Thymidylate Synthase Polymorphism and Microsatellite Instability: Association in Colorectal Cancer

5‐Fluorouracil (5FU) is the main drug used for the treatment of colorectal cancer (CRC) and Thymidilate Synthase (TS) is its target enzyme. TS gene has regulatory tandemly repeated sequences in its 5′ and 3′untraslated region (5′–3′ UTR). CRC often shows a kind of genomic instability called Microsatellite Instability (MSI) that is associated with TS levels and survival. Our data show that the genotype 2R/2R (homozygosity for 2 tandem repeat sequences in the 5′UTR) is more frequently associated with MSI + and lower TS levels. More over we did not find any significant association between the 2R/3R (heterozygosity for 2 and 3 tandem repeat sequences in the 5′UTR) and 3R/3R (homozygosity for 3 tandem repeat sequences in the 5′ UTR) genotypes with the MSI + and MSI ?, while these genotypes were associated with a higher TS expression. As a consequence we can hypothesise that patients bearing CRC with the MSI +, the 2R/2R genotype and with low TS levels could have a better prognosis and they could not be drug resistant.     

Dynamic Modulation of Thymidylate Synthase Gene Expression and Fluorouracil Sensitivity in Human Colorectal Cancer Cells

Biomarkers have revolutionized cancer chemotherapy. However, many biomarker candidates are still in debate. In addition to clinical studies, a priori experimental approaches are needed. Thymidylate synthase (TS) expression is a long-standing candidate as a biomarker for 5-fluorouracil (5-FU) treatment of cancer patients. Using the Tet-OFF system and a human colorectal cancer cell line, DLD-1, we first constructed an in vitro system in which TS expression is dynamically controllable. Quantitative assays have elucidated that TS expression in the transformant was widely modulated, and that the dynamic range covered 15-fold of the basal level. 5-FU sensitivity of the transformant cells significantly increased in response to downregulated TS expression, although being not examined in the full dynamic range because of the doxycycline toxicity. Intriguingly, our in vitro data suggest that there is a linear relationship between TS expression and the 5-FU sensitivity in cells. Data obtained in a mouse model using transformant xenografts were highly parallel to those obtained in vitro. Thus, our in vitro and in vivo observations suggest that TS expression is a determinant of 5-FU sensitivity in cells, at least in this specific genetic background, and, therefore, support the possibility of TS expression as a biomarker for 5-FU-based cancer chemotherapy.     

Ethnic Variation in the Thymidylate Synthase Enhancer Region Polymorphism among Caucasian and Asian Populations

Thymidylate synthase (TS) regulates the production of DNA synthesis precursors and is an important target of cancer chemotherapy. A tandem repeat sequence in a TS promoter enhancer region (TSER) was recently identified. Polymorphic variation affected in vitro expression levels of the gene. We evaluated the influence of ethnicity on TSER genotype. Allele frequency was similar in Caucasian and Southwest Asian subjects. However, homozygous triple repeat subjects were twice as common in Chinese subjects (67%) than in Caucasian subjects (38%). This demonstrates significant ethnic variation in a TS gene regulatory element which may have significant impact on pyrimidine homeostasis and drug therapy.     

胸苷酸合成酶表达调控的分子机制

胸苷酸合成酶(thymidylate synthase,TS)是生物体内催化胸苷酸合成所必需的酶．多年来一直作为肿瘤化疗的重要靶酶。对TS基因调控机制的研究表明：基因扩增、转录、翻译和翻译后过程都参与了TS表达的调控。先前的研究表明：TS可与自身的mRNA结合形成TS-mRNA复合物,使mRNA翻译受阻,5-氟尿嘧啶(5-fluorouracil,5-FU)等抗代谢药物可与TS蛋白结合,结合后的复合物不能与TS mRNA作用,导致体内TS的表达升高,是肿瘤细胞产生抗药性的重要分子机制之一。现对TS基因表达调控研究进展、翻译调控与抗药性产生的分子机制进行综述。     

Thymidylate Synthase Protein and p53 mRNA Form an In Vivo Ribonucleoprotein Complex

