Elevated Thymidylate Synthase Research Articles

Abstract 2000: Role of O-GlcNAc conjugation and TS translational repression in 5-FU/LV resistance in CRC and CF10 response

Abstract Rationale: Fluoropyrimidine drugs (FPs; e.g., 5-FU) are widely used to treat colorectal cancer (CRC), the 3rd leading cause of cancer-related mortality. However, acquired resistance to 5-FU is a principal cause of treatment failure in the metastatic setting. Further, previous studies selected for cells resistant to single agent 5-FU however 5-FU is invariably used clinically in combination with the reduced folate leucovorin (LV). Our studies focus on resistance mechanisms upon treatment with 5-FU+LV in folate-restricted media and test if CF10, a novel polymeric FP being developed in our laboratory displays activity in CRC cells resistant to 5-FU+LV. Experimental: We selected CRC cells (HCT-116, LS174T, HCT15, MC38) resistant to 5-FU+LV by selecting for survival upon culture in folate-restricted media with stepwise increased concentrations of 5-FU+LV over 5 months. Since elevated thymidylate synthase (TS) levels were observed in multiple studies selecting CRC cells for 5-FU resistance, we performed Western blot on cell lysates from parental and (5-FU+LV)-R cells. Since Myc and p53 expression are regulated in part via translational inhibition by TS binding to these mRNAs we also compared Myc and p53 levels in parental and (5-FU+LV)-R cells by Western blot. Further, since TS, Myc, and p53 protein stability are all regulated in part via O-GlcNAc conjugation we tested if OGT levels were changed in (5-FU+LV)-R cells. Resistance factors for CF10 were also determined in (5-FU+LV)-R cells. Results: A surprising result was that basal TS levels were reduced in (5-FU+LV)-R cells relative to the parental cells. We also observed that TS levels were increased in both parental and (5-FU+LV)-R cells by 5-FU+LV treatment however the effect was much greater in (5-FU+LV)-R cells. We observed strongly decreased p53 and Myc levels in the (5-FU+LV)-R cells. CF10 treatment further decreased p53 and Myc levels in parental cells, but increased p53 and further decreased Myc in (5-FU+LV)-R cells. In contrast, 5-FU alone treatment in (5-FU+LV)-R cells increased Myc and decreased p53 levels and further decreased in combination with LV. CF10 retained strong activity in (5-FU+LV)-R cells. Conclusions: Overall, we have validated that cancer cells can be cultured under conditions with human-like folate levels and selected for resistance to 5-FU+LV under folate-restricted conditions. The causes of 5-FU+LV resistance are multi-factorial and include TS overexpression induced by 5-FU+LV treatment that may be mediated by O-GlcNAc conjugation. We demonstrated that CF10 is more potent than 5-FU, and importantly its potency advantage is enhanced in cells selected for 5-FU+LV resistance. Our studies support CF10 translation for treatment of metastatic CRC in patients with progressive disease following treatment with combination therapy that includes 5-FU+LV. Citation Format: Charles C. Okechukwu, Xue Ma, William H. Gmeiner. Role of O-GlcNAc conjugation and TS translational repression in 5-FU/LV resistance in CRC and CF10 response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2000.

Abstract 6178: A novel second-generation nano-fluoropyrimidine to treat metastatic colorectal cancer and overcome 5-fluorouracil resistance

