Biomedical subjects

W C Dewey

Publications and source records attributed to W C Dewey.

At least 19 recordsLinked to original sources

PubMed1995

Radiation-induced apoptosis: relevance to radiotherapy.

Radiation-induced apoptosis is reviewed in terms of: (a) the identification of apoptotic and necrotic cells, (b) observations in vitro and in vivo of radiation-induced apoptosis, (c) genes controlling apoptosis, (d) evidence that the target may be the plasma membrane or nuclear DNA, (e) quantitative comparisons of apoptotic death and reproductive (clonogenic) death, (f) the importance of radiation-induced apoptosis in radiotherapy, and (g) studies of radiation-induced apoptosis that are needed. High priority should be placed on determining the molecular pathways that are important in the expression and modulation of radiation-induced apoptosis. Specifically, the events that modulate the apoptosis that occurs in interphase before the cell can divide should be distinguished from the events before division that modulate the misrepair of DNA damage, that results in chromosomal aberrations observed in mitotic cells, which in turn cause the progeny of the dividing cell with aberrations to die by either apoptosis or necrosis. Then, molecular events that determine whether a cell that divides with or without a chromosomal aberration will produce progeny that apoptose or necrose need to be identified. These considerations are important for determining how modulation of radiation-induced apoptosis will affect the ultimate clonogenic survival, and possibly genomic instability in the surviving progeny.

Animals↗

PubMed1995

Thermotolerant cells possess an enhanced capacity to repair heat-induced alterations to centrosome structure and function.

To study the mechanisms of thermotolerance, the adaptive response by which cells become transiently resistant to killing by heat shock, we have focused on the centrosome, an organelle whose disorganization is closely correlated with thermal killing in Chinese hamster ovary (CHO) cells. Centrosome structure was studied by use of antisera directed against pericentrin, a 220 Kd protein of the pericentriolar material (PCM). Centrosome function was measured in intact cells by performing microtubule regrowth following exposure to the drug nocodazole. Immediately following heating at 45 degrees C for 4-18 min, centrosomal staining by antipericentrin decreased. Thereafter, staining gradually recovered, although abnormal configurations of staining appeared in heated cultures 10-20 h later. In contrast, abnormal patterns of staining rarely developed in thermotolerant cultures. Centriole number was not perturbed by heat, indicating that the heat effect was specific for the PCM. Heat also caused an immediate reduction in the number of microtubules nucleated by the PCM. As for staining by antipericentrin, microtubule nucleation recovered during 3-20 h at 37 degrees C after heating. The immediate, heat-induced decrease in antipericentrin staining or microtubule nucleation was similar in thermotolerant and nontolerant cells. In contrast, the inhibition for both endpoints recovered to control levels much more quickly in thermotolerant cells than in nontolerant cells. Furthermore, new protein synthesis was not required for the recovery of microtubule nucleation. These data show that thermotolerant cells have an enhanced capacity to repair thermal damage to centrosome structure and function, and suggest that a faster rate of recovery prevents disorganization of the PCM that is observed in nontolerant cells several hours after heating.

Adaptation, Physiological↗

PubMed1995

Analysis by pulsed-field gel electrophoresis of DNA double-strand breaks induced by heat and/or X-irradiation in bulk and replicating DNA of CHO cells.

