Andrei Laszlo

Hyperthermia Activates a Subset of Ataxia-Telangiectasia Mutated Effectors Independent of DNA Strand Breaks and Heat Shock Protein 70 Status

All cells have intricately coupled sensing and signaling mechanisms that regulate the cellular outcome following exposure to genotoxic agents such as ionizing radiation (IR). In the IR-induced signaling pathway, specific protein events, such as ataxia-telangiectasia mutated protein (ATM) activation and histone H2AX phosphorylation (gamma-H2AX), are mechanistically well characterized. How these mechanisms can be altered, especially by clinically relevant agents, is not clear. Here we show that hyperthermia, an effective radiosensitizer, can induce several steps associated with IR signaling in cells. Hyperthermia induces gamma-H2AX foci formation similar to foci formed in response to IR exposure, and heat-induced gamma-H2AX foci formation is dependent on ATM but independent of heat shock protein 70 expression. Hyperthermia also enhanced ATM kinase activity and increased cellular ATM autophosphorylation. The hyperthermia-induced increase in ATM phosphorylation was independent of Mre11 function. Similar to IR, hyperthermia also induced MDC1 foci formation; however, it did not induce all of the characteristic signals associated with irradiation because formation of 53BP1 and SMC1 foci was not observed in heated cells but occurred in irradiated cells. Additionally, induction of chromosomal DNA strand breaks was observed in IR-exposed but not in heated cells. These results indicate that hyperthermia activates signaling pathways that overlap with those activated by IR-induced DNA damage. Moreover, prior activation of ATM or other components of the IR-induced signaling pathway by heat may interfere with the normal IR-induced signaling required for chromosomal DNA double-strand break repair, thus resulting in increased cellular radiosensitivity.

Mammalian Rad9 Plays a Role in Telomere Stability, S- and G2-Phase-Specific Cell Survival, and Homologous Recombinational Repair

ABSTRACT The protein products of several rad checkpoint genes of Schizosaccharomyces pombe (rad1+, rad3 +, rad9 +, rad17 +, rad26 +, and hus1 +) play crucial roles in sensing changes in DNA structure, and several function in the maintenance of telomeres. When the mammalian homologue of S. pombe Rad9 was inactivated, increases in chromosome end-to-end associations and frequency of telomere loss were observed. This telomere instability correlated with enhanced S- and G2-phase-specific cell killing, delayed kinetics of γ-H2AX focus appearance and disappearance, and reduced chromosomal repair after ionizing radiation (IR) exposure, suggesting that Rad9 plays a role in cell cycle phase-specific DNA damage repair. Furthermore, mammalian Rad9 interacted with Rad51, and inactivation of mammalian Rad9 also resulted in decreased homologous recombinational (HR) repair, which occurs predominantly in the S and G2 phases of the cell cycle. Together, these findings provide evidence of roles for mammalian Rad9 in telomere stability and HR repair as a mechanism for promoting cell survival after IR exposure.

Evidence for two states of thermotolerance in mammalian cells

The effect of the inhibition of protein synthesis on the development of thermotolerance in Chinese hamster fibroblasts following a brief heat shock or exposure to sodium arsenite has been examined. Under conditions that inhibit protein synthesis by 95 per cent, significant amounts of thermotolerance develop after a brief exposure to 45 degrees C or continuous exposure to 41 degrees C, without the significant accumulation of heat shock proteins. However, no thermotolerance development in cells treated with sodium arsenite was observed if protein synthesis was inhibited. Heated cells which developed thermotolerance in the absence of protein synthesis are subject to the thermal sensitizing action of subsequent exposure to amino acid analogues, while cells which developed thermotolerance with unimpeded protein synthesis are refractory. These results suggest that heat can simultaneously induce two states of thermotolerance, only one of which is dependent on protein synthesis. These two states can be distinguished operationally with respect to their response to amino acid analogue exposure.

The relationship of heat-shock proteins, thermotolerance, and protein synthesis.

