se Chk1 is essential for arresting the cell cycle of p53 defective cells in response to DNA damage including that induced by cyototoxic chemotherapeutic drugs such as gemcitabine and cisplatin. The ability of VER-150548 to abrogate gemcitabine induced S-phase 1355612-71-3 arrest was determined in p53-defective HT29 cells. Following treatment with gemcitabine then VER-150548 plus nocodazole, cells were examined for expression of pH 3 ; a BIBW-2992 structure marker indicative of mitosis. Nocodazole arrests cells in mitosis whilst gemcitabine, in combination with nocodazole, results in S-phase arrest with a low proportion of pH 3 positive mitotic cells. VER-150548 abrogated gemcitabine induced S-phase arrest leading to the accumulation of cells in mitosis with an EC50 of 23 nM. Gemcitabine, camptothecin or cisplatin arrested HT29 cells in either S- or G2-phase and low MPM-2, pPP1a and pH 3 levels). This cell cycle arrest could be abrogated by VER-150548, allowing cells to progress through into mitosis and subsequent trapping by nocodazole. Checkpoint abrogation occurred at concentrations of VER-150548 as low as 100 nM. At higher concentrations, a decrease in mitotic markers was observed reflecting the Aurora kinase inhibitory activity of the molecule. DNA damage induced checkpoint abrogation appeared reliant on the absence of functional p53 as no checkpoint abrogation was observed in the p53 proficient colon carcinoma cell line HCT116. Abrogation of DNA damage induced cell cycle checkpoints by VER-150548 resulted in rapid cell death, as confirmed by the large increase in cells with a DNA content,2N after 24 and 48 hours. Cell death occurred in a dose and time dependent fashion with the greatest cell death occurring after 48 hours. The Chk1 inhibitor PF-477736 similarly abrogated DNA damage induced cell cycle arrest whilst the Aurora inhibitor VX680 was unable to override the DNA damage induced arrest. Combination treatment of camptothecin or cisplatin with VER-150548 resulted in a small fraction of cells with a DNA content.4N. This was noticeably less than those cells treated with VER-150548 alone. The combination treatment induced a DNA content between 4 and 7N and this did not match the 8N DNA profile expected from reduplication following Aurora inhibition. Similarly, in cells treated with DNA damaging agents followed by PF-477736 plus VX680, only a small percentage had a DNA content.4N. Again the DNA content of this fraction of cells varied from 4N to around 7N and did not correspond with the 8N predicted from reduplication. Hoescht nuclear staining of cells treated with camptothecin plus VER- 150548 or PF-477736 indicated a high degree of cells with aberrant nuclear morphology indicative of a high degree of chromosomal abnormalities and damage. An additional checkpoint, the spindle