For a given amount of cell killing, heat alone (10-80 min, 45.5 degrees C) induced very few double-strand breaks (dsbs) compared with X-rays. Furthermore, 10 min at 45.5 degrees C immediately prior to X-rays caused only a 1.3-fold increase in the slope of the X-ray-induced dsb dose-response curve, i.e. 0.67 +/- 0.006 (95% confidence) dsbs/100Mbp/Gy for heated cells compared with 0.53 +/- 0.005 for unheated control cells. However, this same heat treatment caused > 5-fold inhibition in the rate of repair of dsbs induced by 60-Gy X-rays, with the degree of inhibition being much less in thermotolerant (TT) cells than in non-tolerant (NT) cells. This reduced inhibition of repair in TT cells correlated with the more rapid removal of excess nuclear protein from nuclei isolated from TT cells than from NT cells. These results plus a TT ratio of 2-3 for both heat-induced radiosensitization and heat-inhibition of repairing dsbs are consistent with the hypothesis that heat radiosensitization results primarily from heat aggregation of nuclear protein interfering with access of repair enzymes to DNA dsbs. The selective heat-radiosensitization of S-phase cells, however, may result from an increase in radiation-induced dsbs in or near replicating regions. For example, a preferential increase in dsbs in replicating DNA compared with bulk DNA was found following either hyperthermia alone (10-30 min, 45.5 degrees C) or a combined treatment (10 min, 45.5 degrees C before 60 Gy). A 30-min treatment at 45.5 degrees C induced dsbs equivalent to approximately 10 Gy in replicating DNA compared with 3-5 Gy in bulk DNA. When cells were heated immediately before irradiation, the increase in dsbs induced in the replicating DNA by 60 Gy was equivalent to 200 Gy. We hypothesize that the observed 2-fold increase in single-stranded regions in replicating DNA after heat resulted in radiation selectively inducing dsbs at or near the replication fork where the heat-induced increase in single-stranded DNA should occur. Thus, this preferential increase in dsbs in the replicating DNA by heat alone and especially when heat was combined with radiation may explain at least in part, the high sensitivity of S-phase cells to heat killing and heat radiosensitization.

Animals↗

PubMed1995

Methods for the quantification of DNA double-strand breaks determined from the distribution of DNA fragment sizes measured by pulsed-field gel electrophoresis.

Different methods were used for evaluating data for DNA double-strand breaks (DSBs), as obtained by pulsed-field gel electrophoresis (PFGE) after X irradiation of Chinese hamster ovary cells. A total of 60 data points in the dose range of 0 to 116 Gy, along with repair data for 30 and 60 Gy, were analyzed by four methods: (1) percentage of DNA released from the plug, (2) specific size markers (percentage of DNA less than specific sizes, (3) fragment size distributions and (4) shape of the molecular weight (M) distributions. With the last method, both the slope and the intercept of the logarithm of the amount of radioactive DNA/delta M/M plotted as a function of M were used for calculating DSBs/100 Mbp. The slope and the intercept analyses differ in that the former is relatively independent of DNA trapped in the agarose plugs, i.e. cannot be released by doses of 100-150 Gy, whereas the intercept is dependent on the percentage of DNA trapped. Also, calculations of DSBs/100 Mbp for methods 1, 2 and 3 depend on the amount of DNA trapped in the plug. However, the slope method is unreliable for doses below about 20 Gy, and the scatter of data points is much greater than that obtained by the intercept method and by methods 1, 2 and 3. Therefore, the fragment size distribution and the specific size marker methods give the most consistent results, with 0.49 +/- 0.03 (95% CI) (DSBs/100 Mbp)/Gy. With the specific size marker method, however, care must be taken in selection of size markers in relation to the levels of DSBs of interest. Assuming randomly distributed DSBs, all four methods gave essentially the same results; i.e., the dose response was linear with a calculated level of 0.5-0.6 (DSBs/100 Mbp)/Gy, which is the same as 0.47-0.62 determined previously by calibrating with 125IdU.

Animals↗

PubMed1995

Repair of DNA double-strand breaks: errors encountered in the determination of half-life times in pulsed-field gel electrophoresis and neutral filter elution.

For theoretical reasons, it is incorrect to define experimentally the half-life times of DNA double-strand breaks (DSBs) as the half-life time of an amount of DNA. This is illustrated by one example of human DNA, where the half-life for first-order kinetics of the disappearance of DSBs has been assumed to be 10 min. Experimental sources of errors and their influence on experimental results are analyzed. Some experimental situations may lead to serious misinterpretation data. The differential decreases in fractions released (amounts of DNA) as often followed in pulsed-field gel electrophoresis depend on run conditions, background and level of DSB induction and are a function of time itself--a time function that is unrelated to the half-life of DSBs. It is shown that, using the decrease of a measured amount of DNA, one may obtain practically any value for the half-life time.

Animals↗

PubMed1994

A charge-coupled-device camera image analysis system for quantifying DNA distributions in agarose gels after pulsed-field gel electrophoresis.

