• Asian Pacific Journal of Cancer Prevention
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• v.14 no.11
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• pp.6721-6726
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• 2013

Background: Changes in DNA methylation patterns are believed to be early events in hepatocarcinogenesis. A better understanding of methylation states and how they correlate with disease progression will aid in finding potential strategies for early detection of HCC. The aim of our study was to analyze the methylation frequency of tumor suppressor genes, P14, P15, and P73, and a mismatch repair gene (O6MGMT) in HCV related chronic liver disease and HCC to identify candidate epigenetic biomarkers for HCC prediction. Materials and Methods: 516 Egyptian patients with HCV-related liver disease were recruited from Kasr Alaini multidisciplinary HCC clinic from April 2010 to January 2012. Subjects were divided into 4 different clinically defined groups - HCC group (n=208), liver cirrhosis group (n=108), chronic hepatitis C group (n=100), and control group (n=100) - to analyze the methylation status of the target genes in patient plasma using EpiTect Methyl qPCR Array technology. Methylation was considered to be hypermethylated if >10% and/or intermediately methylated if >60%. Results: In our series, a significant difference in the hypermethylation status of all studied genes was noted within the different stages of chronic liver disease and ultimately HCC. Hypermethylation of the P14 gene was detected in 100/208 (48.1%), 52/108 (48.1%), 16/100 (16%) and 8/100 (8%) among HCC, liver cirrhosis, chronic hepatitis and control groups, respectively, with a statistically significant difference between the studied groups (p-value 0.008). We also detected P15 hypermethylation in 92/208 (44.2%), 36/108 (33.3%), 20/100 (20%) and 4/100 (4%), respectively (p-value 0.006). In addition, hypermethylation of P73 was detected in 136/208 (65.4%), 72/108 (66.7%), 32/100 (32%) and 4/100 (4%) (p-value <0.001). Also, we detected O6MGMT hypermethylation in 84/208 (40.4%), 60/108 (55.3%), 20/100 (20%) and 4/100 (4%), respectively (p value <0.001. Conclusions: The epigenetic changes observed in this study indicate that HCC tumors exhibit specific DNA methylation signatures with potential clinical applications in diagnosis and prognosis. In addition, methylation frequency could be used to monitor whether a patient with chronic hepatitis C is likely to progress to liver cirrhosis or even HCC. We can conclude that methylation processes are not just early events in hepatocarcinogenesis but accumulate with progression to cancer.

• Journal of Genetic Medicine
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• v.5 no.1
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• pp.41-46
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• 2008

Purpose : Fragile X syndrome (FXS) is the most common heritable cause of cognitive impairment. FXS is caused by hyperexpansion and hypermethylation of a polymorphic CGG trinucleotide repeat in the 5' untranslated region of the fragile X mental retadation-1(FMR1) gene. Combination of Southern blotting and simple polymerase chain reaction(PCR) amplification of the FMR1 repeat region is commonly used for diagnosis in females. To give a definite diagnosis in a female child suspected of having FXS, we carried out the molecular diagnostic test for FXS using the recently developed Abbott Molecular Fragile X PCR Kit. Methods : The PCR amplification of the FMR1 repeat region was performed using the Abbott Mdecular Fragile X PCR Kit. The amplified products were analyzed by size-separate analysis on 1.5% agarose gels and by DNA fragment analysis using Gene scan. Results : Agarose gel and Gene scan analyses of PCR products of the FMR1 repeat region showed that the patient had two heterozygous alleles with a normal 30 repeats and full mutation of >200 repeats whereas her mother had two heterozygous alleles with the normal 30 repeats and premutation of 108 repeats, suggesting that the premutation of 108 repeats in her mother may have led to the full mutation of >200 repeats in the patient. Conclusion : We diagnosed FXS in a female patient using a simplified molecular diagnostic test. This commercially available diagnostic test for FXS, based on PCR, may be a suitable alternative or complement method to Southern blot analysis and PCR analysis and/or methylation specific(MS)-PCR analysis for the molecular diagnosis of FXS in both males and females.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.4
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• pp.2185-2193
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• 2016

