The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity bein...The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included 1) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, 2) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and 3) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumorselective reduction in MGMT activity exist in human tissue.展开更多
AIM:To investigate whether gene methylation in the peritoneal fluid (PF) predicts peritoneal recurrence in gastric cancer patients.METHODS: The gene methylation of CHFR (checkpoint with forkhead and ring finger domain...AIM:To investigate whether gene methylation in the peritoneal fluid (PF) predicts peritoneal recurrence in gastric cancer patients.METHODS: The gene methylation of CHFR (checkpoint with forkhead and ring finger domains), p16, RUNX3 (runt-related transcription factor 3), E-cadherin, hMLH1 (mutL homolog 1), ABCG2 (ATP-binding cassette, sub-family G, member 2) and BNIP3 (BCL2/adenovirus E1B 19 kDa interacting protein 3) were analyzed in 80 specimens of PF by quantitative methylation-specific polymerase chain reaction (PCR). Eighty patients were divided into 3 groups; Group A (n=35):the depth of cancer invasion was less than the muscularis propria; Group B (n=31): the depth of cancer invasion was beyond the muscularis propria. Both group A and B were diagnosed as no cancer cells in peritoneal cytology and histology; Group C (n=14): disseminated nodule was histologically diagnosed or cancer cells were cytologically defi ned in the peritoneal cavity.RESULTS: The positive rates of methylation in CHFR, E-cadherin and BNIP3 were significantly different among the 3 groups and increased in order of group A, B and C (0%,0% and 21% in CHFR, P<0.05; 20%, 45% and 50% in E-cadherin, P<0.05;26%,35% and 71% in BNIP3, P<0.05). In addition, the multigene methylation rate among CHFR, E-cadherin and BNIP3 was correlated with group A, B and C (9%,19% and 57%, P<0.001). Moreover, the prognosis was analyzed in group B, excluding 3 patients who underwent a non-curative resection. Two of the 5 patients with multigene methylation showed peritoneal recurrence after surgery, while those without or with a single gene methylation did not experience recurrence (P<0.05).CONCLUSION: This study suggested that gene methylation in the PF could detect occult neoplastic cells in the peritoneum and might be a risk factor for peritoneal metastasis.展开更多
AIM： To explore epigenetic changes in the gene encod- ing X chromosome-linked inhibitor of apoptosis-associ- ated factor 1 （XAF1） during esophageal carcinogenesis. METHODS： Methylation status of XAF1 was detected ...AIM： To explore epigenetic changes in the gene encod- ing X chromosome-linked inhibitor of apoptosis-associ- ated factor 1 （XAF1） during esophageal carcinogenesis. METHODS： Methylation status of XAF1 was detected by methylation-specific polymerase chain reaction （MSP） in four esophageal cancer cell lines （KYSE30, KYSE70, BICl and partially methylated in TE3 cell lines）, nine cases of normal mucosa, 72 cases of pri- mary esophageal cancer and matched adjacent tissue. XAF1 expression was examined by semi-quantitative reverse transcriptional polymerase chain reaction and Western blotting before and after treatment with 5-aza- deoxycytidine （5-aza-dc）, a demethylating agent. To investigate the correlation of XAF1 expression and methylation status in primary esophageal cancer, immu- nohistochemistry for XAF1 expression was performed in 32 cases of esophageal cancer and matched adjacent tissue. The association of methylation status and clini-copathological data was analyzed by logistic regression. RESULTS： MSP results were as follows： loss of XAF1 expression was found in three of four esophageal cell lines with promoter region hypermethylation （com- pletely methylated in KYSE30, KYSE70 and BIC1 cell lines and partially in TE3 cells）; all nine cases of normal esophageal mucosa were unmethylated; and 54/72 （75.00%） samples from patients with esophageal can- cer were methylated, and 25/72 （34.70%） matched adjacent tissues were methylated （75.00% vs 34,70%, z2 = 23.5840, P = 0.000）. mRNA level of XAF1 mea- sured with semi-quantitative reverse transcription poly- merase chain reaction was detectable only in TE3 cells, and no expression was detected in KYSE30, KYSE70 or BIC1 cells. Protein expression was not observed in KYSE30 cells by Western blotting before treatment with 5-aza-dc. After treatment, mRNA level of XAF1 was detectable in KYSE30, KYSE70 and BIC1 cells. Protein expression was detected in KYSE30 after treatment with 5-aza-dc. Immunohistochemistry was performed on 32 cases of