A charge-coupled-device camera system was coupled to a personal computer and, with uniformity in illumination and detection (within 4-8%) along each lane, was used for quantifying the distribution of DNA molecules that migrate from the PFGE well (plug) into the lane at distances varying from 1 to 50 mm (with 0.5 mm/pixel). By using a specially designed transmission filter for transmitting 470-725 nm fluorescence from ethidium bromide-stained DNA while eliminating most of the fluorescence (< 400 nm) from the agarose gel, and by using neutral density filters to prevent saturation of the camera, the fluorescence intensity is linearly related to the amount of DNA varying from approximately 0.03 micrograms in a 3-mm-diameter cylindrical plug 5 mm long (equal to background) to approximately 4 micrograms (where ethidium bromide staining saturates). The percentage DNA released from the plug and distribution in the lane (with 1-2 mm resolution) obtained by quantifying DNA fluorescence were not significantly different from the same data obtained by analysis of radioactivity of the same DNA labeled with [3H]dThd. However, scattering of fluorescence from one lane into an adjacent lane 3 mm away and as far as 10 mm from the plug into the lane presented a problem. This problem was overcome by using a form with slots to cover every other lane when the images were obtained and either (1) cutting the lane from the plug and moving it 15 mm away or (2) imaging the intact gel and applying a correction for approximately 7% of the fluorescence from the plug tailing out approximately 10 mm beyond the first 1 mm in the lane. In addition, the following were required: (1) carefully controlled staining and destaining procedures, and (2) a low background that is obtained as an average uniform background in each lane 5 mm beyond where DNA migration stops.

DNA↗

PubMed1993

Cisplatin induced cell killing and chromosomal aberrations in CHO cells: treated during G1 or S phase.

Variation in sensitivity to cisplatin during the cell cycle was studied in synchronous CHO cells treated during G1 or late S phase. The cells were assayed for cell killing, cell-cycle delay, and chromosomal aberrations after they were treated with cisplatin (1-12 micrograms/ml) for 1 h at 37 degrees C. They were either plated for colony survival, or colcemid was added from 12 to 40 h after plating followed by fixation 4 h later for analysis of chromosomal aberrations after the cells completed 1 or 2 cycles (i.e. first or second mitosis). Cells treated with 6 micrograms/ml exhibited about a 10-h delay during the first cycle after treatment during G1 compared with about 3 h during the first cycle and 6 h during the second cycle after treatment during late S. In both cases, cells entering metaphase exhibited predominantly chromatid-type breaks and exchanges. For both cell killing and chromosomal aberrations, the cells in G1 were 1.5-1.6 times more sensitive than those treated in late S, with 1 aberration per cell corresponding to about 37% survival. However, the exchanges and breaks were observed primarily in the first mitosis when cells were treated in G1 compared with the second mitosis when cells were treated in late S. These results suggest that DNA replication opposite cisplatin cross-links in the DNA results in lethal chromosomal aberrations.

Animals↗

PubMed1993

Plasma membrane activities retained after lethal heat shock.

Cultures of Chinese hamster ovary (CHO) cells were examined to determine if heat killing could be attributed to severe damage in the plasma membrane. Three independent transport activities of the plasma membrane were measured. Glucose transport into the cells (measured with the non-metabolizable analogue 3-O-methyl-D-glucose) was stimulated rather than inhibited by heat. Most of the stimulation was found after non-toxic heat doses. Although amino acid transport (measured with the non-metabolizable analogue 2-aminoisobutyric acid) was slightly inhibited by heat, heat-sterilized cells were able to accumulate high intracellular concentrations. Cellular uptake of the nucleoside uridine was unaffected for at least 4 h after heating. In contrast, its incorporation into RNA was immediately inhibited. To further study plasma membrane damage, cells were either heated or treated with drugs which localize to the plasma membrane, ionophore A23187 or amphotericin B. The mode of cell killing by heat was radically different from that of the two drugs: heat-sterilized cells retained a phase-bright morphology and excluded the viability dye trypan blue while drug-killed cells rapidly became phase-dark and absorbed the dye. These results add to a growing list of plasma membrane activities which are retained in heat-sterilized cells, and suggest that the initial thermal damage responsible for cell killing is at an alternate site(s).

Amino Acids↗

PubMed1992

Relationship between thermal tolerance and protein degradation in temperature-sensitive mouse cells.

