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Tumor promoters enhance cap formation in mouse thymocytes

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Abstract

Potent tumor promoters such as 12-O-tetradecanoylphorbol 13-acetate (TPA) and teleocidin, rapidly evoked a dose-dependent stimulation of concanavalin A (Con A)-induced cap formation in mouse T lymphocytes. The effect was reversible upon removal of the drugs. Weaker tumor promoters, phorbol didecanoate, phorbol dibenzoate and iodoacetic acid stimulated capping to a lower extent. Mezerein, a phorbol-related macrocyclic diterpene derivative, which acts as a second-stage promoter was also active in increasing the number of caps. In contrast, 4 alpha-phorbol didecanoate and phorbol which are devoid of tumor promoting activity, did not affect capping. Anti-promoting glucocorticoids inhibited capping stimulation. Flow cytofluorometric analysis of Con A binding has shown that TPA did not modify the lectin binding to surface receptors. TPA-facilitated capping was energy-dependent. Cytochalasin B prevented the TPA-induced response whereas colchicine was ineffective. Phenothiazines fully inhibited the TPA effect, thus suggesting that tumor-promoter-mediated lectin receptor redistribution may be ascribed to the facilitation of a Ca2+-dependent process involving the submembrane actin filaments.

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Quercetin, a ubiquitous flavonoid in plants, inhibited the incorporation of [32P]inorganic phosphate (32Pi) into phospholipid of HeLa cells enhanced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter. Among the flavonoids tested luteolin was the most effective in inhibiting the action of TPA. Studies on structure-activity relationships demonstrated that more hydroxylated flavones and flavonols had stronger inhibitory effects. Quercetin and luteolin also inhibited enhancement of 32Pi-incorporation into phospholipid by dihydroteleocidin B, another potent tumor promoter. The inhibitory effect of quercetin on the biological action of tumor promoters is interesting in relation to the non-carcinogenicity of this flavonoid in animals, in spite of its mutagenicity.
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A detailed kinetic analysis of the distribution of cytoplasmic myosin during the capping of various lymphocytic surface molecules revealed two distinct capping mechanisms. (a) Some cell surface molecules, including immunoglobulin, Fc receptor, and thymus leukemia antigen, all cap spontaneously in a small fraction of lymphocytes during locomotion. Cytoplasmic myosin becomes concentrated in the cytoplasm underlying these spontaneous caps. Exposure to specific antibodies causes all three of these surface molecules to cap rapidly with a concomitant redistribution of cytoplasmic myosin to the area of the cap. These antibodies also stimulate cell locomotion. (b) Other lymphocyte surface molecules, including H2 and Thy.1, do not cap spontaneously. Moreover, exposure to antibodies to these molecules causes them to cap slowly without a redistribution of cytoplasmic myosin or stimulation of cell locomotion. Exposure to concanavalin A gives a response intermediate between these two extremes. We believe that the first type of capping is active and may involve a direct link between the surface molecules and the cytoplasmic contractile apparatus. The second type of capping appears to result simply from aggregation of cross-linked molecules in the plane of the membrane.
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CYTOSKELETAL control of cell surface receptor movements1 has been suggested by several authors2,3 and is compatible with the fluid mosaic model of cell membranes4. Evidence for this hypothesis comes mostly from experiments using drugs which alter the activity of cytoskeletal components3,5,6. We report here co-capping of actin and tubulin with immunoglobulins (Ig) in B lymphocytes from mouse spleen. These findings provide structural evidence for cytoskeletal control of receptors.
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The probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to determine if tumor promoting agents alter cell membranes. The active tumor promoters TPA (12-0-tetra-decanoyl-phorbol-13-acetate), PDD (phorbol-12,13-didecanoate) and PDB (phorbol-12,13-dibenzoate) were found to decrease fluorescence polarization of DPH in rat embryo cells, whereas the inactive tumor promoting compounds phorbol and 4α-PDD failed to induce this change.
