Repression of thermotolerance in Dunning R3327 prostate carcinoma cells by 2-deoxy-glucose.

The transient addition of the cytosolic energy depletor 2-deoxy-glucose to cultures of rat prostate carcinoma cells blunted the induction of Hsp70 protein following exposure to elevated temperatures in a manner that appeared to parallel its effects on energy metabolism. While the reduction in stress-induced heat-shock protein expression by treatment with 2-deoxy-glucose had no effects on the acute loss of cellular viability after exposure to heat, the acquisition of thermotolerance in response to a conditioning stimulus was specifically repressed. Therefore, 2-deoxy-glucose will be a useful tool in the investigation of mechanisms that mediate immediate versus chronic responses to cellular stress, including the specific roles played by members of the heat-shock protein family of proteins. These results might have important implications in the design of protocols for the hyperthermic treatment of tumours.

Estramustine phosphate enhances the effects of hyperthermia and induces the small heat shock protein HSP27 in the human prostate carcinoma cell line PC-3.

The antimicrotubule drug estramustine phosphate (EMP) has been shown to sensitize prostate carcinoma cells to radiation via synchronization at the G2/M phase of the cell cycle. This synchronization may also render cells more sensitive to hyperthermia, providing a rationale for multimodal treatment approaches. We have investigated the effects of EMP and hyperthermia, as well as the regulation of heat shock proteins (HSP) in the PC-3 prostatic carcinoma cell line. Cells were incubated with four doses of EMP for 48 h followed by a 1-h hyperthermia treatment ranging from 41 degrees C to 44 degrees C. Cell cycle distribution at the end of the EMP incubation was investigated by flow cytometry. Cytotoxicity was assessed by colony formation assays. HSP accumulation was investigated by Western immunoblotting. Doses of 1, 5, 10 and 15 microM EMP synchronized 27, 28, 46, and 68% of PC-3 cells at G2/M. With 5, 10 and 15 microM, a sensitizing effect of EMP was assessed at hyperthermic temperatures of 42, 43 and 44 degrees C. EMP did not alter the expression of HSP72, but substantially induced the synthesis of HSP27 in PC-3 cells. Our data show that EMP sensitizes PC-3 cells to hyperthermia induced cytotoxicity. This observation supports the rationale for multimodal treatment approaches in locally advanced prostate cancer.

Selected contribution: Hyperthermia-induced intestinal permeability and the role of oxidative and nitrosative stress.

The purpose of this study was to characterize intestinal permeability changes over a range of physiologically relevant body temperatures in vivo and in vitro. Initially, FITC-dextran (4,000 Da), a large fluorescent molecule, was loaded into the small intestine of anesthetized rats. The rats were then maintained at approximately 37 degrees C or heated over 90 min to a core body temperature of approximately 41, approximately 41.5, or approximately 42.5 degrees C. Permeability was greater in the 42.5 degrees C group compared with the 37, 41, or 41.5 degrees C groups. Histological analysis revealed intestinal epithelial damage in heated groups. Everted intestinal sacs were then used to further characterize hyperthermia-induced intestinal permeability and to study the potential role of oxidative and nitrosative stress. Increased permeability to 4,000-Da FITC-dextran in both small intestinal and colonic sacs was observed at a temperature of 41.5-42 degrees C compared with 37 degrees C, along with widespread intestinal epithelial damage. Administration of antioxidant enzyme mimics or a nitric oxide synthase inhibitor did not reduce permeability due to heat stress, and tissue concentrations of a lipid peroxidation product were not altered by heat stress, suggesting that oxidative and nitrosative stress were not likely mediators of this phenomenon in vitro. In conclusion, hyperthermia produced increased permeability and marked intestinal epithelial damage both in vivo and in vitro, suggesting that thermal disruption of epithelial membranes contributes to the intestinal barrier dysfunction manifested with heat stress.

Effect of heat stress on LPS-induced febrile response in D-galactosamine-sensitized rats.

