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Journal of Cellular Physiology May 2020Podosomes and tight junctions (TJs) are subcellular compartments that both exist in endothelial cells and localize at cell surfaces. In contrast to the...
Podosomes and tight junctions (TJs) are subcellular compartments that both exist in endothelial cells and localize at cell surfaces. In contrast to the well-characterized role of TJs in maintaining cerebrovascular integrity, the specific function of endothelial podosomes remains unknown. Intriguingly, we discovered cross-talk between podosomes and TJs in human brain endothelial cells. Tight junction scaffold proteins ZO-1 and ZO-2 localize at podosomes in response to phorbol-12-myristate-13-acetate treatment. We found that both ZO proteins are essential for podosome formation and function. Rather than being derived from new protein synthesis, podosomal ZO-1 and ZO-2 are relocated from a pre-existing pool found at the peripheral plasma membrane with enhanced physical interaction with cortactin, a known protein marker for podosomes. Sequestration of ZO proteins in podosomes weakens tight junction complex formation, leading to increased endothelial cell permeability. This effect can be further attenuated by podosome inhibitor PP2. Altogether, our data revealed a novel cellular function of podosomes, specifically, their ability to negatively regulate tight junction and endothelial barrier integrity, which have been linked to a variety of cerebrovascular diseases.
Topics: Brain; Cells, Cultured; Endothelial Cells; Humans; Permeability; Podosomes; Protein Multimerization; Protein Transport; Tetradecanoylphorbol Acetate; Tight Junctions; Zonula Occludens-1 Protein; Zonula Occludens-2 Protein
PubMed: 31637713
DOI: 10.1002/jcp.29343 -
Cancer Science Nov 2011Tumor development induced by 7,12-dimethylbenz[a]anthracene (DMBA) plus 12-O-tetradecanoylphorbol-13-acetate (TPA) is a well-characterized model of multistep...
Involvement of retrotransposition of long interspersed nucleotide element-1 in skin tumorigenesis induced by 7,12-dimethylbenz[a]anthracene and 12-O-tetradecanoylphorbol-13-acetate.
Tumor development induced by 7,12-dimethylbenz[a]anthracene (DMBA) plus 12-O-tetradecanoylphorbol-13-acetate (TPA) is a well-characterized model of multistep carcinogenesis. DMBA mutates the Ha-ras gene, whereas TPA promotes the growth of transformed cells by activating cellular signaling molecules. It remains to be clarified how repeated TPA treatment endows transformed cells with autonomous cell growth. Long interspersed nucleotide element-1 (L1) is an endogenous retroelement, and 80-100 copies of L1 function as autonomous mobile elements. Although the L1 retrotransposition (RTP) has been found in various human tumors, implying the possible mobility of L1 during carcinogenesis, little is known about how L1-RTP arises in tumor cells, owing to a lack of experimental models. To dissect the mechanism of L1-RTP during carcinogenesis, we established a line of transgenic mice carrying human L1 and enhanced green fluorescent protein (hL1-EGFP mice) and subjected them to DMBA/TPA-induced skin tumorigenesis. Of 15 skin tumors examined, 13 were positive for L1-RTP; L1-RTP was not detected in normal skin tissues adjacent to the tumors. Moreover, nine L1-RTP-positive tumors were positive for activated Ha-ras, and immunohistochemical analysis revealed cells positive for both L1-RTP and phosphorylated Stat3, a marker of tumor cells. Additional in vivo experiments suggested that L1-RTP occurred during tumor promotion by TPA. This is the first report on the involvement of L1-RTP in chemical carcinogenesis. We propose hL1-EGFP mice as a versatile system for investigating the mode of L1-RTP in tumor development and discuss the possible role of L1-RTP in tumorigenesis.
Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; Carcinogens; Cell Division; Cocarcinogenesis; Drug Synergism; Genes, ras; Green Fluorescent Proteins; Humans; Long Interspersed Nucleotide Elements; Mice; Mice, Inbred C57BL; Mice, Transgenic; Models, Animal; Mutagenesis, Insertional; Neoplasm Proteins; Receptors, Aryl Hydrocarbon; Skin Neoplasms; Tetradecanoylphorbol Acetate; Transfection
PubMed: 21827582
DOI: 10.1111/j.1349-7006.2011.02060.x -
Molecules and Cells 2015Matrix metalloproteinase (MMP)-9 degrades type IV collagen in the basement membrane and plays crucial roles in several pathological implications, including tumorigenesis...
