Thymidine-containing derivatives are considered to be among the most significant derivatives in medicinal chemistry. In this study, we employed a combined computational approach involving density-functional theory (DF...Thymidine-containing derivatives are considered to be among the most significant derivatives in medicinal chemistry. In this study, we employed a combined computational approach involving density-functional theory (DFT) calculations, molecular docking simulations, and absorption, distribution, metabolism, excretion, and toxicity (ADMET) property predictions. Prediction of activity spectra for substances (PASS) revealed promising antiviral, antimicrobial and anti-carcinogenic activities of these thymidine derivatives. Using Gaussian 09, we optimized the molecular structures of the thymidine derivatives to obtain their stable conformations and calculate their electronic properties. Subsequently, molecular docking simulations were performed to explore the binding interactions between the thymidine derivatives and the active site of the Candida albicans (PDB: 1IYL and 2Y7L) proteins. The docking results were evaluated based on docking scores, hydrogen bonding, and hydrophobic interactions and revealed favorable binding interactions between the thymidine derivatives and the proteins, suggesting their potential as antifungal agents. The thermodynamic properties, including binding free energy, enthalpy, and entropy changes were determined to assess the stability and strength of the ligands-protein complexes. The calculated pharmacokinetic parameters, such as ADMET properties, provided insights into the drug-likeness and potential bioavailability of the thymidine derivatives. These results offer a foundation for further experimental investigations and the design of novel antifungal agents targeting Candida albicans infections.展开更多
The mRNA and protein expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) and their relationship with prognosis were investigated. Real-time quantitative RT-P...The mRNA and protein expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) and their relationship with prognosis were investigated. Real-time quantitative RT-PCR (Taqman) was used to detect the mRNA expression of TS, TP and DPD in formalin-fixed and paraffin-embedded 106 samples of epithelial ovarian cancer and 29 normal ovaries. A TATA box-binding protein (TBP) was used as an endogenous reference gene. A relationship between TS, TP, DPD expression and clinicopathologic features was investigated. The protein location and expression of TS, TP and DPD was examined in the same patients by an avidin-biotin-peroxidase immunohistochemistry. TS and TP mRNA expression levels were significantly higher in tumor group than in normal controls, with the average value of TS and TP mRNA being 6.14±0.62 and 0.59±0.06 in tumor tissue, and 0.71±0.14 and 0.16±0.04 in normal tissue, respectively. DPD mRNA expression levels were significantly lower in tumor group (0.11±0.02) than in normal controls (0.38±0.05). There was statistically significant difference in TS and TP mRNA expression levels among different pathological grades and clinical stages (P<0.05), but histological subtype was not significantly associated with TS and TP mRNA expression. DPD gene expression was not significantly associated with any clinicopathological parameters. Immunohistochemistry revealed that TP protein was mainly distributed in nucleus, and TS and DPD mainly in cytoplasm. The protein expression intensity of TS, TP and DPD was coincided with the mRNA expression levels. It was concluded that TS, TP mRNA and protein expression levels were significantly higher in epithelial ovarian cancer, and DPD mRNA and protein expression levels were significantly lower. The expression levels of TS and DPD were related to the patients' prognosis and survival. Combined gene expression levels of TS, TP and DPD represent a new variable to predict the clinical outcome in ovarian cancer. The association of TS, TP and DPD expression levels with survival suggests an importance of these genes for tumor occurrence and progression.展开更多
