Prognostic significance of orotate phosphoribosyltransferase activity in bladder carcinoma

Authors


Abstract

BACKGROUND

5-Fluorouracil (5-FU), an antitumor agent, is used clinically against a variety of malignancies, including bladder carcinoma. 5-FU is a prodrug, and orotate phosphoribosyltransferase (OPRT) is the principal enzyme that converts 5-FU directly into an active antitumor metabolite, 5-fluoro-2′-deoxyuridine 5′-monophosphate. In addition, OPRT is the key enzyme in the de novo DNA and RNA synthetic process. To the authors' knowledge, little is known regarding the significance of OPRT in various malignancies, including bladder carcinoma. The authors analyzed the activity levels of OPRT in 60 bladder carcinomas and evaluated the association between the level of OPRT activity and the stage and grade status of bladder carcinoma. They also examined the prognostic significance of OPRT activity in patients with bladder carcinoma and the correlation between OPRT activity levels in bladder carcinoma cells and the sensitivity of those cells to 5-FU.

METHODS

OPRT activity levels in nonfixed, fresh-frozen specimens of bladder carcinoma and normal bladder were determined enzymatically using a 5-FU phosphorylation assay. The sensitivity of bladder cells to 5-FU was assessed using a microculture tetrazolium dye assay.

RESULTS

The activity levels of OPRT were approximately 7.5-fold higher in bladder carcinoma specimens compared with the activity levels in normal bladder specimens. OPRT activity in muscle-invasive bladder carcinoma was 2-fold higher compared with the activity in superficial bladder carcinoma (classified as Ta and T1). In addition, the activity of OPRT in T1 bladder carcinoma was 2-fold higher compared with the activity in Ta bladder carcinoma. The level of OPRT activity in Grade 3 bladder carcinoma was 6-fold and 2-fold higher compared with the activity in Grade 1 and Grade 2 bladder carcinoma, respectively. Patients who had Ta and T1 bladder carcinoma with low OPRT activity had a longer postoperative tumor free period compared with patients who had bladder carcinoma with high OPRT activity in the 3-year follow-up. There was a positive association between the activity levels of OPRT and thymidylate synthase/thymidine kinase, which are the key enzymes in the de novo/salvage DNA synthetic process. OPRT activity in bladder carcinoma cells was correlated positively with their sensitivity to 5-FU.

CONCLUSIONS

to the authors' knowledge, the current study is the first to demonstrate that OPRT activity levels in bladder carcinoma were higher compared with its activity in the normal bladder tissues and that OPRT activity levels were correlated positively with the stage and grade of bladder carcinoma. In addition, high OPRT activity levels in patients with superficial bladder carcinoma predicted early recurrence and high sensitivity to 5-FU. These results suggest that the level of OPRT activity may be used both as a prognostic parameter and as a predictive indicator for 5-FU efficacy in patients with bladder carcinoma and that OPRT may be a molecular therapeutic target in bladder carcinoma. Cancer 2004;100:723–31. © 2003 American Cancer Society.

5-Fluorouracil (5-FU) is one of the most widely used antitumor chemotherapeutic agents for the treatment of a variety of malignancies, including bladder carcinoma.1, 2 5-FU itself is inactive and requires its intracellular conversion to 5-fluorouridine 5′-monophosphate (FUMP) and, furthermore, to 5-fluoro-2′-deoxyuridine 5′-monophosphate (FdUMP), which exerts its cytotoxic activity through the formation of a ternary complex with thymidylate synthase (TS) and 5,10-methylene-tetrahydrofolate, resulting in inhibition of TS and blockade of the DNA synthetic process.3, 4 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-1-pyrophosphate as a cofactor.5, 6 Previous studies have demonstrated that adenovirus-mediated transduction of the OPRT gene results in marked sensitization of colon, gastric, hepatic, and pancreatic carcinoma cells to 5-FU cytotoxicity.7, 8 Thus, OPRT is an 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 OPRT is an important enzyme in 5-FU cytotoxicity and DNA synthesis, to our knowledge reported data concerning OPRT activity levels in various malignancies, including bladder carcinoma, are limited, and little is known about the significance of OPRT activity in the biology of bladder carcinoma. In the current study, we measured the activity of OPRT in 60 bladder carcinoma cases and evaluated the correlation between the level of OPRT activity and stage and grade status of bladder carcinoma. Furthermore, the prognostic significance of OPRT activity in bladder carcinoma was examined. In addition, we investigated the association between OPRT activity in bladder carcinoma cells and their sensitivity to 5-FU.

