Promoter methylation is associated with the age-dependent loss of N-cadherin in the rat kidney

The cadherins are cell adhesion molecules required for cellular homeostasis, and N-cadherin is the predominant cadherin expressed in proximal tubular epithelial cells in humans and rats. Our laboratory previously reported an age-dependent decrease in renal N-cadherin expression; the levels of N-cadherin mRNA and protein expression decreased in parallel, implicating a transcriptional mechanism in the age-dependent loss of expression ( 19 ). In this study, we examined the hypothesis that promoter hypermethylation underlies the loss of N-cadherin expression in aging rat kidney. We cloned the 5′ flanking region of the rat N-cadherin gene and observed basic promoter activity in a 3,992-bp region localized immediately upstream of the ATG start site. Nucleotide analysis revealed 87% identity with the human N-cadherin minimal promoter region. Consistent with a role for regulation by DNA methylation, we found that a dense CpG island, which spans 1,104 bp (−1,158 to −55), flanks the rat N-cadherin gene; a similar CpG profile was found in the human N-cadherin 5′ flanking region. Methylation-specific PCR analysis demonstrated that the promoter region of N-cadherin is heavily methylated in aged, but not young, rat kidney. Interestingly, the promoter is not methylated in age-matched, calorically restricted animals. In contrast, the promoter region is not methylated in either young or aged rat liver; this corresponds to the finding that aging is not associated with decreased N-cadherin expression in the liver. In addition, N-cadherin expression is markedly induced in NRK-52E cells treated with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine, further suggesting that methylation at CpG in the promoter region may underlie the age-dependent decrease in renal N-cadherin expression.

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Promoter hypermethylation in MLL-r infant acute lymphoblastic leukemia: biology and therapeutic targeting

Blood ◽

10.1182/blood-2009-09-243634 ◽

2010 ◽

Vol 115 (23) ◽

pp. 4798-4809 ◽

Cited By ~ 75

Author(s):

Eric Schafer ◽

Rafael Irizarry ◽

Sandeep Negi ◽

Emily McIntyre ◽

Donald Small ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Dna Methyltransferase ◽

Cpg Island ◽

Lymphoblastic Leukemia ◽

Promoter Hypermethylation ◽

Therapeutic Targeting ◽

Specific Pcr ◽

Genome Wide ◽

Methylation Patterns ◽

Infant Acute Lymphoblastic Leukemia

Abstract Cooperating leukemogenic events in MLL-rearranged (MLL-r) infant acute lymphoblastic leukemia (ALL) are largely unknown. We explored the role of promoter CpG island hypermethylation in the biology and therapeutic targeting of MLL-r infant ALL. The HELP (HpaII tiny fragment enrichment by ligation-mediated polymerase chain reaction [PCR]) assay was used to examine genome-wide methylation of a cohort of MLL-r infant leukemia samples (n = 5), other common childhood ALLs (n = 5), and normals (n = 5). Unsupervised analysis showed tight clustering of samples into their known biologic groups, indicating large differences in methylation patterns. Global hypermethylation was seen in the MLL-r cohort compared with both the normals and the others, with ratios of significantly (P < .001) hypermethylated to hypomethylated CpGs of 1.7 and 2.9, respectively. A subset of 7 differentially hypermethylated genes was assayed by quantitative reverse-transcription (qRT)–PCR, confirming relative silencing in 5 of 7. In cell line treatment assays with the DNA methyltransferase inhibitor (DNMTi) decitabine, MLL-r (but not MLL wild-type cell lines) showed dose- and time-dependent cytotoxicity and re-expression of 4 of the 5 silenced genes. Methylation-specific PCR (MSP) confirmed promoter hypermethylation at baseline, and a relative decrease in methylation after treatment. DNMTi may represent a novel molecularly targeted therapy for MLL-r infant ALL.

