Dark recovery of diploid yeast cells after simultaneous exposure to UV-irradiation and hyperthermia

Quantitative regularities of dark recovery of wild-type diploid yeast cells of Saccharomyces cerevisiae simultaneously treated with UV-light (254 nm) and high temperatures (53-56 degrees C) were studied. Under this combined action, the constant of recovery, which defines the probability of elimination of the UV-radiation induced damage per unit of time, did not depend on the temperature of irradiation. It was shown that both the irreversible component of cell damage and the number of cells that died without division gradually increased as the temperature of exposure increased. It is concluded, on this basis, that the mechanism of synergistic interaction of UV-radiation and hyperthermia is related not to the inhibition of dark recovery itself, but to the increase in the shape of irreversibly damaged cells incapable of recovering from the induced damage.     

Recovery of yeast cells after exposure to ionizing radiation and hyperthermia

Quantitative regularities of recovery of wild-type diploid yeast cells irradiated with gamma-rays (60Co) simultaneously with exposure to high temperatures were studied. It was shown that in conditions of such a combined action the constant of recovery did not depend on the temperature at which the irradiation was carried out. However, with an increase of acting temperature an augmentation in the portion of irreversible component was registered. The analysis of cell inactivation revealed that the augmentation of the irreversible component was accompanied by a continuous increase of cell killing without any postirradiation division after which cells are incapable of recovery. The reproductive death was mainly exerted after ionizing radiation applied alone while in conditions of simultaneous thermoradiation action the interphase killing (cell death without division) predominated. It is concluded on this base that the mechanism of synergistic interaction of ionizing radiation and hyperthermia may be related with cardinal change in mechanisms of cell killing.     

Relation of the radiosensitivity of yeast cells to the LET of the radiation. Experiments with diploid cells

A study was made of the dependence of radiosensitivity D0-1 of diploid yeast cells on linear energy transfer L of radiation, and the obtained results were analyzed in terms of the previously developed model concepts. A diploid-specific repair of radiation damages was shown to be equally effective with different L. This mode of repair had no direct effect on D0-1 (L) function. The observed contribution of the diploid-specific repair to cell radioresistance decreased with increasing L. This, however, was due to physical and geometric factors rather than to the decreased efficiency of diploid-specific repair.     

Prognosis of yeast cells recovery after simultaneous exposure to UV-radiation and hyperthermia

The results of experimental investigations of survival of diploid yeast cells Saccharomyces cerevisiae (strain XS800) after simultaneous exposure to UV-radiation (254 nm) and hyperthermia (53-57 degrees C) have been described. It was shown that the portion of cells capable of recovery in innutrient medium after the action of these agents decreased with the increasing of temperature under which the irradiation was occurred. Mathematical model taking into account the synergistic interaction was suggested for quantitative prediction of irreversible component after combined actions of these agents. A good correspondence between experimental data and model predictions has been demonstrated. The importance of the results obtained for the interpretation of the synergistic interaction mechanisms are discussed.     

The effect of hyperglycemia on the tumor response to irradiation given alone or in combination with hyperthermia

The effect of hyperglycemia (elevated blood glucose level) on the response of a murine tumor to irradiation given alone or in combination with hyperthermia was studied. Tumors were early generation isotransplants of a spontaneous C3H/Sed mouse fibrosarcoma, FSa-II. Single-cell suspensions were transplanted into the foot, and irradiation was given when each tumor reached an average diameter of 7 mm. Following irradiation, the tumor growth time to reach 1000 mm3 was studied and the dose-response curve between the tumor growth time and radiation dose was fitted. Preadministration of glucose increased the size of the hypoxic and chronically hypoxic cell fractions without altering the slope of the dose-response curve where the chronically hypoxic cell fraction is determined as the fraction of cells which were not oxygenated under hyperbaric oxygen conditions. Hyperthermia given prior to irradiation enhanced the tumor response to irradiation, but simultaneously increased the size of the hypoxic and chronically hypoxic cell fractions. Similar results were observed following hyperthermia given after irradiation. When hyperthermia at 43.5 degrees C was given 24 h before irradiation, the size of the hypoxic cell fraction increased with increasing treatment time, while a substantial decrease in the chronically hypoxic cell fraction was observed. Administration of glucose 60 min before hyperthermia further increased the size of the hypoxic cell fraction. Possible mechanisms explaining why glucose administration increases the hypoxic cell fractions are discussed.     