The induction of thermotolerance was studied in a temperature sensitive mouse cell line, ts85, and results were compared with those for the wild-type FM3A cells. At the nonpermissive temperature of 39 degrees C, ts85 cells are defective in the degradation of short-lived abnormal proteins, apparently because of loss of activity of a ubiquitin-activating enzyme. The failure of the ts85 cells to develop thermotolerance to 41-43 degrees C after incubation at the nonpermissive temperature of 39 degrees C correlated with the failure of the cells to degrade short-lived abnormal proteins at 39 degrees C. However, the failure of the ts85 cells to develop thermotolerance to 43 degrees C during incubation at 33 degrees C after either arsenite treatment or heating at 45.5 degrees C for 6 or 10 min did not correlate with protein degradation rates. Although the rate of degrading abnormal protein was reduced after heating at 45.5 degrees C for 10 min, the rates were normal after arsenite treatment or heating at 45.5 degrees C for 6 min. In addition, when protein synthesis was inhibited with cycloheximide both during incubation at 33 degrees C or 39 degrees C and during heating at 41-43 degrees C, resistance to heating was observed, but protein degradation rates at 39 degrees C or 43 degrees C were not altered by the cycloheximide treatment. Therefore, there is apparently no consistent relationship between rates of degrading abnormal proteins and the ability of cells to develop thermotolerance and resistance to heating in the presence of cycloheximide.

Acclimatization↗

PubMed1991

Heat-induced morphological alterations in non-tolerant and thermotolerant cells.

CHO cells were heated at 43 degrees C or 45 degrees C for various durations up to 300 min. Survival values varied from 5 x 10(-1) to 10(-7). Unheated, non-tolerant control cells were compared with cells made thermotolerant (TT) by incubating at 37 degrees C for 6 or 12 h after treatment with either sodium arsenite (100 microM-As) or 45.5 degrees C for 10 min, respectively. Groups also were included in which heat-induced TT cells were heated at 43 degrees C for 5 h immediately before they were challenged at 45 degrees C; in these groups, cycloheximide was sometimes added to inhibit protein synthesis before and/or during heating at 43 degrees C. Morphological alterations were quantified immediately and at various times after heating by using phase-contrast microscopy to determine the percentage of cells that were severely blebbed and rounded. About 800 cells were analysed per datum point. When effects of heat on thermotolerant cells were compared with effects of heat on non-tolerant cells, heat-induced thermotolerance (HTT) was observed by an increase in survival, and by a reduction in the percentage of cells with morphological alteration observed immediately after the challenging heat. After the As treatment, very little thermotolerance was observed for morphological alterations immediately after the challenging heat, although thermotolerance was observed for survival. However, as the cells were incubated for 12 or 24 h at 37 degrees C after the challenging heat treatment, recovery from morphological alterations was observed in the As-TT cells. Possible mechanisms for the difference between HTT and As-TT are discussed.

Adaptation, Physiological↗

PubMed1991

Maintenance of intracellular free Ca2+ homeostasis following lethal heat shock.

We have manipulated the extracellular Ca2+ concentration (1.8 mM in normal Dulbecco's modified Eagle's medium) to test whether the resulting effect on intracellular free Ca2+ homeostasis was similar in heat-sterilized and nonheated mouse NIH-3T3 cells. The responsiveness of the intracellular free Ca2+ concentration to changes in the extracellular Ca2+ concentration was not affected by prior treatment of the cells with trypsin, or by the extracellular Ca2+ concentration during dye loading (indo-1, AM). Rather, the intracellular free Ca2+ concentration was dependent upon the ambient Ca2+ concentration during analysis by flow cytometry. When the extracellular Ca2+ concentration was decreased to 0.017 mM, either before or after a lethal heat shock, the intracellular free Ca2+ concentration (approximately 300 nM) decreased to a similar extent in both heated and control cells (to approximately 30-100 nM). Similarly, when the extracellular Ca2+ concentration was increased to 15.0 mM, either before or after a lethal heat shock, the intracellular free Ca2+ concentration exhibited a quantitatively similar increase in both heated and nonheated cells (to approximately 400-1000 nM). These data indicate that a lethal heat dose does not inhibit the intact cell's ability to maintain intracellular free Ca2+ homeostasis.

