Objective:To evaluate the predictive values of gene expressions of ribonucleotide reductase M1(RRM1) and breast cancer susceptibility gene 1(BRCA1) in peripheral blood from Chinese patients with non-small-cell lung ca...Objective:To evaluate the predictive values of gene expressions of ribonucleotide reductase M1(RRM1) and breast cancer susceptibility gene 1(BRCA1) in peripheral blood from Chinese patients with non-small-cell lung cancer(NSCLC) treated with gemcitabine plus platinum.Methods:Forty Chinese patients with advanced NSCLC were recruited and received gemcitabine 1200 mg/m 2 on Days 1 and 8 plus carboplatin AUC 5 on Day 1.RRM1 and BRCA1 expression levels in peripheral blood were detected by quantitative reverse transcription-polymerase chain reaction(RT-PCR) .Kaplan-Meier survival curve and log-rank test were performed to evaluate the correlation between gene expression and overall survival for these subjects.Results:No correlation was observed between gene expression of RRM1 and that of BRCA1(P>0.05) ,but there was a strong correlation between the expression of RRM1 and the response to chemotherapy(P=0.003) .Subjects with low RRM1 expression levels in peripheral blood had longer sur-vival time than those with high RRM1 expression levels(16.95 vs.12.76 months,log-rank 3.989,P=0.046) .However,no significant association between BRCA1 expression levels and survival time was found(16.80 vs.13.77 months,log-rank 0.830,P=0.362) .Conclusions:Patients with low RRM1 expression levels in peripheral blood have a greater response to chemotherapy and longer survival time.Advanced NSCLC patients with low RRM1 expression levels may benefit from gemcitabine plus platinum therapy.RRM1 mRNA expression in peripheral blood could be used to predict the prognosis of NSCLC treated by gemcitabine and platinum.展开更多
Ribonucleotide reductase(RNR), the rate-limitingenzyme in DNA synthesis, catalyzes reduction of thedifferent ribonucleotides to their corresponding deoxyri-bonucleotides. The crucial role of RNR in DNA synthesishas ma...Ribonucleotide reductase(RNR), the rate-limitingenzyme in DNA synthesis, catalyzes reduction of thedifferent ribonucleotides to their corresponding deoxyri-bonucleotides. The crucial role of RNR in DNA synthesishas made it an important target for the development ofantiviral and anticancer drugs. Taking account of the re-cent developments in this field of research, this reviewfocuses on the role of thioredoxin and glutaredoxin sys-tems in the redox reactions of the RNR catalysis.展开更多
To investigate the dynamics of plasma cAMP/cGMP in patients during cardiac surgery, and its relationship to traumatic stress. Methods: Sixteen patients, aged 19.31 years± 10.4 years , who underwent an open heart ...To investigate the dynamics of plasma cAMP/cGMP in patients during cardiac surgery, and its relationship to traumatic stress. Methods: Sixteen patients, aged 19.31 years± 10.4 years , who underwent an open heart operation with cardiopulmonary bypass (CPB) and hy pother mia were served as subjects. The arterial plasma concentrations of cyclic adenos ine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were measured by radioimmunoassay 2 hours before operation, after heparinization, 20 minutes following CPB, at the end of the operation, and 24 and 72 hours postoperatively, respectiv ely. The patients preoperative blood samples were heparinized and the venous b lood samples of 30 healthy blood donors were taken to measure the levels of cAMP and cGMP as heparin and normal controls separately. Results: There were no statistical difference among the heparin control, preoperative level and normal control. The peak values of cAMP and cGM P occurred during CPB and plasma cAMP levels changed synchronously with intensit ies of operative stimulus to human body. However cGMP level was mainly related t o the operative stimulus to the heart and CPB. The cAMP value was positivel y correlated with the cGMP value (r= 0.6313 , P< 0.001 ).Conclusions: Dynamic variation of plasma cyclic ribonucleotide can be considered as a reference parameter for intensity of traumatic stress.展开更多
AIM:To investigate the roles of the ribonucleotide reductase M2 (RRM2) subunit in colorectal cancer (CRC) and ultraviolet (UV)-induced DNA damage repair. METHODS:Immunohistochemical staining of tissue microarray was p...AIM:To investigate the roles of the ribonucleotide reductase M2 (RRM2) subunit in colorectal cancer (CRC) and ultraviolet (UV)-induced DNA damage repair. METHODS:Immunohistochemical staining of tissue microarray was performed to detect the expression of RRM2. Seven CRC cell lines were cultured and three human colon cancer cell lines, i.e., HCT116, SW480 and SW620, were used. Reverse transcription polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels of RRM2, respectively. Cell proliferation assay, cell cycle analysis were performed. Cell apoptosis was evaluated by double staining with fluorescein isothiocyanate-conjugated Annexin Ⅴ and propidium iodide (PI) usingAnnexin Ⅴ/PI apoptosis kit. The motility and invasion of CRC cells were assessed by the Transwell chamber assay. Cells were irradiated with a 254 nm UV-C lamp to detect the UV sensitivity after RRM2 depletion. RESULTS:Immunohistochemical staining revealed elevated RRM2 levels in CRC tissues. RRM2 overexpression was positively correlated with invasion depth (P < 0.05), poorly differentiated type (P = 0.0051), and tumor node metastasis stage (P = 0.0015). The expression of RRM2 in HCT116 cells was downregulated after transfection, and HCT116 cell proliferation was obviously suppressed compared to control groups (P < 0.05). In the invasion test, the number of cells that passed through the chambers in the RRM2-siRNA group was 81 ± 3, which was lower than that in the negative control (289 ± 7) and blank control groups (301 ± 7.2). These differences were statistically significant (P < 0.01). Our data suggest that RRM2 overexpression may be associated with CRC progression. RRM2 silencing by siRNA may inhibit the hyperplasia and invasiveness of CRC cells, suggesting that RRM2 may play an important role in the infiltration and metastasis of CRC, which is a potential therapeutic strategy in CRC. In addition, RRM2 depletion increased UV sensitivity. CONCLUSION:These findings suggest that RRM2 may be 展开更多
