Cellular responses to hyperthermia (40–46°C): Cell killing and molecular events

Abstract

The goal of this review is to provide a brief introduction to the effects of hyperthermia on cellular structures and physiology. The review focuses on the effects of hyperthermia thought to contribute to the enhancement of cancer therapy namely the mechanisms of cell killing and the sensitization of cells to ionizing radiation or chemotherapeutic agents. Specifically the review addresses four topics: hyperthermia induced cell killing, mathematical models of cell killing, mechanisms of thermal effects in the hyperthermia temperature range and effects on proteins that contribute to resistance to other stresses, i.e., DNA damage. Hyperthermia has significant effects on proteins including unfolding, exposing hydrophobic groups, and aggregation with proteins not directly altered by hyperthermia. Protein aggregation has effects throughout the cell but has a significant impact within the nucleus. Changes in the associations of nuclear proteins particularly those involved in DNA replication cause the stalling of DNA replication forks and lead to the induction of DNA damage such as double strand breaks. It has long been recognized that heat has effects on plasma membrane protein distribution alters the permeability of plasma membranes resulting in a calcium spike and disrupts the mitochondrial membrane potential resulting in the change in the redox status of cells. These effects contribute to the protein unfolding effects of hyperthermia and contribute to effects observed in the nucleus. Thus heat effects on multiple cellular targets can be integrated through global effects on protein folding to affect specific end points such as cell killing and sensitization to additional stresses.

• Hyperthermia
• cell killing
• protein unfolding
• DNA damage

Introduction

The goal of this review is to introduce the reader to the effects of hyperthermia on cellular structures and physiology. Since this field is too broad for a 30-minute talk or a review article of modest length, I have chosen to highlight several aspects of the effects of hyperthermia, focusing on those that contribute to the enhancement of cancer therapy, namely the mechanisms of cell killing and the sensitization of cells to ionizing radiation or chemotherapeutic agents. The potential importance of this subject for cancer treatment has been recently highlighted by Coffey et al. [1]. Specifically, the review addresses four topics: (1) hyperthermia induced cell killing, (2) mathematical models of cell killing, (3) mechanisms of thermal effects in the hyperthermia temperature range and (4) effects on proteins that contribute to resistance to other stresses, for example, DNA damage.

Cell killing

It has been long recognized that hyperthermia in the 40–47°C temperature range kills cells in a reproducible time and temperature dependent manner. Survival curves for temperatures in the 43–47°C range typically show a shoulder with an exponential reduction in clonogenic survival as a function of time at a given temperature [2–4]. In contrast, cell survival curves for temperatures of 42.5°C and below, depending on the cell line, will show a shoulder, an exponential portion of cell killing followed by a plateau in cell killing, due to the development of chronic thermal tolerance. The principal conclusion from these studies is that for hyperthermia, thermal dose is a combination of time and temperature.

Mathematical models of cell killing

Mathematical representation of survival curves can be written by several mathematical treatments including mathematical models. Such models were based on survival curves from radiation. The earliest such equation was based on the multi-target single hit model, resulting in the survival equation: S = 1 − (1 − e^t_T/To)ⁿ, where t_T is time at temperature T and T_o is the activation energy for cell killing at temperature T [5]. In this model the T_o parameter is temperature dependent, and is related to the reciprocal of the absolute temperature by Arrhenius analysis [5]. On the other hand, the parameter, n, did not follow the same function with absolute temperature for all cell lines [6]. An alternate approach was to use a linear quadratic type equation where S = exp (−αt_T − βt_T²). As above, t_T is the time at a given temperature T, while α and β are arbitrary constants. However, α is linear function of the reciprocal of the absolute temperature and β is a linear function of the reciprocal of the absolute temperature squared, according to the Arrhenius analysis [4], [7]. Another model, developed by Mackey et al. [8] utilized the distribution of an arbitrary temperature dependent parameter, which needed to be above a specified threshold for the cell to be clonogenic. This model was able to simulate the development of chronic thermal tolerance and the hypersensitivity of S phase cells, two important aspects of cell killing that were not addressed by the two earlier models. While the Mackey model was based on thermodynamic considerations, evidence for a direct link between the empirical parameters and thermodynamic constants remained elusive. Thus, a cell survival model, completely determined by thermodynamics and the biological response to heat induced changes in protein folding has not been derived from the first principles and is still a tool that is lacking for the field of hyperthermia. Such a model would be important for any improved definition and quantification of thermal dose.

