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Significance of Orotate Phosphoribosyltransferase Activity in Renal Cell Carcinoma
0022-5347/04/1712-0605/0 THE JOURNAL OF UROLOGY® Copyright © 2004 by AMERICAN UROLOGICAL ASSOCIATION
Vol. 171, 605– 610, February 2004 Printed in U.S.A.
DOI: 10.1097/01.ju.0000107833.03488.d2
SIGNIFICANCE OF OROTATE PHOSPHORIBOSYLTRANSFERASE ACTIVITY IN RENAL CELL CARCINOMA YOICHI MIZUTANI,* HIROMI WADA, OSAMU YOSHIDA, MASAKAZU FUKUSHIMA, HIROYUKI NAKANISHI AND TSUNEHARU MIKI From the Department of Urology, Kyoto Prefectural University of Medicine (YM, HN, TM) and Departments of Thoracic Surgery (HW) and Urology (OY), Graduate School of Medicine, Faculty of Medicine, Kyoto University, Kyoto and Cancer Research Laboratory, Taiho Pharmaceutical Co., Ltd. (MF), Saitama, Japan
ABSTRACT
Purpose: The anticancer agent 5-fluorouracil (5-FU) is clinically used against various cancers, including renal cell carcinoma (RCC). It is a prodrug and orotate phosphoribosyltransferase (OPRT) is the principal enzyme that directly converts 5-FU to the active anticancer metabolite 5-fluoro-2⬘-deoxyuridine 5⬘-monophosphate. In addition, OPRT is the key enzyme in the de novo DNA and RNA synthetic process. Little is known about the significance of OPRT in various cancers, including RCC. We investigated the activity of OPRT in 83 RCCs and evaluated the association between the level of OPRT activity and the stage/grade of RCC. The relationship between OPRT activity in RCC cells and their sensitivity to 5-FU was also examined. Materials and Methods: OPRT activity in nonfixed, fresh frozen RCC and normal kidney was determined enzymatically by the 5-FU phosphorylation assay. The sensitivity of RCC cells to 5-FU was assessed by the microculture tetrazolium dye assay. Results: The activity of OPRT was approximately 8.5-fold higher in RCC than in normal kidney. OPRT activity in stage III/IV RCC was 3-fold higher than in stage I/II RCC. The level of OPRT activity in grade 3 RCC was 3-fold higher than that in grade 1/2 cancer. Patients with RCC with low OPRT activity had longer postoperative disease specific survival than those with high activity at 5-year followup. OPRT activity in RCC cells positively correlated with their sensitivity to 5-FU. Conclusions: To our knowledge this is the first study to demonstrate that OPRT activity in RCC was higher than that in normal kidney and OPRT activity positively correlated with RCC stage/ grade. In addition, higher OPRT activity in RCC predicted worse prognosis and higher sensitivity to 5-FU. These results suggest that the level of OPRT activity may be used as a prognostic parameter and predictive indicator for 5-FU efficacy in RCC and OPRT may be a molecular therapeutic target in RCC. KEY WORDS: kidney; carcinoma, renal cell; fluorouracil; orotate phosphoribosyltransferase; tumor markers, biological
The agent 5-fluorouracil (5-FU) is one of the widely used anticancer chemotherapeutic agents for various cancers, including renal cell carcinoma (RCC).1 Alone 5-FU is inactive and it requires intracellular conversion to 5-fluorouridine 5⬘-monophosphate and, furthermore, to 5-fluoro-2⬘deoxyuridine 5⬘-monophosphate (FdUMP). FdUMP exerts cytotoxic activity through the formation of a ternary complex with thymidylate synthase (TS) and 5,10-methylenetetrahydrofolate, resulting in TS inhibition and blockade of the DNA synthetic process.2 Orotate phosphoribosyltransferase (OPRT) is the first limiting enzyme in this 5-FU conversion, leading to the formation of FdUMP in the presence of 5-phosphoribosyl-l-pyrophosphate as a co-factor.3 Previous studies have demonstrated that adenovirus mediated transduction of the OPRT gene results in marked sensitization of colon, gastric, liver and pancreas cancer cells to 5-FU cytotoxicity.4 Thus, OPRT is the important enzyme in 5-FU activation. In addition, OPRT is the rate limiting enzyme in the
de novo process of DNA and RNA synthesis, which converts orotic acid to orotidine 5⬘-phosphate. Although 5-FU is clinically used for RCC and OPRT is an important enzyme in 5-FU cytotoxicity and DNA synthesis, reported data on OPRT activity in RCC are limited. Little is known about the significance of OPRT activity in the biology of RCC. In the current study we measured OPRT activity in 83 RCCs and evaluated the relationship between the level of OPRT activity and stage/grade status of RCC. In addition, we investigated the association between OPRT activity in RCC and sensitivity to 5-FU. MATERIALS AND METHODS
Patients. Surgical specimens were obtained from 83 patients with RCC, including 61 men and 22 women 32 to 83 years old. Histological diagnosis revealed that 74, 8 and 1 patients had clear cell carcinoma, papillary RCC and Bellini duct carcinoma, respectively. Histological classification and staging according to the 1997 TNM classification, fifth edition were T1 to T4 in 56, 12, 11 and 4, N0 to N2 in 80, 1 and 2, M0 and M1 in 74 and 9, stages I to IV in 52, 11, 8 and 12, and G1 to G3 in 11, 55 and 17 cases, respectively. RCC tissues were pathologically examined for viability and absent necrotic areas. Samples of normal kidneys were collected from the same 83 patients with RCC. Specimens were stored frozen at ⫺80C until assay for OPRT, TS and thymidine
Accepted for publication September 26, 2003. Supported by a grant-in-aid from the Japanese Urological Association and Grants-in-Aid 14657410 and 15390496 from the Japanese Ministry of Education, Culture, Sports, Science and Technology. * Correspondence: Department of Urology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 6028566, Japan (telephone: ⫹81-75-251-5595; FAX: ⫹81-75-251-5598; e-mail: [email protected]). 605
