DNA replication is tightly regulated during the S phase of the cell cycle, and the activation of the intra-S-phase checkpoint due to DNA damage usually results in arrest of DNA synthesis. However, the molecular detail...DNA replication is tightly regulated during the S phase of the cell cycle, and the activation of the intra-S-phase checkpoint due to DNA damage usually results in arrest of DNA synthesis. However, the molecular details about the correlation between the checkpoint and regulation of DNA replication are still unclear. To investigate the connections between DNA replication and DNA damage checkpoint, a DNA-damage reagent, tripchlorolide, was applied to CHO (Chinese ovary hamster) cells at early- or middle-stages of the S phase. The early-S-phase treatment with TC significantly delayed the progression of the S phase and caused the phosphorylation of the Chk1 checkpoint protein, whereas the middle-S-phase treatment only slightly slowed down the progression of the S phase. Furthermore, the analysis of DNA replication patterns revealed that replication pattern Ⅱ was greatly prolonged in the cells treated with the drug during the early-S phase, whereas the late-replication patterns of these cells were hardly detected, suggesting that the activation of the intra-S-phase checkpoint inhibits the late-origin firing of DNA replication. We conclude that cells at different stages of the S phase are differentially sensitive to the DNA-damage reagent, and the activation of the intra-Sphase checkpoint blocks the DNA replication progression in the late stage of S phase.展开更多
BACKGROUND Anoctamin 5(ANO5)/transmembrane protein 16E belongs to the ANO/transmembrane protein 16 anion channel family.ANOs comprise a family of plasma membrane proteins that mediate ion transport and phospholipid sc...BACKGROUND Anoctamin 5(ANO5)/transmembrane protein 16E belongs to the ANO/transmembrane protein 16 anion channel family.ANOs comprise a family of plasma membrane proteins that mediate ion transport and phospholipid scrambling and regulate other membrane proteins in numerous cell types.Previous studies have elucidated the roles and mechanisms of ANO5 activation in various cancer types.However,it remains unclear whether ANO5 acts as a plasma membrane chloride channel,and its expression and functions in gastric cancer(GC)have not been investigated.AIM To examine the role of ANO5 in the regulation of tumor progression and clinicopathological significance of its expression in GC.METHODS Knockdown experiments using ANO5 small interfering RNA were conducted in human GC cell lines,and changes in cell proliferation,cell cycle progression,apoptosis,and cellular movement were assessed.The gene expression profiles of GC cells were investigated following ANO5 silencing by microarray analysis.Immunohistochemical staining of ANO5 was performed on 195 primary tumor samples obtained from patients with GC who underwent curative gastrectomy between 2011 and 2013 at our department.RESULTS Reverse transcription-quantitative polymerase chain reaction(PCR)and western blotting demonstrated high ANO5 mRNA and protein expression,respectively,in NUGC4 and MKN45 cells.In these cells,ANO5 silencing inhibited cell proliferation and induced apoptosis.In addition,the knockdown of ANO5 inhibited G1-S phase progression,invasion,and migration.The results of the microarray analysis revealed changes in the expression levels of several cyclin-associated genes,such as CDKN1A,CDK2/4/6,CCNE2,and E2F1,in ANO5-depleted NUGC4 cells.The expression of these genes was verified using reverse transcription-quantitative PCR.Immunohistochemical staining revealed that high ANO5 expression levels were associated with a poor prognosis.Multivariate analysis identified high ANO5 expression as an independent prognostic factor for 5-year survival in patients with GC(P=0.0457).CONCLUSION ANO5 regulates the cell cycle progression by regulating the expression of cyclin-associated genes and affects the prognosis of patients with GC.These results may provide insights into the role of ANO5 as a key mediator in tumor progression and/or promising prognostic biomarker for GC.展开更多
Chromosomal DNA replication is one of the central biological events occurring inside cells.Due to its large size,the replication of genomic DNA in eukaryotes initiates at hundreds to tens of thousands of sites called ...Chromosomal DNA replication is one of the central biological events occurring inside cells.Due to its large size,the replication of genomic DNA in eukaryotes initiates at hundreds to tens of thousands of sites called DNA origins so that the replication could be completed in a limited time.Further,eukaryotic DNA replication is sophisticatedly regulated,and this regulation guarantees that each origin fires once per S phase and each segment of DNA gets duplication also once per cell cycle.The first step of replication initiation is the assembly of pre-replication complex(pre-RC).Since 1973,four proteins,Cdc6/Cdc18,MCM,ORC and Cdt1,have been extensively studied and proved to be pre-RC components.Recently,a novel pre-RC component called Sap1/Girdin was identified.Sap1/Girdin is required for loading Cdc18/Cdc6 to origins for pre-RC assembly in the fission yeast and human cells,respectively.At the transition of G1 to S phase,pre-RC is activated by the two kinases,cyclin-dependent kinase(CDK)and Dbf4-dependent kinase(DDK),and subsequently,RPA,primase-polα,PCNA,topoisomerase,Cdc45,polδ,and polεare recruited to DNA origins for creating two bi-directional replication forks and initiating DNA replication.As replication forks move along chromatin DNA,they frequently stall due to the presence of a great number of replication barriers on chromatin DNA,such as secondary DNA structures,protein/DNA complexes,DNA lesions,gene transcription.Stalled forks must require checkpoint regulation for their stabilization.Otherwise,stalled forks will collapse,which results in incomplete DNA replication and genomic instability.This short review gives a concise introduction regarding the current understanding of replication initiation and replication fork stabilization.展开更多
基金This work was supported by a grant from the National Natural Science Foundation of China(No.30230110)a special grant from the Major State Basic Research Pro-gram of China(No.G1999053901)a grant from the Chinese Academy of Sciences(No.KSCX2-SW-203)to Jia Rui WU.
