Influence of ethidium bromide on hyperthermic modification of bleomycin-DNA interaction

The effect of hyperthermic treatment on the binding of 59Fe-labeled bleomycin to DNA has been studied. Enhanced binding was observed at elevated temperatures. The influence of the DNA-intercalating agent, ethidium bromide, on bleomycin-DNA interaction was also studied and revealed a considerable decrease in this interaction at ethidium bromide levels below 1 microgram/ml. Ethidium bromide was observed to remove the enhanced bleomycin-DNA interaction recorded previously following incubation at hyperthermic temperatures. Synergistic action of bleomycin and hyperthermia on loss of clonogenic ability of HT29R cells is reported. Incubation of cells under hyperthermic conditions with bleomycin in the presence of ethidium bromide removes this synergism, producing a less than additive effect for the action of bleomycin and heat after ethidium bromide effects are taken into account.     

Bleomycin-induced DNA-strand damage in isolated male germ cells

DNA strand damage in isolated male germ cells (MGC) was evaluated after in vitro exposure to bleomycin (BLM), a known genotoxin. The alkaline elution technique was used to determine DNA-strand breaks. Concentration-dependent strand damage was established following exposure to bleomycin for 1 h at 37 degrees C. Exposure at 0 degrees C resulted in an increase in the frequency of strand breaks as compared to those observed at 37 degrees C. Pretreatment of cells with deferoxamine (DM), an iron-selective chelating agent, abolished the DNA damage induced by bleomycin. Isolated male germ cells responded in a predictable and reproducible manner thus supporting their use in mechanistic studies of genotoxicity.     

Hyperthermia blocks DNA processing at the nuclear matrix

The capacity of control and heated HeLa cells to process newly polymerized DNA at the nuclear matrix was measured. DNA which had been pulse-labeled with [3H]thymidine was enriched by a factor of up to 6 at the cell's nuclear matrix. During continuous exposure to [3H]thymidine at 37 degrees C this enrichment for pulse-labeled DNA was reversed with a half-time of 7 min. We interpret this processing of newly replicated DNA to be a distribution of newly polymerized DNA throughout replicon-sized nuclear DNA domains. Both processing of newly polymerized DNA at the nuclear matrix and ligation of replicon clusters into the interphase cell chromosome were halted by incubation of cells at temperatures at or above 43 degrees C. When HeLa cells were pulse-labeled during a 30-min incubation at 45 degrees C and replaced at 37 degrees C, the enrichment for 3H-labeled DNA at the nuclear matrix was reversed with an initial half-time of 4 h. The results indicate that exposure of cells to hyperthermic temperatures blocks ongoing nascent DNA processing at the nuclear matrix and results in a retardation of DNA processing in preheated cells replaced at 37 degrees C.     

Apurinic site induction in the DNA of cells heated at hyperthermic temperatures

The induction of DNA damage in cells heated at hyperthermic (43-48 degrees C) temperatures was determined by alkaline filter elution and alkaline sucrose gradient-sedimentation analysis of cell DNA denatured at pH 13.0. A class of DNA lesion which converted to strand breaks during denaturation of DNA at pH 13.0 was produced randomly throughout the cell DNA at temperatures as low as 43 degrees C. Induction of this lesion occurred with a T0 of 90 and 10 min at 45 and 48 degrees C, respectively. We estimate that these pH 13.0-detectable DNA lesions are produced in the cell DNA with a frequency of approximately 75 and 660 per min of heating at 45 and 48 degrees C, respectively. Since the lesions were quantitatively converted to DNA strand breaks at pH 13.0 with a half-time of 30 min, or less, we suggest that these pH 13.0-detectable DNA lesions are heat-induced, abasic DNA sites. The induction of these lesions does not appear to be directly involved in the initial heat-induced inhibition of DNA synthesis. The presence of these lesions cannot be excluded as an explanation for the long-term inhibition of replicon initiated in heated cells.     

