Hyperthermic Cell Killing Research Articles

The heat-induced γ-H2AX response does not play a role in hyperthermic cell killing

Purpose: The goal of this study was to determine whether the heat-induced formation of γ-H2AX foci is involved in hyperthermic cell killing.Materials and methods: The heat-induced γ-H2AX response was determined in cells exhibiting various degrees of heat sensitivity. The panel of cells tested included cells that are transiently thermotolerant, permanently heat resistant, permanently heat sensitive, and permanently resistant to oxidative stress. Cells exposed to non-thermal environmental conditions that lead to protection from, or sensitization to, heat were also tested. The heat sensitivity of cells in which H2AX was knocked out was also ascertained.Results: The protein synthesis independent state of thermotolerance, but not the protein synthesis dependent state of thermotolerance, was found to be involved in the attenuation of the γ-H2AX response in thermotolerant cells. The initial magnitude of the γ-H2AX response was found to be the same in all cell lines with altered heat sensitivity. Furthermore, no differences in the resolution of γ-H2AX foci were found among the cell lines tested. We also found that H2AX knock-out cells were not more heat sensitive.Conclusions: We conclude that the heat-induced γ-H2AX response does not play a role in heat-induced cell killing, thereby adding further evidence that the heat-induced γ-H2AX foci are not due to DNA double strand breaks.

Role of ATP in the sensitivity to heat and the induction of apoptosis in mammalian cells.

Heat-induced cell death and apoptosis were studied with respect to intracellular ATP. Studies on the relationship between hyperthermic cell-killing at 44 degrees C and cellular ATP levels in four cell lines grown as monolayers and six cell lines grown in suspension showed good correlations between cellular ATP levels and the sensitivity to heat. D(0) values (the dose required to reduce survival in the linear portion of the response by 63%) linearly increased with an increase in cellular ATP levels. No such changes in sensitivity to heat were observed between the cells cultured at different cell densities, regardless of the change in the cellular ATP level. These results suggest that cellular intrinsic ability to supply ATP rather than the level of pooled ATP per se is responsible for the thermal response. Heat-induced apoptosis in L5178Y cells was observed following treatment at 42 degrees C for 70 min, 44 degrees C for 20 min or 47 degrees C for 3 min, which corresponded to surviving fractions of 25, 0.6 and 0.8%, respectively, but not at 47 degrees C for 20 min, indicating that mild heat shock induced apoptosis. 2-deoxyglucose (2DG) and 2,4-dinitrophenol (DNP) increased the sensitivity to heat and affected the mode of cell death. Cells treated with 2DG and DNP (2DG/DNP) were heated at 42 degrees C for 20 min, and then incubated at 37 degrees C for up to 2h in the presence or absence of 2DG/DNP. In the absence of 2DG/DNP, the cellular ATP level recovered to 76% of the control level and DNA ladder formation was observed, whereas in the presence of 2DG/DNP, the cellular ATP level was further decreased (3-7% of the control) and no DNA fragmentation was detected. These results suggest that the inhibition of ATP synthesis is closely associated with the enhancement of sensitivity to heat and that ATP is required for the induction of apoptosis.

Radiofrequency lesioning and chronic spinal pain: A review of current concepts

Abstract Data on the efficacy of radiofrequency lesioning of dorsal root ganglia in the treatment of chronic spinal pain are contradictory. Following a review of the available clinical trials, we will discuss physical effects of radiofrequency and hyperthermia, pathophysiological mechanisms associated with radiofrequency and hyperthermia, mechanisms of hyperthermic cell modulation and cell killing, and a possible parallel of radiofrequency lesioning of dorsal root ganglia with lesioning of the trigeminal ganglion. Next, various hypotheses on the mode of action of radiofrequency lesioning are critically reviewed. Finally, a model is presented based on recent advances in hyperthermia, molecular biology and neurobiology, which improves our insight into the impact of heat on a dorsal root ganglion. Radiofrequency lesioning of a dorsal root ganglion is followed by: (i) direct heat damage of axons and perikarya in a small central necrotic area; (ii) ischemia in a surrounding zone by damage to vascular endotheli...

