A stable O2-resistant cell line: role of lipid peroxidation byproducts in O2-mediated injury

1992 ◽  
Vol 262 (6) ◽  
pp. L748-L756 ◽  
Author(s):  
S. J. Sullivan ◽  
T. D. Oberley ◽  
R. J. Roberts ◽  
D. R. Spitz
Keyword(s):  
Lipid Peroxidation ◽  
Superoxide Dismutase ◽  
Cell Line ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Antioxidant Enzyme Activities ◽  
Enzymatic System ◽  
Glutathione S Transferase ◽  
Toxic Byproduct

HA-1 hamster fibroblasts receiving fresh media every 24 h were continuously passaged in progressively increasing O2 concentrations for 18 mo (designated O2R95). These cells were significantly more resistant than parental HA-1 to clonogenic inactivation mediated by 95% O2 without media replacement. The O2R95 cell line exhibited increases in the activities of catalase (CAT), Mn superoxide dismutase (MnSOD), Cu,Zn superoxide dismutase (Cu,Zn SOD), and glutathione peroxidase (GPx). O2R95 cells demonstrated uniformly distributed increased staining for CAT, MnSOD, Cu,Zn SOD, and GPx proteins, as determined by immunohistochemistry. Cellular resistance to and metabolism of 4-hydroxy-2-nonenal (4HNE), a toxic byproduct of lipid peroxidation implicated in mechanisms of O2 toxicity, was examined in HA-1 and O2R95 cell lines. O2R95 cells were significantly more resistant to 4HNE cytotoxicity, which was accompanied by a significant increase in 4HNE metabolism. O2R95 cells also demonstrated an increase in total glutathione (GSH) and glutathione S-transferase (GST) activity, an enzymatic system believed to be involved with 4HNE metabolism. Furthermore, homogenates from O2R95 cells consumed greater quantities of 4HNE in the presence of NADPH (but not NADH, NAD+, or NADP+), suggesting that an enzyme(s) utilizing NADPH contributes to 4HNE metabolism, resistance to 95% O2 and 4HNE as well as increased total GSH, antioxidant enzyme activities, and NADPH-dependent metabolism of 4HNE, persisted in O2R95 cells for 75 days of growth in 21% O2. These findings are compatible with the hypothesis that aldehydic byproducts of lipid peroxidation contribute to mechanisms of O2 toxicity and the selective pressure exerted by exposure of cells to hyperoxia.(ABSTRACT TRUNCATED AT 250 WORDS).

K562-R As a Model to Study Jak2 in a Non Bcr-Abl Addicted Cell Line

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4412-4412
Author(s):  
Bastianella Perazzona ◽  
Yu-Hsi Lin ◽  
Ralph B. Arlinghaus
Keyword(s):  
Tyrosine Kinase ◽  
Cell Line ◽  
Cell Lines ◽  
K562 Cells ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Janus Kinase ◽  
Cell Fractionation ◽  
Lyn Kinase

Abstract Abstract 4412 Chronic myeloid leukemia (CML) is a hematological disease caused by the fusion protein Bcr-Abl tyrosine kinase. Development of the tyrosine kinase inhibitor Imatinib Mesylate (IM) has significantly improved the long-term survival of early stage CML patients. However, occurrence of drug resistance, permanence of residual disease and recurrence of active leukemia if IM is discontinued, remain problems awaiting solution. Therefore, new therapeutic strategies aimed at targeting alternative signaling pathways or CML progenitor cells that survive IM treatment are needed. We have previously shown that Janus kinase 2 (Jak2) is activated in Bcr-Abl+ cells. We have demonstrated that reduction of Jak2 activity by the Jak2-specific inhibitor TG101209 (TG) or by genetic knock down (Jak2 shRNA and siRNA) in Bcr-Abl+ cell lines, IM-resistant cells and CML blast crisis cell lines resulted in reduced levels of phosphorylation of Tyr177 and of total Bcr-Abl protein. Jak2 inhibition results in diminished activation of the Ras, PI-3 kinase pathways and reduced levels of pTyrSTAT5 (Samanta et al., Leukemia 2011). During these studies we observed that K562 cells and IM-resistant cell line K562-R had different susceptibility to the effect of TG, with K562-R showing increase sensitivity to lower concentrations of TG resulting in faster destabilization of the Bcr-Abl protein. Based on these observations, we hypothesized that increased sensitivity of the K562-R cells was due to the different state of activation of Jak2. In addition, based on recent studies by (Dawson et al., Nat 2009) and by (Rinaldi et al., Blood 2010) we also hypothesized that different levels of Jak2 activation may influence the localization of Jak2 in the cell. We used cell fractionation and western blotting analysis to show that in K562-R cells, active Jak2 is mostly localized in the nucleus with a minor pool found in the cytoplasm. In K562 cells, active Jak2 is equally distributed in both cytoplasmic and nuclear compartment. In addition, immuno-fluorescence confocal analysis of total Jak2 distribution in K562 shows a very organized localization of Jak2 at one pole of the cells but this organization is lost in K562-R and total Jak2 appears uniformly distributed within the cell. K562-R cells were isolated as a Bcr-Abl independent IM resistant cell line that expressed high levels of activated Lyn kinase (Donato et al., Blood 2003). We used K562-R as a model to study the role of Jak2 in a non Bcr-Abl addicted cell line. Since we have previously published that Jak2 up-regulates Lyn kinase activity (Samanta et al., Oncogene 2009), we propose that the higher activation of Jak2 in K562-R is the main driver of oncogenicity and IM resistance and that this cell line may be used to model the role of Jak2 in a cell that is not Bcr-Abl “addicted.” Disclosures: No relevant conflicts of interest to declare.

