UV response of the temperature-conditional rad 54 mutant of the yeast Saccharomyces cerevisiae

The survival of the yeast mutant rad 54-3, which is temperature-conditional for the repair of double-strand breaks, was measured after exposure to UV-light (254 nm) and incubation at 23 degrees C and 36 degrees C. It was found that survival was drastically reduced with incubation at the restrictive temperature. Temperature-shift experiments indicated that repair of UV-induced damage which is controlled by the rad 54 gene proceeds with a half-value-time of about 7 h.     

Thermosensitization by methylglyoxal bis(guanylhydrazone) in Chinese hamster cells

The modification of methylglyoxal bis(guanylhydrazone) (MGBG) by 42 degrees C hyperthermia-and/or radiation-induced cell killing was examined in Chinese hamster V-79 cells. At concentrations of more than 10 microM, cell survival decreased exponentially with increased MGBG exposure times. Cell lethality of MGBG (10 microM) was not specific for cell-cycle phases tested from G1/S through G2. When cells were treated with MGBG (10 microM) for 6 hr and then exposed to 42 degrees C hyperthermia with or without a 24-hr interval, cell survival decreased markedly compared with that for 42 degrees C alone. Cells became thermosensitive after MGBG treatment. Cells exposed to MGBG (10 microM) for 6 hr before or after X irradiation were slightly radiosensitive. When X irradiation was combined with MGBG and 42 degrees C hyperthermia, cells became more radiosensitive. From these results, it is suggested that MGBG may change the intracellular state to sensitize cells to the cytotoxic action(s) of hyperthermia.     

Inhibitors of poly(ADP-ribose) synthetase enhance the cytotoxicity of 42 degrees C and 45 degrees C hyperthermia in cultured Chinese hamster cells

Two inhibitors of poly(ADP-ribose) synthetase, 5-methylnicotinamide and m-methoxybenzamide, enhanced the cytotoxicity of 42 degrees C and 45 degrees C hyperthermia in cultured Chinese hamster V79 cells. The inhibitors showed minimal toxicity for cells treated at 37 degrees C, and did not appreciably alter cellular ATP levels under any of the experimental conditions used. Enhanced cell killing occurred when the inhibitors were added after an acute (5-10 min) 45 degrees C heat shock, and after 50 and 100 min exposures to 42 degrees C. When present during heating at 42 degrees C, the inhibitors reduced the shoulder of the 42 degrees C survival curves but did not appreciably affect the slopes. The results suggest a possible role for poly(ADP-ribose) synthetase in the survival response of V79 cells to hyperthermia.     

Effect of ATP on glucose-6-phosphate dehydrogenase during erythroblast maturation in the rabbit

The glucose-6-phosphate dehydrogenase (G6PD) activity of erythroblasts, separated at different advancing stages of development, shows a marked decline of activity. A proteolytic mechanism, strictly controlled, is likely responsible of this decay, since a sufficient level of enzyme activity still remains in the circulating erythrocyte. In this report we suggest a model that could explain what triggers the mechanism of proteolytic degradation. HPLC analysis of the nucleotide content of erythroblasts and reticulocytes, showed a marked decline of adenine and pyridine nucleotides and of their catabolic products during the cell development. From thermostability tests, at fixed temperature, we have seen that ATP and NADP only, significantly protected the enzyme activity. In this light, we incubated 10 min at increasing temperatures, with and without ATP or NADP lysates of erythroblasts, separated at different stage of development and of reticulocytes. In the absence of nucleotides, we determined for all fractions a T degree break at 42 degrees C. In the presence of NADP all fractions were stabilized with no break point in the range 37-50 degrees C. On the contrary, the presence of ATP caused a progressive shift of the T degrees C break from the most immature erythroblasts (T degree break at 46 degrees C) to the reticulocytes (T degree break at 42 degrees C). Since ATP did not show any protective effect on the reticulocyte enzyme, we hypothesize the presence in these cells of a structurally modified G6PD. Furthermore, these data support our belief that the marked decline of ATP during cellular development, may represent the element responsible for the enzyme modification.     

