Hyperthermic killing and hyperthermic radiosensitization in Chinese hamster ovary cells: effects of pH and thermal tolerance

To quantitatively relate heat killing and heat radiosensitization, asynchronous or G1 Chinese hamster ovary (CHO) cells at pH 7.1 or 6.75 were heated and/or X-irradiated 10 min later. Since no progression of G1 cells into S phase occurred during the heat and radiation treatments, cell cycle artifacts were minimized. However, results obtained for asynchronous and G1 cells were similar. Hyperthermic radiosensitization was expressed as the thermal enhancement factor (TEF), defined as the ratio of the D0 of the radiation survival curve to that of the D0 of the radiation survival curve for heat plus radiation. The TEF increased continuously with increased heat killing at 45.5 degrees C, and for a given amount of heat killing, the amount of heat radiosensitization was the same for both pH's. When cells were heated chronically at 42.4 degrees C at pH 7.4, the TEF increased initially to 2.0-2.5 and then returned to near 1.0 during continued heating as thermal tolerance developed for both heat killing and heat radiosensitization. However, the shoulder (Dq) of the radiation survival curve for heat plus radiation did not manifest thermal tolerance; i.e., it decreased continuously with increased heat killing, independent of temperature, pH, or the development of thermotolerance. These results suggest that heat killing and heat radiosensitization have a target(s) in common (TEF results), along with either a different target(s) or a difference in the manifestation of heat damage (Dq results). For clinical considerations, the interaction between heat and radiation was expressed as (1) the thermal enhancement ratio (TER), which is the dose of X rays alone divided by the dose of X rays combined with heat to obtain an isosurvival, e.g., 10(-4), and (2) the thermal gain factor (TGF), the ratio of the TER at pH 6.75 to the TER at pH 7.4. Since low pH reduced the rate of development of thermal tolerance during heating at low temperatures, low pH enhanced heat killing more at 42-42.5 degrees C than at 45.5 degrees C where thermal tolerance did not develop. Therefore, the increase in the TGF after chronic heating at 42-42.5 degrees C was greater than after acute heating at 45.5 degrees C, due primarily to the increase in heat killing causing an even greater increase in heat radiosensitization. These findings agree with animal experiments suggesting that in the clinic, a therapeutic gain for tumor cells at low pH may be greater for temperatures of 42-42.5 degrees C than of 45.5 degrees C.     

Hyperthermic radiosensitization of thermotolerant Chinese hamster ovary cells

Synchronous G1 cells were given a priming dose of heat (45.5 degrees C for 15 min) and then heated and irradiated 6-120 h later. Compared to heat radiosensitization for cells irradiated 10 min after the priming heat dose (thermal enhancement ratio, TER of 2.6 for a 10-fold reduction in survival), heat radiosensitization 18-24 h after the priming heat dose was less (i.e., TER of 1.6 for radiation at 24 h compared with heat-radiation at 24 h). A thermotolerance ratio (TTR) at 24 h was calculated to be 2.6/1.6 = 1.6. TERs at 100-fold or 1000-fold reduction in survival and ratios of slopes of radiation survival curves also showed that the cells developed a similar amount of thermotolerance for heat radiosensitization at 18-24 h. Furthermore, since the TER for heat radiosensitization increased with heat killing either from the priming heat dose or the second heat dose in a similar manner for single or fractionated doses, the TER for nonthermotolerant and thermotolerant cells was the same when related to the heat damage (i.e., amount of killing from heat alone). When the radiation response of cells heated and irradiated 6-120 h after the priming heat dose was compared with the response of cells receiving radiation only, changes in TER as a function of time after the initial priming heat dose were shown to involve: recovery of heat damage interacting with the subsequent radiation dose, thermotolerance for heat radiosensitization, and redistribution of cells surviving the first heat dose into radioresistant phases of the cell cycle. In fact, redistribution resulted in a minimal TER at 72 h for heat-radiation compared with radiation alone, instead of at 24 h where maximal thermotolerance for heat killing was observed [P. K. Holahan and W. C. Dewey, Radiat. Res. 106, 111 (1986)]. These observations are discussed relative to clinical considerations and similar results reported from in vivo experiments.     

