Substrate phage as a tool to identify novel substrate sequences of proteases.

Combinatorial phage peptide libraries have been used to identify the ligands for specific target molecules. These libraries are also useful for identification of the specific substrates of various proteases. A substrate phage library has a random peptide sequence at the N-terminus of the phage coat protein and an additional tag sequence that enables attachment of the phage to an immobile phase. When these libraries are incubated with a specific enzyme, such as a protease, the uncleaved phage is excluded from the solution with tag-binding macromolecules. This provides a novel approach to define substrate specificity. The aim of this review is to summarize recent progress on the application of the substrate phage technique to identify specific substrates of proteolytic enzymes. As an example, some of our own experimental data on the selection and characterization of substrate sequences for thrombin, a serine protease, and membrane type-1 matrix metalloproteinase (MT1-MMP) will be presented. Using this approach, the canonical consensus substrate sequence for thrombin was deduced from the selected clones. As expected from the collagenolytic activity of MT1-MMP, a collagen-like sequence was identified in the case of MT1-MMP. A more selective substrate sequence for MT1-MMP was identified during a substrate phage screen. The delineation of the substrate specificity of proteases will help to elucidate the enzymatic properties and the physiological roles of these enzymes. Comprehensive screening of very large numbers of potential substrate sequences is possible with substrate phage libraries. Thus, this approach allows novel substrate sequences and previously unknown target molecules to be defined.

gamma-Hydroxybutyric acid and 5-fluorouracil, metabolites of UFT, inhibit the angiogenesis induced by vascular endothelial growth factor.

UFT, a drug composed of uracil and tegafur at the molar ratio of 4:1, is an orally active agent for the treatment of a wide variety of malignant tumours. Using a murine dorsal air sac (DAS) assay, we have previously shown that UFT and its metabolites, gamma-hydroxybutyric acid (GHB) and 5-fluorouracil (5-FU), inhibited the angiogenesis induced by murine renal cell carcinoma. Here we report that UFT was more effective than other fluorinated pyrimidines such as 5-FU and doxifluridine (5'-DFUR) in blocking the angiogenic responses elicited by five human cancer cell lines which produced high levels of vascular endothelial growth factor (VEGF), but no detectable fibroblast growth factor-2 (FGF-2) in vitro. In contrast, UFT was unable to block the angiogenic response to one human gastric cancer cell line which produced both VEGF and FGF-2 in vitro. However, the production or secretion of VEGF by these cells was unaffected by GHB and 5-FU treatment. Interestingly, GHB suppressed the chemotactic migration and tube formation of human umbilical vein endothelial cells (HUVECs) stimulated by VEGF, without inhibiting their DNA synthesis. Since GHB did not affect the FGF-2-driven activities in HUVECs, its action appears to be VEGF-selective. On the other hand, 5-FU inhibited DNA synthesis and migration of HUVECs stimulated by both VEGF and FGF-2, and tube formation driven by VEGF, suggesting that 5-FU is cytotoxic to endothelial cells. The inhibitory effects of 5-FU, and especially those GHB, were reproduced under in vivo condition using the DAS assay. The VEGF-mediated angiogenesis was significantly inhibited by UFT, 5-FU, and especially by GHB. We propose that the selective inhibitory effects of GHB on VEGF-mediated responses of endothelial cells are involved in the anti-angiogenic activity of UFT.

UFT and its metabolites inhibit cancer-induced angiogenesis. Via a VEGF-related pathway.

