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Sample records for phorbol diester-induced apoptosis

  1. Protein kinase C-{beta}, fibronectin, {alpha}{sub 5}{beta}{sub 1}-integrin and tumor necrosis factor-{alpha} are required for phorbol diester-induced apoptosis in human myeloid leukemia cells in human myeloid leukemia cells.

    SciTech Connect

    Laouar, A.; Glesne, D.; Huberman, E.

    2001-12-01

    The human myeloid HL-60 cell line and its cell variant HL-525 were used to study signaling events leading to apoptosis induction by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC) enzymes. Unlike parental cells, HL-525 cells are PKC-{beta} deficient and resistant to PMA-induced apoptosis. These cells regain susceptibility to apoptosis induction after transfection with a PKC-{beta} expression vector. By using this vector and specific neutralizing monoclonal antibodies (mAbs), it was established that PMA-induced apoptosis also called for an interaction between cell-surface {alpha}{sub 5}{beta}{sub 1}-integrin and its deposited ligand fibronectin (FN), which is downstream of PKC-{beta} activation. Experiments with mAbs, the PKC-{beta} vector, and exogenous FN revealed that the next step entailed an interaction between secreted tumor necrosis factor-{alpha} and its type I receptor. By using a sphingomyelinase inhibitor, it was concluded that the subsequent step involved ceramide production. Moreover, a permeable ceramide was effective in inducing apoptosis in both HL-60 and HL-525 cells, and this induction was caspase-1 and/or -4 dependent because an inhibitor of these caspases abrogated the induced apoptosis. Based on these and related differentiation studies, we conclude that the above signaling events, the early ones in particular, are shared with PMA-induced macrophage differentiation in the HL-60 cells. It is likely that once these cells acquire their macrophage phenotype and perform their tasks, they become superfluous and are eliminated from the body by a self-triggered apoptotic process that involves our proposed signaling scheme.

  2. Protection against apoptosis in chicken bursa and thymus cells by phorbol ester in vitro

    SciTech Connect

    Asakawa, J.; Thorbecke, G.J. )

    1991-03-15

    Programmed suicide or apoptosis, due to activation of endogenous nucleases, occurs in immature CD4{sup {minus}}85{sup {minus}} mammalian thymus cells. Like the thymus, the bursa of Fabricius is a site of massive lymphopoiesis accompanied by cell death in vivo. In the present study the authors have, therefore, examined whether chicken bursa and thymus cells exhibit apoptosis. Bursa and thymus cells from SC chickens, 4-10 weeks of age, were incubated for 8-24 hrs with various reagents. Genomic DNA was isolated, electrophoresed in 3% Nusieve agarose gels, and examined for patterns of DNA fragmentation. A laddering of DNA in multiples of 200 base pairs, indicative of apoptosis, was observed with both bursa and thymus cells. These patterns of DNA fragmentation from bursa cells could be prevented by adding phorbol myristic acetate during culture and, more effectively, by PMA plus ionomycin, but not by ionomycin alone or by anti-{mu}. PMA did not affect the patterns of DNA fragmentation seen with spleen cells. Addition of the protein kinase C inhibitor staurosporin inhibited the preventive effect of PMA on apoptosis. PMA also greatly promoted the survival of bursa cells in culture, as assayed by percentage cell death and by {sup 3}H-thymidine incorporation. It is concluded that bursa and thymus cells from the chicken exhibit apoptosis. The data further suggest that protein kinase C activation protects apoptosis in cultured bursa cells.

  3. Prevention of neuronal apoptosis by phorbol ester-induced activation of protein kinase C: blockade of p38 mitogen-activated protein kinase.

    PubMed

    Behrens, M M; Strasser, U; Koh, J Y; Gwag, B J; Choi, D W

    1999-01-01

    Consistent with previous studies on cell lines and non-neuronal cells, specific inhibitors of protein kinase C induced mouse primary cultured neocortical neurons to undergo apoptosis. To examine the complementary hypothesis that activating protein kinase C would attenuate neuronal apoptosis, the cultures were exposed for 1 h to phorbol-12-myristate-13-acetate, which activated protein kinase C as evidenced by downstream enhancement of the mitogen-activated protein kinase pathway. Exposure to phorbol-12-myristate-13-acetate, or another active phorbol ester, phorbol-12,13-didecanoate, but not to the inactive ester, 4alpha-phorbol-12,13-didecanoate, markedly attenuated neuronal apoptosis induced by serum deprivation. Phorbol-12-myristate-13-acetate also attenuated neuronal apoptosis induced by exposure to beta-amyloid peptide 1-42, or oxygen-glucose deprivation in the presence of glutamate receptor antagonists. The neuroprotective effects of phorbol-12-myristate-13-acetate were blocked by brief (non-toxic) concurrent exposure to the specific protein kinase C inhibitors, but not by a specific mitogen-activated protein kinase 1 inhibitor. Phorbol-12-myristate-13-acetate blocked the induction of p38 mitogen-activated protein kinase activity and specific inhibition of this kinase by SB 203580 attenuated serum deprivation-induced apoptosis. c-Jun N-terminal kinase 1 activity was high at rest and not modified by phorbol-12-myristate-13-acetate treatment. These data strengthen the idea that protein kinase C is a key modulator of several forms of central neuronal apoptosis, in part acting through inhibition of p38 mitogen-activated protein kinase regulated pathways.

  4. Apoptosis induced by microtubule disrupting drugs in cultured human lymphoma cells. Inhibitory effects of phorbol ester and zinc sulphate.

    PubMed

    Takano, Y; Okudaira, M; Harmon, B V

    1993-03-01

    The effects of the microtubule disrupting drugs (MDD) vinblastine, vincristine and colchicine on a human lymphoma cell line, BM 13674, were investigated. Twelve hours after administration of vinblastine (10(-3) mg/ml), vincristine (10(-2) mg/ml) or colchicine (10(-2) mg/ml), cell death with the characteristic morphology of apoptosis was observed in 71.6%, 82.2% and 76.9% of the cells respectively. The mode of death was confirmed as apoptotic by the occurrence of internucleosomal DNA cleavage, which was demonstrated by agarose gel electrophoresis. For the purpose of casting light on the mechanism involved, inhibition tests were performed on apoptosis induced by one of these drugs, vinblastine, using a phorbol ester (PDBu), zinc sulphate and cycloheximide. PDBu, an activator of protein kinase C, and zinc sulphate, a putative inhibitor of the endonuclease were thought to be responsible for internucleosomal DNA cleavage; both markedly reduced the induction of apoptosis. The protein synthesis inhibitor cycloheximide, on the other hand, had no inhibitory effect. Moreover, cycloheximide treatment per se enhanced apoptosis. This suggests that new protein synthesis is not required for the execution of vinblastine-induced apoptosis. Such a finding is in accord with recent reports suggesting that the "death program" within many cell types may be primed but unable to proceed due to concomitant production of specific "apoptotic inhibitors". It is suggested that phorbol esters prevent vinblastine-induced apoptosis in the BM 13674 cells by activating one or more of these specific "apoptotic inhibitors", possibly by means of PKC-mediated phosphorylation.

  5. Diacylglycerols mimic phorbol diester induction of leukemic cell differentiation.

    PubMed Central

    Ebeling, J G; Vandenbark, G R; Kuhn, L J; Ganong, B R; Bell, R M; Niedel, J E

    1985-01-01

    Activation of cellular protein kinase C appears to be involved in the mechanism by which phorbol diesters induce differentiation of human myeloid leukemia cells (HL-60). Protein kinase C is thought to be physiologically activated by diacylglycerol derived from receptor-mediated phosphatidylinositol hydrolysis. sn-1,2-diacylglycerols with short saturated acyl side chains (C4-C10) were synthesized and found to be potent activators of protein kinase C partially purified from HL-60 cells. These diacylglycerols were also competitive inhibitors of [3H]phorbol dibutyrate binding to the soluble phorbol diester receptor. The most potent diacylglycerol, sn-1,2-dioctanoylglycerol, displaced greater than 90% of [3H]phorbol dibutyrate from the phorbol diester receptor of intact HL-60 cells. Because of probable cellular metabolism of sn-1,2-dioctanoylglycerol, hourly doses were required to maintain persistent occupancy of the phorbol diester binding site. Treatment of HL-60 cells with either phorbol 12-myristate 13-acetate or sn-1,2-dioctanoylglycerol produced identical phosphoprotein changes. Finally, sn-1,2-dioctanoylglycerol induced differentiation of the HL-60 cells into cells with morphologic characteristics of macrophages. Substitution of the hydroxyl group at position 3 with a hydrogen, chloro, or sulfhydryl moiety inactivated sn-1,2-dioctanoylglycerol. These data strengthen the hypothesis that protein kinase C activation plays a role in macrophage differentiation. Images PMID:3156372

  6. ROCK mediates phorbol ester-induced apoptosis in prostate cancer cells via p21Cip1 up-regulation and JNK.

    PubMed

    Xiao, Liqing; Eto, Masumi; Kazanietz, Marcelo G

    2009-10-23

    It is established that androgen-dependent prostate cancer cells undergo apoptosis upon treatment with phorbol esters and related analogs, an effect primarily mediated by PKCdelta. Treatment of LNCaP prostate cancer cells with phorbol 12-myristate 13-acetate (PMA) causes a strong and sustained activation of RhoA and its downstream effector ROCK (Rho kinase) as well as the formation of stress fibers. These effects are impaired in cells subjected to PKCdelta RNA interference depletion. Functional studies revealed that expression of a dominant negative RhoA mutant or treatment with the ROCK inhibitor Y-27632 inhibits the apoptotic effect of PMA in LNCaP cells. Remarkably, the cytoskeleton inhibitors cytochalasin B and blebbistatin blocked not only PMA-induced apoptosis but also the activation of JNK, a mediator of the cell death effect by the phorbol ester. In addition, we found that up-regulation of the cell cycle inhibitor p21(Cip1) is required for PMA-induced apoptosis and that inhibitors of ROCK or the cytoskeleton organization prevent p21(Cip1) induction. Real time PCR analysis and reporter gene assay revealed that PMA induces p21(Cip1) transcriptionally in a ROCK- and cytoskeleton-dependent manner. p21(Cip1) promoter analysis revealed that PMA induction is dependent on Sp1 elements in the p21(Cip1) promoter but independent of p53. Taken together, our studies implicate ROCK-mediated up-regulation of p21(Cip1) and the cytoskeleton in PKCdelta-dependent apoptosis in prostate cancer cells.

  7. Involvement of phorbol-12-myristate-13-acetate-induced protein 1 in goniothalamin-induced TP53-dependent and -independent apoptosis in hepatocellular carcinoma-derived cells

    SciTech Connect

    Kuo, Kung-Kai; Chen, Yi-Ling; Chen, Lih-Ren; Li, Chien-Feng; Lan, Yu-Hsuan; Chang, Fang-Rong; Wu, Yang-Chang; Shiue, Yow-Ling

    2011-10-01

    The objective was to investigate the upstream apoptotic mechanisms that were triggered by a styrylpyrone derivative, goniothalamin (GTN), in tumor protein p53 (TP53)-positive and -negative hepatocellular carcinoma (HCC)-derived cells. Effects of GTN were evaluated by the flow cytometry, alkaline comet assay, immunocytochemistry, small-hairpin RNA interference, mitochondria/cytosol fractionation, quantitative reverse transcription-polymerase chain reaction, immunoblotting analysis and caspase 3 activity assays in two HCC-derived cell lines. Results indicated that GTN triggered phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1, also known as NOXA)-mediated apoptosis via TP53-dependent and -independent pathways. In TP53-positive SK-Hep1 cells, GTN furthermore induced TP53 transcription-dependent and -independent apoptosis. After GTN treatment, accumulation of reactive oxygen species, formation of DNA double-strand breaks, transactivation of TP53 and/or PMAIP1 gene, translocation of TP53 and/or PMAIP1 proteins to mitochondria, release of cytochrome c from mitochondria, cleavage of caspases and induction of apoptosis in both cell lines were sustained. GTN might represent a novel class of anticancer drug that induces apoptosis in HCC-derived cells through PMAIP1 transactivation regardless of the status of TP53 gene. - Highlights: > Goniothalamin (GTN) induced apoptosis in hepatocellular carcinomas-derived cells. > The apoptosis induced by GTN is PMAIP1-dependent, regardless of TP53 status. > The apoptosis induced by GTN might be TP53 transcription-dependent or -independent. > GTN-induced apoptosis is mitochondria- and caspases-mediated.

  8. Phorbol ester-induced apoptosis of C4-2 cells requires both a unique and a redundant protein kinase C signaling pathway.

    PubMed

    Yin, Lihong; Bennani-Baiti, Nabila; Powell, C Thomas

    2005-02-18

    Phorbol 12-myristate 13-acetate (PMA) potently induces apoptosis of LNCaP human prostate cancer cells. Here, we show that C4-2 cells, androgen-hypersensitive derivatives of LNCaP cells, also are sensitive to PMA-induced apoptosis. Previous reports have implicated activation of protein kinase C (PKC) isozymes alpha and delta in PMA-induced LNCaP apoptosis using overexpression, pharmacological inhibitors, and dominant-negative constructs, but have left unresolved if other isozymes are involved, if there are separate requirements for individual PKC isozymes, or if there is redundancy. We have resolved these questions in C4-2 cells using stable expression of short hairpin RNAs to knock down expression of specific PKC isozymes individually and in pairs. Partial knockdown of PKCdelta inhibited PMA-induced C4-2 cell death almost completely, whereas near-complete knockdown of PKCalpha had no effect. Knockdown of PKCepsilon alone had no effect, but simultaneous knockdown of both PKCalpha and PKCepsilon in C4-2 cells that continued to express normal levels of PKCdelta inhibited PMA-induced apoptosis. Thus, our data indicate that there is an absolute requirement for PKCdelta in PMA-induced C4-2 apoptosis but that the functions of PKCalpha and PKCepsilon in apoptosis induction are redundant, such that either one (but not both) is required. Investigation of PMA-induced events required for LNCaP and C4-2 apoptosis revealed that p38 activation is dependent on PKCdelta, whereas induction of retinoblastoma protein hypophosphorylation requires both PKC signaling pathways and is downstream of p38 activation in the PKCdelta pathway.

  9. Bryostatin 1 Inhibits Phorbol Ester-Induced Apoptosis in Prostate Cancer Cells by Differentially Modulating Protein Kinase C (PKC) δ Translocation and Preventing PKCδ-Mediated Release of Tumor Necrosis Factor-α

    PubMed Central

    von Burstin, Vivian A.; Xiao, Liqing

    2010-01-01

    Bryostatin 1, a macrocyclic lactone that has been widely characterized as an ultrapotent protein kinase C (PKC) activator, displays marked pharmacological differences with the typical phorbol ester tumor promoters. Bryostatin 1 impairs phorbol 12-myristate 13-acetate (PMA)-induced tumor promotion in mice and is in clinical trials as an anticancer agent for a number of hematopoietic malignancies and solid tumors. In this study, we characterized the effect of bryostatin 1 on LNCaP prostate cancer cells, a cellular model in which PKC isozymes play important roles in the control of growth and survival. Although phorbol esters promote a strong apoptotic response in LNCaP cells via PKCδ-mediated release of TNFα, bryostatin 1 failed to trigger a death effect even at high concentrations, and it prevented PMA-induced apoptosis in these cells. Mechanistic analysis revealed that bryostatin 1 is unable to induce TNFα release, and it impairs the secretion of this cytokine from LNCaP cells in response to PMA. Unlike PMA, bryostatin 1 failed to promote the translocation of PKCδ to the plasma membrane. Moreover, bryostatin 1 prevented PMA-induced PKCδ peripheral translocation. Studies using a membrane-targeted PKCδ construct revealed that the peripheral localization of the kinase is a requisite for triggering apoptosis in LNCaP cells, arguing that mislocalization of PKCδ may explain the actions of bryostatin 1. The identification of an antiapoptotic effect of bryostatin 1 may have significant relevance in the context of its therapeutic efficacy. PMID:20516369

  10. Bryostatin 1 inhibits phorbol ester-induced apoptosis in prostate cancer cells by differentially modulating protein kinase C (PKC) delta translocation and preventing PKCdelta-mediated release of tumor necrosis factor-alpha.

    PubMed

    von Burstin, Vivian A; Xiao, Liqing; Kazanietz, Marcelo G

    2010-09-01

    Bryostatin 1, a macrocyclic lactone that has been widely characterized as an ultrapotent protein kinase C (PKC) activator, displays marked pharmacological differences with the typical phorbol ester tumor promoters. Bryostatin 1 impairs phorbol 12-myristate 13-acetate (PMA)-induced tumor promotion in mice and is in clinical trials as an anticancer agent for a number of hematopoietic malignancies and solid tumors. In this study, we characterized the effect of bryostatin 1 on LNCaP prostate cancer cells, a cellular model in which PKC isozymes play important roles in the control of growth and survival. Although phorbol esters promote a strong apoptotic response in LNCaP cells via PKCdelta-mediated release of TNFalpha, bryostatin 1 failed to trigger a death effect even at high concentrations, and it prevented PMA-induced apoptosis in these cells. Mechanistic analysis revealed that bryostatin 1 is unable to induce TNFalpha release, and it impairs the secretion of this cytokine from LNCaP cells in response to PMA. Unlike PMA, bryostatin 1 failed to promote the translocation of PKCdelta to the plasma membrane. Moreover, bryostatin 1 prevented PMA-induced PKCdelta peripheral translocation. Studies using a membrane-targeted PKCdelta construct revealed that the peripheral localization of the kinase is a requisite for triggering apoptosis in LNCaP cells, arguing that mislocalization of PKCdelta may explain the actions of bryostatin 1. The identification of an antiapoptotic effect of bryostatin 1 may have significant relevance in the context of its therapeutic efficacy.

  11. Phorbol diester-induced phosphorylation of nuclear matrix proteins in HL60 promyelocytes. Possible role in differentiation studied by cationic detergent gel electrophoresis

    SciTech Connect

    Macfarlane, D.E.

    1986-05-25

    Immortal HL60 promyelocytes are induced to differentiate to mortal adherent cells by a variety of agents which activate protein kinase C, including 12-O-tetradecanoylphorbol 13-acetate (TPA). In order to investigate the mechanism of this effect, we incubated HL60 cells with (/sup 32/P)orthophosphate with or without TPA and extracted their proteins with the cationic detergent benzyldimethyl-n-hexadecylammonium chloride prior to electrophoresis in a discontinuous polyacrylamide gel system in the first dimension. In this system, proteins migrate toward the cathode as a function of their molecular weight, and they are separated from other radioactive components which can obscure the pattern of protein phosphorylation on sodium dodecyl sulfate (SDS) gels. SDS gel electrophoresis was used in the second dimension, resulting in the clear resolution of a large number of proteins. TPA caused many changes in the pattern of protein phosphorylation in intact cells. Two proteins which prominently increased their incorporation of /sup 32/P were investigated in particular, and they were both found to be retained in the nuclear matrix following successive extraction of cells with Triton, digestion with DNase and RNase, and extraction with 2 M NaCl. These proteins migrated with apparent molecular weights of 80,000 and 33,000 on SDS gels, and are designated NP80 and NP33, respectively. NP80 was half-maximally phosphorylated after 7 min exposure to TPA, and half-maximally phosphorylated by 10 nM TPA. NP80 co-migrated with a faint Coomassie Blue-stained protein, and NP33 co-migrated with a more prominent protein. Several proteins incorporated less /sup 32/P when the cells were exposed to TPA, including one which was extracted from nuclei with the core histones and which co-migrated with histone H2A.

  12. Phorbol diesters and transferrin modulate lymphoblastoid cell transferrin receptor expression by two different mechanisms

    SciTech Connect

    Alcantara, O.; Phillips, J.L.; Boldt, D.H.

    1986-12-01

    Expression of transferrin receptors (TfR) by activated lymphocytes is necessary for lymphocyte DNA synthesis and proliferation. Regulation of TfR expression, therefore, is a mechanism by which the lymphocyte's proliferative potential may be directed and controlled. The authors studied mechanisms by which lymphoblastoid cells modulate TfR expression during treatment with phorbol diesters or iron transferrin (FeTf), agents which cause downregulation of cell surface TfR. Phorbol diester-induced TfR downregulation occurred rapidly, being detectable at 2 min and reaching maximal decreases of 50% by 15 min. It was inhibited by cold but not by agents that destabilize cytoskeletal elements. Furthermore, this downregulation was reversed rapidly by washing or by treatment with the membrane interactive agent, chlorpromazine. In contrast, FeTf-induced TfR downregulation occurred slowly. Decreased expression of TfR was detectable only after 15 min and maximal downregulation was achieved after 60 min. Although FeTf-induced downregulation also was inhibited by cold, it was inhibited in addition by a group of microtubule destabilizing agents (colchicine, vinblastine, podophyllotoxin) or cytochalasin B, a microfilament inhibitor. Furthermore, FeTf-induced downregulation was not reversed readily by washing or by treatment with chlorpromazine. Phorbol diesters cause TfR downregulation by a cytoskeleton-independent mechanism. These data indicate that TfR expression is regulated by two independent mechanisms in lymphoblastoid cells, and they provide the possibility that downregulation of TfR by different mechanisms may result in different effects in these cells.

  13. Specific binding of phorbol ester tumor promoters

    PubMed Central

    Driedger, Paul E.; Blumberg, Peter M.

    1980-01-01

    [20-3H]Phorbol 12,13-dibutyrate bound to particulate preparations from chicken embryo fibroblasts in a specific, saturable, reversible fashion. Equilibrium binding occurred with a Kd of 25 nM; this value is very close to the 50% effective dose (ED50), 50 nM, previously determined for the biological response (induction of fibronectin loss) in growing chicken embryo fibroblasts. At saturation, 1.4 pmol of [20-3H]phorbol 12,13-dibutyrate was bound per mg of protein (approximately 7 × 104 molecules per cell). Binding was inhibited by phorbol 12-myristate 13-acetate (Ki = 2 nM), mezerein (Ki = 180 nM), phorbol 12,13-dibenzoate (Ki = 180 nM), phorbol 12,13-diacetate (Ki = 1.7 μM), phorbol 12,13,20-triacetate (Ki = 39 μM), and phorbol 13-acetate (Ki = 120 μM). The measured Ki values are all within a factor of 3.5 of the ED50 values of these derivatives for inducing loss of fibronectin in intact cells. Binding was not inhibited by the inactive compounds phorbol (10 μg/ml) and 4α-phorbol 12,13-didecanoate (10 μg/ml) or by the inflammatory but nonpromoting phorbol-related diterpene esters resiniferatoxin (100 ng/ml) and 12-deoxyphorbol 13-isobutyrate 20-acetate (100 ng/ml). These data suggest that biological responses to the phorbol esters in chicken embryo fibroblasts are mediated by this binding activity and that the binding activity corresponds to the phorbol ester target in mouse skin involved in tumor promotion. Binding was not inhibited by the nonphorbol promoters anthralin (1 μM), phenol (1 mM), iodoacetic acid (1.7 μM), and cantharidin (75 μM), or by epidermal growth factor (100 ng/ml), dexamethasone acetate (2 μM), retinoic acid (10 μM), or prostaglandin E2 (1 μM). These agents thus appear to act at a target distinct from that of the phorbol esters. PMID:6965793

  14. Nineteen-Step Total Synthesis of (+)-Phorbol

    PubMed Central

    Kawamura, Shuhei; Chu, Hang; Felding, Jakob; Baran, Phil S.

    2016-01-01

    Phorbol, the flagship member of the tigliane diterpene family, has been known for over 80 years and has attracted attention from scores of chemists and biologists due to its intriguing chemical structure and the medicinal potential of phorbol esters.1 Access to useful quantities of phorbol and related analogs has relied upon isolation from natural sources and semisynthesis. Despite relentless efforts spanning 40 years, chemical synthesis has been unable to compete with these strategies due to its sheer complexity and unusual oxidation pattern. In fact, purely synthetic enantiopure phorbol has remained elusive and efforts on the synthetic biology side have not led to even the simplest members of this terpene family. Recently the chemical syntheses of eudesmanes,2 germacrenes,3 taxanes,4,5 and ingenanes6-8 have all benefited from a strategy inspired by the logic of two-phase terpene biosynthesis where powerful C–C bond constructions and C–H bond oxidations go hand in hand. In this manuscript, we show how a two-phase terpene synthesis strategy can be enlisted to achieve the first enantiospecific total synthesis of (+)-phorbol in only 19 steps from the abundant monoterpene (+)-3-carene. The purpose of this route is not to displace isolation/semisynthesis as a means to generate the natural product per se, but rather to enable access to analogs containing unique oxidation patterns that are otherwise inaccessible. PMID:27007853

  15. Phorbol ester phorbol-12-myristate-13-acetate promotes anchorage-independent growth and survival of melanomas through MEK-independent activation of ERK1/2

    SciTech Connect

    Jorgensen, Kjersti; Skrede, Martina; Cruciani, Veronique; Mikalsen, Svein-Ole; Slipicevic, Ana; Florenes, Vivi Ann . E-mail: v.a.florenes@labmed.uio.no

    2005-04-01

    The phorbol ester, phorbol-12-myristate-13-acetate (PMA), an activator of PKCs, is known to stimulate the in vitro growth of monolayer cultures of normal human melanocytes whereas it inhibits the growth of most malignant melanoma cell lines. We examined the effect of PMA on proliferation and survival of melanoma cells grown as multicellular aggregates in suspension (spheroids), and aimed to elucidate downstream targets of PKC signaling. In contrast to monolayer cultures, PMA increased cell proliferation as well as protected melanoma cells from suspension-mediated apoptosis (anoikis). Supporting the importance of PKC in anchorage-independent growth, treatment of anoikis-resistant melanoma cell lines with antisense oligonucleotides against PKC-{alpha}, or the PKC inhibitor Goe6976, strongly induced anoikis. PMA induced activation of ERK1/2, but this effect was not prevented by the MEK inhibitors PD98059 or by U0126. Whereas PD98059 treatment alone led to marked activation of the pro-apoptotic Bim and Bad proteins and significantly increased anoikis, these effects were clearly reversed by PMA. In conclusion, our results indicate that the protective effect of PMA on anchorage-independent survival of melanoma cells at least partly is mediated by MEK-independent activation of ERK1/2 and inactivation of downstream pro-apoptotic effector proteins.

  16. Light induced degradation of phorbol esters.

    PubMed

    Yunping, Bu; Ha, Bui Thi Ngoc; Eunice, Yeo; Chueng, Lo Loong; Yan, Hong

    2012-10-01

    Jatropha curcas (Jatropha) is a tropical shrub that is gaining popularity as a biofuel feedstock plant. Phorbol esters (PEs) are tetracyclic tiglian diterpenoids that are present in Jatropha seeds and other parts of plant. Epidermal cell irritating and cancer promoting PEs not only reduce commercial values of Jatropha seed cake but also cause some safety and environment concerns on PE leaching to soil. A simple bioassay of PE toxicity was conducted by incubating 48 h old brine shrimp (Artemia salina) nauplii with Jatropha oil for 24 h. 1-4% of Jatropha oil (corresponding to PE concentration of 25-100 mg L(-1)) had mortality rate of 5-95%, with LC50 estimated to be 2.7% of oil or 67 mg L(-1) of PE. Jatropha oil was incubated with clay or black soil (autoclaved or non-autoclaved) in the darkness or under sunlight for different periods of time before oil was re-extracted and tested for PE content by HPLC and for remaining toxicity with the brine shrimp bioassay. Under sunlight, PE decreased to non-detectable level within six days. Toxicity reduced to less than 5% mortality rate that is comparable to rapeseed oil control within the same period. In contrast, PE level and toxicity remained little changed when Jatropha oil was incubated in the darkness. Such PE degradation/detoxification was also found independent of the presence of soil or soil microorganisms. We conclude that sunlight directly degrades and detoxifies PEs and this finding should alleviate the concern on long term environmental impact of PE leaching. Copyright © 2012 Elsevier Inc. All rights reserved.

  17. An improved preparation of phorbol from croton oil.

    PubMed

    Pagani, Alberto; Gaeta, Simone; Savchenko, Andrei I; Williams, Craig M; Appendino, Giovanni

    2017-01-01

    Background: Croton oil is the only commercial source of the diterpenoid phorbol (1a), the starting material for the semi-synthesis of various diesters extensively used in biomedical research to investigate cell function and to evaluate in vivo anti-inflammatory activity. While efficient chemoselective esterification protocols have been developed for phorbol, its isolation from croton oil is technically complicated, and involves extensive manipulation of very toxic materials like the oil or its native diterpenoid fraction. Results: The preparation of a crude non-irritant phorboid mixture from croton oil was telescoped to only five operational steps, and phorbol could then be purified by gravity column chromatography and crystallization. Evidence is provided that two distinct phorboid chemotypes of croton oil exist, differing in the relative proportion of type-A and type-B esters and showing different stability to deacylation. Conclusion: The isolation of phorbol from croton oil is dangerous because of the toxic properties of the oil, poorly reproducible because of differences in its phorboid profile, and time-consuming because of the capricious final crystallization step. A solution for these issues is provided, suggesting that the poor-reproducibility of croton oil-based anti-inflammatory assays are the result of poor quality and/or inconsistent composition of croton oil.

  18. Epidermal cell proliferation and promoting ability of phorbol esters.

    PubMed

    Slaga, T J; Scribner, J D; Viaje, A

    1976-11-01

    Dose-response relationships on the abilities of several phorbol ester tumor promoters to promote skin tumors after 7,12-dimethylbenz[a]anthracene initiation and to bring about edema, inflammation, and epidermal hyperplasia were determined in female Charles River CD-1 mice. The promoting ability of the potent synthetic promoter, phorbol-12,13-dioctanoate (PdiC8), was determined over a dose range of 0.1-10 mug/application. Administration of PdiC8 two times weekly at dosages of 4, 6, 8, and 10 mug gave little variation in tumor response. A dose-dependent tumor response occurred at doses of 1-4 mug PdiC8. Only 1 papilloma was observed when PdiC8 was given twice weekly at a dose of 0.1 or 0.5 mug. A similar dose-response relation was observed for the ability of PdiC8 to stimulate epidermal hyperplasia. Investigations of other phorbol esters revealed an excellent correlation between their promoting ability and their ability to induce epidermal hyperplasia; however, that was not the case for compounds outside the phorbol ester series (i.e., acetic acid, cantharidin, and ethylphenylpropiolate).

  19. Phorbol diesters inhibit enzymatic hydrolysis of diacylglycerols in vitro.

    PubMed Central

    Chabbott, H; Cabot, M C

    1986-01-01

    The effect of phorbol 12-myristate 13-acetate (PMA) on diacylglycerol lipase activity was examined in rat serum, tissue, and cellular preparations by using di[14C]oleoylglycerol, [3H]palmitoylacetylglycerol, and membrane-resident phospholipase C-generated diacylglycerols as substrates. These experiments were conducted to address whether phorbol esters can mimic diacylglycerols in interacting with enzymes other than protein kinase C. Serum hydrolysis of palmitoylacetylglycerol, assayed by the formation of [3H]palmitic acid, was inhibited by PMA, 4-O-methyl-PMA, or phorbol 12,13-dibutyrate (in order of decreasing potency). The hydrolysis of palmitoylacetylglycerol was inhibited more than 40% by the addition of PMA at a 1:1 molar ratio with substrate. The inhibition resembled the competitive type, with a Ki of approximately 2.7 microM. PMA in the 10-60 microM range also inhibited hydrolysis of palmitoylacetylglycerol by lipases from rat brain microsomes and by homogenates of C3H/10T1/2 mouse fibroblasts. PMA was likewise inhibitory when assayed in an intramembrane enzyme-substrate milieu in which diacylglycerols were generated, in situ, by treatment of [3H]palmitate-labeled cell homogenates with phospholipase C. Collectively, these data demonstrate that PMA, which is now thought to act by mimicry of diacylglycerols, can inhibit the action of diacylglycerol lipase. It is possible that such a mechanism is linked to the multiplicity of responses elicited by phorbol diesters and that other agents may function by means of enzyme interactions (post-phospholipase C) to influence the levels of the cellular diacylglycerol mediators. PMID:3458169

  20. Properties of the protein kinase C-phorbol ester interaction

    SciTech Connect

    Bazzi, M.D.; Nelsestuen, G.L. )

    1989-04-18

    The properties of the protein kinase C (PKC)-phorbol ester interaction were highly dependent on assay methods and conditions. Binding to cation-exchange materials or adsorption to gel matrices resulted in PKC that was capable of binding phorbol 12,13-dibutyrate (PDBu). The extraneous interactions were eliminated by measuring phorbol ester binding with a gel filtration chromatography assay in the presence of bovine serum albumin (BSA). In the absence of calcium, free PKC did not bind PDBu or phospholipids. Calcium caused structural changes in PKC which enhanced its interaction with surfaces such as the gel chromatography matrix. While BSA prevented this interaction, it did not interfere with PKC association with acidic phospholipids. Interaction of PKC with phospholipid resulted in two forms of membrane-associated PKC. Once PKC was inserted into a phospholipid bilayer, it bound PDBu in the presence and in the absence of Ca{sup 2+}. Calcium enhanced the affinity of PKC-PDBu interaction and decreased the dissociation rate. These results showed that dramatic changes occurred in the in vitro properties of PKC upon the formation of the irreversible PKC-membrane complex. These properties may be related to cellular events that induce formation of the chelator-resistant form of membrane-bound PKC.

  1. Apoptosis in metanephric development

    PubMed Central

    1992-01-01

    During metanephric development, non-polarized mesenchymal cells are induced to form the epithelial structures of the nephron following interaction with extracellular matrix proteins and factors produced by the inducing tissue, ureteric bud. This induction can occur in a transfilter organ culture system where it can also be produced by heterologous cells such as the embryonic spinal cord. We found that when embryonic mesenchyme was induced in vitro and in vivo, many of the cells surrounding the new epithelium showed morphological evidence of programmed cell death (apoptosis) such as condensed nuclei, fragmented cytoplasm, and cell shrinking. A biochemical correlate of apoptosis is the transcriptional activation of a calcium-sensitive endonuclease. Indeed, DNA isolated from uninduced mesenchyme showed progressive degradation, a process that was prevented by treatment with actinomycin- D or cycloheximide and by buffering intracellular calcium. These results demonstrate that the metanephric mesenchyme is programmed for apoptosis. Incubation of mesenchyme with a heterologous inducer, embryonic spinal cord prevented this DNA degradation. To investigate the mechanism by which inducers prevented apoptosis we tested the effects of protein kinase C modulators on this process. Phorbol esters mimicked the effects of the inducer and staurosporine, an inhibitor of this protein kinase, prevented the effect of the inducer. EGF also prevented DNA degradation but did not lead to differentiation. These results demonstrate that conversion of mesenchyme to epithelial requires at least two steps, rescue of the mesenchyme from apoptosis and induction of differentiation. PMID:1447305

  2. Biological responsiveness to the phorbol esters and specific binding of (/sup 3/H)phorbol 12,13-dibutyrate in the nematode Caenorhabditis elegans, a manipulable genetic system

    SciTech Connect

    Lew, K.K.; Chritton, S.; Blumberg, P.M.

    1982-01-01

    Because of its suitability for genetic studies, the nematode Caenorhabditis elegans was examined for its responsiveness to the phorbol esters. Phorbol 12-myristate 13-acetate had three effects. It inhibited the increase in animal size during growth; it decreased the yield of progeny; and it caused uncoordinated movement of the adult. The effects on nematode size, progeny yield, and movement were quantitated. Concentrations of phorbol 12-myristate 13-acetate yielding half-maximal responses were 440, 460, and 170 nM, respectively. As was expected from the biological responsiveness of the nematodes, specific, saturable binding of phorbol ester to nematode extracts was found. (/sup 3/H)phorbol 12,13-dibutyrate bound with a dissociation constant of 26.8 +/- 3.9 nM. At saturation, 5.7 +/- 1.4 pmole/mg protein was bound.

  3. Protein Kinase C Regulates Ionic Conductance in Hippocampal Pyramidal Neurons: Electrophysiological Effects of Phorbol Esters

    NASA Astrophysics Data System (ADS)

    Baraban, Jay M.; Snyder, Solomon H.; Alger, Bradley E.

    1985-04-01

    The vertebrate central nervous system contains very high concentrations of protein kinase C, a calcium-and phospholipid-stimulated phosphorylating enzyme. Phorbol esters, compounds with inflammatory and tumor-promoting properties, bind to and activate this enzyme. To clarify the role of protein kinase C in neuronal function, we have localized phorbol ester receptors in the rat hippocampus by autoradiography and examined the electrophysiological effects of phorbol esters on hippocampal pyramidal neurons in vitro. Phorbol esters blocked a calcium-dependent potassium conductance. In addition, phorbol esters blocked the late hyperpolarization elicited by synaptic stimulation even though other synaptic potentials were not affected. The potencies of several phorbol esters in exerting these actions paralleled their affinities for protein kinase C, suggesting that protein kinase C regulates membrane ionic conductance.

  4. Phorbol esters and neurotransmitter release: more than just protein kinase C?

    PubMed Central

    Silinsky, Eugene M; Searl, Timothy J

    2003-01-01

    This review focuses on the effects of phorbol esters and the role of phorbol ester receptors in the secretion of neurotransmitter substances. We begin with a brief background on the historical use of phorbol esters as tools to decipher the role of the enzyme protein kinase C in signal transduction cascades. Next, we illustrate the structural differences between active and inactive phorbol esters and the mechanism by which the binding of phorbol to its recognition sites (C1 domains) on a particular protein acts to translocate that protein to the membrane. We then discuss the evidence that the most important nerve terminal receptor for phorbol esters (and their endogenous counterpart diacylglycerol) is likely to be Munc13. Indeed, Munc13 and its invertebrate homologues are the main players in priming the secretory apparatus for its critical function in the exocytosis process. PMID:12711617

  5. Phorbol Ester Effects on Neurotransmission: Interaction with Neurotransmitters and Calcium in Smooth Muscle

    NASA Astrophysics Data System (ADS)

    Baraban, Jay M.; Gould, Robert J.; Peroutka, Stephen J.; Snyder, Solomon H.

    1985-01-01

    Stimulation of the phosphatidylinositol cycle by neurotransmitters generates diacylglycerol, an activator of protein kinase C, which may regulate some forms of neurotransmission. Phorbol esters, potent inflammatory and tumorpromoting compounds, also activate protein kinase C. We demonstrate potent and selective effects of phorbol esters on smooth muscle, indicating a role for protein kinase C in neurotransmission. In rat vas deferens and dog basilar artery, phorbol esters synergize with calcium to mimic the contractile effects of neurotransmitters that act through the phosphatidylinositol cycle. In guinea pig ileum and rat uterus, phorbol esters block contractions produced by these neurotransmitters.

  6. Phorbol ester effects on neurotransmission: interaction with neurotransmitters and calcium in smooth muscle.

    PubMed Central

    Baraban, J M; Gould, R J; Peroutka, S J; Snyder, S H

    1985-01-01

    Stimulation of the phosphatidylinositol cycle by neurotransmitters generates diacylglycerol, an activator of protein kinase C, which may regulate some forms of neurotransmission. Phorbol esters, potent inflammatory and tumor-promoting compounds, also activate protein kinase C. We demonstrate potent and selective effects of phorbol esters on smooth muscle, indicating a role for protein kinase C in neurotransmission. In rat vas deferens and dog basilar artery, phorbol esters synergize with calcium to mimic the contractile effects of neurotransmitters that act through the phosphatidylinositol cycle. In guinea pig ileum and rat uterus, phorbol esters block contractions produced by these neurotransmitters. PMID:2857490

  7. Effect of phorbol esters on mitochondrial actions of glucagon

    SciTech Connect

    Cardellach, F.; Moehren, G.; Hoek, J.B.

    1987-05-01

    Glucagon generates different second messenger signals in liver. It increases cAMP levels and elevates cytosolic Ca/sup 2 +/ levels by degradation of polyphosphoinositides. The phorbol ester 12-0-tetradecanoyl phorbol 13-acetate (TPA) inhibits glucagon-induced calcium mobilization, but not cAMP formation. TPA can thus be used to assess the role of Ca/sup 2 +/ and cAMP in the activation of mitochondrial processes. In isolated hepatocytes, glucagon increased the steady state NAD(P)H level, probably by activating mitochondrial Ca/sup 2 +/ dependent dehydrogenases. TPA inhibited the glucagon-induced NAD(P) reduction without affecting phosphorylase activation. The effects of glucagon and TPA on mitochondrial respiratory activity and calcium retention were tested after isolation of the mitochondria from perfused livers. Electron transport rates were increased by 15-25% and calcium retention time was increased four-fold after glucagon treatment. When livers were pretreated with TPA, glucagon had no effect on electron transport activity, but calcium retention was increased by the same factor. The results suggest that glucagon-induced calcium mobilization is required for the stimulation of the respiratory activity but not for the increased capacity to retain a calcium overload in the mitochondria.

  8. Antiallergic Phorbol Ester from the Seeds of Aquilaria malaccensis

    PubMed Central

    Korinek, Michal; Wagh, Vitthal D.; Lo, I-Wen; Hsu, Yu-Ming; Hsu, Hsue-Yin; Hwang, Tsong-Long; Wu, Yang-Chang; Cheng, Yuan-Bin; Chen, Bing-Hung; Chang, Fang-Rong

    2016-01-01

    The Aquilaria malaccensis (Thymelaeaceae) tree is a source of precious fragrant resin, called agarwood, which is widely used in traditional medicines in East Asia against diseases such as asthma. In our continuous search for active natural products, A. malaccensis seeds ethanolic extract demonstrated antiallergic effect with an IC50 value less than 1 µg/mL. Therefore, the present research aimed to purify and identify the antiallergic principle of A. malaccensis through a bioactivity-guided fractionation approach. We found that phorbol ester-rich fraction was responsible for the antiallergic activity of A. malaccensis seeds. One new active phorbol ester, 12-O-(2Z,4E,6E)-tetradeca-2,4,6-trienoylphorbol-13-acetate, aquimavitalin (1) was isolated. The structure of 1 was assigned by means of 1D and 2D NMR data and high-resolution mass spectrometry (HR-MS). Aquimavitalin (1) showed strong inhibitory activity in A23187- and antigen-induced degranulation assay with IC50 values of 1.7 and 11 nM, respectively, with a therapeutic index up to 71,000. The antiallergic activities of A. malaccensis seeds and aquimavitalin (1) have never been revealed before. The results indicated that A. malaccensis seeds and the pure compound have the potential for use in the treatment of allergy. PMID:27007372

  9. Antiallergic Phorbol Ester from the Seeds of Aquilaria malaccensis.

    PubMed

    Korinek, Michal; Wagh, Vitthal D; Lo, I-Wen; Hsu, Yu-Ming; Hsu, Hsue-Yin; Hwang, Tsong-Long; Wu, Yang-Chang; Cheng, Yuan-Bin; Chen, Bing-Hung; Chang, Fang-Rong

    2016-03-21

    The Aquilaria malaccensis (Thymelaeaceae) tree is a source of precious fragrant resin, called agarwood, which is widely used in traditional medicines in East Asia against diseases such as asthma. In our continuous search for active natural products, A. malaccensis seeds ethanolic extract demonstrated antiallergic effect with an IC50 value less than 1 µg/mL. Therefore, the present research aimed to purify and identify the antiallergic principle of A. malaccensis through a bioactivity-guided fractionation approach. We found that phorbol ester-rich fraction was responsible for the antiallergic activity of A. malaccensis seeds. One new active phorbol ester, 12-O-(2Z,4E,6E)-tetradeca-2,4,6-trienoylphorbol-13-acetate, aquimavitalin (1) was isolated. The structure of 1 was assigned by means of 1D and 2D NMR data and high-resolution mass spectrometry (HR-MS). Aquimavitalin (1) showed strong inhibitory activity in A23187- and antigen-induced degranulation assay with IC50 values of 1.7 and 11 nM, respectively, with a therapeutic index up to 71,000. The antiallergic activities of A. malaccensis seeds and aquimavitalin (1) have never been revealed before. The results indicated that A. malaccensis seeds and the pure compound have the potential for use in the treatment of allergy.

  10. Phorbol esters induce multidrug resistance in human breast cancer cells

    SciTech Connect

    Fine, R.L.; Patel, J.; Chabner, B.A.

    1988-01-01

    Mechanisms responsible for broad-based resistance to antitumor drugs derived from natural products (multidrug resistance) are incompletely understood. Agents known to reverse the multidrug-resistant phenotype (verapamil and trifluoperazine) can also inhibit the activity of protein kinase C. When the authors assayed human breast cancer cell lines for protein kinase C activity, they found that enzyme activity was 7-fold higher in the multidrug-resistance cancer cells compared with the control, sensitive parent cells. Exposure of drug-sensitive cells to the phorbol ester phorbol 12,13-dibutyate (P(BtO)/sub 2/) led to an increase in protein kinase C activity and induced a drug-resistance phenotype, whereas exposure of drug-resistant cells to P(BtO)/sub 2/ further increased drug resistance. In sensitive cells, this increased resistance was accomplished by a 3.5-fold increased phosphorylation of a 20-kDa particulate protein and a 35-40% decreased intracellular accumulation of doxorubicin and vincristine. P(BtO)/sub 2/ induced resistance to agents involved in the multidrug-resistant phenotype (doxorubicin and vincristine) but did not affect sensitivity to an unrelated alkylating agent (melphalan). The increased resistance was partially or fully reversible by the calcium channel blocker verapamil and by the calmodulin-antagonist trifluoperazine. These data suggest that stimulation of protein kinase C playus a role in the drug-transport changes in multidrug-resistant cells. This may occur through modulation of an efflux pump by protein phosphorylation.

  11. Identification of the phorbol ester receptor in human and avian erythrocytes

    SciTech Connect

    Kramer, C.M.; Sando, J.J.; Speizer, L.A.

    1986-05-01

    The ability of phorbol esters to inhibit the uptake of a fluorescent glucose analogue in goose but not human erythrocytes is consistent with earlier reports that the human red blood cell lacks the phorbol ester receptor. However, they have located specific phorbol 12,13-dibutyrate binding sites in both human and goose erythrocytes. Human and goose red blood cells contain 2 classes of phorbol ester receptors with similar affinities, however the human erythrocyte contains 1/3 as many phorbol ester receptors as does the goose red blood cell. An additional contrast in the binding of phorbol esters to human and goose red blood cells is the temperature-induced enhancement of binding to goose, but not human erythrocytes. Equilibrium phorbol ester binding to goose red blood cells at 37/sup 0/C is enhanced 3.3 +/- 0.4 times that amount bound at 4/sup 0/C. Equilibrium binding of phorbol esters to human erythrocytes is identical at both temperatures. In vivo and in vitro phosphorylation profiles of C-kinase substrates also differ between the human and goose erythrocyte.

  12. Tumor-promoting phorbol esters effect alkalinization of canine renal proximal tubular cells

    SciTech Connect

    Mellas, J.; Hammerman, M.R.

    1986-03-01

    We have demonstrated the presence of specific receptors for tumor-promoting phorbol esters in the plasma membrane of the canine renal proximal tubular cell. These compounds affect proximal tubular metabolism in vitro. For example, we have shown that they inhibit gluconeogenesis in canine renal proximal tubular segments. Tumor-promoting phorbol esters have been shown to effect alkalinization of non-renal cells, by enhancing Na/sup +/-H/sup +/ exchange across the plasma membrane. To determine whether the actions of tumor-promoting phorbol esters in proximal tubular segments might be mediated by a similar process, we incubated suspensions of segments from dog kidney with these compounds and measured changes in intracellular pH using (/sup 14/C)-5,5-dimethoxazoladine-2-4-dione (DMO) and flow dialysis. Incubation of segments with phorbol 12,13 dibutyrate, but not inactive phorbol ester, 4 ..gamma.. phorbol, effected alkalinization of cells within the segments in a concentration-dependent manner. Alkalinization was dependent upon the presence of extracellular (Na/sup +/) > intracellular (Na/sup +/), was prevented by amiloride and was demonstrable in the presence of SITS. Our findings suggest that tumor-promoting esters stimulate the Na/sup +/-H/sup +/ exchanger known to be present in the brush border membrane of the renal proximal tubular cell. It is possible that the stimulation reflects a mechanism by which phorbol esters affect metabolic processes in these cells.

  13. Phosphatidylinositol 4,5-bisphosphate competitively inhibits phorbol ester binding to protein kinase C

    SciTech Connect

    Chauhan, A.; Cauhan, V.P.S.; Deshmukh, D.S.; Brokerhoff, H. )

    1989-06-13

    Calcium phospholipid dependent protein kinase C (PKC) is activated by diacylglycerol (DG) and by phorbol esters and is recognized to be the phorbol ester receptor of cells; DG displaces phorbol ester competitively from PKC. A phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP{sub 2}), can also activate PKC in the presence of phosphatidylserine (PS) and Ca{sup 2+} with a K{sub PIP{sub 2}} of 0.04 mol %. Preliminary experiments have suggested a common binding site for PIP{sub 2} and DG on PKC. Here, the authors investigate the effect of PIP{sub 2} on phorbol ester binding to PKC in a mixed micellar assay. In the presence of 20 mol % PS, PIP{sub 2} inhibited specific binding of ({sup 3}H)phorbol 12,13-dibutyrate (PDBu) in a dose-dependent fashion up to 85% at 1 mol %. Inhibition of binding was more pronounced with PIP{sub 2} than with DG. Scatchard analysis indicated that the decrease in binding of PDBu in the presence of PIP{sub 2} is the result of an altered affinity for the phorbol ester rather than of a change in maximal binding. The plot of apparent dissociation constants (K{sub d{prime}}) against PIP{sub 2} concentration was linear over a range of 0.01-1 mol % with a K{sub i} of 0.043 mol % and confirmed the competitive nature of inhibition between PDBu and PIP{sub 2}. Competition between PIP{sub 2} and phorbol ester could be determined in a liposomal assay system also. These results indicate that PIP{sub 2}, DG, and phorbol ester all compete for the same activator-receiving region on the regulatory moiety of protein kinase C, and they lend support to the suggestion that PIP{sub 2} is a primary activator of the enzyme.

  14. Phorbol esters alter the expression of lymphocyte membrane proteins

    SciTech Connect

    Reder, A.T.; Antel, J.P.

    1986-03-01

    T cell activation via the T cell receptor (T3-Ti complex) by OKT3 results in modulation of the T3-Ti complex, but does not affect T4, T8, or T11 antigen expression. To study the effect of other T cell activators on these T cell membrane antigens, the authors incubated mononuclear cells for 0-3 days with lectins or pharmacologic agents and stained with monoclonal antibodies to their antigens. The median fluorescence intensity (MFI) was measured with a fluorescence activated cell sorter. Activation of PBL with Con A, PHA, calcium ionophore A23187, or with dbcAMP, isoproterenol, or theophyllin had minimal effects on the MFI of T3, T4, T8, or T11. Phorbol myristate acetate (PMA), a protein kinase C activator which stimulates PBL though an alternate pathway, caused a 90-100% reduction of T3 and T4 MFI, a 25% reduction in T8 MFI, and a 400% increase in T11 MFI after 2 days. Addition of A23187 slightly increased these effects. PMA induced a 2-3-fold increase in cell diameter concomitant with the alterations in membrane antigens. These data suggest that T cell activation through pathways not directly linked to the T cell antigen receptor can result in surface antigen expression different from that which follows activation via the T cell receptor.

  15. Phorbol esters modulate cyclic AMP accumulation in porcine thyroid cells

    SciTech Connect

    Emoto, T.; Kasai, K.; Hiraiwa, M.; Shimoda, S.

    1988-01-01

    In cultured porcine thyroid cells, during 60 min incubation phorbol 12-myristate 13-acetate (PMA) had no effect on basal cyclic AMP accumulation and slightly stimulated cyclic AMP accumulation evoked by thyroid stimulating hormone (TSH) or forskolin. Cholera toxin-induced cyclic AMP accumulation was significantly stimulated by PMA. On the other hand, cyclic AMP accumulation evoked by prostaglandin E/sub 1/ or E/sub 2/ (PGE/sub 1/ and PGE/sub 2/) was markedly depressed by simultaneous addition of PMA. These opposing effects of PMA on cyclic AMP accumulation evoked by PGE and cholera toxin were observed in a dose-related fashion, with half-maximal effect of around 10/sup -9/ M in either case. The almost same effects of PMA on cyclic AMP accumulation in basal and stimulated conditions were also observed in freshly prepared thyroid cells. The present study was performed in the presence of phosphodiesterase inhibitor, 3-iso-butyl-1-methylxanthine (IBMX), indicating that PMA affected adenylate cyclase activity. Therefore, it is suggested that PMA may modulate the production of cyclic AMP in response to different stimuli, possibly by affecting several sites in the adenylate cyclase complex in thyroid cells.

  16. Acute airsacculitis in turkeys inoculated with phorbol myristate acetate.

    PubMed

    Ficken, M D; Barnes, H J

    1990-06-01

    Phorbol myristate acetate (PMA), which induces acute pulmonary injury in mammals, induced acute airsacculitis in turkeys after intra-airsac inoculation of 0.1 mg/kg. Grossly, air sacs contained multifocal to diffuse hemorrhage and edema at postinoculation hours (PIH) 3 and 6. Microscopically, there was multifocal congestion and small thrombocyte aggregates within small blood vessels by PIH 0.5, with a few vessels containing small numbers of marginating heterophils. By PIH 1.5, thrombocyte aggregates were larger and more numerous, and moderate numbers of heterophils were located perivascularly. Erythrocytes and proteinaceous fluid were in air sac interstitium. By PIH 3 and 6, hemorrhage and exudation of proteinaceous fluid had increased, in some instances severely distending the air sac. Ultrastructurally, changes resulting from PMA-induced injury were thrombocyte aggregation and degeneration, air sac epithelial cell vacuolation with separation of interdigitating cell processes, and endothelial cell vacuolar degeneration with loss of vascular integrity. Air sac lavage fluids had mildly increased total cell counts by PIH 1.5, but values returned to baseline by the end of the experiment, indicating lack of cell exudation into the air sac lumen. Circulating leukocyte changes included transient lymphopenia at PIH 3 and marked heterophilia at PIH 6. These results indicate that thrombocytes and/or heterophils are central to the pathogenesis of injury induced in air sacs by PMA and that the air sac responds differently to PMA than to pathogenic bacteria.

  17. Tumor promoting phorbol diesters: substrates for diacylglycerol lipase

    SciTech Connect

    Cabot, M.C.

    1984-08-30

    Enzyme activity in rat serum was examined utilizing the potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and various glycerolipids as substrates. The serum activity was specific for hydrolysis of the long chain tetradecanoate moiety of TPA, hydrolyzed mono- and diacylglycerols, but was not effective against triacylglycerols, cholesterylesters, or phospholipids. Heating the enzyme preparation at 56/sup 0/C for 1 min was dually effective in reducing the hydrolysis of both TPA and dioleoylglycerol by 83-86% of control levels. The potent diacylglycerol lipase inhibitor, RHC 80267, inhibited the hydrolysis of TPA in the 0.2-1.0 ..mu..M range and was also a potent blocker of monoacyl- and diacylglycerol hydrolysis. In substrate competition studies, exogenous unlabeled TPA was added to the (/sup 14/C)dioleoylglycerol-containing reaction mixture, however, this produced an approximate 3-fold stimulation of (/sup 14/)dioleoylglycerol hydrolysis. Although we have not established whether the hydrolysis of TPA and diacylglycerol is the work of one enzyme, the effectiveness of the specific lipase inhibitor, RHC 80267, demonstrates that diacylglycerol lipase can utilize TPA as substrate, a finding never before documented. This point is of interest in light of the theory that phorbol esters act by mimicry of the natural lipid mediator, diacylglycerols. 44 references, 3 figures, 1 table.

  18. Phorbol ester and spontaneous activity in SHR aorta

    SciTech Connect

    Moisey, D.M.; Cox, R.H.

    1986-03-01

    Thoracic aortas (TA) were excised from 6-week old SHR and WKY. 2mm rings were mounted isometrically at optimum preload. Spontaneous rhythmical activity developed in TA from SHR and had a frequency of 3-4/min with varying periods of quiescence between bursts of activity. The spontaneous activity often produced an increase in tension development which was associated with increased frequency of oscillations. Verapamil (10/sup -7/ M) or Ca/sup + +/-free solution added during the contractile phase resulted in an immediate loss of tension and spontaneous activity. Addition of ouabain (10/sup -4/ M) during the contractile phase of spontaneous activity, increased the frequency of oscillations which appeared to fuse into a tetanus. Spontaneous rhythmical activity was infrequently observed in TA from WKY. However, addition of phorbol 12-myristate-13 acetate (TPA), frequently induced spontaneous rhythmic oscillations associated with tension development in TA from WKY. TPA contracted the SHR TA and increased the frequency of oscillations. SHR TA were more sensitive to TPA than WKY. This study demonstrates (1) spontaneous rhythmical activity, independent of agonist stimulation in TA from 6-week old SHR and (2) TPA induced spontaneous oscillatory activity. The mechanism underlying the spontaneous oscillatory activity may involve membrane coupling events and Na-pump difference between SHR and WKY.

  19. Phorbol ester and atrial natriuretic peptide receptor response on vascular smooth muscle.

    PubMed

    Yasunari, K; Kohno, M; Murakawa, K; Yokokawa, K; Horio, T; Takeda, T

    1992-04-01

    At least two types of receptors for natriuretic peptides have been reported: biologically active receptors coupled with guanylate cyclase (atrial natriuretic peptide [ANP]-B receptors) and clearance receptors (ANP-C receptors). To elucidate the role of protein kinase C (PKC) in the regulation of ANP-B receptors, vascular smooth muscle cells in culture were treated with phorbol ester. Incubation with receptor agonists and phorbol ester led to the desensitization of receptor-mediated cyclic guanosine monophosphate (ANP-B receptor response) in rat vascular smooth muscle cells. Although a PKC inhibitor and downregulation of PKC by long-term incubation of cells with phorbol esters blocked the phorbol ester-induced desensitization of the ANP-B receptor response, they did not block the ANP-induced desensitization of the ANP-B receptor response. In addition, when desensitization by phorbol esters was observed, ANP was still capable of desensitization. These observations suggest that the mechanism for regulating ANP-B receptor sensitivity may be both PKC-dependent and PKC-independent and mediated by phorbol esters and ANP, respectively.

  20. Similar effects of phospholipase C and phorbol ester tumor promoters on primary mouse epidermal cells

    SciTech Connect

    Jeng, A.Y.; Lichti, U.; Strickland, J.E.; Blumberg, P.M.

    1985-11-01

    Interaction of tumor promoting phorbol esters with specific high affinity receptors is probably essential for many of the biological responses elicited by these agents. Since diacylglycerols which can be produced enzymatically from phospholipids by phospholipase C are postulated to be the physiological ligands for the phorbol ester receptor, the authors have examined primary cultures of mouse epidermal basal cells exposed to phospholipase C (Clostridium perfringens) for several biological and biochemical responses characteristic of treatment with 12-O-tetradecanoyl-phorbol-13-acetate, the most potent phorbol ester tumor promoter. Formation of diacylglycerols by treatment with phospholipase C was demonstrated by the dose-dependent release of radioactive diacylglycerols in cells prelabeled with (TH)arachidonic acid. Treatment with phospholipase C led to the morphological changes and to the reduction in epidermal growth factor binding (90%) associated with 12-O-tetradecanoylphorbol-13-acetate treatment. Continuous treatment at the same dose led to the induction of the enzymes ornithine decarboxylase and transglutaminase with a time course and extent similar to the inductions by 12-O-tetradecanoylphorbol-13-acetate. Treatment with phospholipase C yielded substantial suppression of the binding affinity of phorbol-12,13-dibutyrate for its receptors without reduction in total number of binding sites, consistent with the production by phospholipase C of a competitive inhibitor of phorbol ester binding.

  1. Effects of phorbol esters on fluid transport and blood flow in the small intestine

    SciTech Connect

    Sjoeqvist, A.; Henderson, L.S.; Fondacaro, J.D.

    1986-07-01

    Studies were designed to examine the effects of phorbol esters on intestinal fluid transport and blood flow in the anesthetized cat and enteropooling in the conscious rat. Intraluminal administration of phorbol ester into a segment of isolated small bowel produced a copious intestinal secretion and a concomitant mesenteric hyperemia in the cat. Net fluid movement in the intestine was converted from absorption in the control state to secretion following phorbol ester administration. Intravenous atropine reduced the phorbol ester-induced secretion by 56%; clonidine abolished the remaining secretory response. In the rat, intragastric administration of phorbol ester produced enteropooling comparable to that of other potent intestinal secretagogues. Since phorbol esters are known to activate protein kinase C, these suggest that activation of protein kinase C in the small intestine may lead to a full secretory response. The evidence suggests that this secretion is accompanied by a metabolic hyperemia. These results suggest that protein kinase C plays an important role in the regulation of intestinal fluid transport.

  2. Phorbol esters potentiate the induction of class I HLA expression by interferon. alpha

    SciTech Connect

    Erusalimsky, J.D.; Kefford, R.F.; Gilmore, D.J.; Milstein, C. )

    1989-03-01

    The authors have studied the effect of phorbol esters on the induction of class I histocompatibility antigen (HLA) expression by interferons (IFNs) in the T-cell line MOLT-4 and in the MOLT-4 mutant YHHH. Addition of IFN-{alpha} to phorbol 12,13-dibutyrate-pretreated MOLT-4 cells causes a >20-fold increase in the expression of class I HLA, as compared to a 4- to 7-fold IFN-{alpha}-induced increase in control cells. Pretreatment with phorbol 12,13-dibutyrate does not alter the class I HLA response to IFN-{gamma} or the responses of other IFN-induced genes. This effect of phorbol 12,13-dibutyrate reproduces in MOLT-4 cells the phenotype of the mutant YHHH, which also displays a selective enhanced class I HLA response to IFN-{alpha}. Pretreatment of YHHH with phorbol 12,13-dibutyrate does not affect any of the responses induced by IFN. These findings suggest the existence of a phorbol ester-sensitive factor, inducible in MOLT-4 and constitutively expressed or modified in YHHH, which operates in the pathway of induction of class I HLA by IFN-{alpha} but not in the pathway used by IFN-{gamma}.

  3. Phorbol esters potentiate the induction of class I HLA expression by interferon alpha.

    PubMed Central

    Erusalimsky, J D; Kefford, R F; Gilmore, D J; Milstein, C

    1989-01-01

    We have studied the effect of phorbol esters on the induction of class I histocompatibility antigen (HLA) expression by interferons (IFNs) in the T-cell line MOLT-4 and in the MOLT-4 mutant YHHH. Addition of IFN-alpha to phorbol 12,13-dibutyrate-pretreated MOLT-4 cells causes a greater than 20-fold increase in the expression of class I HLA, as compared to a 4- to 7-fold IFN-alpha-induced increase in control cells. Pretreatment with phorbol 12,13-dibutyrate does not alter the class I HLA response to IFN-gamma or the responses of other IFN-induced genes. This effect of phorbol 12,13-dibutyrate reproduces in MOLT-4 cells the phenotype of the mutant YHHH, which also displays a selective enhanced class I HLA response to IFN-alpha. Pretreatment of YHHH with phorbol 12,13-dibutyrate does not affect any of the responses induced by IFN. These findings suggest the existence of a phorbol ester-sensitive factor, inducible in MOLT-4 and constitutively expressed or modified in YHHH, which operates in the pathway of induction of class I HLA by IFN-alpha but not in the pathway used by IFN-gamma. Images PMID:2494657

  4. Early Induction of Cyclin D2 Expression in Phorbol Ester–responsive B-1 Lymphocytes

    PubMed Central

    Tanguay, Debra A.; Colarusso, Thomas P.; Pavlovic, Sandra; Irigoyen, Macarena; Howard, Robert G.; Bartek, Jiri; Chiles, Thomas C.; Rothstein, Thomas L.

    1999-01-01

    B-1 lymphocytes represent a distinct B cell subset with characteristic features that include self-renewing capacity and unusual mitogenic responses. B-1 cells differ from conventional B cells in terms of the consequences of phorbol ester treatment: B-1 cells rapidly enter S phase in response to phorbol ester alone, whereas B-2 cells require a calcium ionophore in addition to phorbol ester to trigger cell cycle progression. To address the mechanism underlying the varied proliferative responses of B-1 and B-2 cells, we evaluated the expression and activity of the G1 cell cycle regulator, cyclin D2, and its associated cyclin-dependent kinases (Cdks). Cyclin D2 expression was upregulated rapidly, within 2–4 h, in phorbol ester–stimulated B-1 cells, in a manner dependent on intact transcription/translation, but was not increased in phorbol ester– stimulated B-2 cells. Phorbol ester–stimulated cyclin D2 expression was accompanied by the formation of cyclin D2–Cdk4, and, to a lesser extent, cyclin D2–Cdk6, complexes; cyclin D2– containing complexes were found to be catalytically functional, in terms of their ability to phosphorylate exogenous Rb in vitro and to specifically phosphorylate endogenous Rb on serine780 in vivo. These results strongly suggest that the rapid induction of cyclin D2 by a normally nonmitogenic phorbol ester stimulus is responsible for B-1 cell progression through G1 phase. The ease and rapidity with which cyclin D2 responds in B-1 cells may contribute to the proliferative features of this subset. PMID:10359571

  5. Phorbol myristate acetate and dioctanoylglycerol inhibit transport in rabbit proximal convoluted tubule

    SciTech Connect

    Baum, M.; Hays, S.R. )

    1988-01-01

    The present in vitro microperfusion study examined the effect of protein kinase C activation on transport in the rabbit proximal convoluted tubule (PCT). PCT were perfused with an ultrafiltrate-like solution and were bathed in a serumlike albumin solution. Addition of phorbol 12-myristate 13-acetate, an activator of protein kinase C, inhibited volume absorption from 1.06 {plus minus} 0.10 to 0.77 {plus minus} 0.07 nl{center dot}mm{sup {minus}1}min{sup {minus}1}, and 0.76 {plus minus} 0.14 to 0.48 {plus minus} 0.08 nl{center dot}mm{sup {minus}1}{center dot}min{sup {minus}1}, respectively. Bath phorbol 12-myristate 13-acetate had no effect on volume absorption. In contrast, bath 4{alpha}-phorbol, an inactive phorbol that does not activate protein kinase C, had no effect on J{sub v}. Bath L-{alpha}-dioctanoylglycerol, another known activator of protein kinase C, inhibited volume absorption. A 10-fold lower concentration of L-{alpha}-dioctanoylglycerol had no effect on J{sub v}. Both 5 x 10{sup {minus}8} M phorbol 12-myristate 13-acetate and 10{sup {minus}4} M L-{alpha}-dioctanoylglycerol inhibited glucose, bicarbonate, and chloride transport in the PCT. These data are consistent with protein kinase C activation playing a role in the modulation of proximal tubular transport.

  6. Five new phorbol esters with cytotoxic and selective anti-inflammatory activities from Croton tiglium.

    PubMed

    Wang, Jun-Feng; Yang, Sheng-Hui; Liu, Yan-Qun; Li, Din-Xiang; He, Wei-Jun; Zhang, Xiao-Xiao; Liu, Yong-Hong; Zhou, Xiao-Jiang

    2015-05-01

    Five new phorbol esters, (four phorbol diesters, 1-4, and one 4-deoxy-4α-phorbol diester, 5), as well as four known phorbol esters analogues (6-9) were isolated and identified from the branches and leaves of Croton tiglium. Their structures were elucidated mainly by extensive NMR spectroscopic, and mass spectrometric analysis. Among them, compound (1) was the first example of a naturally occurring phorbol ester with the 20-aldehyde group. Compounds 2-5, and 7-9 showed potent cytotoxicity against the K562, A549, DU145, H1975, MCF-7, U937, SGC-7901, HL60, Hela, and MOLT-4 cell lines, with IC50 values ranging from 1.0 to 43 μM, while none of the compounds exhibited cytotoxic effects on normal human cell lines 293T and LX-2, respectively. In addition, compound 3 exhibited moderate COX-1 and COX-2 inhibition, with IC50 values of 0.14 and 8.5 μM, respectively.

  7. Effect of phorbol and Bryostatin I on chondrogenic expression of chick limb bud, in vitro

    SciTech Connect

    Garrison, J.C.; Pettit, G.R.; Uyeki, E.M.

    1987-10-26

    The present paper describes the effects of PMA (phorbol 12-myristate 13 acetate) on in vitro chondrogenesis in non-passaged, embryonic limb bud cells, relative to the effects of Bryostatin I. This compound also activates C kinase and binds competitively to the phorbol ester receptor, yet does not affect cell differentiation. Levels of PMA as low as 10/sup -7/ M markedly reduced cartilage formation in 4-day cultures, as indicated by nodule count and Alcian blue staining for chondroitin sulfate. Coadministration of Bryostatin I at equimolar concentration prevented the PMA inhibitory effect on chondrocytic expression. This confirms other findings that phorbol activation of C kinase cannot exclusively account for the activity of phorbol on cell expression. Altering the time of PMA exposure demonstrated that PMA inhibited chondrocyte phenotypic expression, rather than cell commitment: early exposure to PMA had little inhibitor effect on the staining index, whereas, exposure from 49-96 h and 0-96 h had moderate and strong inhibitory effects, respectively, on cartilage synthesis. Further research on the phorbol/Bryostatin I interaction should add to their knowledge of the control processes involved in tumor promotion and cell differentiation. 21 references, 3 figures.

  8. Phorbol esters broaden the action potential in CA1 hippocampal pyramidal cells.

    PubMed

    Storm, J F

    1987-03-20

    Intracellular recordings were made from CA1 pyramidal cells in rat hippocampal slices. Single action potentials were elicited by injection of brief current pulses. Bath application of phorbol esters (4 beta-phorbol-12,13-diacetate, 0.3-5 microM; or 4 beta-phorbol-12,13-dibutyrate, 5-10 microM) broadened the action potential in each of the cells tested (n = 9). The broadening reflected slowing of the repolarization, whereas the upstroke of the spike was unchanged. This effect may enhance transmitter release from synaptic terminals, and contribute to enhancement of synaptic transmission through activation of protein kinase C, a mechanism which has been associated with long term potentiation.

  9. Insulin reverses the growth retardation effect of phorbol ester in chicken embryos during organogenesis

    SciTech Connect

    Girbau, M.; Bassas, L.; Roth, J.; de Pablo, F. )

    1989-01-01

    The tumor promoting phorbol esters can affect early embryonic development by causing interference with the normal pathways of cellular growth and differentiation. The present study was designed to: (a) define a time in organogenesis when a vertebrate embryo model, the chicken, was sensitive to the phorbol ester 12-0-tetradecanoil-13-acetate (TPA), and (b) attempt a rescue of the embryos disturbed by TPA with simultaneous addition of insulin. In embryos treated at days 2 and 3 of development, TPA caused dose-dependent mortality. Survivors were biochemically retarded as indicated by their decreased weight, protein, DNA, RNA, total creatine kinase, triglycerides, phospholipids and cholesterol contents. When intermediated doses of TPA were applied together with insulin the embryonic growth disturbance was largely antagonized. These data, generated with an in vivo whole embryo, support the strong link between the mode of action of insulin and signal transduction mechanisms typical of phorbol esters.

  10. Stimulation of dopamine synthesis and activation of tyrosine hydroxylase by phorbol diesters in rat striatum

    SciTech Connect

    Onali, P.; Olianas, M.C.

    1987-03-23

    In rat striatal synaptosomes, 4..beta..-phorbol 12-myristate 13-acetate (PMA) and 4 ..beta..-phorbol 12,13-dibutyrate (PDBu), two activators of Ca/sup 2 +/-phospholipid-dependent protein kinase (protein kinase C) increased dopamine (DA) synthesis measured by following the release of /sup 14/CO/sub 2/ from L-(1-/sup 14/C) tyrosine. Maximal stimulation (21-28% increase of basal rate) was produced by 0.5 ..mu..M PMA and 1 ..mu..M PDBu. 4 ..beta..-Phorbol and 4 ..beta..-phorbol 13-acetate, which are not activators of protein kinase C, were ineffective at 1 ..mu..M. PMA did not change the release of /sup 14/CO/sub 2/ from L-(1-/sup 14/C)DOPA. Addition of 1 mM EGTA to a Ca/sup 2 +/-free incubation medium failed to affect PMA stimulation. KCl (60 mM) enhanced DA synthesis by 25%. Exposure of synaptosomes to either PMA or PDBu prior to KCl addition resulted in a more than additive increase (80-100%) of DA synthesis. A similar synergistic effect was observed when the phorbol diesters were combined with either veratridine or d-amphetamine but not with forskolin and dibutyryl cyclic AMP. Pretreatment of striatal synaptosomes with phorbol diesters produced an activation of tyrosine hydroxylase (TH) associated with a 60% increase of the Vmax and a decrease of the Km for the pterine cofactor 6-methyl-5,6,7,8-tetrahydropterin. These results indicate that protein kinase C participates in the regulation of striatal TH in situ and that its activation may act synergistically with DA releasing agents in stimulating DA synthesis. 37 references, 3 figures, 3 tables.

  11. Specific binding of phorbol ester tumor promoters to intact primary epidermal cells from Sencar mice

    SciTech Connect

    Solanki, V.; Slaga, T.J.

    1981-04-01

    The binding of (20-/sup 3/H)phorbol 12,13-dibutyrate ((/sup 3/H)PDB) to intact living epidermal cells in monolayer culture was characterized. At 37/sup 0/C, the maximum specific (/sup 3/H)PDB binding (binding displaceable by 30 ..mu..M unlabeled PDB) was attained in 15 to 20 min and was followed by a rapid decrease (down regulation) of radioactivity bound to the cells. The activity lost by the cells during this decrease was found in the incubation medium. Prior exposure of cells to phorbol 12-myristate 13-acetate (PMA; 12-O-tetradecanoylphorbol 13-acetate) but not to phorbol for 2 h at 37/sup 0/C caused approx. 55% reduction in the number of measurable binding sites for (/sup 3/H)PDB. The down regulation was temperature sensitive; there was no loss of radioactivity after 1 h at 4/sup 0/C. The specific binding of (/sup 3/H)PDB at 4/sup 0/C reached equilibrium in 15 to 20 min and was saturable and freely reversible. At equilibrium, epidermal cells contained 1.2 x 10/sup 5/ binding sites per cell, and binding sites had a K/sub D/ of 10 nM. Specificity of binding was shown by the observation that the biologically active phorbol esters PMA and 12-deoxyphorbol 13-decanoate inhibited the binding, whereas the inactive parent compound phorbol and the nonphorbol tumor promoter anthralin did not have any effect. The abilities of these compounds to inhibit (/sup 3/H)PDB binding directly correlates with their tumor promoting activities. Epidermal cells exposed to retinoic acid or fluocinolone acetonide for 24 h had similar (/sup 3/H)PDB binding characteristics as untreated cells suggesting that inhibition of tumor promotion induced by these compounds is not mediated through alterations in the phorbol ester binding sites.

  12. Specific binding of phorbol ester tumor promoters to intact primary epidermal cells from Sencar mice.

    PubMed Central

    Solanki, V; Slaga, T J

    1981-01-01

    The binding of [20-3H]phorbol 12,13-dibutyrate ([3H]PDB) to intact living epidermal cells in monolayer culture was characterized. At 37 degrees C, the maximum specific [3H]PDB binding (binding displaceable by 30 microM unlabeled PDB) was attained in 15--20 min and was followed by a rapid decrease (down regulation) of radioactivity bound to the cells. The activity lost by the cells during this decrease was found in the incubation medium. Prior exposure of cells to phorbol 12-myristate 13-acetate (PMA; 12-O-tetradecanoylphorbol 13-acetate) but not to phorbol for 2 hr at 37 degrees C caused approximately 55% reduction in the number of measurable binding sites for [3H]PDB. The down regulation was temperature sensitive; there was no loss of radioactivity after 1 hr at 4 degrees C. The specific binding of [3H]PDB at 4 degrees C reached equilibrium in 15--20 min and was saturable and freely reversible. At equilibrium, epidermal cells contained 1.2 x 10(5) binding sites per cell, and binding sites had a KD of 10 nM. Specificity of binding was shown by the observation that the biologically active phorbol esters PMA and 12-deoxyphorbol 13-decanoate inhibited the binding, whereas the inactive parent compound phorbol and the nonphorbol tumor promoter anthralin did not have any effect. The abilities of these compounds to inhibit [3H]PDB binding directly correlates with their tumor promoting activities. Epidermal cells exposed to retinoic acid or fluocinolone acetonide for 24 hr had similar [3H]PDB binding characteristics as untreated cells suggesting that inhibition of tumor promotion induced by these compounds is not mediated through alterations in the phorbol ester binding sites. PMID:6941309

  13. Ultraviolet exposure of thymocytes: selective inhibition of apoptosis.

    PubMed

    Ojeda, F; Guarda, M I; Lovengreen, C; Hidalgo, M A; Folch, H; Härtel, S; Maldonado, C

    2004-06-01

    To evaluate selective effects of ultraviolet (UV) irradiation on spontaneous and induced apoptosis in freshly extracted mice thymocytes. Cells were exposed to UV radiation with emission peaks of 365 nm (UVA) exposures of 1620-10200 J m(-2), of 312 nm (UVB) exposures of 34-1620 J m(-2) or of 254 nm (UVC) exposures of 1.5-1620 J m(-2), and incubated for 5.5 h with or without hydrocortisone, phorbol-12-myristate-13-acetate or anti-Fas antibody. Additionally, cells were irradiated with gamma-rays (5 Gy) before UVB exposure (408 J m(-2)) at different times. Apoptosis was quantified by DNA fragmentation. Up to an irradiation of 5000 J m(-2), UVA exposure did not show any effect on thymocyte apoptosis, while at 10200 J m(-2) irradiation, considerable DNA fragmentation was observed. In contrast, UVB and UVC irradiation clearly inhibited natural and cortisone-induced apoptosis. Moreover, UVB inhibited apoptosis triggered by phorbol-12-myristate-13-acetate and gamma-irradiation, but not by anti-Fas antibody. The response of mouse thymocytes in culture to UV irradiation strongly depends on the wavelength used. It is suggested that either a survival or an apoptotic pathway occurs depending on the physiological state of the cell, spectral composition of the UV light and cell type. The possible involvement of extracellular signal-regulated kinase and stress-activated protein kinase/c-Jun N-terminal kinase in the apoptotic pathway is discussed.

  14. Interaction between a phorbol ester and dopamine DA1 receptors on vascular smooth muscle.

    PubMed

    Yasunari, K; Kohno, M; Murakawa, K; Yokokawa, K; Horio, T; Takeda, T

    1993-01-01

    The interaction between dopamine DA1 receptors and a phorbol ester was studied to elucidate the role of protein kinase C in the response of this receptor. The in vitro binding of [3H]Sch 23390 to DA1 receptor sites on vascular smooth muscle cells was saturable. The extent of [3H]Sch 23390 binding to phorbol ester-treated cells was increased without any change in the dissociation constant. The production of adenosine 3',5'-cyclic monophosphate (cAMP) in response to DA1 receptor stimulation was enhanced by preincubation of vascular smooth muscle cells with the phorbol ester for 4 h. However, no enhancement was observed when the medium used for preincubation was supplemented with a protein kinase C inhibitor. Direct stimulation of stimulatory guanine nucleotide-binding regulatory protein with 5-guanylylimidodiphosphate and direct stimulation of adenylate cyclase with forskolin produced no significant differences in cyclase levels between phorbol ester-treated and untreated cells. These results suggest that activation of protein kinase C triggers an increase in the membrane expression of DA1 receptors, thereby enhancing receptor-coupled cAMP generation.

  15. Irradiation with narrowband-ultraviolet B suppresses phorbol ester-induced up-regulation of H1 receptor mRNA in HeLa cells.

    PubMed

    Kitamura, Yoshiaki; Mizuguchi, Hiroyuki; Okamoto, Kentaro; Kitayama, Mika; Fujii, Tatsuya; Fujioka, Akira; Matsushita, Toshio; Mukai, Takashi; Kubo, Yoshiaki; Kubo, Nobuo; Fukui, Hiroyuki; Takeda, Noriaki

    2016-01-01

    Conclusion These findings suggest that low dose irradiation with 310 nm NB-UVB specifically suppressed the up-regulation of H1R gene expression without inducing apoptosis and that UVB of shorter or longer wavelength than 310 nm NB-UVB had no such effects. Objective To develop a narrowband-ultraviolet B(NB-UVB) phototherapy for allergic rhinitis, this study investigated the effects of irradiation with NB-UVB at wavelength of 310 nm on phorbol-12-myristate-13-acetate (PMA)-induced up-regulation of histamine H1 receptor (H1R) mRNA in HeLa cells. Methods The mRNA levels of H1R in HeLa cells were measured using real-time RT-PCR. Apoptosis were evaluated with DNA fragmentation assay. Results PMA induced a significant increase in H1R mRNA expression in HeLa cells. Irradiation with 305 nm UVB and 310 nm NB-UVB, but not with 315 nm UVB at doses of 200 and 300 mJ/cm(2) significantly suppressed PMA-induced up-regulation of H1R mRNA. At a dose of 200 mJ/cm(2), irradiation with 305 nm UVB, but not with 310 nm NB-UVB, induced apoptosis, although exposure of the cells to both 305 and 310 nm UVB induced apoptosis at a dose of 300 mJ/cm(2) after PMA treatment in HeLa cells. Conversely, irradiation with 315 nm UVB at doses of 200 and 300 mJ/cm(2) did not induce apoptosis.

  16. Role of the Chemokine MCP-1 in Sensitization of PKC-Mediated Apoptosis in Prostate Cancer Cells

    DTIC Science & Technology

    2010-02-01

    atypical (aPKCs ζ and λ). Only cPKCs and nPKCs are regulated by phorbol esters and diacylglycerol (DAG), a lipid second messenger generated upon...heterogeneous cell populations and aging tissues, such as skin biopsies from older individuals (Itahana et al., 2007). Phorbol Ester–Induced Apoptosis and...senescent human cells in culture and in aging skin in vivo. Proc. Natl. Acad. Sci. USA 92, 9363–9367. Ding, L., Wang, H. M., Lang, W. H., and Xiao, L. (2002

  17. Degradation of Jatropha curcas phorbol esters derived from Jatropha oil cake and their tumor-promoting activity.

    PubMed

    Nakao, Motoyuki; Hasegawa, Go; Yasuhara, Tadashi; Ishihara, Yoko

    2015-04-01

    Large amount of oil cake is generated during biodiesel production from Jatropha seeds. Although Jatropha oil cake is rich in plant nutrients, presence of toxic phorbol esters restricts the usage of oil cake as a fertilizer. The objective of this study is to evaluate the components and tumor promoting activity of phorbol esters in Jatropha oil cake-supplemented soil and plants grown in the treated soil. Contents and their biological activity of Jatropha phorbol esters in soil and plants were sequentially analyzed by high-performance liquid chromatography (HPLC) and in vitro cell transformation assay, respectively. Disappearance of Jatropha phorbol-ester-specific peaks were followed with HPLC during incubation of Jatropha oil cake with soil for five weeks. Along with the degradation of Jatropha phorbol ester in soil, tumor-promoting activity in the sample was also attenuated and ultimately disappeared. Jatropha phorbol esters and tumor promoting activity were not detected from mustard spinach grown in the Jatropha oil cake-supplemented soil. In addition, the esterase KM109 degrades DHPB (see definition below; Jatropha phorbol ester) and reduced its tumor-promoting activity. From these data, we conclude: (1) components and tumor promoting activity of Jatropha phorbol esters in the oil cake disappeared completely by incubation with soil for five-week, (2) Jatropha phorbol esters did not transfer into plants grown in the Jatropha oil cake-supplemented soil, and (3) DHPB can be degraded by esterase from soil bacterium. These observations are useful for utilization of Jatropha oil cake as a fertilizer.

  18. Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding.

    PubMed

    Ikin, Annat F; Causevic, Mirsada; Pedrini, Steve; Benson, Lyndsey S; Buxbaum, Joseph D; Suzuki, Toshiharu; Lovestone, Simon; Higashiyama, Shigeki; Mustelin, Tomas; Burgoyne, Robert D; Gandy, Sam

    2007-12-09

    Shedding of the Alzheimer amyloid precursor protein (APP) ectodomain can be accelerated by phorbol esters, compounds that act via protein kinase C (PKC) or through unconventional phorbol-binding proteins such as Munc13-1. We have previously demonstrated that application of phorbol esters or purified PKC potentiates budding of APP-bearing secretory vesicles at the trans-Golgi network (TGN) and toward the plasma membrane where APP becomes a substrate for enzymes responsible for shedding, known collectively as alpha-secretase(s). However, molecular identification of the presumptive "phospho-state-sensitive modulators of ectodomain shedding" (PMES) responsible for regulated shedding has been challenging. Here, we examined the effects on APP ectodomain shedding of four phorbol-sensitive proteins involved in regulation of vesicular membrane trafficking of APP: Munc13-1, Munc18, NSF, and Eve-1. Overexpression of either phorbol-sensitive wildtype Munc13-1 or phorbol-insensitive Munc13-1 H567K resulted in increased basal APP ectodomain shedding. However, in contrast to the report of Rossner et al (2004), phorbol ester-dependent APP ectodomain shedding from cells overexpressing APP and Munc13-1 wildtype was indistinguishable from that observed following application of phorbol to cells overexpressing APP and Munc13-1 H567K mutant. This pattern of similar effects on basal and stimulated APP shedding was also observed for Munc18 and NSF. Eve-1, an ADAM adaptor protein reported to be essential for PKC-regulated shedding of pro-EGF, was found to play no obvious role in regulated shedding of sAPPalpha. Our results indicate that, in the HEK293 system, Munc13-1, Munc18, NSF, and EVE-1 fail to meet essential criteria for identity as PMES for APP.

