ABSENCE OF AUTOANTIGEN KU IN MATURE HUMAN NEUTROPHILS AND HUMAN PROMYELOCYTIC LEUKEMIA LINE (HL-60) CELLS AND LYMPHOCYTES UNDERGOING APOPTOSIS

The Ku autoantigen is a heterodimer of 70- and 80-kD proteins recogniz ed by autoantibodies from patients with systemic lupus erythematosus a nd related diseases that is the DNA-binding component of a DNA-depende nt protein kinase. The catalytic activity of DNA-dependent protein kin ase is carried by a 350-kD subunit (p350). In light of the recently de scribed role of Ku in repairing double-strand DNA breaks, we investiga ted the regulation of Ku and p350 levels in neutrophils, a terminally differentiated cell type destined to undergo apoptosis. Since the appe arance of double-strand DNA breaks is characteristic of apoptosis, we were interested in the possibility that Ku might oppose programmed cel l death. Analysis of peripheral blood cells by flow cytometry using an ti-Ku and anti-p350 monoclonal antibodies revealed that neutrophils we re unstained, whereas resting (G0) lymphocytes were positive. The abse nce of Ku in mature neutrophils was confirmed by Western blotting and enzyme-linked immunosorbent assay for Ku antigen. In contrast, the hum an promyelocytic leukemia line, HL-60, which undergoes differentiation toward neutrophils after dimethylsulfoxide treatment, was positive fo r Ku and p350. In view of the short lifespan of neutrophils and the pr olonged half-life of Ku and p350 (>5 d), these data suggested that Ku was actively degraded during myeloid differentiation. Analysis of HL-6 0 cells by flow cytometry revealed that Ku staining was bimodal. Cells in G1/G0, S, or G2/M were all stained positively, whereas cells with a subdiploid DNA content characteristic of apoptosis were Ku negative. Similar results were obtained with phytohemagglutin-stimulated human lymphocytes. These data suggest that the Ku antigen is actively degrad ed in both myeloid cells destined to undergo apoptosis and apoptotic l ymphocytes, raising the possibility that degradation of Ku may help to prevent the inappropriate repair of fragmented nuclear DNA during apo ptosis.