Effect of thermotolerance on thermal radiosensitization in hepatoma cells

The interaction between hyperthermia and X irradiation was determined in cultured Reuber H35 hepatoma cells with different states of thermosensitivity. Incubation at 41 degrees C followed by 4-Gy X rays resulted after 2 hr in a stabilization of cell survival for heat or plus X rays, with a maximum synergism factor of 1.6. Thermotolerance did not develop during incubation at 41.7 or 42.5 degrees C. When heat treatment of cells was followed by irradiation, the synergism factor for thermal radiosensitization increased with both the amount of thermal cell killing and the amount of X-ray cell killing; the influence of thermal exposure on the synergism factor was greater than that of the X-ray dose. Cells were made thermotolerant either by incubation at 42.5 degrees C for 30 or 60 min followed by an interval at 37 degrees C, or by continuous incubation at 41 degrees C. In both cases thermotolerance was measured by incubation at 42.5 degrees C. No difference was observed between the maximum thermotolerance achieved with both methods. When cells were irradiated in addition to the second heat treatment, thermal radiosensitization was strongly reduced concomitant with the decreased sensitivity to killing by heat.     

Effects of a perfluorochemical emulsion on the response of BA1112 rat rhabdomyosarcomas to continuous low-dose-rate irradiation

The effects of the combination of a perfluorochemical emulsion (Fluosol DA, 20%) and carbogen (95% O2, 5% CO2) on the response of BA1112 rat rhabdomyosarcomas to continuous low-dose-rate irradiation were examined. Tumors were irradiated locally in unrestrained, unanesthetized rats at a dose rate of 0.98 Gy/h, using a specially designed 241Am irradiator system. Cell survival was measured using a colony formation assay. The tumor cell survival curves were fitted to linear relationships of the form ln S = - alpha D, where alpha for air-breathing rats was 0.104 +/- 0.005 Gy-1, as compared to 0.137 +/- 0.009 Gy-1 for rats treated with Fluosol plus carbogen. The increase in the slope of the survival curve produced by the treatment with Fluosol and carbogen was highly significant with a P value of 0.0015. The radiosensitization factor for the combination of Fluosol/carbogen plus continuous low-dose-rate irradiation was 1.32 +/- 0.11. Slightly less radiosensitization was observed with continuous low-dose-rate irradiation than in previous experiments using acute high-dose-rate irradiation. The diminished sensitization with Fluosol/carbogen during continuous low-dose-rate irradiation probably reflects the intrinsically lower oxygen enhancement ratio (OER) of low-dose/low-dose-rate irradiation, reoxygenation of the tumors during the prolonged treatment times used for continuous low-dose-rate irradiation, and the decrease in the levels of circulating perfluorochemicals during the 30-h irradiations. More importantly, the significant level of radiosensitization observed in the experiments with continuous low-dose-rate irradiation suggests that hypoxic cells persist in BA1112 tumors during continuous low-dose-rate irradiations and that the response of these tumors to continuous low-dose-rate irradiation can be improved by adjunctive treatments which oxygenate these radioresistant hypoxic tumor cells.     

Influence of hyperthermia on gamma-ray-induced mutation in V79 cells

Asynchronously growing V79 cells were assayed for mutation induction following exposure to hyperthermia either immediately before or after being irradiated with 60Co gamma rays. Hyperthermia exposures consisted of either 43.5 degrees C for 30 min or 45 degrees C for 10 min. Each of these heat treatments resulted in a survival level of 42%. For all sequences of combined treatment with hyperthermia and radiation, cell killing by gamma rays was enhanced. Mutation induction by gamma rays was enhanced when heat preceded gamma irradiation, but no increase was observed when heat was given after gamma exposures. Treatment at 45 degrees C for 10 min gave a higher yield in mutants at all gamma doses studied compared to treatment at 43.5 degrees C for 30 min. When heat-treated cells were incubated for different periods before being exposed to gamma rays, thermal enhancement of radiation killing was lost after 24 h. In contrast, only 5-6 h incubation was needed for loss of mutation induction enhancement.     