Treatment with UFT for spontaneous lung metastasis of murine renal carcinoma (RENCA) after resection of the primary tumor has resulted in significant prolongation of the life span of tumor-bearing animals. UFT inhibited the growth of metastatic nodules in the lung, apparently via decreased density of microvessels in the metastatic foci. Subsequent experiments used dorsal air sac assay to directly trace newly forming microvessels. UFT abrogated the process of angiogenesis, induced by the RENCA cells, in a dose-dependent manner. The inhibitory effect appeared to originate from tegafur, a component of UFT, and from its known metabolites: fluorouracil (5-FU), gamma-hydroxybutyric acid (GHB), and gamma-butyrolactone (GBL). The inhibition of angiogenesis by UFT appeared to be a common phenomenon, also observed in other human cancer cell lines characterized by an excessive production of vascular endothelial growth factor (VEGF)--such as gastric, lung, and colon cancers. In vitro analysis revealed that 5-FU and gamma-hydroxybutyric acid regulated VEGF-dependent responses of human umbilical vein endothelial cells. Dorsal air sac assay revealed that UFT, 5-FU, and gamma-hydroxybutyric acid strongly inhibited the angiogenesis induced by recombinant human VEGF. These data suggest that the antiangiogenic activity of UFT is at least partially associated with an ability of the metabolites of UFT to interfere with VEGF-dependent responses of vascular endothelial cells.

The role of thymidine phosphorylase in the pathogenesis of allergic rhinitis.

The activity and distribution of thymidine phosphorylase (TP) in the nasal mucosa of patients with nasal allergy was examined and compared with those in healthy subjects. TP activity was analyzed by spectrophotometry and expression was examined by immunoblotting and immunohistochemical staining using a monoclonal antibody specific to TP. The expression level of TP detected by immunoblotting showed a correlation with the activity detected by spectrophotometry. In nasal mucosa obtained from patients with nasal allergy, the level of TP was significantly higher than that from normal subjects. Eosinophils, basal cells in mucosal epithelium and fibroblasts in nasal mucosa obtained from patients with nasal allergy were stained with anti-TP monoclonal antibody. Strong staining of eosinophils present in nasal discharge was observed. The present results indicate that an increased number of TP-expressing cells, especially eosinophils in nasal mucosa, might be associated with the pathogenesis of nasal allergy.

Suppression of metastasis by thymidine phosphorylase inhibitor.

We developed a novel inhibitor of thymidine phosphorylase (TP), 5-chloro-6-[1-(2-iminopyrrolidinyl) methyl] uracil hydrochloride (TPI), that is about 1000-fold more active than 6-amino-5-chlorouracil, one of the most potent TP inhibitors. TPI inhibited the high chemotactic motility and basement membrane invasion of KB/TP cells, a TP-positive clone transfected with Rous sarcoma virus (RSV)/TP, to the levels seen in KB/CV cells, a control clone transfected with RSV. In nude mice, oral administration of TPI suppressed not only macroscopic liver metastases of highly metastatic KB/TP cells but also the level of human beta-globin as a molecular marker of micrometastases in the livers of the mice. These findings demonstrate that TP plays a key role in the invasiveness and metastasis of TP-expressing solid tumors and suggest that TPI might be a novel antimetastatic agent for blood-borne metastasis.

[Anti-angiogenic activities of UFT and its metabolites, GHB and GBL, in the dorsal air sac (DAS) model in mice].

We investigated the effects of UFT and its metabolites, GHB and GBL, on angiogenesis induced by tumor cells in a dorsal air sac (DAS) assay in mice. Five tumor cell lines (murine renal carcinoma; RENCA, human gastric cancer; 4-1ST, human small-cell lung carcinoma; LX-1, and human colon carcinoma; DLD-1, KM-20C) were used in the DAS assay. In this model, UFT demonstrated a significant anti-angiogenic activity in a dose-dependent manner while 5-FU (19 mg/kg/day) and 5'-DFUR (200 mg/kg/day) were less effective. Moreover, tegafur (FT), a component of UFT, and gamma-hydroxybutyric acid (GHB) and gamma-butyrolactone (GBL), in vivo metabolites of UFT, inhibited angiogenesis induced by RENCA cells. The inhibitory effects of 5-FU, GHB, and GBL on angiogenesis were increased with administration by continuous infusion, providing a suitable pharmacokinetic profile. These results suggest that GHB and GBL are involved in the expression of anti-angiogenic activity of UFT.

Identification of substrate sequences for membrane type-1 matrix metalloproteinase using bacteriophage peptide display library.