Mechanisms of thermal effects in the hyperthermia temperature range

In the hyperthermia temperature range (40–47°C), the main effects at the cellular level that are of relevance to cancer therapy are cell killing and those effects that alter the resistance of cells to radiation and/or chemotherapeutic agents. While it is acknowledged that at the tissue, organ and whole body level there are numerous effects that can contribute to the clinical uses of hyperthermia; this review focuses on effects at the cellular level. Thus, we will discuss cell killing and those effects that alter resistance to other cellular stresses.

Prior to discussing the mechanisms of cell death it is important to consider the types of cell death that are induced by hyperthermia. The types of cell death induced by hyperthermia include heat induced apoptosis [9], mitotic catastrophe secondary to alterations in the proteins that support DNA metabolism [10–11]. The type of cell death induced by heat shock is highly cell-type and temperature dependent [12]. In fact, most cell lines die of a combination of death processes.

The goal of hyperthermia research is to find the molecular mechanisms by which heat kills tumour cells or any cells and the mechanisms by which hyperthermia radiosensitizes cells to radiation or chemotherapeutic agents. A major problem is that hyperthermia causes a large number of macromolecular changes and affects functions in all cellular compartments at temperatures above 43°C. Although significantly fewer macromolecular changes occur in the 40.5° to 42°C range, these changes are still numerous and occur in multiple cellular compartments. The challenge is to determine which molecular changes are critical for the relevant endpoint such as tumour cell killing and sensitization to chemo and radiation therapy. By the late 1980s there was a significant body of literature describing heat effects on DNA, proteins, lipids and other cellular components [13]. Information on the effects of hyperthermia on cellular macromolecules has been growing rapidly since that time, making a comprehensive review almost impossible. However, no clear, detailed mechanism for cell killing or sensitization to other stresses has been established. Nevertheless, the main consensus that has emerged is that the mechanism by which hyperthermia kills cells and/or sensitizes cells to radiation involves the unfolding and subsequent aggregation of proteins [14]. Therefore, one approach would be to focus on how heat affects those proteins that contribute to resistance to other stresses.

Effects on proteins that contribute to resistance to other stresses, for example, DNA damage

Hyperthermia effects on proteins include unfolding and exposing hydrophobic groups, and aggregation of unfolded proteins due to the interactions of the exposed hydrophobic groups (Figure 1, panel A). Aggregation of proteins can include proteins not directly altered by hyperthermia, thereby affecting a larger number of proteins than those directly altered by heat shock. Protein aggregation and unfolding will have effects throughout the cell but should have significant impact within the nucleus because of the large number of proteins and the large amount of DNA packed within it. For example, over 2 m of DNA (5 × 10⁹ base pairs) and a protein mass within the nucleus is from 3 to 6 times that of the mass of DNA packed within the nucleus [15–20]. The second reason that protein unfolding within the nucleus can have a significant impact is that the proteins within the nucleus are critical to managing the proper transcription, replication and repair of DNA [17–20]. Therefore disruptions of these proteins and their function(s) should have critical consequences. Specifically, disruption of the proteins involved in DNA replication leads to replication fork stalling and improper processing of nascent DNA fragments, resulting in chromosome aberrations, genomic instability and improper chromosome segregation and cell death [21–23]. Disruption of proteins involved in DNA repair leads to an increase in unrepaired DNA damage. Ultimately unrepaired DNA damage contributes to genomic instability and results in chromosome aberrations.

Figure 1. Mathematical equations for hyperthermia survival curves. A typical hyperthermia survival curve is illustrated in the left panel. Two equations to describe the curve mathematically are shown on the right. While there is no there is no empirical difference between the equations in terms of fitting the data, there is a difference in the number of model parameters that are simple functions of absolute temperature (see text).

It is important to distinguish the effects, on proteins, of temperatures in the hyperthermic range, 40–47°C from the effects of thermal ablation temperatures (48–60°C) on proteins. Temperatures in the hyperthermic range cause proteins to be unfolded, exposing hydrophobic groups, which can then interact forming aggregates (Figure 1). These heat effects on proteins are, in principle, reversible by the presence of molecular chaperones, such as HSP 70 [24–25]. In the case of thermal ablation, severe protein denaturation is occurring and largely these protein changes are irreversible. The main evidence for protein unfolding aggregation comes from the studies by Jim Lepock using scanning calorimetry [26–27] and the model system proteins [28–29]. Using differential scanning calorimetry, it is possible to detect the endothermic reactions within a cell. The endothermic reactions are due to the denaturation or change of state in proteins in the cell. Peaks in the differential calorimetry curves, reflecting protein unfolding can be seen at temperatures in the 45–55°C range, 57–65°C temperature range, 65–75°C, 85°C and 100°C range. Sensitizers to heat shift the differential scanning calorimetry curves to lower temperatures, whereas thermal protectors shift these curves to higher temperatures [30–31]. One interesting point that comes from studying these endothermic transitions is that a hyperthermia time/temperature combination that reduces survival by 10⁻² can occur when ∼10% of the endothermic cellular transitions have occurred [26]. Thus, the lower temperature transitions have a significant impact on cell survival.