A thymidylate synthase (TS)-ribonucleoprotein (RNP) complex composed of TS protein and the mRNA of the tumor suppressor gene p53 was isolated from cultured human colon cancer cells. RNA gel shift assays confirmed a specific interaction between TS protein and the protein-coding region of p53 mRNA, and in vitro translation studies demonstrated that this interaction resulted in the specific repression of p53 mRNA translation. To demonstrate the potential biological role of the TS protein-p53 mRNA interaction, Western immunoblot analysis revealed nearly undetectable levels of p53 protein in TS-overexpressing human colon cancer H630-R10 and rat hepatoma H35(F/F) cell lines compared to the levels in their respective parent H630 and H35 cell lines. Polysome analysis revealed that the p53 mRNA was associated with higher-molecular-weight polysomes in H35 cells compared to H35(F/F) cells. While the level of p53 mRNA expression was identical in parent and TS-overexpressing cell lines, the level of p53 RNA bound to TS in the form of RNP complexes was significantly higher in TS-overexpressing cells. The effect of TS on p53 expression was also investigated with human colon cancer RKO cells by use of a tetracycline-inducible system. Treatment of RKO cells with a tetracycline derivative, doxycycline, resulted in 15-fold-induced expression of TS protein and nearly complete suppression of p53 protein expression. However, p53 mRNA levels were identical in transfected RKO cells in the absence and presence of doxycycline. Taken together, these findings suggest that TS regulates the expression of p53 at the translational level. This study identifies a novel pathway for regulating p53 gene expression and expands current understanding of the potential role of TS as a regulator of cellular gene expression.     

Polymorphism of the thymidylate synthase gene and thymidylate synthase levels in colon cancer cell lines and different tissues of colorectal cancer patients

In a panel of 18 colon cancer cell lines we found that the thymidylate synthase (TS) genotype was related to TS enzyme activity, but not to TS protein and mRNA levels. In addition, no relation with drug sensitivity was observed. TS genotyping of different tissues from 78 colorectal cancer patients revealed a high level of homology in polymorphic status between normal and malignant tissues and the heterozygous genotype to be the most frequent.     

中国淋巴囊肿病毒胸苷酸合酶基因结构特点及分析

胸苷酸合酶(Thymidylate synthase,TS)是进行DNA合成所必需的酶类,与细胞分化及肿瘤发生密切相关。淋巴囊肿病毒属于虹彩病毒科成员,是能引起百余种淡、海水鱼感染,并产生肿瘤的病毒病原。在已完成中国淋巴囊肿病毒株(Lymphocystis disease virus-China,LCDV-C)基因组序列测定的基础上,本文对位于LCDV-C基因组开放阅读框ORF011L的TS基因结构、及其推定蛋白结构进行了分析。该基因全长858bp,GC含量为28．2％,编码一个长为286aa、分子量为32．7kD、等电点为7．1的推定蛋白,称之为中国淋巴囊肿病毒胸苷酸合酶(LCDV-CTS)。该酶所具有的叶酸结合区在第44和70位氨基酸之间,dUMP结合区在第163和206位氨基酸之间,24个必需氨基酸具有高度保守性。二级结构预测结果显示LCDV-C TS含8个a螺旋,6个β折叠和23个环,表明其具备酶活性分子所有的柔性和可变性结构特征。这是迄今所知在脊椎动物虹彩病毒中唯一含两个完整结合区的TS。对来自包括LCDV在内二十个物种的TS结构进行同源性分析,显示LCDV-C TS被单独分为一枝。进一步对LCDV-C TS可能的起源途径及与宿主的作用等进行了探讨。     

Elevated Levels of Thymidylate Synthase Linked to Neoplastic Transformation of Mammalian Cells

Thymidylate synthase (TS), an enzyme that is essential for DNA synthesis and repair has been identified as an important biomarker for colorectal and other human cancers. The elevated steady-state levels of TS found in many common human malignancies have been thought to represent a secondary event in tumor formation. However, it has recently been demonstrated that the deregulated levels of ectopic TS may also have a causal effect on tumorgenesis since overexpression of human TS transforms immortalized mammalian cells to a malignant phenotype. Since the levels of TS are regulated by E2F-1 and thus are linked to the cell cycle pathway, regulating TS activity may be an important factor for the control of cell cycle progression and for the development of therapeutic strategies and cancer prevention.     

Functional Inactivity and Mutations of p53 Differentially Affect Sensitivity to 5-Fluorouracil and Antifolate Inhibitors of Thymidylate Synthase (TS) by Altering TS Levels in Colorectal Cancer Cells

The role of p53 in altering TS expression and chemosensitivity was studied in colorectal cancer cells with wildtype, mutated, or functionally inactive p53. Cytotoxicity of TS inhibitors was studied by MTT, while PCR, Western blot, and activity assays assessed whether p53 status influenced TS expression. Lovo-175X2 cells showed increased resistance to TS inhibitors and significantly greater than wildtype expression and activity of TS. In contrast, Lovo-273X17 and Lovo-li were more sensitive to TS inhibitors and had reduced TS expression, due either to reduced TS mRNA or altered regulation of TS activity. Thus, functional inactivity and mutations of p53 differentially affect TS, potentially influencing response to TS inhibitor-based treatment.     

