Figure 7. Effects of CaM kinase inhibitors on neutrophil growth and functional activation.
(A) KN93 inhibited the proliferation of both EPRO and SCF ER-Hoxb8 cell lines, whereas the inhibitor did not affect the growth of EML cells. For each assay, cells were diluted to 2 × 105 cells/mL and total numbers of cells that excluded trypan blue after the indicated times were counted using a hemacytometer. Note levels of KN93 used with EPRO cells were two-fold higher than those used in SCF ER-Hoxb8 and EML cells. (B) The CaMKK inhibitor STO-609 significantly reduced the growth profiles of both EPRO and SCF ER-Hoxb8 cells, and inhibited the growth of EML cells. (C) Morphologic maturation of EPRO cells exposed to the CaMK inhibitors KN-62 or KN-93 was assessed by examining cells after 5 days of treatment and counting the number undifferentiated cells with promyelocyte characteristics (e.g. high nuclear to cytoplasmic ratios) versus differentiated cells with kidney shaped or lobulated nuclei. Shown are the results of three independent experiments. P values: KN-62 vs. control, p = 0.001; KN-93 vs. control, p = 0.001. (D) The respiratory burst exhibited by ATRA-induced EPRO or MPRO cells upon PMA stimulation was inhibited by the CaM kinase inhibitor KN-93 (left panel); STO-609 also inhibited the oxidative burst produced by EPRO cells in a dose-dependent fashion. Shown are data from differentiated cells that were incubated with the inhibitors for 24 hours prior to each assay, with data sets given as percentages of peak light units emitted as compared to cells incubated with the diluting reagent. Data shown are the average responses ± SD from three independent experiments.