[Benefits of TS-1 plus leucovorin combination therapy for colorectal cancer: evaluation of therapeutic effect of TS-1 and leucovorin combination therapy using rodent model fed a low folate diet].

We evaluated the therapeutic effect of TS-1, a novel oral fluoropyrimidine, in combination with leucovorin with in vivo experiments using a murine tumor xenograft model fed a low folate diet. The reduced folate levels in the tumors of mice fed a low folate diet were significantly lower than those in the tumors of mice fed a normal diet. In addition, the basal reduced folate levels in the tumors of mice fed a low folate diet were comparable to those in human colorectal cancer tissues. Furthermore, when leucovorin was orally administered, the reduced folate levels in the tumors of mice fed a low folate diet were more than two-fold higher than those of mice fed the normal diet. The leucovorin-induced potentiation of TS-1 antitumor activity was obviously higher in COL-1 tumor-bearing mice fed a low folate diet than in mice fed a normal diet. The remarkable increase in reduced folate levels following the administration of leucovorin contributed to the leucovorin-induced potentiation of TS-1 antitumor activity in this low-folate-diet animal model. These results suggest that rodent models fed a low folate diet are suitable for in vivo evaluation of reduced folate drugs like leucovorin. In this model, the combination of leucovorin with TS-1 resulted in a higher antitumor efficacy than TS-1 alone or the combination of UFT and leucovorin, suggesting that TS-1 and leucovorin combination therapy may be an effective regimen for patients with colorectal cancer.

Structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase.

Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine and cytidine and activates pharmacological ribonucleoside analogs. Here we present the crystal structures of human UCK alone and in complexes with a substrate, cytidine, a feedback inhibitor, CTP or UTP, and with phosphorylation products, CMP and ADP, respectively. Free UCK takes an alpha/beta mononucleotide binding fold and exists as a homotetramer with 222 symmetry. Upon inhibitor binding, one loop region was loosened, causing the UCK tetramer to be distorted. Upon cytidine binding, a large induced fit was observed at the uridine/cytidine binding site, which endows UCK with a strict specificity for pyrimidine ribonucleosides. The first UCK structure provided the structural basis for the specificity, catalysis, and regulation of human uridine-cytidine kinase, which give clues for the design of novel antitumor and antiviral ribonucleoside analogs that inhibit RNA synthesis.

Crystallization and preliminary X-ray analysis of human uridine-cytidine kinase 2.

Uridine-cytidine kinase (UCK), which converts uridine and cytidine to their corresponding monophosphates, is a rate-limiting enzyme involved in the salvage pathway of pyrimidine-nucleotide biosynthesis. Two members of human UCK, named UCK1 and UCK2, were cloned recently and UCK2 was reported to play a crucial role in activating anti-tumour pro-drugs in human cancer cells. Human UCK2 was expressed, purified and crystallized alone or in complex with various ligands. Free UCK and UCK complexes were crystallized in six crystal forms. Form I (ligand-free) belongs to space group P2(1)2(1)2, with unit-cell parameters a = 83.1, b = 93.7, c = 157.1 A. Forms IIa (with CTP), IIb (with UTP) and IIc (with cytidine) belong to space group F222, with unit-cell parameters a = 133.3, b = 247.3, c = 91.6 A (IIa), a = 132.1, b = 247.0, c = 91.5 A (IIb) and a = 136.7, b = 246.3, c = 90.4 A (IIc), respectively. Form III (with ATPgammaS) belongs to space group C222(1), with unit-cell parameters a = 70.3, b = 149.9, c = 117.2 A. Form IV (with cytidine and ATP) belongs to space group C2, with unit-cell parameters a = 89.0, b = 109.7, c = 64.8 A, beta = 95.3 degrees. Diffraction data were collected from these crystals; form IV diffracted to 1.8 A resolution.

Regulation of dihydropyrimidine dehydrogenase gene expression in regenerating mouse liver.