Difference in Ki67 and Thymidylate Synthase Expression in Primary Tumour Compared with Metastatic Nodes in Breast Cancer Patients

Breast cancer is a heterogeneous disease, so therapeutic predictive biological markers need to be identified. To date an accurate evaluation of predictive markers is mainly done at the primary site; however, the main goal of adjuvant therapy for breast cancer is the control of micrometastases. The aim of this study is to assess as therapeutic and/or prognostic marker, the proliferation status of primary tumors and involved nodes as measured by Ki67 and thymidylate synthase (TS) expression, in 30 breast cancer node positive patients. TS is the main target of 5-fluorouracil (5-FU) activity, and its overexpression is one of the mechanisms of 5-FU drug resistance; however, in some studies its absence is responsible for a worse response to 5-FU. Our results show that malignant cells of involved nodes were in a post mitotic phase of the cell cycle, and show a low proliferation index and TS expression, while the primary tumours and controls, were strongly positive. On these basis we can hypothesize that these cells could be less sensitive to 5-FU. Further studies are necessary to identify other mechanisms responsible for their metastasing capability and/or for their aggressiveness.     

斑马鱼胸苷酸合成酶与自身mRNA的相互作用

斑马鱼(zebrafish,Danio rerio)是生物学领域中公认的研究脊椎类生物的模式生物.胸苷酸合成酶(thymidylate synthase,TS)是DNA从头合成的限速酶,多年来一直作为肿瘤化疗的重要靶酶.前期的研究表明,人和大肠杆菌中TS能与自身的mRNA结合,在翻译水平上具有反馈抑制自调控现象.斑马鱼作为药物模型的研究已成为热点研究领域,为了探讨斑马鱼的胸苷酸合成酶的调控规律,以及与人TS的相关性,利用原核表达,纯化获得高均一性斑马鱼TS蛋白,采用凝胶迁移研究了TS和其mRNA的体外结合,采用免疫共沉淀:RT-PCR技术研究了它们在体内的相互作用,实验结果表明,斑马鱼的TS在体内外均与自身的mRNA存在特异性的相互作用.研究说明,斑马鱼和人的TS具有高度生物学过程相关性,为构建斑马鱼抗肿瘤药理模型提供了理论基础.     

Intracellular Thymidylate Synthase Inhibition by Trifluorothymidine in FM3A Cells

Trifluorothymidine (TFT) can be phosphorylated by thymidine kinase (TK) to TFTMP which can inhibit thymidylate synthase (TS), resulting in depletion of thymidine nucleotides. TFT can be degraded by thymidine phosphorylase (TP) which can be inhibited by thymidine phosphorylase inhibitor (TPI). Using the TS in situ Inhibition Assay (TSIA) FM3A breast cancer cells were exposed 4 h or 24 h to TFT and 5‐Fluorouracil (5FU). TS activity reduced to 9% (0.1 µM TFT) and 58% (1 µM 5FU) after 4 h exposure and to 6% (TFT) and 21% (5FU) after 24 h exposure. TPI did not affect TS inhibition by TFT. FM3A cells lacking TK or TS activity (FM3A/TK^?) were far less sensitive to TFT compared to FM3A cells. Conclusion: TFT can be taken up and activated very rapidly by FM3A cancer cells, probably due to favourable TK enzyme properties, and TPI did not influence this.     

Thymidylate synthase polymorphism and microsatellite instability: association in colorectal cancer

5-Fluorouracil (5FU) is the main drug used for the treatment of colorectal cancer (CRC) and Thymidilate Synthase (TS) is its target enzyme. TS gene has regulatory tandemly repeated sequences in its 5' and 3'untraslated region (5'-3' UTR). CRC often shows a kind of genomic instability called Microsatellite Instability (MSI) that is associated with TS levels and survival. Our data show that the genotype 2R/2R (homozygosity for 2 tandem repeat sequences in the 5'UTR) is more frequently associated with MSI+ and lower TS levels. More over we did not find any significant association between the 2R/3R (heterozygosity for 2 and 3 tandem repeat sequences in the 5'UTR) and 3R/3R (homozygosity for 3 tandem repeat sequences in the 5' UTR) genotypes with the MSI+ and MSI-, while these genotypes were associated with a higher TS expression. As a consequence we can hypothesise that patients bearing CRC with the MSI+, the 2R/2R genotype and with low TS levels could have a better prognosis and they could not be drug resistant.     