AIM: To evaluate the role of thymidine phosphorylase (TP) in cholangiocarcinoma using small interfering RNA (siRNA). METHODS: A human cholangiocarcinoma-derived cell line KKU-M139, which has a naturally high level of ...AIM: To evaluate the role of thymidine phosphorylase (TP) in cholangiocarcinoma using small interfering RNA (siRNA). METHODS: A human cholangiocarcinoma-derived cell line KKU-M139, which has a naturally high level of endogenous TP, had TP expression transiently knocked down using siRNA. Cell growth, migration, in vitro angiogenesis, apoptosis, and cytotoxicity were assayed in TP knockdown and wild-type cell lines. RESULTS: TP mRNA and protein expression were decreased by 87.1% ± 0.49% and 72.5% ± 3.2%, respectively, compared with control cells. Inhibition of TP significantly decreased migration of KKU-M139, and suppressed migration and tube formation of human umbilical vein endothelial cells. siRNA also reduced the ability of TP to resist hypoxia-induced apoptosis, while suppression of TP reduced the sensitivity of KKU-M139 to 5-fluorouracil. CONCLUSION: Inhibition of TP may be beneficial in decreasing angiogenesis-dependent growth and migration of cholangiocarcinoma but may diminish the response to 5-fluorouracil chemotherapy.展开更多
Thymidine glycol (5,6-dihydroxy-5,6-dihydrothymidine, Tg) is a major type of oxidative damage in DNA. During chemical oligonucleotide synthesis, Tg residue was incorporated in the different positions of 17 b.p. DNA du...Thymidine glycol (5,6-dihydroxy-5,6-dihydrothymidine, Tg) is a major type of oxidative damage in DNA. During chemical oligonucleotide synthesis, Tg residue was incorporated in the different positions of 17 b.p. DNA duplexes, which differ in one base pair in the internal part. According to UV-melting curves, Tg destabilizes the double helix in a sequence independent manner. In contrast, the localized alterations in duplex structure were shown by CD spectroscopy to depend on the type of base pairs flanking the Tg lesion. Molecular dynamics simulations demonstrate that Tg is partially out of the double helix. For the first time, Tg impact on several site-specific DNA-binding proteins is studied, namely p50 and p65 subunits of nuclear factor kappa-B (NF-κB) and DNA methyltransferase SsoII (M.SsoII). Our results show that p50/p50 and p65/p65 homodimers of NF-κB can tolerate a single Tg residue in the binding site quite well. Nevertheless the homodimers have different affinities to the oxidized κB site depending on the Tg position. M.SsoII can act as a transcription repressor when bound to the regulatory site. M.SsoII demonstrates decreased affinity and lowered methylation efficiency when its methylation site contains Tg in the central position. Single Tg in one half of the regulatory site decreases M.SsoII affinity to the oxidized DNA, whereas Tg presence in both half-sites prevents M.SsoII binding to such ligand.展开更多
Hand-foot syndrome(HFS)is a widely recognized dose-limiting cutaneous toxicity effect of fluoropyrimidine chemotherapy agents that impairs clinical benefits and treatment outcomes.Even though the cause and pathophysio...Hand-foot syndrome(HFS)is a widely recognized dose-limiting cutaneous toxicity effect of fluoropyrimidine chemotherapy agents that impairs clinical benefits and treatment outcomes.Even though the cause and pathophysiology of HFS are relatively widely reported,how the toxicity of fluoropyrimidine translates into persistent inflammation has not been studied.Additionally,prevention and treatment strategies for HFS based on its mechanistic occurrence and development are scarce.In our study,we demonstrated that cGAS-STING signaling pathway-mediated cellular senescence played a critical role in the inflammatory reaction and provided a therapeutic solution for HFS.Mechanistically,DNA damage,as the primary cytotoxic cause,in keratinocytes induces cell cycle arrest,activates the cGAS-STING signaling pathway,and subsequently mediates cellular senescence,ultimately fueling a robust secondary inflammatory response that results in HFS.More importantly,the thymidine prodrug thymidine diacetate was proven to be effective in preventing HFS by compensating for thymidylate deficiency to facilitate the replication and repair of DNA and thus causing the escape from cellular senescence.These data highlight the importance of DNA damage-mediated cellular senescence in the etiology of HFS and provide a potential therapeutic anchor point for fluoropyrimidine-induced HFS.展开更多
Aim:Thynidine phosphorylase(TP)acts as a proangiogenic growth factor which may regulate mammalian Target of Rapamycin(mTOR).We investigated whether the TP substrate thymidine and overexpression of TP affected mTOR sig...Aim:Thynidine phosphorylase(TP)acts as a proangiogenic growth factor which may regulate mammalian Target of Rapamycin(mTOR).We investigated whether the TP substrate thymidine and overexpression of TP affected mTOR signaling by comparing Colo320(TP deficient)cells and its TP-transfected variant(Colo320TP1).Methods:Drug resistance was assessed with the sulforhodamine B assay,protein expression with Western blotting,cell cycle distribution and cell death with Fluorescence-activated cell sorting analysis,and autophagy with immunofluorescence.Results:Colo320 and Colo320TP1 cells had