• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.2034-2042
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• 2015

A one-pot process of enzymatic synthesis of deoxythymidine-5'-triphosphate (5'-dTTP) employing whole cells of recombinant Escherichia coli coexpressing thymidylate kinase (TMKase) and acetate kinase (ACKase) was developed. Genes tmk and ack from E. coli were cloned and inserted into pET28a(+), and then transduced into E. coli BL21 (DE3) to form recombinant strain pTA in which TMKase and ACKase were simultaneously overexpressed. It was found that the relative residual specific activities of TMKase and ACKase, in pTA pretreated with 20 mM ethylene diamine tetraacetic acid (EDTA) at 25℃ for 30 min, were 94% and 96%, respectively. The yield of 5'-dTTP reached above 94% from 5 mM deoxythymidine 5'-monophosphate (5'-dTMP) and 15 mM acetyl phosphate catalyzed with intact cells of pTA pretreated with EDTA. The process was so effective that only 0.125 mM adenosine-5'-triphosphate was sufficient to deliver the phosphate group from acetyl phosphate to dTMP and dTDP.

• Archives of Pharmacal Research
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• v.24 no.4
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• pp.323-326
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• 2001

Previous studies have demonstrated that CW252053, a quinazoline antifolate, exhibits potent inhibitory activity against thymidylate synthase (TS) as well as cytotoxic activity against tumor cell lines in vitro. In this studys, we evaluated the in vivo antitumor efficacy of CW252053 in the mouse tumor model. Female B6D2F$_1$ mice were injected with LY3.7. 2C TK-/- (thymidine kinase deficient mouse Iymphoma) cells into the gastrocnemius muscle. Then, CW252053 was administered twice daily by intraperitoneal injection for 10 days, and tumor growth was monitored daily by leg diameter measurement. All animals in the vehicle, 5-FU, and low dose (30mgmg/kg CW252053 treated groups died between days 12 and 23 because of the tumor burden. In contrast, dosing with 60 mg/kg of CW252053 produced a cure rat against tumor growth of 37.5% and a survival rate of 50%. Even more significantly, a higher dose of CW252053 (120 mg/kg) elicited both a 100% cure rate and a 100% survival rate at the termination of the study, confirming that this compound has very potent in vivo antitumor activity against tumor growth. During the experimental period of this study no signs of toxicity were observed even at the high CW252053 dosage rate of 120 mg/kg.

• Journal of Microbiology and Biotechnology
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• v.15 no.3
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• pp.477-483
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• 2005

Corynebacterium ammoniagenes ATCC 6872, which does not accumulate pyrimidine nucleoside or nucleotide, was metabolically engineered to secrete a large amount of thymidine. Characteristics of 5-fluorouracil resistance ($FU^r$), hydroxyurea resistance ($HU^r$), trimethoprim resistance ($TM^r$), thymidylate phosphorylase deficiency ($deoA^-$), inosine auxotrophy ($ino^-$), 5-fluorocytosine resistance ($FC^r$), thymidine kinase deficiency, and thymidine resistance ($thym^r$) were successively introduced into mutant strains KR3 and DY5T9-5, and shake-flask cultures were able to accumulate 408.1 mg/l and 428.2 mg/l of thymidine, respectively, as a major product. The mutant strains did not accumulate thymine at all and accumulated less than 10 mg/l of other pyrimidine nucleosides, such as cytosine, cytidine, and deoxycytidine, as byproducts.

• Journal of Life Science
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• v.26 no.9
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• pp.1015-1021
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• 2016

5-fluorouracil (5-FU), a pyrimidine analog, is a widely used anticancer drug, which works through irreversible inhibition of thymidylate synthase. In the present study, it was investigated the anti-proliferative effects and molecular mechanisms of 5-FU using Ewing's Sarcoma CHP-100 Cells. The present data indicated that treatment of 5-FU to CHP-100 cells induced a G1 phase arrest of the cell cycle in a time-dependent manner. 5-FU-induced G1 arrest was correlated with the accumulation of the hypophosphorylated form of the retinoblastoma protein (pRB) and association of pRB with the transcription factors E2F-1 and E2F-4. Although 5-FU treatment did affect the levels of cyclin-dependent kinases, the levels of cyclin A and B were markedly down-regulated as compared with the untreated control group. In addition, 5-FU-induced G1 arrest of CHP-100 cells was also associated with the induction of apoptosis, as determined by apoptotic cell morphologies, degradation of poly(ADP-ribose) polymerase and Annexin V staining. Furthermore, 5-FU induced the loss of mitochondrial membrane potential with up-regulated pro-apoptotic Bax expression, down-regulated anti-apoptotic Bcl-2 expression and cytochrome c release from mitochondria to cytosol. Collectively, the data suggest that 5-FU is effective in inducing cell growth reduction and apoptosis, in part, by reducing phosphorylation of pRB and activating mitochondrial dysfunction in CHP-100 cells.