p53-Mediated gene activation in mice at high doses of chronic low-dose-rate γ radiation

The time course of the changes in the expression of p53-mediated genes in vivo after high doses of chronic low-dose-rate γ radiation remains unclear. Here we analyzed peripheral blood cell counts and the expression of p53-mediated genes in the spleens of mice chronically irradiated at low dose rate (0.0167 Gy/h) for 1-40 days. Low-dose-rate irradiation induced p53-dependent chronic decreases in white blood cell (WBC) counts in p53 wild-type mice. Upregulation of p53-mediated genes by low-dose-rate radiation was confirmed in the whole spleen cells from the p53 wild-type mice, while suppressed gene expression was observed in the spleen cells of p53-deficient mice. The expression of p21 and Bax in radiosensitive cells such as T and B lymphocytes from low-dose-rate irradiated mice at 10, 20, and 40 days were increased, although that of Mdm2 in both the lymphocytes was decreased at 20 and 40 days. Moreover, spleen weights for low-dose-rate irradiated mice were decreased at 20 and 40 days. Thus downregulation of Mdm2 in both T and B lymphocytes by low-dose-rate radiation may cause higher p53 activation; further, higher p53 expression may determine the radiosensitivity and cause a reduction in the spleen weights in low-dose-rate irradiated mice. These results indicate that p53 may be chronically activated by low-dose-rate radiation.     

The effect of combined heat and ultrasound on multicellular tumour spheroids

The effect of combined ultrasound and heat treatments on Chinese hamster multicellular spheroids of varying size was investigated using growth rate, single cell survival and ultrastructural damage as endpoints. Ultrasonic irradiation at 37 degrees C had no effect on the growth rate of 200-730 microns spheroids. Similarly there was no effect on the growth rate of 350 microns spheroids when irradiated during a 60 min exposure to 41.5 degrees C. However, spheroids of 200-700 mm diameter showed growth delay when held at 43 degrees C for 1 h. The effect was enhanced with concomitant ultrasound irradiation but was not dependent on spheroid size. When 200 and 400 microns spheroids held at 43 degrees C for 60 min were irradiated with different ultrasonic intensities a dose-dependent decrease in surviving fraction and a dose-dependent increase in growth delay was obtained. When surviving fraction was plotted as a function of growth delay a good correlation was obtained, suggesting that the combination of heat and ultrasound irradiation does not produce cytostasis in the surviving cells of either 200 or 400 microns spheroids. At the ultrastructural level increased cytoplasmic vacuolation was the only result of ultrasonic irradiation at 37 degrees C. Exposure to 43 degrees C for 60 min was required to elicit thermal damage. This took the form of membrane evagination at the spheroid surface, vacuolation of the cytoplasm, grouping of organelles around the periphery of the nucleus, and fragmentation of the nucleolus. These effects were enhanced with concomitant ultrasonic irradiation but other features were also noted, viz. disaggregation of polyribosomes, dilation of the rough endoplasmic reticulum and blebbing of the nuclear membrane. Damage was independent of spheroid size. These results are in agreement with previous data obtained from single-cell studies. Indicating that there is a non-thermal, non-cavitational component to the cell killing in multicellular spheroids resulting from combined heat and ultrasound treatment.     

The rate of progression of radiation-transformed mammary epithelial cells is enhanced after low-dose-rate neutron irradiation

Studies in this laboratory have shown enhancement of the mammary tumorigenic effects of neutron irradiation after low-dose-rate neutron exposures. To investigate possible reasons, a mammary cell system was used which allows quantitation of initiated mammary epithelial cells and examination of the progression of these radiation-altered cells toward the neoplastic phenotype. Female BALB/c mice were irradiated with fission-spectrum neutrons at dose rates of 1 rad/min or 1 rad/day. Twenty-four hours or 16 weeks after irradiation, mammary cells were obtained by enzymatic dissociation. Mammary outgrowths were derived by injection of 10(4) cells into gland-free fat pads of 3-week-old female BALB/c mice. The frequency of ductal dysplasias in outgrowths from cells irradiated at high or low dose rates was similar. Persistence of dysplasias differed markedly. Few of the dysplasias in outgrowths derived from cells irradiated at the high dose rate persisted, while a large fraction of the dysplasias in outgrowths derived from cells irradiated at low dose rate persisted. When cells remained in situ for 16 weeks prior to dissociation a higher frequency of persistent altered cells was also observed in outgrowths derived from cells irradiated at low neutron dose rates. These data suggest that low-dose-rate neutron exposures enhance the probability of progression of carcinogen-altered cells rather than increase the numbers of initiated cells.     