Ways of realizing apoptosis of human lymphocytes induced by UV-light and reactive oxygen species

Changes of DNA structural condition, the level of membrane Fas-receptor expression, caspase-3 functional activity, concentrations of Ca2+, p53 and cytochrome c proteins of human lymphocytes in dynamics of apoptosis development induced by UV-light (240-390 nm) at doses 151, 1510, 3020 J/m2 and reactive oxygen species (superoxide anion-radical, hydroxyl radicals, hydrogen peroxide, singlet oxygen) have been studied. UV-light and reactive oxygen species have been established to induce fragmentation of lymphocyte DNA after 20 h incubation of the modified cells. It has been shown, that the increase in the expression level of membrane death Fas-receptors is observed during 1-5 h after exposure oflymphocytes to UV-light and ROS compared with intact cells. Also revealed is augmentation of lymphocyte caspase-3 functional activity 4 h after generation of singlet oxygen, hydroxyl radical and hydrogen peroxide addition, as well as 8 and 24 and 6 and 8 h after UV-irradiation of the cells at doses 151 and 1510 J/m2, correspondingly. Using DNA-comet method made it possible to tape that DNA damages (single-strand breaks) appear 15-20 min after lymphocyte UV-irradiation at doses 1510 and 3020 J/m and addition of hydrogen peroxide in concentration 10(-6) mol/l (C1 type comet) and reach their maximum 6 h after modification of the cells (C2 and C3 type comets). It has been observed, that 6 h after exposure oflymphocytes to hydrogen peroxide and UV-light at doses 1510 and 3020 J/m2, the p53 level of investigated cells raises. It has also been shown that the higher level of calcium in lymphocyte cytosol in conditions of UV-light exposure (1510 J/m2) and exogenous generation of reactive oxygen species is caused by Ca2+ exit from intracellular depots as a result of activating the components of the phosphoinositide mechanism for transferring information into a cell. Ideas about correlation between alterations of the calcium level and initiation of programmed cellular destruction of human lymphocytes after exposure to UV-irradiation and ROS is proposed. The authors come to the conclusion about the leading role of receptor-mediated (Fas-dependent) caspase- and p53-dependent ways of realizing apoptosis oflymphocytes induced by UV-light at doses 151 and 1510 J/m2 and active oxygen metabolites. The pattern of the possible intracellular events leading to apoptotic destruction of lymphocytes after their UV-irradiation is offered.     

Subcellular localization of glutathione and thermal sensitivity

Chinese hamster ovary (CHO) cells were exposed to various concentrations of diethylmaleate (DEM) during a 42 degrees C incubation to determine if glutathione (GSH) compartmentalization was a factor in modification of thermal sensitivity. Cytoplasmic and mitochondrial GSH were isolated from CHO cells immediately after a hyperthermic treatment consisting of 2 h at 42 degrees C. Under these experimental conditions differential GSH depletion between the cytosol and mitochondrial compartments were observed. For example, 12 microM DEM was needed to deplete cytoplasmic GSH by 50% compared to 24 microM DEM needed to deplete mitochondrial GSH to the same level. Further, an ln-ln plot of the relative cytosolic GSH concentration vs the DEM concentration indicated a linear relationship (slope = -1.0). In contrast, the mitochondrial GSH plot exhibited a shoulder followed by a linear removal (slope = -0.90). Essentially the two linear curves were parallel. Analysis of thermal dose-response curves for cells exposed to between 10 and 100 microM DEM indicated that cell survival was unaffected by the addition of DEM until a critical concentration was surpassed. This threshold response was interpreted to mean that mitochondrial GSH depletion was the limiting factor.     

Effects of m-aminobenzamide on the response of Chinese hamster cells to hyperthermia and/or radiation

The modifying effects of m-aminobenzamide (m-ABA), an inhibitor of poly(ADP-ribose) synthesis, on 42 degrees C hyperthermia- and/or radiation-induced cell killing were examined in Chinese hamster V-79 cells. When cells were exposed to 42 degrees C hyperthermia in combination with m-ABA (10 mM), cell survival decreased compared with that for 42 degrees C hyperthermia alone. Thermosensitizing effects of m-ABA changed with treatments in a decreasing order of during and after heating greater than during heating greater than after heating. Treatments with m-ABA during and/or after X irradiation enhanced radiation-induced cell killing. When cells were exposed to combined treatment with X irradiation, 42 degrees C hyperthermia (60 min), and m-ABA (24 hr), cell survival decreased markedly compared with that for X irradiation alone. However, with both X----42 degrees C and X----42 degrees C----m-ABA, the enhancement ratios (ER), designated as D0 ratio, were similar. These results suggest that the mechanisms of radiosensitization by m-ABA may be similar to those of 42 degrees C hyperthermia.     