Genotoxicity of selenite in diploid yeast

Selenite, a chemical of industrial importance and also an antimutagenic/anticarcinogenic agent, was tested for mutagenic and recombinogenic effects in 2 diploid yeast strains, Saccharomyces cerevisiae BZ 34 and D7. Selenite induced gene conversion and toxicity in BZ 34 and a variety of genetic events, viz. back-mutation, gene conversion, mitotic crossing-over, aberrant colony formation and also toxicity in the D7 strain. In both strains, the genetic effects of selenite showed a peak and a decline during 5 h of treatment while its toxicity increased marginally during 1-5 h. In the BZ 34 strain, the presence of glutathione (GSH) during selenite treatment greatly enhanced the convertogenic and toxic effects of selenite.     

Rad24 is essential for proliferation of diploid cells in fission yeast

The rad24(+) gene of Schizosaccharomyces pombe encodes a ubiquitously expressed 14-3-3 protein. We report here that Deltarad24 cells displayed a defect in diploid colony formation, although they conjugated efficiently. We found that a cumulative deletion of mei2(+) gene almost completely suppressed this defect, and demonstrated using two-hybrid analysis that Rad24 protein directly associates with Mei2 protein by recognizing Ser-438 which is a phosphorylation target of Pat1 kinase. We conclude that constitutive progression to meiosis, caused by lack of Mei2 inhibition due to the absence of Rad24 protein, is the primary cause of the proliferative deficiency observed in Deltarad24 cells.     

Hyperthermic inactivation of diploid yeast and the interaction of damage caused by hyperthermia and ionizing radiation.

Inactivation of diploid yeast by hyperthermia has been studied. DO and Dq decrease with temperature for euoxic and anoxic conditions. The Arrhenius plot shows a break at 52 degrees C; the inactivation energies above and below this temperature are 153 and 94kcal/mol respectively. Hyperthermia (20 min at 51 degrees C) also potentiates the lethal action of gamma rays in diploid yeast cells under both euoxic and anoxic conditions. The interaction between hyperthermic and radiation damage appears to be largely at the sublethal level. The euoxic cells, the hyperthermic potentiation decreases with increasing time between the application of hyperthermia and radiation, being completely lost after 24 hours. However, in the anoxic cells there was no decrease in the hyperthermic potentiation with increasing time interval. These results suggest that yeast cells are capable of repairing hyperthermic sublethal damage, but require oxygen to do so. Thus there is a similarity in the process of repair of sublethal damage caused by ionizing radiation on the one hand and hyperthermia on the other.     

Cellular radiation effects and hyperthermia: Cytokinetic investigations with stationary phase yeast cells

Summary Wild type diploid yeast, Saccharomyces cerevisiae strain 211, was subjected to 250 kV X-rays or 50° C heat treatment for 30 min or to a combination of both. X-ray exposure took place either in air or in nitrogen. Cell number, percentage of budding cells and cell cycle progression was followed for up to 12 h post irradiation. The distribution of cell cycle stages was determined by flow cytofluorometry. All treatments cause a retardation of cell division rate. Hyperthermia leads mainly to a lengthening of G₁, whereas X-rays arrest the cells reversibly in G₂. The effect of the combined treatment appears to be merely additive. No selective action of hyperthermia on hypoxic cells was found.Dedicated to Prof. Dr. A. Schraub on the occasion of his 70th birthday     

Relative competitiveness of haploid and diploid yeast cells growing in a mixed population

Saccharomyces cerevisiae was grown in a rich medium under the conditions of "quasi-continuous" cultivation and, after 200-300 generations, its diploid cells almost completely displaced haploid cells from the original mixed "haploid-diploid" population where the ratio between diploid and haploid strains was either 1:1 or 1:100. The cultivation at 40 degrees C did not change the relative competitive ability of haploids and diploids. When cells were cultivated in a rich medium at 6 degrees C or in a minimal medium at 30 degrees C, none of the strains showed an advantage over others for about 200 generations. Haploid cells had an advantage over diploid cells during "quasi-continuous" growth in the minimal medium at 30 degrees C. When the temperature was elevated to 40 degrees C, diploid cells displaced haploid cells from the mixed population. No advantage was found for diploid or haploid cells grown in a medium with an elevated KCl content (1.5 M). Haploid cells had an advantage over diploid cells when Pichia pinus was cultivated in a minimal medium. The results are discussed using the hypothesis about the diploid phase being fixed in the course of biological evolution.     