Does hyperthermia cause DNA damage?

It has long been recognized that higher temperatures in the 50–60°C range induce DNA damage and denaturation [32–34]. However, early work on DNA damage after hyperthermia in the 40–46°C range, showed that although heat does induce chromosome aberrations in S-phase cells [22], [34], DNA damage could not be detected by direct measurement [36] or when detected could not be attributed to a direct effect on DNA [37]. Thus the effects of hyperthermia on DNA in chromatin have been thought to be not due to direct heat-induced DNA damage, but secondary to heat effects on proteins involved in DNA replication, chromosome segregation and DNA repair [22]. However, this idea has become somewhat controversial in the last year due to a paper reporting the formation of γH2AX foci as a result of hyperthermia and suggesting that these foci were markers for DNA double strand breaks [38]. Since the formation of γ-H2AX foci is more sensitive to radiation dose than the classical DNA damage techniques [39], it was conceivable that these techniques failed to detect DNA damage that was detected as γH2AX foci. To understand this issue it is important to realize that γH2AX is a modification of chromatin that occurs in the vicinity of a DNA double strand break. Therefore, γH2AX foci are, in fact, measuring a phosphorylation change in protein, which, in turn, is thought to reflect the presence of a DNA double strand break. A recent publication [39] showed that heat shock (43°C for 30 or 60 min) induced γH2AX foci but not DNA double strand breaks detectable by pulse field gel electrophoresis. Interestingly, the γH2AX foci formation as the result of heat shock was dependent on the presence of the ATM molecule, whereas in cells lacking ATM, these foci did not occur. In contrast, following ionizing radiation, in which the formation of DNA double strand breaks is well established, γH2AX foci form independently of the presence of the ATM protein [39]. Another striking difference between the γH2AX foci induced by hyperthermia and those induced by ionizing radiation is that those induced by hyperthermia do not support the finding of ⁵³BP1, a molecule that is necessary for the assembly of other proteins required for repair of the double strand break [39]. Interestingly, the γH2AX foci from both hyperthermia and ionizing radiation include the molecule, MDC1 so the conclusion of these studies is that heat induces foci containing γH2AX and MDC1 in the absence of DNA double strand breaks and these foci are inherently different from those which form in response to DNA double strand breaks. However, the exact nature of the differences between ionizing radiation-induced γH2AX foci and those induced by hyperthermia remains to be delineated. From these studies we conclude that hyperthermia does not directly induce DNA damage. However, the fact that heated S-phase cells develop chromosome aberrations suggests that DNA damage (resulting in chromosome aberrations 34,40) could arise secondary to the effects of heat on DNA replication.

Figure 2. Thermal effects on protein folding. Panel A is an illustration of a model for protein unfolding under hyperthermic conditions, in contrast to the conditions that apply for thermal ablation. As part of the evidence for this model, Panel B shows differential scanning calorimetry data. The peaks represent endothermic transitions. (See text for additional evidence.) Panel C shows the integral of all the endothermic transitions. It can be seen that more than 60% of the endothermic cellular transitions occur in the thermal ablation temperature range 60–70°C. In contrast, less than 10% of the transitions have occurred in the hyperthermic range even for temperatures that reduce survival by 10⁻² in less than 15 min [14].

Figure 3. Two disparate heat effects on S-phase progression. Hyperthermia causes a greater delay in the completion of DNA synthesis (Panel B) than in the transition from a type I DNA replication pattern to a type II DNA replication pattern. This differential delay causes mis-timing of DNA replication within S-phase and means that early S and mid S DNA replication factories are synthesizing DNA at the same time indicating a loss of check point control. (From [47], used with permission.)