Membrane type-1 matrix metalloproteinase (MT1-MMP) has been reported to mediate the activation of progelatinase A (proMMP-2) which is associated with tumor invasion and metastasis, and also known to have an ability to digest extracellular matrix components. To clarify substrate specificity of MT1-MMP, we have searched for amino acid sequences cleaved by this protease using the hexamer substrate phage library consisting of a large number of randomized amino acids sequences. The consensus substrate sequences for MT1-MMP were deduced from the selected clones and appeared to be P-X-G/P-L at the P3-P1' sites. Peptide cleavage assay revealed that MT1-MMP preferentially digested a synthetic substrate containing Pro of the P1 position compared to that being substituted with Gly. Our results may have an important implication to identifying new target proteins for MT1-MMP and leading to the design of its selective inhibitors suitable for cancer chemotherapy.

UFT and its metabolites inhibit the angiogenesis induced by murine renal cell carcinoma, as determined by a dorsal air sac assay in mice.

UFT, an anticancer agent that is composed of tegafur (FT) and uracil at a molar ratio of 1:4, is widely used in clinical practice in Japan to treat cancer patients requiring a long-term chemotherapy, and it is associated with few side effects, if any. In this study, we have evaluated the inhibitory effect of UFT against RENCA cell-induced angiogenesis by a dorsal air sac assay. Marked angiogenesis is induced by implantation of a chamber containing RENCA cells into mice. In this model, UFT showed a strong angiogenesis-inhibitory effect, whereas 5-fluorouracil (5-FU) and doxifluridine were less effective. Additional experiments revealed FT to be effective component of UFT; uracil remained ineffective in the inhibition of angiogenesis. Moreover, we have found that gamma-hydroxybutyric acid and gamma-butyrolactone, the metabolites of FT, possess a potent angiogenesis inhibitory effect that is amplified when the compounds are administered by a continuous infusion. This may reflect a transition in blood concentration of each metabolite resulting from the administration of UFT. Similar results were also obtained with respect to 5-FU. It was suggested that UFT has a stronger angiogenesis-inhibitory effect than did other fluorinated pyrimidines, partly due to its pharmacokinetic properties characterized by maintaining of higher and long-lasting blood levels of 5-FU and partly due the inhibitory effects derived from gamma-hydroxybutyric acid and gamma-butyrolactone, UFT-specific metabolites.

The effect of a thymidine phosphorylase inhibitor on angiogenesis and apoptosis in tumors.

Thymidine phosphorylase (TP) is an enzyme involved in the reversible conversion of thymidine to thymine and is identical to an angiogenic factor, platelet-derived endothelial cell growth factor. TP is expressed at higher levels in a wide variety of solid tumors than in the adjacent nonneoplastic tissues. Patients with TP-positive colon and esophageal tumors have a poorer prognosis than those with TP-negative tumors. We have recently synthesized a new TP inhibitor (TPI), 5-chloro-6-[1-(2-iminopyrrolidinyl) methyl] uracil hydrochloride. We investigated the effect of TPI on angiogenesis in KB cells transfected with platelet-derived endothelial cell growth factor cDNA, KB/TP, and a mock transfectant, KB/CV, using the mouse dorsal air sac assay model. We found that KB/TP cells had a higher angiogenic ability than KB/CV cells and that TPI completely suppressed angiogenesis by KB/TP. Furthermore, at a dose of 50 mg/kg/day, TPI considerably decreased the growth rate of KB/TP cells xenografted into nude mice. Microvessel density in KB/TP tumors was higher than that in KB/CV tumors, and TPI did not significantly change the density in either of the tumors. The apoptotic index in KB/TP tumors was significantly lower than that in KB/CV tumors, and TPI significantly increased the apoptotic index in KB/TP tumors but not in KB/CV tumors. These findings, taken together with previous reports, suggest that the expression of TP plays an important role in tumor growth and that TPI suppresses tumor growth by increasing the proportion of apoptotic cells and probably inhibiting angiogenesis.

Expression of thymidine phosphorylase and vascular endothelial cell growth factor in human head and neck squamous cell carcinoma and their different characteristics.

