Thermal stress and the disruption of redox-sensitive signalling and transcription factor activation: possible role in radiosensitization.

In spite of ongoing research efforts, the specific mechanism(s) of heat-induced alterations in the cellular response to ionizing radiation (IR) remain ambiguous, in part because they likely involve multiple mechanisms and potential targets. One such group of potential targets includes a class of cytoplasmic signalling and/or nuclear transcription factors known as immediate early response genes, which have been suggested to perform cytotoxic as well as cytoprotective roles during cancer therapy. One established mechanism regulating the activity of these early response elements involves changes in cellular oxidation/reduction (redox) status. After establishing common alterations in early response genes by oxidative stress and heat exposure, one could infer that heat shock may have similarities to other forms of environmental antagonists that induce oxidative stress. In this review, recent evidence supporting a mechanistic link between heat shock and oxidative stress will be summarized. In addition, the hypothesis that one mechanism whereby heat shock alters cellular responses to anticancer agents (including hyperthermic radiosensitization) is through heat-induced disruption of redox-sensitive signalling factors will be discussed.

Heat shock and the activation of AP-1 and inhibition of NF-kappa B DNA-binding activity: possible role of intracellular redox status.

The early response genes comprising the AP-1 and NF-kappa B transcription factors are induced by environmental stress and thought to modulate responses to injury processes through the induction of target genes. Exposure to heat and ionizing radiation (IR) has been shown to affect signalling machinery involved in AP-1 and NF-kappa B activation. Furthermore, regulation of the signalling pathways leading to the activation of these transcription factors has been linked to changes in intracellular oxidation/reduction (redox) reactions. The hypothesis is proposed that exposure to thermal stress and/or IR might alter metabolic processes impacting upon cellular redox state and thereby modify the activity of redox-sensitive transcription factors such as AP-1 and NF-kappa B. Gel electromobility shift assays (EMSA) demonstrated that heat shock-induced AP-1 DNA-binding activity but inhibited IR-induced activation of NF-kappa B. A time course showed that activation of the AP-1 complex occurs between 4 and 5 h following thermal stress, and inhibition of IR-induced NF-kappa B activation also occurs during this time interval. Using a redox-sensitive fluorescent probe [5-(and -6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate], a shift to 40% less intracellular dye oxidation was observed in HeLa cells 0-4 h post-heat shock (45 degrees C, 15 min) relative to cells held at 37 degrees C. This was followed by a shift to greater dye oxidation between 4 and 12 h after treatment (about 1.8-fold) that returned to control levels by 24 h post-heating. These results show changes in DNA-binding activity closely paralleled apparent heat-induced changes in the intracellular redox state. Taken together, these results provide correlative evidence for disruption of redox-sensitive IR-induced signalling pathways by heat shock and support the hypothesis that this mechanism might play a role in heat-induced alterations in radiation response.

Inhibition of cyclooxygenase-2 with NS-398 and the prevention of radiation-induced transformation, micronuclei formation and clonogenic cell death in C3H 10T1/2 cells.