Hyperthermic enhancement of antibody-complement cytotoxicity against normal mouse B lymphocytes and its relation to capping

Normal mouse B lymphocytes were exposed to water-bath hyperthermia in vitro and examined for susceptibility to antibody-complement (Ab-C) cytotoxicity. Enhancement of Ab-C cytotoxicity was observed during heat treatment at 42 or 43 degrees C. Sensitivity to Ab-C cytotoxicity returned to normal levels by 2-3 hr post exposure to 42 degrees C. No such recovery was observed when cells were preheated at 43 degrees C for 40 min. The mechanism responsible for heat-induced enhancement of Ab-C cytotoxicity may be related to the way heat affects the redistribution of membrane-bound antigen-antibody (Ag-Ab) complexes. To investigate this possibility, cells were preheated at 37, 42, or 43 degrees C. The Ab-C assay was then performed at 37 degrees C immediately or 2.5 hr after hyperthermia. The distribution of Ag-Ab complexes was evaluated by immunofluorescence. A direct correlation was found between the hyperthermic enhancement of Ab-C cytotoxicity and the hyperthermic inhibition of capping, a process where membrane-bound Ag-Ab complexes coalesce into a polar cap on the cell surface. Sensitivity to Ab-C cytotoxicity returned to normal levels when cells restored the ability to cap Ag-Ab complexes following 42 degrees C hyperthermia. Cells heated at 43 degrees C were still sensitive to Ab-C cytotoxicity and did not recover the capping ability even 2.5 hr after heat treatment.     

The effect of hyperthermia on radiation-induced carcinogenesis

Ten groups of mice were exposed to either a single (30 Gy) or multiple (six fractions of 6 Gy) X-ray doses to the leg. Eight of these groups had the irradiated leg made hyperthermic for 45 min immediately following the X irradiation to temperatures of 37 to 43 degrees C. Eight control groups had their legs made hyperthermic with a single exposure or six exposures to heat as the only treatment. In mice exposed to radiation only, the postexposure subcutaneous temperature was 36.0 +/- 1.1 degrees C. Hyperthermia alone was not carcinogenic. At none of the hyperthermic temperatures was the incidence of tumors in the treated leg different from that induced by X rays alone. The incidence of tumors developing in anatomic sites other than the treated leg was decreased in mice where the leg was exposed to hyperthermia compared to mice where the leg was irradiated. A systemic effect of local hyperthermia is suggested to account for this observation. In mice given single X-ray doses and hyperthermia, temperatures of 37, 39, or 41 degrees C did not influence radiation damage as measured by the acute skin reactions. A hyperthermic temperature of 43 degrees C potentiated the acute radiation reaction (thermal enhancement factor 1.1). In the group subjected to hyperthermic temperatures of 37 or 39 degrees C and X rays given in six fractions, the skin reaction was no different from that of the group receiving X rays alone. Hyperthermic temperatures of 41 and 43 degrees C resulted in a thermal enhancement of 1.16 and 1.36 for the acute skin reactions. From Day 50 to Day 600 after treatment, the skin reactions showed regular fluctuations with a 150-day periodicity. Following a fractionated schedule of combined hyperthermia and X rays, late damage to the leg was less than that following X irradiation alone. Mice subjected to X rays and hyperthermic temperatures of 41 and 43 degrees C had a lower median survival time than the mice treated with hyperthermia alone. This effect was not associated with tumor incidence.     

Thermal analysis of CHL V79 cells using differential scanning calorimetry: implications for hyperthermic cell killing and the heat shock response

Differential scanning calorimetry (DSC) was used to assay thermal transitions that might be responsible for cell death and other responses to hyperthermia or heat shock, such as induction of heat shock proteins (HSP), in whole Chinese hamster lung V79 cells. Seven distinct peaks, six of which are irreversible, with transition temperatures from 49.5 degrees C to 98.9 degrees C are detectable. These primarily represent protein denaturation with minor contributions from DNA and RNA melting. The onset temperature of denaturation, 38.7 degrees C, is shifted to higher temperatures by prior heat shock at 43 degrees and 45 degrees C, indicative of irreversible denaturation occurring at these temperatures. Thus, using DSC it is possible to demonstrate significant denaturation in a mammalian cell line at temperatures and times of exposure sufficient to induce hyperthermic damage and HSP synthesis. A model was developed based on the assumption that the rate limiting step of hyperthermic cell killing is the denaturation of a critical target. A transition temperature of 46.3 degrees C is predicted for the critical target in V79 cells. No distinct transition is detectable by DSC at this temperature, implying that the critical target comprises a small fraction of total denaturable material. The short chain alcohols methanol, ethanol, isopropanol, and t-butanol are known hyperthermic sensitizers and ethanol is an inducer of HSP synthesis. These compounds non-specifically lower the denaturation temperature of cellular protein. Glycerol, a hyperthermic protector, non-specifically raises the denaturation temperature for proteins denaturing below 60 degrees C. Thus, there is a correlation between the effect of these compounds on protein denaturation in vivo and their effect on cellular sensitivity to hyperthermia.     

