophosphatidylinositol of a 3H/14C ratio similar to phosphatidylinositol). Furthermore, we did not detect between control and stimulated cells any significant difference in the level of several phospholipase C metabolites including inositol phosphate, diglyceride, and phosphatidic acid. These results suggest that phospholipase C is probably not activated. The formation of lysophosphatidylinositol was also stimulated by thrombin and ionophore A23187, both activators of prostaglandin synthesis. Dexamethasone, a lipase inhibitor, inhibited the appearance of lysophosphatidylinositol, whereas aspirin and low concentrations of indomethacin, the cyclooxygenase inhibitor, did not inhibit. The results presented in ths paper provide evidence that a phospholipase A2-hydrolyzing phosphatidylinositol is activated when intact cells are stimulated for prostaglandin synthesis.

Topics: Animals; Bradykinin; Calcimycin; Cell Transformation, Neoplastic; Cells, Cultured; Enzyme Activation; Kinetics; Mice; Mice, Inbred BALB C; Phosphatidylinositols; Phospholipases; Phospholipases A; Phospholipases A1; Phospholipases A2; Prostaglandins; Prostaglandins E; Thrombin

The Ca2+ ionophore A23187 increases intracellular calcium content in normal thymic cells, while it is without effect on the corresponding neoplastic cell (Ascites thymoma) and on Ehrlich ascites tumour cells. The A23187-induced total cell calcium increase in normal thymocytes takes place both in control and energy-depleted cells, while it is lacking in neoplastic cells. In addition the ionophore stimulates aerobic glycolysis of normal thymocytes, whereas it is ineffective on neoplastic cells. The study of intracellular calcium exchange properties reveals that in normal cells the ionophore A23187 provokes a 60% increase of the exchangeable pool together with a more significant, 4-fold enlargement of the unexchangeable pool. These effects are lacking in cancer cells. The data give rise to interesting considerations concerning the regulation and compartmentalization of calcium in neoplastic cells. The results will be also discussed in relation to the models that predict altered cell calcium metabolism as a cause of cancer cell high aerobic glycolysis and uncontrolled growth.

Topics: Animals; Anti-Bacterial Agents; Calcimycin; Calcium; Carcinoma, Ehrlich Tumor; Cell Membrane Permeability; Cell Transformation, Neoplastic; Mice; Neoplasms, Experimental; Rats; Thymoma; Thymus Gland; Thymus Neoplasms

Concanavalin A (Con A)-mediated red blood cell (RBC) adsorption with the RBC coating method (in which Con A-coated RBC's are adsorbed to fibroblasts) was greatly increased by glutaraldehyde fixation of RBCs before Con A-coating and decreased by the fixation of fibroblasts. On the other hand, RBC adsorption with the fibroblast coating method (in which RBCs are adsorbed to Con A-coated fibroblasts) was decreased by the fixation of RBCs and increased by the fixation of fibroblasts before Con A coating. The fixation of RBCs or fibroblasts after Con A coating did not have these effects. In addition, the fixation of both RBCs and fibroblasts nearly completely abolished RBC adsorption with either method. However, the amount of [3H] Con A binding was not affected by the fixation. RBC adsorption with the fibroblast coating method was also affected by cytochalasin B, colchicine, NaN3 and dibucane treatments of fibroblasts. These drug treatments of fibroblasts, however, did not affect RBC adsorption with the RBC coating method, except cytochalasin B. In addition, the effects of drug treatments of fibroblasts examined occurred nearly to the same extent for nonsenescent, senescent, and transformed cells. Our results suggest that secondary processes after Con A binding, receptor mobility and receptor association with cytoskeletals, play important roles in RBC adsorption, but that the roles do not change with aging or transformation.

Topics: Aldehydes; Azides; Calcimycin; Cell Survival; Cell Transformation, Neoplastic; Colchicine; Concanavalin A; Cytochalasin B; Dibucaine; Erythrocytes; Female; Fibroblasts; Fixatives; Glutaral; Hemadsorption; Humans

Methylcholanthrene-transformed mouse fibroblasts synthesize prostaglandins in response to bradykinin, thrombin, serum, and the ionophore A23187. These agents activate phospholipases, thereby releasing fatty acids from phospholipids. To examine the phospholipid specificity of the phospholipases activated by bradykinin, thrombin, serum, and A23187, cells were labeled with [14C]arachidonic acid and stimulated with these agents in the presence of delipidated bovine serum albumin. Phospholipid classes were resolved by two-dimensional chromatography on silica gel-coated paper. Only phosphatidylinositol and phosphatidylcholine lost radioactivity upon stimulation. To characterize the fatty acid specificity of the phospholipases, cells were incubated with 14C-labeled stearic, oleic, linoleic, eicosatrienoic, or arachidonic acid and then exposed to the stimuli. Bradykinin, thrombin, and serum caused specific release of radioactivity into the medium only from cells labeled with arachidonic acid or eicosatrienoic acid, whereas A23187 caused release from cells labeled with any one of the five fatty acids. We conclude that bradykinin, thrombin, and serum activate phospholipases that specifically hydrolyze arachidonyl and eicosatrienoyl phosphatidylinositol and phosphatidylcholine, whereas A23187 is less specific activator of phospholipases.

Topics: Animals; Anti-Bacterial Agents; Arachidonic Acids; Blood; Bradykinin; Calcimycin; Cell Line; Cell Transformation, Neoplastic; Culture Media; Enzyme Activation; Fibroblasts; Kinetics; Methylcholanthrene; Mice; Phospholipases; Phospholipids; Thrombin

Actively growing Swiss 3T3 cells secret high levels of plasminogen activator which decreases after the cells become confluent. In contrast, simian virus 40-transformed 3T3 cells secrete large amounts of plasminogen activator independent of cell density (Chou, I.-N., O'Donnel, S.P., Black, P.H., and Roblin, R.O. (1977) J. Cell. Physiol. 91, 31-38). These results suggest a correlation between active cell multiplication and plasminogen activator secretion in both 3T3 and simian virus-transformed 3T3 cells. The data reported herein indicate that treatment of both subconfluent and confluent Swiss 3T3 cells with high concentrations of Ca2+ (final 3.0 to 4.9 mM) increases the amounts of both secreted and cell-associated plasminogen activator in a dose-dependent manner. In addition, the ionophore A23187 (19 to 95 nM) in the presence of a normal level of Ca2+ (1.8 mM) stimulates both production and secretion of plasminogen activator from growing 3T3 cells. The Ca2+ stimulation of plasminogen activator production/secretion may be related to the mitogenic effect of Ca2+.

Topics: Calcimycin; Calcium; Cell Transformation, Neoplastic; Cells, Cultured; Plasminogen Activators; Simian virus 40; Stimulation, Chemical

Topics: Agglutination; Calcimycin; Calcium; Cell Membrane; Cell Transformation, Neoplastic; Cells, Cultured; Concanavalin A; Fibroblasts; Metals