  19. Evidence against roles for phorbol binding protein Munc13-1, ADAM adaptor Eve-1, or vesicle trafficking phosphoproteins Munc18 or NSF as phospho-state-sensitive modulators of phorbol/PKC-activated Alzheimer APP ectodomain shedding

    PubMed Central

    Ikin, Annat F; Causevic, Mirsada; Pedrini, Steve; Benson, Lyndsey S; Buxbaum, Joseph D; Suzuki, Toshiharu; Lovestone, Simon; Higashiyama, Shigeki; Mustelin, Tomas; Burgoyne, Robert D; Gandy, Sam

    2007-01-01

    Background Shedding of the Alzheimer amyloid precursor protein (APP) ectodomain can be accelerated by phorbol esters, compounds that act via protein kinase C (PKC) or through unconventional phorbol-binding proteins such as Munc13-1. We have previously demonstrated that application of phorbol esters or purified PKC potentiates budding of APP-bearing secretory vesicles at the trans-Golgi network (TGN) and toward the plasma membrane where APP becomes a substrate for enzymes responsible for shedding, known collectively as α-secretase(s). However, molecular identification of the presumptive "phospho-state-sensitive modulators of ectodomain shedding" (PMES) responsible for regulated shedding has been challenging. Here, we examined the effects on APP ectodomain shedding of four phorbol-sensitive proteins involved in regulation of vesicular membrane trafficking of APP: Munc13-1, Munc18, NSF, and Eve-1. Results Overexpression of either phorbol-sensitive wildtype Munc13-1 or phorbol-insensitive Munc13-1 H567K resulted in increased basal APP ectodomain shedding. However, in contrast to the report of Roßner et al (2004), phorbol ester-dependent APP ectodomain shedding from cells overexpressing APP and Munc13-1 wildtype was indistinguishable from that observed following application of phorbol to cells overexpressing APP and Munc13-1 H567K mutant. This pattern of similar effects on basal and stimulated APP shedding was also observed for Munc18 and NSF. Eve-1, an ADAM adaptor protein reported to be essential for PKC-regulated shedding of pro-EGF, was found to play no obvious role in regulated shedding of sAPPα. Conclusion Our results indicate that, in the HEK293 system, Munc13-1, Munc18, NSF, and EVE-1 fail to meet essential criteria for identity as PMES for APP. PMID:18067682

  20. Constitutive apoptosis in equine peripheral blood neutrophils in vitro

    PubMed Central

    Brazil, Timothy J.; Dixon, Padraic M.; Haslett, Christopher; Murray, Joanna; McGorum, Bruce C.

    2014-01-01

    The aim of this study was to characterise constitutive apoptosis in equine peripheral blood neutrophils, including assessment of factors that potentially modulate neutrophil survival through alteration of the rate of constitutive apoptosis. Cells underwent spontaneous time-dependent constitutive apoptosis when aged in culture for up to 36 h, developing the structural and functional features of apoptosis observed in many cell types, including human neutrophils. Neutrophils undergoing apoptosis also had diminished zymosan activated serum (ZAS)-stimulated chemiluminescence, but maintained responsiveness to phorbol myristate acetate (PMA). The constitutive rate of equine neutrophil apoptosis was promoted by lipopolysaccharide (LPS), tumour necrosis factor α and phagocytosis of opsonised ovine erythrocytes, while it was inhibited by dexamethasone and ZAS (a source of C5a). Formyl-Met-Leu-Phe, leukotriene B4, platelet activating factor and PMA had no demonstrable effect on equine neutrophil apoptosis. There was a difference between equine and human neutrophil apoptosis in response to LPS and the time-dependence of the response to dexamethasone. PMID:25239298

  1. Constitutive apoptosis in equine peripheral blood neutrophils in vitro.

    PubMed

    Brazil, Timothy J; Dixon, Padraic M; Haslett, Christopher; Murray, Joanna; McGorum, Bruce C

    2014-12-01

    The aim of this study was to characterise constitutive apoptosis in equine peripheral blood neutrophils, including assessment of factors that potentially modulate neutrophil survival through alteration of the rate of constitutive apoptosis. Cells underwent spontaneous time-dependent constitutive apoptosis when aged in culture for up to 36 h, developing the structural and functional features of apoptosis observed in many cell types, including human neutrophils. Neutrophils undergoing apoptosis also had diminished zymosan activated serum (ZAS)-stimulated chemiluminescence, but maintained responsiveness to phorbol myristate acetate (PMA). The constitutive rate of equine neutrophil apoptosis was promoted by lipopolysaccharide (LPS), tumour necrosis factor α and phagocytosis of opsonised ovine erythrocytes, while it was inhibited by dexamethasone and ZAS (a source of C5a). Formyl-Met-Leu-Phe, leukotriene B4, platelet activating factor and PMA had no demonstrable effect on equine neutrophil apoptosis. There was a difference between equine and human neutrophil apoptosis in response to LPS and the time-dependence of the response to dexamethasone.

  2. Structural modifications induced by TPA (12-O-tetradecanoyl phorbol-13-acetate) in sea urchin eggs.

    PubMed

    Ciapa, B; Crossley, I; De Renzis, G

    1988-07-01

    We investigated the effect of the phorbol ester TPA (12-O-tetradecanoyl phorbol 13-acetate) on the egg morphology of the sea urchin Arbacia lixula. Our study indicates that TPA alters the cortical region of the egg: the pigment granules migrate toward the surface, while cortical granules detach from the plasma membrane. Cortical granule exocytosis did not occur but the endocytosis process was turned on. Prolonged treatment of the eggs by TPA partially inhibits the cortical granule exocytosis normally triggered by fertilization. We discuss the effects of TPA in terms of its interaction with the Ca2+ pool and cytoskeletal structures. In order to discern the respective roles of pHi and protein kinase C activity in endocytosis process activation, we compared the ultrastructural effects of TPA and ammonia. Finally, the role of pigment vesicles in egg metabolism activation is discussed.

  3. Characterization of a phorbol ester-stimulated S6 kinase from MDCK renal epithelial cells

    SciTech Connect

    Meier, K.E.; Krebs, E.G.

    1987-05-01

    Increased phosphorylation of S6, a 40S ribosomal subunit protein, is observed in mammalian cells in response to growth factors and phorbol esters. The goal of this study was to identify the S6 kinase that is stimulated by phorbol ester treatment of MDCK cells. MDCK clone D1 cells express high levels of protein kinase C(PKC). PKC and S6 kinase activities were measured following DEAE-Sephacel fractionation of cytosol; this procedure separated the two kinase activities. When confluent MDCK-D1 cells were exposed to 100 nM phorbol 12-myristate 13-acetate (PMA), 95% of the total cellular PKC activity became associated with the particulate fraction within 1 hour. Cytosolic S6 kinase activity was maximal by 1 hour and then declined thereafter, preceding any detectable loss of total cellular PKC. The PMA-responsive S6 kinase was partially purified from MDCK-D1 cytosol by consecutive steps of DEAE-Sephacel, ammonium sulfate precipitation, Ultrogel AcA 34, heparin-agarose, and Ultrogel AcA 34. The partially-purified enzyme had an apparent molecular size of approximately 80 kDa. In addition to S6, the enzyme phosphorylated synthetic peptides based on the carboxyl terminal sequence of S6. S6 kinase activity utilized ATP but not GTP, and was inhibited by heparin, NaCl, and ..beta..-glycerophosphate. In conclusion, a phorbol ester-stimulated S6 kinase has been partially purified from an epithelial cell line. This kinase is distinct from PKC.

  4. Mechanisms for cardiac depression induced by phorbol myristate acetate in working rat hearts.

    PubMed Central

    Karmazyn, M.; Watson, J. E.; Moffat, M. P.

    1990-01-01

    1. The effects of the phorbol ester, phorbol myristate acetate (PMA) were examined on function and energy metabolism in the isolated working heart of the rat. 2. At a concentration of 10(-9) M PMA produced a rapid loss in cardiac function in terms of aortic flow rate (AFR) and coronary flow rates (CFR) whereas a similar concentration of 4 alpha-phorbol 12,13-didecanoate was ineffective. At a concentration of 10(-10) M, the PMA-induced depression was more gradual but nevertheless very pronounced with an almost total loss in AFR after 30 min perfusion. The reduction in CFR was more moderate than that observed with respect to AFR. 3. The protein kinase C (PKC) inhibitor (+/-)-1-O-hexadecyl-2-O-acylglycerol significantly attenuated the loss in AFR and CFR following addition of PMA. 4. Two inhibitors of Na+/H+ exchange, amiloride and quinacrine, totally prevented the reduction in AFR. Although the PMA-induced depression in CFR was also attenuated by both amiloride and quinacrine, these effects were not significant, probably reflecting the less pronounced effect of PMA on this parameter. 5. Nifedipine, a dihydropyridine calcium channel blocker reduced PMA toxicity to a similar degree as Na+/N+ exchange inhibition whereas the calcium channel agonist Bay K 8644 was without effect. 6. Tissue content of energy metabolites including high energy phosphates, total adenine nucleotides or lactate were not significantly affected by PMA perfusion. 7. We conclude that PKC activation is necessary for phorbol ester-induced cardiac dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2207502

  5. Phorbol ester induces elevated oxidative activity and alkalization in a subset of lysosomes

    SciTech Connect

    Chen, Chii-Shiarng )

    2002-01-01

    Background: Lysosomes are acidic organelles that play multiple roles in various cellular oxidative activities such as the oxidative burst during cytotoxic killing. It remains to be determined how lysosomal lumen oxidative activity and pH interact and are regulated. Here, I report the use of fluorescent probes to measure oxidative activity and pH of lysosomes in live macrophages upon treatment with the tumor promotor phorbol 12-myristate 13-acetate (PMA), and provide novel insight regarding the regulation of lysosomal oxidative activity and pH. Results: The substrate used to measure oxidative activity was bovine serum albumin covalently coupled to dihydro-2?, 4,5,6,7,7?-hexafluorofluorescein (OxyBURST Green H2HFF BSA). During pulse-chase procedures with live macrophages, this reduced dye was internalized through an endocytic pathway and accumulated in the lysosomes. Oxidation of this compound results in a dramatic increase of fluorescence intensity. By using low-light level fluorescence microscopy, I determined that phorbol ester treatment results in increased oxidative activity and pH elevation in different subsets of lysosomes. Furthermore, lysosomes with stronger oxidative activity tended to exclude the acidotropic lysosomal indicator, and thus exhibit higher alkalinity. Conclusions: Results indicate that there is a regulatory mechanism between lysosomal oxidative activity and pH. Activation of lysosomal Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase by phorbol ester may result in increase of intralysosomal O2?- and H2O2, concurrent with pH elevation due to consumption of H+ and generation of OH-. Furthermore, effect of phorbol ester on elevated oxidative activity and pH is heterogeneous among total lysosomal population. Higher oxidative activity and/or pH are only observed in subsets of lysosomes.

  6. Phorbol ester-stimulated phosphorylation of keratinocyte transglutaminase in the membrane anchorage region.

    PubMed Central

    Chakravarty, R; Rong, X H; Rice, R H

    1990-01-01

    The membrane-bound transglutaminase of cultured keratinocytes became radioactively labelled upon addition of [32P]Pi to the medium. Transglutaminase phosphorylation was also demonstrable using particulate material isolated from cell homogenates. Compatible with mediation of the labelling by protein kinase C, the degree of phosphorylation in intact cells was stimulated approx. 5-fold in 4 h on treatment with the tumour-promoting phorbol ester phorbol 12-myristate 13-acetate, but not by phorbol. The extent of labelling was virtually unaffected by cycloheximide inhibition of protein synthesis, indicating that it arose primarily through turnover of phosphate in the membrane-bound enzyme. Phosphoamino acid analysis detected labelling only of serine residues. Most of the label was removed by trypsin release of the enzyme from the particulate fraction of cell homogenates, which deletes a membrane anchorage region of approximately 10 kDa. Upon trypsin treatment of the enzyme after immunoprecipitation, the phosphate label was recovered in soluble peptide material with a size of several thousand Da or less. Indicative of fragmentation of the membrane anchorage region, this material was separable by h.p.l.c. into two equally labelled peptides. Moreover, when the enzyme was labelled with [3H]palmitate or [3H]myristate, the fatty-acid-labelled peptide material required non-ionic detergent for solubilization and was separable from the phosphate-labelled material by gel filtration. Phorbol ester treatment of cultured keratinocytes in high- or low- Ca2(+)-containing medium was not accompanied by an appreciable protein-synthesis-independent change in transglutaminase activity. Independent of possible alteration of the intrinsic catalytic activity of the enzyme, phosphorylation may well modulate its interaction with substrate proteins, a potential site for physiological regulation. Images Fig. 1. Fig. 3. PMID:1977383

  7. Synergy between phorbol esters, 1-oleyl-2-acetylglycerol, urushiol, and calcium ionophore in eliciting aggregation of marine sponge cells.

    PubMed

    Weissmann, G; Azaroff, L; Davidson, S; Dunham, P

    1986-05-01

    Aggregation of marine sponge cells (Microciona prolifera) resembles stimulus-response coupling of higher organisms in which activation of protein kinase C and movements of intracellular Ca provide twin signals. We now report that activators of protein kinase C (phorbol esters) and ionomycin act synergistically to aggregate sponge cells. Surprisingly--since extracellular Ca is required for integrity of the species-specific aggregation factor--synergistic aggregation proceeded in the complete absence of added extracellular Ca (2.5-20 mM EDTA). The order of activity of phorbol esters and related compounds was that of their effect on protein kinase C (phorbol myristate acetate, phorbol dibutyrate greater than phorbol diacetate much greater than phorbol, 4 alpha-phorbol). 1-Oleyl, 2-acetylglycerol a synthetic activator of protein kinase C, also showed synergy with ionomycin. Phorbol esters and 1-oleyl, 2-acetylglycerol acted in synergy with ionomycin to liberate membrane Ca as detected by decreased fluorescence of chlortetracycline in prelabeled cells. Moreover, urushiol, the toxic principle of poison ivy, but not pentadecanylcatechol, its inert analogue, showed synergy with ionomycin. Synergistic aggregation was inhibited by calmidazolium (10 microM), piroxicam (20-100 microM), and pertussis toxin (20 micrograms/ml). The data not only confirm that marine sponge cell aggregation follows the general sequence of stimulus-response coupling in the cells of higher organisms but also support, in this most ancient of multicellular creatures, the hypothesis that mobilization of intracellular Ca and activation of protein kinase C provide the twin signals for cell activation in the absence of added extracellular Ca.

  8. Phorbol ester induced phosphorylation of the estrogen receptor in intact MCF-7 human breast cancer cells

    SciTech Connect

    Knabbe, C.; Lippman, M.E.; Greene, G.L.; Dickson, R.B.

    1986-05-01

    Recent studies with a variety of cellular receptors have shown that phorbol ester induced phosphorylation modulates ligand binding and function. In this study the authors present direct evidence that the estrogen receptor in MCF-7 human breast cancer cells is a phosphoprotein whose phosphorylation state can be enhanced specifically by phorbol-12-myristate-13-acetate (PMA). Cells were cultured to 6h in the presence of (/sup 32/P)-orthophosphate. Whole cell extracts were immunoprecipitated with a monoclonal antibody (D58) against the estrogen receptor and subjected to SDS-polyacrylamide electrophoresis. Autoradiography showed a specific band in the region of 60-62 kDa which was significantly increased in preparations from PMA treated cells. Phospho-amino acid analysis demonstrated specific phosphorylation of serine and threonine residues. Cholera toxin or forskolin did not change the phosphorylation state of this protein. In a parallel binding analysis PMA led to a rapid decrease of estrogen binding sites. The estrogen induction of both progesterone receptors and growth in semisolid medium was blocked by PMA, whereas the estrogen induction of the 8kDa protein corresponding to the ps2 gene product and of the 52 kDa protein was not affected. In conclusion, phorbol esters can induce phosphorylation of the estrogen receptor. This process may be associated with the inactivation of certain receptor functions.

  9. Effect of phorbol esters on contractile state and calcium flux in cultured chick heart cells

    SciTech Connect

    Leatherman, G.F.; Kim, D.; Smith, T.W.

    1987-07-01

    Phorbol esters are potent tumor promoters that have been widely used in studies of transmembrane signaling because of their ability to activate protein kinase C. To study the effect of phorbol esters (and indirectly, the role of protein kinase C) on the cardiac muscle contractility, the authors examined the effects of phorbol myristate acetate (PMA) on contractile state, transmembrane /sup 45/Ca fluxes, and cytosolic free Ca concentration ((Ca)/sub i/) using spontaneously contracting cultured chick ventricular cells. PMA produced a concentration- and time-dependent decrease in the amplitude of cell motion (half maximum inhibitory concentration) with maximal effect observed at 1 ..mu..M. PMA (1 ..mu..M) reduced /sup 45/Ca uptake rate by 16 /plus minus/ 4% and the size of the rapidly exchangeable Ca pool by 11 /plus minus/ 2%, but did not alter the /sup 45/Ca efflux rate. In fura-2-loaded cells. PMA produced a decrease in (Ca)/sub i/ from 96 /plus minus/ 7 to 72 /plus minus/ 5 nM with a time course similar to that of alteration in contractile amplitude. These results indicate that PMA influences transsarcolemmal Ca uptake, and thus the excitation-contraction process, and suggest that protein kinase C may modulate myocardial Ca homeostassis and contractile state.

  10. Stimulation of 86Rb+ and 32Pi movements in 3T3 cells by prostaglandins and phorbol esters.

    PubMed

    Moroney, J; Smith, A; Tomei, L D; Wenner, C E

    1978-06-01

    The potent tumor promoter tetradecanoyl phorbol acetate (TPA) induces early changes in ion movements analogous to those induced by prostaglandins E1 and F 2alpha. Among the earliest changes induced by TPA is a significant increase in 32Pi incorporation within 15 minutes incubation of TPA (10(-8)-10(-6) M) with post-confluent Swiss 3T3 mouse embryonic fibroblasts. Similarly, the active phorbol ester homolog 4-beta-OH phorbol didecanoate but not the inactive stereoisomeric 4-alpha-OH phorbol didecanoate stimulated 32Pi incorporation. Also, TPA at the above concentrations stimulated 86Rb+ influx shortly after administration. Both fluxes were ouabain-sensitive in accord with the idea that an early effect of TPA is to alter (Na+ + K+)-ATPase activity. Further, prostaglandin E1 (10(-7)-10(-6) M) and prostaglandin F 2alpha (3 X 10(-9)-10(-7) M) caused a similar stimulation of 86Rb+ and 32Pi uptake. The finding that water-soluble prostaglandin F 2alpha also exhibited stimulatory effects indicated that those hormone-induced responses are not mediated by solvent interactions. The similar responses of phorbol esters and prostaglandin derivatives suggests that phorbol esters and prostaglandin derivatives may act at common membrane sites. The finding that stimulatory effects were observed at discrete times in the logarithmic phase of growth suggests that the activation of membrane receptors may be cell-cycle dependent.

  11. Differential role of protein kinase C in desensitization of muscarinic receptor induced by phorbol esters and receptor agonists

    SciTech Connect

    Lai, Wi Sheung.

    1989-01-01

    PKC, a phorbol ester receptor, copurified with specific binding sites of ({sup 3}H)phorbol-12,13,-dibutyrate (({sup 3}H)PDBu). The specific binding of ({sup 3}H)PDBu to intact cells was saturable to a single class of binding sites. The PKC and phorbol ester receptors in N1E-115 cells can be down regulated by prolonged phorbol ester incubation. Phorbol 12-myristate 13-acetate (PMA) suppressed muscarinic receptor-mediated cyclic GMP response in a time-dependent and a concentration-dependent fashion and the suppressive effect of PMA could be attenuated by a protein kinase inhibitor, H-7, as well as by down-regulation of the PKC through long-term incubation with PDBu. Exposure of the cells to the muscarinic agonist carbamylcholine also desensitized subsequent CBC-mediated cyclic GMP response. However, pretreatment with carbamylcholine did not desensitize histamine-induced cyclic GMP formation while treatment with PMA suppressed this histamine-mediated response. Preincubation of the cells with CBC, but not with phorbol ester, resulted in down-regulation of muscarinic receptors. The loss of muscarinic receptors induced by agonist even occurred when the phosphoinositide hydrolysis response was suppressed.

  12. Phorbol esters enhance attachment of NIH/3T3 cells to laminin and type IV collagen substrates

    SciTech Connect

    Kato, Shigemi; Ben, T.L.; De Luca, L.M. )

    1988-11-01

    The effect of phorbol esters on the adhesive properties of NIH/3T3 mouse fibroblasts was investigated using plastic substrates precoated with the extracellular matrix proteins fibronectin, collagen, and laminin. Treatment with phorbol 12-myristate 13-acetate (PMA) enhanced NIH/3T3 cell attachment to laminin and type IV collagen substrates but had little or no effect on attachment to fibronectin and type I collagen substrates. The effect of PMA in enhancing cell attachment to laminin and type IV collagen substrates was dose dependent between 10{sup {minus}9} and 10{sup {minus}7} M. PMA was effective as early as 30 min; the effect reached a maximum at 2 h and decreased gradually. Phorbol 12, 13-dibenzoate and phorbol 12, 13-diacetate were effective but to a lesser extent and phorbol 12-myristate and phorbol 13-acetate showed little or no effect. These results suggest that PMA may enhance NIH/3T3 cell adhesion through effects on laminin and type IV collagen receptors. Retinoic acid, which itself requires at least 6 h to show an effect on attachment, did not have any effect on cell attachment in 2 h and, if anything, slightly inhibited PMA-enhanced cell attachment to laminin and type IV collagen substrates.

  13. Phorbol esters potentiate rapid dopamine release from median eminence and striatal synaptosomes

    SciTech Connect

    Shu, C.; Selmanoff, M.

    1988-06-01

    In the present study, we investigated the ability of phorbol esters to potentiate Ca2+-dependent depolarization-induced release of tritium-labeled dopamine ((3H)DA) from median eminence and striatal synaptosomes. Phorbol esters potentiated (3H)DA release in a concentration-dependent manner in both kinds of dopaminergic nerve terminals and with a potency series similar to that reported for stimulation of protein kinase-C (PKC) activity in other cell systems. Evoked (3H)DA release was increased by 12-O-tetradecanoylphorbol-13-acetate (TPA; 10(-7) M) after 1, 3, 5, and 10 sec of depolarization. The effect of TPA was suppressed by sphingosine, a PKC inhibitor. TPA enhanced (3H)DA release evoked by high K+, veratridine or the Ca2+ ionophore A23187. Phorbol ester potentiation was found to be depolarization dependent, as it was present from 30-75 mM, but not at 5-20 mM external K+. Potentiation was seen at all external Ca2+ concentrations studied between 0.01-3 mM. However, in the absence of external free Ca2+ (i.e. with 0.1 mM EGTA), the phorbol effect was not present. These data indicate that an increase in intrasynaptosomal Ca2+ concentration is necessary for the enhancement of (3H)DA release by phorbol esters to occur. The combination of TPA and the Ca2+ ionophore A23187 does not show the marked synergism observed in some other systems, that is maximal release was not reinstated. This suggests that in dopaminergic nerve terminals, activation of PKC has a modulatory, rather than a mediating, effect on release. Recently, we have shown that hyperprolactinemia stimulated (3H)DA release from median eminence synaptosomes by an external Ca2+-independent mechanism which might involve the PKC pathway. However, in the present work we found that the TPA and PRL effects on evoked (3H)DA release were additive, suggesting that two independent mechanisms are involved.

  14. Tephrosia purpurea alleviates phorbol ester-induced tumor promotion response in murine skin.

    PubMed

    Saleem, M; Ahmed Su; Alam, A; Sultana, S

    2001-02-01

    In recent years, considerable emphasis has been placed on identifying new cancer chemopreventive agents, which could be useful for the human population. Tephrosia purpurea has been shown to possess significant activity against hepatotoxicity, pharmacological and physiological disorders. Earlier we showed that Tephrosia purpurea inhibits benzoyl peroxide-mediated cutaneous oxidative stress and toxicity. In the present study, we therefore assessed the effect of Tephrosia purpurea on 12-O-tetradecanoyl phorbal-13-acetate (TPA; a well-known phorbol ester) induced cutaneous oxidative stress and toxicity in murine skin. The pre-treatment of Swiss albino mice with Tephrosia purpurea prior to application of croton oil (phorbol ester) resulted in a dose-dependent inhibition of cutaneous carcinogenesis. Skin tumor initiation was achieved by a single topical application of 7,12-dimethyl benz(a)anthracene (DMBA) (25 microg per animal per 0.2 ml acetone) to mice. Ten days later tumor promotion was started by twice weekly topical application of croton oil (0.5% per animal per 0.2 ml acetone, v /v). Topical application of Tephrosia purpurea 1 h prior to each application of croton oil (phorbol ester) resulted in a significant protection against cutaneous carcinogenesis in a dose-dependent manner. The animals pre-treated with Tephrosia purpurea showed a decrease in both tumor incidence and tumor yield as compared to the croton oil (phorbol ester)-treated control group. In addition, a significant reduction in TPA-mediated induction in cutaneous ornithine decarboxylase (ODC) activity and [3H]thymidine incorporation was also observed in animals pre-treated with a topical application of Tephrosia purpurea. The effect of topical application of Tephrosia purpurea on TPA-mediated depletion in the level of enzymatic and non-enzymatic molecules in skin was also evaluated and it was observed that topical application of Tephrosia purpurea prior to TPA resulted in the significant recovery of

  15. Conventional protein kinase C isoforms mediate phorbol ester-induced lysophosphatidic acid LPA1 receptor phosphorylation.

    PubMed

    Hernández-Méndez, Aurelio; Alcántara-Hernández, Rocío; Acosta-Cervantes, Germán C; Martínez-Ortiz, Javier; Avendaño-Vázquez, S Eréndira; García-Sáinz, J Adolfo

    2014-01-15

    Using C9 cells stably expressing LPA1 receptors fused to the enhanced green fluorescent protein, it was observed that activation of protein kinase C induced a rapid and strong increase in the phosphorylation state of these receptors. Overnight incubation with phorbol esters markedly decreased the amount of conventional (α, βI, βII and γ) and novel (δ) but not atypical (ζ) immunodetected PKC isoforms, this treatment blocks the action of protein kinase on receptor function and phosphorylation. Bis-indolylmaleimide I a general, non-subtype selective protein kinase C inhibitor, and Gö 6976, selective for the isoforms α and β, were also able to block LPA1 receptor desensitization and phosphorylation; hispidin, isoform β-selective blocker partially avoided receptor desensitization. Expression of dominant-negative protein kinase C α or β II mutants and knocking down the expression of these kinase isozymes markedly decreased phorbol ester-induced LPA1 receptor phosphorylation without avoiding receptor desensitization. This effect was blocked by bis-indolyl-maleimide and Gö 6976, suggesting that these genetic interventions were not completely effective. It was also observed that protein kinase C α and β II isozymes co-immunoprecipitate with LPA1 receptors and that such an association was further increased by cell treatments with phorbol esters or lysophosphatidic acid. Our data suggest that conventional protein kinase C α and β isozymes modulate LPA1 receptor phosphorylation state. Receptor desensitization appears to be a more complex process that might involve additional elements. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Decrease of epidermal histidase activity by tumor-promoting phorbol esters.

    PubMed

    Colburn, N H; Lau, S; Head, R

    1975-11-01

    The potent skin tumor promoter (12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulates epidermal macromolecular synthesis as well as proliferation, but little is known of specific functional aberrations produced by TPA. This report presents results of a study on the effects of TPA on epidermal histidase (L-histidine ammonia lyase), an enzyme found in normal epidermis but not in dermis or in mouse squamous cell carcinomas. Histidase activity was assayed on postmitochondrial supernatants obtained from hairless mouse epidermis after removal by keratotome. Topical TPA treatment at doses active in tumor promotion (1.7 to 17.0 nmoles/application) produced dose-dependent decreases in epidermal histidase specific activity at 19 hr posttreatment. The onset of the decrease occurred at 12 hr with recovery to control level specific activity by 5 days, showing kinetics similar to those obtained for stimulation of DNA synthesis. This decrease in histidase could not be attributed to a general inhibition of soluble protein synthesis or to the appearance of an inhibitor of histidase activity. The strong promoter TPA produced a greater histidase decrease than did the moderate promoter and mitogen 12,13-didecanoyl phorbol at equimolar dose, while phorbol, a nonpromoter and nonmitogen, produced no effects on histidase. The relationship of this histidase depression to tumor promotion and not initiation is further indicated by the finding that (a) Tween 60, a structurally unrelated tumor promotor, also produced a decrease in histidase; and (b) the tumor initiator urethan and an initiating dose of 9,10-dimethybenz(a)anthracene showed no effects on histadase activity.

  17. Phorbol esters inhibit alpha/sub 1/-adrenergic receptor stimulated phosphoinositide hydrolysis and contraction in rat aorta

    SciTech Connect

    Not Available

    1986-03-01

    The mechanisms of pharmacomechanical coupling in vascular tissue are at the present time unclear. The authors and others have proposed that receptor-induced activation of phosphoinositide (PI) hydrolysis may be involved. To investigate this possibility they studied the actions of two biologically active phorbol esters: phorbol dibutyrate (PDB) and phorbol myristate diacetate (PMA) on receptor-stimulated PI hydrolysis in rat aortic rings. They found both PDB (IC/sub 5//sup 0/ approx. 5nM) and PMA (IC/sub 50/ approx. 30 nM) but not 4-..cap alpha..-phorbol (IC32%/sub 0/ > 10,000 nM) inhibited norepinephrine-stimulated PI hydrolysis. In the presence of the calcium channel antagonist nitrendipine, PDB potently inhibited both the phasic and tonic components of norepinephrine-induced vascular contraction. In the presence of 10/sup -7/M nitrendipine, PDB had an IC/sub 50/ for contraction of approximately 10nM. The results thus suggest a functional coupling between ..cap alpha../sub 1/-adrenergic receptor-stimulated PI hydrolysis and vascular contraction. The findings further imply a mode of feed-back regulation in vascular tissue involving phorbol ester and receptor-stimulated PI hydrolysis.

  18. The insulin-like effects of phorbol myristate acetate (PMA) in the isolated fat cell

    SciTech Connect

    Solomon, S.S.; Palazzolo, M. )

    1989-01-01

    Recent data from many laboratories suggest that insulin stimulates diacylglycerol formation. Data presented in this manuscript demonstrate an insulin-like effect of PMA, a tumor promoting agent that mimics the action of diacylglycerol, in isolated adipocytes on; (a) glucose oxidation using uniformly labelled, C-1-labelled and C-6-labelled glucose, (b) epinephrine-induced lipolysis and (c) low Km cAMP phosphodiesterase activity. Additionally, a lipolytic effect of PMA is identified when unopposed by epinephrine. These data not only demonstrate an insulin-like effect of phorbol esters in adipose tissue but they lend support to the concept of diacylglycerol involvement in the mechanism of insulin action.

  19. OSTEOCYTE APOPTOSIS

    PubMed Central

    Jilka, Robert L.; Noble, Brendon; Weinstein, Robert S.

    2012-01-01

    Apoptotic death of osteocytes was recognized over 15 years ago, but its significance for bone homeostasis has remained elusive. A new paradigm has emerged that invokes osteocyte apoptosis as a critical event in the recruitment of osteoclasts to a specific site in response to skeletal unloading, fatigue damage, estrogen deficiency and perhaps in other states where bone must be removed. This is accomplished by yet to be defined signals emanating from dying osteocytes, which stimulate neighboring viable osteocytes to produce osteoclastogenic cytokines. The osteocyte apoptosis caused by chronic glucocorticoid administration does not increase osteoclasts; however, it does negatively impact maintenance of bone hydration, vascularity, and strength. PMID:23238124

  20. Luminol-dependent photoemission from single neutrophil stimulated by phorbol ester and calcium ionophore--role of degranulation and myeloperoxidase

    SciTech Connect

    Suematsu, M.; Oshio, C.; Miura, S.; Suzuki, M.; Houzawa, S.; Tsuchiya, M.

    1988-08-30

    Luminol-dependent photonic burst from phorbol ester-treated single neutrophil was visually investigated by using an ultrasensitive photonic image intensifier microscope. Neutrophils stimulated by phorbol myristate acetate (0.1 microgram/ml) alone produced a negligible level of photonic activities in the presence of luminol (10 micrograms/ml). The additional application of 0.1 microM Ca2+ ionophore A23187 induced explosive changes of photonic burst corresponding to the distribution of neutrophils, and these photonic activities were gradually spread to extracellular space. Sodium azide, which prevents myeloperoxidase activity, inhibited Ca2+ ionophore-induced photonic burst from phorbol ester-treated neutrophil. These findings suggest a prerequisite role of degranulation and myeloperoxidase release in luminol-dependent photoemission from stimulated neutrophils.

  1. Phorbol esters alter adenylate cyclase responses to vasoactive intestinal peptide and forskolin in the GH cell line

    SciTech Connect

    Summers, S.; Florio, T.; Cronin, M.

    1986-05-01

    Activation of protein kinase C with phorbol ester modifies cyclic AMP production in several anterior pituitary cell systems. In the GH cell line from a rat pituitary tumor, exposure to phorbol 12-myristate 13-acetate (PMA: 100 nM) for 30 minutes significantly reduces vasoactive intestinal peptide (VIP: 100 nM) stimulated adenylate cyclase (AC) activity in subsequent membrane preparations to 62 + 4% of control (n = 6 independent studies). In contrast, these same membrane preparations respond to forskolin (1 ..mu..M) with significantly more activity, 130 +/- 6% of controls (n = 6 independent studies). Finally, phorbol ester does not block an inhibitory hormone input into the AC system; somatostatin (100 nM) reduction of VIP-stimulated AC activity is not significantly different in membrane preparations from PMA treated and control cells (n = 3 independent studies). These other findings lead the authors to propose that protein kinase C can modify several sites in the AC complex in anterior pituitary cells.

  2. Studies on glycogen autophagy: effects of phorbol myristate acetate, ionophore A23187, or phentolamine.

    PubMed

    Kalamidas, S A; Kotoulas, O B; Hann, A C

    2002-06-15

    The effects of agents that could manipulate the lysosomal calcium such as phorbol myristate acetate, ionophore A23187, and phentolamine on the lysosomal glycogen degradation were studied by electron microscopy, morphometric analysis, and biochemical assays in newborn rat hepatocytes. Phorbol myristate acetate, which promotes the input of calcium to lysosomes, increased the total volume of autophagic vacuoles and the activity of lysosomal glycogen-hydrolyzing acid alpha 1,4 glucosidase and decreased the fractional volume of undigested glycogen inside the autophagic vacuoles and also decreased the activity of acid mannose 6-phosphatase. Ionophore A23187, which releases lysosomal calcium, produced opposite results in these enzyme activities. Phentolamine, an alpha-adrenergic blocking agent which interferes with the generation of phosphoinositides and may activate the lysosomal calcium uptake pump, increased the total volume of autophagic vacuoles and the activity of lysosomal glycogen-hydrolyzing acid glucosidase and decreased the fractional volume of undigested glycogen inside the autophagic vacuoles. The results of this study constitute evidence that changes in lysosomal calcium may influence certain aspects of autophagy, including the degradation of glycogen inside the autophagic vacuoles. They also support our previous postulate [Kalamidas and Kotoulas (2000a,b) Histol Histopathol 15:29-35, 1011-1018] that stimulation of autophagic mechanisms in newborn rat hepatocytes may be associated with acid mannose 6-phosphatase activity-deficient lysosomes.

  3. Down-modulation of receptors for phorbol ester tumor promoter in primary epidermal cells

    SciTech Connect

    Solanki, V.; Slaga, T.J.

    1982-01-01

    The specific (20-/sup 3/H)phorbol 12,13-dibutyrate ((/sup 3/H)PDBu) binding to intact epidermal cells displayed the phenomenon of down-modulation, i.e., the specific binding of (/sup 3/H)PDBu to its receptors on primary epidermal cells reached a maximum within 1 h and steadily declined thereafter. The apparent down-modulation of radiolabel resulted from a partial loss in the total number of receptors; the affinity of receptors for the ligand was essentially unchanged. A number of agents such as chloroquine, methylamine, or arginine which are known to prevent clustering, down-modulation, and/or internalization of several hormone receptors did not affect the down-modulation of phorbol ester receptors. Furthermore, cycloheximide had no effect either on down-modulation or on the binding capacity of cells. The surface binding capacity of down-modulated cells following a 90-min incubation with unlabeled ligand was almost returned to normal within 1 h. The effect of the antidepressant drug chlorpromazine, which is known to interact with calmodulin, on (/sup 3/H)PDBu binding was also investigated. Our data indicate that the effect of chlorpromazine on (/sup 3/H)PDBu binding is probably unrelated to its calmodulin-binding activity.

  4. Degradation of phorbol 12,13-diacetate in aqueous solution by gamma irradiation

    NASA Astrophysics Data System (ADS)

    Kongmany, Santi; Furuta, Masakazu; Matsuura, Hiroto; Okuda, Shuichi; Imamura, Kiyoshi; Maeda, Yasuaki

    2014-12-01

    Phorbol esters (PEs) are highly toxic compounds that cause skin irritation, inflammation, and tumor promotion upon contact with humans or animals. These compounds are naturally present in Jatropha curcas L. To promote the use of J. curcas seed oil in bio-diesel production industries and reduce environmental concerns, it is necessary to find methods of degrading PEs. In this study, the degradation of phorbol 12,13-diacetate (PDA), as a representative PE, in aqueous solution at a concentration of 10 mg/L by 60Co-γ-irradiation was investigated. The results demonstrate that PDA was effectively degraded by this treatment and the degradation efficiency increased with the absorbed dose within the range of 0.5-3 kGy. Complete degradation of PDA was achieved at a dose of 3 kGy. In the presence of radical scavengers (i.e., methanol, tert-butanol, 2-propanol), reactive species from water radiolysis were scavenged, and significant inhibition of PDA degradation was observed at absorbed doses less than 1 kGy. In the presence of nitrous oxide, the generation of hydroxyl radicals (rad OH) was promoted during gamma irradiation and PDA degradation was drastically enhanced.

  5. Phorbol ester stimulates secretory activity while inhibiting receptor-activated aminopyrine uptake by gastric glands

    SciTech Connect

    Brown, M.R.; Chew, C.S.

    1986-03-05

    Both cyclic AMP-dependent and -independent secretagogues stimulate pepsinogen release, respiration and H/sup +/ secretory activity (AP uptake) in rabbit gastric glands. 12-O-tetradecanoylphorbol-13-acetate (T), a diacyglycerol analog, activates protein kinase C (PKC) and stimulates secretion in many systems. T stimulated respiration and pepsinogen release by glands and increased AP uptake by both glands and purified parietal cells. However, T reduced AP uptake by glands stimulated with carbachol (C) or histamine (H) with an apparent IC/sub 50/ of 1 nM. Preincubation with T for 30 min produced maximum inhibition which was not reversed by removal of T. T accelerated the decline of the transient C peak while the late steady state response to H was most inhibited. H-stimulated AP uptake was also inhibited by 50 ..mu..g/ml 1-oleoyl-2-acetyl-glycerol, a reported PKC activator, but not by the inactive phorbol, 4..cap alpha..-phorbol-12,13-didecanoate. In contrast, T potentiated AP uptake by glands stimulated with submaximal doses of dibutyryl cyclic AMP. These results suggest inhibition by T is a specific effect of PKC activators. The differing effects of T on secretion indicators may result from a dual action of T on receptor and post-receptor intracellular events.

  6. Compartmentation of protein kinase C is regulated by Ca and phorbol esters

    SciTech Connect

    Kanter, J.R.; Brunton, L.L.; Watson, M.J.; Shultz, M.; Speizer, L.A.

    1987-05-01

    In S49 lymphoma cells, both Ca and phorbol esters enhance the association of protein kinase C (PKC) with membranes. Ca alone (greater than or equal to 100 M) or the combination of Ca and a brief (10 min) exposure to phorbol 12-myristate,13-acetate (PMA) results in the association of approximately 40% of PKC with the membrane fraction. The Ca -induced translocation of PKC to membrane can be reversed by 1 mM EGTA. After the extraction of this EGTA-sensitive component of PKC, an integral compartment remains, which can be removed only by detergent (0.3% Triton X-100) solubilization of membranes. They have studied the effects of PMA on the Ca -dependent association of PKC into these two membrane compartments. If intact S49 cells are treated with PMA and fractionated with 100 M Ca , this EGTA-sensitive membrane compartment of PKC is rapidly decreased (t/sub 1/2/ = 2 minutes), and replaced by an increase in integral PKC. Ten minutes after the addition of 100 nM PMA to intact cells, the EGTA-sensitive membrane compartment of PKC activity is decreased from 1102 +/- 34 to 94 +/- 20 pmoles PO transferred/min/mg protein. During this same time interval, the integral compartment of PKC increases from 480 +/- 27 to 1293 +/- 41 pmol/min/mg protein. Their findings are consistent with a PMA-induced conversion of PKC from a loosely-associated (EGTA-sensitive) compartment to an integral membrane compartment.

  7. Enhanced cAMP accumulation by a phorbol ester in cerebral cortical cells

    SciTech Connect

    Beeler, J.F.; Davis, C.W.

    1987-05-01

    Phorbol 12-myristate-13-acetate (PMA) was found to be selective in its ability to alter cAMP accumulations in cultured rat cerebral cortical cells. Basal levels of cAMP in cultured neuronal and nonneuronal cells preincubated in the absence or presence of PMA were 14 pmol/mg protein and 16 pmol/mg protein, respectively. Adenosine increased cAMP levels in a dose-dependent manner. cAMP accumulation in response to low concentrations of adenosine was not significantly altered by pretreatment with PMA but marked potentiation of adenosine elicited accumulations was observed at 10 and 100 ..mu..M adenosine. Longer preincubation with PMA resulted in a decreased ability of PMA to enhance adenosine elicited accumulations of cAMP. PMA did not significantly alter cAMP accumulation by forskolin (FOR) and enhanced norepinephrine stimulated cAMP by only 2-fold. For similarly potentiated adenosine/sub 2/ (A/sub 2/)- receptor elicited accumulation of cAMP which could be further enhanced by PMA. These results suggest that the effects of the phorbol ester are more specific for potentiating adenosine stimulated cAMP accumulation and may occur as a result of a more efficient coupling between the A/sub 2/-receptor, N-protein and adenylate cyclase.

  8. A phorbol ester response element within the human T-cell receptor beta-chain enhancer.

    PubMed Central

    Prosser, H M; Wotton, D; Gegonne, A; Ghysdael, J; Wang, S; Speck, N A; Owen, M J

    1992-01-01

    The activity of the T-cell receptor beta-chain gene enhancer is increased by activators of the protein kinase C pathway during T-cell activation. Analysis of mutant enhancer constructs identified two elements, beta E2 and beta E3, conferring phorbol ester inducibility. Multimerized beta E2 acted in isolation as a phorbol ester-responsive element. Both beta E2 and beta E3, which contain a consensus Ets-binding site, were shown to bind directly to the product of the c-ets-1 protooncogene. Both regions also bound a second factor, core-binding factor. Mutation of the beta E2 Ets site abolished the inducibility of the beta E2 multimer. beta E2 and beta E3 Ets site mutations also profoundly affected activity and inducibility of the enhancer. In contrast, enhancer activity but not its inducibility was affected by mutation of the beta E2 core-binding factor site. Cotransfection studies showed that Ets-1 specifically repressed activity of the multimerized beta E2 element and the complete T-cell receptor beta-chain enhancer. These data show that the T-cell receptor beta-chain enhancer responds to protein kinase C-mediated activation signals via a functional domain, composed of two elements, which contains binding sites for Ets transcription factors and which is negatively regulated by Ets-1. Images PMID:1409722

  9. ACE expression in monocytes is induced by cytokines, phorbol ester and steroid

    SciTech Connect

    Lazarus, D.; Lanzillo, J.; Fanburg, B. )

    1991-03-15

    Angiotensin converting enzyme (ACE) levels are elevated in the serum and peripheral blood monocytes (PBM) of patients with granulomatous diseases. However, the role of ACE in (Mo) physiology and the regulation of the inflammatory response is not well understood. Since Mo can be stimulated to form giant cells using phorbol esters, glucocorticoids or certain inflammatory cytokines, the authors examined production of ACE protein by normal PBM, a Mo-like cell line, THP-1, and a macrophage-like cell line, U937 following stimulation with these agents. Using a sensitive ELISA assay, they found that in U937 cells, expression of ACE protein increased by 3.4 fold with dexamethasone, 3.7. fold with phorbol 12-myristate acetate (PMA), and 5.8 fold with the two agents combined. The cytokines IL-4 and GM-CSF substantially increased ACE expression, by 7.6 and 7.7 fold respectively, with maximal effect at 0.01 U/ml, while IFN-{gamma} and TNF-{alpha} had little effect. Similar results were found with PBM and THP-1 cells. The combination of dexamethasone and PMA also induced homotypic cluster formation in PBM, suggesting a correlation between cell adhesion and ACE production. The authors conclude that ACE expression in monocytes and macrophages is stimulated by low concentration of glucocorticoids and certain inflammatory cytokines. ACE may participate in the initiation and propagation of granulomatous inflammatory processes.

  10. Beta/sub 1/-adrenoceptors in rat hepatoma, desensitization by isoproterenol and phorbol-myristate-acetate

    SciTech Connect

    Garcia-Sainz, J.A.; Alcantara, R.; Hernandez-Sotomayor, S.M.T.; Mas-Oliva, J.

    1989-01-01

    The beta-adrenergic responsiveness of hepatocytes obtained from hypothyroid rats and of a transplantable hepatoma cell line (AS-30D) were studied by measuring the accumulation of cyclic AMP. The potency order for agonists in hepatocytes was: isoproterenol > epinephrine >> norepinephrine whereas in the hepatoma cells the potency order was: isoproterenol > norepinephrine /equivalent to/ epinephrine. The effect of isoproterenol was antagonized in hepatocytes by low concentrations of ICI 118551 and only partially by concentrations of atenolol as high as 100 ..mu..M. In hepatome cells the effect of isoproterenol was inhibited by both antagonists with the potency order atenolol > ICI 118551. These data indicate that in hepatocytes the effect is mediated by beta/sub 2/-adrenoceptors whereas in hepatoma cells it is through beta/sub 1/-adrenoceptors. Preincubation of hepatoma cells with isoproterenol or phorbol-myristate-acetate diminished the subsequent beta-adrenergic responsiveness of the cells. Interestingly, when both isoproterenol and phorbol-myristate-acetate were present during the preincubation the beta-adrenergic desensitization observed was bigger than that induced by any of these agents alone.

  11. An AP-2 element acts synergistically with the cyclic AMP- and Phorbol ester-inducible enhancer of the human proenkephalin gene

    SciTech Connect

    Hyman, S.E.; Comb, M.; Pearlberg, J.; Goodman, H.M.

    1989-01-01

    An enhancer with two DNA elements, one containing the sequence CGTCA, is required for cyclic AMP-and phorbol ester-inducible transcription of the human proenkephalin gene. The authors report that an AP-2 element located adjacent to the enhancer acts synergistically with it to confer maximal response to cyclic AMP and phorbol esters.

  12. [Apoptosis of human leukemic cells induced by topoisomerase I and II inhibitors].

    PubMed

    Solary, E; Dubrez, L; Eymin, B; Bertrand, R; Pommier, Y

    1996-03-01

    Comparison between five human leukemic lines (BV173, HL60, U937, K562, KCL22) suggest that the main determinant of their sensitivity to topoisomerase I (camptothecin) and II (VP-16) inhibitors is their ability to regulate cell cycle progression in response to specific DNA damage, then to die through apoptosis: the more the cells inhibit cell cycle progression, the less sensitive they are. The final pathway of apoptosis induction involves a cytoplasmic signal, active at neutral pH, needing magnesium, sensitive to various protease inhibitors and activated directly by staurosporine. Modulators of intracellular signaling (calcium chelators, calmodulin inhibitors, PKC modulators, kinase and phosphatase inhibitors) have no significant influence upon apoptosis induction. Conversely, apoptosis induction pathway is modified during monocytic differentiation of HL60 cells induced by phorbol esters. Lastly, poly(ADP-ribosyl)ation and chromatine structure should regulate apoptotic DNA fragmentation that is prevented by 3-aminobenzamide and spermine, respectively.

  13. Phorbol ester activation of chloride current in guinea-pig ventricular myocytes.

    PubMed Central

    Shuba, L. M.; Asai, T.; McDonald, T. F.

    1996-01-01

    1. Although earlier studies with phorbol esters indicate that protein kinase C (PKC) may be an important regulator of Cl- current (Icl) in cardiac cells, there is a need for additional quantitative data and investigation of conflicting findings. Our objectives were to measure the magnitude, time course, and concentration-dependence of Icl activated in guinea-pig ventricular myocytes by phorbol 12-myristate 13-acetate (PMA), evaluate its PKC dependence, and examine its modification by external and internal ions. 2. The whole-cell patch clamp technique was used to apply short depolarizing and hyperpolarizing pulses to myocytes superfused with Na(+)-, K(+)-, Ca(2+)-free solution (36 degrees C) and dialysed with Cs+ solution. Stimulation of membrane currents by PMA (threshold < or = 1nM, EC50 approximately equal to 14 nM, maximal 40% increase with > or = 100 nM) plateaued within 6-10 min. 3. PMA-activated current was time-independent, and suppressed by l mM 9-anthracenecarboxylic acid (9-AC). Its reversal potential (Erev) was sensitive to changes in the Cl- gradient, and outward rectification of the current-voltage (I-V) relationship was more pronounced with 30 mM than 140 mM Cl- dialysate. 4. The relative permeability of PMA-activated channels estimated from Erev measurements was I- > Cl- > > aspartate. Channel activation was independent of external Na+. 5. PMA failed to activate Icl in myocytes pretreated with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or dialysed with pCa 10.5 solution. Lack of response to 4 alpha-phorbol 12, 13-didecanoate (alpha PDD) was a further indication of mediation by PKC. 6. Icl induced by 2 microM forskolin was far larger than that induced by PMA, suggesting that endogenous protein kinase A is a much stronger Cl- channel activator than endogenous PKC in these myocytes. 7. The macroscopic properties of PMA-induced Icl appear to be indistinguishable from those of PKA-activated Icl. We discount stimulation of PKA by PMA as an

  14. Heterogeneity of (TH)phorbol 12,13-dibutyrate binding in primary mouse keratinocytes at different stages of maturation

    SciTech Connect

    Dunn, J.A.; Jeng, A.Y.; Yuspa, S.H.; Blumberg, P.M.

    1985-11-01

    Mouse keratinocytes respond heterogeneously to phorbol esters with distinct subpopulations stimulated to proliferate or induced to differentiate. The maturation state of the epidermal cell at the time of exposure may determine its response. The binding of phorbol esters to primary mouse keratinocytes was studied under culture conditions selecting for proliferating cells or differentiating cells. (20-TH)-12-Deoxyphorbol 13-isobutyrate ((TH)-DPB) bound to both types of cells at one class of binding sites. The dissociation constant (Kd) for (TH)DPB in the proliferative cells was 69 nM and the binding at saturation (Bmax) was 1.3 pmol/mg of protein. The corresponding values in the differentiative cells were 96 nM and 1.5 pmol/mg of protein, respectively. In contrast to the results obtained with (TH)DPB, (20-TH)phorbol 12,13-dibutyrate ((TH)PDBU) bound to both cell types in a heterogeneous fashion. The site for (TH)DPB binding seemed to correspond to the higher affinity (TH)PDBU binding site. The major difference in the cells grown in the medium containing 1.2 mM CaCl2 was an increase in the Bmax of the lower affinity binding site with the other three parameters remaining similar. The state of epidermal differentiation thus appears to modulate the amount of the lower affinity binding sites for phorbol esters.

  15. Effects of phorbol 12-myristate 13-acetate and cortisol interaction on steroid-binding capacity in the rat.

    PubMed Central

    Janssens, J P; de Loecker, W

    1979-01-01

    The specificity of the cortisol-receptor protein is examined in plasma and liver cytosol of rats. Phorbol 12-myristate 13-acetate does not inhibit the binding of cortisol to transcortin, nor does it affect the binding capacity of dexamethasone to the intracellular glucocorticoid receptor, but, by interacting with the cortisol molecule, it interferes with hormone-mediated processes in the cell. PMID:534535

  16. A receptor model for tumor promoters: rational superposition of teleocidins and phorbol esters.

    PubMed Central

    Itai, A; Kato, Y; Tomioka, N; Iitaka, Y; Endo, Y; Hasegawa, M; Shudo, K; Fujiki, H; Sakai, S

    1988-01-01

    Four 12-O-tetradecanoyl-13-O-acetylphorbol-type tumor promoters--teleocidin, phorbol ester, aplysiatoxin, and ingenol ester--are superposed in an attempt to understand their common biological activity on the assumption that they may bind to the same receptor site. A method using three-dimensional computer graphics was applied for superposing molecules and receptor mapping. The main feature of the method is that molecules are superposed in terms of spatial arrangement of physical and chemical properties but not in terms of the atomic positions as in conventional methods. This led to successful extraction of common structural features required for potent tumor-promoting activity: two hydrogen donors, a hydrogen acceptor, and a large lipophilic group. Their mutual spatial arrangements are most important for biological activity. Images PMID:3131760

  17. Lymphocyte activation by OKT3: cyclosporine sensitivity and synergism with phorbol ester.

    PubMed Central

    Kay, J E; Benzie, C R

    1986-01-01

    Lymphocyte activation by the mitogenic monoclonal antibody OKT3 is less effective than activation by mitogenic lectins such as phytohaemagglutinin (PHA) and concanavalin A (Con A). Activation by OKT3 is also very sensitive to inhibition by cyclosporine (CSA), which selectively inhibits Ca2+-activated steps in the activation process. In addition, the magnitude of the OKT3 response can be raised to that seen with mitogenic lectins by coincubation with phorbol esters (which activate protein kinase C). These observations suggest that OKT3 may deliver efficiently the Ca2+ signal involved in the initiation of lymphocyte activation, and that the comparatively weak overall response is due to a failure to generate a second signal, probably the activation of protein kinase C, as efficiently as the mitogenic lectins. PMID:3485075

  18. Screening for toxic phorbol esters in jerky pet treat products using LC–MS

    PubMed Central

    Nishshanka, Upul; Jayasuriya, Hiranthi; Chattopadhaya, Chaitali; Kijak, Philip J.; Chu, Pak-Sin; Reimschuessel, Renate; Tkachenko, Andriy; Ceric, Olgica; De Alwis, Hemakanthi G.

    2016-01-01

    Since 2007, the U.S. FDA’s Center for Veterinary Medicine (CVM) has been investigating reports of pets becoming ill after consuming jerky pet treats. Jerky used in pet treats contains glycerin, which can be made from vegetable oil or as a byproduct of biodiesel production. Because some biodiesel is produced using oil from Jatropha curcas, a plant that contains toxic compounds including phorbol esters, CVM developed a liquid chromatography-mass spectrometry (LC–MS) screening method to evaluate investigational jerky samples for the presence of these toxins. Results indicated that the samples analyzed with the new method did not contain Jatropha toxins at or above the lowest concentration tested. PMID:27038400

  19. Acute promyelocytic leukemia mutated to radioresistance suppressed monocyte lineage differentiation by phorbol 12-myristate 13-acetate.

    PubMed

    Monzen, Satoru; Takimura, Kodai; Kashiwakura, Ikuo; Hosokawa, Yoichiro

    2013-09-01

    Induction of myeloid differentiation in radioresistant HL60 cells (Res-HL60) was examined to clarify the developmental mechanism of radioresistant leukemia. Compared to wild-type HL60 cells (Wt-HL60), Res-HL60 were smaller and strongly expressed CD38. Under all-trans retinoic acid (ATRA) stimulation, Res-HL60 continued to proliferate slowly and with similar level of CD11b expression to Wt-HL60. Phorbol 12-myristate 13-acetate (PMA) strongly suppressed proliferation of Res-HL60, downregulated CD14, and affected mRNA expression. These results suggested that the specific myeloid differentiation of Res-HL60 suppressed monocyte lineage by ATRA and PMA occurred through regulation of mRNA expression. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Stimulation of progesterone production by phorbol-12-myristate 13-acetate (PMA) in cultured Leydig tumor cells

    SciTech Connect

    Chaudhary, L.R.; Raju, V.S.; Stocco, D.M.

    1987-05-01

    It has been shown that addition of hCG or c-AMP to cultured Leydig tumor cells (MA-10) increases synthesis of progesterone as the major steroid. To investigate the possible involvement of protein kinase C (PK-C) in the regulation of steroid synthesis, the authors have studied the effect of PMA, an activator of PK-C, on progesterone production in MA-10 cells. The addition of PMA (100 ng/ml) stimulated steroid production whereas 4 -phorbol-12,13-didecanoate, an inactive phorbol ester, did not have any effects. Like hCG and c-AMP, PMA-stimulated progesterone production was inhibited by cycloheximide. hCG-stimulated steroid synthesis was inhibited by PMA. The addition of PMA to MA-10 Leydig cells further increased the c-AMP-stimulated progesterone production. To determine whether c-AMP has a obligatory role in the regulation of steroid production, the effect of adenylate cyclase inhibitor, 9-(tetrahydro-2-furyl)adenine (TFA), was studied on progesterone production in the presence of hCG. At lower dose (17 ng/ml) hCG-stimulated intracellular c-AMP levels and steroid production were inhibited by TFA (300 M). At higher dose of hCG (34 ng/ml) TFA did not inhibit the hCG-stimulated intracellular c-AMP levels, however, progesterone production was inhibited. Results suggest that the action of hCG, c-AMP and PMA in controlling steroidogenesis might be regulated by similar but different mechanisms.

  1. Bovine somatotropin attenuates phorbol ester-induced prostaglandin F2alpha production in bovine endometrial cells.

    PubMed

    Badinga, L; Guzeloglu, A; Thatcher, W W

    2002-03-01

    The recent observation that bovine somatotropin (bST) treatment at a timed insemination improves pregnancy rates in lactating dairy cows raises the possibility that growth hormone (GH) may modulate the endocrine and biochemical cross talk between the conceptus and maternal uterus at the time of pregnancy establishment in cattle. The objective of this study was to characterize the cellular and molecular mechanisms by which exogenous GH affects phorbol ester-induced prostaglandin F2alpha (PGF2alpha) production in cultured bovine endometrial (BEND) cells. Serum-deprived BEND cells were incubated with or without recombinant bovine GH (rbGH), insulin-like growth factor (IGF)-I, recombinant bovine interferon (rbIFN)-tau or a combination of rbGH + rbIFN-tau for 3 h and then treated with phorbol 12,13-dibutyrate (PDBu) for an additional 6 h. Exogenous PDBu increased PGF2alpha secretion and steady-state levels of COX-2 mRNA within 3 h. Priming of BEND cells with rbGH reduced PGF2alpha response to PDBu, whereas cotreatment with IGF-I amplified PDBu induction of PGF2alpha. Preincubation of cell monolayers with rbIFN-tau suppressed PGF2alpha and COX-2 mRNA responses to PDBu. Inhibitory effects of rbGH and rbIFN-tau on PDBu-induced PGF2alpha production were additive. Results provide the first direct evidence that supplemental bST may interact with conceptus-secreted IFN-tau to modulate PGF2alpha secretion at the critical time of maternal recognition of pregnancy.

  2. CA/sup 2 +/-regulation of binding to two phorbol ester receptors

    SciTech Connect

    Jaken, S.; Kiley, S.

    1986-05-01

    The purpose of these experiments was to characterize Ca/sup 2 +/ regulation of phorbol dibutyrate (PDBu) binding to both protein kinase C (PKC) and a previously undescribed PDBu receptor. PKC from rabbit brain cytosol was prepared by anion exchange chromatography. Subsequent chromatography on hydroxylapatite revealed 2 peaks of PDBu binding. The 2nd peak eluted with PKC with approximately 190 mM phosphate; the first peak eluted with an independent kinase activity (OINK) with approximately 100 mM phosphate. Scatchard analysis of binding in the presence of excess EGTA or Ca/sup 2 +/ showed that Ca/sup 2 +/ increased PDBu receptor affinity of the PKC peak from Kd = 46 +/- 10 nM to 1.2 +/- 0.4 nM (all values are means +/- S.E., n = 4). The total amount bound in the presence of excess EGTA was slightly greater (126 +/- 11%). The effect of Ca/sup 2 +/ on OINK affinity was smaller with Kd = 4.4 +/- 0.8 nM in EGTA vs 1.8 +/- 0.6 nM in Ca/sup 2 +/. Binding capacity was increased in the presence of Ca/sup 2 +/ by 127 +/- 21%. The amount of Ca/sup 2 +/ required for these effects on both receptors was in the range of an EGTA:Ca/sup 2 +/ ratio of 1.5. These results may help to define the relationship between PDBu receptor occupancy and cellular responses to phorbol esters, including activation of PKC.

  3. Regulation of osteosarcoma EGF receptor affinity by phorbol ester and cyclic AMP

    SciTech Connect

    Borst, S.E.; Catherwood, B.D. )

    1989-04-01

    We studied the binding and degradation of 125I-labeled epidermal growth factor (EGF) by UMR-106 osteosarcoma cells and the regulation of EGF receptor affinity for EGF by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and by treatments that raise intracellular levels of cyclic AMP. Cell surface binding of (125I)EGF to A431 cells reached a plateau after a 30 minute incubation at 37 degrees C but was undetectable in UMR-106 cells. Degradation of (125I)EGF proceeded at a 50-fold higher rate in A431 cells on a per cell basis, but receptor-bound (125I)EGF was internalized and degraded at a 3.5-fold higher rate by UMR-106 cells on a per receptor basis. At 4 degrees C, (125I)EGF labeled a single class of surface binding sites in the UMR-106 cell. Treatment with TPA at 37 degrees C reduced subsequent cell surface binding of (125I)EGF at 4 degrees C a maximum of 80% with an IC50 of 1.25 ng/ml. Maximal TPA reduction of (125I)EGF binding was observed within 5-15 minutes and was due to a reduction in the affinity of cell surface receptors of (125I)EGF without a change in receptor density. Pretreatment of the cells for 4 h with 30 microM forskolin, 1 mM isobutylmethylxanthine (IBMX) plus 30 microM forskolin, or 1 mM IBMX plus 100 ng/ml parathyroid hormone (PTH) attenuated the loss in (125I)EGF binding caused by a subsequent dose of 10 ng/ml of TPA by 17% (p less than 0.0005), 39% (p less than 0.0002), and 35% (p less than 0.002), respectively.

  4. Inhibition of bone collagen synthesis by the tumor promoter phorbol 12-myristate 13-acetate.

    PubMed

    Feyen, J H; Petersen, D N; Kream, B E

    1988-04-01

    We characterized the effect of the tumor promoter phorbol 12-myristate 13-acetate (PMA) on osteoblast function and DNA synthesis in 21-day-old fetal rat calvaria maintained in organ culture. Protein synthesis was determined by measuring the incorporation of [3H]proline into collagenase-digestible (CDP) and noncollagen protein (NCP), respectively. Alkaline phosphatase activity was assessed as the release of p-nitrophenol from p-nitrophenol phosphate. DNA synthesis was determined by the incorporation of [3H]thymidine into acid-insoluble bone and total DNA content. PMA at 3-100 ng/ml (4-133 nM) caused a dose-related inhibition of collagen synthesis that was observed 6 hours after adding PMA to calvaria. PMA inhibited collagen synthesis in the osteoblast-rich central bone of calvaria but did not alter collagen synthesis in the periosteum. There was little effect of PMA on noncollagen protein synthesis in the central bone or periosteum. Phorbol esters that do not promote tumor formation in vivo did not alter collagen synthesis in calvaria. PMA stimulated prostaglandin E2 (PGE2) production in calvaria, but indomethacin did not alter the inhibitory effect of PMA on bone collagen synthesis. PMA decreased alkaline phosphatase activity measured after 48 hr of culture and increased the incorporation of [3H]thymidine into bone and DNA content after 96 hr of culture. These data indicate that PMA inhibits collagen synthesis and alkaline phosphatase activity, while stimulating DNA synthesis, suggesting that activation of protein kinase C might regulate osteoblast function and bone cell replication.

  5. Effect of phorbol esters on iron uptake in human hematopoietic cell lines

    SciTech Connect

    Testa, U.; Titeux, M.; Louache, F.; Thomopoulos, P.; Rochant, H.

    1984-11-01

    We have investigated the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on iron uptake into human hematopoietic cell lines K562, U937, and HL-60. TPA inhibited both cell growth and iron uptake by these cell lines. This effect was rapid, which is typical of phorbol esters which are biologically active, and it occurred at very low concentrations of TPA. This effect of TPA was dependent upon an inhibition of the transferrin-binding capacity as estimated on intact cells. However, experiments with transferrin binding on cell samples dissolved in 1% Triton X-100 showed that TPA-treated cells exhibited a transferrin-binding capacity similar to that of control cells. On the basis of this result, it is suggested that TPA modified a part of transferrin receptors present in the cells; as a result of this modification, these receptors became unavailable for binding transferrin, but they remained physically present in the cell. Other compounds capable of inducing the differentiation of leukemic cells, such as dimethyl sulfoxide, butyrate, retinoic acid, and 1 alpha,25-dihydroxy-vitamin D3, did not acutely inhibit iron uptake. We also investigated the effect of TPA on transferrin receptors in a cellular system in which phorbol esters stimulate cell proliferation. At 16 X 10(-9) M, TPA markedly stimulated the proliferation of T-lymphocytes. However, in spite of this marked stimulation of cell proliferation, TPA-stimulated lymphocytes exhibited a transferrin-binding capacity much inferior to cells stimulated by other mitogens, such as phytohemagglutinin.

  6. Effects of phorbol ester on cholecystokinin octapeptide-evoked exocrine pancreatic secretion in the rat.

    PubMed Central

    Francis, L P; Camello, P J; Singh, J; Salido, G M; Madrid, J A

    1990-01-01

    1. A comparative study was made of the effect of the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) on cholecystokinin octapeptide-evoked exocrine pancreatic secretion in the anaesthetized rat and isolated permeabilized pancreatic acinar cells. 2. Cholecystokinin octapeptide (CCK8; 0.10-6.40 nmol (kg body weight)-1) induced dose-dependent increases in pancreatic juice flow, total protein output and amylase release in the anaesthetized rat. 3. Administration of TPA (10(-8) mol (kg body weight)-1) in combination with CCK8 resulted in marked attenuation of the CCK8-evoked secretory response. 4. Simultaneous injection of polymyxin B (10(-8) mol (kg body weight)-1), an inhibitor of protein kinase C, with TPA and CCK8 reversed the inhibitory effect of the phorbol ester on CCK8-induced pancreatic juice flow, total protein output and amylase release. 5. In permeabilized rat pancreatic acini CCK8 (10(-13)-10(-9) M) elicited dose-dependent increases in [3H]leucine-labelled protein secretion (3H-labelled protein release). Combining TPA (10(-8) M) with CCK8 resulted in an inhibition of the CCK8-induced 3H-labelled protein release especially at lower concentrations of CCK8. At higher concentrations of CCK8, TPA was unable to inhibit the CCK8-evoked 3H-labelled protein release. Again, polymyxin B reversed the TPA-induced inhibition of CCK8-evoked 3H-labelled protein output. 6. The results indicate that protein kinase C activation may play an important physiological role in modulating the CCK8-evoked secretory response in rat pancreas in vivo and in vitro. PMID:1712842

  7. Effect of phorbol ester on the release of atrial natriuretic peptide from the hypertrophied rat myocardium.

    PubMed Central

    Kinnunen, P.; Taskinen, T.; Järvinen, M.; Ruskoaho, H.

    1991-01-01

    1. To determine the cellular mechanisms of atrial natriuretic peptide (ANP) release from ventricular cardiomyocytes, the secretory and the cardiac effects of a phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), known to stimulate protein kinase C activity in heart cells, were studied in isolated, perfused heart preparations from 2- and 21-month-old Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) rats. TPA was added to the perfusion fluid for 30 min at a concentration of 46 nM after removal of atrial tissue. Additionally, atrial and ventricular levels of immunoreactive ANP (IR-ANP) and ANP mRNA, the distribution of ANP within ventricles as well as the relative contribution of atria and ventricles in the release of ANP were studied. 2. Ventricular hypertrophy that gradually developed in hypertensive rats resulted in remarkable augmentation of ANP gene expression, as reflected by elevated levels of immunoreactive ANP and ANP mRNA. The total amount of IR-ANP in the ventricles of the SHR rats increased 41 fold and ANP mRNA levels 12.9 fold from the age of 2 to 21 months. At the age of 21 months, levels of IR-ANP and ANP mRNA in the ventricles of SHR rats were 5.4 fold and 3.7 fold higher, respectively, than in the normotensive WKY rats. Immunohistochemical studies demonstrated ANP granules within the hypertrophic ventricles of the old SHR rats, but not within normal ventricular tissue. 3. In isolated perfused heart preparations, the severely hypertrophied ventricular tissue of SHR rats after atrialectomy secreted more ANP into the perfusate than did the control hearts.(ABSTRACT TRUNCATED AT 250 WORDS) Images Figure 2 PMID:1826618

  8. Development of a sensitive in vitro assay to quantify the biological activity of pro-inflammatory phorbol esters in Jatropha oil.

    PubMed

    Pelletier, Guillaume; Padhi, Bhaja K; Hawari, Jalal; Sunahara, Geoffrey I; Poon, Raymond

    2015-06-01

    New health safety concerns may arise from the increasing production and use of Jatropha oil, a biodiesel feedstock that also contains toxic, pro-inflammatory, and co-carcinogenic phorbol esters. Based on the exceptional sensitivity of Madin-Darby canine kidney (MDCK) cells to the model phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a robust bioassay was developed to quantify the biological activity of Jatropha phorbol esters directly in oil, without sample extraction. We first verified that the characteristic response of MDCK cells to TPA was also observed following direct exposure to phorbol esters in Jatropha oil. We further confirmed that similarly to TPA, Jatropha oil's phorbol esters can activate protein kinase C (PKC). We then assessed the transcriptional response of MDCK cells to Jatropha oil exposure by measuring the expression of cyclooxygenase-2 (COX-2), a gene involved in inflammatory processes which is strongly upregulated following PKC activation. Based on the parameterization of a TPA dose-response curve, the transcriptional response of MDCK cells to Jatropha oil exposure was expressed in term of TPA toxic equivalent (TEQ), a convenient metric to report the inflammatory potential of complex mixtures. The sensitive bioassay described in this manuscript may prove useful for risk assessment, as it provides a quantitative method and a convenient metric to report the inflammatory potential of phorbol esters in Jatropha oil. This bioassay may also be adapted for the detection of bioactive phorbol esters in other matrices.

  9. Agonist-stimulated alveolar macrophages: apoptosis and phospholipid signaling.

    PubMed

    Lütjohann, J; Spiess, A N; Gercken, G

    1998-08-01

    Bovine alveolar macrophages (BAM) were labeled with [3H]-choline or [3H]-ethanolamine and exposed to quartz dust, metal oxide-coated silica particles, Escherichia coli-derived lipopolysaccharide (LPS) or tumor promotor 12-O-tetradecanoyl phorbol 13-acetate (PMA). The activation of phospholipases A2, C and D (PLA2, PLC and PLD) acting on phosphatidylcholine and phosphatidylethanolamine was determined by high performance liquid chromatography (HPLC) separation and liquid scintillation counting of water- and lipid-soluble phospholipid metabolites. Exposure of BAM to quartz dust, metal oxide-coated silica particles, and LPS led to a transient PLD activation while treatment with PMA caused a prolonged rise in PLD activity. LPS and quartz dust induced a short-term increase of PLC cleavage products. All agonists caused a transient activation of PLA2. To induce apoptosis, BAM were stimulated with C8-ceramide, calcium-ionophore 23187, or gliotoxin. Apoptosis was investigated by qualitative and quantitative methods like flow cytometry, propidium iodide/Hoechst 33258 double staining, Cell Death Detection ELISA, and electrophoretical detection of DNA fragmentation. All three agonists led to apoptosis of BAM in a time- and concentration-dependent manner. After stimulation with gliotoxin an increase in ceramide and a drastic decrease in sphingosine-1-phosphate levels were observed, suggesting an involvement of these sphingolipids in gliotoxin-mediated apoptosis.

  10. Conversion of protein kinase C from a Ca/sup 2 +/-dependent to an independent form of phorbol ester-binding protein by digestion with trypsin

    SciTech Connect

    Huang, K.P.; Huang, F.L.

    1986-08-29

    Tryptic fragments of protein kinase C containing the kinase (45 KDa) and phorbol ester-binding activity (38 KDa) were separated by Mono O column chromatography. The purified phorbol ester-binding fragment exhibits a higher affinity for phosphatidylserine than the native enzyme but comparable Kd for (/sup 3/H)phorbol 12,13-dibutyrate as the native enzyme. This proteolytic fragment binds phorbol ester equally efficient either in the presence or absence of Ca/sup 2 +/ and the addition of the kinase fragment did not restore the Ca/sup 2 +/-requirement for the binding. These results indicate that protein kinase C is composed of two functionally distinct units which can be expressed independently after limited proteolysis with trypsin.

  11. Curcumin does not alter the phorbol ester effect on cell-cell transfer of lucifer yellow CH.

    PubMed

    Pásti, G; Kertai, P; Adány, R

    1995-05-01

    Curcumin, the dietary pigment responsible for the yellow color of curry, has been reported to be a potent inhibitor of tumor promotion in mouse epidermis. Since most tumor promoters inhibit cell-cell communication, we have examined the effect of curcumin on the reduction of gap junctional intercellular communication induced by the phorbol ester phorbol-12,13-dibutyrate (PDBu) in BALB/c 3T3 cells. Treatment of cells with 50 microM curcumin slightly inhibited the dye coupling evaluated by intercellular transfer of a fluorescent dye Lucifer Yellow CH; however, lower concentrations of curcumin did not affect the level of intercellular communication. Addition of 200 nM PDBu caused a rapid reduction of dye coupling, which was not altered by either pretreatment or simultaneous curcumin addition.

  12. Chronic ethanol consumption decreases the phorbol ester binding to membranal but not cytosolic protein kinase C in rat brain.

    PubMed

    Pandey, S C; Dwivedi, Y; Piano, M R; Schwertz, D W; Davis, J M; Pandey, G N

    1993-01-01

    We examined the effect of 60 days of ethanol treatment on protein kinase C (PKC) in membrane and cytosolic fractions of the rat cerebral cortex. Membranal and cytosolic PKC were determined by binding technique using [3H]-phorbol 12,13 dibutyrate (PDBU) as radioligand and phorbol 12-myristate 13-acetate (PMA) as displacer. Chronic ethanol consumption resulted in a decrease in the maximum number of binding sites (Bmax) of [3H]-PDBU binding to membranal PKC without significant change in the apparent dissociation constant (KD) in the rat cortex. We also observed that chronic ethanol consumption had no significant effect on Bmax or KD of [3H]-PDBU binding to cytosolic PKC in the rat cerebral cortex. These results suggest that chronic ethanol consumption leads to the down-regulation of brain PKC associated with membrane but not with cytosol.

  13. Inhibition of alpha interferon but not gamma interferon signal transduction by phorbol esters is mediated by a tyrosine phosphatase.

    PubMed Central

    Petricoin, E; David, M; Igarashi, K; Benjamin, C; Ling, L; Goelz, S; Finbloom, D S; Larner, A C

    1996-01-01

    Previous studies have indicated that the expression of viral oncoproteins, cell transformation, or phorbol ester treatment of cells can inhibit alpha/beta interferon (IFN-alpha/beta)-induced gene expression. The mechanisms by which these promoters of cell growth exert their inhibitory effects vary, but in most instances they involve a disruption of the IFN-alpha/beta-induced transcription complex ISGF3 such that the DNA-binding component of this complex (the 48-kDa ISGF3gamma protein) does not bind to the interferon-stimulated response element (ISRE). In this report, we demonstrated that phorbol ester treatment of human peripheral blood monocytes dramatically inhibits activation of IFN-alpha/B-stimulated early response genes but by a mechanism which does not involve abrogation of the ISRE binding of ISGF3gamma. Phorbol ester treatment of monocytes inhibited IFN alpha-stimulated tyrosine phosphorylation of the transcription factors Stat1alpha, Stat2, and Stat3 and of the tyrosine kinase Tyk2 but had no effect on IFN-gamma activation of Stat1alpha. IFNalpha-stimulated tyrosine phosphorylation of Jak1 and the alpha subunit of the IFN-alpha receptor were unaffected by phorbol 12-myristate 13-acetate (PMA). Moreover, PMA caused the dephosphorylation of Tyk2 but not of Jak1, which was activated by IFN. Pretreatment of cells with vanadate prevented the effects of PMA with regard to PMA-induced Tyk2 dephosphorylation. These observations suggest that PMA exerts its inhibitory effects by activation of a tyrosine phosphatase which selectively regulates Tyk2 but not Jak1 activity. PMID:8657115

  14. Alterations in polyamine levels induced by phorbol diesters and other agents that promote differentiation in human promyelocytic leukemia cells

    SciTech Connect

    Huberman, E.; Weeks, C.; Herrmann, A.; Callaham, M.; Slaga, T.

    1981-02-01

    Polyamine levels were evaluated in human HL-60 promyelocytic leukemia cells after treatment with inducers of terminal differentiation. Differentiation in these cells was determined by increases in the percentage of morphologically mature cells and in lysozyme activity. Treatment of the HL-60 cells with phorbol 12-myristate-13-acetate (PMA), phorbol 12,13-didecanoate or other inducers of terminal differentiation such as dimethylsulfoxide and retinoic acid resulted in increased levels of putrescine. However, no increase in putrescine could be detected after PMA treatment of a HL-60 cell variant that exhibited a decreased susceptibility to PMA-induced terminal differentiation. Similarly, no increase in putrescine was observed with two nontumor-promoters (phorbol 12,13-diacetate and 4-O-methyl-PMA) or with anthralin, a non-phorbol tumor promoter. In addition to enhancing putrescine levels, PMA also increased the amount of spermidine and decreased the amount of spermine. The increase in putrescine and spermidine preceded the expression of the various differentiation markers. Unlike the changes observed in the polyamine levels after PMA treatment, the activities of ornithine and S-adenosylmethionine decarboxylases, which are polyamine biosynthetic enzymes, did not significantly change. ..cap alpha..-Methylornithine and ..cap alpha..-difluoromethylornithine and methylglyoxal bis(guanylhydrazone), which are inhibitors of the polyamine biosynthetic enzymes, did not affect differentiation in control or PMA-treated cells. Because of these observations, we suggest that the change in polyamine levels involve biochemical pathways other than the known biosynthetic ones. By-products of these pathways may perhaps be the controlling factors involved in the induction of terminal differentiation in the HL-60 and other cell types as well.

  15. Noradrenaline, oxymetazoline and phorbol myristate acetate induce distinct functional actions and phosphorylation patterns of α1A-adrenergic receptors.

    PubMed

    Alcántara-Hernández, Rocío; Hernández-Méndez, Aurelio; Romero-Ávila, M Teresa; Alfonzo-Méndez, Marco A; Pupo, André S; Adolfo García-Sáinz, J

    2017-09-06

    In LNCaP cells that stably express α1A-adrenergic receptors, oxymetazoline increased intracellular calcium and receptor phosphorylation, however, this agonist was a weak partial agonist, as compared to noradrenaline, for calcium signaling. Interestingly, oxymetazoline-induced receptor internalization and desensitization displayed greater effects than those induced by noradrenaline. Phorbol myristate acetate induced modest receptor internalization and minimal desensitization. α1A-Adrenergic receptor interaction with β-arrestins (colocalization/coimmunoprecipitation) was induced by noradrenaline and oxymetazoline and, to a lesser extent, by phorbol myristate acetate. Oxymetazoline was more potent and effective than noradrenaline in inducing ERK 1/2 phosphorylation. Mass spectrometric analysis of immunopurified α1A-adrenergic receptors from cells treated with adrenergic agonists and the phorbol ester clearly showed that phosphorylated residues were present both at the third intracellular loop and at the carboxyl tail. Distinct phosphorylation patterns were observed under the different conditions. The phosphorylated residues were: a) Baseline and all treatments: T233; b) noradrenaline: S220, S227, S229, S246, S250, S389; c) oxymetazoline: S227, S246, S381, T384, S389; and d) phorbol myristate acetate: S246, S250, S258, S351, S352, S401, S402, S407, T411, S413, T451. Our novel data, describing the α1A-AR phosphorylation sites, suggest that the observed different phosphorylation patterns may participate in defining adrenoceptor localization and action, under the different conditions examined. Copyright © 2017. Published by Elsevier B.V.

  16. Platelet-derived growth factor mimics phorbol diester action on epidermal growth factor receptor phosphorylation at threonine-654

    SciTech Connect

    Davis, R.J.; Czech, M.P.

    1985-06-01

    Addition of platelet-derived growth factor (PDGF) to quiescent WI-38 human fetal lung fibroblasts mimics the effect of tumor-promoting phorbol diesters to inhibit the high-affinity binding of SVI-labeled epidermal growth factor ( SVI-EGF). PDGF, like phorbol diesters, was found to increase the phosphorylation state of EGF receptors immunoprecipitated from intact fibroblasts that were labeled to equilibrium with (TSP)phosphate. Phosphoamino acid analysis of the EGF receptors indicated that both PDGF and phorbol diesters increased the level of (TSP)phosphoserine and (TSP)phosphothreonine. Phosphopeptide mapping of the EGF receptor demonstrated that PDGF increased the phosphorylation of several sites and induced the phosphorylation of a site that was not observed to be phosphorylated on EGF receptors isolated from control cells. This latter phosphorylation site on the EGF receptor was identified as threonine-654. These results are consistent with the hypothesis that increases in diacylglycerol and CaS levels caused by addition of PDGF to fibroblasts activate protein kinase C and that this kinase, at least in part, mediates the effect of PDGF on the phosphorylation of the EGF receptor. The data further suggest that protein kinase C may play an important role in the regulation of cellular metabolism and proliferation by PDGF.

  17. Effect of ozone on platelet activating factor metabolism in phorbol-differentiated HL60 cells

    SciTech Connect

    Samet, J.M.; Friedman, M. )

    1992-11-01

    The mechanisms of ozone (O3) toxicity in the lung may involve the formation of lipid inflammatory mediators. We have previously demonstrated that exposure to O3 in vitro results in increased accumulation and release of platelet activating factor (PAF) in the macrophage-like cell line HL60 differentiated with phorbol ester (dHL60). In the present study we have examined possible biochemical mechanisms responsible for the O3-induced increase in PAF levels in dHL60 cells. Specifically, we studied the effect of O3 on phospholipase A2 (PLA2), acetyltransferase, acetylhydrolase, and reacylation activities. dHL60 cells were exposed to 1.0 ppm O3 or air alone. O3 exposure was found to significantly decrease dHL60 cell acetylhydrolase activity by 36%. Additional experiments demonstrated that extracellular acetylhydrolase activity, but not intracellular acetylhydrolase activity, was inhibited by O3 exposure of dHL60 cells. O3 exposure resulted in a small (13%) but statistically significant reduction in reacylation activity in dHL60 cells. In addition, a significant (22%) contribution of PLA2 activation to the O3-induced increase in PAF levels was also found. Basal and calcium ionophore-induced acetyltransferase activity was found to be unaffected by exposure of dHL60 cells to O3. These data suggest that in vitro exposure to O3 affects both synthetic and degradative pathways of PAF metabolism in dHL60 cells.

