Conditional Overexpression of the Wild-Type Gsα as the gsp Oncogene Initiates Chronic Extracellularly Regulated Kinase 1/2 Activation and Hormone Hypersecretion in Pituitary Cell Lines

Pevonedistat is a neddylation inhibitor that blocks proteasomal degradation of cullin–RING ligase (CRL) proteins involved in the degradation of short-lived regulatory proteins, including those involved with cell-cycle regulation. We determined the sensitivity and mechanism of action of pevonedistat cytotoxicity in neuroblastoma. Pevonedistat cytotoxicity was assessed using cell viability assays and apoptosis. We examined mechanisms of action using flow cytometry, bromodeoxyuridine (BrDU) and immunoblots. Orthotopic mouse xenografts of human neuroblastoma were generated to assess in vivo anti-tumor activity. Neuroblastoma cell lines were very sensitive to pevonedistat (IC50 136–400 nM). The mechanism of pevonedistat cytotoxicity depended on p53 status. Neuroblastoma cells with mutant (p53MUT) or reduced levels of wild-type p53 (p53si-p53) underwent G2-M cell-cycle arrest with rereplication, whereas p53 wild-type (p53WT) cell lines underwent G0-G1 cell-cycle arrest and apoptosis. In orthotopic neuroblastoma models, pevonedistat decreased tumor weight independent of p53 status. Control mice had an average tumor weight of 1.6 mg + 0.8 mg versus 0.5 mg + 0.4 mg (p < 0.05) in mice treated with pevonedistat. The mechanism of action of pevonedistat in neuroblastoma cell lines in vitro appears p53 dependent. However, in vivo studies using mouse neuroblastoma orthotopic models showed a significant decrease in tumor weight following pevonedistat treatment independent of the p53 status. Novel chemotherapy agents, such as the NEDD8-activating enzyme (NAE) inhibitor pevonedistat, deserve further study in the treatment of neuroblastoma.

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Phosphoglycolate has profound metabolic effects but most likely no role in a metabolic DNA response in cancer cell lines

Biochemical Journal ◽

10.1042/bcj20180435 ◽

2019 ◽

Vol 476 (4) ◽

pp. 629-643 ◽

Cited By ~ 1

Author(s):

Isabelle Gerin ◽

Marina Bury ◽

Francesca Baldin ◽

Julie Graff ◽

Emile Van Schaftingen ◽

...

Keyword(s):

Dna Damage ◽

Cell Lines ◽

Oxidative Dna Damage ◽

Fold Increase ◽

Metabolic Effects ◽

Phosphoglycerate Mutase ◽

Wild Type ◽

Metabolic Disturbances ◽

Phosphoglycolate Phosphatase ◽

Damage Repair

Abstract Repair of a certain type of oxidative DNA damage leads to the release of phosphoglycolate, which is an inhibitor of triose phosphate isomerase and is predicted to indirectly inhibit phosphoglycerate mutase activity. Thus, we hypothesized that phosphoglycolate might play a role in a metabolic DNA damage response. Here, we determined how phosphoglycolate is formed in cells, elucidated its effects on cellular metabolism and tested whether DNA damage repair might release sufficient phosphoglycolate to provoke metabolic effects. Phosphoglycolate concentrations were below 5 µM in wild-type U2OS and HCT116 cells and remained unchanged when we inactivated phosphoglycolate phosphatase (PGP), the enzyme that is believed to dephosphorylate phosphoglycolate. Treatment of PGP knockout cell lines with glycolate caused an up to 500-fold increase in phosphoglycolate concentrations, which resulted largely from a side activity of pyruvate kinase. This increase was much higher than in glycolate-treated wild-type cells and was accompanied by metabolite changes consistent with an inhibition of phosphoglycerate mutase, most likely due to the removal of the priming phosphorylation of this enzyme. Surprisingly, we found that phosphoglycolate also inhibits succinate dehydrogenase with a Ki value of <10 µM. Thus, phosphoglycolate can lead to profound metabolic disturbances. In contrast, phosphoglycolate concentrations were not significantly changed when we treated PGP knockout cells with Bleomycin or ionizing radiation, which are known to lead to the release of phosphoglycolate by causing DNA damage. Thus, phosphoglycolate concentrations due to DNA damage are too low to cause major metabolic changes in HCT116 and U2OS cells.

