calcimycin and Neoplasms

Tumor promoters act on carcinogen-initiated tissues to cause phenotypic expression of malignancy. Phorbol ester tumor promoters, like hormones, act on cells and tissues at nanomolar concentrations, often producing "physiological" effects. These promoters also act on cells to produce what appear to be nonphysiologic or toxic effects. One of the major questions regarding these phenomena is, Which action or actions of promoters are important in phenotypic expression of malignancy?

Topics: Animals; Calcimycin; Calcium; Chromosomes; Concanavalin A; Cyclic GMP; DNA; Egtazic Acid; Humans; Myristates; Neoplasms; Potassium; Protein Kinase C; Protons; Sodium; Tetradecanoylphorbol Acetate

Nitric oxide (NO) is one of the main cytotoxic effector molecules involved in the killing of tumor cells by macrophages. In macrophages, lipopolysaccharide (LPS) alone or in combination with IFN-gamma causes the generation of NO by an inducible form of NO synthase (iNOS). We have previously reported that macrophages from mammary tumor bearers have a downregulation of their NO production leading to a diminished cytotoxic activity. Further studies lead to the isolation and characterization of phosphatidyl serine (PS) as a NO inhibitory factor produced by mammary tumor cells. Pretreatment of macrophages with PS was shown to downregulate their cytotoxic potential and NO production upon stimulation with LPS. Activation of PS-pretreated macrophages with LPS and IFN-gamma resulted in higher levels of NO than those observed with LPS alone, but lower than those of untreated macrophages activated with LPS and IFN-gamma. These results correlated with the levels of iNOS RNA as detected by Northern blot analyses. A study of the expression and binding activity of the transcription factor NF kappa B in macrophages pretreated with PS revealed no differences with untreated macrophages. Investigation of the possible signaling pathways leading to the induction of iNOS revealed that in LPS-stimulated macrophages, increases in internal calcium concentration [Ca2+]i were not observed, while NO was normally produced even under calcium-deprived conditions. In contrast, an effective synergism of IFN-gamma with LPS in the production of NO by macrophages required an optimal increase in [Ca2+]i stimulated by IFN-gamma. This increment in [Ca2+]i was significantly reduced in PS-pretreated macrophages. Further experiments demonstrated that pretreatment of macrophages with PS did not change the normal pattern of tyrosine phosphorylation stimulated by LPS but strikingly inhibited PKC activity. Combinations of LPS and IFN-gamma did not alter the latter result, suggesting that IFN-gamma enhances LPS-induction of iNOS through a pathway other than activation of PKC. Importantly, expression of PKC isozymes in both untreated and PS-pretreated macrophages stimulated with LPS remained constant. Out data suggest that, in tumor bearers, PKC and not NF kappa B is the main target for PS to exert its NO inhibitory action on LPS-activated macrophages. An excess of PS in PS-PKC interaction may be responsible, at least in part, for this type of PKC inhibition. Furthermore, PS also appears to downregulat

Topics: Animals; Calcimycin; Calcium; Female; Gene Expression Regulation, Enzymologic; Interferon-gamma; Ionophores; Lipopolysaccharides; Macrophage Activation; Macrophages, Peritoneal; Male; Mice; Mice, Inbred BALB C; Neoplasms; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase; Phosphatidylserines; Protein Kinase C; RNA, Messenger

The identification of self-renewing and multipotent neural stem cells (NSCs) in the mammalian brain holds promise for the treatment of neurological diseases and has yielded new insight into brain cancer. However, the complete repertoire of signaling pathways that governs the proliferation and self-renewal of NSCs, which we refer to as the 'ground state', remains largely uncharacterized. Although the candidate gene approach has uncovered vital pathways in NSC biology, so far only a few highly studied pathways have been investigated. Based on the intimate relationship between NSC self-renewal and neurosphere proliferation, we undertook a chemical genetic screen for inhibitors of neurosphere proliferation in order to probe the operational circuitry of the NSC. The screen recovered small molecules known to affect neurotransmission pathways previously thought to operate primarily in the mature central nervous system; these compounds also had potent inhibitory effects on cultures enriched for brain cancer stem cells. These results suggest that clinically approved neuromodulators may remodel the mature central nervous system and find application in the treatment of brain cancer.

