Expression of thymidylate synthase in salivary adenoid myoepithelial cells and its clinical significance / 南方医科大学学报

OBJECTIVE@#To evaluate the expression of thymidylate synthase (TS) in myoepithelial cells (MECs) of salivary adenoid tissues and explore its clinical significance.@*METHODS@#Immunohistochemical staining EnVision method was used to detect the expression of TS, P63, Calponin, CK5/6 and S-100 in 32 salivary gland specimens, including 10 non-neoplastic and salivary inflammation specimens, 11 mixed tumor specimens, 5 basal cell carcinoma specimens and 6 adenoid cyst carcinoma specimens. The specificity and sensitivity of TS as a specific molecular marker of salivary muscle epithelial cells were evaluated in comparison with P63, Calponin, CK5/6 and S-100.@*RESULTS@#The expression pattern of TS in all the salivary gland tissue specimens was identical with that of p63. TS and P63 both showed strong immunohistochemical expressions in MECs of salivary adenoid tissue specimens. Calponin, CK5/6, and S-100 showed cytoplasmic/membranous expressions in the MECs. In addition, TS exhibited weak or moderate cytoplasmic expression in a few salivary gland epithelial cells, cancer cells and scattered stromal cells, with negative expression in the cell nuclei. The expression of TS in the MECs of all the salivary adenoid specimens was highly consistent with those of P63, Calponin, CK5/6 and S-100 (>0.05) Except for CK5/6 expression in Salivary inflammation and Salivary gland specimens. Kappa>0.75. The specificity and sensitivity of TS as a molecular marker of MECs were both 100%.@*CONCLUSIONS@#TS is a new specific marker of MECs for differential diagnosis of salivary gland tumors.

Immunohistochemistry Biomarkers Predict Survival in Stage II/III Gastric Cancer Patients: From a Prospective Clinical Trial / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Identification of biomarkers to predict recurrence risk is essential to improve adjuvant treatment strategies in stage II/III gastric cancer patients. This study evaluated biomarkers for predicting survival after surgical resection. MATERIALS AND METHODS: This post-hoc analysis evaluated patients from the CLASSIC trial who underwent D2 gastrectomywith orwithout adjuvant chemotherapy (capecitabine plus oxaliplatin) at the Yonsei Cancer Center. Tumor expressions of thymidylate synthase (TS), excision repair cross-complementation group 1 (ERCC1), and programmed death-ligand 1 (PD-L1) were evaluated by immunohistochemical (IHC) staining to determine their predictive values. RESULTS: Among 139 patients, IHC analysis revealed high tumor expression of TS (n=22, 15.8%), ERCC1 (n=23, 16.5%), and PD-L1 (n=42, 30.2%) in the subset of patients. Among all patients, high TS expression tended to predict poor disease-free survival (DFS; hazard ratio [HR], 1.80; p=0.053), whereas PD-L1 positivity was associated with favorable DFS (HR, 0.33; p=0.001) and overall survival (OS; HR, 0.38; p=0.009) in multivariate Cox analysis. In the subgroup analysis, poor DFS was independently predicted by high TS expression (HR, 2.51; p=0.022) in the adjuvant chemotherapy subgroup (n=66). High PD-L1 expression was associated with favorable DFS (HR, 0.25; p=0.011) and OS (HR, 0.22; p=0.015) only in the surgery-alone subgroup (n=73). The prognostic impact of high ERCC1 expression was not significant in the multivariate Cox analysis. CONCLUSION: This study shows that high TS expression is a predictive factor for worse outcomes on capecitabine plus oxaliplatin adjuvant chemotherapy, whereas PD-L1 expression is a favorable prognostic factor in locally advanced gastric cancer patients.

Association of genetic polymorphis of thymidylate synthase and methylene tetrahydro folate reductase with therapeutic effect of pemetrexed for treating advanced lung adenocarcinoma patients / 实用医学杂志

Objective To investigate the expressions of thymidylate synthase (TS) and methylene tetrahydrofolate reductase (MTHFR) polymorphisms and the therapeutic efficacy of chemotherapy with pemetrexed and platinum in advanced lung adenocarcinoma patients. Methods Fifty-eight patients with advanced lung adenocarcinoma were enrolled in this study. The blood samples from 25 of them were examined for extraction of DNA. The associations of the gene polymorphisms with the chemotherapy efficacy and PFS were analyzed. Results Disease control rate was noted by 38％ and the median time of progression-free survival was 8.1 months among 58 patients.There were 16％, 32％, 52％, and TS genotypes as 2R/2R, 2R/3R and 3R/3R respectively; the difference in the control rate between those with TS gene of 3R/3R and those with TS gene of R/2R+2R/3 R was significant statistically (53.8％ vs. 91.7％, P = 0.046) , but the difference in PFS was statistically insignificant (9.3 vs. 10.4 months, P> 0.05). There were 40％, 52％, 8％, and MTHFR genotypes as CC, CT and TT respectively. The DCR in those with MTHFR CC and C/T + T/T was 70％ and 73.3％, respectively and PFS was 10 months and 9.7 months respectively, showing no significant difference (P> 0.05). Conclusion The TS gene polymorphism is associated with therapeutic effect of pemetrexed for advanced lung adenocarcinoma, but MTHFR is not.

