FANCD2 is involved in DNA damage repair and maintenance of chromosome stability.The purpose of this study was to investigate the expression of FANCD2 in sporadic breast cancer tissues and its association with clinicop...FANCD2 is involved in DNA damage repair and maintenance of chromosome stability.The purpose of this study was to investigate the expression of FANCD2 in sporadic breast cancer tissues and its association with clinicopathological features.A total of 162 Chinese women with invasive breast carcinoma who had no family history in first-degree relatives and 12 normal breast tissues were examined.The expression of FANCD2 was detected by immunohistochemical staining based on a tissue microarray technique.SAS system was used to analyze the data.Twenty-one out of the 162 invasive breast cancers(13%) were negative for FANCD2.The mean percentage of FANCD2 positive cells was significantly lower in breast cancers than in controls(P0.05).It was suggested that FANCD2 may play a critical role in breast carcinogenesis.It may become a valuable and independent marker for identifying women with sporadic breast cancer and evaluating the prognosis.展开更多
目的研究FANCD2 sh RNA干扰对人头颈鳞癌(head and neck squamous cell carcinoma,HNSCC)细胞SIHN-005A化疗敏感度的影响,并初步探讨其可能机制。方法应用前期实验获得的FANCD2基因沉默效应稳定的人HNSCC SIHN-005A细胞,以嘌呤霉素持续...目的研究FANCD2 sh RNA干扰对人头颈鳞癌(head and neck squamous cell carcinoma,HNSCC)细胞SIHN-005A化疗敏感度的影响,并初步探讨其可能机制。方法应用前期实验获得的FANCD2基因沉默效应稳定的人HNSCC SIHN-005A细胞,以嘌呤霉素持续筛选;Western blot检测沉默效应;CCK-8法检测顺铂作用后细胞增殖抑制率;Hoechst法检测细胞凋亡;Western blot检测Cyclin D1、Bax/Bcl-2、HIF-1α蛋白的表达。结果实验组细胞FANCD2蛋白表达明显下降,FANCD2蛋白沉默效率达60.5%。实验组细胞增殖抑制率明显高于阴性对照组和空白对照组,且具有浓度及时间依赖性,实验组细胞对CDDP的IC50值明显低于阴性对照组和空白对照组。FANCD2基因沉默后实验组细胞凋亡率明显增高。加用CDDP后,Cyclin D1、Bcl-2、HIF-1α蛋白表达水平较加用CDDP前明显下调,Bax蛋白明显增强。结论沉默FANCD2可增加SIHN-005A细胞对化疗药物CDDP的敏感度,其机制可能与FANCD2 sh RNA干扰引起Bax/bcl-2、Cyclin D1、HIF-1α的表达变化有关。展开更多
The tumor necrosis factor （TNF）-α/NF-kB-signaling pathway plays a pivotal role in various processes including apoptosis, cellular differentiation, host defense, inflammation, autoimmunity and organogenesis. The com...The tumor necrosis factor （TNF）-α/NF-kB-signaling pathway plays a pivotal role in various processes including apoptosis, cellular differentiation, host defense, inflammation, autoimmunity and organogenesis. The complexity of the TNF-α/NF-kB signaling is in part due to the dynamic protein behaviors of key players in this pathway. In this present work, a dynamic and global view of the signaling components in the nucleus at the early stages of TNF-a/ NF-KB signaling was obtained in HEK293 cells, by a combination of subcellular fractionation and stable isotope la- beling by amino acids in cell culture （SILAC）. The dynamic profile patterns of 547 TNF-α-induced nuclei-associated proteins were quantified in our studies. The functional characters of all the profiles were further analyzed using that Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway annotation. Additionally, many previously unknown effectors of TNF-α/NF-kB signaling were identified, quantified and clustered into differential activation profiles. In- terestingly, levels of Fanconi anemia group D2 protein （FANCD2）, one of the Fanconi anemia family proteins, was found to be increased in the nucleus by SILAC quantitation upon TNF-α stimulation, which was further verified by western blotting and immunofluorescence analysis. This indicates that FANCD2 might be involved in TNF-α/NF-kB signaling through its accumulation in the nucleus. In summary, the combination of subcellular proteomics with quan- titative analysis not only allowed for a dissection of the nuclear TNF-α/NF-kB-signaling pathway, but also provided a systematic strategy for monitoring temporal and spatial changes in cell signaling.展开更多