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.
Topics: Cell Line, Tumor; Cell Proliferation; Cell Survival; Fibrosarcoma; Flavonoids; Gene Expression Regulation, Neoplastic; Humans; I-kappa B Proteins; Kaempferols; Matrix Metalloproteinase 9; NF-KappaB Inhibitor alpha; Phosphorylation; Signal Transduction; Tetradecanoylphorbol Acetate
PubMed: 25518925
DOI: 10.14348/molcells.2015.2229 -
International Journal of Molecular... Mar 2022The Bhas 42 cell transformation assay (Bhas 42 CTA) is the first Organization for Economic Cooperation and Development (OECD)-certificated method used as a specific tool...
The Bhas 42 cell transformation assay (Bhas 42 CTA) is the first Organization for Economic Cooperation and Development (OECD)-certificated method used as a specific tool for the detection of the cell-transformation potential of tumor-promoting compounds, including non-genotoxic carcinogens (NGTxCs), as separate from genotoxic carcinogens. This assay offers the great advantage of enabling the phenotypic detection of oncotransformation. A key benefit of using the Bhas 42 CTA in the study of the cell-transformation mechanisms of tumor-promoting compounds, including non-genotoxic carcinogens, is that the cell-transformation potential of the chemical can be detected directly without treatment with a tumor-initiating compound since Bhas 42 cell line was established by transfecting the v-Ha- gene into a mouse fibroblast cloned cell line. Here, we analyzed the gene expression over time, using DNA microarrays, in Bhas 42 cells treated with the tumor-promoting compound 12--tetradecanoylphorbol-13-acetate (TPA), and NGTxC, with a total of three repeat experiments. This is the first paper to report on gene expression over time during the process of cell transformation with only a tumor-promoting compound. Pathways that were activated or inactivated during the process of cell transformation in the Bhas 42 cells treated with TPA were related not only directly to but also to various pathways in the hallmarks of cancer.
Topics: Animals; BALB 3T3 Cells; Butylated Hydroxyanisole; Carcinogenicity Tests; Carcinogens; Cell Transformation, Neoplastic; Gene Expression; Mice; Tetradecanoylphorbol Acetate
PubMed: 35328637
DOI: 10.3390/ijms23063216 -
International Journal of Implant... May 2022The preparation of platelet-rich fibrin (PRF) requires glass blood collection tubes, and thus, the shortage or unavailability of such tubes has driven clinicians to...
BACKGROUND
The preparation of platelet-rich fibrin (PRF) requires glass blood collection tubes, and thus, the shortage or unavailability of such tubes has driven clinicians to search for suitable substitutes, such as silica-coated plastic tubes. However, we have previously demonstrated the cytotoxicity of silica microparticles (MPs) used in plastic tubes to cultured human periosteal cells. To further establish the effects of silica MPs on inflammation, we examined silica MP-induced changes in a human promyelocytic cell model in vitro.
METHODS
Human promyelocytic HL60 cells were used either without chemical induction or after differentiation induced using phorbol myristate acetate (PMA) or dimethyl sulfoxide. HL60 cells, osteoblastic MG63, and Balb/c mouse cells were treated with silica MPs, and their surface ultrastructure and numbers were examined using a scanning electron microscope and an automated cell counter, respectively. Differentiation markers, such as acid phosphatase, non-specific esterase, and CD11b, were visualized by cytochemical and immunofluorescent staining, and superoxide dismutase (SOD) activity was quantified.
RESULTS
Regardless of SOD activity, silica cytotoxicity was observed in MG63 and Balb/c cells. At sub-toxic doses, silica MPs slightly or moderately upregulated the differentiation markers of the control, PMA-induced monocytic, and dimethyl sulfoxide-induced granulocytic HL60 cells. Although SOD activity was the highest (P < 0.05) in PMA-induced cells, a silica-induced reduction in cell adhesion was observed only in those cells (P < 0.05).
CONCLUSIONS
Silica MP contamination of PRF preparations can potentially exacerbate inflammation at implantation sites. Consequently, unless biomedical advantages can be identified, silica-coated plastic blood collection tubes should not be routinely used for PRF preparations.
