Gene therapy using a liver-targeted AAV vector restores nucleoside and nucleotide homeostasis in a murine model of MNGIE.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in TYMP, enconding thymidine phosphorylase (TP). TP deficiency results in systemic accumulation of thymidine and deoxyuridine, which interferes with mitochondrial DNA (mtDNA) replication and leads to mitochondrial dysfunction. To date, the only treatment available for MNGIE patients is allogeneic hematopoietic stem cell transplantation, which is associated with high morbidity and mortality. Here, we report that AAV2/8-mediated transfer of the human TYMP coding sequence (hcTYMP) under the control of a liver-specific promoter prevents the biochemical imbalances in a murine model of MNGIE. hcTYMP expression was restricted to liver, and a dose as low as 2 × 10(11) genome copies/kg led to a permanent reduction in systemic nucleoside levels to normal values in about 50% of treated mice. Higher doses resulted in reductions to normal or slightly below normal levels in virtually all mice treated. The nucleoside reduction achieved by this treatment prevented deoxycytidine triphosphate (dCTP) depletion, which is the limiting factor affecting mtDNA replication in this disease. These results demonstrate that the use of AAV to direct TYMP expression in liver is feasible as a potentially safe gene therapy strategy for MNGIE.

Thymidine phosphorylase inhibits vascular smooth muscle cell proliferation via upregulation of STAT3.

Dysregulated growth and motility of vascular smooth muscle cells (VSMC) play important role in obstructive vascular diseases. We previously reported that gene transfer of thymidine phosphorylase (TP) into rat VSMC inhibits cell proliferation and attenuates balloon injury induced neointimal hyperplasia; however, the mechanism remains unclear. The current study identified a signaling pathway that mediates effect of TP inhibited VSMC proliferation with a TP activity-dependent manner. Rat VSMC overexpressing human TP gene (C2) or control empty vector (PC) were used. Serum stimulation induced constitutive STAT3 phosphorylation at tyrosine705 in C2 cell but not in PC, which was independent of JAK2 signaling pathway. Inhibition of Src family kinases activity inhibited STAT3 phosphorylation in C2 cells. Lyn activity was higher in C2 cell than in PC. SiRNA based gene knockdown of Lyn significantly decreased serum induced STAT3 phosphorylation in C2 and dramatically increased proliferation of this cell, suggesting that Lyn plays a pivotal role in TP inhibited VSMC proliferation. Unphosphorylated STAT3 (U-STAT3) expression was significantly increased in C2 cells, which may be due to the increased STAT3 transcription. Gene transfection of mouse wild-type or Y705F mutant STAT3 into PC cell or mouse primary cultured VSMC significantly reduced proliferation of these cells, suggesting that overexpression of U-STAT3 inhibits VSMC proliferation. We conclude that Lyn mediates TP induced STAT3 activation, which subsequently contributes to upregulate expression of U-STAT3. The U-STAT3 plays a critical role in inhibiting VSMC proliferation.

Evaluations of biomarkers associated with sensitivity to 5-fluorouracil and taxanes for recurrent/advanced breast cancer patients treated with capecitabine-based first-line chemotherapy.

The aim of the present study was to investigate the gene expression of biomarkers associated with the sensitivity to fluoropyrimidine and taxanes in recurrent/advanced breast cancer patients treated with first-line capecitabine chemotherapy. We evaluated the clinicopathological/prognostic significance of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), class III ß-tubulin (ßIII-tubulin), and stathmin-1 or oncoprotein-18 (STMN1). Formalin-fixed, paraffin-embedded tumor specimens from 42 patients were used for analysis of TS, DPD, TP, ßIII-tubulin, and STMN1 expression with a real-time reverse transcription-PCR technique. Patients were classified into the high-expression and low-expression groups according to the median value of the expression level of each biomarker. There was a significantly longer time to progression (TTP) in the high-TP group (P=0.018). The multivariate analysis revealed that the TP expression (hazard ratio for the low-TP group vs. the high-TP group, 2.873; 95% confidence interval, 1.143-7.223; P=0.025) is independent of prognostic factors for TTP. In the subgroup of patients treated with capecitabine plus taxanes as first-line chemotherapy, TTP was significantly longer in the low-ßIII-tubulin group (P=0.047). The gene expression of TS, DPD, and STMN1 failed to have any significant impact on the outcome. These results provide further evidence that the TP expression may be a prognostic factor in breast cancer patients treated with capecitabine-based first-line chemotherapy, and ßIII-tubulin can be used to predict the outcome of capecitabine in combination with taxanes as first-line chemotherapy. Therefore, these potential biomarkers should be further evaluated.