comparable levels of sensitivity to the mTOR inhibitor rapamycin.Thymidine treatment led to 13-and 50-fold resistance to rapamycin in Colo320 and Colo320TP1 cells,respectively.In Colo320TP1 cells,the thymidine phosphorylase inhibitor(TPI)reversed the thymidine induced resistance to rapamycin,but not in Colo320 cells,indicating a role for TP in the protection.Thymidine increased p70/S6k-phosphorylation(downstream of mTOR)in Colo320TP1,but it was not affected in Colo320.As a mechanism behind resistance,we studied the levels of autophagy and found that,in Colo320TP1 cells,autophagy was highly induced by thymidine-rapamycin,which was decreased by TPI.In addition,the autophagy inhibitor 3-methyl-adenine completely inhibited autophagy and its protection.Conclusion:Rapamycin resistance in TP-expressing cancer cells may therefore be related to thymidine-mediated autophagy activation.展开更多
To explore correlation between the tk gene structure of pseudorabies virus (PRV) and its virulence, to study the effect of the gene mutation on PRV biological properties, and to investigate mechinism of reduced virule...To explore correlation between the tk gene structure of pseudorabies virus (PRV) and its virulence, to study the effect of the gene mutation on PRV biological properties, and to investigate mechinism of reduced virulence, thymidine kinase (TK)-deficient mutant of pseudorabies virus strain Hubei (PRV HB) was isolated by selection for resistance to 5-bromodeoxyuridine. The tk genes of PRV HB and its TK mutant were cloned and sequenced. 1587 base pairs of the tk gene and flanking regions of wild-type (wt) virus were sequenced, which included an open reading frame (ORF) of 1098 bp encoding a protein of 366 amino acids. The ORF contained two 137-bp repeated sequences, which were connected by an adenosine. 1458 bp of the tk and flanking regions of TK- mutant were sequenced. Analysis of the tk gene sequence of TK mutant indicated that one of 137 bp repeated sequence and the connecting adenosine in the tk gene of the wt virus was deleted and a repeated sequence of 8 nucleotides (GCGCGCC) was inserted. All展开更多
The human cytosolic thymidine kinase (TK-C)gene has been localized on chromosome 17 (17q21--22). Morbid anatomy of the human genome has shown that seve?al disease genes are located in the same chromosome region. Our g...The human cytosolic thymidine kinase (TK-C)gene has been localized on chromosome 17 (17q21--22). Morbid anatomy of the human genome has shown that seve?al disease genes are located in the same chromosome region. Our goal is to search for the restriction site polymorphism in the DNA sequence of the human TK-C gene.展开更多
Neutrophils are derived from bone marrow hematopoietic stem cells(HSCs)and are the largest population among circulating white blood cells in humans,acting as the first line of defense against invading pathogens.Whethe...Neutrophils are derived from bone marrow hematopoietic stem cells(HSCs)and are the largest population among circulating white blood cells in humans,acting as the first line of defense against invading pathogens.Whether neutrophils can be generated by transdifferentiation strategies is unknown.Here,we show that thymidine induces the conversion of mouse fibroblasts to neutrophils.Induced neutrophils(iNeus)showed antibacterial effects and did not undergo malignant transformation in vivo.Importantly,iNeu transplantation cured neutropenia in mice in vivo.Mechanistically,thymidine mediates iNeu conversion by enhancing Tet3 activity.Tet3 initiates the expression of the neutrophil fate decision factors Cebpδ and Rfx1 that drive the transdifferentiation of mouse fibroblasts to neutrophils.Therefore,the induction of functional neutrophils by chemicals may provide a potential therapeutic strategy for patients with neutropenia patients and infectious diseases.展开更多
Viral-mediated gene transfer of thymidine kinase ofherpes simplex virus (HSV-tk) has been used to confercytotoxic sensitivity to ganciclovir (GCV) in a variety oftnmor cells. HSV-tk converts GCV into a phosphorylatedc...Viral-mediated gene transfer of thymidine kinase ofherpes simplex virus (HSV-tk) has been used to confercytotoxic sensitivity to ganciclovir (GCV) in a variety oftnmor cells. HSV-tk converts GCV into a phosphorylatedcompound which is toxic for dividing cells by blockingDNA synthesis. Our previous study has shown展开更多