Topics: Animals; Cell Lineage; Dimethyl Sulfoxide; HL-60 Cells; Humans; Inflammation; Leukemia, Promyelocytic, Acute; Mice; Plastics; Silicon Dioxide; Superoxide Dismutase; Tetradecanoylphorbol Acetate
PubMed: 35567654
DOI: 10.1186/s40729-022-00424-4 -
Frontiers in Immunology 2023The infusion of -generated regulatory B cells may represent a promising novel therapeutic approach for a variety of autoimmune and hyperinflammatory conditions including...
INTRODUCTION
The infusion of -generated regulatory B cells may represent a promising novel therapeutic approach for a variety of autoimmune and hyperinflammatory conditions including graft-versus-host disease.
METHODS
Previously, we developed a protocol for the generation of a novel population of regulatory B cells, which are characterized by secretion of enzymatically active granzyme B (). This protocol uses recombinant interleukin 21 (IL-21) and goat-derived F(ab)'2 fragments against the human B cell receptor (anti-BCR). Generally, the use of xenogeneic material for the manufacturing of advanced therapy medicinal products should be avoided to prevent adverse immune reactions as well as potential transmission of so far unknown diseases.
RESULTS
In the present work we demonstrated that phorbol-12-myristate-13-acetate (PMA/TPA), a phorbol ester with a particular analogy to the second messenger diacylglycerol (DAG), is a potent enhancer of IL-21-induced differentiation of pre-activated B cells into . The percentage of after stimulation of pre-activated B cells with IL-21 and PMA/TPA was not significantly lower compared to stimulation with IL-21 and anti-BCR.
DISCUSSION
Given that PMA/TPA has already undergone encouraging clinical testing in patients with certain haematological diseases, our results suggest that PMA/TPA may be a safe and feasible alternative for manufacturing of .
Topics: Humans; B-Lymphocytes, Regulatory; Granzymes; Tetradecanoylphorbol Acetate
PubMed: 37588597
DOI: 10.3389/fimmu.2023.1194880 -
The Journal of Investigative Dermatology Sep 1987Recent advances in the culturing of pigment cells from human beings have made it possible to begin the transplantation of autologous melanocytes into areas of skin that...
Recent advances in the culturing of pigment cells from human beings have made it possible to begin the transplantation of autologous melanocytes into areas of skin that are hypopigmented. In a patient with piebaldism we were able to take pigment cells from a shave biopsy of the normally pigmented skin of the back, expand the cells in culture, and return them to an area devoid of pigment cells and get a perfect take. To grow the cells in culture we used 12-O-tetradecanoyl-phorbol-13-acetate (TPA) as well as cholera toxin and isobutylmethyl xanthine. At this time, one can substitute basic fibroblast growth factor for TPA. The procedure of using autologous pigment cell cultures opens the door for further advances in the treatment of patients who do not have melanocytes in certain areas of the skin, as seen in patients with vitiligo or piebaldism, or as a consequence of severe mechanical or thermal trauma.
Topics: Adult; Cells, Cultured; Humans; Male; Melanocytes; Microscopy, Electron; Pigmentation Disorders; Skin; Tetradecanoylphorbol Acetate
PubMed: 3624895
DOI: 10.1111/1523-1747.ep12470973 -
The Journal of Biological Chemistry Nov 2004Gap junctions are specialized plasma membrane domains enriched in connexin proteins that form channels between adjacent cells. Gap junctions are highly dynamic, and...
Gap junctions are specialized plasma membrane domains enriched in connexin proteins that form channels between adjacent cells. Gap junctions are highly dynamic, and modulation of the connexin turnover rate is considered to play an important role in the regulation of gap junctional intercellular communication. In the present study, we show that the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induces ubiquitination of connexin-43 (Cx43) in IAR20 rat liver epithelial cells. The accelerated ubiquitination of Cx43 in response to TPA occurred concomitantly with Cx43 hyperphosphorylation and inhibition of cell-cell communication via gap junctions. The TPA-induced ubiquitination of Cx43 was mediated via protein kinase C and partly involved the mitogen-activated protein kinase pathway. Following ubiquitination, Cx43 was internalized and degraded. The loss of Cx43 protein was counteracted by ammonium chloride, indicating that acidification of internalized Cx43 gap junctions is a prerequisite for its degradation. Furthermore, the Cx43 degradation was partly counteracted by leupeptin, an inhibitor of cathepsin B, H, and L. Cx43 internalization and subsequent degradation were blocked by inhibitors of the proteasome. Evidence is provided that Cx43 is modified by multiple monoubiquitins rather than a polyubiquitin chain in response to TPA. Moreover, the TPA-induced ubiquitination of Cx43 was blocked by proteasomal inhibitors. Taken together, the data indicate that Cx43 ubiquitination is a highly regulated process. Moreover, the results suggest that the proteasome might play an indirect role in Cx43 degradation by affecting the level of monoubiquitin conjugation and trafficking of Cx43 to endosomal compartments.
