Chemotherapy resistance plays a pivotal role in the prognosis and therapeutic failure of patients with colorectal cancer(CRC).Cisplatin(DDP)-resistant cells exhibit an inherent ability to evade the toxic chemotherapeu...Chemotherapy resistance plays a pivotal role in the prognosis and therapeutic failure of patients with colorectal cancer(CRC).Cisplatin(DDP)-resistant cells exhibit an inherent ability to evade the toxic chemotherapeutic drug effects which are characterized by the activation of slow-cycle programs and DNA repair.Among the elements that lead to DDP resistance,O^(6)-methylguanine(O^(6)-MG)-DNA-methyltransferase(MGMT),a DNA-repair enzyme,performs a quintessential role.In this study,we clarify the significant involvement of MGMT in conferring DDP resistance in CRC,elucidating the underlying mechanism of the regulatory actions of MGMT.A notable upregulation of MGMT in DDP-resistant cancer cells was found in our study,and MGMT repression amplifies the sensitivity of these cells to DDP treatment in vitro and in vivo.Conversely,in cancer cells,MGMT overexpression abolishes their sensitivity to DDP treatment.Mechanistically,the interaction between MGMT and cyclin dependent kinase 1(CDK1)inducing slow-cycling cells is attainted via the promotion of ubiquitination degradation of CDK1.Meanwhile,to achieve nonhomologous end joining,MGMT interacts with XRCC6 to resist chemotherapy drugs.Our transcriptome data from samples of 88 patients with CRC suggest that MGMT expression is co-related with the Wnt signaling pathway activation,and several Wnt inhibitors can repress drug-resistant cells.In summary,our results point out that MGMT is a potential therapeutic target and predictive marker of chemoresistance in CRC.展开更多
Nonhomologous DNA end joining （NHEJ） is the primary pathway for repair of double-strand DNA breaks in human cells and in multicellular eukaryotes. The causes of double-strand breaks often fragment the DNA at the sit...Nonhomologous DNA end joining （NHEJ） is the primary pathway for repair of double-strand DNA breaks in human cells and in multicellular eukaryotes. The causes of double-strand breaks often fragment the DNA at the site of damage, resulting in the loss of information there. NHEJ does not restore the lost information and may resect additional nucleotides during the repair process. The ability to repair a wide range of overhang and damage configurations reflects the flexibility of the nuclease, polymerases, and ligase of NHEJ. The flexibility of the individual components also explains the large number of ways in which NHEJ can repair any given pair of DNA ends. The loss of information locally at sites of NHEJ repair may contribute to cancer and aging, but the action by NHEJ ensures that entire segments of chromosomes are not lost.展开更多
Recent studies revealed the relationship among homologous recombination repair(HRR),androgen receptor(AR),and poly(adenosine diphosphate-ribose)polymerase(PARP);however,the synergy between anti-androgen enzalutamide(E...Recent studies revealed the relationship among homologous recombination repair(HRR),androgen receptor(AR),and poly(adenosine diphosphate-ribose)polymerase(PARP);however,the synergy between anti-androgen enzalutamide(ENZ)and PARP inhibitor olaparib(OLA)remains unclear.Here,we showed that the synergistic effect of ENZ and OLA significantly reduced proliferation and induced apoptosis in AR-positive prostate cancer cell lines.Next-generation sequencing followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed the significant effects of ENZ plus OLA on nonhomologous end joining(NHEJ)and apoptosis pathways.ENZ combined with OLA synergistically inhibited the NHEJ pathway by repressing DNA-dependent protein kinase catalytic subunit(DNA-PKcs)and X-ray repair cross complementing 4(XRCC4).Moreover,our data showed that ENZ could enhance the response of prostate cancer cells to the combination therapy by reversing the anti-apoptotic effect of OLA through the downregulation of anti-apoptotic gene insulin-like growth factor 1 receptor(IGF1R)and the upregulation of pro-apoptotic gene death-associated protein kinase 1(DAPK1).Collectively,our results suggested that ENZ combined with OLA can promote prostate cancer cell apoptosis by multiple pathways other than inducing HRR defects,providing evidence for the combined use of ENZ and OLA in prostate cancer regardless of HRR gene mutation status.展开更多