BACKGROUND: Thymidine phosphorylase (dThdPase) is identical to platelet-derived endothelial cell growth factor (PD-ECGF). dThdPase is known to promote the development of new blood vessels, which are fundamental to tumor growth and metastasis. Vascular endothelial growth factor (VEGF) is a 34-42 kilodalton (kD) protein that induces both angiogenesis and vascular permeability. Proliferating cell nuclear antigen (PCNA) is a 36 kD nuclear protein, and its expression is associated with DNA synthesis and cell proliferation. METHODS: The authors investigated the correlations of dThdPase and VEGF with the growth of head and neck squamous cell carcinoma (HNSCC) in 95 patients by examining PCNA expression as a marker of tumor proliferation. They also retrospectively examined the expression of dThdPase in primary HNSCC and its association with angiogenesis and clinicopathologic findings. RESULTS: Microvessel count was significantly correlated with the expression of VEGF (P = 0.046) but not with dThdPase expression. The expression of PCNA was significantly correlated with dThdPase (P < 0.001) but not VEGF expression. A significant correlation was found between VEGF and dThdPase expression (P = 0.003). Neither dThdPase nor VEGF correlated with clinicopathologic findings, except for the correlation between tumor location and VEGF expression (P 0.020). CONCLUSIONS: These findings suggest that VEGF is involved in angiogenesis in HNSCC. dThdPase may have effects on tumor growth other than angiogenic activity in HNSCC.

Thymidine phosphorylase in human esophageal squamous cell carcinoma.

BACKGROUND: Experimental evidence has shown that thymidine phosphorylase (dThdPase) is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and has angiogenic activity. The enzymatic activity of dThdPase was needed for the angiogenesis by the enzyme. These observations were catalysts for the current study. METHODS: The authors examined retrospectively the expression of the angiogenic factor dThdPase in 163 primary esophageal squamous cell carcinomas and its association with angiogenesis and clinicopathologic findings. To determine whether dThdPase expression was a prognostic factor after adjustment for the established prognostic factors and microvessel count, the authors conducted a survival analysis using the Cox proportional hazards model. RESULTS: dThdPase was expressed significantly more frequently (P < 0.001) in esophageal carcinomas (83 of 163, 50.9%) than in adjacent nonneoplastic esophageal tissue samples (20 of 163, 12.3%). Microvessel counts were significantly higher (P < 0.001) in dThdPase positive carcinomas (18.3+/-6.2) than in dThdPase negative carcinomas (8.2+/-7.5). Significant correlations were observed between dThdPase expression and numerous clinicopathologic findings, including pT, pN, pM categories; lymphatic invasion; venous invasion; and residual tumors. Prognostic variables studied using a Cox hazard regression model confirmed that dThdPase expression was an independent prognostic factor in esophageal squamous cell carcinoma, although pN category was the best predictor of patient survival. CONCLUSIONS: This study indicated that in esophageal squamous cell carcinoma, dThdPase expression is associated with angiogenesis and is an unfavorable prognostic factor. These findings implied that the inhibition of dThdPase would improve the prognoses of some patients with dThdPase positive esophageal tumors.

The expression of thymidine phosphorylase/platelet-derived endothelial cell growth factor is correlated to angiogenesis in breast cancer.

It has been shown that human thymidine phosphorylase (TP) is identical to platelet-derived endothelial cell growth factor and has angiogenic activity. In the present study, the expression of TP was examined in 139 mammary carcinomas and 35 benign mammary disorders using biochemical and immunohistochemical methods. Moreover, in order to evaluate the significance of TP expression in mammary carcinomas, the relationship between vascular density and various clinicopathological factors, including age and menopausal status of patients with a mammary carcinoma, were compared with the size, nodal status, expression of estrogen receptor (ER), progesterone receptor (PgR), c-erbB-2, p53 and TP of a mammary carcinoma. Thymidine phosphorylase expression increased in both the nuclei and cytoplasm of mammary carcinoma cells in comparison to mammary benign disorder cells. The number of microvessels in mammary carcinomas was generally correlated to the number of tumor cells with TP expression in cytoplasm. The number of cells with TP expression in cytoplasm was significantly large in tumors that measured 3-4 cm in diameter, compared with tumors measuring 1-2 and 5-6 cm in diameter. In mammary tumors of 1-4 cm diameter, TP expression and vessel density were significantly high in tumors negative for ER or positive for c-erbB2 and in tumors positive for TP or c-erbB2, respectively; whereas tumors of 5-6 cm in diameter were not modified by any clinicopathological factors. The results indicated that TP plays an important angiogenetic role in mammary carcinomas, especially tumors with a certain progression.

