HAMNO (NSC-111847) is a protein interaction inhibitor of replication protein A (RPA).

RPA:

HAMNO inhibits colony formation in both HNSCC cell lines. The combination of HAMNO with etoposide markedly inhibits colony formation to a greater degree than HAMNO alone. In UMSCC38 cells, HAMNO dose-dependently increased the occurs of pan-nuclear γ-H2AX staining. UMSCC38 and UMSCC11B cells have prominent γ-H2AX staining, particularly after incubation with HAMNO (20 μM). In UMSCC38 and OKF4 cells, HAMNO increased γ-H2AX staining, with the greatest increase in signal occurring in S-phase.

In mice, HAMNO inhibits the progression of UMSCC11B tumors. After treatment with etoposide (20 μM, 2 h), Ser33 of RPA32 (an ATR substrate) is highly phosphorylated, which is reduced with the addition of 2 μM HAMNO and is nearly absent at higher concentrations, demonstrating HAMNO can inhibit RPA32 phosphorylation by ATR.

Cell cycle assessment and γ-H2AX staining are monitored in UMSCC38 and OKF4 cells after 2 h incubation with HAMNO (2, 20, 50 μM) and fixed in 70% ethanol overnight. Cells are washed with PBS and incubated overnight in PBS containing 1% BSA, 10% goat serum, and PS139-H2AX antibodies washed and incubated in goat anti-mouse Alexa Fluor 647 antibody for 30 min at room temperature. Cells are incubated in 50 μg/mL propidium iodide and 100 μg/mL RNase A for 30 min, and 10,000 cells per sample are analyzed.

UMSCC38 and UMSCC11B cells are implanted into 6-week-old female mice by a single subcutaneous injection of tumor cells (2 to 6×105 cells in 100 mL of sterile PBS). The growth rates of tumors are determined by daily monitoring of tumor volume with vernier calipers [tumor volume=1/2(length×width2)]. Once the tumor size reaches 50 mm3, etoposide (10 mg/kg mouse) and HAMNO (2 mg/kg) are administered intraperitoneally every day for 3 days. Tumor size is monitored daily and the volume of the tumor is compared among all experimental groups.