Induction of apoptosis in human colon carcinoma cells HT29 by sublethal cryo-injury: Mediation by cytochrome C release

Cryosurgery is an emerging treatment for human solid tumors, notably colore ctal liver metastasis, Cryosurgical procedures generate a thermal gradient of from at least -50 degreesC at the center of the tumor being treated to a bout 0 degreesC at the periphery. Cell death occurs by necrosis in the cent er, while the peripheral zone of frozen tumor harbors a mix of viable and d ead tissue. In order to understand the mechanisms of cell death and surviva l in this peripheral area at risk for tumor recurrence, we have established an in vitro freezing system that mimics in vivo conditions of sublethal in jury. HT29 colon cancer cells were subjected to freezing temperatures from -6 degreesC to -36 degreesC, thawed at room temperature for 30 min and rewa rmed at 37 degreesC for a period of time. Post-freeze-thaw, cryolytic cells were evaluated by trypan blue exclusive assay. We also identified apoptoti c cells after rewarming by cell shrinkage, nucleic condensation, TUNEL assa y, DNA fragmentation and PARP degradation. The intensity of cryolysis and a poptosis was increased by lowering the freezing temperature. At -36 degrees C, all cells were dead immediately after freeze-thaw. A kinetic analysis of cryo-induced apoptosis showed that the commitment to enter apoptosis occur red right after the freeze-thaw period and lasted less than 8 hr after rewa rming, We further demonstrated that freezing triggers one of the caspase ca scade involved in apoptosis: release of cytochrome c from mitochondria to c ytosol, followed by activation of caspase-9 and degradation of PARP. These results indicate the death of cancer cells under cryo-treatment at subletha l freezing temperature can be attributed 2 different modes, cryolysis as we ll as apoptosis, HT29 cells carrying p53 mutant have very quick response fo r induction of apoptosis by cryo-treatment and contain an intact pathway of caspase cascade. Further studies will address if mechanisms in cells with wild-type p53 will differ. (C) 2001 Wiley-Liss, Inc.