Sensitizing effects of gallium citrate on hyperthermic cell killing in vitro

The lethal effects of gallium citrate in combination with heat were studied using four cell lines, L5178Y, FM3A, P388 and HeLa. Cells were incubated with different concentrations (0.2-2mM) of gallium citrate at 37°C for 24h and heated at a range of temperatures from 40-44°C for various time periods up to 6 h in the absence of gallium citrate. Survival and cell viability were determined by clonogenic assay and the dye-exclusion test, respectively. All of the cell lines tested were insensitive to heat below 41°C, but were very sensitive to heat above 43°C. Gallium citrate was cytotoxic to these cell lines at different levels: P388 and HeLa were far more sensitive than L5178Y and FM3A. The killing effects of heat at 41°C were greatly enhanced by gallium citrate in L5178Y and P388 cells. The Arrhenius analysis for the lethal effect of heat, determined by clonogenic assay, in L5178Y cells showed that the transition temperature was remarkably decreased for the gallium-treated cells from approximately 43°C to 41°C. The mechanism for this decrease in the transition temperature may be attributable to the additional effects of gallium citrate on energy metabolism. Preincubation with 0.05mM gallium citrate at 37°C for 7 days also enhanced heat sensitization at 41°C in L5178Y. This preincubation condition may correspond to the condition for the continuous infusion of gallium that is clinically used for cancer treatment. In contrast, treatment with gallium did not greatly enhance the sensitivity of FM3A or HeLa cells to heat at 41°C, but the effects of gallium were significant.

Stabilization of two families of critical targets for hyperthermic cell killing and acquired thermotolerance of yeast cells.

Hyperthermic cell killing profiles of Saccharomyces cerevisiae cells were biphasic and a shoulder (phase 1) was followed by an exponential killing (phase 2). Assuming that (i) the rate of thermal damage in particular macromolecules or their assemblies limits the rate of hyperthermic cell killing (the critical target model), and (ii) the damages of two families of targets are lethal independently, we built a 'dual critical target model' in order to interpret the biphasic cell killing. Time-courses of temperature-programmed fractional survival were traced for S. cerevisiae cells in exponentially growing phase, heat shocked, and in stationary phase. Non-linear curve-fitting of the time-courses by using the dual critical target model provided the Arrhenius parameters of denaturation of the two families of targets. The cells were killed more slowly in phase 1 than in phase 2. Arrest in stationary phase, not heat shock, stabilizes the family of targets that is critical to phase 1 death. On the other hand, both heat-shock response and arrest in stationary phase stabilizes the other family of targets that, in addition to the previous one, is responsible for phase 2 death.

Stabilization of two families of critical targets for hyperthermic cell killing and acquired thermotolerance of yeast cells

Stabilization of two families of critical targets for hyperthermic cell killing and acquired thermotolerance of yeast cells

Promotion of Heat-Induced Apoptosis in FM3A Cells by Protease Inhibitors

Although it has been shown that proteases may play a positive role in causing apoptosis of some cells, we report here that, on the contrary, protease inhibitors can promote heat-induced apoptosis in FM3A cells. Cysteine protease inhibitor, trans-Epoxy-succinyl-l-leucylamido-(4-guanidino)butane (E-64, 100 μg/ml) and aspartate protease inhibitor, pepstatin-A (100 μg/ml) were used to test hyperthermic effect on FM3A cells and showed remarkable cytotoxicity when they were present in cell suspension during heating at 44 °C. The cytotoxicity was due to promotion of heat-induced apoptosis as judged by DNA agarose electrophoresis. Furthermore, using flow cytometric analysis, we observed a decrease in the G0/G1phase cell and an increase in the S phase cell as well as increased apoptosis after heat shock. E-64 and pepstatin-A exhibited a promotive effect on the changes of cell cycle induced by heat. The data presented suggest that the enhancement of hyperthermic cell killing by protease inhibitors may be related to promotion of heat-induced apoptosis and changes of cell cycle.

