Superior antitumour activity of S-1 in tumours with a high dihydropyrimidine dehydrogenase activity.

To elucidate the mechanism of the enhanced antitumour activity of S-1 (1 M tegafur, 0.4 M 5-chloro-2, 4-dihydroxypyridine, and 1 M potassium oxonate) in terms of the phosphorylation and degradation pathways of 5-fluorouracil (5-FU) metabolism, we investigated tumoral thymidylate synthase (TS) content, dihydropyrimidine dehydrogenase (DPD) activity, the TS inhibition rate (TS-IR), and 5-FU incorporated into RNA (F-RNA) in four human gastric cancer xenografts (MKN-28, MKN-74, GCIY and GT3TKB) and compared the results obtained with S-1 with those obtained with 5-FU and UFT (1 M tegafur, 4 M uracil). 5-FU was administered intraperitoneally (i.p.) to mice at a dose of 50 mg/kg, three times, on days 0, 4 and 8. S-1 and UFT were administered orally at doses of 10 and 24 mg/kg, respectively, once a day, for 9 consecutive days. Antitumour activity was evaluated as the maximum inhibition of tumour growth in each animal. S-1 showed a better antitumour activity than 5-FU and UFT in tumours with a high DPD activity (GCIY and GT3TKB). There were inverse correlations between the antitumour activity and both TS content and DPD activity in the 5-FU and UFT groups. However, no such correlations were observed in the S-1 group. In GCIY and GT3TKB xenografts, TS-IR was significantly higher in the S-1 group than in the 5-FU or UFT groups. In GT3TKB xenografts, the F-RNA level was significantly higher in the S-1 group than in the 5-FU or UFT groups. The superior cytotoxicity of S-1 appears to be attributable to both an increased inhibition of DNA synthesis and an enhanced blockade of RNA function against tumours with a high DPD activity.

Enhancement of the antitumour activity of 5-fluorouracil (5-FU) by inhibiting dihydropyrimidine dehydrogenase activity (DPD) using 5-chloro-2,4-dihydroxypyridine (CDHP) in human tumour cells.

The purpose of this study was to evaluate the use of 5-chloro-2,4-dihydroxypyridine (CDHP), a potent inhibitor of dihydropyrimidine dehydrogenase (DPD), to enhance the antitumour activity of the fluoropyrimidines. In an in vitro study, CDHP did not influence cell proliferation by itself. However, CDHP did inhibit 5-fluorouracil (5-FU) degradation and enhanced 5-FU cytotoxicity in a concentration-dependent manner in two human tumour cell lines (MIAPaCa-2 and HuTu80) with relatively high basal DPD activity. CDHP exhibited a maximum effect at a molar ratio (CDHP:5-FU) of more than 0.2. However, CDHP did not have any effect on 5-FU cytotoxicity in the CAL27 tumour cell line, which has a relatively low basal DPD activity, even at concentrations where the DPD activity is almost completely inhibited. In an in vivo study, the maximal tolerable doses (MTD) of tegafur (FT) and a combination of FT and CDHP at a molar ratio of 1:0.4 (FT/CDHP) for nude mice were determined by oral administration for 14 consecutive days. After a single oral administration of either FT or FT/CDHP at the MTD, the 5-FU serum concentration-time profiles were almost the same for both treatment strategies. When nude mice bearing subcutaneous (s.c.) MIAPaCa-2 cells were treated with either FT or FT/CDHP at the MTD, the FT/CDHP treatment showed a significantly higher antitumour effect than the FT treatment (tumour growth inhibition: FT/CDHP, 51+/-12%; FT, 21+/-25%; P<0.05). However, the host-body weight suppression induced by FT/CDHP and FT was equivalent. These findings suggest that the combination of fluoropyrimidine and CDHP for the treatment of tumours with a high basal DPD elicits a greater antitumour effect than treatment with fluoropyrimidines alone and we suggest that CDHP inhibits the degradation of 5-FU in the tumour.