Expression of thymidine phosphorylase is associated with a poor prognosis in patients with ductal adenocarcinoma of the pancreas.

Thymidine phosphorylase (dThdPase) is identical to platelet-derived endothelial cell growth factor and has angiogenic activity. In this study, we investigated the expression of dThdPase in ductal adenocarcinoma of the pancreas and examined the correlation between dThdPase expression and clinicopathological factors and clinical outcome. dThdPase expression was demonstrated by immunohistochemistry in the cytoplasm of tumor cells in 59% of the 54 patients studied. The expression of dThdPase correlated significantly with a poor prognosis (P=0.013). Significant correlations were also observed between dThdPase expression and extrapancreatic neural plexus invasion and the presence of postoperative hepatic metastases (P=0.05 and 0.03, respectively). The average microvessel count in dThdPase-positive tumors was significantly higher than that in dThdPase-negative tumors (P < 0.0001). These findings suggest that dThdPase expression in pancreatic adenocarcinoma enhances the abilities of tumor invasion and/or metastasis through its angiogenic properties.

Prevention of hypoxia-induced apoptosis by the angiogenic factor thymidine phosphorylase.

The angiogenic factor platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is expressed at higher levels in a wide variety of solid tumors compared to adjacent normal tissues. Patients with PD-ECGF/TP-positive colon and esophageal tumors have a poorer prognosis than those with negative tumors. The expression of PD-ECGF/TP is a prognostic factor independent of microvessel density suggesting that TP has effects on tumor progression independent of its angiogenic activity. Evidence that hypoxia and apoptosis affect tumor growth prompted us to determine whether increased expression of PD-ECGF/TP prevents apoptosis induced by hypoxia. KB/TP cells transfected with a PD-ECGF/TP cDNA were resistant to hypoxia-induced apoptosis. Among the degradation products of thymidine produced by PD-ECGF/TP, 2-deoxy-D-ribose and thymine partially prevented hypoxia-induced apoptosis. The ability of 1 microM 2-deoxy-D-ribose in combination with the same concentration of thymine to prevent hypoxia-induced apoptosis was similar to that of the overexpressed TP in KB cells. A concentration of 1 microM 2-deoxy-L-ribose abrogated the effects of these degradation products of thymidine. These findings suggested that TP can confer resistance to apoptosis induced by hypoxia and the degradation products of thymidine are involved in this resistance. Expression of PD-ECGF/TP may play an important role in the progression of solid tumors, and inhibitors of TP and analogs of the degradation products of thymidine may suppress the growth of tumors by promoting apoptosis.

Correlation between thymidine phosphorylase expression and prognosis in human renal cell carcinoma.

PURPOSE: Thymidine phosphorylase (TP) is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and has angiogenic activity. We examined whether TP expression in renal cell carcinoma (RCC) is associated with microvessel density as a marker of angiogenesis, clinicopathologic characteristics, and outcome. PATIENTS AND METHODS: The enzymatic activity and expression of TP were examined in 18 RCCs and 19 kidney tissues not grossly involved with tumor from 24 patients with 13 paired samples and 11 unpaired samples by spectrophotometry and immunoblotting. The relationship between TP expression and microvessel density was assessed by immunohistochemistry in 133 RCCs. RESULTS: The median enzymatic activity of TP in RCCs was nine fold higher than that in nonneoplastic kidney tissues (P < .001). Similar results were obtained by immunoblot analysis. According to the TP staining profile, tumors were classified as no or low, intermediate, or high TP-expressing tumors. TP positivity was significantly correlated with microvessel density. TP expression was correlated with tumor grade, but there was no significant association between TP expression and other clinicopathologic characteristics. TP expression as a prognostic variable was studied using Cox's proportional hazards model. TP overexpression was an independent prognostic factor (hazards ratio, 3.95; 95% confidence interval, 0.98 to 15.89; P = .039) as were nodal category, metastases category, tumor grade, and venous invasion. CONCLUSION: These findings suggest that TP expression is correlated with microvessel density in RCC and is an unfavorable independent prognostic factor. The future development and characterization of TP inhibitors may provide a novel approach to the therapy of RCC.

