SW620 (5.5×10³ per well) or MDA-MB-231 (5×10³ per well) cells are seeded in 96-well plates and incubated overnight. Cells are dosed for 24 h with a 9-point titration of NSC 613327 ranging from 0.01 to 100 nM with or without a constant dose of AZD-7762 (300 nM). Control wells are dosed with vehicle alone (0.1% DMSO) or 300 nM AZD-7762. After 24 h, medium is removed and AZD-7762 alone is added back to the wells treated previously with AZD-7762 for an additional 24 h. Cells are then incubated in drug-free medium for an additional 72 h. The effect on cell proliferation is determined by MTS assay^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice and Rats^[1]
Male NCr mice and male rnu rats are used. For xenograft models in mice, tumor cells are harvested, pelleted by centrifugation for 5 min, and resuspended in sterile PBS. Cells (3×10³-6×10⁶) are implanted s.c. into the right flank of the mice in a volume of 0.1 to 0.2 mL using a 25-gauge needle. Tumors are allowed to grow to the designated size of 100 to 200 mm³ before the administration of compound. For xenograft models in rats, Cells are harvested, pelleted by centrifugation for 5 min, and resuspended in 50% sterile PBS and 50% Matrigel. Rats receive a 5 Gy whole-body radiation dose 5 days before cell implantation to improve tumor growth. H460-DNp53 cells (1×10⁷) are implanted s.c., into the right flank of the rats in a volume of 0.2 mL using a 25-gauge needle. Tumors are allowed to grow to the designated size of 100 to 200 mm³ before the administration of AZD-7762. AZD-7762 (10 and 20 mg/kg) is administered by i.v. injection via the tail vein. Cyclic schedules are used and treatment ranged from three to five cycles. Each cycle includes administration of a standard agent (NSC 613327 or CPT-11) every 3 days follow by delivery of AZD-7762. Tumor volumes are measured with electronic calipers and calculated.
Mice ^[2]
C57Bl/6 mice are intravenously injected with 2×10⁵ Eμ-Myc B-lymphoma cells in PBS and treated with pharmacological inhibitors from 8 days post-injection. Treatment of mice is continued until an ethical end-point is reached; hunched posture, ruffled fur, enlarged lymph nodes, laboured breathing, weight loss greater than 20% of start body weight and limited mobility or paralysis. AZD7762 is delivered intraperitoneally in 10.3% -hydroxypropyl-β-cyclodextrin in 0.9% saline at 20 mg/kg daily on weekdays.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Zabludoff SD, et al. AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies. Mol Cancer Ther. 2008 Sep;7(9):2955-66.  [Content Brief]

  [2]. Quin J, et al. Inhibition of RNA polymerase I transcription initiation by CX-5461 activates non-canonical ATM/ATR signaling. Oncotarget. 2016 Aug 2;7(31):49800-49818.  [Content Brief]