Background:Apolipoprotein B mRNA editing catalytic polypeptide(APOBEC),an endogenous mutator,induces DNA damage and activates the ataxia telangiectasia and Rad3-related(ATR)-checkpoint kinase 1(Chk1)pathway.Although c...Background:Apolipoprotein B mRNA editing catalytic polypeptide(APOBEC),an endogenous mutator,induces DNA damage and activates the ataxia telangiectasia and Rad3-related(ATR)-checkpoint kinase 1(Chk1)pathway.Although cisplatin-based therapy is the mainstay for muscle-invasive bladder cancer(MIBC),it has a poor survival rate.Therefore,this study aimed to evaluate the efficacy of an ATR inhibitor combined with cisplatin in the treatment of APOBEC catalytic subunit 3B(APOBEC3B)expressing MIBC.Methods:Immunohistochemical staining was performed to analyze an association between APOBEC3B and ATR in patients with MIBC.The APOBEC3B expression in MIBC cell lines was assessed using real-time polymerase chain reaction and western blot analysis.Western blot analysis was performed to confirm differences in phosphorylated Chk1(pChk1)expression according to the APOBEC3B expression.Cell viability and apoptosis analyses were performed to examine the anti-tumor activity of ATR inhibitors combined with cisplatin.Results:There was a significant association between APOBEC3B and ATR expression in the tumor tissues obtained from patients with MIBC.Cells with higher APOBEC3B expression showed higher pChk1 expression than cells expressing low APOBEC3B levels.Combination treatment of ATR inhibitor and cisplatin inhibited cell growth in MIBC cells with a higher APOBEC3B expression.Compared to cisplatin single treatment,combination treatment induced more apoptotic cell death in the cells with higher APOBEC3B expression.Conclusion:Our study shows that APOBEC3B’s higher expression status can enhance the sensitivity of MIBC to cisplatin upon ATR inhibition.This result provides new insight into appropriate patient selection for the effective application of ATR inhibitors in MIBC.展开更多
目的探讨脑胶质瘤共济失调毛细血管扩张突变基因(ATM)及磷脂酰肌醇3-激酶(PI3K)的表达及其意义。方法采用反转录-聚合酶链反应技术检测40例脑胶质瘤组织(WHOⅠ~Ⅱ级25例,WHOⅢ~Ⅳ级15例)和8例非瘤脑组织ATM及PI3K m RNA的表达水...目的探讨脑胶质瘤共济失调毛细血管扩张突变基因(ATM)及磷脂酰肌醇3-激酶(PI3K)的表达及其意义。方法采用反转录-聚合酶链反应技术检测40例脑胶质瘤组织(WHOⅠ~Ⅱ级25例,WHOⅢ~Ⅳ级15例)和8例非瘤脑组织ATM及PI3K m RNA的表达水平。结果与非瘤脑组织相比,胶质瘤组织ATM和PI3K m RNA水平明显增高(P〈0.05),而且随着胶质瘤病理级别增高,ATM和PI3K m RNA水平亦明显增高(P〈0.05)。结论脑胶质瘤ATM、PI3K表达显著增高,且随着病理学分级的升高,其表达水平显著升高。这提示两者在胶质瘤发生、发展过程中扮演重要角色。展开更多
Pim kinases contribute to tumor formation and development of lymphoma,which shows enhanced DNA replication,DNA recombination and repair.Endothelial cells (ECs) express all the three members of Pim kinase gene family.W...Pim kinases contribute to tumor formation and development of lymphoma,which shows enhanced DNA replication,DNA recombination and repair.Endothelial cells (ECs) express all the three members of Pim kinase gene family.We hypothesized that DNA repair gene would regulate Pim ex-pression in ECs.Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium.The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining.The siRNA fragments were synthesized and transfected by using Lipofectamine LTX.The total cellular RNA was extracted from the cells by using Trizol reagent.cDNAs were quantified by semi-quantity PCR.The effects of LY294002 and wortmannin on RNA stability in ECs were also ex-amined.Our data showed that LY294002 and wortmannin,phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors,increased Pim mRNA expression in ECs without altering the mRNA stabil-ity.RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1,respectively.Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs.But etoposide,a nucleoside analogue,which could activate DNA-PKcs and ATM,increased Pim expression in ECs.Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.展开更多