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kinase (TK) activities. These enzyme activities were measured in at least 2 samples derived from each patient. Measurement of OPRT activity in RCC and normal kidney. OPRT activity was determined by the 5-FU phosphorylation assay, as previously described.3 RCC and normal kidney were sonicated in homogenate buffer (50 mM tris-HCl, pH 7.4, and 5 mM 2-mercaptoethanol) at maximum output using a Sonifier Cell Disruptor 350 (SmithKline, London, United Kingdom). They were centrifuged at 105,000 ⫻ gravity at 4C for 60 minutes in a model TL-100 (Beckman, Fullerton, California) ultracentrifuge. The supernatants from each sample were divided into several tubes and frozen at ⫺80C until use. Test supernatant was incubated with 10 M [6-3H] 5-FU (74 kBq), 50 mM tris-HCl, pH 8.0, 50 mM MgCl2, 10 mM NaF and 4 mM phosphoribosylpyrophosphate at 37C for 30 minutes. Incubation was terminated by the addition of 2 M perchloric acid, followed by centrifugation at 3000 rpm for 10 minutes. Supernatant (100 l) was then neutralized with 30 l 2 M KOH solution and 20 l aliquots were subjected to silica gel 60F254 thin layer chromatography (2.5 ⫻ 10 cm and 0.25 mm thick) with a mixture of chloroform, methanol and acetic acid (17:3:1 volume per volume per volume) as the mobile phase. Spots of 5-fluorouridine 5⬘-monophosphate were scraped into vials and extracted with 0.1 ml 4 M HCl. The extracts were mixed with 10 ml ACS-II (Amersham, Buckinghamshire, United Kingdom) scintillation fluid. Radioactivity was measured in a Wallac 1410 (Pharmacia, Uppsala, Sweden) liquid scintillation counter. Protein content of test supernatant was measured using BCA protein assay reagent (Pierce Chemical Co., Rockford, Illinois). OPRT activity was calculated per mg protein. Internal standards were used to compare assays. We analyzed all samples at the same time. This method made it possible to estimate OPRT activity higher than 0.05 pmol/mg protein per minute. Repeat measurements yielded almost the same results. OPRT activity greater than the median value (1.52 pmol/mg protein per minute) was considered high activity. OPRT activity less than the median value was considered low activity. Measurement of TS activity in RCC. TS activity was determined by the FdUMP binding assay combined with gel filtration, as previously described.5, 6 This method made it possible to estimate TS activity higher than 1.0 fmol/mg protein. Measurement of TK activity in RCC. TK activity was measured by determining the conversion of labeled thymidine to labeled nucleotides by the diethylaminoethyl cellulose disc method, as previously described.7, 8 This method made it possible to estimate TK activity higher than 1.0 pmol/mg protein per minute. Tumor cells. Fresh RCC cells derived from 22 patients were separated from surgical specimens for in vitro primary culture, as previously described.9, 10 Histological staging according to the 1997 TNM classification, fifth edition were stages I to IV in 16, 2, 1 and 3, and G1 to G3 in 3, 17 and 2 cases, respectively. ACHN and NC65 human RCC cell lines were maintained in monolayers on plastic dishes in complete medium.9, 10 Cytotoxicity assay. Microculture tetrazolium dye assay was used to determine tumor cell lysis, as previously described.11, 12 Statistical analysis. All determinations were made in triplicate. For statistical analysis Student’s t and Pearson’s correlation tests were used. Postoperative disease specific survival was determined by the Kaplan-Meier method. The Cox-Mantel test was used to establish the statistical difference in survival between patients with high and low OPRT activity. Factors related to disease specific survival in patients with RCC were also assessed by multivariate analysis with p ⱕ0.05 considered significant.
RESULTS
Activity of OPRT in RCC and normal kidney. Figure 1 shows OPRT activity values in RCC and normal kidney in patients with RCC. Mean OPRT activity in RCC was approximately 8.5-fold higher than that in normal kidney. OPRT activity in normal kidney in patients with RCC was similar to that in patients with renal pelvic or ureteral cancer (data not shown). These findings demonstrated that OPRT activity in RCC was significantly higher than in normal kidney. The level of OPRT activity in RCC. We then examined OPRT activity in RCC as a function of histological stage and grade of the disease. OPRT activity was approximately 3-fold higher in T3/4 RCC than that in T1/2 RCC (fig. 2). Furthermore, OPRT activity in M1 RCC was significantly (2.5-fold) higher than in M0 RCC (fig. 3). OPRT activity in stage III/IV RCC was 3-fold higher than in stage I/II RCC (fig. 4). OPRT activity in grade 3 RCC was 3-fold higher than in grade 1/2 cancer (fig. 5). OPRT activity in clear cell RCC was similar to that in papillary RCC (data not shown). These findings showed that OPRT activity correlated positively with RCC stage progression and increased histological grade. Correlation between OPRT activity and postoperative disease specific survival in patients with RCC. Patients with RCC undergoing radical nephrectomy were evaluated for the postoperative clinical course. Postoperative disease specific survival was estimated by Kaplan-Meier analysis. Based on this analysis patients with RCC were divided into 2 groups, namely those with high OPRT activity (greater than the median value) and those with low activity (less than the median value). Patients with low OPRT activity had longer disease specific survival than those with high activity at 5-year followup (fig. 6). Moreover, notably that no patients with RCC with low OPRT activity died in this study. However, multivariate analysis showed that OPRT activity was not an independent prognostic factor in patients with RCC. Relationship between OPRT and TS activities in RCC. TS as well as OPRT is the key enzyme for DNA synthesis in the de novo pathway.13, 14 We examined the association between OPRT and TS activities in RCC. There was a positive corre-
FIG. 1. OPRT activity in RCC and normal kidney was quantitated by 5-FU phosphorylation assay. Asterisk indicates p ⬍0.05 vs normal.
