IDENTIFICATION AND CONDITIONS FOR SELECTIVE EXPRESSION OF MEGAKARYOCYTIC MARKERS IN FRIEND-ERYTHROLEUKEMIA CELLS

Friend murine erythroleukemia cells (MELCs) have been reevaluated in t erms of their nature and potential pathways of differentiation. MELC i nduced with 5 mmol/L hexamethylene bisacetamide (HMBA), in addition to expression of known markers of the erythroid phenotype, were also fou nd to exhibit traits of the megakaryocytic lineage. Erythroid differen tiation was shown by the typical synthesis and accumulation of hemoglo bin (Hb); megakaryoblastoid differentiation of MELCs upon induction wa s shown by increased specific activity of acetylcholinesterase (AChE). Incubation of MELCs with 5 mmol/L HMBA in RPMI supplemented with 1% f etal calf serum (FCS) (instead of the usual 5%), induced cells to sele ctively express high levels of AChE (up to approximate to 170 mU/mg pr otein) with little activation of Hb synthesis (less than 5% B+ cells). The increase in AChE levels was a general phenomenon affecting the wh ole cell population and approached its maximum within 3 days of incuba tion with the inducer. Subsequently, MELCs become committed to termina l division, undergoing growth arrest and expression of the megakaryocy tic phenotype even after the removal of HMBA. There were no appreciabl e changes of basal AChE levels in MELCs that were either made resistan t to HMBA or treated with 0.1 mmol/L hemin that activated differentiat ed erythroid function without commitment. Phorbol 12-myristate 13-acet ate (PMA), known to repress induced Hb synthesis in these cells, did n ot prevent the full increase in AChE when incubated with MELCs 2 days before HMBA addition. HMBA-induced MELCs always underwent AChE increas e that was more or less pronounced depending on the low or high serum content in culture, respectively. Conversely, Hb expression was permit ted only when MELCs were transferred in the late phase or at the end o f commitment from low to high serum media. Variations of FCS content i n culture media proved to be a simple and reliable approach to change the MELC response to inducers and to modulate expression of either meg akaryocytic or mixed erythromegakaryocytic phenotype. These findings s uggested that MELC might be considered, at least, as a bipotential mod el of differentiation to be used for studies on regulation of either m egakaryocytic or erythroid markers and on competition between the two hematopoietic lineages. In this regard, it was intriguing that AChE le vels attained under selective induction (low serum) were always higher than under conditions allowing coexpression of both AChE and Hb (high serum). Moreover, MELCs were also found to bind the specific rat-anti mouse platelet monoclonal antibody 4A5. Flow cytometry experiments hav e shown that both uninduced and especially PMA-treated MELCs were prom pted to express 4A5-positivity, which increased progressively during 4 days of culture. On the contrary, HMBA-induced cultures showed no app reciable binding of the antibody within the same period. Despite diffe rences in the modulation of AChE and platelet antigen, both of these m egakaryocytic markers were expressed constitutively and independently from Hb. The fact that Hb, AChE, and platelet antigen could be elicite d, whether alternatively or concurrently, in the bulk of MELC populati on was strongly against the supposed unipotentiality of these cells. ( C) 1995 by The American Society of Hematology.