Regulation of the 12-o-tetradecanoyl-phorbol-13-acetate-induced inhibition of intercellular communication

Effects of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and protein kinase C (PKC) inhibitors on cell-cell communication were studied in a normal rat liver cell line, clone 9. Communication was observed and quantitated with microspectrofluorometric and image analysis techniques following scrape-loading of the cells with lucifer yellow. Lucifer yellow migrated as far as ten cells away from the scraped edge in control populations. Two minute TPA (25–50 μg/ml) treatment inhibited dye movement such that the dye remained mainly in the cells at the cut edge. The TPA-induced inhibition of cell-cell communication could be partially blocked by 15 min treatment of the cell populations with the PKC inhibitors trifluoperazine (30 μg/ml), staurosporine (2 × 10^?8 or 2 × 10^?6 M), sangivamycin (15 or 200 μM), or a PKC inhibitor peptide (20 μg/ml) scraped in at the same time as lucifer yellow. Normal communication was observed in cultures treated only with PKC inhibitors. Lower concentrations of TPA (50 ng/ml-1 μg/ml) used for 2 min did not inhibit dye communication. Our results demonstrate the phorbol ester-induced interruption of cell-cell communication. The inhibition of PKC by inhibitors eliminates the effect of TPA on communication. Our data are consistent with a role of PKC in the control of junctional communication. © 1993 Wiley-Liss, Inc.     

Okadaic Acid Is a Potent Angiogenesis Inducer

Okadaic acid, which is a non-12- 0 -tetradecanoylphorbol-13-acetate (TPA)-type tumor promoter and an inhibitor of protein phosphatases 1 and 2A, induced angiogenesis in the chorioallantoic membrane of the chick embryo. Its potent angiogenic activity was dose-dependent. The minimum effective dose was 5 fmol/egg and the effective dose for 50% induction was 90 fmol/egg. These results indicated that okadaic acid exhibits angiogenic activity one order of magnitude stronger than that of TPA (reported previously). Moreover, the time-course of angiogenesis induction by okadaic acid was much slower than that by TPA. The difference is consistent with the time-courses of other biochemical and biological activities and also various gene expressions induced by okadaic acid and TPA, indicating that the difference in the time-course is associated with their mechanisms of action. We conclude that okadaic acid induces angiogenesis through a different pathway than does TPA, indicating the existence of a new mechanism of angiogenesis induction.     

Differentiation of human leukemia cells and its usefulness for clinical diagnosis

Various chemical inducers have effects on the induction of terminal differentiation of human myelogenous leukemia cell lines. We studied morphological and functional changes of human leukemia cells freshly obtained from patients using 12-O-tetradecanoyl phorbol-13-acetate (TPA), retinoic acid (RA) or dimethyl sulphoxide (DMSO). The myeloid leukemia cells cultured with TPA became adherent to plastic culture dishes, and then developed macrophage-like morphology with long filamentous pseudopods within 48 h incubation. They showed marked enhancement of the ability to phagocytose latex particles. But these acquired properties did not always parallel each other, suggesting that the mechanism of functional maturation of leukemic cells induced by chemical agents was not identical with that of morphological changes. On the other hand, the lymphoid leukemia cells did not show morphological and functional changes when cultured with the above inducers. It is suggested that exposure of leukemic cells to TPA for relatively short times (12–24 h) may be useful for determining whether they are of myeloid or lymphoid origin. These characteristic changes were also observed in leukemic cells from the myeloid or lymphoid crisis of chronic myelogenous leukemia.     

Differentiation-associated changes in human non-T, non-B leukemia cell lines after treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA)

The ability of TPA to induce stable phenotypic changes that normally serve as markers of differentiation was examined in the four human non-T, non-B cell lines, NALL-1, NALM-16, REH and KM-3. In all four lines, noncytotoxic concentrations of the phorbol ester caused an extensive reduction in the number of cells expressing cALL surface antigen and terminal deoxynucleotidyl transferase. The disappearance of these markers correlated with the loss of cell proliferation. In one of the cell lines, NALL-1, TPA treatment gave rise to a significant increase in Ia-like antigen and antigen T-101, markers which represent more advanced stages of cell maturation. However, surface or cytoplasmic immunoglobins, indicators of mature B cells, were not detectable. Antigen 3A1, specific for myeloid and for T cells, antigen Leu-4, specific for T cells and antigen CM1, specific for monocytes, were also absent. In all cell lines, exposure to TPA resulted in an approximately two-fold increase in acid phosphatase and beta-glucuronidase activity. The emergence of these phenotype changes was not altered upon repeated washing of the TPA-treated cells. These results demonstrate that while TPA is capable of inducing various non-T, non-B cell lines to differentiate to a limited degree, differences exist between the lines in the extent to which they can mature towards the B-cell stage.     

