Freezing induces artificial cleavage of apoptosis-related proteins in human bone marrow cells

The aim of this study was to investigate whether freeze-thawing of freshly isolated human mononuclear bone marrow cells (MNC) influences the integrity of apoptosis-related proteins as determined by immunoblot analyses. Our re sults show that bone marrow is more sensitive to this process than either m yelomonocytoid leukemic P39 or Jurkat T-lymphocyte cell lines. Specifically , bone marrow cells displayed a high level of intrinsic proteolytic activit y in response to a single freeze-thaw cycle, which led to the cleavage of v arious proteins involved in apoptosis cell signaling. This effect was compl etely blocked by the inclusion of broad-spectrum protease inhibitors in the freezing medium and subsequently thawing the cells on ice. Since differenc es in the freezing conditions (- 80 degreesC vs, liquid nitrogen) did not a lter the proteins of interest, we suggest that the thawing process is the c ritical point when proteolytic enzyme activity is elevated. (C) 2000 Elsevi er Science B.V. All rights reserved.