AIM: To evaluate the genetic and epigenetic inactivation mechanism of the RASSF1A tumor suppressor gene at 3p21.3 in extrahepatic cholangiocarcinoma.METHODS: RT-PCR was used to investigate the transcriptional expressi...AIM: To evaluate the genetic and epigenetic inactivation mechanism of the RASSF1A tumor suppressor gene at 3p21.3 in extrahepatic cholangiocarcinoma.METHODS: RT-PCR was used to investigate the transcriptional expressing and re-expression of RASSF1A.RASSF1A mutation was analyzed with SSCP and selective sequencing. PCR was performed to detect the loss of heterozygosity (LOH) at the region of chromosome 3p21.3.Genomic DNA were modificated bisulfite and the frequency of methylation of CpG islands in RASSF1A promoter were evaluated by methylation specific PCR (MS-PCR).RESULTS: In all 48 samples and one cell lines of extrahepatic cholangiocarcinoma, the RASSF1A mutation is rare (6.12%, 3/49), 33 samples (68.75%) and QBC-939cell lines (x2 = 14.270, P= 0.001＜0.01) showed RASSF1A express inactivation with LOH at microsatellite loci D3S4604. Among these 33 samples and QBC-939, 28 of 33 (84.85%) tumor samples and 1 cell lines were methylated for majority of 16 CpGs, the average frequency is 73.42%.CONCLUSION: The data we present suggest that RASSF1A which we have been searching for at 3p21.3may be one of the key tumor suppressor gene and play an important role in the pathogenesis of extrahepatic cholangiocarcinoma, and the promoter methylation and allelic loss are the major mechanism for inactivation of RASSF1A.展开更多
Esophageal squamous cell carcinoma(ESCC) is a prevalent and fatal cancer in China and other Asian countries.Epigenetic silencing of key tumor suppressor genes(TSGs) is critical to ESCC initiation and progression.Recen...Esophageal squamous cell carcinoma(ESCC) is a prevalent and fatal cancer in China and other Asian countries.Epigenetic silencing of key tumor suppressor genes(TSGs) is critical to ESCC initiation and progression.Recently,many novel TSGs silenced by promoter methylation have been identified in ESCC,and these genes further serve as potential tumor markers for high-risk group stratification,early detection,and prognosis prediction.This review summarizes recent discoveries on aberrant promoter methylation of TSGs in ESCC,providing better understanding of the role of disrupted epigenetic regulation in tumorigenesis and insight into diagnostic and prognostic biomarkers for this malignancy.展开更多
Nasopharyngeal carcinoma (NPC) is among the most common malignancies in southern China.Deletion of genomic DNA,which occurs during the complex pathogenesis process for NPC,represents a pivotal mechanism in the inactiv...Nasopharyngeal carcinoma (NPC) is among the most common malignancies in southern China.Deletion of genomic DNA,which occurs during the complex pathogenesis process for NPC,represents a pivotal mechanism in the inactivation of tumor suppressor genes (TSGs).In many circumstances,loss of TSGs can be detected as diagnostic and prognostic markers in cancer.The short arm of chromosome 3 (3p) is a frequently deleted chromosomal region in NPC,with 3p21.1-21.2 and 3p25.2-26.1 being the most frequently deleted minimal regions.In recent years,our research group and others have focused on the identification and characterization of novel target TSGs at 3p,such as RASSF1A,BLU,RBMS3,and CHL1,in the development and progression of NPC.In this review,we summarize recent findings of TSGs at 3p and discuss some of these genes in detail.A better understanding of TSGs at 3p will significantly improve our understanding of NPC pathogenesis,diagnosis,and treatment.展开更多
Gastric cancer is one of the most common malignancies and a leading cause of cancer mortality worldwide.The pathogenesis mechanisms of gastric cancer are still not fully clear.Inactivation of tumor suppressor genes an...Gastric cancer is one of the most common malignancies and a leading cause of cancer mortality worldwide.The pathogenesis mechanisms of gastric cancer are still not fully clear.Inactivation of tumor suppressor genes and activation of oncogenes caused by genetic and epigenetic alterations are known to play significant roles in carcinogenesis.Accumulating evidence has shown that epigenetic silencing of the tumor suppressor genes,particularly caused by hypermethylation of CpG islands in promoters,is critical to carcinogenesis and metastasis.Here,we review the recent progress in the study of methylations of tumor suppressor genes involved in the pathogenesis of gastric cancer.We also briefly describe the mechanisms that induce tumor suppressor gene methylation and the status of translating these molecular mechanisms into clinical applications.展开更多
