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Strategies to expand thymidylate syn...

Felder, Takita M.

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Strategies to expand thymidylate synthase drug resistance in hematopoietic cells.

紀錄類型:	書目-語言資料,印刷品 : Monograph/item
正題名/作者:	Strategies to expand thymidylate synthase drug resistance in hematopoietic cells./
作者:	Felder, Takita M.
面頁冊數:	207 p.
附註:	Advisers: R. Bruce Dunlap; H. Trent Spencer.
Contained By:	Dissertation Abstracts International63-05B.
標題:	Chemistry, Biochemistry. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3052028
ISBN:	049366338X

Strategies to expand thymidylate synthase drug resistance in hematopoietic cells.
Felder, Takita M.

Strategies to expand thymidylate synthase drug resistance in hematopoietic cells. - 207 p.

Advisers: R. Bruce Dunlap; H. Trent Spencer.

Thesis (Ph.D.)--University of South Carolina, 2002.

Thymidylate synthase (TS) methylates deoxyuridylate (dUMP) to deoxythymidylate (dTMP) using methylene tetrahydrofolate as a cofactor. Inhibitors of TS either mimic the folate cofactor (BW1843U89 and Raltitrexed) or the nucleotide (5-fluorouracil). These inhibitors are associated with hematopoeitic toxicities that result in myelosuppression. Drug-resistant variants of thymidylate synthase can be used to protect cells against these toxicities. In efforts to establish the use of TS for these chemoprotection strategies, we used stopped-flow fluorescence and steady-state kinetics to compare the binding of human and ecTS to nucleotides and antifolates. Although the stopped-flow studies identified differences between human and ecTS that could help identify fluoropyrimidine resistant variants, comparing antifolate binding to the two enzymes yielded more distinct differences. We saw an 11-fold lower inhibitory constant for BW1843U89 and Raltitrexed when using ecTS compared to human TS. We then transfected a retroviral vector containing the ecTS cDNA optimized for expression in mammalian cells (OPTecTS) into a TS-deficient cell line to test cellular resistance. Cells expressing OPTecTS displayed 15-fold greater resistance to TS antifolates than human TS. To increase the spectrum of drugs to which resistance could be conferred, genes involved in the metabolism of deoxyuridylate (deoxyuridine triphosphate hydrolase (dUTPase), thymidine kinase (TK), and cytosolic 5<super> ′</super>-nucleotidase (cN-I)) and reduced folates (dihydrofolate reductase (DHFR)) were coexpressed with OPTecTS. The presence of dUTPase increased the resistance of OPTecTS to Raltitrexed from 15- to 20-fold these OPTecTS/dUTPase expressing cells were resistant to the DNA damage caused by BW1843U89 exposure. Cells expressing both OPTecTS and TK were not more resistant than those expressing OPTecTS alone. The OPTecTS/cN-I combination conferred resistance to both BW1843U89 and 2-chlorodeoxyadenosine (2-CdA). Cells expressing this vector were 27-fold more resistant to BW1843U89/2-CdA combinations than those expressing the human TS control. Survival studies of transfected cells and hematopoietic progenitors dually expressing OPTecTS and L22Y DHFR showed high levels of resistance to TS and DHFR-directed antifolates. When the inhibitors, BW1843U89 and TMTX were combined, we observed 78-fold greater resistance than control cells. It is envisioned that retroviral vectors that coexpress OPTecTS with cN-I and L22Y DHFR will be advantageous in protecting patients from side effects associated with combined drug therapies.

ISBN: 049366338XSubjects--Topical Terms:

1017722
Chemistry, Biochemistry.

Strategies to expand thymidylate synthase drug resistance in hematopoietic cells.
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520 $a Thymidylate synthase (TS) methylates deoxyuridylate (dUMP) to deoxythymidylate (dTMP) using methylene tetrahydrofolate as a cofactor. Inhibitors of TS either mimic the folate cofactor (BW1843U89 and Raltitrexed) or the nucleotide (5-fluorouracil). These inhibitors are associated with hematopoeitic toxicities that result in myelosuppression. Drug-resistant variants of thymidylate synthase can be used to protect cells against these toxicities. In efforts to establish the use of TS for these chemoprotection strategies, we used stopped-flow fluorescence and steady-state kinetics to compare the binding of human and ecTS to nucleotides and antifolates. Although the stopped-flow studies identified differences between human and ecTS that could help identify fluoropyrimidine resistant variants, comparing antifolate binding to the two enzymes yielded more distinct differences. We saw an 11-fold lower inhibitory constant for BW1843U89 and Raltitrexed when using ecTS compared to human TS. We then transfected a retroviral vector containing the ecTS cDNA optimized for expression in mammalian cells (OPTecTS) into a TS-deficient cell line to test cellular resistance. Cells expressing OPTecTS displayed 15-fold greater resistance to TS antifolates than human TS. To increase the spectrum of drugs to which resistance could be conferred, genes involved in the metabolism of deoxyuridylate (deoxyuridine triphosphate hydrolase (dUTPase), thymidine kinase (TK), and cytosolic 5<super> ′</super>-nucleotidase (cN-I)) and reduced folates (dihydrofolate reductase (DHFR)) were coexpressed with OPTecTS. The presence of dUTPase increased the resistance of OPTecTS to Raltitrexed from 15- to 20-fold these OPTecTS/dUTPase expressing cells were resistant to the DNA damage caused by BW1843U89 exposure. Cells expressing both OPTecTS and TK were not more resistant than those expressing OPTecTS alone. The OPTecTS/cN-I combination conferred resistance to both BW1843U89 and 2-chlorodeoxyadenosine (2-CdA). Cells expressing this vector were 27-fold more resistant to BW1843U89/2-CdA combinations than those expressing the human TS control. Survival studies of transfected cells and hematopoietic progenitors dually expressing OPTecTS and L22Y DHFR showed high levels of resistance to TS and DHFR-directed antifolates. When the inhibitors, BW1843U89 and TMTX were combined, we observed 78-fold greater resistance than control cells. It is envisioned that retroviral vectors that coexpress OPTecTS with cN-I and L22Y DHFR will be advantageous in protecting patients from side effects associated with combined drug therapies.
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