Study on the correlation between thymidylate synthase expression level and postoperative chemotherapy in gastric cancer / 国际肿瘤学杂志

Objective To investigate the expression of thymidylate synthase (TS),and the correlations with the clinicopathologcal factors and the prognosis of postoperative fluorouracil chemo-therapy in gastric cancer.Methods 42 patients with gastric cancer who underwent radical gastrectomy were collected from April 2011 to April 2014 in Lu'an Hospital of Anhui Medical University,and the chemotherapy regimen was Cisplatin + Capecitabine or Cisplatin + Teggio.The levels of TS in tumor tissues of 42 patients with gastric cancer were detected by immunohistochemical method.The relationships between TS expression level,clinical features and the prognosis of patients treated with fluorouracil chemotherapy were analyzed.Results In 42 patients with gastric cancer,19 patients (45.2％) have TS high expressions,23 patients (54.8％) have TS low expressions.There were no significant correlations between the expression of TS and age,sex,clinical stage,differentiation degree,tumor location,depth of invasion,lymph node metastasis,vascular tumor thrombus and nerve bundle invasion (all P ＞0.05).There were significant differences between low TS expression and high TS expression in median progression-free survival and 3-year survival rate of patients [35 months vs.17 months,73.9％ (17/23) vs.42.1％ (8/19)],and the differences were statistically significant (x2 =4.788,P =0.029;x2 =4.369,P =0.037).In patients with stage Ⅲ gastric cancer,the median progression-free survival of patients with low TS expression was significantly prolonged compared to that with high TS expression (33 months vs.9 months),and the difference was statistically significant (x2 =4.731,P =0.030).Conclusion TS expression levels have no significant correlations with the clinicopathologcal factors,but closely relate to the prognosis of gastric cancer patients treatment with fluorouracil chemotherapy.The chemotherapy prognosis of TS high expression is worse than that of TS low expression in gastric cancer.

Clinical significance of epidermal growth factor receptor and thymidylate synthase expression in primary liver cancer / 中华肝脏病杂志

Objective@#To investigate epidermal growth factor receptor (EGFR) and thymidylate synthase (TS) expression in primary liver cancer, and analyze its clinicopathological features and prognostic significance.@*Methods@#Immunohistochemistry was performed using EnVision method to detect EGFR and TS expression in 41 cases of liver cancer. Correlation coefficient between EGFR and TS was calculated by Spearman method. Fisher's exact probability method or χ2 test was used to analyze the clinicopathological features of EGFR and TS. Kaplan-Meier method was used to calculate the survival rate of patients in conjunction with the log-rank test.COX proportional hazard regression model was used to analyze the prognostic factors of patients. ROC curve was used to analyze the predictive accuracy of EGFR and TS for prognosis.@*Results@#The positive rates of EGFR and TS in liver cancer tissues were 34.15% and 39.02%, respectively. There was a positive correlation between EGFR and TS expressions, and the difference was statistically significant (P < 0.05). EGFR was associated with tumor size and tissue differentiation (P < 0.05) in HCC patients, whereas TS was associated with tissue differentiation (P < 0.05). There was no significant difference in prognostic effect of EGFR on survival rate (P > 0.05). TS prognostic effect on survival rate was statistically significant (P < 0.05). HR of EGFR was 0.210 with 95% CI, 0.052-0.852, P = 0.029; indicating that the risk of death in patients with negative EGFR was 0.210 times higher than that in patients with positive EGFR. HR of TS was 2.496, with 95% CI, 1.325-4.701, P = 0.005, indicating that the risk of death increased by 2.496 times with the same level of EGFR. The area under the EGFR curve was 0.553 and its approximate reference confidence interval was 95% (0.355, 0.751), indicating that EGFR was a risk factor for death and the area under the TS curve was 0.695, and its approximate reference confidence interval was 95% (0.513, 0.878), indicating that TS was a risk factor for death.@*Conclusion@#EGFR and TS were equally expressed in primary liver cancer, and EGFR and TS expressions were positively correlated. EGFR and TS had an effect on the degree of tissue differentiation in patients with liver cancer. EGFR and TS were risk factors for prognosis, and TS may assist EGFR.