Excess protein in nuclei isolated from heat-shocked cells results from a reduced extractability of nuclear proteins

An excellent correlation has been established between the quantity of protein associated with nuclei isolated from heat-shocked cells and the level of hyperthermic cell killing. However, controversy remains about whether increases in nuclear-associated protein result from a heat-induced migration of cytoplasmic proteins into the nucleus or because hyperthermia reduces the solubility of nuclear proteins in the detergent buffers commonly used to isolate nuclei. To address this controversy, the nuclear protein content was measured in whole and detergent-extracted cells before and following hyperthermia. It was found that hyperthermia caused no significant change in the nuclear protein content of whole, unextracted cells, and when fluorescently labeled proteins were microinjected into the cytoplasm no gross change in the selective permeability of the nuclear membrane to soluble proteins was observed during or following hyperthermia. Measurements in extracted cells showed that the detergent buffers removed protein from both the nucleus and cytoplasm of control, nonheated cells and that hyperthermia reduced the extractability of both nuclear and cytoplasmic proteins. The amount of protein found in nuclei isolated from heated cells approached that observed in nuclei within nonheated whole cells as the hyperthermic exposure was increased. Thus, the dose-dependent, two- to threefold increase in the protein content of nuclei isolated from heated cells represents a heat-induced reduction in the extractability of proteins normally present within cell nuclei and does not result from a mass migration of cytoplasmic proteins into the nucleus, although some specific proteins (e.g., the 70 KDa heat shock protein) do migrate to the nucleus following heat shock. Differential scanning calorimetry (DSC) measurements of whole cells, isolated nuclei, cytoplasts, and karyoplasts supported these conclusions and suggested that most of the detergent-insoluble proteins remaining in the nuclei and cytoplasm of heated cells are in their native state. Thus, a relatively small amount of denatured protein may be sufficient to initiate and sustain insoluble protein aggregates comprised of mostly native proteins. Analyses of the DSC data also implied that the previously identified critical target proteins, predicted to have a Tm of 46.0 degrees C, are present in both the nucleus and cytoplasm.

Excess protein in nuclei isolated from heat‐shocked cells results from a reduced extractability of nuclear proteins

An excellent correlation has been established between the quantity of protein associated with nuclei isolated from heat-shocked cells and the level of hyperthermic cell killing. However, controversy remains about whether increases in nuclear-associated protein result from a heat-induced migration of cytoplasmic proteins into the nucleus or because hyperthermia reduces the solubility of nuclear proteins in the detergent buffers commonly used to isolate nuclei. To address this controversy, the nuclear protein content was measured in whole and detergent-extracted cells before and following hyperthermia. It was found that hyperthermia caused no significant change in the nuclear protein content of whole, unextracted cells, and when fluorescently labeled proteins were microinjected into the cytoplasm no gross change in the selective permeability of the nuclear membrane to soluble proteins was observed during or following hyperthermia. Measurements in extracted cells showed that the detergent buffers removed protein from both the nucleus and cytoplasm of control, nonheated cells and that hyperthermia reduced the extractability of both nuclear and cytoplasmic proteins. The amount of protein found in nuclei isolated from heated cells approached that observed in nuclei within nonheated whole cells as the hyperthermic exposure was increased. Thus, the dose-dependent, two- to threefold increase in the protein content of nuclei isolated from heated cells represents a heat-induced reduction in the extractability of proteins normally present within cell nuclei and does not result from a mass migration of cytoplasmic proteins into the nucleus, although some specific proteins (e.g., the 70 KDa heat shock protein) do migrate to the nucleus following heat shock. Differential scanning calorimetry (DSC) measurements of whole cells, isolated nuclei, cytoplasts, and karyoplasts supported these conclusions and suggested that most of the detergent-insoluble proteins remaining in the nuclei and cytoplasm of heated cells are in their native state. Thus, a relatively small amount of denatured protein may be sufficient to initiate and sustain insoluble protein aggregates comprised of mostly native proteins. Analyses of the DSC data also implied that the previously identified critical target proteins, predicted to have a Tm of 46.0°C, are present in both the nucleus and cytoplasm. © 1996 Wiley-Liss, Inc.