OROTATE PHOSPHORIBOSYLTRANSFERASE ACTIVITY IN RENAL CELL CANCER
FIG. 2. OPRT activity in RCC according to T classification was quantitated by 5-FU phosphorylation assay. Asterisk indicates p ⬍0.05 vs T1 and T2. Pound sign indicates p ⬍0.05 vs T1/2.
FIG. 3. OPRT activity in RCC according to M classification was quantitated by 5-FU phosphorylation assay. Asterisk indicates p ⬍0.05 vs M0.
lation between the levels of OPRT and TS activities in RCC (fig. 7). Association between OPRT and TK activities in RCC. OPRT is the key enzyme in the de novo DNA and RNA synthetic process, while TK has a key role in the complimentary or alternative salvage pathway of DNA synthesis.15, 16 We have previously observed that TK activity is in parallel
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FIG. 4. OPRT activity in RCC according to stage grouping was quantitated by 5-FU phosphorylation assay. Asterisk indicates p ⬍0.05 vs stage I and stage II. Pound sign indicates p ⬍0.05 vs stage I/II.
FIG. 5. OPRT activity in RCC according to histologic grade was quantitated by 5-FU phosphorylation assay. Asterisk indicates p ⬍0.05 vs grade 1 and grade 2.
with the stage progression and increase in histologic grade of RCC.8 We examined the association between OPRT and TK activities in RCC. There was a positive correlation between the levels of OPRT and TK activities in RCC (fig. 8). These results suggest that OPRT/TS de novo DNA synthetic enzymes and TK salvage enzyme may be up-regulated in high stage/grade RCC. Correlation between OPRT activity in RCC cells and their
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FIG. 6. OPRT activity and postoperative disease specific survival in patients with RCC undergoing radical nephrectomy, as determined by Kaplan-Meier method. OPRT activity greater than median of 1.52 pmol/mg protein per minute was considered high activity and less than median was considered low activity. At 5-year followup there was significant difference in disease specific survival between 41 patients with low OPRT activity (solid line) and 42 with high OPRT activity (dashed line) (Cox-Mantel test p ⬍0.01).
FIG. 7. There was positive correlation between OPRT and TS activities in 83 RCCs (r ⫽ 0.25, Pearson’s correlation test p ⫽ 0.025).
sensitivity to 5-FU. We examined the association between OPRT activity in RCC cells and their sensitivity to 5-FU in 22 primary cultures derived from surgical specimens that served as targets. Figure 9 shows that there was a positive correlation between the level of OPRT activity in RCC cells and their sensitivity to 5-FU. Similar findings were observed at different doses of 5-FU (data not shown). In addition, oxonic acid, an inhibitor of OPRT, significantly suppressed the sensitivity of RCC cells to 5-FU (see table). These findings suggest that the level of OPRT activity in RCC may be a significant predictive parameter for 5-FU efficacy and it may be possible to use 5-FU for RCC with high OPRT activity. DISCUSSION
In the current study we noted that OPRT activity was up-regulated in RCC compared with normal kidney and the level of OPRT activity positively correlated with the stage progression and increase in grade of RCC. Furthermore, to our knowledge this study is the first to show that patients
FIG. 8. There was positive correlation between OPRT and TK activities in 83 RCCs (r ⫽ 0.33, Pearson’s correlation test p ⫽ 0.0025).
FIG. 9. There was positive correlation between OPRT activity in 22 primary cultured RCC cells used as targets and their sensitivity to 100 M 5-FU (r ⫽ 0.68, Pearson’s correlation test p ⫽ 0.0001). Suppression of RCC cell sensitivity to 5-FU by oxonic acid Drugs 5-FU Oxonic acid 5-FU ⫹ oxonic acid
Mean % 5-FU Cytotoxicity ⫾SD ACHN 37.0 ⫾ 2.0 0.5 ⫾ 0.4 6.0 ⫾ 2.2 (p ⬍0.05 vs 5-FU)
NC65 33.0 ⫾ 3.5 3.3 ⫾ 1.6 22.3 ⫾ 1.3 (p ⬍0.05 vs 5-FU)
with RCC with low OPRT activity had longer disease specific survival compared with those with high activity at 5-year followup. Although we report on a small number of patients during a short-term followup, these findings suggest that OPRT may have an important role in regulating the malignant potential of RCC and it may be of prognostic value in RCC. The current study shows that OPRT activity in RCC was significantly higher than in normal kidney. High OPRT activity in RCC may be a reflection of the rate of cancer cell proliferation. We have previously reported that the level of
OROTATE PHOSPHORIBOSYLTRANSFERASE ACTIVITY IN RENAL CELL CANCER
OPRT activity increases in rapidly growing cells, including tumor cells and normal cells, such as those of the testis.17 These findings suggest that OPRT may be necessary for carcinogenesis as well as cell proliferation in RCC. However, further studies are needed to determine the biological interaction between OPRT and growth modulation of RCC. To our knowledge this study is the first to demonstrate that the level of OPRT activity positively correlates with the increase in stage and grade of RCC, and the level of OPRT activity in RCC predicts the clinical outcome. The precise reasons responsible for this relationship currently remain unclear. Since OPRT is the principal de novo DNA and RNA synthetic enzyme associated with cell division and proliferation, it is reasonable to assume that clones of cells that over express OPRT can grow more easily and rapidly following implantation than those that do not over express OPRT. In addition, the current study shows that OPRT activity positively correlates with the activities of TS and TK, which are the key enzymes for pyrimidine synthesis in the de novo and salvage pathways. These findings suggest that DNA synthesis is enhanced by activation of the de novo and salvage pathways in RCC. Accordingly the inhibition of