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Synthetic Lethality by Lentiviral Short Hairpin RNA Silencing of Thymidylate Kinase and Doxorubicin in Colon Cancer Cells Regardless of the p53 Status

Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan, R.O.C.

Requests for reprints: Zee-Fen Chang, Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, No. 1, Section 1, Jen-Ai Road, Taipei, Taiwan, R.O.C. Fax: 886-2-2395-8904; E-mail: ZFCHANG{at}ha.mc.ntu.edu.tw.

Key Words: TMPK • doxorubicin • lentiviral shRNA

Intracellular supply of dTTP is a highly regulated process and has been a key target for chemotherapeutic drug development. Thymidylate kinase (TMPK) is the key enzyme for dTTP formation in both de novo and salvage pathways. In this study, we used lentiviral-based small hairpin RNA to silence TMPK expression in p53(+/+) and p53(–/–) HCT-116 colon cancer cells. This approach was sufficient to decrease the dTTP pool gradually without affecting p53 expression and generating cytotoxicity. TMPK knockdown significantly increased doxorubicin sensitivity dramatically in p53-proficient, p53-null HCT-116, and LoVo colon cancer cells. The decrease in the dTTP pool using this approach augmented the DNA damage response and enhanced apoptotic induction after exposure to low-dose doxorubicin, leading to cell death. In contrast, silencing of thymidylate synthase which blocks the de novo pathway was incapable of sensitizing p53-null HCT-116 cells to doxorubicin-induced apoptosis because of the compensation by the salvage pathway. Our results suggest the lentiviral delivery of small hairpin RNA targeting TMPK in combination with a low dose of doxorubicin as a new approach to kill colon cancer cells regardless of p53 status. [Cancer Res 2008;68(8):2831–40]