We have previously reported that heat conditioning augments lipopolysaccharide (LPS)-induced fever in rats, which is accompanied by an accumulation of heat shock protein (HSP) in the liver and the reduction of the plasma level of tumor necrosis factor (TNF-alpha) (Kluger MJ, Rudolph K, Soszynski D, Conn CA, Leon LR, Kozak W, Wallen ES, and Moseley PL. Am J Physiol Regulatory Integrative Comp Physiol 273: R858-R863, 1997). In the present study we have tested whether inhibition of protein synthesis in the liver can reduce the effect of this heat conditioning on the LPS-induced febrile response in the rat. D-galactosamine (D-gal) was used to selectively inhibit liver protein synthesis. D-gal (500 mg/kg) or PBS as control was administered intraperitoneally 1 h before heat stress. LPS (50 microg/kg ip) was injected 24 h post-heat exposure. Treatment with D-gal blunted the febrile response to LPS. Moreover, heat-conditioned rats treated first with D-gal and subsequently with LPS demonstrated a profound fall in core temperature 10--18 h post-LPS. A significant increase of serum TNF-alpha accompanied this effect of D-gal on fever. Heat-conditioned animals receiving D-gal showed an inhibition in inducible HSP-70 in the liver. These data support the role of hepatic function in modulating the febrile response to LPS.

Exercise increases serum Hsp72 in humans.

Recent evidence suggests that heat shock proteins (Hsps) may have an important systemic role as a signal to activate the immune system. Since acute exercise is known to induce Hsp72 (the inducible form of the 70-kDa family of Hsp) in a variety of tissues including contracting skeletal muscle, we hypothesized that such exercise would result in the release of Hsp72 from stressed cells into the blood. Six humans (5 males, 1 female) ran on a treadmill for 60 minutes at a workload corresponding to 70% of their peak oxygen consumption. Blood was sampled from a forearm vein at rest (R), 30 minutes during exercise, immediately postexercise (60 minutes), and 2, 8, and 24 hours after exercise. These samples were analyzed for serum Hsp72 protein. In addition, plasma creatine kinase (CK) was measured at these time points as a crude marker of muscle damage. With the exception of the sample collected at 30 minutes, muscle biopsies (n = 5 males) were also obtained from the vastus lateralis at the time of blood sampling and analyzed for Hsp72 gene and protein expression. Serum Hsp72 protein increased from rest, both during and after exercise (0.13 0.10 vs 0.87+/-0.24 and 1.02+/-0.41 ng/mL at rest, 30 and 60 minutes, respectively, P < 0.05, mean SE). In addition, plasma CK was elevated (P < 0.05) 8 hours postexercise. Skeletal muscle Hsp72 mRNA expression increased 6.5-fold (P < 0.05) from rest 2 hours postexercise, and although there was a tendency for Hsp72 protein expression to be elevated 2 and 8 hours following exercise compared with rest, results were not statistically significant. The increase in serum Hsp72 preceded any increase in Hsp72 gene or protein expression in contracting muscle, suggesting that Hsp72 was released from other tissues or organs. This study is the first to demonstrate that acute exercise can increase Hsp72 in the peripheral circulation, suggesting that during stress these proteins may indeed have a systemic role.

Aging reduces adaptive capacity and stress protein expression in the liver after heat stress.

A decline in an organism's ability to cope with stress through acute response protein expression may contribute to stress intolerance with aging. We investigated the influence of aging on stress tolerance and the capacity to synthesize the 70-kDa heat shock protein (HSP70) in young and old rats exposed to an environmental heating protocol. Livers were assessed for injury and HSP70 expression after heat stress by use of immunohistochemical and immunoblotting techniques. The inducible HSP70 response in the cytoplasm and nucleus was markedly reduced with age at several time points over a 48-h recovery period, although senescent rats were able to strongly express HSP70 early in recovery. Older animals had extensive zone-specific liver injury, which corresponded to the diminished HSP70 response observed in these regions, and a significant reduction in thermotolerance compared with their young counterparts. These data highlight the regional nature of stress-induced injury and HSP70 expression in the liver and the impact of aging on these responses. Furthermore, the results suggest a functional link between the age-related decrements in the expression of inducible HSP70 and the pathophysiological responses to heat stress.

Exercise, stress, and the immune conversation.

The role of exercise in meaningful alterations in immune function is unclear. The recent demonstration that stress proteins are potent modulators of immune cell activation and cytokine production, coupled with the role of exercise in stress protein production, suggest that exercise-associated alterations in immune function may be related to exercise associated alterations in the cellular stress response.

