Thymidylate Synthase mRNA Research Articles

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.

Evaluation of sensitivity to 5-FU on the basis of thymidylate synthase (TS)/dihydropyrimidine dehydrogenase(DPD) activity and chromosomal analysis in micro tissue specimens of breast cancer

Preoperative assessment of the anticancer drug sensitivity of tumors plays an important role in the selection of therapy. If evaluation of the 5-FU sensitivity of microtissue specimens obtained by techniques such as core needle biopsy could be performed, the addition of fluorouracil to adriamycin and cyclophosphamide may further enhance response rates. In order to evaluate a simple sensitivity test for the anti-tumor agent 5-fluorouracil (5-FU), we examined whether an assay of a small sample could measure mRNA to predict the activities of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD). In addition, gene abnormalities on chromosomes 1 and 18 corresponding to DPD, TS and the relationships between the gene abnormalities and the amount of mRNA and activity were examined. TS and DPD activity were measured using the fluorodeoxyuridine monophosphate ligand binding assay and radio enzymatic assay, respectively, while mRNA levels were assayed by real-time polymerase chain reaction. Chromosome 1 and 18 aberrations were investigated by fluorescence in situ hybridization (FISH) with centromere probes. TS mRNA and TS activity showed a positive correlation (r=0.518, p=0.0017). TS activity and TS mRNA were significantly higher in the nuclear grade 3 group than in the other groups (p=0.04, p=0.0072, respectively). TS activity and mRNA in tumor tissue tended to decrease in the progesterone receptor positive groups (p=0.059, p=0.066, respectively). There was no correlation between DPD mRNA and DPD activity in tumor tissue (r=0.139, p=0.4423). DPD mRNA was measured as 282.88+/-170.68 copies/cell in tumor tissue and 635.88+/-310.04 copies/cell in normal tissue, and was thus significantly higher in normal tissue (p<0.001). TS mRNA showed a positive correlation with TS activity, suggesting that this method of using small amounts of tissue can replace anti-cancer drug sensitivity tests.

Thymidylate synthase and dihydropyrimidine dehydrogenase gene expression in breast cancer predicts 5-FU sensitivity by a histocultural drug sensitivity test

Thymidylate synthase (TS), Dihydropyrimidine dehydrogenase (DPD) and Thymidine Phosphorylase (TP) gene expressions are reported to be predictive markers for 5-fluorouracil (5-FU) sensitivity in gastrointestinal cancer. However, in breast cancer, it is still controversial whether those molecular markers predict 5-FU sensitivity or not. One possible reason for the difficulty may be the histological heterogeneity in breast cancer specimens. In this study, TS, DPD and TP mRNA expression in 40 breast cancer tumors were semi-quantified separately in cancer cells (Ca), cancerous stroma (Str) and normal glands (Nor) using laser capture microdissection and real time RT-PCR (LCM+RT-PCR). The histoculture drug response assay (HDRA) for 5-FU sensitivity was performed for 22 tumors. TS and TP mRNA expressions were higher in Ca than Str, although DPD gene expression was lower in Ca than Str. The group of high TS and high DPD gene expression in Ca was resistant to 5-FU, and the group of low TS and low DPD gene expression in Ca was sensitive to 5-FU ( P=0.048 chi-square test). TS and DPD mRNA expressions measured using LCM+RT-PCR might be useful predictive markers for 5-FU sensitivity in human breast cancer.

