DNA methylation in 5-aza-2'-deoxycytidine-resistant variants of C3H 10T1/2 C18 cells

1984 ◽  
Vol 4 (10) ◽  
pp. 2098-2102
Author(s):  
E Flatau ◽  
F A Gonzales ◽  
L A Michalowsky ◽  
P A Jones
Keyword(s):  
Cell Line ◽  
Cytosine Methylation ◽  
De Novo ◽  
Lethal Dose ◽  
Cytotoxic Effects ◽  
Drug Treatments ◽  
A Cell ◽  
Mouse Cells ◽  
Low Levels ◽  
Dna Cytosine Methylation

A cell line (T17) was derived from C3H 10T1/2 C18 cells after 17 treatments with increasing concentrations of 5-aza-2'-deoxycytidine. The T17 cell line was very resistant to the cytotoxic effects of 5-aza-2'-deoxycytidine, and the 50% lethal dose for 5-aza-2'-deoxycytidine was ca. 3 microM, which was 30-fold greater than that of the parental C3H 10T1/2 C18 cells. Increased drug resistance was not due to a failure of the T17 cell line to incorporate 5-aza-2'-deoxycytidine into DNA. The cells were also slightly cross-resistant to 5-azacytidine. The percentage of cytosines modified to 5-methylcytosine in T17 cells was 0.7%, a 78% decrease from the level of 3.22% in C3H 10T1/2 C18 cells. The DNA cytosine methylation levels in several clones isolated from the treated lines were on the order of 0.7%, and clones with methylation levels lower than 0.45% were not obtained even after further drug treatments. These highly decreased methylation levels appeared to be unstable, and DNA modification increased as the cells divided in the absence of further drug treatment. The results suggest that it may not be possible to derive mouse cells with vanishingly low levels of 5-methylcytosine and that considerable de novo methylation can occur in cultured lines.

scholarly journals DNA methylation in 5-aza-2'-deoxycytidine-resistant variants of C3H 10T1/2 C18 cells.

1984 ◽  
Vol 4 (10) ◽  
pp. 2098-2102 ◽  
Author(s):  
E Flatau ◽  
F A Gonzales ◽  
L A Michalowsky ◽  
P A Jones
Keyword(s):  
Cell Line ◽  
Cytosine Methylation ◽  
De Novo ◽  
Lethal Dose ◽  
Cytotoxic Effects ◽  
Drug Treatments ◽  
A Cell ◽  
Mouse Cells ◽  
Low Levels ◽  
Dna Cytosine Methylation

A cell line (T17) was derived from C3H 10T1/2 C18 cells after 17 treatments with increasing concentrations of 5-aza-2'-deoxycytidine. The T17 cell line was very resistant to the cytotoxic effects of 5-aza-2'-deoxycytidine, and the 50% lethal dose for 5-aza-2'-deoxycytidine was ca. 3 microM, which was 30-fold greater than that of the parental C3H 10T1/2 C18 cells. Increased drug resistance was not due to a failure of the T17 cell line to incorporate 5-aza-2'-deoxycytidine into DNA. The cells were also slightly cross-resistant to 5-azacytidine. The percentage of cytosines modified to 5-methylcytosine in T17 cells was 0.7%, a 78% decrease from the level of 3.22% in C3H 10T1/2 C18 cells. The DNA cytosine methylation levels in several clones isolated from the treated lines were on the order of 0.7%, and clones with methylation levels lower than 0.45% were not obtained even after further drug treatments. These highly decreased methylation levels appeared to be unstable, and DNA modification increased as the cells divided in the absence of further drug treatment. The results suggest that it may not be possible to derive mouse cells with vanishingly low levels of 5-methylcytosine and that considerable de novo methylation can occur in cultured lines.

scholarly journals DNA Cytosine Methylation in the Bovine Leukemia Virus Promoter Is Associated with Latency in a Lymphoma-derived B-cell Line

2010 ◽  
Vol 285 (25) ◽  
pp. 19434-19449 ◽  
Author(s):  
Valérie Pierard ◽  
Allan Guiguen ◽  
Laurence Colin ◽  
Gaëlle Wijmeersch ◽  
Caroline Vanhulle ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Bovine Leukemia Virus ◽  
Cytosine Methylation ◽  
Leukemia Virus ◽  
Dna Cytosine Methylation ◽  
Virus Promoter

Differential nuclear protein binding to 5-azacytosine-containing DNA as a potential mechanism for 5-aza-2'-deoxycytidine resistance

1987 ◽  
Vol 7 (9) ◽  
pp. 3076-3083
Author(s):  
L A Michalowsky ◽  
P A Jones
Keyword(s):  
Protein Binding ◽  
Cell Line ◽  
Cytosine Methylation ◽  
Nuclear Protein ◽  
Protein Complexes ◽  
Lethal Dose ◽  
Tight Binding ◽  
Cell Types ◽  
Resistant Cell ◽  
Relative Increase

