Yuji Takebayashi

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.

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

PURPOSEThymidine 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 METHODSThe 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.RESULTSThe 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 ...

Expression of Thymidine Phosphorylase in Human Gastric Carcinoma

The activity of thymidine phosphorylase (dThdPase) has heen 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 immnnostaining 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 × 400 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.

The expression of thymidine phosphorylase and thrombomodulin in human colorectal carcinomas

Thymidine phosphorylase (dThdPase) is an enzyme involved in pyrimidine nucleoside metabolism. dThdPase activity is increased in several types of malignant tumors. Recently, we demonstrated that dThdPase is identical to platelet-derived endothelial cell growth factor (PD-ECGF) and that dThdPase has angiogenic activity. We measured dThdPase activity and the level of thrombomodulin (TM) as a marker for endothelial cells in colorectal carcinomas and adjacent normal tissues from 21 patients, and in adenomas from 13 patients. The average dThdPase activity of colorectal carcinomas (11.58 +/- 6.30 nmol/100 micrograms protein/h) was significantly higher than that of adenomas (8.57 +/- 4.14 nmol/100 micrograms protein/h) or normal tissues (4.89 +/- 3.16 nmol/100 micrograms protein/h). In immunohistochemical study, the expression of dThdPase was observed more frequently in colorectal carcinomas than in adenomas or normal mucosas. The amount of TM in colorectal carcinomas (8.32 +/- 5.07 ng/100 micrograms protein) was significantly higher than that of adenomas (4.51 +/- 4.49 ng/100 micrograms protein) or normal tissues (3.51 +/- 2.78 ng/100 micrograms protein). dThdPase activity in human colorectal carcinomas, adenomas and normal tissues was significantly correlated with the expression of TM in these tissues. These results indicate that the expression levels of both dThdPase and TM in colorectal carcinomas are higher than those in colorectal adenomas and normal tissues and suggest that dThdPase may be involved in angiogenesis in human colorectal carcinomas, adenomas and normal tissues.

Expression of the multidrug‐resistance‐associated protein (MRP) gene in human colorectal, gastric and non‐small‐cell lung carcinomas

MRP has been identified as another multidrug‐resistance (MDR) gene and may be involved in an alternative MDR mechanism in some solid tumors. We investigated the expression of MRP mRNA in multidrug‐resistant KB sublines (KB‐8‐5, KB‐C2, C‐A40 and C‐A120), human non‐small‐cell lung carcinomas (NSCLC), gastric and colorectal carcinomas, and compared it with that in drug‐sensitive human KB cells, MRP gene expression was elevated in 8 of 9 (89%) squamous‐cell carcinomas of the lung. Furthermore, MRP expression in 4 squamous‐cell carcinomas (L13, 18, 19 and 20) was more than 3.6 times higher than in KB‐3‐I cells, and the average MRP mRNA expression level of all squamous‐cell carcinomas was significantly higher than that of adenocarcinoma of the lung and of colorectal and gastric carcinomas. These results suggested that the MRP is responsible, at least in part, for drug resistance in some squamous‐cell carcinomas of the lung.

Preoperative assessment of vascularity by color Doppler ultrasonography in human rectal carcinoma.

PURPOSE: Although angiogenesis assessed by immunostaining endothelial cells (microvessel density) is a well-known prognostic factor in a wide variety of human solid tumors, preoperative determination of microvessel density seems to be difficult in rectal carcinoma. Thus, we performed transanal color Doppler ultrasonography in 46 patients with rectal carcinoma to assess preoperative angiogenic status and compare it with microvessel density in surgical specimens. METHODS: Time-averaged maximal velocity, peak systolic velocity, number of vascular points, and vascular point index were conducted by color Doppler ultrasonography in 46 patients with rectal carcinoma. Number of vascular points was defined as the number of vessels with pulsation in the section of tumor. Vascular point index was defined as the average number of vascular points divided by the area assessed by color Doppler ultrasonography in the section of tumor. The profiles of number of vascular points were similar to those assessed by microangiography in five rectal carcinomas. RESULTS: Vascular point index significantly correlated with microvessel density (P<0.0001). No significant correlation was found between microvessel density and time-averaged maximal velocity or peak systolic velocity. Vascular point index was also a better indicator of lymph node metastasis and venous invasion than microvessel density. In addition, 11 of 46 cases with postoperative hematogenous metastasis (23.9 percent) were observed prospectively. Vascular point index may be a best predictor for hematogenous metastasis from rectal carcinoma compared with peak systolic velocity, time-averaged maximal velocity, and microvessel density by receiver operating characteristic analysis. CONCLUSION: These results suggest that preoperative quantification of angiogenesis using color Doppler ultrasonography will provide quick and useful information in the management of rectal carcinoma.

The expression of multidrug resistance protein in human gastrointestinal tract carcinomas

Multidrug resistance protein (MRP) is a membrane phosphoglycoprotein with an Mr of 190,000 that is involved in the non‐P‐glycoprotein mediated multidrug resistance of human tumor cells. The aim of this study was to determine the clinicopathologic relevance of MRP expression in human gastrointestinal tract carcinomas.