Simultaneous detection of variable number tandem repeats, single nucleotide polymorphisms, and allelic imbalance in the thymidylate synthase gene enhancer region using denaturing high-performance liquid chromatography

Polymorphisms in the thymidylate synthase enhancer region (TSER) have been reported to be associated with alterations in thymidylate synthase (TS) mRNA protein levels. The TSER is characterized by the presence of variable double (2R) and triple (3R) number tandem repeats (VNTRs). In addition to VNTRs, single nucleotide polymorphisms (SNPs) and allelic imbalance (AI), including loss of heterozygosity (LOH), have recently been associated with response to 5-fluorouracil (5-FU)-based chemotherapy. The aim of the current study was to develop a specific denaturing high-performance liquid chromatography (DHPLC) method for the rapid detection of these variations in the TSER in clinical samples. DHPLC analysis was validated in parallel with agarose gel electrophoresis (AGE), enzyme digestion, and quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR). The optimized DHPLC method resolved 100% of the known TSER variations, differentiated between homozygous and heterozygous genotypes, and allowed the qualitative and quantitative detection of AI, including LOH, in tumor samples. This DHPLC method was developed to permit the rapid, sensitive, and accurate identification of the TSER genotype (VNTRs, SNPs, and AI) in clinical protocols where response to flouropyrimidines may be correlated with TSER polymorphisms.

Combined therapy of locally advanced rectal adenocarcinoma with capecitabine and concurrent radiotherapy

3720 Background: Concomitant chemoradiotherapy with capecitabine could improve outcome in: patients with locoregionally advanced undisseminated rectal cancer. Methods: Since X/2001 to V/2003 capecitabine and concurrent radiotherapy was applied in locally advanced rectal cancer (cT3–4, and/or N+, M0). 43 patients, 31 men, 12 women, median age 59. Capecitabine 825 mg/m2 twice a daily was given concomitantly with pelvic radiotherapy 1,8 Gy daily up to 45 Gy followed by a boost up to 50,4 Gy. Results: Pathological complete response in 9(21%) pts, minimal microscopic residuum in 7(16%) pts and together 37% pts. with a substantial remission were diagnosed. Thymidylate synthase (TS), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD) and TNFα mRNA/GAPDH mRNA relative levels from tumor and adjacent tissue has been measured by means of Real Time PCR. Before treatment TS mRNA median was higher in tumors (1.7x) than in adjacent mucosa. Changes in mRNA levels in the course of therapy were different in tumor and adjacent non affected mucosa. TS mRNA median level in tumors increased 1,3x in the course of therapy, but median in adjacent mucosa increased 2,3x. Tumor TS mRNA median in the course of therapy was 1.5x higher in responders (pT0,N0 or pR1,N0) than in non responders. Tumor TP mRNA median levels increased 3.09x in all pts, but only 2.63x in patients with remission. There was an 2.56x increase of mucosal TP mRNA in total, but negligible (1.004x) in patients with remissions in the course of therapy. Median tumor DPD mRNA level of non responders is 3.9x higher than in responders before therapy or 3.5x higher in the course of therapy respectively. Tumor TNFα mRNA median level is increased 2.8x in responders but 4.32x in non responders. Conclusions: The concurrent chemoradiotherapy proved to be effective for treatment of locally advanced rectal cancer. The analysis shows the increased level of TS mRNA in tumor tissue and elevation of TP mRNA in tumor during radiotherapy in all pts. But the evaluated markes of fluoropyrimidine metabolism don't correlate with therapeutic response. No significant financial relationships to disclose.

Thymidylate synthase polymorphism in the development of Barrett's associated adenocarcinoma