3T3 Cells↗

PubMed1991

Use of pulsed-field gel electrophoresis to measure X-ray-induced double-strand breaks in DNA substituted with BrdU.

The substitution of BrdU for TdR in the DNA of Chinese hamster ovary cells caused radiosensitization for both cell killing and an increase in the rate of neutral elution of the DNA. However, no radiosensitization was observed for the amount of DNA that migrated from the plug of agarose gels subjected to pulsed-field gel electrophoresis. An unexpected observation, however, was that the migration rate of BrdU-substituted DNA was relatively independent of radiation dose and was much less than that of unsubstituted DNA which migrated at a faster rate as the radiation dose increased. This difference in migration between TdR- and BrdU-labeled DNA was observed only when electrophoresis conditions were optimized for separating DNA molecules from 1 to 7 Mb. Possibly, the increase in negative charge on BrdU-labeled DNA increases the reorientation time during each pulse, with a resulting decrease in rate of migration, or radiation effects on BrdU-labeled DNA may be responsible for the decrease in migration rate.

Animals↗

PubMed1990

Comparison of DMO and flow cytometric methods for measuring intracellular pH and the effect of hyperthermia on the transmembrane pH gradient.

Intracellular pH (pHi) was measured in both unheated and heated cells by the distribution of the weak acid, 5,5-dimethyl-2,4-oxazolidinedione-2-14C (14C-DMO), and by the fluorescence intensity ratio (I530/I630) of the pH sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF), analyzed by flow cytometry (FCM). BCECF-loaded Chinese hamster ovary (CHO) cells were analyzed by FCM after they had incubated in fresh medium at 37 degrees C for 90 min, during which time a decrease in fluorescence ratio stabilized. After stabilization, the pHi determined for CHO cells by the FCM method at pHe values of 6.0-8.1 agreed-within 0.1 pH units with that determined by the 14C-DMO method. There is a pH gradient across the plasma membrane that is not affected by heat. In CHO cells, the gradient, determined by DMO and FCM, is less or greater than pHe by 0.30 and 0.15 pH units at pHe 7.4 and 6.3, respectively, and in NG108-15 cells, the gradient determined by DMO increases to 0.50 pH units at pHe 6.3. Both cells maintained their pH gradients for at least 4 h after heating, although 99.9% of the cells were reproductively dead (survival of 10(-3)) after heating at 45.5 degrees C either at the normal pHe of 7.4 or at a low pHe of 6.4-6.7.

Adolescent↗

PubMed1990

Effects of pH on heat sensitization of mammalian cells with procaine hydrochloride.

The enhancement of heat killing of CHO cells by treatment with 7 mM procaine HCl increased when cells were treated under alkaline conditions. Below a pH of 6.9, very little heat sensitization was observed; however, as the pH was increased to 7.4 and above, considerable heat sensitization occurred. There were no changes in intracellular pH at the beginning of heating that could be responsible for this phenomenon.

Animals↗

PubMed1990

Time-temperature analysis of cell killing of BHK cells heated at temperatures in the range of 43.5 degrees C to 57.0 degrees C.

Baby hamster kidney (BHK) cells were heated at temperatures in the range of 43.5 degrees C to 57.0 degrees C to determine the time-temperature relationship of cell killing. The cells were grown on 0.025 mm thick pieces of mylar to minimize warm-up times. After heating, the cells were plated for the colony formation assay. The endpoints of 1%, 10%, or 90% isosurvival, or the D0 values of the survival curves were used to construct plots of the logarithm of the reciprocol of the exposure time versus the reciprocol of the absolute temperature. The data for each endpoint resulted in a straight line plot, indicating that the time-temperature relationship for cell killing remained constant from 43.5 degrees C to 57.0 degrees C; namely, a 1.8-fold increase in exposure time was required for a 1 degree C decrease in temperature in order to obtain isosurvival. Heated BHK cells were also examined using electron microscopy. The threshold level of altered morphology was the dissociation of polyribosomal structure and the formation of electron-dense granules within the mitochondria. The time-temperature relationship for the induction of this altered morphology was identical to that for the 90% isosurvival endpoint. Hence, the appearance of altered morphology appears to be related to cell killing.

Animals↗

PubMed1990

Calibration of pulsed field gel electrophoresis for measurement of DNA double-strand breaks.

