GLAD-PCR assay of R(5mC)GY sites in the regulatory region of tumor-suppressor genes associated with gastric cancer

At early stages of carcinogenesis, the regulatory regions of some tumor suppressor genes become aberrantly methylated at RCGY sites, which are substrates of DNA methyltransferase Dnmt3. Identification of aberrantly methylated sites in tumor DNA is considered to be the first step in the development of epigenetic PCR test systems for early diagnosis of cancer. Recently, we have developed a GLAD-PCR assay, a method for detecting the R(5mC)GY site in the genome position of interest even at significant excess of DNA molecules with a non-methylated RCGY site in this location. The aim of the present work is to use the GLAD-PCR assay to detect the aberrantly methylated R(5mC)GY sites in the regulatory regions of tumor suppressor genes (brinp1, bves, cacna2d3, cdh11, cpeb1, epha7, fgf2, galr1, gata4, hopx, hs3st2, irx1, lrrc3b, pcdh10, rprm, runx3, sfrp2, sox17, tcf21, tfpi2, wnt5a, zfp82, and znf331) in DNA samples obtained from gastric cancer (GC) tissues. The study of the DNA samples derived from 29 tumor and 25 normal gastric tissue samples demonstrated a high diagnostic potential of the selected RCGY sites in the regulatory regions of the irx1, cacna2d3, and epha7 genes; the total indices of sensitivity and specificity for GC detection being 96.6% and 100%, respectively.

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GLAD-PCR assay of DNA methylation sites in regulatory regions of some tumor-suppressor genes in breast cancer

Siberian Journal of Oncology ◽

10.21294/1814-4861-2021-20-6-41-54 ◽

2022 ◽

Vol 20 (6) ◽

pp. 41-54

Author(s):

N. A. Smetannikova ◽

M. A. Abdurashitov ◽

A. G. Akishev ◽

P. I. Pozdnyakov ◽

E. V. Dubinin ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Suppressor ◽

Tumor Suppressor Genes ◽

Mammary Tissue ◽

Aberrant Methylation ◽

Pcr Assay ◽

Suppressor Genes ◽

Regulatory Regions ◽

Tissue Samples ◽

Diagnostic Potential

Hypermethylation of the RcgY sites is shown for many cancer diseases. such aberrant methylation, suppressing the gene activity, occurs at early stages of carcinogenesis. Recently, using glad-pcR assay, we have detected aberrantly methylated RcgY sites, which can be considered to be epigenetic markers of colorectal, lung, and gastric cancers. in breast cancer, methylation of the regulatory regions of ALX4, BMP2, CCND2, CDH13, CDX1, FOXA1, GALR1, GATA5, GREM1, HIC1, HMX2, HS3ST2, HOXC10, ICAM5, LAMA1, RARB, RASSF1A, RUNX3, RXRG, RYR2, SFRP2, SOX17, TERT, and ZNF613 tumor-suppressor genes is reported. in the present work, we determined aberrantly methylated RcgY sites in the regulatory regions of these genes in dNa preparations from breast cancer tissues. the study of dNa samples from 30 tumor and 22 normal mammary tissue samples demonstrates a high diagnostic potential of selected R(5mc)gY sites in regulatory regions of CCND2, BMP2, GALR1, SOX17, HMX2, and HS3ST2 genes with total index of sensitivity and specificity for R(5mc)gY detection in tumor dNa 90.0 % and 100.0 %, respectively.

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Association Between Cyclin D1 Polymorphism with CpG Island Promoter Methylation Status of Tumor Suppressor Genes in Gastric Cancer

Digestive Diseases and Sciences ◽

10.1007/s10620-010-1206-5 ◽

2010 ◽

Vol 55 (12) ◽

pp. 3449-3457 ◽

Cited By ~ 3

Author(s):

Tomomitsu Tahara ◽

Tomoyuki Shibata ◽

Masakatsu Nakamura ◽

Hiromi Yamashita ◽

Daisuke Yoshioka ◽

...