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Functional Analysis of p53 Binding under Differential Stresses

Molecular and Cellular Biology ◽

10.1128/mcb.00322-06 ◽

2006 ◽

Vol 26 (19) ◽

pp. 7030-7045 ◽

Cited By ~ 44

Author(s):

Adam J. Krieg ◽

Ester M. Hammond ◽

Amato J. Giaccia

Keyword(s):

Chromatin Immunoprecipitation ◽

Target Genes ◽

Cpg Island ◽

P53 Protein ◽

Pcr Analysis ◽

Glutathione Peroxidase 1 ◽

Specific Pcr ◽

Subsequent Effect ◽

P53 Target Genes ◽

Hct116 Cells

ABSTRACT Hypoxia and DNA damage stabilize the p53 protein, but the subsequent effect that each stress has on transcriptional regulation of known p53 target genes is variable. We have used chromatin immunoprecipitation followed by CpG island (CGI) microarray hybridization to identify promoters bound by p53 under both DNA-damaging and non-DNA-damaging conditions in HCT116 cells. Using gene-specific PCR analysis, we have verified an association with CGIs of the highest enrichment (>2.5-fold) (REV3L, XPMC2H, HNRPUL1, TOR1AIP1, glutathione peroxidase 1, and SCFD2), with CGIs of intermediate enrichment (>2.2-fold) (COX7A2L, SYVN1, and JAG2), and with CGIs of low enrichment (>2.0-fold) (MYC and PCNA). We found little difference in promoter binding when p53 is stabilized by these two distinctly different stresses. However, expression of these genes varies a great deal: while a few genes exhibit classical induction with adriamycin, the majority of the genes are unchanged or are mildly repressed by either hypoxia or adriamycin. Further analysis using p53 mutated in the core DNA binding domain revealed that the interaction of p53 with CGIs may be occurring through both sequence-dependent and -independent mechanisms. Taken together, these experiments describe the identification of novel p53 target genes and the subsequent discovery of distinctly different expression phenomena for p53 target genes under different stress scenarios.

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The novel DNA methylation inhibitor zebularine is effective against the development of murine T-cell lymphoma

Blood ◽

10.1182/blood-2005-05-2033 ◽

2006 ◽

Vol 107 (3) ◽

pp. 1174-1177 ◽

Cited By ~ 43

Author(s):

Michel Herranz ◽

Juan Martín-Caballero ◽

Mario F. Fraga ◽

Jesús Ruiz-Cabello ◽

Juana Maria Flores ◽

...

Keyword(s):

Dna Methylation ◽

Dna Methyltransferase ◽

Cpg Island ◽

Promoter Hypermethylation ◽

Dna Hypomethylation ◽

Chemotherapy Drugs ◽

Positron Emission ◽

Methylation Inhibitor ◽

Dna Methylation Inhibitor ◽

T Lymphomas

AbstractGene silencing by CpG island promoter hypermethylation has awakened the interest for DNA demethylating agents as chemotherapy drugs. Zebularine (1-[β-D-ribofuranosil]-1,2-dihydropyrimidin-2-1) has been recently described as a new DNA methylation inhibitor. Here we have studied its effects in a mouse model of radiation-induced lymphomagenesis using nuclear magnetic resonance (NMR) and positron emission tomography (PET). All control animals presented large thymic T lymphomas and died between 4 and 5.5 months. In contrast, 40% (12 of 30) of zebularine-treated animals were still alive after 1 year (Kaplan-Meier P < .001). NMR and PET imaging showed that surviving animals presented a thymus structure/volume similar to normal mice of the same age. Most important, zebularine demonstrated a complete lack of toxicity in nonirradiated control mice. DNA hypomethylation induced by zebularine occurred in association with depletion in extractable DNA methyltransferase 1 protein. Thus, our data support the role of zebularine as a DNA demethylating agent with antitumor activity and little toxicity.