Roles of thymidylate synthase and dihydropyrimidine dehydrogenase in tumor progression and sensitivity to 5-fluorouracil in human gastric cancer.

BACKGROUND: The role of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) enzyme activities in tumor progression and sensitivity to 5-fluorouracil (5-FU) were evaluated. MATERIALS AND METHODS: TS and DPD activities were measured in 81 clinical samples of gastric cancer. TS and DPD activities were determined by 5-fluorodeoxyuridine monophosphate binding assay and by radioenzymatic assay, respectively. Sensitivity to 5-FU was determined by in vitro ATP assay. RESULTS: There was no correlation between TS activity and sensitivity to 5-FU. However, a weak correlation was found between DPD activity and sensitivity to 5-FU. In a subgroup of patients who did not receive adjuvant chemotherapy, overall survival was poorer in patients with high TS activity (p=0.0265). Conversely, in a subgroup of patients who received 5-FU-based adjuvant chemotherapy, overall survival was poorer in patients with high DPD activity (p=0.0465). CONCLUSION: These results suggest that TS has an important role in tumor progression and DPD may be the dominant predictor of 5-FU sensitivity in gastric cancer.

Thymidylate synthase (TS) and ribonucleotide reductase (RNR) may be involved in acquired resistance to 5-fluorouracil (5-FU) in human cancer xenografts in vivo.

A human tumour sub-line resistant to 5-fluorouracil (5-FU) was established by once a day and every 5, with at least 50 administrations of 5-FU to KM12C human colorectal xenografts in nude mice. KM12C tumours treated with 5-FU showed less sensitivity to 5-FU with an inhibition rate (IR) of 7.9%, while non-treated tumours were highly sensitive to 5-FU with an IR of 81.8%. To clarify the mechanism of 5-FU-resistance, the activities of various enzymes and gene expressions involved in the metabolism of 5-FU in both parental and 5-FU-treated KM12C tumours were measured. A 2- to 3-fold increase in thymidylate synthase (TS) activity and 4- to 5-fold decrease in ribonucleotide reductase (RNR) activity were observed in 5-FU-resistant KM12C tumours, while the activities of orotate phosphoribosyltransferase (OPRT) thymidine and uridine phosphorylases (TP,UP) and thymidine kinase (TK) were not markedly changed as a consequence of repeated treatment of KM12C tumours with 5-FU. The expression of TS mRNA was also amplified in accordance with the increased TS activity in a 5-FU-treated tumour sub-line (KM12C/5-FU) compared with that in parental tumours, but changed expressions of both RNR-R1 and RNA-R2 mRNA could not be detected in the 5-FU-resistant tumour sub-line compared with the parental tumours, suggesting possible post-transcriptional regulation of RNR. Moreover, RNR, in addition to TS and OPRT, seemed to be related to the inherent insensitivity to 5-FU in human cancer xenografts. From these results, it may be concluded that RNR activity is one of the acquired or inherent resistant factors, including TS, to 5-FU in human cancer xenografts in vivo.

Screening of differentially expressed genes in 5-fluorouracil-resistant human gastrointestinal tumor cells.

To identify genes differentially expressed in association with resistance to 5-fluorouracil (5FU), an mRNA differential display (DD) analysis was used to compare transcripts from the NUGC-3 human gastric tumor cell line and the NUGC-3 / 5FU / L line, which had acquired 208-fold resistance as a consequence of repeated exposure to escalating concentrations of 5FU. The 110 cDNA fragments differentially expressed in the DD analysis of either the NUGC-3 or NUGC-3 / 5FU / L cells were sequenced and subjected to a homology search, and 29 overexpressed and 22 underexpressed genes were identified in NUGC-3 / 5FU / L as a result. To confirm whether the changes in the gene expression levels in the NUGC-3 / 5FU / L cells were shared by other 5FU-resistant cells, 35 genes were analyzed by northern hybridization in 3 pairs of parent / 5FU-resistant human gastrointestinal tumor cell lines. The analysis revealed 20 overexpressed and 10 underexpressed genes in at least one of the three 5FU-resistant cells as compared with those in the parent cells. Among them, P-glycoprotein, equilibrative nucleoside transporter 1, and methylenetetrahydrofolate dehydrogenase were highly expressed in two of the three 5FU-resistant cells and cytidine deaminase and integrin alpha3 were underexpressed. The acquisition of resistance to 5FU by tumor cells may result from multiple changes in cellular functions.

