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Eukaryotic nuclear replication requires three polymerases (pol), pol α, pol δ, and pol ε. Pols δ and ε contain a 3’→5’ exonuclease (exo) domain that has proofreading capacity. Exos facilitate the removal of misincorporated nucleotides and replace them with the corresponding correct base to preserve accurate DNA replication. Mice that lack exo function are prone to cancer. In addition to the excision of misinserted deoxynucleotides, biochemical studies show that exos also recognize and remove nucleotide analogs from DNA. Gemcitabine (2’,2’-difluorodeoxycytidine), a deoxycytidine analog, has been used effectively to treat various malignancies. Gemcitabine is phosphorylated to a triphosphate, incorporates into DNA, inhibits DNA synthesis, and induces cell death. In vitro assays using DNA primers and pols have demonstrated that gemcitabine is incorporated in the 3’ penultimate position in DNA and is subject to excision by exos. Based on these observations, we hypothesized that gemcitabine-induced cytotoxicity is influenced by the 3’→5’ exonucleolytic activity of DNA pols δ and ε. To test our postulate, we used wild type (WT), pol δ exo-minus (δ-/-), pol ε exo-minus (ε-/-), and double exo-minus (δε-/-) mouse embryonic fibroblast cells with inactivating point mutations in the exo domains of pol δ and pol ε. These point mutations render the cells exo null but allow normal polymerase function. To quantitate the accumulation of analog triphosphate, cells were treated with 3 nM [3H] gemcitabine for 24 hours in the presence of a deaminase inhibitor. These isogenic cells accumulated similar levels of gemcitabine triphosphate; intracellular levels were less than 1 μM. To determine if exo status plays a role in gemcitabine-induced cytotoxicity, growth inhibition assays were performed. Cell growth was inhibited 76% in WT, 82% in δ-/- and ε-/- cells, and 59% in δε-/- cells compared to untreated cells. To identify if the differences in growth inhibition were associated with inhibition of DNA synthesis, [3H] thymidine incorporation was measured after gemcitabine treatment. WT and single exo-minus (δ-/- and ε-/-) cells had similar levels of DNA synthesis inhibition: 76%, 82%, and 82% respectively, suggesting that a single exo is as efficient as exo proficient WT cells. In contrast, in double exo-minus (δε-/-) cells, there was 56% inhibition of DNA synthesis, which was significantly different (p=0.004) than WT or single exo-minus cells. These data suggest that pol δ and pol ε exos may have redundancies in function, and a deficiency in both exos results in a differential effect on the extent of gemcitabine-induced DNA synthesis inhibition and cytotoxicity. Future studies will focus on the number of gemcitabine molecules incorporated in the respective cell lines and the downstream effects of the recognition of analogs by exos.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]