Fig. 3.

Evidence for cellular inhibition of Chk2 by PV1019. A, abbreviated molecular interaction map depicting the phosphorylations of Chk2, Cdc25C, and HDMX after Chk2 activation. The conventions used for the interactions (Kohn, 1999) are as follows: the line with an open circle indicates enzymatic stimulation and phosphorylation, the line with an open arrowhead indicates general stimulation, and the open circle with a line crossing through indicates degradation. For details of the molecular interaction map for Chk2 see Pommier et al. (2006). B, OVCAR-4 cells were exposed to 10 Gy of IR followed by a 1-h incubation in the presence or absence of PV1019 or ABI (preincubated for 1 h). The levels of Chk2 phosphorylated on Ser⁵¹⁶ and total Chk2 were detected by Western blotting. Actin was used as a loading control. C, OVCAR-5 cells were exposed to PV1019 and TPT (or control) for 1 h. The levels of Chk2 phosphorylated on Ser⁵¹⁶ were detected by Western blotting. Actin was used as a loading control. D, OVCAR-5 cells were pretreated with TPT for 1 h, the drug was washed off, and the cells were exposed to PV1019 (or control) for an additional hour. The levels of Chk2 phosphorylated on Ser⁵¹⁶ were detected by Western blotting. Actin was used as loading control. E, MCF7 cells were exposed to either 1 μM TPT for 4 h or 10 Gy of IR followed by a 4-h incubation in the presence or absence of PV1019 (preincubated for 2.5 h). Western blotting using anti-HDMX antibodies was performed to measure the levels of HDMX. Representative gels are depicted, and the quantified levels of HDMX are shown in the graphs. F, MCF7 cells were exposed to 10 Gy of IR followed by a 1-h incubation in the presence or absence of PV1019 (preincubated for 2.5 h). The levels of Chk2 phosphorylated on Ser⁵¹⁶ were detected by Western blotting (top panel). The levels of Cdc25C phosphorylated on Ser⁵¹⁶ were detected by Western blotting (bottom panel). Actin was used as a loading control for each experiment.