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Induction of a trophoblast-like phenotype by hydralazine in the p19 embryonic carcinoma cell line.

ABSTRACT: Chemicals that affect cellular differentiation through epigenetic mechanisms have potential utility in treating a wide range of diseases. Hydralazine decreases DNA methylation in some cell types but its effect on differentiation has not been well explored. After five days of exposure to hydralazine, P19 embryocarcinoma cells displayed a giant cell morphology and were binucleate, indicative of a trophoblast-like morphology. Other trophoblast-like properties included the intermediary filament Troma-1/cytokeratin 8 and the transcription factor Tead4. A decrease in CpG methylation at three sites in the TEAD4 promoter and the B1 repeated sequence was observed. Knocking down expression of Tead4 with siRNA blocked the increase in Troma-1/cytokeratin 8 and over expression of Tead4 induced the expression of Troma-1/cytokeratin 8. Cells treated for 5days with hydralazine were no longer capable of undergoing retinoic acid-mediated neuronal differentiation. An irreversible loss of the pluripotent transcription factor Oct-4 was observed following hydralazine exposure. In summary, hydralazine induces P19 cells to assume a trophoblast-like phenotype by upregulating Tead4 expression through a mechanism involving DNA demethylation.

SUBMITTER: O'Driscoll CM

PROVIDER: S-EPMC5859318 | biostudies-literature | 2013 Mar

REPOSITORIES: biostudies-literature

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Induction of a trophoblast-like phenotype by hydralazine in the p19 embryonic carcinoma cell line.

O'Driscoll Cliona M CM Coulter Jonathan B JB Bressler Joseph P JP

Biochimica et biophysica acta 20121126 3

Chemicals that affect cellular differentiation through epigenetic mechanisms have potential utility in treating a wide range of diseases. Hydralazine decreases DNA methylation in some cell types but its effect on differentiation has not been well explored. After five days of exposure to hydralazine, P19 embryocarcinoma cells displayed a giant cell morphology and were binucleate, indicative of a trophoblast-like morphology. Other trophoblast-like properties included the intermediary filament Trom ...[more]

PMID: 23195226

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Project description:Given the relatively long life of stem cells (SCs), efficient mechanisms of quality control to balance cell survival and resistance to external and internal stress are required. Our objective was to test the relevance of cell quality control mechanisms for SCs maintenance, differentiation and resistance to cell death. We compared cell quality control in P19 stem cells (P19SCs) before and after differentiation (P19dCs). Differentiation of P19SCs resulted in alterations in parameters involved in cell survival and protein homeostasis, including the redox system, cardiolipin and lipid profiles, unfolded protein response, ubiquitin-proteasome and lysosomal systems, and signaling pathways controlling cell growth. In addition, P19SCs pluripotency was correlated with stronger antioxidant protection, modulation of apoptosis, and activation of macroautophagy, which all contributed to preserve SCs quality by increasing the threshold for cell death activation. Furthermore, our findings identify critical roles for the PI3K-AKT-MTOR pathway, as well as autophagic flux and apoptosis regulation in the maintenance of P19SCs pluripotency and differentiation potential.Abbreviations: 3-MA: 3-methyladenine; AKT/protein kinase B: thymoma viral proto-oncogene; AKT1: thymoma viral proto-oncogene 1; ATG: AuTophaGy-related; ATF6: activating transcription factor 6; BAX: BCL2-associated X protein; BBC3/PUMA: BCL2 binding component 3; BCL2: B cell leukemia/lymphoma 2; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CASP3: caspase 3; CASP8: caspase 8; CASP9: caspase 9; CL: cardiolipin; CTSB: cathepsin B; CTSD: cathepsin D; DDIT3/CHOP: DNA-damage inducible transcript 3; DNM1L/DRP1: dynamin 1-like; DRAM1: DNA-damage regulated autophagy modulator 1; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EIF2S1/eIF2α: eukaryotic translation initiation factor 2, subunit alpha; ERN1/IRE1α: endoplasmic reticulum to nucleus signaling 1; ESCs: embryonic stem cells; KRT8/TROMA-1: cytokeratin 8; LAMP2A: lysosomal-associated membrane protein 2A; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NANOG: Nanog homeobox; NAO: 10-N-nonyl acridine orange; NFE2L2/NRF2: nuclear factor, erythroid derived 2, like 2; OPA1: OPA1, mitochondrial dynamin like GTPase; P19dCs: P19 differentiated cells; P19SCs: P19 stem cells; POU5F1/OCT4: POU domain, class 5, transcription factor 1; PtdIns3K: phosphatidylinositol 3-kinase; RA: retinoic acid; ROS: reactive oxygen species; RPS6KB1/p70S6K: ribosomal protein S6 kinase, polypeptide 1; SCs: stem cells; SOD: superoxide dismutase; SHC1-1/p66SHC: src homology 2 domain-containing transforming protein C1, 66 kDa isoform; SOX2: SRY (sex determining region Y)-box 2; SQSTM1/p62: sequestosome 1; SPTAN1/αII-spectrin: spectrin alpha, non-erythrocytic 1; TOMM20: translocase of outer mitochondrial membrane 20; TRP53/p53: transformation related protein 53; TUBB3/betaIII-tubulin: tubulin, beta 3 class III; UPR: unfolded protein response; UPS: ubiquitin-proteasome system.

