Joseph L. Roti

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

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.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.

The effects of hyperthermia on the protein-to-DNA ratio of isolated HeLa cell chromatin.

HeLa cells were heated for a fixed time interval, 30 min, at various temperatures ranging from 40 to 48°C; or heated at a fixed temperature, 45°C, for various times between 7.5 and 90 min. After heating, chromatin was isolated and a heat-dependent increase in the protein to DNA ratio of the chromatin was found. After 30 min of heat at 45°C, the protein to DNA ratio increased 1.57 ± 0.08-fold relative to control. No significant DNA degradation was found with these heat treatments. The increase in protein content was biphasic with increasing time at 45°C or increasing temperature. If cells were incubated at 37°C following hyperthermia at 45°C for either 15 or 30 min, the protein to DNA ratio of control cells was restored to normal within 3 hr following 15 min of heat or 15 hr following 30 min of heat. During the heating and recovery periods, there was no measurable increase in fraction of trypan blue stainable cells. To determine if these additional proteins in isolated chromatin were present in situ, we co...

Measurement of DNA Damage after Exposure to Electromagnetic Radiation in the Cellular Phone Communication Frequency Band (835.62 and 847.74 MHz)

Mouse C3H 10T1/2 fibroblasts and human glioblastoma U87MG cells were exposed to cellular phone communication frequency radiations to investigate whether such exposure produces DNA damage in in vitro cultures. Two types of frequency modulations were studied: frequency-modulated continuous-wave (FMCW), with a carrier frequency of 835.62 MHz, and code-division multiple-access (CDMA) centered on 847.74 MHz. Exponentially growing (U87MG and C3H 10T1/2 cells) and plateau-phase (C3H 10T1/2 cells) cultures were exposed to either FMCW or CDMA radiation for varying periods up to 24 h in specially designed radial transmission lines (RTLs) that provided relatively uniform exposure with a specific absorption rate (SAR) of 0.6 W/kg. Temperatures in the RTLs were monitored continuously and maintained at 37 +/- 0.3 degrees C. Sham exposure of cultures in an RTL (negative control) and 137Cs gamma-irradiated samples (positive control) were included with every experiment. The alkaline comet assay as described by Olive et al. (Exp. Cell Res. 198, 259-269, 1992) was used to measure DNA damage. No significant differences were observed between the test group exposed to FMCW or CDMA radiation and the sham-treated negative controls. Our results indicate that exposure of cultured mammalian cells to cellular phone communication frequencies under these conditions at an SAR of 0.6 W/kg does not cause DNA damage as measured by the alkaline comet assay.

DNA Damage in Rat Brain Cells after In Vivo Exposure to 2450 MHz Electromagnetic Radiation and Various Methods of Euthanasia

The present study was done to confirm the reported observation that low-intensity acute exposure to 2450 MHz radiation causes DNA single-strand breaks (Lai and Singh, Bioelectromagnetics 16, 207-210, 1995). Male Sprague-Dawley rats weighing approximately 250 g were irradiated with 2450 MHz continuous-wave (CW) microwaves for 2 h at a specific absorption rate of 1.2 W/kg in a cylindrical waveguide system (Guy et al., Radio Sci. 14, 63-74, 1979). There was no associated rise in the core body temperature of the rats. After the irradiation or sham treatments, rats were euthanized by either CO2 asphyxia or decapitation by guillotine (eight pairs of animals per euthanasia group). After euthanasia the brains were removed and immediately immersed in cold Ames medium and the cells of the cerebral cortex and the hippocampus were dissociated separately and subjected to the alkaline comet assay. Irrespective of whether the rats were euthanized by CO2 asphyxia or decapitated by guillotine, no significant differences were observed between either the comet length or the normalized comet moment of cells from either the cerebral cortex or the hippocampus of sham-treated rats and those from the irradiated rats. However, the data for the rats asphyxiated with CO2 showed more intrinsic DNA damage and more experiment-to-experiment variation than did the data for rats euthanized by guillotine. Therefore, the guillotine method of euthanasia is the most appropriate in studies relating to DNA damage. Furthermore, we did not confirm the observation that DNA damage is produced in cells of the rat cerebral cortex or the hippocampus after a 2-h exposure to 2450 MHz CW microwaves or at 4 h after the exposure.

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.

Measurement of DNA damage and apoptosis in Molt-4 cells after in vitro exposure to radiofrequency radiation.