Abstract Colorectal cancer (CRC) is the 3rd leading cause of cancer-related mortality. The mortality associated with colon cancer results notably from metastatic disease (mCRC), with a 5-year survival rate for patients with stage IV CRC being < 14%. Despite extensive efforts towards developing personalized therapies, limited success has been achieved and chemotherapeutic regimens that include the fluoropyrimidine (FP) drug 5-fluorouracil (5-FU) are central to patient management. While using 5-FU-based regimens such as FOLFOX and FOLFIRI in combination with biologics confer a survival benefit, their ineffectiveness at promoting long-term survival in a substantial fraction of mCRC patients and associated toxicities limits their use. This underscores the pressing need to develop improved FP drugs. To overcome the limitations of 5-FU that include resistance due to elevated expression of thymidylate synthase (TS) and inefficient anabolic metabolism to FdUMP, the TS-inhibitory/active metabolite, we are developing FP polymers consisting of single-stranded DNA with FdUMP (only active metabolite) as the repeating nucleotide unit. Our therapeutic objective is to initiate a Phase I clinical trial with a new 2nd generation nanoscale FP polymer (CF10) that demonstrates promising anticancer effects in several preclinical models and significantly lower systemic toxicity. We observed that CF10 displayed significantly higher anti-tumor activity than 5-FU in flank and syngeneic tumor models. Additionally, CF10 treatment improved survival (84.5 days vs 32 days; P < 0.0001) relative to 5-FU in an orthotopic HCT-116-luc colorectal cancer mice model that spontaneously metastasized to the liver. Interestingly, a reduction in metastatic tumor burden in the CF10 treatment group led us to hypothesize that CF10 could inhibit the TS-mediated EMT phenotype and pro-metastatic activities in colorectal cancer cells (CRCs). This was supported by increased vimentin, decreased E-cadherin expression, and increased cell migration and invasion in TS-overexpressing CRC cells (“Tet-on” system). Alkaline comet assay, immunofluorescence imaging, and immunoblot identified significantly higher replication stress with CF10 treatment than 5-FU in mouse, rat, and human-established CRC cell lines and primary CRC cells. Strikingly, we also discovered that CF10 attenuates the growth of TS-overexpressing primary colonoids. In conclusion, CF10 induces increased replication stress compared to a similar concentration of conventional drugs. Our findings suggest the significance of elevated TS in CRC metastatic progression and 5-FU resistance and demonstrate that CF10 may be effective at inhibiting CRC metastatic progression. CF10 may be a successful candidate for early-phase clinical trials to treat mCRC. Citation Format: Naresh Sah, Pamela Luna, Chinnadurai Mani, William Gmeiner, Komaraiah Palle. A novel second-generation nano-fluoropyrimidine to treat metastatic colorectal cancer and overcome 5-fluorouracil resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6178.

Thymidylate synthase accelerates Men1-mediated pancreatic tumor progression and reduces survival.

Clinical studies of cancer patients have shown that overexpression or amplification of thymidylate synthase (TS) correlates with a worse clinical outcome. We previously showed that elevated TS exhibits properties of an oncogene and promotes pancreatic neuroendocrine tumors (PanNETs) with a long latency. To study the causal impact of elevated TS levels in PanNETs, we generated a mouse model with elevated human TS (hTS) and conditional inactivation of the Men1 gene in pancreatic islet cells (hTS/Men1-/-). We demonstrated that increased hTS expression was associated with earlier tumor onset and accelerated PanNET development in comparison with control Men1-/- and Men1+/ΔN3-8 mice. We also observed a decrease in overall survival of hTS/Men1+/- and hTS/Men1-/- mice as compared with control mice. We showed that elevated hTS in Men1-deleted tumor cells enhanced cell proliferation, deregulated cell cycle kinetics, and was associated with a higher frequency of somatic mutations, DNA damage, and genomic instability. In addition, we analyzed the survival of 88 patients with PanNETs and observed that high TS protein expression independently predicted worse clinical outcomes. In summary, elevated hTS directly participates in promoting PanNET tumorigenesis with reduced survival in Men1-mutant background. This work will refocus attention on new strategies to inhibit TS activity for PanNET treatment.