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.     

Alteration of poly(ADP-ribose) metabolism by hyperthermia

The intracellular levels of poly(ADP-ribose) in cultured mouse cells were increased in response to hyperthermic treatment (43 degrees C). When hyperthermia was combined with other stressful treatments such as with ethanol and/or an alkylating agent, a dramatic synergistic increase in polymer levels was observed. The effect of hyperthermia did not appear to be related to the presence of DNA strand breaks. A possible involvement of poly(ADP-ribose) metabolism in the general cellular response to environmental stress is suggested.     

Cycloheximide can rescue heat-shocked L cells from death by blocking stress-induced apoptosis.

Cultured mouse L cells undergo apoptosis upon 1 h heat shock at 43 and 45 degrees C. Morphologically characteristic apoptotic cells begin to appear soon after the shock. Immunohistochemistry with anti-transglutaminase antibody shows that in most treated cells the enzyme is induced. Its activation results in the formation of highly cross-linked detergent-resistant apoptotic bodies during recovery. Cycloheximide added during hyperthermic stress inhibits the appearance of apoptotic bodies, showing that heat-shock-induced apoptosis is dependent on protein neosynthesis. The analysis of colony-forming ability of heat-shocked L cells shows a survival of 5% at 43 degrees C and less than 0.02% at 45 degrees C. When protein synthesis is inhibited during heat shock the fraction of surviving cells increases to 23% at 43 degrees C and 0.9% at 45 degrees C. This suggest that part of the cells that die upon heat shock are not heavily damaged and would have survived in the presence of a block in protein synthesis.     

A point mutation that decreases the thermal stability of human interferon gamma.

We have identified a mutation of human gamma-interferon (IFN gamma) causing a temperature-sensitive phenotype. We used a randomized oligonucleotide to mutagenize a synthetic human IFN gamma gene, then screened the resulting mutants produced in Escherichia coli for proteins with altered biological activity. One mutant protein selected for detailed characterization exhibited less than 0.3% of the specific biological activity of native IFN gamma in an antiviral activity assay performed at 37 degrees C. However, the protein bound the human IFN gamma receptor with native efficiency at 4 degrees C. Sequencing the plasmid DNA encoding this protein showed that the mutation changed the lysine residue at amino acid 43 to glutamic acid (IFN gamma/K43E). Site-specific mutagenesis at amino acid 43 showed that this protein's phenotype resulted from positioning a negative charge at position 43. Structural characterization of IFN gamma/K43E using CD demonstrated that the protein had native conformation at 25 degrees C, but assumed an altered conformation at 37 degrees C. IFN gamma/K43E in this altered conformation bound poorly to the IFN gamma receptor at 37 degrees C, providing a rationale for the mutant's decreased antiviral activity.     

Inhibition of repair of radiation-induced DNA damage by thermal shock in Chinese hamster ovary cells

The effect of exposure to elevated temperatures (41-45 degrees C) on the repair of radiation-induced DNA strand breaks was measured in monolayer cultured Chinese hamster ovary (CHO) cells. Prior exposure of cells to temperatures between 43 and 45 degrees C resulted in significant decreases in the rate of repair of DNA damage. Exposure to 45 degrees C for 15 min slowed the rate of DNA repair to 0.17 of the control repair rate. The To for inactivation of DNA repair was observed to be 34, 13 and 6 min at 43, 44 and 45 degrees C, respectively. Stepdown-heating (45 degrees C for 15 min followed by repair at 41 degrees C) resulted in greater inhibition of DNA repair (0.11 of the control rate) than was observed after acute heating alone. Repair at 41 degrees C was observed to proceed in unheated cells at a faster rate than at 37 degrees C. An Arrhenius analysis of the inactivation kinetics of DNA repair between 43 and 45 degrees C indicated an activation energy of 140 kcal mol-1 of protein for the inhibition of DNA repair. In general, the results were inconsistent with either a retardation of the DNA repair rate or an increase in unrepaired DNA lesions being responsible for heat-induced radiosensitization.     

Accumulation of heat shock protein 72 (hsp 72) in postimplantation rat embryos after exposure to various periods of hyperthermia (40 degrees -43 degrees C) in vitro: evidence that heat shock protein 72 is a biomarker of heat-induced embryotoxicity.