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PAR2 Promoter Hypomethylation Regulates PAR2 Gene Expression and Promotes Lung Adenocarcinoma Cell Progression

Computational and Mathematical Methods in Medicine ◽

10.1155/2021/5542485 ◽

2021 ◽

Vol 2021 ◽

pp. 1-10

Author(s):

Kuan Wu ◽

Lei Xu ◽

Ling Cheng

Keyword(s):

Gene Expression ◽

Lung Adenocarcinoma ◽

Promoter Methylation ◽

Dna Methyltransferase ◽

Bioinformatics Analysis ◽

Promoter Hypermethylation ◽

Cellular Level ◽

Specific Pcr ◽

Cancer Occurrence ◽

Cell Progression

Objective. Protease-activated receptor-2 (PAR2) also known as F2RL1 is a G protein-coupled receptor that intimately correlates with cancer occurrence. DNA methylation turns out a vital mechanism regulating gene expression, while PAR2 promoter methylation is proven to be involved in cancer development. Hence, this study attempted to clarify the molecular mechanism by which PAR2 mediates lung adenocarcinoma (LUAD) progression, via identifying the effect of PAR2 promoter methylation on LUAD cell progression. Methods. Associations of PAR2 promoter methylation with PAR2 gene expression and prognosis of LUAD patients were analyzed via bioinformatics analysis. PAR2 promoter methylation and gene expression at the cellular level were measured using methylation-specific PCR, qRT-PCR, and Western blot assays. DNA methyltransferase inhibitor 5-AzadC was used to treat cells to assess PAR2 gene expression alteration. Cell biological behaviors upon PAR2 overexpression were characterized via MTT, wound healing assay, and Transwell assay. Results. Bioinformatics analysis revealed that PAR2 promoter methylation was negatively related to PAR2 gene expression, while PAR2 promoter hypermethylation and low gene expression indicated favorable LUAD prognosis. Besides, it turned out that PAR2 presented upregulated expression and hypomethylated promoter in LUAD cells. Moreover, PAR2 gene expression was elevated in cells treated with 5-AzadC, and the proliferative, migratory, and invasive capabilities of cells with 5-AzadC or high PAR2 gene expression were all enhanced. Conclusion. In sum, PAR2 promoter hypomethylation potentiates LUAD cell progression, in turn affecting the prognosis of LUAD patients.

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Expression of p16 in sporadic primary uveal melanoma.

Acta Biochimica Polonica ◽

10.18388/abp.2002_3796 ◽

2002 ◽

Vol 49 (2) ◽

pp. 377-385 ◽

Cited By ~ 4

Author(s):

Katarzyna Lamperska ◽

Krystyna Mackiewicz ◽

Aldona Kaczmarek ◽

Eliza Kwiatkowska ◽

Maria Starzycka ◽

...

Keyword(s):

Optic Nerve ◽

Uveal Melanoma ◽

Promoter Region ◽

Mixed Type ◽

Cpg Island ◽

Promoter Hypermethylation ◽

Staining Intensity ◽

Histological Type ◽

Silent Mutation ◽

Tumor Invasiveness

Expression of p16 protein, intragenic mutations of CDKN2A and hypermethylation of CDKN2A promoter region in 41 sporadic primary uveal melanomas were studied. There were 2 cases of spindle cell B histological type, 11 of A + B and 28 of mixed type. All melanomas infiltrated sclera but in 28 cases infiltration was superficial while in 13 profound. In 7 cases the tumor infiltrated the optic nerve. Expression of p16 was studied by immunohistochemistry and recorded by assessment of the proportion of positive tumor cells and staining intensity. Results were expressed as staining index (IRS). Intragenic mutations were studied by PCR-SSCP followed by sequencing, while hypermethylation of the promoter region by CpG methylation assay. In 15% of cases less than 10% of melanoma cells were p16 positive, in 70% of cases less than 50% of cells, while in 7% more than 80% of cells stained for p16 (mean IRS for all cases was 4.87 +/- 2.43). In B type the IRS was 8.5 +/- 0.7, in A + B type 6.0 +/- 2.1 and in the mixed type 4.17 +/- 2.43 (differences statistically significant). In melanomas profoundly infiltrating sclera mean IRS was 4.16, while in those infiltrating optic nerve 3.71 (statistically not significant). Analysis of the intragenic mutations revealed in two patients a GAC/GAT substitution in codon 84--a silent mutation. No hypermethylation of the CpG island of the p16 promoter region was found. In conclusion, we found that the degree of p16 expression is related to the histological type of tumor but not to the histological indicators of tumor invasiveness and that intragenic mutations and promoter hypermethylation are not major mechanisms of p16 inactivation in sporadic uveal melanoma.