The multifactorial and multistage pathogenesis of hepatocellular carcinoma(HCC)has fascinated a wide spectrum of scientists for decades.While a number of major risk factors have been identified,their mechanistic roles...The multifactorial and multistage pathogenesis of hepatocellular carcinoma(HCC)has fascinated a wide spectrum of scientists for decades.While a number of major risk factors have been identified,their mechanistic roles in hepatocarcinogenesis still need to be elucidated.Many tumor suppressor genes(TSGs)have been identified as being involved in HCC.These TSGs can be classified into two groups depending on the situation with respect to allelic mutation/loss in the tumors:the recessive TSGs with two required mutated alleles and the haploinsufficient TSGs with one required mutated allele.Hepatitis B virus(HBV)is one of the most important risk factors associated with HCC.Although mice cannot be infected with HBV due to the narrow host range of HBV and the lack of a proper receptor,one advantage of mouse models for HBV/HCC research is the numerous and powerfulgenetic tools that help investigate the phenotypic effects of viral proteins and allow the dissection of the dose-dependent action of TSGs.Here,we mainly focus on the application of mouse models in relation to HBV-associated HCC and on TSGs that act either in a recessive or in a haploinsufficient manner.Discoveries obtained using mouse models will have a great impact on HCC translational medicine.展开更多
AIM:To identify the novel methylation-silenced gene pentraxin 3(PTX3) in esophageal squamous cell carcinoma(ESCC).METHODS:PTX3 mRNA expression was examined in six human ESCC cell lines,one human immortalized normal es...AIM:To identify the novel methylation-silenced gene pentraxin 3(PTX3) in esophageal squamous cell carcinoma(ESCC).METHODS:PTX3 mRNA expression was examined in six human ESCC cell lines,one human immortalized normal esophageal epithelial cell line,primary ESCC tumor tissue,and paired adjacent nontumor tissue using reverse transcription polymerase chain reaction(RTPCR).Semi-quantitative immunohistochemistry was used to examine cellular localisation and protein levels.Methylation specific PCR and bisulphite genomic sequencing were employed to investigate the methylation of the candidate gene.RESULTS:In the majority of ESCC cell lines,we found that PTX3 expression was down-regulated due to gene promoter hypermethylation,which was further confirmed by bisulphite genomic sequencing.Demethylation treatment with 5-aza-2'-deoxycytidine restored PTX3 mRNA expression in ESCC cell lines.Methylation was more common in tumor tissues(85%) than in adjacent nontumor tissues(25%)(P < 0.01).CONCLUSION:PTX3 is down-regulated through promoter hypermethylation in ESCC,and could potentially serve as a biomarker of ESCC.展开更多
Objective: To explore the changes and significance of tumor suppressor gene p53 in primary hepatocellu-lar carcinoma (PHC ) with hepatitis B virus (HBV ) infection. Methods: Tumor tissues and surrounding nontumortissu...Objective: To explore the changes and significance of tumor suppressor gene p53 in primary hepatocellu-lar carcinoma (PHC ) with hepatitis B virus (HBV ) infection. Methods: Tumor tissues and surrounding nontumortissues of sixteen PHC cases were studied by Southern hybridization to detect the state of HBV-DNA in tissues, byimmunohistochemical staining to determine HBsAg, HBxAg and p53 protein, and by PCR directed sequencing toanalyse the point mutation of p53 gene exons 5 to 8. Results: Among the 16 cases. 13 cases were HBV-DNA posi-tive, 10 tumor cases and 13 nontumor tissues cases HBxAg positive, and 9 cases posltive for p53 protein. The se-quencing of p53 gene point mutation was found in 5 cases, only one of which was sited at codon 249 G to T. Con-clusion: The mutation of p53 gene codon 249 is infrequent in HBV related PHC,indicating the accumulation of p53protein in cells may be associated with expression of HBxAg. HBxAg binding to p53 protein and inactivation of p53function play important roles in the development of PHC.展开更多
Objective: To investigate whether deletion of chromosome 17 is involved in the carcinogenesis of primary glioblastoma multiforme and to localize the possible common deletion region in the aforementioned chromosome. Me...Objective: To investigate whether deletion of chromosome 17 is involved in the carcinogenesis of primary glioblastoma multiforme and to localize the possible common deletion region in the aforementioned chromosome. Methods: Polymerase chain reaction-based microsatellite analysis was used to assess loss of heterozygosity (LOH) on chromosome 17 in 20 primary glioblastoma multiforme (GBM). Fifteen fluorescent dye-labeled polymorphic markers were used. Results: Thirteen of twenty (65%) GBM displayed LOH on at least one marker of chromosome 17p. Two tumors showed either LOH or non-informativeness on all markers tested. The most frequent LOH was observed at loci including D17s799 (53.3%), Dl7s1852 (53.8%), Dl7s938 (63.20/o), Dl7s831 (55.6%). The loci D17s831 (on 17pl3) and D17s799-Dl7sl852 (17p11.2-pl2) are distal and proximal to p53 respectively. The frequencies of LOH at all loci examined on chromosome 17q were relatively low (<30%). None of informative loci exhibited microsatellite instability in this study. Conclusion: Loss of genetic material on chromosome 17p may play an important role in the pathogenesis of GBM. Besides the well-known TSG p53 on 17p, other unknown TSCs associated with GBM may be present on the chromosomal regions 17pl3 and 17p11.2-pl2, which are distal and proximal to p53 respectively.展开更多
