Defining the process of cellular injury during freezing, at the molecular level, is important for cryosurgical applications. This work shows changes to both membrane lipids and protein structures within AT-1 Dunning prostate tumor cells after a freezing stress which induced extreme injury and cell death. Cells were frozen in an uncontrolled fashion to -20 or -80°C. Freezing resulted in an increase in the gel to liquid crystalline phase transition temperature (Tm) of the cellular membranes and an increase in the temperature range over which the transition occurred, as determined by Fourier transform infrared spectroscopy (FTIR). Thin layer chromatography (TLC) analysis of total lipid extracts showed free fatty acids (FFA) in the frozen samples, indicating a change in the lipid composition. The final freezing temperature had no effect on the thermotropic response of the membranes or on the FFA content of the lipid fraction. The overall protein secondary structure as determined by FTIR showed only slight changes after freezing to -20°C, in contrast to a strong and apparently irreversible denaturation after freezing to -80°C. Taken together, these results suggest that the decrease in viability between control and frozen cells can be correlated with small changes in the membrane lipid composition and membrane fluidity. In addition, loss of cell viability is associated with massive protein denaturation as observed in cells frozen to -80°C, which was not observed in samples frozen to -20°C.
Bibliographical noteFunding Information:
This work was supported by Grants HL57810 and HL61204 from NIH, and (N660001-00-C-8048) from DARPA. J.C.B. was supported by a semester leave program from the University of Minnesota and NIH R29 CA 75284-01.
- Freeze/thaw injury
- Protein denaturation