MATERIALS AND METHODS

Patients

Surgical specimens were obtained from 60 patients with initial primary bladder carcinoma. They included 44 male patients and 16 female patients who ranged in age from 24–86 years. Histologic diagnoses revealed that all patients had transitional cell carcinoma. The histologic classification and staging according to the TNM classification system were as follows: Ta (n = 20 tumors), T1 (n = 28 tumors), T2 (n = 2 tumors), T3 (n = 8 tumors), T4 (n = 2 tumors), Grade 1 (n = 7 tumors), Grade 2 (n = 26 tumors), and Grade 3 (n = 27 tumors). There were no patients with carcinoma in situ. Samples of histologically normal bladder tissues (hereafter referred to as normal bladder tissues) were excised from 26 patients with bladder carcinoma who underwent radical cystectomy. The specimens were stored frozen at − 80 °C until they were used for the assay of OPRT, TS, and thymidine kinase (TK) activity.

This study was performed after approval by a local Human Investigations Committee. Informed consent was obtained from each patient.

Measurement of OPRT Activity in Bladder Carcinoma and Normal Bladder

The activity of OPRT was determined by using the 5-FU phosphorylation assay, as described previously.5, 6 Bladder carcinoma specimens and normal bladder tissues were sonicated in the homogenate buffer (50 mM Tris-HCl, pH 7.4; 5 mM 2-mercaptoethanol) at maximum output (Sonifier cell disruptor 350; SmithKline, London, U.K.) and centrifuged at × 105,000 g at 4 °C for 60 minutes in an ultracentrifuge (model TL-100; Beckman, Fullerton, CA). The supernatant fluids from each sample were divided into several tubes and were frozen at − 80 °C until they were used.

The test supernatant fluid was incubated with 10 μM [6-3H] 5-FU (74 kBq); 50 mM Tris-HCl, pH 8.0; 50 mM MgCl2; 10 mM NaF; and 4 mM phosphoribosylpyrophosphate at 37 °C for 30 minutes. The incubation was terminated by the addition of 2 M perchloric acid followed by centrifugation at 3000 revolutions per minute (rpm) for 10 minutes. Then, 100 μL of the supernatant fluid were neutralized with 30 μL of 2 M potassium hydroxide solution, and 20-μL aliquots were subjected to silica gel 60F254 thin-layer chromatography (2.5 × 10.0 cm; thickness, 0.25 mm; Merck, Whitehouse Station, NJ) with a mixture of chloroform, methanol, and acetic acid (17:3:1; volume/volume/volume) as the mobile phase. Spots of FUMP were scraped into vials and extracted with 0.1 mL of 4 M HCl. The extracts were mixed with 10 mL of ACS-II scintillation fluid (Amersham, Buckinghamshire, U.K.). The radioactivity was measured in a Wallac 1410 liquid scintillation counter (Pharmacia, Uppsala, Sweden). 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. Repeated measurements yielded nearly the same results. OPRT activity greater than the median value was regarded as high activity, and OPRT activity less than the median value was regarded as low activity.