esophageal cancer and adjacent tissue, and demonstrated XAF1 in the nucleus and cytoplasm. XAF1 staining was found in 20/32 samples of adjacent normal tissue but was present in only 8/32 samples of esophageal cancer tissue （Z2= 9.143, P = 0.002）. XAF1 expression was decreased in cancer samples compared with adjacent tissues. In 32 cases of esophageal can- cer, 24/32 samples were methylated, and 8/32 esopha- geal cancer tissues were unmethylated. XAF1 staining was found in 6/8 samples of unmethylated esophageal cancer and 2/24 samples of methylated esophageal cancer tissue. XAF1 staining was inversely correlated with XAF1 promoter region methylation （Fisher＇s exact test, P = 0.004）. Regarding methylation status and clinicopathological data, no significant differences were found in sex, age, tumor size, tumor stage, or metas- tasis with respect to methylation of XAF1 for the 72 tis- sue samples from patients with esophageal cancer. CONCLUSION： XAF1 is frequently methylated in eso- phageal cancer, and XAF1 expression is regulated by promoter region hypermethylation.展开更多
Background: Oncogenic insulin-like growth factor-II(IGF-II) is overexpressed in hepatocellular carcinoma(HCC). The present study aimed to analyze the dynamic alteration of IGF-II CpG site methylation status and its mo...Background: Oncogenic insulin-like growth factor-II(IGF-II) is overexpressed in hepatocellular carcinoma(HCC). The present study aimed to analyze the dynamic alteration of IGF-II CpG site methylation status and its molecular mechanism in HCC progression. Methods: IGF-II alterations were observed in rat hepatocarcinogenesis models induced by 2-acetylaminofluorene. Liver IGF-II expression was compared by immunohistochemistry or tissue IGF-II specific concentration(nmol/mg protein). Status of human IGF-II promoter 3(P3) or rat IGF-II P2 CpG site methylation was amplified by methylation-specific polymerase chain reaction(MSP). Serum IGF-II levels were quantitatively detected by an enzyme-linked immunosorbent assay. Results: The levels of hepatic IGF-II expression were significantly elevated in the HCC group( P < 0.001). The unmethylation rate of IGF-II P3 CpG sites was 100% in the HCC-, 52.5% in the paracancerous-, and none(0%) in the distal noncancerous-tissues. Abnormal IGF-II expression was related to differentiation degree, tumor invasion, and positive HBV-DNA(all P < 0.001), with a negative correlation between P3 methylation degree and IGF-II expression. There was a positive correlation between liver IGF-II specific concentration and circulating IGF-II level( r = 0.97, P < 0.001). Significantly negative correlation was found between IGF-II P2 CpG site methylation and circulating IGF-II( r s =-0.89, P < 0.001) or liver IGF-II level( r s =-0.84, P < 0.001). Conclusions: The increase of serum IGF-II and the alteration of oncogenic gene IGF-II methylation may be biomarkers for HCC diagnosis and DNA methylation may be the therapeutic target of HCC.展开更多
BACKGROUND: Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-regulated Ser/Thr kinase which is involved in apoptosis. The aberrant methylation of its promoter region CpG islands may be one of the important ...BACKGROUND: Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-regulated Ser/Thr kinase which is involved in apoptosis. The aberrant methylation of its promoter region CpG islands may be one of the important mechanisms of carcinogenesis. We studied the relationship of methylation status and expression of the DAPK gene with the clinical findings in cholangiocarcinoma. METHODS: Target DNA was modified by sodium bisulfite, coverting all unmethylated, but not methylated, cytosines to uracil, and subsequently detected by methylation-specific PCR. Moreover, mRNA expression of the DAPK gene was assessed by RT-PCR. RESULTS: Aberrant methylation of the DAPK gene was detected in 11 (30.6%) of 36 tissue specimens of cholangiocarcinoma, and in 2 (5.6%) of 36 specimens of adjacent normal tissues. DAPK mRNA was not expressed in tumor and adjacent tissues with hypermethylation of the DAPK promoter. There were no statistical differences in the extent of differentiation and invasion, lymph node metastasis or pathologic type between the methylated and unmethylated tissues. CONCLUSIONS: The frequency of DAPK gene methylation in cholangiocarcinoma is high and it may offer an effective means for earlier auxiliary diagnosis of the malignancy. The DAPK gene is probably suppressed by methylation, and it could become resistant to apoptosis and immunological surveillance. The DAPK gene epigenetically affected by methylation may be associated with the carcinogenesis of cholangiocarcinoma.展开更多