Ribonucleotide rcductase （RNR） supplies cellular deoxyribonucleotidc triphosphates （dNTP） pools by converting ribonucleotides to the corresponding deoxy forms using radical-based chemistry. Eukaryotic RNR comprise...Ribonucleotide rcductase （RNR） supplies cellular deoxyribonucleotidc triphosphates （dNTP） pools by converting ribonucleotides to the corresponding deoxy forms using radical-based chemistry. Eukaryotic RNR comprises a and β subunits： u contains the catalytic and ailosteric sites; β houses a diferric-tyrosyl radical cofactor （FeⅢ2-Y· ） that is required to initiates nucleotide reduction in α. Cells have evolved multi-layered mechanisms to regulate RNR level and activity in order to maintain the adequate sizes and ratios of their dNTP pools to ensure high- fidelity DNA replication and repair. The central role of RNR in nucleotide metabolism also makes it a proven target of chemotherapeutics. In this review, we discuss recent progress in understanding the function and regulation of eukaryofic RNRs, with a focus on studies revealing the cellular machineries involved in RNR metaUocofactor biosynthesis and its implication in RNR-targeting therapeutics.展开更多
Aim:Gemcitabine is a frontline agent for locally-advanced and metastatic pancreatic ductal adenocarcinoma(PDAC),but neither gemcitabine alone nor in combination produces durable remissions of this tumor type.We develo...Aim:Gemcitabine is a frontline agent for locally-advanced and metastatic pancreatic ductal adenocarcinoma(PDAC),but neither gemcitabine alone nor in combination produces durable remissions of this tumor type.We developed three PDAC patient-derived xenograft(PDX)models with gemcitabine resistance(gemR)acquired in vivo,with which to identify mechanisms of resistance relevant to drug exposure in vivo and to evaluate novel therapies.Methods:Mice bearing independently-derived PDXs received 100 mg/kg gemcitabine once or twice weekly.Tumors initially responded,but regrew on treatment and were designated gemR.We used immunohistochemistry to compare expression of proteins previously associated with gemcitabine resistance[ribonucleotide reductase subunit M1(RRM1),RRM2,human concentrative nucleoside transporter 1(hCNT1),human equilibrative nucleoside transporter 1(hENT1),cytidine deaminase(CDA),and deoxycytidine kinase(dCK)]in gemR and respective gemcitabine-naïve parental tumors.Results:Parental and gemR tumors did not differ in tumor cell morphology,amount of tumor-associated stroma,or expression of stem cell markers.No consistent pattern of expression of the six gemR marker proteins was observed among the models.Increases in RRM1 and CDA were consistent with in vitro-derived gemR models.However,rather than the expected decreases of hCNT1,hENT1,and dCK,gemR tumors expressed no change in or higher levels of these gemR marker proteins than parental tumors.Conclusion:These models are the first PDAC PDX models with gemcitabine resistance acquired in vivo.The data indicate that mechanisms identified in models with resistance acquired in vitro are unlikely to be the predominant mechanisms when resistance is acquired in vivo.Ongoing work focuses on characterizing unidentified mechanisms of gemR and on identifying agents with anti-tumor efficacy in these gemR models。展开更多
Background The formation and growth of tumors are related to the synthesis of the DNA. The enzyme ribonucleotide reductase （RR） is an enzyme that regulates the total rate of DNA synthesis and thus plays a pivotal ro...Background The formation and growth of tumors are related to the synthesis of the DNA. The enzyme ribonucleotide reductase （RR） is an enzyme that regulates the total rate of DNA synthesis and thus plays a pivotal role in cell growth. Catalytic subunit M2 （RRM2） is the main unit modulating the ribonucleotide reductase enzymatic activity. This study aimed to investigate the expression of RRM2 mRNA and protein in patients with ovarian cancer and its relevance to diagnosis and clinical outcome of the patients. Methods RRM2 mRNA levels and protein expression were detected in 98 ovarian specimens with immunohistochemistry and real-time quantitative polymerase chain reaction （PCR）. Expression of the RRM2 protein and correlation of the RRM2 gene expression with clinical pathological features were analyzed. The Kaplan-Meier test was used for evaluating RRM2 expression and time to progression and survival. The Cox proportional model was used to analyze the risk factors in prognosis of patients. Results Positive RRM2 immunostaining was found in 43 of 62 （69.4%） patients with epithelial ovarian cancer, 10 of 15 （66.7%） patients with borderline neoplasm, 4 of 15 （26.7%） patients with benign growths, and none of the normal group. The RRM2 mRNA levels were significantly over expressed in epithelial ovarian cancer （1.722＋0.639） and borderline ovarian neoplasms （1.365＋0.615）, compared to the normal group （0.678＋0.446） and benign group （0.828_＋0.545）. Patients with ovarian caner in clinical FIGO-stages Ill-IV presented higher RRM2 gene expression than those in clinical FIGO-stages I-I1. Furthermore, the survival of patients with low RRM2 mRNA level was significantly better than patients with high levels （P 〈0.05）. By Cox proportional risk model analysis, the risk of mortality of patients with high level expression of RRM2 mRNA was 2.553 times greater than those with low expression. Conclusion RRM2 expression closely correlates with the development of ovarian tumor and may serve展开更多