Up-regulation of extracellular signal-regulated kinase 1/2-dependent thymidylate synthase and thymidine phosphorylase contributes to cisplatin resistance in human non-small-cell lung cancer cells.

Chemotherapy for advanced human non-small-cell lung cancer (NSCLC) includes platinum-containing compound such as cisplatin in combination with a second- or third-generation cytotoxic agent. 5-Fluorouracil (5-FU) belongs to antimetabolite chemotherapeutics, and its mechanism of cytotoxicity is involved in the inhibition of thymidylate synthase (TS). TS and thymidine phosphorylase (TP) are key enzymes of the pyrimidine salvage pathway. In this study, we have examined the molecular mechanism of TS and TP in regulating drug sensitivity to cisplatin in NSCLC cell lines. Cisplatin could increase the phosphorylation of mitogen-activated protein kinase kinase 1/2 (MKK1/2)-extracellular signal-regulated kinase 1/2 (ERK1/2) and the protein levels of TS and TP through enhancing the protein stability in A549 and H1975 cells. Blocking ERK1/2 activation by MKK1/2 inhibitor [U0126; 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene)] decreased TS and TP protein levels in both cell lines treated with cisplatin. Depletion of endogenous TS or TP expression by specific small interfering RNA transfection significantly increased cisplatin-induced cell death and growth inhibition. Combined treatment with 5-FU could decrease cisplatin-induced ERK1/2 activation and the induction of TS and TP, which subsequently resulted in synergistic cytotoxic effects. Enforced expression of constitutive active MKK1/2 vectors rescued the protein levels of phospho-ERK1/2, TS, and TP, and the cell viability that were decreased by cisplatin and 5-FU combination. In contrast, U0126 enhanced drug sensitivity to cisplatin and/or 5-FU in lung cancer cells. In conclusion, the up-regulation of ERK1/2-dependent TS and TP can protect human lung cancer cells from cisplatin-induced cytotoxicity.

Vascular endothelial growth factor and thymidine phosphorylase expression in salivary gland tumors with distinct metastatic behavior.

BACKGROUND: Metastasis of salivary gland tumors has a negative impact on survival. Angiogenesis and its factors are potential markers for predicting metastasis in different malignant tumors, but this is not the case for salivary gland tumors. METHODS: Salivary gland tumors of distinct biologic behavior were analyzed according to the semiquantitative immunoexpression of vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP). RESULTS: Vascular endothelial growth factor expression was predominantly weak in benign tumors. Weak TP expression was observed in 100% cases of benign tumors and in 74.3% of primary malignant tumors. High VEGF and TP expression levels were significantly associated with primary malignant tumors but not with primary non-metastasizing and primary metastasizing malignant tumors or with subtypes of malignant tumors. CONCLUSIONS: Vascular endothelial growth factor and TP expression levels discriminate benign and malignant tumors but cannot predict metastasis from non-metastasizing tumors.

FK506 inhibition of gliostatin/thymidine phosphorylase production induced by tumor necrosis factor-α in rheumatoid fibroblast-like synoviocytes.