AIM： To detect the MLH1 gene promoter germline- methylation in probands of Chinese hereditary non- polyposis colorectal cancer （HNPCC）, and to evaluate the role of methylation in MLH1 gene promoter and molecular ge...AIM： To detect the MLH1 gene promoter germline- methylation in probands of Chinese hereditary non- polyposis colorectal cancer （HNPCC）, and to evaluate the role of methylation in MLH1 gene promoter and molecular genetics in screening for HNPCC.METHODS： The promoter germline methylation of MLH1 gene was detected by methylation-specific PCR （MSP） in 18 probands from unrelated HNPCC families with high microsatellite-instability （MSI-H） phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. At the same time, 6 kindreds were col- lected with microsatellite-stability （MSS） phenotype but without germline mutations in MSH2, MIH1 and MSH6 genes as controls. The results of MSP were confirmed by clone sequencing. To ensure the reliability of the results, family H65 with nonsense germline mutation at c.2228C 〉 A in MSH2 gene was used as the negative control and the cell line sw48 was used as the known positive control along with water as the blank control. Immunochemical staining of MIH1 protein was performed with Envision two-step method in those patients with aberrant methylation to judge whether the status of MLH1 gene methylation affects the expression of MLH1 protein.RESULTS： Five probands with MIH1 gene promoter methylation were detected in 18 Chinese HNPCC families with MSI-H phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. Two of the five probands from families H10 and H29 displayed exhaustive-methylation, fulfilling the Japanese criteria （JC） and the Amsterdam criteria （AC）, respectively. The other 3 probands presented part-methylation fulfilling the AC. Of the 13 probands with unmethylation phenotype, 8 fulfilled the JC and the Bethesda guidelines （BG）, 5 fulfilled the AC. The rate of aberrant methylation in MLH1 gene in the AC group （22.2%, 4/18） was higher than that in the JC/BG groups （5.6%, 1/18） in all HNPCC families with MSI-H phenotype but without germline mutations in PISH2, PIIH1 and MSH6 genes. However, no proband with methylation in MLH1 gene was found in the families with MSS phenotype and without germline mutations in MSH2, MLH1 and MSH6 genes. No expression of MLH1 protein was found in tumor tissues from two patients with exhaustive-methylation phenotype, whereas positive expression of MLH1 protein was observed in tumor tissues from patients with partial methylation phenotype （excluding family H42 without tumor tissue）, indicating that exhaustive-methylation of MLH1 gene can cause defective expression of MLH1 protein.CONCLUSION： Methylation phenotype of MLH1 gene is correlated with microsatellite phenotype of MMR genes, especially with MSI-H. Exhaustive-methylation of MLH1 gene can silence the expression of MLH1 protein. MLH1 promoter methylation analysis is a promising tool for molecular genetics screening for HNPCC.展开更多
The relationship between hypermethylation of CpG islands in the promoter regions of O^6- methylguanine DNA methyhransferase （MGMT） genes and laryngeal squamous cell carcinoma was explored. Methylation-specific PCR a...The relationship between hypermethylation of CpG islands in the promoter regions of O^6- methylguanine DNA methyhransferase （MGMT） genes and laryngeal squamous cell carcinoma was explored. Methylation-specific PCR and semi-quantitative RT-PCR were used to study the promoter methylation and mRNA expression of the MGMT gene in laryngeal carcinoma tissues, tissues adjacent to the tumor and normal laryngeal tissues. Hypermethylation of MGMT gene was detected in 16 samples of 46 （34.8 %） laryngeal squamous cell carcinoma samples. However, the MGMT hypermethylation was not detected in all tissues adjacent to the tumors and normal tissues. No significant difference in MGMT gene hypermethylation was found in samples with different histological grades （χ^2= 3. 130, P=0. 077） or in samples from patients with different TNM status （χ^2= 3. 957, P=0. 138）. No expression of MGMT mRNA was detected in all hypermethylated laryngeal carcinoma tissues. The expression of MGMT mRNA was detected in all unmethylated laryngeal carcinoma tissues, tissues adjacent to the tumors and normal tissues. It suggests that MGMT gene promoter hypermethylation is associated with MGMT gene transcription loss in laryngeal carcinoma tissues and possibly plays an important role in carcinogenesis of laryngeal tissues.展开更多