Connexin-43 (Cx43) expression in prostate cancer (PCa) cells and the potency of gap junctional intercellular communication (GJIC) in the cells were investigated, with an attempt to elu- cidate the reason why the so-ca...Connexin-43 (Cx43) expression in prostate cancer (PCa) cells and the potency of gap junctional intercellular communication (GJIC) in the cells were investigated, with an attempt to elu- cidate the reason why the so-called "bystander effect" mediated by thymidine kinase (TK) suicide gene therapy on PCa cells is not of significance and to explore the role of GJIC in PCa carcinogenesis. mRNA and protein expression of Cx43 in a PCa cell line PC-3m was detected by re- verse-transcription polymerase chain reaction (RT-PCR) and strapt-avidin-biotin-enzyme complex (SABC) immunohistochemical staining, and inherent GJIC of PC-3m cells was assayed by scrape-loading and dye transfer (SLDT) assay. The expression of Cx43 in human normal and malig- nant prostate tissues was determined by SABC immunohistochemistry as well. It was found that Cx43 mRNA and protein expression in PC-3m cells was slightly reduced as compared with positive controls and the location of Cx43 protein was aberrant in cytoplasm rather than on membrane. As- sessment of paraffin sections demonstrated that the expression of Cx43 protein in PCa cells was ab- normally located and markedly diminished as compared with normal prostatic epithelial ones, dis- playing a negative correlation to the pathological grade (χ2=4.025, P<0.05). Additionally, capacity of inherent GJIC in PC-3m cells was disrupted, which was semi-quantified as (+) or (-). It was indi- cated that both down-regulated expression of Cx43 mRNA and aberrant location of Cx43 protein par- ticipated in the mechanisms leading to deficient GJIC in PC-3m cells. Lack of efficient GJIC is a molecular event, which may contribute not only to limited extent of "bystander effect", but also to initiation and progression of prostatic neoplasm.展开更多
Thymidylate synthase(TS)is a key enzyme in the de novo biosynthesis of thymidine monophosphate,serving as a well-known drug target in chemotherapy against cancers and infectious diseases.Additional to its clinical val...Thymidylate synthase(TS)is a key enzyme in the de novo biosynthesis of thymidine monophosphate,serving as a well-known drug target in chemotherapy against cancers and infectious diseases.Additional to its clinical value,TS is supposed to be a promising drug target in aquatic-disease control.To facilitate designing pathogen-specific TS inhibitors for shrimp-disease control,we report the crystal structures of TS from Litopenaeus vannamei(LvTS)in the apo form,LvTS-dUMP complex and LvTS-dUMP-raltitrexed complex at 2.27Å,1.54Å,and 1.56Åresolution,respectively.LvTS shares a similar fold with known TSs,existing as a dimer in the crystal.The apo LvTS and LvTS-dUMP take an open conformation,and raltitrexed binding induces structural changes into a closed conformation in LvTS-dUMP-raltitrexed.Compared to those in other known TS-dUMP-raltitrexed complexes with the closed conformation,the C-terminal loop in LvTS-dUMP-raltitrexed shifts its position away from the bound raltitrexed;the distance between C6 of dUMP and Sγof the catalytic cysteine is obviously longer than that in the known TS structures with closed conformations,resembling that in the TS structures with open conformations.Other species-specific interactions with dUMP and raltitrexed are also observed.Therefore,LvTS-dUMP-raltitrexed adopts a loosely closed conformation with structural features intermediate between the closed and the open conformations that were reported in other TSs.Our study provides the first crustcean TS structure,and reveals species-specific interactions between TSs and the ligands,which would facilitate designing pathogen-specific TS inhibitors for shrimp-disease control.展开更多
A chemosensitivity test for ovarian cancer using tritiated thymidine incorporation assay was carried out. A dose-response relationship for cisplatin and potentiation of verapamil in increasing vincristine inhibition t...A chemosensitivity test for ovarian cancer using tritiated thymidine incorporation assay was carried out. A dose-response relationship for cisplatin and potentiation of verapamil in increasing vincristine inhibition to ovarian cancer were investigated. A 5- fold increase of cisplatin density converted the tumors which were initially resistance to standard-dose cisplatin Into drug-sensitive ones. Vera-pamil was found to be able to overcome vincristine-resistance of some tumors in vitro. These results suggest that using high dose cisplatin therapy or increasing local drug concentration by using other administration way, we could expect some ovarian cancers that had failed to standard dose cisplatin therapy to be effective. Combination of vincristine with verapamll may be helpful in treating some vincristineresistant cases.展开更多