Thymidine phosphorylase activity required for tumor angiogenesis and growth.

Thymidine phosphorylase (TP) is identical to platelet-derived endothelial cell growth factor (PD-ECGF), and has angiogenic activity. We examined the involvement of TP activity in tumor growth and angiogenesis. KB cells were transfected with wild-type or mutant (L148R) PD-ECGF cDNA, and two sublines with high TP activity, KB/wt4 and KB/wt6, and one subline with no TP activity, KB/L148R, were cloned, respectively. The doubling times of these subclones in vitro were similar to that of KB cells. However, the growth of KB/wt4 and KB/wt6 cells was significantly faster when xenografted into nude mice than that of control cells with no TP activity. The tumors with high TP activity (KB/wt4 and KB/wt6) had significantly more microvessels than those with no TP activity (KB/-, KB/CV and KB/L148R) (P<0.01). These results, taken together with previous reports, suggest that the TP enzyme activity itself is involved in angiogenesis and growth of the KB tumors.

Expression of thymidine phosphorylase and angiogenesis in human ovarian tumor.

Thymidine phosphorylase (dThdPase) is an enzyme involved in pyrimidine nucleoside metabolism. dThdPase activity is increased in various types of malignant tumors. Recently, we demonstrated that dThdPase is identical to platelet-derived endothelial cell growth factor (PD-ECGF), and has angiogenic activity. We measured the dThdPase activity and the level of thrombomodulin (TM), as a marker for endothelial cells, in ovarian carcinomas, benign tumors and normal ovarian tissues. The average dThdPase activity of ovarian carcinomas (10.86+/-6.98 nmol/100 mu g protein/h) was significantly higher than that of benign tumors (4.66+/-3.91 nmol/100 mu g protein/h) or normal tissues (2.52+/-1.90 nmol/100 mu g protein/h). The expression of dThdPase detected by immunoblot analysis was well correlated with dThdPase activity. In an immunohistochemical study, the expression of dThdPase was more frequently observed in ovarian carcinomas than in benign tumors or normal tissues. dThdPase activity in human ovarian carcinomas was significantly correlated with the expression of TM in human ovarian carcinomas. These findings suggest that dThdPase expression is significantly correlated with angiogenesis in ovarian tumors.

Clinicopathologic and prognostic significance of an angiogenic factor, thymidine phosphorylase, in human colorectal carcinoma.

BACKGROUND: Platelet-derived endothelial cell growth factor (PD-ECGF) is known to promote the development of new blood vessels, which are fundamental to tumor growth and metastasis. We previously found that thymidine phosphorylase (dThdPase) and PD-ECGF are the same protein. PURPOSE: We retrospectively examined the expression of dThdPase in primary colorectal carcinomas, its association with angiogenesis and clinicopathologic findings, and its prognostic value. METHODS: Tissues were obtained from the tumors of 163 patients whose colorectal carcinomas were completely removed by surgery. Microvessels assessed by immunostaining endothelial cells for factor VIII were counted on a 400x field in the most active areas of neovascularization within the tumor. We purified the monoclonal antibody against dThdPase and studied the expression of dThdPase in the same serial sections used for the detection of factor VIII. Those who carried out microvessel counting and dThdPase expression assessment had no knowledge of clinicopathologic findings. The significance of dThdPase in the prognosis of patients with colorectal carcinomas was also examined in the survival analysis of mortality follow-up data covering the period between 1984 through 1991. Reported P values are from two-sided tests of statistical significance. RESULTS: The mean microvessel count (+/- standard deviation) in dThdPase-positive colorectal carcinoma specimens (17.5 +/- 7.2) was higher (P < .001) than that in dThdPase-negative carcinoma specimens (9.3 +/- 5.5). The dThdPase positivity was in accordance with the microvessel count. dThdPase positivity showed highly significant statistical associations with tumor size, extent of invasion, lymph node metastasis, lymphatic invasion, and venous invasion. Cox regression analysis revealed that dThdPase expression was prognostic for poor disease outcome after adjustment for Dukes' stage and microvessel count. CONCLUSIONS: These findings suggest that higher levels of dThdPase expression in colorectal carcinomas are associated with more extensive angiogenesis, poor clinical and laboratory findings, and unfavorable clinical outcome. IMPLICATIONS: Inhibition of dThdPase in human colorectal carcinomas might improve prognosis for some patients.