scholarly journals Exosomal lncRNA HNF1A-AS1 affects cisplatin resistance in cervical cancer cells through regulating microRNA-34b/TUFT1 axis

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Xiaoqiong Luo ◽  
Jingxi Wei ◽  
Feng-lian Yang ◽  
Xiao-xia Pang ◽  
Feng Shi ◽  
...  
Keyword(s):  
Cervical Cancer ◽  
Drug Resistance ◽  
Cell Line ◽  
Nude Mice ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Cell Line Hela ◽  
Cervical Cancer Cells ◽  
Line Hela

Abstract Background There is growing evidence of the role of long non-coding RNAs (lncRNAs) in cervical cancer (CC). The objective was to discuss whether exosomal lncRNA HNF1A-AS1 impacted drug resistance in CC via binding to microRNA-34b (miR-34b) and regulating TUFT1 expression. Methods The expression of HNF1A-AS1 in normal cervical epithelial cells, cisplatin (DDP)-sensitive cell line (HeLa/S) and DDP-resistant cell line (HeLa/DDP) cells were detected. HeLa/S and HeLa/DDP cells were interfered with HNF1A-AS1 to determine IC50, proliferation, colony formation and apoptosis of CC cells. The exosomes were isolated and identified. Subcellular localization of HNF1A-AS1, expression of miR-34b and TUFT1 in receptor cells were also verified. The binding site between HNF1A-AS1 and miR-34b, together with miR-34b and TUFT1 were confirmed. Tumorigenic ability of cells in nude mice was also detected. Results HNF1A-AS1 was upregulated in DDP-resistant cell line HeLa/DDP. Silencing HNF1A-AS1 suppressed CC cell proliferation and promoted its apoptosis. HNF1A-AS1 was found to act as a competing endogenous RNA (ceRNA) of miR-34b to promote the expression of TUFT1. Exosomes shuttled HNF1A-AS1 promoted the proliferation and drug resistance of CC cells and inhibited their apoptosis by upregulating the expression of TUFT1 and downregulating miR-34b. Furthermore, suppressed exosomal HNF1A-AS1 in combination with DDP inhibited tumor growth in nude mice. Conclusion Our study provides evidence that CC-secreted exosomes carrying HNF1A-AS1 as a ceRNA of miR-34b to promote the expression of TUFT1, thereby promoting the DDP resistance in CC cells.

scholarly journals Genomic Adaption and Mutational Patterns in a HaCaT Subline Resistant to Alkylating Agents and Ionizing Radiation

2021 ◽  
Vol 22 (3) ◽  
pp. 1146
Author(s):  
Reinhard Ullmann ◽  
Benjamin Valentin Becker ◽  
Simone Rothmiller ◽  
Annette Schmidt ◽  
Horst Thiermann ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Line ◽  
Copy Number ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Chromosomal Translocations ◽  
Dna Copy Number ◽  
Mutational Signatures ◽  
Copy Number Changes ◽  
And Oxidative Stress