Clinical significance of the expression levels of ERCC1, BRCA1 and TS in advanced colorectal cancer / 中国医师进修杂志

Objective To explore the relationship between the expression levels of excision repair cross complementation group 1(ERCC1), breast cancer susceptibility gene 1(BRCA1), thymidylate synthase (TS) mRNA and clinicopathological features, prognosis in advanced colorectal cancer, and the correlation between the expression levels of ERCC1 and BRCA1. Methods The expression levels of ERCC1, BRCA1 and TS mRNA of postoperative paraffin embedded tissue were tested by real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) in 49 advanced colorectal cancer cases. The results were analyzed by χ2 test of the correlation between the expression levels and clinicopathological characteristics. Patients were followed up by clinic or telephone. The prognosis was analyzed by small sample Kaplan-Meier survival analysis and Log-rank time series analysis, and P0.05). The expression level of BRCA1 mRNA had no significant correlation with the above clinical and pathological features (P>0.05) except distant metastasis (P=0.030) and differentiation degree (P=0.002). The expression level of TS mRNA had no significant correlation with the above clinical and pathological features (P>0.05) except distant metastasis (P=0.003). The expression level of ERCC1 and BRCA1 mRNA obviously correlated (P=0.002). The 1 year overall survival rate was 95.92%(47/49);the 2 year overall survival rate was 83.67%(41/49);and the 3 year overall survival rate was 73.47%(36/49). Overall survival and progression-free survival time in ERCC1 mRNA low expression group (47.8, 41.0 months) was higher than that in ERCC1 mRNA low expression group (27.3, 20.0 months) respectively (P=0.001, P=0.001). Overall survival and progression-free survival time in BRCA1 mRNA low expression group (43.7, 42.7 months) was higher than that in BRCA1 mRNA high expression group (29.3, 25.1 months) respectively (P=0.009, 0.006). Overall survival time in TS mRNA low expression group (39.8 months) was higher than that in BRCA1 mRNA high expression group (25.2 months). Conclusions The expression level of ERCC1 mRNA is not correlated with its clinical and pathological characteristics, but with its biological characteristics. BRCA1 and TS levels are correlated with invasion and metastasis. Low levels of ERCC1 and BRCA1 expression have a better prognostic effect on platinum based first-line chemotherapy for advanced colorectal cancer, and they are correlated. Low level of TS also has longer disease-free survival. Three joint detection could be used as a prognostic factor for colorectal cancer chemotherapy.

Chemotherapy resistance of 5-fluorouracil:research advances / 国际药学研究杂志

5-Fluorouracil(5-FU)has been widely used to treat gastrointestinal,head,neck,chest and ovarian malignant tu-mors since 1957. As an analogue of pyrimidine,5-FU plays anti-cancer roles by inhibiting thymioylate synthase and integrating its me-tabolites into DNA and RNA. Although 5-FU is one of the first-line therapeutic compounds for gastrointestinal malignant tumor as a sin-gle drug or in combination with other drugs,its effectiveness is hindered by its low efficiency,which may be due to chemotherapy re-sistance. 5-FU chemotherapy resistance may stem from enzyme abnormality,genetic abnormality and tumor microenvironment. In this paper,we make a review about 5-FU actions and the mechanisms underlying chemotherapy resistance.

The therapeutic efficacy of thymidylate synthase on pemetrexed treatment of lung adenocarcinoma / 中国医师杂志

Objective To investigate thymidylate synthase on pemetrexed treatment of lung adenocarcinoma effect relationship.Methods The 60 patients with lung adenocarcinoma in our hospital from January 2014 to January 2016 were selected as the research subjects.They were treated with pemetrexed.According to the clinical efficacy,they were divided into the effective group (n =27) and ineffective group (n =33) after 3 courses of treatment.The levels of thymidylate synthase (thymidylate synthase,TS),TS mRNA expression,and the expression of TS protein in the tumor tissues of two groups were analyzed by enzyme-linked immunoadsorbent assay (ELISA),fluorescence quantitative polymerase chain reaction (PCR),and immunohistochemistry.The relationship between TS level and pemetrexed in the treatment of lung adenocarcinoma was investigated.Results The level of ST in peripheral blood of the effective group was significantly lower than ineffective group.The objective response rate and protein of ST gene low expression were significantly higher than high expression of ST.Conclusions The level of thymidylate synthase in patients with adenocarcinoma of the lung is related to the therapeutic effect of pemetrexed in the treatment of adenocarcinoma of the lung.It can be used as a molecular marker to evaluate the clinical efficacy of pemetrexed in the treatment of patients with lung adenocarcinoma.