Measurement of TS Activity in Bladder Carcinoma

The activity of TS in bladder carcinoma was determined using the FdUMP-binding assay combined with gel filtration, as described previously.9, 10 Bladder carcinoma tissues were sonicated in the homogenate buffer (50 mM Tris-HCl; 1 mM ethylenediamine tetraacetic acid; and 5 mM MgCl2 [pH 7.4]) at maximum output (Sonifier cell disruptor 350; SmithKline) and centrifuged at × 105,000 g at 4 °C for 60 minutes in a Beckman ultracentrifuge (TL-100). The supernatants from each sample were divided into several tubes and frozen at − 80 °C until they were used.

The test supernatant fluid was incubated with [6-3H] FdUMP and 5,10-CH2-FH4 at 30 °C for 20 minutes; then, the mixture was gel-filtered using a PD-10 column (Pharmacia) to separate TS-bound from free [6-3H] FdUMP. The sample was eluted with phosphate buffered saline, and the total radioactivity of the fractions containing protein was measured. Protein content of the supernatant was measured using the BCA protein assay reagent (Pierce Chemical Company, Rockford, IL). Internal standards were used to compare assays. We analyzed all samples at the same time. This method made it possible to estimate TS activity > 1.0 fmol/mg protein. Repeated measurements yielded nearly the same results.

Measurement of TK Activity in Bladder Carcinoma

The activity of TK in bladder carcinoma was measured by determining the conversion of labeled thymidine to labeled nucleotides using the diethylaminoethanol (DEAE) cellulose-disc method, as described previously.11, 12 Bladder carcinoma samples were homogenized in 4 volumes of 50 mM Tris-HCl, pH 8.0, containing 5 mM 2-mercaptoethanol, 25 mM KCl, and 5mM MgCl2 and were centrifuged at × 105,000 g at 4 °C for 60 minutes in a ultracentrifuge (model TL-100; Beckman). The supernatant fluids from each sample were divided into several tubes and frozen at − 80 °C until they were used.

The reaction mixture, in a total volume of 0.25 mL, was comprised of 50 mM Tris-HCl, pH 8.0; 5 mM MgCl2; 6 mM α-glycerophosphate; 5 mM adenosine triphosphate; 0.05 mM [6-3H] thymidine (0.05 μCi); and the supernatant fluid (0.2 mL). The mixture was incubated for 10 minutes at 37 °C, and the reaction was stopped by heating at 100 °C in a water bath. After centrifugation at 3000 revolutions per minute for 10 minutes, the supernatant fluid (0.1 mL) was applied to a disc of DEAE cellulose that measured 3 cm in dimension. The disc was immersed in 1 mM ammonium formate for 20 minutes. The washing liquid was discarded, and the disc was then washed with distilled water. This procedure was repeated once more. [6-3H] thymidine nucleotides were retained on the disc during this procedure. The dried disc was placed in a vial containing 10 mL of toluene-phosphorus mixture, and its radioactivity was counted. Internal standards were used to compare assays. We analyzed all samples at the same time. This method made it possible to estimate TK activity > 1.0 pmol/mg protein per minute. Repeated measurements yielded almost the same results.

Reagents and Medium

5-FU and oxonic acid were supplied kindly by Taiho Pharmaceutical Company Ltd. (Tokyo, Japan). [6-3H] 5-FU (525 GBq/mmol), [6-3H] FdUMP (625 GBq/mmol), and [6-3H] thymidine (2.41 TBq/mmol) were obtained from Moravek Biochemicals Inc. RPMI-1640 medium (Bio-cult; Gibco, Glasgow, Scotland, United Kingdom) supplemented with 25 mM N-2-hydroxyethyl-piper-az-ine-N′-2-ethane sulphonate (Gibco), 2 mM L-glutamine (Gibco), 1% nonessential amino acid (Gibco), 100 units/mL penicillin (Gibco), 100 μg/mL streptomycin (Gibco), and 10% heat-inactivated fetal bovine serum (Gibco) was used as complete medium.