Hyperthermia enhances cytomegalovirus regulation of HIV-1 and TNF-alpha gene expression.

The immediate-early (IE) genes of human cytomegalovirus (CMV) can be expressed in monocytic cells and are known to regulate viral and cellular genes. Reactivation of human immunodeficiency virus (HIV-1) may be stimulated by a variety of factors including other viruses and inflammatory cytokines. These studies examine the role of hyperthermia and CMV in the regulation of HIV-1 and tumor necrosis factor (TNF)-alpha. THP-1 cells were transfected with the CMV IE genes. HIV-1 and TNF-alpha transcription were assessed with chloramphenicol acetyltransferase promoter constructs. Hyperthermia sufficient to stimulate production of heat shock proteins was used to stimulate the cells. Hyperthermia significantly enhances the effect of CMV IE gene products on the expression of HIV-1 and TNF-alpha. The increases in HIV-1 transcription appear to be in part due to increases in TNF-alpha. Heat shock proteins induced by hyperthermia may play an important role in the viral regulation of monocytic function by CMV.

Effect of estrogen supplementation on exercise thermoregulation in premenopausal women.

This study examined the effects of 3 days of estrogen supplementation (ES) on thermoregulation during exercise in premenopausal (20-39 yr) adult women during the follicular phase of the menstrual cycle. Subjects (11 control, 10 experimental) performed upright cycle ergometer exercise at 60% of maximal O2 consumption in a neutral environment (25 degreesC, 30% relative humidity) for 20 min. Subjects were given placebo (P) or beta-estradiol (2 mg/tablet, 3 tablets/day for 3 days). All experiments were conducted between 6:30 and 9:00 AM after ingestion of the last tablet. Heart rate, forearm blood flow (FBF), mean skin temperature, esophageal temperature (Tes), and forearm sweat rate were measured. Blood analysis for estrogen and progesterone reflected the follicular phase of the menstrual cycle. Maximal O2 consumption (37.1 +/- 6.2 in P vs. 38.4 +/- 6.3 ml. kg-1. min-1 in ES) and body weight-to-surface area ratio (35.58 +/- 2.85 in P vs. 37.3 +/- 2.7 in ES) were similar between groups. Synthesis of 70-kDa heat shock protein was not induced by 3 days of ES. Neither the threshold for sweating (36.97 +/- 0.15 in P vs. 36.90 +/- 0.22 degreesC in ES), the threshold for an increase in FBF (37.09 +/- 0. 22 in P vs. 37.17 +/- 0.26 degreesC in ES), the slope of sweat rate-Tes relationship (0.42 +/- 0.16 in P vs. 0.41 +/- 0.17 in ES), nor the FBF-Tes relationship (10.04 +/- 4.4 in P vs. 9.61 +/- 3.46 in ES) was affected (P > 0.05) by 3 days of ES. We conclude that 3 days of ES by young adult women in the follicular phase of their menstrual cycle have no effect on heat transfer to the skin, heat dissipation by evaporative cooling, or leukocyte synthesis of 70-kDa heat shock protein.

Increased flux of free radicals in cells subjected to hyperthermia: detection by electron paramagnetic resonance spin trapping.

It has been hypothesized that hyperthermia promotes oxygen-centered free radical formation in cells; however, to date there is no direct evidence of this heat-induced increase in oxygen free radical flux. Using electron paramagnetic resonance (EPR) spin trapping, we sought direct evidence for free radical generation during hyperthermia in intact, functioning cells. Rat intestinal epithelial cell monolayers were exposed to 45 degrees C for 20 min, after which the nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was added. Compared to control cells at 37 degrees C, heat-exposed cells had increased free radical EPR signals, consistent with the formation of DMPO/.OH (aN = aH = 14.9 G). These findings indicate that heat increases the flux of cellular free radicals and support the hypothesis that increased generation of oxygen-centered free radicals and the resultant oxidative stress may mediate in part, heat-induced cellular damage.

Effects of combined treatment of chemotherapeutics and hyperthermia on survival and the regulation of heat shock proteins in Dunning R3327 prostate carcinoma cells.

