Objective Vasculogenic mimicry(VM)is a novel vasculogenic process integral to glioma stem cells(GSCs)in glioblastoma(GBM).However,the relationship between VM and ataxia-telangiectasia mutated(ATM)serine/threonine kina...Objective Vasculogenic mimicry(VM)is a novel vasculogenic process integral to glioma stem cells(GSCs)in glioblastoma(GBM).However,the relationship between VM and ataxia-telangiectasia mutated(ATM)serine/threonine kinase activation,which confers chemoradiotherapy resistance,remains unclear.Methods We investigated VM formation and phosphorylated ATM(pATM)levels by CD31/GFAPperiodic acid-Schiff dual staining and immunohistochemical staining in 145 GBM specimens.Glioma stem-like cells(GSLCs)derived from the formatted spheres of U87 and U251 cell lines and their pATM level and VM formation ability were examined using western blot and three-dimensional culture.For the examination of the function of pATM in VM formation by GSLCs,ATM knockdown by shRNAs and deactivated via ATM phosphorylation inhibitor KU55933 were studied.Results VM and high pATM expression occurred in 38.5% and 41.8% of tumors,respectively,and were significantly associated with reduced progression-free and overall survival.Patients with VM-positive GBMs exhibited higher pATM levels(r_(s)=0.425,P=0.01).The multivariate analysis established VM as an independent negative prognostic factor(P=0.002).Furthermore,GSLCs expressed high levels of pATM and formed vascular-like networks in vitro.ATM inactivation or knockdown hindered VM-like network formation concomitant with the downregulation of pVEGFR-2,VE-cadherin,and laminin B2.Conclusion VM may predict a poor GBM prognosis and is associated with pATM expression.We propose that pATM promotes VM through extracellular matrix modulation and VE-Cadherin/pVEGFR-2 activation,thereby highlighting ATM activation as a potential target for enhancing anti-angiogenesis therapies for GBM.展开更多
Objective:Mitotic arrest-deficient protein 1(MAD1)is a kinetochore protein essential for the mitotic spindle checkpoint.Proteomic studies have indicated that MAD1 is a component of the DNA damage response(DDR)pathway....Objective:Mitotic arrest-deficient protein 1(MAD1)is a kinetochore protein essential for the mitotic spindle checkpoint.Proteomic studies have indicated that MAD1 is a component of the DNA damage response(DDR)pathway.However,whether and how MAD1 might be directly involved in the DDR is largely unknown.Methods:We ectopically expressed the wild type,or a phosphorylation-site--mutated form of MAD1 in MAD1 knockdown cells to look for complementation effects.We used the comet assay,colony formation assay,immunofluorescence staining,and flow cytometry to assess the DDR,radiosensitivity,and the G2/M checkpoint.We employed co-immunoprecipitation followed by mass spectrometry to identify MAD1 interacting proteins.Data were analyzed using the unpaired Student'st-test.Results:We showed that MAD1 was required for an optimal DDR,as knocking down MAD1 resulted in impaired DNA repair and hypersensitivity to ionizing radiation(IR).We found that IR-induced serine 214 phosphorylation was ataxia-telangiectasia mutated(ATM)kinase-dependent.Mutation of serine 214 to alanine failed to rescue the phenotypes of MAD1 knockdown cells in response to IR.Using mass spectrometry,we identified a protein complex mediated by MAD1 serine 214 phosphorylation in response to IR.Among them,we showed that KU80 was a key protein that displayed enhanced interaction with MAD1 after DNA damage.Finally,we showed that MAD1 interaction with KU80 required serine 214 phosphorylation,and it was essential for activation of DNA protein kinases catalytic subunit(DNA-PKcs).Conclusions:MAD1 serine 214 phosphorylation mediated by ATM kinase in response to IR was required for the interaction with KU80 and activation of DNA-PKCs.展开更多
Ataxia-telangiectasia mutated(ATM)plays a key role in regulating the cellular response to ionizing radiation.The tumor-suppressor gene ATM,mutations in which cause the human genetic disease ataxia telangiecta-sia,enco...Ataxia-telangiectasia mutated(ATM)plays a key role in regulating the cellular response to ionizing radiation.The tumor-suppressor gene ATM,mutations in which cause the human genetic disease ataxia telangiecta-sia,encodes a key protein kinase that controls the cellular response to double-stranded breaks.Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage response pathways,most notably cell cycle checkpoints.Here,we highlight some of the new developments in thefield in our understanding of the mechanism of activation of ATM and its signaling pathways,explore whether DNA double-strand breaks are the sole activators of ATM and ATM-dependent signaling pathways,and address some of the prominent,unanswered questions related to ATM and its function.The scope of this article is to provide a brief overview of the recent literature on this subject and to raise questions that could be addressed in future studies.展开更多
基金supported by the Natural Science Foundation of Anhui Province(2208085MH250,2308085MH272)National Key Research and Development Program of China(2021YFF1201000)+2 种基金Natural Science Research Project of the Anhui Educational Committee(2023AH040404,2023AH053402)Anhui Provincial Health and Medical Research Project(AHWJ2023A10143)Research Funds of Centre for Leading Medicine and Advanced Technologies of IHM(2023IHM01043)。