The relationship of heat-induced inhibition of protein synthesis (HIIPS) and thermotolerance, the transient ability to survive otherwise lethal heat treatments, was studied in HA-1 Chinese hamster fibroblasts exposed to various treatments. A mild heatshock or exposure to sodium arsenite induced a refractoriness to HIIPS, while exposure to the amino acid analog of proline, azetidine, did not. The development and decay of refractoriness to HIIPS after exposure to heat or sodium arsenite paralleled in the increase and decrease of the rate of synthesis of the heat-shock proteins (HSP), and was associated with neither the persistence of elevated levels of HSP nor the persistence of the thermotolerant state. Refractoriness to HIIPS was not associated with the elevated synthesis of HSP in the presence of amino acid analogs regardless of the mode of induction, indicating a requirement for functional HSP for the effect. The refractoriness to HIIPS was also found in heat-resistant variants of HA-1 cells that express elevated levels of hsp 70, implicating a role for this protein in this process. Our observation establish an unique biological effect associated with the period of elevated synthesis of the HSP, especially the hsp 70.

The relationship between hsp 70 localization and heat resistance.

Using indirect immunofluorescence we have investigated the kinetics of nuclear accumulation and removal of hsp 70 in HA-1 Chinese hamster fibroblasts exposed to elevated temperatures. The kinetics of accumulation of hsp 70 in the nuclei were found to be time/temperature dependent at all temperatures tested (42-45 degrees C). At a given temperature, the fraction of cells manifesting nuclear localization of hsp 70 increased with exposure time. For a given duration of heating, the fraction of cells manifesting nuclear localization of hsp 70 increased with the temperature. The kinetics of the nuclear accumulation of hsp 70 were similar for normal HA-1 cells, their heat-resistant variants, and transiently thermotolerant cells (triggered by prior exposure to a brief heat shock or to sodium arsenite). Upon return to 37 degrees C after heat shock, the kinetics of removal of the hsp 70 associated with the nucleus was dependent on the severity of the initial heat challenge. However, for a given heat dose, the decay of nuclear localization of hsp 70 was more rapid in thermotolerant and heat-resistant cells than in their normal counterparts. These results suggest that the increased levels of hsp 70 associated with the transient or permanently heat-resistant state may play a direct role in restoring and/or repairing heat-induced nuclear and nucleolar alterations associated with heat-induced cell killing. Furthermore, they also suggest that the heat-resistant state may involve ameliorated repair of heat-induced cellular alterations.

The thermoresistant state: Protection from initial damage or better repair?

The induction of and recovery from heat-induced perturbations in several cellular parameters were examined in normal, transiently thermotolerant, and permanently heat-resistant HA-1 Chinese hamster fibroblasts. The initial heat-induced perturbations in total cellular protein synthesis, RNA synthesis, vimentin-containing intermediate filaments, and nuclear protein mass were similar in the three different cell types which display various levels of thermal resistance as determined by clonogenic survival. The posthyperthermia recovery from the heat-induced perturbations in all of the cellular parameters was more rapid in both the permanently heat-resistant cells and in the transiently thermotolerant cells. This response was observed in cells in which transient thermotolerance was induced by either a mild heat shock or exposure to sodium arsenite. The development and decay of the capacity for more rapid recovery from the initial heat-induced perturbations in total cellular protein and RNA synthesis paralleled the development and decay of clonogenic thermotolerance. Overall, these results support the notion that more rapid recovery from similar levels of heat-induced perturbations in various cellular parameters are a salient feature of both the transiently and permanently heat-resistant state.

Heat-induced perturbations of DNA damage signaling pathways are modulated by molecular chaperones.

