Inhibition of Ku heterodimer DNA end binding activity during granulocytic differentiation of human promyelocytic cell lines

The heterodimeric Ku protein (composed of the Ku 86 and Ku 70 sub-units) is a nuclear protein which binds to DNA termini without sequence specificity, Ku is the DNA-targeting component of the large catalytic sub-unit of the D NA-dependent protein kinase complex that is required for the repair of DNA double-strand breaks in mammalian cells. We studied the expression and func tion of Ku/DNA-PK during granulocytic differentiation of two human promyelo cytic cell lines, HL60 and NB4, a process associated to decreased radiation resistance. After 3 days exposure to differentiating agents (either all-tr ans-retinoic acid or DMSO), Ku binding to double stranded (ds)-DNA ends dec lined dramatically whereas Ku protein levels remain unchanged, The nuclear, but not cytoplasmic, fraction of differentiated HL60 cells extracts exhibi ted a heat-sensitive inhibitory activity towards DNA binding of recombinant Ku heterodimer, We further demonstrate that immunoprecipitation of Ku is i mpaired in extracts from differentiated cells by using two antibodies that recognize epitopes within the C-terminus DNA binding domains of Ku 70 and K u 86 proteins, These results favor the hypothesis of a protein interacting with Ku that would prevent DNA binding of heterodimerized Ku protein by ste ric hindrance.