Quantitative assessment of bleomycin-induced poly(ADP-ribosyl)ation in human lymphocytes by immunofluorescence and image analysis

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme that is catalyticall y activated by DNA strand interruptions. It catalyses the covalent modifica tion of proteins with ADP-ribose polymers, using NAD(+) as precursor. Here, we have studied the DNA damage-induced formation of poly(ADP-ribose) in in tact human peripheral blood lymphocytes (PBL) by in-situ immunofluorescence detection. The response of PBL to bleomycin (BLM), which is known to induc e DNA single and double strand breaks, was investigated with regard to poly mer formation. For this purpose, a quantitative approach was developed to a ssess more accurately the immunostaining of polymer formation by computeris ed image analysis. As an application of this new method, we have determined the polymer formation following BLM treatment in quiescent human PBL versu s mitogen activated cells. Quiescent human PBL showed a similar basal immun ostaining for the polymer compared to phytohemagglutinin (PHA)-activated ce lls, expressed as relative mean pixel intensity (RMPI) (1.3+/-0.8 and 2.2+/ -0.9, respectively; P<0.3). After BLM treatment. there was a clear-cut enha ncement of polymer immunostaining, with PHA-activated cells showing signifi cantly higher RMPI than non-activated cells (9.2+/-1.4 and 4.2+/-1.0, respe ctively; P<0.005). As expected, in the presence of the ADP-ribosylation inh ibitor 3-aminobenzamide (3-AB), the RMPI of immunostained polymer was decre ased in both quiescent and PHA-activated PBL to 1.2+/-0.7 and 1.5+/-0.9, re spectively. Our findings reveal (i) that mitogen-stimulated, intact lymphoc ytes show enhanced polymer formation following BLM treatment, and (ii) that our new quantitative immunofluorescence assay coupled with computerised im age analysis is reliable and sensitive enough to detect changes in polymer formation rate. (C) 2000 Elsevier Science B.V. All rights reserved.