An unusual UMP C-5 methylase in nucleoside antibiotic polyoxin biosynthesis

Polyoxin is a group of structurally-related peptidyl nucleoside antibiotics bearing C-5 modifications on the nucleoside skeleton. Although the structural diversity and bioactivity preference of polyoxin are, to some extent, affected by such modifications, the biosynthetic logic for their occurence remains obscure. Here we report the identification of PolB in polyoxin pathway as an unusual UMP C-5 methylase with thymidylate synthase activity which is responsible for the C-5 methylation of the nucleoside skeleton. To probe its molecular mechanism, we determined the crystal structures of PolB alone and in complexes with 5-Br UMP and 5-Br dUMP at 2.15 Å, 1.76 Å and 2.28 Å resolutions, respectively. Loop 1 (residues 117-131), Loop 2 (residues 192-201) and the substrate recognition peptide (residues 94-102) of PolB exhibit considerable conformational flexibility and adopt distinct structures upon binding to different substrate analogs. Consistent with the structural findings, a PolB homolog that harbors an identical function from Streptomyces viridochromogenes DSM 40736 was identified. The discovery of UMP C5-methylase opens the way to rational pathway engineering for polyoxin component optimization, and will also enrich the toolbox for natural nucleotide chemistry.

Expression of excision repair cross-complementing 1, topoisomeraseⅡ, ribonucleotide reductase M1,β3-tubulin and thymidylate synthase in lung cancer / 肿瘤研究与临床

Objective To analyze the expression characteristics of excision repair cross-complementing 1 (ERCC1), topoisomeraseⅡ (TOPOⅡ), ribonucleotide reductase M1 (RRM1), β3-tubulin and thymidylate synthase (TS) in lung cancer and their associations with the pathological types. Methods The immunohistochemical EnVision method was used to determine the expression of ERCC1, TOPOⅡ, RRM1,β3-tubulin and TS in 548 patients who were diagnosed as lung cancer from January 2011 to December 2014. Variance analysis was performed to analyze their expression characteristics among different pathological types and correlation. Results The expression positive rates of ERCC1, TOPOⅡ, RRM1, β3-tubulin and TS were 61.86 % (339/548), 91.06 % (499/548), 62.59 % (343/548), 73.18 % (401/548) and 70.44 % (386/548), respectively. The expression of ERCC1 was weak positive mostly (P<0.05), meanwhile the expression of TOPOⅡ was medium-strong positive mostly (P<0.05). In ERCC1 group, the positive rate of squamous cell carcinoma was higher than that of adenocarcinoma [57.39 % (167/291) vs. 42.61 % (124/291), P=0.000]. In weak positive of TOPOⅡ group, the proportion of adenocarcinoma was higher than that of squamous cell carcinoma [23.58 % (100/137) vs. 8.73 % (37/137), P=0.000]. In medium-strong positive of TOPOⅡ group, the proportion of squamous cell carcinoma was higher than that of adenocarcinoma [47.41 % (201/287/) vs. 20.28%(86/287), P=0.000]. The expressions of ERCC1, TOPOⅡ, RRM1,β3-tubulin and TS were irrelevant (r=0.4, P=0.397). Conclusions The expressions of ERCC1 and TOPOⅡ are higher in squamous cell carcinoma than those in adenocarcinoma. The expression of ERCC1 is weak positive mostly, meanwhile the expression of TOPOⅡis medium-strong positive mostly. There is no correlation between them.

Polimorfismo do gene timidilato sintase e nível plasmático total de homocisteína em um grupo de pacientes turcos com artrite reumatoide: relação com a atividade da doença e toxicidade ao metotrexato / Thymidylate synthase genetic polymorphism and plasma total homocysteine level in a group of Turkish patients with rheumatoid arthritis: relationship with disease activity and methotrexate toxicity

