Thermotolerance as a possible cause of the critical temperature at 43 degrees in mammalian cells.

Most mammalian cell lines appear to have a critical temperature near 43 degrees in their response to hyperthermia. This critical temperature is usually defined as the temperature at which a break occurs in an Arrhenius plot of the rate of cell killing. Below this temperature, thermotolerance (the appearance of a more heat-resistant subpopulation during the survival curve) is also observed. This critical temperature may indicate a real change in the cellular response to heat or may be due to thermotolerance. For example, an apparent (measured) increase in the Do relative to the actual Do of the normal or sensitive population can be due to the following: (a) the presence of a normal (or sensitive) subpopulation and a thermotolerant subpopulation: (b) the induction with time of thermotolerance at a hyperthermic temperature in a defined subpopulation; or (c) the induction of thermotolerance with time in the surviving population. Thus, the critical temperature at 43 degrees may be an artifact due to this increase in the measured Do instead of being due to a change in the mode of inactivation of cells at that temperature. If this is true, the 43 degrees critical temperature should be considered as the maximum temperature at which thermotolerance can be induced and not as an infection point indicating two modes of cell killing.

Membrane lipid fluidity as rate limiting in the concanavalin A-mediated agglutination of pyBHK cells.

The initial rate of concanavalin A-mediated agglutination of polyoma transformed Baby Hamster Kidney (pyBHK) cells follows Arrhenius kinetics. There is a smooth decrease in the agglutination rate from 37 degrees C to 22 degrees C with an activation energy of 11.8 +/- 0.2 kcal/mol in this region. There is a sharp decrease in agglutination rate below 22 degrees C. The addition of 0.1 mM 1,3-di-tert-2-hydroxyl-5-methylbenzene, a lipid perturber, increases the agglutination rate by a factor of two and increases the membrane lipid fluidity as determined by the spin label method. The rotational correlation time of the spin label 2N14 (2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide) was measured. The sum of the enthalpy of activation of rotational diffusion and the enthalpy of activation of translational diffusion is very nearly equal to the enthalpy of activation of agglutination. This is consistent with the rate limiting step of agglutination being receptor diffusion, which is probably limited in pyBHK cells by membrane lipid fluidity.

Site of freeze-thaw damage and cryoprotection by amino acids of the calcium ATPase of sarcoplasmic reticulum.

The Ca2+,Mg(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) is irreversibly inactivated by a freeze-thaw (FT) cycle. The membrane does not become more permeable to calcium after a FT cycle, suggesting that the reduced uptake is due to damage to the Ca2+,Mg(2+)-ATPase. Several amino acids, in addition to standard cryoprotectants provide good protection of calcium uptake against FT damage. The amount of protection given by the amino acids is generally inversely proportional to a measure of hydrophobicity, the mean fractional area loss upon incorporation in globular proteins of the amino acid side chain. Unlike the case for cells, glutamine and dimethyl sulfoxide do not act independently as cryoprotectants for SR calcium ATPase. When the protein is exposed to multiple FT cycles, the amount of inactivation is exponentially proportional to the number of FT cycles. This is true for both protected and unprotected samples. Some SR vesicles fuse during FT. Fusion of vesicles cannot account for the observed inactivation of the enzyme. Fluorescence studies, using intrinsic tryptophan and extrinsic FITC and NCD-4, suggest that FT does not damage the transmembrane region of the Ca2+,Mg(2+)-ATPase or the calcium binding sites, but only the mechanism coupling ATPase activity to calcium translocation. Differential scanning calorimetry (DSC) studies suggest that this region comprises less than 15% of the whole enzyme.

The effects of cotyledon senescence on the composition and physical properties of membrane lipid.

