Sequence analysis of mutational hot spots in the endogenous and transfected colony stimulating factor 1 receptor in gamma‐irradiation induced factor independent subclones of clonal hematopoietic progenitor cell lines

Abstract

AbstractAmino acid substitutions in several proto‐oncogenes and growth factor receptors may be activating mutations. The macrophage‐colony stimulator factor (M‐CSF) receptor (c‐fms) differs from the transforming v‐fms at several sites in the extracellular domain and contains a tyrosine residue at the carboxy terminus which may be involved in the negative regulation of the receptor kinase activity. The engineering of two point mutations in human c‐fms gene (residue 301, leucine to serine; and residue 969, tyrosine to phenylalanine) induces factor independent growth and malignant transformation when expressed in murine cells. Point mutations have also been detected in c‐fms in marrow cells or peripheral blood from patients with myelodysplastic syndromes or acute myelomonocytic leukemias. We have previously shown that murine factor independent cell lines, derived from cocultivation of clonal interleukin‐3 (IL‐3) dependent hematopoietic progenitor cell lines with the gamma‐irradiated marrow stromal cell line, D2XRII, have an upregulated c‐fms expression during an intermediate stage on the way to factor independence, but that fully factor independent subclones do not have an overexpression of c‐fms. In order to determine whether the factor independent phenotype resulted from an activating mutation in c‐fms during the transient phase of overexpression, we examined both intracellular and extracellular domains of c‐fms in irradiation induced factor independent subclones of FDC‐P1 JL26 or 32D c‐fmsm cells. Polymerase chain reaction (PCR) amplification of the regions flanking residues 301 and 973 with sequence analysis by the dideoxy chain‐termination method revealed no detectable mutations at either site. These data show that the factor independence of these cell lines does not result from activating mutations at 301 or 973 of the c‐fms receptor. Thus, the induction of growth factor independence may be attributable to 1) activating mutations at other sites in c‐fms; 2) activation of sites distal to the CSF‐1 receptor in the signal transduction pathway; or 3) a mechanism independent of the c‐fms/M‐CSF pathway. © 1993 Wiley‐Liss, Inc.