AIM: To investigate the expression deficiency of key molecular markers in the homologous recombination pathway. METHODS: Expression loss of breast cancer type 1 susceptibility protein (BRCA1), ataxia telangiectasia mu...AIM: To investigate the expression deficiency of key molecular markers in the homologous recombination pathway. METHODS: Expression loss of breast cancer type 1 susceptibility protein (BRCA1), ataxia telangiectasia mutated (ATM), ATM-Rad3-related (ATR), mediator of DNA damage checkpoint protein 1 (MDC1) and meiotic recombination 11 (Mre11) were correlated with their clinicopathological parameters in gastric cancer (GC). One hundred and twenty treatment-naive GC samples were formalin-fixed and paraffin-embedded into tissue blocks. Two representative cores from each block were extracted and constructed into tissue microarrays. Expression levels of BRCA1, ATM, ATR, MDC1 and Mre11 were determined using immunohistochemical analysis, and correlated with clinical parameters, including age, gender, Lauren subtype, tumor grades, clinical stage and overall survival.RESULTS: Expression loss of BRCA1, ATM, ATR, MDC1, and Mre11 was found in 21.4%, 20.2%, 21.0%, 11.1% and 4.6%, respectively, of interpretable cases. BRCA1 loss was significantly associated with patients of diffused subtype (intestinal vs diffused, 8.2% vs 31.7%, P = 0.001), higher tumor grade (Ⅰ/Ⅱ vs Ⅲ, 10.7% vs 20.5;Ⅰ/Ⅱ vs Ⅳ, 10.7% vs 54.5%, P = 0.047) and advanced clinical stage (Ⅰ/Ⅱ vs Ⅲ, 12.9% vs 16.9%;Ⅰ /Ⅱ vs Ⅳ, 12.9% vs 45.5%, P = 0.006). MDC1 loss was significantly associated with patients of diffused subtype (intestinal vs diffused, 0% vs 19.7%, P = 0.001) and higher tumor grade (Ⅰ/Ⅱ vs Ⅲ, 0% vs 12%;Ⅰ/Ⅱ vs Ⅳ, 0% vs 30.8%, P = 0.012). In addition, the survival time of the patients with expression loss of BRCA1 was significantly shorter than those with positive expression of BRCA1 (2-year survival rate, 32.4% vs 62.8%, P = 0.015). No correlations were found between clinicopathological parameters and expression loss of ATM, ATR and Mre11. CONCLUSION: Our results support the hypothesis that homologous recombination deficiency plays an important role in the progression of gastric carcinoma. Loss of expression of BRCA1 and MDC1 may serve as predictive factors in tumor development or progression in GC patients.展开更多
基金supported by St.Vincent’s Hospital,the Research Institute of Medical Science(Grant Number:SVHR-2021-03).
文摘Background:Apolipoprotein B mRNA editing catalytic polypeptide(APOBEC),an endogenous mutator,induces DNA damage and activates the ataxia telangiectasia and Rad3-related(ATR)-checkpoint kinase 1(Chk1)pathway.Although cisplatin-based therapy is the mainstay for muscle-invasive bladder cancer(MIBC),it has a poor survival rate.Therefore,this study aimed to evaluate the efficacy of an ATR inhibitor combined with cisplatin in the treatment of APOBEC catalytic subunit 3B(APOBEC3B)expressing MIBC.Methods:Immunohistochemical staining was performed to analyze an association between APOBEC3B and ATR in patients with MIBC.The APOBEC3B expression in MIBC cell lines was assessed using real-time polymerase chain reaction and western blot analysis.Western blot analysis was performed to confirm differences in phosphorylated Chk1(pChk1)expression according to the APOBEC3B expression.Cell viability and apoptosis analyses were performed to examine the anti-tumor activity of ATR inhibitors combined with cisplatin.Results:There was a significant association between APOBEC3B and ATR expression in the tumor tissues obtained from patients with MIBC.Cells with higher APOBEC3B expression showed higher pChk1 expression than cells expressing low APOBEC3B levels.Combination treatment of ATR inhibitor and cisplatin inhibited cell growth in MIBC cells with a higher APOBEC3B expression.Compared to cisplatin single treatment,combination treatment induced more apoptotic cell death in the cells with higher APOBEC3B expression.Conclusion:Our study shows that APOBEC3B’s higher expression status can enhance the sensitivity of MIBC to cisplatin upon ATR inhibition.This result provides new insight into appropriate patient selection for the effective application of ATR inhibitors in MIBC.
