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To establish the individualized treatment of patients with colorectal cancer, factors associated with chemotherapeutic effects should be identified. However, to the best of our knowledge, few studies are available on this topic, although it is known that the prognosis of patients and sensitivity to chemotherapy depend on the location of the tumor and that the tumor location is important for individualized treatment. In this study, primary tumors obtained from 1,129 patients with colorectal cancer were used to measure the mRNA expression levels of the following genes associated with the effects of standard chemotherapy for colorectal cancer: 5-fluorouracil (5-FU)-related thymidylate synthase (TYMS), dihydropyrimidine dehydrogenase (DPYD) and thymidine phosphorylase (TYMP); folate-related dihydrofolate reductase (DHFR), folylpolyglutamate synthase (FPGS) and gamma-glutamyl hydrolase (GGH); irinotecan-related topoisomerase I (TOP1); oxaliplatin-related excision repair cross-complementing 1 (ERCC1); biologic agent-related vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR). Large-scale population analysis was performed to determine the association of gene expression with the clinicopathological features, in particular, the location of the colorectal cancer. From the results of our analysis of the mRNA expression of these 10 genes, we noted the strongest correlation between DPYD and TYMP, followed by TYMS and DHFR. The location of the colorectal cancer was classified into 4 regions (the right- and left-sided colon, rectosigmoid and rectum) and was compared with gene expression. A significant difference in all genes, apart from VEGF, was noted. Of the remaining 9 genes, the highest expression of TYMS and DPYD was observed in the right-sided colon; the highest expression of GGH and EGFR was noted in the left-sided colon; the highest expression of DHFR, FPGS, TOP1 and ERCC1 was noted in the rectosigmoid, whereas TYMP expression was approximately equivalent in the right-sided colon and rectum, and higher than that in other locations. The data generated from this study may prove to be useful for the development of individualized chemotherapeutic treatments for patients with colorectal cancer, and will mean that the tumor location is taken into account.

TAS–106 [l–(3–C‐ethynyl‐β‐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.

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‐β‐D‐ribo‐pentofuranosyl)‐cytosine (ECyd, TAS‐106) and 1‐(3‐C‐ethynyl‐p‐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 IC50 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.

A compound containing both CNDP (3‐cyano‐2,6‐dihydroxypyridine), an inhibitor of 5‐fluorouracil (5‐FU) degradation, and EM‐FU (1‐ethoxymethyl‐5‐fluorouracil), a masked form of 5‐FU, was synthesized and named BOF‐A2 (3‐[3‐(6‐benzoyloxy‐3‐cyano‐2‐pyridyloxycarbonyl)benzoyl]‐1‐ethoxymethyl‐5‐fluorouracil). The antitumor activity of BOF‐A2 was investigated in sarcoma‐180‐bearing mice and Yoshida sarcoma‐bearing rats. The ED50 (the dose for 50% inhibition) values of BOF‐A2 were 25 mg/kg against sarcoma‐180 and 15 mg/kg against Yoshida sarcoma. In vitro studies showed that BOF‐A2 was rapidly degraded to EM‐FU and CNDP in homogenates of the liver and small intestine of mice and rats, and in sera of mice, rats and human, and the conversion of EM‐FU to 5‐FU occurred only in the microsomal fraction of rat liver in the presence of NADPH. After oral administration of BOF‐A2 at 15 mg/kg to Yoshida sarcoma‐bearing rats, BOF‐A2 was hydrolyzed to EM‐FU, CNDP and 5‐FU, and their maximum concentrations in the blood were 2000 ng/ml, 300 ng/ml and 40 ng/ml, respectively. Moreover when BOF‐A2 was given at the same dose to tumor‐bearing mice and rats, the 5‐FU levels in the tumor tissue increased much more than those in the blood and persisted for more than 8 h, whereas those in the blood decreased more rapidly. This accumulation and maintenance of a high level of 5‐FU in the tumor tissue are concluded to be related to the high antitumor activity of BOF‐A2.