Apigenin enhances apoptosis induction by 5-fluorouracil through regulation of thymidylate synthase in colorectal cancer cells

Although effective drugs have been developed, including 5-fluorouracil (5-FU), advanced colorectal cancer (CRC) shows low therapeutic sensitivity resulting from the development of 5-FU resistance. Thymidylate synthase (TS) is a target protein of 5-FU, and elevated TS lowers the 5-FU sensitivity of CRC cells. Here, we tested the efficacy of several candidate phytochemicals against human CRC-derived HCT116 cells expressing wild-type tumor suppressor protein P53 and HT29 cells expressing mutant P53. Among them, we found that apigenin enhanced the inhibitory effect of 5-FU on cell viability. In addition, apigenin inhibited the upregulation of TS induced by 5-FU. Apigenin also potentiated 5-FU-induced apoptosis of HCT116 cells and enhanced cell cycle disruption. Furthermore, apigenin increased reactive oxygen species production, intracellular and intramitochondrial Ca2+ concentrations, and mitochondrial membrane potential upon cotreatment with 5-FU. Knockdown of forkhead box protein M, a transcription factor modulating 5-FU sensitivity, enhanced the potentiation of apoptosis by apigenin in HCT116 cells. Moreover, apigenin suppressed TS expression and inhibited the viability of 5-FU-resistant HCT116 cells. Therefore, apigenin may improve the therapeutic efficacy of 5-FU against CRC by suppressing TS, but apoptosis induction is mainly dependent on functional P53.

A French multifactorial prospective study of tumor protein and genetic markers in stage I-III colorectal cancer (CRC): Highlight on molecular characteristics related to mismatch repair (MMR) status.

3596 Background: There is still a need to identify prognostic markers in stage II CRC for setting up adjuvant treatment. The prognostic value of tumor genetic and protein markers was analyzed in CRC patients, as well as relationships between markers. Given the strong prognostic and predictive value of deficient MMR (dMMR), we examined whether dMMR tumors had a distinct protein profile as compared to proficient (pMMR) tumors. Methods: This prospective multicentric study involved 251 stage I-II-III CRC patients with complete surgical resection. Primary end-point was disease free survival (DFS, 60 events, median follow-up 88 months). Biomarkers analyzed on frozen primary tumors were: MMR status (bat 25, bat 26), mutations of KRAS (codons 12-13), BRAF (V600E), PIK3CA (exons 9 and 20), APC (exon 15) and P53 (exons 4-9), CIMP status, ploidy, S-phase fraction, LOH (8p, 17p, 18q), EGFR (ligand-binding assay), VEGFA expression (Elisa), thymidylate synthase (TS) enzyme activity and expression (RT-PCR), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) expressions (RT-PCR). Results: 30 stages I, 116 stages II and 105 stages III were included (FUFOL adjuvant treatment in 30 stages II and 61 stages III). 14% were dMMR. Multivariate Cox analyses showed that tumor staging was the only significant predictor of DFS. Log Rank analyses restricted to stage III showed tendencies for a shorter DFS in KRAS-mutated (p=0.005), BRAF wt (p=0.009) and pMMR tumors (p=0.036). dMMR tumors significantly expressed elevated TS (median 3.1 vs 1.4) and TP (median 5.8 vs 3.5) expression relative to pMMR (p<0.001) and tended to express higher DPD expression (median 14.9 vs 7.9, p=0.027) and EGFR content (median 69 vs 38, p=0.037) relative to pMMR. Conclusions: The present data, suggesting for the first time that both TS (5FU target) and DPD (FU catabolism enzyme) are overexpressed in dMMR tumors, bring strong arguments to explain the resistance of dMMR CRC tumors to FU-based therapy. The fact that dMMR tumors tend to express elevated EGFR levels and are prone to be KRAS wt suggests that anti-EGFR may be a relevant therapy in these patients. Clinical trial information: 1997.CHUNice-948.