Sensitization to cisplatin action by step‐down heating in cddp‐sensitive and ‐resistant cells

Hyperthermia treatment (> or = 43 degrees C) has been shown to be able to (partially) reverse acquired cDDP resistance. However, such heat treatment is difficult to achieve in the clinic. Short pre-treatment at a high temperature (> 42 degrees C), immediately before a treatment at a lower temperature (< 42 degrees C) can enhance the heat toxicity of the lower temperatures. This "step-down heating schedule" was explored for its possible drug-sensitizing potential in in vitro-cultured cDDP-sensitive and -resistant murine and human tumour cells. A 10-min pre-treatment at 44 degrees C enhanced the cytotoxicity of 41 degrees C hyperthermia alone. It also enhanced sensitivity to cDDP when given at 37 degrees C. However, it did not increase the 41 degrees C-induced cDDP sensitization. Thus, no correlation was found between heat kill and cDDP sensitization for step-down heating schedules. The observed effects of step-down heating were comparable in sensitive and in resistant cells, so the step-down heating schedule, unlike the 43 degrees C treatment, did not lead to a decrease of the cDDP-resistance factor. Yet the total cytotoxicity caused by this treatment protocol was 10-fold more than for cDDP with 41 degrees C alone, due to the extra hyperthermic cell killing and the cDDP-sensitizing effect of the pre-treatment. This treatment could have a substantial impact on cDDP efficacy in the clinic even when cDDP resistance has developed.

Protocol for freezing thermotolerant cells and maintaining thermotolerance following thawing.

Two independent laboratories have demonstrated that suspension-grown, Chinese hamster ovary (CHO) cells can be made thermotolerant, frozen and subsequently thawed such that they still express thermotolerance. Thermotolerance was determined as the ability to protect cells against hyperthermic cell killing (colony formation assay) and the ability to reduce protein aggregation within the nuclei of heated cells. Cells were frozen either following development of full or partial thermotolerance. In the former case frozen cells maintained thermotolerance upon thawing and in the latter case cells subsequently developed full thermotolerance following thawing and incubation at 37.0 degrees C. After thawing, frozen cells displayed a temporal course of thermotolerance development and decay that was similar to that for never-frozen cells. Success was obtained using either asynchronous or synchronous cell populations, and the heat sensitivity of the cells was not altered by the freezing procedure. The experimental results demonstrate the plausibility of utilizing a frozen stock of thermotolerant cells to make thermotolerance experiments more convenient.

Formation of tight junctions and desmosomes protects MDCK cells against hyperthermic killing

Cell density is known to modify the survival of mammalian cells exposed to elevated temperatures. We have examined the role that cell-cell contact plays in this phenomenon. The formation of cell-cell contact is carried out by cells' junctional complex, i.e., tight junctions, desmosomes, and gap junctions. Lack of formation of tight junctions and desmosomes, or their opening, could interfere with the functions and structures of cell membrane. Membrane damage is at least partially responsible for cell death at elevated temperatures. MDCK cells with high density plated in low calcium medium form confluent monolayers devoid of the formation of tight junctions and desmosomes but quickly assemble them after Ca2+ restoration. We used MDCK cells and the calcium switch technique to investigate effects of cell-cell contact and, independently, of cell density on hyperthermic cell killing. We found that MDCK cells that formed tight junctions and desmosomes were more resistant to hyperthermic treatment than those that did not. Blocking the formation pathway of tight junctions made cells sensitive to heat. Cells growing at lowdensity showed almost the same survival as did cells at high density in the absence of the formation of tight junctions and desmosomes. The results suggest that the formation of tight junctions and desmosomes play a more important role in determining hyperthermic response than does density per se. The formation of tight junctions and desmosomes appears to protect cells modestly against hyperthermic killing.

L-HISTIDINOL POTENTIATES HYPERTHERMIC CELL-KILLING IN-VITRO

To clarify the effect of L-histidinol on hyperthermic killing of cells, HeLa and mouse sarcoma-180 (S-180) cells were exposed to heat in vitro in the presence of L-histidinol and the clonogenic surviving fraction of the cells was examined. After pretreating the cells with L-histidinol for 4 h, exposure of the cells to the combination of heat at 43-degrees-C and L-histidinol for various times (30 min to 4 h) reduced the surviving fraction more prominently than heat alone. Optimal concentrations to confer effective enhancement of heat on HeLa and S-180 cells were 9 mM and 3 mM, respectively. Those concentrations showed little toxicity of L-histidinol alone. Enhancement of the effect of heat by L-histidinol was observed only at 43-degrees-C, but not at 41 and 42-degrees-C. Flow cytometric DNA analysis was used to examine the cell cycle transit effect of L-histidinol. L-histidinol alone arrested the HeLa cells in G1-phase. Heat treatment at 43-degrees-C led to an accumulation of the cells in G2/M-phase and a decrease in the G1-phase fraction. The effect of combined treatment with heat and L-histidinol was complementary, in which L-histidinol attenuated the accumulation of the cells in G2/M-phase and prevented a decrease in the G1-phase fraction. Thus, L-histidinol has the capacity to potentiate hyperthermic cell killing in vitro by a mechanism that may be related to the cell cycle transit effect.