Background: The pathogenesis of sporadic colorectal cancer (CRC) is influenced by the patient genetic background and environmental factors. Based on prior understanding, these are classified in two major pathways of genetic instability. Microsatellite instability (MSI) and CPG island methylator phenotype (CIMP) are categorized as features of the hypermethylated prototype, and chromosomal instability (CIN) is known to be indicative of the non-hypermethylated category. Secreted frizzled related protein 2 (SFRP2), APC1A in WNT signaling pathway and the DNA repair gene, O6-methylguanine-DNA methyltransferase (MGMT), are frequently hypermethylated in colorectal cancer. Detection of methylated DNA as a biomarker by easy and inexpensive methods might improve the quality of life of patients with CRC via early detection of cancer or a precancerous condition. Aim: To evaluate the rate of SFRP2 and MGMT hypermethylation in both polyp tissue and serum of patients in south Iran as compared with matched control normal population corresponding samples. Materials and Methods: Methylation-specific PCR was used to detect hypermethylation in DNA extracted from 48 polypoid tissue samples and 25 healthy individuals. Results: Of total polyp samples, 89.5% had at least one promoter gene hypermethylation. The most frequent methylated locus was SFRP2 followed by MGMT-B (81.2 and 66.6 percent respectively). Serologic detection of hypermethylation was 95% sensitive as compared with polyp tissue. No hypermethylation was detected in normal tissue and serum and its detection in patients with polyps, especially of serrated type, was specific. Conclusions: Serologic investigation for detection of MGMT-B, SFRP2 hypermethylation could facilitate prioritization of high risk patients for colonoscopic polyp detection and excision.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.11
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• pp.4539-4543
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• 2014

Since the epigenetic alteration in tumor cells can be reversed by the dietary polyphenol quercetin (Q) or butyrate (B) with chemopreventive activity, suggesting that Q or B can be used for chemopreventive as well as therapeutic agent against tumors. In this study the polyphenol flavonoid quercetin (Q) or sodium butyrate (B) suppressed human esophageal 9706 cancer cell growth in dose dependent manner, and Q combined with B (Q+B) could further inhibit Eca9706 cell proliferation than that induced by Q or B alone, compared with untreated control group (C) in MTT assay. The reverse expressions of global DNMT1, $NF-{\kappa}Bp65$, HDAC1 and Cyclin D1 were down-regulated, while expressions of caspase-3 and $p16INK4{\alpha}$ were up-regulated, compared with the C group in immunoblotting; the down-regulated HDAC1-IR (-immunoreactivity) with nuclear translocation, and up-regulated E-cadherin-IR demonstrated in immunocytochemistry treated by Q or B, and Q+B also displayed further negatively and positively modulated effects compared with C group. The order of methylation specific (MS) PCR of $p16INK4{\alpha}$: C>B/Q>Q+B group, while the order of E-cadherin expression level was contrary, Q+B>Q/B>C group. Thus, Q/B, especially Q+B display reverse effect targeting both altered DNA methylation and histone acetylation, acting as histone deacetylase inhibitor mediated via epigenetic-$NF-{\kappa}B$ cascade signaling.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.7
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• pp.4421-4426
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• 2013

Objective: The present study employed 5-aza-2'-deoxycytidine (5-Aza-CdR) to treat non-small cell lung cancer (NSCLC) cell line A549 to investigate the effects on proliferation and expression of the TFPI-2 gene. Methods: Proliferation was assessed by MTT assay after A549 cells were treated with 0, 1, 5, 10 ${\mu}mol/L$ 5-Aza-CdR, a specific demethylating agent, for 24, 48 and 72h. At the last time point cells were also analyzed by flow cytometry (FCM) to identify any change in their cell cycle profiles. Methylation-specific polymerase chain reaction (MSPCR), real time polymerase chain reaction(real-time PCR) and western blotting were carried out to determine TFPI-2 gene methylation status, mRNA expression and protein expression. Results: MTT assay showed that the growth of A549 cells which were treated with 5-Aza-CdR was significantly suppressed as compared with the control group (0 ${\mu}mol/L$ 5-Aza-CdR). After treatment with 0, 1, 5, 10 ${\mu}mol/L$ 5-Aza-CdR for 72h, FCM showed their proportion in G0/G1 was $69.7{\pm}0.99%$, $76.1{\pm}0.83%$, $83.8{\pm}0.35%$, $95.5{\pm}0.55%$ respectively (P<0.05), and the proportion in S was $29.8{\pm}0.43%$, $23.7{\pm}0.96%$, $15.7{\pm}0.75%$, $1.73{\pm}0.45%$, respectively (P<0.05), suggesting 5-Aza-CdR treatment induced G0/G1 phase arrest. MSPCR showed that hypermethylation in the promoter region of TFPI-2 gene was detected in control group (0 ${\mu}mol/L$ 5-Aza-CdR), and demethylation appeared after treatment with 1, 5, 10 ${\mu}mol/L$ 5-Aza-CdR for 72h. Real-time PCR showed that the expression levels of TFPI-2 gene mRNA were $1{\pm}0$, $1.49{\pm}0.14$, $1.86{\pm}0.09$ and $5.80{\pm}0.15$ (P<0.05) respectively. Western blotting analysis showed the relative expression levels of TFPI-2 protein were $0.12{\pm}0.01$, $0.23{\pm}0.02$, $0.31{\pm}0.02$, $0.62{\pm}0.03$ (P<0.05). TFPI-2 protein expression in A549 cells was gradually increased significantly with increase in the 5-Aza-CdR concentration. Conclusions: TFPI-2 gene promoter methylation results in the loss of TFPI-2 mRNA and protein expression in the non-small cell lung cancer cell line A549, and 5-Aza-CdR treatment could induce the demethylation of TFPI-2 gene promoter and restore TFPI-2 gene expression. These findings provide theoretic evidence for clinical treatment of advanced non-small cell lung cancer with the demethylation agent 5-Aza-CdR. TFPI-2 may be one molecular marker for effective treatment of advanced non-small cell lung cancer with 5-Aza-CdR.

