APPLICATION OF BIOTIN, DIGOXIGENIN OR FLUORESCEIN CONJUGATED DEOXYNUCLEOTIDES TO LABEL DNA STRAND BREAKS FOR ANALYSIS OF CELL-PROLIFERATIONAND APOPTOSIS USING FLOW-CYTOMETRY

A flow cytometric method has recently been developed using biotinylate d dUTP (b-dUTP) in a reaction catalyzed by terminal deoxynucleotidyl t ransferase (TdT) to identify the endonuclease-induced DNA strand break s occurring during apoptosis. Counterstaining of DNA makes it possible to relate apoptosis to cell cycle position or DNA index. In the prese nt study, we compared this method with one using digoxigenin-conjugate d dUTP (d-dUTP) to label apoptotic cells. The discrimination of apopto tic from nonapoptotic cells was similar when incorporation of d-dUTP w as compared with b-dUTP. Both techniques resulted in a 20-30 fold incr ease in staining of apoptotic over nonapoptotic cells although somewha t less background fluorescence was observed with the d-dUTP. Direct la beling with fluoresceinated dUTP (f-dUTP) was less sensitive in detect ing DNA strand breaks, but had the advantage of simplicity. The princi ple of labeling DNA strand breaks using TdT was also employed to ident ify DNA replicating cells, To this end, the cells were incubated in th e presence of BrdUrd, then exposed to UV light to selectively photolys e DNA containing the incorporated BrdUrd, DNA strand breaks resulting from the photolysis were then labeled with b-dUTP or d-dUTP. This appr oach is an alternative to immunocytochemical detection of BrdUrd incor poration, but unlike the latter does not require prior DNA denaturatio n, thus can be applied when the denaturation step must be avoided. The method was sensitive enough to recognize DNA synthesizing cells that were incubated with BrdUrd for only 5 min, the equivalent of replicati on of less than 1% of the cell's genome. The discrimination between ap optotic vs. BrdUrd incorporating-cells is based on different extractab ility of DNA following cell fixation. This method can be applied to an alyze both cell proliferation (DNA replication) and death (by apoptosi s) in a single measurement.