Tumor Cells

Fresh bladder carcinoma cells derived from 14 patients were separated from surgical specimens for in vitro primary culture, as described previously.13, 14 Although we tried to make primary cultures from all bladder carcinoma specimens, we succeeded with only 14 specimens. Briefly, cell suspensions were prepared by treating finely minced carcinoma tissues with collagenase (3 mg/mL; Sigma Chemical Company, St. Louis, MO). After washing in RPMI-1640 medium, the cell suspensions were layered on discontinuous gradients consisting of 2 mL 100% Ficoll-Hypaque, 2 mL 80% Ficoll-Hypaque, and 2 mL of 50% Ficoll-Hypaque in 15-mL plastic tubes and were centrifuged at × 400 g for 30 minutes. Lymphocyte-rich mononuclear cells were collected from the 100% interface, and tumor cells and mesothelial cells were collected from the 80% interface. Cell suspensions enriched with tumor cells sometimes were contaminated by monocyte-macrophages, mesothelial cells, or lymphocytes. To eliminate further contamination of host cells, we layered the cell suspensions on discontinuous gradients of 2 mL each of 25%, 15%, and 10% Percoll in complete medium in 15-mL plastic tubes and centrifuged them for 7 minutes at × 25 g at room temperature. Tumor cells that were depleted of lymphoid cells were collected from the bottom, washed, and suspended in complete medium. Tumor cells were > 93% viable on average according to the Trypan blue dye-exclusion test. The tumor cells were maintained in monolayers on plastic dishes in complete medium. The primarily cultured tumor cells were used as target cells for lysis of 5-FU in the microculture tetrazolium dye (MTT) assay. The T24, J82, and HT1197 human bladder carcinoma cell lines were maintained in monolayers on plastic dishes in complete medium.

Cytotoxicity Assay

The MTT assay was used to determine tumor cell lysis, as described previously.15, 16 Briefly, 100 μL of target cell suspension (2 × 104 cells) were added to each well of 96-well, flat-bottom microtiter plates (Corning Glass Works, Corning, NY), and each plate was incubated for 24 hours at 37 °C in a humidified 5% CO2 atmosphere. After incubation, the supernatant fluid was aspirated, tumor cells were washed 3 times with RPMI medium, and 200 μL of drug solution or of complete medium for control specimens were distributed in the 96-well plates. Each plate was incubated for 24 hours at 37 °C. After incubation, 20 μL of MTT working solution (5 mg/mL; Sigma Chemical Company) were added to each culture well, and the cultures were incubated for 4 hours at 37 °C in a humidified 5% CO2 atmosphere. The culture medium was removed from the wells and replaced with 100 μL of isopropanol (Sigma Chemical Company) supplemented with 0.05 N HCl. The absorbance of each well was measured with a microculture plate reader (Immunoreader; Japan Intermed Company Ltd., Tokyo, Japan) at 540 nm. The percent cytotoxicity was calculated using the following formula: percentage cytotoxicity = [1 − (absorbance of experimental wells/absorbance of control wells)] × 100.

Statistical Analysis

All determinations were made in triplicate. For statistical analysis, the Student t test, the Pearson correlation test, and the chi-square test were used. The postoperative tumor free rate was determined using the Kaplan–Meier method. The Cox–Mantel test was used to establish statistically significant differences in the disease free rate between the patients with high OPRT activity and low OPRT activity. Factors related to the postoperative tumor free rate in patients with superficial bladder carcinoma also were analyzed by multivariate analysis. P values ≤ 0.05 were considered significant.

RESULTS

The Level of OPRT Activity in Bladder Carcinoma and in Normal Bladder

The levels of OPRT activity in bladder carcinoma specimens and in normal bladder tissues from patients with bladder carcinoma are summarized in Figure 1. The mean level of OPRT activity in bladder carcinoma was approximately 7.5-fold higher compared with the activity in normal bladder tissues.

Figure 1.