  18. Silver nanoparticles impede phorbol myristate acetate-induced monocyte-macrophage differentiation and autophagy

    NASA Astrophysics Data System (ADS)

    Xu, Yingying; Wang, Liming; Bai, Ru; Zhang, Tianlu; Chen, Chunying

    2015-09-01

    Monocytes/macrophages are important constituents of the innate immune system. Monocyte-macrophage differentiation is not only crucial for innate immune responses, but is also related to some cardiovascular diseases. Silver nanoparticles (AgNPs) are one of the most widely used nanomaterials because of their broad-spectrum antimicrobial properties. However, the effect of AgNPs on the functions of blood monocytes is scarcely reported. Here, we report the impedance effect of AgNPs on THP-1 monocyte differentiation, and that this effect was mediated by autophagy blockade and lysosomal impairment. Firstly, AgNPs inhibit phorbol 12-myristate 13-acetate (PMA)-induced monocyte differentiation by down-regulating both expression of surface marker CD11b and response to lipopolysaccharide (LPS) stimulation. Secondly, autophagy is activated during PMA-induced THP-1 monocyte differentiation, and the autophagy inhibitor chloroquine (CQ) can inhibit this process. Thirdly, AgNPs block the degradation of the autophagy substrate p62 and induce autophagosome accumulation, which demonstrates the blockade of autophagic flux. Fourthly, lysosomal impairments including alkalization and decrease of lysosomal membrane stability were observed in AgNP-treated THP-1 cells. In conclusion, we demonstrate that the impedance of monocyte-macrophage differentiation by AgNPs is mediated by autophagy blockade and lysosomal dysfunction. Our results suggest that crosstalk exists in different biological effects induced by AgNPs.

  19. Potentiation of phorbol ester-induced coronary vasoconstriction in dogs following endothelium disruption

    SciTech Connect

    Roberts, R.B.; Ku, D.D.

    1986-03-05

    In the present study, the effect of phorbol ester, 12-0-tetradecanoylphorbol 13-acetate (TPA), activation of protein kinase C on coronary vascular reactivity was studied in isolated dog coronary arteries. Addition of TPA (10-100 nM) produced a slow, time- and dose-dependent contraction reaching a maximum at approx 2-3 hrs and was essentially irreversible upon washing. Disruption of the endothelium(EC) greatly accelerated the development as well as increase the magnitude of TPA contraction (50-100%). Prior treatment of vessels with phentolamine (1..mu..M), cyproheptadine (1..mu..H) and ibuprofen (1..mu..g/ml) did not alter the TPA contraction. Furthermore, in contrast to previously reported calcium-dependence of TPA contraction in other vessels, complete removal of extracellular calcium (Ca/sub 0/) or addition of 1..mu..M nimodipine after TPA(30nM) resulted in only 32 +/- 4% and 25 +/- 3% reversal of TPA contraction, respectively. Addition of amiloride (10..mu..M to 1mM), however, resulted in a dose-dependent reversal of TPA contraction. The results of the present study indicate that a similar activation of protein kinase C by TPA leads to potent coronary vasoconstriction, which is not completely dependent on Ca/sub 0/. More importantly, these results further support their hypothesis that EC also functions as an inhibitory barrier to prevent circulating vasoconstrictors from exerting their deleterious constrictory effects.

  20. Rapid isolation and purification of phorbol esters from Jatropha curcas by high-speed countercurrent chromatography.

    PubMed

    Hua, Wan; Hu, Huiling; Chen, Fang; Tang, Lin; Peng, Tong; Wang, Zhanguo

    2015-03-18

    In this work, a high-speed countercurrent chromatography (HSCCC) method was established for the preparation of phorbol esters (PEs) from Jatropha curcas. n-Hexane-ethyl acetate-methanol-water (1.5:1.5:1.2:0.5, v/v) was selected as the optimum two-phase solvent system to separate and purify jatropha factor C1 (JC1) with a purity of 85.2%, as determined by HPLC, and to obtain a mixture containing four or five PEs. Subsequently, continuous semipreparative HPLC was applied to further purify JC1 (99.8% as determined by HPLC). In addition, UPLC-PDA and UPLC-MS were established and successfully used to evaluate the isolated JC1 and PE-rich crude extract. The purity of JC1 was only 87.8% by UPLC-UV. A peak (a compound highly similar to JC1) was indentified as the isomer of JC1 by comparing the characteristic UV absorption and MS spectra. Meanwhile, this strategy was also applied to analyze the PE-rich crude extract from J. curcas. It is interesting that there may be more than 15 PEs according to the same quasi-molecular ion peaks, highly similar sequence-specific fragment ions, and similar UV absorption spectrum.

  1. Study of protein modifications induced by phorbol ester tumor promoters in mouse skin

    SciTech Connect

    Nelson, K.G.

    1981-08-01

    The purpose of this study was to determine if the phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) induced any specific changes in mouse epidermal proteins using the high resolution technique of two-dimensional electrophoresis. To accomplish this goal of determining the specificity and possibly the stage in promotion with which these protein changes were associated, epidermal proteins were analyzed (1) after treatment of adult mouse epidermis with several weakly promoting hyperplasiogenic agents, (2) following treatment with TPA in combination with various inhibitors of tumor promotion, (3) in basal kerotinocytes isolated from adult epidermis following treatment with TPA or several weakly promoting agents, and (4) during an initiation-promotion experiment. Evidence was found which indicated that the potent tumor promoter TPA as well as the weakly promoting hyperplasiogenic agents, mezerein, ethylphenylpropiolate (EPP), and mechanical abrasion, induced similar modifications of epidermal proteins, particularly among the keratins. These keratin modifications progressed with time following treatment resulting in a keratin pattern which resembled that of newborn epidermis.

  2. Nanomechanical measurement of adhesion and migration of leukemia cells with phorbol 12-myristate 13-acetate treatment

    PubMed Central

    Zhou, Zhuo Long; Ma, Jing; Tong, Ming-Hui; Chan, Barbara Pui; Wong, Alice Sze Tsai; Ngan, Alfonso Hing Wan

    2016-01-01

    The adhesion and traction behavior of leukemia cells in their microenvironment is directly linked to their migration, which is a prime issue affecting the release of cancer cells from the bone marrow and hence metastasis. In assessing the effectiveness of phorbol 12-myristate 13-acetate (PMA) treatment, the conventional batch-cell transwell-migration assay may not indicate the intrinsic effect of the treatment on migration, since the treatment may also affect other cellular behavior, such as proliferation or death. In this study, the pN-level adhesion and traction forces between single leukemia cells and their microenvironment were directly measured using optical tweezers and traction-force microscopy. The effects of PMA on K562 and THP1 leukemia cells were studied, and the results showed that PMA treatment significantly increased cell adhesion with extracellular matrix proteins, bone marrow stromal cells, and human fibroblasts. PMA treatment also significantly increased the traction of THP1 cells on bovine serum albumin proteins, although the effect on K562 cells was insignificant. Western blots showed an increased expression of E-cadherin and vimentin proteins after the leukemia cells were treated with PMA. The study suggests that PMA upregulates adhesion and thus suppresses the migration of both K562 and THP1 cells in their microenvironment. The ability of optical tweezers and traction-force microscopy to measure directly pN-level cell–protein or cell–cell contact was also demonstrated. PMID:27994457

  3. Insulin and phorbol ester stimulate conductive Na/sup +/ transport through a common pathway

    SciTech Connect

    Civan, M.M.; Peterson-Yantorno, K.; O'Brien, T.G.

    1988-02-01

    Insulin stimulates Na/sup +/ transport across frog skin, toad urinary bladder, and the distal renal nephron. This stimulation reflects an increase in apical membrane Na/sup +/ permeability and a stimulation of the basolateral membrane Na,K-exchange pump. Considerable indirect evidence has suggested that the apical natriferic effect of insulin is mediated by activation of protein kinase C. However, no direct information has been available documenting that insulin and protein kinase C indeed share a common pathway in stimulating Na/sup +/ transport across frog skin. In the present work, the authors have studied the interaction of insulin and phorbol 12-myristate 13-acetate (PMA), a documented activator of protein kinase C. Preincubation of skins with 1,2-dioctanoylglycerol, another activator of protein kinase C, increases baseline Na/sup +/ transport and reduces the subsequent natriferic response to PMA. Preincubation with PMA markedly reduces the subsequent natriferic action of insulin. This effect does not appear to primarily reflect PMA-induced internalization of insulin receptors. The insulin receptors are localized on the basolateral surface of frog skin, but the application of PMA to this surface is much less effective than mucosal treatment in reducing the response to insulin. The current results provide documentation that insulin and protein kinase C share a common pathway in stimulating Na/sup +/ transport across frog skin. The data are consistent with the concept that the natriferic effect of insulin on frog skin is, at least in part, mediated by activation of protein kinase C.

  4. Inhaled nitric oxide exacerbated phorbol-induced acute lung injury in rats.

    PubMed

    Lin, Hen I; Chu, Shi Jye; Hsu, Kang; Wang, David

    2004-01-01

    In this study, we determined the effect of inhaled nitric oxide (NO) on the acute lung injury induced by phorbol myristate acetate (PMA) in isolated rat lung. Typical acute lung injury was induced successfully by PMA during 60 min of observation. PMA (2 microg/kg) elicited a significant increase in microvascular permeability, (measured using the capillary filtration coefficient Kfc), lung weight gain, lung weight/body weight ratio, pulmonary arterial pressure (PAP) and protein concentration of the bronchoalveolar lavage fluid. Pretreatment with inhaled NO (30 ppm) significantly exacerbated acute lung injury. All of the parameters reflective of lung injury increased significantly except PAP (P<0.05). Coadministration of Nomega-nitro-L-arginine methyl ester (L-NAME) (5 mM) attenuated the detrimental effect of inhaled NO in PMA-induced lung injury, except for PAP. In addition, L-NAME (5 mM) significantly attenuated PMA-induced acute lung injury except for PAP. These experimental data suggest that inhaled NO significantly exacerbated acute lung injury induced by PMA in rats. L-NAME attenuated the detrimental effect of inhaled NO.

  5. Protective effect of U74500A on phorbol myristate acetate-induced acute lung injury.

    PubMed

    Chu, Shi-Jye; Chang, Deh-Ming; Wang, David; Lin, Hen-I; Lin, Shih-Hua; Hsu, Kang

    2004-08-01

    1. The present study was designed to determine whether U74500A could ameliorate acute lung injury (ALI) induced by phorbol myristate acetate (PMA) in our rat isolated lung model compared with any amelioration induced by dimethylthiourea (DMTU), superoxide dismutase (SOD) and catalase. 2. Acute lung injury was induced successfully by PMA during 60 min of observation. At 2 microg/kg, PMA elicited a significant increase in microvascular permeability (measured using the capillary filtration coefficient Kfc), lung weight gain, the lung weight/bodyweight ratio, pulmonary arterial pressure and protein concentration of the bronchoalveolar lavage fluid. 3. Pretreatment with 1.5 mg/kg U74500A significantly attenuated ALI; there was no significant increase in any parameters measured, except for pulmonary arterial pressure. The protective effect of U74500A was approximately the same as that of 600 mg/kg DMTU. However, 6000 U/kg SOD, 50,000 U/kg catalase and 6000 U/kg SOD + 50,000 U/kg catalase had no protective effect. 4. These experimental data suggest that U74500A significantly ameliorates ALI induced by PMA in rats.

  6. A pseudosubstrate of PKC inhibits the phorbol dibutyrate (PDBu) effect on permeabilized smooth muscle

    SciTech Connect

    Sullivan, T.S.; Wells, J.N. )

    1991-03-11

    Phorbol esters can induce contraction of vascular smooth muscle and potentiate calcium-induced contractions of permeabilized smooth muscle strips. The authors have used a synthetic peptide inhibitor based on residues 19-31 of PKC (PKC-I) to determine the importance of PKC in the PDBu potentiation of calcium-induced contractions in permeabilized coronary artery smooth muscle. Although peptides similar to PKC-I have been shown to also inhibit MLCK in vitro, MLCK was presumably not inhibited in our system since 30 {mu}M PKC-I alone did not alter the calcium-induced contractions. However, the potentiation of these contractions by 1 {mu}M PDBu was reduced by about 50% in the presence of 10 {mu}M PKC-I, and the potentiation was completely abolished by 30 {mu}M PKC-I. These data indicate that, in this system, PKC is not involved in calcium-induced contractions but that activation of PKC may be the mechanism by which PDBu potentiates calcium-induced contractions in permeabilized coronary artery smooth muscle.

  7. Phorbol ester-mediated desensitization of histamine Hl receptors on a cultured smooth muscle cell line

    SciTech Connect

    Mitsuhashi, M.; Payan, D.G.

    1988-01-01

    The present study was undertaken in order to examine the effect of protein kinase C (PKC) on histamine Hl receptors, (HlR) present on the smooth muscle cell line, DDT/sub 1/MF-2. (/sup 3/H)-pyrilamine binding revealed that specific (/sup 3/H)-pyrilamine binding sites were reduced be pretreatment with 12-O-tetra-decanoylphorbol-13-acetate (TPA), an activator of PKC, but not the Kd. The TPA analogue, 4..cap alpha.. phorbol 12,13-didecanoate, which does not activate PKC, failed to induce down-regulation of HlR. TPA-induced down regulation of HlR was inhibited by pretreatment with 1-(5-Isoquinilinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a PKC inhibitor, in a dose dependent manner. The H-7 analogue, H-8, which is a less potent inhibitor of PKC, but a potent inhibitor of cyclic nucleotide dependent protein kinase, had no effect on HlR. Moreover, treatment with TPA inhibited histamine-induced increases in (Ca/sup 2 +/)/sub i/ in cells loaded with the fluorescent indicator, indo-1. These data suggest that HlR in DDT/sub 1/MF-2 cells were functionally regulated by PKC.

  8. Reactive oxygen species mediate phorbol ester-stimulated cAMP response in human eosinophils.

    PubMed

    Ezeamuzie, Charles I; Taslim, Najla

    2006-08-14

    Recently, we showed that phorbol 12-myristate 13-acetate (PMA) can cause a direct, PKC-dependent, stimulation of intracellular cAMP in human eosinophils. Since PMA also stimulates the release of reactive oxygen species in these cells, we have investigated whether reactive oxygen species are involved in the cAMP response. Provided eosinophils were incubated for <20 min at 37 degrees C before stimulation, PMA potently stimulated cAMP generation that surpassed that of histamine. Pre-treatment of the cells with the NADPH oxidase inhibitors, diphenyleneiodonium (DPI) and apocynin, strongly inhibited the cAMP production induced by PMA, but not that induced by histamine. This treatment also strongly inhibited the release of superoxide anions (O(2)(-)). The cAMP response was also inhibited by pre-treatment with the specific peroxide scavenger, ebselen, but not superoxide dismutase, or NG-nitro-l-arginine methyl ester (L-NAME), thus, suggesting the possible involvement of a peroxide rather than O(2)(-) or nitric oxide (NO). These results reveal a novel involvement of intracellular reactive oxygen species in protein kinase C (PKC)-dependent stimulation of cAMP production in human eosinophils.

  9. Contraction of rat thoracic aorta strips induced by phorbol 12-myristate 13-acetate

    SciTech Connect

    Itoh, H.; Lederis, K.

    1987-02-01

    Phorbol 12-myristate 13-acetate (PMA) induced a slow and progressive increase in tension of rat thoracic aorta strips in the presence of extracellular CaS . Complete relaxation could not be obtained in CaS -free buffer containing 1 mM ethyleneglycol-bis(US -aminoethylether)-N,N'-tetraacetic acid (EGTA) and 10 X M PMA. In the absence of extracellular CaS , PMA (10 X M) induced a small but sustained contraction which was not altered by the addition of another 2 mM EGTA and 3 x 10 V M verapamil. Papaverine (10 U M) relaxed the PMA-induced contraction to the base line, but phentolamine (10 V M), cyproheptadine (10 V M), atropine (10 V M) and tetrodotoxine (10 W M) did not change the contraction. CaS -depleted muscle strips, prepared by four repeated applications of 10 X M norepinephrine in CaS -free buffer, were contracted by 10 X M PMA, but at a lower maximum tension than nontreated strips. The action of PMA on rat aorta strips in CaS -free buffer did not require the presence of the adventitial layer or endothelial cells. These results suggest that PMA may induce activation of protein kinase C and smooth muscle contraction in the absence of extracellular CaS , without an increase in myoplasmic CaS .

  10. Ethanol stimulates superoxide production and inhibits phorbol ester induced superoxide production in alveolar macrophages

    SciTech Connect

    Dorio, R.J.; Hoek, J.B.; Forman, H.J.; Rubin, E.

    1986-05-01

    Ethanol stimulates superoxide (O/sub 2//sup -/) production in rat alveolar macrophages. Increasing the ethanol concentration from 75 to 500 mM produces a linear dose response curve, generating between 10 and 30 pmol O/sub 2//sup -//min/10/sup 6/ cells. Thus, ethanol is a weak agonist of O/sub 2//sup -/ in these cells. Pretreatment with ethanol in the same concentration range results in a dose and time dependent inhibition of O/sub 2//sup -/ production by phorbol-12-myristate-13-acetate (PMA). 100 mM ethanol inhibits PMA (100 ng/ml)-induced O/sub 2//sup -/ production by 60% after 5 minutes and by 80% after 30 minutes of preincubation. At lower concentrations (10-25 mM), however, ethanol causes a synergistic stimulation of PMA-induced O/sub 2//sup -/ production. Preincubation for 15 minutes with 10 mM ethanol results in a 20% increase in PMA-induced O/sub 2//sup -/ production. Synergism between PMA and ethanol is seen at ethanol concentrations which do not result in O/sub 2//sup -/ production by ethanol alone. This synergism is abolished by a 15 minute preincubation of the cells in EGTA. Thus, ethanol acts as a weak agonist for O/sub 2//sup -/ production and interacts significantly with PMA-induced stimulation of O/sub 2//sup -/ production.

  11. ICAM-1-independent adhesion of neutrophils to phorbol ester-stimulated human airway epithelial cells.

    PubMed

    Celi, A; Cianchetti, S; Petruzzelli, S; Carnevali, S; Baliva, F; Giuntini, C

    1999-09-01

    Intercellular adhesion molecule-1 (ICAM-1) is the only inducible adhesion receptor for neutrophils identified in bronchial epithelial cells. We stimulated human airway epithelial cells with various agonists to evaluate whether ICAM-1-independent adhesion mechanisms could be elicited. Phorbol 12-myristate 13-acetate (PMA) stimulation of cells of the alveolar cell line A549 caused a rapid, significant increase in neutrophil adhesion from 11 +/- 3 to 49 +/- 7% (SE). A significant increase from 17 +/- 4 to 39 +/- 6% was also observed for neutrophil adhesion to PMA-stimulated human bronchial epithelial cells in primary culture. Although ICAM-1 expression was upregulated by PMA at late time points, it was not affected at 10 min when neutrophil adhesion was already clearly enhanced. Antibodies to ICAM-1 had no effect on neutrophil adhesion. In contrast, antibodies to the leukocyte integrin beta-chain CD18 totally inhibited the adhesion of neutrophils to PMA-stimulated epithelial cells. These results demonstrate that PMA stimulation of human airway epithelial cells causes an increase in neutrophil adhesion that is not dependent on ICAM-1 upregulation.

  12. Comparison of the hypertrophic effect of phorbol ester, norepinephrine, angiotensin II and contraction on cultured cardiomyocytes

    SciTech Connect

    Allo, S.N.; Carl, L.L.; Morgan, H.E. )

    1991-03-15

    Phorbol 12-myristate 13-acetate (PMA), norepinephrine (NE), angiotensin II (AII) and contraction stimulate cardiomyocyte growth. Differences exist in the time course and extent of protein and RNA accumulation. Cells plated at 4 {times} 10{sup 6} cells/60mm dish and arrested with 50 mM KCl demonstrated no significant growth. Treatment with PMA stimulated growth to a maximum of 17% at 48 h. In contrast, maximal stimulation of growth was 36% at 48 h and 31% at 72 h for contracting and NE treated cells, respectively. Maximal stimulation of the capacity for protein synthesis was 32% for PMA treated cells at 24 h as compared to 59% and 77% for NE treated and contracting cells respectively at 72 h. In support of a primary role for altered capacity in the regulation of protein synthesis, there was a significant correlation between RNA and protein content independent of the stimulus used. AII increased RNA content by 28% at 48h, but had no effect on growth up to 72h. Treatment with staurosporine blocked the stimulation of growth, suggestive of a role for protein kinase C (PKC). However, the inhibition of contraction-induced growth was due in part to a reduction in the rate of contraction. It was concluded that: significant differences existed in the time course of growth stimulation and RNA accumulation, depending on the stimulus; and growth inhibition by staurosporine is suggestive of an important role of PKC in hypertrophic growth induced by these stimuli.

  13. Enhanced histamine production through the induction of histidine decarboxylase expression by phorbol ester in Jurkat cells.

    PubMed

    Nagashima, Yusuke; Kako, Koichiro; Kim, Jun-Dal; Fukamizu, Akiyoshi

    2012-11-01

    Histamine (HA), a mediator of inflammation, type I allergic responses and neurotransmission, is synthesized from L-histidine, the reaction of which is catalyzed by histidine decarboxylase (HDC). HDC has been reported to be induced by various stimuli, not only in mast cells and basophils, but also in T lymphocytes and macrophages. Although its mRNA has been shown to be increased in Jurkat cells when treated with phorbol 12-myristate 13-acetate (TPA), little is known concerning the induced production of HA by HDC. The present study quantified the trace amounts of intracellular HA using ultra-high liquid chromatography in combination with the 6-aminoquinoline carbamate-derivatization technique. To test whether the cellular level of HA is elevated by the induction of HDC in Jurkat cells treated with TPA, the peak corresponding to authentic HA in the cell lysate was fractioned and its molecular weight determined by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight mass spectrometry. The results of this study show that the HA level is increased by the induction of HDC expression by TPA in Jurkat cells. Therefore, this method is useful in elucidating the physiological significance of HA production.

  14. Cyclosporin A inhibits phorbol ester-induced activation of superoxide production in resident mouse peritoneal macrophages.

    PubMed Central

    Chiara, M D; Bedoya, F; Sobrino, F

    1989-01-01

    Peritoneal resident macrophages from mice are sensitive to inhibition by cyclosporin A (CsA) of phorbol 12-myristate 13-acetate (PMA)-stimulated oxidative burst. Inhibition was assessed in terms of superoxide anion (O2.-) and H2O2 production. Key findings were as follows. (a) CsA inhibited in a dose-dependent manner the production of O2.- when cells were stimulated with PMA. CsA did not alter the respiratory burst induced by other stimuli (zymosan, concanavalin A and fMet-Leu-Phe). It was verified that CsA itself had no scavenger effect. (b) A concomitant decrease in H2O2 liberation following CsA exposure was found. This inhibition was observed both in the initial rate of synthesis and in the accumulation after 15 min of incubation. (c) NADPH oxidase activity in the crude supernatant was unaffected by the previous incubation of macrophages with CsA. CsA does not inhibit glucose transport measured as 14CO2 production. (d) The production of O2.- was strongly dependent on the glucose concentration. Sodium oleate also stimulated O2.- production in resident macrophages. These data might be correlated with the inhibitory effect of CsA upon other functions of macrophages. PMID:2557828

  15. Increased phorbol 12,13-dibutyrate (PDBu) receptor function associated with sickle red cell membrane ghosts

    SciTech Connect

    Ramachandran, M.; Nair, C.N.; Abraham, E.C.

    1987-05-01

    The biological receptor for tumor-promoting phorbol esters has been identified as the CaS /phospholipid dependent enzyme, protein kinase C. In the red cell, this enzyme is mainly cytosolic but becomes translocated to the membrane if the cellular CaS is allowed to rise. Since cellular CaS in sickle red cells is high, it was reasoned that this enzyme may become more membrane-bound. In fact, the authors noticed a four-fold increase in the binding of TH-PDBu by membrane ghosts isolated from sickle red cells compared to normal red cells (pmoles PDBu bound/mg protein; normal = 0.3 vs sickle cell = 1.4). Attempts to assay the enzyme directly as phospholipid-activated TSP incorporation into the acid-precipitable membrane proteins also indicated a two-fold increase in the radiolabelling of sickle cell membrane ghosts. Autophosphorylation of membrane proteins and analysis of the phosphorylation profile by SDS-PAGE and autoradiography revealed phosphorylation predominantly of bands 3, 4.1 and 4.9 which are known protein kinase C substrates for the red cell enzyme. The increased membrane-associated protein kinase C in sickle red cells may have a bearing on the altered membrane properties reported in this condition.

  16. Nanomechanical measurement of adhesion and migration of leukemia cells with phorbol 12-myristate 13-acetate treatment.

    PubMed

    Zhou, Zhuo Long; Ma, Jing; Tong, Ming-Hui; Chan, Barbara Pui; Wong, Alice Sze Tsai; Ngan, Alfonso Hing Wan

    The adhesion and traction behavior of leukemia cells in their microenvironment is directly linked to their migration, which is a prime issue affecting the release of cancer cells from the bone marrow and hence metastasis. In assessing the effectiveness of phorbol 12-myristate 13-acetate (PMA) treatment, the conventional batch-cell transwell-migration assay may not indicate the intrinsic effect of the treatment on migration, since the treatment may also affect other cellular behavior, such as proliferation or death. In this study, the pN-level adhesion and traction forces between single leukemia cells and their microenvironment were directly measured using optical tweezers and traction-force microscopy. The effects of PMA on K562 and THP1 leukemia cells were studied, and the results showed that PMA treatment significantly increased cell adhesion with extracellular matrix proteins, bone marrow stromal cells, and human fibroblasts. PMA treatment also significantly increased the traction of THP1 cells on bovine serum albumin proteins, although the effect on K562 cells was insignificant. Western blots showed an increased expression of E-cadherin and vimentin proteins after the leukemia cells were treated with PMA. The study suggests that PMA upregulates adhesion and thus suppresses the migration of both K562 and THP1 cells in their microenvironment. The ability of optical tweezers and traction-force microscopy to measure directly pN-level cell-protein or cell-cell contact was also demonstrated.

  17. Proteasomes play an essential role in thymocyte apoptosis.

    PubMed Central

    Grimm, L M; Goldberg, A L; Poirier, G G; Schwartz, L M; Osborne, B A

    1996-01-01

    Cell death in many different organisms requires the activation of proteolytic cascades involving cytosolic proteases. Here we describe a novel requirement in thymocyte cell death for the 20S proteasome, a highly conserved multicatalytic protease found in all eukaryotes. Specific inhibitors of proteasome function blocked cell death induced by ionizing radiation, glucocorticoids or phorbol ester. In addition to inhibiting apoptosis, these signals prevented the cleavage of poly(ADP-ribose) polymerase that accompanies many cell deaths. Since overall rates of protein degradation were not altered significantly during cell death in thymocytes, these results suggest that the proteasome may either degrade regulatory protein(s) that normally inhibit the apoptotic pathway or may proteolytically activate protein(s) than promote cell death. Images PMID:8670888

  18. Inhibition of Nef- and phorbol ester-induced CD4 degradation by macrolide antibiotics.

    PubMed Central

    Luo, T; Anderson, S J; Garcia, J V

    1996-01-01

    Human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS. The simian immunodeficiency virus (SIV) causes a similar syndrome in macaques. The product of the nef gene of SIV has been shown to be important for virus replication and disease progression in vivo. In vitro, both SIV and HIV Nef downregulate surface expression of CD4 and accelerate total CD4 turnover. The mechanism by which Nef downregulates CD4 has not been established. A current model suggests that Nef enhances cell surface CD4 endocytosis and degradation in lysosomes. However, this was recently challenged when CD4 was found to accumulate in early endosomes of cells expressing Nef. Because inhibition of Nef function might halt virus replication and disease progression, we tested two macrolide antibiotics for their ability to inhibit Nef function. Concanamycin B (ConB) and bafilomycin A1 (BFLA1) are specific inhibitors of acidification of cell endosomes and lysosomes and, unlike other inhibitors, do not affect transport. Although ConB (25 nM) and BFLA1 (100 nM) blocked phorbol myristate acetate- and Nef-induced CD4 degradation in human monocyte U937 cells, CD4 surface expression was not recovered. Instead, CD4 accumulated in lysosomes. To determine if Nef is directly responsible for CD4 degradation or if they bind to each other in a manner similar to Vpu, transcripts of human CD4 and HIV-1 nef were cotranslated in vitro. Our results indicate that under our experimental conditions, Nef does not affect CD4 stability and does not associate with CD4 in this in vitro system. Our data suggest that (i) CD4 downregulation by Nef results in degradation of CD4 in lysosomes, (ii) inhibition of CD4 degradation by macrolide antibiotics does not restore surface expression, and (iii) the inhibition of CD4 expression by Nef appears to be indirect and is likely to involve cellular factors. PMID:8627671

  19. Phorbol myristate acetate and catechol as skin cocarcinogens in SENCAR mice

    SciTech Connect

    Van Duuren, B.L.; Melchionne, S.; Seidman, I.

    1986-09-01

    The enhancement of the carcinogenicity of benzo(a) pyrene (B(a)P) and ..beta..-propiolactone (BPL) by the mouse skin cocarcinogens phorbol myristate acetate (PMA) and catechol were examined in female SENCAR mice, 30 per group. The carcinogen and cocarcinogen were applied simultaneously, three times weekly for 490-560 days. B(a)P and BPL were used at constant doses of 5 and 50 ..mu..g, respectively, in all experiments. PMA was used at three doses, 2.5, 1.0, and 0.5 ..mu..g per application, and catechol was used at one dose, 2 mg per application. Control groups included animals that received carcinogen only, cocarcinogen only, acetone only, and no treatment. The carcinogenicity of B(a)P and BPL were enhanced by the cocarcinogens, particularly in terms of tumor multiplicity. For both carcinogens, the most marked cocarcinogenic effects were observed at the lowest dose of PMA used (0.5 ..mu..g per application). This observation applied for days to first tumor, animals with tumors, tumor multiplicity, and incidence of malignant skin tumors. Catechol applied alone did not induce any tumors; with PMA alone there were significant incidences of benign and malignant tumors, e.g., at a dose of only 0.5 ..mu..g per application, 15 of 30 animals had 28 tumors, 5 of which were squamous carcinomas. In two-stage carcinogenesis experiments with 7,12-dimethylbenz(a)anthracene (DMBA) as initiator and PMA as promoter, SENCAR mice showed a greater susceptibility to tumor induction when compared to ICR/Ha mice used in earlier work. This susceptibility was most notable in terms of rate of tumor appearance and tumor multiplicity.

  20. Dopamine stimulates [3H]phorbol 12,13-dibutyrate binding in cultured striatal cells.

    PubMed

    McMillian, M K; He, X P; Hong, J S; Pennypacker, K R

    1992-04-01

    The effect of dopamine (DA) on the binding of [3H]phorbol 12,13-dibutyrate ([3H]PdBu) in cultured rat striatal cells was examined. DA maximally increased specific [3H]PdBu binding by 70 +/- 10%, an increase comparable to that observed with norepinephrine (NE). This finding suggests that DA activates protein kinase C in cultured striatal cells, because increases in [3H]PdBu binding reflect translocation of protein kinase C. Half-maximal stimulation was observed with 10(-6) M DA. The peak response was observed at 2-3 min after addition of 10(-4) M DA, but [3H]PdBu binding was still increased above basal at 30 min. DA was not acting via an adrenergic receptor. Prazosin (10(-6) M) blocked the response to NE, suggesting mediation by an alpha 1-adrenergic receptor, but had little effect on the response to DA. Conversely, the D1 receptor antagonist SCH-23390 (10(-6) M) blocked the response to DA, but only partially inhibited the response to NE. Morphine (10(-6) M) inhibited the response to DA by 46 +/- 14%, but did not affect significantly the response to NE. The DA effect on [3H]PdBu binding is apparently independent of the increase in cyclic AMP seen on D1 receptor activation. Forskolin, apomorphine, and the D1 agonist SKF-38393 all increased cyclic AMP in striatal cells, but were less effective than DA in stimulating [3H]PdBu binding. The D2 agonist quinpirole was ineffective in stimulating either cyclic AMP or [3H]PdBu binding.

  1. Generation of cytotoxic T lymphocytes (CTL) with phorbol ester and calcium ionophore

    SciTech Connect

    Tuttle, T.M.; Bear, H.D. )

    1991-03-15

    Stimulation of lymphocytes with viable tumor cells can induce cytotoxic T lymphocytes (CTL) against autologous tumor. However, sufficient numbers of tumor cells are not always available for such stimulation, and high dose interleukin-2 (IL-2) is often required for growth. Using the weakly immunogenic methylcholanthrene-induced sarcoma MCA105, the authors demonstrate here that CTLs can be expected by pharmacologic manipulation of protein kinase C (PKC) and intracellular calcium with phorbol dibutyrate (PD-Bu) and ionomycin (Io), respectively. Lymphocytes were obtained from the spleens and ipsilateral popliteal draining lymph nodes (DLN) 10 days after the footpad injection of viable MCA105 tumor cells. The cells were stimulated with autologous tumor and 20U/ml IL-2 for 7 days and then treated with PDBu and Io and expanded in culture with 20U/ml IL-2 for an additional 14 days. The lymphocytes from the spleens and DLNs demonstrated significant expansion and marked cytotoxicity against MCA105. In another regimen, lymphocytes from the DLNs of tumor-bearing mice were stimulated directly with PDBu and Io without prior in vitro exposure to autologous tumor and expanded in culture with 20U/ml IL-2. The expansion of these lymphocytes was 500 fold and the cytotoxicity against MCA 105 remained high. Lymphocytes expanded with PDBu and Io also killed MCA102, but normal spleen cells expanded in the same way had no cytotoxic activity. The authors conclude that PKC activators coupled with calcium ionophores and low-dose IL-2 can generate CTL when little or no antigen is available.

  2. Mapping of QTLs for Seed Phorbol Esters, a Toxic Chemical in Jatropha curcas (L.).

    PubMed

    Amkul, Kitiya; Laosatit, Kularb; Somta, Prakit; Shim, Sangrea; Lee, Suk-Ha; Tanya, Patcharin; Srinives, Peerasak

    2017-08-18

    Jatropha (Jatropha curcas L.) is an oil-bearing plant that has potential to be cultivated as a biodiesel crop. The seed cake after oil extraction has 40-50% protein that can be used in animal feeds. A major limitation in utilizing the cake is the presence of phorbol esters (PE), a heat-tolerant toxic chemical. To identify the quantitative trait loci (QTLs) for PE, we constructed a genetic linkage map from an F₂ population of 95 individuals from a cross "Chai Nat" × "M10" using 143 simple sequence repeat (SSR) markers. M10 is low in seed PE while Chai Nat is high. Seeds from each F₂ individual were quantified for PE content by high performance liquid chromatography. A single marker analysis revealed five markers from linkage group 3 (LG3) and nine markers from LG8 associated with seed PE. Inclusive composite interval mapping identified two QTLs, each on LG3 (qPE3.1) and LG8 (qPE8.1) responsible for the PE. qPE3.1 and qPE8.1 accounted for 14.10%, and 15.49% of total variation in seed PE, respectively. Alelle(s) from M10 at qPE3.1 increased seed PE, while at qPE8.1 decreased seed PE. qPE3.1 is a new loci for PE, while qPE8.1 is the same locus with that reported recently for PE.

  3. Bacterial lipopolysaccharides, phorbol myristate acetate, and zymosan induce the myristoylation of specific macrophage proteins.

    PubMed Central

    Aderem, A A; Keum, M M; Pure, E; Cohn, Z A

    1986-01-01

    We demonstrate stimulus-dependent incorporation of exogenously added [3H]myristic acid into specific macrophage proteins. In control unstimulated cells an 18-kDa protein is the major acylated species. In cells incubated with bacterial lipopolysaccharide (LPS), or its monoacyl glucosamine phosphate derivative, fatty acid is incorporated into proteins with molecular mass of 68 kDa and a doublet of approximately 42-45 kDa. Phorbol 12-myristate 13-acetate (PMA) or a phagocytic stimulus (zymosan) promotes the acylation of a similar array of proteins. However, PMA and zymosan also promote the myristoylation of unique proteins of 92 and 50 kDa. The fatty acid associated with each of the acylated proteins is myristic acid. The myristate is probably linked to the proteins through amide bonds, since it is not released by treatment with hydroxylamine. Palmitate and arachidonate are not incorporated into proteins in the same manner. Temporal analysis revealed that LPS-induced proteins are myristoylated by 30 min, while the 50-kDa protein myristoylated in response to PMA is labeled later. Most myristoylated proteins appear to be associated with the membrane fraction. Macrophages from C3H/HeJ mice, which do not respond to LPS, do not show any LPS-dependent protein acylation. Interestingly, zymosan and PMA induce the myristoylation of the 50-kDa protein in C3H/HeJ macrophages, but not the acylation of the 68-kDa and 42-kDa doublet species. We suggest that myristoylation of specific proteins is an intermediary in the capacity of LPS, PMA, and zymosan to alter macrophage functions such as arachidonic acid metabolism. Images PMID:3461461

  4. Calcium mobilization in permeabilized fibroblasts: effects of inositol trisphosphate, orthovanadate, mitogens phorbol ester, and guanosine triphosphate

    SciTech Connect

    Muldoon, L.L.; Jamieson, G.A. Jr.; Villereal, M.L.

    1987-01-01

    Utilizing a digitonin-permeabilized cell system, the authors have studied the release of calcium from a non-mitochondrial intracellular compartment in cultured human fibroblasts (HSWP cells). Addition of 1 mM MgATP to a monolayer of permeabilized cells in a cytosolic media buffered to 150 nM Ca with EGTA rapidly stimulates /sup 45/Ca uptake, and the subsequent addition of the putative intracellular messenger inositol trisphosphate (InsP/sub 3/) induces rapid release of 85% of the /sup 45/Ca taken up in response to ATP. Mitogenic peptides (bradykinin, vasopressin, epidermal growth factor (EGF), and insulin) and orthovanadate, which are effective in mobilizing intracellular Ca in intact cells, have little or no effect when added alone to permeabilized cells. However, in the presence of GTP these agents stimulate accumulation of inositol phosphates and release Ca from the InsP/sub 3/-sensitive pool. These data suggest that a GTP binding protein is involved in receptor mediated activation of phospholipase C, which leads to release of inositol phosphates. The GTP-dependent release of InsP/sub 3/ and the mobilization of /sup 45/Ca from the intracellular compartment are inhibited by pretreatment of cells, prior to permeabilization, with the protrein kinase C activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA). These results suggest that protein kinase C is involved in down-regulation or inhibition of phospholipase C, or the GTP binding protein responsible for relaying the mitogenic signal from the cell surface receptor to the phospholipase C activity.

  5. The phorbol ester fraction from Jatropha curcas seed oil: potential and limits for crop protection against insect pests.

    PubMed

    Ratnadass, Alain; Wink, Michael

    2012-11-30

    The physic nut shrub, Jatropha curcas (Euphorbiaceae), has been considered as a "miracle tree", particularly as a source of alternate fuel. Various extracts of the plant have been reported to have insecticidal/acaricidal or molluscicidal/anthelminthic activities on vectors of medical or veterinary interest or on agricultural or non-agricultural pests. Among those extracts, the phorbol ester fraction from seed oil has been reported as a promising candidate for use as a plant-derived protectant of a variety of crops, from a range of pre-harvest and post-harvest insect pests. However, such extracts have not been widely used, despite the "boom" in the development of the crop in the tropics during recent years, and societal concerns about overuse of systemic chemical pesticides. There are many potential explanations to such a lack of use of Jatropha insecticidal extracts. On the one hand, the application of extracts potentially harmful to human health on stored food grain, might not be relevant. The problem of decomposition of phorbol esters and other compounds toxic to crop pests in the field needing further evaluation before such extracts can be widely used, may also be a partial explanation. High variability of phorbol ester content and hence of insecticidal activity among physic nut cultivars/ecotypes may be another. Phytotoxicity to crops may be further limitation. Apparent obstacles to a wider application of such extracts are the costs and problems involved with registration and legal approval. On the other hand, more studies should be conducted on molluscicidal activity on slugs and land snails which are major pests of crops, particularly in conservation agriculture systems. Further evaluation of toxicity to natural enemies of insect pests and studies on other beneficial insects such as pollinators are also needed.

  6. The Phorbol Ester Fraction from Jatropha curcas Seed Oil: Potential and Limits for Crop Protection against Insect Pests

    PubMed Central

    Ratnadass, Alain; Wink, Michael

    2012-01-01

    The physic nut shrub, Jatropha curcas (Euphorbiaceae), has been considered as a “miracle tree”, particularly as a source of alternate fuel. Various extracts of the plant have been reported to have insecticidal/acaricidal or molluscicidal/anthelminthic activities on vectors of medical or veterinary interest or on agricultural or non-agricultural pests. Among those extracts, the phorbol ester fraction from seed oil has been reported as a promising candidate for use as a plant-derived protectant of a variety of crops, from a range of pre-harvest and post-harvest insect pests. However, such extracts have not been widely used, despite the “boom” in the development of the crop in the tropics during recent years, and societal concerns about overuse of systemic chemical pesticides. There are many potential explanations to such a lack of use of Jatropha insecticidal extracts. On the one hand, the application of extracts potentially harmful to human health on stored food grain, might not be relevant. The problem of decomposition of phorbol esters and other compounds toxic to crop pests in the field needing further evaluation before such extracts can be widely used, may also be a partial explanation. High variability of phorbol ester content and hence of insecticidal activity among physic nut cultivars/ecotypes may be another. Phytotoxicity to crops may be further limitation. Apparent obstacles to a wider application of such extracts are the costs and problems involved with registration and legal approval. On the other hand, more studies should be conducted on molluscicidal activity on slugs and land snails which are major pests of crops, particularly in conservation agriculture systems. Further evaluation of toxicity to natural enemies of insect pests and studies on other beneficial insects such as pollinators are also needed. PMID:23203190

  7. Cloning and characterization of the major promoter of the human protein kinase C beta gene. Regulation by phorbol esters.

    PubMed

    Obeid, L M; Blobe, G C; Karolak, L A; Hannun, Y A

    1992-10-15

    The expression of the beta isoenzyme for protein kinase C is regulated developmentally and in response to inducers of cell differentiation (such as phorbol esters and 1 alpha,25-dihydroxyvitamin D3). The 5' segment of the gene for protein kinase C beta was cloned from a human leukocyte genomic library in EMBL3 bacteriophage. This segment of the gene (greater than 54 kilobases in length) encompassed the coding sequence for the amino-terminal regulatory domain of the enzyme, the 5'-untranslated region, and the 5'-flanking region. Initiation of transcription was identified by S1 nuclease analysis and confirmed by RNase protection analysis at 197 base pairs 5' of the initiator ATG. Sequence analysis of the 5'-flanking region revealed it to be extremely G+C-rich (> 80%) with many features of a CpG island. Comparison of sequence with known cis-regulatory motifs disclosed a number of potential regulatory elements including an octamer binding motif at -76, Sp1-binding sites at -94 and -63, E boxes at -110, -26, and +18, an AP-1 site at -442, and an AP-2 site at -330. To demonstrate promoter activity, a 630-base pair fragment extending from -587 to +43 was subcloned in front of a promoterless luciferase gene. This fragment was able to drive the expression of luciferase in transient transfections of human hematopoietic cells. Deletion analysis demonstrated that a fragment -111 to +43 was necessary and sufficient for promoter activity; this fragment did not contain TATA or CAAT motifs. The promoter was stimulated 8-20-fold by phorbol esters accounting for the previously observed transcriptional activation of protein kinase C beta. This phorbol ester responsiveness was conferred by the basal promoter (-111 to +43) and was independent of the AP-1 site. These results define a novel mechanism of protein kinase C autoregulation at a transcriptional level.

  8. Effect of phorbol ester and pertussis toxin on the enhancement of noradrenaline release by angiotensin II in mouse atria.

    PubMed Central

    Musgrave, I. F.; Majewski, H.

    1989-01-01

    1. Mouse atria were incubated with [3H]-noradrenaline, and the outflow of radioactivity due to electrical field stimulation (5 Hz, 60 s) was used as an index of noradrenaline release. Angiotensin II (0.01 and 0.1 microM) significantly enhanced the stimulation-induced (S-I) outflow of radioactivity. 2. Phorbol 12-myristate 13-acetate (0.001, 0.03, 0.1 and 1.0 microM), a protein kinase C activating phorbol ester, significantly enhanced the S-I outflow of radioactivity. When angiotensin II (0.1 microM) was present with the concentration of phorbol 12-myristate 13-acetate that was maximally effective in increasing the S-I outflow (0.1 microM), the enhancement of S-I outflow produced by angiotensin II was maintained. 3. Polymyxin B (70 microM), an inhibitor of protein kinase C, significantly inhibited the S-I outflow. Polymyxin B also inhibited the enhancement of the S-I outflow produced by angiotensin II (0.1 microM). 4. In another series of experiments mice were injected with pertussis toxin (1.5 micrograms per mouse), 4 days before their atria were removed. The effectiveness of pertussis toxin pretreatment was determined indirectly using carbachol. Carbachol caused a concentration-dependent fall in both the rate and force of beating of isolated spontaneously beating atria from mice pretreated with vehicle. This effect of carbachol was not seen with atria from mice pretreated with pertussis toxin. 5. Pertussis toxin pretreatment did not alter the enhancement of the S-I outflow of radioactivity produced by angiotensin II (0.01 and 0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2720295

  9. Effects of phorbol esters and secretagogues on nitrobenzylthioinosine binding to nucleoside transporters and nucleoside uptake in cultured chromaffin cells.

    PubMed Central

    Delicado, E G; Sen, R P; Miras-Portugal, M T

    1991-01-01

    Secretagogues inhibited adenosine uptake in chromaffin cells without causing apparent changes in the uptake affinity. The inhibition caused by carbachol, nicotine and acetylcholine reached 50%. This inhibition was reproduced by the action of protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA; 100 nM), phorbol 12,13-dibutyrate (PDBu; 100 nM), dicaproin (10 micrograms/ml) and tricaprylin (10 micrograms/ml), with inhibitions of Vmax. of 18, 20, 37 and 47% respectively. No changes in the affinity of uptake were observed with these effectors. Down-regulation of protein kinase C by phorbol esters decreased the inhibitory effects of carbachol on adenosine uptake. Binding studies with nitrobenzylthioinosine (NBTI) showed a similar decrease in the number of transporters when chromaffin cells were treated with the same effectors used for the uptake studies. The high-affinity dissociation constants showed minor changes with respect to the control. The ratio between maximal uptake capacity and the transporter number per cell was not significantly modified by the action of secretagogues or direct effectors of protein kinase C. The number of high-affinity binding sites for NBTI was decreased in cellular homogenates by the direct action of protein kinase C activators, with staurosporine able to reverse this action. Protein kinase C from bovine brain in the presence of ATP and effectors, decreased the number of high-affinity NBTI-binding sites in purified chromaffin cell plasma membranes. These data suggest the possibility of a molecular modification at the transporter level. PMID:1953658

  10. Identification, activity, and structural studies of peptides incorporating the phorbol ester-binding domain of protein kinase C.

    PubMed Central

    Wender, P A; Irie, K; Miller, B L

    1995-01-01

    The family of homologous enzymes known as protein kinase C (PKC) has been the object of intense interest because of its crucial role in cellular signal transduction. Although considerable information about the activation of PKC has been gained through structure-activity, molecular modeling, and synthetic studies of both natural and designed activators, information about the structure of PKC itself has been limited by its large size and requirement for phospholipid cofactors. Additionally, difficulties in the purification of truncated mutants of PKC have thus far prevented their analysis by nuclear magnetic resonance (NMR) or x-ray crystallographic methods. We describe the identification, synthesis, ligand-binding analysis, cofactor requirements, and preliminary NMR evaluation of two subdomains (peptides B and C) of the regulatory domain of PKC-gamma. Peptides B and C bind [3H]phorbol 12,13-dibutyrate with good affinity (Kd = 6.4 microM and 414 nM, respectively) in the presence of phosphatidylserine. In comparison, the binding affinity of [3H]phorbol 12,13-dibutyrate for PKC was found to be 2.6 nM. Like PKC itself, these peptides also recognize other PKC activators, including dioctanoylglycerol and teleocidin B-4, and exhibit an ability to differentiate phorbol ester from its C-4 epimer. NMR studies of PKC subdomains are also described, indicating that both peptides B and C are well behaved in solution and do not exhibit any concentration-dependent changes. Finally, these studies reveal that peptide B becomes conformationally ordered only in the presence of phospholipid, suggesting that the regulatory domain of PKC itself might be organized for activation only when associated with the lipid bilayer, where its activator (diacylglycerol) is encountered. PMID:7816824

  11. Inhibition of high-affinity gamma-aminobutyric acid uptake in primary astrocyte cultures by phorbol esters and phospholipase C.

    PubMed Central

    Gomeza, J; Casado, M; Gimenez, C; Aragon, C

    1991-01-01

    The effects of phorbol 12-myristate 13-acetate (PMA), a potent activator of protein kinase C (PKC), on high-affinity Na(+)-dependent gamma-aminobutyric acid (GABA) uptake were investigated in primary cultures of neurons and glial cells from rat brain cortex. Incubation of glial cells with PMA led to concentration- and time-dependent decreases in the GABA transport in glial cells. This effect could be completely suppressed by addition of the PKC inhibitor H7. The PMA effects could be mimicked by oleoylacetylglycerol, the diacylglycerol kinase inhibitor R59022 and exogenous phospholipase C. Treatment with PMA did not affect GABA transport in neuronal cells. PMID:1902665

  12. Niacinamide mitigated the acute lung injury induced by phorbol myristate acetate in isolated rat's lungs.

    PubMed

    Lin, Chia-Chih; Hsieh, Nan-Kuang; Liou, Huey Ling; Chen, Hsing I

    2012-03-01

    Phorbol myristate acetate (PMA) is a strong neutrophil activator and has been used to induce acute lung injury (ALI). Niacinamide (NAC) is a compound of B complex. It exerts protective effects on the ALI caused by various challenges. The purpose was to evaluate the protective effects of niacinamide (NAC) on the PMA-induced ALI and associated changes. The rat's lungs were isolated in situ and perfused with constant flow. A total of 60 isolated lungs were randomized into 6 groups to received Vehicle (DMSO 100 μg/g), PMA 4 μg/g (lung weight), cotreated with NAC 0, 100, 200 and 400 mg/g (lung weight). There were 10 isolated lungs in each group. We measured the lung weight and parameters related to ALI. The pulmonary arterial pressure and capillary filtration coefficient (Kfc) were determined in isolated lungs. ATP (adenotriphosphate) and PARP [poly(adenosine diphophate-ribose) polymerase] contents in lung tissues were detected. Real-time PCR was employed to display the expression of inducible and endothelial NO synthases (iNOS and eNOS). The neutrophil-derived mediators in lung perfusate were determined. PMA caused increases in lung weight parameters. This agent produced pulmonary hypertension and increased microvascular permeability. It resulted in decrease in ATP and increase in PARP. The expression of iNOS and eNOS was upregulated following PMA. PMA increased the neutrophil-derived mediators. Pathological examination revealed lung edema and hemorrhage with inflammatory cell infiltration. Immunohistochemical stain disclosed the presence of iNOS-positive cells in macrophages and endothelial cells. These pathophysiological and biochemical changes were diminished by NAC treatment. The NAC effects were dose-dependent. Our results suggest that neutrophil activation and release of neutrophil-derived mediators by PMA cause ALI and associated changes. NO production through the iNOS-producing cells plays a detrimental role in the PMA-induced lung injury. ATP is beneficial

  13. Niacinamide mitigated the acute lung injury induced by phorbol myristate acetate in isolated rat's lungs

    PubMed Central

    2012-01-01

    Background Phorbol myristate acetate (PMA) is a strong neutrophil activator and has been used to induce acute lung injury (ALI). Niacinamide (NAC) is a compound of B complex. It exerts protective effects on the ALI caused by various challenges. The purpose was to evaluate the protective effects of niacinamide (NAC) on the PMA-induced ALI and associated changes. Methods The rat's lungs were isolated in situ and perfused with constant flow. A total of 60 isolated lungs were randomized into 6 groups to received Vehicle (DMSO 100 μg/g), PMA 4 μg/g (lung weight), cotreated with NAC 0, 100, 200 and 400 mg/g (lung weight). There were 10 isolated lungs in each group. We measured the lung weight and parameters related to ALI. The pulmonary arterial pressure and capillary filtration coefficient (Kfc) were determined in isolated lungs. ATP (adenotriphosphate) and PARP [poly(adenosine diphophate-ribose) polymerase] contents in lung tissues were detected. Real-time PCR was employed to display the expression of inducible and endothelial NO synthases (iNOS and eNOS). The neutrophil-derived mediators in lung perfusate were determined. Results PMA caused increases in lung weight parameters. This agent produced pulmonary hypertension and increased microvascular permeability. It resulted in decrease in ATP and increase in PARP. The expression of iNOS and eNOS was upregulated following PMA. PMA increased the neutrophil-derived mediators. Pathological examination revealed lung edema and hemorrhage with inflammatory cell infiltration. Immunohistochemical stain disclosed the presence of iNOS-positive cells in macrophages and endothelial cells. These pathophysiological and biochemical changes were diminished by NAC treatment. The NAC effects were dose-dependent. Conclusions Our results suggest that neutrophil activation and release of neutrophil-derived mediators by PMA cause ALI and associated changes. NO production through the iNOS-producing cells plays a detrimental role in the PMA

  14. Non-small cell lung cancer-derived soluble mediators enhance apoptosis in activated T lymphocytes through an I kappa B kinase-dependent mechanism.

    PubMed

    Batra, Raj K; Lin, Ying; Sharma, Sherven; Dohadwala, Mariam; Luo, Jie; Pold, Mehis; Dubinett, Steven M

    2003-02-01

    T lymphocyte survival is critical for the development and maintenance of an effective host antitumor immune response; however, the tumor environment can negatively impact T-cell survival. Lymphocytes exposed to tumor supernatants (TSNs) were evaluated for apoptosis after mitogen stimulation. TSN was observed to significantly enhance phorbol 12-myristate 13-acetate/ionomycin- and anti-CD3-stimulated lymphocyte apoptosis. Enhanced lymphocyte apoptosis was associated with an impairment of nuclear factor kappa B nuclear translocation and diminished I kappa B alpha degradation. In lymphocytes stimulated after exposure to TSNs, cytoplasmic I kappa B alpha persisted as a result of alterations in I kappa B kinase (IKK) activity. Accordingly, although there were no apparent differences in IKK component concentrations, lymphocytes preexposed to TSNs exhibited markedly reduced IKK activity. We conclude that non-small cell lung cancer-derived soluble factors promote apoptosis in activated lymphocytes by an IKK-dependent pathway.

  15. Mechanism of Hepatocyte Apoptosis

    PubMed Central

    Cao, Lei; Quan, Xi-Bing; Zeng, Wen-Jiao; Yang, Xiao-Ou; Wang, Ming-Jie

    2016-01-01

    Hepatocyte apoptosis plays important roles in both the removal of external microorganisms and the occurrence and development of liver diseases. Different conditions, such as virus infection, fatty liver disease, hepatic ischemia reperfusion, and drug-induced liver injury, are accompanied by hepatocyte apoptosis. This review summarizes recent research on the mechanism of hepatocyte apoptosis involving the classical extrinsic and intrinsic apoptotic pathways, endoplasmic reticulum stress, and oxidative stress-induced apoptosis. We emphasized the major causes of apoptosis according to the characteristics of different liver diseases. Several concerns regarding future research and clinical application are also raised. PMID:28058033

  16. Contribution of nitric oxide synthase to luminol-dependent chemiluminescence generated by phorbol-ester-activated Kupffer cells.

    PubMed Central

    Wang, J F; Komarov, P; Sies, H; de Groot, H

    1991-01-01

    Phorbol 12-myristate 13-acetate-induced luminol chemiluminescence in rat Kupffer cells was doubled by the addition of L-arginine and significantly (up to 70%) inhibited by NG-nitro-L-arginine and NG-monomethyl-L-arginine, competitive inhibitors of L-arginine-dependent nitric oxide (NO) formation. The release of superoxide anion (O2-) by NADPH oxidase was neither affected by L-arginine nor by the inhibitors. Only very slight luminol chemiluminescence was detectable in lipopolysaccharide-pretreated Kupffer cells, a condition in which significant amounts of NO were formed but no O2-. In a cell-free system, significant luminol chemiluminescence only occurred when both authentic NO and the O2-/H2O2- generating system xanthine/xanthine oxidase were present. The results indicate that luminol chemiluminescence in phorbol-ester-activated Kupffer cells largely depends on L-arginine metabolism by NO synthase, requiring the concurrent formation of NO and O2-/H2O2. PMID:1718262

  17. Phorbol esters enhance the Ca/sup + +/-induced translocation of C-kinase in S49 lymphoma cells

    SciTech Connect

    Speizer, L.A.; Kanter, J.R.; Watson, M.J.; Brunton, L.L.

    1986-05-01

    The authors identified specific (/sup 3/H)-phorbol 12,13-dibutyrate ((/sup 3/H)PDB) binding sites in S49 lymphoma cells. (/sup 3/H)PDB binding to intact cells reveals a single class of sites: B/sub max/ = 4.3 pmoles/mg protein (130,000 sites/cell), KD = 31 nM. The time dependence and analog specificity of (/sup 3/H)PDB binding are consistent with its identity as protein kinase C. When S49 cells are fractionated in the presence of 1 mM EGTA, (/sup 3/H)PDB binding sites are largely (91%) cytosolic, with 5% in membranes and 4% in a nuclear fraction. Fractionation in the presence of 1 mM Ca/sup + +/ alters this distribution in favor of membrane-associated receptor: 41% in the cytosol, 38% in membranes and 21% in the nuclear fraction. Cytosolic C-kinase activity parallels these changes in (/sup 3/H)PDB binding: 1 mM EGTA, 490; 1 mM Ca/sup + +/, 155 pmol PO/sub 4/ transferred/min/mg protein. Furthermore, PDB treatment of intact cells enhances the Ca/sup + +/-dependent translocation of C-kinase to membranes (cells homogenized at 1 ..mu..M Ca/sup + +/). The authors hypothesize that phorbol esters increase the Ca/sup + +/ sensitivity of C-kinase for membrane binding and thereby for enzyme activation.

  18. Stimulation of prostaglandin E/sub 2/ production by phorbol esters and epidermal growth factor in porcine thyroid cells

    SciTech Connect

    Kasai, K.; Hiraiwa, M.; Emoto, T.; Akimoto, K.; Takaoka, T.; Shimoda, S.I.

    1987-07-13

    Effects of phorbol esters and epidermal growth factor (EGF) on prostaglandin E/sub 2/ production by cultured porcine thyroid cells were examined. Both phorbol 12-myristate 13-acetate (PMA) and EGF stimulated prostaglandin E/sub 2/ production by the cells in dose related fashion. PMA stimulated prostaglandin E/sub 2/ production over fifty-fold with the dose of 10/sup -7/ M compared with control. EGF (10/sup -7/ M) also stimulated it about ten-fold. The ED/sub 50/ values of PMA and EGF were respectively around 1 x 10/sup -9/ M and 5 x 10/sup -10/ M. Thyroid stimulating hormone (TSH), however, did not stimulate prostaglandin E/sub 2/ production from 1 to 24-h incubation. The release of radioactivity from (/sup 3/H)-arachidonic acid prelabeled cells was also stimulated by PMA and EGF, but not by TSH. These results indicate that both PMA and EGF are potent stimulators of prostaglandin E/sub 2/ production, associated with the activity to stimulate arachidonic acid release in porcine thyroid cells. 36 references, 2 figures, 1 table.

  19. Tumor-promoting phorbol diesters cause the phosphorylation of epidermal growth factor receptors in normal human fibroblasts at threonine-654.

    PubMed Central

    Davis, R J; Czech, M P

    1985-01-01

    The effect of tumor-promoting phorbol diesters to potentiate the action of epidermal growth factor (EGF) on cell proliferation is associated with phosphorylation of EGF receptors, acute depression of EGF binding, and inhibition of EGF receptor tyrosine kinase activity. In the present studies, normal human fibroblasts and A431 carcinoma cells were labeled with [32P]phosphate and treated with and without 10 nM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). The EGF receptors then were isolated by immunoprecipitation and digested with trypsin. Analysis of the labeled receptor phosphopeptides by reversed-phase HPLC revealed that PMA induces the phosphorylation of a unique phosphopeptide containing [32P]phosphothreonine. Comparison of several chemical and physical properties of the 32P-labeled phosphopeptide with the primary structure of the EGF receptor suggested the identify Lys-Arg-Thr(P)-Leu-Arg. This was confirmed by direct demonstration that a synthetic peptide of this structure comigrates during HPLC and electrophoresis with the 32P-labeled phosphopeptide isolated from the EGF receptors of normal human fibroblasts. The phosphorylated site on the peptide corresponds to threonine-654 of the EGF receptor, which is located on the cytoplasmic side of the plasma membrane nine residues distant from the transmembrane domain. These data indicate that phosphorylation of the EGF receptor in human fibroblasts and A431 cells at threonine-654 may regulate the EGF receptor tyrosine kinase activity and the binding of EGF. Images PMID:2984676

  20. Cardiomyocytes from phorbol myristate acetate-activated mesenchymal stem cells restore electromechanical function in infarcted rat hearts

    PubMed Central

    Song, Heesang; Hwang, Hye Jin; Chang, Woochul; Song, Byeong-Wook; Cha, Min-Ji; Lim, Soyeon; Choi, Eun Ju; Ham, Onju; Lee, Chang Youn; Park, Jun-Hee; Lee, Se-Yeon; Choi, Eunmi; Lee, Chungkeun; Lee, Myoungho; Lee, Moon-Hyoung; Kim, Sung-Hou; Jang, Yangsoo; Hwang, Ki-Chul

    2011-01-01

    Despite the safety and feasibility of mesenchymal stem cell (MSC) therapy, an optimal cell type has not yet emerged in terms of electromechanical integration in infarcted myocardium. We found that poor to moderate survival benefits of MSC-implanted rats were caused by incomplete electromechanical integration induced by tissue heterogeneity between myocytes and engrafted MSCs in the infarcted myocardium. Here, we report the development of cardiogenic cells from rat MSCs activated by phorbol myristate acetate, a PKC activator, that exhibited high expressions of cardiac-specific markers and Ca2+ homeostasis-related proteins and showed adrenergic receptor signaling by norepinephrine. Histological analysis showed high connexin 43 coupling, few inflammatory cells, and low fibrotic markers in myocardium implanted with these phorbol myristate acetate-activated MSCs. Infarct hearts implanted with these cells exhibited restoration of conduction velocity through decreased tissue heterogeneity and improved myocardial contractility. These findings have major implications for the development of better cell types for electromechanical integration of cell-based treatment for infarcted myocardium. PMID:21173226

  1. Molecular Basis for Failure of “Atypical” C1 Domain of Vav1 to Bind Diacylglycerol/Phorbol Ester*

    PubMed Central

    Geczy, Tamas; Peach, Megan L.; El Kazzouli, Saïd; Sigano, Dina M.; Kang, Ji-Hye; Valle, Christopher J.; Selezneva, Julia; Woo, Wonhee; Kedei, Noemi; Lewin, Nancy E.; Garfield, Susan H.; Lim, Langston; Mannan, Poonam; Marquez, Victor E.; Blumberg, Peter M.

    2012-01-01

    C1 domains, the recognition motif of the second messenger diacylglycerol and of the phorbol esters, are classified as typical (ligand-responsive) or atypical (not ligand-responsive). The C1 domain of Vav1, a guanine nucleotide exchange factor, plays a critical role in regulation of Vav activity through stabilization of the Dbl homology domain, which is responsible for exchange activity of Vav. Although the C1 domain of Vav1 is classified as atypical, it retains a binding pocket geometry homologous to that of the typical C1 domains of PKCs. This study clarifies the basis for its failure to bind ligands. Substituting Vav1-specific residues into the C1b domain of PKCδ, we identified five crucial residues (Glu9, Glu10, Thr11, Thr24, and Tyr26) along the rim of the binding cleft that weaken binding potency in a cumulative fashion. Reciprocally, replacing these incompatible residues in the Vav1 C1 domain with the corresponding residues from PKCδ C1b (δC1b) conferred high potency for phorbol ester binding. Computer modeling predicts that these unique residues in Vav1 increase the hydrophilicity of the rim of the binding pocket, impairing membrane association and thereby preventing formation of the ternary C1-ligand-membrane binding complex. The initial design of diacylglycerol-lactones to exploit these Vav1 unique residues showed enhanced selectivity for C1 domains incorporating these residues, suggesting a strategy for the development of ligands targeting Vav1. PMID:22351766

  2. Phorbol esters induce intracellular accumulation of the anti-apoptotic protein PED/PEA-15 by preventing ubiquitinylation and proteasomal degradation.

    PubMed

    Perfetti, Anna; Oriente, Francesco; Iovino, Salvatore; Alberobello, A Teresa; Barbagallo, Alessia P M; Esposito, Iolanda; Fiory, Francesca; Teperino, Raffaele; Ungaro, Paola; Miele, Claudia; Formisano, Pietro; Beguinot, Francesco

    2007-03-23

    Phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes (PED/PEA)-15 is an anti-apoptotic protein whose expression is increased in several cancer cells and following experimental skin carcinogenesis. Exposure of untransfected C5N keratinocytes and transfected HEK293 cells to phorbol esters (12-O-tetradecanoylphorbol-13-acetate (TPA)) increased PED/PEA-15 cellular content and enhanced its phosphorylation at serine 116 in a time-dependent fashion. Ser-116 --> Gly (PED(S116G)) but not Ser-104 --> Gly (PED(S104G)) substitution almost completely abolished TPA regulation of PED/PEA-15 expression. TPA effect was also prevented by antisense inhibition of protein kinase C (PKC)-zeta and by the expression of a dominant-negative PKC-zeta mutant cDNA in HEK293 cells. Similar to long term TPA treatment, overexpression of wild-type PKC-zeta increased cellular content and phosphorylation of WT-PED/PEA-15 and PED(S104G) but not of PED(S116G). These events were accompanied by the activation of Ca2+-calmodulin kinase (CaMK) II and prevented by the CaMK blocker, KN-93. At variance, the proteasome inhibitor lactacystin mimicked TPA action on PED/PEA-15 intracellular accumulation and reverted the effects of PKC-zeta and CaMK inhibition. Moreover, we show that PED/PEA-15 bound ubiquitin in intact cells. PED/PEA-15 ubiquitinylation was reduced by TPA and PKC-zeta overexpression and increased by KN-93 and PKC-zeta block. Furthermore, in HEK293 cells expressing PED(S116G), TPA failed to prevent ubiquitin-dependent degradation of the protein. Accordingly, in the same cells, TPA-mediated protection from apoptosis was blunted. Taken together, our results indicate that TPA increases PED/PEA-15 expression at the post-translational level by inducing phosphorylation at serine 116 and preventing ubiquitinylation and proteosomal degradation.

  3. Control of macrophage cell differentiation in human promyelocytic HL-60 leukemia cells by 1,25-dihydroxyvitamin D/sub 3/ and phorbol-12-myristate-13-acetate

    SciTech Connect

    Murao, S.; Gemmell, M.A.; Callaham, M.F.; Anderson, N.L.; Huberman, E.

    1983-10-01

    Human promyelocytic leukemia cells (HL-60) were induced to differentiate into macrophage-like cells in a dose (3 x 10/sup -10/ to 10/sup -7/ M) and time (1 to 6 days)-dependent manner by 1,25-dihydroxyvitamin D/sub 3/ and the tumor promoter, phorbol-12-myristate-13-acetate. Differentiation was determined by an increase in the percentage of morphologically mature cells, in lysozyme and nonspecific esterase activities, and in reactivity with the murine OKM1 monoclonal antibody. Two HL-60 cell variants, designated as R-80 and B-II, were also examined. R-80 cells, which are resistant to induction of cell differentiation by phorbol-12-myristate-13-acetate, also exhibited resistance, although to a lesser degree, to induction of cell differentiation by 1,25-dihydroxyvitamin D/sub 3/. Te resistance to the action of the two compounds is presumably not due to similar binding sites for the two inducers, since 1,25-dihydroxyvitamin D/sub 3/ was unable to compete for the phorbol diester binding sites as measured by (/sup 3/H)phorbol-12,13-dibutyrate binding. B-II cells were resistant to induction of cell differentiation by 1,25-dihydroxyvitamin D/sub 3/, phorbol-12-myristate-13-acetate, retinoic acid, and dimethyl sulfoxide. Two-dimensional electrophoretic analysis of HL-60 cell protein patterns indicated that treatment of the HL-60 cells with 1,25-dihydroxyvitamin D/sub 3/, phorbol-12-myristate-13-acetate, retinoic acid, and dimethyl sulfoxide caused the cells to express various monocyte-macrophage and granulocyte marker proteins. These results indicate that 1,25-dihydroxyvitamin D/sub 3/ induces in the HL-60 cells a phenotype that resembles, but is not identical to, that of peripheral monocytes-macrophages. 40 references, 3 figures, 1 table.

  4. Effects of phorbol ester on contraction, intracellular pH and intracellular Ca2+ in isolated mammalian ventricular myocytes.

    PubMed Central

    MacLeod, K T; Harding, S E

    1991-01-01

    1. We have investigated the actions of certain phorbol esters on the intracellular pH, intracellular Ca2+ and contractility of isolated rat and guinea-pig cardiac myocytes. Intracellular pH was measured using 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and intracellular Ca2+ was measured using Fura-2. 2. Application of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (also called phorbol 12-myristate 13-acetate) (TPA) (which activates protein kinase C) to rat cardiac myocytes significantly increased cell shortening by 116 +/- 34% (n = 8) (p less than 0.02). The rate of change of cell length during contraction (i.e. +dL/dt) increased from 67.2 +/- 8.7 microns/s to 127.7 +/- 14.1 microns/s (n = 7). The rate of change of cell length during relaxation (-dL/dt) increased from 55.8 +/- 7.4 microns/s to 118.9 +/- 12.1 microns/s (n = 7). Time to peak shortening was unchanged. 3. Application of 4 alpha-phorbol 12,13-didecanoate, which does not activate protein kinase C, did not affect rat myocyte contractility. An insignificant decrease in contractility (by 7.5 +/- 7.5%) was observed (n = 5). The positive inotropic effect of TPA may therefore be evoked through an activation of protein kinase C. 4. In rat myocytes we have measured the changes of pHi and contractility (cell shortening) during an alkalosis and acidosis induced by exposure to and subsequent removal of NH4Cl both in the presence and absence of TPA. Recovery times from an acid load were significantly (p less than 0.05) enhanced by 15.1 +/- 6.9% (n = 13) in the presence of TPA. Recovery times of cell shortening were also more rapid (p less than 0.05) by an average of 59.1 +/- 10.6% (n = 5) in the presence of TPA. Recovery times were unchanged in the presence of 4-phorbol 12,13-didecanoate (which does not activate protein kinase C). 5. Since pHi recovery of an isolated myocyte from an acid load is partially inhibited by the presence of 1 mM-amiloride and inhibited by removing extracellular Na

  5. l-Cystathionine Inhibits the Mitochondria-Mediated Macrophage Apoptosis Induced by Oxidized Low Density Lipoprotein

    PubMed Central

    Zhu, Mingzhu; Du, Junbao; Chen, Siyao; Liu, Angie Dong; Holmberg, Lukas; Chen, Yonghong; Zhang, Chunyu; Tang, Chaoshu; Jin, Hongfang

    2014-01-01

    This study was designed to investigate the regulatory role of l-cystathionine in human macrophage apoptosis induced by oxidized low density lipoprotein (ox-LDL) and its possible mechanisms. THP-1 cells were induced with phorbol 12-myristate 13-acetate (PMA) and differentiated into macrophages. Macrophages were incubated with ox-LDL after pretreatment with l-cystathionine. Superoxide anion, apoptosis, mitochondrial membrane potential, and mitochondrial permeability transition pore (MPTP) opening were examined. Caspase-9 activities and expression of cleaved caspase-3 were measured. The results showed that compared with control group, ox-LDL treatment significantly promoted superoxide anion generation, release of cytochrome c (cytc) from mitochondrion into cytoplasm, caspase-9 activities, cleavage of caspase-3, and cell apoptosis, in addition to reduced mitochondrial membrane potential as well as increased MPTP opening. However, 0.3 and 1.0 mmol/L l-cystathionine significantly reduced superoxide anion generation, increased mitochondrial membrane potential, and markedly decreased MPTP opening in ox-LDL + l-cystathionine macrophages. Moreover, compared to ox-LDL treated-cells, release of cytc from mitochondrion into cytoplasm, caspase-9 activities, cleavage of caspase-3, and apoptosis levels in l-cystathionine pretreated cells were profoundly attenuated. Taken together, our results suggested that l-cystathionine could antagonize mitochondria-mediated human macrophage apoptosis induced by ox-LDL via inhibition of cytc release and caspase activation. PMID:25514411

  6. The uremic toxin methylguanidine increases the oxidative metabolism and accelerates the apoptosis of canine neutrophils.

    PubMed

    Bosco, A M; Almeida, B F M; Pereira, P P; Dos Santos, D B; Neto, Á J S; Ferreira, W L; Ciarlini, P C

    2017-03-01

    We investigated the hypothesis that the increased concentration of plasma methylguanidine (MG) increases oxidative metabolism and accelerates apoptosis of neutrophils from dogs with chronic kidney disease (CKD). To achieve this, the levels of MG were quantified in healthy (n=16) and uremic dogs with CKD stage 4 of according to the guidelines of the International Renal Interest Society (IRIS, 2015) (n=16) using high performance liquid chromatography (HPLC). To evaluate the isolated effect of MG on neutrophil oxidative metabolism and apoptosis, neutrophils isolated from 12 healthy dogs were incubated with the highest concentration of plasma MG (0.005g/L) observed in dogs with CKD. Neutrophil oxidative metabolism was assessed by flow cytometry, using the probes hydroethidine for superoxide production and 2',7'-dichlorofluorescein diacetate for hydrogen peroxide production, with or without phorbol myristate acetate (PMA) stimulus. Neutrophil apoptosis and viability were also evaluated in flow cytometer using the Annexin V-PE system, with or without the apoptosis-inducing effect of camptothecin. Uremic dogs presented higher concentrations of MG (p<0.0001), increased oxidative stress and primed neutrophils with higher apoptosis rate. The neutrophil abnormalities observed in vivo were also reproduced in vitro, using cells isolated from healthy dogs and incubated with MG. We obtained strong evidence that in dogs with CKD, increased MG levels contributed to oxidative stress and potentially compromised the non-specific immune response by altering the oxidative metabolism and viability of canine neutrophils.

  7. Active Oxygen Metabolites and Thromboxane in Phorbol Myristate Acetate Toxicity to the Isolated, Perfused Rat Lung.

    NASA Astrophysics Data System (ADS)

    Carpenter, Laurie Jean

    When administered intravenously or intratracheally to rats, rabbits and sheep, phorbol myristate acetate (PMA) produces changes in lung morphology and function are similar to those seen in humans with the adult respiratory distress syndrome (ARDS). Therefore, it is thought that information about the mechanism of ARDS development can be gained from experiments using PMA-treated animals. Currently, the mechanisms by which PMA causes pneumotoxicity are unknown. Results from other studies in rabbits and in isolated, perfused rabbit lungs suggest that PMA-induced lung injury is mediated by active oxygen species from neutrophils (PMN), whereas studies in sheep and rats suggest that PMN are not required for the toxic response. The role of PMN, active oxygen metabolites and thromboxane (TxA_2) in PMA-induced injury to isolated, perfused rat lungs (IPLs) was examined in this thesis. To determine whether PMN were required for PMA to produce toxicity to the IPL, lungs were perfused for 30 min with buffer containing various concentrations of PMA (in the presence or absence of PMN). When concentrations >=q57 ng/ml were added to medium devoid of added PMN, perfusion pressure and lung weight increased. When a concentration of PMA (14-28 ng/ml) that did not by itself cause lungs to accumulate fluid was added to the perfusion medium containing PMN (1 x 10 ^8), perfusion pressure increased, and lungs accumulated fluid. These results indicate that high concentrations of PMA produce lung injury which is independent of PMN, whereas injury induced by lower concentrations is PMN-dependent. To examine whether active oxygen species were involved in mediating lung injury induced by PMA and PMN, lungs were coperfused with the oxygen radical scavengers SOD and/or catalase. Coperfusion with either or both of these enzymes totally protected lungs against injury caused by PMN and PMA. These results suggest that active oxygen species (the hydroxyl radical in particular), mediate lung injury in

  8. [Apoptosis in allergic disease].

    PubMed

    Rojas Ramos, E; Martínez Jiménez, N E; Martínez Aguilar, N E; Garfias Becerra, J

    2000-01-01

    Apoptosis (cell programmed death) it is a mechanism that implicate a physiological suicide, to keep the cellular homeostasis in big amount of tissues. Fas (APO-1; CD95) system is one of the most important cellular responsible via to induce apoptosis on different tissues. Eosinophillia on peripheral blood and tissues are the main characteristics on allergic like asthma. Eosinophil apoptosis is upper regulated in those diseases by IL-5 y GM-CSF. Corticoids, teophyllin and some macrolids have been used like apoptosis inductors on eosinophills, these could be a novel mechanism to promote a better solution on inflammatory allergic diseases.

  9. Effects of Phorbol Esters and Lipopolysaccharide on Endothelial Cell Microfilaments: Laser Scanning Confocal Microscopy and Quantitative Morphometry of Dose Dependent Changes

    DTIC Science & Technology

    1988-11-29

    the same ECmo (1-5 nM) for both biochemical and morphological processes. -PDB was less potent in inducing the disruption of microfilament structure...but the agent was less potent than PMA, with an ECmo of about 80 nM. The agent 4a-phorbol, which is not a tumor-promoter, had no effect on the

  10. Isolation of Phorbol Esters from Euphorbia grandicornis and Evaluation of Protein Kinase C- and Human Platelet-Activating Effects of Euphorbiaceae Diterpenes.

    PubMed

    Tsai, Ju-Ying; Rédei, Dóra; Forgo, Peter; Li, Yu; Vasas, Andrea; Hohmann, Judit; Wu, Chin-Chung

    2016-10-28

    Human platelets contain conventional (α and β) and novel isoforms of PKC (δ and θ), and PKC activation can result in platelet aggregation and secretion reaction that are important for thrombus formation. Several tumor-promoting Euphorbiaceae diterpenes are known to act as direct activators of PKC, but many types of such diterpenes have not been studied as platelet stimulators. In the present study, two new and five known phorbol esters were isolated from Euphorbia grandicornis. Two of the isolated phorbol esters together with compounds representing ingenane, jatrophane, and myrsinane structural types were studied on PKC activation and platelet stimulation. The investigated phorbol esters and ingenane esters induced blood platelet aggregation and ATP secretion. PKC activation was demonstrated by inducing membrane translocation of PKCs, phosphorylation of PKC substrates, and activation of PKC signaling pathways. The PKC-activating effect of the compounds correlated well with their efficacy to cause platelet stimulation. Moreover, by using an isoform-specific PKC inhibitor, it was found that besides conventional PKCs novel PKCs also play a positive role in platelet activation caused by phorbol/ingenane esters, especially in regulating platelet aggregation. The present results suggest that platelets afford a useful model for studying PKC activators of natural origin or their chemical derivatives.

  11. The effect of lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) on whole blood oxidative response as assessed by luminol-amplified chemiluminescence in dairy cows

    USDA-ARS?s Scientific Manuscript database

    The differences between lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) on whole blood oxidative response using luminol-amplified chemiluminescence (CL) are currently unknown in cattle. Luminol-dependent CL measures the amount of reactive oxygen species released from leukocytes a...

  12. Stimulation of Ca2+ efflux from fura-2-loaded platelets activated by thrombin or phorbol myristate acetate.

    PubMed

    Pollock, W K; Sage, S O; Rink, T J

    1987-01-05

    We investigated the restoration of [Ca2+]i in fura-2-loaded human platelets following discharge of internal Ca2+ stores in the absence of external Ca2+. After stimulation by thrombin [Ca2+]i returned from a peak level of 0.6 microM to resting levels within 4 min. When ionomycin discharged the internal stores the recovery was slower with [Ca2+]i still elevated at around 0.5 microM after 5 min. Thrombin added shortly after ionomycin could accelerate the recovery of [Ca2+]i and restore resting levels within 5 min, an effect that was mimicked by phorbol-12-myristate-13-acetate (PMA). Since the continued presence of ionomycin precluded reuptake into the internal stores we conclude that thrombin and PMA stimulate Ca2+ efflux, perhaps via protein kinase C actions on a plasma membrane Ca2+ pump.

  13. Inhibitory action of sphingosine, sphinganine and dexamethasone on glucose uptake: Studies with hydrogen peroxide and phorbol ester

    SciTech Connect

    Murray, D.K.; Hill, M.E.; Nelson, D.H. )

    1990-01-01

    The mechanism of the inhibitory action of glucocorticoids on glucose uptake is incompletely understood. Treatment with corticosteriods of cells in which glucose uptake is stimulated at insulin postbinding and postreceptor sites may clarify the site of the steroid inhibitory action. Hydrogen peroxide, which has been shown to stimulate the insulin receptor tyrosine kinase, and phorbol myristate acetate (PMA) which stimulates protein kinase C were, therefore, used as stimulators of glucose transport in this study. These studies demonstrate that dexamethasone and the sphingoid bases, sphinganine and sphingosine, inhibit glucose uptake that has been stimulated at either the receptor kinase or protein kinase C level in both 3T3-L1 and 3T3-C2 cells. These data confirm glucocorticoid inhibitory action at a post binding level and support the suggestion that some corticosteriod inhibitory effects may be mediated by an action on sphingolipid metabolism.

  14. Detoxification of toxic phorbol esters from Malaysian Jatropha curcas Linn. kernel by Trichoderma spp. and endophytic fungi.

    PubMed

    Najjar, Azhar; Abdullah, Norhani; Saad, Wan Zuhainis; Ahmad, Syahida; Oskoueian, Ehsan; Abas, Faridah; Gherbawy, Youssuf

    2014-02-05

    The presence of phorbol esters (PEs) with toxic properties limits the use of Jatropha curcas kernel in the animal feed industry. Therefore, suitable methods to detoxify PEs have to be developed to render the material safe as a feed ingredient. In the present study, the biological treatment of the extracted PEs-rich fraction with non-pathogenic fungi (Trichoderma harzianum JQ350879.1, T. harzianum JQ517493.1, Paecilomyces sinensis JQ350881.1, Cladosporium cladosporioides JQ517491.1, Fusarium chlamydosporum JQ350882.1, F. chlamydosporum JQ517492.1 and F. chlamydosporum JQ350880.1) was conducted by fermentation in broth cultures. The PEs were detected by liquid chromatography-diode array detector-electrospray ionization mass spectrometry (LC-DAD-ESIMS) and quantitatively monitored by HPLC using phorbol-12-myristate 13-acetate as the standard. At day 30 of incubation, two T. harzianum spp., P. sinensis and C. cladosporioides significantly (p < 0.05) removed PEs with percentage losses of 96.9%-99.7%, while F. chlamydosporum strains showed percentage losses of 88.9%-92.2%. All fungal strains could utilize the PEs-rich fraction for growth. In the cytotoxicity assay, cell viabilities of Chang liver and NIH 3T3 fibroblast cell lines were less than 1% with the untreated PEs-rich fraction, but 84.3%-96.5% with the fungal treated PEs-rich fraction. There was no inhibition on cell viability for normal fungal growth supernatants. To conclude, Trichoderma spp., Paecilomyces sp. and Cladosporium sp. are potential microbes for the detoxification of PEs.

  15. Detoxification of Toxic Phorbol Esters from Malaysian Jatropha curcas Linn. Kernel by Trichoderma spp. and Endophytic Fungi

    PubMed Central

    Najjar, Azhar; Abdullah, Norhani; Saad, Wan Zuhainis; Ahmad, Syahida; Oskoueian, Ehsan; Abas, Faridah; Gherbawy, Youssuf

    2014-01-01

    The presence of phorbol esters (PEs) with toxic properties limits the use of Jatropha curcas kernel in the animal feed industry. Therefore, suitable methods to detoxify PEs have to be developed to render the material safe as a feed ingredient. In the present study, the biological treatment of the extracted PEs-rich fraction with non-pathogenic fungi (Trichoderma harzianum JQ350879.1, T. harzianum JQ517493.1, Paecilomyces sinensis JQ350881.1, Cladosporium cladosporioides JQ517491.1, Fusarium chlamydosporum JQ350882.1, F. chlamydosporum JQ517492.1 and F. chlamydosporum JQ350880.1) was conducted by fermentation in broth cultures. The PEs were detected by liquid chromatography-diode array detector-electrospray ionization mass spectrometry (LC-DAD-ESIMS) and quantitatively monitored by HPLC using phorbol-12-myristate 13-acetate as the standard. At day 30 of incubation, two T. harzianum spp., P. sinensis and C. cladosporioides significantly (p < 0.05) removed PEs with percentage losses of 96.9%–99.7%, while F. chlamydosporum strains showed percentage losses of 88.9%–92.2%. All fungal strains could utilize the PEs-rich fraction for growth. In the cytotoxicity assay, cell viabilities of Chang liver and NIH 3T3 fibroblast cell lines were less than 1% with the untreated PEs-rich fraction, but 84.3%–96.5% with the fungal treated PEs-rich fraction. There was no inhibition on cell viability for normal fungal growth supernatants. To conclude, Trichoderma spp., Paecilomyces sp. and Cladosporium sp. are potential microbes for the detoxification of PEs. PMID:24504029

  16. Increased glucose transport in response to phorbol ester growth factors, and insulin: relationship to phosphorylation of the glucose transporter

    SciTech Connect

    Allard, W.J.; Gibbs, E.M.; Witters, L.A.; Lienhard, G.E.

