APPARENT INHIBITION OF APOPTOSIS BY POLYAMINES AND AMINOTHIOLS IN DNAFRAGMENTATION ASSAYS IS ARTIFACTUAL

We report that, in commonly used DNA fragmentation assays, polyamines and the radioprotective aminothiol WR1065 artifactually depress the de gree of spontaneous or induced cellular apoptosis in two distinct ways , Firstly, in assays utilizing Hoechst 33258 dye to measure apoptotic DNA, both amines quench the fluorescence of low affinity dye/DNA bindi ng resulting in preferential underestimation of DNA in the apoptotic D NA fraction and a resultant underestimation of the extent of DNA fragm entation, Secondly, these amines can cause aggregation and condensatio n of apoptotic DNA, causing anomalous sedimentation under conditions u niversally employed to separate apoptotic from intact DNA in cell lysa tes, This anomalous sedimentation of apoptotic DNA leads to underestim ation of fragmentation in fluorescence assays as well as in agarose ge l assays,We demonstrate that manipulation of the ionic strength of the lysis buffer or lowering the dye concentration ameliorates the effect s of dye quenching in the Hoechst assay, Alternatively, this effect is alleviated by substituting DAPI for Hoechst in this assay, Inclusion of a polyanion to the lysis buffer antagonizes the condensation and an omalous sedimentation of apoptotic DNA observed regardless of which dy e is used in the assay, These studies call into question the validity of previously reported studies suggesting that polyamines and the radi oprotective aminothiol, WR1065, inherently suppress the apoptotic proc ess and underline the need to consider alternative endpoints of apopto sis such as morphology in order to assess effects on cellular apoptosi s of exogenously added agents, particularly di- or polycations.