Resumo Introdução: Relata-se que o polimorfismo do gene timidilato sintase (TS) e a homocisteína têm relação com o metabolismo do metotrexato (MTX), com achados conflitantes. O objetivo deste estudo foi determinar os níveis de homocisteína e a frequência de polimorfismos de repetição tripla (TS3R) e dupla (TS2R) do gene TS em um grupo de pacientes turcos com AR e avaliar sua associação com a toxicidade ao MTX e a atividade da doença. Métodos: Foram incluídos no estudo 64 pacientes com AR e 31 indivíduos no grupo controle, com média de 48,7 ± 12,5 e 46,2 ± 13,4 anos. Foram obtidas as características demográficas e foi registrado o número de pacientes que relataram efeitos adversos ao MTX no grupo AR. Foram analisados os níveis de homocisteína e os polimorfismos TS2R/TS3R. Foi determinada a distribuição de genótipos de acordo com a toxicidade ao MTX e a atividade da doença. Resultados: Os dados demográficos foram semelhantes entre os pacientes e controles. Todos faziam suplementação de ácido fólico a uma dose média de 5 mg/semana. Dos 64 pacientes, 36 apresentaram efeitos adversos ao tratamento com MTX. Encontrou-se uma frequência de polimorfismos TS2R e TS3R semelhante nos grupos AR e controle. Encontrou-se que os polimorfismos TS2R e TS3R eram semelhantes em pacientes com e sem eventos adversos relacionados com o MTX. O nível médio de homocisteína também foi similar em pacientes com e sem polimorfismo do gene TS, mas era mais elevado (12,45 μmol/L vs. 10,7 μmol/L) em pacientes com do que sem efeitos adversos relacionados com o MTX. O nível médio de homocisteína se correlacionou com o VHS no grupo AR. Conclusões: Os níveis de homocisteína podem afetar a atividade da doença e a toxicidade ao MTX, mas os polimorfismos 2 R e 3 R no gene TS não se correlacionaram com a toxicidade ao MTX em pacientes com AR que recebem suplementação de ácido fólico. São necessários mais estudos para esclarecer os polimorfismos em outras enzimas que podem ser responsáveis pela toxicidade ao MTX em pacientes com AR.

Abstract Background: The polymorphism of thymidylate synthase (TS) gene and homocysteine are reported to have a relationship to methotrexate (MTX) metabolism, with conflicting results. The aim of this study was to determine homocysteine levels and the frequency of TS gene triple repeat (TS3R) and double repeat (TS2R) polymorphisms in a group of Turkish RA patients and evaluate its association with MTX toxicity and disease activity. Methods: Sixty-four patients with RA and 31 control subjects with a mean age of 48.7 ± 12.5 and 46.2 ± 13.4 years were enrolled for the study. Demographic characteristics were obtained and a number of patients with MTX-related adverse affects were recorded in the patient group. The homocysteine levels and TS2R/TS3R polymorphisms of the TS gene were analyzed and the distribution of genotypes according to MTX toxicity and disease activity was determined. Results: The demographic properties were similar between the patient and control subjects. Folic acid supplementation with a mean dose of 5 mg folic acid/week was present in all patients. Thirty-six of the 64 patients showed adverse effects to MTX treatment. The respective frequency of TS2R and TS3R polymorphisms was found to be similar in the patient and control groups. TS2R and TS3R gene polymorphisms were found to be similar in patients with and without MTX-related adverse events. The mean homocysteine level was also similar in patients with and without TS gene polymorphism, but was found to be higher (12.45 μmol/L vs 10.7 μmol/L) in patients with MTX-related side effects than in patients without side effects. The mean level of homocysteine was correlated with levels of ESR in the patient group. Conclusions: In conclusion, homocysteine levels might affect the disease activity and toxicity of MTX but 2R and 3R polymorphisms in the TS gene were not related with MTX-related toxicity in RA patients receiving folate supplementation. Further studies are needed to illuminate the polymorphisms in other enzymes that might be responsible for the MTX toxicity in patients suffering from RA.

Polymorphism analysis of thymidylate synthase in 252 colorectal cancer patients / 国际检验医学杂志

Objective To analyze the polymorphism of thymidylate synthase(TS)in colorectal cancer patients,further more,to provide guidance for personalized therapy of colorectal cancer.Methods PCR direct sequencing was used to detect the polymor-phism of TS in 252 patients with colorectal cancer.Results TS genotypes of 252 patients with colorectal cancer were detected total-ly,including 137 male and 115 female.3RG/3RC accounted for the largest proportion in both male and female(36.50% and 36.52%respectively).In female,2RC/3RC and 3RG/3RG both accounted for the second largest proportion(both 18.25%).While in fe-male,3RG/3RC accounted for the second largest proportion(26.09%).If patients were divided according to age groups,in youth patients(n=28),3RG/3RC accounted for the largest proportion(42.86%),and the second was 2RC/3RG(21.43%).In the middle aged patients(n=84),3RG/3RC(45.24%)and 2RC/3RC(16.67%)were the major genotypes.For old patients(n=115),the ma-jor genotypes were 3RG/3RC(36.52%)and 2RC/3RG(16.52%).Conclusion The polymorphism of TS are mainly 3RG/3RC in colorectal cancer patients.

