Department of Molecular Biology, Institute for Biological Research Sinisa Stanković, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia.
Cryobiology. 2010 Jun;60(3):271-80. doi: 10.1016/j.cryobiol.2010.01.005. Epub 2010 Feb 2.
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.
虽然冷冻手术越来越被临床所接受，但我们对低温细胞破坏机制的了解仍不完整。虽然人们普遍认为冷冻损伤的细胞会发生坏死，但最近发现了细胞凋亡的参与。我们对低温导致的肝细胞死亡的研究揭示了内切核酸酶 p23 的激活及其与核基质的新的关联。这一发现强烈提示存在一种程序化的细胞死亡过程。通过检查 p23 激活的机制，我们在这里探讨了冷冻坏死细胞死亡的假定顺序。为此，我们从新鲜肝脏中制备了不含 p23 活性的核蛋白，并与从经历冷冻/解冻的肝脏中分离的蛋白部分孵育，这些蛋白部分被认为含有 p23 激活因子。我们观察到 p23 的激活是一个蛋白水解事件的结果，其中组织蛋白酶 D 起主要作用。在体外孵育核和从冷冻/解冻的肝脏中分离的样品后，观察到核蛋白的不同蛋白水解切割模式。虽然这两个过程都诱导了 p23 的激活，但孵育实验产生了凋亡的蛋白水解特征，而整个肝脏的冷冻/解冻导致了典型的坏死 PARP-1 切割产物和完整的 lamin B。作为一种解释，我们提出了一个假设，即在冷冻之后，细胞有可能通过坏死和凋亡机制死亡，这是基于我们的发现，即暴露于低温的细胞的细胞质中同时含有坏死和凋亡的细胞死亡执行者。
