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
AIM: To study the characteristics of mismatch repair gene mutation of Chinese hereditary non-polyposis colorectal cancer (HNPCC) and hMLH1 gene promoter methylation, and to improve the screening strategy and explore t...AIM: To study the characteristics of mismatch repair gene mutation of Chinese hereditary non-polyposis colorectal cancer (HNPCC) and hMLH1 gene promoter methylation, and to improve the screening strategy and explore the pertinent test methods. METHODS: A systematic analysis of 30 probands from HNPCC families in the north of China was performed by immunohistochemistry, microsatellite instability (MSI), gene mutation and methylation detection. RESULTS: High frequency microsatellite instability occurred in 25 probands (83.3%) of HNPCC family. Loss of hMLH1 and hMSH2 protein expression accounted for 88% of all microsatellite instability. Pathogenic muta-tion occurred in 14 samples and 3 novel mutational sites were discovered. Deletion of exons 1-6, 1-7 and 8 of hMSH2 was detected in 3 samples and no large fragment deletion was found in hMLH1. Of the 30 probands, hMLH1 gene promoter methylation occurred in 3 probands. The rate of gene micromutation detection combined with large fragment deletion detection was 46.7%-56.7%. The rate of the two methods in combination with methylation detection was 63.3%. CONCLUSION: Scientific and rational detection strategy can improve the detection rate of HNPCC. Based on traditional molecular genetics and combined with epigenetics, multiple detection methods can accurately diagnose HNPCC.展开更多
Objective： To discuss the difference between multi-drug resistant cell line H460/Gem and its parental cell NCl-H460 on the basis of establishment of human gemcitabine-resistant cell line H460/Gem so as to elaborate t...Objective： To discuss the difference between multi-drug resistant cell line H460/Gem and its parental cell NCl-H460 on the basis of establishment of human gemcitabine-resistant cell line H460/Gem so as to elaborate the possible mechanisms of gemcitabine resistance. Methods： Human gemcitabine-resistant non-small cell lung cancer cell line H460/Gem was established by 2/3 clinical serous peak concentration gemcitabine intermittent selection from its parental cell human large cell lung carcinoma cell line NCl-H460 which was sensitive to gemcitabine. During the course of inducement, we had monitored their morphology, checked their resistance indexes and resistant pedigree by MTT method, gathered their growth curves and calculated their doubling time, examined their DNA contents and cell cycles by FCM; at the same time, we had measured its expressions of P53, EGFR, c-erb-B-2, PTEN, PCNA, c-myc, VEGF, MDR-1, Bcl-2, nm23, MMP-9, TIMP-1, CD44v6 proteins via immunocytochemistry staining, RRM1 and ERCC1 mRNA by real-time fluorescent quantitative-PCR. Results： The resistance index of H460/Gem＇ cells （the deputy of cells in the process of inducement） to gemcitabine was 1.201, and the cell line also exhibited cross-resistance to paclitaxol, fluorouraci, etoposide, cisplatin and oxaliplatin, but kept sensitivity to vinorelbine and taxotere. The doubling time of H460/Gem＇ cells was longer and figures in G0-G1 phase was decreased than that of NCl-H460 cells. Compared with NCl-H460 cells, H460/Gem＇ cells had achieved TIMP-1 protein expression emerged, nm23 protein expression enhanced, VEGF and MMP-9 protein expressions reduced, and CD44v6, P53 protein expressions vanished, but expressions of EGFR, c-erb-B-2, PTEN, PCNA, c-myc, MDR-1, Bcl-2 proteins and RRM1, ERCC1 mRNA changed trivially. The resistance index of H460/Gem cells to gemcitabine was 1.644, and the ceil line also exhibited cross-resistance to fluorouraci, cisplatin and oxaliplatin, but kept sensitivity to paclitaxol, vinorelbine, taxotere, and etoposide. The doubling time of H460/Gem cells was longer and figures in G0-G1 phase was decreased than those of NCl-H460 cells. The farther studies indicated that, compared with NCl-H460 cells, the expressions of MDR-1, nm23 and Bcl-2 proteins in H460/Gem cells had been enhanced, c-erb-B-2 protein expression emerged, P53, MMP-9 and VEGR protein expression had been weakened, but the changes of PTEN, PCNA, c-myc, TIMP-1, EGFR, CD44v6 protein, RRM1 mRNA and ERCC1 mRNA expressions were trivial. Furthermore, compared with its parental cells, H460/Gem cells were mixed with giant cells of different sizes that were larger and more irregular. Conclusion： The human gemcitabine-resistant non-small cell lung cancer cell line H460/Gem had achieved multi-drug resistance and great changes of biological characters compared with its parental cells. And these changes possibly participated in the formation of multidrug resistance.展开更多