Fanconi anemia(FA),an X-linked genetic or autosomal recessive disease,exhibits complicated pathogenesis.Previously,we detected the mutated Dynein Axonemal Heavy Chain 2(DNAH2)gene in 2 FA cases.Herein,we further inves...Fanconi anemia(FA),an X-linked genetic or autosomal recessive disease,exhibits complicated pathogenesis.Previously,we detected the mutated Dynein Axonemal Heavy Chain 2(DNAH2)gene in 2 FA cases.Herein,we further investigated the potential association between DNAH2 and the homologous recombination repair pathway of FA.The assays of homologous recombination repair,mitomycin C(MMC)sensitivity,immunofluorescence,and ubiquitination modification were performed in U2OS and DR-U2OS cell lines.In MMC-treated U2OS cells,the downregulation of the DNAH2 gene increased the sensitivity of cells to DNA inter-strand crosslinks.We also observed the reduced enrichment of FANCD2 protein to DNA damage sites.Furthermore,the ubiquitination modification level of FANCD2 was influenced by the deficiency of DNAH2.Thus,our results suggest that DNAH2 may modulate the cell homologous recombination repair partially by increasing the ubiquitination and the enrichment to DNA damage sites of FANCD2.DNAH2 may act as a novel co-pathogenic gene of FA patients.展开更多
Objective:To find out how to overcome resistance during multiple myeloma(MM) treatment through establishing a multidrug resistant human multiple myeloma cell line and investigating its biological features.Methods:The ...Objective:To find out how to overcome resistance during multiple myeloma(MM) treatment through establishing a multidrug resistant human multiple myeloma cell line and investigating its biological features.Methods:The parent cell line MOLP-2 was exposed to different concentrations of melphalan and a melphalan-resistant cell line MOLP-2/R was identified by continuous stepwise selection.The cell morphology and growth curves were examined.Protein levels of P-gp, MRP and FANCD2 monoubiquitination were checked by Western blotting.The IC50 of melphalan and resistance index(RI) were detected by MTT assay.Results:A melphalan-resistant cell line MOLP-2/R was finally identified.The RI of MOLP-2/R cells to melphalan was 6.03.Besides melphalan it was cross resistant to other chemotherapeutic agents, including ADM, CTX, DDP and VP-16.The multiplication time was postponed(P < 0.05).Studies showed that FANCD2 protein monoubiquitination was enhanced, but the levels of P-gp and MRP expressions in the MOLP-2/R cells were similar with the parent cells.Conclusion:MOLP-2/R cell line may serve as an ideal model for exploring the mechanism of MDR.Over-expression of FANCD2 protein monoubiquitination might contribute to acquired drug resistance in MOLP-2/R cell line.展开更多
Fanconi anemia(FA)is an autosomal or X-linked recessive disorder characterized by chromosomal instability,bone marrow failure,cancer susceptibility,and a profound sensitivity to agents that produce DNA interstrand cro...Fanconi anemia(FA)is an autosomal or X-linked recessive disorder characterized by chromosomal instability,bone marrow failure,cancer susceptibility,and a profound sensitivity to agents that produce DNA interstrand cross-link(ICL).To date,15 genes have been identified that,when mutated,result in FA or an FA-like syndrome.It is believed that cellular resistance to DNA interstrand cross-linking agents requires all 15 FA or FAlike proteins.Here,we review our current understanding of how these FA proteins participate in ICL repair and discuss the molecular mechanisms that regulate the FA pathway to maintain genome stability.展开更多