Intractable Q fever treated with recombinant gamma interferon.

A 3-year-old boy with Q fever received several kinds of antibiotics including minocycline, but spiking fever and positive PCR of Coxiella burnetii continued for several months. He became asymptomatic and his abnormal laboratory data normalized after the administration of gamma interferon three times a week.

Discrepancies between the gene expression, protein expression, and enzymatic activity of thymidylate synthase and dihydropyrimidine dehydrogenase in human gastrointestinal cancers and adjacent normal mucosa.

The relationships between gene expression, protein expression, and enzymatic activity of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) have not been clarified in tumor and non-tumor tissues of human. In this study, we compared three different parameters by evaluating TS and DPD levels, mRNA expression assessed by RT-PCR, protein expression evaluated by immunohistochemical examination, and enzymatic activity measured by biochemical assay, in the tumor tissue and adjacent normal mucosa of 43 patients with gastrointestinal cancer. TS enzymatic activity in the tumor tissue was significantly higher than in normal tissue in both the stomach (median activity: 81.0 and 38.0 pmol/mg protein, respectively, p=0.012) and the colorectum (49.8 and 30.8, respectively, p=0.023). Similarly, TS mRNA expression in the tumor tissue was significantly higher than in normal tissue in both the stomach (median TS/GAPDH ratio: 6.0 and 2.0, respectively, p=0.009) and the colorectum (3.20 and 0.91, respectively, p=0.001). But no significant differences in DPD activity were observed between the tumor and normal tissue in either stomach (median activity: 41.3 and 41.6 pmol/min/mg protein, respectively) or colorectum (34.9 and 49.0, respectively). On the other hand, DPD mRNA levels in normal tissue were significantly higher than in tumor tissue only in the colorectum (DPD/GAPDH ratio: 0.83 and 0.20, respectively, p=0.003). No linear relationships were found between the enzymatic activity and mRNA expression of TS or DPD either in tumor or normal tissue. Nor were any correlations found between protein expression and either mRNA expression or enzymatic activity for either TS or DPD. These results suggest that tissue TS and DPD levels may vary with differences in the methods used to measure them. These discrepancies must be taken into account when interpreting correlation between TS and DPD levels and clinical outcome.

Dihydropyrimidine dehydrogenase activity and mRNA expression in advanced gastric cancer analyzed in relation to effectiveness of preoperative 5-fluorouracil-based chemotherapy.

Dihydroxypyrimidine dehydrogenase (DPD) is an enzyme involved in degradation and inactivation of 5-fluorouracil (5-FU). The amount of its expression in a tumor is thought to be a factor determining the response of the tumor to 5-FU therapy. We compared DPD activity and DPD mRNA expression in resected tumors between two groups of patients, i.e., a group of 14 patients with advanced gastric cancer who received preoperative chemotherapy (neoadjuvant chemotherapy; NAC) and surgery and a group of 24 patients with advanced gastric cancer who underwent surgery without preoperative chemotherapy. Tumor DPD activity was found to correlate well with tumor DPD mRNA expression. In the surgery alone group, DPD activity decreased significantly as the tumor stage advanced. This change was not observed in the NAC plus surgery group. Neither tumor depth (T factor) nor lymph node metastasis was found to correlate with DPD activity. Patients who responded to preoperative chemotherapy had lower DPD mRNA levels. Based on these results, we anticipate that measurement of DPD expression in clinical specimens may be clinically useful in managing advanced gastric cancer.