Enhancement of 5-fluorouracil-induced cytotoxicity by leucovorin in 5-fluorouracil-resistant gastric cancer cells with upregulated expression of thymidylate synthase

BackgroundElucidation of the mechanisms by which gastric cancer cells acquire resistance to 5-fluorouracil (5FU) may provide important clues to the development of effective chemotherapy for 5FU-resistant gastric cancerMethodsFour 5FU-resistant cell lines (MKN45/5FU, MKN74/5FU, NCI-N87/5FU, and KATOIII/5FU) were established by continuous exposure of the cells to progressively increasing concentrations of 5FU for about 1 year. Then, mRNA expression levels of four genes associated with 5FU metabolism, i.e., thymidylate synthase (TS), dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase, were quantitatively evaluated by real-time reverse transcriptase-polymerase chain reaction. In addition, TS protein expression was measured by Western blot analysis.ResultsAs compared with the parent cell lines, the 5FU-resistant cell lines showed 3.8- to 11.6-fold higher resistance to 5FU, as well as 1.9- to 3.5-fold higher TS mRNA expression and 1.6- to 7.1-fold higher TS protein expression. In contrast, the expressions of other genes did not differ significantly among the cell lines. The cytotoxicity of 5FU was enhanced 2.3- to 2.8 fold by leucovorin (LV) against three of the four 5FU-resistant cell lines.ConclusionsCollectively, LV enhanced the cytotoxicity of 5FU not only against the parent gastric cancer cell lines, but also against the 5FU-resistant cell lines, even those with elevated TS expression levels. These results suggest that clinical studies of a combination of 5FU and LV are warranted in patients who have recurrent gastric cancer after 5FU-based therapy.

Emerging role of thymidylate synthase for the pharmacogenomic selection of patients with thoracic cancer

The use of antifolate drugs, and particularly of pemetrexed, is emerging as a relevant therapeutic strategy for the treatment of some specific thoracic cancers. Pemetrexed is a novel multitargeted antifolate which has been shown in vitro to inhibit at least three different enzymes in the folate pathway: thymidylate synthase (TS), dihydrofolate reductase (DHFR) and glycinamide ribonucleotide formyl transferase (GARFT) of which TS is its main target [1]. These enzymes are involved in the synthesis of nucleotides and interference with their function will ultimately hinder the synthesis of RNA and DNA. Pre-clinical studies have demonstrated the cytotoxic activity of this agent in a broad range of tumor types including non-small cell lung cancer (NSCLC). In vitro studies indicated that tumor cell lines expressing high levels of TS or DHFR have reduced sensitivity to pemetrexed, suggesting that increased expression levels might correlate with reduced clinical efficacy [2]. In NSCLC, which accounts for more than 80% of all lung cancer, two recent independent Phase III trials showed that pemetrexed alone or in combination with cisplatin demonstrated a statistically superior activity in patients with non-squamous histotypes [3, 4]. Although histology has not consistently been associated with clinical outcomes in almost every trial testing the role of platinum-based chemotherapy in advanced NSCLC, it has now emerged as a potential predictive factor due to the evidence of the differential expression of TS between the different tumor histotypes, which seems to play an important role [3]. Preliminary molecular evidence, in fact, has previously shown that TS expression is significantly higher in squamous cell carcinoma compared with adenocarcinoma by means of both Real-Time PCR gene quantification and of immunohistochemistry for the detection of the protein levels [5]. In undifferentiated large cell carcinoma, a subsequent analysis consistently reported significantly higher TS levels in desmocollin 3 (DSC-3)-positive as compared to DSC-3negative tumors (indicating a squamous and adenocarcinoma origin of the tumors, respectively), reporting that lack of DSC-3 immunoreactivity in this histologic subtype is associated with lower TS expression [8]. In the same experimental setting, TS expression levels were tested in 22 cases having a cytological diagnosis of NSCLC with no clear histological definition, and in the corresponding histological samples obtained at the time of subsequent surgical resection. As a result, a significantly high degree of concordance in terms of TS protein expression in matched specimens was detected. This result raises the possibility of using the protein levels, evaluated by immunohistostaining on cytological specimens, to select the patients with unresectable advanced disease to be treated with TS-inhibiting agents. On the other hand, the small cell carcinoma (SCLC) group of patients, comprising approximately 13% of all lung cancers, has shown minimal response to pemetrexed in several studies including a large Phase III study [6]. Again, the explanation for these results may be found at the molecular level, in that SCLC has been shown to have very high TS expression when compared with either well-differentiated pulmonary neuroendocrine tumors or NSCLC [7]. These data once again support the hypothesis that elevated TS expression decreases the response to pemetrexed, and that the determination of TS levels can be used in to exclude those patients who are most likely to be unaffected from the treatment. A clear example of this is found in a very recent retrospective study from our group carried out on malignant pleural mesothelioma (MPM), a disease in which pemetrexed has been previously shown to be clinically active. We showed that TS protein and gene expression levels are significantly correlated with the clinical outcome in a consecutive series of 60 patients with advanced MPM treated with pemetrexed, with or without plat