Epigenetic changes of DNA, including methylation, have long been recognized as key indicators of various diseases, including aging, cancer, and neurological disorders. Biomarker discoveries based on distinct methylati...Epigenetic changes of DNA, including methylation, have long been recognized as key indicators of various diseases, including aging, cancer, and neurological disorders. Biomarker discoveries based on distinct methylation patterns for both hypermethylation and hypomethylation lead the way in discovery of novel diagnosis and treatment targets. Many different approaches are present to detect the level of methylation in whole genome (whole genome bisulfite sequencing, microarray) as well as at specific loci (methylation specific PCR). Cell-free DNA (cf-DNA) found in body fluids like blood provides information about DNA methylation and serves as a less invasive approach for genetic screening. Cell-free DNA and methylation screening technologies, when combined, have the potential to transform the way we approach genetic screening and personalized therapy. These technologies can help enhance disease diagnostic accuracy and inform the development of targeted therapeutics by providing a non-invasive way for acquiring genomic information and identifying disease-associated methylation patterns. We highlight the clinical benefits of using cell-free DNA (cf-DNA) liquid biopsy analysis and available methylation screening technologies that have been crucial in identifying biomarkers for disease from patients using a non-invasive way. Powering such biomarker discoveries are various methods of cf-DNA methylation analysis such as Bisulfite Sequencing and most recently, Methylation-Specific Restriction Enzyme (MSRE-seq) Analysis, paving the way for novel epigenetic biomarker discoveries for more robust diagnosis such as early disease detection, prognosis, monitoring of disease progression and treatment response as well as discovery of novel drug targets.展开更多
O6-Methylguanine-DNA methyltransferase （MGMT） is a cellular DNA repair protein that rapidly reverses alkylation （e.g. methylation） at the O6 position of guanine, thereby neutralizing the cytotoxic effects of alkyl...O6-Methylguanine-DNA methyltransferase （MGMT） is a cellular DNA repair protein that rapidly reverses alkylation （e.g. methylation） at the O6 position of guanine, thereby neutralizing the cytotoxic effects of alkylating agent therapy such as temozolomide （TMZ） and carmustine. It has been shown that epigenetic silencing of the MGMT gene by promoter methylation shuts down gene transcription and reflects a common alteration in primary human tumors leading to MGMT deficiency. Epigenetic silencing of the MGMT gene has been shown to correlate with improved survival in several studies of glioma patients treated with the alkylating agent therapy and has been substantiated in two clinical trials.展开更多
Background Both in vitro and in vivo data have demonstrated the TGFBI gene functions as a putative tumor suppressor and is frequently downregulated in human tumors of different histological types.The hypermethylation ...Background Both in vitro and in vivo data have demonstrated the TGFBI gene functions as a putative tumor suppressor and is frequently downregulated in human tumors of different histological types.The hypermethylation of the TGFBI promoter,as one of the main regulatory mechanisms,is associated with TGFBI silencing.In this study,we used a methylation-specific PCR （MSP） method to evaluate the methylation status of the TGFBI promoter in human leukemias.Methods Real-time RT-PCR and methylation-specific PCR approaches were performed to define the TGFBI expression and promoter methylation in human leukemia call lines and clinical samples.Genomic DNA was isolated from peripheral blood mononuclear cells from leukemia patients,bisulfite-converted,and analyzed by the MSP method.Results Hypermethylation of the TGFBI promoter occurred in leukemia cell lines and demethylation treatment reexpressed TGFBI at a substantially increased level in most of leukemia cell lines tested.Furthermore,a much higher level of CpG island methylation and a significantly lower TGFBI expression were also identified in clinical leukemia samples.Conclusion The results suggest an important role of promoter methylation in regulating TGFBI expression in leukemia,which provides a useful diagnostic marker for clinical management of human leukemias.展开更多
文摘The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included 1) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, 2) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and 3) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumorselective reduction in MGMT activity exist in human tissue.
