IN-SITU STAINING FOR POLY(ADP-RIBOSE) POLYMERASE-ACTIVITY USING AN NAD ANALOG

Poly(ADP-ribose) polymerase (PARP) is a highly abundant nuclear enzyme which metabolizes NAD, in response to DNA strand breakage, to produce chains of poly(ADP-ribose) attached to nuclear proteins. PARP activat ion has been implicated in ischemia/reperfusion injury, but its biolog ical significance is not fully understood. We have modified an existin g in situ method for detection of PARP activity by using an NAD analog ue in which adenine is modified by an ''etheno'' (vinyl) bridge. Ethen o-NAD serves as a PARP substrate in an initial enzymatic reaction; a s pecific antibody to ethenoadenosine is then used in an immunohistochem ical reaction to detect the production of modified poly(ADP-ribose). T he method produces strong and specific labeling of nuclei in which PAR P has been activated, i.e., those in which DNA strand breaks have been produced, and the results can be analyzed by microscopy, flow cytomet ry, or colorimetry. The method is applicable to cultured cells in seve ral formats and to frozen tissue sections. The particular characterist ics of the new method may assist in future in situ studies of PARP act ivation.