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S-phase checkpoint signaling may spare myeloid leukemia cells (ML-1) toxicity resulting in chemotherapy resistance. Understanding this pathway is critical to developing therapeutic regimens that may overcome resistance. Tezacitabine, (E)-2’-Deoxy-2’-(fluoromethylene) cytidine (FMdC) is a S-phase specific nucleoside analogue. FMdC diphosphate inhibition of ribonucleotide reductase and incorporation of FMdC triphosphate into DNA interferes with DNA polymerases, leading to termination of DNA synthesis and stalled replication folks and induces S-phase checkpoint activation in ML-1 cells. The aim of this study was to determine whether S-phase checkpoint activation is critical to leukemia cell resistance to FMdC. When exponentially growing ML-1 cells were exposed to FMdC (4 nM) for 21 hrs and washed free of drug, there was a re-growth delay of 72 hrs shown by growth rate assays. In clonogenic assays, there was negligible decrease (<8%) in colony formation by FMdC treatment alone. However, the rate of colony development was dramatically less in treated cells compared to untreated controls reflecting the observed delay in population growth. Moreover, cells exposed to FMdC alone were found to be in S-phase arrest (70%) compared to controls (30%), shown by propidium iodide staining of DNA and flow cytometry. FMdC-treated cells demonstrated an increase of Chk1 kinase (Ser317, Ser345) phosphorylation (5-fold) with associated phosphorylation of Cdk2 Tyr15 (>10-fold) as seen by immunoblotting. In order to ascertain whether Chk1 kinase activation was important in the cellular effects of FMdC induced cell death, we treated cells with the Chk1 inhibitor, UCN-01 (50 nM) for 3-9 hrs after FMdC treatment (21 hrs). UCN-01 abrogated S-phase arrest in FMdC-treated cells while increasing sub-G1 content. By clonogenic assays, cell number and colony number decreased (82%) compared to controls and to FMdC alone-treated cells, demonstrating the survival benefit of the S-phase checkpoint pathway. To determine whether these findings were due to off-target activity of UCN-01, two new and specific Chk1 inhibitors (Proc AACR 46:396, 2005; abst#1682) were used to treat ML-1 cells with similar results (94% loss of clonogenecity). Thus, we show that ML-1 cells undergo S-phase arrest with associated Chk1 kinase activation without increased cell death in response to the nucleoside analogue FMdC. Additionally, we show that Chk1 kinase is critical in mediating this potential resistance mechanism to FMdC. Dysregulation of the S-phase checkpoint pathway potentiates cell death in ML-1 cells.

[Proc Amer Assoc Cancer Res, Volume 47, 2006]