文摘DNA replication is tightly regulated during the S phase of the cell cycle, and the activation of the intra-S-phase checkpoint due to DNA damage usually results in arrest of DNA synthesis. However, the molecular details about the correlation between the checkpoint and regulation of DNA replication are still unclear. To investigate the connections between DNA replication and DNA damage checkpoint, a DNA-damage reagent, tripchlorolide, was applied to CHO (Chinese ovary hamster) cells at early- or middle-stages of the S phase. The early-S-phase treatment with TC significantly delayed the progression of the S phase and caused the phosphorylation of the Chk1 checkpoint protein, whereas the middle-S-phase treatment only slightly slowed down the progression of the S phase. Furthermore, the analysis of DNA replication patterns revealed that replication pattern Ⅱ was greatly prolonged in the cells treated with the drug during the early-S phase, whereas the late-replication patterns of these cells were hardly detected, suggesting that the activation of the intra-S-phase checkpoint inhibits the late-origin firing of DNA replication. We conclude that cells at different stages of the S phase are differentially sensitive to the DNA-damage reagent, and the activation of the intra-Sphase checkpoint blocks the DNA replication progression in the late stage of S phase.
基金Supported by Japan Society for the Promotion of Science,No.21K08689,No.21K16456,No.20K09016,No.20K09084,No.19K09202 and No.19K09182.
文摘BACKGROUND Anoctamin 5(ANO5)/transmembrane protein 16E belongs to the ANO/transmembrane protein 16 anion channel family.ANOs comprise a family of plasma membrane proteins that mediate ion transport and phospholipid scrambling and regulate other membrane proteins in numerous cell types.Previous studies have elucidated the roles and mechanisms of ANO5 activation in various cancer types.However,it remains unclear whether ANO5 acts as a plasma membrane chloride channel,and its expression and functions in gastric cancer(GC)have not been investigated.AIM To examine the role of ANO5 in the regulation of tumor progression and clinicopathological significance of its expression in GC.METHODS Knockdown experiments using ANO5 small interfering RNA were conducted in human GC cell lines,and changes in cell proliferation,cell cycle progression,apoptosis,and cellular movement were assessed.The gene expression profiles of GC cells were investigated following ANO5 silencing by microarray analysis.Immunohistochemical staining of ANO5 was performed on 195 primary tumor samples obtained from patients with GC who underwent curative gastrectomy between 2011 and 2013 at our department.RESULTS Reverse transcription-quantitative polymerase chain reaction(PCR)and western blotting demonstrated high ANO5 mRNA and protein expression,respectively,in NUGC4 and MKN45 cells.In these cells,ANO5 silencing inhibited cell proliferation and induced apoptosis.In addition,the knockdown of ANO5 inhibited G1-S phase progression,invasion,and migration.The results of the microarray analysis revealed changes in the expression levels of several cyclin-associated genes,such as CDKN1A,CDK2/4/6,CCNE2,and E2F1,in ANO5-depleted NUGC4 cells.The expression of these genes was verified using reverse transcription-quantitative PCR.Immunohistochemical staining revealed that high ANO5 expression levels were associated with a poor prognosis.Multivariate analysis identified high ANO5 expression as an independent prognostic factor for 5-year survival in patients with GC(P=0.0457).CONCLUSION ANO5 regulates the cell cycle progression by regulating the expression of cyclin-associated genes and affects the prognosis of patients with GC.These results may provide insights into the role of ANO5 as a key mediator in tumor progression and/or promising prognostic biomarker for GC.
文摘Chromosomal DNA replication is one of the central biological events occurring inside cells.Due to its large size,the replication of genomic DNA in eukaryotes initiates at hundreds to tens of thousands of sites called DNA origins so that the replication could be completed in a limited time.Further,eukaryotic DNA replication is sophisticatedly regulated,and this regulation guarantees that each origin fires once per S phase and each segment of DNA gets duplication also once per cell cycle.The first step of replication initiation is the assembly of pre-replication complex(pre-RC).Since 1973,four proteins,Cdc6/Cdc18,MCM,ORC and Cdt1,have been extensively studied and proved to be pre-RC components.Recently,a novel pre-RC component called Sap1/Girdin was identified.Sap1/Girdin is required for loading Cdc18/Cdc6 to origins for pre-RC assembly in the fission yeast and human cells,respectively.At the transition of G1 to S phase,pre-RC is activated by the two kinases,cyclin-dependent kinase(CDK)and Dbf4-dependent kinase(DDK),and subsequently,RPA,primase-polα,PCNA,topoisomerase,Cdc45,polδ,and polεare recruited to DNA origins for creating two bi-directional replication forks and initiating DNA replication.As replication forks move along chromatin DNA,they frequently stall due to the presence of a great number of replication barriers on chromatin DNA,such as secondary DNA structures,protein/DNA complexes,DNA lesions,gene transcription.Stalled forks must require checkpoint regulation for their stabilization.Otherwise,stalled forks will collapse,which results in incomplete DNA replication and genomic instability.This short review gives a concise introduction regarding the current understanding of replication initiation and replication fork stabilization.