Human polymorphonuclear leukocytes (PMN) have been reported to completely lack of DNA-dependent protein kinase (DNA-PK) which is composed of Ku protein and the catalytic subunit DNA-PKcs, needed for nonhomologous end-...Human polymorphonuclear leukocytes (PMN) have been reported to completely lack of DNA-dependent protein kinase (DNA-PK) which is composed of Ku protein and the catalytic subunit DNA-PKcs, needed for nonhomologous end-joining (NHEJ) of DNA double-strand breaks. Promyelocytic HL-60 cells express a variant form of Ku resulting in enhanced radiation sensitivity. This raises the question if low efficiency of NHEJ, instrumental for the cellular repair of oxidative damage, is a normal characteristic of myeloid differentiation. Here we confirmed the complete lack of DNAPK in P MN protein extracts, and the expression of the truncated Ku86 variant form in HL-60. However, this degradation of DNA-PK was shown to be due to a DNA-PK-degrading protease in PMN and HL-60. In addition, by using a protease-resistant whole cell assay, both Ku86 and DNA-PKcs could be demonstrated in PMN, suggesting the previously reported absence in PMN of DNA-PK to be an artefact. The levels of Ku86 and DNA-PKcs were much reduced in PMN, as compared with that of the lymphocytes, whereas HL-60 displayed a markedly elevated DNA-PK concentration.In conclusion, our findings provide evidence of reduced, not depleted expression of DNA-PK during the mature stages of myeloid differentiation.展开更多
OBJECTIVE To detect the differences in gene expression of nonhomologous end-joining pathway including Ku70, Ku80, ERCC4, lig4 and DNA-PKcs between human primary gliomas and normal brain tissues, and furthermore, to ex...OBJECTIVE To detect the differences in gene expression of nonhomologous end-joining pathway including Ku70, Ku80, ERCC4, lig4 and DNA-PKcs between human primary gliomas and normal brain tissues, and furthermore, to explore the underlying mechanism for the expression alteration.METHODS The expression levels of Ku70, Ku80, ERCC4, lig4 and DNA-PKcs in 36 specimens of glioma and 12 specimens of normal brain tissue were measured using SYBR green-based real- time quantitative PCR. Methylation of DNA-PKcs was detected through methylation-specific PCR （MSP）.RESULTS There was no significant difference in expression of Ku70, Ku80, ERCC4 and lig4 between human primary gliomas and normal brain tissues （P 〈 0.05）, while DNA-PKcs were significantly up-regulated （P = 0.002）. The expression of DNA- PKcs was significantly higher in patients with grade III and IV diseases compared to patients with grade II disease or in normal brain tissues （P 〈 0.05）. Moreover, glioma tissue showed weaker methvlation than normal brain tissue.CONCLUSION The up-regulation of the DNA-PKcs may be associated with pathogenesis of glioma. Demethylation of DNA- PKcs promoter is an important reason for its up-regulation.展开更多
To improve the performance of yeast cell factories for industrial production,extensive CRISPR-mediated genome editing systems have been applied by artificially creating double-strand breaks(DSBs)to introduce mutations...To improve the performance of yeast cell factories for industrial production,extensive CRISPR-mediated genome editing systems have been applied by artificially creating double-strand breaks(DSBs)to introduce mutations with the assistance of intracellular DSB repair.Diverse strategies of DSB repair are required to meet various demands,including precise editing or random editing with customized gRNAs or a gRNA library.Although most yeasts remodeling techniques have shown rewarding performance in laboratory verification,industrial yeast strain manipulation relies only on very limited strategies.Here,we comprehensively reviewed the molecular mechanisms underlying recent industrial applications to provide new insights into DSB cleavage and repair pathways in both Saccharomyces cerevisiae and other unconventional yeast species.The discussion of DSB repair covers the most frequently used homologous recombination(HR)and nonhomologous end joining(NHEJ)strategies to the less well-studied illegitimate recombination(IR)pathways,such as single-strand annealing(SSA)and microhomology-mediated end joining(MMEJ).Various CRISPR-based genome editing tools and corresponding gene editing efficiencies are described.Finally,we summarize recently developed CRISPR-based strategies that use optimized DSB repair for genome-scale editing,providing a direction for further development of yeast genome editing.展开更多
基金supported by grants from the National Natural Science Foundation of China(Grant Nos.:82003807,82173394)the Shaanxi Province Science Foundation,China(Grant No.:2023-GHZD-19)+1 种基金the Medical Foundation-Clinical Integration Program of Xi'an Jiaotong University,China(Grant No.:YXJLRH2022043)the Xi'an Jiaotong University Free Exploration and Innovation-Teacher Project Foundation,China(Grant No.:xzy012023104).