Background: Gap junctions enable small molecules to diffuse between adjacent cells and have been associated with greater cytotoxicity of radiation and anti-cancer drugs. We investigated?whether this gap junctional int...Background: Gap junctions enable small molecules to diffuse between adjacent cells and have been associated with greater cytotoxicity of radiation and anti-cancer drugs. We investigated?whether this gap junctional intercellular communication (GJIC) affected the cytotoxicity of the classic?ribonucleotide reductase (RR) inhibitor and anti-cancer agent, hydroxyurea (HU). Materials and Methods: We used GJIC-proficient and deficient, connexin 43-expressing WB rat liver epithelial cell lines. We compared HU toxicity by crystal violet assay, effects of the drug on deoxynucleotide pools by HPLC, and ability of GJIC to increase toxicity of HU-resistant cells through a bystander effect in co-culture experiments. Results: GJIC-proficient cells were three- to five-fold more sensitive (IC50?0.1 mM) to HU than GJIC-deficient derivatives (IC50?0.3 - 0.5 mM). This sensitivity depended upon GJIC because treatment of GJIC-proficient cells with the GJIC blocker oleamide decreased HU toxicity by approximately 60% - 80% and restoration of GJIC in GJIC-deficient cells by stable transduction of connexin 32-encoding?Gjb1?increased HU toxicity (IC500.1 mM). The effects were not due to connexin expression?per se?or its localization since all cell lines expressed comparable quantities of connexin 43 that was localized to the plasma membrane. Also HU sensitivity was not related to differential effects on nucleotide metabolism in the cells. Thymidine triphosphate levels increased and deoxyadenosine triphosphate levels decreased similarly (15% - 20%) in GJIC-proficient and deficient cells over 24 h of HU treatment. More importantly, when HU-resistant cells were co-cultured with sensitive cells, the resistant cells were killed only when GJIC?was present. Conclusion: The data suggest that GJIC enhances cytotoxicity and decreases resistance?to HU. These results may be important clinically if GJIC can be enhanced in drug-resistant cells.展开更多
Twenty patients with chronic hepatitis c were investigated during the treatment with interferon to explore the changes of antibodies to HCV (anti-RCV). Anti-HCV was tested with recombinant immunoblot assay (RIBA) by u...Twenty patients with chronic hepatitis c were investigated during the treatment with interferon to explore the changes of antibodies to HCV (anti-RCV). Anti-HCV was tested with recombinant immunoblot assay (RIBA) by using three antigens (C22, C33c, and C100-3) encoded by different regions of HCV genome. The changes of individual anti-HCV and ALT were compared with the change of HCV RNA. The results showed that persistent disappearance of serum HCV RNA was closely related to the changes of anti-C33c (P<0. 01) and anti-C100-3 (P<0. 005), but there was no relation between persistent ALT normality and HCV viremia clearance (P<0. 05). In conclusion, monitoring anti-C33c and anti-C100-3 could indicate the changes or HCV viremia. The normalization of ALT after interferon treatment did not indicate disappearance of HCV viremia.展开更多
Fluorodeoxyglucose positron emission tomography/conlputed tomography （FDG PET/CT） is widely applied in non-small cell lung cancer （NSCLC）. The standardized uptake value （SUV）, a semi-quantitative index, plays an...Fluorodeoxyglucose positron emission tomography/conlputed tomography （FDG PET/CT） is widely applied in non-small cell lung cancer （NSCLC）. The standardized uptake value （SUV）, a semi-quantitative index, plays an essential role in NSCLC tbr diagnosis, staging, and efficacy evaklation. It has been px3posed that the SUV of tumors may correlate with the presence or absence of chemotherapy resistance-associated biomarkers based on studies that have displayed a close correlation between SUV and the expression levels of excision repair cross-complementary Group 1 （ERCC 1 ）1~1 and Tp53-induced glycolysis and apoptosis regulator.121 FDG avidity of NSCLC and ERCC 1 and ribonucleotide reductase subunit M 1 （RRM 1 ） levels have not been as extensively investigated. Based on these findings, we looked tbr correlations among metabolic parameters （SUVm,,. metabolic tumor volume [MTV], and total lesion glycolysis [TLG]） and ERCC1 and RRM1 expression in patients with NSCLC, to investigate whether FDG uptake reflects the presence or absence ofchemoresistance proteins （ERCC1 and RRM 1 ） within tumor cells.展开更多
As virulence-determining genes, RR1 and RR2 encode the small subunit and large subunit of viral ribonucleotide reductase(RR) in pseudorabies virus which have been extensively studied in mice. However,their role in pig...As virulence-determining genes, RR1 and RR2 encode the small subunit and large subunit of viral ribonucleotide reductase(RR) in pseudorabies virus which have been extensively studied in mice. However,their role in pigs has not been adequately investigated. In this study, we deleted RR1 and RR2 genes based on a TK/g E/g I triple gene-deleted pseudorabies virus and tested its efficacy in pigs as a vaccine candidate. The rescued virus showed similar growth properties and plaque size in vitro as its parent strain. In an animal study, the virus could elicit humoral immune responses shown by generation of g B-specific antibodies and virus neutralizing antibodies.However, vaccination could not provide protection against virulent pseudorabies virus challenge since vaccinated pigs showed clinical pseudorabies-specific syndromes. The deficiency in protection may due to the generation of late and low levels of gB antibodies and virus neutralizing antibodies.展开更多
RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1 (HSV-1) RR is composed ...RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1 (HSV-1) RR is composed of two distinct homodimeric subunits encoded by UL39 and UL40,respectively. In this study,we applied siRNAs targeting the UL39 and UL40 genes of HSV-1. We showed that synthetic siRNA silenced effectively and specifically UL39 and UL40 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.展开更多
Cordycepin was the first adenosine analogue used as an anticancer and antiviral agent, which is extracted from Cordyceps militaris and hasn’t been biosynthesized until now. This study was first conducted to verify th...Cordycepin was the first adenosine analogue used as an anticancer and antiviral agent, which is extracted from Cordyceps militaris and hasn’t been biosynthesized until now. This study was first conducted to verify the role of ribonucleotide reductases(RNRs, the two RNR subunits, RNRL and RNRM) in the biosynthesis of cordycepin by over expressing RNRs genes in transformed C. militaris. Quantitative real-time PCR(qRT-PCR) and western blotting results showed that the m RNA and protein levels of RNR subunit genes were significantly upregulated in transformant C. militaris strains compared to the control strain. The results of the HPLC assay indicated that the cordycepin was significantly higher in the C. militaris transformants carrying RNRM than in the wildtype strain, whereas the RNRML was preferentially downregulated. For the C. militaris transformant carrying RNRL, the content of cordycepin wasn’t remarkably changed. Furthermore, we revealed that inhibiting RNRs with Triapine(3-AP) almost abrogated the upregulation of cordycepin. Therefore, our results suggested that RNRM can probably directly participate in cordycepin biosynthesis by hydrolyzing adenosine, which is useful for improving cordycepin synthesis and helps to satisfy the commercial demand of cordycepin in the field of medicine.展开更多
BACKGROUND Ribonucleotide reductase(RR)is a key enzyme in tumor proliferation,especially its subunit-RRM2.Although there are multiple therapeutics for tumors,they all have certain limitations.Given their advantages,tr...BACKGROUND Ribonucleotide reductase(RR)is a key enzyme in tumor proliferation,especially its subunit-RRM2.Although there are multiple therapeutics for tumors,they all have certain limitations.Given their advantages,traditional Chinese medicine(TCM)monomers have become an important source of anti-tumor drugs.Therefore,screening and analysis of TCM monomers with RRM2 inhibition can provide a reference for further anti-tumor drug development.AIM To screen and analyze potential anti-tumor TCM monomers with a good binding capacity to RRM2.METHODS The Gene Expression Profiling Interactive Analysis database was used to analyze the level of RRM2 gene expression in normal and tumor tissues as well as RRM2's effect on the overall survival rate of tumor patients.TCM monomers that potentially act on RRM2 were screened via literature mining.Using AutoDock software,the screened monomers were docked with the RRM2 protein.RESULTS The expression of RRM2 mRNA in multiple tumor tissues was significantly higher than that in normal tissues,and it was negatively correlated with the overall survival rate of patients with the majority of tumor types.Through literature mining,we discovered that berberine,ursolic acid,gambogic acid,cinobufagin,quercetin,daphnetin,and osalmide have inhibitory effects on RRM2.The results of molecular docking identified that the above TCM monomers have a strong binding capacity with RRM2 protein,which mainly interacted through hydrogen bonds and hydrophobic force.The main binding sites were Arg330,Tyr323,Ser263,and Met350.CONCLUSION RRM2 is an important tumor therapeutic target.The TCM monomers screened have a good binding capacity with the RRM2 protein.展开更多
Metalloproteins have inspired chemists for many years to synthesize artificial catalysts that mimic native enzymes.As a complementary approach to studying native enzymes or making synthetic models,biosynthetic approac...Metalloproteins have inspired chemists for many years to synthesize artificial catalysts that mimic native enzymes.As a complementary approach to studying native enzymes or making synthetic models,biosynthetic approach using small and stable proteins to model native enzymes has offered advantages of incorporating non-covalent secondary sphere interactions under physiological conditions.However,most biosynthetic models are restricted to natural amino acids.To overcome this limitation,incorporating unnatural amino acids into the biosynthetic models has shown promises.In this review,we summarize first synthetic,semisynthetic and biological methods of incorporates unnatural amino acids(UAAs)into proteins,followed by progress made in incorporating UAAs into both native metalloproteins and their biosynthetic models to fine-tune functional properties beyond native enzymes or their variants containing natural amino acids,such as reduction potentials of azurin,O_2 reduction rates and percentages of product formation of HCO models in Mb,the rate of radical transport in ribonucleotide reductase(RNR)and the proton and electron transfer pathways in photosystemⅡ(PSⅡ).We also discuss how this endeavour has allowed systematic investigations of precise roles of conserved residues in metalloproteins,such as Metl21 in azurin,Tyr244 that is cross-linked to one of the three His ligands to CuB in HCO,Tyr122,356,730 and 731 in RNR and TyrZ in PSⅡ.These examples have demonstrated that incorporating UAAs has provided a new dimension in our efforts to mimic native enzymes and in providing deeper insights into structural features responsible high enzymatic activity and reaction mechanisms,making it possible to design highly efficient artificial catalysts with similar or even higher activity than native enzymes.展开更多
