scholarly journals High Throughput Screen Identifies the DNMT1 (DNA Methyltransferase-1) Inhibitor, 5-Azacytidine, as a Potent Inducer of PTEN (Phosphatase and Tensin Homolog)

2020 ◽  
Vol 40 (8) ◽  
pp. 1854-1869
Author(s):  
Keith A. Strand ◽  
Sizhao Lu ◽  
Marie F. Mutryn ◽  
Linfeng Li ◽  
Qiong Zhou ◽  
...  
Keyword(s):  
Protein Expression ◽  
High Throughput ◽  
Knockout Mice ◽  
Vascular Remodeling ◽  
Dna Methyltransferase ◽  
Dna Methyltransferase 1 ◽  
Protective Effects ◽  
High Throughput Screen ◽  
Phosphatase And Tensin Homolog ◽  
Tensin Homolog

Objective: Our recent work demonstrates that PTEN (phosphatase and tensin homolog) is an important regulator of smooth muscle cell (SMC) phenotype. SMC-specific PTEN deletion promotes spontaneous vascular remodeling and PTEN loss correlates with increased atherosclerotic lesion severity in human coronary arteries. In mice, PTEN overexpression reduces plaque area and preserves SMC contractile protein expression in atherosclerosis and blunts Ang II (angiotensin II)-induced pathological vascular remodeling, suggesting that pharmacological PTEN upregulation could be a novel therapeutic approach to treat vascular disease. Approach and Results: To identify novel PTEN activators, we conducted a high-throughput screen using a fluorescence based PTEN promoter-reporter assay. After screening ≈3400 compounds, 11 hit compounds were chosen based on level of activity and mechanism of action. Following in vitro confirmation, we focused on 5-azacytidine, a DNMT1 (DNA methyltransferase-1) inhibitor, for further analysis. In addition to PTEN upregulation, 5-azacytidine treatment increased expression of genes associated with a differentiated SMC phenotype. 5-Azacytidine treatment also maintained contractile gene expression and reduced inflammatory cytokine expression after PDGF (platelet-derived growth factor) stimulation, suggesting 5-azacytidine blocks PDGF-induced SMC de-differentiation. However, these protective effects were lost in PTEN-deficient SMCs. These findings were confirmed in vivo using carotid ligation in SMC-specific PTEN knockout mice treated with 5-azacytidine. In wild type controls, 5-azacytidine reduced neointimal formation and inflammation while maintaining contractile protein expression. In contrast, 5-azacytidine was ineffective in PTEN knockout mice, indicating that the protective effects of 5-azacytidine are mediated through SMC PTEN upregulation. Conclusions: Our data indicates 5-azacytidine upregulates PTEN expression in SMCs, promoting maintenance of SMC differentiation and reducing pathological vascular remodeling in a PTEN-dependent manner.

scholarly journals An Ultrasensitive High Throughput Screen for DNA Methyltransferase 1-Targeted Molecular Probes

PLoS ONE ◽  
2013 ◽  
Vol 8 (11) ◽  
pp. e78752 ◽  
Author(s):  
Rebecca L. Fagan ◽  
Meng Wu ◽  
Frédéric Chédin ◽  
Charles Brenner
Keyword(s):  
High Throughput ◽  
Dna Methyltransferase ◽  
Molecular Probes ◽  
Dna Methyltransferase 1 ◽  
High Throughput Screen ◽  
Methyltransferase 1

99 EFFECT OF SUPPRESSION OF BOVINE DNA METHYLTRANSFERASE 1 ON EMBRYONIC DEVELOPMENT AND EXPRESSION OF IMPRINTED GENES

2009 ◽  
Vol 21 (1) ◽  
pp. 150
Author(s):  
P. Wilaiphan ◽  
F. Rings ◽  
M. Hoelker ◽  
E. Tholen ◽  
C. Phatsara ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Protein Expression ◽  
Dna Methyltransferase ◽  
Critical Role ◽  
Apoptotic Index ◽  
Imprinted Genes ◽  
Dna Methyltransferase 1 ◽  
Relative Standard ◽  
Blastocyst Rate ◽  
Methyltransferase 1