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INTERACTION of multivalent ligands with receptors (immuno-globulin E, IgE) on the surface of the mast cell stimulates the calcium-dependent degranulation and secretion of histamine1,2. Secretion is also initiated by application of the calcium carrier substance (ionophore) A23187 (ref. 3), and so the effective common stimulus to this cell (and many others) is the entry of Ca2+ ions into the cytosol. The mechanism of the normal ligand-induced calcium entry process is not well understood4. Certain flavones, notably quercetin, interfere with the activity of membrane transport adenosine triphosphatases (ATPases) including the calcium-dependent ATPase which is associated with exclusion of calcium from the cytosol of cells. They are not simple ATPase inhibitors but probably act by increasing the efficiency of ion translocation, thus reducing the consumption of ATP (refs 5, 6). Also, the flavones (2-phenyl cromones) have some structural resemblance to the widely used anti-allergic drug cromoglycate whose activity may be expressed at the calcium entry pathway7. We examine here the effect of these substances on histamine secretion from mast cells.
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The treatment of human lymphoblastoid cell cultures with the phorbol diester, 12-O-tetradecanoyl-phorbol-13-acetate, a tumour promoter, caused at nM concentration surface structural changes associated with altered adhesion properties. The effect was observed in several cell lines from normal or leukaemic origin. In addition, the tumour promoter induced an early and transitory growth inhibition which was observed in all tested B-characteristic cells. The phorbol diester, 4-O-methyl-phorbol-12,13-didecanoate, which is devoid of tumour-promoting activity, was much less effective in altering cell adhesion properties and cell growth than the active derivative. These observations suggest that lymphoblastoid cells may be a useful model for studying the molecular alterations at the membrane level resulting from the action of tumour-promoting phorbol diesters.
Article
The mechanism of capping of cell surface receptors has been examined by a double fluorescence staining procedure that permitted simultaneous observations of the distribution of a surface-bound ligand together with intracellular actin or myosin. At an early stage in the capping of the T-25 antigen or the H2 histocompatibility antigens on mouse splenic T lymphocytes, or of concanavalin A receptors on HeLa cells, when the specific receptors in question were collected into patches that were distributed over the entire cell surface, the intracellular membrane-associated actin or myosin was also accumulated into patches that were located directly under the receptor patches. These and other results have led us to propose a general molecular mechanism for the process of capping, in which actin and myosin are directly involved. It is suggested that membrane-associated actin is directly or indirectly bound to an integral protein or class of proteins, X, in the plasma membranes of eukaryotic cells. When any receptor in the membrane is aggregated by an external multivalent ligand, the aggregate binds effectively to X, whereas unaggregated receptors do not bind to X. The receptor aggregates, linked to actin (and myosin) through X, are then actively collected into a cap by an analogue of the actin--myosin sliding filament mechanism of muscle contraction.
Article
By means of double fluorescence staining experiments, intracellular alpha-actinin was found to accumulate under caps and patches induced in several cells by a variety of ligands. This phenomenon was demonstrated in lymphocytes and lymphoma cells treated with anti-H-2 sera; spleen lymphocytes treated with concanavalin A or anti-immunoglobulin antibodies, and VSV-infected mouse fibroblast line MC57 treated with antiserum against viral antigens. It occurred during both rapid and slow capping processes, and could be obtained by either direct or indirect ligand-induced redistribution. These observations were carried out on whole cells. For other cytoskeletal proteins such as filamin, tropomyosin and myosin, a similar accumulation under caps was not readily apparent using whole cell mounts, although earlier experiments with frozen-sectioned cells had shown such an enrichment of myosin (as well as actin). The enrichment of alpha-actinin under the clustered surface molecules was already apparent in early stages (patching) of the capping process, with or without 10 mM sodium azide present. Prolonged incubation of the cells with the different ligands resulted in endocytosis of the ligand-receptor complex. alpha-Actinin was not associated with the inernalized complex, however, suggesting that it may dissociate from the patched or capped surface structures at some stage during endocytosis.