Objective： The aim of this study was to observe the efficacy of gemcitabine combined with cisplatin （GP） in advanced non-smaU cell lung cancer （NSCLC） patients with low expression of ribonucleotide reductase 1 （...Objective： The aim of this study was to observe the efficacy of gemcitabine combined with cisplatin （GP） in advanced non-smaU cell lung cancer （NSCLC） patients with low expression of ribonucleotide reductase 1 （RRM1） protein using immunohistochemistry. Methods： RRM1 protein expression in tumor tissue was detected by streptavidin-peroxidase （SP） method of immunohistochemistry. GP regimen （gemcitabine 1000-1250 mg d1, d8, cisplatin 75 mg/m2） was given to advanced NSCLC patients with low expression of RRM1 protein. Results： In the total of 40 patients, these patients with RRM1 low expression performing GP chemotherapy had a good response rate, the objective response rate （ORR） was 47.5% （95% CI, 32.02%- 62.98%）, and the disease control rate （DCR） was 72.5% （95 % CI, 65.44%-79.56%）. ORR is 45.45% （5/11） in the squamous cell carcinoma patients while 48.15% （13/27） in the adenocarcinoma patients. Conclusion： Supedor ORR and DCR were found in advanced NSCLC patients with low expression of RRM1 protein expression performing GP regimen.展开更多
基金Project supported by the Science and Technology Planning Project of Zhejiang Province(No.2009C33165)the Medical Science Research Fund Scheme of Zhejiang Province(No.2008A060),China
文摘Objective:To evaluate the predictive values of gene expressions of ribonucleotide reductase M1(RRM1) and breast cancer susceptibility gene 1(BRCA1) in peripheral blood from Chinese patients with non-small-cell lung cancer(NSCLC) treated with gemcitabine plus platinum.Methods:Forty Chinese patients with advanced NSCLC were recruited and received gemcitabine 1200 mg/m 2 on Days 1 and 8 plus carboplatin AUC 5 on Day 1.RRM1 and BRCA1 expression levels in peripheral blood were detected by quantitative reverse transcription-polymerase chain reaction(RT-PCR) .Kaplan-Meier survival curve and log-rank test were performed to evaluate the correlation between gene expression and overall survival for these subjects.Results:No correlation was observed between gene expression of RRM1 and that of BRCA1(P>0.05) ,but there was a strong correlation between the expression of RRM1 and the response to chemotherapy(P=0.003) .Subjects with low RRM1 expression levels in peripheral blood had longer sur-vival time than those with high RRM1 expression levels(16.95 vs.12.76 months,log-rank 3.989,P=0.046) .However,no significant association between BRCA1 expression levels and survival time was found(16.80 vs.13.77 months,log-rank 0.830,P=0.362) .Conclusions:Patients with low RRM1 expression levels in peripheral blood have a greater response to chemotherapy and longer survival time.Advanced NSCLC patients with low RRM1 expression levels may benefit from gemcitabine plus platinum therapy.RRM1 mRNA expression in peripheral blood could be used to predict the prognosis of NSCLC treated by gemcitabine and platinum.
基金Supported by The Swedish Research Council Medicine,No.3529The Swedish Cancer Society,No.961The Wallenberg Foundation
文摘Ribonucleotide reductase(RNR), the rate-limitingenzyme in DNA synthesis, catalyzes reduction of thedifferent ribonucleotides to their corresponding deoxyri-bonucleotides. The crucial role of RNR in DNA synthesishas made it an important target for the development ofantiviral and anticancer drugs. Taking account of the re-cent developments in this field of research, this reviewfocuses on the role of thioredoxin and glutaredoxin sys-tems in the redox reactions of the RNR catalysis.
文摘To investigate the dynamics of plasma cAMP/cGMP in patients during cardiac surgery, and its relationship to traumatic stress. Methods: Sixteen patients, aged 19.31 years± 10.4 years , who underwent an open heart operation with cardiopulmonary bypass (CPB) and hy pother mia were served as subjects. The arterial plasma concentrations of cyclic adenos ine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were measured by radioimmunoassay 2 hours before operation, after heparinization, 20 minutes following CPB, at the end of the operation, and 24 and 72 hours postoperatively, respectiv ely. The patients preoperative blood samples were heparinized and the venous b lood samples of 30 healthy blood donors were taken to measure the levels of cAMP and cGMP as heparin and normal controls separately. Results: There were no statistical difference among the heparin control, preoperative level and normal control. The peak values of cAMP and cGM P occurred during CPB and plasma cAMP levels changed synchronously with intensit ies of operative stimulus to human body. However cGMP level was mainly related t o the operative stimulus to the heart and CPB. The cAMP value was positivel y correlated with the cGMP value (r= 0.6313 , P< 0.001 ).Conclusions: Dynamic variation of plasma cyclic ribonucleotide can be considered as a reference parameter for intensity of traumatic stress.