A monoclonal antibody to the 72 kDa heat shock protein and Western blot analysis were used to determine the induction, accumulation and turnover of hsp 72 after day 10 rat embryos were exposed to elevated temperatures (40 degrees-43 degrees C) for various lengths of time (2.5 minutes to 18 hours). Embryos exposed to temperatures that exceed the normal culture temperature (37 degrees C) by 4 degrees C or more for as little as 2.5 minutes (43 degrees C) or 15 minutes (41, 42 degrees C) synthesized and accumulated detectable amounts of heat-inducible hsp 72. Hsp 72 could not be detected by Western blot analysis of proteins from embryos cultured at 40 degrees C or below. Once induced, hsp 72 can be detected in embryos for 24-48 hours after they are removed from the hyperthermic conditions and returned to normothermic conditions. Our results also indicate that hsp 72 is induced by all hyperthermic exposures that induce alterations in rat embryo growth and development; therefore, hsp 72 is a potential biomarker for heat-induced embryotoxicity.     

Effect of heat shock on function of frozen/thawed bull spermatozoa

Deposition of spermatozoa in the reproductive tract of hyperthermic cows could conceivably result in sperm damage. Accordingly, a series of experiments tested the effects of heat shock on functional characteristics and free radical production of bull spermatozoa. Viability was reduced slightly by short-term (1 to 3 h) culture at 42 and 43 degrees C as compared with culture at 39 degrees C. There was no effect of culture at 42 degrees C on the ability of spermatozoa to undergo swim-up or of 42 degrees C on the percentage of motile spermatozoa. However, exposure to 41 degrees C for 3 h reduced percentage of motile sperm, 41 and 42 degrees C reduced sperm velocity and 43 degrees C decreased the proportion of spermatozoa undergoing swim-up. In other experiments, there was no effect of heat shock (41 or 42 degrees C for 1 to 3 h) on DNA integrity, presence of intact acrosomes, or fertilizing ability of the spermatozoa. Superoxide production by spermatozoa was higher at 42 degrees C than at 39 or 41 degrees C, but there was no detectable hydrogen peroxide production at any temperature. The antioxidant, glutathione, tended to improve the ability of spermatozoa to undergo swim-up at 39 degrees C but not at 43 degrees C. Taken together, these results suggest that heat shock of a magnitude similar to that seen in vivo (41 to 42 degrees C) has little effect on sperm functions that affect fertilizing capability.     

The occurrence of DNA strand breaks after hyperthermic treatments of mammalian cells with and without radiation

Strand breaks were detected in the DNA of Ehrlich ascites cells as well as in HeLa S3 cells directly after 1-5 hr at 43-45 degrees C by the use of the unwinding in high salt/hydroxylapatite method. The strand breaks found could not be attributed to the decay of incorporated tritiated thymidine. When the cells were incubated at 37 degrees C after the hyperthermic treatments, the amount of strand breaks formed remained at a constant level. Hyperthermia inhibited the repair of "radiation-induced" strand breaks. The repair curves obtained this way show a heat-dose-dependent decrease of the relative weight of the fast component of repair. Similar repair curves of "radiation-induced" strand breaks could be obtained by mixing heat inactivated and vital control cells prior to irradiation. In the latter case, however, the DNA repair was inhibited to a greater extent for identical levels of cell survival. The possible underlying molecular mechanisms are discussed.     

A systematic approach toward the analysis of drug-DNA interactions using Raman spectroscopy: the binding of metal-free bleomycins A(2) and B(2) to calf thymus DNA

Bleomycins A(2) and B(2) are the two active components in the antineoplastic drug Blenoxane. DNA is targeted by this drug in cancer cells and the mode of action of this drug involves DNA binding. Ambiguity exists as to the way in which bleomycin binds to DNA. Raman spectroscopy was used to examine both calf thymus DNA and a bleomycin/DNA complex at two temperatures. A curvefitting technique was applied to these spectra for a spectral region obscured by many overlapping bands associated with the nucleotide bases in order to derive information about frequencies, bandwidths, and intensities of the vibrational modes in this region. This allowed identification and analysis of bands associated with specific assigned nucleotide base residues. Upon binding of bleomycin, several significant changes in bandwidth, intensities, and frequencies relative to uncomplexed DNA were observed consistently at both higher (30 degrees C) and lower (19 degrees C) temperature. The data presented here support at least a partial intercalation mode of binding for bleomycin that is temperature dependent and more pronounced at the more physiologically relevant temperature of 30 degrees C.     