    1986-05-01

    The authors have examined the relationship between the increase in glucose transport induced by phorbol myristate acetate (PMA), EGF, PDGF, and insulin and the phosphorylation state of the glucose transporter in human fibroblasts. To assay transport, cells were cultured in medium with 10% serum for 5 days and then for 2 days in phosphate-free medium with 5% serum. Exposure to each agonist stimulated transport, as measured by the uptake of /sup 3/H-2-deoxyglucose over a 2 min period. Values for maximal percent stimulation, time needed to reach maximal stimulation, and concentration required to achieve half-maximal stimulation were as follows: PMA, 80%, 30 min, 2 nM; EGF, 30%, 10 min, 0.2 nM; Insulin, 45%, 10 min, 17 nM. In the case of PDGF, uptake was stimulated 65% by treatment with 0.7 or 1.4 nM for 20 min. Phosphorylation of the glucose transporter was measured in cells cultured for 5-7 days in medium with 10% serum and exposed to 670 ..mu..Ci/ml /sup 32/P/sub i/ for 100 min. The agonist was then added at a saturating dose for 20 min, and the glucose transporter was immunoprecipitated from cell lysates using a monoclonal antibody. Under these conditions, no basal phosphorylation of the transporter was detected, and only phorbol ester stimulated significant incorporation of phosphate into the transport protein. Experiments are currently in progress to quantitate transporter phosphorylation under conditions identical to those used for the assay of transport. These results suggest that while the transporter is a substrate for protein kinase C in vivo, phosphorylation of the transporter is not required for increased transport in response to growth factors and insulin.

  17. A fluorescence microplate assay using yopro-1 to measure apoptosis: application to HL60 cells subjected to oxidative stress.

    PubMed

    Plantin-Carrenard, E; Bringuier, A; Derappe, C; Pichon, J; Guillot, R; Bernard, M; Foglietti, M J; Feldmann, G; Aubery, M; Braut-Boucher, F

    2003-04-01

    A new one-step labeling procedure using the membrane permeant fluorescent probe yopro-1 in association with fluorescence microtitration for the rapid determination of apoptosis is reported. Programmed cell death was induced by the pro-apoptotic agents etoposide and staurosporine, and measured in nonadherent HL60 cells and adherent phorbol 12-myristate 13-acetate (PMA)-treated HL60 cells. Cell viability was controlled by trypan blue exclusion and calcein-AM staining. To confirm results of fluorescence microplate assay, apoptosis was measured by flow cytometry analysis using the same fluorescent probe, and results showed corresponding data between both procedures. Development of apoptosis was confirmed by the presence of PARP (poly(ADP-ribose) polymerase cleavage and nuclear DAPI (4,6-diamidino-2-phenylindole) staining, two well-known methods used to investigate apoptosis. The fluorescence microplate assay was also applied to measure apoptosis in cells exposed to an oxidative stress induced by tert-butylhydroperoxide (t-BHP), and results confirmed the potential of the fluorescence microplate assay in measuring events of apoptosis, especially in adherent, cultured, living cells.

  18. Some phorbol esters might partially resemble bryostatin 1 in their actions on LNCaP prostate cancer cells and U937 leukemia cells.

    PubMed

    Kedei, Noemi; Lubart, Emanuel; Lewin, Nancy E; Telek, Andrea; Lim, Langston; Mannan, Poonam; Garfield, Susan H; Kraft, Matthew B; Keck, Gary E; Kolusheva, Sofiya; Jelinek, Raz; Blumberg, Peter M

    2011-05-16

    Phorbol 12-myristate 13-acetate (PMA) and bryostatin 1 are both potent protein kinase C (PKC) activators. In LNCaP human prostate cancer cells, PMA induces tumor necrosis factor alpha (TNFα) secretion and inhibits proliferation; bryostatin 1 does not, and indeed blocks the response to PMA. This difference has been attributed to bryostatin 1 not localizing PKCδ to the plasma membrane. Since phorbol ester lipophilicity influences PKCδ localization, we have examined in LNCaP cells a series of phorbol esters and related derivatives spanning some eight logs in lipophilicity (logP) to see if any behave like bryostatin 1. The compounds showed marked differences in their effects on proliferation and TNFα secretion. For example, maximal responses for TNFα secretion relative to PMA ranged from 97 % for octyl-indolactam V to 24 % for phorbol 12,13-dibenzoate. Dose-response curves ranged from monophasic for indolactam V to markedly biphasic for sapintoxin D. The divergent patterns of response, however, correlated neither to lipophilicity, to plasma membrane translocation of PKCδ, nor to the ability to interact with model membranes. In U937 human leukemia cells, a second system in which PMA and bryostatin 1 have divergent effects, viz. PMA but not bryostatin 1 inhibits proliferation and induces attachment, all the compounds acted like PMA for proliferation, but several induced a reduced level or a biphasic dose-response curve for attachment. We conclude that active phorbol esters are not all equivalent. Depending on the system, some might partially resemble bryostatin 1 in their behavior; this encourages the concept that bryostatin-like behavior may be obtained from other structural templates. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Some phorbol esters may partially resemble bryostatin 1 in their actions on LNCaP prostate cancer cells and U937 leukemia cells

    PubMed Central

    Kedei, Noemi; Lubart, Emanuel; Lewin, Nancy E.; Telek, Andrea; Lim, Langston; Mannan, Poonam; Garfield, Susan H.; Kraft, Matthew B.; Keck, Gary E.; Kolusheva, Sofiya; Jelinek, Raz; Blumberg, Peter M.

    2012-01-01

    Phorbol 12-myristate 13-acetate (PMA) and bryostatin 1 are both potent protein kinase C (PKC) activators. In LNCaP human prostate cancer cells, PMA induces tumor necrosis factor alpha (TNFα) secretion and inhibits proliferation; bryostatin 1 does not and itself blocks the response to PMA. This difference has been attributed to bryostatin 1 not localizing PKCδ to the plasma membrane. Since phorbol ester liphophilicity influences PKCδ localization, we have examined in LNCaP cells a series of phorbol esters and related derivatives spanning some 8 logs in lipophilicity (LogP) to see if any behave like bryostatin 1. The compounds showed marked differences in their effects on proliferation and TNFα secretion. For example, maximal responses for TNFα secretion relative to PMA ranged from 97% for octyl-indolactam V to 24% for phorbol 12,13-dibenzoate. Dose response curves ranged from monophasic for indolactam V to markedly biphasic for sapintoxin D. The divergent patterns of response, however, correlated neither with lipophilicity, with plasma membrane translocation of PKCδ, or with the ability to interact with model membranes. In U937 human leukemia cells, a second system in which PMA and bryostatin 1 have divergent effects, viz. PMA but not bryostatin 1 inhibits proliferation and induces attachment, all the compounds acted like PMA for proliferation but several induced a reduced level or a biphasic dose response curve for attachment. We conclude that active phorbol esters are not all equivalent. Depending on the system, some may partially resemble bryostatin 1 in their behavior, encouraging the concept that bryostatin-like behavior may be obtained from other structural templates. PMID:21542090

  20. Comparison of transcriptional response to phorbol ester, bryostatin 1, and bryostatin analogs in LNCaP and U937 cancer cell lines provides insight into their differential mechanism of action.

    PubMed

    Kedei, N; Telek, A; Michalowski, A M; Kraft, M B; Li, W; Poudel, Y B; Rudra, A; Petersen, M E; Keck, G E; Blumberg, P M

    2013-02-01

    Bryostatin 1, like the phorbol esters, binds to and activates protein kinase C (PKC) but paradoxically antagonizes many but not all phorbol ester responses. Previously, we have compared patterns of biological response to bryostatin 1, phorbol ester, and the bryostatin 1 derivative Merle 23 in two human cancer cell lines, LNCaP and U937. Bryostatin 1 fails to induce a typical phorbol ester biological response in either cell line, whereas Merle 23 resembles phorbol ester in the U937 cells and bryostatin 1 in the LNCaP cells. Here, we have compared the pattern of their transcriptional response in both cell lines. We examined by qPCR the transcriptional response as a function of dose and time for a series of genes regulated by PKCs. In both cell lines bryostatin 1 differed primarily from phorbol ester in having a shorter duration of transcriptional modulation. This was not due to bryostatin 1 instability, since bryostatin 1 suppressed the phorbol ester response. In both cell lines Merle 23 induced a pattern of transcription largely like that of phorbol ester although with a modest reduction at later times in the LNCaP cells, suggesting that the difference in biological response of the two cell lines to Merle 23 lies downstream of this transcriptional regulation. For a series of bryostatins and analogs which ranged from bryostatin 1-like to phorbol ester-like in activity on the U937 cells, the duration of transcriptional response correlated with the pattern of biological activity, suggesting that this may provide a robust platform for structure activity analysis.

  1. Comparison of transcriptional response to phorbol ester, bryostatin 1, and bryostatin analogues in LNCaP and U937 cancer cell lines provides insight into their differential mechanism of action

    PubMed Central

    Kedei, N.; Telek, A.; Michalowski, A.M.; Kraft, M.B.; Li, W.; Poudel, Y.B.; Rudra, A.; Petersen, M.E.; Keck, G.E.; Blumberg, P.M.

    2012-01-01

    Bryostatin 1, like the phorbol esters, binds to and activates protein kinase C (PKC) but paradoxically antagonizes many but not all phorbol ester responses. Previously, we have compared patterns of biological response to bryostatin 1, phorbol ester, and the bryostatin 1 derivative Merle 23 in two human cancer cell lines, LNCaP and U937. Bryostatin 1 fails to induce a typical phorbol ester biological response in either cell line, whereas Merle 23 resembles phorbol ester in the U937 cells and bryostatin 1 in the LNCaP cells. Here, we have compared the pattern of their transcriptional response in both cell lines. We examined by qPCR the transcriptional response as a function of dose and time for a series of genes regulated by PKCs. In both cell lines bryostatin 1 differed primarily from phorbol ester in having a shorter duration of transcriptional modulation. This was not due to bryostatin 1 instability, since bryostatin 1 suppressed the phorbol ester response. In both cell lines Merle 23 induced a pattern of transcription largely like that of phorbol ester although with a modest reduction at later times in the LNCaP cells, suggesting that the difference in biological response of the two cell lines to Merle 23 lies downstream of this transcriptional regulation. For a series of bryostatins and analogues which ranged from bryostatin 1-like to phorbol ester-like in activity on the U937 cells, the duration of transcriptional response correlated with the pattern of biological activity, suggesting that this may provide a robust platform for structure activity analysis. PMID:23146662

  2. α-tomatine inhibits growth and induces apoptosis in HL-60 human myeloid leukemia cells

    PubMed Central

    HUANG, HUARONG; CHEN, SHAOHUA; VAN DOREN, JEREMIAH; LI, DONGLI; FARICHON, CHELSEA; HE, YAN; ZHANG, QIUYAN; ZHANG, KUN; CONNEY, ALLAN H; GOODIN, SUSAN; DU, ZHIYUN; ZHENG, XI

    2015-01-01

    α-tomatine is a glycoalkaloid that occurs naturally in tomatoes (Lycopersicon esculentum). In the present study, the effects of α-tomatine on human myeloid leukemia HL-60 cells were investigated. Treatment of HL-60 cells with α-tomatine resulted in growth inhibition and apoptosis in a concentration-dependent manner. Tomatidine, the aglycone of tomatine had little effect on the growth and apoptosis of HL-60 cells. Growth inhibition and apoptosis induced by α-tomatine in HL-60 cells was partially abrogated by addition of cholesterol indicating that interactions between α-tomatine and cell membrane-associated cholesterol may be important in mediating the effect of α-tomatine. Activation of nuclear factor-κB by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate failed to prevent apoptosis in HL-60 cells treated with α-tomatine. In animal experiments, it was found that treatment of mice with α-tomatine inhibited the growth of HL-60 xenografts in vivo. Results from the present study indicated that α-tomatine may have useful anti-leukemia activities. PMID:25625536

  3. α-Tomatine inhibits growth and induces apoptosis in HL-60 human myeloid leukemia cells.

    PubMed

    Huang, Huarong; Chen, Shaohua; Van Doren, Jeremiah; Li, Dongli; Farichon, Chelsea; He, Yan; Zhang, Qiuyan; Zhang, Kun; Conney, Allan H; Goodin, Susan; Du, Zhiyun; Zheng, Xi

    2015-06-01

    α‑Tomatine is a glycoalkaloid that occurs naturally in tomatoes (Lycopersicon esculentum). In the present study, the effects of α‑tomatine on human myeloid leukemia HL‑60 cells were investigated. Treatment of HL‑60 cells with α‑tomatine resulted in growth inhibition and apoptosis in a concentration‑dependent manner. Tomatidine, the aglycone of tomatine had little effect on the growth and apoptosis of HL‑60 cells. Growth inhibition and apoptosis induced by α‑tomatine in HL‑60 cells was partially abrogated by addition of cholesterol indicating that interactions between α‑tomatine and cell membrane‑associated cholesterol may be important in mediating the effect of α‑tomatine. Activation of nuclear factor‑κB by the phorbol ester, 12‑O‑tetradecanoylphorbol‑13‑acetate failed to prevent apoptosis in HL‑60 cells treated with α‑tomatine. In animal experiments, it was found that treatment of mice with α‑tomatine inhibited the growth of HL‑60 xenografts in vivo. Results from the present study indicated that α‑tomatine may have useful anti‑leukemia activities.

  4. Apoptosis and cancer mechanisms.

    PubMed

    Pan, H; Yin, C; Van Dyke, T

    1997-01-01

    For nearly two decades, studies in the cancer research field focussed on identifying genes that act as positive and negative regulators of cell growth. Only relatively recently was it recognized that the regulation of cell death (apoptosis) is also an important modulator of tumorigenesis. At least two genes linked to human cancers, BCL2 and TP53, have been shown to regulate apoptosis. The correlation between apoptosis modulating genes and human tumours raises an important question as to how dysregulation of apoptosis contributes to neoplastic transformation and malignant cell growth. Cell culture studies have clearly demonstrated that TP53 can induce and BCL2 can suppress apoptosis in response to various stimuli. Studies of mammalian viruses, which possess mechanisms for both inducing and evading apoptosis, have also extended our understanding of this process. On the basis of such findings, several animal models have been developed which begin to address the role of apoptosis regulation in tumorigenesis. This chapter discusses those animal models, focussing on bcl-2 (and its relatives) and p53.

  5. Lysosomes in apoptosis.

    PubMed

    Ivanova, Saska; Repnik, Urska; Bojic, Lea; Petelin, Ana; Turk, Vito; Turk, Boris

    2008-01-01

    Lysosomes are specialized organelles for protein recycling and as such are involved in the terminal steps of autophagy. However, it has become evident that lysosomes also play an important role in the progression of apoptosis. This latter function seems to be dependent on lysosomal proteases, which need to be released into the cytosol for apoptosis to be efficient. Among the lysosomal proteases, the most abundant are the cysteine cathepsins and the aspartic protease cathepsin D, which seem to be the major apoptosis mediators. This chapter reviews the methods used to study lysosomes and lysosomal proteases.

  6. Apoptosis in pneumovirus infection.

    PubMed

    van den Berg, Elske; van Woensel, Job B M; Bem, Reinout A

    2013-01-23

    Pneumovirus infections cause a wide spectrum of respiratory disease in humans and animals. The airway epithelium is the major site of pneumovirus replication. Apoptosis or regulated cell death, may contribute to the host anti-viral response by limiting viral replication. However, apoptosis of lung epithelial cells may also exacerbate lung injury, depending on the extent, the timing and specific location in the lungs. Differential apoptotic responses of epithelial cells versus innate immune cells (e.g., neutrophils, macrophages) during pneumovirus infection can further contribute to the complex and delicate balance between host defense and disease pathogenesis. The purpose of this manuscript is to give an overview of the role of apoptosis in pneumovirus infection. We will examine clinical and experimental data concerning the various pro-apoptotic stimuli and the roles of apoptotic epithelial and innate immune cells during pneumovirus disease. Finally, we will discuss potential therapeutic interventions targeting apoptosis in the lungs.

  7. Apoptosis in Pneumovirus Infection

    PubMed Central

    van den Berg, Elske; van Woensel, Job B.M.; Bem, Reinout A.

    2013-01-01

    Pneumovirus infections cause a wide spectrum of respiratory disease in humans and animals. The airway epithelium is the major site of pneumovirus replication. Apoptosis or regulated cell death, may contribute to the host anti-viral response by limiting viral replication. However, apoptosis of lung epithelial cells may also exacerbate lung injury, depending on the extent, the timing and specific location in the lungs. Differential apoptotic responses of epithelial cells versus innate immune cells (e.g., neutrophils, macrophages) during pneumovirus infection can further contribute to the complex and delicate balance between host defense and disease pathogenesis. The purpose of this manuscript is to give an overview of the role of apoptosis in pneumovirus infection. We will examine clinical and experimental data concerning the various pro-apoptotic stimuli and the roles of apoptotic epithelial and innate immune cells during pneumovirus disease. Finally, we will discuss potential therapeutic interventions targeting apoptosis in the lungs. PMID:23344499

  8. Neurofibromin and Neuronal Apoptosis

    DTIC Science & Technology

    2006-07-01

    role of familial pheochromocytoma genes, including succinate dehydrogenase (SDH) and Nf1, in modulating neuronal apoptosis following neurotrophin...gene products, in Nf1-/- sensory and sympathetic neurons; this work will also have relevance to the biology of familial pheochromocytoma . "So what...Schlisio, S. (2005). Neuronal apoptosis linked to EglN3 prolyl hydroxylase and familial pheochromocytoma genes: Developmental culling and cancer. Cancer

  9. Epidermal growth factor (EGF)-stimulated inositol phosphate formation in hepatocytes is abolished by pertussis toxin and phorbol esters

    SciTech Connect

    Johnson, R.M.; Garrison, J.C.

    1987-05-01

    The EGF-stimulated rise in intracellular Ca/sup 2 +/ (Ca/sup 2 +/)/sub i/ and Ca/sup 2 +/-dependent protein phosphorylation events in isolated hepatocytes are blocked by pertussis toxin and phorbol ester pretreatment. The present study characterized the EGF-stimulated formation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P/sub 3/) and inositol 1,3,4-trisphosphate (Ins(1,3,4)P/sub 3/) in hepatocytes using HPLC methodology to separate the InsP/sub 3/ isomers. Both 66 nM EGF and 10 nM angiotensin II (ANG II) caused a rapid increase in the Ins(1,4,5)P/sub 3/ isomer although EGF-stimulated formation was smaller. At a concentration of ANG II (0.1 nM) which gave an equivalent rise in (Ca/sup 2 +/)/sub i/ as 66 nM EGF, the kinetics and magnitude of Ins(1,4,5)P/sub 3/ formation were similar. EGF or ANG II-stimulated formation of the Ins(1,3,4)P/sub 3/ isomer was more gradual and increased beyond the level of Ins(1,4,5)P/sub 3/ after 60 sec. The initial EGF and ANG II-stimulated increase in both InsP/sub 3/ isomers was not affected by removing external Ca/sup 2 +/ with a 10-fold excess of EGTA. Pretreatment of rats with pertussis toxin for 72 hrs blocked the ability of EGF to increase Ins(1,4,5)P/sub 3/ but did not affect the increase due to ANG II. Three main pretreatment of cells with 1 ..mu..g/ml phorbol 12-myristate-13-acetate (PMA) also inhibited the EGF-stimulated Ins(1,4,5)P/sub 3/ formation. PMA slightly attenuated Ins(1,4,5)P/sub 3/ formation stimulated by 0.1 nM ANG II but not enough to affect the Ca/sup 2 +/ signal. These data suggest that the signal transduction system used by EGF receptors to increase Ins (1,4,5)P/sub 3/ in hepatocytes is somehow different from that used by ANG II receptors.

  10. Effect of oestrogen on T cell apoptosis in patients with systemic lupus erythematosus

    PubMed Central

    Kim, W-U; Min, S-Y; Hwang, S-H; Yoo, S-A; Kim, K-J; Cho, C-S

    2010-01-01

    Defective control of T cell apoptosis is considered to be one of the pathogenetic mechanisms in systemic lupus erythematosus (SLE). Oestrogen has been known to predispose women to SLE and also to exacerbate activity of SLE; however, the role of oestrogen in the apoptosis of SLE T cells has not yet been documented. In this study, we investigated the direct effect of oestrogen on the activation-induced cell death of T cells in SLE patients. The results demonstrated that oestradiol decreased the apoptosis of SLE T cells stimulated with phorbol 12-myristate 13-acetate (PMA) plus ionomycin in a dose-dependent manner. In addition, oestradiol down-regulated the expression of Fas ligand (FasL) in activated SLE T cells at the both protein and mRNA levels. In contrast, testosterone increased FasL expression dose-dependently in SLE T cells stimulated with PMA plus ionomycin. The inhibitory effect of oestradiol on FasL expression was mediated through binding to its receptor, as co-treatment of tamoxifen, an oestrogen receptor inhibitor, completely nullified the oestradiol-induced decrease in FasL mRNA expression. Moreover, pre-treatment of FasL-transfected L5178Y cells with either oestradiol or anti-FasL antibody inhibited significantly the apoptosis of Fas-sensitive Hela cells when two types of cells were co-cultured. These data suggest that oestrogen inhibits activation-induced apoptosis of SLE T cells by down-regulating the expression of FasL. Oestrogen inhibition of T cell apoptosis may allow for the persistence of autoreactive T cells, thereby exhibiting the detrimental action of oestrogen on SLE activity. PMID:20529085

  11. The bryostatin 1 A-ring acetate is not the critical determinant for antagonism of phorbol ester-induced biological responses.

    PubMed

    Keck, Gary E; Li, Wei; Kraft, Matthew B; Kedei, Noemi; Lewin, Nancy E; Blumberg, Peter M

    2009-06-04

    The contribution of the A-ring C(7) acetate to the function of bryostatin 1 has been investigated through synthesis and biological evaluation of an analogue incorporating this feature into the bryopyran core structure. No enhanced binding affinity for protein kinase C (PKC) was observed, relative to previously characterized analogues lacking the C(7) acetate. Functional assays showed biological responses characteristic of those induced by the phorbol ester PMA and distinctly different from those observed with bryostatin 1.

  12. Structural insights into the interactions of phorbol ester and bryostatin complexed with protein kinase C: a comparative molecular dynamics simulation study.

    PubMed

    Thangsunan, Patcharapong; Tateing, Suriya; Hannongbua, Supa; Suree, Nuttee

    2016-07-01

    Protein kinase C (PKC) isozymes are important regulatory enzymes that have been implicated in many diseases, including cancer, Alzheimer's disease, and in the eradication of HIV/AIDS. Given their potential clinical ramifications, PKC modulators, e.g. phorbol esters and bryostatin, are also of great interest in the drug development. However, structural details on the binding between PKC and its modulators, especially bryostatin - the highly potent and non-tumor promoting activator for PKCs, are still lacking. Here, we report the first comparative molecular dynamics study aimed at gaining structural insight into the mechanisms by which the PKC delta cys2 activator domain is used in its binding to phorbol ester and bryostatin-1. As anticipated in the phorbol ester binding, hydrogen bonds are formed through the backbone atoms of Thr242, Leu251, and Gly253 of PKC. However, the opposition of H-bond formation between Thr242 and Gly253 may cause the phorbol ester complex to become less stable when compared with the bryostatin binding. For the PKC delta-bryostatin complex, hydrogen bonds are formed between the Gly253 backbone carbonyl and the C30 carbomethoxy substituent of the ligand. Additionally, the indole Nε1 of the highly homologous Trp252 also forms an H-bond to the C20 ester group on bryostatin. Backbone fluctuations also suggest that this latter H-bond formation may abrogate the transient interaction between Trp252 and His269, thus dampening the fluctuations observed on the nearby Zn(2+)-coordinating residues. This new dynamic fluctuation dampening model can potentially benefit future design of new PKC modulators.

  13. Neutrophil beta-adrenergic receptor responses are potentiated by acute exposure to phorbol ester without changes in receptor distribution or coupling

    SciTech Connect

    Kilfeather, S.A.; Stein, M.; O'Malley, K. )

    1991-01-01

    Exposure to the phorbol ester, phorbol 12-myristate, 13-acetate for 10 minutes enhanced cyclic AMP accumulation in human neutrophils under basal conditions and in response to the beta-adrenergic receptor agonist isoproterenol (ISO, 1{mu}M) and the adenylate cyclase activator forskolin (FSK, 10mM). Potentiation of responses to ISO by PMA was dose-dependent between 0.1 and 100nM PMA. The diacylglycerol analogue, 1-oleoyl-2-actylgylcerol (OAG) (50 {mu}M) also elevated beta-receptor responses, but 4beta-phorbol (100nM), lacking the capacity to activate PMA, was ineffective. Short-term exposure to the peptide n-formylmethionine leucyl-phenylalanine (FMLP, 1 {mu}M) also elevated neutrophil cyclic AMP accumulation. All potentiating effects of PMA on cyclic AMP production were inhibited by the protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H{sub 7}). PMA had no apparent effect on beta-receptor agonist-affinity, distribution between cell-surface and internalized compartments, or the capacity of ISO to induce beta-receptor internalization. Responses to FSK or ISO in terms of fold-stimulation of basal cyclic AMP accumulation int he presence of PMA were not elevated by PMA.

  14. Cross-linking of surface Ig receptors on murine B lymphocytes stimulates the expression of nuclear tetradecanoyl phorbol acetate-response element-binding proteins

    SciTech Connect

    Chiles, T.C.; Liu, J.L.; Rothstein, T.L. )

    1991-03-15

    Cross-linking of sIg on primary B lymphocytes leads to increased nuclear DNA-binding activity specific for the tetradecanoyl phorbol acetate-response element (TRE), as judged by gel mobility shift assays. Stimulation of B cells to enter S phase of the cell cycle by treatment with the combination of phorbol ester plus calcium ionophore also stimulated nuclear TRE-binding activity within 2 h, with maximal expression at 4 h; however, phorbol ester and calcium ionophore were not as effective in stimulating binding activity when examined separately. Stimulated nuclear expression of TRE-binding activity appears to require protein synthesis. Fos- and Jun/AP-1-related proteins participate directly in the identified nucleoprotein complex, as shown by the ability of c-fos- and c-jun-specific antisera to either alter or completely abolish electrophoretic migration of the complex in native gels. Further, UV photo-cross-linking studies identified two major TRE-binding protein species, whose sizes correspond to TRE-binding proteins derived from HeLa cell nuclear extracts. The results suggest that in primary B cells nuclear TRE-binding activity represents a downstream signaling event that occurs subsequent to changes in protein kinase C activity and intracellular Ca2+ but that can be triggered physiologically through sIg.

  15. Double minute chromatin bodies and other chromosome alterations in human myeloid HL-60 leukemia cells susceptible or resistant to induction of differentiation by phorbol-12-myristate-13-acetate

    SciTech Connect

    Au, W.W.; Callaham, M.F.; Workman, M.L.; Huberman, E.

    1983-12-01

    An analysis of the chromosomal karyotype of the human promyelocytic HL-60 leukemia cell line and of a number of its sublines that exhibit varying degrees of resistance to induction of differentiation by phorbol-12-myristate-13-acetate was conducted. The HL-60 cell line and the derived sublines contained two consistent marker chromosomes (9p- and t(10;13)), which suggested that they have a common and possibly clonal origin. HL-60 cells that are susceptible to phorbol-12-myristate-13-acetate-induced cell differentiation contained double minute chromatine bodies. The sublines with different degrees of resistance showed a corresponding sequential reduction of double minute chromatin bodies in metaphase cells. This loss of double minute chromatin bodies was not associated with an appearance of homogeneously staining chromosomal regions. Resistant and susceptible HL-60 cell differed also in a number of other chromosomal alteration, including gains or losses involving chromosomes 5, 8, 11, 13, 16, and 17. Thus, it is suggested that acquisition of resistance to phorbol-12-myristate-13-acetate-induced cell differentiation in the HL-60 cells may involve one or more of the above chromosomal changes.

  16. Application of time-of-flight mass spectrometry for screening of crude glycerins for toxic phorbol ester contaminants.

    PubMed

    Herath, Kithsiri; Girard, Lauren; Reimschuessel, Renate; Jayasuriya, Hiranthi

    2017-03-01

    Since 2007, the U.S. Food and Drug Administration (FDA) has received numerous complaints of pet illnesses that may be related to the consumption of jerky pet treats. Many of those treats include glycerin as an ingredient. Glycerin can be made directly from oils such as palm seed oil, but can also be derived from the seed oil of toxic Jatropha plant during biodiesel production. If crude glycerin from biodiesel production from Jatropha curcas is used in the manufacture of animal feed, toxic tigliane diterpene phorbol esters (PEs), namely Jatropha factors (JFs), may be present and could lead to animal illnesses. Considering the numerous uses of glycerin in consumer products there is a need for a rapid method to screen crude glycerin for JF toxins and other PE contaminants. We describe the development of an ultra-high pressure liquid chromatography/quadrupole time of flight (UHPLC/Q-TOF) method for screening crude glycerin for PEs. An exact mass database, developed in-house, of previously identified PEs from Jatropha curcas as well as putative compounds was used to identify possible contaminants.

  17. Expression of the human B-cell surface protein CD20: alteration by phorbol 12-myristate 13-acetate

    SciTech Connect

    Valentine, M.A.; Cotner, T.; Gaur, L.; Torres, R.; Clark, E.A.

    1987-11-01

    The monoclonal antibody 1F5 recognizes human B-cell surface protein CD20 and can activate resting B cells; with this antibody the authors found CD20 to be a 35/37-kDa non-disulfide-linked protein. The protein has a pI of 7.5-8.0 and is phosphorylated in B-cell lines, tonsillar B cells, and peripheral blood B cells. Both CD20 surface expression and phosphorylation are increased on buoyant tonsillar B cells activated in vivo. Because phorbol 12-myristate 13-acetate (PMA) supports the activation signal initiated by monoclonal antibody 1F5, they studied the effect of PMA on CD20 expression. After brief incubation with mitogenic levels of PMA, the number of dense tonsillar B cells positive for CD20 protein transiently decreased. Paradoxically, the cells remaining positive had more surface CD20 than did control cells, and these remaining surface CD20 molecules were hyperphosphorylated. Furthermore, PMA not only induced phosphorylation of CD20 protein on Raji cells but also increased the internalization of CD20 molecules; both phosphorylation and internalization of CD20 molecules were decreased with the protein kinase C inhibitor palmitoyl carnitine. Conditions that increase CD20 phosphorylation are shown also to increase surface mobility of the molecule, suggesting that CD20 protein internalization may be a critical early event for B-cell entry into the G/sub 1/ phase of the cell cycle.

  18. ERK2-Pyruvate Kinase Axis Permits Phorbol 12-Myristate 13-Acetate-induced Megakaryocyte Differentiation in K562 Cells*

    PubMed Central

    Chaman, Noor; Iqbal, Mohammad Askandar; Siddiqui, Farid Ahmad; Gopinath, Prakasam; Bamezai, Rameshwar N. K.

    2015-01-01

    Metabolic changes that contribute to differentiation are not well understood. Overwhelming evidence shows the critical role of glycolytic enzyme pyruvate kinase (PK) in directing metabolism of proliferating cells. However, its role in metabolism of differentiating cells is unclear. Here we studied the role of PK in phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation in human leukemia K562 cells. We observed that PMA treatment decreased cancer-type anabolic metabolism but increased ATP production, along with up-regulated expression of two PK isoforms (PKM2 and PKR) in an ERK2-dependent manner. Interestingly, silencing of PK (PKM2 and PKR) inhibited PMA-induced megakaryocytic differentiation, as revealed by decreased expression of megakaryocytic differentiation marker CD61 and cell cycle behavior. Further, PMA-induced ATP production reduced greatly upon PK silencing, suggesting that PK is required for ATP synthesis. In addition to metabolic effects, PMA treatment also translocated PKM2, but not PKR, into nucleus. ERK1/2 knockdowns independently and together suggested the role of ERK2 in the up-regulation of both the isoforms of PK, proposing a role of ERK2-PK isoform axis in differentiation. Collectively, our findings unravel ERK2 guided PK-dependent metabolic changes during PMA induction, which are important in megakaryocytic differentiation. PMID:26269597

  19. ERK2-Pyruvate Kinase Axis Permits Phorbol 12-Myristate 13-Acetate-induced Megakaryocyte Differentiation in K562 Cells.

    PubMed

    Chaman, Noor; Iqbal, Mohammad Askandar; Siddiqui, Farid Ahmad; Gopinath, Prakasam; Bamezai, Rameshwar N K

    2015-09-25

    Metabolic changes that contribute to differentiation are not well understood. Overwhelming evidence shows the critical role of glycolytic enzyme pyruvate kinase (PK) in directing metabolism of proliferating cells. However, its role in metabolism of differentiating cells is unclear. Here we studied the role of PK in phorbol 12-myristate 13-acetate (PMA)-induced megakaryocytic differentiation in human leukemia K562 cells. We observed that PMA treatment decreased cancer-type anabolic metabolism but increased ATP production, along with up-regulated expression of two PK isoforms (PKM2 and PKR) in an ERK2-dependent manner. Interestingly, silencing of PK (PKM2 and PKR) inhibited PMA-induced megakaryocytic differentiation, as revealed by decreased expression of megakaryocytic differentiation marker CD61 and cell cycle behavior. Further, PMA-induced ATP production reduced greatly upon PK silencing, suggesting that PK is required for ATP synthesis. In addition to metabolic effects, PMA treatment also translocated PKM2, but not PKR, into nucleus. ERK1/2 knockdowns independently and together suggested the role of ERK2 in the up-regulation of both the isoforms of PK, proposing a role of ERK2-PK isoform axis in differentiation. Collectively, our findings unravel ERK2 guided PK-dependent metabolic changes during PMA induction, which are important in megakaryocytic differentiation.

  20. Effects of an endogenous nitric oxide synthase inhibitor on phorbol myristate acetate-induced acute lung injury in rats.

    PubMed

    Lin, Hen I; Chu, Shi Jye; Wang, David; Chen, Hsing I; Hsu, Kang

    2003-01-01

    1. In the present study, we determined whether the endogenous nitric oxide (NO) synthase (NOS) inhibitor Nomega-nitro-l-arginine methyl ester (l-NAME) could ameliorate the acute lung injury (ALI) induced by phorbol myristate acetate (PMA) in rat isolated lung. 2. Typical ALI was induced successfully by PMA during 60 min of observation. At 2 micro g/kg, PMA elicited a significant increase in microvascular permeability (measured using the capillary filtration coefficient Kfc), lung weight gain, lung weight/bodyweight ratio, pulmonary arterial pressure (PAP) and protein concentration of bronchoalveolar lavage fluid. 3. Pretreatment with the NOS inhibitor l-NAME (5 mmol/L) significantly attenuated ALI. None of the parameters reflective of lung injury showed significant increase, except for PAP (P < 0.001). The addition of l-arginine (4 mmol/L) blocked the protective effective of l-NAME. Pretreatment with l-arginine exacerbated PMA-induced lung injury. 4. These data suggest that l-NAME significantly ameliorates ALI induced by PMA in rats, indicating that endogenous NO plays a key role in the development of lung oedema in PMA-induced lung injury.

  1. beta. -Endorphin and related peptides suppress phorbol myristate acetate-induced respiratory burst in human polymorphonuclear leukocytes

    SciTech Connect

    Diamant, M.; Henricks, P.A.J.; Nijkamp, F.P.; de Wied, D. )

    1989-01-01

    In the present study, the immunomodulatory effect of {beta}-endorphin ({beta}-E) and shorter pro-opiomelancortin (POMC) fragments was evaluated by assessing their influence on respiratory burst in human polymorphonuclear leukocytes (PMN). The effect of the peptides on phorbol myristate acetate (PMA)-stimulated production of reactive oxygen metabolites was measured in a lucigenin-enhanced chemiluminescence (CL) assay. Both POMC peptides with opiate-like activity and their non-opioid derivatives were tested. With the exception of {alpha}-E, PMA-stimulated respiratory burst was suppressed by all POMC fragments tested. A U-shaped dose-response relation was observed. Doses lower than 10{sup {minus}17}M and higher than 10{sup {minus}8}M were without effect. {beta}-E and dT{beta}E both suppressed PMA-induced oxidative burst in human PMN at physiological concentrations. {gamma}-E and dT{gamma}E proved to be less potent inhibitors, reaching maximal effect at higher concentrations. DE{gamma}E exerted an even less pronounced but still significant suppressive effect at the concentration of 10{sup {minus}10}M. None of the endorphins tested was shown to affect resting oxidative metabolism in the PMN. The modulatory effects of the opioid peptides could not be blocked by the opioid antagonist naloxone.

  2. 4-Methylumbelliferone inhibits the phosphorylation of hyaluronan synthase 2 induced by 12-O-tetradecanoyl-phorbol-13-acetate.

    PubMed

    Kuroda, Yoshiyuki; Kasai, Kosuke; Nanashima, Naoki; Nozaka, Hiroyuki; Nakano, Manabu; Chiba, Mitsuru; Yoneda, Masahiko; Nakamura, Toshiya

    2013-04-01

    The effect of 4-methylumbelliferone (MU), a hyaluronan synthase-suppressor, on O-linked β-Nacetylglucosaminylation (O-GlcNAcylation) was investigated in cultured human skin fibroblasts, and we found that MU stimulated O-GlcNAcylation of the cellular proteins. Since O-GlcNAcylation affects protein phosphorylation via Ser/Thr kinases, we examined the effect of MU on both the phosphorylation of hyaluronan synthase 2 (HAS2) and hyaluronan production. The cells were cultured in the presence or absence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and MU independently or in combination. The protein fraction of each cell culture was extracted and divided into 2 parts-phosphorylated and non-phosphorylated fractions-by immobilized metal-affinity chromatography. The hyaluronan level in the medium was determined by an ELISA-like assay. Addition of MU decreased the level of hyaluronan in the medium and that of HAS2 in the phosphorylated protein fraction. On the contrary, the addition of TPA increased the levels of both of them. Interestingly, the combination of TPA and MU lowered the levels of them in treated cells as compared to those in untreated control cells. These results suggest that TPA activated protein kinase C (PKC), which stimulates the phosphorylation of HAS2, and increased hyaluronan production. Further, MU may inhibit the phosphorylation of HAS2 by PKC through the stimulation of O-GlcNAcylation.

  3. Regulation of thyroid peroxidase activity by thyrotropin, epidermal growth factor and phorbol ester in porcine thyroid follicles cultured in suspension

    SciTech Connect

    Kasai, Kikuo; Hiraiwa, Masaki; Emoto, Tatsushi; Hattori, Yoshiyuki; Shimoda, Shin-Ichi ); Ohmori, Takeshi; Koizumi, Narumi; Hosoya, Toichiro )

    1989-01-01

    The activity of thyroid peroxidase (TPO) in porcine follicles cultured for 96 h in suspension with five hormones (5H) still attained over 50% of that in the freshly isolated follicles. On the other hand, the activity in those cultured with 5H + TSH (6H) was several times higher than that cultured with 5H after 96 h, although an initial decrease of TPO activity during the first 24 h of culture was observed in both conditions. The ability of follicles to metabolize iodide when cultured with 6H for 96 h was also several times higher than that of those cultured with 5H. The half-maximal dose of TSH for stimulation of TPO activity and iodide metabolism was 0.03 - 0.04 mU/ml and the effect was mediated by cAMP. These results indicate that in porcine thyroid follicles in primary suspension culture, TPO activity as well as the ability of iodide metabolism is induced by chronic TSH stimulation. In addition, epidermal growth factor and phorbol 12-myristate 13-acetate completely inhibited TSH stimulation on both activities and also basal (5H) activity of iodide metabolism.

  4. Effects of inorganic iodide, epidermal growth factor and phorbol ester on hormone synthesis by porcine thyroid follicles cultured in suspension

    SciTech Connect

    Kasai, Kikuo; Ichimura, Kenichi; Banba, Nobuyuki; Emoto, Tatsushi; Hiraiwa, Masaki; Hishinuma, Akira; Hattori, Yoshiyuki; Shimoda, Shinichi ); Yamaguchi, Fumihiko; Hosoya, Toichiro )

    1992-01-01

    Porcine thyroid follicles cultured in suspension for 96 h synthesized and secreted thyroid hormones in the presence of thyrotropin (TSH). The secretion of newly synthesized hormones was assessed by determining in the contents of thyroxine (T{sub 4}) and triiodothyronine (T{sub 3}) in the media and by paperchromatographic analysis of {sup 125}I-labeled hormones in the media where the follicles were cultured in the presence and absence of inhibitors of hormone synthesis. The hormone synthesis and secretion was modified by exogenously added NaI. The maximal response was obtained at 1 {mu}M. Thyroid peroxidase (TPO) activity in the cultured follicles with TSH for 96 h was dose-dependently inhibited by NaI. One hundred {mu}M and NaI completely inhibited TSH-induced TPO activity. Moreover, both epidermal growth factor and phorbol 12-myristate 13-acetate inhibited de novo hormone synthesis. An induction of TPO activity by TSH was also inhibited by either agent. These data provide direct evidences that thyroid hormone synthesis is regulated by NaI as well as TSH at least in part via regulation of TPO activity and also that both EGF and PMA are inhibitory on thyroid hormone formation.

  5. Phorbol 12-myristate 13-acetate prevents isoproterenol-induced morphological change in cultured vascular smooth muscle cells

    SciTech Connect

    Nabika, Toru; Chaldakov, G.N.; Nara, Yasuo; Endo, Jiro; Yamori, Yukio )

    1988-10-01

    The effect of phorbol 12-myristate 13-acetate (PMA) on isoproterenol (ISO)- and dibutyryl cAMP (dBcAMP)-induced morphological change and cytoskeletal reorganization was studied in cultured vascular smooth muscle cells (VSMC) using the fluorescence staining of actin and microtubules. The treatment of VSMC with 1.0 {mu}M of ISO or with 1.0 mM of dBcAMP for 90 min induced the disruption of actin-containing stress fibers followed by cytoplasmic arborization. The addition of 100 nM of PMA prevented both the destruction of actin fibers and cell arborization induced either by ISO or by dBcAMP. These results indicated that the inhibition of arborization by PMA was mediated through the activation of protein kinase C. Colchicine at 5.0 {mu}M also had an inhibitory effect on ISO- and dBcAMP-induced cell arborization. However, immunofluorescence studies revealed that colchicine but not PMA elicited the reorganization of microtubules, suggesting that the effect of PMA was mediated through a mechanism different from that of colchicine. The observations indicated that the morphology of VSMC was regulated through the alteration of cytoskeletal organization induced by cAMP-mediated and by protein kinase C-dependent systems.

  6. Activation of protein kinase C by phorbol ester increases red blood cell scramblase activity and external phosphatidylserine.

    PubMed

    Barber, Latorya A; Palascak, Mary B; Qi, Xiaoyang; Joiner, Clinton H; Franco, Robert S

    2015-11-01

    Externalization of phosphatidylserine (PS) is thought to contribute to sickle cell disease (SCD) pathophysiology. The red blood cell (RBC) aminophospholipid translocase (APLT) mediates the transport of PS from the outer to the inner RBC membrane leaflet to maintain an asymmetric distribution of PL, while phospholipid scramblase (PLSCR) equilibrates PL across the RBC membrane, promoting PS externalization. We previously identified an association between PS externalization level and PLSCR activity in sickle RBC under basal conditions. Other studies showed that activation of protein kinase C (PKC) by PMA (phorbol-12-myristate-13-acetate) causes increased external PS on RBC. Therefore, we hypothesized that PMA-activated PKC stimulates PLSCR activity in RBC and thereby contributes to increased PS externalization. In the current studies, we show that PMA treatment causes immediate and variable PLSCR activation and subsequent PS externalization in control and sickle RBC. While TfR+ sickle reticulocytes display some endogenous PLSCR activity, we observed a robust activation of PLSCR in sickle reticulocytes treated with PMA. The PKC inhibitor, chelerythrine (Chel), significantly inhibited PMA-dependent PLSCR activation and PS externalization. Chel also inhibited endogenous PLSCR activity in sickle reticulocytes. These data provide evidence that PKC mediates PS externalization in RBC through activation of PLSCR.

  7. Phorbol ester-inducible T-cell-specific expression of variant mouse mammary tumor virus long terminal repeats

    SciTech Connect

    Theunissen, H.J.M.; Paardekooper, M.; Maduro, L.J.; Michalides, R.J.A.M.; Nusse, R. )

    1989-08-01

    Acquired proviruses of mouse mammary tumor virus (MMTV) in T-cell leukemias of male GR mice have rearrangements in the U3 region of their long terminal repeats (LTR). In contrast to the endogenous nonrearranged MMTV proviruses, these mutated copies are highly expressed in leukemic T cells. To investigate whether the sequence alterations in the LTR are responsible for the high expression of rearranged MMTV proviruses, the authors made constructs in which normal and variant LTRs drive the bacterial reporter gene chloramphenicol acetyltransferase (CAT). Two different rearranged LTRs were used, one containing a 420-base-pair (bp) deletion (L13) and another carrying a 456-bp deletion plus an 82-bp insertion (L42). These constructs were transfected into murine (GRSL) and human (MOLT-4) T-cell lines that either had or had not been treated with phorbol ester (12-O-tetradecanoylphorbol-13-acetate (TPA)). In GRSL cells, the L13-LTR-CAT construct showed transcriptional activity that was further enhanced by TPA. In MOLT-4 cells, both variant LTRs were active, but only after stimulation with TPA. In contrast, normal(N)-LTR-CAT constructs were not expressed, irrespective of TPA addition. They conclude that the LTR rearrangements generate TPA responsiveness and contribute to T-cell-specific expression of MMTV variants.

  8. Modulation of Purinergic Neuromuscular Transmission by Phorbol Dibutyrate is Independent of Protein Kinase C in Murine Urinary Bladder

    PubMed Central

    Silinsky, E. M.

    2012-01-01

    Parasympathetic control of murine urinary bladder consists of contractile components mediated by both muscarinic and purinergic receptors. Using intracellular recording techniques, the purinergic component of transmission was measured as both evoked excitatory junctional potentials (EJPs) in response to electrical field stimulation and spontaneous events [spontaneous EJPs (sEJPs)]. EJPs, but not sEJPs, were abolished by the application of the Na+ channel blocker tetrodotoxin and the Ca2+ channel blocker Cd2+. Both EJPs and sEJPs were abolished by the application of the P2X1 antagonist 8,8′-[carbonylbis(imino-4,1-phenylenecarbonylimino-4,1-phenylenecarbonylimino)]bis-1,3,5-naphthalenetrisulfonic acid hexasodium salt (NF279). Application of phorbol dibutyrate (PDBu) increased electrically evoked EJP amplitudes with no effect on mean sEJP amplitudes. Similar increases in EJP amplitudes were produced by PDBu in the presence of either the nonselective protein kinase inhibitor staurosporine or the specific protein kinase C (PKC) inhibitor 2-[1-(3-dimethylaminopropyl)indol-3-yl]-3-(indol-3-yl) maleimide (GF109203X). These results suggest that PDBu increases the purinergic component of detrusor transmission through increasing neurogenic ATP release via a PKC-independent mechanism. PMID:22547572

  9. Modulation by cyclic AMP and phorbol myristate acetate of cephaloridine-induced injury in rat renal cortical slices.

    PubMed

    Kohda, Y; Gemba, M

    2001-01-01

    Intracellular signaling pathways of cAMP and protein kinase C (PKC) have been suggested to modulate the generation of free radicals. We investigated the effects of cAMP and phorbol myristate acetate (PMA), a PKC activator, on cephaloridine (CER)-induced renal cell injury, which has been reported to be due to the generation of free radicals. Incubation of rat renal cortical slices with CER resulted in increases in lipid peroxidation and lactate dehydrogenase (LDH) release and in decreases in gluconeogenesis and p-aminohippurate (PAH) accumulation in rat renal cortical slices, suggesting free radical-induced injury in slices exposed to CER. A derivative of cAMP ameliorated not only the increase in lipid peroxidation but also the renal cell damage induced by CER. This amelioration by a cAMP derivative of lipid peroxidation and renal cell damage caused by CER was blocked by KT 5720, a protein kinase A (PKA) inhibitor. Lipid peroxidation and the indices of cell injury were increased by PMA. PMA also enhanced CER-induced lipid peroxidation and cell damage in the slices. This enhancement by PMA of CER-induced injury was blocked by H-7, a PKC inhibitor. These results indicated that intracellular signaling pathways of cAMP and PKC modulate free radical-mediated nephrotoxicity induced by CER.

  10. Dmrt1 Expression Is Regulated by Follicle-Stimulating Hormone and Phorbol Esters in Postnatal Sertoli Cells*

    PubMed Central

    CHEN, JIANG KAI; HECKERT, LESLIE L.

    2006-01-01

    Dmrt1 is a recently described gene that is expressed exclusively in the testis and is required for postnatal testis differentiation. Here we describe the expression of Dmrt1 in postnatal rat testis and Sertoli cells. RNase protection analysis was used to examine Dmrt1 messenger RNA (mRNA) levels in intact testis during postnatal development and in primary cultures of Sertoli cells under various culture conditions. We show that Dmrt1 mRNA levels rise significantly beginning approximately 10 days after birth and remain elevated until after the third postnatal week. Thereafter, mRNA levels drop coincident with the proliferation of germ cells in the testis. In freshly isolated Sertoli cells, Dmrt1 mRNA levels were robust but decreased significantly when the cells were placed in culture for 24 h. Treatment of Sertoli cells with either FSH or 8-bromo-cAMP resulted in a significant rise in Dmrt1 mRNA levels. This cAMP response was sensitive to treatment with the transcriptional inhibitor actinomycin D but not to the translational inhibitor cycloheximide. The cAMP-dependent rise in Dmrt1 mRNA also required activation of protein kinase A, as mRNA induction was sensitive to the inhibitor H89. Studies also show that Dmrt1 expression was inhibited by phorbol esters (PMA) but only modestly effected by serum. PMID:11181532

  11. Morphine enhances macrophage apoptosis.

    PubMed

    Singhal, P C; Sharma, P; Kapasi, A A; Reddy, K; Franki, N; Gibbons, N

    1998-02-15

    Laboratory data indicate that morphine decreases the numbier of peritoneal and alveolar macrophages (Mphi) and compromises their phagocytic capability for immune complexes and bacteria. We hypothesize that morphine decreases the number of, as well as compromises the phagocytic capability of, Mphi by programming their death. We studied the effect of morphine on Mphi apoptosis in vivo as well as in vitro. Peritoneal Mphi harvested from morphine-treated rats showed DNA fragmentation. Morphine enhanced murine Mphi (J 774.16) apoptosis in a dose-dependent manner. Human monocytes treated with morphine showed a classic ladder pattern in gel electrophoretic and end-labeling studies. Morphine promoted nitric oxide (NO) production both under basal and LPS-activated states. N(G)-nitro-L-arginine methyl ester (L-NAME) and N(G)-monomethyl-L-arginine monoacetate (L-NMMA), inhibitors of NO synthase, attenuated the morphine-induced generation of NO by Mphi. Morphine also enhanced Mphi mRNA expression of inducible NO synthase (iNOS). Since morphine-induced Mphi apoptosis was inhibited by L-NAME and L-NMMA, it appears that morphine-induced Mphi apoptosis may be mediated through the generation of NO. Morphine promoted the synthesis of Bax and p53 proteins by Mphi. Moreover, IL-converting enzyme (ICE)-1 inhibitor attenuated morphine-induced Mphi apoptosis. These studies suggest that morphine activates the induction phase of the apoptotic pathway through accumulation of p53. The effector phase of morphine-induced apoptosis appears to proceed through the accumulation of Bax and activation of ICE-1. The present study provides a basis for a hypothesis that morphine may be directly compromising immune function by promoting Mphi apoptosis in patients with opiate addiction.

  12. Spaceflight Associated Apoptosis

    NASA Technical Reports Server (NTRS)

    Ichiki, Albert T.; Gibson, Linda A.; Allebban, Zuhair

    1996-01-01

    Lymphoid tissues have been shown to atrophy in rats flown on Russian spaceflights. Histological examination indicated evidence for cell degradation. Lymphoid tissues from rats flown on Spacelab Life Sciences-2 mission were analyzed for apoptosis by evidence of fragmented lymphocytes, which could be engulfed by macrophages, or DNA strand breaks using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. Apoptosis was not detected in the thymus and spleen collected inflight or from the synchronous ground rats but was detected in the thymus, spleen and inguinal lymph node of the flight animals on recovery. These results indicate that the apoptosis observed in the lymphatic tissues of the rats on recovery could have been induced by the gravitational stress of reentry, corroborating the findings from the early space-flight observations.

  13. Glutathione and apoptosis

    PubMed Central

    Circu, Magdalena L.; Yee Aw, Tak

    2011-01-01

    Apoptosis or programmed cell death represents a physiologically conserved mechanism of cell death that is pivotal in normal development and tissue homeostasis in all organisms. As a key modulator of cell functions, the most abundant non-protein thiol, glutathione (GSH), has important roles in cellular defense against oxidant aggression, redox regulation of proteins thiols and maintaining redox homeostasis that is critical for proper function of cellular processes, including apoptosis. Thus, a shift in the cellular GSH-to-GSSG redox balance in favour of the oxidized species, GSSG, constitutes an important signal that could decide the fate of a cell. The current review will focus on three main areas: (1) general description of cellular apoptotic pathways, (2) cellular compartmentation of GSH and the contribution of mitochondrial GSH and redox proteins to apoptotic signalling and (3) role of redox mechanisms in the initiation and execution phases of apoptosis. PMID:18671159

  14. Effect of tannic acid, resveratrol and its derivatives, on oxidative damage and apoptosis in human neutrophils.

    PubMed

    Zielińska-Przyjemska, Małgorzata; Ignatowicz, Ewa; Krajka-Kuźniak, Violetta; Baer-Dubowska, Wanda

    2015-10-01

    In this study we compared the antioxidant and DNA protective activity of tannic acid and stilbene derivatives, resveratrol, 3,5,4(')-trimethoxystilbene (TMS) and pterostilbene in human neutrophils stimulated to oxidative burst by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in relation to apoptosis induction. All polyphenols within the concentration range 1-100 μM reduced the intracellular ROS and H2O2 production in the TPA-stimulated cells. Tannic acid was the most effective polyphenol in protection against DNA damage induced by TPA. In the resting neutrophils resveratrol and to lesser extent other polyphenols increased DNA damage and increased the level of p53. Pretreatment of the TPA-stimulated cells with tannic acid or stilbenes led to the induction of apoptosis. The most significant effect was observed as a result of treatment with TMS and resveratrol. These compounds appeared the most effective inducers of p53 in the TPA-challenged neutrophils, what may suggest that pro-apoptotic activity of these stilbenes might be related to p53 activation. Overall, the results of our present study demonstrate that tannic acid and stilbenes modulate the ROS production, ultimately leading to cell apoptosis in human neutrophils stimulated to oxidative burst. In resting neutrophils they exhibit pro-oxidant activity, which is accompanied by p53 induction.

  15. The biochemistry of apoptosis.

    PubMed

    Hengartner, M O

    2000-10-12

    Apoptosis--the regulated destruction of a cell--is a complicated process. The decision to die cannot be taken lightly, and the activity of many genes influence a cell's likelihood of activating its self-destruction programme. Once the decision is taken, proper execution of the apoptotic programme requires the coordinated activation and execution of multiple subprogrammes. Here I review the basic components of the death machinery, describe how they interact to regulate apoptosis in a coordinated manner, and discuss the main pathways that are used to activate cell death.

  16. Soya isoflavones suppress phorbol 12-myristate 13-acetate-induced COX-2 expression in MCF-7 cells.

    PubMed

    Lau, Tak Yi; Leung, Lai K

    2006-07-01

    Epidemiological studies indicate that Asian women have a lower incidence of breast cancer compared with their counterparts in the West, and soya consumption has been suggested as a contributory factor. Clinical and animal studies have revealed that cyclooxygenase-2 (COX-2) expression is associated with a risk of breast cancer. In the present study, we investigated the effect of soya isoflavones on the expression of COX-2 in the breast cell line MCF-7. Genistein, daidzein and equol were found to inhibit COX-2 expression induced by phorbol 12-myristate 13-acetate (PMA). Similar findings were observed in the COX-2 protein analysis. In order to study transcriptional control, a fragment of the 5'-flanking region of the hCOX-2 gene was amplified and inserted into a firefly luciferase reporter plasmid. The reporter assay indicated that the transactivation of the hCOX-2 promoter was induced by PMA, and activity was inhibited with the co-administration of genistein, daidzein or equol. An activator protein-1 (AP-1)/cyclic AMP response element binding protein (CREB) binding site (-59/-53) was identified in hCOX-2 promoter, and this could be critical in PMA-induced COX-2 expression. Truncation reporter plasmids with (-70/-36) and without (-51/-36) AP-1/CREB were constructed for subsequent analysis. The results revealed that the hCOX-2 promoter transactivation suppressed by isoflavone could be dependent on AP-1/CREB binding. Nonetheless, this study illustrated that the soya isoflavones reduced COX-2 expression, which could be important in the post-initiation events of breast carcinogenesis.

  17. Phorbol ester stimulation of RasGRP1 regulates the sodium-chloride cotransporter by a PKC-independent pathway

    PubMed Central

    Ko, Benjamin; Joshi, Leena M.; Cooke, Leslie L.; Vazquez, Norma; Musch, Mark W.; Hebert, Steven C.; Gamba, Gerardo; Hoover, Robert S.

    2007-01-01

    The sodium-chloride cotransporter (NCC) is the principal salt-absorptive pathway in the mammalian distal convoluted tubule (DCT) and is the site of action of one of the most effective classes of antihypertensive medications, thiazide diuretics. We developed a cell model system to assess NCC function in a mammalian cell line that natively expresses NCC, the mouse DCT (mDCT) cell line. We used this system to study the complex regulation of NCC by the phorbol ester (PE) 12-O-tetradecanoylphorbol-13-acetate (TPA), a diacylglycerol (DAG) analog. It has generally been thought that PEs mediate their effects on transporters through the activation of PKC. However, there are at least five other DAG/PE targets. Here we describe how one of those alternate targets of DAG/PE effects, Ras guanyl-releasing protein 1 (RasGRP1), mediates the PE-induced suppression of function and the surface expression of NCC. Functional assessment of NCC by using thiazide-sensitive 22Na+ uptakes revealed that TPA completely suppresses NCC function. Biotinylation experiments demonstrated that this result was primarily because of decreased surface expression of NCC. Although inhibitors of PKC had no effect on this suppression, MAPK inhibitors completely prevented the TPA effect. RasGRP1 activates the MAPK pathway through activation of the small G protein Ras. Gene silencing of RasGRP1 prevented the PE-mediated suppression of NCC activity, the activation of the H-Ras isoform of Ras, and the activation of ERK1/2 MAPK. This finding confirmed the critical role of RasGRP1 in mediating the PE-induced suppression of NCC activity through the stimulation of the MAPK pathway. PMID:18077438

  18. Protein kinase C-associated kinase (PKK) mediates Bcl10-independent NF-kappa B activation induced by phorbol ester.

    PubMed

    Muto, Akihiro; Ruland, Jürgen; McAllister-Lucas, Linda M; Lucas, Peter C; Yamaoka, Shoji; Chen, Felicia F; Lin, Amy; Mak, Tak W; Núñez, Gabriel; Inohara, Naohiro

    2002-08-30

    Protein kinase C-associated kinase (PKK) is a recently described kinase of unknown function that was identified on the basis of its specific interaction with PKC beta. PKK contains N-terminal kinase and C-terminal ankyrin repeats domains linked to an intermediate region. Here we report that the kinase domain of PKK is highly homologous to that of two mediators of nuclear factor-kappa B (NF-kappa B) activation, RICK and RIP, but these related kinases have different C-terminal domains for binding to upstream factors. We find that expression of PKK, like RICK and RIP, induces NF-kappa B activation. Mutational analysis revealed that the kinase domain of PKK is essential for NF-kappa B activation, whereas replacement of serine residues in the putative activation loop did not affect the ability of PKK to activate NF-kappa B. A catalytic inactive PKK mutant inhibited NF-kappa B activation induced by phorbol ester and Ca(2+)-ionophore, but it did not block that mediated by tumor necrosis factor alpha, interleukin-1 beta, or Nod1. Inhibition of NF-kappa B activation by dominant negative PKK was reverted by co-expression of PKC beta I, suggesting a functional association between PKK and PKC beta I. PKK-mediated NF-kappa B activation required IKK alpha and IKK beta but not IKK gamma, the regulatory subunit of the IKK complex. Moreover, NF-kappa B activation induced by PKK was not inhibited by dominant negative Bimp1 and proceeded in the absence of Bcl10, two components of a recently described PKC signaling pathway. These results suggest that PKK is a member of the RICK/RIP family of kinases, which is involved in a PKC-activated NF-kappa B signaling pathway that is independent of Bcl10 and IKK gamma.

  19. Induction of phosphorylation and cell surface redistribution of acetylcholine receptors by phorbol ester and carbamylcholine in cultured chick muscle cells

    PubMed Central

    1988-01-01

    We have investigated the mechanisms regulating the clustering of nicotinic acetylcholine receptor (AChR) on the surface of cultured embryonic chick muscle cells. Treatment of these cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, was found to cause a rapid dispersal of AChR clusters, as monitored by fluorescence microscopy of cells labeled with tetramethylrhodamine-conjugated alpha-bungarotoxin. The loss of AChR clusters was not accompanied by an appreciable change in the amount of AChR on the surface of these cells, as measured by the specific binding of [125I]Bgt. Analysis of the phosphorylation pattern of immunoprecipitable AChR subunits showed that the gamma- and delta- subunits are phosphorylated by endogenous protein kinase activity in the intact muscle cells, and that the delta-subunit displays increased phosphorylation in response to TPA. Structural analogues of TPA which do not stimulate protein kinase C have no effect on AChR surface topography or phosphorylation. Exposure of chick myotubes to the cholinergic agonist carbamylcholine was found to cause a dispersal of AChR clusters with a time course similar to that of TPA. Like TPA, carbamylcholine enhances the phosphorylation of the delta-subunit of AChR. The carbamylcholine-induced redistribution and phosphorylation of AChR is blocked by the nicotinic AChR antagonist d-tubocurarine. TPA and carbamylcholine have no effect on cell morphology during the time- course of these experiments. These findings indicate that cell surface topography of AChR may be regulated by phosphorylation of its subunits and suggest a mechanism for dispersal of AChR clusters by agonist activation. PMID:3417778

  20. Phorbol 12-myristate 13-acetate induces protein kinase ceta-specific proliferative response in astrocytic tumor cells.

    PubMed

    Hussaini, I M; Karns, L R; Vinton, G; Carpenter, J E; Redpath, G T; Sando, J J; VandenBerg, S R

    2000-07-21

    Protein kinase C (PKC) activation has been implicated in cellular proliferation in neoplastic astrocytes. The roles for specific PKC isozymes in regulating this glial response, however, are not well understood. The aim of this study was to characterize the expression of PKC isozymes and the role of PKC-eta expression in regulating cellular proliferation in two well characterized astrocytic tumor cell lines (U-1242 MG and U-251 MG) with different properties of growth in cell culture. Both cell lines expressed an array of conventional (alpha, betaI, betaII, and gamma) and novel (theta and epsilon) PKC isozymes that can be activated by phorbol myristate acetate (PMA). Another novel PKC isozyme, PKC-eta, was only expressed by U-251 MG cells. In contrast, PKC-delta was readily detected in U-1242 MG cells but was present only at low levels in U-251 MG cells. PMA (100 nm) treatment for 24 h increased cell proliferation by over 2-fold in the U-251 MG cells, whereas it decreased the mitogenic response in the U-1242 MG cells by over 90%. When PKC-eta was stably transfected into U-1242 MG cells, PMA increased cell proliferation by 2.2-fold, similar to the response of U-251 MG cells. The cell proliferation induced by PMA in both the U-251 MG and U-1242-PKC-eta cells was blocked by the PKC inhibitor bisindolylmaleimide (0.5 micrometer) and the MEK inhibitor, PD 98059 (50 micrometer). Transient transfection of wild type U-251 with PKC-eta antisense oligonucleotide (1 micrometer) also blocked the PMA-induced increase in [(3)H]thymidine incorporation. The data demonstrate that two glioblastoma lines, with functionally distinct proliferative responses to PMA, express different novel PKC isozymes and that the differential expression of PKC-eta plays a determining role in the different proliferative capacity.

  1. Calcium ionophore and phorbol ester activation of proliferation and. gamma. -IFN production by neonatal mononuclear cells (MNCs)

    SciTech Connect

    Bryson, Y.J.; Kuhls, T.L.; Pineda, E.

    1986-03-01

    Human neonatal MNCs have a dissociation between prolif. and ..gamma..-IFN prod. Although cord MNCs display normal-high prolif. following lectin stim., ..gamma..-IFN prod. is greatly diminished compared to adult MNCs. Increasing data support a 2-stimuli requirement for human T-cell activation as noted in the T-cell line Jurkat as well as in peripheral T-cells. They have compared prolif. and ..gamma..-IFN responses of cord and adult MNCs to the calcium ionophore A23187, phorbol myristate acetate (PMA), PHA and their combinations. Cord and adult MNCs had similar prolif. responses to A23187, PMA and PHA. PMA alone acted as a weak mitogen compared to PHA. Optimal A23187 alone caused very low amts of prolif. Either PMA or A23187 suppressed PHA-stim. prolif. while A23187 augmented PMA-induced prolif. A23187, PMA or PHA alone prod. ..gamma..-IFN in adult but not cord MNCs. The addition of PMA or A23187 augmented the PHA-induced ..gamma..-IFN prod. in both cord and adult MNCs (6..-->..80 IU vs 240..-->..480 IU resp). When combined, A23187 and PMA stim. optimal and comparable amts of ..gamma..-IFN in adult and cord MNCs (480 IU). From these findings they conclude that although the stimuli for ..gamma..-IFN and prolif. may be similar, there is an absolute requirement for 2 stimuli (PMA/A23187) for ..gamma..-IFN prod. by cord cells and optimal prod. in adult MNCs. The defect of ..gamma..-IFN prod. observed in PHA stim. neonatal MNCs can be corrected using a calcium ionophore and protein kinase C activator.

  2. 4α-phorbol 12,13-didecanoate activates cultured mouse dorsal root ganglia neurons independently of TRPV4

    PubMed Central

    Alexander, R; Kerby, A; Aubdool, AA; Power, AR; Grover, S; Gentry, C; Grant, AD

    2013-01-01

    Background and Purpose The Ca2+-permeable cation channel TRPV4 is activated by mechanical disturbance of the cell membrane and is implicated in mechanical hyperalgesia. Nerve growth factor (NGF) is increased during inflammation and causes mechanical hyperalgesia. 4α-phorbol 12,13-didecanoate (4αPDD) has been described as a selective TRPV4 agonist. We investigated NGF-induced hyperalgesia in TRPV4 wild-type (+/+) and knockout (–/–) mice, and the increases in [Ca2+]i produced by 4αPDD in cultured mouse dorsal root ganglia neurons following exposure to NGF. Experimental Approach Withdrawal thresholds to heat, von Frey hairs and pressure were measured in mice before and after systemic administration of NGF. Changes in intracellular Ca2+ concentration were measured by ratiometric imaging with Fura-2 in cultured DRG and trigeminal ganglia (TG) neurons during perfusion of TRPV4 agonists. Key Results Administration of NGF caused a significant sensitization to heat and von Frey stimuli in TRPV4 +/+ and –/– mice, but only TRPV4 +/+ mice showed sensitization to noxious pressure. 4αPDD stimulated a dose-dependent increase in [Ca2+]i in neurons from +/+ and –/– mice, with the proportion of responding neurons and magnitude of increase unaffected by the genotype. In contrast, the selective TRPV4 agonist GSK1016790A failed to stimulate an increase in intracellular Ca2+ in cultured neurons. Responses to 4αPDD were unaffected by pretreatment with NGF. Conclusions and Implications TRPV4 contributes to mechanosensation in vivo, but there is little evidence for functional TRPV4 in cultured DRG and TG neurons. We conclude that 4αPDD activates these neurons independently of TRPV4, so it is not appropriate to refer to 4αPDD as a selective TRPV4 agonist. PMID:22928864

  3. Multiple effects of phorbol esters on hormone-sensitive adenylate cyclase activity in S49 lymphoma cells

    SciTech Connect

    Bell, J.D.; Brunton, L.L.

    1987-06-01

    In S49 lymphoma cells, 12-O-tetradecanoyl phorbol-13-acetate (TPA) enhances adenylate cyclase activity and doubles cAMP accumulation in response to ..beta..-adrenergic stimulation at 37/sup 0/C, putatively via the action of protein kinase C. at 27/sup 0/C, TPA has the opposite effect, inhibiting cAMP production in response to isoproterenol by approx. 25%. TPA also inhibits the response to prostaglandin E/sub 1/ (PGE/sub 1/), another stimulant of hormone-sensitive adenylate cyclase in these cells, by 30% at 37/sup 0/C and almost 50% at 27/sup 0/C. In contrast, TPA enhances responses to forskolin and cholera toxin at both 27 and 37/sup 0/C. In membranes from cells treated with TPA, PGE/sub 1/-stimulated adenylate cyclase activity is inhibited by 50%, whereas the catalytic activity stimulated by NaF or forskolin is enhanced. TPA reduces the potency of both PGE/sub 1/ and isoproterenol for cAMP generation by 50%. TPA causes a similar decrease in ..beta..-adrenergic agonist affinity with no reduction in the density of either antagonist of agonist binding sites in wild type cells and in cells lacking the ..cap alpha..-subunit of the stimulatory transducer protein (G/sub s/) (cyc/sup -/) or lacking functional receptor G/sub s/ coupling (UNC). Therefore, TPA has at least three functionally distinct effects on hormone-sensitive adenylate cyclase in S49 cells. The authors conclude that multiple and opposing effects of TPA on hormone-sensitive adenylate cyclase occur simultaneously within the same cell, affecting the responses to several agonists differently. In addition, the data offer a mechanism by which a cell can achieve heterogeneous efficacies to hormones that activate adenylate cyclase.

  4. Enhancement of adenylate cyclase activity by phorbol ester: effects on the inhibitory pathway in S49 lymphoma cells

    SciTech Connect

    Bell, J.D.; Brunton, L.L.

    1986-05-01

    12-0-tetradecanoylphorbol-13-acetate (TPA) enhances the apparent V/sub max/ of adenylate cyclase (AC) in S49 lymphoma cells. This effect does not result from an increased rate of activation of the catalytic subunit by the stimulatory GTP binding transducer protein (G/sub s/). In wild type (WT) membranes this enhancement seems to involve a GTP binding protein since TPA enhances forskolin-stimulated AC activity by 30% in the presence of GTP (10 ..mu..M) or Gpp(NH)p (1 ..mu..M) but not in the absence of guanine nucleotide. The authors obtain comparable results in the cyc- variant that lacks the GTP binding subunit of G/sub s/ responsible for stimulating AC, suggesting the importance of a different GTP binding protein. Blockade of the activity of the inhibitory GTP binding protein (G/sub i/) by high concentrations of Mg/sup + +/ (approx.100 mM) or Mn/sup + +/ (approx.1 mM) abolishes the effect of TPA to enhance AC activity in WT membranes. The time course of Gpp(NH)p-mediated inhibition of AC reveals a characteristic lag prior to steady state, indicative of the rate of G/sub i/ activation; TPA increases this lag 3-4 fold. The authors conclude that reduction in the rate of activation of G/sub i/ by guanine nucleotide is one mechanism by which phorbol esters enhance guanine nucleotide-dependent activity of AC, hypothetically via the phosphorylation of G/sub i/ by protein kinase C.

  5. Differential regulation by phorbol ester of formyl-methionyl peptide and leukotriene B sub 4 receptors on human neutrophils

    SciTech Connect

    Goldman, D.W.; Chung, S.; Richards, S. )

    1991-03-15

    Activation of protein kinase C (PKC) with suboptimal does of phorbol myristyl acetate (PMA) will increase fMP receptor expression with parallel potentiation of superoxide generation. PMA-induced changes in leukotriene B{sub 4} (LTB{sub 4}) receptor expression were assessed in parallel with fMP receptor expression to determine if these two independent receptor classes are regulated in a similar manner by PKC. The relative density of fMP receptors was assessed by flow cytometry. The relative density of receptors for LTB{sub 4} was quantitated by incubating 2 {times} 10{sup 6} Ns with 10nM({sup 3}H)-LTB{sub 4} and determining the amount of radioactivity bound after filtration on glass fiber filters. Incubation of N with 10ng/mL PMA induced a 3.2-fold increase in fMP receptor expression by 5 min which was sustained for up to 15 min. In contrast, LTB{sub 4} receptor density decreased by 36% within 5 min. in response to 10 ng/mL PMA. Staurosporine, a potent antagonist of PKC, had no effect of fMP receptor expression but markedly enhanced LTB{sub 4} receptor expression by 1.7-fold at 200nM. PKC acts to decrease the surface expression of LTB{sub 4} receptors in contrast to the enhancement of fMP receptor expression, suggesting in contrast to the enhancement of fMP receptor expression, suggesting that potentiation of N function by PMA may be stimulus-specific.

  6. Role of Daxx in Apoptosis

    DTIC Science & Technology

    2002-07-01

    protection against cancer . We previously identified a pro-apoptotic protein Daxx (1). Daxx can be found in large amount in the promyelocytic leukemia protein...Words) Control of the activation of apoptosis is important in protection against cancer . PML oncogenic domains (PODs) are subnuclear macromolecular...apoptosis, and may help identify new approaches to modulate apoptosis for cancer therapy. 14. SUBJECT TERMS 15. NUMBER OF PAGES apoptosis, cancer , PML

  7. Induction of bcl-2 expression by phosphorylated CREB proteins during B-cell activation and rescue from apoptosis.

    PubMed Central

    Wilson, B E; Mochon, E; Boxer, L M

    1996-01-01

    Engagement of surface immunoglobulin on mature B cells leads to rescue from apoptosis and to proliferation. Levels of bcl-2 mRNA and protein increase with cross-linking of surface immunoglobulin. We have located the major positive regulatory region for control of bcl-2 expression in B cells in the 5'-flanking region. The positive region can be divided into an upstream and a downstream regulatory region. The downstream regulatory region contains a cyclic AMP-responsive element (CRE). We show by antibody supershift experiments and UV cross-linking followed by denaturing polyacrylamide gel electrophoresis that both CREB and ATF family members bind to this region in vitro. Mutations of the CRE site that result in loss of CREB binding also lead to loss of functional activity of the bcl-2 promoter in transient-transfection assays. The presence of an active CRE site in the bcl-2 promoter implies that the regulation of bcl-2 expression is linked to a signal transduction pathway in B cells. Treatment of the mature B-cell line BAL-17 with either anti-immunoglobulin M or phorbol 12-myristate 13-acetate leads to an increase in bcl-2 expression that is mediated by the CRE site. Treatment of the more immature B-cell line, Ramos, with phorbol esters rescues the cells from calcium-dependent apoptosis. bcl-2 expression is increased following phorbol ester treatment, and the increased expression is dependent on the CRE site. These stimuli result in phosphorylation of CREB at serine 133. The phosphorylation of CREB that results in activation is mediated by protein kinase C rather than by protein kinase A. Although the CRE site is necessary, optimal induction of bcl-2 expression requires participation of the upstream regulatory element, suggesting that phosphorylation of CREB alters its interaction with the upstream regulatory element. The CRE site in the bcl-2 promoter appears to play a major role in the induction of bcl-2 expression during the activation of mature B cells and during

  8. Is Phosphorylation of the α1 Subunit at Ser-16 Involved in the Control of Na,K-ATPase Activity by Phorbol Ester–activated Protein Kinase C?

    PubMed Central

    Féraille, Eric; Béguin, Pascal; Carranza, Maria-Luisa; Gonin, Sandrine; Rousselot, Martine; Martin, Pierre-Yves; Favre, Hervé; Geering, Käthi

    2000-01-01

    The α1 subunit of Na,K-ATPase is phosphorylated at Ser-16 by phorbol ester-sensitive protein kinase(s) C (PKC). The role of Ser-16 phosphorylation was analyzed in COS-7 cells stably expressing wild-type or mutant (T15A/S16A and S16D-E) ouabain-resistant Bufo α1 subunits. In cells incubated at 37°C, phorbol 12,13-dibutyrate (PDBu) inhibited the transport activity and decreased the cell surface expression of wild-type and mutant Na,K-pumps equally (∼20–30%). This effect of PDBu was mimicked by arachidonic acid and was dependent on PKC, phospholipase A2, and cytochrome P450-dependent monooxygenase. In contrast, incubation of cells at 18°C suppressed the down-regulation of Na,K-pumps and revealed a phosphorylation-dependent stimulation of the transport activity of Na,K-ATPase. Na,K-ATPase from cells expressing α1-mutants mimicking Ser-16 phosphorylation (S16D or S16E) exhibited an increase in the apparent Na affinity. This finding was confirmed by the PDBu-induced increase in Na sensitivity of the activity of Na,K-ATPase measured in permeabilized nontransfected COS-7 cells. These results illustrate the complexity of the regulation of Na,K-ATPase α1 isozymes by phorbol ester-sensitive PKCs and reveal 1) a phosphorylation-independent decrease in cell surface expression and 2) a phosphorylation-dependent stimulation of the transport activity attributable to an increase in the apparent Na affinity. PMID:10637289

  9. Convergent assembly of highly potent analogues of bryostatin 1 via pyran annulation: bryostatin look-alikes that mimic phorbol ester function.

    PubMed

    Keck, Gary E; Kraft, Matthew B; Truong, Anh P; Li, Wei; Sanchez, Carina C; Kedei, Noemi; Lewin, Nancy E; Blumberg, Peter M

    2008-05-28

    Highly potent bryostatin analogues which contain the complete bryostatin core structure have been synthesized using a pyran annulation approach as a key strategic element. The A ring pyran was assembled using a pyran annulation reaction between a C1-C8 hydroxy allylsilane and an aldehyde comprising C9-C13. This pyran was transformed to a new hydroxy allylsilane and then coupled with a preformed C ring aldehyde subunit in a second pyran annulation, with concomitant formation of the B ring. This tricyclic intermediate was elaborated to bryostatin analogues which displayed nanomolar to subnanomolar affinity for PKC, but displayed properties indistinguishable from a phorbol ester in a proliferation/attachment assay.

  10. Gadolinium induces macrophage apoptosis.

    PubMed

    Mizgerd, J P; Molina, R M; Stearns, R C; Brain, J D; Warner, A E

    1996-02-01

    Gadolinium (Gd) suppresses reticuloendothelial functions in vivo by unknown mechanisms. In vitro exposure of rat alveolar macrophages to GdCl3.6H20 caused cell death, as measured by trypan blue permeability, in both dose- and time-dependent fashions. Even a 10-min exposure to Gd caused significant cell death by 24 h. The morphology of Gd-treated cells, pyknosis and karyorrhexis prior to loss of membrane integrity, suggested apoptosis. Upon flow cytometric examination, Gd-treated propidium iodide-excluding cells demonstrated light scatter changes characteristic of apoptotic cells (decreased forward and increased right angle scatter). Gel electrophoresis of DNA from Gd-treated macrophages clearly showed the ladder pattern unique to apoptotic cells. Electron-dense structures containing Gd were observed via electron spectroscopic imaging within phagosomes and also within nuclei (associated with condensed chromatin). Gadolinium, endocytosed by macrophages and distributed to nuclei, causes apoptosis of macrophages in vitro.

  11. Genetic conflict and apoptosis.

    PubMed

    LeGrand, E K

    2001-01-01

    The benefits of apoptosis in the removal of unnecessary, damaged, or dangerous cells are dependent on the altruism resulting from the absence of genetic conflict between genes in cells. However, this altruism can be exploited by self-promoting or ultra-selfish genes. These self-promoting genes can be endogenous, as with neoplasia or germ cell mutations, or exogenous, as with cellular pathogens. The fundamental flaw of apoptosis is that its development and maintenance as a system is constantly opposed by the emergence of self-promoting genes. Since apoptotically impaired cells cannot be relied on to kill themselves, apoptotic input from other cells is required for controlling self-promoting genes. Certain unique features of germ cell development, such as linkage by cytoplasmic bridges and the requirement for granulosa or Sertoli cells, appear to serve this requirement for control of self-promoting genes.

  12. Mortalin, Apoptosis, and Neurodegeneration

    PubMed Central

    Londono, Carolina; Osorio, Cristina; Gama, Vivian; Alzate, Oscar

    2012-01-01

    Mortalin is a highly conserved heat-shock chaperone usually found in multiple subcellular locations. It has several binding partners and has been implicated in various functions ranging from stress response, control of cell proliferation, and inhibition/prevention of apoptosis. The activity of this protein involves different structural and functional mechanisms, and minor alterations in its expression level may lead to serious biological consequences, including neurodegeneration. In this article we review the most current data associated with mortalin’s binding partners and how these protein-protein interactions may be implicated in apoptosis and neurodegeneration. A complete understanding of the molecular pathways in which mortalin is involved is important for the development of therapeutic strategies for cancer and neurodegenerative diseases. PMID:24970131

  13. Evading apoptosis in cancer.

    PubMed

    Fernald, Kaleigh; Kurokawa, Manabu

    2013-12-01

    Carcinogenesis is a mechanistically complex and variable process with a plethora of underlying genetic causes. Cancer development comprises a multitude of steps that occur progressively starting with initial driver mutations leading to tumorigenesis and, ultimately, metastasis. During these transitions, cancer cells accumulate a series of genetic alterations that confer on the cells an unwarranted survival and proliferative advantage. During the course of development, however, cancer cells also encounter a physiologically ubiquitous cellular program that aims to eliminate damaged or abnormal cells: apoptosis. Thus, it is essential that cancer cells acquire instruments to circumvent programmed cell death. Here we discuss emerging evidence indicating how cancer cells adopt various strategies to override apoptosis, including amplifying the antiapoptotic machinery, downregulating the proapoptotic program, or both. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Evading apoptosis in cancer

    PubMed Central

    Fernald, Kaleigh

    2013-01-01

    Carcinogenesis is a mechanistically complex and variable process with a plethora of underlying genetic causes. Cancer development consists of a multitude of steps that occur progressively starting with initial driver mutation(s), to tumorigenesis, and ultimately metastasis. During these transitions, cancer cells accumulate a series of genetic alterations that confer upon the cells an unwarranted survival and proliferative advantage. During the course of development, however, cancer cells also encounter a physiologically ubiquitous cellular program that aims to eliminate damaged or abnormal cells: Apoptosis. Thus, it is essential that cancer cells acquire instruments to circumvent programmed cell death. Here we discuss emerging evidence indicating how cancer cells adopt various strategies to override apoptosis including amplifying the anti-apoptotic machinery, downregulating the pro-apoptotic program, or both. PMID:23958396

  15. Inhibitory effect of inositol hexaphosphate on metalloproteinases transcription in colon cancer cells stimulated with phorbol-12-myristate 13-acetate.