Methods for pharmacokinetic modulation of the plasma 5‐fluorouracil (5‐FU) level to increase antitumor activity during continuous venous infusion (CVI) of low doses of 5‐FU were examined in Yoshida sarcoma‐bearing rats. These methods were additional infusion of 5‐FU for a short period (4 h) or oral administration of LIFT or Tegafur during long‐term CVI of 5‐FU that alone gave a plasma 5‐FU level of about 50 ng/ml. The antitumor effect on Yoshida sarcoma was markedly potentiated when an additive dose of 5‐FU combined with 3‐cyano‐2,6‐dihydroxypyridine (CNDP), a potent inhibitor of 5‐FU degradation, giving a plasma level of about 500 ng/ml, was infused for 4 h. A similar increase in the antitumor effect was observed with oral administration of a conventional dose of UFT during CVI of 5‐FU without CNDP, giving a plasma level of 30 to 60 ng/ml. These results suggest that the antitumor effect of CVI of 5‐FU can be potentiated by pharmacokinetic modulation of the 5‐FU concentration in the blood.

The antitumor ribonucleoside analogues 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)cytosine (ECyd) and 1-(3-C-ethynyl-beta-D-ribo-pentofuranosyl)uracil (EUrd), first synthesized in 1995, have strong antitumor activity against human cancer xenografts without severe side effects. Here, we studied the antitumor mechanisms of ECyd and EUrd using mouse mammary tumor FM3A cells in vitro and the mechanism of selective cytotoxicity of ECyd using human tumor xenografts in nude rats in vivo. In FM3A cells, ECyd and EUrd were rapidly phosphorylated to ECyd 5'-triphosphate (ECTP) and EUrd 5'-triphosphate (EUTP), which strongly inhibiting RNA synthesis. Cells treated with EUrd were later found to contain both EUTP and ECTP, and ECTP accumulated as the final product. Probably the uracil moieties of EUrd derivatives were efficiently converted to cytosine moieties in the cells. EUrd and its derivatives were minor metabolites in the cells treated with ECyd, so cytidine forms probably were not converted to uridine forms at the nucleoside or nucleotide stage. The ultimate metabolite of ECyd and EUrd, ECTP, is stable in cultured cells with a half-life of at least 3 days, so ECyd and EUrd are on a \"closed\" metabolic pathway to ECTP. These characteristics of ECyd and EUrd may be important for their antitumor activity. ECyd had strong and selective antitumor activity against the human tumor xenografts. ECyd-phosphorylating activity (uridine/cytidine kinase) in the xenografts was higher than that in the organs of the rats. This finding may account for the strong activity with mild side effects. ECyd and EUrd may be a new kind of antitumor nucleoside analogue for clinical use.

We examined the effects of dosage schedule on antitumor activity in vitro and in vivo to determine the optimal administration schedule for a new nucleoside antimetabolite l‐(3‐C‐ethynyl‐β‐D‐ribo‐pentofuranosyl)cytosine (ECyd, TAS‐106). The cytotoxicity of TAS‐106 in vitro against human tumors was evaluated at three drug exposure periods. TAS‐106 exhibited fairly potent cytotoxicity even with 4 h exposure, and nearly equivalent and sufficiently potent cytotoxicity with 24 and 72 h exposures. These results suggest that long‐term exposure to TAS‐106 will not be required to achieve maximal cytotoxicity. The antitumor activity of TAS‐106 in vivo was compared in nude rat models bearing human tumors on three administration schedules, once weekly, 3 tunes weekly, and 5 tunes weekly for 2 or 4 consecutive weeks. TAS‐106 showed strong antitumor activity without serious toxicity on all three schedules, but the antitumor activity showed no obvious schedule‐dependency in these models. When tumor‐bearing nude rats were given a single i.v. dose of [3H]TAS‐106, tumor tissue radioactivity tended to remain high for longer periods of time as compared to the radioactivity in various normal tissues. Furthermore, when the metabolism of TAS‐106 in the tumor was examined, it was found that TAS‐106 nucleotides (including the active metabolite, the triphosphate of TAS‐106) were retained at high concentrations for prolonged periods. These pharmacodynamic features of TAS‐106 may explain the strong antitumor activity without serious toxicity, observed on intermittent administration schedules, in nude rat models with human tumors. We therefore consider TAS‐106 to be a promising compound which merits further investigation in patients with solid tumors.