基金Supported by Fund from the Department of Surgery,Saga University Faculty of Medicine
文摘AIM:To investigate whether gene methylation in the peritoneal fluid (PF) predicts peritoneal recurrence in gastric cancer patients.METHODS: The gene methylation of CHFR (checkpoint with forkhead and ring finger domains), p16, RUNX3 (runt-related transcription factor 3), E-cadherin, hMLH1 (mutL homolog 1), ABCG2 (ATP-binding cassette, sub-family G, member 2) and BNIP3 (BCL2/adenovirus E1B 19 kDa interacting protein 3) were analyzed in 80 specimens of PF by quantitative methylation-specific polymerase chain reaction (PCR). Eighty patients were divided into 3 groups; Group A (n=35):the depth of cancer invasion was less than the muscularis propria; Group B (n=31): the depth of cancer invasion was beyond the muscularis propria. Both group A and B were diagnosed as no cancer cells in peritoneal cytology and histology; Group C (n=14): disseminated nodule was histologically diagnosed or cancer cells were cytologically defi ned in the peritoneal cavity.RESULTS: The positive rates of methylation in CHFR, E-cadherin and BNIP3 were significantly different among the 3 groups and increased in order of group A, B and C (0%,0% and 21% in CHFR, P<0.05; 20%, 45% and 50% in E-cadherin, P<0.05;26%,35% and 71% in BNIP3, P<0.05). In addition, the multigene methylation rate among CHFR, E-cadherin and BNIP3 was correlated with group A, B and C (9%,19% and 57%, P<0.001). Moreover, the prognosis was analyzed in group B, excluding 3 patients who underwent a non-curative resection. Two of the 5 patients with multigene methylation showed peritoneal recurrence after surgery, while those without or with a single gene methylation did not experience recurrence (P<0.05).CONCLUSION: This study suggested that gene methylation in the PF could detect occult neoplastic cells in the peritoneum and might be a risk factor for peritoneal metastasis.
基金Supported by Grants from the National Basic Research Program(973 Program), No. 2012CB934002, 2010CB912802National Key Scientific Instrument Special Programme of China, No.2011YQ03013405National Science Foundation of China,No. 81121004, 81071953 and 81161120432
文摘AIM： To explore epigenetic changes in the gene encod- ing X chromosome-linked inhibitor of apoptosis-associ- ated factor 1 （XAF1） during esophageal carcinogenesis. METHODS： Methylation status of XAF1 was detected by methylation-specific polymerase chain reaction （MSP） in four esophageal cancer cell lines （KYSE30, KYSE70, BICl and partially methylated in TE3 cell lines）, nine cases of normal mucosa, 72 cases of pri- mary esophageal cancer and matched adjacent tissue. XAF1 expression was examined by semi-quantitative reverse transcriptional polymerase chain reaction and Western blotting before and after treatment with 5-aza- deoxycytidine （5-aza-dc）, a demethylating agent. To investigate the correlation of XAF1 expression and methylation status in primary esophageal cancer, immu- nohistochemistry for XAF1 expression was performed in 32 cases of esophageal cancer and matched adjacent tissue. The association of methylation status and clini-copathological data was analyzed by logistic regression. RESULTS： MSP results were as follows： loss of XAF1 expression was found in three of four esophageal cell lines with promoter region hypermethylation （com- pletely methylated in KYSE30, KYSE70 and BIC1 cell lines and partially in TE3 cells）; all nine cases of normal esophageal mucosa were unmethylated; and 54/72 （75.00%） samples from patients with esophageal can- cer were methylated, and 25/72 （34.70%） matched adjacent tissues were methylated （75.00% vs 34,70%, z2 = 23.5840, P = 0.000）. mRNA level of XAF1 mea- sured with semi-quantitative reverse transcription poly- merase chain reaction was detectable only in TE3 cells, and no expression was detected in KYSE30, KYSE70 or BIC1 cells. Protein expression was not observed in KYSE30 cells by Western blotting before treatment with 5-aza-dc. After treatment, mRNA level of XAF1 was detectable in KYSE30, KYSE70 and BIC1 cells. Protein expression was detected in KYSE30 after treatment with 5-aza-dc. Immunohistochemistry was performed on 32 cases of esophageal cancer and adjacent tissue, and demonstrated XAF1 in the nucleus and cytoplasm. XAF1 staining was found in 20/32 samples of adjacent normal tissue but was present in only 8/32 samples of esophageal cancer tissue （Z2= 9.143, P = 0.002）. XAF1 expression was decreased in cancer samples compared with adjacent tissues. In 32 cases of esophageal can- cer, 24/32 samples were methylated, and 8/32 esopha- geal cancer tissues were unmethylated. XAF1 staining was found in 6/8 samples of unmethylated esophageal cancer and 2/24 samples of methylated esophageal cancer tissue. XAF1 staining was inversely correlated with XAF1 promoter region methylation （Fisher＇s exact test, P = 0.004）. Regarding methylation status and clinicopathological data, no significant differences were found in sex, age, tumor size, tumor stage, or metas- tasis with respect to methylation of XAF1 for the 72 tis- sue samples from patients with esophageal cancer. CONCLUSION： XAF1 is frequently methylated in eso- phageal cancer, and XAF1 expression is regulated by promoter region hypermethylation.
基金supported partly by grants from the National Natural Science Foundation(81673241,81702419,81872738,81873915)the Jiangsu Medical Science(BE2016698)&Graduate Innovation(KYCX17_1934)the International S.&T.Coopera-tion Program(2013DFA32150)of China
文摘Background: Oncogenic insulin-like growth factor-II(IGF-II) is overexpressed in hepatocellular carcinoma(HCC). The present study aimed to analyze the dynamic alteration of IGF-II CpG site methylation status and its molecular mechanism in HCC progression. Methods: IGF-II alterations were observed in rat hepatocarcinogenesis models induced by 2-acetylaminofluorene. Liver IGF-II expression was compared by immunohistochemistry or tissue IGF-II specific concentration(nmol/mg protein). Status of human IGF-II promoter 3(P3) or rat IGF-II P2 CpG site methylation was amplified by methylation-specific polymerase chain reaction(MSP). Serum IGF-II levels were quantitatively detected by an enzyme-linked immunosorbent assay. Results: The levels of hepatic IGF-II expression were significantly elevated in the HCC group( P < 0.001). The unmethylation rate of IGF-II P3 CpG sites was 100% in the HCC-, 52.5% in the paracancerous-, and none(0%) in the distal noncancerous-tissues. Abnormal IGF-II expression was related to differentiation degree, tumor invasion, and positive HBV-DNA(all P < 0.001), with a negative correlation between P3 methylation degree and IGF-II expression. There was a positive correlation between liver IGF-II specific concentration and circulating IGF-II level( r = 0.97, P < 0.001). Significantly negative correlation was found between IGF-II P2 CpG site methylation and circulating IGF-II( r s =-0.89, P < 0.001) or liver IGF-II level( r s =-0.84, P < 0.001). Conclusions: The increase of serum IGF-II and the alteration of oncogenic gene IGF-II methylation may be biomarkers for HCC diagnosis and DNA methylation may be the therapeutic target of HCC.
基金The study was supported by a grant from the Provincial Outstanding Youth Foundation of Shandong, China (No. 2005BS02008).