PURPOSE: Abnormally high levels of the cyclooxygenase (COX)-2 isozyme as well as the prostaglandin metabolites produced by the COX pathway have been observed in a variety of malignancies, including cancers of the skin, pancreas, colon, breast, cervix, prostate, and head and neck. Furthermore, exogenous genotoxic agents, including ionizing radiation (IR), have been shown to induce cellular transformation and to elevate COX-2 activity, whereas exposure to agents that specifically inhibit COX-2 activity have been shown to inhibit transformation. These data suggest a possible role of COX-2 both in IR-mediated cellular transformation processes and cell death. MATERIALS AND METHODS: C3H 10T1/2 and/or HeLa cells were treated with N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS-398) and/or exposed to IR. Following treatment, cells were assayed for neoplastic transformation, clonogenicity, growth rates, cell cycle distribution, micronuclei formation and DNA damage by established methodologies. Statistical tests were performed on data as described. RESULTS: In the present study, experiments in normal murine fibroblast C3H 10T1/2 cells demonstrated that the chemical inhibition of COX-2 activity with moderate doses of NS-398 abrogated IR-induced transformation events by fourfold and protected irradiated C3H 10T1/2 cells from clonogenic cell death. Considering that these doses of NS-398 had no significant effect on cellular proliferation or cell cycle distribution in C3H 10T1/2 cells, the results suggest that inhibition of COX-2 either increases DNA repair or prevents the accumulation of DNA damage. In supplemental experiments, treatment with NS-398 caused a 1.5-fold reduction in IR-induced micronuclei formation and a significant decrease in DNA damage. CONCLUSIONS: These results suggest a role for COX-2 inhibitors in the normal tissue response to IR when administered at therapeutically achievable doses and therefore may have clinical implications for radiation oncology patients in the prevention of IR-induced malignancy.

Indomethacin lowers the threshold thermal exposure for hyperthermic radiosensitization and heat-shock inhibition of ionizing radiation-induced activation of NF-kappaB.

PURPOSE: It is well established that salicylate and several other non-steroidal anti-inflammatory agents (NSAID), including indomethacin, can activate the heat-shock response, albeit at high concentrations. This is significant since heat shock significantly alters the cellular cytotoxic response to ionizing radiation (IR). It was previously shown that heat shock, as well as NSAIDs, inhibits IR-induced activation of NF-kappaB and that NF-kappaB protects against IR-induced cytotoxicity. Hence, it is hypothesized that pretreatment with indomethacin before heating will lower the temperature and heating times required to inhibit the activation of NF-kappaB and induce significant hyperthermic radiosensitization. MATERIALS AND METHODS: Experiments were performed in HeLa cell lines and the DNA-binding activity was determined by EMSA. Cellular radiosensitivity was determined by clonogenic assay. RESULTS: HeLa cells pretreated with indomethacin showed a decrease in the temperature-time combination necessary to inhibit IR-induction of NF-kappaB DNA binding. In addition, clonogenic cell survival assays using identical conditions showed an indomethacin dose-dependent enhancement of hyperthermic radiosensitization. Thus, similar concentrations of indomethacin both lowered the threshold thermal exposure to inhibit activation of NF-kappaB DNA-binding and increased the sensitivity of tumour cells to hyperthermic radiosensitization-induced cytotoxicity. In HeLa cells treated with N-alpha-tosylphenylalanyl-chloromethyl ketone (TPCK), a serine protease inhibitor that blocks activation of NF-kappaB, an increase in radiosensitivity was observed. Interestingly, no additional cell killing was observed when heat shock was added to cells treated with TPCK before IR, suggesting a possible common cytotoxic pathway. CONCLUSIONS: The results demonstrate that indomethacin lowers the temperature-time conbination necessary to induce several physiological processes associated with the heat-shock response. Furthermore, NSAID may be potential adjuvants in improving the clinical effectiveness of hyperthermia in radiation therapy.

Indomethacin-induced radiosensitization and inhibition of ionizing radiation-induced NF-kappaB activation in HeLa cells occur via a mechanism involving p38 MAP kinase.

