Thymidylate synthase (TS) is the only de novo source of thymidylate (dTMP) for DNA synthesis and repair. Drugs targeting TS protein are a mainstay in cancer treatment but off-target effects and toxicity limit their use. Cytosolic thymidine kinase (TK1) and mitochondrial thymidine kinase (TK2) contribute to an alternative dTMP-producing pathway, by salvaging thymidine from the tumour milieu, and may modulate resistance to TS-targeting drugs. We have previously shown that TS antisense molecules (oligodeoxynucleotides, ODNs, and small interfering siRNA, siRNA) sensitize tumour cells, both in vitro and in vivo, to TS targeting drugs. As both TS and TKs contribute to cellular dTMP, we hypothesized that TKs mediate resistance to the capacity of TS siRNA to sensitize tumour cells to TS-targeting drugs. Downregulation of TKs with siRNA enhanced the capacity of TS siRNA to sensitize tumour cells to traditional TS protein-targeting drugs (5FUdR and pemetrexed). Combined downregulation of these enzymes is an attractive strategy to enhance TS-targeted anticancer therapy. TK2 can phosphorylate both thymidine and deoxycytidine to generate dTMP and dCMP, precursors for dTTP and dCTP, respectively. dCTP negatively regulates deoxycytidine kinase (dCK), another enzyme that phosphorylates deoxycytidine as well as the anticancer drug gemcitabine. Antisense knockdown of TK2 could reduce TK2-produced dCMP, thus decreasing dCTP levels and inhibition of dCK, and lead to increased dCK activity, gemcitabine activation, and anticancer effectiveness. Given the substrate promiscuity of TK2, we hypothesized that: (1) TK2 can mediate human tumour cell resistance to gemcitabine, (2) antisense downregulation of TK2 can overcome that resistance, and (3) TK2 siRNA-induced drug sensitization results in mitochondrial damage. siRNA downregulation of TK2 expression sensitized MCF7 and HeLa cells to gemcitabine, but did not sensitize A549 cells (low TK2 expresser). Treatment with TK2 siRNA and gemcitabine: 1) decreased mitochondrial redox status, 2) decreased mitochondrial DNA (mtDNA:nDNA ratio), and 3) decreased mitochondrial activity. This is the first demonstration of a direct role for TK2 in gemcitabine resistance, or any independent role in cancer drug resistance, and further distinguishes TK2 from other dTMP-producing enzymes
