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 shRNA 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 by 10-fold in both p53 proficient and null cells and enhanced apoptotic induction by low dosage of doxorubicin treatment.The decreaseof dTTP pool by this approach augmentsthe DNA damage lesion in response to doxorubicin,leading to apoptosis independent of p53.