Hereditary non-polyposis colorectal carcinoma （HNPCC） is an autosomal dominant disorder associated with colorectal and endometrial cancer and a range of other tumor types. Germline mutations in the DNA mismatch repa...Hereditary non-polyposis colorectal carcinoma （HNPCC） is an autosomal dominant disorder associated with colorectal and endometrial cancer and a range of other tumor types. Germline mutations in the DNA mismatch repair （MMR） genes, particularly MLH1, MEH2, and MEH5, underlie this disorder. The vast majority of these HNPCC-associated mutations have been proven, or assumed, given the family history of cancer, to be transmitted through several generations. To the best of our knowledge, only a single case of a de novo germline MMR gene mutation （in MEH2） has been reported till now. Here, we report a patient with a de novo mutation in MLH1. We identified a MLH1 Q701X truncating mutation in the blood lymphocytes of a male who had been diagnosed with rectal cancer at the age of 35. His family history of cancer was negative for the first- and second-degree relatives. The mutation could not be detected in the patient＇s parents and sibling and paternity was confirmed with a set of highly polymorphic markers. Non-penetrance and small family size is the common explanation of verified negative family histories of cancer in patients with a germline MMR gene mutation. However, in addition to some cases explained by non-paternity, de novo germline mutations should be considered as a possible explanation as well. As guidelines that stress not to restrict MMR gene mutation testing to patients with a positive family history are more widely introduced, more cases of de novo MMR gene germline mutations may be revealed.展开更多
Virus-induced gene silencing is regarded as a powerful and efficient tool for the analysis of gene function in plants because it is simple, rapid and transformation-free. It has been used to perform both forward and r...Virus-induced gene silencing is regarded as a powerful and efficient tool for the analysis of gene function in plants because it is simple, rapid and transformation-free. It has been used to perform both forward and reverse genetics to identify plant functional genes. Many viruses have been developed into virus-induced gene silencing vectors and gene functions involved in development, biotic and abiotic stresses, metabolism, and cellular signaling have been reported. In this review, we discuss the development and application of virus-induced gene silencing in plant functional genomics.展开更多
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.
基金Supported by Beijing Natural Science Foundation, No. 7062064
文摘AIM: To study the characteristics of mismatch repair gene mutation of Chinese hereditary non-polyposis colorectal cancer (HNPCC) and hMLH1 gene promoter methylation, and to improve the screening strategy and explore the pertinent test methods. METHODS: A systematic analysis of 30 probands from HNPCC families in the north of China was performed by immunohistochemistry, microsatellite instability (MSI), gene mutation and methylation detection. RESULTS: High frequency microsatellite instability occurred in 25 probands (83.3%) of HNPCC family. Loss of hMLH1 and hMSH2 protein expression accounted for 88% of all microsatellite instability. Pathogenic muta-tion occurred in 14 samples and 3 novel mutational sites were discovered. Deletion of exons 1-6, 1-7 and 8 of hMSH2 was detected in 3 samples and no large fragment deletion was found in hMLH1. Of the 30 probands, hMLH1 gene promoter methylation occurred in 3 probands. The rate of gene micromutation detection combined with large fragment deletion detection was 46.7%-56.7%. The rate of the two methods in combination with methylation detection was 63.3%. CONCLUSION: Scientific and rational detection strategy can improve the detection rate of HNPCC. Based on traditional molecular genetics and combined with epigenetics, multiple detection methods can accurately diagnose HNPCC.
