Protein kinase C during differentiation of human promyelocytic leukemia cell line, HL‐60

Abstract

Protein kinase C @'KC) from human promyeiocytic leukemia HL-60 cells can be resolved into three fractions (peak a, b and c) by hydroxyapatite coiumn ch~mato~phy.Peak a and c enzymes are indistin~i~able from the brain type II PKC having @ @I and ~I)-~~~ and type III having e-sequence, respectively. Peak 6 enzyme is a previously unidentified PKC subspecies that has enxymological properties subtly different from type I (having y-sequence), type II and type III PKC. Upon treatment of AL-60 cells with 1 pM retinoic acid, this peak b enxyme is decreased dramatically within 24 h, whilst peak u enzyme (J-PKC) is increased, and peek c (c+PKC) enzyme is slightly decreased within 48 h. The result implies that the PKC subspecies in HL-60 cells have distinct functions during cell differentiation.

Sustained activation of protein kinase C is essential to HL-60 cell differentiation to macrophage.

“…HL-60 cells contain the a-and (B-subspecies of PKC and, additionally, a structurally unidentified subspecies as described (9). The addition of PMA to the cell suspension caused rapid translocation and a subsequent decrease in enzyme activity as shown in Fig.…”

“…PKCs in the soluble and particulate membrane fractions were separated by centrifugation after disruption of the cells, and the subspecies were analyzed by hydroxyapatite column chromatography under the conditions specified earlier (8). The enzyme was assayed by measuring incorporation of radioactive phosphate of [y-32P]ATP into a synthetic oligopeptide, MBP-(4-14) (myelin basic protein, residues 4-14), which serves as a specific substrate for PKC, as described (9).…”

Differentiation of HL60 promyelocytic cells is promoted by a ‘differentiation enhancing factor’ produced by erythroleukemia cells

“…The procedure used was essentially that described by Hashimoto et al [13]. HL60 cells were lysed and the bulk of PKC activity was collected as a single peak on a DEAE-cellulose column chromatography [13] and submitted to hydroxyapatite (HA) chromatography under the conditions described in [14].…”

“…HL60 cells were lysed and the bulk of PKC activity was collected as a single peak on a DEAE-cellulose column chromatography [13] and submitted to hydroxyapatite (HA) chromatography under the conditions described in [14]. PKC isozymes were identified by their effector requirements [14] and by their position on HA chromatography, using as a standard the PKC isozyme activities present in rat brain [14].…”

“…1A, bar 3, full bar) and PMA at 1 nM (Fig. 1B, bar 3 The various PKC isoforms were identified in the HA-chromatography by the elution positions, using the rat brain ~-and fl-PKC isoforms as standards [13]. The level of each PKC isozyme was calculated from the area of the eluted peaks and plotted as the percentage of the starting values (% of original).…”