Binuclear cells induced by acridine orange in giant cell tumor of bone

We have recently found the presence of many binuclear cells among isolated and smeared cells in giant cell tumor of the bone (GCT). These binuclear ce lls ale possibly associated with the formation of multinuclear cells. There fore, this study was undertaken to clarify the mechanism of binucleation in GCT, using primary culture cells exposed to acridine orange (AO) which is a fluorescent vital staining dye for the cytoplasm and nucleus and which in hibits mitosis. The cells were isolated from explants of fresh tumor materi als obtained from two GCT patients (GCT1 and GCT2). These cells were cultur ed in Dulbecco's modified Eagle medium (DMEM) with 10% Fetal calf serum (FC S). After exposure to 0.5 mug/ml AO, for 0, 6, 24, 45, 96 and 144 hows the following parameters were investigated; 1) cell growth rate (GR); 2) freque ncy of hyperdiploid cells (%HDC) by DNA cytofluorometry; 3) mitotic index ( MI); 4) BrdU labeling index (LI); 5) frequency of binuclear cells (%BNC). C ompared to the control cells which were cultured in AO-free medium, the GR of both GCT cells exposed to AO was remarkably inhibited. The MI was 0 from 24 to 144 hours. The %HDC was increased at 24 hours and was maintained hig h until 144 hours. The LI was temporarily increased at 6 hours, brit was de creased at 48 hours. The %BNC was gradually increased. AO inhibited DNA syn thesis and cell mitotic activity in cultured GCT cells and it finally cause d inhibition of cell growth. However; the frequencies of G2 arrest cells an d binuclear cells were increased. These results suggested that the binuclea r cells in GCT may be formed from G2 arrest cells by amitotic nuclear divis ion, but not by mitosis without cytoplasmic division, ol by cell fusion.