Objective: To investigate whether deletion ofchromosome 3 is involved in the carcinogenesis of primary glioblastoma multiforme (GBM) and to localize the possible common deletion region in the aforementionedchromosome....Objective: To investigate whether deletion ofchromosome 3 is involved in the carcinogenesis of primary glioblastoma multiforme (GBM) and to localize the possible common deletion region in the aforementionedchromosome. Methods: PCR based microsatellite polymorphism analyses were performed to detect loss of heterozygosity (LOH). Twenty-three loci on chromosome were examined in 20 cases of GBM. Fluorescence-labeled primers and Perkin Elmer 377 DNA Sequencer wereapplied. Results: 50% informative cases of GBM displayed LOH on chromosome 3. 50% of informative casesdisplayed LOH on 3q and 35% on 3p. 25.6% ofinformative loci showed LOH in our series, in whichfrequent LOH were observed in the chromosomal regionfrom loci D3S1614 (42.9%) to D3S1565 (35.3%) on 3q24-27 and at loci D3S1569 (35.3%) on 3q22-23 and D3S1289(33.3%) on 3p14.1-14.3. Conclusion: Loss of geneticmaterial on chromosome 3 may play an important part ithe tumorigenesis of GBM. The chromosomal regionsfrom loci D3S1614 to D3S1565 on 3q24-27 and at loci D3S1569 on 3q22-23 and D3S1289 on 3p14.1-14.3 are potential sites for novel tumor suppressor genes associated with GBM.展开更多
AIM: To study the candidate tumor suppressor genes (TSG) on chromosome 4p by detecting the high frequency of loss of heterozygosity (LOH) in sporadic colorectal carcinoma in Chinese patients.METHODS: Seven fluorescent...AIM: To study the candidate tumor suppressor genes (TSG) on chromosome 4p by detecting the high frequency of loss of heterozygosity (LOH) in sporadic colorectal carcinoma in Chinese patients.METHODS: Seven fluorescent labeled polymorphic microsatellite markers were analyzed in 83 cases of colorectal carcinoma and matched normal tissue DNA by PCR. PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan 3.7 and Genotype 3.7 software were used for LOH scanning and analysis. The same procedure was performed by the other six microsatellite markers spanning D4S3013 locus to make further detailed deletion mapping. Comparison between LOH frequency and clinicopathological factors was performed by χ2 test.RESULTS: Data were collected from all informative loci. The average LOH frequency on 4p was 24.25%, and 42.3% and 35.62% on D4S405 and D4S3013 locus, respectively. Adjacent markers of D4S3013 displayed a low LOH frequency (< 30%) by detailed deletion mapping. Significant opposite difference was observed between LOH frequency and tumor diameter on D4S412 and D4S1546 locus (0% vs 16.67%, P = 0.041; 54.55% vs 11.11%, P = 0.034, respectively). On D4S403 locus, LOH was significantly associated with tumor gross pattern (11.11%, 0, 33.33%, P = 0.030). No relationship was detected on other loci compared with clinicopathologial features.CONCLUSION: By deletion mapping, two obvious high frequency LOH regions spanning D4S3013 (4p15.2) and D4S405 (4p14) locus are detected. Candidate TSG, which is involved in carcinogenesis and progression of sporadic colorectal carcinoma on chromosome 4p, may be located between D4S3017 and D4S2933 (about 1.7 cm).展开更多
To observe the relationship between tumor suppressor gene p16 expression and ovarian cancer occurrence and development. Metbods: Using ABC immunohistochemistry method, we investigated the expression of p16 in 72 cases...To observe the relationship between tumor suppressor gene p16 expression and ovarian cancer occurrence and development. Metbods: Using ABC immunohistochemistry method, we investigated the expression of p16 in 72 cases of ovarian neoplasm. Results: The positive rates of p16 in malignant, benign, borderline tumors and normal ovarian tissue were 7. 89%, 60.00%, 66. 67% and 83. 33%, respectively (P<0.01). In the cases whose tumors were more malignant and poorly differentiated, and who relapsed and died, the positive stainings were not discovered. Conclusiou: p16 is well related with the occurrence and development of malignant ovarian tumor.展开更多
FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expression showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investig...FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expression showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investigate the biological function of FUS1 protein, FUS1 cDNA from MRC-5 cells was amplified by RT-PCR and cloned into prokaryotic expression vector pQE-30. The recombinant expression plasmids were transformed into M15 strain and grown at 20℃ or 37℃. SDS–PAGE analysis revealed that the accumulation of the recombinant protein FUS1 (rFUS1) in inclusion body forms reached maxium amount when induced with 0.5 mM IPTG for 5 h at 37℃. The inclusion bodies were solubilized in 2M urea and purified by a 6 ×His tagged affinity column under denaturing condition. The purified rFUS1 was identified by electrospray ionization-mass spectrometry (ESI-MS) and tested for purity by HPLC chromatography. The purified rFUS1 proteins were then used to immunize rabbits to obtain anti-human FUS1 polyclonal antibodies, which were suitable to detect both the recombinant exogenous FUS1 and the endogenous FUS1 from tissues and cells by western blot and immunohistochemistry, Available purified rFUS1 proteins and self-prepared polyclonal antibodies against FUS1 may provide effective tools for further studies on biological function and application of FUS1.展开更多
