Table 4

Effect of HKI-272 on HER-2-signaling pathways and cell cycle regulationa

Signaling markerIC50 (nm)
Phospho-HER-25
Phospho-EGFRb3
Phospho-MAPK2
Phospho-Akt2
Cyclin D19
% cells in S phase2
  • a In HER-2 and EGFR phosphorylation assays, cells (BT474 and A431, respectively) were incubated with various concentrations of HKI-272 for 3 h at 37°C. For evaluation of downstream signaling markers, BT474 cells were incubated with HKI-272 for 12–16 h at 37°C. Protein extracts were analyzed by immunoblotting using phospho-tyrosine, phospho-MAPK, phospho-Akt, or cyclin D1 antibodies. Antibodies to HER-2, EGFR, MAPK, and Akt were used to correct for protein loading. Actin antibodies were used to normalize loading for cyclin D1. Blots were quantified by densitometric scanning. Concentration of HKI-272 (nm), which inhibits phosphorylation (protein levels for cyclin D1) by 50%, is given. For cell cycle analysis, BT474 cells were treated with HKI-272 for 12–16 h. Untreated and treated cells were pulse labeled for 30 min with bromodeoxyuridine to label cells in S phase. Uptake of label was quantified using bromodeoxyuridine antibodies/FITC conjugates by flow cytometry. The concentration of HKI-272, which decreased the number of cells in S phase by 50%, is given.

  • b EGFR, epidermal growth factor receptor; MAPK, mitogen-activated protein kinase.