Background Fanconi anemia is a severe congenital disorder associated with mutations in a cluster of genes responsible for DNA repair.Arriving at an accurate and timely diagnosis can be difficult in cases of Fanconi an...Background Fanconi anemia is a severe congenital disorder associated with mutations in a cluster of genes responsible for DNA repair.Arriving at an accurate and timely diagnosis can be difficult in cases of Fanconi anemia with atypical clinical features.It is very important to increase the rate of accurate diagnosis for such cases in a clinical setting.The purpose of this study is to explore the clinical diagnosis of Fanconi anemia in children with atypical clinical features.Methods Six cases of Fanconi anemia with atypical clinical features were enrolled in the study,and their clinical features were recorded,their FANCA gene transcription was assessed by RT-PCR,and FANCA mutations and the ubiquitination of FANCD2 protein were analyzed using DNA sequencing and western blotting respectively.Results All six cases showed atypical clinical features including no apparent deformities,lack of response to immune therapy,and progressively increasing bone marrow failure.They also have significantly increased fetal hemoglobin,negative mitomycin-induced fracture test results,and carry a FANCA gene missense mutation.Single protein ubiquitination of FANCD2 was not observed in those patients.Conclusion The combination of clinical features,FANCA pathogenic gene mutation genotype and the absence of FANCD2 protein ubiquitination are helpful in the accurate and timely diagnosis of Fanconi anemia in children.展开更多
Next-generation sequencing technology has been widely utilized for the diagnosis of Fanconi anemia(FA).However,mixed cell sequencing and chimerism of FA patients may lead to unconfirmed genetic subtypes.Herein,we intr...Next-generation sequencing technology has been widely utilized for the diagnosis of Fanconi anemia(FA).However,mixed cell sequencing and chimerism of FA patients may lead to unconfirmed genetic subtypes.Herein,we introduced two novel diagnostic methods,including single-cell sequencing and capillary nano-immunoassay.One FA case with FANCM c.4931G>A p.R1644Q and FANCD1 c.6325G>A p.V2109I was studied.The DNA of 28 cells was amplified and eight types of cells were observed after Sanger sequencing.There were two homozygous mutations(FANCM/FANCD1).Furthermore,the capillary nano-immunoassay was conducted to analyze the expression profile of FA-associated proteins.Abnormal FANCM and FANCD1 expressions simultaneously existed.This case was thus diagnosed as FA-D1/FA-M dual subtype.Compared with mixed cell sequencing,single-cell sequencing data shows more accuracy for the FA subtype evaluation,while the capillary nano-immunoassay is a good method to detect the expression profile of abnormal or modified FA protein.展开更多
文摘FANCD2 is involved in DNA damage repair and maintenance of chromosome stability.The purpose of this study was to investigate the expression of FANCD2 in sporadic breast cancer tissues and its association with clinicopathological features.A total of 162 Chinese women with invasive breast carcinoma who had no family history in first-degree relatives and 12 normal breast tissues were examined.The expression of FANCD2 was detected by immunohistochemical staining based on a tissue microarray technique.SAS system was used to analyze the data.Twenty-one out of the 162 invasive breast cancers(13%) were negative for FANCD2.The mean percentage of FANCD2 positive cells was significantly lower in breast cancers than in controls(P0.05).It was suggested that FANCD2 may play a critical role in breast carcinogenesis.It may become a valuable and independent marker for identifying women with sporadic breast cancer and evaluating the prognosis.
