Induction of Heat Shock Protein Synthesis in Murine Tumors during the Development of Thermotolerance1

The function of one or more heat shock proteins (HSPs) may be to confer protection of cells against thermal damage. We examined the induction kinetics of thermotolerance and the synthesis of HSPs in murine tumor models. Squamous cell carcinomas (SCC VII/SF) or radiation-induced fibrosarcomas (RIF) were implanted in the flanks of C3H mice. These flank tumors were first exposed to an elevated temperature (41°45°C)for a fixed duration, for example, 43°Cfor 15 min. Some of the tumors were excised immediately, and tumor cell suspen sions were made. The other mice with tumors were returned to the cages and left undisturbed for various times up to 72 h before being sacrificed. Again, tumors were then removed and tumor cell suspensions were prepared. These tumor cells were either challenged with a second heat treatment at 45°Cin vitro or labeled with [^SJmethionine at 37°Cin vitro. The tumor cell survival after the combined heat treatments was measured using the in vitro cloning assay. The cellular proteins were analyzed by oneor two-dimensional gel electrophoresis. We found that mild heat shock induced thermotolerance in murine tumors, a result consistent with those of others. The kinetics of induction and decay of thermotolerance depended on the temperature and duration of the priming treatment. Mild heat shock also enhanced the rate of synthesis and accumulation of some HSPs during the development of thermotolerance. For example, after an initial treatment at 43°Cfor 15 min, the rates of synthesis of HSPs with molecular weights 68,000,70,000, and 87,000 were greatly enhanced in SCC VII/SF tumors when compared to unheated controls. Qualitatively similar results were seen with radiationinduced fibrosarcoma tumors. The rate of synthesis of M, 68,000 to 70,000 HSPs reached maximum value (300% of control value) 2 to 4 h after heat shock and decreased to the control value 6 to 24 h later. On the other hand, the rate of synthesis of actin, a major structural cellular protein, remained relatively constant throughout the 72 h of experiments. We then determined the relationship between the synthesis and accumulation of these HSPs and the expression of thermotolerance in murine tumors after a priming heat treatment. The data indicate that the levels of Mr 68,000 to 70,000 HSPs correlate well with thermotolerance. Thus, it appears likely that the measurement of levels of these HSPs can be used as an assay to determine the degree of thermotolerance in tissues during fractionated hyperthermia as applied in the clinic. INTRODUCTION Mammalian cells, when exposed to a nonlethal heat treatment, have the ability to acquire a transient resistance to a subsequent heat challenge. This phenomenon has been termed thermotol erance (1-3). The induction of thermotolerance, its development, and its subsequent decay, both in vitro and in vivo, have been studied by many investigators using various cell lines (1, 3, 45), various rodent tumors (6-12) and normal tissues (13-19). Even though different endpoints were used in these studies and the experimental designs sometimes did not allow for differentia tion between recovery from the heat-induced sublethal damage and development of thermotolerance, the results from the in vivo studies were qualitatively similar to those from in vitro studies. The molecular mechanisms of thermotolerance are not well understood. It has been suggested that HSPs3 may play a role that is related to heat-induced thermal protection in Drosophila and yeast (20, 21). HSPs are a family of proteins the synthesis of which is either induced or enhanced by heat shock or other environmental stresses (22). This group of proteins is induced in a wide variety of biological systems ranging from Drosophila to yeast and mammalian cells (20-27). Many investigators, in the past few years, have performed experiments to determine the relationship between thermotolerance and HSP synthesis using various mammalian cell lines. So far, most studies show a good temporal relationship between the development of thermotoler ance and enhanced synthesis of HSPs (25,26,28-30); although there are reports that suggest that correlation is not universal (30, 31 ). Recently, we have examined the quantitative relation ship between cell survivals after a 45°C,45-min heat treatment and the levels of HSPs in transient thermotolerant Chinese hamster fibroblasts and in their stable heat-resistant variants (32). These results show a good correlation between the levels of HSP with a molecular weight around 70,000 (HSP 70) and cells' thermal sensitivity. During decay of tolerance when the concentration of HSP 70 decreases, cells also become much more sensitive to thermal stress. These data indicate that the level of HSP 70 appears to be a good predictor of thermal response in vitro. However, no similar data are available from animal tumor models. We report here on experiments designed to study the effects of hyperthermia (41 °-45°C)on the induction of thermotolerance and on the profiles of protein synthesis in murine tumor models, a squamous cell carcinoma (SCC VII/SF), and a RIF. Specifically, we examined whether the synthesis of HSPs can be induced or enhanced in murine tumors after a nonlethal heat treatment. We also investigated the kinetics of the development of thermotol erance by doing survival experiments and the time patterns of synthesis of HSPs and other cellular proteins during the devel opment of thermotolerance using gel electrophoresis techniques. Finally, we determined the relationship between HSP synthesis 1This work was supported by NIH Grant CA-31397. 2To whom requests for reprints should be addressed, at Radiation Oncology Research Laboratory, CED-200, Department of Radiation Oncology, University of California, San Francisco, CA 94143. Received 9/7/84; revised 4/3/85; accepted 4/18/85. 3The abbreviations used are: HSP, heat shock protein; HSP 70, heat shock protein of molecular weight 70,000 (other heat shock proteins are similarly desig nated); MEM, minimal essential medium; SCC VII/SF, squamous cell carcinoma; RIF, radiation-induced fibrosarcoma; SDS, sodium dodecyl sulfate. CANCER RESEARCH VOL. 45 AUGUST 1985