IMMUNOCYTOMETRIC DETERMINATION OF THE ALPHA-ISOFORM AND BETA-ISOFORM OF HUMAN DNA TOPOISOMERASE-II - INFLUENCE OF DIFFERENT FIXATIVES

The immunoreactivity of the alpha and beta isoforms (170 and 180 kDa, respectively) of human DNA topoisomerase II (topo II) toward specific monoclonal antibodies (MoAbs), was investigated in HeLa cells. The ext ent of antigen preservation achieved with the dehydrating fixatives, e thanol and acetone, was compared with that obtained with the cross-lin king agent paraformaldehyde. Binding of each MoAb to the relative isoz yme was detected by indirect immunofluorescence labelling and quantifi ed by flow cytometry. The amount of antigen detectable was calculated by normalizing the mean fluorescence intensity of samples incubated wi th specific MoAbs, to the respective background fluorescence. For both isozymes, acetone provided the highest immunofluorescence/background ratio. A strong reduction in the immunoreactivity of the 180-kDa isofo rm was observed after ethanol, and to a minor extent after paraformald ehyde fixation, while reactivity of the 170-kDa isoform was not signif icantly affected. Cell cycle distribution of each protein was assessed by dual-parameter analysis of immunofluorescence vs, DNA content. No evident difference in the cell cycle distribution of each isozyme was found with each fixation protocol. At the morphological level, the pat tern of immunofluorescence showed that the localization of each isozym e was very similar for all these fixatives, These results confirm the great sensitivity of the 180-kDa isoform to degradation, and indicate that fixation has to be carefully chosen in order to determine the rel ative amount of topo II isoforms with immunocytometric techniques.