Inhibition of repair of X-ray-induced DNA damage by heat: the role of hyperthermic inhibition of DNA polymerase alpha activity

HeLa S3 cells growing in suspension have been used to investigate possible mechanisms underlying the inhibitory action of hyperthermia (44 degrees C) on the repair of DNA strand breaks as caused by a 6-Gy X-irradiation treatment. The role of hyperthermic inactivation of DNA polymerase alpha was investigated using the specific DNA polymerase alpha inhibitor, aphidicolin. It was found that both heat and aphidicolin (greater than or equal to 2 micrograms ml-1) could decrease DNA repair rates in a dose-dependent way. When the applications of heat and aphidicolin were combined, each at nonmaximal doses, no full additivity in effects was observed on DNA repair rates. When the heat and radiation treatment were separated in time by postheat incubation at 37 degrees C, restoration to normal repair kinetics was observed within 8 h after hyperthermia. When heat was combined with aphidicolin addition, restoration of the aphidicolin effect to control level was also observed about 8 h after hyperthermia. It is suggested that although DNA polymerase alpha seems to be involved in the repair of X-ray-induced DNA damage, and although this enzyme is partially inactivated by heat, other forms of heat damage have to be taken into account to explain the observed repair inhibition.     

Effects of gamma radiation and hyperthermia on DNA repair synthesis and the level of NAD+ in cultured human mononuclear leukocytes

DNA repair has been investigated, estimated by unscheduled DNA synthesis (UDS) and the cellular NAD+ pool, after exposing human mononuclear leukocytes to hyperthermia and gamma radiation separately and in combination. It was found that gamma radiation induced a decline in UDS with increasing temperature through the temperature region studied (37-45 degrees C). At 42.5 degrees C the gamma-ray-induced UDS was reduced to about 70% of that at 37 degrees C. Following gamma-ray damage the NAD+ pool dropped to about 20% of control values. Without hyperthermic treatment the cells completely recovered to the original level within 5 hr. Moderate hyperthermia (42.5 degrees C for 45 min) followed by gamma-ray damage altered the kinetics so that even after 8 hr the NAD+ pool had recovered to only 70% of the original level. After heat treatment at 44 degrees C for 45 min prior to gamma radiation the cells did not recover at all, presumably because of the cytotoxic effects from the combined treatment.     

Effect of hyperthermia on DNA loop-size in HeLa S3 cells

Nuclear matrices of heated and non-heated HeLa S3 cells were isolated and average DNA loop-sizes were compared. Heat treatment (30 min at 45 degrees C) resulted in an ultimate survival level of the cells of about 10 per cent. The loop-size determinations were done on nuclear material isolated from the cells directly after heat treatment. In the nuclear matrices isolated from the heated cells about 1.8 times more protein was bound as compared to the matrices from control cells. Enzymatic analysis using DNase I digestion, followed by centrifugation on neutral sucrose gradients, was performed. Also, halo visualization was combined with autoradiography. Both methods revealed no gross alterations in DNA loop-sizes. The possible function of DNA loop organization in the effect of hyperthermic interference with DNA-related processes is discussed.     

Effects of hyperthermia on xanthine oxidase activity and glutathione levels in the perfused rat liver

The hepatotoxic effects of hyperthermic liver perfusion were investigated in male Fischer 344 rat livers. Perfusions were carried out at 37, 41, 42, 42.5, and 43 degrees C for 2 hr. During the 2 hr, the perfusate was analyzed for activity of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), N-acetyl-beta-glucosaminidase (NAG), and glutathione (GSH), oxidized glutathione (GSSG), allantoin, and potassium. After perfusion, each liver was homogenized and analyzed for total xanthine oxidase (XO) activity, percentage type-D and type-O XO, and total GSH content. Perfusate AST, LDH, NAG, and potassium levels were increased significantly with time and were significantly different in all hyperthermic perfusions from the 37 degrees C perfusion values by the end of the perfusion. Perfusate GSH + GSSG levels were increased significantly in all hyperthermic perfusions after 60 min. Liver GSH levels were significantly lowered following perfusion at hyperthermic temperatures. There was a temperature-dependent increase in the percentage of XO in the type-O form following perfusion at hyperthermic temperatures, which was strongly and positively correlated with the loss of hepatic GSH. These data support the hypothesis that hyperthermic toxicity to the liver is the result of oxidative stress brought about by conversion of XO to the type-O form.