9528 Background: Thymidylate synthase (TS), one of the rate-limiting enzymes in the de novo synthesis of DNA, has a unique 28bp tandemly repeated sequence in the promoter region. Patients who are homozygous for the double repeat (2R/2R) of this sequence have been reported to have lower TS expression and better outcome from chemotherapy than patients who are homozygous for the triple repeat (3R/3R). However, loss of heterozygosity (LOH) at the TS locus during tumor development can cause the heterozygous (2R/3R) patients to acquire either the 2R/loss or the loss/3R genotype in their cancer cells, which could affect tumor TS expression as well as clinical outcome. The aim of this study was 1) to verify the relationship between TS mRNA expression and TS genotype in Barrett's associated adenocarcinoma (BA), and 2) to analyze the frequency and timing of LOH at the TS locus in BA and its precursory lesion such as intestinal metaplasia (IM) and high grade dysplasia (HGD). Methods: Sixty-six patients (including 21 with IM, 6 with HGD and 23 with BA) were included in this study. Biopsies were obtained during endoscopy from: (1) normal squamous epithelium at 20cm (HNE); (2) normal squamous epithelium 3cm above the gastroesophageal junction (LNE); (3) IM, HGD or BA. All samples were analyzed for the TS genotype and TS mRNA expression using a quantitative real-time RT-PCR method after laser-capture microdissection. Results: TS mRNA expression from HNE tissue of 3R/3R genotype patients (n=8) was significantly higher than that of 2R/2R genotype patients (1.8-fold; n=14; p=0.0024) and that of 2R/3R genotype patients (1.4-fold; n=44; p=0.0282). Out of 44 patients with heterozygous (2R/3R) genotype in the HNE, no patient lost an allele in the LNE. However, 10 of 18 patients with IM had LOH (55.6%), as had 4/6 patients with HGD (66.7%) and 9/17 patients with BA (52.9%). Conclusions: Our results demonstrate 1) an association between TS genotype and TS expression in normal squamous epithelium and 2) that LOH at the TS locus is a frequent and early event in the IM-BA sequence, and thus tumor and normal genotypes will not be the same in these heterozygous patients. No significant financial relationships to disclose.

Assessment of multiple markers for association with response rate (RR) and failure-free survival (FFS) in patients with advanced colorectal cancer (CRC) treated with chemotherapy in the MRC CR08 (FOCUS) randomized trial

9506 Background: We have assessed potential prognostic and predictive markers of response to fluorouracil (FU) in advanced CRC patients randomized 4:1:1 to FU alone, FU+oxaliplatin, or FU+irinotecan. Methods: Pathology specimens were retrieved from 846 patients in the FOCUS trial. 85% were from the primary site. Normal and tumor DNA and RNA were extracted and tissue microarrays were made for immunohistochemistry (IHC). The following factors were assessed for effect on FFS and RR to first-line therapy: age; performance status; primary site; liver metastases; serum ALP; grade; mucoid status; IHC for hMLH1, hMSH2, P53, SMAD4, dUTPase, DPD, thymidine phosphorylase (TP), thymidylate synthase (TS); TS mRNA expression; DNA polymorphisms in TS (TSER, TS1494), hMLH1 (–93 G→A), MTHFR (677 C→T). Results: Overall RR and median FFS to first-line therapy were 42% and 8 months respectively. The table shows markers with significant relationships to RR and FFS. Together markers explained 6% of the variability in outcome, with the IHC/histology markers contributing most. DNA polymorphisms showed no clear association with outcomes. Similar associations of marker status were found for patients treated with FU alone or FU+oxaliplatin/irinotecan. Using logistic regression, a predictive algorithm could separate tumors into groups with RR from 15% to 50% for treatment with FU alone and 25% to 70% for treatment with FU+oxaliplatin/irinotecan. Conclusions: Significant differences in RR have been identified for mucoid status, TS IHC and dUTPase IHC scores allowing development of a predictive algorithm for response to FU-containing chemotherapy. Associations with modest differences in FFS were also identified. Ongoing work is looking at markers specific for oxaliplatin or irinotecan benefit. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Aventis

Assessment of multiple markers for association with response rate (RR) and failure-free survival (FFS) in patients with advanced colorectal cancer (CRC) treated with chemotherapy in the MRC CR08 (FOCUS) randomized trial