OPRT, TS and TK activities may provide a therapeutic means of preventing the growth of RCC. OPRT activity correlated with the stage and the grade of RCC in this study but the SD of OPRT activity was high. Therefore, it is unclear whether we are dealing with a coincidental observation with an elevated level of enzyme that may or may not be causative of more aggressive biological behavior or simply be one of many that are changed in more aggressive cancers. Increased levels of OPRT activity was observed in higher stage/grade RCC. Since OPRT activity was calculated per mg protein, higher stage/grade RCC may have higher production of OPRT per cell rather than there being more cells in higher stage/grade RCC. The overall response rate of immunotherapy and/or chemotherapy for RCC has improved. However, RCC metastasis and recurrence remain major problems. Therefore, new therapeutic approaches are required for patients. The upregulation of OPRT activity in RCC compared with normal kidney identifies OPRT as a molecular therapeutic target. Since OPRT is the first limiting enzyme that converts 5-FU to 5-fluoro-2⬘-uridine 5-monophosphate, leading to the active anticancer metabolite FdUMP,3 our observation that elevated OPRT activity in RCC was associated with high 5-FU sensitivity may be of potential clinical importance in the management of RCC. In addition, oxonic acid, an inhibitor of OPRT, decreased 5-FU cytotoxicity. Thus, chemoimmunotherapy including 5-FU may be effective for RCC with high OPRT activity. Furthermore, enhancing OPRT activity may provide a therapeutic means of augmenting 5-FU efficacy for RCC. Accordingly OPRT gene therapy may be an option for overcoming 5-FU resistance. OPRT activity in RCC was 8.5-fold higher than in normal kidney. The activity of OPRT in RCC cells positively correlated with their sensitivity to 5-FU. Therefore, the high ratio of cancer-to-normal OPRT activity may contribute to the favorable differential between the anticancer effect and the adverse effect of 5-FU. Thus, a higher degree of 5-FU sensitivity may occur in cancer tissues compared with normal tissues. TS is the target enzyme of 5-FU. Several studies and our previous observation suggest that high TS activity may be related to a favorable response to 5-FU.6, 16 Most administered 5-FU is degraded through the catabolic pathway with dihydropyrimidine dehydrogenase (DPD).18, 19 Our previous report has demonstrated that DPD activity in RCC and bladder cancer inversely correlates with sensitivity to 5-FU.12, 20 These findings suggest that TS and DPD activities as well as OPRT activity in RCC may be an important predictive indi-
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cator for 5-FU efficacy. Therefore, the measurement of TS and DPD activities as well as OPRT activity may be necessary for the accurate evaluation of efficacy of 5-FU containing chemotherapy. The data in this communication show that the level of OPRT activity in metastatic RCC was significantly higher than in nonmetastatic RCC. Since OPRT activity is important for activating 5-FU, metastatic RCC is supposed to be sensitive to 5-FU. However, 5-FU has not been found to be particularly effective for metastatic RCC. A reason may be that the mechanisms responsible for 5-FU resistance in cancer cells are multifactorial.2 CONCLUSIONS
The current study shows that OPRT activity in RCC positively correlates with histological stage and grade, and low OPRT activity is a good prognostic sign. Furthermore, elevated OPRT activity is associated with a high response to 5-FU. These findings suggest that the assessment of OPRT activity may be useful in the management of RCC. Since the level of OPRT activity could be used as a prognostic parameter in patients with RCC and a predictive indicator for 5-FU efficacy against RCC, the accurate prediction of prognosis and 5-FU efficacy may help select patients for more intensive surgical or immunochemotherapy approaches including 5-FU. However, further studies are needed to determine the regulatory effects of OPRT activity in RCC. Oxonic acid and 5-FU were provided by Taiho Pharmaceutical Co., Ltd., Tokyo, Japan. REFERENCES
1. Bukowski, R. M.: Natural history and therapy of metastatic renal cell carcinoma: the role of interleukin-2. Cancer, 80: 1198, 1997 2. Longley, D. B., Harkin, D. P. and Johnston, P. G.: 5-Fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer, 3: 330, 2003 3. Shirasaka, T., Shimamoto, Y. and Fukushima, M.: Inhibition by oxonic acid of gastrointestinal toxicity of 5-fluorouracil without loss of its antitumor activity in rats. Cancer Res, 53: 4004, 1993 4. Kanai, F., Kawakami, T., Hamada, H., Sadata, A., Yoshida, Y., Tanaka, T. et al: Adenovirus-mediated transduction of Escherichia coli uracil phosphoribosyltransferase gene sensitizes cancer cells to low concentrations of 5-fluorouracil. Cancer Res, 58: 1946, 1998 5. Mizutani, Y., Wada, H., Ogawa, O., Yoshida, O., Fukushima, M., Nonomura, N. et al: Prognostic significance of thymidylate synthase activity in bladder carcinoma. Cancer, 92: 510, 2001 6. Mizutani, Y., Wada, H., Yoshida, O., Fukushima, M., Nonomura, M., Nakao, M. et al: Significance of thymidylate synthase activity in renal cell carcinoma. Clin Cancer Res, 9: 1453, 2003 7. Mizutani, Y., Wada, H., Yoshida, O., Fukushima, M., Kamoi, K. and Miki, T.: Prognostic significance of thymidine kinase activity in bladder carcinoma. Cancer, 95: 2120, 2002 8. Mizutani, Y., Wada, H., Yoshida, O., Fukushima, M., Nakao, M. and Miki, T.: Significance of thymidine kinase activity in renal cell carcinoma. J Urol, 169: 706, 2003 9. Wu, X. X., Mizutani, Y., Kakehi, Y., Yoshida, O. and Ogawa, O.: Enhancement of Fas-mediated apoptosis in renal cell carcinoma cells by adriamycin. Cancer Res, 60: 2912, 2000 10. Mizutani, Y., Bonavida, B., Koishihara, Y., Akamatsu, K., Ohsugi, Y. and Yoshida, O.: Sensitization of human renal cell carcinoma cells to cis-diamminedichloroplatinum (II) by antiinterleukin-6 monoclonal antibody or anti-interleukin-6receptor monoclonal antibody. Cancer Res, 55: 590, 1995 11. Mizutani, Y., Nakanishi, H., Yoshida, O., Fukushima, M., Bonavida, B. and Miki, T.: Potentiation of the sensitivity of renal cell carcinoma cells to TRAIL-mediated apoptosis by subtoxic concentrations of 5-fluorouracil. Eur J Cancer, 38: 167, 2002 12. Mizutani, Y., Wada, H., Yoshida, O., Fukushima, M., Nakanishi, H., Nakao, M. et al: Significance of dihydropyrimidine dehy-