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Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

Molecular and Cellular Biology ◽

10.1128/mcb.8.10.4185 ◽

1988 ◽

Vol 8 (10) ◽

pp. 4185-4189 ◽

Cited By ~ 7

Author(s):

J A Greenspan ◽

F M Xu ◽

R L Davidson

Keyword(s):

Cell Line ◽

Cell Lines ◽

Dna Sequences ◽

Mammalian Cells ◽

Molecular Mechanisms ◽

Shuttle Vector ◽

Ethyl Methanesulfonate ◽

Wild Type ◽

Single Base ◽

Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

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CYP1A1 and CYP1A2 expression: Comparing ‘humanized’ mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

Toxicology and Applied Pharmacology ◽

10.1016/j.taap.2009.03.001 ◽

2009 ◽

Vol 237 (1) ◽

pp. 119-126 ◽

Cited By ~ 21

Author(s):

Shigeyuki Uno ◽

Kaori Endo ◽

Yuji Ishida ◽

Chise Tateno ◽

Makoto Makishima ◽

...

Keyword(s):

Cell Lines ◽

Wild Type ◽

Humanized Mouse ◽

Mouse Hepatoma ◽

Human And Mouse ◽

Mouse Lines

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DNA tumor virus oncoproteins and retinoblastoma gene mutations share the ability to relieve the cell's requirement for cyclin D1 function in G1.

The Journal of Cell Biology ◽

10.1083/jcb.125.3.625 ◽

1994 ◽

Vol 125 (3) ◽

pp. 625-638 ◽

Cited By ~ 176

Author(s):

J Lukas ◽

H Müller ◽

J Bartkova ◽

D Spitkovsky ◽

A A Kjerulff ◽

...

Keyword(s):

Cell Cycle ◽

Complex Formation ◽

Cyclin D1 ◽

Cell Lines ◽

T Antigen ◽

Regulatory Function ◽

Retinoblastoma Gene ◽

Checkpoint Control ◽

Wild Type ◽

In Cells

The retinoblastoma gene product (pRB) participates in the regulation of the cell division cycle through complex formation with numerous cellular regulatory proteins including the potentially oncogenic cyclin D1. Extending the current view of the emerging functional interplay between pRB and D-type cyclins, we now report that cyclin D1 expression is positively regulated by pRB. Cyclin D1 mRNA and protein is specifically downregulated in cells expressing SV40 large T antigen, adenovirus E1A, and papillomavirus E7/E6 oncogene products and this effect requires intact RB-binding, CR2 domain of E1A. Exceptionally low expression of cyclin D1 is also seen in genetically RB-deficient cell lines, in which ectopically expressed wild-type pRB results in specific induction of this G1 cyclin. At the functional level, antibody-mediated cyclin D1 knockout experiments demonstrate that the cyclin D1 protein, normally required for G1 progression, is dispensable for passage through the cell cycle in cell lines whose pRB is inactivated through complex formation with T antigen, E1A, or E7 oncoproteins as well as in cells which have suffered loss-of-function mutations of the RB gene. The requirement for cyclin D1 function is not regained upon experimental elevation of cyclin D1 expression in cells with mutant RB, while reintroduction of wild-type RB into RB-deficient cells leads to restoration of the cyclin D1 checkpoint. These results strongly suggest that pRB serves as a major target of cyclin D1 whose cell cycle regulatory function becomes dispensable in cells lacking functional RB. Based on available data including this study, we propose a model for an autoregulatory feedback loop mechanism that regulates both the expression of the cyclin D1 gene and the activity of pRB, thereby contributing to a G1 phase checkpoint control in cycling mammalian cells.