Topics: Animals; Cell Survival; Cells, Cultured; Mice; Molecular Structure; Neoplasms; Neurons; Pharmaceutical Preparations; Sensitivity and Specificity; Stem Cells

The human transforming growth factor-alpha (TGF-alpha) gene is thought to contain five introns and six exons, encoding a transmembrane precursor (proTGF-alpha) from which the mature polypeptide is released by proteolytic cleavage. We identified a novel 32-nucleotide exon (exon alpha) within intron 5 and an alternative splice acceptor site in exon 6, splitting exon 6 into two segments: 6A and 6B. Therefore, in addition to wild type (wt) proTGF-alpha mRNA, which skips exon alpha, two novel proTGF-alpha variants are produced: Variant I (VaI), skipping exons alpha and 6A, and Variant II (VaII) which includes exon alpha and skips exon 6A. The only significant difference between variant and wt proTGF-alpha proteins is that the two wt carboxyl-terminal valines are replaced in the variants by five or four other amino acids, respectively. Both variant TGF-alpha mRNAs were readily detected in human keratinocytes and tumor-derived cell lines. Their protein products were cleaved as efficiently as wt TGF-alpha in response to the calcium ionophore A23187. However, both variants (but not wt) reduced serum requirements for proliferation in CHO cells. In addition, VaII-expressing CHO cells (not VaI or wt) formed foci in monolayer cultures. These results suggest that variant TGF-alpha precursors induce autonomous growth.

Topics: Amino Acid Substitution; Animals; Calcimycin; Calcium; CHO Cells; Cricetinae; Cricetulus; Exons; Humans; Introns; Ionophores; Keratinocytes; Molecular Sequence Data; Neoplasms; Protein Processing, Post-Translational; Recombinant Fusion Proteins; RNA Splicing; RNA, Messenger; RNA, Neoplasm; Serine Endopeptidases; Transforming Growth Factor alpha; Tumor Cells, Cultured; Valine

Adhesion of tumor cells (TC) to endothelial cells (EC) is necessary for movement of TC out of the interstitium to form metastatic deposits. This interaction may be influenced by proadhesive molecules such as lipoxygenase products of arachidonic acid metabolism. We studied the effect of inflammatory stimuli, A23187 calcium ionophore, n-formyl-methionyl-leucine-phenylalanine (FMLP) and phorbol myristate acetate (PMA) on TC-EC interaction. Adherence of metastatic breast tumor cell line (MCF-7), choriocarcinoma cell line (JEG-3), and non metastatic pituitary cell (GH-3) were assayed as the number of radiolabeled TC attached to EC (cpm/well). TC and EC were incubated with A23187, FMLP, and PMA for varying time periods. Lipoxygenase products (LTB4, 5-HETE) were measured under basal and stimulated conditions using RP-HPLC and RIA. There were no differences in basal adherence of TC lines to EC. When EC were incubated with stimuli, there were significant increases in the numbers of MCF-7 and JEG-3 cells adherent to EC compared to GH-3. Light and phase contrast microscopy confirmed that TC were attached to EC. Upon stimulation, GH-3 preferentially produced prostaglandins (PGI1(2)) while MCF-7 and JEG-3 produced lipoxygenase products (LTB4 and 5-HETE). Pre-incubation of MCF-7 and JEG-3 with the lipoxygenase inhibitor nordihydroguiaretic acid resulted in partial inhibition of adhesion to EC. Our data strongly indicate a role for lipoxygenase products of arachidonic acid in adherence of TC to EC.

Topics: 5,8,11,14-Eicosatetraynoic Acid; Animals; Arachidonic Acid; Calcimycin; Cattle; Cell Adhesion; Endothelium, Vascular; Humans; Lipoxygenase; Lipoxygenase Inhibitors; Masoprocol; N-Formylmethionine Leucyl-Phenylalanine; Neoplasm Metastasis; Neoplasms; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