Article
The probe, 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to determine if tumor promoting agents alter cell membranes. The active tumor promoters TPA (12-0-tetra-decanoyl-phorbol-13-acetate), PDD (phorbol-12,13-didecanoate) and PDB (phorbol-12,13-dibenzoate) were found to decrease fluorescence polarization of DPH in rat embryo cells, whereas the inactive tumor promoting compounds phorbol and 4α-PDD failed to induce this change.
Article
An analysis of the inhibition by concanavalin A of the mobility of lymphocyte surface receptors is used to construct an hypothesis on membrane receptor-cytoplasmic interactions. It is proposed that binding of multivalent lectins alters the interaction of an assembly of colchicine-binding proteins with lectin receptors and other receptors, and reciprocally that the state of the colchicine-binding assembly alters the mobility and distribution of surface receptors on the cell membrane. Observations of the effect of colchicine and related drugs on the inhibition of receptor mobility by concanavalin A lend support to this hypothesis. The proposed model has several implications for studies of the initial events of mitogenesis in lymphocytes as well as for cell-cell interactions in general.
Article
Human lymphoblastoid cells transformed by Epstein-Barr virus aggregated rapidly in the presence of tumor-promoting phorbol esters and dihydroteleocidin B. Cell aggregation was almost complete after incubation for 6 hours. In amounts of a few ng, they induced significant aggregation. Their abilities to aggregate cells could be measured quantitatively and correlated well with their effects in promoting skin tumors.
Article
The effects of fluocinolone acetonide (FA), retinoic acid (RA), and tosylphenylalanine chloromethyl ketone (TPCK) on two-stage promotion after 7,12-dimethylbenz[a]-anthracene (DMBA) initiation in female Sencar mice were investigated. The two-stage promotion protocol was achieved by twice weekly applications of 2 microgram of 12-O-tetradecanoylphorbol 13-acetate (TPA) for 2 weeks (stage I) followed by twice weekly applications of mezerein for 18 weeks (stage II). Separately stage I and II do not cause any tumors to develop after DMBA initiation. FA was found to be a potent inhibitor of stages I and II but to a greater degree for stage I than for stage II. RA was ineffective in stage I but was a potent inhibitor of stage II; TPCK specifically inhibited stage I but not stage II. FA and TPCK effectively counteract the appearance of the dark basal keratinocytes, whereas RA has no effect. These results provide additional evidence for the importance of dark basal keratinocytes in stage I of promotion and indicate that most of the other biochemical and morphological responses normally associated with promotion (such as polyamines) are actually associated with stage II of promotion.
Article
The effects of nonpromoting and weakly promoting diterpenes on skin tumor promotion by 12-O-tetradecanoylphorbol 13-acetate (TPA) were investigated. When phorbol and phorbol 12,13-diacetate (both nonpromoting) were given simultaneously with TPA after 7,12-dimethylbenz[a]-anthracene (DMBA) initiation in female mice, they had no effect on TPA promotion. However, the nonpromoter 4-O-methyl-TPA and the weak promoter mezerein were found to inhibit TPA promotion in a dose-dependent manner when given simultaneously with TPA. Because mezerein was found to be an effective inhibitor of TPA promotion when given simultaneously and because it induces many biological responses similar to those to TPA, the capacity of mezerein to act as an incomplete promoter in a two-stage promotion protocol was also investigated. Twice-weekly applications of 1,2, or 5 mug of TPA for 2 weeks after DMBA initiation produced 0, 0, and 0.5 papilloma per mouse, respectively, at 20 weeks. When the twice-weekly applications of TPA for 2 weeks were followed by twice-weekly treatments with 2 mug of mezerein for 18 weeks, the number of papillomas per mouse was 2.2, 3.5, and 9.0, respectively. Twice-weekly applications of 2 mug of TPA for 2 weeks followed by twice-weekly treatments with 1, 2, or 4 mug of mezerein for 18 weeks produced 2.1, 3.5, and 6.8 papillomas per mouse, respectively, in DMBA-treated mice. Twice-weekly doses as high as 40 mug of 4-O-methyl-TPA were not effective in producing tumors when given after a limited treatment with TPA; however, 4-O-methyl-TPA had weak activity as a first-stage promoter. The results suggest that although mezerein by itself is a weak promoter and mimics TPA in many biochemical and morphological effects it is a potent second-stage promoter in a two-stage promotion regimen.
