scholarly journals Nanotoxic Effects of Silver Nanoparticles on Normal HEK-293 Cells in Comparison to Cancerous HeLa Cell Line

2021 ◽  
Vol Volume 16 ◽  
pp. 753-761 ◽  
Author(s):  
Xiongwei Liu ◽  
Kuizhong Shan ◽  
Xiaxia Shao ◽  
Xianqing Shi ◽  
Yun He ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Hela Cell ◽  
Cell Line ◽  
Hela Cell Line ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

scholarly journals Stimulation of Intracellular Free Calcium in GH3 Cells byγ 3-Melanocyte-Stimulating Hormone. Involvement of a Novel Melanocortin Receptor?

Endocrinology ◽  
2001 ◽  
Vol 142 (1) ◽  
pp. 257-266 ◽  
Author(s):  
Lies Langouche ◽  
Morad Roudbaraki ◽  
Katrien Pals ◽  
Carl Denef
Keyword(s):  
Cell Line ◽  
Messenger Rna ◽  
Intracellular Free Calcium ◽  
Gh3 Cells ◽  
Free Calcium ◽  
Camp Accumulation ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Gh3 Cell

Abstract The melanocortin (MC) γ3MSH is a peptide that can be generated from the N-terminal domain of POMC and is believed to signal through the MC3 receptor. We recently showed that it induces a sustained rise in intracellular free calcium levels ([Ca2+]i) in a subpopulation of pituitary cells, particularly in the lactosomatotroph lineage. In the present study we report that γ3MSH and some analogs increase [Ca2+]i in the GH- and PRL-secreting GH3 cell line and evaluate on the basis of pharmacological experiments and gene expression studies which MC receptor may be involved. A dose as low as 1 pm γ3MSH induced an oscillating[ Ca2+]i increase in a significant percentage of GH3 cells. Increasing the dose recruited an increasing number of responding cells; a maximum was reached at 0.1 nm. γ2MSH,α MSH, and NDP-αMSH displayed a similar effect. SHU9119, an MC3 and MC4 receptor antagonist, and an MC5 receptor agonist, did not affect the number of cells showing a [Ca2+]i rise in response to γ3MSH. SHU9119 had also no effect when added alone. MTII, a potent synthetic agonist of the MC3, MC4, and MC5 receptor as well as an N-terminally extended recombinant analog of γ3MSH showed low potency in increasing [Ca2+]i in GH3 cells, but high potency in stimulating cAMP accumulation in HEK 293 cells stably transfected with the MC3 receptor. In contrast, a peptide corresponding to the γ2MSH sequence of POMC-A of Acipenser transmontanus increased [Ca2+]i in GH3 cells, but was about 50 times less potent than γ2- or γ3MSH in stimulating cAMP accumulation in the MC3 receptor expressing HEK 293 cells. By means of RT-PCR performed on a RNA extract from GH3 cells, the messenger RNA of the MC2, MC3, and MC4 receptor was undetectable, but messenger RNA of the MC5 receptor was clearly present. These data suggest that the GH3 cell line does not mediate the effect of γ3MSH through the MC3 receptor. The involvement of the MC5 receptor is unlikely, but cannot definitely be excluded. The findings animate the hypothesis that there exists a second, hitherto unidentified, MC receptor that displays high affinity for γ3MSH.

scholarly journals Regulation of GPR54 Signaling by GRK2 and β-Arrestin

2009 ◽  
Vol 23 (12) ◽  
pp. 2060-2074 ◽  
Author(s):  
Macarena Pampillo ◽  
Natasha Camuso ◽  
Jay E. Taylor ◽  
Jacob M. Szereszewski ◽  
Maryse R. Ahow ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Cell Line ◽  
Molecular Mechanisms ◽  
Control Cell ◽  
Cytoplasmic Tail ◽  
Intracellular Loop ◽  
Membrane Expression ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells

Abstract Kisspeptin and its receptor, GPR54, are major regulators of the hypothalamic-pituitary-gonadal axis as well as regulators of human placentation and tumor metastases. GPR54 is a Gq/11-coupled G protein-coupled receptor (GPCR), and activation by kisspeptin stimulates phosphatidy linositol 4, 5-biphosphate hydrolysis, Ca2+ mobilization, arachidonic acid release, and ERK1/2 MAPK phosphorylation. Physiological evidence suggests that GPR54 undergoes agonist-dependent desensitization, but underlying molecular mechanisms are unknown. Furthermore, very little has been reported on the early events that regulate GPR54 signaling. The lack of information in these important areas led to this study. Here we report for the first time on the role of GPCR serine/threonine kinase (GRK)2 and β-arrestin in regulating GPR54 signaling in human embryonic kidney (HEK) 293 cells, a model cell system for studying the molecular regulation of GPCRs, and genetically modified MDA MB-231 cells, an invasive breast cancer cell line expressing about 75% less β-arrestin-2 than the control cell line. Our study reveals that in HEK 293 cells, GPR54 is expressed both at the plasma membrane and intracellularly and also that plasma membrane expression is regulated by cytoplasmic tail sequences. We also demonstrate that GPR54 exhibits constitutive activity, internalization, and association with GRK2 and β- arrestins-1 and 2 through sequences in the second intracellular loop and cytoplasmic tail of the receptor. We also show that GRK2 stimulates the desensitization of GPR54 in HEK 293 cells and that β-arrestin-2 mediates GPR54 activation of ERK1/2 in MDA-MB-231 cells. The significance of these findings in developing molecular-based therapies for treating certain endocrine-related disorders is discussed.

Glyco-engineered HEK 293-F cell lines for the production of therapeutic glycoproteins with human N-glycosylation and improved pharmacokinetics

Glycobiology ◽  
2021 ◽  
Author(s):  
Rico Uhler ◽  
Ruth Popa-Wagner ◽  
Mario Kröning ◽  
Anja Brehm ◽  
Paul Rennert ◽  
...  
Keyword(s):  
Cell Line ◽  
Therapeutic Proteins ◽  
Coagulation Factor ◽  
Factor Vii ◽  
Hek 293 ◽  
Knock Out ◽  
Hek 293 Cells ◽  
Coagulation Factor Vii ◽  
293 Cells

Abstract N-glycosylated proteins produced in human embryonic kidney 293 (HEK 293) cells often carry terminal N-acetylgalactosamine (GalNAc) and only low levels of sialylation. On therapeutic proteins, such N-glycans often trigger rapid clearance from the patient bloodstream via efficient binding to asialoglycoprotein receptor (ASGP-R) and mannose receptor (MR). This currently limits the use of HEK 293 cells for therapeutic protein production. To eliminate terminal GalNAc, we knocked-out GalNAc transferases B4GALNT3 and B4GALNT4 by CRISPR/Cas9 in FreeStyle 293-F cells. The resulting cell line produced a coagulation factor VII-albumin fusion protein without GalNAc but with increased sialylation. This glyco-engineered protein bound less efficiently to both the ASGP-R and MR in vitro and it showed improved recovery, terminal half-life and area under the curve in pharmacokinetic rat experiments. By overexpressing sialyltransferases ST6GAL1 and ST3GAL6 in B4GALNT3 and B4GALNT4 knock-out cells, we further increased factor VII-albumin sialylation; for ST6GAL1 even to the level of human plasma-derived factor VII. Simultaneous knock-out of B4GALNT3 and B4GALNT4, and overexpression of ST6GAL1 further lowered factor VII-albumin binding to ASGP-R and MR. This novel glyco-engineered cell line is well-suited for the production of factor VII-albumin and presumably other therapeutic proteins with fully human N-glycosylation and superior pharmacokinetic properties.

Overloading and removal of N-glycosylation targets on human acetylcholinesterase: effects on glycan composition and circulatory residence time

2002 ◽  
Vol 363 (3) ◽  
pp. 619-631 ◽  
Author(s):  
Theodor CHITLARU ◽  
Chanoch KRONMAN ◽  
Baruch VELAN ◽  
Avigdor SHAFFERMAN
Keyword(s):  
Structural Analysis ◽  
Cell Line ◽  
Residence Time ◽  
Residence Times ◽  
Post Translational Modifications ◽  
Hek 293 ◽  
Hek 293 Cells ◽  
293 Cells ◽  
Mean Residence Times ◽  
Enzyme Species