The pulsed field gel electrophoresis (PFGE) assay was calibrated for the measurement of X-ray-induced DNA double-strand breaks in Chinese hamster ovary (CHO) cells. Calibration was conducted by incorporating [125I]deoxyuridine into DNA, which induces one double-strand break for every disintegration that occurs in frozen cells. Based on the percentage of the DNA migrating into the gel, the number of breaks/dalton/Gy was estimated to be (9.3 +/- 1.0) x 10(-12). This value is close to (10 to 12) x 10(-12) determined by neutral filter elution using similar cell lysis procedures at 24 degrees C and at pH 8.0. Also, the estimate is in good agreement with the value of (11.7 +/- 2) x 10(-12) breaks/dalton/Gy as measured in Ehrlich ascites tumour cells using the neutral sucrose gradient method (Blöcher 1988), and (6 to 9) x 10(-12) breaks/dalton/Gy as measured in mouse L and Chinese hamster V79 cells using neutral filter elution (Radford and Hodgson 1985).

Calibration↗

PubMed1990

Differences in thermotolerance induced by heat or sodium arsenite: cell killing and inhibition of protein synthesis.

Chinese hamster ovary (CHO) cells became thermotolerant after treatment with either heat for 10 min at 45.5 degrees C or incubation in 100 microM sodium arsenite for 1 h at 37 degrees C. Thermotolerance was tested using heat treatment at 45 degrees C or 43 degrees C administered 6-12 h after the inducing agent. At 45 degrees C thermotolerance ratios at 10(-2) isosurvival levels were 4.2 and 3.8 for heat and sodium arsenite, respectively. Recovery from heat damage as measured by resumption of protein synthesis was more rapid in heat-induced thermotolerant cells than in either sodium arsenite-induced thermotolerant cells or nonthermotolerant cells. Differences in inhibition of protein synthesis between heat-induced thermotolerant cells and sodium arsenite-induced thermotolerant cells were also evident after test heating at 43 degrees C for 5 h. At this temperature heat-induced thermotolerant cells were protected immediately from inhibition of protein synthesis, whereas sodium arsenite-induced thermotolerant cells, while initially suppressed, gradually recovered within 24 h. Furthermore, adding cycloheximide during the thermotolerance development period greatly inhibited sodium arsenite-induced thermotolerance (SF less than 10(-6] but not heat-induced thermotolerance (SF = 1.7 X 10(-1] when tested with 43 degrees C for 5 h. Our results suggest that both the development of thermotolerance and the thermotolerant state for the two agents, while similar in terms of survival, differed significantly for several parameters associated with protein synthesis.

Animals↗

PubMed1990

Noninvolvement of the heat-induced increase in the concentration of intracellular free Ca2+ in killing by heat and induction of thermotolerance.

Mouse C3H 10T1/2 cells exhibited a two- to threefold increase in the concentration of free Ca2+ during heating at 45 degrees C. The increase was maximal for a heat dose which was still in the shoulder region of the survival curve. The increase was fully reversible in heat-sterilized cells. By changing the concentration of extracellular Ca2+, it was possible to modulate the concentration of intracellular free Ca2+ in heated cells. Lowering the extracellular concentration to 0.03 mM reduced the baseline concentration of intracellular free Ca2+, and prevented it from increasing in heated cells to a level exceeding that of nonheated cells incubated in medium containing 2.0 or 5.0 mM Ca2+. Raising the concentration of extracellular Ca2+ to 15.0 mM raised the baseline, and resulted in a heat-induced increase in free Ca2+ which was twofold higher than that of cells heated in medium containing 2.0 or 5.0 mM Ca2+. An elevated concentration of intracellular free Ca2+ during and after heating did not potentiate thermal killing, nor did a reduced concentration during and after heating mitigate killing. Furthermore, the data argue against a heat-induced increase in free Ca2+ to some threshold level, which potentiates cell killing by some other parameter. In addition, cells heat-shocked in either 0.03 or 5.0 mM extracellular Ca2+, and then incubated in the same concentration for 12 h at 37 degrees C, developed quantitatively similar amounts of tolerance to a second heating. The data suggest that the concentration of intracellular free Ca2+ does not play a critical role in thermal killing or the induction and development of thermotolerance.

Acclimatization↗