文摘目的探讨脑胶质瘤共济失调毛细血管扩张突变基因(ATM)及磷脂酰肌醇3-激酶(PI3K)的表达及其意义。方法采用反转录-聚合酶链反应技术检测40例脑胶质瘤组织(WHOⅠ~Ⅱ级25例,WHOⅢ~Ⅳ级15例)和8例非瘤脑组织ATM及PI3K m RNA的表达水平。结果与非瘤脑组织相比,胶质瘤组织ATM和PI3K m RNA水平明显增高(P〈0.05),而且随着胶质瘤病理级别增高,ATM和PI3K m RNA水平亦明显增高(P〈0.05)。结论脑胶质瘤ATM、PI3K表达显著增高,且随着病理学分级的升高,其表达水平显著升高。这提示两者在胶质瘤发生、发展过程中扮演重要角色。
基金supported by grants from the National Natural Science Foundation of China (No. 30900631)Hubei Provincial Department of Education (No. Q20102101 andD20082406)
文摘Pim kinases contribute to tumor formation and development of lymphoma,which shows enhanced DNA replication,DNA recombination and repair.Endothelial cells (ECs) express all the three members of Pim kinase gene family.We hypothesized that DNA repair gene would regulate Pim ex-pression in ECs.Human umbilical vein endothelial cells (HUVECs) were isolated and maintained in M199 culture medium.The cellular distribution of Pim-3 in ECs was determined by immunofluorescent staining.The siRNA fragments were synthesized and transfected by using Lipofectamine LTX.The total cellular RNA was extracted from the cells by using Trizol reagent.cDNAs were quantified by semi-quantity PCR.The effects of LY294002 and wortmannin on RNA stability in ECs were also ex-amined.Our data showed that LY294002 and wortmannin,phosphatidylinositol 3-kinase (PI3K) and PI3K-like kinase inhibitors,increased Pim mRNA expression in ECs without altering the mRNA stabil-ity.RNA interference (RNAi) targeting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) increased mRNA expression of Pim-3 and Pim-1,respectively.Silencing of Akt decreased Pim-1 instead of Pm-2 and Pim-3 gene expression in ECs.But etoposide,a nucleoside analogue,which could activate DNA-PKcs and ATM,increased Pim expression in ECs.Our study indicates that the expression of Pim kinases is physiologically related to DNA-PKcs and ATM in ECs.
文摘AIM: To investigate the expression deficiency of key molecular markers in the homologous recombination pathway. METHODS: Expression loss of breast cancer type 1 susceptibility protein (BRCA1), ataxia telangiectasia mutated (ATM), ATM-Rad3-related (ATR), mediator of DNA damage checkpoint protein 1 (MDC1) and meiotic recombination 11 (Mre11) were correlated with their clinicopathological parameters in gastric cancer (GC). One hundred and twenty treatment-naive GC samples were formalin-fixed and paraffin-embedded into tissue blocks. Two representative cores from each block were extracted and constructed into tissue microarrays. Expression levels of BRCA1, ATM, ATR, MDC1 and Mre11 were determined using immunohistochemical analysis, and correlated with clinical parameters, including age, gender, Lauren subtype, tumor grades, clinical stage and overall survival.RESULTS: Expression loss of BRCA1, ATM, ATR, MDC1, and Mre11 was found in 21.4%, 20.2%, 21.0%, 11.1% and 4.6%, respectively, of interpretable cases. BRCA1 loss was significantly associated with patients of diffused subtype (intestinal vs diffused, 8.2% vs 31.7%, P = 0.001), higher tumor grade (Ⅰ/Ⅱ vs Ⅲ, 10.7% vs 20.5;Ⅰ/Ⅱ vs Ⅳ, 10.7% vs 54.5%, P = 0.047) and advanced clinical stage (Ⅰ/Ⅱ vs Ⅲ, 12.9% vs 16.9%;Ⅰ /Ⅱ vs Ⅳ, 12.9% vs 45.5%, P = 0.006). MDC1 loss was significantly associated with patients of diffused subtype (intestinal vs diffused, 0% vs 19.7%, P = 0.001) and higher tumor grade (Ⅰ/Ⅱ vs Ⅲ, 0% vs 12%;Ⅰ/Ⅱ vs Ⅳ, 0% vs 30.8%, P = 0.012). In addition, the survival time of the patients with expression loss of BRCA1 was significantly shorter than those with positive expression of BRCA1 (2-year survival rate, 32.4% vs 62.8%, P = 0.015). No correlations were found between clinicopathological parameters and expression loss of ATM, ATR and Mre11. CONCLUSION: Our results support the hypothesis that homologous recombination deficiency plays an important role in the progression of gastric carcinoma. Loss of expression of BRCA1 and MDC1 may serve as predictive factors in tumor development or progression in GC patients.