Evidence that a novel human differentiation-inhibiting protein blocks the dimethyl sulfoxide-induced differentiation of erythroleukemia cells by inhibiting the activation of membrane protein kinase C.

We have previously reported (J. P. Durkin et al., Blood, 79: 1161-1171, 1992) the isolation of a human differentiation-inhibiting protein (DIP) which selectively inhibits and blocks the differentiation of erythroid burst-forming unit progenitor cells in bone marrow colony assay, and the dimethyl sulfoxide (DMSO)-induced differentiation of cultured murine erythroleukemia (MEL) cells. DIP blocks MEL cell differentiation directly, without affecting the ability of the cells to proliferate. In the present study, DIP (at < 1 ng/ml) inhibited MEL cell differentiation only when added to the culture medium within 1 h after DMSO induction, indicating that it blocked an early, critical step in erythroleukemia cell differentiation. The protein kinase C (PKC) inhibitor H-7 also maximally inhibited the differentiation of MEL cells during this same period following induction, suggesting that DIP may have blocked an early PKC-dependent process. Indeed, DIP was found to abolish a transient increase in membrane PKC activity which was triggered in MEL cells within 10-30 min after DMSO addition. This increase in membrane PKC activity resulted from the activation of an inactive pool of PKC residing on membranes, and not from the translocation of cytosolic PKC to membranes. DMSO also stimulated membrane PKC activity and differentiation in human erythroleukemia cells and HL-60 myeloid leukemia cells. As was the case with MEL cells, DIP prevented the early activation of PKC and the differentiation of human erythroleukemia cells. However, it did not inhibit the early increase in PKC activity in HL-60 cells or the subsequent differentiation of these cells. These results suggest that DIP blocks erythroleukemia cell differentiation by inhibiting an early and critical activation of inactive membrane PKC.