Project description:BackgroundSilver nanoparticles (AgNP), the most popular nano-compounds, possess unique properties. Albizia adianthifolia (AA) is a plant of the Fabaceae family that is rich in saponins. The biological properties of a novel AgNP, synthesized from an aqueous leaf extract of AA (AA(AgNP)), were investigated on A549 lung cells. Cell viability was determined by the MTT assay. Cellular oxidative status (lipid peroxidation and glutathione (GSH) levels), ATP concentration, caspase-3/-7, -8 and -9 activities were determined. Apoptosis, mitochondrial (mt) membrane depolarization (flow cytometry) and DNA fragmentation (comet assay) were assessed. The expression of CD95 receptors, p53, bax, PARP-1 and smac/DIABLO was evaluated by flow cytometry and/or western blotting.ResultsSilver nanoparticles of AA caused a dose-dependent decrease in cell viability with a significant increase in lipid peroxidation (5-fold vs. control; p?=?0.0098) and decreased intracellular GSH (p?=?0.1184). A significant 2.5-fold decrease in cellular ATP was observed upon AA(AgNP) exposure (p?=?0.0040) with a highly significant elevation in mt depolarization (3.3-fold vs. control; p?<?0.0001). Apoptosis was also significantly higher (1.5-fold) in AA(AgNP) treated cells (p?<?0.0001) with a significant decline in expression of CD95 receptors (p?=?0.0416). Silver nanoparticles of AA caused a significant 2.5-fold reduction in caspase-8 activity (p?=?0.0024) with contrasting increases in caspase-3/-7 (1.7-fold vs. control; p?=?0.0180) and -9 activity (1.4-fold vs. control; p?=?0.0117). Western blots showed increased expression of smac/DIABLO (4.1-fold) in treated cells (p?=?0.0033). Furthermore, AA(AgNP) significantly increased the expression of p53, bax and PARP-1 (1.2-fold; p?=?0.0498, 1.6-fold; p?=?0.0083 and 1.1-fold; p?=?0.0359 respectively).ConclusionData suggests that AA(AgNP) induces cell death in the A549 lung cells via the mt mediated intrinsic apoptotic program. Further investigation is required to potentiate the use of this novel compound in cancer therapy trials.

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Induction of a trophoblast-like phenotype by hydralazine in the p19 embryonic carcinoma cell line.

Publications

Induction of a trophoblast-like phenotype by hydralazine in the p19 embryonic carcinoma cell line.

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