[Expression of recombinant human dihydropyrimidine dehydrogenase and its application to the preparation of anti-DPD antibodies for immunochemical detection].

Dihydropyrimidine dehydrogenase (DPD) is a key enzyme in the catabolism of 5-fluorouracil (5-FU), and its expression in a tumor is thought to reduce the efficacy of 5-FU against the tumor. To detect a DPD molecule by immunoblotting and/or immunohistochemical methods, we attempted to prepare highly specific antibodies against recombinant human DPD (rhDPD) expressed in the baculovirus-expression system using hDPD cDNA. The expressed rhDPD protein was found to retain its entire molecular form and to show a high 5-FU-degrading activity equivalent to that of human liver DPD. Using this recombinant protein, both monoclonal and polyclonal antibodies to ehDPD were generated and their specificities, relationship to enzyme activity and the possibility of immunohistochemical measurement of tumoral DPD expression were investigated. The results revealed that anti-rhDPD monoclonal antibodies recognized only human DPD, while anti-rhDPD polyclonal antibodies reacted with both human and rodent DPD. The DPD content in 26 tumor cells, estimated by immunoblotting, was closely related to the 5-FU-degrading activities in those cells (r = 0.874). Moreover, immunohistochemical evaluation of tumor cellular DPD expression using our anti-rhDPD antibodies revealed that tumor cells expressing high levels of DPD showed strongly positive staining, but not those expressing low level or no DPD. These results suggest that immunochemical detection of tumoral DPD expression using our anti-rhDPD antibodies may be a means to predict the clinical response to 5-FU-based chemotherapy.

Dihydropyrimidine dehydrogenase and messenger RNA levels in gastric cancer: possible predictor for sensitivity to 5-fluorouracil.

We investigated the correlation between tumor sensitivity to 5-fluorouracil (5-FU) and enzymatic activities of thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) in human gastric cancer specimens. Forty-one patients with advanced gastric cancer gave informed consent and were enrolled in the study. Biopsy specimens of gastric cancer were obtained preoperatively through gastrofiberscopy and used to determine TS and DPD messenger RNA (mRNA) levels. TS and DPD enzyme activity and mRNA levels were also measured in resected tumor tissue samples obtained after surgical resection. TS and DPD activity were measured using the TS-binding assay and a radioenzymatic assay, respectively, while mRNA levels were measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), with co-amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal standard. 5-FU sensitivity of resected tumor specimens was measured by the tetrazolium-based colorimetric assay (MTT assay). Both TS and DPD mRNA levels correlated well between biopsied and resected tumor specimens. A statistically significant correlation was also observed between mRNA levels in biopsied specimens and enzymatic activities in resected specimens. DPD levels significantly correlated with 5-FU sensitivity, such that high DPD activity and high DPD mRNA levels resulted in low sensitivity to 5-FU. In contrast, no correlation was observed between TS activity or TS mRNA levels and 5-FU sensitivity. We conclude that tumor DPD mRNA level, as assessed from biopsy specimens obtained by gastrofiberscopy, may be a useful indicator in predicting tumor sensitivity to 5-FU in patients with gastric cancer.

Experimental postoperative adjuvant chemotherapy by UFT using primary tumor amputation model.