Keyword(s):

Gastric Cancer ◽

Tumor Suppressor ◽

Cyclin D1 ◽

Promoter Methylation ◽

Tumor Suppressor Genes ◽

Cpg Island ◽

Methylation Status ◽

Suppressor Genes ◽

Promoter Methylation Status

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Targeting DNA methylation for treating triple-negative breast cancer

Pharmacogenomics ◽

10.2217/pgs-2019-0078 ◽

2019 ◽

Vol 20 (16) ◽

pp. 1151-1157 ◽

Cited By ~ 3

Author(s):

Jia Yu ◽

Jacqueline Zayas ◽

Bo Qin ◽

Liewei Wang

Keyword(s):

Breast Cancer ◽

Dna Methylation ◽

Tumor Suppressor ◽

Triple Negative Breast Cancer ◽

Targeted Therapies ◽

Tumor Suppressor Genes ◽

Dna Methyltransferase ◽

Triple Negative ◽

Suppressor Genes ◽

Dna Methyltransferase Inhibitors

Triple-negative breast cancer (TNBC) accounts for 15–20% of all invasive breast cancers and tends to have aggressive histological features and poor clinical outcomes. Unlike, estrogen receptor- or HER2-positive diseases, TNBC patients currently lack the US FDA-approved targeted therapies. DNA methylation is a critical mechanism of epigenetic modification. It is well known that aberrant DNA methylation contributes to the malignant transformation of cells by silencing critical tumor suppressor genes. DNA methyltransferase inhibitors reactivate silenced tumor suppressor genes and result in tumor growth arrest, with therapeutic effects observed in patients with hematologic malignancies. The antitumor effect of these DNA methyltransferase inhibitors has also been explored in solid tumors, especially in TNBC that currently lacks targeted therapies.

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Antitumor Activity of Cda-2, An Extract from Unine, in a High-Risk Myelodysplastic Syndrome Cells in Vitro

Blood ◽

10.1182/blood.v112.11.5099.5099 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5099-5099

Author(s):

Lin Qiu ◽

Xiao-dan Wang ◽

Bing-hong Han ◽

Zhao-min Zhan ◽

Zhang Bo-long ◽

...

Keyword(s):

Cell Differentiation ◽

High Risk ◽

Tumor Suppressor ◽

Myelodysplastic Syndrome ◽

Tumor Suppressor Genes ◽

Dna Methyltransferase ◽

Dependent Manner ◽

Suppressor Genes ◽

Specific Pcr

Abstract DNA methyltransferase inhibitors (DNMTI), including 5-azacytidine and 5-aza-2′- deoxycytidine, are a new class of epigenetic drug, which exhibit higher response rates in myelodysplastic syndrome (MDS) patients. Cell differentiation agent (CDA-2) is a kind of urine extracts, which contains several DNMTIs. A phase IV clinical trials for MDS showed total response rate is 69.22%. In the present study, we investigated the mechanism of CDA-2 on MDS using high-risk MDS cell line namely MuTz-1. MTT assay results showed that CDA-2 significantly inhibit the cell growth at a dose and time-dependent manner. Flow cytometer anlyasis showed that this growth inhibition was remarkblely associated with cycle arrest in G1-phase, but not associated with apoptosis. In addition, CDA-2 increased the expression of CD11b/CD14, a pair markers representing cell differentiation. we found the spectrum of hypermethylated tumor suppressor genes (TIMP3, CDKN2B, CHFR, CD44, RASSF1, TP73, IGSF4, CDH13 and DAPK) in MuTz-1 cells by Methylation-Specific Multiplex ligation-dependent Probe amplification (MS-MLPA), but the hypermethylation of these genes were remarkable decreased, as well as the expressions of DNA methyltransferase genes DNMT1 and DNMT3B at mRNA and protein level were downregulated in the treatment for 3 days with CDA-2. Also, we used CDA-2 for treatment of three MDS patients, whose several tumor suppressor genes are hypermethylated. After tour weeks of treatment, all the hypermethylation genes were undetected, part of this result was verified by methylation specific PCR (MSP) and bisulphite sequencing. In conclusion, our results demonstrated that CDA-2 may be an effective agent targeting hepermethylated tumor suppressor genes on MDS.