基金Capital Medical Developmental Foundation (No. 2003-3028)
文摘Objective： To discuss the difference between multi-drug resistant cell line H460/Gem and its parental cell NCl-H460 on the basis of establishment of human gemcitabine-resistant cell line H460/Gem so as to elaborate the possible mechanisms of gemcitabine resistance. Methods： Human gemcitabine-resistant non-small cell lung cancer cell line H460/Gem was established by 2/3 clinical serous peak concentration gemcitabine intermittent selection from its parental cell human large cell lung carcinoma cell line NCl-H460 which was sensitive to gemcitabine. During the course of inducement, we had monitored their morphology, checked their resistance indexes and resistant pedigree by MTT method, gathered their growth curves and calculated their doubling time, examined their DNA contents and cell cycles by FCM; at the same time, we had measured its expressions of P53, EGFR, c-erb-B-2, PTEN, PCNA, c-myc, VEGF, MDR-1, Bcl-2, nm23, MMP-9, TIMP-1, CD44v6 proteins via immunocytochemistry staining, RRM1 and ERCC1 mRNA by real-time fluorescent quantitative-PCR. Results： The resistance index of H460/Gem＇ cells （the deputy of cells in the process of inducement） to gemcitabine was 1.201, and the cell line also exhibited cross-resistance to paclitaxol, fluorouraci, etoposide, cisplatin and oxaliplatin, but kept sensitivity to vinorelbine and taxotere. The doubling time of H460/Gem＇ cells was longer and figures in G0-G1 phase was decreased than that of NCl-H460 cells. Compared with NCl-H460 cells, H460/Gem＇ cells had achieved TIMP-1 protein expression emerged, nm23 protein expression enhanced, VEGF and MMP-9 protein expressions reduced, and CD44v6, P53 protein expressions vanished, but expressions of EGFR, c-erb-B-2, PTEN, PCNA, c-myc, MDR-1, Bcl-2 proteins and RRM1, ERCC1 mRNA changed trivially. The resistance index of H460/Gem cells to gemcitabine was 1.644, and the ceil line also exhibited cross-resistance to fluorouraci, cisplatin and oxaliplatin, but kept sensitivity to paclitaxol, vinorelbine, taxotere, and etoposide. The doubling time of H460/Gem cells was longer and figures in G0-G1 phase was decreased than those of NCl-H460 cells. The farther studies indicated that, compared with NCl-H460 cells, the expressions of MDR-1, nm23 and Bcl-2 proteins in H460/Gem cells had been enhanced, c-erb-B-2 protein expression emerged, P53, MMP-9 and VEGR protein expression had been weakened, but the changes of PTEN, PCNA, c-myc, TIMP-1, EGFR, CD44v6 protein, RRM1 mRNA and ERCC1 mRNA expressions were trivial. Furthermore, compared with its parental cells, H460/Gem cells were mixed with giant cells of different sizes that were larger and more irregular. Conclusion： The human gemcitabine-resistant non-small cell lung cancer cell line H460/Gem had achieved multi-drug resistance and great changes of biological characters compared with its parental cells. And these changes possibly participated in the formation of multidrug resistance.
文摘Hereditary non-polyposis colorectal carcinoma （HNPCC） is an autosomal dominant disorder associated with colorectal and endometrial cancer and a range of other tumor types. Germline mutations in the DNA mismatch repair （MMR） genes, particularly MLH1, MEH2, and MEH5, underlie this disorder. The vast majority of these HNPCC-associated mutations have been proven, or assumed, given the family history of cancer, to be transmitted through several generations. To the best of our knowledge, only a single case of a de novo germline MMR gene mutation （in MEH2） has been reported till now. Here, we report a patient with a de novo mutation in MLH1. We identified a MLH1 Q701X truncating mutation in the blood lymphocytes of a male who had been diagnosed with rectal cancer at the age of 35. His family history of cancer was negative for the first- and second-degree relatives. The mutation could not be detected in the patient＇s parents and sibling and paternity was confirmed with a set of highly polymorphic markers. Non-penetrance and small family size is the common explanation of verified negative family histories of cancer in patients with a germline MMR gene mutation. However, in addition to some cases explained by non-paternity, de novo germline mutations should be considered as a possible explanation as well. As guidelines that stress not to restrict MMR gene mutation testing to patients with a positive family history are more widely introduced, more cases of de novo MMR gene germline mutations may be revealed.
基金supported by the National Transgenic Research Projects of China (Grant No. 2009ZX08009-026B)
文摘Virus-induced gene silencing is regarded as a powerful and efficient tool for the analysis of gene function in plants because it is simple, rapid and transformation-free. It has been used to perform both forward and reverse genetics to identify plant functional genes. Many viruses have been developed into virus-induced gene silencing vectors and gene functions involved in development, biotic and abiotic stresses, metabolism, and cellular signaling have been reported. In this review, we discuss the development and application of virus-induced gene silencing in plant functional genomics.