Thermal radiosensitization and thermotolerance in cultured cells from a murine mammary carcinoma

Cultured murine mammary carcinoma cells M8013 could be made thermotolerant by a priming heat treatment, 30 min at 43 degrees C, applied 5 h prior to subsequent heat treatment. The sensitivity of non-tolerant and thermotolerant cells to either radiation or heat combined with radiation was investigated. Analysis of survival curves with respect to D0 and N showed that thermotolerance had no influence on the radiation sensitivity of the cells. Thermal enhancement of radiation effects (in combined heat/irradiation treatments) was however reduced as a result of thermotolerance. When thermal enhancement ratios were (D0) plotted as a function of the cell killing effects of heat treatment alone, thermotolerance did not seem to have any influence. This latter observation suggests that thermotolerance modifies the effectiveness of the heat treatment for heat-induced cell lethality and radiosensitization equally. Comparison of our in vitro results with several in vivo data on normal tissues suggest that the reduction in 'effective' treatment temperature which has been observed in the in vivo studies as a result of thermotolerance may be explained by equal modification of the effects of heat by thermotolerance both for its direct effects and the radiosensitization.     

Inhibition of gamma-ray dose-rate effects by D2O and inhibitors of poly(ADP-ribose) synthetase in cultured mammalian L5178Y cells

Effects of deuterium oxide (D2O) and 3-aminobenzamide, an inhibitor of poly(ADP-ribose) synthetase, on cell proliferation and survival were studied in cultured mammalian L5178Y cells under growing conditions and after acute and low-dose-rate irradiation at about 0.1 to 0.4 Gy/hr of gamma rays. Growth of irradiated and unirradiated cells was inhibited by 45% D2O but not by 3-aminobenzamide at 10 mM, except for treatments longer than 30 hr. The presence of these agents either alone or in combination during irradiation at low dose rates suppressed almost totally the decrease in cell killing due to the decrease in dose rate. The D2O did not inhibit the radiation-induced increase in poly(ADP-ribose) synthesis as measured by the incorporation of [14C]NAD into the acid insoluble fraction, contrary to 3-aminobenzamide. Among other inhibitors tested, theobromine and theophylline were found to be effective in eliminating the dose-rate effects of gamma rays. Possible mechanisms underlying the inhibition are discussed.     

Hyperthermia in a differentiating murine erythroleukemia cell line: cell killing by heat and radiation

The survival response of Friend erythroleukemia cells (a differentiating cell system) to heat and radiation has been examined. The Friend erythroleukemia cells (FELC) were more heat and radiation sensitive than V79 cells, and the heat and radiation survival curves possessed shoulders, showing the ability of the cells to accumulate sublethal damage. Thermal tolerance was expressed after prolonged heating at 41.0-42.0 degrees C. Thermal radiosensitization by heating at 42.0 or 45.0 degrees C was greatest for simultaneous heat and radiation treatments, and recovery occurred when the cells were incubated at 37 degrees C between the heat and radiation or radiation and heat treatments. Arrhenius analysis of the FELC heat survival data showed that the curve for thermal inactivation possessed a break at about 43.0 degrees C and that the thermal inactivation energies above and below the break point were comparable to those for V79 cells and other cell lines reported in the literature.     

