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.

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.

scholarly journals Progressive increases in the methylation status and heterochromatinization of the myoD CpG island during oncogenic transformation

1994 ◽  
Vol 14 (9) ◽  
pp. 6143-6152
Author(s):  
W M Rideout ◽  
P Eversole-Cire ◽  
C H Spruck ◽  
C M Hustad ◽  
G A Coetzee ◽  
...  
Keyword(s):  
Cell Line ◽  
Structural Changes ◽  
De Novo ◽  
Cpg Island ◽  
Methylation Status ◽  
S Phase ◽  
Common Mechanism ◽  
Oncogenic Transformation ◽  
Putative Promoter ◽  
De Novo Methylation

Alterations in DNA methylation patterns are one of the earliest and most common events in tumorigenesis. Overall levels of genomic methylation often decrease during transformation, but localized regions of increased methylation have been observed in the same tumors. We have examined changes in the methylation status of the muscle determination gene myoD, which contains a CpG island, as a function of oncogenic transformation. This CpG island underwent de novo methylation during immortalization of 10T1/2 cells, and progressively more sites became methylated during the subsequent transformation of the cells to oncogenicity. The greatest increase in methylation occurred in the middle of the CpG island in exon 1 during transformation. Interestingly, no methylation was apparent in the putative promoter of myoD in either the 10T1/2 cell line or its transformed derivative. The large number of sites in the CpG island that became methylated during transformation was correlated with heterochromatinization of myoD as evidenced by a decreased sensitivity to cleavage of DNA in nuclei by MspI. A site in the putative promoter also became insensitive to MspI digestion in nuclei, suggesting that the chromatin structural changes extended beyond the areas of de novo methylation. Unlike Lyonized genes on the inactive X chromosome, whose timing of replication is shifted to late S phase, myoD replicated early in S phase in the transformed cell line. Methylation analysis of myoD in DNAs from several human tumors, which presumably do not express the gene, showed that hypermethylation also frequently occurs during carcinogenesis in vivo. Thus, the progressive increase in methylation of myoD during immortalization and transformation coinciding with a change in chromatin structure, as illustrated by the in vitro tumorigenic model, may represent a common mechanism in carcinogenesis for permanently silencing the expression of genes which can influence cell growth and differentiation.

scholarly journals Progressive increases in the methylation status and heterochromatinization of the myoD CpG island during oncogenic transformation.

1994 ◽  
Vol 14 (9) ◽  
pp. 6143-6152 ◽  
Author(s):  
W M Rideout ◽  
P Eversole-Cire ◽  
C H Spruck ◽  
C M Hustad ◽  
G A Coetzee ◽  
...  
Keyword(s):  
Cell Line ◽  
Structural Changes ◽  
De Novo ◽  
Cpg Island ◽  
Methylation Status ◽  
S Phase ◽  
Common Mechanism ◽  
Oncogenic Transformation ◽  
Putative Promoter ◽  
De Novo Methylation

Alterations in DNA methylation patterns are one of the earliest and most common events in tumorigenesis. Overall levels of genomic methylation often decrease during transformation, but localized regions of increased methylation have been observed in the same tumors. We have examined changes in the methylation status of the muscle determination gene myoD, which contains a CpG island, as a function of oncogenic transformation. This CpG island underwent de novo methylation during immortalization of 10T1/2 cells, and progressively more sites became methylated during the subsequent transformation of the cells to oncogenicity. The greatest increase in methylation occurred in the middle of the CpG island in exon 1 during transformation. Interestingly, no methylation was apparent in the putative promoter of myoD in either the 10T1/2 cell line or its transformed derivative. The large number of sites in the CpG island that became methylated during transformation was correlated with heterochromatinization of myoD as evidenced by a decreased sensitivity to cleavage of DNA in nuclei by MspI. A site in the putative promoter also became insensitive to MspI digestion in nuclei, suggesting that the chromatin structural changes extended beyond the areas of de novo methylation. Unlike Lyonized genes on the inactive X chromosome, whose timing of replication is shifted to late S phase, myoD replicated early in S phase in the transformed cell line. Methylation analysis of myoD in DNAs from several human tumors, which presumably do not express the gene, showed that hypermethylation also frequently occurs during carcinogenesis in vivo. Thus, the progressive increase in methylation of myoD during immortalization and transformation coinciding with a change in chromatin structure, as illustrated by the in vitro tumorigenic model, may represent a common mechanism in carcinogenesis for permanently silencing the expression of genes which can influence cell growth and differentiation.