The phospholipid content of rough and smooth microsomal fractions from cotyledons of germinating bean declines as the tissue becomes senescent. Both types of membrane contain comparable proportions of three major phospholipids, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol, which collectively comprise about 90% of the total. This proportionality does not change appreciably during senescence. Only small quantities of lysophosphatides were noted at all stages of senescence. The unsaturated:saturated fatty acid ratio for total extracted lipid declined only slightly in both membrane systems, but pronounced differences in this ratio were observed among the major phospholipids of the membranes. The most striking alteration in lipid composition with advancing senescence was an increase in the sterol:phospholipid ratio; this rose by about 50% for rough microsomes and 400% for smooth microsomes. For both types of membrane the patterns of change in this ratio correlated with previously reported changes in bulk lipid transition temperature, suggesting that the increase in sterol level may contribute to changes in phase behaviour of the membranes during senescence. Arrhenius plots of rotational correlation times for the electron spin label 2,2-dimethyl-5-dodecyl-5-methyloxazolidine-N-oxide (2N14) partitioned into the membrane lipid showed an increase in viscosity with advancing senescence and a corresponding increase in activation energy for both types of membrane. These changes in activation energy and viscosity correlated closely with the increase in sterol:phospholipid ratio. However, no phase transitions were detectable between temperatures of 2 and 55 degrees C despite the fact that transitions from a lipid-crystalline to gel state are detectable within this temperature range by wide angle X-ray diffraction.

X-ray sensitivity of synchronized Chinese hamster cells irradiated during hypoxia.

Synchronized Chinese hamster cells were irradiated in air and in nitrogen at various points in the cell cycle. The irradiations were carried out after flushing with air or nitrogen with the medium removed from the mono-layer of cells. Under these conditions the dose-modifying factor, or oxygen enhancement ratio, was between 2.0 and 2.3 for survival in asynchronous cells. The variation in x-ray sensitivity evident as the cell progresses through its cycle was not differentially affected by its state of oxygenation at the time of irradiation. The x-ray age-response curves for irradiation in air and in nitrogen were similar at each point, except for the dose-modifying factor. This was true not only for the cells of a normal short generation time (10 hours) subline of the V79 line but also for a longer generation time (with longer GC period) subline derived from a "small colony". The variation in radiosensitivity as the cell progresses through its cycle must therefore be due to factors other than change in oxygen tension within the cell. The fact that the same variation in x-ray sensitivity with age exists for hypoxic cells as for well-oxygenated cells has a bearing on the radiotherapy of tumors which contain cells at low oxygen tensions.

Characterization and radiation response of a heat-resistant variant of V79 cells.

A thermoresistant variant of the established cell line V79-S171-W1 was isolated after treatment with nitrosoguanidine and repeated heat treatments at 42.6 to 43 degrees C, and showed an enhanced ability to survive at 42.6, 43.5, and 44.5 degrees C. The rates of inactivation of the normal and heat-resistant lines differed by approximately a factor of 2 over this temperature range. This level of thermoresistance was stable for the first 80 doublings, but was lost by 120 doublings. This may have been due to a reversion to the normal V79 line since there was no continuous selection pressure and the thermoresistant variant, which was designated at HR7, had a longer average doubling time. Transient thermotolerance was induced in both the V79 and HR7 cells by a 10-min exposure to 44.5 degrees C. After 3 hr incubation at 37 degrees C, both cell lines had an identical sensitivity to further exposure to 44.5 degrees C. Thus the long-term thermoresistance of the HR7 cells may be due to a permanent induction of a low level of thermotolerance. The (ionizing) radiation survival curves and the ability to repair sublethal radiation damage were identical for the thermoresistant variant and the parent cell line.

Protein degradation in CHL V79 cells during and after exposure to 43 degrees C.

Cellular protein degradation during and following hyperthermia should be altered due to increased enzymatic activity at elevated temperatures, inhibition of protein synthesis, and denaturation of proteins. We have previously demonstrated by differential scanning calorimetry that approximately 1-2% of total CHL V79 cellular protein denatures during a 10- to 15-min exposure to 43 degrees C (J. E. Lepock et al., J. Cell. Physiol. 137, 14-24 (1988)). Proteolysis was measured during and after exposure to 43 degrees C. The decay curves of the degradation of [3H]Leu-labeled proteins are fit well by a double exponential; however, each component is the sum of the decay curves of a large number of proteins, probably with a distribution of rates of degradation. At 37 degrees C a fast-decaying component (T1/2 congruent to 1.3 h), representing short-term proteins, and a slow-decaying component (T1/2 congruent to 50 h), representing long-term proteins, are observed. At 43 degrees C the rate of degradation of the fast-decaying component is stimulated three- to fivefold (to T1/2 = 0.27-0.45 h). After return to 37 degrees C, the rate of degradation of the slow-decaying component is depressed twofold (to T1/2 = 109-141 h). The period of depression is dose dependent (i.e., time at 43 degrees C) and recovers at approximately the same time as resumption of protein synthesis and growth. Overall stimulation of degradation lasts for approximately 15 min at 43 degrees C and, coupled with an inhibition of synthesis, leads to the loss of at least a small percentage of total cellular protein. It is likely that the initial stimulated degradation is in part due to increased substrate in the form of denatured protein, further supporting the denaturation of proteins during hyperthermia.