Sulfur mustard (SM) is a chemical warfare agent that can damage DNA via alkylation and oxidative stress. Because of its genotoxicity, SM is cancerogenic and the progenitor of many chemotherapeutics. Previously, we developed an SM-resistant cell line via chronic exposure of the popular keratinocyte cell line HaCaT to increasing doses of SM over a period of 40 months. In this study, we compared the genomic landscape of the SM-resistant cell line HaCaT/SM to its sensitive parental line HaCaT in order to gain insights into genetic changes associated with continuous alkylation and oxidative stress. We established chromosome numbers by cytogenetics, analyzed DNA copy number changes by means of array Comparative Genomic Hybridization (array CGH), employed the genome-wide chromosome conformation capture technique Hi-C to detect chromosomal translocations, and derived mutational signatures by whole-genome sequencing. We observed that chronic SM exposure eliminated the initially prevailing hypotetraploid cell population in favor of a hyperdiploid one, which contrasts with previous observations that link polyploidization to increased tolerance and adaptability toward genotoxic stress. Furthermore, we observed an accumulation of chromosomal translocations, frequently flanked by DNA copy number changes, which indicates a high rate of DNA double-strand breaks and their misrepair. HaCaT/SM-specific single-nucleotide variants showed enrichment of C > A and T > A transversions and a lower rate of deaminated cytosines in the CpG dinucleotide context. Given the frequent use of HaCaT in toxicology, this study provides a valuable data source with respect to the original genotype of HaCaT and the mutational signatures associated with chronic alkylation and oxidative stress.

scholarly journals Effect of Cross-Sex Hormonal Replacement on Antioxidant Enzymes in Rat Retroperitoneal Fat Adipocytes

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Israel Pérez-Torres ◽  
Verónica Guarner-Lans ◽  
Alejandra Zúñiga-Muñoz ◽  
Rodrigo Velázquez Espejel ◽  
Alfredo Cabrera-Orefice ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Superoxide Dismutase ◽  
Antioxidant Enzymes ◽  
Glutathione Peroxidase ◽  
Glutathione Reductase ◽  
Enzymatic Activities ◽  
Glutathione S Transferase ◽  
Control Groups ◽  
Intact Female ◽  
Hormonal Replacement

We report the effect of cross-sex hormonal replacement on antioxidant enzymes from rat retroperitoneal fat adipocytes. Eight rats of each gender were assigned to each of the following groups: control groups were intact female or male (F and M, resp.). Experimental groups were ovariectomized F (OvxF), castrated M (CasM), OvxF plus testosterone (OvxF + T), and CasM plus estradiol (CasM + E2) groups. After sacrifice, retroperitoneal fat was dissected and processed for histology. Adipocytes were isolated and the following enzymatic activities were determined: Cu-Zn superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR). Also, glutathione (GSH) and lipid peroxidation (LPO) were measured. In OvxF, retroperitoneal fat increased and adipocytes were enlarged, while in CasM rats a decrease in retroperitoneal fat and small adipocytes are observed. The cross-sex hormonal replacement in F rats was associated with larger adipocytes and a further decreased activity of Cu-Zn SOD, CAT, GPx, GST, GR, and GSH, in addition to an increase in LPO. CasM + E2exhibited the opposite effects showing further activation antioxidant enzymes and decreases in LPO. In conclusion, E2deficiency favors an increase in retroperitoneal fat and large adipocytes. Cross-sex hormonal replacement in F rats aggravates the condition by inhibiting antioxidant enzymes.

Platinum pyridinecarboxaldimine complexes containing boronate esters

2004 ◽  
Vol 82 (12) ◽  
pp. 1692-1699 ◽  
Author(s):  
Hanni A Darwish ◽  
Stephen J Scales ◽  
Jennifer L Horton ◽  
Liliya G Nikolcheva ◽  
Haiwen Zhang ◽  
...  
Keyword(s):  
Ovarian Carcinoma ◽  
Key Words ◽  
Cell Line ◽  
Platinum Complexes ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Boronate Esters ◽  
Human Ovarian Carcinoma

Condensation of 2-pyridinecarboxaldehydes with 2-, 3-, and 4-H2NC6H4Bpin (pin = 1,2-O2C2Me4) gave the corresponding boron-containing pyridinecarboxaldimines (N–NBpin). Addition of these ligands to [PtCl2(coe)]2 (coe = cis-cyclooctene) gave complexes of the type cis-PtCl2(N–NBpin) in moderate yields. The platinum complexes have been examined for their potential cytotoxicities against OV2008 (human ovarian carcinoma) and the analogous cisplatin-resistant cell line C13. Key words: boronate esters, pyridinecarboxaldimines, cytotoxicity, platinum, boron.

An immortalized drug-resistant cell line established from 12–13-day mouse embryos for the propagation of human embryonic stem cells

2006 ◽  
Vol 74 (4) ◽  
pp. 160-166 ◽  
Author(s):  
Shiro Iuchi ◽  
Meytha Marsch-Moreno ◽  
Cristina Velez-DelValle ◽  
Karen Easley ◽  
Walid Kuri-Harcuch ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Line ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Mouse Embryos ◽  
Drug Resistant ◽  
Drug Resistant Cell
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