We evaluated the postoperative adjuvant chemotherapy by UFT using the primary tumor amputation-pulmonary metastasis model. When Lewis lung carcinoma (LLC) primary tumors on the hind foot pad grew palpable, they were amputated on two different days. In experiment (A) (earlier amputation model), micrometastases were detected on the day of amputation only by the histopathological examination. In the experiment (B) (later amputation model), nodules could be determined even by necropsy. Long-term (60-day) consecutive administration of UFT (22 mg/kg/day), which produced no body weight loss, markedly prolonged the survival period in experiment (A) (ILS: over 118%), 1 of the 15 mice being cured. UFT had a relatively weak but significant effect (67% of ILS) in schedule (B). Using the same model, we examined the inhibitory effect of UFT (2-week administration) on the number of metastatic nodules. A significant decrease of metastatic nodules was observed by UFT with both amputation schedules, but its effect was superior with schedule (A). In the same model using Colon 26 PMF-15, UFT markedly prolonged the survival period of mice (150% of ILS) and significantly decreased the metastatic nodules (86% inhibition). The dose of UFT used was relatively low, and did not significantly inhibit the growth of large tumors. However, the sensitivity to the micrometastases was high. These findings suggest that the postoperative adjuvant chemotherapy by the long-term consecutive administration of UFT would be effective for clinical cancer especially in curatively resected cases.

Relationship between intratumoral dihydropyrimidine dehydrogenase activity and gene expression in human colorectal cancer.

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme for 5-fluorouracil catabolism. In this study, both the enzymatic activity and mRNA level of DPD were estimated in the tumor tissue and adjacent normal mucosa of 51 patients with colorectal cancer. Although no significant difference in enzymatic activity was observed between tumor tissue and normal mucosa (70.4 and 70.7 pmol/min/mg protein, respectively), the mRNA level in normal mucosa was significantly higher than that in tumor tissue (1.37 and 0.39, respectively; P<0.01). A linear relationship was noted between DPD activity and the DPD mRNA level in cancerous tissue (r(s) = 0.714, P<0.001). Thus, the DPD mRNA level as determined by reverse transcription-PCR can be used to indicate the DPD activity of colorectal cancers.

Bioremediation of trichloroethylene and cis-1,2-dichloroethylene-contaminated groundwater by methane-utilizing bacteria.

Experimental studies on the bioremediation of groundwater contaminated with low concentration trichloroethylene (TCE) and cis1,2-dichloroethylene (DCE) were performed with two sets of bioreactors. Reactors No. 1 and No. 2 were operated without and with methane supplement, respectively. No inoculum was used. The concentrations of TCE and DCE in the effluent and the off gas from reactor No. 2 were much lower than those from reactor No. 1. When air and an H2O2 solution were supplied to reactor No. 2, concentrations of TCE and DCE in the effluent and the off gas were lower than the lowest detectable limit. The population of methane-utilizing bacteria in reactor No. 2 was 1,000 times higher than that in groundwater or in the effluent from reactor No. 1. These methane-utilizing bacteria were apparently attributable to the treatment of TCE.

Some characteristics of bacteria found in a bioreactor to treat trichloroethylene-contaminated groundwater.

A mixture of bacteria, having a methane-utilizing ability, was separated from a bioreactor supplied with air and methane gas. The bioreactor was operated to treat trichloroethylene (TCE)-contaminated groundwater. The mixture was composed of an obligate methane-utilizing bacterium and a heterotroph, identified as Methylomonas methanica and Pseudomonas sp., respectively. The mixed culture of these two strains removed TCE. In addition, it appeared that a cooperative metabolic interaction of these strains enabled Meth.methanica to maintain the TCE degradation ability.

Dihydropyrimidine dehydrogenase activity and messenger RNA level may be related to the antitumor effect of 5-fluorouracil on human tumor xenografts in nude mice.