Thymidylate synthase expression and response to irinotecan/5-fluorouracil therapy in advanced colorectal cancer (CRC).

e14123 Background: Molecular diagnostics are at the forefront of cancer therapy. Defining cellular profiles of malignancies facilitates selecting the most efficacious chemotherapy regimen. Thymidylate synthase (TS) is a critical enzyme in DNA synthesis and the target of 5-fluorouracil (5FU). Previous studies have identified an inverse relationship between tumoral TS expression and 5FU efficacy in colorectal cancer. Saltz et al described irinotecan (CPT-11) as an effective agent even in patients with high TS levels when combined with 5FU. Noda et al confirmed the relationship in 27 Japanese patients treated with IFL (5FU, CPT-11 and leucovorin). This association highlights the ability to treat patients with high TS expression using 5FU and CPT-11 combinations. Our study was designed to confirm these findings. Methods: TS gene expression was measured from metastatic tumors (primary tumor tissue was used if metastatic was not available) via RT-PCR extraction of formalin-fixed, paraffin-embedded samples. No clear cutoff exists for high and low TS expression; therefore, 3 separate cutoffs were assessed as previously published in the literature. A total of 36 patients treated with an CPT-11/5FU based regimen for advanced colorectal cancer were evaluated. Fifteen tissue blocks had limited tissue and analysis was not done. Seventeen patients had full follow-up data and are included in this interim analysis. The TS gene expression was then correlated to the response of the CPT-11- 5FU based regimen. Primary endpoints included clinical benefit (CR+PR+SD) and time to treatment failure (TTF). Results: Clinical benefit and TTF were found to be comparable in patients treated with an CPT-11-5FU combination regardless of TS expression (Table). Conclusions: This study provides additional data confirming that CPT-11-5FU combination therapies for advanced CRC can be effective even in patients with elevated TS mRNA values across all three reported TS cutoff values. TS ≥ 1.6 TS < 1.6 P value TS ≥ 2.76 TS < 2.76 P value TS ≥ 4 TS < 4 P value CB (%) 50 100 0.10 50 77.8 0.34 50 63.6 0.60 PD (%) 50 0 50 22.2 50 36.4 TTF (weeks) 44.3 16.8 0.93 40.9 34.8 0.72 32.0 39.9 0.66 Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Response Genetics

Abstract A4: Identification of novel histone deacetylase inhibitor-regulated drug targets and a phase I/II trial of vorinostat in combination with 5-fluorouracil in metastatic colorectal cancer