基金Abbreviations： TNF （tumor necrosis factor） SILAC （stable isotope label- ing by amino acids in cell culture）+4 种基金 LC-MS/MS （liquid chromatography tandem mass spectrometry） LTQ-Orbitrap （linear ion trap/Orbitrap） HPRD （Human Protein Reference Database） SMARC （SWI/SNF-related matrix-associated actin-dependent regulator chromatin） KEGG （Kyoto Encyclopedia of Genes and Genomes）Acknowledgment We would like to thank Dr/an Scott （Department of Molecular Genetics, University of Toronto）, Dr Chengjian Tu （Vanderbilt Medical Center, Vanderbilt University） and Ya-Wen Tian （Key Laboratory of Systems Biology, Shanghai Institutes for Biologi- cal Sciences） for critical reading of the manuscript. This work was supported by the National Natural Science Foundation of China （30425021, 30521005）, Basic Research Foundation （2006CB910700）, CAS Project （KSCX2-YW-R-106, KSCX1- YW-02） and High-technology Project （2007AA02Z334）.
文摘The tumor necrosis factor （TNF）-α/NF-kB-signaling pathway plays a pivotal role in various processes including apoptosis, cellular differentiation, host defense, inflammation, autoimmunity and organogenesis. The complexity of the TNF-α/NF-kB signaling is in part due to the dynamic protein behaviors of key players in this pathway. In this present work, a dynamic and global view of the signaling components in the nucleus at the early stages of TNF-a/ NF-KB signaling was obtained in HEK293 cells, by a combination of subcellular fractionation and stable isotope la- beling by amino acids in cell culture （SILAC）. The dynamic profile patterns of 547 TNF-α-induced nuclei-associated proteins were quantified in our studies. The functional characters of all the profiles were further analyzed using that Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway annotation. Additionally, many previously unknown effectors of TNF-α/NF-kB signaling were identified, quantified and clustered into differential activation profiles. In- terestingly, levels of Fanconi anemia group D2 protein （FANCD2）, one of the Fanconi anemia family proteins, was found to be increased in the nucleus by SILAC quantitation upon TNF-α stimulation, which was further verified by western blotting and immunofluorescence analysis. This indicates that FANCD2 might be involved in TNF-α/NF-kB signaling through its accumulation in the nucleus. In summary, the combination of subcellular proteomics with quan- titative analysis not only allowed for a dissection of the nuclear TNF-α/NF-kB-signaling pathway, but also provided a systematic strategy for monitoring temporal and spatial changes in cell signaling.
基金This work was partially supported by the National Key Research and Development Program of China(2016YFC0901503,2018YFA0107801)the Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences,CIFMS(2017-I2M-3-015)+1 种基金the National Natural Science Foundation of China(81500156,81170470,81970149)and Tianjin Natural Science Foundation Project(20JCYBJC00470).
文摘Fanconi anemia(FA),an X-linked genetic or autosomal recessive disease,exhibits complicated pathogenesis.Previously,we detected the mutated Dynein Axonemal Heavy Chain 2(DNAH2)gene in 2 FA cases.Herein,we further investigated the potential association between DNAH2 and the homologous recombination repair pathway of FA.The assays of homologous recombination repair,mitomycin C(MMC)sensitivity,immunofluorescence,and ubiquitination modification were performed in U2OS and DR-U2OS cell lines.In MMC-treated U2OS cells,the downregulation of the DNAH2 gene increased the sensitivity of cells to DNA inter-strand crosslinks.We also observed the reduced enrichment of FANCD2 protein to DNA damage sites.Furthermore,the ubiquitination modification level of FANCD2 was influenced by the deficiency of DNAH2.Thus,our results suggest that DNAH2 may modulate the cell homologous recombination repair partially by increasing the ubiquitination and the enrichment to DNA damage sites of FANCD2.DNAH2 may act as a novel co-pathogenic gene of FA patients.