Article
Dihydroteleocidin B, which is a derivative of teleocidin from Streptomyces, showed potent tumor-promoting activity in vivo when painted on mouse skin. Although the chemical structure of dihydroteleocidin B is entirely different from those of phorbol esters, the tumor-promoting activity of dihydroteleocidin B was found to be comparable to that of 12-O-tetradecanoylphorbol 13-acetate (TPA) in vivo. Teleocidin from Streptomyces and lyngbyatoxin A and debromoaplysiatoxin from the marine blue-green alga Lyngbya majuscula induced ornithine decarboxylase activity when painted on mouse skin, their effects being similar to those of dihyroteleocidin B and TPA. 13-cis-Retinoic acid inhibited this ornithine decarboxylase induction when painted on the skin 1 hr before these natural products. These three compounds produced adhesion of human promyelocytic leukemia cells (HL-60) to the flasks and inhibited differentiation of Friend erythroleukemia cells induced by dimethyl sulfoxide. The in vitro biological potencies of teleocidin and lyngbyatoxin A were almost as great as those of dihydroteleocidin B and TPA, but that of debromoaplysiatoxin was much weaker.
Article
Capping provides a rapid assay for the transduction of 1 type of membrane signal generated by the cross-linking of cell surface receptors. The order of the steps comprising this signal was determined by employing reversible inhibitors of lymphocyte surface Ig capping in a sequential incubation protocol. The results demonstrated that surface Ig cross-linking leads to capping by a linear series of discrete events. Although the steps inhibited by the calcium ionophore A23187 and the tranquilizer chlorpromazine could not be distinguished, other agents also thought to influence calcium distribution in the cell acted at different steps. The order of inhibited steps was shown to be: 1) hydrocortisone; 2) calcium ionophore or chlorpromazine; 3) cytochalasins; 4) dibucaine; 5) propranolol; 6) fluoride; 7) azide. These results suggest a model wherein cross-linked membrane Ig aggregates engage the preassembled microfilament system by means of a calcium-dependent linkage. Further calcium redistribution within the cell then leads to an energy-consuming contractile event.
Article
One of the best models for studying the control of mammalian cell growth and proliferation is the response of lymphocytes to mitogenic agents1-4, which stimulate growth, DNA synthesis and division. How mitogens work remains obscure, although it has been hypothesized that they increase cytoplasmic free calcium, [Ca2+]i, as a trigger for the cascade of intracellular processes necessary for proliferation3-6. However, lymphocyte [Ca2+]i has not previously been measurable. A new technique7 for loading a novel Ca2+-specific indicator8 into the cytoplasm of intact small cells has now made possible the first direct measurements of [Ca2+]i in mouse thymocytes and pig node lymphocytes. We show here that lectins known to stimulate T cells raise average [Ca2+]1 approximately twofold within a few minutes. Deprivation of external Ca2+or elevation of cyclic AMP, conditions known to inhibit mitogenesis, prevented the [Ca2+]i response. Rises in [Ca2+]i were accompanied by hyper-polarization of the membrane potential, apparently due to a Ca2+-activated K+conductance. The co-carcinogen 12-0-tetradecanoylphorbol-13-acetate (TPA) seems to stimulate cell functions normally activated by Ca2+.