Abstract Histone deacetylase inhibitors (HDACi) represent a novel class of agents that are undergoing clinical evaluation in colorectal cancer (CRC). Previously, we identified that HDACi including vorinostat (Vor) and LBH589 induced downregulation of the 5-FU target enzyme thymidylate synthase (TS) resulting in the sensitization of CRC cells to 5-FU. There were two goals in the present study: Firstly to identify additional genes in CRC cells that are differentially regulated by two clinically advanced HDACi, Vor and LBH589, furthering our understanding of their mechanism of action and to identify potential targets for novel HDACi-based drug combinations. Secondly, to extend our preclinical observations and evaluate the safety and feasibility of combining Vor with 5-FU in a phase I/II trial in patients with metastatic CRC (mCRC) and elevated intratumoral TS who had failed 5-FU-based therapy. HCT116 and HT29 CRC cells treated with Vor and LBH589 were analyzed using the Illumina Human-6 V2 BeadChip array. Differentially expressed genes (DEGs) were subject to hierarchical clustering, venn diagram analysis and interpreted using Ingenuity® Pathway Analysis. Results from microarray analysis were validated by quantitative PCR (qPCR) and analyzed in a panel of 8 CRC cell lines in vitro and in HCT116 and HT29 xenograft models. Bioinformatic analysis of the microarray results revealed significant similarity in DEGs following treatment with the two HDACi tested within each cell line. However, analysis of in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 DEGs modulated by both Vor and LBH589 were identified in both CRC cell lines analyzed and further validated in a panel of 8 CRC cell lines and in HCT116 and HT29 xenograft models treated with LBH589. This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel HDACi-based drug combinations in CRC. We concurrently conducted a Phase I/II clinical trial to determine the safety and feasibility of combining Vor with 5-FU in patients with mCRC and elevated intratumoral TS. Patients who had failed all standard therapeutic options were eligible. Intratumoral TS mRNA expression and peripheral blood mononuclear cell (PBMC) histone acetylation were measured before and after 6 consecutive days of Vor treatment at 400 mg PO daily. 5-FU/LV were given on days 6 and 7 and repeated every 2 wks, along with continuous daily Vor. Ten patients were enrolled and three dose levels were explored in the phase I portion of the study. Two dose-limiting toxicities (DLTs) were observed at the starting dose level, resulting in de-escalation to levels −1 and −2. Given the occurrence of two DLTs at each of the dose levels, we were unable to establish a maximum tolerated dose (MTD). Two patients achieved significant disease stabilization for 4 and 6 months. Grade 3 and 4 toxicities included fatigue, thrombocytopenia and mucositis. Intratumoral TS downregulation ≥50% was observed in one patient only. Acetylation of histone 3 was observed in PBMCs following Vor treatment. The study failed to establish a MTD and was terminated. The presence of PBMC histone acetylation indicates biological activity of Vor, however, consistent reductions in intratumoral TS mRNA were not observed. Alternate Vor dose-scheduling may alleviate the toxicity, achieve optimal TS downregulation and improve efficacy. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A4

A novel approach to thymidylate synthase as a target for cancer chemotherapy.

Tumor cell resistance to fluoropyrimidines and other inhibitors of thymidylate synthase (TS) is a serious problem often associated with increased intracellular TS. Clinically, another problem that arises from the use of TS inhibitors is toxicity, which develops, in part, because normal cells may be adversely affected by doses of inhibitor that do not impact tumor cells. To circumvent this problem, we have devised a new strategy called enzyme-catalyzed therapeutic activation (ECTA), which takes advantage of overexpressed TS to enzymatically generate cytotoxic moieties preferentially in tumor cells. We show herein that tumor cells expressing elevated levels of TS are preferentially sensitive to NB1011, a phosphoramidate derivative of (E)-5-(2-bromovinyl)-2'-deoxyuridine. We find support for the proposed mechanism of NB1011 in the following results: 1) positive relationship between TS protein level and sensitivity to NB1011 in engineered HT1080 tumor cells, designed to express defined levels of TS protein; 2) NB1011 activity is enhanced on tumor cells which express endogenous elevated TS; 3) cytotoxicity of NB1011 is blocked by raltitrexed (Tomudex); 4) NB1011 selection of TS-overexpressing MCF7TDX tumor cells results in recovery of cell populations and clones with diminished TS levels (and restored sensitivity to raltitrexed). A preliminary comparison of TS mRNA levels in multiple normal tissues versus colon tumor samples suggests that selective tumor cytotoxicity of NB1011 may be possible in the clinical setting. Because NB1011 cytotoxicity is dependent upon activation by TS, its proposed mechanism of action is distinct from current TS-targeted drugs, which require inhibition of TS to be effective.