Effect of cycloheximide on heat-induced cell killing, radiosensitization, and loss of cellular DNA polymerase activities in Chinese hamster ovary cells

A whole-cell assay technique for DNA polymerase alpha and beta was used to measure the activities of both enzymes in Chinese hamster ovary cells after hyperthermic treatment at 43 degrees C in the presence or absence of 10 micrograms/ml cycloheximide (CHM). In the same experiments, the effect of CHM on heat killing and heat radiosensitization was also investigated. CHM treatment before and during heating protected the cells for all three end points, i.e., heat-induced cell killing, radiosensitization, and loss of cellular DNA polymerase activities.     

Differences in heat-induced cell killing as determined in three mammalian cell lines do not correspond with the extent of heat radiosensitization

Three different cell lines, Ehrlich ascites tumour (EAT) cells, HeLa S3 cells and LM mouse fibroblasts, were used to investigate whether or not the extent of heat killing (44 degrees C) and heat radio-sensitization (44 degrees C before 0-6 Gy X-irradiation) are related. Although HeLa cells were the most heat-resistant cell line and showed the least heat radiosensitization, we found that the most heat-sensitive EAT cells (D0, EAT = 8.0 min; D0, LM = 10.0 min; D0, HeLa = 12.5 min) showed less radiosensitization than the more heat-resistant LM fibroblasts (TERHeLa less than TEREAT less than TERLM). Therefore, it is concluded that the routes leading to heat-induced cell death are not identical to those determining heat radiosensitization. Furthermore the inactivation of DNA polymerase alpha and beta activities by heat seemed not to correlate with heat survival alone but showed a positive relationship to heat radiosensitization. The possibility of these enzymes being a determinant in heat radiosensitization is discussed.     

Identification of Mre11 as a target for heat radiosensitization

Thermal radiosensitization is believed to be mediated by an inhibition of double-strand break (DSB) repair, but the exact mechanism of radiosensitization remains to be elucidated. Previously, we demonstrated that proteins of the Mre11/Rad50/Nbs1 complex (MRN) translocate from the nucleus to the cytoplasm in cells have that been heated or heated and then irradiated; this finding led us to propose that heat radiosensitization was due at least in part to translocation of MRN. In the current study, we used leptomycin B to inhibit MRN translocation in heated, irradiated cells, but we found that heat radiosensitization was not altered. Thus enhanced radiosensitivity was not attributed to translocation of MRN proteins. To determine which of the MRN subunits contributed to heat radiosensitization, we compared the extent of heat radiosensitization in wild-type cells with that of cells hypomorphic for Mre11 or Nbs1 or cells in which the level of Rad50 was suppressed. We found that neither Nbs1 nor Rad50 is involved in heat radiosensitization, because a similar amount of heat radiosensitization was observed in cells deficient in those proteins compared to cells expressing normal levels. However, heat radiosensitization was not observed in A-TLD1 cells deficient in Mre11. Measurement of exonuclease activity of purified Mre11 heated at 42.5°C or 45.5°C indicated that the protein is very heat-labile. Immunoprecipitation of Mre11 from heated HeLa cells also revealed that hsp70 associates with Mre11 and that this association is maintained long after heating. Taken together, these findings implicate Mre11 as a target for heat radiosensitization and suggest that heat radiosensitization and inhibition of DSB repair may be mediated by heat-induced conformational changes in Mre11.     

Ultrastructural study of mitochondrial damage in CHO cells exposed to hyperthermia

A unique direct-view stereo electron microscope technique was used to visualize the structure and three-dimensional distributions of mitochondria in CHO cells in situ following hyperthermic treatments. Aberrations induced by various heating regimens were recorded. The protocol included a trypsin digestion that may have enhanced the expression of the initial heat damage. The developed damage was observed as increasing levels of mitochondrial distortion, swelling, and dissociation. Minimal damage was induced at 42 degrees C for exposures of up to 4 h, while significant damage was induced at 43 degrees C for exposures of more than 30 min and at 45 degrees C for exposures of more than 10 min. For moderate exposures, a partial recovery of mitochondrial integrity was observed when the heat treatment was followed by incubation at 37 degrees C for 24 h. Mitochondrial damage was related to the heat dose in that increasing treatment temperature resulted in greater damage, but when compared to cell survival the damage did not parallel cell killing under all time-temperature conditions.     