Using cytotoxicity and thymidine uptake assays, we investigated the effects of human recombinant in-terleukin-2 (rIL-2) on the induction of lympholine-activated killer (LAK) activity and cellular proliferation in sple...Using cytotoxicity and thymidine uptake assays, we investigated the effects of human recombinant in-terleukin-2 (rIL-2) on the induction of lympholine-activated killer (LAK) activity and cellular proliferation in splenocytes and thymocytes from human fetuses (18-22 weeks). We observed that fetal splenocytes and thymocytes incubated with low doses of rIL-2 (10-100 U ml) developed broad antitumor activity (LAK activity) although the kinetics and magnitudes of the responses were different. It indicated the LAK precursors are present in fetal spleen and thymus. Further, rIL-2 induced a strong proliferative response in splenocytes, but not in thymocytes. On the basis of the findings, we conclude that the responses of fetal splenocytes and thymocytes to IL-2 are different.展开更多
文摘Thymidine-containing derivatives are considered to be among the most significant derivatives in medicinal chemistry. In this study, we employed a combined computational approach involving density-functional theory (DFT) calculations, molecular docking simulations, and absorption, distribution, metabolism, excretion, and toxicity (ADMET) property predictions. Prediction of activity spectra for substances (PASS) revealed promising antiviral, antimicrobial and anti-carcinogenic activities of these thymidine derivatives. Using Gaussian 09, we optimized the molecular structures of the thymidine derivatives to obtain their stable conformations and calculate their electronic properties. Subsequently, molecular docking simulations were performed to explore the binding interactions between the thymidine derivatives and the active site of the Candida albicans (PDB: 1IYL and 2Y7L) proteins. The docking results were evaluated based on docking scores, hydrogen bonding, and hydrophobic interactions and revealed favorable binding interactions between the thymidine derivatives and the proteins, suggesting their potential as antifungal agents. The thermodynamic properties, including binding free energy, enthalpy, and entropy changes were determined to assess the stability and strength of the ligands-protein complexes. The calculated pharmacokinetic parameters, such as ADMET properties, provided insights into the drug-likeness and potential bioavailability of the thymidine derivatives. These results offer a foundation for further experimental investigations and the design of novel antifungal agents targeting Candida albicans infections.
基金supported by a grant from the National Natural Sciences Foundation of China (No. 30973184)
文摘The mRNA and protein expression of thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) and their relationship with prognosis were investigated. Real-time quantitative RT-PCR (Taqman) was used to detect the mRNA expression of TS, TP and DPD in formalin-fixed and paraffin-embedded 106 samples of epithelial ovarian cancer and 29 normal ovaries. A TATA box-binding protein (TBP) was used as an endogenous reference gene. A relationship between TS, TP, DPD expression and clinicopathologic features was investigated. The protein location and expression of TS, TP and DPD was examined in the same patients by an avidin-biotin-peroxidase immunohistochemistry. TS and TP mRNA expression levels were significantly higher in tumor group than in normal controls, with the average value of TS and TP mRNA being 6.14±0.62 and 0.59±0.06 in tumor tissue, and 0.71±0.14 and 0.16±0.04 in normal tissue, respectively. DPD mRNA expression levels were significantly lower in tumor group (0.11±0.02) than in normal controls (0.38±0.05). There was statistically significant difference in TS and TP mRNA expression levels among different pathological grades and clinical stages (P<0.05), but histological subtype was not significantly associated with TS and TP mRNA expression. DPD gene expression was not significantly associated with any clinicopathological parameters. Immunohistochemistry revealed that TP protein was mainly distributed in nucleus, and TS and DPD mainly in cytoplasm. The protein expression intensity of TS, TP and DPD was coincided with the mRNA expression levels. It was concluded that TS, TP mRNA and protein expression levels were significantly higher in epithelial ovarian cancer, and DPD mRNA and protein expression levels were significantly lower. The expression levels of TS and DPD were related to the patients' prognosis and survival. Combined gene expression levels of TS, TP and DPD represent a new variable to predict the clinical outcome in ovarian cancer. The association of TS, TP and DPD expression levels with survival suggests an importance of these genes for tumor occurrence and progression.