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DNA Methylation Changes Following 5-Azacitidine Treatment in Patients with Myelodysplastic Syndrome.

Blood ◽

10.1182/blood.v114.22.2791.2791 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2791-2791

Author(s):

Huong Thi Thanh Tran ◽

Hee Nam Kim ◽

Yeo-Kyeoung Kim ◽

Jae-Sook Ahn ◽

Il-Kwon Lee ◽

...

Keyword(s):

Dna Methylation ◽

Tumor Suppressor ◽

Tumor Suppressor Genes ◽

Dna Methyltransferase ◽

Conflicts Of Interest ◽

Control Group ◽

Disease Pathogenesis ◽

Suppressor Genes ◽

Clinical Responses ◽

Dependent Probe

Abstract Abstract 2791 Poster Board II-767 Gene silencing by promoter methylation is as potent as functional inactivating of tumor suppressor genes by mutations. DNA methyltransferase inhibitor, 5-azacytidine (AC) and 5-aza-2 -deoxycitidine (DAC), which is proved to be effective in myelodysplastic syndromes (MDS) can induce re-expression in cancer; however their mechanism remains controversial. 25 tumor suppressor genes by MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification) were analyzed in 44 MDS patients treated Vidaza® (5-azacitidine, AC). Hypermethylation of at least one gene was detected in 9/44 patients (20.5%), including four genes CDKN2B, FHIT, ESR1 and IGSF4. Interestingly, of 9 hypermethylated patients, 8 patients showed demethylation in concordance with their clinical responses after three to five cycles AC treatment. Lack or decrease methylation was observed in four patients with hematological improvements. Persistence methylation was observed in four others who became AML transformation or no response after treatment, especially reinforcing methylated gene in a case progressed to leukemia later. Our study also founds out IGSF4 gene hypermethylation in MDS as a first report. Additionally, mRNA expression of CDKN2B, IGSF4, and ESR1 in MDS were significantly lower than those in the control group (p < 0.05). Our results suggest that the methylation changes of specific genes contributes to disease pathogenesis and might present a molecular marker that can be used to monitor the efficacy of AC treatment in MDS. Disclosures: No relevant conflicts of interest to declare.

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Hypermethylation of tumor suppressor genes in gastric cancer: associations with demographic and clinicopathological characteristics

International Journal of Research in Medical Sciences ◽

10.18203/2320-6012.ijrms20161783 ◽

2016 ◽

pp. 2185-2192

Author(s):

Binnur Bagci ◽

Kursat Karadayi ◽

Gokhan Bagci ◽

Hatice Ozer ◽

Ersin Tuncer ◽

...

Keyword(s):

Gastric Cancer ◽

Tumor Suppressor ◽

Tumor Suppressor Genes ◽

Clinicopathological Characteristics ◽

Suppressor Genes

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Hypermethylation of the Putative Tumor-suppressor Genes DCC, p51/63 and O6-methylguanine-DNA Methyltransferase (MGMT) and Loss of Their Expressions in Cell Lines of Hematological Malignancies

Journal of Nippon Medical School ◽

10.1272/jnms.72.270 ◽

2005 ◽

Vol 72 (5) ◽

pp. 270-277 ◽

Cited By ~ 2

Author(s):

Kazutaka Nakayama ◽

Koiti Inokuchi ◽

Kazuo Dan

Keyword(s):

Tumor Suppressor ◽

Cell Lines ◽

Tumor Suppressor Genes ◽

Dna Methyltransferase ◽

Hematological Malignancies ◽

Suppressor Genes ◽

Methylguanine Dna Methyltransferase ◽

Putative Tumor Suppressor

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Increased Number of CpG Island Hypermethylation in Tumor Suppressor Genes of Non-Neoplastic Gastric Mucosa Correlates with Higher Risk of Gastric Cancer

Digestion ◽

10.1159/000252766 ◽

2010 ◽

Vol 82 (1) ◽

pp. 27-36 ◽

Cited By ~ 17

Author(s):

Tomomitsu Tahara ◽

Tomoyuki Shibata ◽

Masakatsu Nakamura ◽

Hiromi Yamashita ◽

Daisuke Yoshioka ◽

...