文摘AIM:To investigate the roles of the ribonucleotide reductase M2 (RRM2) subunit in colorectal cancer (CRC) and ultraviolet (UV)-induced DNA damage repair. METHODS:Immunohistochemical staining of tissue microarray was performed to detect the expression of RRM2. Seven CRC cell lines were cultured and three human colon cancer cell lines, i.e., HCT116, SW480 and SW620, were used. Reverse transcription polymerase chain reaction and Western blotting were performed to determine the mRNA and protein expression levels of RRM2, respectively. Cell proliferation assay, cell cycle analysis were performed. Cell apoptosis was evaluated by double staining with fluorescein isothiocyanate-conjugated Annexin Ⅴ and propidium iodide (PI) usingAnnexin Ⅴ/PI apoptosis kit. The motility and invasion of CRC cells were assessed by the Transwell chamber assay. Cells were irradiated with a 254 nm UV-C lamp to detect the UV sensitivity after RRM2 depletion. RESULTS:Immunohistochemical staining revealed elevated RRM2 levels in CRC tissues. RRM2 overexpression was positively correlated with invasion depth (P < 0.05), poorly differentiated type (P = 0.0051), and tumor node metastasis stage (P = 0.0015). The expression of RRM2 in HCT116 cells was downregulated after transfection, and HCT116 cell proliferation was obviously suppressed compared to control groups (P < 0.05). In the invasion test, the number of cells that passed through the chambers in the RRM2-siRNA group was 81 ± 3, which was lower than that in the negative control (289 ± 7) and blank control groups (301 ± 7.2). These differences were statistically significant (P < 0.01). Our data suggest that RRM2 overexpression may be associated with CRC progression. RRM2 silencing by siRNA may inhibit the hyperplasia and invasiveness of CRC cells, suggesting that RRM2 may play an important role in the infiltration and metastasis of CRC, which is a potential therapeutic strategy in CRC. In addition, RRM2 depletion increased UV sensitivity. CONCLUSION:These findings suggest that RRM2 may be
文摘Ribonucleotide rcductase （RNR） supplies cellular deoxyribonucleotidc triphosphates （dNTP） pools by converting ribonucleotides to the corresponding deoxy forms using radical-based chemistry. Eukaryotic RNR comprises a and β subunits： u contains the catalytic and ailosteric sites; β houses a diferric-tyrosyl radical cofactor （FeⅢ2-Y· ） that is required to initiates nucleotide reduction in α. Cells have evolved multi-layered mechanisms to regulate RNR level and activity in order to maintain the adequate sizes and ratios of their dNTP pools to ensure high- fidelity DNA replication and repair. The central role of RNR in nucleotide metabolism also makes it a proven target of chemotherapeutics. In this review, we discuss recent progress in understanding the function and regulation of eukaryofic RNRs, with a focus on studies revealing the cellular machineries involved in RNR metaUocofactor biosynthesis and its implication in RNR-targeting therapeutics.
文摘Aim:Gemcitabine is a frontline agent for locally-advanced and metastatic pancreatic ductal adenocarcinoma(PDAC),but neither gemcitabine alone nor in combination produces durable remissions of this tumor type.We developed three PDAC patient-derived xenograft(PDX)models with gemcitabine resistance(gemR)acquired in vivo,with which to identify mechanisms of resistance relevant to drug exposure in vivo and to evaluate novel therapies.Methods:Mice bearing independently-derived PDXs received 100 mg/kg gemcitabine once or twice weekly.Tumors initially responded,but regrew on treatment and were designated gemR.We used immunohistochemistry to compare expression of proteins previously associated with gemcitabine resistance[ribonucleotide reductase subunit M1(RRM1),RRM2,human concentrative nucleoside transporter 1(hCNT1),human equilibrative nucleoside transporter 1(hENT1),cytidine deaminase(CDA),and deoxycytidine kinase(dCK)]in gemR and respective gemcitabine-naïve parental tumors.Results:Parental and gemR tumors did not differ in tumor cell morphology,amount of tumor-associated stroma,or expression of stem cell markers.No consistent pattern of expression of the six gemR marker proteins was observed among the models.Increases in RRM1 and CDA were consistent with in vitro-derived gemR models.However,rather than the expected decreases of hCNT1,hENT1,and dCK,gemR tumors expressed no change in or higher levels of these gemR marker proteins than parental tumors.Conclusion:These models are the first PDAC PDX models with gemcitabine resistance acquired in vivo.The data indicate that mechanisms identified in models with resistance acquired in vitro are unlikely to be the predominant mechanisms when resistance is acquired in vivo.Ongoing work focuses on characterizing unidentified mechanisms of gemR and on identifying agents with anti-tumor efficacy in these gemR models。
文摘Background The formation and growth of tumors are related to the synthesis of the DNA. The enzyme ribonucleotide reductase （RR） is an enzyme that regulates the total rate of DNA synthesis and thus plays a pivotal role in cell growth. Catalytic subunit M2 （RRM2） is the main unit modulating the ribonucleotide reductase enzymatic activity. This study aimed to investigate the expression of RRM2 mRNA and protein in patients with ovarian cancer and its relevance to diagnosis and clinical outcome of the patients. Methods RRM2 mRNA levels and protein expression were detected in 98 ovarian specimens with immunohistochemistry and real-time quantitative polymerase chain reaction （PCR）. Expression of the RRM2 protein and correlation of the RRM2 gene expression with clinical pathological features were analyzed. The Kaplan-Meier test was used for evaluating RRM2 expression and time to progression and survival. The Cox proportional model was used to analyze the risk factors in prognosis of patients. Results Positive RRM2 immunostaining was found in 43 of 62 （69.4%） patients with epithelial ovarian cancer, 10 of 15 （66.7%） patients with borderline neoplasm, 4 of 15 （26.7%） patients with benign growths, and none of the normal group. The RRM2 mRNA levels were significantly over expressed in epithelial ovarian cancer （1.722＋0.639） and borderline ovarian neoplasms （1.365＋0.615）, compared to the normal group （0.678＋0.446） and benign group （0.828_＋0.545）. Patients with ovarian caner in clinical FIGO-stages Ill-IV presented higher RRM2 gene expression than those in clinical FIGO-stages I-I1. Furthermore, the survival of patients with low RRM2 mRNA level was significantly better than patients with high levels （P 〈0.05）. By Cox proportional risk model analysis, the risk of mortality of patients with high level expression of RRM2 mRNA was 2.553 times greater than those with low expression. Conclusion RRM2 expression closely correlates with the development of ovarian tumor and may serve
文摘Background: Gap junctions enable small molecules to diffuse between adjacent cells and have been associated with greater cytotoxicity of radiation and anti-cancer drugs. We investigated?whether this gap junctional intercellular communication (GJIC) affected the cytotoxicity of the classic?ribonucleotide reductase (RR) inhibitor and anti-cancer agent, hydroxyurea (HU). Materials and Methods: We used GJIC-proficient and deficient, connexin 43-expressing WB rat liver epithelial cell lines. We compared HU toxicity by crystal violet assay, effects of the drug on deoxynucleotide pools by HPLC, and ability of GJIC to increase toxicity of HU-resistant cells through a bystander effect in co-culture experiments. Results: GJIC-proficient cells were three- to five-fold more sensitive (IC50?0.1 mM) to HU than GJIC-deficient derivatives (IC50?0.3 - 0.5 mM). This sensitivity depended upon GJIC because treatment of GJIC-proficient cells with the GJIC blocker oleamide decreased HU toxicity by approximately 60% - 80% and restoration of GJIC in GJIC-deficient cells by stable transduction of connexin 32-encoding?Gjb1?increased HU toxicity (IC500.1 mM). The effects were not due to connexin expression?per se?or its localization since all cell lines expressed comparable quantities of connexin 43 that was localized to the plasma membrane. Also HU sensitivity was not related to differential effects on nucleotide metabolism in the cells. Thymidine triphosphate levels increased and deoxyadenosine triphosphate levels decreased similarly (15% - 20%) in GJIC-proficient and deficient cells over 24 h of HU treatment. More importantly, when HU-resistant cells were co-cultured with sensitive cells, the resistant cells were killed only when GJIC?was present. Conclusion: The data suggest that GJIC enhances cytotoxicity and decreases resistance?to HU. These results may be important clinically if GJIC can be enhanced in drug-resistant cells.
