calcimycin and Skin-Neoplasms

The pathways of oxidant generation in mouse epidermis were investigated by 32P-postlabeling analysis of diastereomeric DNA adducts derived from oxidation of (7S,8S)-dihydroxy-7,8-dihydrobenzo(a)pyrene ((+)-BP-7,8-diol). The pattern of deoxynucleoside-3'-5'-bis-phosphate adducts in epidermal scrapings from female CD-1 mice indicated that cytochrome P-450 was the major oxidant. When animals were pretreated with the tumor-promoting phorbol ester, tetradecanoyl phorbol acetate (TPA), 24 h before coadministration of TPA and (+)-BP-7,8-diol, the pattern of DNA adducts indicated that peroxyl radicals made a major contribution to (+)-BP-7,8-diol epoxidation. Peroxy radical-dependent epoxidation was maximal when the time between the 2 TPA administrations was 24-72 h. No increase in radical-derived adducts was observed when the non-tumor-promoting phorbol ester 4-O-methyl-TPA was substituted for TPA. The calcium ionophore A23187 stimulated radical generation when substituted for the first, but not the second, TPA treatment. The antiinflammatory steroid fluocinolone acetonide inhibited (-)-anti-BPDE-DNA adduct formation when coadministered with the first but not the second TPA treatment. In contrast, all-trans-retinoic acid inhibited (-)-anti-BPDE-DNA adduct formation when coadministered with the second but not the first TPA treatment. These findings demonstrate that tumor promoting phorbol esters stimulate oxygen radical generation in mouse skin and that radical generation is blocked by inhibitors of tumor promotion.

Topics: Animals; Calcimycin; Carcinogens; Dihydroxydihydrobenzopyrenes; DNA; Female; Mice; Mice, Inbred Strains; Oxidants; Phorbol Esters; Skin; Skin Neoplasms; Structure-Activity Relationship

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

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

Recent data suggest that normal tissue mast cells can express functional receptors for IgG under certain conditions. However, little is known about IgG receptor expression and functional consequences in mast cell neoplasms.. In this study, neoplastic mast cells were obtained from a dog with cutaneous mastocytoma (CM-MC) and from a dog with visceral mastocytoma (VI-MC). Both cell populations were characterized morphologically and functionally.. Most cells proliferated constantly in suspension without particular supplements. Doubling times of CM-MC and VI-MC were 52.2 and 27.5 h, respectively. Both cell types were sensitive to formalin fixation, did not contain heparin and were tryptase and chymase positive. Electron microscopy showed fine granules with electron-dense content in both cell populations. The total histamine content of CM-MC and VI-MC was 0.25 and 0.10 pg/cell, respectively. Calcium ionophore A23187 and substance P induced dose-dependent histamine release, whereas compound 48/80 had no effect. Most significantly, both cell types, when sensitized with monomeric dog IgG, released histamine upon stimulation by anti-dog IgG.. Dog mastocytoma-derived cells may be useful to study the regulation of neoplastic mast cell growth and differentiation, as well as IgG receptor-mediated activation in neoplastic mast cells. Further research is required to clarify the pathophysiological significance of constitutive expression of IgG receptors in neoplastic (canine) mast cells.

Topics: Animals; Calcimycin; Cell Line; Chymases; Dog Diseases; Dogs; Female; Histamine Release; Immunoglobulin G; Intestinal Neoplasms; Ionophores; Male; Mast Cells; Mast-Cell Sarcoma; Microscopy, Electron; Serine Endopeptidases; Skin Neoplasms; Substance P; Tryptases; Tumor Cells, Cultured

We have measured the levels of thioredoxin, thioredoxin reductase and glutaredoxin enzyme activity in mouse skin following topical application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a protein kinase C (PKC) activator and tumor promoter. The specific activity of thioredoxin and thioredoxin reductase in extracts from normal epidermis increased by 40 and 50%, respectively, after single or multiple application of TPA. Multiple applications (twice per week for 2 weeks) of TPA increased glutaredoxin activity by >300%. Induction of the proteins lasted several days. Other PKC activators, like 12-O-retinoylphorbol 13-acetate, mezerein, 1-oleoyl-2-acetylglycerol and the calcium ionophore A23187, also induced all the enzyme activities. Phorbol and 4-O-methyl-12-O-tetradecanoylphorbol-13-acetate, weak activators of PKC, selectively induced the thioredoxin system only and did not influence glutaredoxin activity. Multiple applications of TPA to tumor initiated (7,12-dimethyl[a]benzanthracene-treated) skin resulted in elevated levels of both the thioredoxin and glutaredoxin systems when examined 6 days after the last phorbol ester treatment. Induction of thioredoxin, thioredoxin reductase and glutaredoxin activities by TPA and calcium ionophores may play a general role in the epigenetic mechanism of tumor promotion via thiol redox control mechanisms.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Calcimycin; Calcium; Carcinogens; Cocarcinogenesis; Diglycerides; Diterpenes; Enzyme Activation; Enzyme Induction; Epidermis; Female; Fluocinolone Acetonide; Gene Expression Regulation; Glutaredoxins; Glutathione; Ionophores; Mice; Oxidation-Reduction; Oxidoreductases; Phorbol Esters; Protein Kinase C; Proteins; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate; Thioredoxin-Disulfide Reductase; Thioredoxins; Tosylphenylalanyl Chloromethyl Ketone; Tretinoin

