A series of heat-resistant mutants selected from a murine tumor cell line, RIF-1, display a markedly increased and stable resistance to heat shock. The mutant cell lines were analyzed for differences that may explain their increased resistance. Membrane lipid analysis showed no change in cholesterol content but an increase in the proportion of saturated fatty acids in the phospholipid fraction. Two-dimensional gel analysis revealed a generally increased constitutive synthesis of several major heat shock proteins (HSP), including HSP90, 68, 60, and 28. In addition, a new protein in the 70-kilodalton region is present in the resistant lines.The new protein has a lower isoelectric point than the constitutive HSP70 does, is only weakly induced by heat shock, and is immunologically cross-reactive with other members of the HSP70 family. After heat shock, the mutants display increases in HSP similar to those seen in the wild-type cells and they develop further transient tolerance to heat. Analysis of these mutants may help in understanding the function of HSP, both in normal growth and after heat shock.The heat shock proteins (HSP), or stress proteins, are synthesized in response to a variety of stresses apart from heat, including ethanol, arsenite, transition metals, release from anoxia, and mutagens (31). The molecular basis for this transient alteration in gene expression is the subject of intensive investigation. In tissue culture and in mouse tumor systems, the appearance of HSP is accompanied by the development of thermotolerance or transient resistance to subsequent heating. The close temporal matching of the appearance and decay of thermotolerance and HSP both in tissue culture (25,30,48) and in vivo (29) has led to the hypothesis that HSP function to protect cells from heat damage. However, an increasing number of examples of thermotolerance in the apparent absence of elevated levels of HSP (11,20,41,50) has raised some doubts about this proposed function. At the very least, these results suggest that cells have alternative methods for developing thermotolerance that do not involve the HSP.One possible alternative mechanism that has received considerable attention is an alteration of the cellular membranes to a more thermostable form (7). Heat increases the fluidity of membranes (28), leading to loss of their selective permeability and possibly to cell death. The importance of the plasma membrane in heat damage is indicated in the synergistic interaction of the membrane-active drug, amphotericin B, with hyperthermia (17) and the enhanced uptake of drugs such as adriamycin in cells held at elevated temperatures (43 to 45°C) (43). An aggregation of membrane proteins in the plasma membrane occurs immediately after heat shock in cells destined to die (42). Several integral membrane proteins, such as the ouabain-sensitive Na+, K' -ATPase (1, 8), the Na+-dependent amino acid transport protein (34), the epidermal growth factor receptor (35), and the insulin receptor (9) all show diminished activity after hyperthermic...

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An enzyme-linked immunosorbent assay (ELISA) for measurement of levels of heat shock proteins 73 and 72 (HSP73/72) in cultured cells and tissues is described. The assay involves detection of HSP73/72 in cell homogenates in 96-well plates using a specific monoclonal antibody. The assay has been used to explore the relationship between the amount of HSP73/72 in a cell and its response to heat shock, both before and after the development of thermotolerance. Six mammalian cell lines with differing responses to heat were characterized with respect to their response to heat treatments at 44 degrees C and concentrations of HSP73/72. Contrary to the widely expressed idea that the amount of HSP73/72 dictates the degree of heat resistance, no positive correlation between levels of HSP73/72 and heat resistance was found for the six lines tested here: if one particular line, a mutant selected for heat resistance, was excluded from the analysis, there was a negative correlation between HSP73/72 levels and heat resistance. A different result was, however, obtained when thermotolerant (transiently resistant) cells were compared to control cells. Here, we found a good correlation between the extent of thermotolerance and the amount of HSP73/72, suggesting that an increase in HSP73/72 level is important for the development of thermotolerance. The validity of the ELISA technique was checked using a second method for quantifying levels of HSP73/72. This involved uniform radiolabeling of cellular proteins, separation on two-dimensional gels and radioscanning to quantify radioactivity in each protein. The second technique is more powerful in that different isoforms of HSP73/72 can be distinguished, but it is more difficult to perform, is more labour intensive and requires an expensive device for gel scanning. The results using the second technique agreed well with those from the immunoassay and indicated that the level of the highly inducible HSP72 correlated best with the extent of thermotolerance.

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