Topics: Animals; Carcinogens; Connexin 43; Down-Regulation; Gap Junctions; Proteasome Inhibitors; Rats; Tetradecanoylphorbol Acetate; Ubiquitin
PubMed: 15371442
DOI: 10.1074/jbc.M402006200 -
Folia Histochemica Et Cytobiologica 2008In vitro studies have shown that human myeloid leukemia cell lines: HL60 and NB4 can be stimulated to differentiation by various agents, for example, all-trans retinoic...
In vitro studies have shown that human myeloid leukemia cell lines: HL60 and NB4 can be stimulated to differentiation by various agents, for example, all-trans retinoic acid (ATRA) and phorbol 12-myristate 13-acetate (PMA). The purpose of this study was to investigate whether differentiation of HL60 and NB4 leukemia cell lines induced by ATRA and PMA alters their drug sensitivity. The differentiation along the neutrophil lineage (upon stimulation with ATRA) and along the monocyte/macrophage lineage (upon stimulation with PMA) was proved by decreased proliferative potential of cells, changes in their morphology, increased ability for NBT reduction and increased expression of CD11b and CD14 cell surface markers. The effect of drugs: cytosine arabinoside, daunorubicin, mitoxantrone and etoposide was examined by Alamar Blue test (proliferation and survival rates), as well as by evaluation of cell smears stained with Hoechst 33342 (apoptotic index). Differentiation resulted in the change of drug sensitivity in both cell lines: the differentiation along the neutrophil pathway (after stimulation with ATRA) increased sensitivity to cytosine arabinoside and mitoxantrone but decreased sensitivity to etoposide; the differentiation along the monocyte/macrophage pathway (induced by PMA) resulted in the decreased sensitivity of both cell lines to all drugs tested. In conclusion, we have shown that ATRA- and PMA-mediated differentiation of HL60 and NB4 cell lines results in the changes of their drug sensitivity. Our data may provide a contribution to a strategy aimed at a rational combination of differentiating agents and conventional anticancer drugs.
Topics: Antineoplastic Agents; Cell Differentiation; Cell Line, Tumor; HL-60 Cells; Humans; Tetradecanoylphorbol Acetate; Time Factors; Tretinoin
PubMed: 19056536
DOI: 10.2478/v10042-008-0080-x -
International Journal of Molecular... Apr 2022Megakaryocytes are large hematopoietic cells present in the bone marrow cavity, comprising less than 0.1% of all bone marrow cells. Despite their small number,...
Megakaryocytes are large hematopoietic cells present in the bone marrow cavity, comprising less than 0.1% of all bone marrow cells. Despite their small number, megakaryocytes play important roles in blood coagulation, inflammatory responses, and platelet production. However, little is known about changes in gene expression during megakaryocyte maturation. Here we identified the genes whose expression was changed during K562 leukemia cell differentiation into megakaryocytes using an Affymetrix GeneChip microarray to determine the multifunctionality of megakaryocytes. K562 cells were differentiated into mature megakaryocytes by treatment for 7 days with phorbol 12-myristate 13-acetate, and a microarray was performed using RNA obtained from both types of cells. The expression of 44,629 genes was compared between K562 cells and mature megakaryocytes, and 954 differentially expressed genes (DEGs) were selected based on a p-value < 0.05 and a fold change >2. The DEGs was further functionally classified using five major megakaryocyte function-associated clusters—inflammatory response, angiogenesis, cell migration, extracellular matrix, and secretion. Furthermore, interaction analysis based on the STRING database was used to generate interactions between the proteins translated from the DEGs. This study provides information on the bioinformatics of the DEGs in mature megakaryocytes after K562 cell differentiation.
Topics: Acetates; Cell Differentiation; Computational Biology; Humans; K562 Cells; Megakaryocytes; Microarray Analysis; Myristic Acid; Phorbols; Tetradecanoylphorbol Acetate; Thrombopoiesis
PubMed: 35457039
DOI: 10.3390/ijms23084221