9506 Background: We have assessed potential prognostic and predictive markers of response to fluorouracil (FU) in advanced CRC patients randomized 4:1:1 to FU alone, FU+oxaliplatin, or FU+irinotecan. Methods: Pathology specimens were retrieved from 846 patients in the FOCUS trial. 85% were from the primary site. Normal and tumor DNA and RNA were extracted and tissue microarrays were made for immunohistochemistry (IHC). The following factors were assessed for effect on FFS and RR to first-line therapy: age; performance status; primary site; liver metastases; serum ALP; grade; mucoid status; IHC for hMLH1, hMSH2, P53, SMAD4, dUTPase, DPD, thymidine phosphorylase (TP), thymidylate synthase (TS); TS mRNA expression; DNA polymorphisms in TS (TSER, TS1494), hMLH1 (–93 G→A), MTHFR (677 C→T). Results: Overall RR and median FFS to first-line therapy were 42% and 8 months respectively. The table shows markers with significant relationships to RR and FFS. Together markers explained 6% of the variability in outcome, with the IHC/histology markers contributing most. DNA polymorphisms showed no clear association with outcomes. Similar associations of marker status were found for patients treated with FU alone or FU+oxaliplatin/irinotecan. Using logistic regression, a predictive algorithm could separate tumors into groups with RR from 15% to 50% for treatment with FU alone and 25% to 70% for treatment with FU+oxaliplatin/irinotecan. Conclusions: Significant differences in RR have been identified for mucoid status, TS IHC and dUTPase IHC scores allowing development of a predictive algorithm for response to FU-containing chemotherapy. Associations with modest differences in FFS were also identified. Ongoing work is looking at markers specific for oxaliplatin or irinotecan benefit. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Aventis

A 6 bp polymorphism in the thymidylate synthase gene causes message instability and is associated with decreased intratumoral TS mRNA levels.

A 6 bp deletion polymorphism in the thymidylate synthase (TS) gene was investigated in order to determine its function. A luciferase system was used to investigate the function of the 6 bp/1494 polymorphism in vitro. A group of 43 patients with colorectal carcinoma were evaluated for the 6 bp/1494 polymorphism and for intratumoral TS mRNA levels in vivo. The 3'UTR of TS containing the +6 bp polymorphism resulted in an approximate 35% decrease in luciferase activity and mRNA levels, while the TS-3'UTR bearing the -6 bp deletion resulted in an approximate 70% decrease in luciferase activity and mRNA levels. The TS-3'UTR construct containing the -6 bp/1494 deletion also had a higher rate of message degradation compared to the +6 bp/1494 construct. Individuals homozygous for the insertion (+6 bp/+6 bp) had significantly higher TS mRNA levels compared to individuals that were homozygous for the deletion (-6 bp/-6 bp) (P < 0.007). We determined the frequency of the -6 bp/1494 deletion polymorphism to be 41% in non-Hispanic whites, 26% in Hispanic whites, 52% in African-Americans and 76% in Singapore Chinese. These results suggest that the -6 bp/1494 deletion polymorphism in the 3'UTR of TS is associated with decreased mRNA stability in vitro and lower intratumoral TS expression in vivo. Further, the 6 bp/1494 polymorphism varies greatly within different ethnic populations and is in linkage disequilibrium with the TS 5' tandem repeat enhancer polymorphism. Taken together, these data suggest that the 6 bp/1494 polymorphism may be a useful screening tool in predicting TS mRNA expression.

Thymidylate synthase and dihydropyrimidine dehydrogenase mRNA levels in tumor tissues and the efficacy of 5-fluorouracil in patients with non-small-cell lung cancer

We examined 116 stage I–IIIA non-small-cell lung cancer (NSCLC) patients for intra-tumoral expression of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) using TaqMan reverse transcription polymerase chain reaction (RT-PCR) assay to clarify the correlation between gene expression and the efficacy of 5-fluorouracil (5-FU) in patients with NSCLC. Patients who were administered 5-FU alone after surgery comprised the 5-FU group ( n=30), and those who underwent only surgery comprised the control group ( n=86). When dichotomized at the mean TS and DPD mRNA level, patients with low-DPD tumors who were administered 5-FU had a significantly better prognosis than those who did not receive adjuvant treatment ( p=0.041). In addition, in the 5-FU group, 10 patients with both low-TS and low-DPD tumors have not had any relapse, whereas 8 of the 20 patients with either high-TS or high-DPD tumors developed distant metastasis after surgery. Based on these results, the quantitation of TS and DPD mRNA levels may predict the efficacy of 5-FU after surgery for patient with NSCLC.