    PubMed

    Kapral, Małgorzata; Wawszczyk, Joanna; Hollek, Andrzej; Dymitruk, Dominika; Weglarz, Ludmiła

    2012-01-01

    Inositol hexaphosphate (IP6) is a naturally occurring phytochemical, found in abundance in cereals, legumes and other high-fiber-content diets. IP6 has shown promising efficacy against a wide range of cancers. Its anti-cancer activity involves anti-proliferative, pro-apoptotic and anti-metastatic effects. Both matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs), are implicated in tumor growth, metastasis, and angiogenesis. Phorbol-12-myristate 13-acetate (PMA) is a well-known inflammatory stimulator and tumor promoter that activates PKC and increases the invasiveness of various types of cancer cells by activating MMPs. The aim of the present study was to examine the influence of IP6 on the expression of selected MMPs, i.e., MMP-1, -2, -3, -9, 10, -13 and their TIMP-1 and -2 in unstimulated and PMA-stimulated colon cancer cell line Caco-2. Quantification of genes expression in Caco-2 cells treated with 100 ng/mL of PMA, 2.5 mM of IP6 and both for 6 and 12 h was carried out using real time QRT-PCR technique. Stimulation of cells with PMA resulted in an up-expression of MMP-2, MMP-3, MMP-9, MMP-10, MMP-13 and TIMP-1 mRNAs and decrease in MMP-1 gene expression. The quantity of TIMP-2 transcript was reduced by PMA. A significant decrease in MMP-2, MMP-3, MMP-10, MMP-13, and TIMP-1 expression in response to IP6 was observed. IP6 down-regulated MMP-9 transcription induced by PMA and decreased the level of both MMP-2 and MMP-3 mRNAs in PMA-stimulated cells. Caco-2 treated with both PMA and IP6 showed a significant decrease in MMP-1 expression in comparison to PMA-stimulated cells. The results of this study show that PMA can modulate MMP and TIMP genes transcription in colon cancer cells Caco-2. IP6 exerts an influence of basal mRNA expression of some MMPs and their tissue inhibitors and down-regulates MMP-1, MMP-2, MMP-3 and MMP-9 in cells treated with PMA. IP6 could be an effective anti-metastatic agent that suppresses expression of MMP genes at

  16. Apoptosis in canine distemper.

    PubMed

    Moro, L; de Sousa Martins, A; de Moraes Alves, C; de Araújo Santos, F G; dos Santos Nunes, J E; Carneiro, R A; Carvalho, R; Vasconcelos, A C

    2003-01-01

    Canine distemper is a systemic viral disease characterized by immunosuppression followed by secondary infections. Apoptosis is observed in several immunosuppressive diseases and its occurrence on canine distemper in vivo has not been published. In this study, the occurrence of apoptosis was determined in lymphoid tissues of thirteen naturally infected dogs and nine experimentally inoculated puppies. Healthy dogs were used as negative controls. Samples of lymph nodes, thymus, spleen and brain were collected for histopathological purposes. Sections, 5 microm thick, of retropharingeal lymph nodes were stained by HE, Shorr, Methyl Green-Pyronin and TUNEL reaction. Shorr stained sections were further evaluated by morphometry. Canine distemper virus nucleoprotein was detected by immunohistochemistry. Retropharingeal lymph nodes of naturally and experimentally infected dogs had more apoptotic cells per field than controls. In addition, DNA from thymus of infected dogs were more fragmented than controls. Therefore, apoptosis is increased in lymphoid depletion induced by canine distemper virus and consequently play a role in the immunosuppression seen in this disease.

  17. Apoptosis, fibrosis and senescence.

    PubMed

    Portilla, Didier

    2014-01-01

    Fibrosis is a major hallmark of progressive kidney disease. The cellular mechanisms that lead to kidney tissue fibrosis are complex and include, for example, increased inflammation, increased oxidative stress, and proximal tubule cell death in the form of apoptosis or senescence. Recent studies have identified TWEAK, a tumor necrosis factor-like weak inducer of apoptosis, as a novel cytokine that mediates kidney inflammation in models of renal fibrosis. Inhibition of apoptosis via TWEAK inhibition has been shown to reduce kidney fibrosis. Recent studies using lineage tracing suggest that interstitial pericytes/perivascular fibroblasts differentiate into myofibroblasts and undergo proliferative expansion during fibrosis. Furthermore, increased expression of nuclear peroxisome proliferator-activated receptor-α in proximal tubules can directly reduce increased expression of transforming growth factor-β1 and interstitial inflammation in models of renal fibrosis, which suggests preservation of proximal tubule cell metabolism and integrity represents an important new therapeutic target. In this review, the current evidence and potential molecular mechanisms involved in the development of kidney fibrosis are discussed. 2014 S. Karger AG, Basel.

  18. Beauveria attenuates asthma by inhibiting inflammatory response and inducing lymphocytic cell apoptosis

    PubMed Central

    Zhang, Jingying; Zhou, Xianmei; Zhu, Jiping

    2016-01-01

    The present study aimed to investigate the role of beauveria (BEA) in asthma. We investigated the cytotoxic effect of BEA on the proliferation of inflammatory cells and secretion of inflammatory mediators both in-vitro and in-vivo. In in-vitro studies, BEA inhibited lymphocytic cell proliferation and the proliferation of lymphocytic cells induced by Phorbol-12-myristate-13-acetate (PMA). We used ELISA to test the effects of BEA on the secretion of inflammatory factors including tumor necrosis factor-alpha (TNF-α), interleukin-12 (IL-12) and interferon-gamma (IFN-γ). Flow cytometry was used to evaluate the influence of BEA on cell apoptosis. The effect of BEA on the cell numbers of eosinophils, lymphocytes, macrophages, neutrophils and other cells in mouse bronchoalveolar lavage fluid (BALF) was also evaluated. The expression of apoptosis related molecules Bax, Caspase-3 and Bcl-2 was examined by Western blotting analysis. Our results indicated that BEA played a protective role in asthma. BEA inhibited lymphocytic cell proliferation and secretion of inflammatory mediators. BEA promoted cell apoptosis, stimulated the expression of Bax and Caspase-3 and inhibited Bcl-2 protein expression in a dose-dependent manner. In in-vivo experiments, BEA reduced the cell number of eosinophils, lymphocytes, macrophages, neutrophils and other cells in mouse BALF. BEA inhibited secretion of inflammatory mediators, stimulated expression of Bax and Caspase-3, and inhibited expression of Bcl-2 in mouse lung tissue dose-dependently. All together, our results indicated that BEA could attenuate asthma by inhibiting inflammatory response and induce apoptosis of inflammatory cells. PMID:27801673

  19. Epidermal growth factor (EGF) stimulated Ca/sup 2 +/ mobilization in hepatocytes is abolished by phorbol esters, pertussis toxin and partial hepatectomy

    SciTech Connect

    Johnson, R.M.; Garrison, J.C.

    1986-05-01

    EGF has been demonstrated to increase free intracellular Ca/sup 2 +/ levels in isolated hepatocytes putatively by generation of the second messenger inositol trisphosphate (IP/sub 3/). Pretreatment of cells with phorbol 12-myristate 13-acetate (PMA) inhibited the EGF (66 nM) stimulated Ca/sup 2 +/ response as measured by quin2. Inhibition by PMA was maximal within 3 min and was concentration dependent (IC/sub 50/ = 13.5 nM). Four other active phorbol ester analogues blocked the Ca/sup 2 +/ response while inactive analogues did not. EGF was unable to increase intracellular Ca/sup 2 +/ levels in hepatocytes isolated from rats treated with pertussis toxin for 72 hrs. Neither PMA nor toxin pretreatment was able to inhibit the Ca/sup 2 +/ response to angiotensin II (Ang II). In hepatocytes isolated 24 hrs after partial hepatectomy, the Ca/sup 2 +/ response to EGF (as measured by phosphorylase activity, EC/sub 50/ = 5 nM) was completely abolished and remained attenuated for 7 days post-hepatectomy. The Ca/sup 2 +/ response to Ang II in this model system was also blunted but required 3 days for development of the full effect and within 7 days full activity is nearly restored. The results suggest that fundamental differences exist in the transduction mechanisms used by these two Ca/sup 2 +/-linked hormones to mobilize intracellular Ca/sup 2 +/ (and putatively increase IP/sub 3/ formation).

  20. Cyclic AMP-dependent protein kinase regulates basal and cyclic AMP-stimulated but not phorbol ester-stimulated transcription of the tyrosine hydroxylase gene.

    PubMed

    Kim, K S; Tinti, C; Song, B; Cubells, J F; Joh, T H

    1994-09-01

    To define the precise role of cyclic AMP (cAMP)-dependent protein kinase (PKA) in transcriptional regulation of the tyrosine hydroxylase (TH) gene, we performed transient cotransfection analyses of a reporter construct containing the upstream 2,400 bp sequence of the rat TH gene with expression plasmids encoding a heat-stable specific inhibitor of PKA (PKI), a mutant regulatory subunit of PKA, or the catalytic subunit of PKA. Inhibition of PKA activity by expression of either PKI or mutant regulatory subunit blocked cAMP-stimulated induction and reduced basal transcription of the TH-reporter construct. Expression of the catalytic subunit of PKA induced the expression of the TH-reporter construct up to 50-fold in a dose-dependent manner. Primer extension analysis confirmed that PKA-mediated induction of TH-reporter expression occurred at the correct transcription initiation site. Expression of PKI did not affect induction following phorbol ester treatment, suggesting that PKA and protein kinase C (PKC) induce TH transcription by independent mechanisms. Finally, a double mutation within the cAMP response element (CRE) of TH2400-CAT diminished its basal and forskolin-stimulated transcription to the level of the promoterless plasmid, pBLCAT3, but did not alter the induction following treatment with phorbol ester, indicating that the CRE is not required for PKC-mediated transcriptional induction. Our results indicate that PKA, via the CRE, plays a crucial role for basal and cAMP-inducible transcription of the TH gene.

  1. Point mutations in the Moloney murine leukemia virus enhancer identify a lymphoid-specific viral core motif and 1,3-phorbol myristate acetate-inducible element.

    PubMed Central

    Speck, N A; Renjifo, B; Hopkins, N

    1990-01-01

    The transcriptional enhancer of the Moloney murine leukemia virus (MoMLV) is organized as a 75-base-pair repeat, and in each copy of the repeat there are multiple binding sites for nuclear factors. We have introduced point mutations into each of the known nuclear factor-binding sites in the MoMLV enhancer, in both copies of the direct repeat, and have analyzed the transcriptional activity conferred by the mutated enhancers by transient-expression assays in both hematopoietic and nonhematopoietic cell lines. Mutation of individual binding sites in the MoMLV enhancer has moderate effects (less than 2-fold to 20-fold) on transcription in six independent cell lines. Several mutations decreased transcription from the MoMLV enhancer ubiquitously (the leukemia virus factor b site and the glucocorticoid response element), whereas others affected transcription specifically in lymphoid cell lines (core motif) or, more significantly, in fibroblasts (nuclear factor 1 site). The transcriptional activity of the MoMLV enhancer can be induced 8- to 10-fold by 1,3-phorbol myristate acetate in Jurkat T cells. Mutations in any of three adjacent binding sites (leukemia virus factor b and c sites and the core motif) within a 28-base-pair region in the center of the direct repeat sequence of the MoMLV enhancer completely attenuate the response to 1,3-phorbol myristate acetate. Images PMID:2104942

  2. Protein kinase C phosphorylates topoisomerase II: topoisomerase activation and its possible role in phorbol ester-induced differentiation of HL-60 cells

    SciTech Connect

    Sahyoun, N.; Wolf, M.; Besterman, J.; Hsieh, T.S.; Sander, M.; LeVine H. III; Chang, K.J.; Cuatrecasas, P.

    1986-03-01

    DNA topoisomerase II from Drosophila was phosphorylated effectively by protein kinase C. With a K/sub m/ of about 100 nM, the reaction was rapid, occurring at 4/sup 0/C as well as at 30/sup 0/C and requiring as little as 0.6 ng of the protein kinase per 170 ng of topoisomerase. About 0.85 mol of phosphate could be incorporated per mol of topoisomerase II, with phosphoserine as the only phospho amino acid produced. The reaction was dependent on Ca/sup 2 +/ and phosphatidylserine and was stimulated by phorbol esters. Calmodulin-dependent protein kinase II, but not cyclic AMP-dependent protein kinase, was also able to phosphorylate the topoisomerase. Phosphorylation of topoisomerase II by protein kinase C resulted in appreciable activation of the topoisomerase, suggesting that it may represent a possible target for the regulation of nuclear events by protein kinase C. This possibility is supported by the finding that the phorbol ester-induced differentiation of HL-60 cells was blocked by the topoisomerase II inhibitors novobiocin and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), but not by the inactive analog o-AMSA.

  3. Differential effect of bryostatin 1 and phorbol 12-myristate 13-acetate on HOP-92 cell proliferation is mediated by down-regulation of protein kinase Cdelta.

    PubMed

    Choi, Sung Hee; Hyman, Tehila; Blumberg, Peter M

    2006-07-15

    Bryostatin 1 is currently in clinical trials as a cancer chemotherapeutic agent. Although bryostatin 1, like phorbol 12-myristate 13-acetate (PMA), is a potent activator of protein kinase C (PKC), it induces only a subset of those responses induced by PMA and antagonizes others. We report that, in the HOP-92 non-small cell lung cancer line, bryostatin 1 induced a biphasic proliferative response, with maximal proliferation at 1 to 10 nmol/L. This biphasic response mirrored a biphasic suppression of the level of PKCdelta protein, with maximal suppression likewise at 1 to 10 nmol/L bryostatin 1. The typical phorbol ester PMA, in contrast to bryostatin 1, had no effect on the level of PKCdelta and modest suppression of cell proliferation, particularly evident at later treatment times. Flow cytometric analysis revealed changes in the fraction of cells in the G0-G1 and S phases corresponding to the effects on proliferation. Cells overexpressing PKCdelta exhibited a lower rate of cell proliferation compared with control untreated cells and showed neither a proliferative response nor a loss of PKCdelta in response to bryostatin 1. Conversely, treatment with PKCdelta small interfering RNA significantly increased the cellular growth compared with controls. We conclude that the differential effect on cellular proliferation induced by bryostatin 1 compared with PMA reflects the differential suppression of PKCdelta.

  4. Modulation of survival and proliferation of BSC-1 cells through changes in spreading behavior caused by the tumor-promoting phorbol ester TPA.

    PubMed

    Shiba, Y; Kanno, Y

    1989-12-01

    The effect of a tumor-promoting phorbol ester on spreading behavior was investigated to clarify the involvement of the interactions between cells and substratum in the maintenance of cell viability and the control of cell proliferation. BSC-1 cells did not spread and lost cell viability after a 24-h incubation in the absence of calf serum. Addition of calf serum initially induced radial spreading and then polarized spreading, with the formation on stress fibers and focal contact-like structure, and enhanced survival. Vitronectin also induced both radial spreading and polarized spreading, and enhanced cell survival. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced radial spreading with actin ribbons in the absence of serum. It improved the survival of cells attached to the substratum, but not in suspension. TPA suppressed polarized spreading, formation of stress fibers and of focal contact-like structure, and cell proliferation, in the presence of serum. Phorbol did not have any effect. These results suggest that enhancement of radial spreading and inhibition of polarized spreading of BSC-1 cells by TPA are closely related to the enhancement of cell survival and inhibition of cell growth.

  5. Changes in the migratory properties of neural crest and early crest-derived cells in vivo following treatment with a phorbol ester drug.

    PubMed

    Sears, R; Ciment, G

    1988-11-01

    In previous work, we found that the phorbol ester drug 12-O-tetradecanoyl phorbol acetate (TPA) reversed the developmental restriction of melanogenesis that normally occurs in neural crest-derived Schwann cell precursors around embryonic Day 5 of quail development. That is, TPA treatment of dorsal root ganglia (DRG) from 7-day quail embryos caused Schwann cell precursors to regain the ability to give rise to melanocytes. In this paper, we examine other long-term effects of TPA on the differentiative and migratory properties of neural crest and crest-derived DRG cells, using heterospecific grafting methods. We report that TPA treatment in culture increased the extent of cell migration following grafting into host embryos, including some ectopic migration into the central nervous system and other locations. TPA did not, however, seem to change the fate of these crest-derived cells, except that some DRG cells underwent pigmentation, as had been observed previously. Interestingly, graft cells associated with peripheral nerves were found to be exclusively unpigmented, whereas graft cells found in all other locations, including the central nervous system, were both pigmented and unpigmented. This suggests that peripheral nerves may act in a fashion antagonistic to the effects of TPA. These findings are consistent with the notion that TPA treatment causes early crest-derived cells to regain developmental properties lost with developmental age.

  6. Pathophysiological Significance of Hepatic Apoptosis

    PubMed Central

    Wang, Kewei; Lin, Bingliang

    2013-01-01

    Apoptosis is a classical pathological feature in liver diseases caused by various etiological factors such as drugs, viruses, alcohol, and cholestasis. Hepatic apoptosis and its deleterious effects exacerbate liver function as well as involvement in fibrosis/cirrhosis and carcinogenesis. An imbalance between apoptotic and antiapoptotic capabilities is a prominent characteristic of liver injury. The regulation of apoptosis and antiapoptosis can be a pivotal step in the treatment of liver diseases. PMID:27335822

  7. The beetroot component betanin modulates ROS production, DNA damage and apoptosis in human polymorphonuclear neutrophils.

    PubMed

    Zielińska-Przyjemska, Małgorzata; Olejnik, Anna; Kostrzewa, Artur; Łuczak, Michał; Jagodziński, Paweł P; Baer-Dubowska, Wanda

    2012-06-01

    The aim of this study was to evaluate the effect of betanin, one of the beetroot major components, on ROS production, DNA damage and apoptosis in human resting and stimulated with phorbol 12-myristate13-acetate polymorphonuclear neutrophils, one of the key elements of the inflammatory response. Incubation of neutrophils with betanin in the concentration range 2-500 µM resulted in significant inhibition of ROS production (by 15-46%, depending on the ROS detection assay). The antioxidant capacity of betanin was most prominently expressed in the chemiluminescence measurements. This compound decreased also the percentage of DNA in comet tails in stimulated neutrophils, but only at the 24 h time point. In resting neutrophils an increased level of DNA in comet tails was observed. Betanin did not affect the activity of caspase-3, in resting neutrophils, but significantly enhanced the enzyme activity in stimulated neutrophils. The western blot analysis showed, however, an increased level of caspase-3 cleavage products as a result of betanin treatment both in resting and stimulated neutrophils. The results indicate that betanin may be responsible for the effect of beetroot products on neutrophil oxidative metabolism and its consequences, DNA damage and apoptosis. The dose and time dependent effects on these processes require further studies.

  8. Regulation of the salvage pathway of deoxynucleotides synthesis in apoptosis induced by growth factor deprivation.

    PubMed Central

    Oliver, F J; Collins, M K; López-Rivas, A

    1996-01-01

    Here we describe changes in dNTP metabolism that precede DNA fragmentation in a model of apoptosis driven by deprivation of the cytokine interleukin 3 (IL-3). In haemopoietic BAF3 cells, IL-3 withdrawal leads to a rapid decrease in the size of dATP, dTTP and dGTP pools without affecting dCTP levels. This imbalance in dNTP pools precedes DNA fragmentation and is accompanied by down-regulation of enzymes controlling the de novo and salvage pathways of dNTP synthesis, ribonucleotide reductase and thymidine kinase (TK) respectively. Readdition of IL-3 results in a rapid, protein synthesis-independent restoration of normal dNTP pools, enhanced TK activity and increased precursor incorporation through the salvage pathway. Up-regulation of TK activity after IL-3 readdition is prevented by the protein kinase C (PKC) inhibitor staurosporin, but not by tyrosine kinase inhibitors. Furthermore activation of PKC by phorbol esters mimics the stimulatory effect of IL-3 on TK activity, suggesting that PKC might be involved in regulating this effect. These results indicate that regulation by IL-3 of the salvage pathway of dNTP synthesis plays a role in the maintenance of cellular dNTP pool balance and suggests that alterations in dNTP metabolism after IL-3 deprivation could be a relevant event in the commitment of haemopoietic cells to apoptosis. PMID:8687383

  9. Phorbol ester and A23187 have additive but mechanistically separate effects on vasopressin action in rabbit collecting tubule.

    PubMed Central

    Ando, Y; Jacobson, H R; Breyer, M D

    1988-01-01

    Activation of protein kinase C (PKC) and elevation of intracellular calcium ion concentration ([Ca++]i) result from phosphatidylinositol biphosphate (PIP2) breakdown. We previously demonstrated that PKC activation inhibits arginine vasopressin (AVP)-induced osmotic water flow in rabbit cortical collecting tubule (CCT) perfused in vitro at 37 degrees C. To estimate the potential significance of PIP2 turnover as a modulator of water transport in this nephron segment, we examined the effect of Ca on AVP action and explored the mechanisms of action of PKC and increased [Ca++]i. In rabbit CCTs perfused at 37 degrees C, pretreatment with bath A23187 (2 x 10(-8) M, 2 x 10(-6) M), a Ca ionophore, almost totally suppressed AVP (10 microU/ml)-induced peak hydraulic conductivity (Lp). The suppression by 2 x 10(-8) M A23187 was as potent as that by 2 x 10(-6) M A23187, and significant even when it was administered 10 min after AVP. When phorbol myristate acetate (PMA, 10(-9) M), a PKC activator, and A23187 (2 x 10(-8) M) were placed in the bath simultaneously, the combined suppressive effect on peak Lp was greater than that of either inhibitor alone. However, the mechanisms of inhibition by PMA and A23187 were different. While both 10(-7) and 10(-9) M PMA suppression are primarily post-cAMP, A23187 predominantly suppressed a pre-cAMP step: 10(-4) M chlorophenylthio-cAMP-induced peak Lp was not affected by 2 x 10(-8) M A23187, and only partially inhibited by 2 x 10(-6) M A23187. The PMA (10(-7) M) suppression of AVP-induced peak Lp was totally reversed by bath staurosporine (10(-7) M), a PKC inhibitor, but not attenuated by either bath indomethacin (5 x 10(-6) M) or low Ca (1-2 x 10(-6) M) bath medium. In contrast, the A23187 (2 x 10(-8) M) suppression of the peak Lp was not affected by staurosporine, but was significantly reversed by indomethacin or low Ca bath medium. We conclude: (a) Elevation of [Ca++]i, as well as activation of PKC, suppresses the hydroosmotic effect of

  10. Effect of PMA-induced protein kinase C activation on development and apoptosis in early zebrafish embryos.

    PubMed

    Hrubik, Jelena; Glisic, Branka; Samardzija, Dragana; Stanic, Bojana; Pogrmic-Majkic, Kristina; Fa, Svetlana; Andric, Nebojsa

    2016-12-01

    Protein kinase C (PKC) isoforms have been implicated in several key steps during early development, but the consequences of xenobiotic-induced PKC activation during early embryogenesis are still unknown. In this study, zebrafish embryos were exposed to a range of phorbol 12-myristate 13-acetate (PMA) concentrations (0-200μg/L) at different time points after fertilization. Results showed that 200μgPMA/L caused development of yolk bags, cardiac edema, slow blood flow, pulsating blood flow, slow pulse, elongated heart, lack of tail fins, curved tail, and coagulation. PMA exposure decreased survival rate of the embryos starting within the first 24h and becoming more pronounced after prolonged exposure (96h). PMA increased the number of apoptotic cells in the brain region as demonstrated by acridine orange staining and caused up-regulation of caspase 9 (casp9) and p53 up-regulated modulator of apoptosis (puma) mRNA in whole embryos. PMA caused oxidative stress in the embryos as demonstrated by decreased mRNA expression of catalase and superoxide dismutase 2. Inhibition of Pkc with GF109203X improved overall survival rate, reduced apoptosis in the brain and decreased expression of casp9 and puma in the PMA-exposed embryos. However, Pkc inhibition neither prevented development of deformities nor reversed oxidative stress in the PMA-exposed embryos. These data suggest that direct over-activation of Pkc during early embryogenesis of zebrafish is associated with apoptosis and decreased survival rate of the embryos.

  11. Apoptosis Resistance in Endometriosis

    PubMed Central

    Salmassi, Ali; Acar-Perk, Bengi; Schmutzler, Andreas G.; Koch, Kerstin; Püngel, Frank; Jonat, Walter; Mettler, Liselotte

    2011-01-01

    Introduction In a cytological analysis of endometriotic lesions neither granulocytes nor cytotoxic T-cells appear in an appreciable number. Based on this observation we aimed to know, whether programmed cell death plays an essential role in the destruction of dystopic endometrium. Disturbances of the physiological mechanisms of apoptosis, a persistence of endometrial tissue could explain the disease. Another aspect of this consideration is the proliferation competence of the dystopic mucous membrane. Methods Endometriotic lesions of 15 patients were examined through a combined measurement of apoptosis activity with the TUNEL technique (terminal deoxyribosyltransferase mediated dUTP Nick End Labeling) and the proliferation activity (with the help of the Ki-67-Antigens using the monoclonal antibody Ki-S5). Results Twelve out of 15 women studied showed a positive apoptotic activity of 3-47% with a proliferation activity of 2-25% of epithelial cells. Therefore we concluded that the persistence of dystopic endometrium requires proliferative epithelial cells from middle to lower endometrial layers. Conclusion A dystopia misalignment of the epithelia of the upper layers of the functionalism can be rapidly eliminated by apoptotic procedures. PMID:23678417

  12. Cl(-) channels in apoptosis.

    PubMed

    Wanitchakool, Podchanart; Ousingsawat, Jiraporn; Sirianant, Lalida; MacAulay, Nanna; Schreiber, Rainer; Kunzelmann, Karl

    2016-10-01

    A remarkable feature of apoptosis is the initial massive cell shrinkage, which requires opening of ion channels to allow release of K(+), Cl(-), and organic osmolytes to drive osmotic water movement and cell shrinkage. This article focuses on the role of the Cl(-) channels LRRC8, TMEM16/anoctamin, and cystic fibrosis transmembrane conductance regulator (CFTR) in cellular apoptosis. LRRC8A-E has been identified as a volume-regulated anion channel expressed in many cell types. It was shown to be required for regulatory and apoptotic volume decrease (RVD, AVD) in cultured cell lines. Its presence also determines sensitivity towards cytostatic drugs such as cisplatin. Recent data point to a molecular and functional relationship of LRRC8A and anoctamins (ANOs). ANO6, 9, and 10 (TMEM16F, J, and K) augment apoptotic Cl(-) currents and AVD, but it remains unclear whether these anoctamins operate as Cl(-) channels or as regulators of other apoptotic Cl(-) channels, such as LRRC8. CFTR has been known for its proapoptotic effects for some time, and this effect may be based on glutathione release from the cell and increase in cytosolic reactive oxygen species (ROS). Although we find that CFTR is activated by cell swelling, it is possible that CFTR serves RVD/AVD through accumulation of ROS and activation of independent membrane channels such as ANO6. Thus activation of ANO6 will support cell shrinkage and induce additional apoptotic events, such as membrane phospholipid scrambling.

  13. Lovastatin induces platelet apoptosis.

    PubMed

    Zhao, Qing; Li, Ming; Chen, Mengxing; Zhou, Ling; Zhao, Lili; Hu, Renping; Yan, Rong; Dai, Kesheng

    2016-03-01

    Statins are widely used in the prevention of atherosclerosis and treatment of coronary artery disease because of pleiotropic effects on thrombosis. Thrombocytopenia and hemorrhage occurred in some statin-treated patients, but the reason remains unclear. In the current study, we show that lovastatin dose-dependently induces depolarization of mitochondrial inner transmembrane potential, leading to up-regulation of Bak, down-regulation of Bcl-XL, and activation of caspase-3/8/9. Lovastatin treatment did not increase the surface expression of P-selectin or PAC-1 binding but led to strongly reduced collagen- and thrombin-induced platelet aggregation. The integrin αIIbβ3 antagonist, RGDS, inhibited lovastatin-induced apoptosis in both human platelets and Chinese hamster ovary (CHO) cells stably expressing integrin αIIbβ3. The number of circulating platelets in mice was significantly reduced after intraperitoneal injections with lovastatin. Taken together, these data indicate that lovastatin induced caspase-dependent platelet apoptosis. Lovastatin does not incur platelet activation, whereas impairs platelet function and reduces circulating platelets in vivo, suggesting the possible pathogenesis of thrombocytopenia and hemorrhage in patients treated with statins. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. BARC: A Novel Apoptosis Regulator

    DTIC Science & Technology

    2004-07-01

    turnover is normally achieved through programmed cell death , also known as apoptosis. Effects in apoptosis occur in breast cancers and other types of...malignancies, making tumor cells difficult to kill by chemotherapy, hormonal therapy, and radiation. Restoring function of cell death pathways is a strategy...These findings provide new insights into cell death regulation in breast cancer.

  15. Human fibroblast growth factor 1 gene expression in vascular smooth muscle cells is modulated via an alternate promoter in response to serum and phorbol ester.

    PubMed Central

    Chotani, M A; Payson, R A; Winkles, J A; Chiu, I M

    1995-01-01

    We have previously isolated the human FGF-1 gene in order to elucidate the molecular basis of its gene expression. The gene spans over 100 kbp and encodes multiple transcripts expressed in a tissue- and cell-specific manner. Two variants of FGF-1 mRNA (designated FGF-1.A and 1.B), which differ in their 5' untranslated region, were identified in our laboratory. Recently, two novel variants of FGF-1 mRNA (designated FGF-1.C and 1.D) have been isolated. In this study we used RNase protection assays to demonstrate expression of FGF-1.D mRNA in human fibroblasts and vascular smooth muscle cells and to show that promoter 1D has multiple transcription start sites. A single-strand nuclease-sensitive region has also been identified in the promoter 1D region that may have implications in chromatin conformation and transcriptional regulation of this promoter. Using Northern blot hybridization analyses, a previous study demonstrated a significant increase of FGF-1 mRNA levels in cultured saphenous vein smooth muscle cells in response to serum and phorbol ester. Here we confirm these results by RNase protection analysis and show that FGF-1.C mRNA is significantly increased in response to these stimuli. RNase protection assays indicate that promoter 1C has one major start site. The phorbol ester effect suggests that a protein kinase C-dependent signalling pathway may be involved in this phenomenon. Our results point to a dual promoter usage of the FGF-1 gene in vascular smooth muscle cells. Thus, normal growing cells primarily utilize promoter 1D. In contrast, quiescent cells, when exposed to serum or phorbol ester, utilize a different FGF-1 promoter, namely promoter 1C. Overall, these phenomena suggest mechanisms for increased production of FGF-1 that may play a role in inflammatory settings, wound healing, tissue repair, and neovascularization events and processes via autocrine and paracrine mechanisms. Our findings suggest that different FGF-1 promoters may respond to

  16. Apoptosis and the Airway Epithelium

    PubMed Central

    White, Steven R.

    2011-01-01

    The airway epithelium functions as a barrier and front line of host defense in the lung. Apoptosis or programmed cell death can be elicited in the epithelium as a response to viral infection, exposure to allergen or to environmental toxins, or to drugs. While apoptosis can be induced via activation of death receptors on the cell surface or by disruption of mitochondrial polarity, epithelial cells compared to inflammatory cells are more resistant to apoptotic stimuli. This paper focuses on the response of airway epithelium to apoptosis in the normal state, apoptosis as a potential regulator of the number and types of epithelial cells in the airway, and the contribution of epithelial cell apoptosis in important airways diseases. PMID:22203854

  17. Apoptosis Evaluation by Electrochemical Techniques.

    PubMed

    Yin, Jian; Miao, Peng

    2016-03-04

    Apoptosis has close relevance to pathology, pharmacology, and toxicology. Accurate and convenient detection of apoptosis would be beneficial for biological study, clinical diagnosis, and drug development. Based on distinct features of apoptotic cells, a diversity of analytical techniques have been exploited for sensitive analysis of apoptosis, such as surface plasmon resonance, electrochemical methods, flow cytometry, and some imaging assays. Among them, the features of simplicity, easy operation, low cost, and high sensitivity make electrochemical techniques powerful tools to investigate electron-transfer processes of in vitro biological systems. In this contribution, a general overview of current knowledge on various technical approaches for apoptosis evaluation is provided. Furthermore, recently developed electrochemical biosensors for detecting apoptotic cells and their advantages over traditional methods are summarized. One of the main considerations focuses on designing the recognition elements based on various biochemical events during apoptosis.

  18. High ACSL5 Transcript Levels Associate with Systemic Lupus Erythematosus and Apoptosis in Jurkat T Lymphocytes and Peripheral Blood Cells

    PubMed Central

    2011-01-01

    Background Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease in which increased apoptosis and decreased apoptotic cells removal has been described as most relevant in the pathogenesis. Long-chain acyl-coenzyme A synthetases (ACSLs) have been involved in the immunological dysfunction of mouse models of lupus-like autoimmunity and apoptosis in different in vitro cell systems. The aim of this work was to assess among the ACSL isoforms the involvement of ACSL2, ACSL4 and ACSL5 in SLE pathogenesis. Findings With this end, we determined the ACSL2, ACSL4 and ACSL5 transcript levels in peripheral blood mononuclear cells (PBMCs) of 45 SLE patients and 49 healthy controls by quantitative real time-PCR (q-PCR). We found that patients with SLE had higher ACSL5 transcript levels than healthy controls [median (range), healthy controls = 16.5 (12.3–18.0) vs. SLE = 26.5 (17.8–41.7), P = 3.9×10 E-5] but no differences were found for ACSL2 and ACSL4. In in vitro experiments, ACSL5 mRNA expression was greatly increased when inducing apoptosis in Jurkat T cells and PBMCs by Phorbol-Myristate-Acetate plus Ionomycin (PMA+Io). On the other hand, short interference RNA (siRNA)-mediated silencing of ACSL5 decreased induced apoptosis in Jurkat T cells up to the control levels as well as decreased mRNA expression of FAS, FASLG and TNF. Conclusions These findings indicate that ACSL5 may play a role in the apoptosis that takes place in SLE. Our results point to ACSL5 as a potential novel functional marker of pathogenesis and a possible therapeutic target in SLE. PMID:22163040

  19. Apoptosis in Primary Hyperparathyroidism.

    PubMed

    Segiet, Oliwia Anna; Mielańczyk, Łukasz; Piecuch, Adam; Michalski, Marek; Tyczyński, Szczepan; Brzozowa-Zasada, Marlena; Deska, Mariusz; Wojnicz, Romuald

    2017-03-31

    Primary hyperparathyroidism (PHPT) is defined by inappropriate elevation of parathormone, caused by parathyroid hyperplasia, also known as multi-gland disease (MGD), parathyroid adenoma (PA), or parathyroid carcinoma (PC). Although several studies have already been conducted, there is a lack of a definite diagnostic marker, which could unambiguously distinguish MGD from PA or PC. The accurate and prompt diagnosis has the key meaning for effective treatment and follow-up. This review paper presents the role of apoptosis in PHPT. The comparison of the expression of Fas, TRAIL, BCL-2 family members, p53 in MGD, PA, and PC, among others, was described. The expression of described factors varies among proliferative lesions of parathyroid gland; therefore, these could serve as additional markers to assist in the diagnosis.

  20. Galangin and kaempferol suppress phorbol-12-myristate-13-acetate-induced matrix metalloproteinase-9 expression in human fibrosarcoma HT-1080 cells.

    PubMed

    Choi, Yu Jung; Lee, Young Hun; Lee, Seung-Taek

    2015-01-01

    Matrix metalloproteinase (MMP)-9 degrades type IV collagen in the basement membrane and plays crucial roles in several pathological implications, including tumorigenesis and inflammation. In this study, we analyzed the effect of flavonols on MMP-9 expression in phorbol-12-myristate-13-acetate (PMA)-induced human fibrosarcoma HT-1080 cells. Galangin and kaempferol efficiently decreased MMP-9 secretion, whereas fisetin only weakly decreased its secretion. Galangin and kaempferol did not affect cell viability at concentrations up to 30 μM. Luciferase reporter assays showed that galangin and kaempferol decrease transcription of MMP-9 mRNA. Moreover, galangin and kaempferol strongly reduce IκBα phosphorylation and significantly decrease JNK phosphorylation. These results indicate that galangin and kaempferol suppress PMA-induced MMP-9 expression by blocking activation of NF-κB and AP-1. Therefore, these flavonols could be used as chemopreventive agents to lower the risk of diseases involving MMP-9.

  1. System-wide analysis of the transcriptional network of human myelomonocytic leukemia cells predicts attractor structure and phorbol-ester-induced differentiation and dedifferentiation transitions.

    PubMed

    Sakata, Katsumi; Ohyanagi, Hajime; Sato, Shinji; Nobori, Hiroya; Hayashi, Akiko; Ishii, Hideshi; Daub, Carsten O; Kawai, Jun; Suzuki, Harukazu; Saito, Toshiyuki

    2015-02-06

    We present a system-wide transcriptional network structure that controls cell types in the context of expression pattern transitions that correspond to cell type transitions. Co-expression based analyses uncovered a system-wide, ladder-like transcription factor cluster structure composed of nearly 1,600 transcription factors in a human transcriptional network. Computer simulations based on a transcriptional regulatory model deduced from the system-wide, ladder-like transcription factor cluster structure reproduced expression pattern transitions when human THP-1 myelomonocytic leukaemia cells cease proliferation and differentiate under phorbol myristate acetate stimulation. The behaviour of MYC, a reprogramming Yamanaka factor that was suggested to be essential for induced pluripotent stem cells during dedifferentiation, could be interpreted based on the transcriptional regulation predicted by the system-wide, ladder-like transcription factor cluster structure. This study introduces a novel system-wide structure to transcriptional networks that provides new insights into network topology.

  2. System-wide analysis of the transcriptional network of human myelomonocytic leukemia cells predicts attractor structure and phorbol-ester-induced differentiation and dedifferentiation transitions

    NASA Astrophysics Data System (ADS)

    Sakata, Katsumi; Ohyanagi, Hajime; Sato, Shinji; Nobori, Hiroya; Hayashi, Akiko; Ishii, Hideshi; Daub, Carsten O.; Kawai, Jun; Suzuki, Harukazu; Saito, Toshiyuki

    2015-02-01

    We present a system-wide transcriptional network structure that controls cell types in the context of expression pattern transitions that correspond to cell type transitions. Co-expression based analyses uncovered a system-wide, ladder-like transcription factor cluster structure composed of nearly 1,600 transcription factors in a human transcriptional network. Computer simulations based on a transcriptional regulatory model deduced from the system-wide, ladder-like transcription factor cluster structure reproduced expression pattern transitions when human THP-1 myelomonocytic leukaemia cells cease proliferation and differentiate under phorbol myristate acetate stimulation. The behaviour of MYC, a reprogramming Yamanaka factor that was suggested to be essential for induced pluripotent stem cells during dedifferentiation, could be interpreted based on the transcriptional regulation predicted by the system-wide, ladder-like transcription factor cluster structure. This study introduces a novel system-wide structure to transcriptional networks that provides new insights into network topology.

  3. Effect of phorbol 12-myristate 13-acetate on function and gene expression of P-glycoprotein in adriamycin-resistant K562/ADM cells.

    PubMed

    Li, Yuhua; Bi, Huichang; Zhong, Guoping; Huang, Ling; Li, Gelin; Xia, Yanzhe; Chen, Xiao; Huang, Min

    2013-01-01

    Multidrug resistance (MDR) is a critical issue during chemotherapy of cancers. Phorbol 12-myristate 13-acetate (PMA), a diester of phorbol, is a typical activator of protein kinase C (PKC). In the present study, we investigated the effect of PMA on MDR and P-glycoprotein (P-gp) gene expression in K562/ADM cells. 3-(4,5-dimethylthiazol-2-thiazolyl)-2,5-diphenyltetrazolium bromide assay was used to assess adriamycin (Adr)-induced cytotoxicity towards K562/ADM cells in the absence or presence of PMA. The intracellular accumulation of Adr was measured by determining the mean fluorescence intensity. The effect of PMA on P-gp activity was investigated by rhodamine-123 accumulation and efflux experiment. Protein expression and mRNA expression of P-gp in K562/ADM cells were determined by Western blot analysis and real-time qPCR, respectively. Adr-induced cytotoxicity towards K562/ADM cells was significantly decreased by PMA at 5 μmol/l. Furthermore, intracellular Adr-associated mean fluorescence intensity was attenuated by 53.8% 1 h after exposure to PMA at 5 μmol/l compared with the control group (p < 0.05). A dose-dependent decrease of intracellular rhodamine-123 and increase of efflux activity of P-gp were also observed in K562/ADM cells incubation with PMA. In addition, P-gp mRNA and protein expression were significantly induced by PMA. Activation of PKC pathway by PMA can significantly induce expression and activity of P-gp, and thus decrease intracellular Adr level and strengthen MDR in K562/ADM cells. © 2013 S. Karger AG, Basel.

  4. Targeting of FAK Ser910 by ERK5 and PP1δ in non-stimulated and phorbol ester-stimulated cells

    PubMed Central

    Villa-Moruzzi, Emma

    2007-01-01

    Ser910 of FAK (focal adhesion kinase) was phosphorylated in fibroblasts treated with the phorbol ester PMA and dephosphorylated by PP1δ (protein phosphatase 1δ), as indicated by shRNA (small-hairpin RNA) gene silencing. Ser910 of FAK was reported previously to be an ERK (extracellular-signal-regulated kinase) 1/2 target in cells treated with phorbol esters. In contrast, various approaches, including the use of the MEK (mitogen-activated protein kinase/ERK kinase) inhibitors UO126 and CI-1040 to inhibit ERK1/2 pointed to the involvement of ERK5. This hypothesis was confirmed by: (i) shRNA ERK5 gene silencing, which resulted in complete pSer910 loss in non-stimulated and PMA-stimulated cells; (ii) direct phosphorylation of recombinant FAK by ERK5; and (iii) ERK5 activation by PMA. PMA stimulation and ERK5 silencing in MDA-MB 231 and MDA-MB 361 breast cancer cells indicated Ser910 targeting by ERK5 also in these cells. Given the proximity of Ser910 to the FAT (focal adhesion targeting) regulatory domain of FAK, cell proliferation and morphology were investigated in FAK−/− cells expressing S910A mutant FAK. The cell growth rate decreased and exposure to PMA induced peculiar morphological changes in cells expressing S910A, with respect to wild-type FAK, suggesting a role for Ser910 in these processes. The present study indicates, for the first time, the phosphorylation of Ser910 of FAK by ERK5 and its dephosphorylation by PP1δ, and suggested a role for Ser910 in the control of cell shape and proliferation. PMID:17692050

  5. Tyrosine hydroxylase is activated and phosphorylated at different sites in rat pheochromocytoma PC 12 cells treated with phorbol ester and forskolin

    SciTech Connect

    Tachikawa, E.; Tank, A.W.; Weiner, D.H.; Mosimann, W.F.; Yanagihara, N.; Weiner, N.

    1986-03-01

    The effects of phorbol ester (4..beta..-phorbol, 12..beta..-myristate, 13..cap alpha..-acetate; TPA), an activator of Ca/sup + +//phospholipid-dependent protein kinase (PK-C), and forskolin, which stimulates adenylate cyclase and cyclic AMP-dependent protein kinase (cAMP-PK), on the activation and phosphorylation of tyrosine hydroxylase (TH) in rat pheochromocytoma (PC 12) cells were examined. Incubation of the cells with TPA (0.01-1 ..mu..M) or forskolin (0.01-0.1 ..mu..M) produces increases in activation and phosphorylation of TH in a concentration-dependent manner. The stimulatory effects of TPA are dependent on extracellular Ca/sup + +/ and are inhibited by pretreatment of the cells with trifluoperazine (TFP). The effects of forskolin are independent of Ca/sup + +/ and are not inhibited by TFP. In cells treated with forskolin, the time course of the increase in cAMP correlates with the increases in TH activity and phosphorylation. cAMP levels do not increase in cells treated with TPA. There is an increase in the phosphorylation of only one tryptic phosphopeptide derived from TH in cells treated with either forskolin or TPA. The peptide phosphorylated in TPA-treated cells exhibits different elution characteristics on HPLC from that in forskolin-treated cells. The authors conclude that TH in PC 12 cells is phosphorylated on different sites by cAMP-PK and PK-C. Phosphorylation of either of these sites is associated with enzyme activation.

  6. Apoptosis in Gingival Overgrowth Tissues

    PubMed Central

    Kantarci, A.; Augustin, P.; Firatli, E.; Sheff, M.C.; Hasturk, H.; Graves, D.T.; Trackman, P.C.

    2010-01-01

    Variations in the balance between cell proliferation and apoptosis could contribute to the etiology of gingival overgrowth. The aim of this study was to test the hypothesis that, in fibrotic gingival lesions, fibroblast proliferation is stimulated and apoptosis is decreased. Apoptotic index, caspase 3 expression, the proliferative index, FOXO1 expression, and histological inflammation were measured in situ. Analysis of data showed that apoptosis decreased in all forms of gingival overgrowth examined (p < 0.05), and inflammation caused a small but significant increase compared with non-inflamed tissues (p < 0.05). The greatest decrease of apoptosis occurred in the most fibrotic tissues. Cell proliferation was elevated in all forms of gingival overgrowth tested, independent of inflammation (p < 0.05). To identify potential mechanisms of transcriptional regulation of apoptosis, we assessed FOXO1 and caspase 3 expression levels and found them to correlate well with diminished apoptosis. Analysis of data suggests that increased fibroblast proliferation and a simultaneous decrease in apoptosis contribute to gingival overgrowth. PMID:17720861

  7. Effects of 12-O-tetradecanoyl-phorbol-13-acetate [corrected] and sodium lauryl sulfate on the production and expression of cytokines and proto-oncogenes in photoaged and intrinsically aged human keratinocytes.

    PubMed

    Suh, D H; Youn, J I; Eun, H C

    2001-11-01

    Skin aging may be divided into photoaging and intrinsic aging. The purpose of this study was to investigate the effects of 12-O-tetradecanoyl-phorbol-13-acetate and sodium lauryl sulfate on the production and expression of cytokines and proto-oncogenes in photoaged and intrinsically aged skin, compared with young skin. Keratinocytes were taken from newborns, young adults in their twenties, and from the forearm and thigh of volunteers in their fifties and seventies. Interleukin-1alpha and -6, and interleukin-1 receptor antagonist, c-fos and c-myc were measured after cultured keratinocytes had been treated with 12-O-tetradecanoyl-phorbol-13-acetate and sodium lauryl sulfate. There has been no report concerning the dependence of cytokine production by sodium lauryl sulfate upon photoaging and intrinsic aging. This study also involves the first investigation of the effects of aging on c-myc expression by 12-O-tetradecanoyl-phorbol-13-acetate treatment. Cytokine production decreased markedly with age. These results suggest the progressive decline of cellular function with age. The ratio of cytokine production in the irritant-treated group compared with that in the control group showed a different pattern in photoaging and intrinsic aging. With the significant difference between photoaging and intrinsic aging, T/C ratio decreased in interleukin-1alpha and interleukin-1 receptor antagonist upon aging, whereas it increased in interleukin-6. S/C ratio was uniquely elevated on photoaged skin in the 50 y age group. It is suggested that photoaged skin shows an exaggerated reaction to surfactant. Compared with the control, c-fos expression in 12-O-tetradecanoyl-phorbol-13-acetate-treated keratinocytes decreased with age in the thigh, but increased in the photoaged skin of forearm. The increased c-fos expression in 12-O-tetradecanoyl-phorbol-13-acetate-treated keratinocytes could be relevant for the predisposition of photoaged keratinocytes to malignant transformation.

  8. PMA induces androgen receptor downregulation and cellular apoptosis in prostate cancer cells.

    PubMed

    Itsumi, Momoe; Shiota, Masaki; Yokomizo, Akira; Takeuchi, Ario; Kashiwagi, Eiji; Dejima, Takashi; Inokuchi, Junichi; Tatsugami, Katsunori; Uchiumi, Takeshi; Naito, Seiji

    2014-08-01

    Phorbol 12-myristate 13-acetate (PMA) induces cellular apoptosis in prostate cancer cells, the growth of which is governed by androgen/androgen receptor (AR) signaling, but the mechanism by which PMA exerts this effect remains unknown. Therefore, in this study, we investigated the mechanistic action of PMA in prostate cancer cells with regard to AR. We showed that PMA decreased E2F1 as well as AR expression in androgen-dependent prostate cancer LNCaP cells. Furthermore, PMA activated JNK and p53 signaling, resulting in the induction of cellular apoptosis. In LNCaP cells, androgen deprivation and a novel anti-androgen enzalutamide (MDV3100) augmented cellular apoptosis induced by PMA. Moreover, castration-resistant prostate cancer (CRPC) C4-2 cells were more sensitive to PMA compared with LNCaP cells and were sensitized to PMA by enzalutamide. Finally, the expression of PKC, E2F1, and AR was diminished in PMA-resistant cells, indicating that the gain of independence from PKC, E2F1, and AR functions leads to PMA resistance. In conclusion, PMA exerted its anti-cancer effects via the activation of pro-apoptotic JNK/p53 and inhibition of pro-proliferative E2F1/AR in prostate cancer cells including CRPC cells. The therapeutic effects of PMA were augmented by androgen deletion and enzalutamide in androgen-dependent prostate cancer cells, as well as by enzalutamide in castration-resistant cells. Taken together, PMA derivatives may be promising therapeutic agents for treating prostate cancer patients including CRPC patients.

  9. Regulation of Apoptosis by Inhibitors of Apoptosis (IAPs)

    PubMed Central

    Berthelet, Jean; Dubrez, Laurence

    2013-01-01

    Abstract Inhibitors of Apoptosis (IAPs) are a family of proteins with various biological functions including regulation of innate immunity and inflammation, cell proliferation, cell migration and apoptosis. They are characterized by the presence of at least one N-terminal baculoviral IAP repeat (BIR) domain involved in protein-protein interaction. Most of them also contain a C-terminal RING domain conferring an E3-ubiquitin ligase activity. In drosophila, IAPs are essential to ensure cell survival, preventing the uncontrolled activation of the apoptotic protease caspases. In mammals, IAPs can also regulate apoptosis through controlling caspase activity and caspase-activating platform formation. Mammalian IAPs, mainly X-linked IAP (XIAP) and cellular IAPs (cIAPs) appeared to be important determinants of the response of cells to endogenous or exogenous cellular injuries, able to convert the survival signal into a cell death-inducing signal. This review highlights the role of IAP in regulating apoptosis in Drosophila and Mammals. PMID:24709650

  10. Methylselenium and Prostate Cancer Apoptosis

    DTIC Science & Technology

    2005-02-01

    15. NUMBER OF PAGES Selenium, methylselenol , prostate cancer chemoprevention, apoptosis 15 16. PRICE CODE 17. SECURITY CLASSIFICATION 18. SECURITY...that Methylselenol has been implicated as an active metabolite inhibit one or more steps in the natural history of prostate for the anticancer effect of...in PCa chemoprevention and therapy activity) for their apoptosis responses to the methylselenol by selenium compounds (8). Methylselenol has been impli

  11. [Apoptosis: cellular and clinical aspects].

    PubMed

    Løvschall, H; Mosekilde, L

    1997-04-01

    Removal of damaged cells is essential for the maintenance of life in multicellular organisms. The process of self destruction, apoptosis, eliminates surplus or damaged cells as part of the pathophysiological defence system. Apoptosis is essential in structural and functional organogenesis during embryological development. The physiological regulation of tissue kinetics is a product of both cell proliferation and cell death. Internal and external regulatory stimuli regulate the balance between apoptosis and mitosis by genetic interaction. Apoptosis is characterized by condensation of chromatine as a result of DNA degradation, formation of blebs in the plasma and nuclear membranes, condensation of cytoplasma, formation of vesicular apoptotic bodies, and phagocytosis by neighbouring cells without inflammatory response. A number of observations indicate that programmed cell death plays an important role in the regulation of cytofunctional homeostasis and defense against accumulation of damaged cells, eg with DNA alterations. Dysregulation of the apoptotic gene program, eg by mutations, may not only lead to loss or degeneration of tissue, but also to hyperproliferative and tumorigenic disorders. New evidence indicates that apoptosis regulation is important both in aging processes and diseases such as: neuropathies, immunopathies, viral infections, cancer, etc. Pharmacological intervention designed to modulate apoptosis seems to raise new possibilities in the treatment of disease.

  12. In vitro effects of beetroot juice and chips on oxidative metabolism and apoptosis in neutrophils from obese individuals.

    PubMed

    Zielińska-Przyjemska, Małgorzata; Olejnik, Anna; Dobrowolska-Zachwieja, Agnieszka; Grajek, Włodzimierz

    2009-01-01

    Oxidative stress and inflammation are involved in the development of obesity. Beetroot (Beta vulgaris var. rubra) is a food ingredient containing betalain pigments that show antioxidant activity. The in vitro effect of beetroot juice and chips on oxidative metabolism and apoptosis in neutrophils from obese individuals has been investigated. Fifteen obese women (aged 45 +/- 9 years, BMI >30 kg/m2) and nine healthy controls (women, aged 29 +/- 11 years, BMI = 22.2 +/- 1.6 kg/m2) were examined. The investigated products were used as concentrates and after transport and digestion in an artificial gastrointestinal tract. Neutrophil oxidant production, in response to phorbol 12-myristate 13-acetate, was characterized by luminol-dependent chemiluminescence and a flow cytometric dichlorofluorescin oxidation assay. Caspase-3 activity, a marker of apoptosis, was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Neutrophils from obese individuals had a significantly higher ROS production compared with the controls (p < 0.05). Beetroot products inhibited neutrophil oxidative metabolism in a concentration-dependent manner. Also observed were the pro-apoptotic effects of beetroot at a concentration range of 0.1-10% in 24 h culture of stimulated neutrophils. These natural products (in both the liquid and solid state) have antioxidant and antiinflammatory capacity, and could be an important adjunct in the treatment of obesity.

  13. Phenotypes and phorbol ester-induced differentiation of human histiocytic lymphoma cell lines (U-937 and SU-DHL-1) and Reed-Sternberg cells.

    PubMed

    Hsu, S M; Hsu, P L

    1986-02-01

    Hodgkin's mononuclear cells, Reed-Sternberg (H-RS) cells, and U-937 and SU-DHL-1 histocytic cell lines were induced to differentiate by phorbol ester in cultures. The phenotypes of cells were determined by a panel of antibodies specific for monocytes, histiocytes, and interdigitating reticulum cells. Before induction, SU-DHL-1 cells and H-RS cells expressed similar markers, such as HeFi-1, 2H9, 1A2, and 1E9. In addition, SU-DHL-1 cells were also stained by Tac and Leu M5. Other monocyte markers, including OK M1, Co Mo2, BRL Mo1, BRL Mo2, and Leu M3 were consistently negative in both types of cells. After induction, SU-DHL-1 cells conserved the same phenotype, but H-RS cells became negative for HeFi-1, 1A2, and 2H9. The U-937 cells expressed Leu M1 and Co Mo2 and became positive for Leu M5, OK M1, Co Mo2, BRL Mo2, 2H9, and 1E9 after phorbol ester induction. The U-937 cells did not express HeFi-1 or 1A2. The marker expression of H-RS cells, SU-DHL-1 cells, and U-937 cells were compared with those of histiocytes or interdigitating reticulum cells in lymphoid tissues and with neoplastic cells in true histiocytic lymphoma and malignant histiocytosis. It is concluded that SU-DHL-1, U-937, and H-RS cells are derived from or most closely related to fixed histiocytes, free histiocytes, and interdigitating reticulum cells, respectively. Our study further confirms the diagnosis of SU-DHL-1 as true histiocytic lymphoma but reveals that U-937 is a case of malignant histiocytosis rather than the previously diagnosed histiocytic lymphoma. The phenotypes and induction properties of SU-DHL-1 cells are quite different from those of U-937 cells, which suggests that true histiocytic lymphoma and malignant histiocytosis are two distinct disease entities.

  14. Effects of chlorella on activities of protein tyrosine phosphatases, matrix metalloproteinases, caspases, cytokine release, B and T cell proliferations, and phorbol ester receptor binding.

    PubMed

    Cheng, Fong-Chi; Lin, Atsui; Feng, Jin-Jye; Mizoguchi, Toru; Takekoshi, Hideo; Kubota, Hitoshi; Kato, Yoko; Naoki, Yo

    2004-01-01

    A Chlorella powder was screened using 52 in vitro assay systems for enzyme activity, receptor binding, cellular cytokine release, and B and T cell proliferation. The screening revealed a very potent inhibition of human protein tyrosine phosphatase (PTP) activity of CD45 and PTP1C with 50% inhibitory concentration (IC(50)) values of 0.678 and 1.56 microg/mL, respectively. It also showed a moderate inhibition of other PTPs, including PTP1B (IC(50) = 65.3 microg/mL) and T-cell-PTP (114 microg/mL). Other inhibitory activities and their IC(50) values included inhibition of the human matrix metalloproteinases (MMPs) MMP-1 (127 microg/mL), MMP-3 (185 microg/mL), MMP-7 (18.1 microg/mL), and MMP-9 (237 microg/mL) and the human peptidase caspases caspase 1 (300 microg/mL), caspase 3 (203 microg/mL), caspase 6 (301 microg/mL), caspase 7 (291 microg/mL), and caspase 8 (261 microg/mL), as well as release of the cytokines interleukin (IL)-1 (44.9 microg/mL), IL-2 (14.8 microg/mL), IL-4 (49.2 microg/mL), IL-6 (34.7 microg/mL), interferon-gamma (31.6 microg/mL), and tumor necrosis factor-alpha (11 microg/mL) from human peripheral blood mononuclear cells. Chlorella also inhibited B cell proliferation (16.6 microg/mL) in mouse splenocytes and T cell proliferation (54.2 microg/mL) in mouse thymocytes. The binding of a phorbol ester, phorbol 12,13-dibutyrate, to its receptors was also inhibited by Chlorella with an IC(50) of 152 microg/mL. These results reveal potential pharmacological activities that, if confirmed by in vivo studies, might be exploited for the prevention or treatment of several serious pathologies, including inflammatory disease and cancer.

  15. Phenotypes and phorbol ester-induced differentiation of human histiocytic lymphoma cell lines (U-937 and SU-DHL-1) and Reed-Sternberg cells.

    PubMed Central

    Hsu, S. M.; Hsu, P. L.

    1986-01-01

    Hodgkin's mononuclear cells, Reed-Sternberg (H-RS) cells, and U-937 and SU-DHL-1 histocytic cell lines were induced to differentiate by phorbol ester in cultures. The phenotypes of cells were determined by a panel of antibodies specific for monocytes, histiocytes, and interdigitating reticulum cells. Before induction, SU-DHL-1 cells and H-RS cells expressed similar markers, such as HeFi-1, 2H9, 1A2, and 1E9. In addition, SU-DHL-1 cells were also stained by Tac and Leu M5. Other monocyte markers, including OK M1, Co Mo2, BRL Mo1, BRL Mo2, and Leu M3 were consistently negative in both types of cells. After induction, SU-DHL-1 cells conserved the same phenotype, but H-RS cells became negative for HeFi-1, 1A2, and 2H9. The U-937 cells expressed Leu M1 and Co Mo2 and became positive for Leu M5, OK M1, Co Mo2, BRL Mo2, 2H9, and 1E9 after phorbol ester induction. The U-937 cells did not express HeFi-1 or 1A2. The marker expression of H-RS cells, SU-DHL-1 cells, and U-937 cells were compared with those of histiocytes or interdigitating reticulum cells in lymphoid tissues and with neoplastic cells in true histiocytic lymphoma and malignant histiocytosis. It is concluded that SU-DHL-1, U-937, and H-RS cells are derived from or most closely related to fixed histiocytes, free histiocytes, and interdigitating reticulum cells, respectively. Our study further confirms the diagnosis of SU-DHL-1 as true histiocytic lymphoma but reveals that U-937 is a case of malignant histiocytosis rather than the previously diagnosed histiocytic lymphoma. The phenotypes and induction properties of SU-DHL-1 cells are quite different from those of U-937 cells, which suggests that true histiocytic lymphoma and malignant histiocytosis are two distinct disease entities. Images Figure 2 Figure 4 Figure 6 PMID:3511721

  16. Ric-8A gene deletion or phorbol ester suppresses tumorigenesis in a mouse model of GNAQQ209L-driven melanoma

    PubMed Central

    Patel, B R; Tall, G G

    2016-01-01

    The heterotrimeric G protein α subunit oncogenes GNAQ or GNA11 carry Q209X or R183X activating mutations and are present with ~90% frequency in human uveal melanomas. Forced expression of GNAQ/11Q209L in melanocytes is sufficient to drive metastatic melanoma in immune-compromised mice. No known drugs directly target these oncogenic G proteins. Ric-8A is the molecular chaperone that selectively folds Gαq/i/13 subunits. Targeting Ric-8A serves as a rational, yet unexplored approach to reduce the functional abundance of oncogenic Gαq/11 in order to blunt cancer signaling. Here, using mouse melanocyte cell graft tumorigenesis models, we determined that Ric-8A genetic ablation attenuated the abundance and melanoma-driving potential of Gαq-Q209L. A new conditional Ric-8AFlox/Flox; Rosa-CreER+/− mouse strain was derived and used as a tissue source to culture an immortalized, tamoxifen-inducible Ric-8A knockout melanocyte cell line that required 12-O-tetradecanoylphorbol-13-acetate (TPA, phorbol ester) for growth. The cell line failed to grow tumors when grafted into immune-compromised mice regardless of Ric-8A expression. Stable expression of human GNAQQ209L, but not GNAQWT in the cell line promoted TPA-independent cell proliferation, and upon cell grafting in mice, the initiation and robust growth of darkly-pigmented melanoma tumors. Deletion of Ric-8A in GNAQQ209L cells restored TPA-dependent growth, reduced Gαq-Q209L below detectable levels and completely mitigated tumorigenesis from primary or secondary cell line grafts. Interestingly, TPA treatment of cultured GNAQQ209L cells or host animals grafted with GNAQQ209L cells also sharply reduced Gαq-Q209L abundance and tumorigenic capacity. Finally, tumorigenesis initiated from GNAQQ209L cell grafts, followed by host mouse systemic tamoxifen treatment to delete Ric-8A in the grafted cells completely abrogated GNAQQ209L-driven tumor progression unless a stable human RIC-8A transgene was used to rescue the floxed

  17. Betulinic acid protects against ischemia/reperfusion-induced renal damage and inhibits leukocyte apoptosis.

    PubMed

    Ekşioğlu-Demiralp, Emel; Kardaş, E Riza; Ozgül, Seçkin; Yağci, Tayfur; Bilgin, Hüseyin; Sehirli, Ozer; Ercan, Feriha; Sener, Göksel

    2010-03-01

    The possible protective effect of betulinic acid on renal ischemia/reperfusion (I/R) injury was studied. Wistar Albino rats were unilaterally nephrectomized and subjected to 45 min of renal pedicle occlusion followed by 6 h of reperfusion. Betulinic acid (250 mg/kg, i.p.) or saline was administered at 30 min prior to ischemia and immediately before the reperfusion. Creatinine, blood urea nitrogen (BUN), lactate dehydrogenase (LDH) and TNF-alpha as well as the oxidative burst of neutrophil and leukocyte apoptosis were assayed in blood samples. Malondialdehyde (MDA), glutathione (GSH) levels, Na(+), K(+)-ATPase and myeloperoxidase (MPO) activities were determined in kidney tissue which was also analysed microscopically. I/R caused significant increases in blood creatinine, BUN, LDH and TNF-alpha. In the kidney samples of the I/R group, MDA levels and MPO activity were increased significantly, however, GSH levels and Na(+), K(+)-ATPase activity were decreased. Betulinic acid ameliorated the oxidative burst response to both formyl-methionyl-leucyl-phenylalanine (fMLP) and phorbol 12-myristate 13-acetate (PMA) stimuli, normalized the apoptotic response and most of the biochemical indices as well as histopathological alterations induced by I/R. In conclusion, these data suggest that betulinic acid attenuates I/R-induced oxidant responses, improved microscopic damage and renal function by regulating the apoptotic function of leukocytes and inhibiting neutrophil infiltration.

  18. Proteolytic cleavage of protein kinase Cmu upon induction of apoptosis in U937 cells.

    PubMed

    Häussermann, S; Kittstein, W; Rincke, G; Johannes, F J; Marks, F; Gschwendt, M

    1999-12-03

    Treatment of U937 cells with various apoptosis-inducing agents, such as TNFalpha and beta-D-arabinofuranosylcytosine (ara-C) alone or in combination with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), bryostatin 1 or cycloheximide, causes proteolytic cleavage of protein kinase Cmu (PKCmu) between the regulatory and catalytic domain, generating a 62 kDa catalytic fragment of the kinase. The formation of this fragment is effectively suppressed by the caspase-3 inhibitor Z-DEVD-FMK. In accordance with these in vivo data, treatment of recombinant PKCmu with caspase-3 in vitro results also in the generation of a 62 kDa fragment (p62). Treatment of several aspartic acid to alanine mutants of PKCmu with caspase-3 resulted in an unexpected finding. PKCmu is not cleaved at one of the typical cleavage sites containing the motif DXXD but at the atypical site CQND378/S379. The respective fragment (amino acids 379-912) was expressed in bacteria as a GST fusion protein (GST-p62) and partially purified. In contrast to the intact kinase, the fragment does not respond to the activating cofactors TPA and phosphatidylserine and is thus unable to phosphorylate substrates effectively.

  19. Placental apoptosis in recurrent miscarriage.

    PubMed

    Atia, Tarek A

    2017-09-01

    Apoptosis is an interactive and dynamic biological process involved in all phases of embryogenesis. We aimed to study the effect of placental apoptosis on recurrent miscarriage (RM). Placental tissue samples were collected from 40 women with RM (study group) and 30 women with sporadic spontaneous abortion (control group). Samples were prepared and stained immunohistochemically with markers for both the apoptotic protein (p53) and anti-apoptotic Bcl-2 antibodies. Our results showed that expression of the apoptotic (p53) protein was significantly increased in the placental tissues of the RM group (p = 0.003). By contrast, the expression of anti-apoptotic (Bcl-2) antibodies was significantly increased in the placental tissues of the control group (p = 0.025). We concluded that placental apoptosis plays a crucial role in pregnancy continuation. However, increased p53 expression in placental tissue in early pregnancy could negatively affect pregnancy continuation. Copyright © 2017. Published by Elsevier Taiwan.

  20. Effect of Dark Chocolate Extracts on Phorbol 12-Myristate 13-Acetate-Induced Oxidative Burst in Leukocytes Isolated by Normo-Weight and Overweight/Obese Subjects

    PubMed Central

    Ioannone, Francesca; Sacchetti, Giampiero; Serafini, Mauro

    2017-01-01

    Oxidative and inflammatory stress represents a major risk factor for cardiovascular disease (CVD) in overweight and obese subjects. Between the different plant foods, chocolate has been shown to decrease CVD risk due to its antioxidant and anti-inflammatory properties. However, as we recently showed in epidemiological studies, meta-analyses, and human trials, dietary antioxidants resulted more effective in subjects characterized by an ongoing oxidative stress, than in healthy people. Aim of this work was to investigate the effect of different concentrations of chocolate phenolic extract (CPE) on in vitro free radical production, stimulated by phorbol 12-myristate 13-acetate (PMA), in leukocytes extracted from blood of normo-weight and overweight/obese subjects. Neutrophils from overweight/obese group had a significantly higher free radical production compared to the normo-weight group. In neutrophils, the lowest CPE concentration significantly reduced free radical production in overweight/obese group only, and higher CPE concentrations were effective in both groups. In monocytes, the CPE concentration that was significantly effective in reducing free radical production was lower in overweight/obese subjects than in normo-weight subjects. Chocolate polyphenol extracts inhibit oxidative burst in human neutrophils and monocytes with a higher efficiency in subjects characterized by an unphysiological oxidative/inflammatory stress, such as overweight and obese. Results of this study provide further evidence about a differential role of dietary antioxidant strictly related to the “stress” condition of the subjects. PMID:28649567

  1. Semi-preparative HPLC separation followed by HPLC/UV and tandem mass spectrometric analysis of phorbol esters in Jatropha seed.

    PubMed

    Kongmany, Santi; Hoa, Truong Thi; Hanh, Le Thi Ngoc; Imamura, Kiyoshi; Maeda, Yasuaki; Boi, Luu Van

    2016-12-01

    Phorbol esters (PEs) are well known as the main toxic compounds in Jatropha curcas Linnaeus (JCL), the seed oil of which has been considered as a major feedstock for the production of biodiesel. In the present study, we investigated a series of PEs extracted from JCL seed kernels with methanol (MeOH), and identified more than seven components contained in the PEs. The isolation of main five components of a series of PEs was revised using a semi-preparative reversed phase HPLC analysis of ODS-3 column. The five peaks of components were successfully isolated, and peaks of J2, J3, J5, and J7 were assigned to be Jatropha factors C1, C2, C3, and C4/5, but J6 was a mixture of Jatropha factor C6 and its isomer based on the data of UV and LC-MS/MS, and J2 was identified using (1)H NMR analysis. By characterization using LC-MS/MS analysis, all components of a series of PEs were elucidated to be the 12-deoxy-16-hydroxyphorbol esters composed of isomeric form of dicarboxylic groups with same m/z value of 380.

  2. Sp1 involvement in the 4beta-phorbol 12-myristate 13-acetate (TPA)-mediated increase in resistance to methotrexate in Chinese hamster ovary cells.

    PubMed

    Noé, V; Alemany, C; Nicolás, M; Ciudad, C J

    2001-06-01

    4beta-Phorbol 12-myristate 13-acetate (TPA) increases the number of colonies resistant to methotrexate (MTX), mainly by amplification of the dihydrofolate reductase (dhfr) locus. We showed previously that inhibition of protein kinase C (PKC) prevents this resistance. Here, we studied the molecular changes involved in the development of TPA-mediated MTX resistance in Chinese hamster ovary (CHO) cells. TPA incubation increased the expression and activity of DHFR. Because Sp1 controls the dhfr promoter, we determined the effect of TPA on the expression of Sp1 and its binding to DNA. TPA incubation increased Sp1 binding and the levels of Sp1 protein. The latter effect was due to an increase in Sp1 mRNA. Dephosphorylation of nuclear extracts from control or TPA-treated cells reduced the binding of Sp1. Stable transfectants of PKCalpha showed increased Sp1 binding, and when treated with MTX, developed a greater number of resistant colonies than control cells. Seventy-five percent of the isolated colonies showed increased copy number for the dhfr gene. Transient expression of PKCalpha increased DHFR activity. Over-expression of Sp1 increased resistance to MTX, and inhibition of Sp1 binding by mithramycin decreased this resistance. We conclude that one mechanism by which TPA enhances MTX resistance, mainly by gene amplification, is through an increase in Sp1 expression which leads to DHFR activation.

  3. Phorbol ester stimulates membrane association of protein kinase C and inhibits spontaneous Ca/sup 2 +/ dependent sarcoplasmic reticulum Ca/sup 2 +/ release in rat cardiac cells

    SciTech Connect

    Capogrossi, M.C.; Kaku, T.; Filburn, C.H.; Pelto, D.J.; Hansford, R.G.; Lakatta, E.G.

    1986-03-01

    Spontaneous oscillatory Ca/sup 2 +/ release from sarcoplasmic reticulum (SR) occurs in rat cardiac myocytes at hyperpolarized membrane potentials and is manifested as contractile waves (W). W frequency varies with SR functional status and cell Ca/sup 2 +/ loading. In myocyte suspensions (Hepes buffer, 37/sup 0/C (Ca/sup 2 +/) = 1.0mM) phorbol myristate acetate, PMA, (10/sup -7/ M) increased protein kinase C activity in membranes as a fraction of total (PKCAM) fivefold with a t 1/2 of < 30 sec (n = 3) and decreased W frequency in individual myocytes (n = 8). This effect varied directly and linearly with baseline W frequency, r = .94, p < .001). Dioctanoyl glycerol (10 ..mu.. M) had a similar effect on W. The PMA effect to decrease W frequency could be a direct one on SR or result from a reduction in cell Ca/sup 2 +/. The time course of PKCAM change is sufficiently rapid for it to mediate the effect on W. Thus, enhanced PKCAM may exert negative feedback control on Ca/sup 2 +/ mobilization during ..cap alpha..-adrenergic stimulation.

  4. Differentiation of neuroblastoma cells by phorbol esters and insulin-like growth factor 1 is associated with induction of retinoic acid receptor beta gene expression.

    PubMed

    Perez-Juste, G; Aranda, A

    1999-09-23

    The retinoic acid (RA) receptor beta isoform (RARbeta) plays an important role in RA-induced differentiation of human neuroblastoma. In this study we show that insulin-like growth factor 1 (IGF-1) and tetradecanoyl phorbol acetate (TPA) induce RARbeta gene expression in neuroblastoma SH-SY5Y cells. IGF-1 and TPA caused a marked induction of RARbeta2 promoter activity and had a synergistic effect with RA that also upregulates transcription. The effect of RA is mediated by two RA responsive elements (RAREs), whereas the IGF-1 and TPA actions are independent of the RAREs and map to sequences that overlap the TATA box. These results suggest that the signaling pathways stimulated by TPA and IGF-1 could modify the components assembled at the core RARbeta2 promoter and activate transcription. Expression of RasVal12 mimics the effect of IGF-1 and TPA on the promoter, and a dominant negative Ras mutant abrogates activation. A dominant negative Raf also blocks activation showing that the Ras-Raf pathway mediates stimulation of the RARbeta2 promoter. Our results show that neuronal differentiation induced by non-retinoid agents that activate Ras is accompanied by increased transcription of the RARbeta gene.

  5. Phorbol 12-Myristate 13-Acetate Enhances Long-Term Potentiation in the Hippocampus through Activation of Protein Kinase Cδ and ε

    PubMed Central

    Kim, Eung Chang; Lee, Myeong Jong; Shin, Sang Yep; Seol, Geun Hee; Han, Seung Ho; Yee, Jaeyong; Kim, Chan

    2013-01-01

    Many intracellular proteins and signaling cascades contribute to the sensitivity of N-methyl-D-aspartate receptors (NMDARs). One such putative contributor is the serine/threonine kinase, protein kinase C (PKC). Activation of PKC by phorbol 12-myristate 13-acetate (PMA) causes activation of extracellular signal-regulated kinase (ERK) and promotes the formation of new spines in cultured hippocampal neurons. The purpose of this study was to examine which PKC isoforms are responsible for the PMA-induced augmentation of long-term potentiation (LTP) in the CA1 stratum radiatum of the hippocampus in vitro and verify that this facilitation requires NMDAR activation. We found that PMA enhanced the induction of LTP by a single episode of theta-burst stimulation in a concentration-dependent manner without affecting to magnitude of baseline field excitatory postsynaptic potentials. Facilitation of LTP by PMA (200 nM) was blocked by the nonspecific PKC inhibitor, Ro 31-8220 (10µM); the selective PKCδ inhibitor, rottlerin (1µM); and the PKCε inhibitor, TAT-εV1-2 peptide (500 nM). Moreover, the NMDAR blocker DL-APV (50µM) prevented enhancement of LTP by PMA. Our results suggest that PMA contributes to synaptic plasticity in the nervous system via activation of PKCδ and/or PKCε, and confirm that NMDAR activity is required for this effect. PMID:23440225

  6. A Metabolic Shift toward Pentose Phosphate Pathway Is Necessary for Amyloid Fibril- and Phorbol 12-Myristate 13-Acetate-induced Neutrophil Extracellular Trap (NET) Formation*

    PubMed Central

    Azevedo, Estefania P.; Rochael, Natalia C.; Guimarães-Costa, Anderson B.; de Souza-Vieira, Thiago S.; Ganilho, Juliana; Saraiva, Elvira M.; Palhano, Fernando L.; Foguel, Debora

    2015-01-01

    Neutrophils are the main defense cells of the innate immune system. Upon stimulation, neutrophils release their chromosomal DNA to trap and kill microorganisms and inhibit their dissemination. These chromatin traps are termed neutrophil extracellular traps (NETs) and are decorated with granular and cytoplasm proteins. NET release can be induced by several microorganism membrane components, phorbol 12-myristate 13-acetate as well as by amyloid fibrils, insoluble proteinaceous molecules associated with more than 40 different pathologies among other stimuli. The intracellular signaling involved in NET formation is complex and remains unclear for most tested stimuli. Herein we demonstrate that a metabolic shift toward the pentose phosphate pathway (PPP) is necessary for NET release because glucose-6-phosphate dehydrogenase (G6PD), an important enzyme from PPP, fuels NADPH oxidase with NADPH to produce superoxide and thus induce NETs. In addition, we observed that mitochondrial reactive oxygen species, which are NADPH-independent, are not effective in producing NETs. These data shed new light on how the PPP and glucose metabolism contributes to NET formation. PMID:26198639

  7. Nylon wool adherent accessory cell can augment CTL to MOPC 104E in the presence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA)

    SciTech Connect

    Miura, T.; Ghanta, V.K.; Hiramoto, R.

    1986-03-01

    An active tumor-promoting agent, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) is effective in generating cytotoxic lymphocytes toward the weakly antigenic tumor, MOPC 104E plasmacytoma. Enhanced stimulation of non-adherent spleen lymphocytes (NSC), from which plastic adherent cells were removed, was observed at a concentration of 2 and 10 ng/ml of TPA. This enhancing effect of TPA was obtained at the NSC responder:MOPC 104E stimulator cell ratios of 10:1 and 100:1. T-cells enriched by passage through nylon wool columns from normal or in vivo primed spleens were not responsive to TPA, even after reconstitution with plastic adherent cells. These observations suggest that the generation of cytotoxic lymphocytes by TPA is augmented by an accessory cell present in NSC, which is effectively removed by passage through nylon wool column. The nylon wool adherent accessory cell differs from the plastic adherent macrophages in that the plastic adherent cells were not able to augment the activity of T-cells with TPA.

  8. Anti-edema effects of brown seaweed (Undaria pinnatifida) extract on phorbol 12-myristate 13-acetate-induced mouse ear inflammation.

    PubMed

    Khan, Mohammed Nurul Absar; Yoon, Seung-Je; Choi, Jae-Suk; Park, Nam Gyu; Lee, Hyung-Ho; Cho, Ji-Young; Hong, Yong-Ki

    2009-01-01

    The brown seaweed Undaria pinnatifida (Harvey) Suringar is used in traditional medicine to treat fever, urination problems, lumps and swelling, and as a dietary supplement for post-childbirth women. We examined the anti-inflammatory activities of the seaweed. The methanol extract of the seaweed was active against mouse ear edema induced by phorbol myristate acetate (PMA), with an IC(50) of 10.3 mg/ml. The extract reduced the edema to a half-maximal level when applied at the concentration of 40 mg/ml within 3 hours before or 2 hours after application of PMA. Extract taken from the blade section of the seaweed demonstrated the highest activity. The Northern form of U. pinnatifida was more active than the Southern form. In the analgesic test, the methanol extract suppressed the acetic acid-induced writhing response, with an IC(50) of 0.48 g/kg body weight. The extract also demonstrated antipyretic activity in yeast-induced hyperthermic mice. Activity-related constituents were arachidonic, eicosapentaenoic, and stearidonic acids.

  9. Dynamic membrane-cytoskeletal interactions: specific association of integrin and talin arises in vivo after phorbol ester treatment of peripheral blood lymphocytes.

    PubMed Central

    Burn, P; Kupfer, A; Singer, S J

    1988-01-01

    Members of the family of transmembrane integral membrane proteins called integrins have been implicated in forming attachments to actin microfilaments of the cytoskeleton. These attachments are thought to involve one or more intervening peripheral membrane proteins linked to integrin. To detect such possible linkages in vivo, the integrin molecules on the surfaces of intact chicken peripheral blood lymphocytes were collected into caps by cross-linking with specific antibodies, and the capped cells were examined by double immunofluorescence to determine whether particular cytoskeletal proteins were co-collected with the integrin. With resting lymphocytes, the capping of integrin did not result in any detectable redistribution of either talin, vinculin, or alpha-actinin inside the cells. However, if the capping was carried out upon the addition of phorbol 12-myristate 13-acetate (PMA) to the cells, then talin, but not vinculin or alpha-actinin, was found associated with the integrin caps. PMA is known to activate protein kinase C. These results suggest that after, but not before, PMA stimulation of intact cells, talin becomes linked either directly or indirectly with integrin, reflecting the formation of a membrane-cytoskeletal association that is metabolically regulated. Images PMID:3124107

  10. Insights on profiling of phorbol, deoxyphorbol, ingenol and jatrophane diterpene esters by high performance liquid chromatography coupled to multiple stage mass spectrometry.

    PubMed

    Nothias-Scaglia, Louis-Félix; Schmitz-Afonso, Isabelle; Renucci, Franck; Roussi, Fanny; Touboul, David; Costa, Jean; Litaudon, Marc; Paolini, Julien

    2015-11-27

    This paper reports our effort to develop a comprehensive HPLC-MS(n)-based dereplication strategy for phorbol ester (PE), deoxyphorbol ester (dPE) and ingenol ester (IE) profiling in plant extracts. This strategy is composed of two sequential analysis exploiting specific hybrid triple quadrupole/linear ion trap instrument modes. A first run was performed using a multiple reaction monitoring (MRM) mode targeting fragmentation of PE and dPE/IE coupled with the acquisition of MS(2) spectrum for the ions at m/z 311 and m/z 313, respectively. A second run was then completed based on precursor ion scan mode (PIS) and automatic MS(2) acquisition for each quasimolecular ion. The developed approach was used to investigate ten Euphorbia extracts showing bioactivity against chikungunya virus replication. Experiments allowed partial annotation of three dPE/IE but no PE was detected. Results suggested that other types of diterpene esters displayed PE- and dPE/IE-like fragmentations. The study of jatrophane ester (JE) standards by CID fragmentation using low and high resolution mass spectrometry confirmed this hypothesis, highlighting challenges and difficulties of diterpene esters profiling within plant extracts. Nonetheless, the present LC-MS(n) method can be easily adapted to profile other types of diterpene esters.

  11. Identification of cis-acting sequences responsible for phorbol ester induction of human serum amyloid A gene expression via a nuclear factor kB-like transcription factor

    SciTech Connect

    Edbrooke, M.R.; Cheshire, J.K.; Woo, P.; Burt, B.W.

    1989-05-01

    The authors have analyzed the 5'-flanking region of one of the genes coding for the human acute-phase protein, serum amyloid A (SAA). They found that SAA mRNA could be increased fivefold in transfected cells by treatment with phorbol 12-myristate 13-acetate (PMA). To analyze this observation further, they placed a 265-base-pair 5' SAA fragment upstream of the reporter chloramphenicol acetyltransferase (CAT) gene and transfected this construct into HeLa cells. PMA treatment of these transient transfectants resulted in increased CAT expression. Nuclear proteins from PMA-treated HeLa cells bound to this DNA fragment, and methylation interference analysis showed that the binding was specific to the sequence GGGACTTTCC (between -82 and -91), a sequence previously described by others as the binding site for the nuclear factor NF/kappa/B. In a cotransfection competition experiment, they could abolish PMA-induced CAT activity by using cloned human immunodeficiency virus long-terminal-repeat DNA containing the NF/kappa/B-binding sequence. The same long-terminal-repeat DNA containing mutant NF/kappa/B-binding sequences did not affect CAT expression, which suggested that binding by an NF/kappa/B-like factor is required for increased SAA transcription.

  12. Fluorescent redox dyes. 1. Production of fluorescent formazan by unstimulated and phorbol ester- or digitonin-stimulated Ehrlich ascites tumor cells.

    PubMed

    Stellmach, J

    1984-01-01

    The reduction of a new series of tetrazolium salts to red fluorescent formazans by Ehrlich ascites tumor cells is described. The qualitative effect on this reaction of two cell surface-active compounds and of six exogenous electron carriers was investigated by varying the incubation conditions. After incubation of Ehrlich ascites cells with the new colourless, water soluble 5-cyan-2.3-ditolyltetrazolium salts, bright red water-insoluble formazan crystals on the cell surface can be observed under fluorescence microscopy. The production of formazan is enhanced by 12-0-tetradecanoyl-phorbol-13-acetate (TPA) or digitonin (DIG), two potent stimulators of oxygen consumption or by the electron carriers phenazine methosulphate (PMS), 1-methoxy-phenazine methosulphate (MPMS), meldola blue (MB), methylene blue (MTB), and 2.6-dichlorindophenol (DCIP). These results provide further evidence for the existence of redox enzymes bound to the plasma membrane of intact ascites cells and for a free radical mechanism of tetrazolium salt reduction. The fluorescence property of the new redox dyes offers the advantage of high sensitivity. Moreover, their greater homogeneity relative to the commonly used di-tetrazolium salts lowers the chances of misinterpretations due to impurities. The possible application of these new mono-tetrazolium salts to cytochemical investigations of oxidative metabolic reactions is discussed.

  13. Effects of antiinflammatory agents on mouse skin tumor promotion, epidermal DNA synthesis, phorbol ester-induced cellular proliferation, and production of plasminogen activator.

    PubMed

    Viaje, A; Slaga, T J; Wigler, M; Weinstein, I B

    1977-05-01

    The antinflammatory ateroids fluocinoine acetonide, fluocinonide, and fluclorolone acetonide were found to be very effectiveinhibitory agents of mouse skin tumor promotion. These steroids also drastically inhibited epidermal DNA synthesis and epidermal cellular proliferation induced by a phorbal ester tumor promoter. In addition, these compounds were potent inhibitors, of plasminogen activator production in tumor cell cultures. The clinically used non-steroidal antiinflammatory agents oxyphenbutazone, indomethacin, and Seclazone also inhibite tumor promotion but were much less effective. Although these agents are useful against inflammatory disorders in general when given p.o., in our studies they had little effect on inflammation and epidermal cellular proliferation induced by a phorbol ester tumor promoter when given topically. The afore mentioned nonsteroidal antiinflammatory agents also had little effect on epidermal DNA synthesis. Oxyphenbutazone and indomethacin were less potent inhibitors of plasminogen activator production in tumor cells than were the antiinflammatory steroids, and Seclazone produced a negligible inhibition. There is, therefore, a general correlation in the potencies of a series of steroidal antiinflammatory agents for inhibition of tumor promotion and their ability to inhibit plasminogen activator production by tumor cell cultures and epidermal DNA synthesis.

