The role of protein kinase C in 1,25 -dihydroxyvitamin D(3)-induced differentiation of HL-60 promyelocytic leukemia cells.

This thesis research characterizes the role of protein kinase C in 1,25-dihydroxyvitamin D$\sb3$ (1,25-(OH)$\sb2$D$\sb3$) induced HL-60 promyelocytic leukemia cell differentiation. A dose dependent increase in ($\sp3$H) phorbol-12,13-dibutyrate (PDBu) binding to HL-60 cells was observed within 12 h of 1,25-(OH)$\sb2$D$\sb3$ treatment. ($\sp3$H) PDBu binding was 200% of control after 24 h, but not different from control after 48 h 1,25(OH)$\sb2$D$\sb3$ treatment. The increased ($\sp3$H) PDBu binding following 1,25(OH)$\sb2$D$\sb3$ treatment involved a two-fold increase in B$\sb{\rm max}$, was dependent on protein synthesis, and was seen in cell homogenates and in intact cells. Studies to determine the requirement for PKC in 1,25(OH)$\sb2$D$\sb3$ induced differentiation of HL-60 cells have found that 1,25(OH)$\sb2$D$\sb3$ induced differentiation was inhibited up to 80% in a dose dependent manner by protein kinase inhibitors H-7 (IC$\sb{50}$ = 15 uM) and HA1004 (IC$\sb{50}$ = 170 uM). The relative potency of these inhibitors suggested that PKC is important in 1,25(OH)$\sb2$D$\sb3$ induced differentiation. The nuclear phosphoproteins whose phosphorylation was regulated by 1,25(OH)$\sb2$D$\sb3$ were also characterized. Using both in vitro and in vivo phosphorylation assays, ten nuclear phosphoproteins were detected that show alterations in phosphorylation within 6 to 40 h of 1,25-(OH)$\sb2$D$\sb3$ treatment, designated p66, p45, p36, p33, p32, p27, p22, p19, p18 and p17. P66, p36, and p19 were phosphorylated in a calcium and phospholipid dependent manner. P66 was identified as lamin B, p17 as histone H2b, and p18 as histone H3. The DNA-cellulose binding ability, resolution on 2-D SDS-PAGE, and salt extraction from nuclei were examined for the 1,25-(OH)$\sb2$D$\sb3$ sensitive nuclear phosphoproteins. To determine if any of these 1,25-(OH)$\sb2$D$\sb3$ regulated phosphoproteins might interact with a transcriptional regulatory element within the c-myc gene, the binding of nuclear proteins to the c-myc intron element (MIE) was assayed. Following 1,25-(OH)$\sb2$D$\sb3$ treatment, p32 binding to the MIE increased by 2-3 fold within 12 h. This increase in binding corresponded to increased phosphorylation of p32, and did not occur with all regions of DNA tested. This thesis research has characterized a role for PKC in 1,25-(OH)$\sb2$D$\sb3$-induced HL-60 cell differentiation.PhDPharmacologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/103110/1/9303779.pdfDescription of 9303779.pdf : Restricted to UM users only