DNA methyltransferase 1 (DNMT1) is believed to be involved in DNA methylation, which is a well-characterized epigenetic modulator shown to have essential functions in germ line and embryonic genome imprinting. This study was conducted to investigate the consequences of suppressing and inhibiting DNMT1 on the development, level of apoptosis, and expression of imprinted genes in pre-implantation bovine embryos. In vitro-produced zygotes were categorized into 4 groups; namely, those injected with Smartpool siRNA (SpsiRNA; Dharmacon Inc., Chicago, IL) (n = 800), 5aza-2′-deoxycytidine (5-AZA; Sigma, St. Louis, MO) (n = 864), nuclease-free water (n = 850), and uninjected control (n = 755). The mRNA expression data were generated using RT-PCR based on the relative standard curve method employing glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a normalizer. Apoptotic index (API) was calculated by dividing the number of apoptotic cells to total cell number. The proportion of 2-, 4-, 8-cell and 2-, 4-, 8-, and 16-cell embryos was assessed 48 and 72 h, respectively, post-micro injection (pmi), whereas blastocyst rate was assessed at Day 8 pmi. Data on embryonic development and the relative mRNA abundance were analyzed using ANOVA followed by a multiple pair-wise mean comparison using Tukey test. The proportion of 2-, 4-, and 8-cell embryos at 48 h pmi was not significant among treatment groups. However, the proportion of the 8-cell embryos was significantly lower (P < 0.05) in SpsiRNA (16.3 ± 4.5) and 5-AZA injected groups (17.7 ± 4.9) compared with water-injected (26.8 ± 2.9) and uninjected controls (30.7 ± 6.2). The lowest total blastocyst rate (P < 0.05) was observed in the 5-AZA treatment group (16.9 ± 4.9) compared with SpsiRNA (23.4 ± 2.1) and water-injected (24.1 ± 5.3) and uninjected controls (29.4 ± 2.1). Microinjection of SpsiRNA reduced the target mRNA by 80 and 50% in 8-cell and blastocyst stage embryos, respectively, compared with uninjected control, and the protein expression level was also reduced at 8-cell embryos as confirmed by Western blotting. Injection of 5-AZA had no significant effect on mRNA or protein expression. The greatest API (P < 0.05) was found in SpsiRNA (4.2 ± 2.0) and 5-AZA (4.1 ± 1.7) injected groups compared with water-injected (2.8 ± 2.1) and uninjected controls (2.9 ± 2.3). The relative expression study also showed that microinjection of SpsiRNA and 5-AZA increased the expression of IGF2 (by 67 and 55%), IGF2R (29 and 30%), and IGFPB-4 (22 and 24%), respectively, compared with uninjected control, without affecting the expression of both IGF2R and IGFPB-4. In conclusion, suppression of DNMT1 resulted in lower proportion of 8-cell embryos, reduced blastocyst rate, and increased apoptotic index, and affected the expression of some imprinted genes, demonstrating a critical role of this gene in bovine embryonic development.

Reduced PTEN Protein Expression and Its Prognostic Implications in Canine and Feline Mammary Tumors

2009 ◽  
Vol 46 (5) ◽  
pp. 860-868 ◽  
Author(s):  
L. Ressel ◽  
F. Millanta ◽  
E. Caleri ◽  
V. M. Innocenti ◽  
A. Poli
Keyword(s):  
Protein Expression ◽  
Lymphatic Vessel ◽  
Mammary Tumors ◽  
Lymphatic Vessel Invasion ◽  
Pten Protein ◽  
Phosphatase And Tensin Homolog ◽  
Mammary Carcinomas ◽  
Tensin Homolog ◽  
Vessel Invasion ◽  
Distant Organ

Phosphatase and tensin homolog (PTEN) belongs to the group of gatekeeper tumor suppressor genes and is involved in multiple mechanisms leading to cellular defense against neoplastic transformation and progression. Twenty-four dogs and 17 cats were submitted to a 2-year follow-up study, and clinicopathologic features were recorded and compared with immunohistochemical PTEN staining. PTEN-negative status occurred in 33% of canine and 76% of feline mammary carcinomas. In canine mammary carcinomas, there was a significant ( P < .05) correlation between loss of PTEN protein expression and simple carcinoma histotype, lymphatic vessel invasion, lymph node metastases, distant organ metastases, tumor dedifferentiation, tumor recurrence, and shorter overall survival. In feline mammary tumors, a significant correlation between loss of PTEN protein expression and lymphatic vessel invasion was found. Loss of PTEN expression could be a useful prognostic marker in canine mammary carcinomas.

scholarly journals The molecular dynamics of long noncoding RNA control of transcription in PTEN and its pseudogene

2017 ◽  
Vol 114 (37) ◽  
pp. 9942-9947 ◽  
Author(s):  
Nicholas Lister ◽  
Galina Shevchenko ◽  
James L. Walshe ◽  
Jessica Groen ◽  
Per Johnsson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Structure ◽  
Noncoding Rna ◽  
Dna Methyltransferase ◽  
Regulation Of Gene Expression ◽  
Control Of Gene Expression ◽  
Phosphatase And Tensin Homolog ◽  
Tensin Homolog ◽  
Sequence Components ◽  
Antisense Lncrnas

RNA has been found to interact with chromatin and modulate gene transcription. In human cells, little is known about how long noncoding RNAs (lncRNAs) interact with target loci in the context of chromatin. We find here, using the phosphatase and tensin homolog (PTEN) pseudogene as a model system, that antisense lncRNAs interact first with a 5′ UTR-containing promoter-spanning transcript, which is then followed by the recruitment of DNA methyltransferase 3a (DNMT3a), ultimately resulting in the transcriptional and epigenetic control of gene expression. Moreover, we find that the lncRNA and promoter-spanning transcript interaction are based on a combination of structural and sequence components of the antisense lncRNA. These observations suggest, on the basis of this one example, that evolutionary pressures may be placed on RNA structure more so than sequence conservation. Collectively, the observations presented here suggest a much more complex and vibrant RNA regulatory world may be operative in the regulation of gene expression.

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