Objective: To clone tumor suppressor gene Fhit from normal human appendix’s tissue and construct its cloning vector. Methods: Extract human total RNA with TRIzol reagent and amplification the Fhit gene with reverse t...Objective: To clone tumor suppressor gene Fhit from normal human appendix’s tissue and construct its cloning vector. Methods: Extract human total RNA with TRIzol reagent and amplification the Fhit gene with reverse transcription-PCR (RT-PCR), then clone Fhit into a pGEM-T easy cloning vector, lastly identify the pGEM-Fhit vector with polymerase chain reaction (PCR), endonuclease digestion and DNA sequencing. Results: Fhit complementary DNA (cDNA) was cloned into pGEM-T vector, and DNA sequencing was the same as reported. Conclusions: The cloning vector pGEM-Fhit was constructed successfully and builds up a good foundation going deep into the study of Fhit.展开更多
Breast cancer is the leading cause for mortality among women worldwide.Dysregulation of oncogenes and tumor suppressor genes is the major reason for the cause of cancer.Understanding these genes will provide clues and...Breast cancer is the leading cause for mortality among women worldwide.Dysregulation of oncogenes and tumor suppressor genes is the major reason for the cause of cancer.Understanding these genes will provide clues and insights about their regulatory mechanism and their interplay in cancer.In the present study,an attempt is made to compare the functional characteristics and interactions of oncogenes and tumor suppressor genes to understand their biological role.431 breast cancer samples from seven publicly available microarray datasets were collected and analysed using GEO2R tool.The identified 416 differentially expressed genes were classified into five gene sets as oncogenes(OG),tumor suppressor genes(TSG),druggable genes,essential genes and other genes.The gene sets were subjected to various analysis such as enrichment analysis(viz.,GO,Pathways,Diseases and Drugs),network analysis,calculation of mutation frequencies and Guanine-Cytosine(GC)content.From the results,it was observed that the OG were having high GC content as well as high interactions than TSG.Moreover,the OG are found to have frequent mutations than TSG.The enrichment analysis results suggest that the oncogenes are involved in positive regulation of cellular protein metabolic process,macromolecule biosynthetic process and majorly in cell cycle and focal adhesion pathway in cancer.It was also found that these oncogenes are involved in other diseases such as skin diseases and viral infections.Collagenase,paclitaxel and docetaxel are some of the drugs found to be enriched for oncogenes.展开更多
AIM To analyse cumulative loss of heterozygosity (LOH) of chromosomal regions and tumor suppressor genes in hepatocellular carcinomas (HCCs) from 20 southern African blacks. METHODS p53, RB1, BRCA1, BRCA2, WT1 and E c...AIM To analyse cumulative loss of heterozygosity (LOH) of chromosomal regions and tumor suppressor genes in hepatocellular carcinomas (HCCs) from 20 southern African blacks. METHODS p53, RB1, BRCA1, BRCA2, WT1 and E cadherin genes were analysed for LOH, and p53 gene was also analysed for the codon 249 mutation, in tumor and adjacent non tumorous liver tissues using molecular techniques and 10 polymorphic microsatellite markers. RESULTS p53 codon 249 mutation was found in 25% of the subjects, as was expected, because many patients were from Mozambique, a country with high aflatoxin B 1 exposure. LOH was found at the RB1, BRCA2 and WT1 loci in 20%(4/*!20) of the HCCs, supporting a possible role of these genes in HCC. No LOH was evident in any of the remaining genes. Reports of mutations of p53 and RB1 genes in combination, described in other populations, were not confirmed in this study. Change in microsatellite repeat number was noted at 9/*!10 microsatellite loci in different HCCs, and changes at two or more loci were detected in 15%(3/*!20) of subjects. CONCLUSION We propose that microsatellite/genomic instability may play a role in the pathogenesis of a subset of HCCs in black Africans.展开更多
In addition to the tumor suppressor genes such as Rb and p53, it has been found that some molecules of the same class named CKI (cyclin-dependent kinase inhibitor) also play an important role in the inhibition of tumo...In addition to the tumor suppressor genes such as Rb and p53, it has been found that some molecules of the same class named CKI (cyclin-dependent kinase inhibitor) also play an important role in the inhibition of tumorigenesis and the tumor progression. In the KIP and INK4 families of CKIs, p15 shares extensive homology with p16. Findings in many tumors and their cell lines show that the inactivation of p15 (deletion, mutation, rearrangement, etc.) is very frequent, and inactive p15 is involved in the progress of some tumors. These studies provide evidence that the p15 is a new tumor suppressor gene. Furthermore, the research on the molecular mechanism of p15 in regulation of cell proliferation shows that p15 can inhibit the growth of some kinds of tumor cells, and p15 is the mediator of TGF-β-induced cell arrest. Investigations on p15 in cell differentiation suggest that increased p15 is related to the change of malignant phenotype. These results supply clues for further interpretation about the展开更多
