Fesoterodine was not effective at disrupting PKC b sub cellular localization

AML3 cells, the kinase was potently phosphorylated suggesting high PKC b activity levels. However, enzastaurin up to 10 mM concentrations appeared to have little if any effect on PKC b phosphorylation levels in either cell line . While enzastaurin displays high specificity for PKC b, the drug can inhibit PKC a at higher concentrations. The reported IC50 Aloin for PKC a is 0.039 mM. While little phosphorylation of PKC a is detected in OCI AML3 cells, there is potent phosphorylation of the kinase in HL60 cells. As shown in Figure 4, enzastaurin potently blocked PKC a phosphorylation in both cell lines. At least in OCI AML3 cells, the drug appears to suppress expression of the kinase at the highest dose used . These findings suggest that PKC a might be a possible target for the drug in AML cells.
ENZASTAURIN BLOCKS MEMBRANE LOCALIZATION OF PKC a BUT NOT PKC b IN AML CELL LINES While phosphorylation of PKC is not truly indicative LY-231514 clinical trial of its activation state, lipid activation of PKC results in its localization to cellular membranes . We examined the effect of enzastaurin on PKC a and PKC b sub cellular localization in OCI AML3 cells using immunofluorescence microscopy. Cells were treated with 1 or 10 mM drug or vehicle for 24 h. PKC a or PKC b was visualized using rabbit polyclonal antibody against each kinase and fluorescent labeled donkey anti rabbit antibody . DAPI was included to visualize nuclei . As shown in Figure 5, both PKC a and PKC b are distributed among intra cellular membranes in vehicle treated control cells.
Enzastaurin at 1 mM suppressed localization of PKC a while the drug at even 10 mM was not effective at disrupting PKC b sub cellular localization Everolimus structure . These results suggest that enzastaurin can inhibit activation of PKC a but not PKC b in OCI AML3 cells. ENZASTAURIN BLOCKS BCL2 PHOSPHORYLATION It has been demonstrated in hematopoietic and acute leukemia cells that BCL2 phosphorylation at serine asenapine structure 70 is required for the antiapoptotic molecule’s full and potent suppression of apoptosis . Physiologic BCL2 kinases that promote survival include PKC a and ERK . OCI AML3 cells exhibit robust basal levels of phosphorylated BCL2 and phosphorylation of the antiapoptotic molecule positively promotes resistance to chemotherapeutic drugs including the BH3 small molecule inhibitor ABT737 .
To determine the effect of enzastaurin on BCL2 phosphorylation, OCI AML3 cells were treated with 10 mM enzastaurin for 3 h during metabolic labeling with liberty 32P orthophosphate and the phosphorylation status of BCL2 was examined following immunoprecipitation . Western analysis demonstrates that roughly equivalent levels of BCL2 protein were immunoprecipitated from untreated cells and cells treated with the drug. While BCL2 was highly phosphorylated in OCI AML3 cells, phosphorylation of the protein was nearly completely inhibited by enzastaurin . Enzastaurin potently blocked both PKC a and ERK in OCI AML3 cells , thus it is not surprising that the drug is effective at blocking BCL2 phosphorylation. To determine if enzastaurin suppresses phosphorylation of BCL2 at serine 70 , we used a phospho serine 70 BCL2 specific antibody to examine BCL2 phosphorylation by Western analysis in cell lysates of OCI AML3 cells treated with enzastaurin .