Enhancement of cell killing by induction of apoptosis after treatment with mild hyperthermia at 42 degrees C and cisplatin.

Ohtsubo, T., Igawa, H., Saito, T., Matsumoto, H., Park, H. J., Song, C. W., Kano, E. and Saito, H. Enhancement of Cell Killing by Induction of Apoptosis after Treatment with Mild Hyperthermia at 42 degrees C and Cisplatin. Radiat. Res. 156, 103-109 (2001).We examined the interactive effects of cisplatin (1.0 microg/ml) combined with hyperthermia on cell killing and on the induction of apoptosis in IMC-3 human maxillary carcinoma cells. The cytotoxic effects of hyperthermia on IMC-3 cells at 44 degrees C were greater than at 42 degrees C, as has been reported for many other cells. The induction of apoptosis, DNA fragmentation and poly(ADP-ribose) polymerase cleavage were greater after hyperthermia at 44 degrees C for 30 min compared with treatment at 42 degrees C for 105 min, even though both of these heat doses were isoeffective in reducing cell survival to 50%. Treatment with cisplatin at 37 degrees C for up to 120 min did not result in cytotoxicity or the induction of apoptosis. The enhancement ratio for treatment with cisplatin at 42 degrees C was greater than that at 44 degrees C. More apoptosis was induced after the treatment with cisplatin at 42 degrees C compared to treatment with cisplatin at 44 degrees C. Taking these findings together, the combination of cisplatin and hyperthermia at 42 degrees C appeared to be more effective than cisplatin with hyperthermia at 44 degrees C for the induction of apoptosis in IMC-3 cells.     

Relationship between cell survival and heat-stress protein synthesis in a Drosophila cell line

Heat-stress protein (hsp) kinetics and clonogenic survival were studied at 33, 37 and 42 degrees C in a continuous Drosophila cell line, WR69-DM-1. Induction and repression of hsp were temperature-dependent and independently modulated. The subsequent cell-survival curves were complex; however, survival generally decreased in a time- and temperature-dependent manner during continuous heating at 33, 37 or 42 degrees C. Constant 33 degrees C heating induced five hsp at 90, 72, 70, 24 and 19 kilodaltons (kDa). A 30 min 33 degrees C heat dose led to thermotolerance after 1, 3 or 6 h incubations at 28 degrees C. The hsp synthesized after this dose were quickly repressed, suggesting the cells were able to respond to this stress. Increasing the challenge temperature to 37 degrees C induced three additional hsp at 34, 22 and 14 kDa, but hsp synthesis did not lead to thermotolerance over the 6 h interval. The number and intensity of hsp synthesized was higher and repression was much slower than at 33 degrees C. Heating at 42 degrees C inhibited all protein synthesis, and thermotolerance was not observed. Direct survival data are critical to understanding the role and function of hsp in Drosophila thermotolerance since the relevance of information on number and kinetics of hsp synthesis and their subsequent localization is dubious without it.     

Heat-induced changes in the membrane fluidity of Chinese hamster ovary cells measured by flow cytometry.

Flow cytometry was used to measure the fluorescence polarization of the lipid probe trimethylammonium-diphenylhexatriene as an indicator of plasma membrane fluidity of Chinese hamster ovary (CHO) cells heated under various conditions. Fluorescence polarization was measured at room temperature about 25 min after heating. When cells were heated for 45 min at temperatures above 42 degrees C, fluorescence polarization decreased progressively, signifying an increase in plasma membrane fluidity. The fluorescence polarization of cells heated at 42 degrees C for up to 55 h was nearly the same as for unheated control populations, despite a reduction in survival. The fluorescence polarization of cells heated at 45 degrees C decreased progressively with heating time, which indicated a progressive increase in membrane fluidity. The fluorescence polarization distributions broadened and skewed toward lower polarization values for long heating times at 45 degrees C. Thermotolerant cells resisted changes in plasma membrane fluidity when challenged with subsequent 45 degrees C exposures. Heated cells were sorted on the basis of their position in the fluorescence polarization distribution and plated to determine survival. The survival of cells which were subjected to various heat treatments and then sorted from high or low tails of the fluorescence polarization histograms was not significantly different. These results show that hyperthermia causes persistent changes in the membrane fluidity of CHO cells but that membrane fluidity is not directly correlated with cell survival.     