5-fluorouracil (5-FU) is one of the most commonly used agents in treatment of the cancer. The administered 5-FU is degraded mainly in the liver by dihydropyrimidine dehydrogenase (DPD), which is the initial rate-limiting enzyme in the catabolic pathway of pyrimidine. This enzyme activity in the tumors has been shown to correlate with the effectiveness of 5-FU against tumors. Therefore, to understand the regulation of DPD gene expression is critical in cancer chemotherapy. Since several studies suggested correlation of DPD activity with the cell proliferation rate we studied the DPD gene expression during liver regeneration. DPD enzyme activity decreased rapidly [24 h after partial hepatectomy (PH): 57% of the control] and remained low until 72 h after PH. Protein content also decreased after PH, however, the lowest level (43.2+/-6.3% of control) was reached only 48 h after PH. The DPD mRNA decreased rapidly to about 20% of control 24 h after PH and then recovered to the control level 72 h after PH. The present results indicate that the DPD gene expression is regulated first at the mRNA level when the hepatocytes enter the cell cycle. The protein content of DPD changed in proportion to the level of DPD mRNA with a 24-h lag, suggesting very little regulation at the translational level. There was a discrepancy between the DPD enzyme activity and the protein content, 24 and 72 h after PH, suggesting that the enzyme activity is modulated also by modification of the protein, and the cell proliferation rate is one of the factors that influences the modification.

Sensitivity of human cancer cells to the new anticancer ribo-nucleoside TAS-106 is correlated with expression of uridine-cytidine kinase 2.

TAS-106 [1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine] is a new anticancer ribo-nucleoside with promising antitumor activity. We have previously presented evidence suggesting that the TAS-106 sensitivity of cells is correlated with intracellular accumulation of the triphosphate of TAS-106, which may be affected both by cellular membrane transport mechanisms and uridine-cytidine kinase (UCK) activity. Since the presence of a UCK family consisting of two members, UCK1 and UCK2, has recently been reported in human cells, we investigated the relation between expression of UCK1 and UCK2 at both the mRNA and protein levels and UCK activity (TAS-106 phosphorylation activity) in a panel of 10 human cancer cell lines. Measurement of UCK activity in these cell lines revealed that it was well correlated with the cells' sensitivity to TAS-106. In addition, the mRNA or protein expression level of UCK2 was closely correlated with UCK activity in these cell lines, but neither the level of expression of UCK1 mRNA nor that of protein was correlated with enzyme activity. We therefore compared the protein expression level of UCK2 in several human tumor tissues and the corresponding normal tissues. Expression of UCK2 protein was barely detectable in 4 of the 5 human tumor tissues, but tended to be high in the pancreatic tumor tissue. It could not be detected at all in any of the normal tissues. Thus, expression of UCK2 appeared to be correlated with cellular sensitivity to TAS-106, and it may contribute to the tumor-selective cytotoxicity of TAS-106.

Cellular and biochemical mechanisms of the resistance of human cancer cells to a new anticancer ribo-nucleoside, TAS-106.

We have established variants of DLD-1 human colon carcinoma and HT-1080 human fibrosarcoma cells resistant to the new anticancer ribo-nucleosides, 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)-cytosine (ECyd, TAS-106) and 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)uracil (EUrd). Both variants were shown to have decreased (3- to 24-fold decrease) uridine-cytidine kinase (UCK) activity, and exhibited cross-resistance to EUrd and TAS-106. Based on the IC(50) values determined by chemosensitivity testing, a 41- to 1102-fold resistance to TAS-106 was observed in the resistant cells. TAS-106 concentration-dependently inhibited RNA synthesis, while its effect on DNA synthesis was negligible. The degree of resistance (14- to 3628-fold resistance) calculated from the inhibition of RNA synthesis tended to be close to the degree of chemoresistance of tested cells to TAS-106. The experiments on the intracellular metabolism of TAS-106 in the parental cells revealed a rapid phosphorylation to its nucleotides, particularly the triphosphate (ECTP), its major active metabolite. The amount of TAS-106 transported into the resistant cells was markedly reduced and the intracellular level of ECTP was decreased from 1/19 to below the limit of detection; however, the unmetabolized TAS-106 as a percentage of the total metabolite level was high as compared with the parental cells. The ratio of the intracellular level of ECTP between parental and resistant cells tended to approximate to the degree of resistance calculated from the inhibitory effect on RNA synthesis. These results indicate that the TAS-106 sensitivity of cells is correlated with the intracellular accumulation of ECTP, which may be affected by both the cellular membrane transport mechanism and UCK activity.