Keyword(s):

Gastric Cancer ◽

Gastric Mucosa ◽

Tumor Suppressor ◽

Tumor Suppressor Genes ◽

Cpg Island ◽

Suppressor Genes

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Identification of Epigenetically Silenced Tumor Suppressor Genes in Myeloid Disorders Leads to the Identification of α-Catenin as a Target Gene in 5q- Syndrome.

Blood ◽

10.1182/blood.v104.11.2565.2565 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2565-2565

Author(s):

J. C. Desmond ◽

S. D. Raynaud ◽

L. C. Jones ◽

W. K. Hofmann ◽

T. Haferlach ◽

...

Keyword(s):

Tumor Suppressor ◽

Tumor Suppressor Genes ◽

Cytosine Methylation ◽

Cpg Island ◽

Hot Spot ◽

Pcr Analysis ◽

Chromosome 5 ◽

Suppressor Genes ◽

Regulatory Regions ◽

Myeloid Metaplasia

Abstract CpG islands in the 5′ regulatory regions of genes are generally protected from cytosine methylation as methylation in a promoter can result in transcriptional silencing of the associated gene. Failure of a cell to prevent methylation in the promoter regions of tumor suppressor genes contributes to the onset and progression of cancers. The demethylating agent 5-aza-2′deoxycytidine (DAC) and the histone deacetylase inhibitor suberoyl anilide bishydroxamide (SAHA) possess potent antitumorigenic properties against myeloid disorders. Understanding the alterations of the transcriptome mediated by these drugs should prove vital in uncovering potential tumor suppressor genes epigenetically silenced in myeloid disorders. To this end, we used DAC and SAHA to induce expression of methylated genes in the CD34+ AML cell line KG-1. Expression levels of over 22,000 genes were compared between normal CD34+ cells and treated and untreated KG-1 cells using Affymetrix HG-U133A GeneChip® micoarrays. Statistical analyses revealed 76 genes constitutively expressed in normal CD34+ stem cells, absent in KG-1 cells but whose expression was induced in KG-1 after drug treatment. 39 (51%) of these genes harbored a CpG island in their 5′ regulatory regions, representing potentially methylated tumor suppressor genes in AML. To fit the tumor suppressor paradigm, we hypothesized that any gene possessing antitumorigenic properties would not be expressed in a number of AML patient samples. We examined the expression level of our 39 genes in 120 AML patient samples using microarray analyses. 20 patients belonging to each of the following AML karyotypic groups were analyzed: t(8;21), t(15:17), inv(16), 11q23/MLL, complex and normal karyotpye. Of special note were 8 genes, whose expression was markedly diminished in a subset of patients across all AML karyotypes examined: DAR22, TFIIS, EH-3, ENO2, MXA, DRAL, ASTML and MG50. These represent strong candidates for tumor suppressor genes in AML. Unsupervised clustering analyses using our original 39 genes were performed upon microarray data obtained from patients with myeloproliferative disease (MPD). A subset of 10 genes discriminated between granulocyte samples obtained from healthy donors and those obtained from a subset of agnogenic myeloid metaplasia, essential thronbocythemia and polycythemia vera patients. One of these genes, α-catenin, is located at q31 of chromosome 5, a hot spot for deletion in MDS and AML. α-catenin was expressed in all 120 primary AML samples, including those harboring deletions in chromosome 5. However, Real Time PCR analysis of 32 MDS patients harboring a 5q deletion in the region of α-catenin showed a marked decrease in expression of this gene compared to 20 non 5q- MDS patients. Neighboring genes in the deleted region of 5q did not show as marked a decrease in expression, suggesting loss of expression of both α-catenin alleles in these patients. These findings imply a double hit mechanism in 5q- MDS, where loss of one allele of α-catenin through deletion is supplemented by epigenetic silencing (directly or indirectly) of the second allele. In summary, we have uncovered groups of genes that may be involved in the pathogenesis of AML and various MPDs by virtue of their transcriptional repression through epigenetic events. Importantly, we have identified α-catenin as a key gene on chromosome 5, whose expression is lost in 5q- MDS.

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