基金Supported by The Thailand Research Fund through The Royal Golden Jubilee PhD Program Grant No. PHD/0037/2544 for Thanasai J and Limpaiboon T and grants-in-aid from the Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Thailand, and from the Ministry of Education, Sports, Science, Culture and Technology, Japan
文摘AIM: To evaluate the role of thymidine phosphorylase (TP) in cholangiocarcinoma using small interfering RNA (siRNA). METHODS: A human cholangiocarcinoma-derived cell line KKU-M139, which has a naturally high level of endogenous TP, had TP expression transiently knocked down using siRNA. Cell growth, migration, in vitro angiogenesis, apoptosis, and cytotoxicity were assayed in TP knockdown and wild-type cell lines. RESULTS: TP mRNA and protein expression were decreased by 87.1% ± 0.49% and 72.5% ± 3.2%, respectively, compared with control cells. Inhibition of TP significantly decreased migration of KKU-M139, and suppressed migration and tube formation of human umbilical vein endothelial cells. siRNA also reduced the ability of TP to resist hypoxia-induced apoptosis, while suppression of TP reduced the sensitivity of KKU-M139 to 5-fluorouracil. CONCLUSION: Inhibition of TP may be beneficial in decreasing angiogenesis-dependent growth and migration of cholangiocarcinoma but may diminish the response to 5-fluorouracil chemotherapy.
文摘Thymidine glycol (5,6-dihydroxy-5,6-dihydrothymidine, Tg) is a major type of oxidative damage in DNA. During chemical oligonucleotide synthesis, Tg residue was incorporated in the different positions of 17 b.p. DNA duplexes, which differ in one base pair in the internal part. According to UV-melting curves, Tg destabilizes the double helix in a sequence independent manner. In contrast, the localized alterations in duplex structure were shown by CD spectroscopy to depend on the type of base pairs flanking the Tg lesion. Molecular dynamics simulations demonstrate that Tg is partially out of the double helix. For the first time, Tg impact on several site-specific DNA-binding proteins is studied, namely p50 and p65 subunits of nuclear factor kappa-B (NF-κB) and DNA methyltransferase SsoII (M.SsoII). Our results show that p50/p50 and p65/p65 homodimers of NF-κB can tolerate a single Tg residue in the binding site quite well. Nevertheless the homodimers have different affinities to the oxidized κB site depending on the Tg position. M.SsoII can act as a transcription repressor when bound to the regulatory site. M.SsoII demonstrates decreased affinity and lowered methylation efficiency when its methylation site contains Tg in the central position. Single Tg in one half of the regulatory site decreases M.SsoII affinity to the oxidized DNA, whereas Tg presence in both half-sites prevents M.SsoII binding to such ligand.
基金supported by the Youth Thousand Talents Program of China,start-up grants from the Shanghai Jiao Tong University(No.WF220408211)supported by grants from the State Key Laboratory of Oncogenes and Related Genes(No.90-17-02)at Shanghai Jiao Tong Universityfrom the Interdisciplinary Program of Shanghai Jiao Tong University(China)(No.YG2017MS18).
文摘Hand-foot syndrome(HFS)is a widely recognized dose-limiting cutaneous toxicity effect of fluoropyrimidine chemotherapy agents that impairs clinical benefits and treatment outcomes.Even though the cause and pathophysiology of HFS are relatively widely reported,how the toxicity of fluoropyrimidine translates into persistent inflammation has not been studied.Additionally,prevention and treatment strategies for HFS based on its mechanistic occurrence and development are scarce.In our study,we demonstrated that cGAS-STING signaling pathway-mediated cellular senescence played a critical role in the inflammatory reaction and provided a therapeutic solution for HFS.Mechanistically,DNA damage,as the primary cytotoxic cause,in keratinocytes induces cell cycle arrest,activates the cGAS-STING signaling pathway,and subsequently mediates cellular senescence,ultimately fueling a robust secondary inflammatory response that results in HFS.More importantly,the thymidine prodrug thymidine diacetate was proven to be effective in preventing HFS by compensating for thymidylate deficiency to facilitate the replication and repair of DNA and thus causing the escape from cellular senescence.These data highlight the importance of DNA damage-mediated cellular senescence in the etiology of HFS and provide a potential therapeutic anchor point for fluoropyrimidine-induced HFS.