• Journal of Applied Biological Chemistry
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• v.48 no.3
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• pp.113-119
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• 2005

O-methylation mediated by O-methyltransferases (OMTs) is a common modification in natural product biosynthesis and contributes to diversity of secondary metabolites. OMTs use phenylpropanoids, flavonoids, other phenolics and alkaloids as substrates, and share common domains for S-adenosyl-L-methionine (AdoMet) and substrate binding. We searched Arabiposis genome and found 17 OMTs genes (AtOMTs). AdoMet- and substrate-binding sites were predicted. AdoMet binding domain of AtOMTs is highly conserved, while substrate-binding domain is diverse, indicating use of different substrates. In addition, expressions of six AtOMT genes in response to UV and in different tissues were investigated using real-time quantitative reverse transcriptase-polymerase chain reaction. All the AtOMTs investigated were expressed under normal growth condition and most, except AtOMT10, were induced after UV illumination. AtOMT1 and AtOMT8 were expressed in all the tissues, whereas AtOMT10 showed flower-specific expression. Analysis of these AtOMT gene expressions could provide some clues on AtOMT involvement in the cellular processes.

• YAKHAK HOEJI
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• v.49 no.6
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• pp.511-517
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• 2005

Low sensitivity to anticancer drugs such as 5-fluorouracil (5-FU) has been associated with decreased expression of genes involved in cell proliferation, apoptosis and metastasis. Recently, it has been shown that the expression levels of some of these genes are reduced by transcription inhibition due to epigenetic silencing on CpG islands. Therefore, epigenetic therapy has been proposed, where epigenetic silencing is repressed with DNA methyltransferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors alone or in combination with other chemotherapeutic agents. The aim of our study was to evaluate the combination effect of 5-FU and its association with the status of epigenetic silencing using methylation-specific PCR of $p14^{ARF}$ when given with S-aza-2'-deoxycytidine (5-aza-dC), a DNMT inhibitor and depsipeptide, an HDAC inhibitor in DLD-1 human colorectal cancer cells. The combination of 5-aza-dC with depsipeptide showed a synergism and induced unmethylation of $p14^{ARF}$. However, triplet combination of 5-aza-dc/depsipeptide and 5-FU resulted in antagonistic effects and abrogated unmethylation of $p14^{ARF}$. These results suggest that unfavorable interaction of 5-aza-dC/depsipeptide with 5-FU in DLD-1 cells may be related with the failure in repression of epigenetic silencing, which warrants further investigation.