The v-src oncogene may not be responsible for the increased radioresistance of hematopoietic progenitor cells expressing v-src.

Infection of the IL-3-dependent, myeloid progenitor cell line 32D cl 3 with murine retroviruses that contain either the wild-type or a temperature-sensitive mutant v-src can render these cells growth-factor independent. These cells also became resistant to gamma irradiation administered at the low-dose rate of 0.05 Gy/min, which is used clinically. The v-src-dependent nature of resistance to gamma irradiation was examined by studying four clones of 32D cl 3 cells that had been infected with a retrovirus carrying the tsLA31A mutant of v-src. The tyrosine-specific kinase activity of this mutant is dramatically reduced at the nonpermissive temperature of 39 degrees C. Cells transformed by v-src and grown at either 34 or 39 degrees C, in the presence or absence of IL-3, demonstrated a significantly higher D0 compared to parental cells examined under identical conditions. In addition, expression of v-src abrogated the synergistic killing effect of heat and gamma irradiation. The D0 of parental 32D cl 3 cells kept at 39 degrees C after gamma irradiation was reduced significantly compared to the D0 of these cells kept at 34 degrees C. This contrasts with data from 32D cl 3 cells infected with either the wild-type v-src or the temperature-sensitive mutant, neither exhibited a synergistic effect in the D0 at either 34 or 39 degrees C. Therefore, while continuous expression of a v-src gene product is required for maintenance of the growth-factor-independent state, v-src does not appear to be responsible for the increased gamma-radiation resistance of these cells at low dose rate.     

Thermal adaptation in CHO cells at 40 degrees C: the influence of growth conditions and the role of heat shock proteins

The kinetics of thermal adaptation at the nonlethal temperature of 40 degrees C was studied in CHO (Chinese hamster ovary) cells in vitro. Thermal resistance, demonstrated as an increase in mean 45 degrees C killing time or as an increase in the shoulder of the 45 degrees C survival curve, was fully developed by 2 h. Control cells in early logarithmic phase were more heat sensitive than those in stationary phase. Corresponding 45 degrees C killing time frequency distributions were unimodal with an increase in mean killing time from early logarithmic to stationary phase. Cells which were thermally adapted at 40 degrees C for 6 h had biphasic 45 degrees C killing time frequency distributions, and as cells progressed from early logarithmic to stationary phase the heat-sensitive subpopulation progressively declined. Exposure to 40 degrees C produced a 30% increase in total protein synthesis. Proteins with molecular weights 72, 89, and 109 kDa which correspond to those induced by lethal heat shock were synthesized at 40 degrees C, but there was no close temporal correlation between the development of heat resistance at 40 degrees C and synthesis of the heat shock proteins. Cycloheximide (100 micrograms/ml) reduced the mean 45 degrees C killing time but did not totally prevent the development of heat resistance at 40 degrees C.     

DNA repair defect in AT cells and their hypersensitivity to low-dose-rate radiation

Ataxia telangiectasia (AT) and normal cells immortalized with the human telomerase gene were irradiated in non-proliferative conditions with high- (2 Gy/min) or low-dose-rate (0.3 mGy/min) radiation. While normal cells showed a higher resistance after irradiation at a low dose rate than a high dose rate, AT cells showed virtually the same survival after low- and high-dose-rate irradiation. Although the frequency of micronuclei induced by low-dose-rate radiation was greatly reduced in normal cells, it was not reduced significantly in AT cells. The number of gamma-H2AX foci increased in proportion to the dose in both AT and normal cells after high-dose-rate irradiation. Although few gamma-H2AX foci were observed after low-dose-rate irradiation in normal cells, significant and dose-dependent numbers of gamma-H2AX foci were observed in AT cells even after low-dose-rate irradiation, indicating that DNA damage was not completely repaired during low-dose-rate irradiation. Significant phosphorylation of ATM proteins was detected in normal cells after low-dose-rate irradiation, suggesting that the activation of ATM plays an important role in the repair of DNA damage during low-dose-rate irradiation. In conclusion, AT cells may not be able to repair some fraction of DNA damage and are severely affected by low-dose-rate radiation.