Survival and DNA repair in ultraviolet-irradiated haploid and diploid cultured frog cells.

Survival and repair of DNA following ultraviolet (254-nm) radiation have been investigated in ICR 2A, a cultured cell line from haploid embryos of the grassfrog, Rana pipiens. Survival curves from cells recovering in the dark gave mean lethal dose value (Do) in the range 1.5--1.7 Jm-2 for both haploid and diploid cell stocks. The only significant difference observed between haploids and diploids was in the extent of the shoulder at low fluence (Dq), the value for exponentially multiplying diploid cells (3.0 Jm-2) being higher than that found for haploids (1.2 Jm-2). Irradiation of cultures reversibly blocked in the G1 phase of the cell cycle gave survival-curve coefficients indistinguishable between haploids and diploids. Post-irradiation exposure to visible light restored colony-forming capacity and removed chromatographically estimated pyrimidine dimers from DNA at the same rates. After fluences killing 90% of the cells, complete restoration of survival was obtained after 60-min exposure to 500 foot-candles, indicating that in this range lethality is entirely photoreversible and therefore attributable to pyrimidine dimers in DNA. Dimer removal required illumination following ultraviolet exposure, intact cells and physiological temperature, implying that the photoreversal involved DNA photolyase activity. Excision-repair capacity was slight, since no loss of dimers could be detected chromatographically during up to 48 h incubation in the dark and since autoradiographically detected "unscheduled DNA synthesis" was limited to a 2-fold increase saturated at 10 Jm-2. These properties make ICR 2A frog cells useful to explore how DNA-repair pathways influence mutant yield.     

Survival of Paracoccidioides brasiliensis yeast cells under microaerophilic conditions

The ability of P. brasiliensis yeast cells to withstand microaerophilic conditions was investigated in a liquid medium distributed in tall columns in screw-capped tubes. Young cells of three isolates were inoculated on top of the medium, and the tubes were incubated aerobically and anaerobically at 36 degrees C for 28 days. The viability of cells that had sedimented to the bottoms of the tubes was studied by fluorescent microscopy and by their capacity to resume growth when transferred to fresh medium under continuous agitation. The proportion of viable cells in the sediments diminished with time of incubation. However, after 28 days, 27% of the cells were still viable and fully capable of active growth when placed under adequate aeration. On the other hand, drastic reduction of oxygen access elicited an accelerated death rate, with no survival after 7 days of incubation.     

Single-gene deletions that restore mating competence to diploid yeast

Using the Saccharomyces cerevisiae MATa/MATalpha ORF deletion collection, homozygous deletion strains were identified that undergo mating with MATa or MATalpha haploids. Seven homozygous deletions were identified that confer enhanced mating. Three of these, lacking CTF8, CTF18, and DCC1, mate at a low frequency with either MATa or MATalpha haploids. The products of these genes form a complex involved in sister chromatid cohesion. Each of these strains also exhibits increased chromosome loss rates, and mating likely occurs due to loss of one copy of chromosome III, which bears the MAT locus. Three other homozygous diploid deletion strains, ylr193cDelta/ylr193cDelta, yor305wDelta/yor305wDelta, and ypr170cDelta/ypr170cDelta, mate at very low frequencies with haploids of either or both mating types. However, an ist3Delta/ist3Delta strain mates only with MATa haploids. It is shown that IST3, previously linked to splicing, is required for efficient processing of the MATa1 message, particularly the first intron. As a result, the ist3Delta/ist3Delta strain expresses unbalanced ratios of Matalpha to Mata proteins and therefore mates with MATa haploids. Accordingly, mating in this diploid can be repressed by introduction of a MATa1 cDNA. In summary, this study underscores and elaborates upon predicted pathways by which mutations restore mating function to yeast diploids and identifies new mutants warranting further study.     