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drogenase activity in renal cell carcinoma. Eur J Cancer, 39: 541, 2003 Danenberg, P. V.: Thymidylate synthetase—a target enzyme in cancer chemotherapy. Biochem Biophys Acta, 473: 73, 1977 Navalgund, L. G., Rossana, C., Muench, A. J. and Johnson, L. F.: Cell cycle regulation of thymidylate synthetase gene expression in cultured mouse fibroblasts. J Biol Chem, 255: 7386, 1980 O’Neil, K. L., Grigsby, R. V. and Fairbairn, D. W.: Thymidine kinase: the future in breast cancer prognosis. Breast, 4: 79, 1995 Foekens, J. A., Romain, S., Look, M. P., Martin, P. M. and Klijn, J. G.: Thymidine kinase and thymidylate synthase in advanced breast cancer: response to tamoxifen and chemotherapy. Cancer Res, 61: 1421, 2001
17. Ikenaka, K., Fukushima, M., Nakamura, H., Okamoto, M., Shirasaka, T. and Fujii, S.: Metabolism of pyrimidine nucleotides in various tissues and tumor cells from rodents. Gann, 72: 590, 1981 18. Diasio, R. B., and Lu, Z.: Dihydropyrimidine dehydrogenase activity and fluorouracil chemotherapy. J Clin Oncol, 12: 2239, 1994 19. Heggie, G. D., Sommadossi, J. P., Cross, D. S., Huster, W. J. and Diasio, R. B.: Clinical pharmacokinetics of 5-fluorouracil and its metabolites in plasma, urine, and bile. Cancer Res, 47: 2203, 1987 20. Mizutani, Y., Wada, H., Fukushima, M., Yoshida, O., Ukimura, O., Kawauchi, A. et al: The significance of dihydropyrimidine dehydrogenase (DPD) activity in bladder cancer. Eur J Cancer, 37: 569, 2001
Vol. 171, 605– 610, February 2004 Printed in U.S.A.
DOI: 10.1097/01.ju.0000107833.03488.d2
SIGNIFICANCE OF OROTATE PHOSPHORIBOSYLTRANSFERASE ACTIVITY IN RENAL CELL CARCINOMA YOICHI MIZUTANI,* HIROMI WADA, OSAMU YOSHIDA, MASAKAZU FUKUSHIMA, HIROYUKI NAKANISHI AND TSUNEHARU MIKI From the Department of Urology, Kyoto Prefectural University of Medicine (YM, HN, TM) and Departments of Thoracic Surgery (HW) and Urology (OY), Graduate School of Medicine, Faculty of Medicine, Kyoto University, Kyoto and Cancer Research Laboratory, Taiho Pharmaceutical Co., Ltd. (MF), Saitama, Japan
ABSTRACT
Purpose: The anticancer agent 5-fluorouracil (5-FU) is clinically used against various cancers, including renal cell carcinoma (RCC). It is a prodrug and orotate phosphoribosyltransferase (OPRT) is the principal enzyme that directly converts 5-FU to the active anticancer metabolite 5-fluoro-2⬘-deoxyuridine 5⬘-monophosphate. In addition, OPRT is the key enzyme in the de novo DNA and RNA synthetic process. Little is known about the significance of OPRT in various cancers, including RCC. We investigated the activity of OPRT in 83 RCCs and evaluated the association between the level of OPRT activity and the stage/grade of RCC. The relationship between OPRT activity in RCC cells and their sensitivity to 5-FU was also examined. Materials and Methods: OPRT activity in nonfixed, fresh frozen RCC and normal kidney was determined enzymatically by the 5-FU phosphorylation assay. The sensitivity of RCC cells to 5-FU was assessed by the microculture tetrazolium dye assay. Results: The activity of OPRT was approximately 8.5-fold higher in RCC than in normal kidney. OPRT activity in stage III/IV RCC was 3-fold higher than in stage I/II RCC. The level of OPRT activity in grade 3 RCC was 3-fold higher than that in grade 1/2 cancer. Patients with RCC with low OPRT activity had longer postoperative disease specific survival than those with high activity at 5-year followup. OPRT activity in RCC cells positively correlated with their sensitivity to 5-FU. Conclusions: To our knowledge this is the first study to demonstrate that OPRT activity in RCC was higher than that in normal kidney and OPRT activity positively correlated with RCC stage/ grade. In addition, higher OPRT activity in RCC predicted worse prognosis and higher sensitivity to 5-FU. These results suggest that the level of OPRT activity may be used as a prognostic parameter and predictive indicator for 5-FU efficacy in RCC and OPRT may be a molecular therapeutic target in RCC. KEY WORDS: kidney; carcinoma, renal cell; fluorouracil; orotate phosphoribosyltransferase; tumor markers, biological
The agent 5-fluorouracil (5-FU) is one of the widely used anticancer chemotherapeutic agents for various cancers, including renal cell carcinoma (RCC).1 Alone 5-FU is inactive and it requires intracellular conversion to 5-fluorouridine 5⬘-monophosphate and, furthermore, to 5-fluoro-2⬘deoxyuridine 5⬘-monophosphate (FdUMP). FdUMP exerts cytotoxic activity through the formation of a ternary complex with thymidylate synthase (TS) and 5,10-methylenetetrahydrofolate, resulting in TS inhibition and blockade of the DNA synthetic process.2 Orotate phosphoribosyltransferase (OPRT) is the first limiting enzyme in this 5-FU conversion, leading to the formation of FdUMP in the presence of 5-phosphoribosyl-l-pyrophosphate as a co-factor.3 Previous studies have demonstrated that adenovirus mediated transduction of the OPRT gene results in marked sensitization of colon, gastric, liver and pancreas cancer cells to 5-FU cytotoxicity.4 Thus, OPRT is the important enzyme in 5-FU activation. In addition, OPRT is the rate limiting enzyme in the