The levels of orotate phosphoribosyltransferase (OPRT) activity in bladder carcinoma and in normal bladder were quantitated using the 5-fluorouracil phosphorylation assay described in the text. An asterisk indicates P < 0.05 versus normal bladder. SE: standard error.

The Level of OPRT Activity in Bladder Carcinoma

We then examined OPRT activity in bladder carcinoma as a function of histologic stage and grade of the disease. The activity levels of OPRT were 2-fold higher in muscle-invasive bladder carcinoma compared with the levels in Ta and T1 superficial bladder carcinoma (Fig. 2). However, there were no statistically significant differences in OPRT activity levels between T2–T4 tumors and T1 tumors (P = 0.1). OPRT activity levels in T1 bladder carcinoma were 2-fold higher compared with the levels in Ta carcinoma. Significantly, OPRT activity levels in Grade 3 bladder carcinoma were 6-fold and 2-fold higher compared with the levels in Grade 1 and Grade 2 tumors, respectively (Fig. 3). Furthermore, OPRT activity levels in Grade 2 bladder carcinoma were 3-fold higher compared with the levels in Grade 1 tumors. These findings showed that OPRT activity levels were correlated positively with stage progression and increased histologic grade of bladder carcinoma.

Figure 2.

The levels of orotate phosphoribosyltransferase (OPRT) activity according to histologic stage of bladder carcinoma. The levels of OPRT activity in bladder carcinoma were quantitated using the 5-fluorouracil phosphorylation assay described in the text. An asterisk indicates P < 0.05 versus Stage Ta; a pound sign indicates P < 0.05 versus Stage Ta and T1. SE: standard error.

Figure 3.

The levels of orotate phosphoribosyltransferase (OPRT) activity according to histologic grade of bladder carcinoma. The levels of OPRT activity in bladder carcinoma were quantitated using the 5-fluorouracil phosphorylation assay described in the text. An asterisk indicates P < 0.05 versus Grade 1; a pound sign indicates P < 0.05 versus Grade 2. SE: standard error.

Correlation between OPRT Activity Levels and the Postoperative Tumor Free Rate in Patients with Ta and T1 Bladder Carcinoma

Forty-eight patients with Ta and T1 bladder carcinoma who underwent transurethral resection were evaluated for their postoperative clinical course. Because these bladder tumors were initial primary bladder carcinomas, the patients did not receive additional treatment, such as intravesical therapy. The postoperative tumor free period was estimated in a Kaplan–Meier analysis. Based on that analysis, patients with superficial bladder carcinoma (Ta and T1) were divided into two groups: patients with high OPRT activity levels (greater than the median value) and patients with low OPRT activity levels (less than the median value). Patients who had low OPRT activity levels had longer tumor free intervals compared with patients who had high OPRT activity levels in the 3-year follow-up (Fig. 4). Only 1 of 24 patients (4%) with superficial bladder carcinoma who had low OPRT activity levels had recurrent bladder carcinoma after undergoing transurethral resection. In contrast, 19 of 24 patients (79%) with high OPRT activity levels had recurrent carcinoma. There were no statistically significant differences observed in age, gender, or histologic grade of bladder carcinoma between patients with high and low OPRT activity levels (Table 1). These results suggest that the level of OPRT activity may be used as an independent prognostic indicator in patients with Ta and T1 bladder carcinoma and that low OPRT activity may be considered a good prognostic sign. In addition, multivariate analysis confirmed the these data (data not shown). There was no correlation with OPRT activity and progression to higher grade or stage in patients who developed recurrent tumors (data not shown).

Figure 4.

Relation between the level of orotate phosphoribosyltransferase (OPRT) activity and the postoperative tumor free period in patients with Ta and T1 bladder carcinoma. The postoperative tumor free period for patients with Ta and T1 bladder carcinoma who underwent transurethral resection was determined by the Kaplan–Meier method. OPRT activity levels greater than the median value (1.05 pmol/mg protein per minute) were regarded as high, and OPRT activity levels less than the median value were regarded as low. There was a significant difference in disease free interval between the following two groups in the 3-year follow-up (P < 0.01; Cox–Mantel test); solid line, 24 patients with low OPRT activity; dashed line, 24 patients with high OPRT activity.