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TRIB2 Gene Overexpression Is Associated with Adult Minimally Differentiated Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v118.21.4628.4628 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4628-4628

Author(s):

Crescenzio Francesco Minervini ◽

Valentina Buttiglione ◽

Nicoletta Coccaro ◽

Luciana Impera ◽

Luisa Anelli ◽

...

Keyword(s):

Bone Marrow ◽

Promoter Region ◽

Conflicts Of Interest ◽

Cpg Island ◽

Expression Profiles ◽

Restriction Enzymes ◽

Pcr Analysis ◽

Gene Overexpression ◽

Fish Analysis ◽

Qrt Pcr

Abstract Abstract 4628 Background. Tribbles homolog gene 2 (TRIB2) is a pseudokinase gene belonging to a three member gene family. Tribbles gene was first identified in Drosophila melanogaster where is involved in the regulation of morphogenesis and mitosis. Likewise, mammalian homologous genes of Tribbles (TRIB1, TRIB2, TRIB3) promotes the degradation of specific transcription factors and interacts with several cell signaling mediators and modulators. Recently it has been demonstrated that TRIB2 was able to induce AML in bone marrow transplanted mouse by inducing C/EBPα proteasome-dependent degradation. Moreover, gene expression profiles data from AML patients revealed that patients carrying C/EBPα mutations clustered with those patients with up-regulated TRIB2.Aims. To test the hypothesis that TRIB2 expression was related to a differentiation degree in AML, we evaluated several AML cases based on FAB cytotype. Patients and Methods. We performed quantitative Real Time-PCR (qRT-PCR) analysis on AML patients (15 AML-M0, 14 AML-M2, 5 AML-M3, 2 M4 and 3 M5b bone marrow aspirate samples) to measure the expression level of TRIB2 in different FAB AML subtype. Four healthy bone marrows were used as reference samples. qRT-PCR was conduct using SYBR green chemistry and specific primers for TRIB2 transcript and for two housekeeping genes (B2M and IPO8) previously tested for their stability and efficiency. All AML sample were tested by conventional cytogenetic analysis and by FISH with TRIB2 specific probe. We performed, also, methylation analysis of a CpG island located in the TRIB2 promoter region by methylation sensitive restriction enzyme (MSRE) and qPCR. Briefly, DNA samples were digested with MSRE and were quantified by qPCR using specific primer pairs surrounding restriction enzymes recognition sites. Results. qRT-PCR experiments revealed that TRIB2 expression was higher (from 3 to 20 fold) in AML-M0 respect to the AML-M2 (p= 0.02), AML-M3 (p=0.01), AML-M5 (p=0.006) FAB subtypes, and references (p=0.003), respectively. Moreover, the AML-M0 cases showed a TRIB2 overexpression compared to that observed in the two AML-M4 cases (about 4 fold changes) but this difference there was not statistically significant probably because of M4 cases paucity in our series. Therefore qRT-PCR displayed a progressive decreasing TRIB2 expression along FAB subtypes. Conventional cytogenetic and FISH analysis did not show any kind of rearrangement involving TRIB2 gene. MSRE experiments revealed an higher methylation degree of a CpG island located in the TRIB2 promoter region in AML-M0 cases respect to the others FAB subtypes; moreover, methylation was significantly correlated with TRIB2 expression (r = 0.9; p = 0.0002). Conclusions. Our data showed that the TRIB2 gene overexpression was associated to AML-M0. In our cases TRIB2 dysregulation was not due to gene amplification as in other cancers. Surprisingly we observed that AML-M0 samples showed an higher methylation degree in the TRIB2 promoter region. Usually methylation cause gene silencing but our results showed that TRIB2 promoter region methylation was associated to the overexpression of the gene. Maybe methylation inhibits binding of some unknown transcriptional repressor as already seen for hTERT gene in others tumors cells. In conclusion, our data revealed that TRIB2 dysregulation seems to be linked to AML-M0 phenotype. Further studies are needed to clarify the reasons for this association. Disclosures: No relevant conflicts of interest to declare.