de novo process of DNA and RNA synthesis, which converts orotic acid to orotidine 5⬘-phosphate. Although 5-FU is clinically used for RCC and OPRT is an important enzyme in 5-FU cytotoxicity and DNA synthesis, reported data on OPRT activity in RCC are limited. Little is known about the significance of OPRT activity in the biology of RCC. In the current study we measured OPRT activity in 83 RCCs and evaluated the relationship between the level of OPRT activity and stage/grade status of RCC. In addition, we investigated the association between OPRT activity in RCC and sensitivity to 5-FU. MATERIALS AND METHODS
Patients. Surgical specimens were obtained from 83 patients with RCC, including 61 men and 22 women 32 to 83 years old. Histological diagnosis revealed that 74, 8 and 1 patients had clear cell carcinoma, papillary RCC and Bellini duct carcinoma, respectively. Histological classification and staging according to the 1997 TNM classification, fifth edition were T1 to T4 in 56, 12, 11 and 4, N0 to N2 in 80, 1 and 2, M0 and M1 in 74 and 9, stages I to IV in 52, 11, 8 and 12, and G1 to G3 in 11, 55 and 17 cases, respectively. RCC tissues were pathologically examined for viability and absent necrotic areas. Samples of normal kidneys were collected from the same 83 patients with RCC. Specimens were stored frozen at ⫺80C until assay for OPRT, TS and thymidine
Accepted for publication September 26, 2003. Supported by a grant-in-aid from the Japanese Urological Association and Grants-in-Aid 14657410 and 15390496 from the Japanese Ministry of Education, Culture, Sports, Science and Technology. * Correspondence: Department of Urology, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyo-ku, Kyoto 6028566, Japan (telephone: ⫹81-75-251-5595; FAX: ⫹81-75-251-5598; e-mail: [email protected]). 605
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kinase (TK) activities. These enzyme activities were measured in at least 2 samples derived from each patient. Measurement of OPRT activity in RCC and normal kidney. OPRT activity was determined by the 5-FU phosphorylation assay, as previously described.3 RCC and normal kidney were sonicated in homogenate buffer (50 mM tris-HCl, pH 7.4, and 5 mM 2-mercaptoethanol) at maximum output using a Sonifier Cell Disruptor 350 (SmithKline, London, United Kingdom). They were centrifuged at 105,000 ⫻ gravity at 4C for 60 minutes in a model TL-100 (Beckman, Fullerton, California) ultracentrifuge. The supernatants from each sample were divided into several tubes and frozen at ⫺80C until use. Test supernatant was incubated with 10 M [6-3H] 5-FU (74 kBq), 50 mM tris-HCl, pH 8.0, 50 mM MgCl2, 10 mM NaF and 4 mM phosphoribosylpyrophosphate at 37C for 30 minutes. Incubation was terminated by the addition of 2 M perchloric acid, followed by centrifugation at 3000 rpm for 10 minutes. Supernatant (100 l) was then neutralized with 30 l 2 M KOH solution and 20 l aliquots were subjected to silica gel 60F254 thin layer chromatography (2.5 ⫻ 10 cm and 0.25 mm thick) with a mixture of chloroform, methanol and acetic acid (17:3:1 volume per volume per volume) as the mobile phase. Spots of 5-fluorouridine 5⬘-monophosphate were scraped into vials and extracted with 0.1 ml 4 M HCl. The extracts were mixed with 10 ml ACS-II (Amersham, Buckinghamshire, United Kingdom) scintillation fluid. Radioactivity was measured in a Wallac 1410 (Pharmacia, Uppsala, Sweden) liquid scintillation counter. Protein content of test supernatant was measured using BCA protein assay reagent (Pierce Chemical Co., Rockford, Illinois). OPRT activity was calculated per mg protein. Internal standards were used to compare assays. We analyzed all samples at the same time. This method made it possible to estimate OPRT activity higher than 0.05 pmol/mg protein per minute. Repeat measurements yielded almost the same results. OPRT activity greater than the median value (1.52 pmol/mg protein per minute) was considered high activity. OPRT activity less than the median value was considered low activity. Measurement of TS activity in RCC. TS activity was determined by the FdUMP binding assay combined with gel filtration, as previously described.5, 6 This method made it possible to estimate TS activity higher than 1.0 fmol/mg protein. Measurement of TK activity in RCC. TK activity was measured by determining the conversion of labeled thymidine to labeled nucleotides by the diethylaminoethyl cellulose disc method, as previously described.7, 8 This method made it possible to estimate TK activity higher than 1.0 pmol/mg protein per minute. Tumor cells. Fresh RCC cells derived from 22 patients were separated from surgical specimens for in vitro primary culture, as previously described.9, 10 Histological staging according to the 1997 TNM classification, fifth edition were stages I to IV in 16, 2, 1 and 3, and G1 to G3 in 3, 17 and 2 cases, respectively. ACHN and NC65 human RCC cell lines were maintained in monolayers on plastic dishes in complete medium.9, 10 Cytotoxicity assay. Microculture tetrazolium dye assay was used to determine tumor cell lysis, as previously described.11, 12 Statistical analysis. All determinations were made in triplicate. For statistical analysis Student’s t and Pearson’s correlation tests were used. Postoperative disease specific survival was determined by the Kaplan-Meier method. The Cox-Mantel test was used to establish the statistical difference in survival between patients with high and low OPRT activity. Factors related to disease specific survival in patients with RCC were also assessed by multivariate analysis with p ⱕ0.05 considered significant.