A number of tumor suppressor and tumor-related genes exhibit promoter hypermethylation with resultant gene silencing in human cancers.The frequencies of methylation differ among genes and genomic regions within CpG is...A number of tumor suppressor and tumor-related genes exhibit promoter hypermethylation with resultant gene silencing in human cancers.The frequencies of methylation differ among genes and genomic regions within CpG islands in different tissue types.Hypermethylation initially occurs at the edge of CpG islands and spreads to the transcription start site before ultimately shutting down gene expression.When the degree of methylation was quantitatively evaluated in neoplastic and non-neoplastic gastric epithelia using DNA microarray analysis,highlevel methylation around the transcription start site appeared to be a tumor-specific phenomenon,although multiple tumor suppressor genes became increasingly methylated with patient age in non-neoplastic gastric epithelia.Quantitative analysis of DNA methylation is a promising method for both cancer diagnosis and risk assessment.展开更多
基金Supported by the National High Technology Research and Development Program of China (863 Program), No. 2002AA214061
文摘AIM: To evaluate the genetic and epigenetic inactivation mechanism of the RASSF1A tumor suppressor gene at 3p21.3 in extrahepatic cholangiocarcinoma.METHODS: RT-PCR was used to investigate the transcriptional expressing and re-expression of RASSF1A.RASSF1A mutation was analyzed with SSCP and selective sequencing. PCR was performed to detect the loss of heterozygosity (LOH) at the region of chromosome 3p21.3.Genomic DNA were modificated bisulfite and the frequency of methylation of CpG islands in RASSF1A promoter were evaluated by methylation specific PCR (MS-PCR).RESULTS: In all 48 samples and one cell lines of extrahepatic cholangiocarcinoma, the RASSF1A mutation is rare (6.12%, 3/49), 33 samples (68.75%) and QBC-939cell lines (x2 = 14.270, P= 0.001＜0.01) showed RASSF1A express inactivation with LOH at microsatellite loci D3S4604. Among these 33 samples and QBC-939, 28 of 33 (84.85%) tumor samples and 1 cell lines were methylated for majority of 16 CpGs, the average frequency is 73.42%.CONCLUSION: The data we present suggest that RASSF1A which we have been searching for at 3p21.3may be one of the key tumor suppressor gene and play an important role in the pathogenesis of extrahepatic cholangiocarcinoma, and the promoter methylation and allelic loss are the major mechanism for inactivation of RASSF1A.
基金supported by NSFC Joint Research Fund for Hong Kong and Macao Young Scholars(No.30928012)National Natural Science Foundation of China(No.81071634,81172582,and 30801344)Shenzhen Science Fund for Distinguished Young Scholars(No.JC201005270328A)
文摘Esophageal squamous cell carcinoma(ESCC) is a prevalent and fatal cancer in China and other Asian countries.Epigenetic silencing of key tumor suppressor genes(TSGs) is critical to ESCC initiation and progression.Recently,many novel TSGs silenced by promoter methylation have been identified in ESCC,and these genes further serve as potential tumor markers for high-risk group stratification,early detection,and prognosis prediction.This review summarizes recent discoveries on aberrant promoter methylation of TSGs in ESCC,providing better understanding of the role of disrupted epigenetic regulation in tumorigenesis and insight into diagnostic and prognostic biomarkers for this malignancy.
文摘Nasopharyngeal carcinoma (NPC) is among the most common malignancies in southern China.Deletion of genomic DNA,which occurs during the complex pathogenesis process for NPC,represents a pivotal mechanism in the inactivation of tumor suppressor genes (TSGs).In many circumstances,loss of TSGs can be detected as diagnostic and prognostic markers in cancer.The short arm of chromosome 3 (3p) is a frequently deleted chromosomal region in NPC,with 3p21.1-21.2 and 3p25.2-26.1 being the most frequently deleted minimal regions.In recent years,our research group and others have focused on the identification and characterization of novel target TSGs at 3p,such as RASSF1A,BLU,RBMS3,and CHL1,in the development and progression of NPC.In this review,we summarize recent findings of TSGs at 3p and discuss some of these genes in detail.A better understanding of TSGs at 3p will significantly improve our understanding of NPC pathogenesis,diagnosis,and treatment.
基金supported by grants from National Natural Science Foundation of China(No.30770920 and 81071651)Zhejiang Provincial Natural Science Foundation of China(No.R2100213,2009C33142,Z2090056 and WKJ2009-2-028)973 Project(No.2010CB834300)
文摘Gastric cancer is one of the most common malignancies and a leading cause of cancer mortality worldwide.The pathogenesis mechanisms of gastric cancer are still not fully clear.Inactivation of tumor suppressor genes and activation of oncogenes caused by genetic and epigenetic alterations are known to play significant roles in carcinogenesis.Accumulating evidence has shown that epigenetic silencing of the tumor suppressor genes,particularly caused by hypermethylation of CpG islands in promoters,is critical to carcinogenesis and metastasis.Here,we review the recent progress in the study of methylations of tumor suppressor genes involved in the pathogenesis of gastric cancer.We also briefly describe the mechanisms that induce tumor suppressor gene methylation and the status of translating these molecular mechanisms into clinical applications.