Heat is one of the most potent radiosensitizers known. Several randomized trials have shown that hyperthermia is a good adjuvant for radiotherapy at several different cancer sites. However, the mechanism(s) involved in the interaction of heat and radiation that lead to radiosensitization remain to be elucidated. In this report, we have determined that heat induces perturbations in some of the earliest events in the cellular response to DNA damage induced by ionizing radiation. We studied the effect of heat on the formation of complexes containing gamma-H2AX/MDC1/53BP1 in heated-irradiated cells. We found that the formation of this complex was delayed in heated-irradiated cells, in a heat but not radiation dose-dependent manner. The length of the heat-induced delay of complex formation was attenuated in thermotolerant and heat radiosensitization-resistant cells. The length of the delay of gamma-H2AX/MDC1/53BP1 complex formation correlated with the magnitude of heat radiosensitization and was modulated by the molecular chaperone Hsc70. Heat radiosensitization was attenuated in 53BP1-null cells, implying that the delay of the formation of the gamma-H2AX/MDC1/53BP1 complex plays a role in heat radiosensitization. Heat also induced a delay of events in the DNA damage response that are downstream from 53BP1. Our results support the notion that heat-induced perturbations in the earliest events of the cellular response to ionizing radiation-induced DNA damage play a role in heat radiosensitization.

Novel chemical enhancers of heat shock increase thermal radiosensitization through a mitotic catastrophe pathway.

Radiation therapy combined with adjuvant hyperthermia has the potential to provide outstanding local-regional control for refractory disease. However, achieving therapeutic thermal dose can be problematic. In the current investigation, we used a chemistry-driven approach with the goal of designing and synthesizing novel small molecules that could function as thermal radiosensitizers. (Z)-(+/-)-2-(1-Benzenesulfonylindol-3-ylmethylene)-1-azabicyclo[2.2.2]octan-3-ol was identified as a compound that could lower the threshold for Hsf1 activation and thermal sensitivity. Enhanced thermal sensitivity was associated with significant thermal radiosensitization. We established the structural requirements for activity: the presence of an N-benzenesulfonylindole or N-benzylindole moiety linked at the indolic 3-position to a 2-(1-azabicyclo[2.2.2]octan-3-ol) or 2-(1-azabicyclo[2.2.2]octan-3-one) moiety. These small molecules functioned by exploiting the underlying biophysical events responsible for thermal sensitization. Thermal radiosensitization was characterized biochemically and found to include loss of mitochondrial membrane potential, followed by mitotic catastrophe. These studies identified a novel series of small molecules that represent a promising tool for the treatment of recurrent tumors by ionizing radiation.

The Heat-Shock Factor is not Activated in Mammalian Cells Exposed to Cellular Phone Frequency Microwaves

Abstract Laszlo, A., Moros, E. G., Davidson, T., Bradbury, M., Straube, W. and Roti Roti, J. The Heat-Shock Factor is not Activated in Mammalian Cells Exposed to Cellular Phone Frequency Microwaves. Radiat. Res. 164, 163–172 (2005). There has been considerable interest in the biological effects of exposure to radiofrequency electromagnetic radiation, given the explosive growth of cellular telephone use, with the possible induction of malignancy being a significant concern. Thus the determination of whether nonthermal effects of radiofrequency electromagnetic radiation contribute to the process leading to malignancy is an important task. One proposed pathway to malignancy involves the induction of the stress response by exposures to cell phone frequency microwaves. The first step in the induction of the stress response is the activation of the DNA-binding activity of the specific transcription factor involved in this response, the heat-shock factor (HSF). The DNA-binding activity of HSF in hamster, mouse and human cells was determined after acute and continuous exposures to frequency domain multiple access (FDMA)- or code domain multiple access (CDMA)-modulated microwaves at low (0.6 W/kg) or high (∼5 W/kg) SARs at frequencies used for mobile communication. The DNA-binding activity of HSF was monitored using a gel shift assay; the calibration of this assay indicated that an increase of ∼10% in the activation of the DNA-binding activity of HSF after a 1°C increase in temperature could be detected. We failed to detect any increase in the DNA-binding ability of HSF in cultured mammalian cells as a consequence of any exposure tested, within the sensitivity of our assay. Our results do not support the notion that the stress response is activated as a consequence of exposure to microwaves of frequencies associated with mobile communication devices.

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.