Effect of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate and its nonpromoting analogue 4-O-methyl-TPA on dorsal dermal melanocytes of the Syrian golden hamster (Mesocricetus auratus)

Summary The effect of the tumor promoter TPA and its inactive structural analogue 4-O-methyl-TPA on the induction of dorsal skin melanosis in the normal Syrian golden hamster and on the promotion of melanomas in DMBA-initiated animals was investigated. Both phenomena were observed in TPA-treated hamsters but could not be detected after exposure of animals to 4-O-methyl-TPA. In contrast to results obtained with a variety of other laboratory animals, neither TPA nor 4-O-methyl-TPA were able to induce epidermal hyperplasia of hamster dorsal skin.Abbreviations DMBA 7,12-dimethylbenz(a)anthracene - TPA 12-O-tetradecanoylphorbol-13-acetate     

Inhibition of macrophage-induced tumor cell cytostasis and cytolysis by tumor-promoting phorbol diesters

The effects of 3 tumor-promoting phorbol diesters and the corresponding inactive polyol phorbol on cytostatic and cytolytic activities of activated murine peritoneal macrophages toward target tumor cells derived from skin cells of the same species were examined. In both cases the diesters, without showing concurrent toxicity, considerably suppressed in dose-related fashion, the activity of the macrophages at concentrations active in promotion in vivo, being active even in nanogram quantities. The order of activity among different diesters for cytostasis or cytolysis was the same as that observed toward tumor promotion in mouse skin. The non-promoter phorbol was inactive in all instances. These findings concur with proposals for an important role for abrogation of normal antitumor defense mechanisms in promotion of mouse skin carcinogenesis by active phorbol diesters.     

The Tumor Promoter Phorbol-12,13-Dibutyrate [P(BU)2] Stimulates Cytotoxic Activity of Human Blood Lymphocytes

The tumor promoters 12-13-phorbol-dibutyrate, P(Bu)2, and 12-0-tetradecanoylphorbol13-acetate, TPA, were shown to augment the cytotoxic potential of human blood lymphocytes with low cell density. In kinetics experiments the enhancing effect was preceded by an initial suppression lasting for about 2 hours. Admixture of mononuclear adherent cells abrogated the P(Bu)2 effect in a dose dependent way. P(Bu)2 altered the sensitivity of K562 cells to the cytotoxic effect. Short term pretreatment increased the sensitivity, but after longer pretreatment the cells became resistant. The results show that tumor promoters can influence the cytolytic system at different levels. By acting directly on the lymphocytes they potentiate the lytic function. When mixed mononuclear populations are used, this effect may be counteracted via activation of the suppressive functions of monocytes. In addition, the target cell sensitivity can also be modulated. As a result, the final outcome of phorbol treatment depends on the strength, kinetics and the mode of its effects on the interactants.     

Genotoxicity testing of cigarette-smoke condensate in the SCE and HGPRT assays with V79 Chinese hamster cells

The genotoxicity of cigarette-smoke condensate (CSC) was investigated using V79 Chinese hamster fibroblasts in the sister-chromatid exchange (SCE) and HGPRT (hypoxanthine-guanine-phosphoribosyl-transferase) tests. CSC was negative in the SCE test both with and without metabolic activation (co-cultivation with chick-embryo primary hepatocytes). In the HGPRT test no direct mutagenicity of CSC was observed but after metabolic activation there was a considerable increase in the number of mutants. Comparison of different metabolizing systems showed that non-induced chick hepatocytes, liver homogenate from non-induced chick embryos and liver homogenate from rats pretreated with Aroclor 1254 generated similar numbers of mutants in cells treated with CSC. In addition the capacity of CSC to inhibit metabolic co-operation between V79 cells was studied. A dose-related increase in the inhibition of metabolic co-operation was observed.     

Protein kinase C in melanoma

Protein kinase C (PKC) is activated by diacylglycerol generated by receptor-mediated hydrolysis of membrane phospholipids to mediate signals for cell growth and plays as a target of tumor-promoting phorbol esters in malignant transformation. PKC is a family of enzymes and their expression profiles have been examined in the normal melanocytes and melanoma cells, and studies have been carried out on the functions of PKC isoforms in proliferation, transformation, and metastasis of melanoma cells. Here, we summarize current knowledge of the expression and possible roles of the PKC family in melanoma in comparison with those of normal melanocytes.     

Protein kinase C isoforms as specific targets for modulation of vascular smooth muscle function in hypertension

Vascular contraction is an important determinant of the peripheral vascular resistance and blood pressure. The mechanisms underlying vascular smooth muscle (VSM) contraction and the pathological changes that occur in hypertension have been the subject of numerous studies and interpretations. Activation of VSM by vasoconstrictor stimuli at the cell surface causes an increase in [Ca(2+)](i), Ca(2+)-dependent activation of myosin light chain (MLC) kinase, MLC phosphorylation, actin-myosin interaction and VSM contraction. Additional signaling pathways involving Rho-kinase and protein kinase C (PKC) may increase the myofilament force sensitivity to [Ca(2+)](i) and MLC phosphorylation, and thereby maintain vascular contraction. PKC is a particularly intriguing protein kinase as it comprises a family of Ca(2+)-dependent and Ca(2+)-independent isoforms, which have different tissue and subcellular distribution, and undergo differential translocation during cell activation. PKC translocation to the cell surface may trigger a cascade of protein kinases, such as mitogen-activated protein kinase (MAPK) and MAPK kinase (MEK) that ultimately interact with the contractile myofilaments and cause VSM contraction. Also, PKC translocation to the nucleus may promote VSM growth and proliferation. Increased PKC expression and activity have been identified in several forms of hypertension. The subcellular location of PKC may determine the state of VSM activity, and may be useful in the diagnosis/prognosis of hypertension. Vascular PKC isoforms may represent specific targets for modulation of VSM hyperactivity, and isoform-specific PKC inhibitors may be useful in treatment of Ca(2+) antagonist-resistant forms of hypertension.