基金Supported by Research grants from the Ministry of Science and Technology(MOST)in Taiwan,No.NSC99-2628-B-010-001-MY3,MOST 103-2321-B-010-003,MOST 103-2633-H-010-001,MOST 103-2633-B-400-002 and MOST104-3011-B-010-001a grant from the Ministry of Education,Aim for the Top University Plan
文摘The multifactorial and multistage pathogenesis of hepatocellular carcinoma(HCC)has fascinated a wide spectrum of scientists for decades.While a number of major risk factors have been identified,their mechanistic roles in hepatocarcinogenesis still need to be elucidated.Many tumor suppressor genes(TSGs)have been identified as being involved in HCC.These TSGs can be classified into two groups depending on the situation with respect to allelic mutation/loss in the tumors:the recessive TSGs with two required mutated alleles and the haploinsufficient TSGs with one required mutated allele.Hepatitis B virus(HBV)is one of the most important risk factors associated with HCC.Although mice cannot be infected with HBV due to the narrow host range of HBV and the lack of a proper receptor,one advantage of mouse models for HBV/HCC research is the numerous and powerfulgenetic tools that help investigate the phenotypic effects of viral proteins and allow the dissection of the dose-dependent action of TSGs.Here,we mainly focus on the application of mouse models in relation to HBV-associated HCC and on TSGs that act either in a recessive or in a haploinsufficient manner.Discoveries obtained using mouse models will have a great impact on HCC translational medicine.
基金Supported by National High Technology Research and Development Program of China (863 Program),No. 2007AA02Z4Z4China Postdoctoral Science Foundation,No. 20090460394Beijing Municipal Natural Science Foundation,No. 7072022
文摘AIM:To identify the novel methylation-silenced gene pentraxin 3(PTX3) in esophageal squamous cell carcinoma(ESCC).METHODS:PTX3 mRNA expression was examined in six human ESCC cell lines,one human immortalized normal esophageal epithelial cell line,primary ESCC tumor tissue,and paired adjacent nontumor tissue using reverse transcription polymerase chain reaction(RTPCR).Semi-quantitative immunohistochemistry was used to examine cellular localisation and protein levels.Methylation specific PCR and bisulphite genomic sequencing were employed to investigate the methylation of the candidate gene.RESULTS:In the majority of ESCC cell lines,we found that PTX3 expression was down-regulated due to gene promoter hypermethylation,which was further confirmed by bisulphite genomic sequencing.Demethylation treatment with 5-aza-2'-deoxycytidine restored PTX3 mRNA expression in ESCC cell lines.Methylation was more common in tumor tissues(85%) than in adjacent nontumor tissues(25%)(P < 0.01).CONCLUSION:PTX3 is down-regulated through promoter hypermethylation in ESCC,and could potentially serve as a biomarker of ESCC.
文摘Objective: To explore the changes and significance of tumor suppressor gene p53 in primary hepatocellu-lar carcinoma (PHC ) with hepatitis B virus (HBV ) infection. Methods: Tumor tissues and surrounding nontumortissues of sixteen PHC cases were studied by Southern hybridization to detect the state of HBV-DNA in tissues, byimmunohistochemical staining to determine HBsAg, HBxAg and p53 protein, and by PCR directed sequencing toanalyse the point mutation of p53 gene exons 5 to 8. Results: Among the 16 cases. 13 cases were HBV-DNA posi-tive, 10 tumor cases and 13 nontumor tissues cases HBxAg positive, and 9 cases posltive for p53 protein. The se-quencing of p53 gene point mutation was found in 5 cases, only one of which was sited at codon 249 G to T. Con-clusion: The mutation of p53 gene codon 249 is infrequent in HBV related PHC,indicating the accumulation of p53protein in cells may be associated with expression of HBxAg. HBxAg binding to p53 protein and inactivation of p53function play important roles in the development of PHC.
文摘Objective: To investigate whether deletion of chromosome 17 is involved in the carcinogenesis of primary glioblastoma multiforme and to localize the possible common deletion region in the aforementioned chromosome. Methods: Polymerase chain reaction-based microsatellite analysis was used to assess loss of heterozygosity (LOH) on chromosome 17 in 20 primary glioblastoma multiforme (GBM). Fifteen fluorescent dye-labeled polymorphic markers were used. Results: Thirteen of twenty (65%) GBM displayed LOH on at least one marker of chromosome 17p. Two tumors showed either LOH or non-informativeness on all markers tested. The most frequent LOH was observed at loci including D17s799 (53.3%), Dl7s1852 (53.8%), Dl7s938 (63.20/o), Dl7s831 (55.6%). The loci D17s831 (on 17pl3) and D17s799-Dl7sl852 (17p11.2-pl2) are distal and proximal to p53 respectively. The frequencies of LOH at all loci examined on chromosome 17q were relatively low (<30%). None of informative loci exhibited microsatellite instability in this study. Conclusion: Loss of genetic material on chromosome 17p may play an important role in the pathogenesis of GBM. Besides the well-known TSG p53 on 17p, other unknown TSCs associated with GBM may be present on the chromosomal regions 17pl3 and 17p11.2-pl2, which are distal and proximal to p53 respectively.