Gliostatin/thymidine phosphorylase (GLS/TP) is known to have angiogenic and arthritogenic activities. The purpose of this study was to determine the inhibitory effects of FK506 (tacrolimus) on GLS production in rheumatoid arthritis (RA). We investigated the modulation of serum GLS by FK506 therapy and the effect of FK506 on the production of GLS in fibroblast-like synoviocytes (FLSs). Serum samples were collected from 11 RA patients with active disease at baseline and after 12 weeks of FK506 treatment. Serum concentrations of GLS and matrix metalloproteinase (MMP)-3 were measured by ELISA and found to be down-regulated in responders evaluated with a disease activity score. Patient FLSs were cultured and stimulated by tumor necrosis factor (TNF)-α with or without FK506. The expression levels of GLS were determined using reverse transcription-polymerase chain reaction (RT-PCR) and enzyme immunoassay and shown to be significantly increased. GLS levels in TNF-α-stimulated FLSs were reduced by FK506 treatment. Our data show a novel mechanism for the action of physiological concentrations of FK506 in RA that regulates the production of GLS in FLSs.

[Improved sensitivity of gastric carcinoma cells to fluorouracil-related drugs by transfection of thymidine phosphorylase gene].

OBJECTIVE: To investigate the relationship between the expression of thymidine phosphorylase (TP) and the sensitivity of gastric carcinoma to 5-fluorouracil (5-FU) and its prodrugs. METHODS: Gastric carcinoma cell line AGS was transfected with recombinant plasmid pEGFP-N1-TP or control plasmid pEGFP-N1 by lipofectamin 2000. The expression of green fluorescence labeled protein was observed under fluorescence microscope. Positive clones AGS-p and AGS-pTP were selected by G418 treatment. Expression of TP protein and mRNA was detected by immunocytochemistry and RT-PCR, respectively. Drug sensitivity to 5-FU and its prodrugs was assessed by MTT assay. RESULTS: Cell clones with the expression of green fluorescent protein (AGS-p) and a clone with TP and green fluorescent fusion protein (AGS-pTP) were established. Immunostaining of TP protein was strongly positive in AGS-pTP and negative in AGS-p and AGS. The expression of TP mRNA was significantly higher in AGS-pTP (0.8090 ± 0.0450) than that in AGS (0.0490 ± 0.0046) and AGS-p (0.0610 ± 0.0069; P < 0.01). The sensitivity to doxifluridine and capecitabine in AGS-pTP was significantly increased, as compared with that in AGS-p. IC50 values of AGS-pTP to doxifluridine and capecitabine were estimated 1.7 folds and 2.2 folds as much as that of AGS-p, respectively. The sensitivity to 5-FU was not different between AGS-pTP and AGS-p. CONCLUSIONS: Enhancement of TP expression improves the sensitivity of gastric carcinoma cells to doxifluridine and capecitabine. But it does not affect the sensitivity to 5-FU.

Comparison of thymidine phosphorylase expression and prognostic factors in gallbladder and bile duct cancer.

BACKGROUND: Biliary tract cancers have limitations in information about different location-related pathogenesis and clinico-pathological characteristics. The goal of this study was to investigate anatomical site-related similarities and differences in biliary tract cancers and to assess the expression and clinical significance of functional proteins such as p53, cyclin D1, survivin, thymidine phosphorylase, and ERCC1. METHODS: One hundred and sixty-one patients with biliary tract adenocarcinomas, who underwent curative or palliative surgery in a single institution between October 1994 and December 2003 were evaluated, retrospectively. The level of protein expression of p53, cyclin D1, survivin, thymidine phosphorylase, and ERCC1 was assessed by immunohistochemistry. RESULTS: With respect to clinico-pathological characteristics, gallbladder cancer was more frequent in women, and bile duct cancer was more common in men. Perineural invasion was more common in bile duct cancer. Recurrence as a distant metastasis was more common in gallbladder cancer. Immunohistochemical analysis revealed that thymidine phosphorylase expression was significantly higher in gallbladder cancer than in bile duct cancer. Positive thymidine phosphorylase and p53 staining were associated with an advanced stage. Differentiation, vascular invasion, perineural invasion, lymphatic invasion, lymph node metastasis, and TNM stage independently predicted poor prognosis in biliary tract cancer. These correlations were seen more clearly in gallbladder cancer. The immunohistochemical staining patterns of p53, cyclin D1, survivin, thymidine phosphorylase, and ERCC1 showed no prognostic significance in biliary tract cancers. CONCLUSIONS: We concluded that gallbladder and bile duct cancers are considered to be separate diseases with different clinico-pathological characteristics and prognostic factors. In addition, we hypothesize that high expression of thymidine phosphorylase by gallbladder cancer results in a higher response rate to capecitabine by gallbladder cancer than bile duct cancer.

