HL-60 CELL-DIFFERENTIATION INDUCED BY PHORBOL-ESTERS AND 12-DEOXYPHORBOL-ESTERS

The human promyelocytic leukaemia cell (HL-60) undergoes differentiati on into a macrophage-like form when exposed to both tumour promoting-a nd non-promoting phorbol esters. We have investigated the effect of th e two non-promoting phorbol esters, 12-deoxyphorbol-13-O-phenylacetate (Dopp) and 12-deoxyphorbol-13-O-phenylacetate-20-acetate (Doppa) on H L-60 cultures, and compared them,vith the tumour promoter 12-O-tetrade canoylphorbol-13-acetate (TPA). All phorbol esters tested were found t o be able to stop HL-60 proliferation and induce cell adherence and mo rphological changes characteristic of differentiation. TPA, fully diff erentiating at 1 nM, was more potent than Dopp and Doppa, which requir ed 100 nM for full differentiation effects within the 4 day study. Dop pa initially appeared weaker than Dopp at inhibiting incorporation of thymidine, the earliest effect studied, but we were able to detect rap id C-20 deacylation of Doppa, converting it to Dopp, using an HPLC pro tocol presented here. A detailed study of this thymidine incorporation inhibition showed that both TPA (10 nM or greater) and Dopp (500 nM o r greater) have very similar time courses, with 50% inhibition occurri ng at similar to 12 h, in contrast to Doppa which had a significantly delayed time course at all doses tested. Exposure tests indicated that Dopp and Doppa could be washed from the cells much more easily than T PA. The data presented here strongly support the notion that the metab olic conversion of Doppa to Dopp by HL-60 cells was necessary to media te its differentiating effects. Since protein kinase C (PKC)-beta(1), present in HL-60 cells, has been found to be the only PKC isotype acti vated so far in vitro by Doppa, our results suggest that activation of this isotype is not sufficient to drive HL-60 differentiation in vivo .