Table 1. Comparison of Characteristics of Ta and T1 Superficial Bladder Carcinoma in Patients with High and Low Levels of Orotate Phosphoribosyltransferase Activity
CharacteristicNo. of patients
OPRT activityTotal (n = 48)
High (n = 24)Low (n = 24)
  • OPRT: orotate phosphoribosyltransferase; SD: standard deviation.

  • a

    Not significant (chi-square test).

  • b

    Not significant (Student t test).

Gendera   
 Male171835
 Female7613
Mean age ± SD (years)b65.5 ± 13.065.5 ± 15.965.5 ± 14.5
Histologic gradea   
 Grade 1167
 Grade 2131124
 Grade 310717

Relation between OPRT and TS Activity Levels in Bladder Carcinoma

TS and OPRT are the key enzymes for DNA synthesis in the de novo pathway.17, 18 We also examined the association between OPRT and TS activity levels in bladder carcinoma. There was a positive correlation between the levels of OPRT and TS activity in bladder carcinoma (Fig. 5). However, this correlation was weak.

Figure 5.

Relation between the levels of orotate phosphoribosyltransferase (OPRT) activity and thymidylate synthase (TS) activity in bladder carcinoma. There was a positive correlation between OPRT and TS activity levels in bladder carcinoma (n = 59 patients; r = 0.33; P = 0.01; Pearson correlation test).

Association between OPRT and TK Activity Levels in Bladder Carcinoma

OPRT is the key enzyme in the de novo DNA and RNA synthetic process, whereas TK plays a key role in the complimentary or alternative salvage pathway of DNA synthesis.19, 20 We previously demonstrated that TK activity is in parallel with disease stage progression and increased histologic grade in bladder carcinoma and renal cell carcinoma.11, 12 In the current study, we examined the association between OPRT and TK activity levels in bladder carcinoma. There was a positive correlation between the levels of OPRT and TK activity in bladder carcinoma (Fig. 6), but the correlation was relatively weak. These results suggest that DNA synthesis in high-stage/high-grade bladder carcinoma is enhanced simultaneously by both the de novo pathway and the salvage pathway.

Figure 6.

Relation between the levels of orotate phosphoribosyltransferase (OPRT) activity and thymidine kinase (TK) activity in bladder carcinoma. There was a positive correlation between OPRT and TK activity levels in bladder carcinoma (n = 55 patients; r = 0.46; P = 0.0005; Pearson correlation test).

Correlation between OPRT Activity Levels in Bladder Carcinoma Cells and their Sensitivity to 5-FU

OPRT is the first-limiting enzyme in 5-FU activation.5, 6 We also examined the association between OPRT activity levels in bladder carcinoma cells and the sensitivity of those cells to 5-FU. Fourteen primary cultures derived from surgical specimens were used as targets. The findings illustrated in Figure 7 demonstrate that there was a positive correlation between the level of OPRT activity in bladder carcinoma cells and the sensitivity of those cells to 5-FU. Similar findings were observed with different doses of 5-FU (data not shown). In addition, oxonic acid, an inhibitor of OPRT, significantly suppressed the sensitivity of bladder carcinoma cells to 5-FU (Table 2). These findings suggest that the level of OPRT activity in bladder carcinoma may be a significant predictive parameter for 5-FU efficacy and that it may be possible to use 5-FU for the treatment of patients who have bladder carcinoma with high OPRT activity.

Figure 7.