Cytotoxicity and mutagencity of hyperthermia for diploid human lymphoblasts

Time dependent survival of diploid human lymphoblasts has been measured at 42, 43, 44, 45, 46 and 47°C. D_O was found to be 1500, 23, 4, 2, 1 and 0.6 min, respectively. Mutation to 6-thioguanine resistance was measured at 45°C and found to increase monotonically with time. Thus, relatively mild heat trauma can result in genetic change in somatic human cells.     

Cellulosic ethanol production by combination of cellulase-displaying yeast cells

As an effort to find suitable endoglucanases to generate cellulolytic yeast strains, two fungal endoglucanases, Thermoascus aurantiacus EGI and Trichoderma reesei EGII, and two bacterial endoglucanases, Clostridium thermocellum CelA and CelD, were expressed on the yeast surface, and their surface expression levels, pH- and temperature-dependent enzyme activities, and substrate specificities were analyzed. T. aurantiacus EGI showed similar patterns of pH- and temperature-dependent activities to those of T. reesei EGII which has been widely used due to its high enzyme activity. Although EGII showed higher carboxymethyl cellulose (CMC) degradation activity than EGI, EGI showed better activity toward phosphoric acid swollen cellulose (PASC). For ethanol production from PASC, we combined three types of yeast cells, each displaying T. aurantiacus EGI, T. reesei CBHII (exoglucanase) and Aspergillus aculeatus BGLI (β-glucosidase), instead of co-expressing these enzymes in a single cell. In this system, ethanol production can be easily optimized by adjusting the combination ratio of each cell type. A mixture of cells with the optimized EGI:CBHII:BGLI ratio of 6:2:1 produced 1.3 fold more ethanol (2.1 g/l) than cells composed of an equal amount of each cell type, suggesting the usefulness of this system for cellulosic ethanol production.     

Antitumor activity of hyperthermia alone or in combination with cisplatin and melphalan in primary cultures of human malignant melanoma

The effects of heat and the interaction between hyperthermia and alkylating agents, such as cisplatin (CDDP) and melphalan (L-PAM) in human malignant melanoma biopsies have been investigated by a short-term assay based upon the inhibition of 3H-thymidine incorporation. Cell suspensions from 50 cutaneous and lymph nodal metastases were heated at 40.5 degrees C or at 42 degrees C for 1 h. There were significant antiproliferative effects due to heat in 10% of the tumors exposed to 40.5 degrees C and 34% to 42 degrees C. Thermal resistance was evident in 73% (at 40.5 degrees C) and 54% (at 43 degrees C) of tumors, and there was significant enhancement of cell growth in 17% and 12% of tumors. The combined effects of hyperthermia and drugs were studied on 36 tumors. Cell suspensions were exposed to different concentrations of CDDP or L-PAM for 1 h at 40.5 degrees C and 42 degrees C. Synergy between heat and CDDP was observed in 7% of cases treated with the lowest drug dose and 38% of cases treated with the highest (40.5 degrees C), with only a slight increase in the frequency of synergy at 42 degrees C. Synergy between heat and L-PAM was also observed in 12% to 44% of tumors at 42 degrees C as a function of drug concentration.     

Survival and recovery of yeast cells after combined treatment with ionizing radiation and heat

Cell survival, recovery kinetics and inactivation forms after successive and simultaneous treatments with gamma rays (60Co) and high temperatures were studied in diploid yeast cells capable of recovery. Both the extent and the rate of the recovery were shown to be greatly decreased with increase in the duration of heat treatment (60 degrees C) followed by radiation and with increase in exposure temperature after simultaneous treatment with heat and radiation. A quantitative approach describing the recovery process was used to estimate the probability of recovery per unit time and the irreversible component of damage after the combined treatment with heat and radiation. It was shown that the probability of recovery was independent of the conditions of the treatment with heat and radiation, while the irreversible component gradually increased as a function of the duration of heat treatment (60 degrees C) after sequential treatment with heat and radiation and as a function of the exposure temperature after simultaneous treatment with heat and radiation. The increase in the irreversible component was accompanied by an increase in cell death without postirradiation division. It is concluded on this basis that the synergistic interaction of ionizing radiation and hyperthermia in yeast cells is not related to the impairment of the recovery capacity itself and that it may be attributed to an increased yield of irreversible damage.