The correlations of the 5‐fluorouracil (5‐FU) level in the plasma and the duration of continuous 5‐FU infusion with the antitumor activity of 5‐FU on Yoshida sarcomas in rats were examined. The circadian variation in the plasma level of 5‐FU during continuous infusion was prevented by treatment with 3‐cyano‐2,6‐dihydroxypyridine (CNDP), which strongly inhibits 5‐FU degradation. On continuous venous infusion of 2 to 30 mg/kg of 5‐FU over 24 h with CNDP at a molar ratio of 1:10 into normal rats, the 5‐FU level in the blood was linearly proportional to the dose of 5‐FU. The optimum schedule for antitumor activity on Yoshida sarcomas in rats was found to be infusion of 5‐FU at 5 mg/kg over 24 h for 6 consecutive days, which gave a plasma 5‐FU level of 176 ng/ml. Continuous infusion of 5‐FU to give a plasma level of 300 ng/ml for 6 consecutive days from day 5 after implantation of tumor cells, when the tumors weighed about 1.0 g, resulted in complete regression of the tumors in all rats.

Using a protocol that recapitulates both meiosis and oocyte growth in vitro , the authors induce mouse pluripotent stem cells to differentiate into fully functional oocytes that can be fertilized and generate viable offspring, thereby recapitulating the full mammalian female germline cycle in a dish. A mouse female germline in vitro This study reports the successful production of functional mouse eggs entirely in culture, a long-standing goal in the fields of stem cell biology and regenerative medicine. From mouse pluripotent stem cells, the authors generated functional oocytes in vitro that can be fertilized and generate fully viable offspring. Their protocol recapitulates both meiosis and oocyte growth, and the authors are also able to isolate pluripotent stem cell lines from the in-vitro -generated eggs after fertilization, thereby recapitulating the full mammalian female germline cycle in a dish. The female germ line undergoes a unique sequence of differentiation processes that confers totipotency to the egg 1 , 2 . The reconstitution of these events in vitro using pluripotent stem cells is a key achievement in reproductive biology and regenerative medicine. Here we report successful reconstitution in vitro of the entire process of oogenesis from mouse pluripotent stem cells. Fully potent mature oocytes were generated in culture from embryonic stem cells and from induced pluripotent stem cells derived from both embryonic fibroblasts and adult tail tip fibroblasts. Moreover, pluripotent stem cell lines were re-derived from the eggs that were generated in vitro , thereby reconstituting the full female germline cycle in a dish. This culture system will provide a platform for elucidating the molecular mechanisms underlying totipotency and the production of oocytes of other mammalian species in culture.

BackgroundPost-ERCP pancreatitis (PEP) with trans-papillary approach remains a major issue, and the multi-factorial etiology can lead to the development of unpredictable PEP. Therefore, the early identification of PEP is highly desirable to assist with the health cost containment, the reduction in unnecessary admissions, earlier appropriate primary care, and intensive care for preventing progression of severe pancreatitis. This study aimed to establish a simplified predictive scoring system for PEP.MethodsBetween January 1, 2012, and December 31, 2019, 3362 consecutive trans-papillary ERCP procedures were retrospectively analyzed. Significant risk factors were extracted by univariate, multivariate, and propensity score analyses, and the probability of PEP in the combinations of each factor were quantified using propensity score analysis. The results were internally validated using bootstrapping resampling.ResultsIn the scoring system with four stratifications using combinations of only five extracted risk factors, the very high-risk group showed 28.79% (95% confidence interval [CI], 18.30%–41.25%; P < 0.001) in the predicted incidence rate of PEP, and 9.09% (95% CI, 3.41%–18.74%; P < 0.001) in that of severe PEP; although the adjusted prevalence revealed 3.74% in PEP and 0.90% in severe PEP, respectively. The prediction model had an area under the curve of 0.86 (95% CI, 0.82–0.89) and the optimism-corrected model as an internal validation had an area under the curve of 0.81 (95% CI, 0.77–0.86).ConclusionsWe established and validated a simplified predictive scoring system for PEP using five risk factors immediately after ERCP to assist with the early identification of PEP.