Although ionizing radiation (IR) activates multiple cellular factors that vary depending on dose and tissue specificity, the activation of NF-kappaB appears to be a well-conserved response in tumor cells exposed to IR. Recently, it also has been demonstrated that nonsteroidal anti-inflammatory agents inhibit tumor necrosis factor and interleukin-1-induced NF-kappaB activation and act as radiosensitizing agents. These observations reinforce the growing notion that NF-kappaB may be a protective cellular factor responding to the cytotoxicity of IR and other damaging stimuli. As such, we addressed the idea and mechanism that NF-kappaB is a downstream target of the nonsteroidal anti-inflammatory agent indomethacin and is involved in the process of radiosensitization. In this study, we report that indomethacin inhibited IR-induced activation of NF-kappaB and sensitized HeLa cells to IR-induced cytotoxicity at similar concentrations. Pretreatment of HeLa cells with SB 203580, a pyridinyl imidazole compound that specifically inhibits p38 mitogen-activated protein kinase (MAPK), abrogated the ability of indomethacin to inhibit IR-induced activation of NF-kappaB and diminished the indomethacin radiosensitizing effect. In addition, the transient genetic activation of p38(MAPK) inhibited IR induction of NF-kappaB gene expression in the absence of indomethacin. Finally, permanently transfected cell lines genetically unable to activate NF-kappaB, because of expression of a dominant negative I-kappaBalpha gene, demonstrated increased sensitivity to IR-induced cytotoxicity. Taken together, these results suggest that p38 MAPK is a target involved in indomethacin-induced radiosensitization and that NF-kappaB may be one downstream target in this process.

Increased activator protein 1 activity as well as resistance to heat-induced radiosensitization, hydrogen peroxide, and cisplatin are inhibited by indomethacin in oxidative stress-resistant cells.

It has been established that tumor cells develop resistance to a variety of therapeutic agents after multiple exposures to these agents/drugs. Many of these therapeutic agents also appear to increase the activity of transcription factors, such as activator protein 1 (AP-1), believed to be involved in cellular responses to oxidative stress. Therefore, we hypothesized that cellular resistance to cancer therapeutic agents may involve the increased activity of transcription factors that govern resistance to oxidative stress, such as AP-1. To investigate this hypothesis, a previously characterized cisplatin, hyperthermia, and oxidative stress-resistant Chinese hamster fibroblast cell line, OC-14, was compared to the parental HA-1 cell line. Electrophoretic mobility shift and Western blot assays performed on extracts isolated from OC-14 cells demonstrated a 10-fold increase in constitutive AP-1 DNA-binding activity as well as increased constitutive c-Fos and c-Jun immunoreactive protein relative to HA-1 cells. Treatment of OC-14 cells with indomethacin inhibited constitutive increases in AP-1 DNA-binding activity and c-Fos/c-Jun-immunoreactive protein levels. Clonogenic survival assays demonstrated that pretreatment with indomethacin, at concentrations that inhibited AP-1 activity, significantly reduced the resistance of OC-14 cells to heat-induced radiosensitization, hydrogen peroxide, and cisplatin. These results demonstrate a relationship between increases in AP-1 DNA-binding activity and increased cellular resistance to cancer therapeutic agents and oxidative stress that is inhibited by indomethacin. These results support the hypothesis that inhibition of AP-1 activity with nonsteroidal anti-inflammatory drugs, such as indomethacin, may represent a useful adjuvant to cancer therapy.

Thioredoxin nuclear translocation and interaction with redox factor-1 activates the activator protein-1 transcription factor in response to ionizing radiation.

Thioredoxin (TRX) is a cytoplasmic, redox-sensitive signaling factor believed to participate in the regulation of nuclear transcription factors mediating cellular responses to environmental stress. Activation of the activator protein (AP)-1 transcription factor is thought to be mediated in part by redox-sensitive interactions between the nuclear signaling protein redox factor-1 (Ref-1) and TRX. In this study, the role of TRX and Ref-1 in the activation of the AP-1 complex was examined in HeLa and Jurkat cell lines exposed to ionizing radiation (IR). After exposure to IR, nuclear levels of immunoreactive TRX increased, accompanied by an increase in AP-1 DNA binding activity. It was shown that a physical interaction between Ref-1 and TRX occurs within the nucleus and is enhanced after exposure to IR. Furthermore, TRX immunoprecipitated from irradiated cells was capable of activating AP-1 DNA binding activity in nonirradiated nuclear extracts. In addition, immunodepletion of Ref-1 from nuclear extracts demonstrated that the increase in AP-1 DNA binding activity after IR was also dependent upon the presence of Ref-1 from irradiated cells. Finally, the ability of both TRX and Ref-1 from irradiated cells to stimulate AP-1 DNA binding in nonirradiated nuclear extracts was abolished by chemical oxidation and restored by chemical reduction. These results indicate that, in response to IR, TRX and Ref-1 undergo changes in redox state that contribute to the activation of AP-1 DNA binding activity. These experiments suggest that a redox-sensitive signaling pathway leading from TRX to Ref-1 to the AP-1 complex participates in the up-regulation of DNA binding activity in response to ionizing radiation.