Enhancement of cell killing and increase in cytosolic calcium concentration by combined treatments with hyperthermia and TMB-8 in mouse mammary carcinoma FM3A cells

Mouse mammary carcinoma FM3A cells were exposed 42°C or 44°C hyperthermia for 60 min in a culture medium with or without 8-(N,N-diethylamino) octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8) at a concentration of 100 μM. Cell survival, cytosolic calcium ion concentration, [Ca 2+] c, and influx of 45Ca 2+ were investigated to examine the cardinal role of intracellular Ca 2+ in hyperthermic cell killing. The [Ca 2+] c in a single cell was measured by a digital image analyzing technique using Fura-2. TMB-8 enhanced cell killing, increase in [Ca 2+] c, and influx of 45Ca 2+ induced by both hyperthermic treatments. In contrast, TMB-8 abolished the increase in [Ca 2+] c induced by hyperthermia at 42°C and 44°C in the absence of extracellular Ca 2+. These results suggest that TMB-8, an agent usually called an inhibitor of intracellular calcium mobilization at 37°C or lower, enhances Ca 2+ entry from the extracellular space at 42°C or 44°C and, in turn, promotes cell killing under hyperthermic conditions.

Synergistic action of calcium-ionophores and hyperthermia is best interpreted as thermal enhancement of calcium toxicity.

It has been shown that no relation exists between [Ca2+]i and hyperthermic cell killing, although heat-induced increase of [Ca2+]i can be observed in some cell lines. When ionophores are used, dose-dependent rises in [Ca2+]i may be found. Beyond a certain threshold of ionophore-induced increases in [Ca2+]i, cells may be killed. Different threshold levels of [Ca2+]i exist in different cell lines. Hyperthermia can act synergistically with calcium ionophores to potentiate cell killing. Since there is no causal relation between [Ca2+]i and heat toxicity, this synergism can be explained as heat enhanced Ca2+ toxicity. In the current report, it is shown that both ionophore-induced Ca2+ toxicity (37 degrees C) and its potentiation by heat are dependent on extracellular calcium and related to sustained increases in [Ca2+]i. With ionomycin concentrations up to 15 microM, no increase in [Ca2+]i was seen in cells maintained in medium without Ca2+. Ionomycin effects on intracellular compartments were absent, and the drug seemed to act solely on the level of the plasmamembrane. Also, the synergism of heat and ionomycin appeared to act at the plasmamembrane, because depletion of extracellular calcium completely abolished this synergistic effect. The data presented are also discussed in the light of controversies existing in the literature for the role of calcium in hyperthermic cell killing.

Hyperthermia, intracellular free calcium and calcium ionophores.

It is shown that heat-induced increase of intracellular calcium does not correlate with hyperthermic cell killing. Six different cell lines were investigated; in four (EAT, HeLa S3, L5178Y-R and L5178Y-S) heat treatments killing 90% of the cells did not affect the levels of intracellular free calcium ([Ca2+]i). In one cell line (3T3) a heat-induced increase in [Ca2+]i was observed. LM cells showed a heat-induced increase of the ratio of the fluorescent signals, but this may be explained by Fura-2 leakage out of the cells. Calcium ionophores are used to address the question whether rises in [Ca2+]i might cause cell killing. To investigate the existence of sensitization to Ca2+ toxicity by heat, ionophore treatments are combined with hyperthermia. Both ionophores used, A23187 and ionomycin, cause cell killing corresponding with increases in [Ca2+]i at 37 degrees C in EAT cells. In HeLa S3 cells, substantial increases in [Ca2+]i due to the action of ionomycin were observed without corresponding cell killing. This indicates the presence of a threshold concentration of [Ca2+]i in HeLa S3 cells before the treatment becomes toxic. Both ionophores show synergism with hyperthermia for cell killing as well as at the level of increased [Ca2+]i. The synergistic action may be explained as thermal enhancement of calcium toxicity.