RESULTS
Activity of OPRT in RCC and normal kidney. Figure 1 shows OPRT activity values in RCC and normal kidney in patients with RCC. Mean OPRT activity in RCC was approximately 8.5-fold higher than that in normal kidney. OPRT activity in normal kidney in patients with RCC was similar to that in patients with renal pelvic or ureteral cancer (data not shown). These findings demonstrated that OPRT activity in RCC was significantly higher than in normal kidney. The level of OPRT activity in RCC. We then examined OPRT activity in RCC as a function of histological stage and grade of the disease. OPRT activity was approximately 3-fold higher in T3/4 RCC than that in T1/2 RCC (fig. 2). Furthermore, OPRT activity in M1 RCC was significantly (2.5-fold) higher than in M0 RCC (fig. 3). OPRT activity in stage III/IV RCC was 3-fold higher than in stage I/II RCC (fig. 4). OPRT activity in grade 3 RCC was 3-fold higher than in grade 1/2 cancer (fig. 5). OPRT activity in clear cell RCC was similar to that in papillary RCC (data not shown). These findings showed that OPRT activity correlated positively with RCC stage progression and increased histological grade. Correlation between OPRT activity and postoperative disease specific survival in patients with RCC. Patients with RCC undergoing radical nephrectomy were evaluated for the postoperative clinical course. Postoperative disease specific survival was estimated by Kaplan-Meier analysis. Based on this analysis patients with RCC were divided into 2 groups, namely those with high OPRT activity (greater than the median value) and those with low activity (less than the median value). Patients with low OPRT activity had longer disease specific survival than those with high activity at 5-year followup (fig. 6). Moreover, notably that no patients with RCC with low OPRT activity died in this study. However, multivariate analysis showed that OPRT activity was not an independent prognostic factor in patients with RCC. Relationship between OPRT and TS activities in RCC. TS as well as OPRT is the key enzyme for DNA synthesis in the de novo pathway.13, 14 We examined the association between OPRT and TS activities in RCC. There was a positive corre-
FIG. 1. OPRT activity in RCC and normal kidney was quantitated by 5-FU phosphorylation assay. Asterisk indicates p ⬍0.05 vs normal.
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FIG. 2. OPRT activity in RCC according to T classification was quantitated by 5-FU phosphorylation assay. Asterisk indicates p ⬍0.05 vs T1 and T2. Pound sign indicates p ⬍0.05 vs T1/2.
FIG. 3. OPRT activity in RCC according to M classification was quantitated by 5-FU phosphorylation assay. Asterisk indicates p ⬍0.05 vs M0.
lation between the levels of OPRT and TS activities in RCC (fig. 7). Association between OPRT and TK activities in RCC. OPRT is the key enzyme in the de novo DNA and RNA synthetic process, while TK has a key role in the complimentary or alternative salvage pathway of DNA synthesis.15, 16 We have previously observed that TK activity is in parallel
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FIG. 4. OPRT activity in RCC according to stage grouping was quantitated by 5-FU phosphorylation assay. Asterisk indicates p ⬍0.05 vs stage I and stage II. Pound sign indicates p ⬍0.05 vs stage I/II.
FIG. 5. OPRT activity in RCC according to histologic grade was quantitated by 5-FU phosphorylation assay. Asterisk indicates p ⬍0.05 vs grade 1 and grade 2.
with the stage progression and increase in histologic grade of RCC.8 We examined the association between OPRT and TK activities in RCC. There was a positive correlation between the levels of OPRT and TK activities in RCC (fig. 8). These results suggest that OPRT/TS de novo DNA synthetic enzymes and TK salvage enzyme may be up-regulated in high stage/grade RCC. Correlation between OPRT activity in RCC cells and their
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FIG. 6. OPRT activity and postoperative disease specific survival in patients with RCC undergoing radical nephrectomy, as determined by Kaplan-Meier method. OPRT activity greater than median of 1.52 pmol/mg protein per minute was considered high activity and less than median was considered low activity. At 5-year followup there was significant difference in disease specific survival between 41 patients with low OPRT activity (solid line) and 42 with high OPRT activity (dashed line) (Cox-Mantel test p ⬍0.01).
FIG. 7. There was positive correlation between OPRT and TS activities in 83 RCCs (r ⫽ 0.25, Pearson’s correlation test p ⫽ 0.025).
sensitivity to 5-FU. We examined the association between OPRT activity in RCC cells and their sensitivity to 5-FU in 22 primary cultures derived from surgical specimens that served as targets. Figure 9 shows that there was a positive correlation between the level of OPRT activity in RCC cells and their sensitivity to 5-FU. Similar findings were observed at different doses of 5-FU (data not shown). In addition, oxonic acid, an inhibitor of OPRT, significantly suppressed the sensitivity of RCC cells to 5-FU (see table). These findings suggest that the level of OPRT activity in RCC may be a significant predictive parameter for 5-FU efficacy and it may be possible to use 5-FU for RCC with high OPRT activity. DISCUSSION
In the current study we noted that OPRT activity was up-regulated in RCC compared with normal kidney and the level of OPRT activity positively correlated with the stage progression and increase in grade of RCC. Furthermore, to our knowledge this study is the first to show that patients
FIG. 8. There was positive correlation between OPRT and TK activities in 83 RCCs (r ⫽ 0.33, Pearson’s correlation test p ⫽ 0.0025).