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Mutation at autosomal loci of Chinese hamster ovary cells: involvement of a high-frequency event silencing two linked alleles

Molecular and Cellular Biology ◽

10.1128/mcb.3.7.1172-1181.1983 ◽

1983 ◽

Vol 3 (7) ◽

pp. 1172-1181

Author(s):

W E Bradley

Keyword(s):

Cell Lines ◽

Chinese Hamster Ovary ◽

High Frequency ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Low Frequency ◽

Class Ii ◽

Class I ◽

Wild Type ◽

Ovary Cells

Two classes of cell lines heterozygous at the galactokinase (glk) locus have been isolated from Chinese hamster ovary cells. Class I, selected by plating nonmutagenized wild-type cells at low density in medium containing 2-deoxygalactose at a partially selective concentration, underwent subsequent mutation to the glk-/- genotype at a low frequency (approximately 10(-6) per cell), which was increased by mutagenesis. Class II heterozygotes, isolated by sib selection from mutagenized wild-type cells, had a higher spontaneous frequency of mutation to the homozygous state (approximately 10(-4) per cell), which was not affected by mutagenesis. About half of the glk-/- mutants derived from a class II heterozygote, but not the heterozygote itself, were functionally hemizygous at the syntenic thymidine kinase (tk) locus. Similarly, a tk+/- heterozygote with characteristics analogous to the class II glk+/- cell lines underwent high-frequency mutation to tk-/-, and most of these mutants, but not the tk+/- heterozygote, were functionally hemizygous at the glk locus. A model is proposed, similar to that for the mutational events at the adenine phosphoribosyl transferase locus (W. E. C. Bradley and D. Letovanec, Somatic Cell Genet. 8:51-66, 1982), of two different events, high and low frequency, being responsible for mutation at either of the linked loci tk and glk. The low-frequency event may be a point mutation, but the high-frequency event, in many instances, involves coordinated inactivation of a portion of a chromosome carrying the two linked alleles. Class II heterozygotes would be generated as a result of a low-frequency event at one allele, and class I heterozygotes would be generated by a high-frequency event. Supporting this model was the demonstration that all class I glk+/- lines examined were functionally hemizygous at tk.

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Extracellular pH alkalinization by Cl−/HCO3− exchanger is crucial for TASK2 activation by hypotonic shock in proximal cell lines from mouse kidney

AJP Renal Physiology ◽

10.1152/ajprenal.00132.2006 ◽

2007 ◽

Vol 292 (2) ◽

pp. F628-F638 ◽

Cited By ~ 16

Author(s):

S. L'Hoste ◽

H. Barriere ◽

R. Belfodil ◽

I. Rubera ◽

C. Duranton ◽

...

Keyword(s):

Cell Lines ◽

Regulatory Volume Decrease ◽

Inhibitory Effect ◽

Buffering Capacity ◽

Mouse Kidney ◽

Wild Type ◽

Cytosolic Ph ◽

Hypotonic Shock ◽

External Ph ◽

Volume Decrease

We have previously shown that K+-selective TASK2 channels and swelling-activated Cl− currents are involved in a regulatory volume decrease (RVD; Barriere H, Belfodil R, Rubera I, Tauc M, Lesage F, Poujeol C, Guy N, Barhanin J, Poujeol P. J Gen Physiol 122: 177–190, 2003; Belfodil R, Barriere H, Rubera I, Tauc M, Poujeol C, Bidet M, Poujeol P. Am J Physiol Renal Physiol 284: F812–F828, 2003). The aim of this study was to determine the mechanism responsible for the activation of TASK2 channels during RVD in proximal cell lines from mouse kidney. For this purpose, the patch-clamp whole-cell technique was used to test the effect of pH and the buffering capacity of external bath on Cl− and K+ currents during hypotonic shock. In the presence of a high buffer concentration (30 mM HEPES), the cells did not undergo RVD and did not develop outward K+ currents (TASK2). Interestingly, the hypotonic shock reduced the cytosolic pH (pHi) and increased the external pH (pHe) in wild-type but not in cftr −/− cells. The inhibitory effect of DIDS suggests that the acidification of pHi and the alkalinization of pHe induced by hypotonicity in wild-type cells could be due to an exit of HCO3−. In conclusion, these results indicate that Cl− influx will be the driving force for HCO3− exit through the activation of the Cl−/HCO3− exchanger. This efflux of HCO3− then alkalinizes pHe, which in turn activates TASK2 channels.

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Growth Properties of Neural Cell Lines Immortalized with the Tsa58 Allele of Sv40 Large T Antigen

Cell Transplantation ◽

10.1177/096368979700600306 ◽

1997 ◽

Vol 6 (3) ◽

pp. 231-238 ◽

Cited By ~ 5

Author(s):

M.E. Truckenmiller ◽

Ora Dillon-Carter ◽

Carlo Tornatore ◽

Henrietta Kulaga ◽

Hidetoshi Takashima ◽

...

