Proteolytic events in cryonecrotic cell death: Proteolytic activation of endonuclease P23

Although cryosurgery is attaining increasing clinical acceptance, our understanding of the mechanisms of cryogenic cell destruction remains incomplete. While it is generally accepted that cryoinjured cells die by necrosis, the involvement of apoptosis was recently shown. Our studies of liver cell death by cryogenic temperature revealed the activation of endonuclease p23 and its de novo association with the nuclear matrix. This finding is strongly suggestive of a programmed-type of cell death process. The presumed order underlying cryonecrotic cell death is addressed here by examining the mechanism of p23 activation. To that end, nuclear proteins that were prepared from fresh liver, which is devoid of p23 activity, were incubated with protein fractions isolated from liver exposed to freezing/thawing that possessed a presumed p23 activation factor. We observed that the activation of p23 was the result of a proteolytic event in which cathepsin D played a major role. Different patterns of proteolytic cleavage of nuclear proteins after in vitro incubation of nuclei and in samples isolated from frozen/thawed liver were observed. Although both processes induced p23 activation, the incubation experiments generated proteolytic hallmarks of apoptosis, while freezing/thawing of whole liver resulted in typical necrotic PARP-1 cleavage products and intact lamin B. As an explanation we offer a hypothesis that after freezing, cells possess the potential to die through necrotic as well as apoptotic mechanisms, based on our finding that the cytosol of cells exposed to cryogenic temperatures contains both necrotic and apoptotic executors of cell death.