文摘Objective: To investigate whether deletion ofchromosome 3 is involved in the carcinogenesis of primary glioblastoma multiforme (GBM) and to localize the possible common deletion region in the aforementionedchromosome. Methods: PCR based microsatellite polymorphism analyses were performed to detect loss of heterozygosity (LOH). Twenty-three loci on chromosome were examined in 20 cases of GBM. Fluorescence-labeled primers and Perkin Elmer 377 DNA Sequencer wereapplied. Results: 50% informative cases of GBM displayed LOH on chromosome 3. 50% of informative casesdisplayed LOH on 3q and 35% on 3p. 25.6% ofinformative loci showed LOH in our series, in whichfrequent LOH were observed in the chromosomal regionfrom loci D3S1614 (42.9%) to D3S1565 (35.3%) on 3q24-27 and at loci D3S1569 (35.3%) on 3q22-23 and D3S1289(33.3%) on 3p14.1-14.3. Conclusion: Loss of geneticmaterial on chromosome 3 may play an important part ithe tumorigenesis of GBM. The chromosomal regionsfrom loci D3S1614 to D3S1565 on 3q24-27 and at loci D3S1569 on 3q22-23 and D3S1289 on 3p14.1-14.3 are potential sites for novel tumor suppressor genes associated with GBM.
基金Supported by The National Natural Science Foundation of China, No. 30080016 and No. 30470977
文摘AIM: To study the candidate tumor suppressor genes (TSG) on chromosome 4p by detecting the high frequency of loss of heterozygosity (LOH) in sporadic colorectal carcinoma in Chinese patients.METHODS: Seven fluorescent labeled polymorphic microsatellite markers were analyzed in 83 cases of colorectal carcinoma and matched normal tissue DNA by PCR. PCR products were eletrophoresed on an ABI 377 DNA sequencer. Genescan 3.7 and Genotype 3.7 software were used for LOH scanning and analysis. The same procedure was performed by the other six microsatellite markers spanning D4S3013 locus to make further detailed deletion mapping. Comparison between LOH frequency and clinicopathological factors was performed by χ2 test.RESULTS: Data were collected from all informative loci. The average LOH frequency on 4p was 24.25%, and 42.3% and 35.62% on D4S405 and D4S3013 locus, respectively. Adjacent markers of D4S3013 displayed a low LOH frequency (< 30%) by detailed deletion mapping. Significant opposite difference was observed between LOH frequency and tumor diameter on D4S412 and D4S1546 locus (0% vs 16.67%, P = 0.041; 54.55% vs 11.11%, P = 0.034, respectively). On D4S403 locus, LOH was significantly associated with tumor gross pattern (11.11%, 0, 33.33%, P = 0.030). No relationship was detected on other loci compared with clinicopathologial features.CONCLUSION: By deletion mapping, two obvious high frequency LOH regions spanning D4S3013 (4p15.2) and D4S405 (4p14) locus are detected. Candidate TSG, which is involved in carcinogenesis and progression of sporadic colorectal carcinoma on chromosome 4p, may be located between D4S3017 and D4S2933 (about 1.7 cm).
文摘To observe the relationship between tumor suppressor gene p16 expression and ovarian cancer occurrence and development. Metbods: Using ABC immunohistochemistry method, we investigated the expression of p16 in 72 cases of ovarian neoplasm. Results: The positive rates of p16 in malignant, benign, borderline tumors and normal ovarian tissue were 7. 89%, 60.00%, 66. 67% and 83. 33%, respectively (P<0.01). In the cases whose tumors were more malignant and poorly differentiated, and who relapsed and died, the positive stainings were not discovered. Conclusiou: p16 is well related with the occurrence and development of malignant ovarian tumor.
文摘FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expression showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investigate the biological function of FUS1 protein, FUS1 cDNA from MRC-5 cells was amplified by RT-PCR and cloned into prokaryotic expression vector pQE-30. The recombinant expression plasmids were transformed into M15 strain and grown at 20℃ or 37℃. SDS–PAGE analysis revealed that the accumulation of the recombinant protein FUS1 (rFUS1) in inclusion body forms reached maxium amount when induced with 0.5 mM IPTG for 5 h at 37℃. The inclusion bodies were solubilized in 2M urea and purified by a 6 ×His tagged affinity column under denaturing condition. The purified rFUS1 was identified by electrospray ionization-mass spectrometry (ESI-MS) and tested for purity by HPLC chromatography. The purified rFUS1 proteins were then used to immunize rabbits to obtain anti-human FUS1 polyclonal antibodies, which were suitable to detect both the recombinant exogenous FUS1 and the endogenous FUS1 from tissues and cells by western blot and immunohistochemistry, Available purified rFUS1 proteins and self-prepared polyclonal antibodies against FUS1 may provide effective tools for further studies on biological function and application of FUS1.