Biochemical characterization of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase from Leishmania ( Viannia ) and its evaluation as a drug target / Caracterización bioquímica de la enzima bifuncional dihidrofolato reductasa-timidilato sintasa de Leishmania ( Viannia ) y su evaluación como blanco molecular

Introduction. Dihydrofolate reductase (DHFR) has been used successfully as a drug target in the area of anti-bacterial, anti-cancer and anti-malarial therapy. Although this bifunctional enzyme is also a potential drug target for treatment of leishmaniasis, there have been no reports on its efficacy against Leishmania ( Viannia ) species . Materials and methods. The gene encoding the bifunctional DHFR and thymidylate synthase (TS) of Le. (V.) braziliensis was isolated and expressed in E. coli. The enzyme was purified and characterized. The inhibitory effects of antifolates and four aporphine alkaloids on its activity were evaluated. Results. The full-length gene consists of a 1560-bp open reading frame encoding a 58 kDa translated peptide containing DHFR and TS domains linked together in a single polypeptide chain. The recombinant DHFR-TS enzyme revealed K m and V max values of 55.35 ± 4.02 µ M (mean ± SE) and 0.02 ± 5.34 x 10 -4 µ M/min respectively for dihydrofolic acid (H 2 F). The Le. braziliensis rDHFR-TS have Ki values for antimicrobial antifolates in the µM range. Methotrexate (MTX) was a more-potent inhibitor of enzymatic activity ( Ki = 22.0 µM) than trimethoprim ( Ki = 33 µM) and pyrimethamine ( Ki = 68 µM). These Ki values are significantly lower than those obtained for the aporphine alkaloids. Conclusion. The results of the study show the inhibitory effect of antifolate drugs on enzymatic activity, indicating that Le. braziliensis rDHFR-TS could be a model to studying antifolate compounds as potential antiprotozoal drugs.

Introducción. La dihidrofolato reductasa (DHFR) se ha utilizado como blanco molecular en tratamientos antibacterianos, anticancerígenos y antipalúdicos. También, actúa como blanco molecular en Leishmania ; sin embargo, no existen reportes de la enzima bifuncional en especies de Leishmania ( Viannia ). Materiales y métodos. Se ha aislado y expresado en Escherichia coli el gen que codifica para la enzima bifuncional DHFR y la timidilato-sintasa (TS) de Leishmania braziliensis . La enzima recombinante se purificó y caracterizó, y se evaluó el efecto inhibitorio de algunos antifolatos, así como de cuatro alcaloides aporfínicos. Resultados. El gen se compone de aproximadamente 1.560 pb y codifica un péptido de 58 kDa que contiene los dominios DHFR y TS ligados en una sola cadena polipeptídica. La enzima recombinante DHFR-TS, utilizando el dihidrofolato (H2F) como sustrato, presentó valores de K m y V max de 55,35 ± 4,02 (media ± el error estándar de la media) y de 0,02 ± 5,34 x 10 -4 , respectivamente. La enzima rDHFR-TS de L. braziliensis presentó valores de Ki para los antifolatos en el rango de micras. El metotrexato fue el inhibidor más potente de la actividad enzimática ( Ki =22,0 mM) en comparación del trimetoprim ( Ki =33 mM) y la pirimetamina ( Ki =68 mM). Estos valores de Ki son significativamente más bajos en comparación con los obtenidos para los alcaloides aporfínicos. Conclusión. Los resultados muestran el efecto inhibitorio de los antifolatos sobre la actividad enzimática, lo cual indica que la rDHFR-TS de L. braziliensis podría ser un modelo para estudiar moléculas antiprotozoarias potenciales.

Expression of glutathione-S-transferase-π, P-glycoprotein, topoisomerase Ⅱ and thymidylate synthase in gastric adenocarcinoma tissues and their clinical significances / 肿瘤研究与临床

Objective To explore the expression of glutathione-S-transferase-π(GST-π),P-glycoprotein (P-gp),topoisomerase Ⅱ (Topo Ⅱ) and thymidylate synthase (TS) in gastric adenocarcinoma tissues and the clinical significance.Methods Envision method of immunohistochemistry was used to detect the expression of GST-π,P-gp,Topo Ⅱ and TS in sample of 64 gastric adenocarcinoma tissues and 12 normal gastric mucosas,and their corresponding clinical data were comprehensive analyzed.Results The expression positive rates of GST-π,P-gp,Topo Ⅱand TS were respectively 76.6 ％(49/64),64.1％ (41/64),84.4 ％ (50/64) and 53.1％ (34/64),that were all higher than in gastric mucosa [8.3 ％ (1/12),8.3 ％(1/12),16.7 ％ (2/12),0],the differences were statistically significant respectively (P ＜ 0.01).Their positive expression rates were closely relevant to the degree of differentiation (P ＜ 0.05),but not to the patients' gender,age,tumour size,clinical staging,invasive depth and lymph node metastasis (P ＞ 0.05).The expressions of GST-π,P-gp and TS in high and middle differentiation adenocarcinoma were high.er than in low differentiation,but the expression of Topo Ⅱ in high and middle differentiation adenocarcinoma was lower than in low differentiation (P ＜ 0.05).Conclusion GST-π,P-gp,Topo Ⅱ and TS are over-expressed in gastric adenocarcinoma,which is related to the multidrug resistance of gastric adenocarcinoma.The joint detection of the expression of GST-π,P-gp,Topo Ⅱ and TS in gastric adenocarcinoma can be looked as an important symbol for guiding its chemotherapy drug and prognosis.