Lapatinib,a Dual EGFR and HER2 Tyrosine Kinase Inhibitor,Downregulates Thymidylate Synthase by Inhibiting the Nuclear Translocation of EGFR and HER2

Background

Epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) has been shown to exert a synergistic antitumor effect when combined with fluoropyrimidine. This synergy may be attributable to the downregulation of thymidylate synthase (TS), which is frequently overexpressed in fluoropyrimidine-resistant cancer cells. However, the molecular mechanism underlying the downregulation of TS has yet to be clearly elucidated.

Methodology and Principal Findings

In this study, we demonstrate that lapatinib, a dual TKI of EGFR and HER2 downregulates TS via inhibition of the nuclear translocation of EGFR and HER2. From our cDNA microarray experiments, we determined that a variety of nucleotide synthesis-related genes, including TS, were downregulated with lapatinib, and this was apparent in HER2-amplified cells. Targeted and pharmacologic inhibition assays confirmed that the dual inhibition of EGFR and HER2 is required for the more effective reduction of TS as compared to what was observed with gefitinib or trasutuzumab alone. Additionally, we determined that co-transfected EGFR and HER2 activate the TS gene promoter more profoundly than do either EGFR or HER2 alone. The translocation of EGFR and HER2 into the nucleus and the subsequent activation of the TS promoter were inhibited by lapatinib.

Conclusions and Significance

These results demonstrate that lapatinib inhibits the nuclear translocation of EGFR and HER2 and downregulates TS, thus sensitizing cancer cells to fluoropyrimidine.     

Polymorphism of the Thymidylate Synthase Gene of Pneumocystis carinii from Different Host Species

Pneumocystis carinii is an opportunistic agent found in the lung of various mammals which often causes severe pneumonia in immunocompromised humans, especially in AIDS patients. In the past several years significant additions have been made to the collection of knowledge we have concerning the genetic diversity of P. carinii. These additions provide new understanding of Pneumocystis transmission and the effect of possible reservoirs of Pneumocystis in the various species. In this study, a 400-bp fragment of the thymidylate synthase (TS) gene of P. carinii has been amplified by PCR from 43 parasite isolates obtained from 4 mammalian host species: rat, mouse, rabbit and human. A probe selected from the TS gene sequence of rat-derived P. carinii was hybridized with the amplified products from rat- and mouse-derived P. carinii, but not with rabbit or human P. carinii DNA. Restriction profiles were performed on amplified fragments from all isolates, and the 4 nucleotide sequences of the TS gene fragment amplifed from rat, mouse, rabbit and human P. carinii were determined. Differences were detected in the gene fragment in P. carinii isolates from the 4 host species; however no difference was revealed in P. carinii isolates within a single host species, whatever the host strain or its geographic origin. Thus, the sequence differences of the P. carinii TS gene appeared as host-species specific. A specific probe which recognized all human P. carinii isolates was defined.     

Association of Thymidylate Synthase Polymorphisms with Acute Pancreatitis and/or Peripheral Neuropathy in HIV-Infected Patients on Stavudine-Based Therapy

Background

Low expression thymidylate synthase (TS) polymorphism has been associated with increased stavudine triphosphate intracellular (d4T-TP) levels and the lipodystrophy syndrome. The use of d4T has been associated with acute pancreatitis and peripheral neuropathy. However, no relationship has ever been proved between TS polymorphisms and pancreatitis and/or peripheral neuropathy.

Methods

We performed a case-control study to assess the relationship of TS and methylene-tetrahydrofolate reductase (MTHFR) gene polymorphisms with acute pancreatitis and/or peripheral neuropathy in patients exposed to d4T. Student’s t test, Pearson’s correlations, one-way ANOVA with Bonferroni correction and stepwise logistic regression analyses were done.