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.

QSCR Analysis of Cytotoxicity of 6-Fluoro-3-(4H-1,2,4-triazol-3-yl)quinolin-4(1H)-ones on Chinese Hamster Ovary Cell Line: Design of REPUBLIC1986

2021 ◽  
Vol 28 ◽  
Author(s):  
Shikha Joon ◽  
Rajeev K. Singla ◽  
Bairong Shen ◽  
Mohammad Amjad Kamal
Keyword(s):  
Cell Line ◽  
Antitumor Agents ◽  
De Novo ◽  
Human Cancer ◽  
Structural Parameters ◽  
Chinese Hamster ◽  
Ovary Cell ◽  
Contour Plot ◽  
Computer Assisted ◽  
Line Design

Background: 6-Fluoro-3-(4H-1,2,4-triazol-3-yl)quinolin-4(1H)-ones are promising antitumor agents with enormous data on their profound cytotoxic effects on the human cancer cell lines. Objectives: We sought to perform a Quantitative structure cytotoxicity relationship (QSCR) analysis of a series of previously reported fluoroquinolone analogues using computer-assisted multiple regression analysis and investigate the cytotoxicity-inducing structural parameters among these congeners. Methods: The dataset was segregated into training and test sets of 6-Fluoro-3-(4H-1,2,4-triazol-3-yl)quinolin-4(1H)-ones by using a random selection method embedded in Vlife MDS 4.6 software and subjected to QSCR analysis. Next, cross-validation of the generated QSCR models was performed along with the external test set prediction. Finally, the data was analyzed, and contour plots were developed to deduce the cytotoxicity-inducing structural parameters among these congeners using Minitab® software. Results: The validated QSCR model exhibited a statistically significant predictive value of 92.27 percent. Our QSCR model revealed a direct proportionality between hydrogen counts and cytotoxicity and exclusion of sulphur and nitrogen with lesser crowding of cyclopropyl rings in future potential 6-Fluoro-3-(4H-1,2,4-triazol-3-yl)quinolin-4(1H)-one analogues. Based on the QSCR model predictions and contour plot analysis, the de novo REPUBLIC1986 molecule provided the best hit with predicted IC50 (µM) of 0.45 against CHO cell line and is amenable to salt formation crucial for anti-ovarian cancer activity. Conclusion: These findings suggest the relevancy of the developed QSCR model in designing novel, potent, and safer anti-cancer drugs with 6-Fluoro-3-(4H-1,2,4-triazol-3-yl)quinolin-4(1H)-ones as seed compounds.

scholarly journals Infection with Human Immunodeficiency Virus Type 1 Upregulates DNA Methyltransferase, Resulting in De Novo Methylation of the Gamma Interferon (IFN-γ) Promoter and Subsequent Downregulation of IFN-γ Production

1998 ◽  
Vol 18 (9) ◽  
pp. 5166-5177 ◽  
Author(s):  
Judy A. Mikovits ◽  
Howard A. Young ◽  
Paula Vertino ◽  
Jean-Pierre J. Issa ◽  
Paula M. Pitha ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
De Novo ◽  
Methylation Status ◽  
Immunodeficiency Virus ◽  
Ifn Γ ◽  
Hiv 1 ◽  
De Novo Methylation