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SUV39H1-Mediated DNMT1 is Participated in the Epigenetic Regulation of Smad3 in Cervical Cancer

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200721110016 ◽

2020 ◽

Vol 20 ◽

Author(s):

Li Zhang ◽

Sijuan Tian ◽

Minyi Zhao ◽

Ting Yang ◽

Shimin Quan ◽

...

Keyword(s):

Cervical Cancer ◽

Cell Lines ◽

Epigenetic Regulation ◽

Promoter Region ◽

Methylation Status ◽

Regulation Mechanism ◽

Methylation Specific Pcr ◽

Specific Pcr ◽

Immune Factor ◽

Cancer Tissues

Background: Smad3 is a pivotal intracellular mediator for participating in the activation of multiple immune signal pathway. Objective: The epigenetic regulation mechanism of the positive immune factor Smad3 in cervical cancer remains unknown. Therefore, the epigenetic regulation on Smad3 is investigated in this study. Methods: The methylation status of SMAD3 was detected by Methylation-specific PCR (MS-PCR) and Quantitative Methylation-specific PCR (MS-qPCR) in cervical cancer tissues and cell lines. The underlying molecular mechanisms of SUV39H1-DNMT1-Smad3 regulation was elucidated using cervical cancer cell lines containing siRNA or/and overexpression system. Confirmation of the regulation of DNMT1 by SUV39H1 used Chromatin immunoprecipitation-qPCR (ChIP-qPCR). The statistical methods used for comparing samples between groups were paired t tests and one-way ANOVAs. Results: H3K9me3 protein which regulated by SUV39H1 directly interacts with the DNMT1 promoter region to regulate its expression in cervical cancer cells, resulting in the reduce expression of the downstream target gene DNMT1. In addition, DNMT1 mediates the epigenetic modulation of the SMAD3 gene by directly binding to its promoter region. The depletion of DNMT1 effectively restores the expression of Smad3 in vitro. Moreover, in an in vivo assay, the expression profile of SUV39H1-DNMT1 was found to correlate with Smad3 expression in accordance with the expression at the cellular level. Notably, the promoter region of SMAD3 was hypermethylated in cervical cancer tissues, and this hypermethylation inhibits the subsequent gene expression. Conclusion: These results indicate that SUV39H1-DNMT1 is a crucial Smad3 regulatory axis in cervical cancer. SUV39H1-DNMT1 axis may provide a potential therapeutic target for the treatment of cervical cancer.

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Abnormal promoter DNA hypermethylation of the integrin, nidogen, and dystroglycan genes in breast cancer

Scientific Reports ◽

10.1038/s41598-021-81851-y ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Vladimir V. Strelnikov ◽

Ekaterina B. Kuznetsova ◽

Alexander S. Tanas ◽

Viktoria V. Rudenko ◽

Alexey I. Kalinkin ◽

...