BACKGROUND: Hyperthermia can enhance the clinical response of chemotherapeutic agents in prostate cancer, but optimal sequencing of this combination therapy needs to be developed. Given the role of heat shock proteins (HSPs) in the development of resistance (thermotolerance) to subsequent hyperthermic stresses as well as to certain chemotherapeutics, the study of HSP regulation is important in the establishment of effective schedules in multimodal treatment strategies. METHODS: In this study we evaluated the effects of the chemotherapeutic agents cisplatin, 5-fluorouracil, and adriamycin in combination with hyperthermia. (43 degrees C, 1 h) on clonogenic survival and inducible HSP70 regulation in Dunning rat adenocarcinoma of the prostate. HSP70 was analyzed by Western blot and by measuring beta-galactosidase produced by cells stably transfected with a gene construct containing the E. coli beta-galactosidase gene driven by the Drosophila HSP70 promoter. RESULTS: Colony formation assays revealed a sensitizing effect of hyperthermia when simultaneously combined with each chemotherapeutic agent, resulting in a potentiated cytotoxicity compared to subsequenced treatments. Thermotolerant cells showed a significantly better survival when treated with adriamycin alone, but also when each chemotherapeutic agent was combined with hyperthermia. This enhanced survival was correlated with inducible HSP70 accumulation. The chemotherapeutics modified the HSP70 promoter activation induced by hyperthermia, suggesting changes in the development of cellular thermotolerance. CONCLUSIONS: Our data reveal synergistic cytotoxic effects of the synchronous application of chemotherapeutic agents and hyperthermia on this model of prostate cancer. Furthermore, they demonstrate that the induction of HSPs in thermotolerant cells, as measured by HSP70 induction, results in a modulation the chemotherapeutic-mediated cytotoxicity. Therefore, HSP70 is a useful marker of cellular resistance in multimodal approaches combining hyperthermia and chemotherapeutic agents in the treatment of locally advanced prostate carcinoma.

Oxidants differentially regulate the heat shock response.

Cells, animals, and humans respond to hyperthermia through the synthesis of a family of proteins termed heat shock proteins (HSPs). Because hyperthermic stress may also result in mitochondrial uncoupling and the generation of reactive oxygen species, we wondered whether oxidant stress was sufficient to increase cellular levels of HSP70. HSP70 was detected in cells heated or treated with menadione but not in those treated with hydrogen peroxide or xanthine/xanthine oxidase. We speculate that oxidant stress from menadione exposure is qualitatively different from exposure from hydrogen peroxide or xanthine/xanthine oxidase.

Effect of heat stress on LPS-induced fever and tumor necrosis factor.

Exposure to heat stress leads to both short-term and long-term effects on morbidity. Male rats were exposed to a high ambient temperature of 40 degrees C, which resulted in biotelemetered core body temperature rising to approximately 42 degrees C. This treatment led to a marked enhancement in lipopolysaccharide (LPS)-induced fever at 24 h after exposure to heat stress. The increase in fever was accompanied by a significant suppression in the circulating concentration of tumor necrosis factor. Heat-shock protein-70 measured in liver was elevated by the heat exposure (but not further elevated by the injection of LPS). An enhanced fever to LPS and other inflammatory stimuli found in heat-stressed human subjects could explain the apparent increase in susceptibility to disease.

Beta-galactosidase as a marker of HSP70 promoter induction in Dunning R3327 prostate carcinoma cells.

Hyperthermia is known to improve the response of tumors to radiation or chemotherapeutic treatment when combined in multimodal strategies. The cellular response to hyperthermia is associated with the synthesis of heat shock proteins (HSP). To study the stress response in prostate cancer we have developed a clone of Dunning R3327 rat prostate carcinoma cells stably transfected with a gene construct containing the E. coli beta-galactosidase gene driven by the Drosophila HSP70 promoter. The measurement of beta-galactosidase serves as a rapid and semiquantitative assay of HSP70 gene activation. The Dunning cell clone showed evidence of incorporation of the HSP70/beta-galactosidase construct within the genomic DNA by Southern blot analysis. When compared to mock-transfected control cells, the clone showed minimal baseline beta-galactosidase activity, which significantly increased following a hyperthermic stress. The time course of beta-galactosidase elevation following heat stress paralleled the time course of cellular HSP70 elevation by Western blot analysis. These stably transfected Dunning R3327 cells may provide a useful tool to study the effects of hyperthermia, radiation, and chemotherapeutic agents on the cellular stress response and in the establishment of HSP70 as a marker of cellular resistance in the multimodal treatment of prostate cancer.