Effects of pre- and post-thaw cell-to-cell contact and trypsin on survival of freeze-thaw damaged mammalian cells.

When multicellular spheroids, which simulate small bits of tissue, are exposed to a freeze-thaw (FT) cycle, the survival of the individual cells in the spheroid is higher if the cells of the spheroid are trypsinized and plated as single cells immediately after thawing than if the spheroid is allowed to remain intact for 4 hr and then trypsinized for plating. The results imply either that cell-to-cell contact inhibits repair of potentially lethal damage (PLD) or that accumulation of additional lethal or sublethal damage during the post-thaw period for cells in contact is taking place. Pre- and post-FT trypsinization of single cells indicate that trypsin does not enhance repair of PLD caused by a FT cycle.

Protective effects of amino acids against freeze-thaw damage in mammalian cells.

Amino acids were tested for their effectiveness as cryoprotectants. From the results of this study, the mean fractional area loss of amino acid residues upon incorporation in globular proteins, a measure of hydrophobicity, is generally inversely proportional to the freeze-thaw protection by these free amino acids. However, the pattern of protection ("fingerprint") of cells by various amino acids is different from that of the enzymes liver alcohol dehydrogenase and calcium ATPase of the sarcoplasmic reticulum. Furthermore, unlike the case with these enzymes, for cells glutamine is the best cryoprotective agent of the amino acids tested.

Factors influencing survival of mammalian cells exposed to hypothermia. VI. Effects of prehypothermic hypoxia followed by aerobic or hypoxic storage at various hypothermic temperatures.

The Arrhenius plot of inactivation (killing) rates of V-79 Chinese hamster cells exposed to hypothermia in air-equilibrated (aerobic) medium contains a break at about 8 degrees C, which corresponds to the minimum inactivation rate, implying that there are distinct hypothermic damage mechanisms above (range I, 8 to 25 degrees C) and below (range II, 0 to 8 degrees C) 8 degrees C. Prehypothermic hypoxia (PHH) for 75 min at room temperature sensitizes cells to subsequent aerobic hypothermia at both 5 and 10 degrees C (range II and I). However, PHH followed by severe hypoxia (0.03 microM oxygen in the medium) protected cells during 10 degrees C (range I) storage by increasing the shoulder, but not the slope, of the cell survival curve compared to the PHH plus 10 degrees C aerobic hypothermia case. On the other hand, PHH plus severe hypoxia during 5 degrees C storage (range II) protected cells by decreasing the slope, but not the shoulder, of the cell survival curve compared to the PHH plus 5 degrees C aerobic hypothermia case. Furthermore, PHH plus severe hypoxia during 5 degrees C storage was not significantly worse than aerobic storage without PHH at 5 degrees C. With or without severe hypoxia, 10 degrees C storage is preferable to 5 degrees C storage in this cell line. Extrapolated to organ storage, the results may imply that if warm ischemia (PHH) has occurred, subsequent hypoxic hypothermic perfusion storage may be preferable to aerobic hypothermic perfusion storage.

The radiation response of cultured mammalian V79-S171 cells exposed to a wide concentration range of sulphate salt solutions.

The radiation response of Chinese hamster cells (V79) exposed to a wide concentration range of Li2SO4, Na2SO4 or K2SO4 has been examined and compared with the radiation response of cells treated in an identical manner with LiCl, NaCl, or KCl solutions. At hypotonic salt concentrations, cells were radiosensitized by both the chloride and sulphate salts. At high salt concentrations, approximately greater than 0.9 M, a radioprotective effect was observed with both chloride and sulphate salts. At intermediate salt concentrations from about 0.2 to 0.9 M, the cells that were treated with the sulphate salt solutions were radioprotected; cells treated with chloride salt solutions were radiosensitized. The difference in radiation response was attributed to the difference in anions for the two types of salts used.