文摘Chemotherapy resistance plays a pivotal role in the prognosis and therapeutic failure of patients with colorectal cancer(CRC).Cisplatin(DDP)-resistant cells exhibit an inherent ability to evade the toxic chemotherapeutic drug effects which are characterized by the activation of slow-cycle programs and DNA repair.Among the elements that lead to DDP resistance,O^(6)-methylguanine(O^(6)-MG)-DNA-methyltransferase(MGMT),a DNA-repair enzyme,performs a quintessential role.In this study,we clarify the significant involvement of MGMT in conferring DDP resistance in CRC,elucidating the underlying mechanism of the regulatory actions of MGMT.A notable upregulation of MGMT in DDP-resistant cancer cells was found in our study,and MGMT repression amplifies the sensitivity of these cells to DDP treatment in vitro and in vivo.Conversely,in cancer cells,MGMT overexpression abolishes their sensitivity to DDP treatment.Mechanistically,the interaction between MGMT and cyclin dependent kinase 1(CDK1)inducing slow-cycling cells is attainted via the promotion of ubiquitination degradation of CDK1.Meanwhile,to achieve nonhomologous end joining,MGMT interacts with XRCC6 to resist chemotherapy drugs.Our transcriptome data from samples of 88 patients with CRC suggest that MGMT expression is co-related with the Wnt signaling pathway activation,and several Wnt inhibitors can repress drug-resistant cells.In summary,our results point out that MGMT is a potential therapeutic target and predictive marker of chemoresistance in CRC.
文摘Nonhomologous DNA end joining （NHEJ） is the primary pathway for repair of double-strand DNA breaks in human cells and in multicellular eukaryotes. The causes of double-strand breaks often fragment the DNA at the site of damage, resulting in the loss of information there. NHEJ does not restore the lost information and may resect additional nucleotides during the repair process. The ability to repair a wide range of overhang and damage configurations reflects the flexibility of the nuclease, polymerases, and ligase of NHEJ. The flexibility of the individual components also explains the large number of ways in which NHEJ can repair any given pair of DNA ends. The loss of information locally at sites of NHEJ repair may contribute to cancer and aging, but the action by NHEJ ensures that entire segments of chromosomes are not lost.
基金This work was supported by the National Natural Science Foundation of China(82002718)the Jiangsu Natural Science Foundation(BK20191077)Jiangsu Province Hospital(the First Affiliated Hospital of Nanjing Medical University)Clinical Capacity Enhancement Project(JSPH-MC-2021-12).
文摘Recent studies revealed the relationship among homologous recombination repair(HRR),androgen receptor(AR),and poly(adenosine diphosphate-ribose)polymerase(PARP);however,the synergy between anti-androgen enzalutamide(ENZ)and PARP inhibitor olaparib(OLA)remains unclear.Here,we showed that the synergistic effect of ENZ and OLA significantly reduced proliferation and induced apoptosis in AR-positive prostate cancer cell lines.Next-generation sequencing followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed the significant effects of ENZ plus OLA on nonhomologous end joining(NHEJ)and apoptosis pathways.ENZ combined with OLA synergistically inhibited the NHEJ pathway by repressing DNA-dependent protein kinase catalytic subunit(DNA-PKcs)and X-ray repair cross complementing 4(XRCC4).Moreover,our data showed that ENZ could enhance the response of prostate cancer cells to the combination therapy by reversing the anti-apoptotic effect of OLA through the downregulation of anti-apoptotic gene insulin-like growth factor 1 receptor(IGF1R)and the upregulation of pro-apoptotic gene death-associated protein kinase 1(DAPK1).Collectively,our results suggested that ENZ combined with OLA can promote prostate cancer cell apoptosis by multiple pathways other than inducing HRR defects,providing evidence for the combined use of ENZ and OLA in prostate cancer regardless of HRR gene mutation status.