Heat shock inhibits radiation-induced activation of NF-kappaB via inhibition of I-kappaB kinase.

Radiation stimulates signaling cascades that result in the activation of several transcription factors that are believed to play a central role in protective response(s) to ionizing radiation (IR). It is also well established that heat shock alters the regulation of signaling cascades and transcription factors and is a potent radiosensitizing agent. To explore the hypothesis that heat disrupts or alters the regulation of signaling factors activated by IR, the effect of heat shock on IR-induced activation of NF-kappaB was determined. Irradiated HeLa cells demonstrated transient increases in NF-kappaB DNA binding activity and NF-kappaB protein nuclear localization. In addition, irradiated cells demonstrated increased I-kappaB phosphorylation and decreased I-kappaBalpha cytoplasmic protein levels, corresponding temporally with the increase of NF-kappaB DNA binding. Heat shock prior to IR inhibited the increase in NF-kappaB DNA binding activity, nuclear localization of NF-kappaB, and the phosphorylation and subsequent degradation of I-kappaB. I-kappaB kinase (IKK) immunoprecipitation assays demonstrated an increase in IKK catalytic activity in response to IR that was inhibited by pretreatment with heat. Kinetic experiments determined that heat-induced inhibition of NF-kappaB activation in response to IR decayed within 5 h after heating. Furthermore, pretreatment with cycloheximide, to block de novo protein synthesis, did not alter heat shock inhibition of IR induction of NF-kappaB. These experiments demonstrate that heat shock transiently inhibits IR induction of NF-kappaB DNA binding activity by preventing IKK activation and suggests a mechanism independent of protein synthesis.

Reduced attention-related negative potentials in schizophrenic adults.

Event-related potentials were recorded from outpatient adult schizophrenics receiving maintenance doses of neuroleptics and from normal control subjects during performance of a reaction time task and a complex visual discrimination task, the Span of Apprehension. Difference potentials were computed to isolate endogenous activity associated with the processing demands of the Span task. Schizophrenics produce significantly less early endogenous negative activity than do normal subjects. This processing-related negativity reflects pattern matching activity to an attentional trace during the serial scan of the visual icon. We previously reported an identical reduction in processing-related negativity in childhood-onset schizophrenia, suggesting that this deficit is age independent. Both frontal contingent negative variation and an early frontal P3 were larger in the schizophrenics than in normal subjects, suggesting an inappropriate mobilization of nonspecific attentional resources. A later posterior P3 was significantly smaller in schizophrenics than in normal subjects.

Nitrite as antidote for acute hydrogen sulfide intoxication?

The detoxification of hydrogen sulfide (H2S) by a heme catalyzed oxidation was examined as part of an on-going study of H2S toxicity. Interlocking O2 absorption and sulfide depletion data indicate that both oxyhemoglobin and methemoglobin are effective catalytic agents. Although the latter is more efficacious, the life time of excess sulfide in the presence of oxygen and either of the above is of the order of minutes. It has also been established that the formation of methemoglobin following nitrite administration occurs preferentially under oxygen poor conditions. Under an atmospheric or oxygen enriched environment, which favors sulfide depletion, the nitrite retards sulfide oxidation. Thus nitrite as an antidote for acute H2S intoxication can only be effective within the first few minutes after the exposure, at which time resuscitation and/or ventilation of the victim is likely to produce conditions in which the nitrite actually slows sulfide removal.