Overexpression of E2F-1 in Lung and Liver Metastases of Human Colon Cancer is Associated with Gene Amplification

We have shown previously that metastatic tumors of human colorectal cancer (CRC) in lung as compared to liver have high levels of thymidylate synthase (TS) mRNA expression correlated with high levels of E2F-1 mRNA expression. We now report that Comparative Genomic Hybridization (CGH) and DNA PCR analyses of lung and liver metastases of human colon cancer show frequent gains in the region of chromosome 20q and have an increase in gene copy number of E2F-1. Six of 48 lung and liver metastases of human CRC (12.5%) had less than or equal to 1.5 fold increase in gene copies while the remainder (87.5%) had greater than 1.5 fold increase. The mean fold increase in E2F-1 gene copy number in the primary CRC samples (2.76, n=9) is lower than that seen in the metastatic CRC tumors (mean value 3.5 fold, n=48). Primary CRC tumors had significantly higher gene copy number for E2F-1 (mean E2F-1 gene copy number was 2.76 fold, n=9) than matched adjacent normal (mean =2.16, n=9, p=0.06 using paired t test). In as much as TS is transcriptionally regulated by E2F-1, these results provide an explanation for the high levels of TS mRNA noted in some tumor samples.

Thymidylate synthase as an oncogene: A novel role for an essential DNA synthesis enzyme

Thymidylate synthase (TS) is an E2F1-regulated enzyme that is essential for DNA synthesis and repair. TS protein and mRNA levels are elevated in many human cancers, and high TS levels have been correlated with poor prognosis in patients with colorectal, breast, cervical, bladder, kidney, and non-small cell lung cancers. In this study, we show that ectopic expression of catalytically active TS is sufficient to induce a transformed phenotype in mammalian cells as manifested by foci formation, anchorage independent growth, and tumor formation in nude mice. In contrast, comparable levels of two TS mutants carrying single point mutations within the catalytic domain had no transforming activity. In addition, we show that overexpression of TS results in apoptotic cell death following serum removal. These data demonstrate that TS exhibits oncogene-like activity and suggest a link between TS-regulated DNA synthesis and the induction of a neoplastic phenotype.

Associations between polymorphisms in the thymidylate synthase gene, the expression of thymidylate synthase mRNA and the microsatellite instability phenotype of colorectal cancer

Microsatellite instability (MSI) is a characteristic feature of up to 15% of colorectal cancers (CRC) and is associated with better response to adjuvant chemotherapy with 5-fluorouracil (5-FU). In this study we have investigated the association between the MSI status and the mRNA expression as well as the polymorphisms of the cellular target of 5-FU therapy, thymidylate synthase. Polymorphisms in the 3'- and the 5'-UTR of the TS gene were determined by a PCR assay in 53 colorectal cancer tissues. TS mRNA was quantified by real-time RT-PCR. Data were correlated with the MSI phenotype. There was neither a significant correlation between the polymorphisms in the TS gene and the MSI phenotype nor between the mRNA expression and MSI status. CRC with a 3R/3R or 2R/3R genotype showed a significantly higher TS mRNA expression than those with 2R/2R genotype (p=0.001 and p=0.026, respectively). No association was seen between the polymorphism of the 3'-UTR and mRNA expression. From our results, we conclude that there is no association between MSI status and TS expression. Samples containing the 3R/3R or 2R/3R genotype of TS seem to have higher mRNA levels perhaps due to a higher mRNA stability. Polymorphic variants of the 3'-UTR do not influence the TS mRNA level. Genotyping of the 5'-UTR and quantitation of TS mRNA levels might serve as predictors for the response to 5-FU based chemotherapy.

