In vivo tumor model

All experiments involving live animals were carried out in strict accordance with the recommendations stated in the Guide for the Care and Use of Laboratory Animals, as adopted and promulgated by the National Institutes of Health. The protocol (#11034) was reviewed and approved by the City of Hope Institutional Animal Care and Use Committee. A breeding colony of ES1^e/SCID mice, originally provided by Dr. Philip M. Potter of the St. Jude Children's Research Hospital, was maintained at the City of Hope. SK-N-BE(2)c and SK-N-AS neuroblastoma cells were suspended in Matrigel (BD Biosciences) at 5 × 10⁷/ml and 1 × 10⁸/ml, respectively, after they were harvested and washed twice in PBS. MDA-MB-468 breast cancer cells and H82 small cell lung cancer cells were suspended in Matrigel at 2 × 10⁷/ml after they were harvested, washed, and mixed with Matrigel in the same manner. 0.1 ml of suspended cells was subcutaneously injected into the right flank ES1^e/SCID mice. For each xenograft model, mice were randomly divided into two groups, each receiving a daily dose of 40 mg/kg AOH1160 or an equivalent amount of vehicle by gavage throughout the entire experiment starting on the fifth day after tumor cell injection. Mice were monitored twice weekly for any sign of side effects. The weight of the animals was measured as an indicator of compound toxicity. At the end of the experiment, tumors were isolated from sacrificed mice and analyzed by immunohistochemistry staining with antibodies specific for phosphor-Chk1 and γH2A.X as described (25).