文摘Objective Vasculogenic mimicry(VM)is a novel vasculogenic process integral to glioma stem cells(GSCs)in glioblastoma(GBM).However,the relationship between VM and ataxia-telangiectasia mutated(ATM)serine/threonine kinase activation,which confers chemoradiotherapy resistance,remains unclear.Methods We investigated VM formation and phosphorylated ATM(pATM)levels by CD31/GFAPperiodic acid-Schiff dual staining and immunohistochemical staining in 145 GBM specimens.Glioma stem-like cells(GSLCs)derived from the formatted spheres of U87 and U251 cell lines and their pATM level and VM formation ability were examined using western blot and three-dimensional culture.For the examination of the function of pATM in VM formation by GSLCs,ATM knockdown by shRNAs and deactivated via ATM phosphorylation inhibitor KU55933 were studied.Results VM and high pATM expression occurred in 38.5% and 41.8% of tumors,respectively,and were significantly associated with reduced progression-free and overall survival.Patients with VM-positive GBMs exhibited higher pATM levels(r_(s)=0.425,P=0.01).The multivariate analysis established VM as an independent negative prognostic factor(P=0.002).Furthermore,GSLCs expressed high levels of pATM and formed vascular-like networks in vitro.ATM inactivation or knockdown hindered VM-like network formation concomitant with the downregulation of pVEGFR-2,VE-cadherin,and laminin B2.Conclusion VM may predict a poor GBM prognosis and is associated with pATM expression.We propose that pATM promotes VM through extracellular matrix modulation and VE-Cadherin/pVEGFR-2 activation,thereby highlighting ATM activation as a potential target for enhancing anti-angiogenesis therapies for GBM.
基金This work was supported by the National Natural Science Foundation of China(Grant Nos.81672743 and 81974464)Beijing Tianjin Hebei Basic Research Cooperation Project(Grant No.19JCZDJC64500(Z))+4 种基金Shenzhen Basic Research Project(Grant No.JCYJ20160331114230843)Tianjin Municipal Health Commission(Grant Nos.2015KR11 and 2013KG134)Tianjin Municipal Science and Technology Bureau(Grant No.18JCYBJC27800)US NIH grant RO 1 CAI33093,the Alabama Innovation Fund of the United Statesthe Tianjin Medical University Cancer Institute and Hospital Innovation Fund(Grant No.1803)。
文摘Objective:Mitotic arrest-deficient protein 1(MAD1)is a kinetochore protein essential for the mitotic spindle checkpoint.Proteomic studies have indicated that MAD1 is a component of the DNA damage response(DDR)pathway.However,whether and how MAD1 might be directly involved in the DDR is largely unknown.Methods:We ectopically expressed the wild type,or a phosphorylation-site--mutated form of MAD1 in MAD1 knockdown cells to look for complementation effects.We used the comet assay,colony formation assay,immunofluorescence staining,and flow cytometry to assess the DDR,radiosensitivity,and the G2/M checkpoint.We employed co-immunoprecipitation followed by mass spectrometry to identify MAD1 interacting proteins.Data were analyzed using the unpaired Student'st-test.Results:We showed that MAD1 was required for an optimal DDR,as knocking down MAD1 resulted in impaired DNA repair and hypersensitivity to ionizing radiation(IR).We found that IR-induced serine 214 phosphorylation was ataxia-telangiectasia mutated(ATM)kinase-dependent.Mutation of serine 214 to alanine failed to rescue the phenotypes of MAD1 knockdown cells in response to IR.Using mass spectrometry,we identified a protein complex mediated by MAD1 serine 214 phosphorylation in response to IR.Among them,we showed that KU80 was a key protein that displayed enhanced interaction with MAD1 after DNA damage.Finally,we showed that MAD1 interaction with KU80 required serine 214 phosphorylation,and it was essential for activation of DNA protein kinases catalytic subunit(DNA-PKcs).Conclusions:MAD1 serine 214 phosphorylation mediated by ATM kinase in response to IR was required for the interaction with KU80 and activation of DNA-PKCs.
基金support of the National Natural Science Foundation of China(Grant No.20872095)was gratefully acknowledged.
文摘Ataxia-telangiectasia mutated(ATM)plays a key role in regulating the cellular response to ionizing radiation.The tumor-suppressor gene ATM,mutations in which cause the human genetic disease ataxia telangiecta-sia,encodes a key protein kinase that controls the cellular response to double-stranded breaks.Activation of ATM results in phosphorylation of many downstream targets that modulate numerous damage response pathways,most notably cell cycle checkpoints.Here,we highlight some of the new developments in thefield in our understanding of the mechanism of activation of ATM and its signaling pathways,explore whether DNA double-strand breaks are the sole activators of ATM and ATM-dependent signaling pathways,and address some of the prominent,unanswered questions related to ATM and its function.The scope of this article is to provide a brief overview of the recent literature on this subject and to raise questions that could be addressed in future studies.