Comparative survival of probiotic lactobacilli spray-dried in the presence of prebiotic substances

AIMS: Probiotic milk-based formulations were spray-dried with various combinations of prebiotic substances in an effort to generate synbiotic powder products. METHODS AND RESULTS: To examine the effect of growth phase and inclusion of a prebiotic substance in the feed media on probiotic viability during spray-drying, Lactobacillus rhamnosus GG was spray-dried in lag, early log and stationary phases of growth in reconstituted skim milk (RSM) (20% w/v) or RSM (10% w/v), polydextrose (PD) (10% w/v) mixture at an outlet temperature of 85-90 degrees C. Stationary phase cultures survived best (31-50%) in both feed media and were the most stable during powder storage at 4-37 degrees C over 8 weeks, with 30-140-fold reductions in cell viability at 37 degrees C in RSM and PD/RSM powders, respectively. Stationary phase Lact. rhamnosus GG was subsequently spray-dried in the presence of the prebiotic inulin in the feed media, composed of RSM (10% w/v) and inulin (10% w/v), and survival following spray-drying was of the order 7.1-43%, while viability losses of 20,000-90,000-fold occurred in these powders after 8 weeks' storage at 37 degrees C. Survival of the Lactobacillus culture after spray-drying in powders produced using PD (20% w/v) or inulin (20% w/v) as the feed media was only 0.011-0.45%. To compare different probiotic lactobacilli during spray-drying, stationary phase Lact. rhamnosus E800 and Lact. salivarius UCC 500 were spray-dried using the same parameters as for Lact. rhamnosus GG in either RSM (20% w/v) or RSM (10% w/v) and PD (10% w/v). Lact. rhamnosus E800 experienced approx. 25-41% survival, yielding powders containing approximately 10(9) CFU g(-1), while Lact. salivarius UCC 500 performed poorly, experiencing over 99% loss in viability during spray-drying in both feed media. In addition to the superior survival of Lact. rhamnosus GG after spray-drying, both strains experienced higher viability losses (570-700-fold) during storage at 37 degrees C over 8 weeks compared with Lact. rhamnosus GG. CONCLUSIONS: Stationary phase cultures were most suitable for the spray-drying process, while lag phase was most susceptible. The presence of the prebiotics PD and inulin did not enhance viability during spray-drying or powder storage. SIGNIFICANCE AND IMPACT OF THE STUDY: High viability (approximately 10(9) CFU g(-1)) powders containing probiotic lactobacilli in combination with prebiotics were developed, which may be useful as functional food ingredients for the manufacture of probiotic foods.     

Vitrification of ECV304 cell suspensions using solutions containing propane-1,2-diol and trehalose

In this paper, we report on the suitability of solutions containing propane-1,2-diol (propylene glycol, PD), sugars, and salts for the vitrification of the human cell line, ECV304. Cooling (at 10 degrees C/min) and rewarming (at 80 degrees C/min) were at rates that are practicable for the tissues to be studied later. Under these conditions, 45% PD in phosphate-buffered saline (PBS) sometimes froze during cooling and always devitrified during rewarming but both events were avoided if the PBS salts were replaced by an osmotically equivalent concentration of sucrose or trehalose. The effect of such solutions on cells was evaluated using a cell culture assay in which the number of cells recovered after 3 days of culture was divided by the number cells plated, giving a cell multiplication factor or CMF. In the absence of PD the cells tolerated a low-salt concentration in solutions that were made isotonic with sugars, but they recovered poorly when 45% PD was also present. Trehalose gave significantly better recovery than sucrose. When 39% PD and 15% trehalose were included in a low-salt vehicle solution (LSV) that contained approximately 5% of the total salt concentration of PBS (this solution was designated LSV/39/15), the cells exhibited approximately 40% of untreated control CMF following exposure for 9min. LSV/39/15 vitrifies with a glass transition temperature of -102 degrees C, does not devitrify when warmed at 80 degrees C/min, and has suitable dielectric properties for uniform and rapid dielectric heating. An improved method for adding and removing LSV/39/15 gave a CMF of approximately 55% of untreated controls. Using this method, 1.0ml suspensions of ECV304 cells was cooled to, and stored briefly at, -120 degrees C and then rewarmed by immersion in a 37 degrees C water bath ( approximately 75 degrees C/min). The CMF of the cooled samples was similar to that of the exposure-only controls, approximately 50% of the untreated control CMF in both cases.     