Correlation between the levels of orotate phosphoribosyltransferase (OPRT) activity in bladder carcinoma (Ca) cells and the sensitivity of those cells to 5-fluorouracil (5-FU). Fourteen primarily cultured bladder carcinoma cells were used as targets. There was a positive correlation between the level of OPRT activity in bladder carcinoma cells and their sensitivity to 5-FU (10 μM) (n = 14 patients; r = 0.88; P = 0.0001; Pearson correlation test).

Table 2. Suppression of the Sensitivity of Bladder Carcinoma Cell Lines to 5-Fluorouracil by Oxonic Acid
AgentMean ± SD cytotoxicity of 5-FU against (%)a
HT1197T24J82
  • SD: standard deviation; 5-FU: 5-fluorouracil.

  • a

    The direct cytotoxic effect of 5-FU (10 μM) with or without oxonic acid (10 μM) on

  • b

    HT1197, T24, and J82 bladder carcinoma cell lines was assessed with a 1-day microculture tetrazolium dye assay.

  • P < 0.05 vs. 5-FU.

5-FU21.3 ± 2.928.3 ± 2.931.7 ± 3.7
Oxonic acid2.4 ± 1.02.0 ± 0.11.7 ± 1.8
5-FU and oxonic acid9.3 ± 1.5b12.0 ± 0.1b16.0 ± 2.6b

DISCUSSION

In the current study, we demonstrated that OPRT activity was up-regulated in bladder carcinoma compared with normal bladder and that the level of OPRT activity was correlated positively with both the progression of disease stage and the increase in tumor grade of bladder carcinoma. Furthermore, to our knowledge, this study is the first to show that patients with superficial bladder carcinoma who had low OPRT activity had a longer tumor free interval compared with patients who had high OPRT activity in the 3-year follow-up. Although small numbers of patients were reported during a short-term follow-up, those findings suggest that OPRT may play an important role in regulating the malignant potential of bladder carcinoma and may be of prognostic value in patients with bladder carcinoma.

The current study showed that the activity of OPRT in bladder carcinoma was significantly higher compared with OPRT activity in normal bladder. High OPRT activity in bladder carcinoma may be a reflection of the rates of tumor cell proliferation. We previously reported that the level of OPRT activity increased in rapidly growing cells, including tumor cells and normal cells, such as testis.21 Those findings suggest that OPRT may be necessary for carcinogenesis as well as cell proliferation in bladder carcinoma. However, further studies are needed to determine the biologic interaction between OPRT and growth modulation of bladder carcinoma.

This study is the first study to demonstrate that the level of OPRT activity is correlated positively with the stage and grade of bladder carcinoma and that the level of OPRT activity in superficial bladder carcinoma predicts clinical outcome. Currently, the precise reasons responsible for this correlation remain unclear. Because 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 overexpress OPRT can grow more easily and rapidly after implantation compared with clones that do not overexpress OPRT. In addition, the current study has shown that OPRT activity is correlated positively with the activity levels of TS and TK, which are the key enzymes for DNA synthesis in the de novo and salvage pathways, respectively. These findings suggest that DNA synthesis in both the de novo and salvage pathways is up-regulated in high-stage/high-grade bladder carcinoma. Accordingly, simultaneous inhibition of the activity of OPRT, TS, and TK may provide therapeutic means of preventing growth and recurrence in patients with bladder carcinoma.

Although OPRT activity was significantly higher in T2–T4 tumors, the activity observed in T1 tumors was not different from the activity observed in higher stage tumors. This raises interesting questions regarding the superficial nature of T1 disease, which has been somewhat controversial. Although Ta tumors infrequently progress to muscle invasion, T1 tumors often progress.22, 23

Figure 3 demonstrates that OPRT activity was higher in higher grade bladder carcinomas. However, this was not true in Ta and T1 bladder carcinomas (Table 1).