Heat shock proteins and heat adaptation of the whole organism.

Adaptation to heat may occur through acclimatization or thermotolerance; however, the linkage of these phenomena is poorly understood. The importance of heat shock proteins (HSPs) in thermotolerance and differences in their accumulation in organisms adapted to the heat suggest a role for HSPs in acclimatization as well. The role of HSPs in heat adaptation of the whole organism and the interrelationships among heat adaptation, endotoxin tolerance, and cytokine resistance through HSPs are reviewed.

Hospital privileges and the Americans with Disabilities Act.

The Americans with Disabilities Act provides protection for equal access to services and employment opportunities for qualified persons with disabilities. Because hospital privileges are essential to physician practice, this article investigates the role of the Americans With Disabilities Act in acquiring and maintaining these privileges. Recent findings from the Act's case law support the concept that: 1) an employer employee relationship exists between the hospital and the non-staff physician for purposes of the Act's analysis, and thus, 2) the Act is applicable to questions of hospital privileges for non-staff physicians. Suggestions for ensuring patient welfare while maintaining compliance with the Act are discussed.

Differential effects of exercise and heat stress on liver HSP70 accumulation with aging.

Previous reports have suggested that the heat shock response to passive heating may be blunted by aging. However, during exertional heating, factors in addition to elevated temperature may amplify the degree of stress compared with hyperthermia alone. The purpose of this study was to compare the pattern of accumulation of the highly inducible 72-kDa heat shock protein (HSP72) in liver tissue of mature (12-mo-old) and senescent (24-mo-old) male Fischer 344 rats after either passive or exertional heat stress. A euthermic control group was exposed to an ambient temperature (Ta) of 25 degrees C for 4.5 h. A passive heating (heat) group was exposed to an Ta of 42 degrees C until colonic temperature (Tco) reached 41 degrees C. An exertional heating (exercise) group performed intermittent moderate-intensity treadmill exercise (similar absolute intensities for the two age groups) at an Ta of 32 degrees C until Tco reached 41 degrees C. Heating rates were similar in the heat and exercise groups (approximately 0.08 degrees C/min). Rats in both the heat and exercise groups were maintained at a Tco of 41 degrees C for an additional 30 min and subsequently returned to an Ta of 25 degrees C for 3 h. Liver HSP72 accumulation was increased in mature rats after both the heat (+192% vs. control) and exercise (+292%) protocols. In contrast, the senescent rats demonstrated no significant increase in inducible HSP70 with heating but a large increase with exercise (+232%; P < 0.01 compared with control and heat groups). These data suggest that the blunted heat shock protein response to heating observed with aging is not a result of the inability to produce inducible HSP72 because older rats had an robust response to exertional hyperthermia.

Effect of radiation and hyperthermia on prostate tumor cells with induced thermal tolerance and the correlation with HSP70 accumulation.

We have investigated the effects of ionizing radiation and hyperthermia on Dunning prostate carcinoma cells after the administration of a conditioning stress (either radiation or hyperthermia) and correlated the presence or absence of a survival advantage to the accumulation of the family of 70,000 kD heat shock proteins (HSP70). Fifty percent lethal dose (LD(50)) determinations of hyperthermia and radiation were determined by clonogenic cell survival. The LD(50) for hyperthermia was 44°C for 1 hour, LD(50) for high energy ionizing radiation was 500 rads. We confirmed that Dunning tumor cells preconditioned with hyperthermia (43°C for 1 hour) are resistant at 10 and 24 hours to a subsequent lethal heat stress (44°C for 1 hour). No radiation tolerance was noted at 10 or 24 hours after a conditioning heat stress of 43°C for 1 hour. We also found no thermal resistance at 10 or 24 hours after radiation conditioning (500 rads). The inducible HSP70 (HSP72) was present 8 and 12 hours after hyperthermia but had decayed by 24 hours. No increase in HSP72 accumulation occurred after radiation. These data suggest that radiation and hyperthermia injure cells in two distinct manners and may explain the observed synergy in the treatment regimens that include combination hyperthermia and radiation. Our results should be useful in creating models in the treatment of carcinoma of the prostate with combined radiation and hyperthermia therapy.