Figure 5. PJ34 inhibits NuMA and tankyrase1 polyADP-ribosylation in cancer cells.

(A) Inhibition of tankyrase1 modification measured by its shifted pI. Treatment with PJ34 (20 μM, 27 h) attenuated the shift in the isoelectric point of negatively charged tankyrase1 in lung cancer (A549), triple negative breast cancer (MDA-MB-231) and glioblastoma (U87) cancer cells. No similar shift of the isoelectric point of tankyrase1 was induced by the non-phenanthrene PARP1 inhibitor ABT888 (20 μM, 27 h). PolyADP-ribosylated PARP1 was similarly suppressed by both PARP1 inhibitors, PJ34 (20 μM) and ABT888 (20 μM). Representative results of 3 experiments in each cell type are displayed. (B) The binding of tankyrase1 to γ-tubulin or NuMA was measured by co-immunoprecipitation. Their binding in MDA-MB-231 cells was not affected by treatment with PJ34 (20 μM, 27 h). The binding of tankyrase1 to kinesin HSET/kifC1 was impaired. Representative results of 3 experiments are displayed. (C) [³²P]polyADP-ribosylation of NuMA bound to tankyrase1 was inhibited after treatment of human cancer cells MDA-MB-231 with PJ34 (20 μM, 30 min). Binding of NuMA to tankyrase1 was measured by dot blot analysis (Methods). [³²P]polyADP-ribosylated NuMA bound to tankyrase1 was trapped by tankyrase1 antibody on nitrocellulose membrane, auto-radiographed and immunolabeled. Results were repeated in 3 different experiments.