Hyperthermia classic article commentary: ‘Re-induction of hsp70 synthesis: An assay for thermotolerance’ by Gloria C. Li and Johnson Y. Mak,International Journal of Hyperthermia1989;5:389-403

Abstract

Of the many heat shock proteins (HSPs), hsp70 appears to correlate best with heat resistance, either permanent or transient. We have investigated various approaches to quantify the concentration of hsp70, and examined the relationship between hsp70 and cells’ thermal sensitivity during the development and decay of thermotolerance in model systems. Specifically, experiments were performed to determine the possibility of using the rate of synthesis of hsp70 after a second test heat shock to predict the kinetics of thermotolerance in tumor cells in vitro and in animal tumor models. We found that the cells’ ability to re-initiate hsp70 synthesis in response to the test heat shock inversely correlated with retained thermotolerance. These data suggest the level of hsp70 in thermotolerant cells regulates the rate of synthesis of additional hsp70 in response to the subsequent heat challenge. Furthermore, the results showed that the rate of re-induction of hsp70 synthesis after a test heat shock can be used as a rapid measure of retained thermotolerance. This study suggests an approach for quantifying the level of retained thermotolerance during fractionated hyperthermia.

• Hyperthermia
• HSP70
• thermotolerance
• in vivo

One of the most interesting aspects of thermal biology is the response of heated cells to subsequent heat challenges. Mammalian cells, when exposed to a non-lethal heat shock, have the ability to acquire a transient resistance to subsequent exposures at elevated temperature, a phenomenon termed thermotolerance 1,2. Thermotolerance has been observed in a variety of organisms and cells, including mammalian cell lines, tumors and normal tissues 3–20. Initially, the mechanism for the development of thermotolerance was not well understood, but emerging experimental evidence suggested that protein synthesis may play a role in its manifestation 21. On the molecular level, heat shock activates a specific set of genes, so called heat shock genes, and results in the preferential synthesis of heat shock proteins 5,18–20. The heat shock response, specifically the regulation, expression and functions of heat shock proteins, has been extensively studied in the past decades and has attracted the attention of a wide spectrum of investigators ranging from molecular and cell biologists to radiation, hyperthermia and medical oncologists 5,18–20. There is much data supporting the hypothesis that heat shock proteins, and particularly the 70 kDa isoform hsp70, play important roles in modulating cellular response to heat shock, and are involved in the development of thermotolerance 22–25.

In the treatment of cancer, hyperthermia has been shown to improve the local control of superficial tumors when combined with radiotherapy 26. However, the optimum periodicity of hyperthermia treatments was uncertain because of the potential influence of thermotolerance. Studies examining the kinetics of thermotolerance have been performed in rodent model systems, both in vitro and in vivo, and using both normal and tumor tissues 3,8,13,15,17,27–29. These studies have shown that thermotolerance is a transient, non-inheritable phenomenon, but that its magnitude and induction kinetics depends on the initial heat dose, and that its decay kinetics is variable and depends on the tissue type. Although similar studies have not been systematically performed in human tumors, the potential importance of thermotolerance in modulating thermal dose and negatively impacting clinical response has been recognized and discussed by several authors 26,30. Therefore, it was highly important to develop an assay which could assess the presence and severity of thermotolerance so that fractionated hyperthermia treatment could be optimized. In other words, the motivation for performing the studies that led to the above referenced article 31 derived from the need to develop a rapid assay to monitor hsp70 and the level of retained thermotolerance during a course of fractionated hyperthermia. The feasibility studies were first performed in vitro 31, then validated in animal model systems 32,33, and finally translated into clinical trials. The series of events associated with this translational and clinical research are briefly described in the following paragraphs.

First, in vitro experiments were performed in Li's laboratory to examine the possibility of using the rate of synthesis of hsp70 after a second test heat shock as an approach for quantifying the level of retained thermotolerance during fractionated hyperthermia. Specifically, using a murine tumor (SQ-1) and a human tumor cell line (HCT-8), Li and Mak studied the relationship between the retained thermotolerance after fractionated heat doses and the cells’ ability to re-initiate synthesis of hsp70 in response to an additional test heat dose in vitro 31. The working hypotheses behind this study are: (1) when tolerance is at its maximum, the cells’ ability to re-initiate synthesis of hsp70 in response to a second heat shock is less than that of control cells; and (2) when tolerance gradually decays, the cells’ ability to re-initiate synthesis of hsp70 in response to a second test heat shock will return to that of control cells. The hypotheses were first tested in vitro. Monolayers of cells were exposed to a first heat treatment (e.g., 41°C for 4 h) and then incubated at 37°C for 0–72 h. At various times after the first heat treatment, cells were either challenged with a 45°C, 45-min heat shock to assess the residual thermotolerance by colony formation assay, or labeled with ³⁵S-methionine before or after an additional test heat dose (e.g., 43.5°C, 15 min). The experimental results showed that the cells’ ability to re-initiate hsp70 synthesis in response to heat shock inversely correlated with retained thermotolerance, suggesting that the level of hsp70 in thermotolerant cells regulates the rate of synthesis of additional hsp70 in response to the subsequent heat challenge. Furthermore, the results showed that the rate of re-induction of hsp70 synthesis after a test heat shock can be used as a rapid measure of retained thermotolerance.