Keyword(s):

Cpg Island ◽

Strong Association ◽

Cell Activity ◽

Promoter Hypermethylation ◽

Normal Breast ◽

Breast Carcinomas ◽

Her2 Positive ◽

A Genome ◽

Extracellular Matrix Components ◽

Integrin Α4

AbstractCell transmembrane receptors and extracellular matrix components play a pivotal role in regulating cell activity and providing for the concerted integration of cells in the tissue structures. We have assessed DNA methylation in the promoter regions of eight integrin genes, two nidogen genes, and the dystroglycan gene in normal breast tissues and breast carcinomas (BC). The protein products of these genes interact with the basement membrane proteins LAMA1, LAMA2, and LAMB1; abnormal hypermethylation of the LAMA1, LAMA2, and LAMB1 promoters in BC has been described in our previous publications. In the present study, the frequencies of abnormal promoter hypermethylation in BC were 13% for ITGA1, 31% for ITGA4, 4% for ITGA7, 39% for ITGA9, 38% for NID1, and 41% for NID2. ITGA2, ITGA3, ITGA6, ITGB1, and DAG1 promoters were nonmethylated in normal and BC samples. ITGA4, ITGA9, and NID1 promoter hypermethylation was associated with the HER2 positive tumors, and promoter hypermethylation of ITGA1, ITGA9, NID1 and NID2 was associated with a genome-wide CpG island hypermethylated BC subtype. Given that ITGA4 is not expressed in normal breast, one might suggest that its abnormal promoter hypermethylation in cancer is non-functional and is thus merely a passenger epimutation. Yet, this assumption is not supported by our finding that it is not associated with a hypermethylated BC subtype. ITGA4 acquires expression in a subset of breast carcinomas, and methylation of its promoter may be preventive against expression in some tumors. Strong association of abnormal ITGA4 hypermethylation with the HER2 positive tumors (p = 0.0025) suggests that simultaneous presence of both HER2 and integrin α4 receptors is not beneficial for tumor cells. This may imply HER2 and integrin α4 signaling pathways interactions that are yet to be discovered.

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MODL-15. THE COMBINATION TREATMENT OF PARP INHIBITOR AND TMZ, OR DAG WILL BE PROMISING TREATMENT IN SF8628

Neuro-Oncology ◽

10.1093/neuonc/noaa222.589 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii414-iii414

Author(s):

Shigeo Ohba ◽

Yuichi Hirose

Keyword(s):

Combination Treatment ◽

Dna Methyltransferase ◽

Excision Repair ◽

Parp Inhibitor ◽

Adenosine Diphosphate ◽

Promoter Hypermethylation ◽

Methylguanine Dna Methyltransferase ◽

Dna Crosslinks ◽

Promising Treatment ◽

Base Excision

Abstract Diffuse midline glioma, H3 K27M-mutant (DMG) is a newly defined entity. The prognosis of DMG is poor. Because surgical resection is often incomplete for DMG, radiotherapy and chemotherapy are important. Temozolomide (TMZ) is an alkylating agent that adds a methyl group to DNA (O6-guanine, N7-guanine, and N3-adenine). TMZ-induced cytotoxicity is mainly derived from O6-methylguanine, which is repaired by O6-methylguanine DNA methyltransferase (MGMT). It has been reported that most of DMG lacked MGMT promoter hypermethylation, which is thought to contribute to less effectiveness of TMZ to DMG. The purpose of the study is to explore the way to inhibit the proliferation of DMG. A DMG cell line, SF8628, was used for the experiments. SF8628 had the expression of MGMT and was revealed to be resistant to TMZ. Because N7-methylguanine and N3-methyladenine are repaired via base excision repair, poly (adenosine diphosphate-ribose) polymerase (PARP) inhibitor combined with TMZ was considered to be effective to suppress the proliferation of SF8628. As expected, PARP inhibitor enhanced TMZ-induced cytotoxicity in SF8628. Dianhydrogalactiol (DAG) is a bifunctional DNA-targeting agent forming N7-alkylguanine and inter-strand DNA crosslinks. DAG reduced the clonogenicity of SF8628. Moreover, inhibition of homologous recombination enhanced the DAG-induced cytotoxicity in SF8629. The combination treatment of PARP inhibitor and TMZ, or DAG were revealed to be promising treatments in SF8628.

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