  14. Defective responses of transformed keratinocytes to terminal differentiation stimuli. Their role in epidermal tumour promotion by phorbol esters and by deep skin wounding.

    PubMed Central

    Parkinson, E. K.

    1985-01-01

    Epidermal tumourigenesis can be achieved in rodents by the application of a single subthreshold dose of a carcinogen (initiation) followed by repeated applications of a tumour promoter such as 12-0-tetradecanoyl phorbol, 13-acetate (TPA). TPA induces terminal differentiation in the majority of epidermal keratinocytes in vitro. However, transformed keratinocytes respond weakly to this terminal differentiation signal, and it is suggested that this property allows initiated cells and their progeny to obtain a selective advantage over their normal counterparts during promotion of papilloma formation by TPA. New data are reviewed which suggest that a putative wound hormone TGF-beta has similar differential effects on normal and transformed epithelial cells to those of TPA. It is proposed that the release of TGF-beta from platelets following deep skin wounding may be an explanation as to why wounding is a promoting stimulus but milder forms of epidermal injury are not. Weakly promoting hyperplasiogenic agents are also discussed within the context of a selection theory of tumour promotion. PMID:2415144

  15. Distribution of Spinal Sensitization Evoked by Inflammatory Pain Using Local Spinal Cord Glucose Utilization Combined with (3) H-Phorbol 12,13-Dibutyrate Binding in Rats.

    PubMed

    Seiko, Yasuda; Kozo, Ishikawa; Yoshihiro, Matsumoto; Toru, Ariyoshi; Hironori, Sasaki; Yuika, Ida; Yasutake, Iwanaga; Hae-Kyu, Kim; Osamu, Nakanishi; Toshizo, Ishikawa

    2013-01-01

    Aims. Hyperalgesia following tissue injury is induced by plasticity in neurotransmission. Few investigators have considered the ascending input which activates the superficial of spinal cord. The aim was to examine neurotransmission and nociceptive processing in the spinal cord after mustard-oil (MO) injection. Both in vitro and in vivo autoradiographs were employed for neuronal activity and transmission in discrete spinal cord regions using the (14)C-2-deoxyglucose method and (3)H-phorbol 12,13-dibutyrate ((3)H-PDBu) binding sites. Methods. To quantify the hyperalgesia evoked by MO, the flinching was counted for 60 min after MO (20%, 50 μL) injection in Wistar rats. Simultaneous determination of (14)C-2-deoxyglucose and (3)H-PDBu binding was used for a direct observation of neuronal/metabolic changes and intracellular signaling in the spinal cord. Results. MO injection evoked an increase in flinching for 60 min. LSCGU significantly increased in the Rexed I-II with (3)H-PDBu binding in the ipsilateral side of spinal cord. Discussion. We clearly demonstrated that the hyperalgesia is primarily relevant to increased neuronal activation with PKC activation in the Rexed I-II of the spinal cord. In addition, functional changes such as "neuronal plasticity" may result in increased neuronal excitability and a central sensitization.

  16. Distribution of Spinal Sensitization Evoked by Inflammatory Pain Using Local Spinal Cord Glucose Utilization Combined with 3H-Phorbol 12,13-Dibutyrate Binding in Rats

    PubMed Central

    Seiko, Yasuda; Kozo, Ishikawa; Yoshihiro, Matsumoto; Toru, Ariyoshi; Hironori, Sasaki; Yuika, Ida; Yasutake, Iwanaga; Hae-Kyu, Kim; Osamu, Nakanishi; Toshizo, Ishikawa

    2013-01-01

    Aims. Hyperalgesia following tissue injury is induced by plasticity in neurotransmission. Few investigators have considered the ascending input which activates the superficial of spinal cord. The aim was to examine neurotransmission and nociceptive processing in the spinal cord after mustard-oil (MO) injection. Both in vitro and in vivo autoradiographs were employed for neuronal activity and transmission in discrete spinal cord regions using the 14C-2-deoxyglucose method and 3H-phorbol 12,13-dibutyrate (3H-PDBu) binding sites. Methods. To quantify the hyperalgesia evoked by MO, the flinching was counted for 60 min after MO (20%, 50 μL) injection in Wistar rats. Simultaneous determination of 14C-2-deoxyglucose and 3H-PDBu binding was used for a direct observation of neuronal/metabolic changes and intracellular signaling in the spinal cord. Results. MO injection evoked an increase in flinching for 60 min. LSCGU significantly increased in the Rexed I-II with 3H-PDBu binding in the ipsilateral side of spinal cord. Discussion. We clearly demonstrated that the hyperalgesia is primarily relevant to increased neuronal activation with PKC activation in the Rexed I-II of the spinal cord. In addition, functional changes such as “neuronal plasticity” may result in increased neuronal excitability and a central sensitization. PMID:27335874

  17. Human anti-peptidoglycan-IgG-mediated opsonophagocytosis is controlled by calcium mobilization in phorbol myristate acetate-treated U937 cells

    PubMed Central

    Rah, So-Young; An, Jang-Hyun; Kurokawa, Kenji; Kim, Uh-Hyun; Lee, Bok Luel

    2015-01-01

    Recently, we demonstrated that human serum amyloid P component (SAP) specifically recognizes exposed bacterial peptidoglycan (PGN) of wall teichoic acid (WTA)-deficient Staphylococcus aureus ΔtagO mutant cells and then induces complement-independent phagocytosis. In our preliminary experiments, we found the existence of human serum immunoglobulins that recognize S. aureus PGN (anti-PGNIgGs), which may be involved in complement-dependent opsonophagocytosis against infected S. aureus cells. We assumed that purified serum anti-PGN-IgGs and S. aureus ΔtagO mutant cells are good tools to study the molecular mechanism of anti-PGN-IgG-mediated phagocytosis. Therefore, we tried to identify the intracellular molecule(s) that is involved in the anti-PGN-IgG-mediated phagocytosis using purified human serum anti-PGN-IgGs and different S. aureus mutant cells. Here, we show that anti-PGN-IgG-mediated phagocytosis in phorbol myristate acetate-treated U937 cells is mediated by Ca2+ release from intracellular Ca2+ stores and anti-PGN-IgGdependent Ca2+ mobilization is controlled via a phospholipase Cγ-2-mediated pathway. [BMB Reports 2015; 48(1): 36-41] PMID:24856825

  18. Classification of signals for blocking apoptosis in vascular endothelial cells.

    PubMed

    Hase, M; Araki, S; Kaji, K; Hayashi, H

    1994-10-01

    The survival and death of human umbilical vascular endothelial cells in culture are affected by several factors, such as fibroblast growth factor (FGF), serum, phorbol ester (TPA), and vanadate. In order to identify common aspects of the various signal-transduction processes during the course of apoptotic or programmed cell death, we designed experiments to distinguish between these factors in terms of the pathway that is responsible for the processing of each stimulus. We found, for example, that the effect of removal of FGF was specifically overcome by the addition of the phorbol ester. Our results indicated that two distinct pathways were operative, one specific for signal transduction initiated by FGF and phorbol ester and another specific for signal transduction initiated by serum and vanadate. These two pathways merged down-stream of the individual signal-processing pathways.

  19. The mechanism of PDT-induced apoptosis

    NASA Astrophysics Data System (ADS)

    Cai, Xiongwei; Liu, Timon C.; Ding, Xin-Min; Gu, Ying; Liu, Fan-Guang; Liu, Song-Hao

    2003-12-01

    Photodynamic therapy (PDT) can induce apoptosis in many cancer cells in vitro and in tumors in vivo. Cells become more oxidation with PDT, and maintain differentiation and proliferation, go apoptosis and necrosis with the increase of reactive oxygen species (ROS) concentration. ROS can induce apoptosis through mitochondria by inhibiting respiration chain or oxidative phosphorylation or damaging mitochondrial membrane. ROS can initiate apoptosis through endoplamic reticulum(ER) by opening Ca2+ channel or starting unfold protein response (UPR). ROS can also induce apoptosis through Golgi by producing ganglioside GD3 by use of ceramide, which induces apoptosis by activating caspase-3, JNK and p38 MAPK. It can also induce apoptosis by activating Bip (mitochondria-dependant) or preocaspase-12 (mitochondria- independent) or inhibiting protein synthesizing. There are so complicated cross-talking among different signal pathways or organnells that we think PDT-induced apoptosis is mediated by multiplex pathways and excessive levels in a refined network.

  20. Oxidative stress, superoxide production, and apoptosis of neutrophils in dogs with chronic kidney disease

    PubMed Central

    Silva, Adriana Carolina Rodrigues Almeida; de Almeida, Breno Fernando Martins; Soeiro, Carolina Soares; Ferreira, Wagner Luis; de Lima, Valéria Marçal Félix; Ciarlini, Paulo César

    2013-01-01

    Oxidative stress is a key component in the immunosuppression of chronic kidney disease (CKD), and neutrophil function may be impaired by oxidative stress. To test the hypothesis that in uremic dogs with CKD, oxidative stress is increased and neutrophils become less viable and functional, 18 adult dogs with CKD were compared with 15 healthy adult dogs. Blood count and urinalysis were done, and the serum biochemical profile and plasma lipid peroxidation (measurement of thiobarbituric acid reactive substances) were determined with the use of commercial reagents. Plasma total antioxidant capacity (TAC) was measured with a spectrophotometer and commercial reagents, superoxide production with a hydroethidine probe, and the viability and apoptosis of neutrophils with capillary flow cytometry and the annexin V-PE system. The plasma concentrations of cholesterol (P = 0.0415), creatinine (P < 0.0001), and urea (P < 0.0001) were significantly greater in the uremic dogs than in the control dogs. The hematocrit (P = 0.0004), urine specific gravity (P = 0.015), and plasma lipid peroxidation (P < 0.0001) were significantly lower in the dogs that were in late stages of CKD than in the control group. Compared with those isolated from the control group, neutrophils isolated from the CKD group showed a higher rate of spontaneous (0.10 ± 0.05 versus 0.49 ± 0.09; P = 0.0033; median ± standard error of mean) and camptothecin-induced (18.53 ± 4.06 versus 44.67 ± 4.85; P = 0.0066) apoptosis and lower levels of superoxide production in the presence (1278.8 ± 372.8 versus 75.65 ± 86.6; P = 0.0022) and absence (135.29 ± 51.74 versus 41.29 ± 8.38; P = 0.0138) of phorbol-12-myristate-13-acetate stimulation. Thus, oxidative stress and acceleration of apoptosis occurs in dogs with CKD, the apoptosis diminishing the number of viable neutrophils and neutrophil superoxide production. PMID:24082406

  1. Sall2 is required for proapoptotic Noxa expression and genotoxic stress-induced apoptosis by doxorubicin

    PubMed Central

    Escobar, D; Hepp, M I; Farkas, C; Campos, T; Sodir, N M; Morales, M; Álvarez, C I; Swigart, L; Evan, G I; Gutiérrez, J L; Nishinakamura, R; Castro, A F; Pincheira, R

    2015-01-01

    The Sall2 transcription factor is deregulated in several cancers; however, little is known about its cellular functions, including its target genes. Recently, we demonstrated that p53 directly regulates Sall2 expression under genotoxic stress. Here, we investigated the role of Sall2 in the context of cellular response to genotoxic stress. In addition, we further examined the Sall2-p53 relationship during genotoxic stress in primary mouse embryo fibroblasts (MEFs), which are derived from Sall2 knockout mice separately, or in combination with the p53ERTAM knock-in mice. We found that the levels of Sall2 mRNA and protein are dynamically modulated in response to doxorubicin. At early times of stress, Sall2 is downregulated, but increases under extension of the stress in a p53-independent manner. Based on caspase-3/7 activities, expression of cleaved poly (ADP-ribose) polymerase, expression of cleaved caspase-3 and induction of proapoptotic proteins, Sall2 expression was correlated with cellular apoptosis. Consequently, Sall2−/− MEFs have decreased apoptosis, which relates with increased cell viability in response to doxorubicin. Importantly, Sall2 was required for apoptosis even in the presence of fully activated p53. Searching for putative Sall2 targets that could mediate its role in apoptosis, we identified proapoptotic NOXA/PMAIP1 (phorbol-12-myristate-13-acetate-induced protein 1). We demonstrated that Sall2 positively regulates Noxa promoter activity. Conserved putative Sall2-binding sites at the NOXA promoter were validated in vitro by electrophoretic mobility shift assay and in vivo by ChIP experiments, identifying NOXA as a novel Sall2 target. In agreement, induction of Noxa protein and mRNA in response to doxorubicin was significantly decreased in Sall2−/− MEFs. In addition, studies in leukemia Jurkat T cells support the existence of the Sall2/Noxa axis, and the significance of this axis on the apoptotic response to doxorubicin in cancer cells. Our

  2. Oxidative stress, superoxide production, and apoptosis of neutrophils in dogs with chronic kidney disease.

    PubMed

    Silva, Adriana Carolina Rodrigues Almeida; de Almeida, Breno Fernando Martins; Soeiro, Carolina Soares; Ferreira, Wagner Luis; de Lima, Valéria Marçal Félix; Ciarlini, Paulo César

    2013-04-01

    Oxidative stress is a key component in the immunosuppression of chronic kidney disease (CKD), and neutrophil function may be impaired by oxidative stress. To test the hypothesis that in uremic dogs with CKD, oxidative stress is increased and neutrophils become less viable and functional, 18 adult dogs with CKD were compared with 15 healthy adult dogs. Blood count and urinalysis were done, and the serum biochemical profile and plasma lipid peroxidation (measurement of thiobarbituric acid reactive substances) were determined with the use of commercial reagents. Plasma total antioxidant capacity (TAC) was measured with a spectrophotometer and commercial reagents, superoxide production with a hydroethidine probe, and the viability and apoptosis of neutrophils with capillary flow cytometry and the annexin V-PE system. The plasma concentrations of cholesterol (P = 0.0415), creatinine (P < 0.0001), and urea (P < 0.0001) were significantly greater in the uremic dogs than in the control dogs. The hematocrit (P = 0.0004), urine specific gravity (P = 0.015), and plasma lipid peroxidation (P < 0.0001) were significantly lower in the dogs that were in late stages of CKD than in the control group. Compared with those isolated from the control group, neutrophils isolated from the CKD group showed a higher rate of spontaneous (0.10 ± 0.05 versus 0.49 ± 0.09; P = 0.0033; median ± standard error of mean) and camptothecin-induced (18.53 ± 4.06 versus 44.67 ± 4.85; P = 0.0066) apoptosis and lower levels of superoxide production in the presence (1278.8 ± 372.8 versus 75.65 ± 86.6; P = 0.0022) and absence (135.29 ± 51.74 versus 41.29 ± 8.38; P = 0.0138) of phorbol-12-myristate-13-acetate stimulation. Thus, oxidative stress and acceleration of apoptosis occurs in dogs with CKD, the apoptosis diminishing the number of viable neutrophils and neutrophil superoxide production.

  3. APOPTOSIS IN WHOLE MOUSE OVARIES

    EPA Science Inventory

    Apoptosis in Whole Mouse Ovaries
    Robert M. Zucker Susan C. Jeffay and Sally D. Perreault
    Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, 27711.

  4. Methylselenium and Prostate Cancer Apoptosis

    DTIC Science & Technology

    2003-02-01

    dependent apoptosis execution in PCa cells. We have in the reporting period refined a methylselenol generation system based on methioninase with... methylselenol , methylselenium 16. PRICE CODE 17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION 20. LIMITATION OF... methylselenol generation system based on methioninase with selenomethionine as substrate (Wang et al, Mol. Carcinogenesis, 2002, appendix 1) and

  5. Methylselenium and Prostate Cancer Apoptosis

    DTIC Science & Technology

    2008-02-01

    methylselenol , prostate cancer chemoprevention, apoptosis 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT 18. NUMBER OF PAGES 19a. NAME OF...considered an immediate precursor to the in vivo active anticancer selenium metabolite methylselenol , greatly sensitized HRPCa cells to undergo

  6. Methylselenium and Prostate Cancer Apoptosis

    DTIC Science & Technology

    2006-02-01

    will lay ground work for future validation studies in vivo and translation from the bench to the bedside. 15. SUBJECT TERMS Selenium, methylselenol ...Results: The methylselenol precursor methylseleninic acid (MSeA) increased the apoptosis potency of SN38, etoposide or paclitaxel by several folds

  7. Pancreatic carcinogenesis: apoptosis and angiogenesis.

    PubMed

    Onizuka, Shinya; Kawakami, Shunsuke; Taniguchi, Ken; Fujioka, Hikaru; Miyashita, Kosei

    2004-04-01

    Apoptosis and angiogenesis are critical biologic processes that are altered during carcinogenesis. Both apoptosis and angiogenesis may play an important role in pancreatic carcinogenesis. Despite numerous advances in the diagnosis and treatment of pancreatic cancer, its prognosis remains dismal and a new therapeutic approach is much needed. Recent research has revealed that apoptosis and angiogenesis are closely interrelated. Several reports show that a tumor suppresser gene that is expressed in pancreatic carcinoma and related to malignant potential can induce apoptosis and also inhibit angiogenesis. At present, it is generally accepted that tumor growth in cancers, including pancreatic cancer, depends on angiogenesis. We have identified 2 new angiogenesis inhibitors from a conditioned medium of human pancreatic carcinoma cell line (BxPC-3): antiangiogenic antithrombin III (aaAT-III) and vitamin D binding protein-macrophage activating factor (DBP-maf). These molecules were able to regress tumors in severe combined immunodeficiency disease (SCID) mice, demonstrating potent inhibition of endothelial cell proliferation. Moreover, the angiogenesis inhibitors induced tumor dormancy in the animal model. These results suggest that antiangiogenic therapy using angiogenesis inhibitors may become a new strategy for treatment of pancreatic cancer in the near future.

  8. [Apoptosis in chronic lymphocytic leukemia].

    PubMed

    Giordano, M

    2000-01-01

    Chronic lymphocytic leukemia of B cells (B-CLL) is the most prevalent leukemia in the Occidental Hemisphere. It is characterized by a progressive accumulation of monoclonal CD5+ B lymphocytes, with low amounts of surface Ig. Most B-CLL cells are arrested in the G0 phase of the cell cycle; therefore their accumulation in vivo appears to result from the inhibition of apoptosis which has been attributed to over-expression of the anti-apoptotic protein Bcl-2. When cultured in vitro, spontaneous apoptosis occurs, suggesting the existence in vivo of survival-promoting factors. We here show that non-malignant leukocytes, particularly monocytes and NK cells, are able to inhibit B-CLL cells apoptosis, at least in part, through the release of soluble factors. Neutralizing antibodies directed to interferon-gamma or IL-4 only partially abolish the protecting effects of accessory cells suggesting that they are not the main cytokines involved. Increased apoptosis of B-CLL cells is not associated with modifications in the expression of Bcl-2, Fas or Fas ligand. Considering that B-CLL is associated to autoimmune phenomena and recurrent infections due to hypogammaglobulinemia, it should be interesting to correlate the activation of immune responses with disease progression.

  9. APOPTOSIS IN WHOLE MOUSE OVARIES

    EPA Science Inventory

    Apoptosis in Whole Mouse Ovaries
    Robert M. Zucker Susan C. Jeffay and Sally D. Perreault
    Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, 27711.

  10. Methods for determining Myc-induced apoptosis.

    PubMed

    Lu, Dan; Littlewood, Trevor D

    2013-01-01

    Although many oncoproteins promote cell growth and proliferation, some also possess the potential to induce cell death by apoptosis. Deregulated expression of the myc oncogene promotes apoptosis in both cultured cells and in some tissues in vivo. Here we describe techniques to detect Myc-induced apoptosis in vitro using flow cytometry and microscopy and in vivo using immunohistochemical staining.

  11. Ca(2+) movement and apoptosis induced by deltamethrin in Madin-Darby canine kidney canine renal tubular cells.

    PubMed

    Liu, Fang-Jin; Chou, Chiang-Ting; Cheng, Jin Shiung; Chang, Hong-Tai; Liang, Wei-Zhe; Kuo, Chun-Chi; Kuo, Soong-Yu; Kuo, Daih-Huang; Shieh, Pochuen; Chang, Fang-Rong; Jan, Chung-Ren

    2015-01-01

    This study explored the effect of deltamethrin, a pesticide, on free Ca(2+) concentration [Ca(2+)]i, viability, and apoptosis in Madin-Darby canine kidney (MDCK) canine renal tubular cells. Deltamethrin at concentrations between 10μM and 40μM evoked [Ca(2+)]i rises in a concentration-dependent manner. The Ca(2+) entry was inhibited by nifedipine, econazole, phorbol 12-myristate 13-acetate, and SKF96365. Treatment with the endoplasmic reticulum Ca(2+) pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) in a Ca(2+)-free medium abolished deltamethrin-induced [Ca(2+)]i rise. Treatment with deltamethrin also abolished BHQ-induced [Ca(2+)]i rise. Inhibition of phospholipase C (PLC) activity with U73122 abolished deltamethrin-evoked [Ca(2+)]i rise. Deltamethrin killed cells at 30-60μM in a concentration-dependent manner. The cytotoxic effect of deltamethrin was not reversed by prechelating cytosolic Ca(2+) with the acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Annexin V/propidium iodide staining data suggest that 30-50μM deltamethrin induced apoptosis. Together, in MDCK renal tubular cells, deltamethrin induced [Ca(2+)]i rises that involved Ca(2+) entry through protein kinase C-mediated store-operated Ca(2+) channels, and PLC-dependent Ca(2+) release from the endoplasmic reticulum. Deltamethrin also induced Ca(2+)-independent cell death that might involve apoptosis. Copyright © 2014. Published by Elsevier Taiwan.

  12. Distinct PKC isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbol ester in RAW264.7 macrophages

    PubMed Central

    Lin, Wan-W; Chen, Bin C

    1998-01-01

    The modulatory effects of protein kinase C (PKC) on the activation of cytosolic phospholipase A2 (cPLA2) and adenylyl cyclase (AC) have recently been described. Since the signalling cascades associated with these events play critical roles in various functions of macrophages, we set out to investigate the crosstalk between PKC and the cPLA2 and AC pathways in mouse RAW 264.7 macrophages and to determine the involvement of individual PKC isoforms. The cPLA2 and AC pathways were studied by measuring the potentiation by the phorbol ester PMA of ionomycin-induced arachidonic acid (AA) release and prostagladin E1 (PGE1)-stimulated cyclic AMP production, respectively.PMA at 1 μM caused a significant increase in AA release both in the presence (371%) and absence (67%) of ionomycin induction, while exposure of RAW 264.7 cells to PMA increased PGE1 stimulation of cyclic AMP levels by 208%.Treatment of cells with staurosporine and Ro 31-8220 inhibited the PMA-induced potentiation of both AA release and cyclic AMP accumulation, while Go 6976 (an inhibitor of classical PKC isoforms) and LY 379196 (a specific inhibitor of PKCβ) inhibited the AA response but failed to affect the enhancement of the cyclic AMP response by PMA.Long term pretreatment of cells with PMA abolished the subsequent effect of PMA in potentiating AA release, but only inhibited the cyclic AMP response by 42%.Neither PD 98059, an inhibitor of MEK, nor genistein, an inhibitor of tyrosine kinases, had any effect on the ability of PMA to potentiate AA or cyclic AMP production.The potentiation of AA release, but not of cyclic AMP formation, by PMA was sensitive to inhibition by wortmannin. This effect was unrelated to the inhibition of PKC activation as deduced from the translocation of PKC activity to the cell membrane.Western blot analysis revealed the presence of eight PKC isoforms (α, βI, βII, δ, ε, μ λ and ξ) in RAW 264.7 cells and PMA was shown to induce the translocation of the α, βI, βII,

  13. Transplacental arsenic plus postnatal 12-O-teradecanoyl phorbol-13-acetate exposures associated with hepatocarcinogenesis induce similar aberrant gene expression patterns in male and female mouse liver

    SciTech Connect

    Liu Jie . E-mail: Liu6@niehs.nih.gov; Xie Yaxiong; Merrick, B. Alex; Shen Jun; Ducharme, Danica M.K.; Collins, Jennifer; Diwan, Bhalchandra A.; Logsdon, Daniel; Waalkes, Michael P.

    2006-06-15

    Our prior work shows that in utero arsenic exposure alone is a complete transplacental carcinogen, producing hepatocellular carcinoma in adult male offspring but not in females. In a follow-up study to potentially promote arsenic-initiated tumors, mice were exposed to arsenic (85 ppm) from gestation day 8 to 18 and then exposed to 12-O-teradecanoyl phorbol-13-acetate (TPA), a well-known tumor promoter after weaning. The dermal application of TPA (2 {mu}g/0.1 ml acetone, twice/week for 21 weeks) after transplacental arsenic did not further increase arsenic-induced liver tumor formation in adult males but significantly increased liver tumor formation in adult females. Thus, for comparison, liver tumors and normal liver samples taken from adult male and female mice at necropsy were analyzed for aberrant gene/protein expression by microarray, real-time RT-PCR and Western blot analysis. Arsenic/TPA treatment resulted in increased expression of {alpha}-fetoprotein, k-ras, c-myc, estrogen receptor-{alpha}, cyclin D1, cdk2na, plasminogen activator inhibitor-1, cytokeratin-8, cytokeratin-18, glutathione S-transferases and insulin-like growth factor binding proteins in liver and liver tumors from both male and female mice. Arsenic/TPA also decreased the expression of BRCA1, betaine-homocysteine methyltransferase, CYP7B1, CYP2F2 and insulin-like growth factor-1 in normal and cancerous livers. Alterations in these gene products were associated with arsenic/TPA-induced liver tumors, regardless of sex. Thus, transplacental arsenic plus postnatal TPA exposure induced similar aberrant gene expression patterns in male and female mouse liver, which are persistent and potentially important to the mechanism of arsenic initiation of hepatocarcinogenesis.

  14. Differential effects of histone deacetylase inhibitors on phorbol ester- and TGF-beta1 induced murine tissue inhibitor of metalloproteinases-1 gene expression.

    PubMed

    Young, David A; Billingham, Olivia; Sampieri, Clara L; Edwards, Dylan R; Clark, Ian M

    2005-04-01

    Expression of the tissue inhibitor of metalloproteinases-1 (Timp-1) gene can be induced by either phorbol myristate acetate (PMA) or transforming growth factor beta1 (TGF-beta1), although the signalling pathways involved are not clearly defined. Canonically, histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) or sodium butyrate (NaB) increase total cellular histone acetylation and activate expression of susceptible genes. Remarkably, PMA and TGF-beta1 stimulation of Timp-1 show a differential response to TSA or NaB. TSA or NaB potentiate PMA-induced Timp-1 expression but repress TGF-beta1-induced Timp-1 expression. The repression of TGF-beta1-induced Timp-1 by TSA was maximal at 5 ng.mL(-1), while for the superinduction of PMA-induced Timp-1 expression, the maximal dose is > 500 ng x mL(-1) TSA. A further HDACi, valproic acid, did not block TGF-beta1-induced Timp-1 expression, demonstrating that different HDACs impact on the induction of Timp-1. For either PMA or TGF-beta1 to induce Timp-1 expression, new protein synthesis is required, and the induction of AP-1 factors closely precedes that of Timp-1. The effects of the HDACi can be reiterated in transient transfection using Timp-1 promoter constructs. Mutation or deletion of the AP-1 motif (-59/-53) in the Timp-1 promoter diminishes PMA-induction of reporter constructs, however, the further addition of TSA still superinduces the reporter. In c-Jun-/- cells, PMA still stimulates Timp-1 expression, but TSA superinduction is lost. Transfection of a series of Timp-1 promoter constructs identified three regions through which TSA superinduces PMA-induced Timp-1 and we have demonstrated specific protein binding to two of these regions which contain either an avian erythroblastosis virus E26 (v-ets) oncogene homologue (Ets) or Sp1 binding motif.

  15. Micromanipulation of adhesion of phorbol 12-myristate-13-acetate-stimulated T lymphocytes to planar membranes containing intercellular adhesion molecule-1.

    PubMed Central

    Tözeren, A; Mackie, L H; Lawrence, M B; Chan, P Y; Dustin, M L; Springer, T A

    1992-01-01

    This paper presents an analytical and experimental methodology to determine the physical strength of cell adhesion to a planar membrane containing one set of adhesion molecules. In particular, the T lymphocyte adhesion due to the interaction of the lymphocyte function associated molecule 1 on the surface of the cell, with its counter-receptor, intercellular adhesion molecule-1 (ICAM-1), on the planar membrane, was investigated. A micromanipulation method and mathematical analysis of cell deformation were used to determine (a) the area of conjugation between the cell and the substrate and (b) the energy that must be supplied to detach a unit area of the cell membrane from its substrate. T lymphocytes stimulated with phorbol 12-myristate-13-acetate (PMA) conjugated strongly with the planar membrane containing purified ICAM-1. The T lymphocytes attached to the planar membrane deviated occasionally from their round configuration by extending pseudopods but without changing the size of the contact area. These adherent cells were dramatically deformed and then detached when pulled away from the planar membrane by a micropipette. Detachment occurred by a gradual decrease in the radius of the contact area. The physical strength of adhesion between a PMA-stimulated T lymphocyte and a planar membrane containing 1,000 ICAM-1 molecules/micron 2 was comparable to the strength of adhesion between a cytotoxic T cell and its target cell. The comparison of the adhesive energy density, measured at constant cell shape, with the model predictions suggests that the physical strength of cell adhesion may increase significantly when the adhesion bonds in the contact area are immobilized by the actin cytoskeleton. Images FIGURE 2 FIGURE 4 FIGURE 5 FIGURE 8 FIGURE 9 PMID:1358239

  16. Activation of glucose transport in skeletal muscle by phospholipase C and phorbol ester. Evaluation of the regulatory roles of protein kinase C and calcium

    SciTech Connect

    Henriksen, E.J.; Rodnick, K.J.; Holloszy, J.O. )

    1989-12-25

    It has been hypothesized on the basis of studies on BC3H-1 myocytes that diacylglycerol generation with activation of protein kinase C (PKC) is involved in the stimulation of glucose transport in muscle by insulin. In the present study, we used the rat epitrochlearis muscle to evaluate the possibility that PKC activity mediates the stimulation of glucose transport by insulin in mammalian skeletal muscle. Phospholipase C from Clostridium perfringens (PLC-Cp), which generates diacylglycerol from membrane phospholipids, and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) induced increases in glucose transport activity (assessed using 3-O-methylglucose transport) that were approximately 80 and approximately 20% as great, respectively, as that induced by a maximal insulin stimulus. PLC-Cp and PMA both caused a approximately 2-fold increase in membrane-associated PKC activity. In contrast, insulin did not affect PKC activity. These findings argue against a role of diacylglycerol-mediated PKC activation in the stimulation of skeletal muscle glucose transport by insulin. They also show that the BC3H-1 myocyte is not a good model for studying regulation of glucose transport in skeletal muscle. Neither the submaximal nor maximal effects of PLC-Cp and insulin on glucose transport were additive, suggesting that PLC-Cp interferes with insulin action. The maximal effects of PLC-Cp and hypoxia or muscle contractions were also not additive. However, the submaximal effects of hypoxia and PLC-Cp were completely additive. These findings raise the possibility that PLC-Cp stimulates glucose transport by the exercise/hypoxia-activated, not the insulin-activated, pathway in skeletal muscle.

  17. The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus.

    PubMed

    Lund, Maria E; To, Joyce; O'Brien, Bronwyn A; Donnelly, Sheila

    2016-03-01

    The human monocytic cell line, THP-1, is the most widely used model for primary human monocytes/macrophages. This is because, following differentiation using phorbol 12-myristate 13-acetate (PMA), THP-1 cells acquire a macrophage-like phenotype, which mimics, in many respects, primary human macrophages. Despite the widespread use of THP-1 cells in studies elucidating macrophage responses to inflammatory stimuli, as well as the development and screening of potential therapeutics, there is currently no standardised protocol for the reliable differentiation of THP-1 monocytes to a macrophage phenotype using PMA. Consequently, reports using THP-1 cells have demonstrated significant phenotypic and functional differences between resultant THP-1 macrophage populations, which are largely attributable to the varying PMA differentiation methods used. Thus, to guarantee consistency and reproducibility between studies, and to ensure the relevance of THP-1 cells as an appropriate model for primary human macrophages, it is crucial to develop a standardised protocol for the differentiation of THP-1 macrophages. Accordingly, we compared the function and phenotype of THP-1 macrophages generated using the range of published PMA differentiation protocols, specifically in response to the pro-inflammatory stimulus, lipopolysaccharide (LPS). Our results demonstrated that the function of the resultant THP-1 macrophage populations, as determined by tumour necrosis factor (TNF) secretion in response to LPS stimulation, varied significantly, and was dependent upon the concentration of PMA used to stimulate the differentiation of monocytes, and the period of rest following PMA exposure. These data indicate that exposure of monocytic THP-1 cells to 25 nM PMA over 48 h, followed by a recovery period of 24h in culture in the absence of PMA, was the optimal protocol for the differentiation of THP-1 cells.

  18. Phorbol 12-myristate 13-acetate-induced endocytosis of the Na-K-2Cl cotransporter in MDCK cells is associated with a clathrin-dependent pathway.

    PubMed

    Mykoniatis, Andreas; Shen, Le; Fedor-Chaiken, Mary; Tang, Jun; Tang, Xu; Worrell, Roger T; Delpire, Eric; Turner, Jerrold R; Matlin, Karl S; Bouyer, Patrice; Matthews, Jeffrey B

    2010-01-01

    In secretory epithelial cells, the basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) plays a major role in salt and fluid secretion. Our laboratory has identified NKCC1 surface expression as an important regulatory mechanism for Cl(-) secretion in the colonic crypt cell line T84, a process also present in native human colonic crypts. We previously showed that activation of protein kinase C (PKC) by carbachol and phorbol 12-myristate 13-acetate (PMA) decreases NKCC1 surface expression in T84 cells. However, the specific endocytic entry pathway has not been defined. We used a Madin-Darby canine kidney (MDCK) cell line stably transfected with enhanced green fluorescent protein (EGFP)-NKCC1 to map NKCC1 entry during PMA exposure. At given times, we fixed and stained the cells with specific markers (e.g., dynamin II, clathrin heavy chain, and caveolin-1). We also used chlorpromazine, methyl-beta-cyclodextrin, amiloride, and dynasore, blockers of the clathrin, caveolin, and macropinocytosis pathways and the vesicle "pinchase" dynamin, respectively. We found that PMA caused dose- and time-dependent NKCC1 endocytosis. After 2.5 min of PMA exposure, approximately 80% of EGFP-NKCC1 endocytic vesicles colocalized with clathrin and approximately 40% colocalized with dynamin II and with the transferrin receptor, the uptake of which is also mediated by clathrin-coated vesicles. We did not observe significant colocalization of EGFP-NKCC1 endocytic vesicles with caveolin-1, a marker of the caveolae-mediated endocytic pathway. We quantified the effect of each inhibitor on PMA-induced EGFP-NKCC1 endocytosis and found that only chlorpromazine and dynasore caused significant inhibition compared with the untreated control (61% and 25%, respectively, at 2.5 min). Together, these results strongly support the conclusion that PMA-stimulated NKCC1 endocytosis is associated with a clathrin pathway.

  19. Phorbol 12-myristate 13-acetate-induced endocytosis of the Na-K-2Cl cotransporter in MDCK cells is associated with a clathrin-dependent pathway

    PubMed Central

    Mykoniatis, Andreas; Shen, Le; Fedor-Chaiken, Mary; Tang, Jun; Tang, Xu; Worrell, Roger T.; Delpire, Eric; Turner, Jerrold R.; Matlin, Karl S.

    2010-01-01

    In secretory epithelial cells, the basolateral Na+-K+-2Cl− cotransporter (NKCC1) plays a major role in salt and fluid secretion. Our laboratory has identified NKCC1 surface expression as an important regulatory mechanism for Cl− secretion in the colonic crypt cell line T84, a process also present in native human colonic crypts. We previously showed that activation of protein kinase C (PKC) by carbachol and phorbol 12-myristate 13-acetate (PMA) decreases NKCC1 surface expression in T84 cells. However, the specific endocytic entry pathway has not been defined. We used a Madin-Darby canine kidney (MDCK) cell line stably transfected with enhanced green fluorescent protein (EGFP)-NKCC1 to map NKCC1 entry during PMA exposure. At given times, we fixed and stained the cells with specific markers (e.g., dynamin II, clathrin heavy chain, and caveolin-1). We also used chlorpromazine, methyl-β-cyclodextrin, amiloride, and dynasore, blockers of the clathrin, caveolin, and macropinocytosis pathways and the vesicle “pinchase” dynamin, respectively. We found that PMA caused dose- and time-dependent NKCC1 endocytosis. After 2.5 min of PMA exposure, ∼80% of EGFP-NKCC1 endocytic vesicles colocalized with clathrin and ∼40% colocalized with dynamin II and with the transferrin receptor, the uptake of which is also mediated by clathrin-coated vesicles. We did not observe significant colocalization of EGFP-NKCC1 endocytic vesicles with caveolin-1, a marker of the caveolae-mediated endocytic pathway. We quantified the effect of each inhibitor on PMA-induced EGFP-NKCC1 endocytosis and found that only chlorpromazine and dynasore caused significant inhibition compared with the untreated control (61% and 25%, respectively, at 2.5 min). Together, these results strongly support the conclusion that PMA-stimulated NKCC1 endocytosis is associated with a clathrin pathway. PMID:19864322

  20. Phorbol ester attenuates the KCl-induced increase in (Ca/sup 2 +/) and inhibits spontaneous sarcoplasmic reticulum Ca/sup 2 +/ release, in rat cardiac myocytes

    SciTech Connect

    Hansford, R.G.; Capogrossi, M.C.; Kaku, T.; Pelto, D.J.; Filburn, C.H.; Lakatta, E.G.

    1986-03-01

    Partial membrane depolarization induced by increasing the KCl concentration of the medium bathing cardiac myocytes leads to an increase in cell (Ca/sup 2 +/), and accelerates the frequency of spontaneous contractile waves (W) caused by periodic sarcoplasmic reticulum (SR) Ca/sup 2 +/ release. In suspensions of myocytes bathed in 1.0mM Ca/sup 2 +/ at 37 (pH 7.4) and loaded with the fluorescent Ca/sup 2 +/ - indicator Fura-2, by incubation with 2 ..mu..M acetoxymethyl ester for 30 min, the addition of KCl to raise (K/sup +/) from 5 to 30 mM is associated with a rapid (< 10 sec) increase in fluorescence, corresponding to an increased cell (Ca/sup 2 +/). Prior exposure (3 min) to 10/sup -7/ M phorbol myristate acetate (PMA) diminishes this response to 44 +/- 10% of that in control suspensions (n = 9). Under the same conditions W frequency (min/sup -1/) in individual cells in 30 mM KCl averaged 8.3 +/- 0.6. Addition of PMA abolished W within 1 min. Diacylglycerol (10 ..mu..M L..cap alpha..-1,2-dioctanoylglycerol, di C8) had a similar effect on W frequency. The thesis is that PMA attenuates cell Ca/sup 2 +/ overload and its associated potentiation of spontaneous SR Ca/sup 2 +/ oscillations. In view of the efficacy of PMA and di C8, it is suggested that the effect is mediated by protein kinase c, and it may involve an alteration in the intracellular distribution of this enzyme.

  1. Modification of fos proteins: phosphorylation of c-fos, but not v-fos, is stimulated by 12-tetradecanoyl-phorbol-13-acetate and serum.

    PubMed Central

    Barber, J R; Verma, I M

    1987-01-01

    We have investigated the covalent modification of the proteins encoded by the murine fos proto-oncogene (c-fos) and that of the corresponding gene product of FBJ murine osteosarcoma virus (v-fos). Both proteins are posttranslationally processed in the cell, resulting in forms with lower electrophoretic mobilities than that of the initial translation product on sodium dodecyl sulfate-polyacrylamide gels. Treatment with alkaline phosphatase indicates that most, if not all, of this electrophoretic shift is due to phosphoesterification of both proteins. These phosphoryl groups stoichiometrically modify the v-fos and c-fos proteins on serine residues and turn over rapidly in vivo in the presence of protein kinase inhibitors (half-life, less than 15 min). Direct quantitative comparison of steady-state labeling studies with L-[35S]methionine and [32P]phosphate reveals that the c-fos protein is four- to fivefold more highly phosphorylated than the v-fos protein is. Comparison of tryptic fragments from [32P]phosphate-labeled proteins indicates that although the two proteins have several tryptic phosphopeptides in common, the c-fos protein contains unique major tryptic phosphopeptides that the v-fos protein lacks. These unique sites of c-fos phosphorylation have been tentatively localized to the carboxy-terminal 20 amino acid residues of the protein. Phosphorylation of the c-fos protein, but not the v-fos protein, can be stimulated at least fivefold in vivo by the addition of either 12-tetradecanoyl-phorbol-13-acetate or serum. This increase in the steady-state degree of phosphorylation of c-fos appears to be independent of protein kinase C since phosphorylation is Ca2+ and diacylglycerol independent. The possible role of phosphorylation of these proteins in cellular transformation is discussed. Images PMID:3110603

  2. Phorbol ester-treated human acute myeloid leukemia cells secrete G-CSF, GM-CSF and erythroid differentiation factor into serum-free media in primary culture.

    PubMed

    Scher, W; Eto, Y; Ejima, D; Den, T; Svet-Moldavsky, I A

    1990-12-10

    Upon treatment with the phorbol ester, tetradecanoylphorbol 13-acetate (PMA), peripheral mononuclear blood cells from patients with acute myeloid leukemia secrete into serum-free cell-conditioned media (PMA-CCM) at least three distinct nondialysable 'hematopoietic' factors: granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage-colony-stimulating factor (GM-CSF) and erythroid differentiation factor (EDF, activin A). G-CSF was identified by its stimulation of [3H]thymidine incorporation into a G-CSF-responsive cell line, NSF-60, and the inhibition of its stimulation by a G-CSF-specific monoclonal antibody (MAB). GM-CSF was identified by its stimulation of [3H]thymidine incorporation into a GM-CSF-responsive line, TALL-101, and the inhibition of its stimulation by a GM-CSF-specific MAB. EDF was identified by its ability to stimulate erythroid differentiation in mouse erythroleukemia cell lines, its identical retention times to those of authentic EDF on three successive reverse-phase HPLC columns and characterization of its penultimate N-terminal residue as leucine which is the same as that of authentic EDF. Both authentic EDF and the erythroid-stimulating activity in PMA-CCM were found to act synergistically with a suboptimal inducing concentration of a well-studied inducing agent, dimethyl sulfoxide, in inducing erythroid differentiation. In addition, a fourth activity was observed in PMA-CCM: normal human fetal bone marrow cell-proliferation stimulating activity (FBMC-PSA). FBMC-PSA was identified by its ability to stimulate the growth of granulocytes and macrophages in FBMC suspension cultures, which neither recombinant G-CSF or GM-CSF were found to do.

  3. Thymoquinone inhibits phorbol ester-induced activation of NF-κB and expression of COX-2, and induces expression of cytoprotective enzymes in mouse skin in vivo

    SciTech Connect

    Kundu, Joydeb Kumar; Liu, Lijia; Shin, Jun-Wan; Surh, Young-Joon

    2013-09-06

    Highlights: •Thymoquinone inhibits phorbol ester-induced COX-2 expression in mouse skin. •Thymoquinone attenuates phosphorylation of IκBα and DNA binding of NF-κB in mouse skin. •Thymoquinone inhibits phosphorylation of p38 MAP kinase, JNK and Akt in mouse skin. •Thymoquinone induces the expression of cytoprotective proteins in mouse skin. -- Abstract: Thymoquinone (TQ), the active ingredient of Nigella sativa, has been reported to possess anti-inflammatory and chemopreventive properties. The present study was aimed at elucidating the molecular mechanisms of anti-inflammatory and antioxidative activities of thymoquinone in mouse skin. Pretreatment of female HR-1 hairless mouse skin with TQ attenuated 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced expression of cyclooxygenase-2 (COX-2). TQ diminished nuclear translocation and the DNA binding of nuclear factor-kappaB (NF-κB) via the blockade of phosphorylation and subsequent degradation of IκBα in TPA-treated mouse skin. Pretreatment with TQ attenuated the phosphorylation of Akt, c-Jun-N-terminal kinase and p38 mitogen-activated protein kinase, but not that of extracellular signal-regulated kinase-1/2. Moreover, topical application of TQ induced the expression of heme oxygenase-1, NAD(P)H-quinoneoxidoreductase-1, glutathione-S-transferase and glutamate cysteine ligase in mouse skin. Taken together, the inhibitory effects of TQ on TPA-induced COX-2 expression and NF-κB activation, and its ability to induce the expression of cytoprotective proteins provide a mechanistic basis of anti-inflammatory and antioxidative effects of TQ in hairless mouse skin.

  4. Topical application of a platelet activating factor receptor agonist suppresses phorbol ester-induced acute and chronic inflammation and has cancer chemopreventive activity in mouse skin.

    PubMed

    Sahu, Ravi P; Rezania, Samin; Ocana, Jesus A; DaSilva-Arnold, Sonia C; Bradish, Joshua R; Richey, Justin D; Warren, Simon J; Rashid, Badri; Travers, Jeffrey B; Konger, Raymond L

    2014-01-01

    Platelet activating factor (PAF) has long been associated with acute edema and inflammatory responses. PAF acts by binding to a specific G-protein coupled receptor (PAF-R, Ptafr). However, the role of chronic PAF-R activation on sustained inflammatory responses has been largely ignored. We recently demonstrated that mice lacking the PAF-R (Ptafr-/- mice) exhibit increased cutaneous tumorigenesis in response to a two-stage chemical carcinogenesis protocol. Ptafr-/- mice also exhibited increased chronic inflammation in response to phorbol ester application. In this present study, we demonstrate that topical application of the non-hydrolysable PAF mimetic (carbamoyl-PAF (CPAF)), exerts a potent, dose-dependent, and short-lived edema response in WT mice, but not Ptafr -/- mice or mice deficient in c-Kit (c-KitW-sh/W-sh mice). Using an ear inflammation model, co-administration of topical CPAF treatment resulted in a paradoxical decrease in both acute ear thickness changes associated with a single PMA application, as well as the sustained inflammation associated with chronic repetitive PMA applications. Moreover, mice treated topically with CPAF also exhibited a significant reduction in chemical carcinogenesis. The ability of CPAF to suppress acute and chronic inflammatory changes in response to PMA application(s) was PAF-R dependent, as CPAF had no effect on basal or PMA-induced inflammation in Ptafr-/- mice. Moreover, c-Kit appears to be necessary for the anti-inflammatory effects of CPAF, as CPAF had no observable effect in c-KitW-sh/W-sh mice. These data provide additional evidence that PAF-R activation exerts complex immunomodulatory effects in a model of chronic inflammation that is relevant to neoplastic development.

  5. Cell-type-specific activity of the human papillomavirus type 18 upstream regulatory region in transgenic mice and its modulation by tetradecanoyl phorbol acetate and glucocorticoids.

    PubMed Central

    Cid, A; Auewarakul, P; Garcia-Carranca, A; Ovseiovich, R; Gaissert, H; Gissmann, L

    1993-01-01

    The upstream regulatory region (URR) of human papillomavirus type 18 (HPV-18) harbors transcriptional promoter and enhancer elements which are thought to determine the cell-type specificity of the virus. In order to study the regulation of HPV-18 expression in vivo, we constructed transgenic mice carrying the bacterial lacZ gene under the control of the HPV-18 URR. Analysis of beta-galactosidase activity by histochemical staining of tissue sections of four independent transgenic mice showed that the viral promoter was specifically active in epithelial cells within a variety of organs (e.g., tongue, ovary, uterus, testis, and small intestine). Very strong staining was observed in newborn transgenic mice in contrast to a weak activity found during fetal life. Determination of beta-galactosidase activity in crude extracts from tissues of three lines of transgenic mice proved to be a useful tool for a quantitative analysis of transgene expression. In mice from two different transgenic lines treated with dexamethasone such measurements revealed a biphasic effect of the hormone on the activity of the enzyme in the stratified epithelium of the tongue (transient increase followed by a decrease). Northern (RNA) blot analysis showed similar changes in beta-galactosidase mRNA in that tissue. Treatment with tetradecanoyl phorbol acetate (TPA) led to a twofold increase in both enzymatic activity and mRNA levels. Finally, combined treatments with dexamethasone and TPA showed that both factors interfered with each other in their respective effects on transgene expression, suggesting that a cross-talk mechanism between transcription factors could be involved in the regulation of the HPV-18 URR. Images PMID:8411377

  6. Epidermal growth factor and phorbol myristate acetate increase expression of the mRNA for cytosolic phospholipase A2 in glomerular mesangial cells.

    PubMed Central

    Maxwell, A P; Goldberg, H J; Tay, A H; Li, Z G; Arbus, G S; Skorecki, K L

    1993-01-01

    We have previously shown that phospholipase A2 (PLA2) activity is rapidly activated by epidermal growth factor (EGF) and phorbol 12-myristate 13-acetate (PMA) in renal mesangial cells and other cell systems in a manner that suggests a covalent modification of the PLA2 enzyme(s). This PLA2 activity is cytosolic (cPLA2) and is distinct from secretory forms of PLA2, which are also stimulated in mesangial cells in response to cytokines and other agonists. However, longer-term regulation of cPLA2 in renal cells may also occur at the level of gene expression. Cultured rat mesangial cells were used as a model system to test the effects of EGF and PMA on the regulation of cPLA2 gene expression. EGF and PMA both produced sustained increases in cPLA2 mRNA levels, with a parallel increase in enzyme activity over time. Inhibition of protein synthesis by cycloheximide increased basal cPLA2 mRNA accumulation in serum-starved mesangial cells, and the combination of EGF and cycloheximide resulted in super-induction of cPLA2 gene expression compared with EGF alone. Actinomycin D treatment entirely abrogated the effect of EGF on cPLA2 mRNA accumulation. These findings suggest that regulation of cPLA2 is achieved by factors controlling gene transcription and possibly mRNA stability, in addition to previously characterized posttranslational modifications. Images Figure 1 Figure 2 PMID:8240289

  7. Antioxidant and Antiradical Activities of Manihot esculenta Crantz (Euphorbiaceae) Leaves and Other Selected Tropical Green Vegetables Investigated on Lipoperoxidation and Phorbol-12-myristate-13-acetate (PMA) Activated Monocytes

    PubMed Central

    Tsumbu, Cesar N.; Deby-Dupont, Ginette; Tits, Monique; Angenot, Luc; Franck, Thierry; Serteyn, Didier; Mouithys-Mickalad, Ange

    2011-01-01

    Abelmoschus esculentus (Malvaceae), Hibiscus acetosella (Malvaceae), Manihot esculenta Crantz (Euphorbiaceae) and Pteridium aquilinum (Dennstaedtiaceae) leaves are currently consumed as vegetables by migrants from sub-Saharan Africa living in Western Europe and by the people in the origin countries, where these plants are also used in the folk medicine. Manihot leaves are also eaten in Latin America and some Asian countries. This work investigated the capacity of aqueous extracts prepared from those vegetables to inhibit the peroxidation of a linoleic acid emulsion. Short chain, volatile C-compounds as markers of advanced lipid peroxidation were measured by gas chromatography by following the ethylene production. The generation of lipid hydroperoxides, was monitored by spectroscopy using N-N′-dimethyl-p-phenylene-diamine (DMPD). The formation of intermediate peroxyl, and other free radicals, at the initiation of the lipid peroxidation was investigated by electron spin resonance, using α-(4-pyridyl-1-oxide)-N-tert-butylnitrone as spin trap agent. The ability of the extracts to decrease the cellular production of reactive oxygen species (ROS) in “inflammation like” conditions was studied by fluorescence technique using 2′,7′-dichlorofluorescine-diacetate as fluorogenic probe, in a cell model of human monocytes (HL-60 cells) activated with phorbol ester. Overall the extracts displayed efficient concentration-dependent inhibitory effects. Their total polyphenol and flavonoid content was determined by classic colorimetric methods. An HPLC-UV/DAD analysis has clearly identified the presence of some polyphenolic compounds, which explains at least partially the inhibitions observed in our models. The role of these plants in the folk medicine by sub-Saharan peoples as well as in the prevention of oxidative stress and ROS related diseases requires further consideration. PMID:22254126

  8. PP2B-mediated Dephosphorylation of c-Jun C Terminus Regulates Phorbol Ester-induced c-Jun/Sp1 Interaction in A431 Cells

    PubMed Central

    Chen, Ben-Kuen; Huang, Chi-Chen; Chang, Wei-Chiao; Chen, Yun-Ju; Kikkawa, Ushio; Nakahama, Ken-ichi; Morita, Ikuo

    2007-01-01

    The c-Jun/Sp1 interaction is essential for growth factor- and phorbol 12-myristate 13-acetate (PMA)-induced genes expression, including human 12(S)-lipoxygenase, keratin 16, cytosolic phospholipase A2, p21WAF1/CIP1, and neuronal nicotinic acetylcholine receptor β4. Here, we examined the mechanism underlying the PMA-induced regulation on the interaction between c-Jun and Sp1. We found that treatment of cells with PMA induced a dephosphorylation at the C terminus of c-Jun at Ser-243 and a concomitant inhibition of PP2B by using PP2B small interfering RNA, resulting in reduction of PMA-induced gene expression as well as the c-Jun/Sp1 interaction. The c-Jun mutant TAM-67-3A, which contains three substitute alanines at Thr-231, Ser-243, and Ser-249 compared with TAM-67, binds more efficaciously with Sp1 and is about twice as efficacious as TAM-67 in inhibiting the PMA-induced activation of the 12(S)-lipoxygenase promoter. Importantly, PP2B not only dephosphorylates the c-Jun at Ser-243 but also interacts with c-Jun in PMA-treated cells. PMA stimulates the association of the PP2B/c-Jun/Sp1 complex with the promoter. These findings indicate the dephosphorylation of c-Jun C terminus is required for the c-Jun/Sp1 interaction and reveal that PP2B plays an important role in regulating c-Jun/Sp1 interaction in PMA-induced gene expression. PMID:17215518

  9. Phorbol ester and B cell-stimulatory factor synergize to induce B-chronic lymphocytic leukemia cells to simultaneous immunoglobulin secretion and DNA synthesis.

    PubMed

    Carlsson, M; Matsson, P; Rosén, A; Sundström, C; Tötterman, T H; Nilsson, K

    1988-11-01

    This paper discusses the response of two B cell-type chronic lymphocytic leukemia (B-CLL) clones, 173 and 183, to the phorbol ester TPA combined with a B cell-stimulatory factor (BSF) derived from a T helper cell hybridoma (MP6). Previous studies with 173 and 183 cells have consistently shown that TPA alone induces differentiation but no proliferation. However, when the two clones were exposed to TPA plus BSF-MP6, not only differentiation but also DNA synthesis was observed. Compared with TPA exposure alone, the fraction of cells with induced lymphoblastoid-plasmacytoid morphology increased and Ig secretion was enhanced. By a 1-hr TPA pulse followed by BSF-MP6, the DNA synthesis was further augmented, but less maturation was observed. T cell and monocyte removal, using cell sorting, showed that the DNA synthesis induced was independent of these cell types, also under serum-free conditions. Quantitation of several cell cycle-associated surface Ags showed that the 4F2, Ba, Bac-1, and cD23 Ags increased while the CD37 decreased in expression upon addition of BSF-MP6. We conclude that B-CLLs are inducible by TPA and BSF-MP6 not only to differentiation, but also to DNA synthesis even under serum-free conditions in vitro. The results furthermore suggest that the very low proliferation activity in B-CLL tumors in vivo may reflect a relative deficiency of proper growth and differentiation factors or a subnormal response of B-CLL cells to such factors.

  10. Effects of phorbol 12,13-diacetate and its influence on spasmogenic responses in normal and sensitized guinea-pig trachea.

    PubMed

    De Diego, A; Cortijo, J; Villagrasa, V; Perpiñá, M; Esplugues, J; Morcillo, E J

    1995-09-01

    We have studied the effects of phorbol 12,13-diacetate (PDA) and its influence on a variety of spasmogenic responses in trachea isolated from normal and sensitized guinea-pigs. Tracheal preparations were denuded of epithelium, treated with indomethacin (2.8 microM), and cooled to 20 degrees C. In these experimental conditions, tracheal strips contracted to PDA (0.1 nM-1 microM). Contractions to PDA (1 microM) were greater in sensitized tissues. In normal trachea, contractions to PDA (0.1 microM) were depressed by H-7, 1-(5-isoquinolinyl-sulphonyl)-2-methylpiperazine, (50 microM), amiloride (10 microM), verapamil (10 microM) and Ca(2+)-free exposure. Similar effects were obtained in sensitized trachea except that PDA-induced contraction was resistant to verapamil and Ca(2+)-free exposure. Cooling (20 degrees C) of normal trachea substantially depressed the response to CaCl2 (in K(+)-depolarized tissues), KCl, histamine and 5-hydroxytryptamine without affecting the spasm induced by acetylcholine. This inhibitory effect of cooling was not observed in sensitized trachea. PDA (0.1 microM) did not affect spasmogenic responses at 37 degrees C but counteracted the inhibitory effect of cooling in normal trachea. PDA had no effect on sensitized tissues. PDA (0.1-1 microM) did not alter Ca(2+)-induced contraction of skinned normal and sensitized trachea. These results support the hypothesis that intracellularly stored Ca2+ plays an important role in the activation of sensitized tracheal muscle.

  11. Involvement of the antioxidative property of morusin in blocking phorbol ester-induced malignant transformation of JB6 P(+) mouse epidermal cells.

    PubMed

    Cheng, Pai-Shan; Hu, Chao-Chin; Wang, Chau-Jong; Lee, Yean-Jang; Chung, Wei-Chia; Tseng, Tsui-Hwa

    2017-02-25

    Chemoprevention has been acknowledged as an important and practical strategy for managing cancer. We have previously synthesized morusin, a prenylated flavonoid that exhibits anti-cancer progression activity. In the present study, we evaluated the anti-cancer promotion potential of morusin by using the mouse epidermal JB6 P(+) cell model. Extensive evidence shows that tumor promotion by phorbol esters is due to the stimulation of reactive oxygen species (ROS). Therefore, the effect of morusin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ROS production was assessed. Noncytotoxic concentrations of morusin were found to dose-dependently reduce TPA-induced ROS production. Moreover, morusin inhibited TPA-induced activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB) activation, which can mediate cell proliferation and malignant transformation. Furthermore, morusin inhibited the TPA upregulation of cyclooxygenase 2 (COX-2), which may be regulated by AP-1 and NF-κB. In addition, noncytotoxic concentrations of morusin reduced the TPA-promoted cell growth of JB6 P(+) cells and inhibited TPA-induced malignant properties, such as cytoskeletal rearrangement and cell migration of JB6 P(+) cells. Similar to the effects of glutathione (GSH) pretreatment, morusin inhibited TPA-induced expression of N-cadeherin and vimentin, which are malignant cell surface proteins. Finally, morusin treatment dose-dependently suppressed the TPA-induced anchorage-independent cell transformation of JB6 P(+) cells. In conclusion, our results evidence that morusin possesses anti-cancer promotion potential because of its antioxidant property, which mediates multiple transformation-associated gene expression.

  12. Phorbol ester and bryostatin differentially regulate the hydrolysis of phosphatidylethanolamine in Ha-ras- and raf-oncogene-transformed NIH 3T3 cells.

    PubMed

    Kiss, Z; Rapp, U R; Pettit, G R; Anderson, W B

    1991-06-01

    Previously it was reported that transformation of NIH 3T3 fibroblast by the Ha-ras, v-src, v-fms, and A-raf oncogenes decreased the stimulatory effects of phorbol 12-myristate 13-acetate (PMA; 'TPA'), an activator of protein kinase C (PKC), on the phosphorylation of an endogenous 80 kDa substrate and on 86Rb uptake [Wolfman, Wingrove, Blackshear & Macara (1987) J. Biol. Chem. 262, 16546-16552], as well as on sphingomyelin synthesis [Kiss, Rapp & Anderson (1988) FEBS Lett. 240, 221-226]. Here, we investigated how transformation affects the PMA-stimulated hydrolysis of phosphatidylethanolamine (PtdEtn), a recently characterized mechanism which may contribute to the generation of the second messengers phosphatidic acid and 1,2-diacylglycerol. The effects of PMA were compared with those of bryostatin, a non-tumour-promoter activator of PKC. Transformation of NIH 3T3 cells with Ha-ras, v-raf, or A-raf enhanced the stimulatory effect of PMA on the phospholipase D-mediated hydrolysis of PtdEtn. On the other hand, the effects of bryostatin on PtdEtn hydrolysis were only slightly increased, if at all, in cells transformed with these oncogenes. In crude membrane preparations isolated from these transformed cells, PMA, but not bryostatin, enhanced the combined stimulatory effects of ATP and the GTP analogue guanosine 5'-[gamma-thio]triphosphate on phospholipase D-mediated PtdEtn hydrolysis. The PKC inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine inhibited the stimulatory effect of PMA only in intact cells. These results indicate that transformation of cells by certain oncogenes differentially affects phospholipase D-mediated hydrolysis of PtdEtn induced by PMA and bryostatin, suggesting that the action of PMA might involve two different mechanisms.

  13. Phorbol myristate acetate and Bryostatin 1 rescue IFN-gamma inducibility of MHC class II molecules in LS1034 colorectal carcinoma cell line

    PubMed Central

    Kudinov, Yuri; Wiseman, Charles L; Kharazi, Alexander I

    2003-01-01

    Background The expression of major histocompatibility complex class II (MHCII) antigens in both mouse and human tumors is rare, and these antigens are not easily inducible by IFN-gamma (IFNg). Since MHCII may play an important role in the development of host antitumor immune response, we explored the possibility of restoring MHCII inducibility in several IFNg-resistant tumor cell lines using protein kinase C (PKC) agonists phorbol myristate acetate (PMA) or Bryostatin. Results Tumor cells were co-cultured with various concentrations of PMA and IFNg for 48 hr. The expression of MHCII antigens and receptors IFNgR1 and IFNgR2 was determined by flow cytometry. We showed that the presence of as little as 0.1 ng/ml of PMA in tissue culture restored the ability of weakly inducible LS1034 colon carcinoma cells to express MHCII in response to IFNg (100 – 10,000 IU/ml) in a dose-dependent manner. Likewise, Bryostatin 1, as low as 10 ng/ml produced a 5–6 fold upregulation of MHCII. The effect of PMA was not observed in two other poorly responding cell lines, MSTO-211H mesothelioma and HepG2 hepatocellular carcinoma, and was abrogated by relatively high concentrations of PKC inhibitors staurosporine (100 nM) and GF 109203X (1,000 nM). Both surface and intracellular staining of all cell lines with antibodies against IFNgR1 and IFNgR2 failed to detect any increase in IFNg receptor expression following incubation with PMA. Conclusion In this study we showed that IFNg-inducibility of MHCII antigens in weakly inducible LS1034 colorectal carcinoma cell line can be rescued by concomitant incubation with PKC agonists. Bryostatin 1 may be considered for further investigation of IFNg-dependent MHCII induction in resistant tumors in vivo. PMID:12787470

  14. Apoptosis in virus infection dynamics models

    PubMed Central

    Fan, Ruili; Dong, Yueping; Huang, Gang; Takeuchi, Yasuhiro

    2014-01-01

    In this paper, on the basis of the simplified two-dimensional virus infection dynamics model, we propose two extended models that aim at incorporating the influence of activation-induced apoptosis which directly affects the population of uninfected cells. The theoretical analysis shows that increasing apoptosis plays a positive role in control of virus infection. However, after being included the third population of cytotoxic T lymphocytes immune response in HIV-infected patients, it shows that depending on intensity of the apoptosis of healthy cells, the apoptosis can either promote or comfort the long-term evolution of HIV infection. Further, the discrete-time delay of apoptosis is incorporated into the pervious model. Stability switching occurs as the time delay in apoptosis increases. Numerical simulations are performed to illustrate the theoretical results and display the different impacts of a delay in apoptosis. PMID:24963975

  15. [Apoptosis modulation by human papillomavirus].

    PubMed

    Jave-Suárez, Luis Felipe; Ratkovich-González, Sarah; Olimón-Andalón, Vicente; Aguilar-Lemarroy, Adriana

    2015-01-01

    One of the most important processes to keep the homeostasis in organisms is the apoptosis, also called programmed cell death. This mechanism works through two pathways: The intrinsic or mitochondrial, which responds to DNA damage and extern agents like UV radiation; and the extrinsic or receptor-mediated, which binds to their ligands to initiate the apoptotic trail. The evasion of apoptosis is one of the main causes of cellular transformation to malignity. Many viruses had shown capacity to modify the apoptotic process; among them is the human papillomavirus, which, by means of its oncoproteins, interferes in pathways, reacting with the receptors and molecules and participating in the death mechanism. This creates ideal conditions for cancer development.

  16. Apoptosis in Cryopreserved Eukaryotic Cells.

    PubMed

    Savitskaya, M A; Onishchenko, G E

    2016-05-01

    This review considers apoptosis mechanisms that have been revealed in cryopreserved cells and which can be controlled using different chemical agents, thereby improving the viability of cells after their return to normal conditions. The role of oxidative stress as of the most significant damaging factor is discussed, as well as the reasonability of including antioxidants into cryopreservation/thawing protocols as independent agents or in combination with other compounds.

  17. Apoptosis in human retinal degenerations.

    PubMed

    Xu, G Z; Li, W W; Tso, M O

    1996-01-01

    This paper examined the role of apoptosis in human retinal degenerations including pathologic myopia, age-related macular degeneration, serous retinal detachment, retinal lattice, and paving stone degenerations. Thirty-seven enucleated human eyes with 1 of the above-mentioned retinal degenerations were studied by histopathology and by TdT-mediated biotin-dUTP nicked-end labelling (TUNEL) technique. Tunnel labelling characteristic DNA fragmentation of apoptosis was observed in photoreceptor cells in 2 of the 4 eyes with pathologic myopia and in 4 of 16 eyes with age-related macular degeneration, 2 of which were exudative and 2 of which were atrophic. However, only a few scattered photoreceptor cells were labelled in 4 of 8 eyes with serous retinal detachment secondary to malignant melanoma of the choroid. Moreover, none of the photoreceptors cells in the 4 eyes with retinal lattice degeneration and 6 eyes with retinal paving stone degeneration were labelled. Apoptosis is 1 of the important pathways of photoreceptor cell degeneration in pathologic myopia and age-related macular degeneration.

  18. Role of Ras-related C3 botulinum toxin substrate 2 (Rac2), NADPH oxidase and reactive oxygen species in diallyl disulphide-induced apoptosis of human leukaemia HL-60 cells.

    PubMed

    Yi, Lan; Ji, Xiao-Xia; Tan, Hui; Lin, Min; Tang, Yi; Wen, Ling; Ma, Yan-Hua; Su, Qi

    2010-12-01

    1. Diallyl disulphide (DADS) has potential as a chemopreventive and therapeutic agent. Previous studies have reported that Ras-related C3 botulinum toxin substrate 2 (Rac2), a regulatory subunit of the NADPH oxidase complex, is upregulated in DADS-induced apoptosis in human leukaemia HL-60 cells. The aim of the present study was to investigate the role of Rac2, NADPH oxidase and reactive oxygen species (ROS) in DADS-induced apoptosis. 2. Expression of the Rac2 gene along with that of five other genes of NADPH oxidase subunits were in HL-60 cells measured by Sybergreen quantitative real-time polymerase chain reaction. RNA interference was used to test the effect of Rac2. Protein expression was evaluated using western blot analysis and ROS levels were measured by 2',7'-dichlorofluorescein diacetate (DCFH-DA) fluorescence. DNA fragmentation and flow cytometry analysis were used to detect apoptotic cells. 3. Levels of Rac2 gene and protein were significantly upregulated and NADPH oxidase was activated in DADS-induced apoptosis. Pretreatment of HL-60 cells with small interfering (si) RNAs to inhibit Rac2 blocked DADS-induced apoptosis. Diallyl disulphide-induced intracellular ROS production was increased in phorbol myristate acetate-stimulated cells, but decreased in Rac2 siRNA-treated cells. In Rac2 siRNA-treated cells, activator protein-1 and caspase 3 levels decreased, c-myc protein levels were increased and p38 protein levels were unchanged compared with Rac2-competent, DADS-treated cells. 4. These results demonstrate that NADPH oxidase is the main source of DADS-induced ROS. In addition, Rac2 selectively activates the c-Jun N-terminal kinase pathway, but not the p38 pathway, in DADS-induced apoptosis. So, Rac2, NADPH oxidase and ROS have a critical role in DADS-induced apoptosis in human leukaemia HL-60 cells.

  19. Carvacrol-induced [Ca2+]i rise and apoptosis in human glioblastoma cells.

    PubMed

    Liang, Wei Zhe; Lu, Cheng Hsien

    2012-05-15

    This study examined whether the essential oil component carvacrol altered cytosolic free Ca(2+) level ([Ca(2+)](i)) and viability in human glioblastoma cells. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Cell viability was measured by detecting reagent WST-1. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry. Carvacrol at concentrations of 400-1000 μM induced a [Ca(2+)](i) rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca(2+). Carvacrol-induced Ca(2+) signal was not altered by nifedipine, econazole, SK&F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca(2+) was removed, incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol-induced [Ca(2+)](i) rise. Incubation with carvacrol also abolished thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished carvacrol-induced [Ca(2+)](i) rise. At concentrations of 200-800 μM, carvacrol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N--tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that carvacrol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. At concentrations of 200, 400 and 600 μM, carvacrol induced production of ROS. In human glioblastoma cells, carvacrol induced a [Ca(2+)](i) rise by inducing phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via protein kinase C-sensitive, non store-operated Ca(2+) channels. Carvacrol induced cell death that might involve ROS-mediated apoptosis. Crown Copyright © 2012. Published by Elsevier Inc. All rights reserved.

  20. The mechanism of honokiol-induced intracellular Ca(2+) rises and apoptosis in human glioblastoma cells.

    PubMed

    Liang, Wei-Zhe; Chou, Chiang-Ting; Chang, Hong-Tai; Cheng, Jin-Shiung; Kuo, Daih-Huang; Ko, Kuang-Chung; Chiang, Ni-Na; Wu, Ru-Fang; Shieh, Pochuen; Jan, Chung-Ren

    2014-09-25

    Honokiol, an active constituent of oriental medicinal herb Magnolia officinalis, caused Ca(2+) mobilization and apoptosis in different cancer cells. In vivo, honokiol crossed the blood-brain or -cerebrospinal fluid barrier, suggesting that it may be an effective drug for the treatment of brain tumors, including glioblastoma. This study examined the effect of honokiol on intracellular Ca(2+) concentration ([Ca(2+)]i) and apoptosis in DBTRG-05MG human glioblastoma cells. Honokiol concentration-dependently induced a [Ca(2+)]i rise. The signal was decreased partially by removal of extracellular Ca(2+). Honokiol-triggered [Ca(2+)]i rise was not suppressed by store-operated Ca(2+) channel blockers (nifedipine, econazole, SK&F96365) and the protein kinase C (PKC) activator phorbol 12-myristate 13 acetate (PMA), but was inhibited by the PKC inhibitor GF109203X. GF109203X-induced inhibition was not altered by removal of extracellular Ca(2+). In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished honokiol-induced [Ca(2+)]i rise. Conversely, incubation with honokiol abolished TG or BHQ-induced [Ca(2+)]i rise. Inhibition of phospholipase C (PLC) with U73122 abolished honokiol-induced [Ca(2+)]i rise. Honokiol (20-80μM) reduced the cell viability, which was not reversed by prechelating cytosolic Ca(2+) with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester). Honokiol (20-60μM) enhanced reactive oxygen species (ROS) production, decreased mitochondrial membrane potential, released cytochrome c, and activated caspase-9/caspase-3. Together, honokiol induced a [Ca(2+)]i rise by inducing PLC-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via PKC-dependent, non store-operated Ca(2+) channels. Moreover, honokiol activated the mitochondrial pathway of apoptosis in DBTRG-05MG human glioblastoma cells. Copyright © 2014

  1. Specific detection of intramitochondrial superoxide produced by either cell activation or apoptosis by employing a newly developed cell-permeative lucigenin derivative, 10,10'-dimethyl-9,9'-biacridinium bis(monomethyl terephthalate).

    PubMed

    Sasaki, Soichiro; Yamada, Sachiko; Iwamura, Michiko; Kobayashi, Yoshiro

    2013-12-01

    Here we developed a new cell-permeative lucigenin derivative, 10,10'-dimethyl-9,9'-biacridinium bis(monomethyl terephthalate) (MMT), to detect intracellular superoxide production. Both MMT and lucigenin were specific to superoxide among reactive oxygen species tested. Although lucigenin barely penetrated into cells, MMT accumulated in mitochondria in a variety of cells such as neutrophils. By employing MMT, we found that, upon activation of neutrophils with phorbol myristate acetate, superoxide was generated extracellularly as well as intramitochondrially and that such intramitochondrial superoxide production was dependent on oxidative phosphorylation. We also found that, during apoptosis, superoxide was gradually produced in mitochondria in association with phosphatidylserine exposure and that the kinetics of superoxide production was very heterogeneous at the single-cell level. Thus this study demonstrates that MMT could serve as a specific probe for intramitochondrial superoxide in either activated or apoptotic cells.

  2. Mammalian target of rapamycin inhibitors, temsirolimus and torin 1, attenuate stemness-associated properties and expression of mesenchymal markers promoted by phorbol-myristate-acetate and oncostatin-M in glioblastoma cells.

    PubMed

    Chandrika, Goparaju; Natesh, Kumar; Ranade, Deepak; Chugh, Ashish; Shastry, Padma

    2017-03-01

    The phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathway is crucial for tumor survival, proliferation, and progression, making it an attractive target for therapeutic intervention. In glioblastoma, activated mammalian target of rapamycin promotes invasive phenotype and correlates with poor patient survival. A wide range of mammalian target of rapamycin inhibitors are currently being evaluated for cytotoxicity and anti-proliferative activity in various tumor types but are not explored sufficiently for controlling tumor invasion and recurrence. We recently reported that mammalian target of rapamycin inhibitors-rapamycin, temsirolimus, torin 1, and PP242-suppressed invasion and migration promoted by tumor necrosis factor-alpha and phorbol-myristate-acetate in glioblastoma cells. As aggressive invasion and migration of tumors are associated with mesenchymal and stem-like cell properties, this study aimed to examine the effect of mammalian target of rapamycin inhibitors on these features in glioblastoma cells. We demonstrate that temsirolimus and torin 1 effectively reduced the constitutive as well as phorbol-myristate-acetate/oncostatin-M-induced expression of mesenchymal markers (fibronectin, vimentin, and YKL40) and neural stem cell markers (Sox2, Oct4, nestin, and mushashi1). The inhibitors significantly abrogated the neurosphere-forming capacity induced by phorbol-myristate-acetate and oncostatin-M. Furthermore, we demonstrate that the drugs dephosphorylated signal transducer and activator transcription factor 3, a major regulator of mesenchymal and neural stem cell markers implicating the role of signal transducer and activator transcription factor 3 in the inhibitory action of these drugs. The findings demonstrate the potential of mammalian target of rapamycin inhibitors as "stemness-inhibiting drugs" and a promising therapeutic approach to target glioma stem cells.

  3. Human papillomavirus oncoproteins and apoptosis (Review)

    PubMed Central

    JIANG, PEIYUE; YUE, YING

    2014-01-01

    The aim of this study was to review the literature and identify the association between human papillomavirus (HPV) oncoproteins and apoptosis. HPV-associated apoptosis may be primarily blocked by a number of oncoproteins, including E5, E6 and E7. E5 protein protects cells from tumor necrosis factor-associated apoptosis; the oncoprotein E6 predominantly inhibits apoptosis through the p53 pathway; and oncoprotein E7 is involved in apoptosis activation and inhibition. In addition, HPV oncoproteins are involved in activating or repressing the transcription of E6/E7. In conclusion, HPV oncoproteins, including E5, E6 and E7 protein, may interfere with apoptosis via certain regulatory principles. PMID:24348754

  4. Activation of p38 and JNK MAPK pathways abrogates requirement for new protein synthesis for phorbol ester mediated induction of select MMP and TIMP genes.

    PubMed

    Sampieri, Clara L; Nuttall, Robert K; Young, David A; Goldspink, Deborah; Clark, Ian M; Edwards, Dylan R

    2008-03-01

    The human matrix metalloproteinase (MMP) gene family includes 24 genes whose regulated expression, together with that of four tissue inhibitors of metalloproteinases (TIMPs), is essential in tissue remodelling and cell signalling. Quantitative real-time-PCR (qPCR) analysis was used to evaluate the shared and unique patterns of control of these two gene families in human MRC-5 and WI-38 fibroblasts in response to the protein kinase C (PKC) activator phorbol-12-myristate-13-acetate (PMA). The requirement for ongoing translation was analysed using three protein synthesis inhibitors, anisomycin, cycloheximide and emetine. PMA induced MMP1, 3, 8, 9, 10, 12, 13, 14 and TIMP1 and TIMP3 RNAs after 4-8 h, and induction of all except MMP9 and TIMP3 was blocked by all protein synthesis inhibitors. However, even though all inhibitors effectively blocked translation, PMA-induction of MMP9 and TIMP3 was blocked by emetine but was insensitive to cycloheximide and anisomycin. Anisomycin alone induced MMP9 and TIMP3, along with MMP25 and MMP19. The extracellular signal-regulated kinases (ERKs)-1/2 were strongly activated by PMA, while anisomycin activated the c-Jun N-terminal kinase (JNK) and p38 pathways, and cycloheximide activated p38, but emetine had no effect on the stress-activated mitogen-activated protein kinase (MAPK) pathways. The involvement of the p38 and JNK pathways in the selective effects of anisomycin and cycloheximide on MMP/TIMP expression was supported by use of pharmacological inhibitors. These data confirm that most inducible MMPs and TIMP1 behave as "late" activated, protein synthesis-dependent genes in fibroblasts. However, the requirement of protein synthesis for PMA-induction of MMPs and TIMPs is not universal, since it is abrogated for MMP9 and TIMP3 by stimulation of the stress-activated MAPK pathways. The definition of clusters of co-regulated genes among the two gene families will aid in bioinformatic dissection of control mechanisms.

  5. Metalloproteinase inhibitors for the disintegrin-like metalloproteinases ADAM10 and ADAM17 that differentially block constitutive and phorbol ester-inducible shedding of cell surface molecules.

    PubMed

    Ludwig, Andreas; Hundhausen, Christian; Lambert, Millard H; Broadway, Neil; Andrews, Robert C; Bickett, D Mark; Leesnitzer, M Anthony; Becherer, J David

    2005-03-01

    The transmembrane metzinkin-proteases of the ADAM (a disintegrin and a metalloproteinase)-family ADAM10 and ADAM 17 are both implicated in the ectodomain shedding of various cell surface molecules including the IL6-receptor and the transmembrane chemokines CX3CL1 and CXCL16. These molecules are constitutively released from cultured cells, a process that can be rapidly enhanced by cell stimulation with phorbol esters such as PMA. Recent research supports the view that the constitutive cleavage predominantly involves ADAM10 while the inducible one is mediated to a large extent by ADAM17. We here describe the discovery of hydroxamate compounds with different potency against ADAM10 and ADAM17 and different ability to block constitutive and inducible cleavage of IL6R, CX3CL1 and CXCL16 by the two proteases. By screening a number of hydroxamate inhibitors for the inhibition of recombinant metalloproteinases, a compound was found inhibiting ADAM10 with more than 100-fold higher potency than ADAM17, which may be explained by an improved fit of the compound to the S1' specificity pocket of ADAM10 as compared to that of ADAM17. In cell-based cleavage experiments this compound (GI254023X) potently blocked the constitutive release of IL6R, CX3CL1 and CXCL16, which was in line with the reported involvement of ADAM10 but not ADAM17 in this process. By contrast, the compound did not affect the PMA-induced shedding, which was only blocked by GW280264X, a potent inhibitor of ADAM17. As expected, GI254023X did not further decrease the residual release of CX3CL1 and CXCL16 in ADAM10-deficient cells verifying that the compound's effect on the constitutive shedding of these molecules was exclusively due to the inhibition of ADAM10. Thus, GI254023X may by of use as a preferential inhibitor of constitutive shedding events without effecting the inducible shedding in response to agonists acting similar to PMA.

  6. Down-regulation of P2U-purinergic nucleotide receptor messenger RNA expression during in vitro differentiation of human myeloid leukocytes by phorbol esters or inflammatory activators.

    PubMed

    Martin, K A; Kertesy, S B; Dubyak, G R

    1997-01-01

    HL-60 human promyelocytic leukocytes express G protein-coupled P2U-purinergic nucleotide receptors (P2UR or P2Y2R) that activate inositol phospholipid hydrolysis and Ca24 mobilization in response to ATP or UTP. We examined the expression of functional P2UR and P2UR mRNA levels during in vitro differentiation of HL-60 cells by dibutyryl-cAMP (Bt2cAMP), which induces a granulocyte/neutrophil phenotype, or by phorbol-12-myristate-13-acetate (PMA), which induces a monocyte/macrophage phenotype. Both P2UR function and P2UR mRNA levels were only modestly attenuated during granulocytic differentiation by Bt2cAMP. In contrast, P2UR function, as assayed by either Ca2+ mobilization or inositol trisphosphate generation, was greatly reduced in PMA-differentiated cells. This inhibition of P2UR function was strongly correlated with PMA-induced decreases in P2UR mRNA levels, as assayed by Northern blot analysis or reverse transcription-polymerase chain reaction-based quantification. Although PMA induced an early, transient up-regulation of P2UR mRNA, this was rapidly followed by a sustained decrease in P2UR mRNA to a level 5-10-fold lower than that in undifferentiated HL-60 cells. The half-life of the P2UR transcript in HL-60 cells was approximately 60 min, and this was not affected by acute exposure (< or = 4 hr) to Bt2cAMP or PMA. PMA down-regulated P2UR mRNA in THP-1 monocytes and HL-60 granulocytes but not in A431 human epithelial cells or human keratinocytes. P2UR mRNA was also down-regulated in THP-1 monocytes differentiated into inflammatory macrophages by gamma-interferon and endotoxin. These data indicate that myeloid leukocytes possess tissue-specific mechanisms for the rapid modulation of P2UR expression and function during differentiation and inflammatory activation.

  7. Phorbol ester and epidermal growth factor enhance the expression of two inducible prostaglandin H synthase genes in rat tracheal epithelial cells.

    PubMed

    Hamasaki, Y; Kitzler, J; Hardman, R; Nettesheim, P; Eling, T E

    1993-07-01

    Previous studies from our laboratory suggested that phorbol 12-myristate 13-acetate (TPA) stimulates prostaglandin E2 (PGE2) production by inducing de novo synthesis of prostaglandin H synthase (PHS) in a rat tracheal cell line. We report here an extension of this work to further elucidate the mechanisms by which TPA (and epidermal growth factor) stimulates PGE2 production. We used the rat tracheal cell line EGV6, which has a lower basal level of PGE2 production and responds to TPA and EGF stimulation with a much greater increase in PGE2 synthesis than the previously used cell line, Incubation of EGV6 cultures with TPA or EGF resulted in a time- and dose-dependent increase in PGE2 synthesis up to 40-fold and 6-fold, respectively. Serum also stimulated PGE2 synthesis, while bombesin, retinoic acid, and bacterial lipopolysaccharide did not. PHS protein levels in microsomal preparations from the cells were estimated by Western analysis. Antibodies raised against murine PHS-2 cross reacted with the EGV-6 PHS while several antibody preparations that react with PHS-1 from ram or mouse reacted poorly with the cellular preparation. TPA treatment increased the de novo synthesis of PHS-2 while dexamethasone treatment reduced the response to TPA. Northern blot analysis of mRNA from EGV6 cultures using a ram PHS cDNA revealed a 2.8- and a 4.5- to 4.9-kb (designated 4.9 kb) transcript. Treatment with TPA or EGF increased the expression of both transcripts and this effect was further enhanced by cyclohexamide. To further define the PHS mRNA species of EGV6 cells, two well-characterized murine PHS cDNA probes were used. The constitutive murine PHS cDNA probe hybridized only with the 2.8-kb transcript, and the inducible murine PHS cDNA hybridized only with the 4.9-kb transcript. The rates of induction as well as degradation of the 4.9-kb PHS mRNA were much more rapid than those of the 2.8-kb mRNA species. Dexamethasone partially inhibited the induction of both PHS transcripts by

  8. The Involvement of Specific PKC Isoenzymes in Phorbol Ester-Mediated Regulation of Steroidogenic Acute Regulatory Protein Expression and Steroid Synthesis in Mouse Leydig Cells

    PubMed Central

    Manna, Pulak R.; Soh, Jae-Won; Stocco, Douglas M.