文摘Twenty patients with chronic hepatitis c were investigated during the treatment with interferon to explore the changes of antibodies to HCV (anti-RCV). Anti-HCV was tested with recombinant immunoblot assay (RIBA) by using three antigens (C22, C33c, and C100-3) encoded by different regions of HCV genome. The changes of individual anti-HCV and ALT were compared with the change of HCV RNA. The results showed that persistent disappearance of serum HCV RNA was closely related to the changes of anti-C33c (P<0. 01) and anti-C100-3 (P<0. 005), but there was no relation between persistent ALT normality and HCV viremia clearance (P<0. 05). In conclusion, monitoring anti-C33c and anti-C100-3 could indicate the changes or HCV viremia. The normalization of ALT after interferon treatment did not indicate disappearance of HCV viremia.
文摘Fluorodeoxyglucose positron emission tomography/conlputed tomography （FDG PET/CT） is widely applied in non-small cell lung cancer （NSCLC）. The standardized uptake value （SUV）, a semi-quantitative index, plays an essential role in NSCLC tbr diagnosis, staging, and efficacy evaklation. It has been px3posed that the SUV of tumors may correlate with the presence or absence of chemotherapy resistance-associated biomarkers based on studies that have displayed a close correlation between SUV and the expression levels of excision repair cross-complementary Group 1 （ERCC 1 ）1~1 and Tp53-induced glycolysis and apoptosis regulator.121 FDG avidity of NSCLC and ERCC 1 and ribonucleotide reductase subunit M 1 （RRM 1 ） levels have not been as extensively investigated. Based on these findings, we looked tbr correlations among metabolic parameters （SUVm,,. metabolic tumor volume [MTV], and total lesion glycolysis [TLG]） and ERCC1 and RRM1 expression in patients with NSCLC, to investigate whether FDG uptake reflects the presence or absence ofchemoresistance proteins （ERCC1 and RRM 1 ） within tumor cells.
基金supported by grant from Major Science and Technology Program in Henan Province (131100110200)Innovation Scientists and Technicians Troop Construction Projects of Henan Province (142101510001)+1 种基金Talents Plan for Scientific and Technological Innovation in Henan Province (144200510002)Science and Technology Innovation team in Henan Province (C20130005)
文摘As virulence-determining genes, RR1 and RR2 encode the small subunit and large subunit of viral ribonucleotide reductase(RR) in pseudorabies virus which have been extensively studied in mice. However,their role in pigs has not been adequately investigated. In this study, we deleted RR1 and RR2 genes based on a TK/g E/g I triple gene-deleted pseudorabies virus and tested its efficacy in pigs as a vaccine candidate. The rescued virus showed similar growth properties and plaque size in vitro as its parent strain. In an animal study, the virus could elicit humoral immune responses shown by generation of g B-specific antibodies and virus neutralizing antibodies.However, vaccination could not provide protection against virulent pseudorabies virus challenge since vaccinated pigs showed clinical pseudorabies-specific syndromes. The deficiency in protection may due to the generation of late and low levels of gB antibodies and virus neutralizing antibodies.
基金The Nation "863" Program of China(2006AA02A226)The Joint Funds of National Science Foundation of China (U0632010)+2 种基金The State KeyLaboratory of Phytochemistry and Plant Resources in West ChinaChinese Academy of Sciences (O807B11211, O807E21211)"211 grant of MOE"
文摘RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1 (HSV-1) RR is composed of two distinct homodimeric subunits encoded by UL39 and UL40,respectively. In this study,we applied siRNAs targeting the UL39 and UL40 genes of HSV-1. We showed that synthetic siRNA silenced effectively and specifically UL39 and UL40 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.