Role of Membrane Components in Thermal Injury of Cells and Development of Thermotolerance

Exposure of cells to hyperthermia induces a transient resistance to subsequent heat treatment. The specific mechanisms responsible for hyperthermic cell killing and thermotolerance development are not well understood. It seems that heat may induce at least two different states of thermotolerance, of which one is dependent on protein synthesis. The expression of thermotolerance may include multiple cytoplasmic and membrane components. A number of studies have indicated that membranes play an important role in governing the thermal injury of cells. It seems, therefore, that heat denatured plasma membrane proteins may be a potential target for thermal stress and a trigger for the induction of thermotolerance. The localization of heat shock proteins in the plasma membrane and the suggestion of thermal resistance in enucleate erythrocytes support this suggestion. However, a direct relationship between the plasma membrane and hyperthermic killing or development of thermotolerance has not been found.

Effects of tamoxifen and protein kinase C inhibitors on hyperthermic cell killing

The effects of the anti-estrogen, tamoxifen, and the protein kinase C inhibitor, 1-(5-isoquinolinyisulfonyl)2-methylpiperazine (H7), on hyperthermic cytotoxicity were studied. Three cell lines were used, a human colon cancer cell line (HT-29), a human mammary carcinoma cell line (MCF-7), and Chinese hamster V79 lung fibroblasts. With all three cell lines, tamoxifen at concentrations greater than 7.5 μM during heating or with a 3-hr exposure prior to heating significantly sensitized cells to heat. When cells were preincubated with 10–20 μM tamoxifen for 1–2 hr at 37°C prior to heat treatment, washed free of extracellular tamoxifen, heated to generate thermoresistance, and examined 18 hr later for thermoresistance, tamoxifen treated HT-29 and MCF-7 cells were significantly more heat sensitive than thermotolerant controls not previously exposed to tamoxifen. In contrast, the degree of induced thermoresistance of V79 cells was unchanged after tamoxifen treatment. H7, but not its structural analogue and low affinity protein kinase C inhibitor, HA1004, also sensitized cells to heat. Neither H7 nor HA1004 had any apparent effect on the degree of heat-induced thermoresistance in the three cell lines tested.

Cycloheximide increases the thermostability of proteins in Chinese hamster ovary cells

Protein denaturation resulting from temperatures between 42.0°C and 50°C has been observed and implicated as the lethal lesion for hyperthermic cell killing. A logical corollary is that protection against hyperthemic killing requires stabilization of cellular proteins against thermal denaturation. To test this, Chinese hamster ovary cells were treated with the heat protector cycloheximide and then subjected to differential scanning calorimetry to measure protein denaturation. Cycloheximide stabilized proteins that denatured between 42°C and 52°C in control cells by increasing their transition (denaturation) temperature by an average of 1.3°C. In addition, cycloheximide reduced the cytotoxicity of actinomycin D and adriamycin, suggesting that protein stabilization protects cells against stresses other than hyperthermia.

Apoptosis induced by mild hyperthermia in human and murine tumour cell lines: A study using electron microscopy and DNA gel electrophoresis

Mild hyperthermia is known to enhance apoptosis in a range of normal and neoplastic cell populations. Studies of tumours previously shown to respond to heating in this manner might be expected to provide insights not only into the mechanism of hyperthermic cell killing, but also into the apoptotic process in general. In the present study, cell death induced by 43 degrees C heating for 30 min in two human Burkitt's lymphoma lines, BM 13674 and WW1, and in murine mastocytoma P-815 x 2.1 was found to be exclusively apoptotic in type, identification being based on light and electron microscopic appearances and on the presence of internucleosomal cleavage of DNA into fragments that are multiples of 180-200 base pairs, which was demonstrated by agarose gel electrophoresis. The heat-induced apoptosis was prevented by the presence of zinc sulphate, an inhibitor of the endonuclease considered to be responsible for the DNA cleavage, but was not suppressed by the protein synthesis inhibitor cycloheximide. The findings question the validity of the widely held view that active protein synthesis is an invariable prerequisite for the execution of apoptosis. It is suggested that an inositol triphosphate-mediated increase in cytosolic Ca2+, resulting from limited membrane damage, might be the critical event responsible for activation of apoptosis by mild hyperthermia.