Article
The early response by lymphocytes to challenge with cell surface-directed ligand, such as antibody or concanavalin A (Con A), involves the clustering of initially diffuse ligand-receptor complexes into patches, followed by the gathering of these patches into a cap over one region of the cell and their endocytosis1-3. Using cells of the human lymphoblastoid line WiL2, we have previously shown that receptor-mediated endocytosis occurring at caps involves clathrin-coated vesicles and that the formation of such vesicles is sensitive to drugs such as trifluoperazine dihydrochloride (TFP) that affect calmodulin (CaM), the calcium-dependent regulatory protein of cells3. By indirect immunofluorescence, we demonstrate here that in WiL2, before surface-directed ligand challenge, CaM is distributed diffusely in the cell. On challenge, concurrent with capping of cell-surface receptors for Con A, CaM localization becomes concentrated in regions of the cytoplasm just below the cap. We show that CaM redistribution is sensitive to TFP and dependent on Ca2+ in the external milieu. Capping is mostly unaffected by TFP or removal of external Ca2+. Cytochalasin D (CD), on the other hand, blocks capping completely, but does not prevent the initial redistribution of CaM. These results are consistent with a functional role for CaM in clathrin recruitment to the cell surface beneath ligand-receptor complexes and an initial Ca2+ requiring, microfilament-independent event in receptor-mediated endocytosis.
Article
Intermediate filaments (IF) constitute a major cytoplasmic filamentous network of higher eukaryotic cells that is distinct from actin and myosin microfilaments or microtubules. Although structurally similar, these filaments are formed by chemically and antigenically different proteins. Vimentin is the major IF polypeptide of mesenchymal cells and cultured non-mesenchymal cell lines. Recently, we have characterized a monoclonal IgM antibody from a patient with Waldenström's macroglobulinaemia which is directed against vimentin. Using this monoclonal antibody, we have shown by direct immunofluorescence that intermediate filaments of human B and T lymphocytes consist of vimentin. In cells exposed to colcemid, the intermediate filaments retracted into a juxtanuclear aggregate ('coli') characteristic of vimentin filaments. As most components of the cytoskeleton, especially actin and myosin, have been implicated in the capping phenomenon, we investigated the effect of capping of either beta 2-microglobulin or membrane immunoglobulins on the organization of the intermediate filament network. We report that capping of these surface molecules induced the redistribution of vimentin just beneath the cap. When colcemid-treated cells were allowed to cap, the location of the cap always coincided with the coil, suggesting that the anchorage point of intermediate filaments is situated within the uropod.
Article
12-O-Tetradecanoylphorbol-13-acetate (TPA), an effective tumor promoter in mouse skin and comitogen in bovine lymphocytes, rapidly stimulates concanavalin A-mediated cap formation in the latter cells. The ability of different phorbol derivatives to facilitate the capping reaction correlates well with their potencies as lymphocyte comitogens and as tumor-promoting agents. This effect of TPA on capping in bovine lymphocytes, which is apparent within min, is neither mimicked nor altered by dibutyryl cyclic adenosine 3':5'-monophosphate or cyclic guanosine 3':5'-monophosphate. Cytochalasin D, a microfilament-disrupting agent, inhibits cap formation, thereby suggesting the participation of microfilaments in the response. Benzoyl tyrosine ethyl ester selectively inhibits the TPA-stimulated cap formation, whereas benzoyl tyrosinamide is inactive. A comparison of related amino acid derivatives reveals that their activities are dependent on the nature of both the amino acid side chain and the carboxyl end blocking group. Trifluoperazine and N-(6-aminohexyl)-5-chloronaphthalenesulfonamide, known inhibitors of the calmodulin-dependent processes, also selectively block the TPA-stimulated cap formation, whereas trifluoperazine sulfoxide, a less effective calmodulin antagonist, is relatively inactive. These data suggest that the stimulation of capping by TPA involves the activation of a calmodulin-dependent process which may also be regulated by the function of an esterase.
Structure activity relationship in diterpene esters irritant and cocarcinogenic to mouse skin in carcinogenesis
  • H Hecker
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