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

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

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

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

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

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

Differentiation of cultured keratinocytes is regulated by the Ca2+ concentration of the culture medium. Below 0.1 mM Ca2+, a monolayer of basal cells is formed which fully differentiates in response to a rise in medium Ca2+. A role for protein kinase C in this differentiation program has been suggested because phorbol esters induce epidermal differentiation in cells grown in reduced Ca2+ medium, and exogenously added phospholipase C (which increases cellular diacylglycerol) mimics phorbol ester action. These findings suggested that the external Ca2+ signal may lead to protein kinase C activation via stimulation of cellular phospholipase C activity. The effect of the external Ca2+ signal on phospholipase C was studied in cultures prelabeled with [3H]-inositol. Within 2 min after addition of Ca2+ to 1 mM, an increase in inositol phosphates was measured. This correlated with a decrease in radiolabeled phosphoinositides, suggesting that these were the source of the increased inositol phosphates. After 3 h in 1 mM Ca2+ medium, each of the inositol phosphates remained increased to 130-140% of control levels. Inositol phosphate metabolism in neoplastic epidermal cells was quantitatively similar to normal cells in response to the Ca2+ signal. Stimulation of phosphatidylinositol (PIP) metabolism appears to be mediated by a rise in intracellular free Ca2+ because Ca2+ ionophores A23187 and ionomycin also cause a similar rise in inositol phosphate levels. Phorbol esters did not increase PIP turnover but instead stimulated phosphatidylcholine metabolism. The induction of epidermal differentiation by phorbol esters was enhanced by ionomycin, suggesting that both protein kinase C activation, elevation of intracellular calcium and PIP turnover were important components of the signal for epidermal differentiation. These results demonstrate that the second messenger system for Ca2+-mediated keratinocyte differentiation may be through a direct effect on phospholipase C activity.

Topics: Animals; Calcimycin; Calcium; Cell Differentiation; Cells, Cultured; Choline; Epidermal Cells; Epidermis; Ethers; Inositol; Ionomycin; Keratins; Mice; Skin Neoplasms; Tetradecanoylphorbol Acetate

The effects of promoter treatments prior to initiation on subsequent promotion by mezerein were examined in SENCAR mice. Groups of mice received two applications of various complete as well as first and second stage promoters given at various time intervals prior to initiation ranging from 3 days to 10 weeks. The mice were then initiated with 2 micrograms of 7,12-dimethylbenz[a]anthracene (DMBA) followed 2 weeks later by twice-weekly treatments with 2 micrograms of mezerein. The papilloma response in mice, receiving pretreatments with 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA) either 3 days, 1, 2, 3 or 5 weeks before initiation, was similar to that seen when TPA was given after initiation during stage I of promotion followed by stage II of promotion with mezerein (4-5 papillomas per mouse in all groups). Surprisingly, pretreatment with the stage II promoter, mezerein (2 micrograms), either 2 or 5 weeks prior to initiation, also gave papilloma responses similar to that induced with the standard two-stage promotion protocol (4.7 and 6.4 papillomas per mouse, respectively). The papilloma response was less than that in the standard two-stage promotion protocol when pretreatments with the stage I promoter A23187 (80 micrograms/mouse) were given either 2 or 5 weeks before initiation (2.6 and 2.3 papillomas per mouse, respectively). However, a repeat experiment (currently in progress) with a higher dose of A23187 (160 micrograms/mouse) given 2 weeks prior to initiation indicates that it is more effective than the 80 micrograms dose. When the time interval between pretreatment and initiation was increased to 10 weeks, the papilloma response with TPA and A23187 pretreatment was reduced to below two papillomas per mouse and with mezerein pretreatment to below three papillomas per mouse, indicating the effect was reversible. Histological changes in epidermis of mice which received two applications of these compounds correlated with the tumor response. In this regard, treatment with two applications of TPA and mezerein resulted in an epidermal hyperplasia of similar magnitude (epidermal thickness of 53.5 +/- 1.5 and 50.0 +/- 1.1 microns, respectively). The hyperplasia produced by treatment with two applications of 80 micrograms A23187 (39.4 +/- 1.8 microns) was significantly less. The ability of pretreatments with benzoyl peroxide (20 mg) and chrysarobin (50 micrograms) to affect the subsequent promoting activity of mezerein was also examined.(ABSTRAC