Effects of hyperthermia on DNA replication and cell cycle progression

It is recognized that heat has numerous affects on DNA replication. The pioneering work of Dewey et al. [34] showed that S-phase cells are hypersensitive to heat induced killing and that this killing correlates with chromosome aberrations [5] [21–22], which, as noted above, are induced when cells are heated in S-phase. At clinically achievable temperatures ≤42.5°C the major component of cell killing will be the killing of S-phase cells [40–42]. For example when cells are continuously heated at 41°C, the prominent observed effect is an S-phase block, after 24 h at 41.5°C, 80% of the cells occur in S-phase and cells will enter mitosis prior to the completion of DNA replication and this process will result in cell death by mitotic catastrophe [43]. Interestingly, the hypersensitivity of S-phase cells to heat shock appears to be due to potentially lethal lesions, i.e. lesions that can be repaired unless they are fixed to lethal lesions before repair is complete. Specifically, if a cell in S-phase can be prevented from making DNA following heat shock for a long enough period of time (2–6 h), the thermal sensitivity of S-phase cells is essentially the same as a non-S-phase cell [44]. Thus, the thermal damage responsible for S-phase hypersensitivity can be essentially completely reversed if it can be repaired. Conversely DNA replication appears to fix the potentially lethal lesions into lethal ones. In other words DNA replication in the presence of the potentially lethal lesion renders it lethal, while reversibly arresting DNA replication allows time for the lesion to be repaired. The time course for repair of this potential lethal damage is 2–6 h, depending on the severity of the damage. For example, after a 15 min, 45°C heat shock, the hypersensitivity of S-phase cells can be reversed by blocking DNA synthesis for 2 h [44]. However, after a 30 min at 45°C heat shock, the hypersensitivity of S cells can only be reversed after 6 h of inhibition of DNA synthesis [44]. Thus, the thermal sensitivity of S-phase cells is due to a condition that can be repaired. DNA synthesis is delayed long enough for the repair to be completed.

Figure 4. Effects on hyperthermia on DNA Supercoiling Ability. (a) illustrates a model of how hyperthermia induced MAR DNA binding can impact DNA supercoiling and the recognition of DNA damage. In the absence of the heat-induced MAR binding proteins single-strand DNA breaks will inhibit DNA supercoil rewinding, but when these proteins bind MAR DNA the effects of these breaks on DNA supercoiling are masked, see (b) which shows some of the supporting data in which DNA damage induced by ionizing radiation is masked in heated cells. The excess halo diameter is illustrated in (c), showing the approximate point at which the difference between control and irradiated samples were measured and added to estimate the difference between the curves. Note that in the samples from heated and irradiated cells the inhibition of DNA supercoil rewinding is reversed due to the masking of the DNA damage. Panel C reprinted from [39].

The role of DNA replication fork stalling secondary to the enhanced protein binding in DNA replication complexes as a mechanism for the hypersensitivity of S-phase cells to hyperthermia

To gain insight into the fixing of heat-induced lethal lesions by DNA replication, we considered the effects of hyperthermia on DNA replication. Nuclear DNA replication is coordinated in time and space and associated with the nuclear matrix. Nuclear DNA is replicated in large protein comp-lexes, called replication factories. These replication factories have different organizations within the nucleus depending on whether the cell is in early, mid or late S-phase [45–46]. For example, actively replicating DNA in early S-phase, DNA replication complexes can be detected (by pulse-labelling with BUdR and staining with a fluorescently labelled antibody to DNA containing BU) as small foci distributed throughout the nucleus, called a type I DNA replication pattern [44], [46]. In mid S-phase active replication complexes are detected in the nuclear periphery and observed in larger aggregates than the foci representative of early S-phase. The mid-S phase replication patterns are called a type II DNA replication pattern [44], [46]. In late S-phase the active replication complexes occur in even larger aggregates, called a type III DNA replication pattern [44], [46]. Hyperthermia can disrupt the timing of the transitions between the Type I, II, and III patterns [45]. Specifically, if one compares the heat induced delay in transition from Type I to Type II DNA replication patterns, one sees a lack of dose dependent delay and a rather short delay of about 2 h, whereas in the case of overall DNA synthesis, this delay is heat-dose dependent and can take up to 12 h for a cell that has been heated at 45°C for 30 min to replicate half of its DNA [47]. This result suggests that the cells were initiating DNA replication complexes scheduled to start in mid S-phase cells prior to the completion of replication by the factories that are scheduled to make DNA in early S. To verify this idea, a double pulse labelled technique was used, using iodo-deoxyuridine and chloro-deoxyuridine to distinguish the actively replicating complexes at two different times. In unheated cells, if the time between the labels is 4 h the labels are completely separated, showing that Type II DNA replication factories do not initiate DNA synthesis until almost all of the Type I replication factories have completed DNA replication [47]. We were able to show that if the labels were separated by 6.5 h, the second label picked up both Type I and Type II DNA replication complexes, showing that Type II DNA replication factories had initiated DNA synthesis while most of the Type I replication factories were still replicating DNA [47]. Thus, heat shock induces a disruption of the timing and coordination of DNA replication as cells progressed through S.