To examine the validity of this approach in vivo, Li evaluated the relationship between the retained thermotolerance and the re-initiation of hsp70 synthesis in a murine squamous cell carcinoma (SCC VII/SF) animal model system after fractionated heat doses 32,33. The tumors implanted in the flanks of the C3H mice were first exposed to elevated temperatures (41°C, 60 min). After the first heat treatment in situ, some of the tumors were excised immediately, and a single-cell suspension was made. The other mice with preheated flank tumors were left undisturbed for various times up to 96 h before being killed. The tumors were then excised and single-cell suspensions prepared. Cells were either challenged with a 45°C treatment to assess the residual thermotolerance by colony-formation assay, or labelled with ³⁵S-methionine before or after an additional test heat dose in vitro (e.g., 43°C for 15 min). Similar to the in vitro finding, Li's laboratory found that in the murine tumor model the tumor cells’ ability to re-initiate hsp70 synthesis inversely correlated with the retained thermotolerance 32,33. Therefore, the determination of the relative rate of re-induction of hsp70 in response to a second heat shock may be a rapid and reliable assay to predict the retained thermotolerance during fractionated hyperthermia.

While the motivation for performing the studies described above was clear, it was uncertain as to whether the technique, though validated in pre-clinical studies, would be translated into clinical implementation. Specifically, would this rapid assay to monitor hsp70 and the level of retained thermotolerance be applied clinically during a course of fractionated hyperthermia? Thus, it was tremendously gratifying when the UCSF hyperthermia clinical team led by Phillips, Sneed and Marquez undertook the study to determine if hsp70 could be used as an assay to predict the presence of retained thermotolerance in human tumors undergoing clinical hyperthermia treatment. Specifically, Marquez et al. 34 performed a clinical study to determine if the re-initiation of hsp70 synthesis could be used as an assay to predict the presence of retained thermotolerance in human tumors. Tissue samples were obtained from patients undergoing hyperthermia and assayed for hsp70 synthesis. Eight patients with advanced, persistent or recurrent malignant tumors had open-ended thermometry catheters placed into the lesion being heated. Through these catheters, tissue samples were obtained using the fine-needle aspiration technique. Attempts were made to obtain samples before and after the first three heat treatments. Some samples were labeled immediately with ³⁵S-methionine at 37°C for 4–8 h, others were given a test heat dose in vitro and then labeled. The protein synthesis profile was analyzed by gel electrophoresis and autoradiography. Their study showed that the baseline ratios of hsp70 were generally <1, indicating the low level of constitutive synthesis of hsp70 without heating. When the 72- or 120-h post-heat-shocked samples were challenged with a second heat stress in vitro, and the hsp70 ratios measured, all of the specimens showed an increase in the hsp70 ratio following the second heat challenge, indicating that thermotolerance had decayed.

The molecular rationale behind this approach has slowly emerged over the years since we developed this assay. Hsp70 expression has been shown to depend on a temperature-dependent transcription factor – heat shock factor 1 (HSF1) 35,36. The properties of this factor suggest an elegant and quite simple mechanism that underlies our assay. HSF1 turns out to be regulated by feedback control; its own products, the HSPs inhibit its activity 37,38. Thus the elevated levels of HSP in thermotolerant cells feed back on the synthesis mechanism and prevent further HSP expression. However, when HSP levels fade away during recovery from heat shock, HSF1 regains its ability to respond to heat and ‘re-induction of hsp70 synthesis’ occurs. Our assay is thus a highly sensitive assay for the activity state of HSF1 and the levels of free hsp70 in cells. To further elucidate the function and its participation as a transcriptional activator of hsp70 under normal or thermal stress conditions in vitro and in vivo, HSF1-deficient mice and cells were generated 39. These studies showed that heat-induced hsp70 expression in mouse tissues is entirely controlled by HSF1 and that cells derived from hsf1^−/− mice lack the ability to develop thermotolerance 39.

The above described an interesting journey. It began with basic scientific investigation of a phenomenon, progressed to the design of studies to characterize it and to understand its underlying molecular mechanism, to the development of techniques for its quantification, and finally to translational and clinical research, i.e., applying what was developed in the laboratory to the clinic. It may have been serendipity and the alignment of stars, but regardless, the requisite components have to come together to the same place at the same time, i.e., scientists and clinicians who share common interests and work well together to bridge the laboratory and the clinic. From these endeavors we learned the importance of combining basic and clinical research in advancing cancer care. In the laboratory, both in vitro and in vivo studies were required, to demonstrate the tumor cells’ ability to re-initiate hsp70 synthesis inversely correlated with the retained thermotolerance. This showed that determining the relative rate of re-induction of hsp70 in response to a second heat shock may constitute a rapid and reliable assay to predict the retained thermotolerance during fractionated hyperthermia. To translate this to a clinical setting, however, the test must be highly sensitive, i.e., requiring only a modest amount of tumor tissue that is obtainable with fine needle aspiration. In the clinical study it was demonstrated that that it was indeed possible to obtain tissue specimens from hyperthermia patients sequentially and in a safe and practical manner, and that the rate of hsp70 synthesis can be measured in a variety of human tumors. Furthermore, the persistence of thermotolerance in the clinical setting can be shown by the inability to re-induce hsp70 synthesis.

In conclusion, we are gratified to be able to provide an example of the collaborative effort between laboratory and clinical scientists that resulted in improving our understanding of both the scientific aspect and the clinical application of hyperthermia for the treatment of human cancer.

Declaration of interest: The authors state that there are no actual or potential conflicts of interest regarding the contents of this manuscript. The authors alone are responsible for the content and writing of the paper.