FIG. 9. There was positive correlation between OPRT activity in 22 primary cultured RCC cells used as targets and their sensitivity to 100 M 5-FU (r ⫽ 0.68, Pearson’s correlation test p ⫽ 0.0001). Suppression of RCC cell sensitivity to 5-FU by oxonic acid Drugs 5-FU Oxonic acid 5-FU ⫹ oxonic acid
Mean % 5-FU Cytotoxicity ⫾SD ACHN 37.0 ⫾ 2.0 0.5 ⫾ 0.4 6.0 ⫾ 2.2 (p ⬍0.05 vs 5-FU)
NC65 33.0 ⫾ 3.5 3.3 ⫾ 1.6 22.3 ⫾ 1.3 (p ⬍0.05 vs 5-FU)
with RCC with low OPRT activity had longer disease specific survival compared with those with high activity at 5-year followup. Although we report on a small number of patients during a short-term followup, these findings suggest that OPRT may have an important role in regulating the malignant potential of RCC and it may be of prognostic value in RCC. The current study shows that OPRT activity in RCC was significantly higher than in normal kidney. High OPRT activity in RCC may be a reflection of the rate of cancer cell proliferation. We have previously reported that the level of
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OPRT activity increases in rapidly growing cells, including tumor cells and normal cells, such as those of the testis.17 These findings suggest that OPRT may be necessary for carcinogenesis as well as cell proliferation in RCC. However, further studies are needed to determine the biological interaction between OPRT and growth modulation of RCC. To our knowledge this study is the first to demonstrate that the level of OPRT activity positively correlates with the increase in stage and grade of RCC, and the level of OPRT activity in RCC predicts the clinical outcome. The precise reasons responsible for this relationship currently remain unclear. Since OPRT is the principal de novo DNA and RNA synthetic enzyme associated with cell division and proliferation, it is reasonable to assume that clones of cells that over express OPRT can grow more easily and rapidly following implantation than those that do not over express OPRT. In addition, the current study shows that OPRT activity positively correlates with the activities of TS and TK, which are the key enzymes for pyrimidine synthesis in the de novo and salvage pathways. These findings suggest that DNA synthesis is enhanced by activation of the de novo and salvage pathways in RCC. Accordingly the inhibition of OPRT, TS and TK activities may provide a therapeutic means of preventing the growth of RCC. OPRT activity correlated with the stage and the grade of RCC in this study but the SD of OPRT activity was high. Therefore, it is unclear whether we are dealing with a coincidental observation with an elevated level of enzyme that may or may not be causative of more aggressive biological behavior or simply be one of many that are changed in more aggressive cancers. Increased levels of OPRT activity was observed in higher stage/grade RCC. Since OPRT activity was calculated per mg protein, higher stage/grade RCC may have higher production of OPRT per cell rather than there being more cells in higher stage/grade RCC. The overall response rate of immunotherapy and/or chemotherapy for RCC has improved. However, RCC metastasis and recurrence remain major problems. Therefore, new therapeutic approaches are required for patients. The upregulation of OPRT activity in RCC compared with normal kidney identifies OPRT as a molecular therapeutic target. Since OPRT is the first limiting enzyme that converts 5-FU to 5-fluoro-2⬘-uridine 5-monophosphate, leading to the active anticancer metabolite FdUMP,3 our observation that elevated OPRT activity in RCC was associated with high 5-FU sensitivity may be of potential clinical importance in the management of RCC. In addition, oxonic acid, an inhibitor of OPRT, decreased 5-FU cytotoxicity. Thus, chemoimmunotherapy including 5-FU may be effective for RCC with high OPRT activity. Furthermore, enhancing OPRT activity may provide a therapeutic means of augmenting 5-FU efficacy for RCC. Accordingly OPRT gene therapy may be an option for overcoming 5-FU resistance. OPRT activity in RCC was 8.5-fold higher than in normal kidney. The activity of OPRT in RCC cells positively correlated with their sensitivity to 5-FU. Therefore, the high ratio of cancer-to-normal OPRT activity may contribute to the favorable differential between the anticancer effect and the adverse effect of 5-FU. Thus, a higher degree of 5-FU sensitivity may occur in cancer tissues compared with normal tissues. TS is the target enzyme of 5-FU. Several studies and our previous observation suggest that high TS activity may be related to a favorable response to 5-FU.6, 16 Most administered 5-FU is degraded through the catabolic pathway with dihydropyrimidine dehydrogenase (DPD).18, 19 Our previous report has demonstrated that DPD activity in RCC and bladder cancer inversely correlates with sensitivity to 5-FU.12, 20 These findings suggest that TS and DPD activities as well as OPRT activity in RCC may be an important predictive indi-
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cator for 5-FU efficacy. Therefore, the measurement of TS and DPD activities as well as OPRT activity may be necessary for the accurate evaluation of efficacy of 5-FU containing chemotherapy. The data in this communication show that the level of OPRT activity in metastatic RCC was significantly higher than in nonmetastatic RCC. Since OPRT activity is important for activating 5-FU, metastatic RCC is supposed to be sensitive to 5-FU. However, 5-FU has not been found to be particularly effective for metastatic RCC. A reason may be that the mechanisms responsible for 5-FU resistance in cancer cells are multifactorial.2 CONCLUSIONS
The current study shows that OPRT activity in RCC positively correlates with histological stage and grade, and low OPRT activity is a good prognostic sign. Furthermore, elevated OPRT activity is associated with a high response to 5-FU. These findings suggest that the assessment of OPRT activity may be useful in the management of RCC. Since the level of OPRT activity could be used as a prognostic parameter in patients with RCC and a predictive indicator for 5-FU efficacy against RCC, the accurate prediction of prognosis and 5-FU efficacy may help select patients for more intensive surgical or immunochemotherapy approaches including 5-FU. However, further studies are needed to determine the regulatory effects of OPRT activity in RCC. Oxonic acid and 5-FU were provided by Taiho Pharmaceutical Co., Ltd., Tokyo, Japan. REFERENCES
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