Expression of ERCC1 and TS in non-small cell lung cancer and its relationship with platinum chemosensitivity / 中国医师杂志

Objective To detect the excision repair cross-complementing gene 1 (ERCC1) and thymidylate of the acid synthase (TS) in non-small cell lung cancer (NSCLC) and its adjacent tissue,and investigate the relationship of the expression of ERCC1 and TS with the clinical characteristics of NSCLC and prognosis for NSCLC individual therapy to provide experimental basis.Methods The protein expression levels of ERCC1 and TS in 50 cases of postoperative NSCLC cancer and adjacent tissue were detected by immunohistochemical method and the relationship among the expression of ERCC1,TS,and overall survival of patients with NSCLC Phase (OS),disease progression time (TTP),the median OS,and median TTP was analyzed.Results (1)There was an obvious difference between the expression of ERCC1,TS in cancer and paraneoplastic tissue of NSCLC,which had statistically significance (64.00％ vs 20.00％,x2 =19.87,P ＜ 0.01 ;48.00％ vs 24.00％,x2 =6.25,P ＜ 0.05) ; (2)The continued investigation in the patients who received postoperative cisplatin or carboplatin chemotherapy showed that the 0S of negative expression of ERCC1 was significantly longer than the positive one (19.10 vs 10.00 months;x2 =8.133,P =0.002),so was median TTP (15.30 vs 9.00 months; x2 =7.410,P =0.003).The median 0S of the negative expression of TS,was significantly longer than the positive one (17.80 vs 11.00 months,x2 =7.001,P =0.008),so was median TTP (11.40 vs 6.80 months; x2 =5.884,P =0.026).Conclusions ERCC1 and TS protein may become sensitive predictors of platinum chemosensitivity for NSCLC patients ; the detection of combined with ERCC1 and TS would contribute to the selection of individualized treatment programs for NSCLC.

Role of carbon in crystal structures of wild-type and mutated form of dihydrofolate reductase-thymidylate synthase of plasmodium falciparum.

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an important target of antimalarial drugs. A characteristic set of mutations in this bifunctional enzymatic protein leads to reduced competitive drug binding at the enzyme's active site. The carbon content distribution study at mutational sites and along the amino acid sequence of this important protein was carried out using carbon analysis tool CARBANA. The mutational sites at residues 16, 51, 59, 108, and 164 were investigated. The study reveals that the carbon content and distribution of A16V and S108N mutants is shifting towards a normal distribution of the carbon content which is symmetrical about 0.3145, conforming to the value for stable and ordered protein. The study also reveals that carbon distribution of PfDHFR-TS mutant protein is maintained at 31.45% of carbon all along the sequence. The hydrophobicity of the entire sequences also balances quite well at the optimum position and carbon is the only element contributing towards this stability. Thus, the study of carbon distribution in mutations of PfDHFR-TS is the most significant step towards understanding the biological features which can provide possible approaches for the design of new drugs to overcome antifolate resistance.

Association between variable number of tandem repeat polymorphism of thymidylate synthase gene and efficacy of pemetrexed in the treatment of advanced non-small cell lung cancer / 肿瘤