基金the Natural Sciences Foundation of China Science (Nos.81872959, 81373920,30801522)Sichuan Province Youth Innovation Team Fund (No.19CXTD0055)China Scholarship Fund (No. 201708570027)。
文摘Cordycepin was the first adenosine analogue used as an anticancer and antiviral agent, which is extracted from Cordyceps militaris and hasn’t been biosynthesized until now. This study was first conducted to verify the role of ribonucleotide reductases(RNRs, the two RNR subunits, RNRL and RNRM) in the biosynthesis of cordycepin by over expressing RNRs genes in transformed C. militaris. Quantitative real-time PCR(qRT-PCR) and western blotting results showed that the m RNA and protein levels of RNR subunit genes were significantly upregulated in transformant C. militaris strains compared to the control strain. The results of the HPLC assay indicated that the cordycepin was significantly higher in the C. militaris transformants carrying RNRM than in the wildtype strain, whereas the RNRML was preferentially downregulated. For the C. militaris transformant carrying RNRL, the content of cordycepin wasn’t remarkably changed. Furthermore, we revealed that inhibiting RNRs with Triapine(3-AP) almost abrogated the upregulation of cordycepin. Therefore, our results suggested that RNRM can probably directly participate in cordycepin biosynthesis by hydrolyzing adenosine, which is useful for improving cordycepin synthesis and helps to satisfy the commercial demand of cordycepin in the field of medicine.
基金Supported by Nanchong City School’s Science and Technology Strategic Cooperation,China,No.20SXQT0304Research and Development Project Plan of Affiliated Hospital of North Sichuan Medical College,China,No.2020ZD003.
文摘BACKGROUND Ribonucleotide reductase(RR)is a key enzyme in tumor proliferation,especially its subunit-RRM2.Although there are multiple therapeutics for tumors,they all have certain limitations.Given their advantages,traditional Chinese medicine(TCM)monomers have become an important source of anti-tumor drugs.Therefore,screening and analysis of TCM monomers with RRM2 inhibition can provide a reference for further anti-tumor drug development.AIM To screen and analyze potential anti-tumor TCM monomers with a good binding capacity to RRM2.METHODS The Gene Expression Profiling Interactive Analysis database was used to analyze the level of RRM2 gene expression in normal and tumor tissues as well as RRM2's effect on the overall survival rate of tumor patients.TCM monomers that potentially act on RRM2 were screened via literature mining.Using AutoDock software,the screened monomers were docked with the RRM2 protein.RESULTS The expression of RRM2 mRNA in multiple tumor tissues was significantly higher than that in normal tissues,and it was negatively correlated with the overall survival rate of patients with the majority of tumor types.Through literature mining,we discovered that berberine,ursolic acid,gambogic acid,cinobufagin,quercetin,daphnetin,and osalmide have inhibitory effects on RRM2.The results of molecular docking identified that the above TCM monomers have a strong binding capacity with RRM2 protein,which mainly interacted through hydrogen bonds and hydrophobic force.The main binding sites were Arg330,Tyr323,Ser263,and Met350.CONCLUSION RRM2 is an important tumor therapeutic target.The TCM monomers screened have a good binding capacity with the RRM2 protein.
基金supported by the US National Science Foundation(CHE-1413328)the Major State Basic Research Program of China(2015CB856203)+1 种基金the National Natural Science Foundation of China (21325211,31500641)theTianjinMunicipalGrant(13ZCZDSY04800, 14ZCZDSY00059,14JCYBJC43400)
文摘Metalloproteins have inspired chemists for many years to synthesize artificial catalysts that mimic native enzymes.As a complementary approach to studying native enzymes or making synthetic models,biosynthetic approach using small and stable proteins to model native enzymes has offered advantages of incorporating non-covalent secondary sphere interactions under physiological conditions.However,most biosynthetic models are restricted to natural amino acids.To overcome this limitation,incorporating unnatural amino acids into the biosynthetic models has shown promises.In this review,we summarize first synthetic,semisynthetic and biological methods of incorporates unnatural amino acids(UAAs)into proteins,followed by progress made in incorporating UAAs into both native metalloproteins and their biosynthetic models to fine-tune functional properties beyond native enzymes or their variants containing natural amino acids,such as reduction potentials of azurin,O_2 reduction rates and percentages of product formation of HCO models in Mb,the rate of radical transport in ribonucleotide reductase(RNR)and the proton and electron transfer pathways in photosystemⅡ(PSⅡ).We also discuss how this endeavour has allowed systematic investigations of precise roles of conserved residues in metalloproteins,such as Metl21 in azurin,Tyr244 that is cross-linked to one of the three His ligands to CuB in HCO,Tyr122,356,730 and 731 in RNR and TyrZ in PSⅡ.These examples have demonstrated that incorporating UAAs has provided a new dimension in our efforts to mimic native enzymes and in providing deeper insights into structural features responsible high enzymatic activity and reaction mechanisms,making it possible to design highly efficient artificial catalysts with similar or even higher activity than native enzymes.
文摘Objective： The aim of this study was to observe the efficacy of gemcitabine combined with cisplatin （GP） in advanced non-smaU cell lung cancer （NSCLC） patients with low expression of ribonucleotide reductase 1 （RRM1） protein using immunohistochemistry. Methods： RRM1 protein expression in tumor tissue was detected by streptavidin-peroxidase （SP） method of immunohistochemistry. GP regimen （gemcitabine 1000-1250 mg d1, d8, cisplatin 75 mg/m2） was given to advanced NSCLC patients with low expression of RRM1 protein. Results： In the total of 40 patients, these patients with RRM1 low expression performing GP chemotherapy had a good response rate, the objective response rate （ORR） was 47.5% （95% CI, 32.02%- 62.98%）, and the disease control rate （DCR） was 72.5% （95 % CI, 65.44%-79.56%）. ORR is 45.45% （5/11） in the squamous cell carcinoma patients while 48.15% （13/27） in the adenocarcinoma patients. Conclusion： Supedor ORR and DCR were found in advanced NSCLC patients with low expression of RRM1 protein expression performing GP regimen.