ABSTRACT The immune response to pathogens is regulated by a delicate balance of cytokines. The dysregulation of cytokine gene expression, including interleukin-12, tumor necrosis factor alpha, and gamma interferon (IFN-γ), following human retrovirus infection is well documented. One process by which such gene expression may be modulated is altered DNA methylation. In subsets of T-helper cells, the expression of IFN-γ, a cytokine important to the immune response to viral infection, is regulated in part by DNA methylation such that mRNA expression inversely correlates with the methylation status of the promoter. Of the many possible genes whose methylation status could be affected by viral infection, we examined the IFN-γ gene as a candidate. We show here that acute infection of cells with human immunodeficiency virus type 1 (HIV-1) results in (i) increased DNA methyltransferase expression and activity, (ii) an overall increase in methylation of DNA in infected cells, and (iii) the de novo methylation of a CpG dinucleotide in the IFN-γ gene promoter, resulting in the subsequent downregulation of expression of this cytokine. The introduction of an antisense methyltransferase construct into lymphoid cells resulted in markedly decreased methyltransferase expression, hypomethylation throughout the IFN-γ gene, and increased IFN-γ production, demonstrating a direct link between methyltransferase and IFN-γ gene expression. The ability of increased DNA methyltransferase activity to downregulate the expression of genes like the IFN-γ gene may be one of the mechanisms for dysfunction of T cells in HIV-1-infected individuals.

scholarly journals De novo methylation of CpG island sequences in human fibroblasts overexpressing DNA (cytosine-5-)-methyltransferase.

1996 ◽  
Vol 16 (8) ◽  
pp. 4555-4565 ◽  
Author(s):  
P M Vertino ◽  
R W Yen ◽  
J Gao ◽  
S B Baylin
Keyword(s):  
Enzyme Activity ◽  
De Novo ◽  
Cpg Island ◽  
Human Fibroblasts ◽  
Methylation Status ◽  
Cpg Islands ◽  
Parental Cell Line ◽  
Direct Role ◽  
Cpg Island Methylation ◽  
De Novo Methylation

Recent studies showing a correlation between the levels of DNA (cytosine-5-)-methyltransferase (DNA MTase) enzyme activity and tumorigenicity have implicated this enzyme in the carcinogenic process. Moreover, hypermethylation of CpG island-containing promoters is associated with the inactivation of genes important to tumor initiation and progression. One proposed role for DNA MTase in tumorigenesis is therefore a direct role in the de novo methylation of these otherwise unmethylated CpG islands. In this study, we sought to determine whether increased levels of DNA MTase could directly affect CpG island methylation. A full-length cDNA for human DNA MTase driven by the cytomegalovirus promoter was constitutively expressed in human fibroblasts. Individual clones derived from cells transfected with DNA MTase (HMT) expressed 1- to 50-fold the level of DNA MTase protein and enzyme activity of the parental cell line or clones transfected with the control vector alone (Neo). To determine the effects of DNA MTase overexpression on CpG island methylation, we examined 12 endogenous CpG island loci in the HMT clones. HMT clones expressing > or = 9-fold the parental levels of DNA MTase activity were significantly hypermethylated relative to at least 11 Neo clones at five CpG island loci. In the HMT clones, methylation reached nearly 100% at susceptible CpG island loci with time in culture. In contrast, there was little change in the methylation status in the Neo clones over the same time frame. Taken together, the data indicate that overexpression of DNA MTase can drive the de novo methylation of susceptible CpG island loci, thus providing support for the idea that DNA MTase can contribute to tumor progression through CpG island methylation-mediated gene inactivation.

scholarly journals Assessment of epigenetic methylation changes in hop (Humulus lupulus) plants obtained by meristem culture

2020 ◽  
Vol 56 (No. 4) ◽  
pp. 159-164
Author(s):  
Josef Patzak ◽  
Alena Henychová ◽  
Petr Svoboda ◽  
Ivana Malířová
Keyword(s):  
De Novo ◽  
Molecular Genetic ◽  
Methylation Status ◽  
Hierarchical Cluster ◽  
Humulus Lupulus ◽  
Field Conditions ◽  
Meristem Culture ◽  
The Individual ◽  
De Novo Methylation