Thymidine phosphorylase expression in metastatic sites is predictive for response in patients with colorectal cancer treated with continuous oral capecitabine and biweekly oxaliplatin.

The primary objective of this study was to determine the activity and safety profile of biweekly oxaliplatin combined with continuous oral capecitabine in the first-line treatment of metastatic colorectal cancer. A secondary endpoint was to investigate the correlation between thymidylate synthase and thymidine phosphorylase (TP) expression in metastatic tissues and tumor response. Forty-one patients received oral capecitabine 1331 mg/m every day combined with intravenous oxaliplatin 85 mg/m every 2 weeks. The overall response rate was 58.5% [95% confidence interval (CI): 43.3-73.6%], the median progression-free survival 9.4 months (95% CI: 7.7-11.2 months) and the median survival 22.3 months (95% CI: 16.1-27.5 months). There were no grade 4 toxicities, and grade 3 toxicity was also uncommon. High TP expression in metastatic tissue was significantly associated with response to treatment (P=0.019), and also with a trend towards a better median progression-free survival and overall survival compared with patients expressing low TP (P=0.056; P=0.073). This study suggests that biweekly oxaliplatin and continuous oral capecitabine is an active and well-tolerated chemotherapy regimen in the first-line treatment of metastatic colorectal cancer. Moreover, these findings add to a growing body of evidence that patients with high levels of intratumoral TP expression are the ideal candidates for capecitabine-based chemotherapy.

Impact of thymidine phosphorylase-expressing macrophages for surgical margin in partial nephrectomy.

OBJECTIVES: We investigated the relationship between the surgical margin in partial nephrectomy (PN) and thymidine phosphorylase (TP)-expressing macrophages in peritumoral tissue of renal cell carcinoma (RCC). METHODS: In 46 patients who underwent radical nephrectomy, we measured TP protein levels in tumor tissue, peritumoral tissue and normal tissue, and conducted immunohistochemical staining for TP and macrophages. In addition, we prospectively conducted PN with a 5-mm margin in 11 patients with pT1a RCC. RESULTS: The TP protein level and TP-positive macrophages were correlated with T classification, histological grade, mode of infiltration and venous invasion. However, for pT1 RCC, TP-positive macrophages in pT1a were significantly lower than in pT1b (p = 0.0140), while there was no significant difference in TP protein levels between pT1a and pT1b. No surgical margin was positive in 11 patients who underwent PN with a 5-mm margin, and no patient had local recurrence or distant metastasis during follow-up. CONCLUSIONS: The TP protein level and TP-positive macrophages in the peritumor area are thought to be associated with tumor progression in RCC, while a similar relationship was not found in pT1a RCC. These data suggest that a 5-mm margin might be safe to reduce the risk of local recurrence when PN is performed for treatment of solitary pT1a RCC.

[The thymidine phosphorylase as the platelet-derived endothelial cell growth factor of endometrial cancer]. / Fosforylaza tymidynowa jako plytkopochodny czynnik wzrostowy komórek sródblonka raka endometrium.