文摘Objective: To clone tumor suppressor gene Fhit from normal human appendix’s tissue and construct its cloning vector. Methods: Extract human total RNA with TRIzol reagent and amplification the Fhit gene with reverse transcription-PCR (RT-PCR), then clone Fhit into a pGEM-T easy cloning vector, lastly identify the pGEM-Fhit vector with polymerase chain reaction (PCR), endonuclease digestion and DNA sequencing. Results: Fhit complementary DNA (cDNA) was cloned into pGEM-T vector, and DNA sequencing was the same as reported. Conclusions: The cloning vector pGEM-Fhit was constructed successfully and builds up a good foundation going deep into the study of Fhit.
文摘Breast cancer is the leading cause for mortality among women worldwide.Dysregulation of oncogenes and tumor suppressor genes is the major reason for the cause of cancer.Understanding these genes will provide clues and insights about their regulatory mechanism and their interplay in cancer.In the present study,an attempt is made to compare the functional characteristics and interactions of oncogenes and tumor suppressor genes to understand their biological role.431 breast cancer samples from seven publicly available microarray datasets were collected and analysed using GEO2R tool.The identified 416 differentially expressed genes were classified into five gene sets as oncogenes(OG),tumor suppressor genes(TSG),druggable genes,essential genes and other genes.The gene sets were subjected to various analysis such as enrichment analysis(viz.,GO,Pathways,Diseases and Drugs),network analysis,calculation of mutation frequencies and Guanine-Cytosine(GC)content.From the results,it was observed that the OG were having high GC content as well as high interactions than TSG.Moreover,the OG are found to have frequent mutations than TSG.The enrichment analysis results suggest that the oncogenes are involved in positive regulation of cellular protein metabolic process,macromolecule biosynthetic process and majorly in cell cycle and focal adhesion pathway in cancer.It was also found that these oncogenes are involved in other diseases such as skin diseases and viral infections.Collagenase,paclitaxel and docetaxel are some of the drugs found to be enriched for oncogenes.
文摘AIM To analyse cumulative loss of heterozygosity (LOH) of chromosomal regions and tumor suppressor genes in hepatocellular carcinomas (HCCs) from 20 southern African blacks. METHODS p53, RB1, BRCA1, BRCA2, WT1 and E cadherin genes were analysed for LOH, and p53 gene was also analysed for the codon 249 mutation, in tumor and adjacent non tumorous liver tissues using molecular techniques and 10 polymorphic microsatellite markers. RESULTS p53 codon 249 mutation was found in 25% of the subjects, as was expected, because many patients were from Mozambique, a country with high aflatoxin B 1 exposure. LOH was found at the RB1, BRCA2 and WT1 loci in 20%(4/*!20) of the HCCs, supporting a possible role of these genes in HCC. No LOH was evident in any of the remaining genes. Reports of mutations of p53 and RB1 genes in combination, described in other populations, were not confirmed in this study. Change in microsatellite repeat number was noted at 9/*!10 microsatellite loci in different HCCs, and changes at two or more loci were detected in 15%(3/*!20) of subjects. CONCLUSION We propose that microsatellite/genomic instability may play a role in the pathogenesis of a subset of HCCs in black Africans.
基金This study was supported by grants from the National Natural Science Foundation of China （No. 30700813, No. 30470977, No, 30080016）, Fundamental Key Science Foundation of Science and Technology Commission of Shanghai Municipality （No. 05JC14029）, Public Scientific Sesearch Platform of Hospital of Grade A at the Tertiary Level of Shanghai （No. SHDC12007704） and Youth Science Foundation of Shanghai Health Bureau （No. 034Y03）.Acknowledgments： We thank CapitalBio Corporation for performing microarray scanning and Jacqueline Ramirez, Keith Mitchilson for editorial comments.
文摘In addition to the tumor suppressor genes such as Rb and p53, it has been found that some molecules of the same class named CKI (cyclin-dependent kinase inhibitor) also play an important role in the inhibition of tumorigenesis and the tumor progression. In the KIP and INK4 families of CKIs, p15 shares extensive homology with p16. Findings in many tumors and their cell lines show that the inactivation of p15 (deletion, mutation, rearrangement, etc.) is very frequent, and inactive p15 is involved in the progress of some tumors. These studies provide evidence that the p15 is a new tumor suppressor gene. Furthermore, the research on the molecular mechanism of p15 in regulation of cell proliferation shows that p15 can inhibit the growth of some kinds of tumor cells, and p15 is the mediator of TGF-β-induced cell arrest. Investigations on p15 in cell differentiation suggest that increased p15 is related to the change of malignant phenotype. These results supply clues for further interpretation about the
文摘A number of tumor suppressor and tumor-related genes exhibit promoter hypermethylation with resultant gene silencing in human cancers.The frequencies of methylation differ among genes and genomic regions within CpG islands in different tissue types.Hypermethylation initially occurs at the edge of CpG islands and spreads to the transcription start site before ultimately shutting down gene expression.When the degree of methylation was quantitatively evaluated in neoplastic and non-neoplastic gastric epithelia using DNA microarray analysis,highlevel methylation around the transcription start site appeared to be a tumor-specific phenomenon,although multiple tumor suppressor genes became increasingly methylated with patient age in non-neoplastic gastric epithelia.Quantitative analysis of DNA methylation is a promising method for both cancer diagnosis and risk assessment.