文摘Aim:Thynidine phosphorylase(TP)acts as a proangiogenic growth factor which may regulate mammalian Target of Rapamycin(mTOR).We investigated whether the TP substrate thymidine and overexpression of TP affected mTOR signaling by comparing Colo320(TP deficient)cells and its TP-transfected variant(Colo320TP1).Methods:Drug resistance was assessed with the sulforhodamine B assay,protein expression with Western blotting,cell cycle distribution and cell death with Fluorescence-activated cell sorting analysis,and autophagy with immunofluorescence.Results:Colo320 and Colo320TP1 cells had comparable levels of sensitivity to the mTOR inhibitor rapamycin.Thymidine treatment led to 13-and 50-fold resistance to rapamycin in Colo320 and Colo320TP1 cells,respectively.In Colo320TP1 cells,the thymidine phosphorylase inhibitor(TPI)reversed the thymidine induced resistance to rapamycin,but not in Colo320 cells,indicating a role for TP in the protection.Thymidine increased p70/S6k-phosphorylation(downstream of mTOR)in Colo320TP1,but it was not affected in Colo320.As a mechanism behind resistance,we studied the levels of autophagy and found that,in Colo320TP1 cells,autophagy was highly induced by thymidine-rapamycin,which was decreased by TPI.In addition,the autophagy inhibitor 3-methyl-adenine completely inhibited autophagy and its protection.Conclusion:Rapamycin resistance in TP-expressing cancer cells may therefore be related to thymidine-mediated autophagy activation.
文摘To explore correlation between the tk gene structure of pseudorabies virus (PRV) and its virulence, to study the effect of the gene mutation on PRV biological properties, and to investigate mechinism of reduced virulence, thymidine kinase (TK)-deficient mutant of pseudorabies virus strain Hubei (PRV HB) was isolated by selection for resistance to 5-bromodeoxyuridine. The tk genes of PRV HB and its TK mutant were cloned and sequenced. 1587 base pairs of the tk gene and flanking regions of wild-type (wt) virus were sequenced, which included an open reading frame (ORF) of 1098 bp encoding a protein of 366 amino acids. The ORF contained two 137-bp repeated sequences, which were connected by an adenosine. 1458 bp of the tk and flanking regions of TK- mutant were sequenced. Analysis of the tk gene sequence of TK mutant indicated that one of 137 bp repeated sequence and the connecting adenosine in the tk gene of the wt virus was deleted and a repeated sequence of 8 nucleotides (GCGCGCC) was inserted. All
基金Project supported by the National Natural Science Foundation of China
文摘The human cytosolic thymidine kinase (TK-C)gene has been localized on chromosome 17 (17q21--22). Morbid anatomy of the human genome has shown that seve?al disease genes are located in the same chromosome region. Our goal is to search for the restriction site polymorphism in the DNA sequence of the human TK-C gene.
基金supported by the National Key R&D Program of China (2020YFA0803501,2019YFA0508501,2021YFF0702802)the National Natural Science Foundation of China (82130088,31930036,81921003,31871494,92042302,91940305,32070533,81772646)+2 种基金the Beijing Natural Science Foundation (5192018)the Biological Resources Program of Chinese Academy of Sciences (KFJ-BRP-017)the Strategic Priority Research Programs of the Chinese Academy of Sciences (XDB19030203).
文摘Neutrophils are derived from bone marrow hematopoietic stem cells(HSCs)and are the largest population among circulating white blood cells in humans,acting as the first line of defense against invading pathogens.Whether neutrophils can be generated by transdifferentiation strategies is unknown.Here,we show that thymidine induces the conversion of mouse fibroblasts to neutrophils.Induced neutrophils(iNeus)showed antibacterial effects and did not undergo malignant transformation in vivo.Importantly,iNeu transplantation cured neutropenia in mice in vivo.Mechanistically,thymidine mediates iNeu conversion by enhancing Tet3 activity.Tet3 initiates the expression of the neutrophil fate decision factors Cebpδ and Rfx1 that drive the transdifferentiation of mouse fibroblasts to neutrophils.Therefore,the induction of functional neutrophils by chemicals may provide a potential therapeutic strategy for patients with neutropenia patients and infectious diseases.