To gain insight into the heat induced lesion that is repairable in the absence of DNA replication, we considered the effects of hyperthermia on selected proteins involved in DNA replication. If one looks at the tightness of binding of DNA replication proteins within the factories and to the nuclear matrix, one can see that hyperthermia has induced a solubility change in the proteins involved in DNA replication [47–48]. In other words, binding of these proteins is tighter, their associations are less fluid and presumably their ability to function is impaired. These solubility changes can be measured under different conditions of heat-dose responses and recovery times in the absence of DNA replication and in this particular study, cells were given various heat exposures and either allowed to continue to synthesize DNA immediately or held under conditions that arrest DNA synthesis for 4 h and then allowed to continue DNA synthesis. Alternatively, cells were given either a 15 min at 45°C or 30 min at 45°C heat shock and kept from continuing DNA synthesis for various time intervals. At the end of each heat exposure and DNA synthesis arrest period, cell survival and the binding of selected DNA replication proteins to the nuclear matrix were measured. Note that tighter binding to the nuclear matrix reflected the binding of proteins in DNA replication complexes. For proteins involved in DNA replication, binding to the nuclear matrix correlated with cell killing, whereas such binding of proteins not involved in DNA replication did not correlate with cell killing suggesting that the repairable, potentially lethal lesion is tighter binding of proteins throughout the DNA replication factories [43], [46]. Thus, it is likely that the protein binding causes an ineffective DNA replication complex, resulting in stalling of DNA replication forks, which can create DNA double strand breaks. It is known that DNA replication forks create double strand breaks and that homologous recombination is required to repair such breaks [49]. So the question then becomes: in S-phase cells, is γH2AX foci higher in S phase cells as opposed to non-S phase cells? Recent studies [37], [39] have found that S phase cells have a higher number of γH2AX foci compared to G1 or G2 cells for the same heat exposure. Thus, it seems that γH2AX foci in S phase cells consist of a combination of heat-induced foci per se, and to foci formed as a result of DNA double strand breaks secondary to stalled DNA replication forks. This issue should be an attractive one for further research because the story is highly suggestive, but few of the details are available at present.

Interestingly, other effects of hyperthermia on cell cycle progression occur in addition to the effects of heating of S-phase. After 15 min at 45°C, when the cells are cultured at 37°C, there is a transient G₂ block which lasts about 10–15 h after heat shock [50]. Although few cells not in the S-phase are killed by this temperature, death appears to be related to failure to complete G₂ in the cells heated by S-phase. After a 30 min at 45°C heat shock, again recovering at 37°C, there is a long lasting G₂ and late S-phase block that persists for 14–35 h after the heat shock. Again cell death appears to be related to failure in exiting late G₂ [51].

Figure 5. Heat-induced mitochondrial damage and oxidative stress. (a) shows an overview of heat effects on mitochondria leading to a burst of reactive oxygen radicals (ROS). (b) shows novel functionalized indoles. (c) shows the effects of VJ112-OH in combination with 41°C hyperthermia. (d) shows that increasing the burden of ROS destabilizes proteins as measured by differential scanning calorimetry using a model system, in which the redox status of a C++ dependent ATPase had been altered so that the effects of redox status on the endothermic transitions characteristic of unfolding could be observed.

Figure 6. An illustration integrating the examples of hyperthermia effects discussed in the text. (a) is a simplified drawing of the effects described. (b) is a statement of an integrating model linking effects in the plasma membrane and the mitochondria to protein effects in the nucleus, cell killing and radio- or chemo- sensitization.

Effects on proteins associated with the nuclear matrix

It is known that heat induces a significant increase in proteins bound to the nuclear matrix [52–56]. [29], [57] The nuclear matrix is defined as an organization structure within the nucleus that organizes the DNA into domains and supports most DNA related functions, such as transcription and replication [17–20]. Aberrant protein binding to this structure has the potential to disrupt a large number of nuclear functions. Transcription and RNA processing, as well as DNA replication are known to be inhibited by heat shock [58–59]. [21]The approaches that have been used to detect the effects of hyperthermia on proteins associated with the nuclear matrix are alterations in protein solubility [52–56], [29], [57] protein-DNA interactions [60], flow cytometry [61], the effects of heat shock on DNA supercoiling ability [62–64] and differential scanning calorimetry [14]. Lepock and Warters have shown that using differential scanning calorimetry on isolated nuclei and isolated nuclear matrix preparations that the nuclear matrix is among the more heat sensitive organelles showing significant endothermic changes as low as 40°C [14]. If we look at the additional proteins that bind nuclear matrix associated DNA after heat shock, these proteins appear to mask DNA strand breaks delaying repair and alter DNA supercoiling changes that may be a critical trigger for DNA repair [65] and are known to be critical for DNA replication and transcription [66].