Dissociation of 68,000 Mr heat shock protein synthesis from thermotolerance expression in rat fibroblasts

Rat embryonic fibroblasts growing exponentially at either 35, 37, or 39 degrees C were exposed to 42 degrees C for times up to 6 hr. Cell survival was unaffected by this heat shock in cultures growing at 39 degrees C but survival was decreased in a temperature dependent manner in cells growing at 37 or 35 degrees C. Exposure to 42 degrees C of cells previously adapted to 35 or 37 degrees C resulted in the induction of heat shock proteins (hsps) with apparent molecular weights of 68,000 (hsp 68), 70,000 (hsp 70), and 89,000 (hsp 89); cells previously adapted to 39 degrees C expressed all hsps except hsp 68. Inasmuch as the synthesis of certain hsps may function to protect cells from thermal damage, these data indicate that hsp 68 may not be required for this adaptation-related thermotolerant survival response. Hsp 68 may only be expressed in cells destined to die.     

Enhanced reactivation and mutagenesis after transfection of carcinogen-treated monkey kidney cells with UV-irradiated simian virus 40 (SV40) DNA

Monkey kidney cells, either untreated or pretreated with UV-light at 254 nm or mitomycin C, were transfected 24 hours later with the intact or UV-irradiated DNA from the thermosensitive tsB201 simian virus 40 mutant unable to grow at 41 degrees C. The survival of the viral progeny obtained from the UV-irradiated DNA is increased in pretreated cells compared to the survival of the viral progeny obtained in untreated cells. Irradiation of the viral DNA enhances the reversion frequency of the viral progeny towards a wild type phenotype able to grow at 41 degrees C. Pretreatment of the cells with UV or mitomycin C does not increase the reversion frequency.     

Physiological characteristics of Salmonella typhimurium treated with penicillin]

Growing bacteria of the two strains of Salmonella typhimurium differing in the sensitivity levels to UV-light formed multinuclear non-septal filaments in the penicillin-containing nutrient medium. The maximum number of the lifefull filaments was formed by the 4th hour of incubation in the beaf-peptone broth at a temperature of 37 degrees C in the presence of 5 gamma/ml of penicillin. The strains exposed to penicillin were less sensitive to UV-light. Exclusion of penicillin from the nutrient medium resulted in a new division of the filamentous cells and reduction of the initial UV-light sensitivity level. It was concluded that the low UV-light sensitivity level of the filaments induced by penicillin was associated with their multinuclear state.     

Radiosensitive Drosophila lines. VIII. The effect of the mus (201)Gl gene of Drosophila melanogaster on the sensitivity of the flies to ultraviolet rays in the imago stage

The effect of UV-light on survival of adult flies of the mutant line mus(2) 201G1 sensitive to methyl methanesulfonate (MMS) was studied. One day old flies were irradiated by UV-light and the survival was determined depending on the UV dose, the genotype and the sex of irradiated flies. High sensitivity of mutant flies was shown in the imaginal stage when division of somatic cells was absent. Moreover, the differences in sensitivity to UV-light were observed depending on the sex of flies. We suggest that the mutation mus(2)201G1 blocking the excision repair affects undivisional cells of the imaginal stage of Drosophila melanogaster.     