文摘Viral-mediated gene transfer of thymidine kinase ofherpes simplex virus (HSV-tk) has been used to confercytotoxic sensitivity to ganciclovir (GCV) in a variety oftnmor cells. HSV-tk converts GCV into a phosphorylatedcompound which is toxic for dividing cells by blockingDNA synthesis. Our previous study has shown
文摘Connexin-43 (Cx43) expression in prostate cancer (PCa) cells and the potency of gap junctional intercellular communication (GJIC) in the cells were investigated, with an attempt to elu- cidate the reason why the so-called "bystander effect" mediated by thymidine kinase (TK) suicide gene therapy on PCa cells is not of significance and to explore the role of GJIC in PCa carcinogenesis. mRNA and protein expression of Cx43 in a PCa cell line PC-3m was detected by re- verse-transcription polymerase chain reaction (RT-PCR) and strapt-avidin-biotin-enzyme complex (SABC) immunohistochemical staining, and inherent GJIC of PC-3m cells was assayed by scrape-loading and dye transfer (SLDT) assay. The expression of Cx43 in human normal and malig- nant prostate tissues was determined by SABC immunohistochemistry as well. It was found that Cx43 mRNA and protein expression in PC-3m cells was slightly reduced as compared with positive controls and the location of Cx43 protein was aberrant in cytoplasm rather than on membrane. As- sessment of paraffin sections demonstrated that the expression of Cx43 protein in PCa cells was ab- normally located and markedly diminished as compared with normal prostatic epithelial ones, dis- playing a negative correlation to the pathological grade (χ2=4.025, P<0.05). Additionally, capacity of inherent GJIC in PC-3m cells was disrupted, which was semi-quantified as (+) or (-). It was indi- cated that both down-regulated expression of Cx43 mRNA and aberrant location of Cx43 protein par- ticipated in the mechanisms leading to deficient GJIC in PC-3m cells. Lack of efficient GJIC is a molecular event, which may contribute not only to limited extent of "bystander effect", but also to initiation and progression of prostatic neoplasm.
基金Supported by the National Natural Science Foundation of China(Nos.31572660,31872600)the“1000 Talents Program”,and the Qingdao Innovation Leadership Project(No.18-1-2-12-zhc)。
文摘Thymidylate synthase(TS)is a key enzyme in the de novo biosynthesis of thymidine monophosphate,serving as a well-known drug target in chemotherapy against cancers and infectious diseases.Additional to its clinical value,TS is supposed to be a promising drug target in aquatic-disease control.To facilitate designing pathogen-specific TS inhibitors for shrimp-disease control,we report the crystal structures of TS from Litopenaeus vannamei(LvTS)in the apo form,LvTS-dUMP complex and LvTS-dUMP-raltitrexed complex at 2.27Å,1.54Å,and 1.56Åresolution,respectively.LvTS shares a similar fold with known TSs,existing as a dimer in the crystal.The apo LvTS and LvTS-dUMP take an open conformation,and raltitrexed binding induces structural changes into a closed conformation in LvTS-dUMP-raltitrexed.Compared to those in other known TS-dUMP-raltitrexed complexes with the closed conformation,the C-terminal loop in LvTS-dUMP-raltitrexed shifts its position away from the bound raltitrexed;the distance between C6 of dUMP and Sγof the catalytic cysteine is obviously longer than that in the known TS structures with closed conformations,resembling that in the TS structures with open conformations.Other species-specific interactions with dUMP and raltitrexed are also observed.Therefore,LvTS-dUMP-raltitrexed adopts a loosely closed conformation with structural features intermediate between the closed and the open conformations that were reported in other TSs.Our study provides the first crustcean TS structure,and reveals species-specific interactions between TSs and the ligands,which would facilitate designing pathogen-specific TS inhibitors for shrimp-disease control.
文摘A chemosensitivity test for ovarian cancer using tritiated thymidine incorporation assay was carried out. A dose-response relationship for cisplatin and potentiation of verapamil in increasing vincristine inhibition to ovarian cancer were investigated. A 5- fold increase of cisplatin density converted the tumors which were initially resistance to standard-dose cisplatin Into drug-sensitive ones. Vera-pamil was found to be able to overcome vincristine-resistance of some tumors in vitro. These results suggest that using high dose cisplatin therapy or increasing local drug concentration by using other administration way, we could expect some ovarian cancers that had failed to standard dose cisplatin therapy to be effective. Combination of vincristine with verapamll may be helpful in treating some vincristineresistant cases.
文摘Using cytotoxicity and thymidine uptake assays, we investigated the effects of human recombinant in-terleukin-2 (rIL-2) on the induction of lympholine-activated killer (LAK) activity and cellular proliferation in splenocytes and thymocytes from human fetuses (18-22 weeks). We observed that fetal splenocytes and thymocytes incubated with low doses of rIL-2 (10-100 U ml) developed broad antitumor activity (LAK activity) although the kinetics and magnitudes of the responses were different. It indicated the LAK precursors are present in fetal spleen and thymus. Further, rIL-2 induced a strong proliferative response in splenocytes, but not in thymocytes. On the basis of the findings, we conclude that the responses of fetal splenocytes and thymocytes to IL-2 are different.
