Fig. 6. Proliferative potential of Namalwa endopolyploid cells after irradiation. (A) Two channel cyclin B1-FITC/PI flow cytometry was performed on Namalwa cells for the first 4 days after irradiation. These data revealed an accumulation of cyclin B1 in G2 arrested 4C cells on day 1 after irradiation. However, on day 4 the amount of cyclin B1-positive cells decreased to below the control values for the 4C fraction, but comprised 41% of the 8C fraction. (B) Cyclin B1 and -H2AX double staining was performed on fixed Namalwa cells 3 days post-irradiation and assessed by immunofluorescent microscopy. These studies revealed the localisation of cyclin B1 (green) in the cytoplasm of large polyploid cell nuclei, indicating that many of these cells are maintained in a G2-like state. -H2AX (red) was excluded from a proportion of these cells (arrows). Rare polyploid cells undergoing mitosis (M) were -H2AX negative. Ao indicates an apoptotic cell with aggregated -H2AX label. DNA was counterstained with DAPI (blue). (C) Identical cyclin B1 and -H2AX double staining was performed on untreated Namalwa cells. (D) DNA image cytometry for multipolar anaphase on day 4 after 10 Gy irradiation. Cytospun cells were fixed, processed and the DNA stained with Toluidine Blue to allow stoichiometric measurement of DNA content by light microscopy and image analysis. Integral optical density (IOD) values, presented for each pole, are relevant to 2C DNA content in control G1 cells. Metaphases (arrow) had double IOD values corresponding to 4C. (E) A frame from a time-lapse video showing the division of a tetraploid Namalwa cell (arrow) on day 7 after irradiation (bottom); division of a diploid untreated 4C cell in culture was taken as a control (top). Scale bars: 40 µm.