Abstract Hook, G. J., Zhang, P., Lagroye, I., Li, L., Higashikubo, R., Moros, E. G., Straube, W. L., Pickard, W. F., Baty, J. D. and Roti Roti, J. L. Measurement of DNA Damage and Apoptosis in Molt-4 Cells after In Vitro Exposure to Radiofrequency Radiation. Radiat. Res. 161, 193–200 (2004). To determine whether exposure to radiofrequency (RF) radiation can induce DNA damage or apoptosis, Molt-4 T lymphoblastoid cells were exposed with RF fields at frequencies and modulations of the type used by wireless communication devices. Four types of frequency/modulation forms were studied: 847.74 MHz code-division multiple-access (CDMA), 835.62 MHz frequency-division multiple-access (FDMA), 813.56 MHz iDEN® (iDEN), and 836.55 MHz time-division multiple-access (TDMA). Exponentially growing cells were exposed to RF radiation for periods up to 24 h using a radial transmission line (RTL) exposure system. The specific absorption rates used were 3.2 W/kg for CDMA and FDMA, 2.4 or 24 mW/kg for iDEN, and 2.6 or 26 mW/kg for TDMA. The temperature in the RTLs was maintained at 37°C ± 0.3°C. DNA damage was measured using the single-cell gel electrophoresis assay. The annexin V affinity assay was used to detect apoptosis. No statistically significant difference in the level of DNA damage or apoptosis was observed between sham-treated cells and cells exposed to RF radiation for any frequency, modulation or exposure time. Our results show that exposure of Molt-4 cells to CDMA, FDMA, iDEN or TDMA modulated RF radiation does not induce alterations in level of DNA damage or induce apoptosis.

Nuclear matrix as a target for hyperthermic killing of cancer cells

The nuclear matrix organizes nuclear DNA into operational domains in which DNA is undergoing replication, transcription or is inactive. The proteins of the nuclear matrix are among the most thermal labile proteins in the cell, undergoing denaturation at temperatures as low as 43-45 degrees C, i.e. relevant temperatures for the clinical treatment of cancer. Heat shock-induced protein denaturation results in the aggregation of proteins to the nuclear matrix. Protein aggregation with the nuclear matrix is associated with the disruption of many nuclear matrix-dependent functions (e.g. DNA replication, DNA transcription, hnRNA processing, DNA repair, etc.) and cell death. Heat shock proteins are believed to bind denatured proteins and either prevents aggregation or render aggregates more readily dissociable. While many studies suggest a role for Hsp70 in heat resistance, we have recently found that nuclear localization/delocalization of Hsp70 and its rate of synthesis, but not its amount, correlate with a tumor cells ability to proliferate at 41.1 degrees C. These results imply that not only is the nuclear matrix a target for the lethal effects of heat, but it also is a target for the protective, chaperoning and/or enhanced recovery effects of heat shock proteins.

Cytogenetic Studies in Human Blood Lymphocytes Exposed In Vitro to Radiofrequency Radiation at a Cellular Telephone Frequency (835.62 MHz, FDMA)

Abstract Vijayalaxmi, Pickard, W. F., Bisht, K. S., Leal, B. Z., Meltz, M. L., Roti Roti, J. L., Straube, W. L. and Moros, E. G. Cytogenetic Studies in Human Blood Lymphocytes Exposed In Vitro to Radiofrequency Radiation at a Cellular Telephone Frequency (835.62 MHz, FDMA). Freshly collected peripheral blood samples from four healthy human volunteers were diluted with RPMI 1640 tissue culture medium and exposed in sterile T-75 tissue culture flasks in vitro for 24 h to 835.62 MHz radiofrequency (RF) radiation, a frequency employed for customer-to-base station transmission of cellular telephone communications. An analog signal was used, and the access technology was frequency division multiple access (FDMA, continuous wave). A nominal net forward power of 68 W was used, and the nominal power density at the center of the exposure flask was 860 W/m2. The mean specific absorption rate in the exposure flask was 4.4 or 5.0 W/kg. Aliquots of diluted blood that were sham-exposed or exposed in vitro to an acute dose of 1.50 Gy of γ radiation were used as negative or positive controls. Immediately after the exposures, the lymphocytes were stimulated with a mitogen, phytohemagglutinin, and cultured for 48 or 72 h to determine the extent of genetic damage, as assessed from the frequencies of chromosomal aberrations and micronuclei. The extent of alteration in the kinetics of cell proliferation was determined from the mitotic indices in 48-h cultures and from the incidence of binucleate cells in 72-h cultures. The data indicated no significant differences between RF-radiation- and sham-exposed lymphocytes with respect to mitotic indices, incidence of exchange aberrations, excess fragments, binucleate cells, and micronuclei. In contrast, the response of the lymphocytes exposed to γ radiation was significantly different from both RF-radiation- and sham-exposed cells for all of these indices. Thus, under the experimental conditions tested, there is no evidence for the induction of chromosomal aberrations and micronuclei in human blood lymphocytes exposed in vitro for 24 h to 835.62 MHz RF radiation at SARs of 4.4 or 5.0 W/kg.

Excision of X-ray-induced thymine damage in chromatin from heated cells.

Experiments were performed to distinguish between two possible modes of hyperthermia-induced inhibition of thymine base damage excision from the DNA of CHO cells: (1) heat denaturation of excision enzyme(s) or (2) heat-induced alteration of the substrate for damage excision (chromatin). While hyperthermia (45/sup 0/C, 15 min) had no apparent effect on the capacity of the excision enzymes to excise damage from DNA it had a dramatic effect (ca. 80% inhibition) on the ability of chromatin to serve as a substrate for unheated enzymes. These results suggest that hyperthermia-induced radiosensitization of CHO cells may be due primarily to lesions in the cellular chromatin.