Down-regulation of thymidylate synthase expression and its steady-state mRNA by oxaliplatin in colon cancer cells.

Recently, evidence has accumulated that weekly 24-h infusion of high-dose 5-fluorouracil (5-FU) with leucovorin (LV, folinic acid) biochemical modulation may improve the response rates compared with the bolus 5-FU regimens in colorectal cancer (CRC). Combining the infusional 5-FU/LV (iFL) regimens with oxaliplatin or irinotecan is widely adopted to further improve treatment efficacy. Either oxaliplatin-iFL or irinotecan-iFL may achieve an overall response rate of more than 50% in the first-line treatment. Intriguingly, in the salvage treatment for metastatic CRC patients who had failed iFL, only oxaliplatin-iFL may achieve a response rate of about 13-25%. In contrast, oxaliplatin alone or irinotecan-iFL had a very low response rate of 5% or less. To test if the oxaliplatin may reverse the iFL-related 5-FU resistance in CRC, we used DLD-1 colon adenocarcinoma cells as the in vitro study model. First, we revealed that oxaliplatin and 5-FU act synergistically on DLD-1 cells by MTT cytotoxicity assay and median drug effect analysis. Second, we treated the DLD-1 cells with serial concentrations of oxaliplatin (0.1-10 microM). Oxaliplatin treatment results in down-regulation of free thymidylate synthase (TS) protein expression by Western blotting. Further, we analyzed the TS mRNA level by reverse transcription and real-time quantitative polymerase chain reaction assay. Oxaliplatin treatment results in down-regulation of the TS mRNA level up to 40% (mean +/- SD of ratio to reference control = 0.60 +/- 0.21, range 0.42-0.84). In this study, our data provide important information explaining the reason why the combination of oxaliplatin and 5-FU results in a better objective response in 5-FU-resistant patients than oxaliplatin alone does. Our data also suggest that TS down-regulation happens at the transcriptional level. TS modulation and down-regulation had, thus, shed light on the useful potential strategy to achieve objective responses in 5-FU-resistant CRC patients.

A longterm follow-up study of thymidylate synthase as a predictor for survival of patients with liver tumours receiving hepatic arterial infusion chemotherapy

Aims. Thymidylate synthase (TS) is a key-enzyme for DNA synthesis and targeted by fluoropyrimidines (FPs). High TS ratios are associated with resistance to systemic FP-based chemotherapy. The aim of this study was to report the influence of TS ratios on primary tumour response to FP-based HAI and long-term follow-up of patients with isolated non-resectable liver tumours in part from a previously published study. Methods. Fifty-one consecutive patients with liver tumours receiving HAI with available tumour tissue for TS quantitation were studied between 1991 and 2001. Liver metastases were from colorectal origin in 41 patients and other primary sites in 6 patients. Four patients had primary liver cancers. Tumour tissue was obtained at laparotomy for the intraarterial infusion device implantation. TS mRNA quantitation was performed by RT-PCR using β-actin as internal standard. Results. The median TS ratio was 2.2 with high variation among tumours ranging from 0.1 to 27. Twenty-two out of 51 patients responded to HAI. The median TS ratio of the responders was 1.6 and more than two-fold lower than the ratio of the non-responders with 3.3 ( p<0.01). In the subgroup with TS≤3.0, 18 out of 29 patients responded. In the subgroup with TS>3.0 only four out of 22 patients responded. No patients with very high TS ratios ≥4.5 ( n=13) responded to HAI. Median survival was 20 months (range: 3–109). Patients with TS-ratios ≤3.0 showed, with 55%, a higher 2-year survival rate than patients with TS-ratios >3.0 with 27%. Conclusion. TS seems to be a predictive and prognostic factor for patients with isolated non-resectable liver tumours receiving FP-based HAI. Patients with very high TS ratios do not seem to benefit from FP-based HAI.

Small interfering double-stranded RNAs as therapeutic molecules to restore chemosensitivity to thymidylate synthase inhibitor compounds.