文摘BACKGROUND: Death-associated protein kinase (DAPK) is a Ca2+/calmodulin-regulated Ser/Thr kinase which is involved in apoptosis. The aberrant methylation of its promoter region CpG islands may be one of the important mechanisms of carcinogenesis. We studied the relationship of methylation status and expression of the DAPK gene with the clinical findings in cholangiocarcinoma. METHODS: Target DNA was modified by sodium bisulfite, coverting all unmethylated, but not methylated, cytosines to uracil, and subsequently detected by methylation-specific PCR. Moreover, mRNA expression of the DAPK gene was assessed by RT-PCR. RESULTS: Aberrant methylation of the DAPK gene was detected in 11 (30.6%) of 36 tissue specimens of cholangiocarcinoma, and in 2 (5.6%) of 36 specimens of adjacent normal tissues. DAPK mRNA was not expressed in tumor and adjacent tissues with hypermethylation of the DAPK promoter. There were no statistical differences in the extent of differentiation and invasion, lymph node metastasis or pathologic type between the methylated and unmethylated tissues. CONCLUSIONS: The frequency of DAPK gene methylation in cholangiocarcinoma is high and it may offer an effective means for earlier auxiliary diagnosis of the malignancy. The DAPK gene is probably suppressed by methylation, and it could become resistant to apoptosis and immunological surveillance. The DAPK gene epigenetically affected by methylation may be associated with the carcinogenesis of cholangiocarcinoma.
基金Supported by Shanghai Medical Development Fund for Major Projects, No. 05III004Shanghai Pujiang Projects for Talents, No. 06PJ14019
文摘AIM： To detect the MLH1 gene promoter germline- methylation in probands of Chinese hereditary non- polyposis colorectal cancer （HNPCC）, and to evaluate the role of methylation in MLH1 gene promoter and molecular genetics in screening for HNPCC.METHODS： The promoter germline methylation of MLH1 gene was detected by methylation-specific PCR （MSP） in 18 probands from unrelated HNPCC families with high microsatellite-instability （MSI-H） phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. At the same time, 6 kindreds were col- lected with microsatellite-stability （MSS） phenotype but without germline mutations in MSH2, MIH1 and MSH6 genes as controls. The results of MSP were confirmed by clone sequencing. To ensure the reliability of the results, family H65 with nonsense germline mutation at c.2228C 〉 A in MSH2 gene was used as the negative control and the cell line sw48 was used as the known positive control along with water as the blank control. Immunochemical staining of MIH1 protein was performed with Envision two-step method in those patients with aberrant methylation to judge whether the status of MLH1 gene methylation affects the expression of MLH1 protein.RESULTS： Five probands with MIH1 gene promoter methylation were detected in 18 Chinese HNPCC families with MSI-H phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. Two of the five probands from families H10 and H29 displayed exhaustive-methylation, fulfilling the Japanese criteria （JC） and the Amsterdam criteria （AC）, respectively. The other 3 probands presented part-methylation fulfilling the AC. Of the 13 probands with unmethylation phenotype, 8 fulfilled the JC and the Bethesda guidelines （BG）, 5 fulfilled the AC. The rate of aberrant methylation in MLH1 gene in the AC group （22.2%, 4/18） was higher than that in the JC/BG groups （5.6%, 1/18） in all HNPCC families with MSI-H phenotype but without germline mutations in PISH2, PIIH1 and MSH6 genes. However, no proband with methylation in MLH1 gene was found in the families with MSS phenotype and without germline mutations in MSH2, MLH1 and MSH6 genes. No expression of MLH1 protein was found in tumor tissues from two patients with exhaustive-methylation phenotype, whereas positive expression of MLH1 protein was observed in tumor tissues from patients with partial methylation phenotype （excluding family H42 without tumor tissue）, indicating that exhaustive-methylation of MLH1 gene can cause defective expression of MLH1 protein.CONCLUSION： Methylation phenotype of MLH1 gene is correlated with microsatellite phenotype of MMR genes, especially with MSI-H. Exhaustive-methylation of MLH1 gene can silence the expression of MLH1 protein. MLH1 promoter methylation analysis is a promising tool for molecular genetics screening for HNPCC.