文摘Human polymorphonuclear leukocytes (PMN) have been reported to completely lack of DNA-dependent protein kinase (DNA-PK) which is composed of Ku protein and the catalytic subunit DNA-PKcs, needed for nonhomologous end-joining (NHEJ) of DNA double-strand breaks. Promyelocytic HL-60 cells express a variant form of Ku resulting in enhanced radiation sensitivity. This raises the question if low efficiency of NHEJ, instrumental for the cellular repair of oxidative damage, is a normal characteristic of myeloid differentiation. Here we confirmed the complete lack of DNAPK in P MN protein extracts, and the expression of the truncated Ku86 variant form in HL-60. However, this degradation of DNA-PK was shown to be due to a DNA-PK-degrading protease in PMN and HL-60. In addition, by using a protease-resistant whole cell assay, both Ku86 and DNA-PKcs could be demonstrated in PMN, suggesting the previously reported absence in PMN of DNA-PK to be an artefact. The levels of Ku86 and DNA-PKcs were much reduced in PMN, as compared with that of the lymphocytes, whereas HL-60 displayed a markedly elevated DNA-PK concentration.In conclusion, our findings provide evidence of reduced, not depleted expression of DNA-PK during the mature stages of myeloid differentiation.
文摘OBJECTIVE To detect the differences in gene expression of nonhomologous end-joining pathway including Ku70, Ku80, ERCC4, lig4 and DNA-PKcs between human primary gliomas and normal brain tissues, and furthermore, to explore the underlying mechanism for the expression alteration.METHODS The expression levels of Ku70, Ku80, ERCC4, lig4 and DNA-PKcs in 36 specimens of glioma and 12 specimens of normal brain tissue were measured using SYBR green-based real- time quantitative PCR. Methylation of DNA-PKcs was detected through methylation-specific PCR （MSP）.RESULTS There was no significant difference in expression of Ku70, Ku80, ERCC4 and lig4 between human primary gliomas and normal brain tissues （P 〈 0.05）, while DNA-PKcs were significantly up-regulated （P = 0.002）. The expression of DNA- PKcs was significantly higher in patients with grade III and IV diseases compared to patients with grade II disease or in normal brain tissues （P 〈 0.05）. Moreover, glioma tissue showed weaker methvlation than normal brain tissue.CONCLUSION The up-regulation of the DNA-PKcs may be associated with pathogenesis of glioma. Demethylation of DNA- PKcs promoter is an important reason for its up-regulation.
基金supported by the National Key Research and Development Program of China(No.2021YFC2101203)the General project of National Natural Science Foundation of China(No.22078021).
文摘To improve the performance of yeast cell factories for industrial production,extensive CRISPR-mediated genome editing systems have been applied by artificially creating double-strand breaks(DSBs)to introduce mutations with the assistance of intracellular DSB repair.Diverse strategies of DSB repair are required to meet various demands,including precise editing or random editing with customized gRNAs or a gRNA library.Although most yeasts remodeling techniques have shown rewarding performance in laboratory verification,industrial yeast strain manipulation relies only on very limited strategies.Here,we comprehensively reviewed the molecular mechanisms underlying recent industrial applications to provide new insights into DSB cleavage and repair pathways in both Saccharomyces cerevisiae and other unconventional yeast species.The discussion of DSB repair covers the most frequently used homologous recombination(HR)and nonhomologous end joining(NHEJ)strategies to the less well-studied illegitimate recombination(IR)pathways,such as single-strand annealing(SSA)and microhomology-mediated end joining(MMEJ).Various CRISPR-based genome editing tools and corresponding gene editing efficiencies are described.Finally,we summarize recently developed CRISPR-based strategies that use optimized DSB repair for genome-scale editing,providing a direction for further development of yeast genome editing.
文摘黑曲霉(Aspergillus niger)是一种重要的工业生产菌株,被广泛地应用于生产酶制剂和有机酸,但仍需要进行基因组改造提高它的应用潜力。CRISPR/Cas9技术是一种被广泛采用的黑曲霉基因组编辑技术,但由于需要在基因组中整合选择标记或基因编辑效率还有待提高,影响了其在工业菌株改造中的应用。本研究建立了一种基于CRISPR/Cas9技术的高效无选择标记的基因编辑方法。首先,利用5S rRNA启动子启动sgRNA的表达,构建了一个含有AMA1(autonomously maintained in Aspergillus)复制起始片段的sgRNA和Cas9共表达质粒;同时通过敲除kusA基因构建非同源末端连接(non-homologous end joining pathway,NHEJ)修复缺陷的高效同源重组菌株;最后利用含有AMA1片段质粒的不稳定性,通过无抗平板传代丢失含有sgRNA和Cas9共表达质粒。利用该方法,在采用同源臂长度仅为20 bp的无选择标记供体DNA进行基因编辑时,基因编辑效率可达到100%。该方法为黑曲霉基因功能的研究和细胞工厂的构建奠定了基础。
