An activating mutation in the Kit receptor abolishes the stroma requirement for growth of ELM erythroleukemia cells, but does not prevent their differentiation in response to erythropoietin

We have previously shown that murine ELM erythroleukemia cells can only be grown in vitro in the presence of a stromal feeder layer, or alternatively stem cell factor (SCF), without which they differentiate. When grown in the presence of SCF, ELM cells can still differentiate in response to erythrop oietin (Epo), but growth on stroma prevents this. We previously isolated a stroma-independent ELM variant, ELM-I-1, that is also defective in Epo-indu ced differentiation, We show here that this variant has an activating mutat ion in the Kit receptor, converting aspartic acid 814 to histidine, Express ion of the mutant receptor in stroma-dependent ELM-D cells causes growth fa ctor-independent proliferation and also gives the cells a selective advanta ge, in terms of proliferation rate and clonegenicity, compared with ELM-D c ells grown in optimal amounts of SCF. Expression of the mutant receptor in ELM-D cells also prevents spontaneous differentiation, but not differentiat ion induced by Epo. Analysis of mitogenic signaling pathways in these cells shows that the mutant receptor induces constitutive activation of p42/p44 mitogen-activated protein kinases. It also selectively inhibits the express ion of p66Shc but not the p46/p52 Shc isoforms (as did treatment of ELM cel ls with SCF), which is of interest, because p66Shc is known to play an inhi bitory role in growth factor signaling. (C) 1998 by The American Society of Hematology.