RNA interference is a post-transcriptional mechanism by which double-stranded RNA specifically silence expression of a corresponding gene. Small interfering double-stranded RNA (siRNA) of 21-23 nucleotides can induce the process of RNA interference. Studies from our laboratory have shown that translation of thymidylate synthase (TS) mRNA is controlled by its own protein end-product TS in a negative autoregulatory manner. Disruption of this process gives rise to increased synthesis of TS and leads to the development of cellular drug resistance to TS-targeted compounds. As a strategy to inhibit TS expression at the mRNA level, siRNAs were designed to target nucleotides 1058-1077 on human TS mRNA. Transfection of TS1058 siRNA into human colon cancer RKO cells resulted in a dose-dependent inhibition of TS expression with an IC(50) value of 10 pM but had no effect on the expression of alpha-tubulin or topoisomerase I. Inhibition of TS expression by TS1058 was maximal at 48 h and remained suppressed for up to 5 days. Pretreatment of RKO cells with TS1058 siRNA suppressed TS protein induction following exposure to raltitrexed. In addition, TS1058 restored chemosensitivity of the resistant RKO-HTStet cell line to various TS inhibitor compounds. On treatment with TS1058, IC(50) values for raltitrexed, 1843U89, and 5-fluoro-2'-deoxyuridine decreased by approximately 15-16-fold. These studies suggest that TS-targeted siRNAs are effective inhibitors of TS expression and may have therapeutic potential by themselves or as chemosensitizers in combination with TS inhibitor compounds.

Trichinella spiralis thymidylate synthase: cDNA cloning and sequencing, and developmental pattern of mRNA expression.

The persistent expression of thymidylate synthase activity has previously been demonstrated not only in adult forms, but also in non-developing muscle larvae of Trichinella spiralis and T. pseudospiralis, pointing to an unusual pattern of cell cycle regulation, and prompting further studies on the developmental pattern of T. spiralis thymidylate synthase gene expression. The enzyme cDNA was cloned and sequenced, allowing the characterization of a single open reading frame of 307 amino acids coding for a putative protein of 35,582 Da molecular weight. The amino acid sequence of the parasite enzyme was analysed, the consensus phylogenetic tree built and its stability assessed. The aa sequence identity with thymidylate synthase was confirmed by the enzymatic activity of the recombinant protein expressed in E. coli. As compared with the enzyme purified from muscle larvae, it showed apparently similar Vmax value, but higher Km(app) values desscribing interactions with dUMP (28.8 microM vs. 3.9 microM) and (6RS,alphaS)-N(5,10)-methylenetetrahydrofolate (383 microM vs. 54.7 microM). With the coding region used as a probe, thymidylate synthase mRNA levels, relative to 18S rRNA, were found to be similar in muscle larvae, adult forms and newborn larvae, in agreement with muscle larvae cells being arrested in the cell cycle.

Gene expression of 5-fluorouracil metabolic enzymes in primary colorectal cancer and corresponding liver metastasis.