OBJECTIVES: The aim of the study was to assess the correlation between the activity of thymidine phosphorylase (TP) and the platelet derived-endothelial cell growth factor (PD-ECGF) expression in endometrial carcinoma. METHODS: The study group consisted of 40 tissue samples taken from patients with endometrial carcinoma, who underwent surgery in First Clinic of Gynecology and Oncologic Gynecology of Medical University in Lodz. The control tissue samples were taken from patients who were operated on for non-oncologic reason. The activity of TP was measured by the spectrophotometric method in the cytosol of tumor cells, and the immunohistochemical staining of PD-ECGF was performed in the same tumors. The results of TP activity were compared with the microvessel density (MD) assessed by immunohistochemical analysis and with clinico-pathological features like tumor grade and FIGO stage. RESULT: A positive correlation between the enzyme activity and expression of TP/PD-ECGF protein was found. Moreover a significantly higher TP activity was confirmed in malignant tumors from endometrial cancer patients when compared to the controls. A positive correlation between the enzyme activity and MD was also stated, but there was no connection to the grade of tumors and FIGO stage. Since the TP activity proved to be related to PD-ECGF expression and angiogenesis, we can state that TP seems to be an active form of PD-ECGF growth factor in endometrial carcinoma. This is in agreement with the results of many publications on other malignancies. The proper modulation of this activity may be useful in adjuvant therapies.

Inhibition of thymidine phosphorylase expression by Hsp90 inhibitor potentiates the cytotoxic effect of salinomycin in human non-small-cell lung cancer cells.

Salinomycin is a polyether ionophore antibiotic having anti-tumorigenic property in various types of cancer. Elevated thymidine phosphorylase (TP) levels, a key enzyme in the pyrimidine nucleoside salvage pathway, are associated with an aggressive disease phenotype and poor prognoses. Heat shock protein 90 (Hsp90) is a ubiquitous molecular chaperone that is responsible for the stabilization and maturation of many oncogenic proteins. In this study, we report whether Hsp90 inhibitor 17-AAG could enhance salinomycin-induced cytotoxicity in NSCLC cells through modulating TP expression in two non-small-cell lung cancer (NSCLC) cell lines, A549 and H1975. We found that salinomycin increased TP expression in a MKK3/6-p38 MAPK activation manner. Knockdown of TP using siRNA or inactivation of p38 MAPK by pharmacological inhibitor SB203580 enhanced the cytotoxic and growth inhibition effects of salinomycin. In contrast, enforced expression of MKK6E (a constitutively active form of MKK6) reduced the cytotoxicity and cell growth inhibition of salinomycin. Moreover, Hsp90 inhibitor 17-AAG enhanced cytotoxicity and cell growth inhibition of salinomycin in NSCLC cells, which were associated with down-regulation of TP expression and inactivation of p38 MAPK. Together, the Hsp90 inhibition induced TP down-regulation involved in enhancing the salinomycin-induced cytotoxicity in A549 and H1975 cells.

Adventitial delivery of platelet-derived endothelial cell growth factor gene prevented intimal hyperplasia of vein graft.