Topics: Animals; Calcimycin; Diterpenes; Drug Synergism; Female; Hyperplasia; Mice; Papilloma; Skin; Skin Neoplasms; Terpenes; Tetradecanoylphorbol Acetate

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

The extracellular matrix of the dermis is subject to severe alterations during tumor promotion with phorbol esters in mouse skin. The metabolic changes also involve general stimulation of protein synthesis and most specifically an increase of collagen synthesis. During chronic treatment with the tumor promoting phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) and 12-O-retinoylphorbol-13-acetate (RPA) increased protein synthesis was observed that did not occur during treatment with the non-promoting mitogens 4-O-methyl-TPA and Ca-ionophore A 23187. Relative collagen synthesis measured as the ratio of radioactivities in hydroxyproline and proline or as the proportion of total radioactivity in pepsin resistant material was elevated, too, but not sufficiently to substitute for TPA-induced collagen loss. In contrast collagen degradation caused by the non-promoting irritant A 23187 is followed by an immediate, substantial increase of collagen synthesis. When TPA treatment was discontinued after a few applications insufficient for tumor development rapid resynthesis of collagen took place. Therefore we assume that continued phorbol ester application not only caused connective tissue damage but also prevents the repair of that damage. This effect seems to be promoter specific and contributes to the disruption of dermal-epidermal interactions during tumor promotion.

Topics: Animals; Calcimycin; Collagen; Female; Mice; Phorbol Esters; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate

Chronic treatment with the tumor-promoting phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) and 12-O-retinoylphorbol-13-acetate (RPA) causes permanently increased levels of active collagenolytic enzymes in the dermis and leads to a stable reduction of dermal collagen content. Non-promoting skin mitogens like the Ca-ionophore A 23187 or the 4-O-methylether of TPA, while being active stimulators of collagenolytic enzymes, do not support chronic collagen degradation throughout the experimental period. On the other hand, TPA-induced collagen degradation is not necessarily influenced by inhibition of tumor promotion. Fluocinolone acetonid (FA), an inhibitor preventing not only tumor development but also chronic inflammation and the establishment of a stationary hyperplasia, has been compared with retinoic acid (RA) which has no influence on either the inflammatory reaction or hyperplasia. While FA inhibited the dermal effects of TPA almost completely, RA at a dose that prevented tumor development by 80% had no effect whatsoever in this respect. Therefore, we conclude that both epidermal proliferation and inflammation are accompanied by collagenolytic reactions in the dermis. During chronic treatment sustained collagenolysis correlates with inflammation and/or the establishment of a stationary hyperplasia. Like these it can be regarded as a necessary but insufficient condition of tumor promotion (second stage).

Topics: Animals; Calcimycin; Cell Division; Collagen; Female; Fluocinolone Acetonide; Mice; Phorbol Esters; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate; Tretinoin

Stimulation of quiescent fibroblasts to growth by polypeptide growth factors is accompanied by the rapid induction of c-fos and c-myc proto-oncogenes. In contrast to fibroblasts, A431 cells respond to epidermal growth factor (EGF) with a decreased growth rate. Here we report that, in spite of its growth inhibitory effect, EGF rapidly induces transient expression of c-fos mRNA, followed by the synthesis of nuclear c-fos protein. In addition, EGF treatment resulted in elevated levels of c-myc expression. Practically identical results were obtained with variant A431 clones that are resistant to the inhibitory effect of EGF on cell proliferation. These observations suggest that in A431 cells c-fos and c-myc induction is a primary consequence of growth factor-receptor interaction. Indeed, efficient induction of both genes was also observed with cyanide bromide-cleaved EGF, which has previously been shown to be non-mitogenic but able to trigger early events induced by EGF. We observed strong induction of c-fos and to a lesser extent of c-myc also by TPA, and by the calcium ionophore A23187, indicating an important role for kinase C in proto-oncogene activation by growth factors.

Topics: Calcimycin; Cell Division; Cell Line; Epidermal Growth Factor; Humans; Oncogenes; Protein Biosynthesis; Protein Kinase C; Protein Kinases; Proto-Oncogene Mas; Skin Neoplasms; Tetradecanoylphorbol Acetate

Topics: Calcimycin; Cell Division; Cocarcinogenesis; Dinoprost; Dinoprostone; Epidermal Cells; Hyperplasia; Indomethacin; Prostaglandins E; Prostaglandins F; Skin; Skin Neoplasms; Structure-Activity Relationship; Tetradecanoylphorbol Acetate