文摘Objective:To find out how to overcome resistance during multiple myeloma(MM) treatment through establishing a multidrug resistant human multiple myeloma cell line and investigating its biological features.Methods:The parent cell line MOLP-2 was exposed to different concentrations of melphalan and a melphalan-resistant cell line MOLP-2/R was identified by continuous stepwise selection.The cell morphology and growth curves were examined.Protein levels of P-gp, MRP and FANCD2 monoubiquitination were checked by Western blotting.The IC50 of melphalan and resistance index(RI) were detected by MTT assay.Results:A melphalan-resistant cell line MOLP-2/R was finally identified.The RI of MOLP-2/R cells to melphalan was 6.03.Besides melphalan it was cross resistant to other chemotherapeutic agents, including ADM, CTX, DDP and VP-16.The multiplication time was postponed(P < 0.05).Studies showed that FANCD2 protein monoubiquitination was enhanced, but the levels of P-gp and MRP expressions in the MOLP-2/R cells were similar with the parent cells.Conclusion:MOLP-2/R cell line may serve as an ideal model for exploring the mechanism of MDR.Over-expression of FANCD2 protein monoubiquitination might contribute to acquired drug resistance in MOLP-2/R cell line.
基金supported in part by grants from the China’s Fundamental Research Funds for the Central Universitiesthe National Natural Science Foundation of China(Grant No.31071243)the Natural Science Foundation of Zhejiang Province(Grant R2110569)(to J.H.).
文摘Fanconi anemia(FA)is an autosomal or X-linked recessive disorder characterized by chromosomal instability,bone marrow failure,cancer susceptibility,and a profound sensitivity to agents that produce DNA interstrand cross-link(ICL).To date,15 genes have been identified that,when mutated,result in FA or an FA-like syndrome.It is believed that cellular resistance to DNA interstrand cross-linking agents requires all 15 FA or FAlike proteins.Here,we review our current understanding of how these FA proteins participate in ICL repair and discuss the molecular mechanisms that regulate the FA pathway to maintain genome stability.
文摘Background Fanconi anemia is a severe congenital disorder associated with mutations in a cluster of genes responsible for DNA repair.Arriving at an accurate and timely diagnosis can be difficult in cases of Fanconi anemia with atypical clinical features.It is very important to increase the rate of accurate diagnosis for such cases in a clinical setting.The purpose of this study is to explore the clinical diagnosis of Fanconi anemia in children with atypical clinical features.Methods Six cases of Fanconi anemia with atypical clinical features were enrolled in the study,and their clinical features were recorded,their FANCA gene transcription was assessed by RT-PCR,and FANCA mutations and the ubiquitination of FANCD2 protein were analyzed using DNA sequencing and western blotting respectively.Results All six cases showed atypical clinical features including no apparent deformities,lack of response to immune therapy,and progressively increasing bone marrow failure.They also have significantly increased fetal hemoglobin,negative mitomycin-induced fracture test results,and carry a FANCA gene missense mutation.Single protein ubiquitination of FANCD2 was not observed in those patients.Conclusion The combination of clinical features,FANCA pathogenic gene mutation genotype and the absence of FANCD2 protein ubiquitination are helpful in the accurate and timely diagnosis of Fanconi anemia in children.
基金This work was partially supported by grants from the National Natural Science Foundation of China(81670112 and 31571380)CAMS Innovation Fund for Medical Sciences(CIFMS,2016-I2M-1-002)The National Key Research and Development Program of China(2016YFC0901503).
文摘Next-generation sequencing technology has been widely utilized for the diagnosis of Fanconi anemia(FA).However,mixed cell sequencing and chimerism of FA patients may lead to unconfirmed genetic subtypes.Herein,we introduced two novel diagnostic methods,including single-cell sequencing and capillary nano-immunoassay.One FA case with FANCM c.4931G>A p.R1644Q and FANCD1 c.6325G>A p.V2109I was studied.The DNA of 28 cells was amplified and eight types of cells were observed after Sanger sequencing.There were two homozygous mutations(FANCM/FANCD1).Furthermore,the capillary nano-immunoassay was conducted to analyze the expression profile of FA-associated proteins.Abnormal FANCM and FANCD1 expressions simultaneously existed.This case was thus diagnosed as FA-D1/FA-M dual subtype.Compared with mixed cell sequencing,single-cell sequencing data shows more accuracy for the FA subtype evaluation,while the capillary nano-immunoassay is a good method to detect the expression profile of abnormal or modified FA protein.