BACKGROUND: Platelet-derived endothelial cell growth factor (PD-ECGF), also known as thymidine phosphorylase (TP) reportedly inhibits vascular smooth muscle cells (VSMCs) migration and proliferation. We hypothesized that adventitial administration of the PD-ECGF/TP gene will suppress intimal hyperplasia and prevent vein graft failure. METHODS: The study used 68 female rabbits. Rabbit jugular vein was autogenously transplanted into carotid artery with a cuff anastomotic technique. To define vascular wall gene transfer efficiency, poloxamer hydrogel (20%) containing plasmid vector encoding the LacZ gene and different concentrations of trypsin (0%, 0.1%, 0.25%, and 0.5%, n = 5 for each group) was applied to the adventitia of the vein graft. Gene transfer efficiency was evaluated 7 days later by X-gal staining. An additional 48 rabbits received poloxamer hydrogel (20%) containing 0.25% trypsin and the human PD-ECGF/TP gene, LacZ gene, or saline. Intima thickness was evaluated at 2 and 8 weeks after grafting (n = 8 for each group at each time point). Transgene expression was examined by reverse transcriptase-polymerase chain reaction, immunoblotting assay, and immunohistochemical staining. Immunohistochemical staining was also used to determine VSMC proliferation, heme oxygenase-1 expression, and macrophage infiltration. RESULTS: Incorporation of trypsin into the poloxamer hydrogel significantly increased vessel wall gene transfer. Trypsin at 0.25% and 0.5% resulted in higher gene transfer at the same level without effecting intimal hyperplasia and inflammation; thus, trypsin at 0.25% concentration was used for subsequent experiments. Compared with the LacZ and saline groups, grafts receiving the PD-ECGF/TP gene significantly reduced intimal thickness at 2 and 8 weeks after treatment. The ratio of proliferative VSMC was lower in PD-ECGF/TP treated grafts. Histologic examination of the PD-ECGF/TP transgene grafts demonstrated high expression of heme oxygenase-1, which has been reported to inhibit VSMC proliferation, suggesting that heme oxygenase-1 may be important in the inhibition effect of PD-ECGF/TP on VSMC. No neoplastic or morphologic changes were found in the remote organs. CONCLUSIONS: A safe and highly efficient gene transfer method was developed by using poloxamer hydrogel and a low concentration of trypsin. Neointimal hyperplasia was significantly reduced by adventitial application of the PD-ECGF/TP gene to the vein graft. Our data suggest that adventitial delivery of the PD-ECGF/TP gene after grafting may be promising method for preventing vein graft failure.

Decreased expression of thymidine phosphorylase/platelet-derived endothelial cell growth factor in basal cell carcinomas.

Thymidine phosphorylase (TP)/platelet-derived endothelial cell growth factor is associated with tumor angiogenesis. We evaluated the TP mRNA and protein expression in basal cell carcinomas (BCC) and in various skin tumors including numerous BCC histological simulants. Immunohistochemistry was performed on 99 paraffin sections of formalin-fixed skin tumors using monoclonal antibodies (mAb) against TP. TP mRNA levels were measured by real time RT-PCR in whole BCCs (wBCC) and laser capture microdissected (LCM) BCC tumor cells. TP immunostaining was negative in all BCC variants and in most of the benign trichogeneic tumors studied. By contrast, TP was constantly immunodetected in actinic keratosis (AK), squamous cell carcinomas (SCC), syringomatous carcinomas (SC), basosquamous carcinomas (BSC) and melanomas. TP mRNA levels were low and statistically not different in wBCC and normal skin but were strongly downregulated in LCM-BCC as compared with LCM-normal epidermis. We concluded that (i) anti-TP mAb is an useful marker to differentiate BCC from AK, SCC, BSC and SC but not from trichoblastic tumors, (ii) the lack of TP protein expression in BCC tumoral cells is linked to transcriptional regulatory mechanisms, (iii) the low TP mRNA levels in whole BCC may be related to the low intra-tumoral microvessel density, the slow growth and the very low metastatic potential of these tumors.

Efficacy of laser capture microdissection plus RT-PCR technique in analyzing gene expression levels in human gastric cancer and colon cancer.