On a factor present in tumor cells, capable of decreasing thermotolerance in mice

Comparison was made of the capacity of tumor cells of three different cell lines to decrease the thermotolerance in mice. Tumor cells used were that of three cell lines of mastocytoma, FMA1 and FMA3, and of Ehrlich's ascites tumor, EAT. Temperature for the assay of thermotolerance of the animal was 42.0 +/- 0.2 degrees C in the core body. Thermotolerance of animal was expressed with LD50, 42 degrees C. Tumor cells were transplanted at a dose of 10(5) cells per mouse. In the animals transplanted with tumor cells of every three cell lines, the minimal value of the LD50, 42 degrees C was obtained one or two days after the transplantation. Thermotolerance ratios (TTRs) calculated with the minimal values of LD50, 42 degrees C were 0.54, 0.19 and 0.62 for the FMA1, FMA3 and EAT cells, respectively. The thermotolerance decreasing effect of the FMA3 cells was kept unchanged even after destruction of the cells by suspending in distilled water and repeated freezing and thawing. But it disappeared partially after heating the cells for 10 min at 90 degrees C, and completely after the heating the cells for 60 min at the same temperature.     

DNA lesions in hyperthermic cell killing: effects of thermotolerance, procaine, and erythritol

When HeLa S3 cells were subjected to 45 degrees C hyperthermia, DNA lesions were detected by the use of the alkaline unwinding/hydroxylapatite method. The number of lesions formed was not affected when the cells were made thermotolerant by either an acute (15 min 44 degrees C + 5 h 37 degrees C) or a chronic (5 h 42 degrees C) pretreatment before 45 degrees C hyperthermia. The presence of 10 mM procaine (heat sensitizer) or 0.5 M erythritol (heat protector) during hyperthermia also had no effect on the rate of formation of heat-induced alkali labile DNA lesions. These observations do not support a concept where DNA lesions are considered to be the ultimate cause of hyperthermic cell killing. Both drugs, however, influenced the rate of repair of radiation-induced strand breaks when present during preirradiation heat treatment. We conclude that the initial number of heat-induced alkali labile DNA lesions is not directly related to cell survival. It cannot be excluded, however, that differences in posthyperthermic repair of these lesions may lead to a positive correlation between residual DNA damage and survival after the different experimental conditions.     

Effects of hyperthermia on DNA interstrand crosslinking after treatment with BCNU in 9L rat brain tumor cells

The effects of hyperthermia (42 degrees C) on 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-mediated DNA interstrand crosslink formation were investigated in 9L rat brain tumor cells using the technique of alkaline elution. When cells were treated with 60 microM BCNU for 1 hr at 37 degrees C and incubated for 6 hr in drug-free medium at 42 degrees C, there was a 50% increase in crosslinking; and when cells were treated at 42 degrees C and incubated at 37 degrees C, there was a 45% increase in crosslinking compared with the results for cells treated and incubated at 37 degrees C. When cells were treated and incubated at 42 degrees C, there was a 129% increase in DNA crosslinking. The same relative order of results was found for cell survival. These results suggest that hyperthermia can increase DNA interstrand crosslink formation and the consequent cell death through two independent mechanisms: an increase in the amount of initial alkylation because of the increased rate of hydrolysis of BCNU at higher temperatures, and the effect of heat on DNA structure that leads to an increase in the number of crosslinks formed.     

Thermotolerance and thermosensitization in CHO and R1H cells: a comparative study

In CHO and R1H cells thermotolerance was induced by a pre-incubation at 40 degrees C, by an acute heat shock at 43 degrees C followed by a time interval at 37 degrees C, and during continuous heating at 42 degrees C. Thermotolerance, which was tested at 43 degrees C, primarily causes an increase in D0 of the heat-response curve. The degree of maximum thermotolerance was found to be generally more pronounced in CHO than in R1H cells, but the time interval at 37 degrees C, as well as at 40 degrees C, to reach this maximum level was the same in both cell lines. CHO and R1H cells could be sensitized to 40 degrees C by a pre-treatment at 43 degrees C. When compared for the same survival rate after pre-treatment at 43 degrees C alone the degree of thermosensitization was about the same in both cell lines. In either cell line thermosensitization was found to be suppressed when cells were made thermotolerant by a previous incubation at 40 degrees C for 16 hours.