ANALYSIS OF PHORBOL ESTER STIMULATED HUMAN MEGAKARYOCYTE DEVELOPMENT

Abstract

The events that occur during the terminal maturation of human megakaryocytes are poorly characterized. To examine these events, a recently characterized human megakaryocytic cell line (EST-IU, Cancer Res. 46: 2155-2159, 1986) was exposed to 12-0-tetradecanoyl-phorbol-13-acetate (TPA), as well as 2 non-transforming phorbol esters (4 alpha phorbol and 4 beta phorbol 12 alpha, 13 alpha diacetate) at the identical concentrations. Morphologic changes, including cellular attachment to untreated plastic or glass, occurred within 4 hrs of treatment with TPA. Treatment of EST-IU cells with either of the 2 non-transforming phorbols (4-alpha phorbol, or 4-beta phorbol, 12-beta, 13-alpha diacetate) failed to change morphology, DNA content, or expression of surface membrane glycoproteins or alpha-granule constituents when compared to control cells. TPA treatment resulted, however, in j^rofound changes in adherence to plastic by the EST-IU cells, with an obvious dose-response relationship. At a 5 × 10-8 M TPA, cellular attachment was noted as early as 4 hours following treatment, agd was complete by 16 hours, at which time > 95% of treated cells were attached. Following TPA treatment at 5 × 10-8 M, a number of morphologic changes occurred, including marked cellular flattening, the appearance of extensive cytoplasmic budding, and the development of numerous filopodia. Cells treated with either of the non-transforming phorbols as assessed by propidium iodide staining and flow cytometric analysis failed to exhibit a change in ploidy, although TPA reproducibly altered this parameter of megakaryocyte development. Cells treated with 10-9 M TPA have approximately the same proportion of cells in the 4N and 8N peaks as control cells. Following exposure to 10-9 M and 10-8 M TPA, there was an apparent shift of cells out of the 4N peak to 8N and 16N levels, and even the appearance of a small percentage of 32N cells. The DNA content of TPA-treated cells was also assessed by Feulgen staining and microdensitome try. Those cells (5%) which failed to adhere following TPA treatment were analyzed separately, and showed a very different ploidy distribution than the adherent cell population. Over 85% of adherent cells have a ploidy > 16N, with some cells attaining the 128N level. Treatment of cells with either of the 2 non-transforming phorbols failed to affect the expression of Factor V, Factor VIIIrRAg, beta-throraboglobulin, fibrinogen, or platelet glycoproteins. Cells treated with 5 × 10-8 M TPA similarly do not significantly increse the expression of Factor V, fibrinogen, or beta-thromoglobulin over that observed in control cells. The expression of both Factor VIIIrRAg and platelet glycoproteins however, increase in TPA-treated cells. A similar increase in the expression of platelet glycoprotein Ilb/IIIA using the mouse monoclonal C17 was also observed. Those cells that express the highest levels of Factor VIII:RAg and platelet glycoproteins following phorbol treatment also demonstrated the highest ploidy levels and also are the largest cells as measured by forward angle light scatter during flow cytometry.These studies indicate that TPA treatment of EST-IU cells initiates a cascade of events characterized by cellular adherence, increases in cell size and DNA content, and enhanced expression of platelet glycoproteins and Factor VIIIrRAg. These events appear to occur in concert and closely resemble information that is available concerning maturation of normal rodent and human megakaryocytes. Although it is important to emphasize that EST-IU cells are leukemic and thus intrinsically different from normal human megakaryocytes, their availability and dynamic responses to TPA will provide an appropriate cellular model with which to study megakaryocyte maturation.