Prognostic significance of thymidylate synthase activity in bladder carcinoma.

5-fluorouracil (5-FU) is an anticancer agent clinically used against various cancers including bladder carcinoma. 5-FU inhibits thymidylate synthase (TS) and blocks DNA synthesis. TS is the key enzyme in the catalysis of the methylation from deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate. Little is known about the significance of TS in bladder carcinoma. The authors investigated the activity of TS in 82 bladder cancers and prognostic significance of the levels of TS and/or activities of dihydropyrimidine dehydrogenase (DPD), an important enzyme in the degradation of 5-FU. The levels of TS and DPD activities in nonfixed, fresh, frozen, bladder carcinoma and normal bladder specimens were determined biochemically by the FdUMP binding assay and the 5-FU degradation assay, respectively. The activity of TS was 10-fold higher in bladder carcinoma compared with normal bladder. TS activity in muscle-invasive bladder carcinoma was threefold higher than that in Ta and T1 cancer. In addition, the activity of TS in T1 bladder carcinoma was threefold higher than that in Ta cancer. The level of TS activity in Grade 3 bladder carcinoma was 4.5-fold and 3.5-fold higher than that in Grade 1 and Grade 2 cancers, respectively. Patients with Ta and T1 bladder carcinoma with low TS activity had a longer postoperative tumor-free period than those with high activity in the 2-year follow-up. Patients with Ta and T1 bladder carcinoma with high or low TS activity were divided into four subgroups: high or low DPD activity subgroups. Patients with low TS activity and high DPD activity had the longest postoperative disease-free period among the 4 subgroups during the 2-year follow-up. To the authors' knowledge, the current study is the first study that demonstrated that the level of TS activity correlates with both the progression of the stage and the increase of the grade of bladder carcinoma and that elevated TS activity predicts early recurrence in Ta and T1 bladder carcinoma. These results suggested that elevated TS activity might have been associated with a higher chance of progression and recurrence of bladder carcinoma in the patients who participated in this study.

Relationships between resistance to cisplatin and antifolates in sensitive and resistant tumour cell lines

Possible relationships between tumour resistance to cisplatin and the folate-based thymidylate synthase (TS) inhibitors, CB3717 and ZD1694 (tomudex), have been investigated in vitro using a panel of tumour cell lines (predominantly human ovarian), either parental or possessing acquired resistance to cisplatin or ZD1694. Across eight parent human tumour cell lines, ZD1694 was the most potent drug (mean ic 50 of 1.9 × 10 −8 M), being over 250 times as potent as its prototype CB3717 (mean ic 50 of 4.8 × 10 −6 M). In five pairs of acquired cisplatin-resistant human tumour cell lines (three ovarian, one cervical and one testicular) which encompass all of the main known mechanisms of platinum drug resistance, ZD1694, CB3717 and the DHFR inhibitor, methotrexate, all exhibited non-cross-resistance. The cervical line, HX/155cisR, showed collateral sensitivity to ZD1694, CB3717, 5-fluorouracil (FUra) and fluorodeoxyuridine (FdUrd). One cell line, A2780cisR, showed a low level of cross-resistance to FUra (resistance factor, RF, of 1.5) and FdUrd (RF of 3.8). A2780cisR, in common with two other cisplatin-resistant lines, did not possess elevated TS activity compared with its parent. Cisplatin retained activity in four acquired ZD1694-resistant cell lines (encompassing reduced folate transport, elevated TS and defective polyglutamation mechanisms of resistance). Furthermore, combinations of ZD1694 with each of the platinum-based drugs, cisplatin, carboplatin and the recently introduced orally administrable, JM216, all showed additive growth inhibitory effects by median effect analysis. These data suggest that the tumour inhibitory properties of the recently introduced highly potent TS inhibitor, ZD1694, and cisplatin, and, moreover, their respective mechanisms of resistance, do not overlap. Therefore, these drugs may be considered for combination in the clinic.