As we have reported, calcium ionophore A23187 activates macrophages for tumor cell killing, and the activated macrophages produced a soluble cytotoxic factor (M phi-CF) that is similar, if not identical, to tumor necrosis factor. Based on these observations, we have investigated whether calcium is involved in the activation mediated by another potent macrophage activator, namely lipopolysaccharide (LPS). We first showed that A23187 caused uptake of extracellular calcium-45 by macrophage monolayers, whereas LPS did not. Because in this system rapid changes would not have been detected, several other approaches also have been used. We have examined the effect of depleting extracellular calcium by using medium containing no added calcium, supplemented with 1 mM EGTA. In no case did depletion result in decreased M phi-CF production by LPS-treated macrophages. Measurements using the fluorescent intracellular calcium indicator Quin 2 have also been performed. The calcium ionophore ionomycin caused a rapid change in the intracellular Quin 2 signal. LPS, even at a concentration in vast excess of that required to activate the macrophages, caused no change in the signal during a 2-hr period. If the macrophages were loaded with high doses of Quin 2 or another intracellular chelator, TMB-8, M phi-CF production decreased and cytotoxic activity was impaired. These data indicate that one or more of the processes involved in M phi-CF production does require calcium, but that activation mediated by LPS occurs without the influx of extracellular calcium or redistribution of intracellular calcium.

Topics: Aminoquinolines; Animals; Calcimycin; Calcium; Cytotoxicity, Immunologic; Cytotoxins; Gallic Acid; Lipopolysaccharides; Macrophage Activation; Macrophages; Mice; Mice, Inbred Strains; Neoplasms

The effects of phorbol ester application and of other mitogenic treatments on the activity of 3',5'-cyclic-nucleotide phosphodiesterase were investigated in dorsal mouse epidermis in vivo. Local treatment with either the weak tumor promoter phorbol 12,13-dibenzoate or the strong promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) increased the activity of the high affinity enzyme (Km = 4 microM). The enzymic changes began within the first hour after application, and lasted for about 5 days. maximal stimulations of approximately 300--400% were reached after 3--6 h with TPA application, whereas with phorbol dibenzoate the maximum could only be reached after 1--2 days. TPA stimulation of the enzyme depended on doses within the range of 0.2 to 20 nmol and could be completely prevented by cycloheximide, but not by 5-azacytidine, actinomycin D, 5,8,11,14-eicosatetraynoic acid or indomethacin. No evidence could be found for cAMP participation in enzyme induction. An increase in enzyme activity could also be observed after other mitogenic treatments such as local application of the weakly promoting phorbol esters C14:4-phorbol acetate ("Ti8") and 4.O-methyl-TPA, or of the non-promoting divalent cation ionophore A 23187, as well as after treatment with a depilatory cream. Skin massage or removal of the horny layer, which also stimulate mitosis, did not evoke a significant increase in enzyme activity. No apparent correlation exists between the hyperplasiogenic and tumor-promoting effectiveness of a manipulation and its effect on epidermal 3',5'-cyclic-nucleotide phosphodiesterase.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Calcimycin; Dose-Response Relationship, Drug; Epidermis; Female; Hyperplasia; Mice; Mitosis; Neoplasms; Phorbol Esters; Phorbols; Skin; Structure-Activity Relationship

Ca ion, ionophore 23187 and the membrane labilizing agents triton X-100, trypsin and phospholipase C promoted benign hyperplastic lesions (BHLs) but only rarely advanced tumors in hamster cheek pouch mucosa, which had been initiated with 7,12 dimethylbenz (a) anthracene. Retinyl acetate (RA) and croton oil markedly promoted both BHLs and advanced tumors. These results suggest that pathways modulated by intracellular Ca cause initiation-stabilized release from growth control, thus the promotion of BHLs, whereas additional mechanisms are required for the progression of BHLs to more advanced tumors. Some Ca-modulated systems which could be involved in promotion of BHLs are discussed in the light of published literature.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Calcimycin; Calcium; Cheek; Cricetinae; Female; Hyperplasia; Membranes; Mesocricetus; Neoplasms; Neoplasms, Experimental

Normal human lymphocytes and human tumor cell lines were labeled with an apolar fluorescent probe (1, 6-diphenyl-1, 3, 5-hexatriene), and fluoresence polarization (P) values were determined. The arguments presented in this study suggest that although P values represent an intricate average of all labeled hydrophobic regions of the cell (phospholipid bilayers, membrane proteins and enzymes, etc.), to a large extent, interactions of the probe with membrane proteins are of primary importance. The lower P values obtained with the tumor cell lines, compared with the normal lymphocytes, were interpreted as indicating alterations in either the structure or concentration of a membrane associated protein(s).

Topics: Animals; Calcimycin; Cell Membrane; Diphenylhexatriene; Humans; Leukemia; Lymphocytes; Membrane Proteins; Mice; Mice, Inbred CBA; Neoplasm Proteins; Neoplasms; Spectrometry, Fluorescence