BACKGROUND:A growing body of evidence suggests that many tumors are initiated by both epigenetic abnormalities and gene mutations,which promote tumor progression. Epigenetic abnormalities include changes in DNA methyl...BACKGROUND:A growing body of evidence suggests that many tumors are initiated by both epigenetic abnormalities and gene mutations,which promote tumor progression. Epigenetic abnormalities include changes in DNA methylation and in the modification of histones.This study aimed to assess the status of methylation in the CpG island(CGI)of the tumor necrosis factor receptor superfamily member 10c(TNFRSF10C) with combined bisulfite restriction analysis(COBRA)and to evaluate its role in the progression of pancreatic cancer(PC). METHODS:The methylation status of four PC cell lines was assessed using COBRA and/or bisulfite genomic sequencing (BGS).Changes in methylation and TNFRSF10C expression in PC cell lines before and after treatment with 5-aza-2’-deoxycytidine(5-aza-dC)and/or trichostatin A(TSA)were assessed by BGS and real-time RT-PCR.Apoptosis in the four cell lines was tested by flow cytometry(FCM)and TUNEL assay. RESULTS:The methylation status of the TNFRSF10C promoter was assessed in PC cells(BxPC-3:68.84±8.71%;CFPAC-1:0; PANC-1:96.77±4.57%;SW1990:54.97±7.33%)with the COBRA assay,which was confirmed by the results of BGS.After treatment with 5-aza-dC and/or TSA,apoptosis was induced in PC cells to different degrees,and the levels of TNFRSF10C transcriptional expression in the PC cell lines(except CFPAC-1) increased markedly after 5-aza-dC treatment. CONCLUSIONS:A high frequency of CGI methylation in the TNFRSF10C promoter results in inactivation of the gene and enhancement of tumor growth in most PC cell lines(except CFPAC-1).Inactivation of TNFRSF10C by CGI hypermethylation can play an important role in PC progression and be potentially useful as a diagnostic marker and a new therapeutic approach for PC.展开更多
10C12 is an anticoagulant antibody identified from a phage display single-chain Fv human antibody library. It can be directed at the calcium-stabilized Gla domain of Factor-IX, an important coagulation factor in intri...10C12 is an anticoagulant antibody identified from a phage display single-chain Fv human antibody library. It can be directed at the calcium-stabilized Gla domain of Factor-IX, an important coagulation factor in intrinsic pathway of blood coagulation cascade, and interfere with membrane anchoring of Factor IX, thus inhibiting blood coagulation function. 10C12 has been demonstrated as an effective anti-coagulant in attenuating thrombosis in several different animal models. Here, we report the crystal structure of the Fab fragment of 10C12. The crystal contains two Fab molecules in the asymmetric unit with identical conformation, forming a lattice with large cavities. In addition, comparison of this free Fab with the antigen-bound structure of 10C12 shows no change in CDR conformations and the relative disposition of the variable subunits of H and L chains, suggesting the rigid conformation of this 10C12 Fab and a lock-and-key mechanism of antibody-antigen recognition for 10C12.展开更多
基金supported by a grant from the National Natural Science Foundation of China(30471691)
文摘BACKGROUND:A growing body of evidence suggests that many tumors are initiated by both epigenetic abnormalities and gene mutations,which promote tumor progression. Epigenetic abnormalities include changes in DNA methylation and in the modification of histones.This study aimed to assess the status of methylation in the CpG island(CGI)of the tumor necrosis factor receptor superfamily member 10c(TNFRSF10C) with combined bisulfite restriction analysis(COBRA)and to evaluate its role in the progression of pancreatic cancer(PC). METHODS:The methylation status of four PC cell lines was assessed using COBRA and/or bisulfite genomic sequencing (BGS).Changes in methylation and TNFRSF10C expression in PC cell lines before and after treatment with 5-aza-2’-deoxycytidine(5-aza-dC)and/or trichostatin A(TSA)were assessed by BGS and real-time RT-PCR.Apoptosis in the four cell lines was tested by flow cytometry(FCM)and TUNEL assay. RESULTS:The methylation status of the TNFRSF10C promoter was assessed in PC cells(BxPC-3:68.84±8.71%;CFPAC-1:0; PANC-1:96.77±4.57%;SW1990:54.97±7.33%)with the COBRA assay,which was confirmed by the results of BGS.After treatment with 5-aza-dC and/or TSA,apoptosis was induced in PC cells to different degrees,and the levels of TNFRSF10C transcriptional expression in the PC cell lines(except CFPAC-1) increased markedly after 5-aza-dC treatment. CONCLUSIONS:A high frequency of CGI methylation in the TNFRSF10C promoter results in inactivation of the gene and enhancement of tumor growth in most PC cell lines(except CFPAC-1).Inactivation of TNFRSF10C by CGI hypermethylation can play an important role in PC progression and be potentially useful as a diagnostic marker and a new therapeutic approach for PC.
基金the grants from Fujian Explore Grant for Young Scientists (2007J0294)CAS (KSCX2-YW-R-082)+1 种基金NNSFC (30430190, 30625011)MOST (2007CB914304 and 2006AA02A313)
文摘10C12 is an anticoagulant antibody identified from a phage display single-chain Fv human antibody library. It can be directed at the calcium-stabilized Gla domain of Factor-IX, an important coagulation factor in intrinsic pathway of blood coagulation cascade, and interfere with membrane anchoring of Factor IX, thus inhibiting blood coagulation function. 10C12 has been demonstrated as an effective anti-coagulant in attenuating thrombosis in several different animal models. Here, we report the crystal structure of the Fab fragment of 10C12. The crystal contains two Fab molecules in the asymmetric unit with identical conformation, forming a lattice with large cavities. In addition, comparison of this free Fab with the antigen-bound structure of 10C12 shows no change in CDR conformations and the relative disposition of the variable subunits of H and L chains, suggesting the rigid conformation of this 10C12 Fab and a lock-and-key mechanism of antibody-antigen recognition for 10C12.