The activity and expression of thymidine phosphorylase in human solid tumours.

Thymidine phosphorylase (dThdPase) is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and has angiogenic activity. Since dThdPase seems to have an important role in angiogenesis of tumours, we measured the activity and expression of dThdPase in various tumours and the adjacent non-neoplastic tissues. We assayed dThdPase activity by spectrophotometric means, and the expression of dThdPase was examined by immunoblotting and by immunohistochemical staining using a monoclonal antibody against dThdPase. In the oesophagus, stomach, colorectum, pancreas, and lung, dThdPase activity in carcinomas was significantly higher (P < 0.05) than that in the adjacent non-neoplastic tissues. The expression level of dThdPase detected by immunoblotting correlated well with the activity of dThdPase. In the oesophagus, stomach, colorectum, gall bladder, pancreas and lung, the proportion of dThdPase-positive tumours was significantly higher (P < 0.05 or 0.01) than that of the dThdPase-positive adjacent normal tissues. In oesophageal, gastric colorectal and lung carcinomas, the proportion of dThdPase positivity in advanced carcinomas was significantly higher (P < 0.01) than that in early carcinomas. Tumour-infiltrative macrophages or lymphocytes in the lymph node, alveolar macrophages and Kupffer cells expressed high levels of dThdPase. The results indicate that dThdPase activity and expression level in many tumours are higher than those in the adjacent non-neoplastic tissues, and that dThdPase may have an important role in the proliferation of these solid tumours.

Expression of thymidine phosphorylase in human gastric carcinoma.

The activity of thymidine phosphorylase (dThdPase) has been reported to increase in several types of malignant tumors. Experimental evidence has shown that dThdPase is identical to platelet-derived endothelial cell growth factor, and that dThdPase has angiogenic activity. We examined the expression of dThdPase to investigate whether the expression of dThdPase correlates with angiogenesis, clinicopathologic features and the prognosis of patients with human gastric carcinomas. Microvessels were assessed by immunostaining endothelial cells for factor VIII. We counted microvessels in the tumors of 158 patients whose tumors were completely removed surgically. Microvessels were counted in a x400 field in the most active areas of neovascularization. We purified a monoclonal antibody (TMA-1) against dThdPase and studied the expression of dThdPase using TMA-1 in the same serial sections as those used for the detection of factor VIII. The correlation between angiogenesis and dThdPase, and the clinicopathological significance of dThdPase, in patients with gastric carcinoma were examined. The positive expression of dThdPase was more frequent (P < 0.001) in gastric carcinomas (67/158, 43.4%) than that in normal tissues (12/158, 7.6%). The average microvessel count in dThdPase-positive gastric carcinomas was higher (P < 0.001) than that in dThdPase-negative carcinomas. The percentage of gastric carcinoma cells expressing dThdPase was significantly correlated with the microvessel count (P < 0.001). Further, the average size of dThdPase-positive carcinomas was significantly larger (P < 0.001) than that of negative carcinomas and the mean microvessel count in dThdPase-positive gastric carcinomas was also significantly higher (P < 0.001) than that in dThdPase-negative carcinomas. There was a significant correlation between the positive expression of dThdPase and microvessel count (P < 0.001) or lymph node metastasis (P = 0.013) by multivariate logistic analysis. Further, patients with dThdPase-positive carcinoma showed a significantly worse prognosis than those with dThdPase-negative carcinoma overall and in stage III. These findings indicate that the expression of dThdPase in gastric carcinomas is related to progression and metastasis, and this enzyme affects the prognosis of some patients with the disease.