We investigated the correlation between tumor sensitivity to 5-fluorouracil (5-FU) and the enzymatic activity and mRNA levels of thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) using human tumor xenografts in nude mice. Three gastric carcinoma xenografts (SC-1-NU, St-4, and H-111), two colon carcinoma xenografts (Co-4 and Col-3-JCK), one pancreatic carcinoma xenograft (PAN-3-JCK), and one breast carcinoma xenograft (MX-1) were inoculated into nude mice. When the resultant tumors reached 100-300 mg, 5-FU was administered i.p. at a dose of 60 mg/kg in a schedule of three times every 4 days, and the antitumor activity of 5-FU was evaluated as the relative mean tumor weight in treated mice compared to control mice. Xenografts were also inoculated into untreated nude mice. When tumors weighed 200-300 mg, tumor tissues were resected for measurement of tumoral TS and DPD. TS and DPD activities were detected by the TS-binding assay and a radioenzymatic assay, respectively. mRNA levels were measured by semiquantitative reverse transcription-PCR, with glyceraldehyde-3-phosphate dehydrogenase coamplified as an internal standard. TS and DPD activities ranged from 84.7 to 775.5 fmol/mg protein and from not detectable to 79.7 pmol/min/mg protein, respectively. TS and DPD mRNA levels ranged from 0.51 to 9.90 and from not detectable to 0.93, respectively. The enzymatic activities of TS and DPD were correlated with observed mRNA levels. DPD levels were significantly correlated with 5-FU sensitivity, with high DPD activity and high DPD mRNA level resulting in low sensitivity to 5-FU. In contrast, no correlation between TS level and 5-FU sensitivity was observed. Tumoral DPD activity and DPD mRNA level may be useful indicators in predicting the antitumor activity of 5-FU.

Thymidylate synthetase and dihydropyrimidine dehydrogenase levels in gastric cancer.

The measurement of thymidylate synthetase (TS) and dihydropyrimidine dehydrogenase (DPD) enzymatic activities and mRNA levels in tumors may be useful in predicting tumor sensitivity to 5-fluorouracil (5-FU). Forty-one patients with advanced gastric cancer gave informed consent and were enrolled in this study. Biopsy specimens of gastric cancer were obtained preoperatively through gastrofiberscopy and used to determine TS and DPD mRNA levels. We also measured TS and DPD enzymatic activities and mRNA levels in surgically resected gastric cancer samples, as well as in adjacent normal gastric mucosa. TS and DPD activities were measured using the TS-binding assay and a radioenzymatic assay, respectively, while mRNA levels were measured by semi-quantitative reverse transcription-PCR (RT-PCR) co-amplified with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal standard. In resected tumor specimens, TS and DPD activities ranged from 7.1 to 176.6 fmol/mg protein and from 3.6 to 99.8 pmol/min/mg protein, respectively, while TS and DPD mRNA levels ranged from 0.50 to 21.12 and from 0.014 to 7:22, respectively. There were no significant correlations between TS/DPD levels and other clinicopathological factors, except for low DPD mRNA levels in undifferentiated carcinoma. Both TS activity and mRNA levels were significantly higher in tumor tissues compared to normal adjacent mucosa. In contrast, there was no significant difference between tumoral and non-tumoral DPD activity, although tumor tissue showed significantly lower DPD mRNA levels than non-tumoral tissue. High tumoral TS mRNA levels in preoperative biopsy specimens from patients with stage III/IV was associated with poor survival outcome after surgery compared with patients with low tumoral TS mRNA levels. In contrast, DPD levels had no influence on prognosis. We conclude that high tumoral TS levels and low tumoral DPD mRNA may indicate the selective cytotoxicity of 5-FU on gastric cancer, and that tumoral TS mRNA levels may be a prognostic factor for patients with stage III/IV gastric cancer.

Preclinical antitumor efficacy of S-1: a new oral formulation of 5-fluorouracil on human tumor xenografts.