Changes in the associations of nuclear proteins and protein DNA interactions and proteins binding to the nuclear matrix do not occur in isolation from other heat effects within the cell. It has long been recognized that heat has effects on plasma membrane protein distribution and alters the permeability of plasma membranes resulting in a calcium spike [67–70]. There are also significant disruptions of mitochondrial membrane potential resulting in the change in the redox status of cells [71–72]. It has been shown by Mike Freeman that this change in redox status can sensitize cells to hyperthermia [73–74]. This work further showed that the mitochondrial membrane depolarization and the resulting burst of reactive oxygen species (ROS) i.e. oxygen radicals alter protein stability rendering proteins sensitive to heat-induced protein unfolding. The altered protein stability can destabilize proteins within the nucleus disrupting DNA repair, DNA replication and DNA segregation. DNA double strand break formation secondary to heat-induced DNA replication fork stalling can lead to lethal lesions. There is also evidence that inhibition of DNA replication coupled with heat induced defects in checkpoint signalling leads to incomplete DNA replication upon entry into mitosis, which leads to mitotic catastrophe. This is a simple integrated model of the killing of S-phase cells by heat that explains the correlation between cell killing and chromosome aberrations [13]. Many aspects of this model have been verified over the years [22], [74]. Further support from the model has been derived from recently developed thermal sensitizing compounds based on indomethacin analogs [76]. These compounds have been shown to sensitize the mitochondrial membrane to heat induced disruption and at the same time decrease the temperatures for thermal destabilization of proteins, alter the binding of DNA repair proteins and reduce the temperature required to cause a delay in the assembly of DNA repair complexes (Laszlo and Freeman, unpublished). The mechanisms of these compounds appear to be consistent with the idea that heat effects on membranes, mitochondria and nuclear proteins are interrelated. The above considerations do not explain how heat alters viability in G1, and G2 cells, which remains to be addressed in detail.

Redistribution of proteins from the nucleus

In contrast to inducing tighter binding of a large number of proteins throughout the nucleus, hyperthermia can also loosen the binding of certain nuclear proteins and drive them out of the nucleus. Two examples of this effect are the DNA repair protein, Mre11, and the nucleolar protein, nucleolin (NCL). Temperatures of 41°C or higher will drive Mre11 out of the nucleus and lead to a subsequent sensitization to ionizing radiation [77–80]. This effect could be related to heat dissociating Mre11 from its functional complex with other DNA repair proteins [81]. As evidence for this possibility, heat induces an association between Mre11 and HSP70 [81]. In the case of NCL, heat induces dissociation between NCL and its functional partner nucleophosmin (NPM) and an association with the DNA replication protein, RPA [82]. This complex will then be exported from the nucleus. It has been suggested that this is a mechanism to prevent DNA replication, as RPA is needed for replication [82]. As discussed above, DNA replication can result in the fixing of potential lethal lesions to lethal lesions and it is speculated that the RPA/NCL binding mechanism may prevent heated cells from entering S phase, where such potential lethal lesions could be fixed. These considerations indicate that heat can disrupt the functions of large protein complexes by either or both tightening the some proteins throughout the complex or by decreasing the association of a critical member of the complex. These individual circumstances then become important areas for hyperthermic research.

Summary

The challenge of understanding of how hyperthermia induces cell killing, inhibits DNA repair and sensitizes cells to chemotherapeutic agents represents an ongoing challenge, because of the numerous protein changes induced by hyperthermia and the fact that it is likely that a small fraction of these changes will be relevant to the endpoint of concern to the investigator. However, much progress has been made in the understanding of hyperthermia effects on cellular processes. Thus, research on the mechanisms of hyperthermic effects becomes more sophisticated as better understanding of cellular processes of interest to the investigator become available. A better understanding of how hyperthermia alters cellular processes relevant to therapeutic endpoints will facilitate a more effective employment of hyperthermia as a therapeutic adjuvant.