文摘The relationship between hypermethylation of CpG islands in the promoter regions of O^6- methylguanine DNA methyhransferase （MGMT） genes and laryngeal squamous cell carcinoma was explored. Methylation-specific PCR and semi-quantitative RT-PCR were used to study the promoter methylation and mRNA expression of the MGMT gene in laryngeal carcinoma tissues, tissues adjacent to the tumor and normal laryngeal tissues. Hypermethylation of MGMT gene was detected in 16 samples of 46 （34.8 %） laryngeal squamous cell carcinoma samples. However, the MGMT hypermethylation was not detected in all tissues adjacent to the tumors and normal tissues. No significant difference in MGMT gene hypermethylation was found in samples with different histological grades （χ^2= 3. 130, P=0. 077） or in samples from patients with different TNM status （χ^2= 3. 957, P=0. 138）. No expression of MGMT mRNA was detected in all hypermethylated laryngeal carcinoma tissues. The expression of MGMT mRNA was detected in all unmethylated laryngeal carcinoma tissues, tissues adjacent to the tumors and normal tissues. It suggests that MGMT gene promoter hypermethylation is associated with MGMT gene transcription loss in laryngeal carcinoma tissues and possibly plays an important role in carcinogenesis of laryngeal tissues.
文摘Epigenetic changes of DNA, including methylation, have long been recognized as key indicators of various diseases, including aging, cancer, and neurological disorders. Biomarker discoveries based on distinct methylation patterns for both hypermethylation and hypomethylation lead the way in discovery of novel diagnosis and treatment targets. Many different approaches are present to detect the level of methylation in whole genome (whole genome bisulfite sequencing, microarray) as well as at specific loci (methylation specific PCR). Cell-free DNA (cf-DNA) found in body fluids like blood provides information about DNA methylation and serves as a less invasive approach for genetic screening. Cell-free DNA and methylation screening technologies, when combined, have the potential to transform the way we approach genetic screening and personalized therapy. These technologies can help enhance disease diagnostic accuracy and inform the development of targeted therapeutics by providing a non-invasive way for acquiring genomic information and identifying disease-associated methylation patterns. We highlight the clinical benefits of using cell-free DNA (cf-DNA) liquid biopsy analysis and available methylation screening technologies that have been crucial in identifying biomarkers for disease from patients using a non-invasive way. Powering such biomarker discoveries are various methods of cf-DNA methylation analysis such as Bisulfite Sequencing and most recently, Methylation-Specific Restriction Enzyme (MSRE-seq) Analysis, paving the way for novel epigenetic biomarker discoveries for more robust diagnosis such as early disease detection, prognosis, monitoring of disease progression and treatment response as well as discovery of novel drug targets.
文摘O6-Methylguanine-DNA methyltransferase （MGMT） is a cellular DNA repair protein that rapidly reverses alkylation （e.g. methylation） at the O6 position of guanine, thereby neutralizing the cytotoxic effects of alkylating agent therapy such as temozolomide （TMZ） and carmustine. It has been shown that epigenetic silencing of the MGMT gene by promoter methylation shuts down gene transcription and reflects a common alteration in primary human tumors leading to MGMT deficiency. Epigenetic silencing of the MGMT gene has been shown to correlate with improved survival in several studies of glioma patients treated with the alkylating agent therapy and has been substantiated in two clinical trials.
基金This study was supported by the National Basic Research Program of China （973 Program, No. 2013CB911000） and the National Nature Science Foundation of China （No. 30971602, 81272929）.Acknowledgements： We thank Dr. Chi Zhenfen for help with cell culture.
文摘Background Both in vitro and in vivo data have demonstrated the TGFBI gene functions as a putative tumor suppressor and is frequently downregulated in human tumors of different histological types.The hypermethylation of the TGFBI promoter,as one of the main regulatory mechanisms,is associated with TGFBI silencing.In this study,we used a methylation-specific PCR （MSP） method to evaluate the methylation status of the TGFBI promoter in human leukemias.Methods Real-time RT-PCR and methylation-specific PCR approaches were performed to define the TGFBI expression and promoter methylation in human leukemia call lines and clinical samples.Genomic DNA was isolated from peripheral blood mononuclear cells from leukemia patients,bisulfite-converted,and analyzed by the MSP method.Results Hypermethylation of the TGFBI promoter occurred in leukemia cell lines and demethylation treatment reexpressed TGFBI at a substantially increased level in most of leukemia cell lines tested.Furthermore,a much higher level of CpG island methylation and a significantly lower TGFBI expression were also identified in clinical leukemia samples.Conclusion The results suggest an important role of promoter methylation in regulating TGFBI expression in leukemia,which provides a useful diagnostic marker for clinical management of human leukemias.