Results

Forty-three cases and 129 controls were studied. Eight patients (18.6%) had acute pancreatitis, and 35 (81.4%) had peripheral neuropathy. Prior AIDS was more frequent in cases than in controls (OR = 2.36; 95%CI 1.10–5.07, P = 0.0247). L7ow expression TS and MTHFR genotype associated with increased activity were more frequent in patients with acute pancreatitis and/or peripheral neuropathy than in controls (72.1% vs. 46.5%, OR = 2.97; 95%CI: 1.33–6.90, P = 0.0062, and 79.1% vs. 56.6%, OR = 2.90, 95%CI: 1.23–7.41, P = 0.0142, respectively). Independent positive or negative predictors for the development of d4T-associated pancreatitis and/or peripheral neuropathy were: combined TS and MTHFR genotypes (reference: A+A; P = 0.002; OR_A+B = 0.34 [95%CI: 0.08 to 1.44], OR_B+A = 3.38 [95%CI: 1.33 to 8.57], OR_B+B = 1.13 [95%CI: 0.34 to 3.71]), nadir CD4 cell count >200 cells/mm³ (OR = 0.38; 95%CI: 0.17–0.86, P = 0.021), and HALS (OR = 0.39 95%CI: 0.18–0.85, P = 0.018).

Conclusions

Low expression TS plus a MTHFR genotype associated with increased activity is associated with the development of peripheral neuropathy in d4T-exposed patients.     

Thymidylate synthase enhancer region polymorphism not related to susceptibility to acute lymphoblastic leukemia in the Kashmir population

Thymidylate synthase (TS) is a crucial enzyme in folate metabolism and plays a vital role in DNA synthesis and repair. The most common polymorphism in TS is a unique double (2R) or triple (3R) 28-bp tandem repeat sequence in the enhancer region of the TS gene (TSER). This genetic variation in TSER has been widely investigated and has been implicated as a risk factor for the development of various cancers, including acute lymphoblastic leukemia. It has also been found to influence sensitivity to anti-cancer drugs, such as methotrexate. We evaluated this polymorphism in acute lymphoblastic leukemia patients in the Kashmir population. In order to determine whether a double (2R2R) versus a triple (3R3R) 28-bp tandem repeat in the TSER modulates risk for acute lymphoblastic leukemia, 72 acute lymphoblastic leukemia cases and 144 age and gender matched, unrelated healthy individuals from the Kashmir region of India were evaluated for this polymorphism by PCR and direct sequencing. We found the frequency of the TS 2R allele to be 32.6 and 26.0%, in cases and controls, respectively. The TS 2R/2R genotype was found to be present in 15.27% of the cases and 9.72% of the controls, the 2R/3R variant in 34.72% of the cases and 32.63% of the controls, and the 3R/3R genotype in 50.0% of the cases and 57.63% of the controls. There was a significant association between the TS 2R/2R genotype and gender of acute lymphoblastic leukemia patients with males harboring the 2R/2R genotype exhibiting a higher risk of developing acute lymphoblastic leukemia than females (P = 0.009) We concluded that the TSER polymorphism appears not to be a risk factor for susceptibility to acute lymphoblastic leukemia in the Kashmir population.     

Thymidylate synthase predicts for clinical outcome in invasive breast cancer

Thymidylate synthase (TS) is a major target of 5-fluorouracil (5-FU) and dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme in the degradation of 5-FU. Whether TS or DPD could be used as valuable parameters for 5-FU sensitivity in clinical patients are largely unknown. We analyzed TS and DPD expression in breast carcinomas to evaluate the clinicopathological significance of these enzymes in patients with invasive breast cancer receiving 5-FU-based chemotherapy. A total of 197 patients with invasive ductal carcinoma were included in our study. Both the TS and DPD expression were analyzed using immunohistochemical method for all the surgical samples. Sixty-three out of 197 (31.97%) patients are positive for TS expression, and 77 out of 197 (39.09%) patients are positive for DPD expression. TS expression was not correlated with DPD expression. Patients with TS-positivity had aggressive phenotype including large tumor size, low differentiation and nodal metastasis. DPD expression is not related with phenotype or prognosis. Multivariate analysis demonstrated that TS expression was an independent prognostic factor for both disease-free and overall survival. The current study demonstrated that TS but not DPD expression was associated with both progression and prognosis in breast cancer receiving 5-FU-based chemotherapy. TS expression in the primary tumor might be useful as a predictive parameter for the efficacy of 5-FU-based chemotherapy for breast cancer.     

Probing the Thymidylate Synthase Active site with Bisubstrate Analog Inhibitors

Abstract

Earlier reports on the enhancement of folate analog binding to thymidylate synthase (TS) by N¹⁰-propargylation prompted the synthesis of a bisubstrate analog containing an 8-deaza-N¹⁰-propargyl-5,6,7,8-tetrahydrofolate moiety coupled through a methylene linkage to 2′-deoxyuridylate.