In vitro meristem cultures have been used for the production of hop (Humulus lupulus L.) virus-free rootstocks worldwide, because multipropagation is considered to preserve the genetic stability of the produced plantlet. Nevertheless, in vitro tissue cultures can cause genetic and epigenetic changes. Therefore, we studied the genetic and epigenetic variability of Saaz Osvald’s clones, Sládek and Premiant cultivars on the DNA methylation level by methylation-sensitive amplification polymorphism (MSAP). In vitro propagated plants, acclimatised glasshouse rootstocks as well as derived mericlones and control plants under field conditions were used for the analyses. A total of 346 clearly and highly reproducible amplified products were detected in the MSAP analyses within the studied hop plants. We found 16 polymorphic products (4.6% of products) and 64 products with methylation changes (18.5% of products) in the analyses. The demethylation events were comparable to the de novo methylation events. Most demethylation changes were found in the in vitro plants, but only a few of them were found in the derived mericlones under field conditions. In contrast, the de novo methylation changes persisted in the acclimatised plants under glasshouse or field conditions. A hierarchical cluster analysis was used for the evaluation of the molecular genetic variability within the individual samples. The dendrogram showed that the individual samples of the same variety, more or less, clustered together. Because the methylation status varied during the virus-free rootstock production process, we suppose that de/methylation process is a natural tool of epigenetics and evolution in vegetatively propagated plants.

scholarly journals Free Radical Scavenging Properties and Induction of Apoptotic Effects of FA Fraction Obtained after Proteolysis of Bioactive Peptides from Microalgae Synechococcus sp. VDW

2019 ◽  
Vol 57 (3) ◽  
pp. 358-368 ◽  
Author(s):  
Rutairat Suttisuwan ◽  
Saranya Phunpruch ◽  
Tanatorn Saisavoey ◽  
Papassara Sangtanoo ◽  
Nuttha Thongchul ◽  
...  
Keyword(s):  
Cell Line ◽  
Bioactive Peptides ◽  
De Novo ◽  
Human Cancer ◽  
Radical Scavenging Activity ◽  
Radical Scavenging ◽  
Peptide Sequencing ◽  
Cytotoxic Activities ◽  
Scavenging Activity ◽  
Synechococcus Sp

This study aims to determine the antioxidant activity of bioactive peptides derived from Synechococcus sp. VDW cells cultured for 21 days. Synechococcus sp. VDW protein hydrolysates were prepared with trypsin and purified by ultrafiltration with molecular mass cut-off membranes of 10, 5 and 3 kDa. The M<3 kDa (FA) fraction had the highest 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,2’-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities, with IC50 values of (11.5±0.3) and (13.6±0.2) µg/mL, respectively. The FA fraction was separated by reversed phase HPLC to yield four subfractions (F1–4). The F4 subfraction showed the highest maximum ABTS radical scavenging activity (3.55±0.61) % and it was selected for further analysis by electrospray ionisation quadrupole time-of-flight mass spectrometry (ESI-Q-TOF-MS/MS) based on de novo peptide sequencing. Five antioxidant peptides were identified, of which AILESYSAGKTK had the highest ABTS radical scavenging activity. Furthermore, the FA fraction showed high cytotoxic activities against human cancer-derived cell lines, especially the colon cancer cell line (SW620) with an IC50 value of (106.6±21.5) µg/mL, but not the untransformed Wi38 cell line. The FA fraction activated the apoptotic pathway in SW620 cells after treatment for 24, 48 and 72 h, with the highest activities of caspases-3, -8 and -9 being observed after treatment for 72 h. These findings suggested that microalgae Synechococcus sp. VDW may be used to develop natural anticancer drugs.

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