Keyword(s):

Amino Acid ◽

Cell Line ◽

Cell Lines ◽

T Antigen ◽

Temperature Sensitive ◽

Permissive Temperature ◽

Large T Antigen ◽

Wild Type ◽

Sv40 Large T Antigen ◽

Growth Properties

In vitro growth properties of three CNS-derived cell lines were compared under a variety of culture conditions. The M213-20 and J30a cell lines were each derived from embryonic CNS culture with the temperature-sensitive (ts) allele of SV40 large T antigen, tsA58, while the A7 cell line was immortalized using wild-type SV40 large T antigen. Cells immortalized with tsA58 SV40 large T proliferate at the permissive temperature, 33° C, while growth is expected to be suppressed at the nonpermissive temperature, 39.5°C. Both the M213-20 and J30a cell lines were capable of proliferating at 39.5°C continuously for up to 6 mo. All three cell lines showed no appreciable differences in growth rates related to temperature over a 7-day period in either serum-containing or defined serum-free media. The percentage of cells in S-phase of the cell cycle did not decrease or was elevated at 39.5°C for all three cell lines. After 3 wk at 39.5°C, the three cell lines also showed positive immunostaining using two monoclonal antibodies reacting with different epitopes of SV40 large T antigen. Double strand DNA sequence analyses of a 300 base pair (bp) fragment of the large T gene from each cell line, which included the ts locus, revealed mutations in both the J30a and M213-20 cell lines. The J30a cell line ts mutation had reverted to wild type, and two additional loci with bp substitutions with predicted amino acid changes were also found. While the ts mutation of the M213-20 cells was retained, an additional bp substitution with a predicted amino acid change was found. The A7 cell line sequence was identical to the reference wild-type sequence. These findings suggest that (a) nucleic acid sequences in the temperature-sensitive region of the tsA58 allele of SV40 large T are not necessarily stable, and (b) temperature sensitivity of cell lines immortalized with tsA58 is not necessarily retained.

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MicroRNA-181a Inhibits Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Progression by Repressing CARD11

Journal of Oncology ◽

10.1155/2019/9832956 ◽

2019 ◽

Vol 2019 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Danxia Zhu ◽

Cheng Fang ◽

Wenting He ◽

Chen Wu ◽

Xiaodong Li ◽

...

Keyword(s):

B Cell ◽

Binding Site ◽

Cell Lines ◽

Expression Vector ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Wild Type ◽

Protein Levels ◽

Large B Cell Lymphoma ◽

Large B Cell

We investigated the role of miR-181a in diffuse large B-cell lymphoma (DLBCL) and its potential target genes. miR-181a levels were lower in activated B-cell- (ABC-) like DLBCL cells than that in germinal center B-cell- (GCB-) like DLBCL cells. Overexpression of miR-181a in ABC-like DLBCL cell lines (OCI-LY10 and U2932) resulted in G0/G1 cell cycle arrest, increased apoptosis, and decreased invasiveness. miRNA target prediction programs (miRanda, TargetScan, and miRDB) identified caspase recruitment domain-containing protein 11 (CARD11) as a putative miR-181a target. CARD11 mRNA and protein levels were higher in the ABC-like DLBCL than that in GCB-like DLBCL. Moreover, CARD11 mRNA and protein levels were downregulated in the OCI-LY10 and U2932 cell lines overexpressing miR-181a. Dual luciferase reporter assays confirmed the miR-181a binding site in the CARD11 3′UTR region. OCI-LY10 and U2932 cells transfected with a CARD11 expression vector encoding miR-181a with a mutated binding site showed higher CARD11 protein levels, cell viability, G2/M phase cells, and invasiveness compared to those transfected with a wild-type CARD11 expression vector. Nude mice xenografted with OCI-LY10 cells with overexpressed wild-type miR-181a generated smaller tumors compared to those with overexpressed mutated binding site of CARD11 3′UTR and miR-181a. These results indicate that miR-181a inhibits ABC-like DLBCL by repressing CARD11.

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