BACKGROUND: Thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase gene expressions are reported to be valid predictive markers for 5-fluorouracil sensitivity to gastrointestinal cancer. For more reliable predictability, their expressions in cancer cells and stromal cells in the cancerous tissue (cancerous stroma) have been separately investigated using laser capture microdissection. METHODS: Thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, and orotate phosphoribosyltransferase mRNA in cancer cells and cancerous stroma from samples of 47 gastric and 43 colon cancers were separately quantified by reverse transcription polymerase chain reaction after laser capture microdissection. RESULTS: In both gastric and colon cancers, thymidylate synthase and orotate phosphoribosyltransferase mRNA expressions were higher (p < 0.0001, p <0.0001 respectively in gastric cancer and P = 0.0002, p < 0.0001 respectively in colon cancer) and dihydropyrimidine dehydrogenase mRNA expressions were lower in cancer cells than in cancerous stroma (P = 0.0136 in gastric cancer and p < 0.0001 in colon cancer). In contrast, thymidine phosphorylase mRNA was higher in cancer cells than in cancerous stroma in gastric cancer (p < 0.0001) and lower in cancer cells than in cancerous stroma in colon cancer (P = 0.0055). CONCLUSION: By using this method, we could estimate gene expressions separately in cancer cells and stromal cells from colon and gastric cancers, in spite of the amount of stromal tissue. Our method is thought to be useful for accurately evaluating intratumoral gene expressions.

Suppression of thymidine phosphorylase expression by promoter methylation in human cancer cells lacking enzyme activity.

PURPOSE: Thymidine phosphorylase (TP, EC 2.4.2.4) activity varies in different human cancer cell lines. Nevertheless, little is known about the regulatory mechanisms of TP expression in such cancers. Promoter methylation of dinucleotide cytosine-guanine (CpG) sites is a known mechanism of reversible gene expression silencing. METHODS: TP promoter methylation was investigated in five cancer cell lines (SKBR-3, 786-O, HT-29, MDA-231, DLD-1). TP mRNA levels were determined by real-time quantitative PCR. The degree of methylation was identified by bisulfite sequencing. Minimal TP promoter activity was determined by Luciferase reporter assays. DNA-protein interactions were evaluated by electrophoretic mobility shift assays. RESULTS: SKBR-3 cells exhibited the highest TP expression, 786-O, HT-29, and MDA-231 cells exhibited intermediate TP expression, while DLD-1 cells did not express TP as demonstrated by TP mRNA, protein, and enzyme activity levels. SKBR-3 lacked methylation in the TP promoter, intron 1 and exon 1 regions, while DLD-1 showed extensive methylation. Treatment of DLD-1 and SKBR-3 with the methylation-inhibitor, 5-aza-2'-deoxycytidine (5-aza-2dC), resulted in a concentration-dependent increase in TP mRNA and protein levels in DLD-1 but not SKBR-3 cells. Trichostatin-A treatment, a histone deacetylase inhibitor, improved the 5-aza-2dC-induced TP re-activation. Electrophoretic mobility shift assays demonstrated that methylation significantly inhibits transcription factor binding. Supershift analyses suggest that the Sp1 and Sp3 (to a lesser degree) transcription factors have a role in the regulation of TP expression. CONCLUSIONS: These findings suggest that TP promoter methylation is a mechanism for down-regulation of TP expression in cancer cells and may have implications in modulating prognosis of cancer patients.

5-Fluorouracil-related gene expression in hepatic artery infusion-treated patients with hepatic metastases from colorectal carcinomas.

AIM: To predict the therapeutic efficacy of hepatic arterial infusion (HAI) with 5-fluorouracil (5FU) for patients with liver metastases from colorectal carcinomas, 5FU-related gene expressions were examined in primary colorectal carcinomas. PATIENTS AND METHODS: Thirty-eight patients with liver metastases from colorectal carcinoma received HAI of 5FU. The expressions of the mRNAs for thymidine synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP), and oroteta phophoribosyl transferase (OPRT) in primary colorectal carcinomas were measured by RT-PCR. RESULTS: The response rate was 52.6% (20/38). The overall median survival time was 29.1 months. DPD and TP expression was significantly higher in the progressive disease (PD) group than in the complete response (CR) or partial response (PR) group (p = 0.032, p = 0.014), respectively. The levels of DPD and TP mRNAs showed a significant correlation (r = 0.76, p = 0.0001). CONCLUSION: The expression of DPD and TP mRNAs in primary colorectal carcinomas was significantly predictive of the therapeutic response to 5FU HAI.