A clonal cell line (56-42) that was stably and exclusively resistant to the toxic effects of the antileukemic agent 5-aza-2'-deoxycytidine (5-aza-CdR) was derived from C3H 10T1/2 C18 cells after multiple treatments with 5-aza-CdR. The 50% lethal dose of 5-aza-CdR for these cells was 1.3 microM, which was 15-fold greater than that for the parental cells. Cell line 56-42 was slightly cross-resistant to the ribo-analog 5-azacytidine, but was sensitive to the nucleoside analog 1-beta-D-arabinofuranosylcytosine and to colcemid. Both parental and resistant cell lines incorporated equimolar amounts of 5-aza-CdR into DNA. Resistance was therefore not due to decreased activation, increased detoxification, or reduced incorporation of the drug. The overall level of cytosine methylation in the resistant clone was 80% lower than the level in the sensitive cells. Therefore, the potential number of hemimethylated sites created by the incorporation of equivalent amounts of 5-aza-CdR into the DNA of the two cell types was much greater in the sensitive cells. Furthermore, 5-azacytosine-substituted DNA from the sensitive cells bound 100% more nuclear protein in the form of highly stable complexes. The incorporation of 5-aza-CdR opposite methylated cytosine residues in DNA of the sensitive cells thus resulted in increased nuclear protein binding at hemimethylated sites. This relative increase in tight-binding protein complexes was shown to occur in living cells and may well disrupt replication and transcription and instigate cell death. The differential binding of proteins to hypomethylated, azacytosine-containing DNA may thus mediate a novel mechanism of drug resistance.

Glucocorticoids regulate surfactant protein synthesis in a pulmonary adenocarcinoma cell line

1989 ◽  
Vol 257 (6) ◽  
pp. L385-L392 ◽  
Author(s):  
M. A. O'Reilly ◽  
A. F. Gazdar ◽  
J. C. Clark ◽  
T. J. Pilot-Matias ◽  
S. E. Wert ◽  
...  
Keyword(s):  
Cell Line ◽  
Northern Blot Analysis ◽  
De Novo ◽  
Surfactant Protein ◽  
Surfactant Proteins ◽  
Cell Content ◽  
A Cell ◽  
The Media ◽  
Junctional Complexes ◽  
Pulmonary Surfactant Proteins

Synthesis of pulmonary surfactant proteins SP-A, SP-B, and SP-C was demonstrated in a cell line derived from a human adenocarcinoma of the lung. The cells contained numerous lamellar inclusion bodies and formed organized groups of cells containing well-developed junctional complexes and apical microvillous membranes. Synthesis of SP-A was detected in the cells by enzyme-linked immunoabsorbent assay and by immunoprecipitation of [35S]methionine-labeled protein. SP-A was identified as an Mr 31,000-36,000 polypeptide containing asparagine-linked carbohydrate. Northern blot analysis detected SP-A mRNA of 2.2 kb. Dexamethasone (1-10 nM) enhanced the relative abundance of SP-A mRNA. Despite stimulation of SP-A mRNA, intracellular SP-A content was unaltered or inhibited by dexamethasone. SP-B and SP-C mRNAs and synthesis of the SP-B and SP-C precursors were markedly induced by dexamethasone. ProSP-B was synthesized and secreted primarily as an Mr 42,000-46,000 polypeptide. Proteolysis of the proSP-B resulted in the generation of endoglycosidase F-sensitive Mr = 19,000-21,000 and 25,000-27,000 peptides, which were detected both intra- and extracellularly. SP-C proprotein of Mr = 22,000 and smaller SP-C fragments were detected intracellularly but were not detected in the media. Mature forms of SP-B (Mr = 8,000) and SP-C (Mr = 4,000) were not detected. Glucocorticoids directly enhance the relative synthesis and mRNA of the surfactant proteins SP-A, SP-B, and SP-C. Discrepancies among SP-A mRNA, its de novo synthesis, and cell content suggest that glucocorticoid may alter both pre- and posttranslational factors modulating SP-A expression.

Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer

1994 ◽  
Vol 14 (6) ◽  
pp. 4225-4232
Author(s):  
A N Magewu ◽  
P A Jones
Keyword(s):  
Cell Line ◽  
Cytosine Methylation ◽  
De Novo ◽  
Human Cancer ◽  
Hot Spot ◽  
Methylation Status ◽  
Osteogenic Sarcoma ◽  
P53 Gene ◽  
Alu Sequence ◽  
De Novo Methylation

Cytosine methylation at CpG dinucleotides is thought to cause more than one-third of all transition mutations responsible for human genetic diseases and cancer. We investigated the methylation status of the CpG dinucleotide at codon 248 in exon 7 of the p53 gene because this codon is a hot spot for inactivating mutations in the germ line and in most human somatic tissues examined. Codon 248 is contained within an HpaII site (CCGG), and the methylation status of this and flanking CpG sites was analyzed by using the methylation-sensitive enzymes CfoI (GCGC) and HpaII. Codon 248 and the CfoI and HpaII sites in the flanking introns were methylated in every tissue and cell line examined, indicating extensive methylation of this region in the p53 gene. Exhaustive treatment of an osteogenic sarcoma cell line, TE85, with the hypomethylating drug 5-aza-2'-deoxycytidine did not demethylate codon 248 or the CfoI sites in intron 6, although considerable global demethylation of the p53 gene was induced. Constructs containing either exon 7 alone or exon 7 and the flanking introns were transfected into TE85 cells to determine whether de novo methylation would occur. The presence of exon 7 alone caused some de novo methylation to occur at codon 248. More extensive de novo methylation of the CfoI sites in intron 6, which contains an Alu sequence, occurred in cells transfected with a vector containing exon 7 and flanking introns. With longer time in culture, there was increased methylation at the CfoI sites, and de novo methylation of codon 248 and its flanking HpaII sites was observed. These de novo-methylated sites were also resistant to 5-aza-2'-deoxycytidine-induced demethylation. The frequent methylation of codon 248 and adjacent Alu sequence may explain the enhanced mutability of this site as a result of the deamination of the 5-methylcytosine.

Non-centrosomal microtubule formation and measurement of minus end microtubule dynamics in A498 cells

1997 ◽  
Vol 110 (19) ◽  
pp. 2391-2401 ◽  
Author(s):  
A.M. Yvon ◽  
P. Wadsworth
Keyword(s):  
Cell Line ◽  
De Novo ◽  
Human Kidney ◽  
Light Level ◽  
Pause Duration ◽  
Living Cells ◽  
Live Cells ◽  
Microtubule Organizing Center ◽  
Organizing Center ◽  
A Cell

Experiments performed on a cell line (A498) derived from a human kidney carcinoma revealed non-centrosomal microtubules in the peripheral lamella of many cells. These short microtubules were observed in glutaraldehyde-fixed cells by indirect immunofluorescence, and in live cells injected with rhodamine-labeled tubulin. The non-centrosomal microtubules were observed to form de novo in living cells, and their complete disassembly was also observed. Low-light-level fluorescence microscopy, coupled to imaging software, was utilized to record and measure the dynamic behavior of both ends of the non-centrosomal microtubules in these cells. For each, the plus end was differentiated from the minus end using the ratio of their transition frequencies and by measuring total assembly at each end. For comparative purposes, dynamics of the plus ends of centrosomally nucleated microtubules were also analyzed in this cell line. Our data reveal several striking differences between the plus and minus ends. The average pause duration was nearly 4-fold higher at the minus ends; the percentage of time spent in pause was 92% at the minus ends, compared to 55% at plus ends. Dynamicity was decreased 4-fold at the minus ends, and the average number of events per minute was reduced from 7.0 at the plus end to 1.5 at the minus ends. The minus ends also showed a 6-fold decrease in frequency of catastrophe over the plus ends. These data demonstrate that in living cells, microtubules can form at sites distant from the perinuclear microtubule organizing center, and once formed, non-centrosomal microtubules can persist for relatively long periods.

scholarly journals Digital Karyotyping for Rapid Authentication of Cell Lines

2019 ◽  
Author(s):  
Ahmed Ibrahim Samir Khalil ◽  
Anupam Chattopadhyay ◽  
Amartya Sanyal
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
De Novo ◽  
Simulated Data ◽  
Read Depth ◽  
Cancer Cell Lines ◽  
Scientific Data ◽  
Cell Line Authentication ◽  
A Cell

Abstract Background The widespread concern about genetic drift and cross-contamination of cell lines calls for a pressing need for their authentication. The current genetic techniques for authentication are time-consuming and require specific documentary standard and laboratory protocols. Given the fact that whole-genome sequencing (WGS) data are readily available, read depth (RD)-based computational analyses has allowed the estimation of genetic profiles of cell lines. Results We propose WGS-derived aneuploidy profiling as a prototype of digital karyotyping for authentication of cancer cell lines. Here, we describe a Python-based software AStra for de novo estimation of the genome-wide aneuploidy profile, the copy number of every genomic loci, from raw WGS reads. We demonstrated that aneuploidy profile offers a unique signature that can distinguish the clonal variants (strains) of a cell line. We evaluated our approach using simulated data and variety of cancer cell lines. We further showed that cell lines exhibit distinct aneuploidy patterns which corroborate well with the experimental observations. Conclusions AStra is a simple, user-friendly, and free tool that provides the elementary information about the chromosomal aneuploidy for cell line authentication. AStra provides an analytical and visualization platform for rapid and easy comparison between different cell lines/strains. We recommend AStra for rapid first-pass quality assessment of scientific data that employ cancer cell lines. AStra is an open source software and is available at https://github.com/AISKhalil/AStra.