Optimization of post-translational modifications was shown to affect the ability of recombinant human acetylcholinesterase (rHuAChE) produced in HEK-293 cells to be retained in the circulation for prolonged periods of time [Kronman, Velan, Marcus, Ordentlich, Reuveny and Shafferman (1995) Biochem. J. 311, 959–967; Chitlaru, Kronman, Zeevi, Kam, Harel, Ordentlich, Velan and Shafferman (1998) Biochem. J. 336, 647–658; Chitlaru, Kronman, Velan and Shafferman (2001) Biochem. J. 354, 613–625]. To evaluate the possible contribution of the number of appended N-glycans in determining the pharmacokinetic behaviour of AChE, a series of sixteen recombinant human AChE glycoforms, differing in their number of appended N-glycans (2, 3, 4 or 5 glycans), state of assembly (dimeric or tetrameric) and terminal glycan sialylation (partially or fully sialylated) were generated. Extensive structural analysis of N-glycans demonstrated that the various glycan types associated with all the different rHuAChE glycoforms are essentially similar both in structure and abundance, and that production of the various glycoforms in the sialyltransferase-overexpressing 293ST-2D6 cell line resulted in the generation of enzyme species that carry glycans sialylated to the same extent. Pharmacokinetic profiling of the rHuAChE glycoforms in their fully tetramerized and sialylated state clearly demonstrated that circulatory longevity correlated directly with the number of attached N-glycans (mean residence times for rHuAChE glycoforms harbouring 2, 3, and 4 glycans = 200, 740, and 1055min respectively). This study provides evidence that glycan loading, together with N-glycan terminal processing and enzyme subunit oligomerization, operate in a hierarchical and concerted manner in determining the pharmacokinetic characteristics of AChE.

Identification and Expression Analysis of CD73 Inhibitors in Cervical Cancer

2020 ◽  
Vol 16 ◽  
Author(s):  
Jamshed Iqbal ◽  
Ayesha Basharat ◽  
Sehrish Bano ◽  
Syed Mobasher Ali Abid ◽  
Julie Pelletier ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Western Blot ◽  
Hela Cell ◽  
Cell Line ◽  
Cell Apoptosis ◽  
Immunofluorescence Staining ◽  
Cell Cycle Analysis ◽  
Hela Cell Line ◽  
Cell Line Hela

Aims: The present study was conducted to examine the inhibitory effects of synthesized sulfonylhydrazones on the expression of CD73 (ecto-5′-NT). Background: CD73 (ecto-5′-NT) represents the most significant class of ecto-nucleotidases which are mainly responsible for dephosphorylation of adenosine monophosphate to adenosine. Inhibition of CD73 played an important role in the treatment of cancer, autoimmune disorders, precancerous syndromes, and some other diseases associated with CD73 activity. Objective: Keeping in view the significance of CD73 inhibitor in the treatment of cervical cancer, a series of sulfonylhydrazones (3a-3i) derivatives synthesized from 3-formylchromones were evaluated. Methods: All sulfonylhydrazones (3a-3i) were evaluated for their inhibitory activity towards CD73 (ecto-5′-NT) by the malachite green assay and their cytotoxic effect was investigated on HeLa cell line using MTT assay. Secondly, most potent compound was selected for cell apoptosis, immunofluorescence staining and cell cycle analysis. After that, CD73 mRNA and protein expression were analyzed by real-time PCR and Western blot. Results: Among all compounds, 3h, 3e, 3b, and 3c were found the most active against rat-ecto-5′-NT (CD73) enzyme with IC50 (µM) values of 0.70 ± 0.06 µM, 0.87 ± 0.05 µM, 0.39 ± 0.02 µM and 0.33 ± 0.03 µM, respectively. These derivatives were further evaluated for their cytotoxic potential against cancer cell line (HeLa). Compound 3h and 3c showed the cytotoxicity at IC50 value of 30.20 ± 3.11 µM and 86.02 ± 7.11 µM, respectively. Furthermore, compound 3h was selected for cell apoptosis, immunofluorescence staining and cell cycle analysis which showed promising apoptotic effect in HeLa cells. Additionally, compound 3h was further investigated for its effect on expression of CD73 using qRT-PCR and western blot. Conclusion: Among all synthesized compounds (3a-3i), Compound 3h (E)-N'-((6-ethyl-4-oxo-4H-chromen-3-yl) methylene)-4-methylbenzenesulfonohydrazide was identified as most potent compound. Additional expression studies conducted on HeLa cell line proved that this compound successfully decreased the expression level of CD73 and thus inhibiting the growth and proliferation of cancer cells.

Molecular events after antisense inhibition of hMSH2 in a HeLa cell line

1998 ◽  
Vol 418 (2-3) ◽  
pp. 61-71 ◽  
Author(s):  
Ying Qian ◽  
Yingnian Yu ◽  
Xingruo Cheng ◽  
Jianhong Luo ◽  
Haiyang Xie ◽  
...  
Keyword(s):  
Hela Cell ◽  
Cell Line ◽  
Antisense Inhibition ◽  
Hela Cell Line ◽  
Molecular Events
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