S-1 is a new oral formulation of 5-fluorouracil (5-FU) consisted of 1M tegafur, 0.4M 5-chloro-2,4-dihydroxypyridine that inhibits a degradation of 5-FU, and 1M potassium oxonate that regulates the phosphorylation of 5-FU in the gastrointestinal tract, and has shown excellent antitumor efficacy against various murine tumors in rodents, compared to the oral tegafur-based antitumor drug, UFT (1M tegafur plus 4M uracil), which is used clinically in Japan. To assess the possibility of clinically using S-1, we investigated the antitumor effect of S-1 on various human solid tumor xenografts in athymic rats and mice. In the nude rat system, S-1 was significantly effective against all 12 tumor xenografts tested when its minimum toxic dose (15 mg/kg) was administered for 14 days. Three tumors, stomach (H-81), colon (KM12C) and breast (H-31) markedly regressed in response to treatment with S-1 but not with UFT. The antitumor potency of S-1 was weak against human tumors xenografted into nude mice and likely similar to that of UFT. The reason of the discrepancy in the efficacy of S-1 between rats and mice was found to be that the 5-FU levels in the blood and tumor tissue of rats after oral administration of S-1 persisted much longer than in mice, and this prolonged maintenance of plasma 5-FU levels was significantly related to the potent antitumor activity of S-1. In conclusion, the results of this study suggested that based on its biological and pharmacokinetic characteristics, oral S-1 should be active against various human cancers.

Antitumor effect of S-1 on DMH induced colon cancer in rats.

The aim of this study was to establish an autochthonous colon cancer model in the rat as an in vivo secondary screen for the general evaluation of new anticancer agents against colorectal cancer, and also to evaluate practically the antitumor activity of 1M tegafur-0.4M 5- chloro-2,4-dihydroxypyridine-1M potassium oxonate(S-1), a new p.o. fluoropyrimidine. Thirty-two Sprague-Dawley rats received dimethlhydrazine(40 mg/kg) s.c. once weekly for 10 weeks to induce colon cancer.20 weeks after beginning the carcinogen treatment, a barium enema was performed to visualize tumors. The animals were divided into a control group and S-1 treatment group. After 5 weeks of treatment, the barium enema was repeated. The mean doubling time of 24 tumors in the control group was 19.0 + 8.4 (SD) days. Response to S-1 was judged as effective when the doubling time exceeded 35.8 days, calculated from the mean + 2SDs in the control group. The response rate of S-1 was 55%, 34% of the tumors were decreased in size after treatment. This figure was higher than that of clinically-used 5-fluorouracil(5-FU) derivatives; 5-FU;6%, Tegafur(FT):6%, 1M tegafur-4M uracil(UFT):14%, reported in our previous study. An autochthonous colon cancer model is useful to evaluate the clinical therapeutic efficacy of drugs for colorectal cancer, and S-1 is expected to have a high therapeutic effect on human colorectal cancer.

Relationship between protein levels and gene expression of dihydropyrimidine dehydrogenase in human tumor cells during growth in culture and in nude mice.

Protein levels and gene expression of dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme for degradation of 5-fluorouracil, were studied in two human tumor cell lines (fibrosarcoma HT-1080 and pancreatic carcinoma MIAPaCa-2) in various growth phases of the cultured cells and of tumor xenografts implanted into nude mice. DPD catalytic activity and DPD protein content in cytosolic preparations were determined by means of radioenzymatic assay and western blot analysis, respectively. Relative DPD mRNA expression was determined by using a semi-quantitative reverse transcription-polymerase chain reaction in which glyceraldehyde-3-phosphate dehydrogenase mRNA was used as an internal standard. DPD activity and protein content in cultures of both cell lines increased in proportion to cell density (DPD activities ranged from undetectable to 84 pmol/min/mg protein in the HT-1080 cells and from undetectable to 335 pmol/min/mg protein in the MIAPaCa-2 cells). DPD mRNA levels, on the other hand, tended to decrease slightly during cell growth. DPD activity and protein content in HT-1080 tumor xenografts increased during growth in proportion to tumor weight (DPD activities ranged from 7 to 131 pmol/min/mg protein), but DPD mRNA levels did not correlate with tumor weight. DPD activity and protein content in MIAPaCa-2 tumor xenografts did not change much, and seemed to have already plateaued, since the tumors were small (weighing about 30 mg). These findings suggest that DPD protein expression during tumor growth is controlled at the post-transcriptional level.