    2011-01-01

    Protein kinase C (PKC) is a multigene family of serine/threonine kinases. PKC is involved in regulating adrenal and gonadal steroidogenesis; however, the functional relevance of the different PKC isoenzymes remains obscure. In this study, we demonstrate that MA-10 mouse Leydig tumor cells express several PKC isoforms to varying levels and that the activation of PKC signaling, by phorbol 12-myristate 13-acetate (PMA) elevated the expression and phosphorylation of PKCα, -δ, -ε, and -μ/protein kinase D (PKD). These responses coincided with the expression of the steroidogenic acute regulatory (StAR) protein and progesterone synthesis. Targeted silencing of PKCα, δ, and ε and PKD, using small interfering RNAs, resulted in deceases in basal and PMA-mediated StAR and steroid levels and demonstrated the importance of PKD in steroidogenesis. PKD was capable of controlling PMA and cAMP/PKA-mediated synergism involved in the steroidogenic response. Further studies pointed out that the regulatory events effected by PKD are associated with cAMP response element-binding protein (CREB) and c-Jun/c-Fos-mediated transcription of the StAR gene. Chromatin immunoprecipitation studies revealed that the activation of phosphorylated CREB, c-Jun, and c-Fos by PMA was correlated with in vivo protein-DNA interactions and the recruitment of CREB-binding protein, whereas knockdown of PKD suppressed the association of these factors with the StAR promoter. Ectopic expression of CREB-binding protein enhanced the trans-activation potential of CREB and c-Jun/c-Fos in StAR gene expression. Using EMSA, a −83/−67-bp region of the StAR promoter was shown to bind PKD-transfected MA-10 nuclear extract in a PMA-responsive manner, targeting CREB and c-Jun/c-Fos proteins. These findings provide evidence for the presence of multiple PKC isoforms and demonstrate the molecular events by which selective isozymes, especially PKD, influence PMA/PKC signaling involved in the regulation of the

  9. Involvement of phospholipase D and protein kinase C in phorbol ester and fatty acid stimulated turnover of phosphatidylcholine and phosphatidylethanolamine in neural cells.

    PubMed

    Cook, H W; Ridgway, N D; Byers, D M

    1998-02-05

    Hydrolysis of phosphatidylcholine (PtdCho) can provide lipid second messengers involved in sustained signal transduction. Four neural-derived cell lines (C6 rat glioma; N1E-115 mouse and SK-N-MC and SK-N-SH human neuroblastoma) express different protein kinase C (PKC) isoforms and differentially respond to 4beta-12-O-tetradecanoylphorbol-13-acetate (beta-TPA)-stimulation of PtdCho synthesis. We examined involvement of PLD and PKC in the hydrolysis and resynthesis of PtdCho and phosphatidylethanolamine stimulated by beta-TPA, bryostatin (a non-phorbol PKC activator) and oleic acid (18:1n-9) in the four cell lines. beta-TPA or bryostatin produced similar enhancement of [3H]Cho incorporation, loss of stimulated synthesis after down regulation of PKC, and activation of PLD. In C6 cells, staurosporine (STS) and bis-indolylmaleimide (BIM) only partially inhibited basal and beta-TPA-stimulated PLD activity measured as choline or ethanolamine release; phosphatidylbutanol formation after prelabeling with [9,10-3H]18:1n-9, [9,10-3H]myristic acid (14:0), [1-14C]eicosapentaenoic acid (20:5n-3) or 1-O-[alkyl-1', 2-3H]-sn-glyceryl-3-phosphorylcholine gave similar results. STS at >200 nM activated PLD in the presence or absence of beta-TPA. In SK-N-SH cells where PtdCho synthesis was stimulated by beta-TPA or bryostatin, no effect of these agents on PLD was observed. 18:1n-9 stimulated PtdCho synthesis and, to a lesser extent, hydrolysis by PLD both with and without beta-TPA present. Fatty acids had no effect on PKC activities and down regulation of PKC with beta-TPA enhanced fatty acid stimulation of PtdCho synthesis. Thus, activation of PLD hydrolysis preceding resynthesis is involved in the stimulatory effects of beta-TPA on PtdCho synthesis in some but not all of these neural derived cells. Further, PLD hydrolysis of PtdCho and PtdEtn appear to have differing aspects of regulation. Fatty acid regulation of PtdCho synthesis occurs independent of PKC activation. Accordingly

  10. Autophagy and apoptosis in planarians.

    PubMed

    González-Estévez, Cristina; Saló, Emili

    2010-03-01

    Adult planarians are capable of undergoing regeneration and body remodelling in order to adapt to physical damage or extreme environmental conditions. Moreover, most planarians can tolerate long periods of starvation and during this time, they shrink from an adult size to, and sometimes beyond, the initial size at hatching. Indeed, these properties have made them a classic model to study stem cells and regeneration. Under such stressful conditions, food reserves from the gastrodermis and parenchyma are first used up and later the testes, copulatory organs and ovaries are digested. More surprisingly, when food is again made available to shrunken individuals, they grow back to adult size and all their reproductive structures reappear. These cycles of growth and shrinkage may occur over long periods without any apparent impairment to the individual, or to its future maturation and breeding capacities. This plasticity resides in a mesoderm tissue known as the parenchyma, which is formed by several differentiated non-proliferating cell types and only one mitotically active cell type, the neoblasts, which represent approximately 20-30% of the cells in the parenchyma. Neoblasts are generally thought to be somatic stem-cells that participate in the normal continuous turnover of all cell types in planarians. Hence, planarians are organisms that continuously adapt their bodies (morphallaxis) to different environmental stresses (i.e.: injury or starvation). This adaptation involves a variety of processes including proliferation, differentiation, apoptosis and autophagy, all of which are perfectly orchestrated and tightly regulated to remodel or restore the body pattern. While neoblast biology and body re-patterning are currently the subject of intense research, apoptosis and autophagy remain much less studied. In this review we will summarize our current understanding and hypotheses regarding where and when apoptosis and autophagy occur and fulfil an essential role in

  11. Mycobacterium tuberculosis effectors interfering host apoptosis signaling.

    PubMed

    Liu, Minqiang; Li, Wu; Xiang, Xiaohong; Xie, Jianping

    2015-07-01

    Tuberculosis remains a serious human public health concern. The coevolution between its pathogen Mycobacterium tuberculosis and human host complicated the way to prevent and cure TB. Apoptosis plays subtle role in this interaction. The pathogen endeavors to manipulate the apoptosis via diverse effectors targeting key signaling nodes. In this paper, we summarized the effectors pathogen used to subvert the apoptosis, such as LpqH, ESAT-6/CFP-10, LAMs. The interplay between different forms of cell deaths, such as apoptosis, autophagy, necrosis, is also discussed with a focus on the modes of action of effectors, and implications for better TB control.

  12. Apoptosis and Molecular Targeting Therapy in Cancer

    PubMed Central

    Hassan, Mohamed; Watari, Hidemichi; AbuAlmaaty, Ali; Ohba, Yusuke; Sakuragi, Noriaki

    2014-01-01

    Apoptosis is the programmed cell death which maintains the healthy survival/death balance in metazoan cells. Defect in apoptosis can cause cancer or autoimmunity, while enhanced apoptosis may cause degenerative diseases. The apoptotic signals contribute into safeguarding the genomic integrity while defective apoptosis may promote carcinogenesis. The apoptotic signals are complicated and they are regulated at several levels. The signals of carcinogenesis modulate the central control points of the apoptotic pathways, including inhibitor of apoptosis (IAP) proteins and FLICE-inhibitory protein (c-FLIP). The tumor cells may use some of several molecular mechanisms to suppress apoptosis and acquire resistance to apoptotic agents, for example, by the expression of antiapoptotic proteins such as Bcl-2 or by the downregulation or mutation of proapoptotic proteins such as BAX. In this review, we provide the main regulatory molecules that govern the main basic mechanisms, extrinsic and intrinsic, of apoptosis in normal cells. We discuss how carcinogenesis could be developed via defective apoptotic pathways or their convergence. We listed some molecules which could be targeted to stimulate apoptosis in different cancers. Together, we briefly discuss the development of some promising cancer treatment strategies which target apoptotic inhibitors including Bcl-2 family proteins, IAPs, and c-FLIP for apoptosis induction. PMID:25013758

  13. Apoptosis in cancer: from pathogenesis to treatment

    PubMed Central

    2011-01-01

    Apoptosis is an ordered and orchestrated cellular process that occurs in physiological and pathological conditions. It is also one of the most studied topics among cell biologists. An understanding of the underlying mechanism of apoptosis is important as it plays a pivotal role in the pathogenesis of many diseases. In some, the problem is due to too much apoptosis, such as in the case of degenerative diseases while in others, too little apoptosis is the culprit. Cancer is one of the scenarios where too little apoptosis occurs, resulting in malignant cells that will not die. The mechanism of apoptosis is complex and involves many pathways. Defects can occur at any point along these pathways, leading to malignant transformation of the affected cells, tumour metastasis and resistance to anticancer drugs. Despite being the cause of problem, apoptosis plays an important role in the treatment of cancer as it is a popular target of many treatment strategies. The abundance of literature suggests that targeting apoptosis in cancer is feasible. However, many troubling questions arise with the use of new drugs or treatment strategies that are designed to enhance apoptosis and critical tests must be passed before they can be used safely in human subjects. PMID:21943236

  14. Apoptosis-Dependent and Apoptosis-Independent Functions Bim in Prostate Cancer Cells

    DTIC Science & Technology

    2004-03-01

    AD_ Award Number: DAMD17-03-1-0146 TITLE: Apoptosis-Dependent and Apoptosis-Independent Functions of Bim in Prostate Cancer Cells PRINCIPAL...FUNDING NUMBERS Apoptosis-Dependent and Apoptosis-Independent Functions of DAMD17-03-1-0146 Bim in Prostate Cancer Cells 6. A UTHORs) Junwei Liu, M.D...extended cell survival have been implicated in prostate cancer (PCa) development and progression. We recently found that Bim , a BH3-only pro

  15. Apoptosis in oral lichen planus.

    PubMed

    Neppelberg, E; Johannessen, A C; Jonsson, R

    2001-10-01

    Apoptotic cell death may be a contributory cause of basal cell destruction in oral lichen planus (OLP). Therefore. the purpose of this study was to investigate the rate of apoptosis in OLP and the expression of two proteins (FasR and FasL) regulating this process. Biopsies from 18 patients with histologically diagnosed OLP were investigated, with comparison to normal oral mucosa of healthy persons. For visualisation of DNA fragmentation, the TUNEL method was used. In order to characterise the infiltrating cell population (CD3. CD4, CD8) and expression of FasR and FasL, we used an immunohistochemical technique. The results showed that T cells dominated in the subepithelial cell infiltrate. Within the epithelium the apoptotic cells were confined to the basal cell layer, and more apoptotic cells were seen in areas with basal cell degeneration and atrophic epithelium. There was a prominent expression of FasR/FasL in OLP. with a rather uniform distribution throughout the inflammatory cell infiltrate. In the epithelium, the FasR/FasL expression was more abundant in the basal cell area compared to the suprabasal cell layer. In conclusion, apoptosis within the epithelium is significantly increased in situ in OLP compared to normal oral mucosa, and seems to be related to the epithelial thickness.

  16. Differentiation and apoptosis in pilomatrixoma.

    PubMed

    Ishige, Toshiyuki; Kikuchi, Kentaro; Miyazaki, Yuji; Hara, Hiroyuki; Yoshino, Atsuo; Terui, Tadashi; Katayama, Yoichi; Kusama, Kaoru; Nemoto, Norimichi

    2011-02-01

    We carried out a histopathologic study of pilomatrixoma, a benign skin tumor, and also examined apoptosis and hair differentiation with the aim to understand the presence of amorphous debris and cyst formation in the tumor. Among 16 cases of pilomatrixoma examined, 11 were at the early regressive stage and 5 were at the late regressive stage according to the classification by Kaddu et al. In the former cases, tumor nests were basically composed of basophilic, transitional, and shadow cells. Cyst formation was evident in all cases and squamoid epithelium was observed in 4 cases at the early regressive stage. Amorphous debris was found in all cases including those at the late regressive stage. Immunohistochemical analysis revealed positive reaction products for β-catenin and Lef-1 in basophilic and transitional cells, although their distribution differed. Immunoreactivity for β-catenin was observed in the lower transitional cells, whereas immunoreactivity for Lef-1 was also evident in the upper transitional cells. Positive reactions for hair keratins were found in the cytoplasm of transitional and shadow cells, but not in the amorphous debris. Examination by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) method revealed positive reactions in transitional and some shadow cells. These results suggest that in pilomatrixoma, production of hair keratin and induction of apoptosis may occur at the same time, and that unlike the normal hair follicle irregular expression of β-catenin and Lef-1 results in the appearance of amorphous debris and cyst formation.

  17. Apoptosis in mammalian oocytes: a review.

    PubMed

    Tiwari, Meenakshi; Prasad, Shilpa; Tripathi, Anima; Pandey, Ashutosh N; Ali, Irfan; Singh, Arvind K; Shrivastav, Tulsidas G; Chaube, Shail K

    2015-08-01

    Apoptosis causes elimination of more than 99% of germ cells from cohort of ovary through follicular atresia. Less than 1% of germ cells, which are culminated in oocytes further undergo apoptosis during last phases of oogenesis and depletes ovarian reserve in most of the mammalian species including human. There are several players that induce apoptosis directly or indirectly in oocytes at various stages of meiotic cell cycle. Premature removal of encircling granulosa cells from immature oocytes, reduced levels of adenosine 3',5'-cyclic monophosphate and guanosine 3',5'-cyclic monophosphate, increased levels of calcium (Ca(2+)) and oxidants, sustained reduced level of maturation promoting factor, depletion of survival factors, nutrients and cell cycle proteins, reduced meiotic competency, increased levels of proapoptotic as well as apoptotic factors lead to oocyte apoptosis. The BH3-only proteins also act as key regulators of apoptosis in oocyte within the ovary. Both intrinsic (mitochondria-mediated) as well as extrinsic (cell surface death receptor-mediated) pathways are involved in oocyte apoptosis. BID, a BH3-only protein act as a bridge between both apoptotic pathways and its cleavage activates cell death machinery of both the pathways inside the follicular microenvironment. Oocyte apoptosis leads to the depletion of ovarian reserve that directly affects reproductive outcome of various mammals including human. In this review article, we highlight some of the important players and describe the pathways involved during oocyte apoptosis in mammals.

  18. Apoptosis in chronic tonsillitis and tonsillar hypertrophy.

    PubMed

    Önal, Merih; Yılmaz, Taner; Bilgiç, Elif; Müftüoğlu, Sevda Fatma; Kuşçu, Oğuz; Günaydın, Rıza Önder

    2015-02-01

    Chronic tonsillitis is the persistent inflammation of the tonsillar tissue that occurs due to recurrent, acute or subclinical infection. The recurrent and chronic inflammation of palatine tonsils sometimes results in hypertrophy. Apoptosis provides an important balance between lymphocytes in tonsillar lymphoid tissue. The aim of this study is to investigate the apoptosis in tonsillar diseases. 43 patients with chronic tonsilitis and tonsillar hypertrophy underwent tonsillectomy. The specimens were examined immunohistochemically for apoptosis. Tonsils were assembled into groups according to their size. Specimens were compared for their apoptotic cell count. The apoptosis difference between the tonsil size groups is not statistically significant (p>0.05). However, when the study group was divided into two at age 6, the difference was not statistically significant for patients at and below 6 years of age; but, the difference was statistically significant for patients above 6 years of age (p<0.05). The comparison of apoptosis in microcompartments of tonsil tissue (intrafollicular, interfollicular, subepithelial and intraepithelial) between tonsil size stages and between chronic tonsillitis and tonsillar hypertrophy groups revealed no statistical significance (p>0.05). There was a statistically significant positive correlation between intrafollicular and interfollicular, interfollicular and intraepithelial & subepithelial and intraepithelial areas (p<0.05). In the light of these findings, it was concluded that apoptosis played a role in the tonsillar hypertrophy and atrophy. Apoptosis functioned to balance lymphocyte proliferation in tonsil tissue. The association of apoptosis with tonsillar hypertrophy seemed to be age-dependent. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  19. Apoptosis in immune-mediated diseases

    PubMed Central

    Sankari, S. Leena; Babu, N. Aravindha; Rajesh, E.; Kasthuri, M.

    2015-01-01

    Apoptosis plays a significant role in both the physiological and pathological process. A dysfunctional apoptotic system can lead to either excessive removal or prolonged survival of cells. Therefore, dysregulation is involved in the pathogenesis of a variety of immunological diseases. The present review aims to provide an overview regarding role of apoptosis in immune-mediated disease. PMID:26015710

  20. Proteases in Fas-mediated apoptosis.

    PubMed

    Zhivotovsky, B; Burgess, D H; Schlegel, J; Pörn, M I; Vanags, D; Orrenius, S

    1997-01-01

    Involvement of a unique family of cysteine proteases in the multistep apoptotic process has been documented. Cloning of several mammalian genes identifies some components of this cellular response. However, it is currently unclear which protease plays a role as a signal and/or effector of apoptosis. We summarize contributions to the data concerning proteases in Fas-mediated apoptosis.

  1. THE ROLE OF APOPTOSIS IN NEUROTOXICOLOGY.

    EPA Science Inventory

    The role of apoptosis in neurodegeneration in developing animals and in adults has become increasingly apparent in the past ten years. Normal apoptosis occurs in the CNS from the embryonic stage through senescence, with different cells in each region of the nervous system having ...

  2. Local anesthetics induce human renal cell apoptosis.

    PubMed

    Lee, H Thomas; Xu, Hua; Siegel, Cory D; Krichevsky, Igor E

    2003-01-01

    Renal cell apoptosis contributes significantly to the pathogenesis of acute renal failure. Local anesthetics induce apoptosis in neuronal and lymphocytic cell lines. We examined the effects of chronic (48 h) local anesthetic treatment (lidocaine, bupivacaine and tetracaine) on human proximal tubular (HK-2) cells. Apoptosis induction was assessed by detecting poly(ADP)-ribose polymerase fragmentation, caspase activation, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, DNA laddering and by cellular morphology. Cell death was quantified by measuring neutral red dye uptake and lactate dehydrogenase released into the cell culture medium. All 3 local anesthetics caused concentration-dependent cell death, induced HK-2 cell apoptosis and potentiated TNF-alpha induced apoptosis. Local anesthetics induced HK-2 cell apoptosis by activation of caspases 3, 6, 7, 8 and 9. ZVAD-fmk, a pan-caspase inhibitor, blocked the local anesthetic induced HK-2 cell apoptosis. Local anesthetics also inhibited the activities of anti-apoptotic kinases protein kinase B (Akt) and extracellular signal regulated mitrogen-activated protein kinase. Local anesthetic's pro-apoptotic effects are independent of sodium channel inhibition as tetrodotoxin, a selective voltage-gated sodium channel blocker, failed to mimic local anesthetic-mediated induction or potentiation of HK-2 cell apoptosis. We conclude that local anesthetics induce human renal cell apoptotic signaling by caspase activation and via inhibition of pro-survival signaling pathways.

  3. Apoptosis in acquired and genetic hearing impairment

    PubMed Central

    de Beeck, Ken Op; Schacht, Jochen; Van Camp, Guy

    2012-01-01

    Apoptosis is an important physiological process. Normally, a healthy cell maintains a delicate balance between pro- and anti-apoptotic factors, allowing it to live and proliferate. It is thus not surprising that disturbance of this delicate balance may result in disease. It is a well known fact that apoptosis also contributes to several acquired forms of hearing impairment. Noise-induced hearing loss is the result of prolonged exposure to excessive noise, triggering apoptosis in terminally differentiated sensory hair cells. Moreover, hearing loss caused by the use of therapeutic drugs such as aminoglycoside antibiotics and cisplatin potentially may result in the activation of apoptosis in sensory hair cells leading to hearing loss due to the “ototoxicity” of the drugs. Finally, apoptosis is a key contributor to the development of presbycusis, age-related hearing loss. Recently, several mutations in apoptosis genes were identified as the cause of monogenic hearing impairment. These genes are TJP2, DFNA5 and MSRB3. This implies that apoptosis not only contributes to the pathology of acquired forms of hearing impairment, but also to genetic hearing impairment as well. We believe that these genes constitute a new functional class within the hearing loss field. Here, the contribution of apoptosis in the pathology of both acquired and genetic hearing impairment is reviewed. PMID:21782914

  4. THE ROLE OF APOPTOSIS IN NEUROTOXICOLOGY.

    EPA Science Inventory

    The role of apoptosis in neurodegeneration in developing animals and in adults has become increasingly apparent in the past ten years. Normal apoptosis occurs in the CNS from the embryonic stage through senescence, with different cells in each region of the nervous system having ...

  5. Apoptosis in 7-hydroxystaurosporine-treated T lymphoblasts correlates with activation of cyclin-dependent kinases 1 and 2.

    PubMed

    Wang, Q; Worland, P J; Clark, J L; Carlson, B A; Sausville, E A

    1995-08-01

    7-Hydroxystaurosporine (UCN-01) is a potent inhibitor of protein kinase C (PKC) isozymes alpha, beta, and gamma [Seynaeve et al., Mol. Pharmacol, 45: 1207-1214, 1994] that also has antitumor effects in vivo. To determine whether inhibition of PKC can be related to inhibition of cell growth with induction of apoptosis, we compared the effects of UCN-01 to those of the highly selective bisindolylmaleimide PKC antagonist GF 109203X in leukemic T-cell lines. Both compounds potently inhibited PKC activity when added to T-cell membrane preparations and reversed phorbol ester-induced c-fos gene expression in intact cells. However, whereas UCN-01 potently inhibited growth of Jurkat, Molt-3, Molt-4, and Hut-78 cells (IC50 = 20-65 nM, irreversible after 24 h of exposure), GF 109203X had IC50s for cell growth of 3.6-5.0 muM. Less than 3 h after addition, UCN-01 but not GF 109203X-treated cells displayed loss of cells with G2-M DNA content, appearance of a hypodiploid DNA fraction, and evidence of internucleosomal DNA fragmentation. Six h after treatment, cells appeared to accumulate with S-phase DNA content. These effects correlated with selective UCN-01 but not GF 109203X-induced decrease in total and tyrosine phosphorylation of cyclin-dependent kinases (cdks) 1 and 2, and with increases in the histone H1 kinase activities of cdk1 and cdk2. UCN-01 was relatively less potent in inhibition of properly activated cdk1 and cdk2 when added in vitro to H1 kinase assays (IC50 = 1000 and 600 nM, respectively). We conclude that inhibition of PKC alone is not sufficient to account for the actions of UCN-01 and are led to the hypothesis that inappropriate cdk activation either correlates with or actually mediates cell growth inhibition with apoptosis in T lymphoblasts exposed to UCN-01.

  6. Expression of alternatively spliced interleukin-1 receptor accessory protein mRNAs is differentially regulated during inflammation and apoptosis.

    PubMed

    Jensen, Liselotte E; Whitehead, Alexander S

    2003-08-01

    Two alternative splice variants of the interleukin-1 receptor accessory protein (IL-1RAcP) mRNA are known. Membrane-bound IL-1RAcP (mIL-1RAcP) promotes intracellular interleukin-1 (IL-1) signalling whereas soluble IL-1RAcP (sIL-1RAcP) is probably an inhibitor of IL-1 signalling. Here we establish that sIL-1RAcP mRNA levels increase 16-fold in response to phorbol esters in the human hepatoma cell line HepG2 via a mechanism that depends on de novo protein synthesis. Following exposure of cells to UV light, a potent inducer of apoptosis, mIL-1RAcP mRNA is rapidly down-regulated and a new steady-state level established briefly before a gradual return to pretreatment levels. Following treatment with staurosporine, also an inducer of apoptosis, mIL-1RAcP mRNA levels steadily decrease through 72 h, with little change in sIL-1RAcP mRNA levels. A novel alternative splice variant, sIL-1RAcP-beta, was identified. Its sequence indicates that sIL-1RAcP-beta is secreted and has a unique second half of the third immunoglobulin (Ig) domain. The dramatic changes in levels of IL-1RAcP mRNAs suggest important functions in regulating sensitivity to IL-1 during stress and may play a role in oncogenic processes that are engaged during chronic inflammation.

  7. Regulation of apoptosis by heat shock proteins.

    PubMed

    Kennedy, Donna; Jäger, Richard; Mosser, Dick D; Samali, Afshin

    2014-05-01

    Thermotolerance, the acquired resistance of cells to stress, is a well-established phenomenon. Studies of the key mediators of this response, the heat shock proteins (HSPs), have led to the discovery of the important roles played by these proteins in the regulation of apoptotic cell death. Apoptosis is critical for normal tissue homeostasis and is involved in diverse processes including development and immune clearance. Apoptosis is tightly regulated by both proapoptotic and antiapoptotic factors, and dysregulation of apoptosis plays a significant role in the pathophysiology of many diseases. In the recent years, HSPs have been identified as key determinants of cell survival, which can modulate apoptosis by directly interacting with components of the apoptotic machinery. Therefore, manipulation of the HSPs could represent a viable strategy for the treatment of diseases. Here, we review the current knowledge with regard to the mechanisms of HSP-mediated regulation of apoptosis. © 2014 International Union of Biochemistry and Molecular Biology.

  8. Cancer Therapy Due to Apoptosis: Galectin-9

    PubMed Central

    Fujita, Koji; Iwama, Hisakazu; Oura, Kyoko; Tadokoro, Tomoko; Samukawa, Eri; Sakamoto, Teppei; Nomura, Takako; Tani, Joji; Yoneyama, Hirohito; Morishita, Asahiro; Himoto, Takashi; Hirashima, Mitsuomi; Masaki, Tsutomu

    2017-01-01

    Dysregulation of apoptosis is a major hallmark in cancer biology that might equip tumors with a higher malignant potential and chemoresistance. The anti-cancer activities of lectin, defined as a carbohydrate-binding protein that is not an enzyme or antibody, have been investigated for over a century. Recently, galectin-9, which has two distinct carbohydrate recognition domains connected by a linker peptide, was noted to induce apoptosis in thymocytes and immune cells. The apoptosis of these cells contributes to the development and regulation of acquired immunity. Furthermore, human recombinant galectin-9, hG9NC (null), which lacks an entire region of the linker peptide, was designed to resist proteolysis. The hG9NC (null) has demonstrated anti-cancer activities, including inducing apoptosis in hematological, dermatological and gastrointestinal malignancies. In this review, the molecular characteristics, history and apoptosis-inducing potential of galectin-9 are described. PMID:28045432

  9. Cancer Therapy Due to Apoptosis: Galectin-9.

    PubMed

    Fujita, Koji; Iwama, Hisakazu; Oura, Kyoko; Tadokoro, Tomoko; Samukawa, Eri; Sakamoto, Teppei; Nomura, Takako; Tani, Joji; Yoneyama, Hirohito; Morishita, Asahiro; Himoto, Takashi; Hirashima, Mitsuomi; Masaki, Tsutomu

    2017-01-01

    Dysregulation of apoptosis is a major hallmark in cancer biology that might equip tumors with a higher malignant potential and chemoresistance. The anti-cancer activities of lectin, defined as a carbohydrate-binding protein that is not an enzyme or antibody, have been investigated for over a century. Recently, galectin-9, which has two distinct carbohydrate recognition domains connected by a linker peptide, was noted to induce apoptosis in thymocytes and immune cells. The apoptosis of these cells contributes to the development and regulation of acquired immunity. Furthermore, human recombinant galectin-9, hG9NC (null), which lacks an entire region of the linker peptide, was designed to resist proteolysis. The hG9NC (null) has demonstrated anti-cancer activities, including inducing apoptosis in hematological, dermatological and gastrointestinal malignancies. In this review, the molecular characteristics, history and apoptosis-inducing potential of galectin-9 are described.

  10. Apoptosis inducers in chronic lymphocytic leukemia

    PubMed Central

    Billard, Christian

    2014-01-01

    Chronic lymphocytic leukemia (CLL) is characterized by a typical defect in apoptosis and is still an incurable disease. Numerous apoptosis inducers have been described. These synthetic compounds and natural products (mainly derived from plants) display antileukemic properties in vitro and in vivo and some have even been tested in the clinic in CLL. They act through several different mechanisms. Most of them involve proteins of the Bcl-2 family, which are the key regulators in triggering the mitochondrial pathway of caspase-dependent apoptosis. Thus, the Mcl-1/Noxa axis appeared as a target. Here I overview natural and synthetic apoptosis inducers and their mechanisms of action in CLL cells. Opportunities for developing novel, apoptosis-based therapeutics are presented. PMID:24525395

  11. Autophagy and apoptosis: where do they meet?

    PubMed

    Mukhopadhyay, Subhadip; Panda, Prashanta Kumar; Sinha, Niharika; Das, Durgesh Nandini; Bhutia, Sujit Kumar

    2014-04-01

    Autophagy and apoptosis are two important cellular processes with complex and intersecting protein networks; as such, they have been the subjects of intense investigation. Recent advances have elucidated the key players and their molecular circuitry. For instance, the discovery of Beclin-1's interacting partners has resulted in the identification of Bcl-2 as a central regulator of autophagy and apoptosis, which functions by interacting with both Beclin-1 and Bax/Bak respectively. When localized to the endoplasmic reticulum and mitochondria, Bcl-2 inhibits autophagy. Cellular stress causes the displacement of Bcl-2 from Beclin-1 and Bax, thereby triggering autophagy and apoptosis, respectively. The induction of autophagy or apoptosis results in disruption of complexes by BH3-only proteins and through post-translational modification. The mechanisms linking autophagy and apoptosis are not fully defined; however, recent discoveries have revealed that several apoptotic proteins (e.g., PUMA, Noxa, Nix, Bax, XIAP, and Bim) modulate autophagy. Moreover, autophagic proteins that control nucleation and elongation regulate intrinsic apoptosis through calpain- and caspase-mediated cleavage of autophagy-related proteins, which switches the cellular program from autophagy to apoptosis. Similarly, several autophagic proteins are implicated in extrinsic apoptosis. This highlights a dual cellular role for autophagy. On one hand, autophagy degrades damaged mitochondria and caspases, and on the other hand, it provides a membrane-based intracellular platform for caspase processing in the regulation of apoptosis. In this review, we highlight the crucial factors governing the crosstalk between autophagy and apoptosis and describe the mechanisms controlling cell survival and cell death.

  12. Differential expression of thrombospondin, collagen, and thyroglobulin by thyroid-stimulating hormone and tumor-promoting phorbol ester in cultured porcine thyroid cells.

    PubMed

    Bellon, G; Chaqour, B; Antonicelli, F; Wegrowski, J; Claisse, D; Haye, B; Borel, J P

    1994-07-01

    In the present study, we have investigated the potential regulation of thyroglobulin (Tg) and extracellular matrix components synthesis by thyroid-stimulating hormone (TSH) and tetradecanoyl phorbol-13-acetate (TPA) on thyroid cells. Porcine thyroid cells isolated by trypsin-EGTA digestion of thyroid glands were maintained in serum containing medium on poly (L-lysine)-coated dishes. Cells differentiated into follicular or vesicular-like structures were distinguished by their ability to organify Na[125I] and to respond to TSH stimulation. After an incubation of the cells with radiolabeled proline or methionine, two major proteins were identified, p450-480 and p290 (so named because of their molecular masses). Tg (p290) synthesis was demonstrated by the synthesis of [131I]-labeled polypeptides with electrophoretic properties identical to those of authentic Tg molecules. P450-480 resolved to M(r) 190,000 under reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) conditions. It was identified as thrombospondin by its reactivity with a monoclonal anti-human thrombospondin and by peptide sequencing of some of its tryptic fragments that displayed identity to thrombospondin I. Collagen synthesis was demonstrated by the formation of radioactive hydroxyproline and by the synthesis of pepsin-resistant polypeptides ranging from M(rs) 120,000 to 200,000. When the cells were cultured in the presence of 100 nM TPA, the culture medium contents of thrombospondin and collagen were increased by 2.7 and 1.6-fold, respectively, whereas Tg content was decreased by a factor 3.9. In contrast, the acute treatment of control cells with TPA induced a decrease in both Tg and collagen content by factors 3.0 and 1.5, respectively, and an increase in thrombospondin content by a factor 2.5. In the presence of 100 nM TPA, TSH (1 mU/ml) did not counteract the stimulating effect of TPA on extracellular matrix components synthesis. In contrast, when cells were cultured in the

  13. Phytosphingosine induced mitochondria-involved apoptosis.

    PubMed

    Nagahara, Yukitoshi; Shinomiya, Takahisa; Kuroda, Sachiko; Kaneko, Naoki; Nishio, Reiji; Ikekita, Masahiko

    2005-02-01

    Sphingolipids are putative intracellular signal mediators in cell differentiation, growth inhibition, and apoptosis. Sphingosine, sphinganine, and phytosphingosine are structural analogs of sphingolipids and are classified as long-chain sphingoid bases. Sphingosine and sphinganine are known to play important roles in apoptosis. In the present study, we examined the phytosphingosine-induced apoptosis mechanism, focusing on mitochondria in human T-cell lymphoma Jurkat cells. Phytosphingosine significantly induced chromatin DNA fragmentation, which is a hallmark of apoptosis. Enzymatic activity measurements of caspases revealed that caspase-3 and caspase-9 are activated in phytosphingosine-induced apoptosis, but there is little activation of caspase-8 suggesting that phytosphingosine influences mitochondrial functions. In agreement with this hypothesis, a decrease in DeltaPsi(m) and the release of cytochrome c to the cytosol were observed upon phytosphingosine treatment. Furthermore, overexpression of mitochondria-localized anti-apoptotic protein Bcl-2 prevented phytosphingosine apoptotic stimuli. Western blot assays revealed that phytosphingosine decreases phosphorylated Akt and p70S6k. Dephosphorylation of Akt was partially inhibited by protein phosphatase inhibitor OA and OA attenuated phytosphingosine-induced apoptosis. Moreover, using a cell-free system, phytosphingosine directly reduced DeltaPsi(m). These results indicate that phytosphingosine perturbs mitochondria both directly and indirectly to induce apoptosis.

  14. Posttraumatic Chondrocyte Apoptosis in the Murine Xiphoid.

    PubMed

    Davis, Christopher G; Eisner, Eric; McGlynn, Margaret; Shelton, John M; Richardson, James; Borrelli, Joseph; Chen, Christopher C T

    2013-10-01

    To demonstrate posttraumatic chondrocyte apoptosis in the murine xiphoid after a crush-type injury and to ultimately determine the pathway (i.e., intrinsic or extrinsic) by which chondrocytes undergo apoptosis in response to mechanical injury. The xiphoids of adult female wild-type mice were injured with the use of a modified Kelly clamp. Postinjury xiphoid cartilage was analyzed via 3 well-described independent means of assessing apoptosis in chondrocytes: hematoxylin and eosin staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, and activated caspase-3 staining. Injured specimens contained many chondrocytes with evidence of apoptosis, which is characterized by cell shrinkage, chromatin condensation, nuclear fragmentation, and the liberation of apoptotic bodies. There was a statistically significant increase in the number of chondrocytes undergoing apoptosis in the injured specimens as compared with the uninjured specimens. Chondrocytes can be stimulated to undergo apoptosis as a result of mechanical injury. These experiments involving predominantly cartilaginous murine xiphoid in vivo establish a baseline for future investigations that employ the genetic and therapeutic modulation of chondrocyte apoptosis in response to mechanical injury.

  15. Semaphorins as mediators of neuronal apoptosis.

    PubMed

    Shirvan, A; Ziv, I; Fleminger, G; Shina, R; He, Z; Brudo, I; Melamed, E; Barzilai, A

    1999-09-01

    Shrinkage and collapse of the neuritic network are often observed during the process of neuronal apoptosis. However, the molecular and biochemical basis for the axonal damage associated with neuronal cell death is still unclear. We present evidence for the involvement of axon guidance molecules with repulsive cues in neuronal cell death. Using the differential display approach, an up-regulation of collapsin response mediator protein was detected in sympathetic neurons undergoing dopamine-induced apoptosis. A synchronized induction of mRNA of the secreted collapsin-1 and the intracellular collapsin response mediator protein that preceded commitment of neurons to apoptosis was detected. Antibodies directed against a conserved collapsin-derived peptide provided marked and prolonged protection of several neuronal cell types from dopamine-induced apoptosis. Moreover, neuronal apoptosis was inhibited by antibodies against neuropilin-1, a putative component of the semaphorin III/collapsin-1 receptor. Induction of neuronal apoptosis was also caused by exposure of neurons to semaphorin III-alkaline phosphatase secreted from 293EBNA cells. Anti-collapsin-1 antibodies were effective in blocking the semaphorin III-induced death process. We therefore suggest that, before their death, apoptosis-destined neurons may produce and secrete destructive axon guidance molecules that can affect their neighboring cells and thus transfer a "death signal" across specific and susceptible neuronal populations.

  16. The role of apoptosis in respiratory diseases.

    PubMed

    Pierce, Janet D; Pierce, Jana; Stremming, Stephanie; Fakhari, Mahtab; Clancy, Richard L

    2007-01-01

    The purpose of this article is to define apoptosis and describe how this cellular pathway is relevant to the pathogenesis of different respiratory diseases. This will assist clinical nurse specialists in understanding how new drugs and therapies inhibit and stimulate apoptotic pathways. Clinical nurse specialists need to expand their knowledge concerning the role of apoptosis so that they can better expand their spheres of influence. The 4 stages of apoptosis are discussed, as well as the various apoptotic pathways involved with asthma, emphysema, and acute respiratory distress syndrome that promote and inhibit apoptosis in patients. It is crucial for clinical nurse specialists to know what apoptosis is and how it relates to different pathophysiologic states. The challenge facing clinical nurse specialists is how to be kept informed and current concerning molecular and cellular mechanisms that are important in the practice setting. Strategies needed to maintain expertise include acquiring new knowledge, developing new skills, and changing attitudes about molecular biology. Apoptosis must become a significant part of any health professionals' continuing educational program because it has been recognized as the pathway to most any disease. Clinical nurse specialists who understand apoptosis and its pathways can use this knowledge to aid in the prevention and treatment of respiratory diseases.

  17. Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-ßI and prooxidant enzyme NADPH oxidase.

    PubMed

    Shao, Beili; Bayraktutan, Ulvi

    2014-01-01

    Blood-brain barrier disruption represents a key feature in hyperglycaemia-aggravated cerebral damage after an ischaemic stroke. Although the underlying mechanisms remain largely unknown, activation of protein kinase C (PKC) is thought to play a critical role. This study examined whether apoptosis of human brain microvascular endothelial cells (HBMEC) might contribute to hyperglycaemia-evoked barrier damage and assessed the specific role of PKC in this phenomenon. Treatments with hyperglycaemia (25 mM) or phorbol myristate acetate (PMA, a protein kinase C activator, 100 nM) significantly increased NADPH oxidase activity, O2 (•-) generation, proapoptotic protein Bax expression, TUNEL-positive staining and caspase-3/7 activities. Pharmacological inhibition of NADPH oxidase, PKC-a, PKC-ß or PKC-ßI via their specific inhibitors and neutralisation of O2 (•-) by a cell-permeable superoxide dismutase mimetic, MnTBAP normalised all the aforementioned increases induced by hyperglycaemia. Suppression of these PKC isoforms also negated the stimulatory effects of hyperglycaemia on the protein expression of NADPH oxidase membrane-bound components, Nox2 and p22-phox which determine the overall enzymatic activity. Silencing of PKC-ßI gene through use of specific siRNAs abolished the effects of both hyperglycaemia and PMA on endothelial cell NADPH oxidase activity, O2 (•-) production and apoptosis and consequently improved the integrity and function of an in vitro model of human cerebral barrier comprising HBMEC, astrocytes and pericytes. Hyperglycaemia-mediated apoptosis of HBMEC contributes to cerebral barrier dysfunction and is modulated by sequential activations of PKC-ßI and NADPH oxidase.

  18. Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-ßI and prooxidant enzyme NADPH oxidase

    PubMed Central

    Shao, Beili; Bayraktutan, Ulvi

    2014-01-01

    Blood–brain barrier disruption represents a key feature in hyperglycaemia-aggravated cerebral damage after an ischaemic stroke. Although the underlying mechanisms remain largely unknown, activation of protein kinase C (PKC) is thought to play a critical role. This study examined whether apoptosis of human brain microvascular endothelial cells (HBMEC) might contribute to hyperglycaemia-evoked barrier damage and assessed the specific role of PKC in this phenomenon. Treatments with hyperglycaemia (25 mM) or phorbol myristate acetate (PMA, a protein kinase C activator, 100 nM) significantly increased NADPH oxidase activity, O2•- generation, proapoptotic protein Bax expression, TUNEL-positive staining and caspase-3/7 activities. Pharmacological inhibition of NADPH oxidase, PKC-a, PKC-ß or PKC-ßI via their specific inhibitors and neutralisation of O2•- by a cell-permeable superoxide dismutase mimetic, MnTBAP normalised all the aforementioned increases induced by hyperglycaemia. Suppression of these PKC isoforms also negated the stimulatory effects of hyperglycaemia on the protein expression of NADPH oxidase membrane-bound components, Nox2 and p22-phox which determine the overall enzymatic activity. Silencing of PKC-ßI gene through use of specific siRNAs abolished the effects of both hyperglycaemia and PMA on endothelial cell NADPH oxidase activity, O2•- production and apoptosis and consequently improved the integrity and function of an in vitro model of human cerebral barrier comprising HBMEC, astrocytes and pericytes. Hyperglycaemia-mediated apoptosis of HBMEC contributes to cerebral barrier dysfunction and is modulated by sequential activations of PKC-ßI and NADPH oxidase. PMID:24936444

  19. Metadherin facilitates podocyte apoptosis in diabetic nephropathy

    PubMed Central

    Liu, Wen-Ting; Peng, Fen-Fen; Li, Hong-Yu; Chen, Xiao-Wen; Gong, Wang-Qiu; Chen, Wen-Jing; Chen, Yi-Hua; Li, Pei-Lin; Li, Shu-Ting; Xu, Zhao-Zhong; Long, Hai-Bo

    2016-01-01

    Apoptosis, one of the major causes of podocyte loss, has been reported to have a vital role in diabetic nephropathy (DN) pathogenesis, and understanding the mechanisms underlying the regulation of podocyte apoptosis is crucial. Metadherin (MTDH) is an important oncogene, which is overexpressed in most cancers and responsible for apoptosis, metastasis, and poor patient survival. Here we show that the expression levels of Mtdh and phosphorylated p38 mitogen-activated protein kinase (MAPK) are significantly increased, whereas those of the microRNA-30 family members (miR-30s) are considerably reduced in the glomeruli of DN rat model and in high glucose (HG)-induced conditionally immortalized mouse podocytes (MPC5). These levels are positively correlated with podocyte apoptosis rate. The inhibition of Mtdh expression, using small interfering RNA, but not Mtdh overexpression, was shown to inhibit HG-induced MPC5 apoptosis and p38 MAPK pathway, and Bax and cleaved caspase 3 expression. This was shown to be similar to the effects of p38 MAPK inhibitor (SB203580). Furthermore, luciferase assay results demonstrated that Mtdh represents the target of miR-30s. Transient transfection experiments, using miR-30 microRNA (miRNA) inhibitors, led to the increase in Mtdh expression and induced the apoptosis of MPC5, whereas the treatment with miR-30 miRNA mimics led to the reduction in Mtdh expression and apoptosis of HG-induced MPC5 cells in comparison with their respective controls. Our results demonstrate that Mtdh is a potent modulator of podocyte apoptosis, and that it represents the target of miR-30 miRNAs, facilitating podocyte apoptosis through the activation of HG-induced p38 MAPK-dependent pathway. PMID:27882943

  20. Modeling heterogeneous responsiveness of intrinsic apoptosis pathway

    PubMed Central

    2013-01-01

    Background Apoptosis is a cell suicide mechanism that enables multicellular organisms to maintain homeostasis and to eliminate individual cells that threaten the organism’s survival. Dependent on the type of stimulus, apoptosis can be propagated by extrinsic pathway or intrinsic pathway. The comprehensive understanding of the molecular mechanism of apoptotic signaling allows for development of mathematical models, aiming to elucidate dynamical and systems properties of apoptotic signaling networks. There have been extensive efforts in modeling deterministic apoptosis network accounting for average behavior of a population of cells. Cellular networks, however, are inherently stochastic and significant cell-to-cell variability in apoptosis response has been observed at single cell level. Results To address the inevitable randomness in the intrinsic apoptosis mechanism, we develop a theoretical and computational modeling framework of intrinsic apoptosis pathway at single-cell level, accounting for both deterministic and stochastic behavior. Our deterministic model, adapted from the well-accepted Fussenegger model, shows that an additional positive feedback between the executioner caspase and the initiator caspase plays a fundamental role in yielding the desired property of bistability. We then examine the impact of intrinsic fluctuations of biochemical reactions, viewed as intrinsic noise, and natural variation of protein concentrations, viewed as extrinsic noise, on behavior of the intrinsic apoptosis network. Histograms of the steady-state output at varying input levels show that the intrinsic noise could elicit a wider region of bistability over that of the deterministic model. However, the system stochasticity due to intrinsic fluctuations, such as the noise of steady-state response and the randomness of response delay, shows that the intrinsic noise in general is insufficient to produce significant cell-to-cell variations at physiologically relevant level of

  1. [Endothelial cell apoptosis in erectile dysfunction].

    PubMed

    Jiang, Rui

    2012-10-01

    Erectile dysfunction (ED) is one of the most common male diseases, which seriously affects the patient's quality of life. The risk factors of ED include aging, diabetes, hypertension, hyperlipidemia, and unhealthy lifestyle, and its exact mechanism remains unclear. The apoptosis of endothelial cells in the corpus cavernosum penis may reduce NOS activity, block NO synthesis, and affect penile erection, and the mechanisms of their apoptosis vary with different causes of ED. This article updates the relationship between the apoptosis of endothelial cells and the development of ED.

  2. The Role of Mitochondria in Apoptosis*

    PubMed Central

    Wang, Chunxin; Youle, Richard J.

    2016-01-01

    Mitochondria play key roles in activating apoptosis in mammalian cells. Bcl-2 family members regulate the release of proteins from the space between the mitochondrial inner and outer membrane that, once in the cytosol, activate caspase proteases that dismantle cells and signal efficient phagocytosis of cell corpses. Here we review the extensive literature on proteins released from the intermembrane space and consider genetic evidence for and against their roles in apoptosis activation. We also compare and contrast apoptosis pathways in Caenorhabditis elegans, Drosophila melanogaster, and mammals that indicate major mysteries remaining to be solved. PMID:19659442

  3. Order of application determines the interaction between phorbol esters and GTP-gamma-S in dorsal raphe neurons: evidence that the effect of 5-HT is modified upstream of the G protein Ca channel interaction.

    PubMed

    Chen, Y; Penington, N J

    1997-05-01

    Phorbol esters activating protein kinase C (PKC) partially uncouple the inhibitory effect of serotonin (5-HT) from serotonergic neuron Ca2+ current. Presently the site of action of PKC is not known and may be the receptor, G protein, or ion channel. We recorded Ca2+ current from acutely isolated neurons with the use of the patch-clamp technique to study the site of action of PKC. Activation of the G protein with internal guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S) occluded the response to 5-HT, but unexpectedly this effect was not reversed by the addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) despite the voltage-dependent reversal of the effect of GTP-gamma-S by long depolarizing steps to +80 mV. PMA was, however, able to partially reverse 5-HT-induced inhibition of Ca2+ current. The rate of reinhibition of the Ca2+ current (related to the concentration of activated G proteins) by GTP-gamma-S after the addition of PMA at -50 mV was identical to the rate when only GTP-gamma-S was present. By contrast, when cells were exposed first to PMA, and then GTP-gamma-S was perfused into the cell, GTP-gamma-S lost about half of its ability to activate the G protein. The rate of reinhibition of the Ca2+ current by internal GTP-gamma-S was also reduced in cells pretreated with PMA. The original result in which PMA did not reverse the action of GTP-gamma-S suggested that the channel was not the functional site of action of PMA, nor was the site on the G protein that binds to the channel, but it did not rule out the receptor. When the receptor was bypassed, after prior PKC activation, it was found that direct activation of the G protein by a nonhydrolyzable analogue of GTP was reduced; taken as a whole, this indicates that in dorsal raphe, and perhaps other neurons, the site of the critical phosphorylation may be on the G protein and possibly at the GTP binding site.

  4. Protein kinase C promotes apoptosis in LNCaP prostate cancer cells through activation of p38 MAPK and inhibition of the Akt survival pathway.

    PubMed

    Tanaka, Yuichi; Gavrielides, M Veronica; Mitsuuchi, Yasuhiro; Fujii, Teruhiko; Kazanietz, Marcelo G

    2003-09-05

    Activation of protein kinase C (PKC) by phorbol esters or diacylglycerol mimetics induces apoptosis in androgen-dependent prostate cancer cells, an effect that involves both the activation of the classic PKC alpha and the novel PKC delta isozymes (Fujii, T., García-Bermejo, M. L., Bernabó, J. L., Caamaño, J., Ohba, M., Kuroki, T., Li, L., Yuspa, S. H., and Kazanietz, M. G. (2000) J. Biol. Chem. 275, 7574-7582 and Garcia-Bermejo, M. L., Leskow, F. C., Fujii, T., Wang, Q., Blumberg, P. M., Ohba, M., Kuroki, T., Han, K. C., Lee, J., Marquez, V. E., and Kazanietz, M. G. (2002) J. Biol. Chem. 277, 645-655). In the present study we explored the signaling events involved in this PKC-mediated effect, using the androgen-dependent LNCaP cell line as a model. Stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) leads to the activation of ERK1/2, p38 MAPK, and JNK in LNCaP cells. Here we present evidence that p38 MAPK, but not JNK, mediates PKC-induced apoptosis. Because LNCaP cells have hyperactivated Akt function due to PTEN inactivation, we examined whether this survival pathway could be affected by PKC activation. Interestingly, activation of PKC leads to a rapid and reversible dephosphorylation of Akt, an effect that was prevented by the pan-PKC inhibitor GF109302X and the cPKC inhibitor Gö6976. In addition, the diacylglycerol mimetic agent HK654, which selectively stimulates PKC alpha in LNCaP cells, also induced the dephosphorylation of Akt in LNCaP cells. Inactivation of Akt function by PKC does not involve the inhibition of PI3K, and it is prevented by okadaic acid, suggesting the involvement of a phosphatase 2A in PMA-induced Akt dephosphorylation. Finally, we show that, when an activated form of Akt is delivered into LNCaP cells by either transient transfection or adenoviral infection, the apoptotic effect of PMA is significantly reduced. Our results highlight a complex array of signaling pathways regulated by PKC isozymes in LNCaP prostate cancer cells

  5. [The inhibitory effect of As₂O₃ combined with phorbol ester on the proliferation of Kasumi-1 cells and its mechanism].

    PubMed

    Yuan, Fangfang; Xu, Jinxia; Mi, Ruihua; Fan, Ruihua; Yin, Qingsong; Wei, Xudong

    2014-06-01

    To investigate the inhibitory effect of arsenic trioxide (As₂O₃) combined with tetradecanoylphorbol acetate (TPA) on the proliferation of Kasumi-1 cell line and its mechanism. Kasumi-1 cells were treated with 200 nmol/L TPA, different concentrations of As₂O₃ alone and combined with 200 nmol/L TPA. The proliferative inhibition rates were determined with CCK-8. Annexin V was adopted to detect apoptosis. Colony formation assay was used to determine the cloning efficiency. Flow cytometry was used to detect the cell differentiation and cell cycle changes. Western blot was employed to detect the expression of P38 and p-P38. The proliferation inhibition rates of Kasumi-1 cells by TPA combined with different concentrations of As₂O₃ (0.2, 2.0 and 20.0 mmol/L)for 48 h were (25.56 ± 7.29)%, (60.63 ± 6.64)%, and (73.37 ± 2.15)%, the apoptosis rates were (61.65 ± 2.62)%, (75.39 ± 1.04)%, and (89.95 ± 1.46)%, and the colony formation rates were (76.17 ± 2.06)%, (38.50 ± 1.87)%, and (18.53 ± 2.20)%, respectively, compared with the different concentrations of As₂O₃ alone groups, the difference was statistically significant (P<0.05). Cells treated with both TPA and As₂O₃ expressed more CD11b antigens compared with the cells exposed to As₂O₃ alone. TPA treated Kasumi-1 cells were arrested at G1 phase compared with the control group, while As₂O₃ increased the percentage of Kasumi-1 cells in the G2 phase. Combination treatment increased the expression of p-P38 of Kasumi-1 cells compared with the cells exposed to As₂O₃ alone. TPA can enhance the effect of As₂O₃ on inducing apoptosis and regulating cell cycle, thereby enhancing its anti-leukemia effect.

  6. MicroRNA-1 promotes apoptosis of hepatocarcinoma cells by targeting apoptosis inhibitor-5 (API-5).

    PubMed

    Li, Dong; Liu, Yu; Li, Hua; Peng, Jing-Jing; Tan, Yan; Zou, Qiang; Song, Xiao-Feng; Du, Min; Yang, Zheng-Hui; Tan, Yong; Zhou, Jin-Jun; Xu, Tao; Fu, Zeng-Qiang; Feng, Jian-Qiong; Cheng, Peng; chen, Tao; Wei, Dong; Su, Xiao-Mei; Liu, Huan-Yi; Qi, Zhong-Chun; Tang, Li-Jun; Wang, Tao; Guo, Xin; Hu, Yong-He; Zhang, Tao

    2015-01-02

    Although microRNA-1 (miR-1) is a known liver cancer suppressor, the role of miR-1 in apoptosis of hepatoma cells has remained largely unknown. Our study shows that ectopic miR-1 overexpression induced apoptosis of liver hepatocellular carcinoma (HepG2) cells. Apoptosis inhibitor 5 (API-5) was found to be a potential regulator of miR-1 induced apoptosis, using a bioinformatics approach. Furthermore, an inverse relationship between miR-1 and API-5 expression was observed in human liver cancer tissues and adjacent normal liver tissues. Negative regulation of API-5 expression by miR-1 was demonstrated to promote apoptosis of HepG2 cells. Our study provides a novel regulatory mechanism of miR-1 in the apoptosis of hepatoma cells. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  7. Myocardial apoptosis in heart disease: does the emperor have clothes?

    PubMed

    Jose Corbalan, J; Vatner, Dorothy E; Vatner, Stephen F

    2016-05-01

    Since the discovery of a novel mechanism of cell death that differs from traditional necrosis, i.e., apoptosis, there have been numerous studies concluding that increased apoptosis augments myocardial infarction and heart failure and that limiting apoptosis protects the heart. Importantly, the vast majority of cells in the heart are non-myocytes with only roughly 30 % myocytes, yet almost the entire field studying apoptosis in the heart has disregarded non-myocyte apoptosis, e.g., only 4.7 % of 423 studies on myocardial apoptosis in the past 3 years quantified non-myocyte apoptosis. Accordingly, we reviewed the history of apoptosis in the heart focusing first on myocyte apoptosis, followed by the history of non-myocyte apoptosis in myocardial infarction and heart failure. Apoptosis of several of the major non-myocyte cell types in the heart (cardiac fibroblasts, endothelial cells, vascular smooth muscle cells, macrophages and leukocytes) may actually be responsible for affecting the severity of myocardial infarction and heart failure. In summary, even though it is now known that the majority of apoptosis in the heart occurs in non-myocytes, very little work has been done to elucidate the mechanisms by which non-myocyte apoptosis might be responsible for the adverse effects of apoptosis in myocardial infarction and heart failure. The goal of this review is to provide an impetus for future work in this field on non-myocyte apoptosis that will be required for a better understanding of the role of apoptosis in the heart.

  8. Targeting apoptosis in acute tubular injury.

    PubMed

    Ortiz, Alberto; Justo, Pilar; Sanz, Ana; Lorz, Corina; Egido, Jesús

    2003-10-15

    Recent research has shown that apoptosis and its regulatory mechanisms contribute to cell number regulation in acute renal failure. Acute tubular necrosis is the most frequent form of parenchymal acute renal failure. The main causes are ischemia-reperfusion, sepsis and nephrotoxic drugs. Exogenous factors such as nephrotoxic drugs and bacterial products, and endogenous factors such as lethal cytokines promote tubular cell apoptosis. Such diverse stimuli engage intracellular death pathways that in some cases are stimulus-specific. We now review the role of apoptosis in acute renal failure, the potential molecular targets of therapeutic intervention, the therapeutic weapons to modulate the activity of these targets and the few examples of therapeutic intervention on apoptosis.

  9. Molecular mechanisms of UV-induced apoptosis.

    PubMed

    Kulms, D; Schwarz, T

    2000-10-01

    Sunburn cells, single standing cells with typical morphologic features occurring in UV-exposed skin, have been recognized as keratinocytes undergoing apoptosis following UV irradiation. Induction of apoptosis following UV exposure appears to be a protective mechanism, getting rid off severely damaged cells that bear the risk of malignant transformation. UV-mediated apoptosis is a highly complex process in which different molecular pathways are involved. These include DNA damage, activation of the tumor suppressor gene p53, triggering of cell death receptors either directly by UV or by autocrine release of death ligands, mitochondrial damage and cytochrome C release. Detailed knowledge about the interplay between these pathways will increase our understanding of photocarcinogenesis. This review briefly discusses recent findings concerning the molecular mechanisms underlying UV-induced apoptosis.

  10. [Apoptosis and thymocyte development (epithelial cells as inducers of thymocyte apoptosis)].

    PubMed

    Iarilin, A A; Bulanova, E G; Sharova, N I; Budagian, V M

    1998-01-01

    Apoptosis, together with proliferation, is a main factor of selection of the clones of developing T-lymphocytes: the clones not supported by positive selection are subject to apoptosis and apoptosis accounts for discarding of potentially autoaggressive clones, i.e., for negative selection in the thymus and peripheral lymphoid tissue. Realization of apoptosis at different stages of the development of T-lymphocytes depends to a varying extent on Fas, Bcl-2, p53, and other regulators. The dendritic cells are the main cell type, the contact with determines apoptosis of T-lymphocytes. A possible role of the epithelial cells was shown in few models (on murine cells) and was not practically studied. We obtained a line of epithelial cells of the human thymus cells HTSC, cocultivation with which induces apoptosis of immature thymocytes and blood T-cells activated by mitogens. Development of apoptosis is suppressed by inhibitors of protein and RNA synthesis, chelators Ca2+, ions Zn2+, and factors destroying the cytoskeleton components. In this model, interaction of pairs of molecules CD4-HLA class II and LFA-1-ICAM-1. When in contact with the HTSC cells, the thymocytes of mice mutant for Fas-receptor (line MRL.lpr) are subject to apoptosis, but when this receptor is present, it affects the development of apoptosis.

  11. Expression of syntaxin 1C, an alternative splice variant of HPC-1/syntaxin 1A, is enhanced by phorbol-ester stimulation in astroglioma: participation of the PKC signaling pathway.

    PubMed

    Nakayama, Takahiro; Mikoshiba, Katsuhiko; Yamamori, Tetsuo; Akagawa, Kimio

    2003-02-11

    Syntaxin 1C is an alternative splice variant of HPC-1/syntaxin 1A; the latter participates in neurotransmitter release and is assigned to the gene domain responsible for Williams' syndrome (WS). It is expressed in the soluble fraction extracted from human astroglioma cell lines T98G and U87MG. Quantitative immunoblot and indirect immunofluorescence analyses revealed that the expression of syntaxin 1C was upregulated by phorbol 12-myristate 13-acetate (PMA), but not by forskolin. A protein kinase C (PKC) inhibitor suppressed this enhancement. These results suggest that syntaxin 1C expression is regulated via the PKC signal pathway. This is the first report of a signal transduction system that directly affects the expression of syntaxin protein.

  12. Molecular Mechanisms and Apoptosis in Pdt

    NASA Astrophysics Data System (ADS)

    Krammer, Barbara; Verwanger, Thomas

    2010-04-01

    Photodynamic Therapy (PDT) is a successful new therapy for malignant and non-malignant diseases. It is based on the activation of a photosensitizing dye by visible light in the target tissue, followed by production of cytotoxic substances. The article gives a short overview on the field of PDT with main focus on molecular mechanisms and apoptosis. It includes photodynamic principles, clinical application and procedures, biological effects, molecular mechanisms of damage processing and apoptosis.

  13. Apoptosis in amphibian organs during metamorphosis

    PubMed Central

    Ishizuya-Oka, Atsuko; Hasebe, Takashi; Shi, Yun-Bo

    2012-01-01

    During amphibian metamorphosis, the larval tissues/organs rapidly degenerate to adapt from the aquatic to the terrestrial life. At the cellular level, a large quantity of apoptosis occurs in a spatiotemporally-regulated fashion in different organs to ensure timely removal of larval organs/tissues and the development of adult ones for the survival of the individuals. Thus, amphibian metamorphosis provides us a good opportunity to understand the mechanisms regulating apoptosis. To investigate this process at the molecular level, a number of thyroid hormone (TH) response genes have been isolated from several organs of Xenopus laevis tadpoles and their expression and functional analyses are now in progress using modern molecular and genetic technologies. In this review, we will first summarize when and where apoptosis occurs in typical larva-specific and larval-to-adult remodeling amphibian organs to highlight that the timing of apoptosis is different in different tissues/organs, even though all are induced by the same circulating TH. Next, to discuss how TH spatiotemporally regulates the apoptosis, we will focus on apoptosis of the X. laevis small intestine, one of the best characterized remodeling organs. Functional studies of TH response genes using transgenic frogs and culture techniques have shown that apoptosis of larval epithelial cells can be induced by TH either cell-autonomously or indirectly through interactions with extracellular matrix (ECM) components of the underlying basal lamina. Here, we propose that multiple intra- and extracellular apoptotic pathways are coordinately controlled by TH to ensure massive but well-organized apoptosis, which is essential for the proper progression of amphibian metamorphosis. PMID:20238476

  14. Modulation of apoptosis by cancer chemopreventive agents.

    PubMed

    D'Agostini, Francesco; Izzotti, Alberto; Balansky, Roumen M; Bennicelli, Carlo; De Flora, Silvio

    2005-12-11

    A review of almost 2000 studies showed that the large majority of 39 putative cancer chemopreventive agents induced "spontaneous" apoptosis. Inhibition of the programmed cell death triggered by a variety of stimuli was consistently reported only with ascorbic acid, alpha-tocopherol, and N-acetylcysteine (NAC). We performed experimental studies in rodents exposed to cigarette smoke, either mainstream (MCS) or environmental (ECS), and UV-A/B-containing light. The nonsteroidal anti-inflammatory drug sulindac did not affect the apoptotic process in the skin of light-exposed mice and in the lungs of ECS-exposed mice. Likewise, 5,6-benzoflavone, indole-3-carbinol, 1,2-dithiole-3-thione and oltipraz failed to modulate apoptosis in the respiratory tract of ECS-exposed rats. Phenethyl isothiocyanate further enhanced the frequency of apoptosis in pulmonary alveolar macrophages and bronchial epithelial cells, and upregulated several genes in the lung of ECS-exposed rats. Both individually and in combination with oltipraz, NAC inhibited apoptosis in the respiratory tract of rats exposed either to MCS or ECS. Moreover, NAC attenuated the ECS-related overexpression of proapoptotic genes and normalized the levels of proapoptotic proteins in rat lung. The transplacental administration of NAC to mice considerably attenuated gene overexpression in the liver of fetuses exposed to ECS throughout pregnancy. Inhibition of apoptosis by chemopreventive agents reflects their ability to counteract certain upstream signals, such as genotoxic damage, redox imbalances, and other forms of cellular stress that trigger apoptosis. On the other hand, enhancement of apoptosis is a double-edged sword, since it represents a protective mechanism in carcinogenesis but may contribute to the pathogenesis of other degenerative diseases. We suggest that stimulation of apoptosis by so many chemopreventive agents, as reported in the literature, may often reflect the occurrence of toxic effects at high doses.

  15. Mitochondrial Ceramide and the Induction of Apoptosis

    PubMed Central

    Siskind, Leah J.

    2007-01-01

    In most cell types, a key event in apoptosis is the release of proapoptotic intermembrane space proteins from mitochondria to the cytoplasm. In general, it is the release of these intermembrane space proteins that is responsible for the activation of caspases and DNases that are responsible for the execution of apoptosis. The mechanism for the increased permeability of the mitochondrial outer membrane during the induction phase of apoptosis is currently unknown and highly debated. This review will focus on one such proposed mechanism, namely, the formation of ceramide channels in the mitochondrial outer membrane. Ceramides are known to play a major regulatory role in apoptosis by inducing the release of proapoptotic proteins from the mitochondria. As mitochondria are known to contain the enzymes responsible for the synthesis and hydrolysis of ceramide, there exists a mechanism for regulating the level of ceramide in mitochondria. In addition, mitochondrial ceramide levels have been shown to be elevated prior to the induction phase of apoptosis. Ceramide has been shown to form large protein permeable channels in planar phospholipid and mitochondrial outer membranes. Thus, ceramide channels are good candidates for the pathway with which proapoptotic proteins are released from mitochondria during the induction phase of apoptosis. PMID:16167171

  16. Yeast as a model to study apoptosis?

    PubMed

    Fleury, Christophe; Pampin, Mathieu; Tarze, Agathe; Mignotte, Bernard

    2002-02-01

    Programmed cell death (PCD) serves as a major mechanism for the precise regulation of cell numbers, and as a defense mechanism to remove unwanted and potentially dangerous cells. Despite the striking heterogeneity of cell death induction pathways, the execution of the death program is often associated with characteristic morphological and biochemical changes termed apoptosis. Although for a long time the absence of mitochondrial changes was considered as a hallmark of apoptosis, mitochondria appear today as the central executioner of programmed cell death. This crucial position of mitochondria in programmed cell death control is not due to a simple loss of function (deficit in energy supplying), but rather to an active process in the regulation of effector mechanisms. The large diversity of regulators of apoptosis in mammals and their numerous interactions complicate the analysis of their individual functions. Yeast, eukaryotic but unicellular organism, lack the main regulators of apoptosis (caspases, Bcl-2 family members, ...) found in mammals. This absence render them a powerful tool for heterologous expression, functional studies, and even cloning of new regulators of apoptosis. Great advances have thus been made in our understanding of the molecular mechanisms of Bcl-2 family members interactions with themselves and other cellular proteins, specially thanks to the two hybrid system and the easy manipulation of yeast (molecular biology and genetics). This review will focus on the use of yeast as a tool to identify new regulators and study function of mammalian apoptosis regulators.

  17. DPI induces mitochondrial superoxide-mediated apoptosis.

    PubMed

    Li, Nianyu; Ragheb, Kathy; Lawler, Gretchen; Sturgis, Jennie; Rajwa, Bartek; Melendez, J Andres; Robinson, J Paul

    2003-02-15

    The iodonium compounds diphenyleneiodonium (DPI) and diphenyliodonium (IDP) are well-known phagocyte NAD(P)H oxidase inhibitors. However, it has been shown that at high concentrations they can inhibit the mitochondrial respiratory chain as well. Since inhibition of the mitochondrial respiratory chain has been shown to induce superoxide production and apoptosis, we investigated the effect of iodonium compounds on mitochondria-derived superoxide and apoptosis. Mitochondrial superoxide production was measured on both cultured cells and isolated rat-heart submitochondrial particles. Mitochondria function was examined by monitoring mitochondrial membrane potential. Apoptotic pathways were studied by measuring cytochrome c release and caspase 3 activation. Apoptosis was characterized by detecting DNA fragmentation on agarose gel and measuring propidium iodide- (PI-) stained subdiploid cells using flow cytometry. Our results showed that DPI could induce mitochondrial superoxide production. The same concentration of DPI induced apoptosis by decreasing mitochondrial membrane potential and releasing cytochrome c. Addition of antioxidants or overexpression of MnSOD significantly reduced DPI-induced mitochondrial damage, cytochrome c release, caspase activation, and apoptosis. These observations suggest that DPI can induce apoptosis via induction of mitochondrial superoxide. DPI-induced mitochondrial superoxide production may prove to be a useful model to study the signaling pathways of mitochondrial superoxide.

  18. Estrogens sensitize anterior pituitary gland to apoptosis.

    PubMed

    Pisera, D; Candolfi, M; Navarra, S; Ferraris, J; Zaldivar, V; Jaita, G; Castro, M G; Seilicovich, A

    2004-10-01

    Tissue homeostasis results from a balance between cell proliferation and cell death by apoptosis. Estradiol affects proliferation as well as apoptosis in hormone-dependent tissues. In the present study, we investigated the apoptotic response of the anterior pituitary gland to lipopolysaccharide (LPS) in cycling female rats, and the influence of estradiol in this response in ovariectomized (OVX) rats. The OVX rats were chronically estrogenized with implanted Silastic capsules containing 1 mg of 17beta-estradiol (E2). Cycling or OVX and E2-treated rats were injected with LPS (250 microg/rat ip). Apoptosis was determined by the terminal deoxynucleotidyl-mediated dUTP nick-end labeling (TUNEL) method in sections of the anterior pituitary gland and spleen. Chronic estrogenization induced apoptosis in the anterior pituitary gland. Acute endotoxemia triggered apoptosis of cells in the anterior pituitary gland of E2-treated rats but not of OVX rats. No differences were observed in the apoptotic response to LPS in spleen between OVX and E2-treated rats. The apoptotic response of the anterior pituitary to LPS was variable along the estrous cycle, being higher at proestrus than at estrus or diestrus I. Approximately 75% of the apoptotic cells were identified as lactotropes by immunofluorescence. In conclusion, our results indicate that estradiol induces apoptosis and enables the proapoptotic action of LPS in the anterior pituitary gland. Also, our study suggests that estrogens may be involved in anterior pituitary cell renewal during the estrous cycle, sensitizing lactotropes to proapoptotic stimuli.

  19. Effect of the pesticide, deltamethrin, on Ca2+ signaling and apoptosis in OC2 human oral cancer cells.

    PubMed

    Chi, Chao-Chuan; Chou, Chiang-Ting; Liang, Wei-Zhe; Jan, Chung-Ren

    2014-01-01

    Deltamethrin is a synthetic pyrethroid insecticide used extensively in pest control. Although deltamethrin has been shown to induce cytosolic free Ca(2+) concentration ([Ca(2+)]i) rises and apoptosis in different cancer cells, there is no information concerning the effects of deltamethrin on oral cancer. This study explored the effects of deltamethrin on [Ca(2+)]i and viability in OC2 human oral cancer cells. Deltamethrin, at concentrations of 5-10 μM, increased [Ca(2+)]i in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). Deltamethrin-induced [Ca(2+)]i rise was not inhibited by econazole, SK&F96365, phorbol 12-myristate 13 acetate (PMA) or GF109203X, but was inhibited by nifedipine. In Ca(2+)-free medium, 10-μM deltamethrin pretreatment inhibited the [Ca(2+)]i rise induced by the endoplasmic reticulum Ca(2+) pump inhibitor, 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with BHQ inhibited deltamethrin-induced [Ca(2+)]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with phospholipase C (PLC) inhibitor U73122 did not suppress deltamethrin-induced Ca(2+) release. At concentrations between 20 and 100 μM, deltamethrin killed cells in a concentration-dependent manner. The cytotoxic effect of deltamethrin was not reversed by prechelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl. Deltamethrin-induced cell death was not caused by a preceding [Ca(2+)]i rise. Annexin V/propidium iodide staining data suggest that deltamethrin (40-60 μM) induced apoptosis in a concentration-dependent manner. To conclude, in OC2 cells, deltamethrin evoked a [Ca(2+)]i rise by inducing PLC-independent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry by nifedipine-sensitive Ca(2+) channels. Further, deltamethrin induced Ca(2+)-independent cell death might involve apoptosis.

  20. CD40 expressed on thymic epithelial cells provides costimulation for proliferation but not for apoptosis of human thymocytes.

    PubMed

    Ruggiero, G; Martinez Cáceres, E; Voordouw, A; Noteboom, E; Graf, D; Kroczek, R A; Spits, H

    1996-05-15

    Human thymic epithelial cells express CD40, so we examined the possible role of CD40 in activation of thymocytes. We observed that both CD4+CD8- and CD4-CD8+ thymocytes proliferate after stimulation by anti-CD3 mAb in the presence of cultured thymic epithelial cells. Costimulation of CD4+ thymocytes by thymic epithelial cells is partly inhibited by an anti-CD40 mAb, but this mAb has no effect on costimulation of CD8+ thymocytes. The selective costimulatory ability of CD40 for CD4+ thymocytes was confirmed in experiments in which thymocytes were stimulated with anti-CD3 in the presence of murine P815 cells transfected with CD40 cDNA. The level of costimulation induced by P815-CD40 was comparable with that induced by P815 cells expressing CD80 (B7.1). Treatment of thymocytes with the Ca2+ ionophore ionomycin and the phorbol ester PMA or with anti-CD3 mAb resulted in up-regulation of the CD40 ligand, suggesting that this molecule is involved in CD40-mediated costimulation of human thymocytes. Costimulation of thymocytes by CD80 strongly increased anti-CD3-induced death of fetal thymocytes. In contrast, costimulation by CD40 did not increase anti-CD3-mediated apoptosis of these thymocytes. To confirm that CD40 does not affect anti-CD3-induced cell death, we established a variant of the Jurkat T leukemic cell line that constitutively expresses CD40L and analyzed the sensitivity of this cell line for activation-induced apoptosis. In contrast to CD80, CD40 failed to increase anti-CD3-mediated apoptosis in CD40L+ Jurkat cells, whereas both CD40 and CD80 strongly increased IL-2 production induced by anti-CD3. These findings suggest that costimulation by CD40 is involved in clonal expansion of CD4+ thymocytes but not in activation-induced cell death.

  1. Apoptosis-Dependent and Apoptosis-Independent Functions of Bim in Prostate Cancer Cells

    DTIC Science & Technology

    2005-03-01

    Independent Functions of Bim in Prostate Cancer Cells PRINCIPAL INVESTIGATOR: Dr. Dean Tang...SUBTITLE 5a. CONTRACT NUMBER Apoptosis-Dependent and Apoptosis-Independent Functions of Bim in Prostate Cancer Cells 5b. GRANT NUMBER DAMD17-03-1...Unlimited 13. SUPPLEMENTARY NOTES Original contains colored plates: ALL DTIC reproductions will be in black and white. 14. ABSTRACT

  2. Drosophila p53 isoforms differentially regulate apoptosis and apoptosis-induced proliferation.

    PubMed

    Dichtel-Danjoy, M-L; Ma, D; Dourlen, P; Chatelain, G; Napoletano, F; Robin, M; Corbet, M; Levet, C; Hafsi, H; Hainaut, P; Ryoo, H D; Bourdon, J-C; Mollereau, B

    2013-01-01

    Irradiated or injured cells enter apoptosis, and in turn, promote proliferation of surrounding unaffected cells. In Drosophila, apoptotic cells have an active role in proliferation, where the caspase Dronc and p53 induce mitogen expression and growth in the surrounding tissues. The Drosophila p53 gene structure is conserved and encodes at least two protein isoforms: a full-length isoform (Dp53) and an N-terminally truncated isoform (DΔNp53). Historically, DΔNp53 was the first p53 isoform identified and was thought to be responsible for all p53 biological activities. It was shown that DΔNp53 induces apoptosis by inducing the expression of IAP antagonists, such as Reaper. Here we investigated the roles of Dp53 and DΔNp53 in apoptosis and apoptosis-induced proliferation. We found that both isoforms were capable of activating apoptosis, but that they each induced distinct IAP antagonists. Expression of DΔNp53 induced Wingless (Wg) expression and enhanced proliferation in both 'undead cells' and in 'genuine' apoptotic cells. In contrast to DΔNp53, Dp53 did not induce Wg expression in the absence of the endogenous p53 gene. Thus, we propose that DΔNp53 is the main isoform that regulates apoptosis-induced proliferation. Understanding the roles of Drosophila p53 isoforms in apoptosis and in apoptosis-induced proliferation may shed new light on the roles of p53 isoforms in humans, with important implications in cancer biology.

  3. Apoptosis predominates in nonmyocytes in heart failure.

    PubMed

    Park, Misun; Shen, You-Tang; Gaussin, Vinciane; Heyndrickx, Guy R; Bartunek, Jozef; Resuello, Ranillo R G; Natividad, Filipinas F; Kitsis, Richard N; Vatner, Dorothy E; Vatner, Stephen F

    2009-08-01

    The goal of this investigation was to determine the distribution of myocardial apoptosis in myocytes and nonmyocytes in primates and patients with heart failure (HF). Almost all clinical cardiologists and cardiovascular investigators believe that myocyte apoptosis is considered to be a cardinal sign of HF and a major factor in its pathogenesis. However, with the knowledge that 75% of the number of cells in the heart are nonmyocytes, it is important to determine whether the apoptosis in HF is occurring in myocytes or in nonmyocytes. We studied both a nonhuman primate model of chronic HF, induced by rapid pacing 2-6 mo after myocardial infarction (MI), and biopsies from patients with ischemic cardiomyopathy. Dual labeling with a cardiac muscle marker was used to discriminate apoptosis in myocytes versus nonmyocytes. Left ventricular ejection fraction decreased following MI (from 78% to 60%) and further with HF (35%, P < 0.05). As expected, total apoptosis was increased in the myocardium following recovery from MI (0.62 cells/mm(2)) and increased further with the development of HF (1.91 cells/mm(2)). Surprisingly, the majority of apoptotic cells in MI and MI + HF, and in both the adjacent and remote areas, were nonmyocytes. This was also observed in myocardial biopsies from patients with ischemic cardiomyopathy. We found that macrophages contributed the largest fraction of apoptotic nonmyocytes (41% vs. 18% neutrophils, 16% fibroblast, and 25% endothelial and other cells). Although HF in the failing human and monkey heart is characterized by significant apoptosis, in contrast to current concepts, the apoptosis in nonmyocytes was eight- to ninefold greater than in myocytes.

  4. Apoptosis predominates in nonmyocytes in heart failure

    PubMed Central

    Park, Misun; Shen, You-Tang; Gaussin, Vinciane; Heyndrickx, Guy R.; Bartunek, Jozef; Resuello, Ranillo R. G.; Natividad, Filipinas F.; Kitsis, Richard N.; Vatner, Dorothy E.; Vatner, Stephen F.

    2009-01-01

    The goal of this investigation was to determine the distribution of myocardial apoptosis in myocytes and nonmyocytes in primates and patients with heart failure (HF). Almost all clinical cardiologists and cardiovascular investigators believe that myocyte apoptosis is considered to be a cardinal sign of HF and a major factor in its pathogenesis. However, with the knowledge that 75% of the number of cells in the heart are nonmyocytes, it is important to determine whether the apoptosis in HF is occurring in myocytes or in nonmyocytes. We studied both a nonhuman primate model of chronic HF, induced by rapid pacing 2–6 mo after myocardial infarction (MI), and biopsies from patients with ischemic cardiomyopathy. Dual labeling with a cardiac muscle marker was used to discriminate apoptosis in myocytes versus nonmyocytes. Left ventricular ejection fraction decreased following MI (from 78% to 60%) and further with HF (35%, P < 0.05). As expected, total apoptosis was increased in the myocardium following recovery from MI (0.62 cells/mm2) and increased further with the development of HF (1.91 cells/mm2). Surprisingly, the majority of apoptotic cells in MI and MI + HF, and in both the adjacent and remote areas, were nonmyocytes. This was also observed in myocardial biopsies from patients with ischemic cardiomyopathy. We found that macrophages contributed the largest fraction of apoptotic nonmyocytes (41% vs. 18% neutrophils, 16% fibroblast, and 25% endothelial and other cells). Although HF in the failing human and monkey heart is characterized by significant apoptosis, in contrast to current concepts, the apoptosis in nonmyocytes was eight- to ninefold greater than in myocytes. PMID:19465551

  5. Chondrocyte Apoptosis in the Pathogenesis of Osteoarthritis

    PubMed Central

    Hwang, Hyun Sook; Kim, Hyun Ah

    2015-01-01

    Apoptosis is a highly-regulated, active process of cell death involved in development, homeostasis and aging. Dysregulation of apoptosis leads to pathological states, such as cancer, developmental anomalies and degenerative diseases. Osteoarthritis (OA), the most common chronic joint disease in the elderly population, is characterized by progressive destruction of articular cartilage, resulting in significant disability. Because articular cartilage depends solely on its resident cells, the chondrocytes, for the maintenance of extracellular matrix, the compromising of chondrocyte function and survival would lead to the failure of the articular cartilage. The role of subchondral bone in the maintenance of proper cartilage matrix has been suggested as well, and it has been proposed that both articular cartilage and subchondral bone interact with each other in the maintenance of articular integrity and physiology. Some investigators include both articular cartilage and subchondral bone as targets for repairing joint degeneration. In late-stage OA, the cartilage becomes hypocellular, often accompanied by lacunar emptying, which has been considered as evidence that chondrocyte death is a central feature in OA progression. Apoptosis clearly occurs in osteoarthritic cartilage; however, the relative contribution of chondrocyte apoptosis in the pathogenesis of OA is difficult to evaluate, and contradictory reports exist on the rate of apoptotic chondrocytes in osteoarthritic cartilage. It is not clear whether chondrocyte apoptosis is the inducer of cartilage degeneration or a byproduct of cartilage destruction. Chondrocyte death and matrix loss may form a vicious cycle, with the progression of one aggravating the other, and the literature reveals that there is a definite correlation between the degree of cartilage damage and chondrocyte apoptosis. Because current treatments for OA act only on symptoms and do not prevent or cure OA, chondrocyte apoptosis would be a valid

  6. CASPASE CONTROL: PROTAGONISTS OF CANCER CELL APOPTOSIS

    PubMed Central

    Fiandalo, M.V.; Kyprianou, N.

    2013-01-01

    Emergence of castration-resistant metastatic prostate cancer is due to activation of survival pathways, including apoptosis suppression and anoikis resistance, and increased neovascularization. Thus targeting of apoptotic players is of critical significance in prostate cancer therapy since loss of apoptosis and resistance to anoikis are critical in aberrant malignant growth, metastasis and conferring therapeutic failure. The majority of therapeutic agents act through intrinsic mitochondrial, extrinsic death receptor pathways or endoplasmic reticulum stress pathways to induce apoptosis. Current therapeutic strategies target restoring regulatory molecules that govern the pro-survival pathways such as PTEN which regulates AKT activity. Other strategies focus on reactivating the apoptotic pathways either by down-regulating anti-apoptotic players such as BCL-2 or by up-regulating pro-apoptotic protein families, most notably, the caspases. Caspases are a family of cystine proteases which serve critical roles in apoptotic and inflammatory signaling pathways. During tumorigenesis, significant loss or inactivation of lead members in the caspase family leads to impairing apoptosis induction, causing a dramatic imbalance in the growth dynamics, ultimately resulting in aberrant growth of human cancers. Recent exploitation of apoptosis pathways towards re-instating apoptosis induction via caspase re-activation has provided new molecular platforms for the development of therapeutic strategies effective against advanced prostate cancer as well as other solid tumors. This review will discuss the current cellular landscape featuring the caspase family in tumor cells and their activation via pharmacologic intervention towards optimized anti-cancer therapeutic modalities. This article is part of a Special Issue entitled “Apoptosis: Four Decades Later”. PMID:23070001

  7. Sodium nitroprusside induces apoptosis of rabbit chondrocytes

    NASA Astrophysics Data System (ADS)

    Liang, Qian; Wang, Xiao-Ping; Chen, Tong-Sheng

    2013-02-01

    Osteoarthritis (OA) is characterized by a slowly progressing degradation of the matrix and destruction of articular cartilage. Apoptosis of chondrocyte is accounted for the mechanism of OA. Nitric oxide (NO), as a stimulus, has been shown to induce chondrocyte apoptosis by activating the matrix metalloproteinases (MMPs), increasing the expression of cyclooxygenase 2 (COX-2) and the level of prostaglandin E2 (PGE2), inhibiting the proteoglycan synthesis and type II collagen expression. In this study, sodium nitroprusside (SNP) was administered to be the NO donor to explore the mechanism of NO-induced apoptosis of rabbit chondrocytes obtained from six weeks old New Zealand rabbits. CCK-8 assay revealed the inhibitory effect of SNP on cell viability. We used flow cytometry (FCM) to assess the form of cell death by Annexin-V/propidium iodide (PI) double staining, and evaluate the change of mitochondrial membrane potential (ΔΨm). We found that the SNP induced chondrocyte apoptosis in a dose- and time-dependent manner and an observable reduction of ΔΨm. In conclusion, our findings indicate that SNP induces apoptosis of rabbit chondrocytes via a mitochondria-mediated pathway.