Objective: To investigate the association between variable number of tandem repeat (VNTR) polymorphism in 5'-untranslation region (5'-UTR) of thymidylate synthase (TS ) gene and the efficacy of pemetrexed-based treatment in non-small cell lung cancer (NSCLC). Methods: Peripheral blood samples were collected from 90 patients with advanced NSCLC and the genomic DNAs were extracted from these blood samples. The VNTR polymorphism in 5'-UTR of TS gene was detected by fluorescent labeled PCR and multiplex short tandem repeat polymerase chain reaction in combination with capillary electrophoresis. Results: The homozygosis (3R/3R and 2R/2R) and heterozygosis (2R/3R and 3R/4R) of VNTR polymorphism in 5'-UTR of TS gene were not associated with the efficacy of pemetrexed. The progression-free survivals, disease control rates and the objective response rates in patients harboring homozygosis (3R/3R and 2R/2R) and heterozygosis (2R/3R and 3R/4R) were 112 and 94 d (P = 0.549), 64.3% and 67.6% (P = 0.745), and 12.5% and 8.8% (P = 0.591), respectively. The patients harboring 2R alleles had similar clinical outcomes as compared with those harboring 3R alleles (progression-free survival: P = 0.518; disease control rate: P = 0.631; objective response rate: P = 0.541). Conclusion: TS gene VNTR polymorphism may not be associated with clinical outcomes of advanced NSCLC treated with pemetrexed. Copyright © 2012 by TUMOR.

A practical approach for assessing chemosensitivity in colorectal cancer cell lines by comparative analysis of cell viability and thymidylate synthase mRNA expression / 대한외과학회지

PURPOSE: The purpose of this study is to suggest a probable problem in chemosensitivity tests performed in practice and to speculate on practicable measures for more accurate chemosensitivity evaluation. METHODS: Three colorectal cancer cells (RSC, RRC1, and RRC2) were treated with 5-fluorouracil (5-FU). Inhibition percentage (%inhibition) of cancer cells and relative quantitation of thymidylate synthase (TS) mRNA were measured on day 2, day 5 after replacement of 70% media on day 2, day 7, and day 3 after replacement of all media on day 7. Doses that produced 50% inhibition (Dm) were calculated to evaluate drug effect. Relative quantitation of TS mRNA and correlations between TS mRNA levels and 5-FU concentrations were analyzed. RESULTS: RRC1 was more resistant than RRC2 on day 7, but Dm value of RRC2 increased three days after replacement of media from 12.3 to 18.1. Mean TS mRNA levels of RSC on D2 and D7 were significantly lower than those of RRC1 and RRC2, respectively (P = 0.004, P = 0.004 on D2; P = 0.010, P = 0.006 on D7). TS mRNA levels in RRC1 were significantly reversely correlated with 5-FU concentrations on day 2 (correlation coefficient = -0.867, P = 0.015). On the other hand, correlations were not significant in RRC2 (r = 0.067). CONCLUSION: Evaluating %inhibition of cancer cells at one point in chemosensitivity tests seems to be inadequate in determining chemotherapeutic regimens. Multilateral approaches, such as trials evaluating cancer cell survival before and after media replacement and correlations between TS mRNA levels and 5-FU concentrations, needs to be implemented for the practical application of chemosensitivity tests.

Expression of thymidylate synthase in bladder urothelial carcinoma and its prognostic value / 中国综合临床

Objective To investigate the expression of thymidylate synthase (TS) in bladder urothelial carcinoma(BUC) and its clinical significance, and to evaluate its prognostic value, thus to help predict the prognosis and choose therapy protocal. Methods The expression of TS was assessed by immunohistochemistry in 54 BUC and 15 normal bladder specimens, and all these clinical data were retrospectively analyzed after surgery. Expression of TS,clinicopathologic findings and two prognostic variances (relapse-free survival (RFS) and overall survival (OS)) were determined. The prognostic value of TS was estimated by survival curve and Cox proportional hazards model. Results Immunohistochemistry showed that 25 of 54 BUC cases (46. 30%) had high TS expression. In these cases,there was 29. 41%o (5/17) for G1 ,46. 43% (13/28) for G2,77. 78% (7/9) for G3 ;21.43% (3/14) for Ta, 26. 32% (5/19) for T1, 86. 67% ( 13/15 ) for T2,66.67% (2/3) for T3,66.67% (2/3) for T4. Expression of TS was positively associated with the grade and stage of carcinorma ( Kruskal - Wallis H, P = 0. 0 3 and P ＜ 0. 0 0 1 ). The recurrence and case - fatality of patients with high TS expression was 60. 00% (15/25) and 44. 00% (11/25) ,respectively. The survival analysis and Cox regression analysis showed that expression of TS were independent predictor for recurrence ( RR = 2. 65,95% CI was 0.80～8.81,P＜0.05) anddeath (RR=1.42,95%CIwas0. 36～5.58,P＜0. 05). Conclusions The probability of BUC progress and recurrence increases with the increasing of TS expression. Detection of TS expressing level can contribute to select appropriate treatment and follow-up schema.

Relationship between the Expression of Thymidylate Synthase,Thymidine Phosphorylase and Dihydropyrimidine Dehydrogenase and Survival in Epithelial Ovarian Cancer / 华中科技大学学报（医学）（英德文版）

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,TE 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.