• Journal of Microbiology and Biotechnology
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• v.27 no.1
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• pp.189-196
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• 2017

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with formation of Kaposi's sarcoma, multicentric Castleman's disease, and primary effusion lymphoma. Replication and transcription activator (RTA) genes are expressed upon reactivation of KSHV, which displays a biphasic life cycle consisting of latent and lytic replication phases. RTA protein expression results in KSHV genome amplification and successive viral lytic gene expression. Transcriptional activity of viral lytic genes is regulated through epigenetic modifications. In Raji cells latently infected with Epstein-Barr virus, various modifications, such as acetylation and methylation, have been identified at specific lysine residues in histone H4 during viral reactivation, supporting the theory that expression of specific lytic genes is controlled by histone modification processes. Data obtained from chromatin immunoprecipitation and quantitative real-time PCR analyses revealed alterations in the H4K8ac and H4K20me3 levels at lytic gene promoters during reactivation. Our results indicate that H4K20me3 is associated with the maintenance of latency, while H4K8ac contributes to KSHV reactivation in infected TREx BCBL-1 RTA cells.

• Journal of Plant Biotechnology
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• v.37 no.4
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• pp.517-528
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• 2010

Rice seed maturation and germination involve drastic changes in water and nutrient transport, in which tonoplast aquaporins may play an important role. In the present study, gene expression profiles of 10 tonoplast intrinsic proteins (TIP) from rice were investigated by RT-PCR during seed development and germination. OsTIP3;1 and OsTIP3;2 were specifically expressed in mature seeds. Their transcript level rapidly decreased after onset of seed germination and gene expression was induced by ABA treatment. In contrast, expression of OsTIP2;1 and OsTIP4;3 was not seed specific as transcripts were found in vegetative tissues as well. Their respective transcript levels decreased at an early stage of seed development, whereas they increased at a later stage of seed germination and elongation of embryonic roots and shoots. When seed germination was inhibited by various stress conditions and ABA, expression of OsTIP2;1 and OsTIP4;3 was completely suppressed. In contrast, the expression level of OsTIP2;2 rapidly increased after seed imbibition and the transcript level was maintained under conditions inhibiting seed germination. These results implicate that tissue specific and developmental transcriptional regulation of OsTIPs in rice seeds depends on their specific function. In addition, OsTIPs can be discriminated by different potential phosphorylation and methylation sites in their protein structures. OsTIP3;1 and OsTIP3;2 possess unique phosphorylation signatures at their N-terminal domain, loop B and loop E, respectively. OsTIP2;1 and OsTIP4;3 have a potential methylation site at their Nterminal domain. This suggests that activity of specific tonoplast aquaporins may be regulated by post-translational modification as well as by transcriptional control.

• Tuberculosis and Respiratory Diseases
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• v.64 no.4
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• pp.285-292
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• 2008

Background: A diagnosis of malignant pleural effusion is clinically important, as the prognosis of lung cancer patients with malignant pleural effusion is poor. The diagnosis will be difficult if a cytological test is negative. This study was performed to investigate whether the detection of hypermethylation of the p16 (CDKN2A) and retinoic acid receptor b2 (RARB2) genes in pleural fluid is useful for a diagnosis of malignant pleural effusion. Methods: Pleural effusion was collected from 43 patients and was investigated for the aberrant promoter methylation of the RARB2 and CDKN2A genes by use of methylation-specific PCR. Results were compared with findings from a pleural biopsy and from pleural fluid cytology. Results: Of 43 cases, 17 cases of pleural effusion were due to benign diseases, and 26 cases were from lung cancer patients with malignant pleural effusion. Hypermethylation of the RARB2 and CDKN2A genes was not detected in the case of benign diseases, independent of whether or not the patients had ever smoked. In 26 cases of malignant pleural effusion, hypermethylation of RARB2, CDKN2A or either of these genes was detected in 14, 5 and 15 cases, respectively. The sensitivities of a pleural biopsy, pleural fluid cytology, hypermethylation of RARB2, hypermethylation of CDKN2A, or hypermethylation of either of the genes were 73.1%, 53.8%, 53.8%, 19.2%, and 57.7%, respectively; negative predictive values were 70.8%, 58.6%, 58.6%, 44.7%, and 60.7%, respectively. If both genes are considered together, the sensitivity and negative predictive value was lower than that for a pleural biopsy, but higher than that for pleural fluid cytology. The sensitivity of hypermethylation of the RARB2 gene for malignant pleural effusion was lower in small cell lung cancers than in non-small cell lung cancers. Conclusion: These results demonstrate that detection of hypermethylation of the RARB2 and CDKN2A genes showed a high specificity, and sensitivity was higher than for pleural fluid cytology. With a better understanding of the pathogenesis of lung cancer according to histological types at the molecular level, and if appropriate genes are selected for hypermethylation testing, more precise results may be obtained.