Increased sensitivity to vincristine of MDR cells by the leukotriene D4 receptor antagonist, ONO-1078

The leukotriene D4 (LTD4) receptor antagonist, 4-oxo-8-[p-(4-phenylbutyloxy)benzoylamino]-2-(tetrazol-5-yl) -4H-1-benzopyran hemihydrate (ONO-1078) is used for the treatment of allergic asthma and other immediate hypersensitivity diseases. We examined the effect of ONO-1078 on the sensitivity to vincristine (VCR) of MRP overexpressing multidrug-resistant CV60 and its parental drug-sensitive KB-3-1 cell lines. The sensitivity to VCR of KB-3-1 and CV60 cells was increased 13- and 15-fold, respectively, by ONO-1078 at the maximum non-toxic concentration (100 microM). The VCR sensitivity of multidrug-resistant KB-C2 cells that overexpressed P-gp was increased 2.6-fold by ONO-1078. The accumulation of VCR in KB-3-1, CV60 and KB-C2 cells was significantly increased by ONO-1078. The efflux of VCR from KB-3-1 cells was not inhibited, but that from CV60 cells was enhanced compared with that from KB-3-1 cells and was partially inhibited by ONO-1078. ONO-1078 competitively inhibited the ATP-dependent [3H]LTC4 uptake in membrane vesicles isolated from CV60 cells. These findings suggest that ONO-1078 inhibits the transporting activity of MRP and that ONO-1078 increases the sensitivity to VCR of KB-3-1 cells by increasing the VCR uptake in the cells.

The correlation of thymidine phosphorylase activity with the expression of interleukin 1α, interferon α and interferon γ in human colorectal carcinoma

Abstract Thymidine phosphorylase (dThdPase) is an angiogenic enzyme and seems to be related to an angiogenesis in human colorectal carcinoma. The incidence of dThdPase-positive cells was significantly correlated with microvessel count in 21 human colorectal carcinomas. Interleukin 1α (IL-1α), tumor necrosis factor α (TNF-α), interferon α (IFN-α) interferon γ (IFN-γ) induce dThdPase activity in human cancer cell lines. To study whether this phenomenon occurs in the human colorectal carcinomas, we examined the correlation between dThdPase activity and the expression levels of IL-1α, TNF-α, IFN-α and IFN-γ in colorectal carcinoma tissues. dThdPase activity was assayed by the methods of Friedkin and Robert, and the expression level of IL-1α, TNF-α, IFN-α and IFN-γ was determined by ELISA. dThdPase activity was significantly correlated with the amount of IL-1α (n = 19, r = 0.347, P = 0.0001), INF-α (n = 18, r = 0.717, P = 0.0008), and IFN-γ (n = 4, r = 0.9777, P = 0.0234) in human colorectal carcinomas. However, the dThdPase activity was not correlated with the amount of TNF-α (n = 21, r = 0.235, P = 0.2682). These results suggested that the expression levels of IL-1α, IFN-α and IFN-γ are correlated with dThdPase activity in human colorectal carcinomas and that these cytokines may cause angiogenesis by inducing the expression of dThdPase.

Expression of the multidrug resistance-associated protein (MRP) gene in urothelial carcinomas.

The intrinsic or acquired resistance of urothelial cancer to chemotherapy is one major obstacle to successful treatment. Generally, the expression level of P‐glycoprotein in urothelial cancer is low, so we accordingly investigated the expression of multidrug resistance‐associated protein (MRP). We examined the expression of MRP mRNA by means of slot‐blotting samples of 11 renal pelvic and/or ureteral tumors, 33 bladder tumors, one lung metastasis from a ureter tumor, 7 non‐cancerous urothelia from patients with transitional‐cell carcinoma (TCC) and one urothelium from a patient with renal‐cell carcinoma (RCC). We also estimated, by Southern blotting, whether or not the MRP gene was amplified in clinical specimens that overexpressed MRP mRNA. MRP was detected immunohistochemically using a polyclonal antibody against MRP. In all, 5 of 11 renal pelvic and/or ureter tumors (45.5%), 17 of 33 bladder tumors (51.5%) and 4 of 7 non‐cancerous urothelia of TCC patients (57.1%) expressed more than 2‐fold the MRP mRNA levels of drug‐sensitive human KB cells. There was no significant difference in the MRP mRNA level between primary and recurrent tumors. Low‐grade urothelial carcinomas (G1 and G2 TCCs) expressed significantly higher levels of MRP mRNA than the high‐grade G3 TCC. The MRP gene was not amplified in urothelial carcinomas, irrespective of their expression levels of MRP mRNA. Immunohistochemically, MRP was located mainly on the plasma membrane, but also detected on the cytoplasm of cancer cells. MRP may be one mechanism responsible for intrinsic drug resistance in low‐grade urothelial cancer.