5-Fluorouracil-related gene expression in primary sites and hepatic metastases of colorectal carcinomas.

UNLABELLED: The aim of this study was to investigate the correlation of the mRNA expressions of 5-fluorouracil (5FU)-related genes in the primary sites and liver metastases of colorectal carcinomas. PATIENTS AND METHODS: Patients with liver metastases from colorectal carcinomas were included (n = 43). The expression ratios to beta-actin of mRNA of thymidine synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and oroteta phosphoribosyl transferase (OPRT) were measured in primary and liver metastases of colorectal carcinomas by laser-captured microdissection and real time PCR. RESULTS: The ratios for the expression of TS, DPD, TP and OPRT mRNAs were significantly correlated between paired primary sites and liver metastases. The mRNA expression ratios of DPD and TP showed a significant correlation both in primary sites and in liver metastases. CONCLUSION: Enzymes of the primary colorectal carcinomas can be used in predicting the therapeutic efficacy of 5FU against liver metastases.

Changes of gene expression of thymidine phosphorylase, thymidylate synthase, dihydropyrimidine dehydrogenase after the administration of 5'-deoxy-5-fluorouridine, paclitaxel and its combination in human gastric cancer xenografts.

BACKGROUND: Although a variety of combination chemotherapies has been tested in gastric carcinoma, the most effective chemotherapeutic regimen and the precise mechanisms underlying anticancer agent combination have not yet been sufficiently elucidated. MATERIALS AND METHODS: Experimental chemotherapy was performed using human gastric carcinoma xenografts, MKN-45 and TMK-1, to examine the anticancer effects and gene expressions of the enzymes involved in 5-fluorouracil metabolism, thymidine phosphorylase (dThdPase), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD). Nude mice were treated with 5'-deoxy-5-fluorouridine (5'-dFUrd), or paclitaxel alone or in combination. The in vivo antitumor effects on gene expressions of the enzymes were examined using the quantitative real-time RT-PCR method. RESULTS: The combined use of 5'-dFUrd and paclitaxel showed additive to synergistic antitumor effects on both gastric cancer xenografts. There were significant differences of the gene expressions of dThdPase, TS, and DPD between the xenografts. The expression of dThdPase mRNA was consistently up-regulated by the administration of paclitaxel, while no constant direction of TS mRNA and DPD mRNA change was found in the xenografts. CONCLUSION: A synergistic antitumor effect of the combined administration of 5'-dFUrd and paclitaxel was found in gastric cancer xenografts and up-regulation of dThdPase mRNA may be an important underlying mechanism especially in tumors with high gene expression of this enzyme.

Importance of thymidine phosphorylase, dihydropyrimidine dehydrogenase and thymidylate synthase expression at the invasive front of T3 rectal cancer as prognostic factors.

BACKGROUND/AIMS: We evaluated a relationship between postoperative recurrence and thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD) and thymidylate synthase (TS) expression at the invasive front of T3 rectal cancer. METHODOLOGY: This study was conducted on 61 patients with T3 rectal cancer who underwent surgically curative resection between 1998 and 2002. Paraffin-embedded sections of these patients were immunostained for TP, DPD and TS. Relationship between expression level of the three factors and postoperative recurrence were evaluated. RESULTS: There was no relationship between expression of DPD or TS in the tumor cells and recurrences. Although no relationship was present between expression of TP in the stromal cells around the invasive front of the tumor and postoperative recurrences, there was a strong correlation between expression of TP in the invasive front of the tumor and postoperative recurrence. Moreover, by multivariate logistic regression analysis, TP expression in the tumor cells was the only independent contributory factor for postoperative recurrences (p = 0.021) with an odds ratio of 8.27. CONCLUSIONS: TP expression at the invasive front of the tumor may be an important prognostic factor for T3 rectal cancer, and patients with such a condition may benefit from intensive chemotherapy.