The overall response rate to chemotherapy against bladder carcinoma has improved. However, metastasis and recurrence of bladder carcinoma remain major problems. Therefore, new therapeutic approaches are required for patients with bladder carcinoma. The dramatic up-regulation of OPRT activity in bladder carcinoma compared with normal bladder identifies OPRT as a molecular therapeutic target. Because OPRT is the first-limiting enzyme that converts 5-FU to FUMP, leading to an active antitumor metabolite (FdUMP),5, 6 our observation that elevated OPRT activity in bladder carcinoma was associated with high 5-FU sensitivity may be of potential clinical importance in the management of patients with bladder carcinoma. In addition, oxonic acid, an inhibitor of OPRT, reduced 5-FU cytotoxicity. Thus, chemotherapy that includes 5-FU may be effective against bladder carcinoma with high OPRT activity. Furthermore, enhancement of OPRT activity may provide a therapeutic means of augmentation of 5-FU efficacy against bladder carcinoma. Accordingly, OPRT gene therapy may be one of the options to overcome 5-FU resistance in patients with bladder carcinoma.

OPRT activity in bladder carcinoma was 7.5-fold higher compared with OPRT activity in normal bladder. The activity of OPRT in bladder carcinoma cells was correlated positively with their sensitivity to 5-FU. Therefore, the high ratio of carcinoma/normal OPRT activity may contribute to the favorable differential between antitumor effects and adverse effects of 5-FU. Thus, a higher degree of 5-FU sensitivity may occur in tumor tissues compared with 5-FU sensitivity in normal tissues.

TS is the target enzyme of 5-FU. Several studies and our previous observations suggest that high TS activity may be related to a favorable response to 5-FU.10, 20, 24 Most of the administered 5-FU is degraded through the catabolic pathway with dihydropyrimidine dehydrogenase (DPD).25, 26 Our previous reports demonstrated that DPD activity in renal cell carcinoma and bladder carcinoma is correlated inversely with the sensitivity to 5-FU.16, 27 Those findings suggest that TS and DPD activity levels, as well as OPRT activity levels, in bladder carcinoma may be important predictive indicators for 5-FU efficacy. Therefore, the measurement of TS and DPD activity levels, as well as OPRT activity levels, may be necessary for the accurate evaluation of efficacy of 5-FU-containing chemotherapy.

5-FU is not used routinely for bladder carcinoma chemotherapy, and most trials have included only patients with superficial disease.1, 2 Although there have been trials conducted using 5-FU-based regimens, those regimens have been used mainly as second-line therapy or in bladder-preservation protocols along with cisplatin for radiosensitization. Hence, currently, the value of measuring OPRT activity in patients with bladder carcinoma may be limited.

The data in this report have demonstrated that the level of OPRT activity is correlated positively with the stage and grade of bladder carcinoma and with the sensitivity of bladder carcinoma cells to 5-FU. Bladder carcinoma is relatively resistant to 5-FU. In contrast, colon carcinoma traditionally is a 5-FU-sensitive carcinoma. Previous reports demonstrated that colon carcinomas with high cell proliferative activity show high OPRT activity and that high OPRT activity in colon carcinoma predicts high sensitivity to 5-FU.28, 29 In addition, transfection of the OPRT gene sensitizes colon carcinoma cells to 5-FU.7 These findings suggest that similar results obtained in bladder carcinoma are observed in colon carcinoma.

The results of the current study demonstrated that OPRT activity in bladder carcinoma was correlated positively with histologic stage and grade and that low OPRT activity was a good prognostic sign. Furthermore, elevated levels of OPRT activity in bladder carcinoma were associated with high response to 5-FU. These findings suggest that the assessment of OPRT activity may be useful in the management of bladder carcinoma. Because the level of OPRT activity may be used both as a prognostic parameter in patients with bladder carcinoma and as a predictive indicator for 5-FU efficacy against bladder carcinoma, the accurate prediction of prognosis and 5-FU efficacy may help select patients for more intensive surgical or chemotherapeutic approaches, including 5-FU. However, further studies will be needed to determine the regulatory effects of OPRT activity in patients with bladder carcinoma.

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