We observed a gradual decrease in global histoneH3 Ser10 phosphorylation, with almost INNO-406 complete dephosphorylation occurring at concentrations of 5 M and higher. We also found that concentrations of SP600125 ranging from 0.1 to 20 M over a 6 h period did not significantly alter the number of viable cells compared with the control. To examine whether SP600125 exerted its effect reversibly, cells were maintained for 1 h in a medium containing 10 M SP600125 and then switched to a medium without inhibitor for various time periods. Total cell extracts were subjected to Western blotting using anti phospho Ser10 histone H3. As shown in Fig. 1B, Ser10 phosphorylation increased after SP600125 was removed and by 6 h had reached the level in untreated cells. We further examined the effect of SP600125 on histone H3 Ser10 phosphorylation in other cell types, i.
e, human cervical carcinoma HeLa cells and human prostate carcinoma PC 3 cells. In both cell types, PD173074 SP600125 caused a dramatic reduction in histone H3 Ser10 phosphorylation compared with an untreated control. SP600125 dependent hypophosphorylation of histone H3 Ser10 is independent of mitogen activated protein kinases . The rapid loss of histone H3 Ser10 phosphorylation by SP600125 possibly indicated that the inhibitor effect on histone kinase was direct, as indirect feedback regulation is expected to take a longer time. We wished to explore the association between histone H3 Ser10 phosphorylation loss and the p46 54JNK inhibition in HepG2 cells. To address this question, we first evaluated the efficacy of p46 54JNK inhibition by SP600125 in a standard immunoprecipitation based kinase assay.
HepG2 cells were pretreated for 1 h with different concentrations of SP600125, and then briefly exposed to interleukin 1 to stimulate JNK activity. The addition of SP600125 led to a dose dependent inhibition of IL 1 stimulated phosphorylation of c Jun, with a 50 inhibitory concentration of about 8 M, consistent with earlier demonstrations showing 50 inhibitory concentrations in the range of 5 to 15 M for p46 54JNK inhibition in different mammalian cell lines. A comparison of the kinetics of a reduction in histone H3 Ser10 phosphorylation and p46 54JNK inhibition by SP600125 suggests that SP600125 dependent loss of histone H3 Ser10 phosphorylation is possibly independent of the p46 54JNK pathway. A close match would be expected if SP600125 mediated its effect by inhibiting this kinase.
We further evaluated the consequence of p46 54JNK inhibition on histone H3 Ser10 phosphorylation by inhibiting p46 54JNK with another pharmacological inhibitor. JNK 1 peptide , a cell permeative peptide that inhibits the activation of this pathway, was employed for the experiments shown in Fig. 2C and D. HepG2 cells were treated with different doses of JNK 1, and histone H3 Ser10 phosphorylation and p46 54JNK activity were determined under parallel conditions, as described in Materials and Methods. Treatment with JNK 1 inhibitor had no effect on histone H3 Ser10 phosphorylation even at 30 M, even though SP600125 resulted in the almost complete inhibition of endogenous p46 54JNK activity at 20 M, further supporting the notion that the effect of SP600125 on histone H3 Ser10 phosphorylation is not a direct consequence of p46 54JNK inhibition.