1-oleoyl-2-acetylglycerol and hexamethylene-bisacetamide

Previous studies have suggested a role for protein kinase C (PKC) during induction of murine erythroleukemia cell (MELC) differentiation by hexamethylene bisacetamide (HMBA) (Melloni, E., Pontremoli, S., Viotti, P. L., Patrone, M., Marks, P. A., and Rifkind, R. A. (1989) J. Biol. Chem. 264, 18414-18418). The present studies assess the effect of HMBA on the content of 1,2-diacylglycerol (DG), the physiologic activator of PKC, in MELC variants. Exposure of parental Sc9 cells to HMBA induced a rapid rise and fall in DG content. The DG level increased within seconds from 225 pmol.10(6) cells-1 to a maximum of 305 pmol.10(6) cells-1 at 5 min. Thereafter, DG content fell reaching control levels at 30 min and 46% of control at 4 h. Similar DG elevations were detected in HMBA-resistant, phorbol ester-resistant, and vincristine-resistant MELC lines. Early DG elevation was followed by the characteristic rapid fall in both the phorbol ester-resistant and vincristine-resistant lines, both of which differentiate rapidly in response to HMBA. In contrast, in an HMBA-resistant MELC the DG level failed to fall for at least 10 h. Selection of HMBA-resistant cells for vincristine resistance restores both HMBA sensitivity and the rapid fall in DG content. Addition of a synthetic DG, 1-oleyl-2-acetyl glycerol (OAG), along with HMBA and every 2 h for the next 48 h blocked differentiation, as measured by accumulation of benzidine-reactive cells or by the commitment assay in methyl-cellulose. However, if addition of OAG was delayed for just a few minutes, until endogenous DG levels began to fall, differentiation was no longer inhibited. Rapid elevation of DG content is the earliest reported event during HMBA action and a subsequent fall in the DG content appears to be a critical step in the process of commitment to terminal differentiation.

Topics: Acetamides; Animals; Diglycerides; Drug Resistance; Erythropoiesis; Genetic Variation; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured; Vincristine

Recent studies have shown that diacylglycerols mimic several effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on cultured cells and suggest that diacylglycerols are the endogenous functional analogues of phorbol esters. However, all such studies have been of short duration, and although a single application of diacylglycerol induces these effects, tumour promotion usually requires long and repeated application of tumour-promoting agents. Here we investigated the effect of 1-oleoyl 2-acetyl glycerol (OAG) on differentiation of Friend erythroleukaemia cells (FELC), since TPA must be present continuously to inhibit differentiation in this system throughout the experiment. We also studied the effects of OAG in both TPA-sensitive and TPA-resistant clones, to investigate whether OAG and TPA have a similar mode of action. We found a dose-dependent inhibition of differentiation by OAG in the TPA-sensitive clone, but not in the TPA-resistant clone. Repeated treatment was necessary to achieve these results; almost complete inhibition could be obtained when OAG was applied 7 times/day for 3.5 days at 3 micrograms/ml per application, whereas a single application of OAG at a higher dose had no effect. Protein kinase C, which is believed to be the common target of OAG and TPA, was present in both TPA-sensitive and TPA-resistant FELC clones. These results suggest that OAG mimics the long-term effect of TPA and that the mechanism by which FELC clones resist TPA and OAG is not a lack or functional deficiency of protein kinase C.

Topics: Acetamides; Animals; Cell Adhesion; Cell Differentiation; Diglycerides; Dose-Response Relationship, Drug; Drug Resistance; Friend murine leukemia virus; Glycerides; Leukemia, Erythroblastic, Acute; Phorbols; Protein Kinase C; Tetradecanoylphorbol Acetate