scholarly journals A Sub-Clone of RAW264.7-Cells Form Osteoclast-Like Cells Capable of Bone Resorption Faster than Parental RAW264.7 through Increased De Novo Expression and Nuclear Translocation of NFATc1

2020 ◽  
Vol 21 (2) ◽  
pp. 538 ◽  
Author(s):  
Laia Mira-Pascual ◽  
Anh N. Tran ◽  
Göran Andersson ◽  
Tuomas Näreoja ◽  
Pernilla Lång
Keyword(s):  
Cell Line ◽  
Nuclear Translocation ◽  
De Novo ◽  
Cathepsin K ◽  
Raw264.7 Cells ◽  
Large Numbers ◽  
Single Cell Cloning ◽  
Before And After ◽  
Low Levels ◽  
Rankl Stimulation

The murine macrophage cell line RAW264.7 is extensively used as a progenitor to study osteoclast (OC) differentiation. RAW264.7 is a heterogeneous cell line, containing sub-clones with different abilities to form OCs. The aim of this study was to identify characteristics within the heterogeneous RAW264.7 cells that define sub-clones with an augmented ability to form bone-resorbing OCs (H9), as well as sub-clones representing non-OCs (J8). RAW264.7 sub-clones were isolated by single cell cloning. Selection was based on TRAP/cathepsin K expression in sub-clone cultures without added RANKL. Sub-clones before and after differentiation with RANKL were assayed for multiple OC-characteristics. Sub-clone H9 cells presented a higher expression of OC-markers in cultures without added RANKL compared to the parental RAW264.7. After 6 days of RANKL stimulation, sub-clone H9 cells had equal expression levels of OC-markers with RAW264.7 and formed OCs able to demineralize hydroxyapatite. However, sub-clone H9 cells displayed rapid differentiation of OC already at Day 2 compared to Day 4 from parental RAW264.7, and when cultured on plastic and on bone they were more efficient in resorption. This rapid differentiation was likely due to high initial expression/nuclear translocation of OC master transcription factor, NFATc1. In contrast to H9, J8 cells expressed initially very low levels of OC-markers, and they did not respond to RANKL-stimulation by developing OC-characteristics/OC-marker expression. Hence, H9 is an additional clone suitable for experimental setup requiring rapid differentiation of large numbers of OCs.

scholarly journals Ubiquitous and tenacious methylation of the CpG site in codon 248 of the p53 gene may explain its frequent appearance as a mutational hot spot in human cancer.

1994 ◽  
Vol 14 (6) ◽  
pp. 4225-4232 ◽  
Author(s):  
A N Magewu ◽  
P A Jones
Keyword(s):  
Cell Line ◽  
Cytosine Methylation ◽  
De Novo ◽  
Human Cancer ◽  
Hot Spot ◽  
Methylation Status ◽  
Osteogenic Sarcoma ◽  
P53 Gene ◽  
Alu Sequence ◽  
De Novo Methylation

Cytosine methylation at CpG dinucleotides is thought to cause more than one-third of all transition mutations responsible for human genetic diseases and cancer. We investigated the methylation status of the CpG dinucleotide at codon 248 in exon 7 of the p53 gene because this codon is a hot spot for inactivating mutations in the germ line and in most human somatic tissues examined. Codon 248 is contained within an HpaII site (CCGG), and the methylation status of this and flanking CpG sites was analyzed by using the methylation-sensitive enzymes CfoI (GCGC) and HpaII. Codon 248 and the CfoI and HpaII sites in the flanking introns were methylated in every tissue and cell line examined, indicating extensive methylation of this region in the p53 gene. Exhaustive treatment of an osteogenic sarcoma cell line, TE85, with the hypomethylating drug 5-aza-2'-deoxycytidine did not demethylate codon 248 or the CfoI sites in intron 6, although considerable global demethylation of the p53 gene was induced. Constructs containing either exon 7 alone or exon 7 and the flanking introns were transfected into TE85 cells to determine whether de novo methylation would occur. The presence of exon 7 alone caused some de novo methylation to occur at codon 248. More extensive de novo methylation of the CfoI sites in intron 6, which contains an Alu sequence, occurred in cells transfected with a vector containing exon 7 and flanking introns. With longer time in culture, there was increased methylation at the CfoI sites, and de novo methylation of codon 248 and its flanking HpaII sites was observed. These de novo-methylated sites were also resistant to 5-aza-2'-deoxycytidine-induced demethylation. The frequent methylation of codon 248 and adjacent Alu sequence may explain the enhanced mutability of this site as a result of the deamination of the 5-methylcytosine.

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