Expression of thymidylate synthase (TS) and the 5-fluorouracil (5-FU) metabolic enzymes, including dihydropyrimidine dehydrogenase (DPD), orotate phosphoribosyl transferase (OPRT), thymidine phosphorylase (TP), and uridine phosphorylase (UP), has been reported to be associated with the sensitivity to 5-FU-based chemotherapy in colorectal cancer. We evaluated the correlation of the expression of these genes between primary tumors and corresponding liver metastases. The mRNA levels of TS, DPD, OPRT, TP, and UP were measured by real-time quantitative RT-PCR in samples from 23 consecutive patients with both primary colorectal adenocarcinoma and liver metastasis. The DPD, OPRT, TP, and UP mRNA levels were significantly higher in liver metastases than in primary tumor (expression in relation to that of beta-actin mRNA: 0.42 vs 0.16, P=0.00053; 1.4 vs 0.92, P=0.016; 23 vs 11, P=0.00014; 0.36 vs 0.25, P=0.0026; respectively). However, the TS mRNA level did not differ significantly between liver metastases than primary tumor (0.20 vs 0.16, P=0.28). No correlation was observed for any gene between primary tumor and liver metastases. In both primary tumor and liver metastasis, the TS mRNA levels correlated significantly with the OPRT mRNA level (primary rS=0.83, P=0.00000081; liver metastasis rS=0.49, P=0.017), while the DPD mRNA level correlated significantly with the TP mRNA level rS=0.81, P=0.0000024; rS=0.63, P=0.0014; respectively). The differential gene expression of 5-FU metabolic enzymes between primary colorectal cancer and corresponding liver metastases should be taken into consideration when estimating the sensitivity to 5-FU-based chemotherapy in colorectal cancer. The gene expression of TS and OPRT, which are involved in de novo pyrimidine synthesis, and that of DPD and TP, may be coregulated.

Correlation of thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase with sensitivity of gastrointestinal cancer cells to 5-fluorouracil and 5-fluoro-2'-deoxyuridine.

To determine the expression levels of three metabolic enzymes of fluoropyrimidines: thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) in seven human gastrointestinal cancer cell lines, and to compare the enzyme levels with the sensitivity to 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FdUrd). TS, TP and DPD mRNA levels were assessed by semi-quantitative RT-PCR, TP and DPD protein contents were measured by ELISA. Fifty percent inhibitory concentrations of growth (IC50), representing the sensitivity to drugs, were determined by MTT assay. IC50 values ranged from 1.28 to 12.26 microM for 5-FU, and from 5.02 to 24.21 microM for FdUrd, respectively. Cell lines with lower DPD mRNA and protein levels tended to be more sensitive to 5-FU (P<0.05), but neither TS nor TP correlated with 5-FU IC50 (P>0.05). Only TS mRNA level was sharply related with FdUrd sensitivity (P<0.05), but TP and DPD were not (P>0.05). A correlation was found between mRNA and protein levels of DPD (P<0.05), but not TP (P<0.05). DPD and TS enzyme levels may be useful indicators in predicting the antitumor activity of 5-FU or FdUrd, respectively.

Intratumoral induction of thymidylate synthase mRNA by 5-FU in colorectal cancer patients: association with survival

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in 5-fluorouracil (5-FU) catabolism. Thymidylate synthase (TS) is inhibited to form an inactive ternary complex by 5-fluoro-dUMP and is considered to be a target enzyme of 5-FU treatment. Two enzymes, DPD and TS, have been reported to be major determinants of individual sensitivity to 5-FU, and it has been reported that TS mRNA levels are modified by 5-FU treatment. We investigated their impact on treatment efficacy in colorectal cancer patients. TS and DPD mRNA levels, which correlated to the corresponding enzyme activities, were quantified in tumor tissues before and after treatment in 40 advanced colorectal cancer patients who had been treated with Doxifluridine (5'-DFUR) for 14 days before surgery. Furthermore inter-individual variations of TS mRNA levels after 5-FU treatment were found, and the individual TS induction varied between patients (0.2-2.4). Increased TS mRNA levels were found in 19 out of 40 cases. The samples were divided into two groups according to their TS mRNA induction (< or >1; TS/beta-actin ratio after treatment divided by values prior treatment) and compared with tumor reduction and survival. TS and DPD mRNA levels in tumor biopsies before treatment were not related to 5-FU responses by histological evaluations in this study. However the efficacy of 5-FU treatment was enhanced in patients with no or low TS mRNA induction (odds ratio: 6.2, p<0.05). Furthermore, longer periods of survival were observed in the group without increased TS mRNA levels. These findings suggest that TS mRNA was induced by 5-FU treatment, and the overall induction level varied between individuals. Therefore, the estimation of TS mRNA induction may be useful to predict the efficacy of 5-FU treatment.