  8. Apoptosis of beta cells in diabetes mellitus.

    PubMed

    Anuradha, Rachakatla; Saraswati, Mudigonda; Kumar, Kishore G; Rani, Surekha H

    2014-11-01

    Diabetes mellitus is a multifactorial metabolic disorder characterized by hyperglycemia. Apoptosis in beta cells has been observed in response to diverse stimuli, such as glucose, cytokines, free fatty acids, leptin, and sulfonylureas, leading to the activation of polyol, hexosamine, and diacylglycerol/protein kinase-C (DAG/PKC) pathways that mediate oxidative and nitrosative stress causing the release of different cytokines. Cytokines induce the expression of Fas and tumor necrosis factor-alpha (TNF-α) by activating the transcription factor, nuclear factor-κb, and signal transducer and activator of transcription 1 (STAT-1) in the β cells in the extrinsic pathway of apoptosis. Cytokines produced in beta cells also induce proapoptotic members of the intrinsic pathway of apoptosis. The genetic alterations in apoptosis signaling machinery and the pathogenesis of diabetes include Fas, FasL, Akt, caspases, calpain-10, and phosphatase and tensin homolog (Pten). The other gene products that are involved in diabetes are nitric oxide synthase-2 (NOS2), small ubiquitin-like modifier (SUMO), apolipoprotein CIII (ApoCIII), forkhead box protein O1 (FOXO1), and Kruppel-like zinc finger protein Gli-similar 3 (GLIS3). The gene products having antiapoptotic nature are Bcl-2 and Bcl-XL. Epigenetic mechanisms play an important role in type I and type II diabetes. Further studies on the apoptotic genes and gene products in diabetics may be helpful in pharmacogenomics and individualized treatment along with antioxidants targeting apoptosis in diabetes.

  9. Evaluation of Apoptosis in Immunotoxicity Testing

    PubMed Central

    Nagarkatti, Mitzi; Rieder, Sadiye Amcaoglu; Vakharia, Dilip; Nagarkatti, Prakash S.

    2014-01-01

    Immunotoxicity testing is important in determining the toxic effects of chemical substances, medicinal products, airborne pollutants, cosmetics, medical devices, and food additives. The immune system of the host is a direct target of these toxicants, and the adverse effects include serious health complications such as susceptibility to infections, cancer, allergic reactions, and autoimmune diseases. One way to investigate the harmful effects of different chemicals is to study apoptosis in immune cell populations. Apoptosis is defined as the programmed cell death, and in general, this process helps in development and maintains homeostasis. However, in the case of an insult by a toxicant, apoptosis of the immune cells can lead to immunosuppression resulting in the development of cancer and the inability to fight infections. Apoptosis is characterized by cell shrinkage, nuclear condensation, changes in cell membrane and mitochondria, DNA fragmentation into 200 base oligomers, and protein degradation by caspases. Various methods are employed in order to investigate apoptosis. These methods include direct measurement of apoptotic cells with flow cytometry and in situ labeling, as well as RNA, DNA, and protein assays that are indicative of apoptotic molecules. PMID:19967519

  10. Estrogen Regulation of Apoptosis in Osteoblasts

    PubMed Central

    Bradford, Peter G; Gerace, Ken V; Roland, Renée L; Chrzan, Brian G

    2010-01-01

    Dysregulated apoptosis is a critical failure associated with prominent degenerative diseases including osteoporosis. In bone, estrogen deficiency has been associated with accelerated osteoblast apoptosis and susceptibility to osteoporotic fractures. Hormone therapy continues to be an effective option for preventing osteoporosis and bone fractures. Induction of apoptosis in G-292 human osteoblastic cells by exposure to etoposide or the inflammatory cytokine TNFα promoted acute caspase-3/7 activity and this increased activity was inhibited by pretreatment with estradiol. Etoposide also increased the expression of a battery of apoptosis-promoting genes and this expression was also inhibited by estradiol. Among the apoptotic genes whose expression was inhibited by estradiol was ITPR1, which encodes the type 1 InsP3R. InsP3Rs are intracellular calcium channels and key proapoptotic mediators. Estradiol via estrogen receptor β1 suppresses ITPR1 gene transcription in G-292 cells. These analyses suggest that an underlying basis of the beneficial activity of estrogens in combating osteoporosis may involve the prevention of apoptosis in osteoblasts and that a key event in this process is the repression of apoptotic gene expression and inhibition of caspase-3/7. PMID:19426747

  11. Caspase activation inhibits proteasome function during apoptosis.

    PubMed

    Sun, Xiao-Ming; Butterworth, Michael; MacFarlane, Marion; Dubiel, Wolfgang; Ciechanover, Aaron; Cohen, Gerald M

    2004-04-09

    The ubiquitin/proteasome system regulates protein turnover by degrading polyubiquitinated proteins. To date, all studies on the relationship of apoptosis and the proteasome have emphasized the key role of the proteasome in the regulation of apoptosis, by virtue of its ability to degrade regulatory molecules involved in apoptosis. We now demonstrate how induction of apoptosis may regulate the activity of the proteasome. During apoptosis, caspase activation results in the cleavage of three specific subunits of the 19S regulatory complex of the proteasome: S6' (Rpt5) and S5a (Rpn10), whose role is to recognize polyubiquitinated substrates of the proteasome, and S1 (Rpn2), which with S5a and S2 (Rpn1) holds together the lid and base of the 19S regulatory complex. This caspase-mediated cleavage inhibits the proteasomal degradation of ubiquitin-dependent and -independent cellular substrates, including proapoptotic molecules such as Smac, so facilitating the execution of the apoptotic program by providing a feed-forward amplification loop.

  12. BASP1 Promotes Apoptosis in Diabetic Nephropathy

    PubMed Central

    Sanchez-Niño, Maria Dolores; Sanz, Ana Belen; Lorz, Corina; Gnirke, Andrea; Rastaldi, Maria Pia; Nair, Viji; Egido, Jesus; Ruiz-Ortega, Marta

    2010-01-01

    Apoptosis contributes to the development of diabetic nephropathy (DN), but the mechanisms that lead to diabetes-induced cell death are not fully understood. Here, we combined a functional genomics screen for cDNAs that induce apoptosis in vitro with transcriptional profiling of renal biopsies from patients with DN. Twelve of the 138 full-length cDNAs that induced cell death in human embryonic kidney cells matched upregulated mRNA transcripts in tissue from human DN. Confirmatory screens identified induction of BASP1 in tubular cross sections of human DN tissue. In vitro, apoptosis-inducing conditions such as serum deprivation, high concentrations of glucose, and proinflammatory cytokines increased BASP1 mRNA and protein in human tubular epithelial cells. In normal cells, BASP1 localized to the cytoplasm, but in apoptotic cells, it colocalized with actin in the periphery. Overexpression of BASP1 induced cell death with features of apoptosis; conversely, small interfering RNA (siRNA)-mediated knockdown of BASP1 protected tubular cells from apoptosis. Supporting possible involvement of BASP1 in renal disease other than DN, we also observed significant upregulation of renal BASP1 in spontaneously hypertensive rats and a trend toward increased tubulointerstitial BASP1 mRNA in human hypertensive nephropathy. In summary, a combined functional genomics approach identified BASP1 as a proapoptotic factor in DN and possibly also in hypertensive nephropathy. PMID:20110383

  13. [6]-Gingerol induces caspase-dependent apoptosis and prevents PMA-induced proliferation in colon cancer cells by inhibiting MAPK/AP-1 signaling.

    PubMed

    Radhakrishnan, E K; Bava, Smitha V; Narayanan, Sai Shyam; Nath, Lekshmi R; Thulasidasan, Arun Kumar T; Soniya, Eppurathu Vasudevan; Anto, Ruby John

    2014-01-01

    We report mechanism-based evidence for the anticancer and chemopreventive efficacy of [6]-gingerol, the major active principle of the medicinal plant, Ginger (Zingiber officinale), in colon cancer cells. The compound was evaluated in two human colon cancer cell lines for its cytotoxic effect and the most sensitive cell line, SW-480, was selected for the mechanistic evaluation of its anticancer and chemopreventive efficacy. The non-toxic nature of [6]-gingerol was confirmed by viability assays on rapidly dividing normal mouse colon cells. [6]-gingerol inhibited cell proliferation and induced apoptosis as evidenced by externalization of phosphatidyl serine in SW-480, while the normal colon cells were unaffected. Sensitivity to [6]-gingerol in SW-480 cells was associated with activation of caspases 8, 9, 3 &7 and cleavage of PARP, which attests induction of apoptotic cell death. Mechanistically, [6]-gingerol down-regulated Phorbol Myristate Acetate (PMA) induced phosphorylation of ERK1/2 and JNK MAP kinases and activation of AP-1 transcription factor, but had only little effects on phosphorylation of p38 MAP kinase and activation of NF-kappa B. Additionally, it complemented the inhibitors of either ERK1/2 or JNK MAP kinase in bringing down the PMA-induced cell proliferation in SW-480 cells. We report the inhibition of ERK1/2/JNK/AP-1 pathway as a possible mechanism behind the anticancer as well as chemopreventive efficacy of [6]-gingerol against colon cancer.

  14. PRR7 Is a Transmembrane Adaptor Protein Expressed in Activated T Cells Involved in Regulation of T Cell Receptor Signaling and Apoptosis*

    PubMed Central

    Hrdinka, Matouš; Dráber, Peter; Štěpánek, Ondřej; Ormsby, Tereza; Otáhal, Pavel; Angelisová, Pavla; Brdička, Tomáš; Pačes, Jan; Hořejší, Václav; Drbal, Karel

    2011-01-01

    Transmembrane adaptor proteins (TRAPs) are important organizers and regulators of immunoreceptor-mediated signaling. A bioinformatic search revealed several potential novel TRAPs, including a highly conserved protein, proline rich 7 (PRR7), previously described as a component of the PSD-95/N-methyl-d-aspartate receptor protein complex in postsynaptic densities (PSD) of rat neurons. Our data demonstrate that PRR7 is weakly expressed in other tissues but is readily up-regulated in activated human peripheral blood lymphocytes. Transient overexpression of PRR7 in Jurkat T cell line led to gradual apoptotic death dependent on the WW domain binding motif surrounding Tyr-166 in the intracellular part of PRR7. To circumvent the pro-apoptotic effect of PRR7, we generated Jurkat clones with inducible expression of PRR7 (J-iPRR7). In these cells acute induction of PRR7 expression had a dual effect. It resulted in up-regulation of the transcription factor c-Jun and the activation marker CD69 as well as enhanced production of IL-2 after phorbol 12-myristate 13-acetate (PMA) and ionomycin treatment. On the other hand, expression of PRR7 inhibited general tyrosine phosphorylation and calcium influx after T cell receptor cross-linking by antibodies. Moreover, we found PRR7 constitutively tyrosine-phosphorylated and associated with Src. Collectively, these data indicate that PRR7 is a potential regulator of signaling and apoptosis in activated T cells. PMID:21460222

  15. Apoptosis in the aged dog brain.

    PubMed

    Kiatipattanasakul, W; Nakamura, S; Hossain, M M; Nakayama, H; Uchino, T; Shumiya, S; Goto, N; Doi, K

    1996-09-01

    Apoptosis similar to that seen in Alzheimer's disease patients was found in the brain of aged dogs by the TUNEL method of detecting in situ DNA fragmentation. Apoptosis was observed in both neurons and glial cells, and was morphologically characterized by round and swollen cytoplasm and aggregated nuclear chromatin, although these changes were slight. Neurons and astrocytes in the gray matter and oligodendrocytes in the white matter were affected. The number of ApopTag-positive brain cells increased slightly with age, but was not correlated to the number of senile plaques. A good correlation between the number of ApopTag-positive cells and the dementia index was clearly found. The present study indicates that brain cell apoptosis could account for dementia in aged dogs and suggested that aged dogs may be useful as a simplified animal model for Alzheimer's disease in man.

  16. Apoptosis in Drosophila: which role for mitochondria?

    PubMed

    Clavier, Amandine; Rincheval-Arnold, Aurore; Colin, Jessie; Mignotte, Bernard; Guénal, Isabelle

    2016-03-01

    It is now well established that the mitochondrion is a central regulator of mammalian cell apoptosis. However, the importance of this organelle in non-mammalian apoptosis has long been regarded as minor, mainly because of the absence of a crucial role for cytochrome c in caspase activation. Recent results indicate that the control of caspase activation and cell death in Drosophila occurs at the mitochondrial level. Numerous proteins, including RHG proteins and proteins of the Bcl-2 family that are key regulators of Drosophila apoptosis, constitutively or transiently localize in mitochondria. These proteins participate in the cell death process at different levels such as degradation of Diap1, a Drosophila IAP, production of mitochondrial reactive oxygen species or stimulation of the mitochondrial fission machinery. Here, we review these mitochondrial events that might have their counterpart in human.

  17. Control of apoptosis by Drosophila DCAF12.

    PubMed

    Hwangbo, Dae-Sung; Biteau, Benoit; Rath, Sneha; Kim, Jihyun; Jasper, Heinrich

    2016-05-01

    Regulated Apoptosis (Programmed Cell Death, PCD) maintains tissue homeostasis in adults, and ensures proper growth and morphogenesis of tissues during development of metazoans. Accordingly, defects in cellular processes triggering or executing apoptotic programs have been implicated in a variety of degenerative and neoplastic diseases. Here, we report the identification of DCAF12, an evolutionary conserved member of the WD40-motif repeat family of proteins, as a new regulator of apoptosis in Drosophila. We find that DCAF12 is required for Diap1 cleavage in response to pro-apoptotic signals, and is thus necessary and sufficient for RHG (Reaper, Hid, and Grim)-mediated apoptosis. Loss of DCAF12 perturbs the elimination of supernumerary or proliferation-impaired cells during development, and enhances tumor growth induced by loss of neoplastic tumor suppressors, highlighting the wide requirement for DCAF12 in PCD.

  18. Cerulein Pancreatitis: Oxidative Stress, Inflammation, and Apoptosis

    PubMed Central

    2008-01-01

    Cerulein pancreatitis is similar to human edematous pancreatitis, manifesting with dysregulation of digestive enzyme production and cytoplasmic vacuolization, the death of acinar cells, edema formation, and infiltration of inflammatory cells into the pancreas. Reactive oxygen species are involved in nuclear factor-κB activation, cytokine expression, apoptosis and pathogenesis of pancreatitis. There is recent evidence that cerulein activates NADPH oxidase, which is a major source of reactive oxygen species during inflammation and apoptosis in pancreatic acinar cells. In addition, the Janus kinase/signal transducer and activator of transcription pathway has been suggested as being involved in inflammatory signaling in the pancreas. This review discusses the involvement of oxidative stress in inflammation and apoptosis in pancreatic acinar cells stimulated with cerulein as an in vitro model of pancreatitis. PMID:20485614

  19. Death penalty for keratinocytes: apoptosis versus cornification.

    PubMed

    Lippens, S; Denecker, G; Ovaere, P; Vandenabeele, P; Declercq, W

    2005-11-01

    Homeostasis implies a balance between cell growth and cell death. This balance is essential for the development and maintenance of multicellular organisms. Homeostasis is controlled by several mechanisms including apoptosis, a process by which cells condemned to death are completely eliminated. However, in some cases, total destruction and removal of dead cells is not desirable, as when they fulfil a specific function such as formation of the skin barrier provided by corneocytes, also known as terminally differentiated keratinocytes. In this case, programmed cell death results in accumulation of functional cell corpses. Previously, this process has been associated with apoptotic cell death. In this overview, we discuss differences and similarities in the molecular regulation of epidermal programmed cell death and apoptosis. We conclude that despite earlier confusion, apoptosis and cornification occur through distinct molecular pathways, and that possibly antiapoptotic mechanisms are implicated in the terminal differentiation of keratinocytes.

  20. Apoptosis and Necrosis in the Liver

    PubMed Central

    Guicciardi, Maria Eugenia; Malhi, Harmeet; Mott, Justin L.; Gores, Gregory J.

    2013-01-01

    Because of its unique function and anatomical location, the liver is exposed to a multitude of toxins and xenobiotics, including medications and alcohol, as well as to infection by hepatotropic viruses, and therefore, is highly susceptible to tissue injury. Cell death in the liver occurs mainly by apoptosis or necrosis, with apoptosis also being the physiologic route to eliminate damaged or infected cells and to maintain tissue homeostasis. Liver cells, especially hepatocytes and cholangiocytes, are particularly susceptible to death receptor-mediated apoptosis, given the ubiquitous expression of the death receptors in the organ. In a quite unique way, death receptor-induced apoptosis in these cells is mediated by both mitochondrial and lysosomal permeabilization. Signaling between the endoplasmic reticulum and the mitochondria promotes hepatocyte apoptosis in response to excessive free fatty acid generation during the metabolic syndrome. These cell death pathways are partially regulated by microRNAs. Necrosis in the liver is generally associated with acute injury (i.e., ischemia/reperfusion injury) and has been long considered an unregulated process. Recently, a new form of “programmed” necrosis (named necroptosis) has been described: the role of necroptosis in the liver has yet to be explored. However, the minimal expression of a key player in this process in the liver suggests this form of cell death may be uncommon in liver diseases. Because apoptosis is a key feature of so many diseases of the liver, therapeutic modulation of liver cell death holds promise. An updated overview of these concepts is given in this article. PMID:23720337

  1. Calcium and apoptosis: facts and hypotheses.

    PubMed

    Rizzuto, Rosario; Pinton, Paolo; Ferrari, Davide; Chami, Mounia; Szabadkai, György; Magalhães, Paulo J; Di Virgilio, Francesco; Pozzan, Tullio

    2003-11-24

    Although longstanding experimental evidence has associated alterations of calcium homeostasis to cell death, only in the past few years the role of calcium in the signaling of apoptosis has been extensively investigated. In this review, we will summarize the current knowledge, focusing on (i) the effect of the proteins of the Bcl-2 family on ER Ca2+ levels, (ii) the action of the proteolytic enzymes of apoptosis on the Ca2+ signaling machinery, (iii) the ensuing alterations on the signaling patterns of extracellular stimuli, and (iv) the intracellular targets of 'apoptotic' Ca2+ signals, with special emphasis on the mitochondria and cytosolic Ca2+-dependent enzymes.

  2. Stress response and apoptosis in pro- and antiinflammatory macrophages.

    PubMed

    Malyshev, I Yu; Kruglov, S V; Bakhtina, L Yu; Malysheva, E V; Zubin, M; Norkin, M

    2004-08-01

    We showed that stress response and apoptosis in macrophages depend on the phenotype of their secretory activity and specific biological and physical characteristics of the factor inducing stress-response or apoptosis.

  3. Soy Metabolites, Isoflavones in Cell Growth and Apoptosis

    DTIC Science & Technology

    2000-07-01

    causes cell cycle arrest and induces apoptosis . To fully test our original hypothesis, we proposed three specific aims containing five tasks of which...435 breast cancer cells, regulates the expression of cell cycle and apoptosis -related genes, and induces apoptosis through a p53-independent pathway...These molecular alterations may be the molecular mechanism(s) by which genistein induces cell growth inhibition and apoptosis in breast cancer cells

  4. A novel method for detection of apoptosis

    SciTech Connect

    Zagariya, Alexander M.

    2012-04-15

    There are two different Angiotensin II (ANG II) peptides in nature: Human type (ANG II) and Bovine type (ANG II*). These eight amino acid peptides differ only at position 5 where Valine is replaced by Isoleucine in the Bovine type. They are present in all species studied so far. These amino acids are different by only one atom of carbon. This difference is so small, that it will allow any of ANG II, Bovine or Human antibodies to interact with all species and create a universal method for apoptosis detection. ANG II concentrations are found at substantially higher levels in apoptotic, compared to non-apoptotic, tissues. ANG II accumulation can lead to DNA damage, mutations, carcinogenesis and cell death. We demonstrate that Bovine antiserum can be used for universal detection of apoptosis. In 2010, the worldwide market for apoptosis detection reached the $20 billion mark and significantly increases each year. Most commercially available methods are related to Annexin V and TUNNEL. Our new method based on ANG II is more widely known to physicians and scientists compared to previously used methods. Our approach offers a novel alternative for assessing apoptosis activity with enhanced sensitivity, at a lower cost and ease of use.

  5. Fluorescence spectroscopy to assess apoptosis in myocardium

    NASA Astrophysics Data System (ADS)

    Ranji, Mahsa; Matsubara, Muneaki; Grosso, Michael A.; Jaggard, Dwight L.; Chance, Britton; Gorman, Robert C.; Gorman, Joseph H., III

    2007-02-01

    Apoptosis induced mitochondrial destruction and dysfunction has been shown to play an important role in the pathogenesis of both acute cardiac ischemia-reperfusion injury and chronic myocardial infarction-induced ventricular remodeling. Unfortunately this understanding has not translated into effective therapeutic strategies for either condition-mostly due to an inability to assess mitochondrial dysfunction/apoptosis effectively in humans. All current measures of apoptosis are pseudo-quantitative and require invasive tissue biopsy. Our group has developed an optical, non-tissue destructive catheter based device that allows the quantitative regional assessment of this pathological process in vivo. This instrument has been designed to acquire fluorescence signals of intrinsic mitochondrial fluorophores, Nicotinamide Adenine Dinucleotide (NAD) and Flavoprotein (FP). The normalized ratio of these fluorophores (FP/FP+NADH) called the redox ratio, is an indicator of the in vivo mitochondrial dysfunction. 1-3 We have demonstrated in a rabbit reperfusion model of apoptotic myocyte injury that this redox ratio is drastically increased which is consistent with profound apoptosis-induced "unhinging" of the mitochondrial respiratory function.

  6. Signaling Pathways in Cardiac Myocyte Apoptosis

    PubMed Central

    Xia, Peng; Liu, Yuening

    2016-01-01

    Cardiovascular diseases, the number 1 cause of death worldwide, are frequently associated with apoptotic death of cardiac myocytes. Since cardiomyocyte apoptosis is a highly regulated process, pharmacological intervention of apoptosis pathways may represent a promising therapeutic strategy for a number of cardiovascular diseases and disorders including myocardial infarction, ischemia/reperfusion injury, chemotherapy cardiotoxicity, and end-stage heart failure. Despite rapid growth of our knowledge in apoptosis signaling pathways, a clinically applicable treatment targeting this cellular process is currently unavailable. To help identify potential innovative directions for future research, it is necessary to have a full understanding of the apoptotic pathways currently known to be functional in cardiac myocytes. Here, we summarize recent progress in the regulation of cardiomyocyte apoptosis by multiple signaling molecules and pathways, with a focus on the involvement of these pathways in the pathogenesis of heart disease. In addition, we provide an update regarding bench to bedside translation of this knowledge and discuss unanswered questions that need further investigation. PMID:28101515

  7. Apoptosis regulates notochord development in Xenopus

    PubMed Central

    Malikova, Marina; Van Stry, Melanie

    2009-01-01

    The notochord is the defining characteristic of the chordate embryo, and plays critical roles as a signaling center and as the primitive skeleton. In this study we show that early notochord development in Xenopus embryos is regulated by apoptosis. We find apoptotic cells in the notochord beginning at the neural groove stage and increasing in number as the embryo develops. These dying cells are distributed in an anterior to posterior pattern that is correlated with notochord extension through vacuolization. In axial mesoderm explants, inhibition of this apoptosis causes the length of the notochord to approximately double compared to controls. In embryos however, inhibition of apoptosis decreases the length of the notochord and it is severely kinked. This kinking also spreads from the anterior with developmental stage such that by the tadpole stage, the notochord lacks any recognizable structure, although notochord markers are expressed in a normal temporal pattern. Extension of the somites and neural plate mirror that of the notochord in these embryos, and the somites are severely disorganized. These data indicate that apoptosis is required for normal notochord development during the formation of the anterior-posterior axis, and its role in this process is discussed. PMID:17920580

  8. The Interferon Stimulated Gene 54 Promotes Apoptosis*

    PubMed Central

    Stawowczyk, Marcin; Van Scoy, Sarah; Kumar, K. Prasanna; Reich, Nancy C.

    2011-01-01

    The ability of interferons (IFNs) to inhibit viral replication and cellular proliferation is well established, but the specific contribution of each IFN-stimulated gene (ISG) to these biological responses remains to be completely understood. In this report we demonstrate that ISG54, also known as IFN-induced protein with tetratricopeptide repeats 2 (IFIT2), is a mediator of apoptosis. Expression of ISG54, independent of IFN stimulation, elicits apoptotic cell death. Cell death and apoptosis were quantified by propidium iodide uptake and annexin-V staining, respectively. The activation of caspase-3, a key mediator of the execution phase of apoptosis, was clearly apparent in cells expressing ISG54. The anti-apoptotic B cell lymphoma-xl (Bcl-xl) protein inhibited the apoptotic effects of ISG54 as did the anti-apoptotic adenoviral E1B-19K protein. In addition, ISG54 was not able to promote cell death in the absence of pro-apoptotic Bcl family members, Bax and Bak. Analyses of binding partners of ISG54 revealed association with two homologous proteins, ISG56/IFIT1 and ISG60/IFIT3. In addition, ISG60 binding negatively regulates the apoptotic effects of ISG54. The results reveal a previously unidentified role of ISG54 in the induction of apoptosis via a mitochondrial pathway and shed new light on the mechanism by which IFN elicits anti-viral and anti-cancer effects. PMID:21190939

  9. Biophotonic probing of macromolecular transformations during apoptosis

    PubMed Central

    Pliss, Artem; Kuzmin, Andrey N.; Kachynski, Aliaksandr V.; Prasad, Paras N.

    2010-01-01

    We introduce here multiplex nonlinear optical imaging as a powerful tool for studying the molecular organization and its transformation in cellular processes, with the specific example of apoptosis. Apoptosis is a process of self-initiated cell death, critically important for physiological regulation and elimination of genetic disorders. Nonlinear optical microscopy, combining the coherent anti-Stokes Raman scattering (CARS) microscopy and two-photon excited fluorescence (TPEF), has been used for analysis of spatial distribution of major types of biomolecules: proteins, lipids, and nucleic acids in the cells while monitoring their changes during apoptosis. CARS imaging revealed that in the nuclei of proliferating cells, the proteins are distributed nearly uniformly, with local accumulations in several nuclear structures. We have found that this distribution is abruptly disrupted at the onset of apoptosis and is transformed to a progressively irregular pattern. Fluorescence recovery after photobleaching (FRAP) studies indicate that pronounced aggregation of proteins in the nucleoplasm of apoptotic cells coincides with a gradual reduction in their mobility. PMID:20615987

  10. Apoptosis: getting rid of the bodies.

    PubMed

    Fadok, V A; Henson, P M

    1998-09-24

    Cells that die by apoptosis need to be removed before lysis to preserve tissue integrity and function. Recent studies have identified components of the uptake machinery used by phagocytes, but much remains to be learnt, particularly about the recognition mechanisms and their coupling to the uptake machinery.

  11. Delayed neutrophil apoptosis in chronic periodontitis patients.

    PubMed

    Gamonal, J; Sanz, M; O'Connor, A; Acevedo, A; Suarez, I; Sanz, A; Martínez, B; Silva, A

    2003-07-01

    Neutrophil cells constitute the first defense barrier against the oral bacterial challenge in the periodontium. Reduction of neutrophils could impair this response against periopathogenic bacteria such as Porphyromonas gingivalis. Our previous work implicates the apoptosis of neutrophils in the pathogenesis of periodontitis. We now demonstrate that granulocyte monocyte-colony stimulating factor (GM-CSF) present in the gingival crevicular fluid (GCF) and secreted during the immune response reduces the apoptosis of neutrophils. In this study, the presence of GM-CSF and tumor necrosis factor-alpha (TNF-alpha) in GCF was determined in samples obtained from adult patients with periodontitis and from control subjects with clinically healthy gingiva. GCF was collected for 30 s using Periopaper(R) strips, and cytokines were quantified by ELISA. We used ex vivo culture of gingival tissue biopsies for 2 and 4 days in the presence of GM-CSF. Apoptosis was determined using the terminal TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique, and expression of Bax by immunohistochemistry. The presence of GM-CSF and TNF-alpha was detected in the majority of sites from periodontal patients (83.3% and 63.3%, respectively), presenting a total amount of 27.65 and 42.38 pg, respectively. GM-CSF reduces the neutrophil apoptosis determined by double staining with TUNEL and myeloperoxidase and by a reduction of Bax expression. These findings suggest a novel mechanism by which neutrophils specifically accumulate in adult patients with periodontitis.

  12. Excitotoxins in neuronal apoptosis and necrosis.

    PubMed

    Nicotera, P; Lipton, S A

    1999-06-01

    Neuronal loss is common to many neurodegenerative diseases. Although necrosis is a common histopathologic feature observed in neuropathologic conditions, evidence is increasing that apoptosis can significantly contribute to neuronal demise. The prevalence of either type of cell death, apoptosis or necrosis, and the relevance for the progression of disease is still unclear. The debate on the occurrence and prevalence of one or the other type of death in pathologic conditions such as stroke or neurotoxic injury may in part be resolved by the proposal that different types of cell death within a tissue reflect either partial or complete execution of a common death program. Apoptosis is an active process of cell destruction, characterized morphologically by cell shrinkage, chromatin aggregation with extensive genomic fragmentation, and nuclear pyknosis. In contrast, necrosis is characterized by cell swelling, linked to rapid energy loss, and generalized disruption of ionic and internal homeostasis. This swiftly leads to membrane lysis, release of intracellular constituents that evoke a local inflammatory reaction, edema, and injury to the surrounding tissue. During the past few years, our laboratories have studied the signals and mechanisms responsible for induction or prevention of apoptosis/necrosis in neuronal injury and this is the subject of this review.

  13. Oligodendroglial degeneration in distemper: apoptosis or necrosis?

    PubMed

    Schobesberger, M; Zurbriggen, A; Summerfield, A; Vandevelde, M; Griot, C

    1999-03-01

    Canine distemper virus (CDV) causes a multifocal demyelinating disease in dogs. It was previously shown that the initial demyelinating lesions are directly virus induced since a correlation between the occurrence of demyelination and CDV replication in white matter cells was observed. During the course of infection oligodendrocytes undergo distinct morphological alterations, partly due to a restricted CDV infection of these cells, and eventually disappear from the lesions. This phenomenon has been described in vivo as well as in vitro. However, the reason for the morphological alterations and the following oligodendroglial depletion remained unclear. Since virus infection can induce cell death, it was investigated whether apoptosis or necrosis plays a role in the pathogenesis of demyelination in canine distemper. In brain tissue sections from dogs with acute distemper apoptotic cells were not detected within the demyelinating lesions using morphological and biochemical cell death criteria. In chronic distemper, apoptotic cells - presumably inflammatory cells - were seen within the perivascular cuffs. These in vivo findings were correlated to the in vitro situation using CDV-infected primary dog brain cell cultures as well as Vero cells. Infection with culture-adapted CDV lead to massive necrosis but not to apoptosis. After infection with virulent CDV neither apoptosis nor necrosis was a predominant feature in either culture system. These findings suggest that virus-induced demyelination in canine distemper is not the direct consequence of apoptosis or necrosis. It is speculated that another mechanism must be responsible for the observed morphological alterations of oligodendrocytes, ultimately leading to demyelination.

  14. Mechanisms of vanilloid-induced apoptosis.

    PubMed

    Hail, Numsen

    2003-06-01

    Chemical compounds that contain the vanillyl moiety (4-hydroxy-3-methoxybenzyl) are collectively classified as vanilloids. Vanilloid phytochemicals can be found in a variety of sources, some of which are routinely consumed by humans throughout the world. The dietary and/or medicinal use of vanilloids may be effective in inhibiting or reversing carcinogenesis, which has sparked a considerable interest in these compounds as potential chemopreventive or chemotherapeutic agents. Certain vanilloids are also valuable as pharmacological tools for investigating neurobiology, and have been proven effective in alleviating neurogenic pain and inflammation. Recently several vanilloids have demonstrated the ability to induce apoptosis in various cell types. Vanilloids can interact with proteins and membranes to initiate pleiotropic effects, some of which are potentially cytotoxic. Certain vanilloids bind to cation channels on nociceptive sensory neurons to regulate Ca(2+) uptake, which can promote neurotoxicity resulting in apoptosis and necrosis. Furthermore, some vanilloids appear to interfere with enzymatic processes in the plasma membrane and the mitochondria by functioning as coenzyme Q antagonist. This can promote reactive oxygen species production and/or the disruption of redox homeostasis resulting in apoptosis. This review will examine the cellular targets, cytotoxic effects, and the downstream effector mechanisms associated with vanilloid-induced apoptosis.

  15. The modulation of apoptosis by oncogenic viruses

    PubMed Central

    2013-01-01

    Transforming viruses can change a normal cell into a cancer cell during their normal life cycle. Persistent infections with these viruses have been recognized to cause some types of cancer. These viruses have been implicated in the modulation of various biological processes, such as proliferation, differentiation and apoptosis. The study of infections caused by oncogenic viruses had helped in our understanding of several mechanisms that regulate cell growth, as well as the molecular alterations leading to cancer. Therefore, transforming viruses provide models of study that have enabled the advances in cancer research. Viruses with transforming abilities, include different members of the Human Papillomavirus (HPV) family, Hepatitis C virus (HCV), Human T-cell Leukemia virus (HTLV-1), Epstein Barr virus (EBV) and Kaposi’s Sarcoma Herpesvirus (KSHV). Apoptosis, or programmed cell death, is a tightly regulated process that plays an important role in development and homeostasis. Additionally, it functions as an antiviral defense mechanism. The deregulation of apoptosis has been implicated in the etiology of diverse diseases, including cancer. Oncogenic viruses employ different mechanisms to inhibit the apoptotic process, allowing the propagation of infected and damaged cells. During this process, some viral proteins are able to evade the immune system, while others can directly interact with the caspases involved in apoptotic signaling. In some instances, viral proteins can also promote apoptosis, which may be necessary for an accurate regulation of the initial stages of infection. PMID:23741982

  16. THE ROLE OF APOPTOSIS IN NEUROTOXICOLOGY

    EPA Science Inventory

    Apoptosis, a form of programmed cell death, occurs in the nervous system throughout development, but with a preponderance of cell death occurring during the prenatal and perinatal periods. Aberrant periods of increased or decreased cell death, induced by toxicants in air, water,...

  17. Measuring apoptosis in mammals in vivo.

    PubMed

    Newbold, Andrea; Martin, Ben P; Cullinane, Carleen; Bots, Michael

    2014-11-03

    Apoptosis is a mode of cell death that is essential in multicellular organisms for the removal of superfluous, damaged, or potentially dangerous cells during development, infection, or normal tissue homeostasis. To prevent inflammation, cells undergoing apoptosis produce "find-me" signals that trigger the recruitment of phagocytes, which clear the apoptotic cells on recognition of "eat-me" signals. Despite the loss of billions of cells per day by apoptosis in the human body, the number of apoptotic cells found in healthy tissue is surprisingly low and reflects the efficiency of this process. However, in certain conditions (e.g., in cancer cells responding to chemotherapy), the number of apoptotic cells is too high to be efficiently cleared by phagocytes, and apoptotic cells can be observed. In these situations, the detection of apoptosis may be helpful in monitoring disease progression as well as in predicting the responses of tumors to anticancer therapies. Here we introduce various methods for monitoring apoptotic cells in vivo using a murine model of B-cell lymphoma and a solid tumor xenograft.

  18. Neuronal remodeling and apoptosis require VCP-dependent degradation of the apoptosis inhibitor DIAP1

    PubMed Central

    Rumpf, Sebastian; Lee, Sung Bae; Jan, Lily Yeh; Jan, Yuh Nung

    2011-01-01

    The regulated degeneration of axons or dendrites (pruning) and neuronal apoptosis are widely used during development to determine the specificity of neuronal connections. Pruning and apoptosis often share similar mechanisms; for example, developmental dendrite pruning of Drosophila class IV dendritic arborization (da) neurons is induced by local caspase activation triggered by ubiquitin-mediated degradation of the caspase inhibitor DIAP1. Here, we examined the function of Valosin-containing protein (VCP), a ubiquitin-selective AAA chaperone involved in endoplasmic reticulum-associated degradation, autophagy and neurodegenerative disease, in Drosophila da neurons. Strong VCP inhibition is cell lethal, but milder inhibition interferes with dendrite pruning and developmental apoptosis. These defects are associated with impaired caspase activation and high DIAP1 levels. In cultured cells, VCP binds to DIAP1 in a ubiquitin- and BIR domain-dependent manner and facilitates its degradation. Our results establish a new link between ubiquitin, dendrite pruning and the apoptosis machinery. PMID:21343367

  19. Drosophila p53 isoforms differentially regulate apoptosis and apoptosis-induced proliferation

    PubMed Central

    Dichtel-Danjoy, M-L; Ma, D; Dourlen, P; Chatelain, G; Napoletano, F; Robin, M; Corbet, M; Levet, C; Hafsi, H; Hainaut, P; Ryoo, H D; Bourdon, J-C; Mollereau, B

    2013-01-01

    Irradiated or injured cells enter apoptosis, and in turn, promote proliferation of surrounding unaffected cells. In Drosophila, apoptotic cells have an active role in proliferation, where the caspase Dronc and p53 induce mitogen expression and growth in the surrounding tissues. The Drosophila p53 gene structure is conserved and encodes at least two protein isoforms: a full-length isoform (Dp53) and an N-terminally truncated isoform (DΔNp53). Historically, DΔNp53 was the first p53 isoform identified and was thought to be responsible for all p53 biological activities. It was shown that DΔNp53 induces apoptosis by inducing the expression of IAP antagonists, such as Reaper. Here we investigated the roles of Dp53 and DΔNp53 in apoptosis and apoptosis-induced proliferation. We found that both isoforms were capable of activating apoptosis, but that they each induced distinct IAP antagonists. Expression of DΔNp53 induced Wingless (Wg) expression and enhanced proliferation in both ‘undead cells' and in ‘genuine' apoptotic cells. In contrast to DΔNp53, Dp53 did not induce Wg expression in the absence of the endogenous p53 gene. Thus, we propose that DΔNp53 is the main isoform that regulates apoptosis-induced proliferation. Understanding the roles of Drosophila p53 isoforms in apoptosis and in apoptosis-induced proliferation may shed new light on the roles of p53 isoforms in humans, with important implications in cancer biology. PMID:22898807

  20. Participation of chloroplasts in plant apoptosis.

    PubMed

    Samuilov, Vitaly D; Lagunova, Elena M; Kiselevsky, Dmitry B; Dzyubinskaya, Elena V; Makarova, Yana V; Gusev, Mikhail V

    2003-01-01

    Mitochondria are known to participate in the initiation of programmed cell death (PCD) in animals and in plants. The role of chloroplasts in PCD is still unknown. We describe a new system to study PCD in plants; namely, leaf epidermal peels. The peel represents a monolayer consisting of cells of two types: phototrophic (guard cells) and chemotrophic (epidermal cells). The peels from pea (Pisum sativum L.) leaves were treated by cyanide as an inducer of PCD. We found an apoptosis-enhancing effect of illumination on chloroplast-containing guard cells, but not on chloroplastless epidermal cells. Antioxidants and anaerobiosis prevented the CN(-)-induced apoptosis of cells of both types in the dark and in the light. On the other hand, methyl viologen and menadione known as ROS-generating reagents as well as the Hill reaction electron acceptors (BQ, DAD, TMPD, or DPIP) that are not oxidized spontaneously by O2 were shown to prevent the CN(-)-induced nucleus destruction in guard cells. Apoptosis of epidermal cells was potentiated by these reagents, and they had no influence on the CN- effect. The light-dependent activation of CN(-)-induced apoptosis of guard cells was suppressed by DCMU, stigmatellin or DNP-INT, by a protein kinase inhibitor staurosporine as well as by cysteine and serine protease inhibitors. The above data suggest that apoptosis of guard cells is initiated upon a combined action of two factors, i.e., ROS and reduced plastoquinone of the photosynthetic electron transfer chain. As to reduction of ubiquinone in the mitochondrial respiratory chain, it seems to be antiapoptotic for the guard cell.

  1. Noscapine induces apoptosis in human glioma cells by an apoptosis-inducing factor-dependent pathway.

    PubMed

    Newcomb, Elizabeth W; Lukyanov, Yevgeniy; Smirnova, Iva; Schnee, Tona; Zagzag, David

    2008-07-01

    Previously, we identified noscapine as a small molecule inhibitor of the hypoxia-inducible factor-1 pathway in hypoxic human glioma cells and human umbilical vein endothelial cells. Noscapine is a nontoxic ingredient in cough medicine currently used in clinical trials for patients with non-Hodgkin's lymphoma or chronic lymphocytic leukemia to assess antitumor efficacy. Here, we have evaluated the sensitivity of four human glioma cell lines to noscapine-induced apoptosis. Noscapine was a potent inhibitor of proliferation and inducer of apoptosis. Induction of apoptosis was associated with activation of the c-jun N-terminal kinase signaling pathway concomitant with inactivation of the extracellular signal regulated kinase signaling pathway and phosphorylation of the antiapoptotic protein Bcl-2. Noscapine-induced apoptosis was associated with the release of mitochondrial proteins apoptosis-inducing factor (AIF) and/or cytochrome c. In some glioma cell lines, only AIF release occurred without cytochrome c release or poly (ADP-ribose) polymerase cleavage. Knock-down of AIF decreased noscapine-induced apoptosis. Our results suggest the potential importance of noscapine as a novel agent for use in patients with glioblastoma owing to its low toxicity profile and its potent anticancer activity.

  2. Andrographolide suppresses thymic stromal lymphopoietin in phorbol myristate acetate/calcium ionophore A23187-activated mast cells and 2,4-dinitrofluorobenzene-induced atopic dermatitis-like mice model

    PubMed Central

    Li, Chun-xiao; Li, Hua-guo; Zhang, Hui; Cheng, Ru-hong; Li, Ming; Liang, Jian-ying; Gu, Yan; Ling, Bo; Yao, Zhi-rong; Yu, Hong

    2016-01-01

    Background Atopic dermatitis (AD) is one of the most common inflammatory cutaneous diseases. Thymic stromal lymphopoietin (TSLP) has been demonstrated to be an important immunologic factor in the pathogenesis of AD. The production of TSLP can be induced by a high level of intracellular calcium concentration and activation of the receptor-interacting protein 2/caspase-1/NF-κB pathway. Andrographolide (ANDRO), a natural bicyclic diterpenoid lactone, has been found to exert anti-inflammatory effects in gastrointestinal inflammatory disorders through suppressing the NF-κB pathway. Objective To explore the effect of ANDRO on the production of TSLP in human mast cells and AD mice model. Methods We utilized enzyme-linked immunosorbent assay, real-time reverse transcription polymerase chain reaction analysis, Western blot analysis, and immunofluorescence staining assay to investigate the effects of ANDRO on AD. Results ANDRO ameliorated the increase in the intracellular calcium, protein, and messenger RNA levels of TSLP induced by phorbol myristate acetate/calcium ionophore A23187, through the blocking of the receptor-interacting protein 2/caspase-1/NF-κB pathway in human mast cell line 1 cells. ANDRO, via oral or local administration, also attenuated clinical symptoms in 2,4-dinitrofluorobenzene-induced AD mice model and suppressed the levels of TSLP in lesional skin. Conclusion Taken together, ANDRO may be a potential therapeutic agent for AD through suppressing the expression of TSLP. PMID:26929603

  3. Diacylglycerol generated by exogenous phospholipase C activates the mitogen-activated protein kinase pathway independent of Ras- and phorbol ester-sensitive protein kinase C: dependence on protein kinase C-zeta.

    PubMed Central

    van Dijk, M; Muriana, F J; van Der Hoeven, P C; de Widt, J; Schaap, D; Moolenaar, W H; van Blitterswijk, W J

    1997-01-01

    The role of diacylglycerol (DG) formation from phosphatidylcholine in mitogenic signal transduction is poorly understood. We have generated this lipid at the plasma membrane by treating Rat-1 fibroblasts with bacterial phosphatidylcholine-specific phospholipase C (PC-PLC). This treatment leads to activation of mitogen-activated protein kinase (MAPK). However, unlike platelet-derived growth factor (PDGF) or epidermal growth factor (EGF), PC-PLC fails to activate Ras and to induce DNA synthesis, and activates MAPK only transiently (<45 min). Down-regulation of protein kinase C (PKC) -alpha, -delta and -epsilon isotypes has little or no effect on MAPK activation by either PC-PLC or growth factors. However, Ro 31-8220, a highly selective inhibitor of all PKC isotypes, including atypical PKC-zeta but not Raf-1, blocks MAPK activation by PDGF and PC-PLC, but not that by EGF, suggesting that atypical PKC mediates the PDGF and PC-PLC signal. In line with this, PKC-zeta is activated by PC-PLC and PDGF, but not by EGF, as shown by a kinase assay in vitro, using biotinylated epsilon-peptide as a substrate. Furthermore, dominant-negative PKC-zeta inhibits, while (wild-type) PKC-zeta overexpression enhances MAPK activation by PDGF and PC-PLC. The results suggest that DG generated by PC-PLC can activate the MAPK pathway independent of Ras and phorbol-ester-sensitive PKC but, instead, via PKC-zeta. PMID:9169602

  4. Dual Stimulus-Dependent Effect of Oenothera paradoxa Extract on the Respiratory Burst in Human Leukocytes: Suppressing for Escherichia coli and Phorbol Myristate Acetate and Stimulating for Formyl-Methionyl-Leucyl-Phenylalanine

    PubMed Central

    Burzynska-Pedziwiatr, Izabela; Bukowiecka-Matusiak, Malgorzata; Wojcik, Marzena; Machala, Waldemar; Bienkiewicz, Malgorzata; Spolnik, Grzegorz; Danikiewicz, Witold; Wozniak, Lucyna Alicja

    2014-01-01

    Although a growing body of evidence suggests that plant polyphenols can modulate human immune responses, their simultaneous action on monocyte and neutrophil oxidative burst is currently poorly understood. Based on the hypothesis that various polyphenols contained in plant extracts might affect the oxidative burst of phagocytes, we evaluated the effects of ethanolic O. paradoxa extract polyphenols on monocyte and neutrophil oxidative burst in vitro activated by different stimuli, including opsonized bacteria E. coli, phorbol 12-myristate 13-acetate (PMA), and formyl-methionyl-leucyl-phenylalanine (fMLP). Samples were analyzed by the dihydrorhodamine flow cytometry assay. Our results showed that the extract repressed significantly and dose-dependently reactive oxygen species production in both cell types stimulated with E. coli and PMA (P < 0.05) and its inhibitory efficiency was stimulus- and cell-type-dependent. Interestingly, there was significant stimulatory effect of the extract on bursting phagocytes induced by fMLP (P < 0.05). Additionally, several flavonoids and phenolic compounds as well as penta-galloyl-β-(D)-glucose (PGG), the representative of hydrolyzable tannins, were identified in the 60% extract by high-performance liquid chromatography (HPLC) coupled to electrospray ionization in negative ion mode. In summary, the ethanolic O. paradoxa extract, rich in flavonoids and phenolic compounds, exhibits dual stimulus-dependent effect on the respiratory burst in human leukocytes; hence, it might affect immune responses in humans. PMID:25298860

  5. The opposing effects of calmodulin, adenosine 5 prime -triphosphate, and pertussis toxin on phorbol ester induced inhibition of atrial natriuretic factor stimulated guanylate cyclase in SK-NEP-1 cells

    SciTech Connect

    Sekiya, M.; Frohlich, E.D.; Cole, F.E. )

    1991-01-01

    In the present study, we investigated the effects of calmodulin, adenosine 5{prime}-triphosphate (ATP) and pertussis toxin (PT) on phorbol ester (PMA) induced inhibition of ANF-stimulated cyclic GMP formation in cells from the human renal cell line, SK-NEP-1. PMA inhibited ANF-stimulated guanylate cyclase activity in particulate membranes by about 65%. Calmodulin reversed this inhibition in a dose dependent manner. ATP potentiated Mg++ but not Mn++ supported guanylate cyclase activity. In PMA treated membranes, ATP potentiating effects were abolished. PMA also inhibited ANF-stimulated cGMP accumulation, but pretreatment with PT prevented this PMA inhibition. PT did not affect basal or ANF-stimulated cGMP accumulation. In conclusion, these results demonstrated that PMA inhibited ANF stimulation of particulate guanylate cyclase in opposition to the activating effects of calmodulin or ATP in SK-NEP-1 cells. The protein kinase C inhibitory effects appeared to be mediated via a PT-sensitive G protein.

  6. The stimulation of superoxide anion production in guinea-pig peritoneal macrophages and neutrophils by phorbol myristate acetate, opsonized zymosan and IgG2-containing soluble immune complexes.

    PubMed Central

    Baxter, M A; Leslie, R G; Reeves, W G

    1983-01-01

    The kinetics of superoxide anion production in guinea-pig peritoneal macrophages and neutrophils were determined following in vitro stimulation with phorbol myristate acetate (PMA), opsonized zymosan (OZ) and soluble immune complexes of guinea-pig IgG2 (SIC). Superoxide production was recorded as chemiluminescence (CL) arising from the reductive cleavage of lucigenin. With PMA, both macrophages and neutrophils displayed a two-phase response consisting of a rapid initial burst of CL, which preceded ligand ingestion, followed by a plateau in the CL response which persisted for more than 30 min. By contrast, OZ induced a slow progressive increase in CL in both phagocytes which was consistent with the development of an oxidative burst concomitant with ingestion. The phagocytes differed in their responses to SIC, the macrophages displaying CL kinetics similar to those observed with PMA, whereas the neutrophils responded in the manner observed with OZ. The relationship between disparity in the patterns of macrophage and neutrophil CL responses to SIC and differences in their expression of Fc receptors for IgG2 (Coupland & Leslie, 1983) is discussed. PMID:6299935

  7. Effects of phorbol esters and site-directed mutations on proteolytic processing of a cell surface precursor to human macrophage colony-stimulating factor (M-CSF, CSF-1)

    SciTech Connect

    Rettenmier, C.W.; Stein, J. Children's Hospital, Los Angeles, CA Case Western Reserve Univ., Cleveland, OH )

    1991-03-11

    Soluble forms of DSF-1 are generated by proteolytic cleavage of membrane-bound glycoprotein precursors. In eukaryotic expression systems, a 4 kilobase (kb) human cDNA encodes a 522 amino acid DSF-1 precursor which is rapidly processed within the cell to yield an efficiently secreted form of the growth factor. By contrast, an alternatively spliced 1.6 kb cDNA encodes a 224 amino acid precursor stably expressed on the cell surface where it is slowly and inefficiently cleaved to release soluble human CSF-1; this plasma membrane-bound precursor is biologically active for stimulating CSF-1-dependent cells. Treatment with phorbol ester (PMA) accelerated proteolytic processing of the cell surface CSF-1 precursor, resulting in a 30-fold increase in the recovery of soluble growth factor within 60 min. This enhanced cleavage was mediated by a cellular protease which is possibly the same enzyme responsible for the normally slow rate of processing and whose activity is stimulated by PMA activation of protein kinase C. Two mutations were introduced near the proteolytic cleavage site of the precursor. Substitution of the only basic amino acid in the vicinity had no effect on processing. However, deletion of a 6 amino acid segment in the region reduced the rate of cleavage about six-fold in the absence or presence of PMA.

  8. Morphofunctional study of 12-O-tetradecanoyl-13-phorbol acetate (TPA)-induced differentiation of U937 cells under exposure to a 6 mT static magnetic field.

    PubMed

    Dini, Luciana; Dwikat, Majdi; Panzarini, Elisa; Vergallo, Cristian; Tenuzzo, Bernadetta

    2009-07-01

    This study deals with the morphofunctional influence of 72 h exposure to a 6 mT static magnetic field (SMF) during differentiation induced by 50 ng/ml 12-O-tetradecanoyl-13-phorbol acetate (TPA) in human leukaemia U937 cells. The cell morphology of U937 cells was investigated by optic and electron microscopy. Specific antibodies and/or molecules were used to label CD11c, CD14, phosphatidylserine, F-actin and to investigate the distribution and activity of lysosomes, mitochondria and SER. [Ca(2+)](i) was evaluated with a spectrophotometer. The degree of differentiation in SMF-exposed cells was lower than that of non-exposed cells, the difference being exposure time-dependent. SMF-exposed cells showed cell shape and F-actin modification, inhibition of cell attachment, appearance of membrane roughness and large blebs and impaired expression of specific macrophagic markers on the cell surface. The intracellular localization of SER and lysosomes was only partially affected by exposure. A significant localization of mitochondria with an intact membrane potential at the cell periphery in non-exposed, TPA-stimulated cells was observed; conversely, in the presence of SMF, mitochondria were mainly localised near the nucleus. In no case did SMF exposure affect cell viability. The sharp intracellular increase of [Ca(2+)](i) could be one of the causes of the above-described changes.

  9. The effect of alpha-tocopherol on the synthesis, phosphorylation and activity of protein kinase C in smooth muscle cells after phorbol 12-myristate 13-acetate down-regulation.

    PubMed

    Clément, S; Tasinato, A; Boscoboinik, D; Azzi, A

    1997-06-15

    Previous work had established that, in smooth muscle cells, alpha-tocopherol negatively regulates protein kinase C by preventing its activation [Tasinato, A., Boscoboinik, D., Bartoli, G. M., Maroni, P. & Azzi, A. (1995) Proc. Natl Acad. Sci. USA 92, 12190-12194]. In this study, the mechanism by which this event takes place has been analyzed. The regulation by alpha-tocopherol of protein kinase C expression, activity and phosphorylation has been followed during the synthesis of protein kinase C after its down-regulation by phorbol 12-myristate 13-acetate. The data show that protein kinase C isoenzyme alpha is synthesised significantly more (30% 72 h after down-regulation) in the presence of alpha-tocopherol. However, its activity is significantly less (45% diminution) and its phosphorylation state is also decreased (60% diminution). The effect of alpha-tocopherol appears not to be shared by the analogue beta-tocopherol, provided with similar radical-scavenging properties. The data are interpreted in terms of a diminution of protein kinase C phosphorylation, specifically caused by alpha-tocopherol, resulting in a decreased enzyme specific activity.

  10. Biomarkers of Chondrocyte Apoptosis and Autophagy in Osteoarthritis

    PubMed Central

    Musumeci, Giuseppe; Castrogiovanni, Paola; Trovato, Francesca Maria; Weinberg, Annelie Martina; Al-Wasiyah, Mohammad K.; Alqahtani, Mohammed H.; Mobasheri, Ali

    2015-01-01

    Cell death with morphological and molecular features of apoptosis has been detected in osteoarthritic (OA) cartilage, which suggests a key role for chondrocyte death/survival in the pathogenesis of OA. Identification of biomarkers of chondrocyte apoptosis may facilitate the development of novel therapies that may eliminate the cause or, at least, slow down the degenerative processes in OA. The aim of this review was to explore the molecular markers and signals that induce chondrocyte apoptosis in OA. A literature search was conducted in PubMed, Scopus, Web of Science and Google Scholar using the keywords chondrocyte death, apoptosis, osteoarthritis, autophagy and biomarker. Several molecules considered to be markers of chondrocyte apoptosis will be discussed in this brief review. Molecular markers and signalling pathways associated with chondroycte apoptosis may turn out to be therapeutic targets in OA and approaches aimed at neutralizing apoptosis-inducing molecules may at least delay the progression of cartilage degeneration in OA. PMID:26334269

  11. Cold-inducible RNA-binding protein inhibits neuron apoptosis through the suppression of mitochondrial apoptosis.

    PubMed

    Zhang, Hai-Tao; Xue, Jing-Hui; Zhang, Zhi-Wen; Kong, Hai-Bo; Liu, Ai-Jun; Li, Shou-Chun; Xu, Dong-Gang

    2015-10-05

    Cold-inducible RNA-binding protein (CIRP) is induced by mild hypothermia in several mammals, but the precise mechanism by which CIRP mediates hypothermia-induced neuroprotection remains unknown. We aimed to investigate the molecular mechanisms by which CIRP protects the nervous system during mild hypothermia. Rat cortical neurons were isolated and cultured in vitro under mild hypothermia (32°C). Apoptosis was measured by annexin V and propidium iodide staining, visualized by flow cytometry. Neuron ultrastructure was visualized by transmission electron microscopy. CIRP overexpression and knockdown were achieved via infection with pL/IRES/GFP-CIRP and pL/shRNA/F-CIRP-A lentivirus. RT(2) Profiler PCR Array Pathway Analysis and western blotting were used to evaluate the effects of CIRP overexpresion/knockdown on the neurons׳ transcriptome. Neuron late apoptosis was significantly reduced at day 7 of culture by 12h hypothermia, but neuron ultrastructure remained relatively intact. RT(2) Profiler PCR Array Pathway Analysis of 84 apoptosis pathway-associated factors revealed that mild hypothermia and CIRP overexpression induce similar gene expression profiles, specifically alterations of genes implicated in the mitochondrial apoptosis pathway. Mild hypothermia-treated neurons up-regulated 12 and down-regulated 38 apoptosis pathway-associated genes. CIRP-overexpressing neurons up-regulated 15 and down-regulated 46 genes. CIRP-knocked-down hypothermia-treated cells up-regulated 9 and down-regulated 40 genes. Similar results were obtained at the protein level. In conclusion, CIRP may inhibit neuron apoptosis through the suppression of the mitochondria apoptosis pathway during mild hypothermia.

  12. Embryo apoptosis identification: Oocyte grade or cleavage stage?

    PubMed

    Bakri, Noraina Mohd; Ibrahim, Siti Fatimah; Osman, Nurul Atikah; Hasan, Nurhaslina; Jaffar, Farah Hanan Fathihah; Rahman, Zulaiha Abdul; Osman, Khairul

    2016-01-01

    Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced.

  13. Cancer gene therapy targeting cellular apoptosis machinery.

    PubMed

    Jia, Lin-Tao; Chen, Si-Yi; Yang, An-Gang

    2012-11-01

    The unraveling of cellular apoptosis machinery provides novel targets for cancer treatment, and gene therapy targeting this suicidal system has been corroborated to cause inflammation-free autonomous elimination of neoplastic cells. The apoptotic machinery can be targeted by introduction of a gene encoding an inducer, mediator or executioner of apoptotic cell death or by inhibition of anti-apoptotic gene expression. Strategies targeting cancer cells, which are achieved by selective gene delivery, specific gene expression or secretion of target proteins via genetic modification of autologous cells, dictate the outcome of apoptosis-based cancer gene therapy. Despite so far limited clinical success, gene therapy targeting the apoptotic machinery has great potential to benefit patients with threatening malignancies provided the availability of efficient and specific gene delivery and administration systems.

  14. Apoptosis and APC in colorectal tumorigenesis.

    PubMed Central

    Morin, P J; Vogelstein, B; Kinzler, K W

    1996-01-01

    Tumors result from disruptions in the homeostatic mechanisms that regulate cell birth and cell death. In colon cancer, one of the earliest manifestation of this imbalance is the formation of polyps, caused by somatic and inherited mutations of the adenomatous polyposis coli (APC) tumor suppressor gene in both humans and mice. While the importance of APC in tumorigenesis is well documented, how it functions to prevent tumors remains a mystery. Using a novel inducible expression system, we show that expression of APC in human colorectal cancer cells containing endogenous inactive APC alleles results in a substantial diminution of cell growth. Further evaluation demonstrated that this was due to the induction of cell death through apoptosis. These results suggest that apoptosis plays a role not only in advanced tumors but also at the very earliest stages of neoplasia. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8755583

  15. MYC and the Control of Apoptosis

    PubMed Central

    McMahon, Steven B.

    2014-01-01

    MYC expression is tightly correlated with cell-cycle progression in normal tissues, whereas unchecked MYC expression is among the most prominent hallmarks of the hyperproliferation associated with most forms of cancer. At first glance it might seem counterintuitive that MYC is also among the most robust agents of programmed cell death (apoptosis) in mammalian cells. However it is clearly beneficial for a multicellular organism to have a mechanism for triggering death in cells that express potentially oncogenic levels of MYC. Decades of intense study have begun to provide an understanding of the mechanisms that regulate MYC’s seemingly split personality. Key features of MYC-induced apoptosis will be discussed here along with examples of how our understanding of this pathway might be exploited for the therapeutic benefit of cancer patients. PMID:24985130

  16. Autophagy and apoptosis dysfunction in neurodegenerative disorders.

    PubMed

    Ghavami, Saeid; Shojaei, Shahla; Yeganeh, Behzad; Ande, Sudharsana R; Jangamreddy, Jaganmohan R; Mehrpour, Maryam; Christoffersson, Jonas; Chaabane, Wiem; Moghadam, Adel Rezaei; Kashani, Hessam H; Hashemi, Mohammad; Owji, Ali A; Łos, Marek J

    2014-01-01

    Autophagy and apoptosis are basic physiologic processes contributing to the maintenance of cellular homeostasis. Autophagy encompasses pathways that target long-lived cytosolic proteins and damaged organelles. It involves a sequential set of events including double membrane formation, elongation, vesicle maturation and finally delivery of the targeted materials to the lysosome. Apoptotic cell death is best described through its morphology. It is characterized by cell rounding, membrane blebbing, cytoskeletal collapse, cytoplasmic condensation, and fragmentation, nuclear pyknosis, chromatin condensation/fragmentation, and formation of membrane-enveloped apoptotic bodies, that are rapidly phagocytosed by macrophages or neighboring cells. Neurodegenerative disorders are becoming increasingly prevalent, especially in the Western societies, with larger percentage of members living to an older age. They have to be seen not only as a health problem, but since they are care-intensive, they also carry a significant economic burden. Deregulation of autophagy plays a pivotal role in the etiology and/or progress of many of these diseases. Herein, we briefly review the latest findings that indicate the involvement of autophagy in neurodegenerative diseases. We provide a brief introduction to autophagy and apoptosis pathways focusing on the role of mitochondria and lysosomes. We then briefly highlight pathophysiology of common neurodegenerative disorders like Alzheimer's diseases, Parkinson's disease, Huntington's disease and Amyotrophic lateral sclerosis. Then, we describe functions of autophagy and apoptosis in brain homeostasis, especially in the context of the aforementioned disorders. Finally, we discuss different ways that autophagy and apoptosis modulation may be employed for therapeutic intervention during the maintenance of neurodegenerative disorders.

  17. HIV-1 protease-induced apoptosis

    PubMed Central

    2014-01-01

    Background Apoptosis is one of the presumptive causes of CD4+ T cell depletion during HIV infection and progression to AIDS. However, the precise role of HIV-1 in this process remains unexplained. HIV-1 protease (PR) has been suggested as a possible factor, but a direct link between HIV-1 PR enzymatic activity and apoptosis has not been established. Results Here, we show that expression of active HIV-1 PR induces death in HeLa and HEK-293 cells via the mitochondrial apoptotic pathway. This conclusion is based on in vivo observations of the direct localization of HIV-1 PR in mitochondria, a key player in triggering apoptosis. Moreover, we observed an HIV-1 PR concentration-dependent decrease in mitochondrial membrane potential and the role of HIV-1 PR in activation of caspase 9, PARP cleavage and DNA fragmentation. In addition, in vitro data demonstrated that HIV-1 PR mediates cleavage of mitochondrial proteins Tom22, VDAC and ANT, leading to release of AIF and Hsp60 proteins. By using yeast two-hybrid screening, we also identified a new HIV-1 PR interaction partner, breast carcinoma-associated protein 3 (BCA3). We found that BCA3 accelerates p53 transcriptional activity on the bax promoter, thus elevating the cellular level of pro-apoptotic Bax protein. Conclusion In summary, our results describe the involvement of HIV-1 PR in apoptosis, which is caused either by a direct effect of HIV-1 PR on mitochondrial membrane integrity or by its interaction with cellular protein BCA3. PMID:24886575

  18. Statin-induced apoptosis and skeletal myopathy.

    PubMed

    Dirks, Amie J; Jones, Kimberly M

    2006-12-01

    Over 100 million prescriptions were filled for statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) in 2004. Statins were originally developed to lower plasma cholesterol in patients with hypercholesterolemia and are the most effective drugs on the market in doing so. Because of the discovered pleiotropic effects of statins, the use has expanded to the treatment of many other conditions, including ventricular arrythmias, idiopathic dilated cardiomyopathy, cancer, osteoporosis, and diabetes. The elderly population is growing. Therefore, it is estimated that the number of statin users will also increase. Fortunately, the use of statins is relatively safe with few side effects. Myopathy is the most common side effect with symptoms ranging from fatigue, weakness, and pain to symptoms associated with rhabdomyolysis which is a life-threatening condition. The development of statin-induced rhabdomyolysis is rare occurring in approximately 0.1% of patients; however, the occurrence of less severe symptoms is underreported and may be 1-5% or more. Physical exercise appears to increase the likelihood for the development of myopathy in patients taking statins. It is thought that as many as 25% of statin users who exercise may experience muscle fatigue, weakness, aches, and cramping due to statin therapy and potentially dismissed by the patient and physician. The mechanisms causing statin-induced myopathy have not been elucidated; however, research efforts suggest that apoptosis of myofibers may contribute. The mitochondrion is considered a regulatory center of apoptosis, and therefore its role in the induction of apoptosis will be discussed as well as the mechanism of statin-induced apoptosis and myopathy.

  19. The role of apoptosis in blepharoptosis

    PubMed Central

    Şahlı, E; Hoşal, B M; Zilelioğlu, G; Dinçer, N; Tezel, G G

    2013-01-01

    Purpose The purpose of this study is to evaluate the role of apoptosis in the pathogenesis of blepharoptosis. Patients and methods Forty-five eyelids of 43 consecutive patients (16 female, 27 males) that underwent levator resection surgery for ptosis correction were included in the study. Twenty-six of the eyelids had congenital myogenic ptosis and 19 had aponeurotic ptosis. Levator palpebrae superioris function and height of the vertical palpebral fissure were measured in all patients. After levator resection surgery, the distal part of the levator aponeurosis was fixed and sent for evaluation. Apoptotic cells were detected using Apop Tag Plus Peroxidase In Situ Apoptosis Detection Kit. Results The mean levator palpebrae superioris function was 8.4 mm (range 5–10 mm) in congenital ptosis group and 12.1 mm (range 10–17 mm) in the aponeurotic ptosis group. The mean height of the vertical palpebral fissure in patients with congenital ptosis and aponeurotic ptosis were 6.5 mm (range 5–9 mm) and 6.1 mm (3–9 mm), respectively. The mean apoptotic index of congenital ptosis and aponeurotic ptosis were 27.3 (16–39) and 29.8 (18–41), respectively. There was no statistically significant difference between congenital and aponeurotic ptosis groups in a mean apoptotic index (P<0.05). Apoptotic index was not correlated with age, levator palpebrae superioris function, palpebral fissure height, and lid crease height in two groups. Conclusion We found no statistically significant difference between two subtypes of blepharoptosis regarding apoptosis. According to this study, apoptosis seems to have no significant role in the development of aponeurotic blepharoptosis. PMID:23598678

  20. The role of apoptosis in blepharoptosis.

    PubMed

    Şahlı, E; Hoşal, B M; Zilelioğlu, G; Dinçer, N; Tezel, G G

    2013-07-01

    The purpose of this study is to evaluate the role of apoptosis in the pathogenesis of blepharoptosis. Forty-five eyelids of 43 consecutive patients (16 female, 27 males) that underwent levator resection surgery for ptosis correction were included in the study. Twenty-six of the eyelids had congenital myogenic ptosis and 19 had aponeurotic ptosis. Levator palpebrae superioris function and height of the vertical palpebral fissure were measured in all patients. After levator resection surgery, the distal part of the levator aponeurosis was fixed and sent for evaluation. Apoptotic cells were detected using Apop Tag Plus Peroxidase In Situ Apoptosis Detection Kit. The mean levator palpebrae superioris function was 8.4 mm (range 5-10 mm) in congenital ptosis group and 12.1 mm (range 10-17 mm) in the aponeurotic ptosis group. The mean height of the vertical palpebral fissure in patients with congenital ptosis and aponeurotic ptosis were 6.5 mm (range 5-9 mm) and 6.1 mm (3-9 mm), respectively. The mean apoptotic index of congenital ptosis and aponeurotic ptosis were 27.3 (16-39) and 29.8 (18-41), respectively. There was no statistically significant difference between congenital and aponeurotic ptosis groups in a mean apoptotic index (P<0.05). Apoptotic index was not correlated with age, levator palpebrae superioris function, palpebral fissure height, and lid crease height in two groups. We found no statistically significant difference between two subtypes of blepharoptosis regarding apoptosis. According to this study, apoptosis seems to have no significant role in the development of aponeurotic blepharoptosis.

  1. Resistance of Actin to Cleavage during Apoptosis

    NASA Astrophysics Data System (ADS)

    Song, Qizhong; Wei, Tie; Lees-Miller, Susan; Alnemri, Emad; Watters, Dianne; Lavin, Martin F.

    1997-01-01

    A small number of cellular proteins present in the nucleus, cytosol, and membrane fraction are specifically cleaved by the interleukin-1β -converting enzyme (ICE)-like family of proteases during apoptosis. Previous results have demonstrated that one of these, the cytoskeletal protein actin, is degraded in rat PC12 pheochromocytoma cells upon serum withdrawal. Extracts from etoposide-treated U937 cells are also capable of cleaving actin. It was assumed that cleavage of actin represented a general phenomenon, and a mechanism coordinating proteolytic, endonucleolytic, and morphological aspects of apoptosis was proposed. We demonstrate here that actin is resistant to degradation in several different human cells induced to undergo apoptosis in response to a variety of stimuli, including Fas ligation, serum withdrawal, cytotoxic T-cell killing, and DNA damage. On the other hand, cell-free extracts from these cells and the ICE-like protease CPP32 were capable of cleaving actin in vitro. We conclude that while actin contains cleavage sites for ICE-like proteases, it is not degraded in vivo in human cells either because of lack of access of these proteases to actin or due to the presence of other factors that prevent degradation.

  2. Lithium protects ethanol-induced neuronal apoptosis

    SciTech Connect

    Zhong Jin . E-mail: jizhong@iupui.edu; Yang Xianlin; Yao Weiguo; Lee Weihua

    2006-12-01

    Lithium is widely used for the treatment of bipolar disorder. Recent studies have demonstrated its neuroprotective effect. Ethanol is a potent neurotoxin that is particularly harmful to the developing nervous system. In this study, we evaluated lithium's neuroprotection against ethanol-induced apoptosis. Transient exposure of infant mice to ethanol caused apoptotic cell death in brain, which was prevented significantly by administering a low dose of lithium 15 min later. In cultured cerebellar granule neurons, ethanol-induced apoptosis and activation of caspase-3/9, both of which were prevented by lithium. However, lithium's protection is not mediated by its commonly known inhibition of glycogen synthase3{beta}, because neither ethanol nor lithium has significant effects on the phosphorylation of Akt (ser473) or GSK3{beta} (ser9). In addition, the selective GSK-3{beta} inhibitor SB-415286 was unable to prevent ethanol-induced apoptosis. These data suggest lithium may be used as a potential preventive measure for ethanol-induced neurological deficits.

  3. Increased small intestinal apoptosis in coeliac disease.

    PubMed Central

    Moss, S F; Attia, L; Scholes, J V; Walters, J R; Holt, P R

    1996-01-01

    BACKGROUND: Coeliac disease (CD) mucosa is flattened despite epithelial hyperproliferation. AIMS: To establish mechanisms of cell loss in CD. PATIENTS: 14 controls, 17 active CD patients, and 16 maintained with gluten free diet. METHODS: Programmed cell death was examined in small intestinal biopsy specimens by staining fragmented DNA using terminal uridine deoxynucleotidyl nick end labelling (TUNEL), in comparison with haematoxylin and eosin stained adjacent sections. Double staining with anti-CD45 antibodies determined the origin of apoptotic cells. Apoptosis was graded from 1-3 (< 5, 5-20, > 20% respectively). Proliferating cells, immunostained by Ki-67 (MIB-1) antibody, were counted. RESULTS: Apoptotic cells were seen rarely by haematoxylin and eosin but more readily by TUNEL. In controls, 1.4 +/- 0.2% of epithelial cells were apoptotic (mean grade 1.1), mainly located in the upper villus. In active CD, frequent apoptotic cells were distributed throughout the crypt-villus unit (mean grade 2.4), decreasing after treatment to 1.1 (p < 0.001) even when still histologically abnormal. CD45 antibodies rarely stained apoptotic cells in active CD. The number of TUNEL positive cells correlated with proliferating cell number (p < 0.001). CONCLUSION: Enterocyte apoptosis is greatly increased in untreated CD, correlates with proliferation, and falls to normal with a gluten free diet, before histological improvement. Increased apoptosis may be responsible for villous atrophy in CD. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9038662

  4. Parvovirus B19-Induced Apoptosis of Hepatocytes

    PubMed Central

    Poole, Brian D.; Karetnyi, Yuory V.; Naides, Stanley J.

    2004-01-01

    Parvovirus B19 (B19 virus) can persist in multiple tissues and has been implicated in a variety of diseases, including acute fulminant liver failure. The mechanism by which B19 virus induces liver failure remains unknown. Hepatocytes are nonpermissive for B19 virus replication. We previously reported that acute fulminant liver failure associated with B19 virus infection was characterized by hepatocellular dropout. We inoculated both primary hepatocytes and the hepatocellular carcinoma cell line Hep G2 with B19 virus and assayed for apoptosis by using annexin V staining. Reverse transcriptase PCR analysis and immunofluorescence demonstrated that B19 virus was able to infect the cells and produce its nonstructural protein but little or no structural capsid protein. Infection with B19 virus induced means of 28% of Hep G2 cells and 10% of primary hepatocytes to undergo apoptosis, which were four- and threefold increases, respectively, over background levels. Analysis of caspase involvement showed that B19 virus-inoculated cultures had a significant increase in the number of cells with active caspase 3. Inhibition studies demonstrated that caspases 3 and 9, but not caspase 8, are required for B19 virus-induced apoptosis. PMID:15220451

  5. Simulating cell apoptosis induced sinus node dysfunction.

    PubMed

    Kharche, Sanjay; Beling, John; Biktasheva, Irina V; Zhang, Henggui; Biktashev, Vadim N

    2013-01-01

    Sinus node dysfunction (SND) is correlated to the pacemaker sinoatrial node (SAN) cell apoptosis. This study explores the effect of such a dysfunctional SAN on electrical propagation into neighboring atrial tissue. The Fenton Karma model was extended to simulate mouse SAN and atrial cell action potentials. The cell models were incorporated into a 2D model consisting of a central SAN region surrounded by atrial tissue. The intercellular gap junctional coupling, as quantified by the diffusion constant, was estimated to give conduction speeds as observed in mouse atrial tissue. The size of mouse SAN pacemaking region was estimated using the 2D model. In multiple simulations, the effects of an increasing proportion of apoptotic pacemaker cells on atrial tissue pacing were simulated and quantified. The SAN size that gave a basal mouse atrial cycle length (ACL) of 295 ms was found to be 0.6 mm in radius. At low pacemaker cell apoptosis proportion, there was a drastic increase of ACL. At modest increase in the number of apoptotic cells, bradycardia was observed. The incidence of sinus arrest was also found to be high. When the number of apoptotic cells were 10% of the total number of pacemaking cells, all pacemaking was arrested. Phenomenological models have been developed to study mouse atrial electrophysiology and confirm experimental findings. The results show the significance of cell apoptosis as a major mechanism of SND.

  6. Alcohol and Apoptosis: Friends or Foes?

    PubMed Central

    Rodriguez, Ana; Chawla, Karan; Umoh, Nsini A.; Cousins, Valerie M.; Ketegou, Assama; Reddy, Madhumati G.; AlRubaiee, Mustafa; Haddad, Georges E.; Burke, Mark W.

    2015-01-01

    Alcohol abuse causes 79,000 deaths stemming from severe organ damage in the United States every year. Clinical manifestations of long-term alcohol abuse on the cardiac muscle include defective contractility with the development of dilated cardiomyopathy and low-output heart failure; which has poor prognosis with less than 25% survival for more than three years. In contrast, low alcohol consumption has been associated with reduced risk of cardiovascular disease, however the mechanism of this phenomenon remains elusive. The aim of this study was to determine the significance of apoptosis as a mediating factor in cardiac function following chronic high alcohol versus low alcohol exposure. Adult rats were provided 5 mM (low alcohol), 100 mM (high alcohol) or pair-fed non-alcohol controls for 4–5 months. The hearts were dissected, sectioned and stained with cresyl violet or immunohistochemically for caspase-3, a putative marker for apoptosis. Cardiomyocytes were isolated to determine the effects of alcohol exposure on cell contraction and relaxation. High alcohol animals displayed a marked thinning of the left ventricular wall combined with elevated caspase-3 activity and decreased contractility. In contrast, low alcohol was associated with increased contractility and decreased apoptosis suggesting an overall protective mechanism induced by low levels of alcohol exposure. PMID:26610584

  7. Role of nuclear bodies in apoptosis signalling.

    PubMed

    Krieghoff-Henning, Eva; Hofmann, Thomas G

    2008-11-01

    Promyelocytic leukemia nuclear bodies (PML NBs) are dynamic macromolecular multiprotein complexes that recruit and release a plethora of proteins. A considerable number of PML NB components play vital roles in apoptosis, senescence regulation and tumour suppression. The molecular basis by which PML NBs control these cellular responses is still just beginning to be understood. In addition to PML itself, numerous further tumour suppressors including transcriptional regulator p53, acetyl transferase CBP (CREB binding protein) and protein kinase HIPK2 (homeodomain interacting protein kinase 2) are recruited to PML NBs in response to genotoxic stress or oncogenic transformation and drive the senescence and apoptosis response by regulating p53 activity. Moreover, in response to death-receptor activation, PML NBs may act as nuclear depots that release apoptotic factors, such as the FLASH (FLICE-associated huge) protein, to amplify the death signal. PML NBs are also associated with other nuclear domains including Cajal bodies and nucleoli and share apoptotic regulators with these domains, implying crosstalk between NBs in apoptosis regulation. In conclusion, PML NBs appear to regulate cell death decisions through different, pathway-specific molecular mechanisms.

  8. HIV increases HCV-induced hepatocyte apoptosis

    PubMed Central

    Jang, Jae Young; Shao, Run-Xuan; Lin, Wenyu; Weinberg, Ethan; Chung, Woo Jin; Tsai, Wei Lun; Zhao, Hong; Goto, Kaku; Zhang, Leiliang; Mendez-Navarro, Jorge; Jilg, Nikolaus; Peng, Lee F.; Brockman, Mark A.; Chung, Raymond T.

    2010-01-01

    Background and Aims HCV related liver disease is one of the most important complications in persons with HIV, with accelerated fibrosis progression in coinfected persons compared to those with HCV alone. We hypothesized that HIV coinfection increases HCV related hepatocyte apoptosis and that HCV and HIV influence TRAIL signaling in hepatocytes. Methods We analyzed the effect of HIV on JFH1-infected Huh 7.5.1 cells. Apoptosis was measured by Caspase-Glo 3/7 assay and Western blot for cleaved PARP. TRAIL, TRAIL receptor 1 (DR4) and 2 (DR5) mRNA and protein levels were assessed by real-time PCR and Western blot. We also investigated activation of caspase pathways using caspase inhibitors and assessed expression of Bid and cytochrome C. Results We found increased caspase 3/7 activity and cleaved PARP in JFH1 HCV-infected Huh7.5.1 cells in the presence of heat-inactivated HIV compared to Huh7.5.1 cells infected with JFH1 or exposed to heat-inactivated HIV alone. Both DR4 and DR5 mRNA and protein expression were increased in JFH1-infected cells in the presence of inactivated HIV compared to Huh7.5.1 cells infected with JFH1 or exposed to heat-inactivated HIV alone. Pancaspase, Caspase-8, and caspase-9 inhibition blocked apoptosis induced by HCV, inactivated HIV and HCV plus inactivated HIV. A caspase-9 inhibitor blocked apoptosis induced by HCV, HIV and HCV-HIV comparably to pancaspase and caspase-8 inhibitors. HCV induced the activation of Bid cleavage and cytochrome C release. The addition of HIV substantially augmented this induction. Conclusions Our findings indicate that hepatocyte apoptosis is increased in the presence of HCV and HIV compared to HCV or HIV alone, and that this increase is mediated by DR4 and DR5 up-regulation. They provide an additional mechanism for the observed accelerated liver disease progression observed in HCV-HIV coinfection. PMID:21146890

  9. Host and Viral Factors in HIV-Mediated Bystander Apoptosis

    PubMed Central

    Garg, Himanshu; Joshi, Anjali

    2017-01-01

    Human immunodeficiency virus (HIV) infections lead to a progressive loss of CD4 T cells primarily via the process of apoptosis. With a limited number of infected cells and vastly disproportionate apoptosis in HIV infected patients, it is believed that apoptosis of uninfected bystander cells plays a significant role in this process. Disease progression in HIV infected individuals is highly variable suggesting that both host and viral factors may influence HIV mediated apoptosis. Amongst the viral factors, the role of Envelope (Env) glycoprotein in bystander apoptosis is well documented. Recent evidence on the variability in apoptosis induction by primary patient derived Envs underscores the role of Env glycoprotein in HIV disease. Amongst the host factors, the role of C-C Chemokine Receptor type 5 (CCR5), a coreceptor for HIV Env, is also becoming increasingly evident. Polymorphisms in the CCR5 gene and promoter affect CCR5 cell surface expression and correlate with both apoptosis and CD4 loss. Finally, chronic immune activation in HIV infections induces multiple defects in the immune system and has recently been shown to accelerate HIV Env mediated CD4 apoptosis. Consequently, those factors that affect CCR5 expression and/or immune activation in turn indirectly regulate HIV mediated apoptosis making this phenomenon both complex and multifactorial. This review explores the complex role of various host and viral factors in determining HIV mediated bystander apoptosis. PMID:28829402

  10. Scavenger receptor BI and HDL regulate thymocyte apoptosis in sepsis

    PubMed Central

    Guo, Ling; Zheng, Zhong; Ai, Junting; Howatt, Deborah A.; Mittelstadt, Paul R.; Thacker, Seth; Daugherty, Alan; Ashwell, Jonathan D.; Remaley, Alan T.; Li, Xiang-An

    2014-01-01

    Objective Thymocyte apoptosis is a major event in sepsis; however, how this process is regulated remains poorly understood. Approach and Results Septic stress induces glucocorticoids (GC) production which triggers thymocyte apoptosis. Here, we used scavenger receptor BI (SR-BI) null mice, which are completely deficient in inducible GC (iGC) in sepsis, to investigate the regulation of thymocyte apoptosis in sepsis. Cecal ligation and puncture (CLP) induced profound thymocyte apoptosis in SR-BI+/+ mice, but no thymocyte apoptosis in SR-BI−/− mice due to lack of iGC. Unexpectedly, supplementation of GC only partly restored thymocyte apoptosis in SR-BI−/− mice. We demonstrated that HDL is a critical modulator for thymocyte apoptosis. SR-BI+/+ HDL significantly enhanced GC-induced thymocyte apoptosis but SR-BI−/− HDL had no such activity. Further study revealed that SR-BI+/+ HDL modulates GC-induced thymocyte apoptosis via promoting glucocorticoid receptor translocation, but SR-BI−/− HDL loses such regulatory activity. To understand why SR-BI−/− HDL loses its regulatory activity, we analyzed HDL cholesterol contents. There was 3-fold enrichment of unesterified cholesterol in SR-BI−/− HDL compared with SR-BI+/+ HDL. Normalization of unesterified cholesterol in SR-BI−/− HDL by probucol administration or LCAT expression restored GC-induced thymocyte apoptosis, and incorporating unesterified cholesterol into SR-BI+/+ HDL rendered SR-BI+/+ HDL dysfunctional. Using lckCre-GRfl/fl mice in whom thymocytes lack CLP-induced thymocyte apoptosis, we showed that lckCre-GRfl/fl mice were significantly more susceptible to CLP-induced septic death than GRfl/fl control mice, suggesting that GC-induced thymocyte apoptosis is required for protection against sepsis. Conclusions The findings in this study reveal a novel regulatory mechanism of thymocyte apoptosis in sepsis by SR-BI and HDL. PMID:24603680

  11. Phorbol ester stimulates ethanolamine release from the metastatic basal prostate cancer cell line PC3 but not from prostate epithelial cell lines LNCaP and P4E6

    PubMed Central

    Schmitt, J; Noble, A; Otsuka, M; Berry, P; Maitland, N J; Rumsby, M G

    2014-01-01

    Background: Malignancy alters cellular complex lipid metabolism and membrane lipid composition and turnover. Here, we investigated whether tumorigenesis in cancer-derived prostate epithelial cell lines influences protein kinase C-linked turnover of ethanolamine phosphoglycerides (EtnPGs) and alters the pattern of ethanolamine (Etn) metabolites released to the medium. Methods: Prostate epithelial cell