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D C Shrieve

Publications and source records attributed to D C Shrieve.

54 records · Page 3Linked to original sources
PubMed1986

Combined effect of buthionine sulfoximine and cyclophosphamide upon murine tumours and bone marrow.

Two and four treatments of 5 mmol kg-1 of buthionine sulfoximine (BSO) at an interval of 12 h depleted the glutathione (GSH) content in NFSa tumours of C3H/He mice, respectively, to 24.0 and 1.78 percent of the untreated controls. BSO pre-treatments every 12 h enhanced the cytotoxicity of cyclophosphamide (CYC) towards artificial lung micrometastases of NFSa tumours giving enhancement ratios (ERs) ranging from 1.75 to 1.83 and from 2.41 to 2.73, for two and four BSO pretreatments respectively. Large ERs were obtained at low CYC doses (high cell survival). Four BSO pre-treatments at an interval of 12 h did not increase the cytotoxicity of CYC to bone marrow stem cells. Our results suggest a clinical applicability of the combination of BSO and CYC.

Animals↗
PubMed1986

Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: some applications to radiation and drug resistance in vitro and in vivo.

An assay using a bimane derivative has been developed to detect free glutathione (GSH) in individual viable cells by flow cytometry. Monochlorobimane [syn-(ClCH2CH3)-1,5-diazabicycla[3.30]acta-3,6-diene-2,8-dio ne], itself nonfluorescent, reacts with GSH to form a highly fluorescent derivative. High pressure liquid chromatography analysis showed that, using specific staining conditions, the only low molecular weight fluorescent derivative formed in Chinese hamster ovary cells was that formed with GSH. Very little reaction with protein sulfhydryls was observed. Rates of GSH depletion in Chinese hamster ovary cells exposed to diethylmaleate were essentially the same, whether measured by relative fluorescence intensity, by flow cytometry or by enzymatic assay on cellular extracts. This method was shown to be useful for measurement of GSH resynthesis, uptake, and depletion by prolonged hypoxia and misonidazole treatment. Since measurements are made on individual cells, cell-to-cell variation and populational heterogeneity in GSH content are revealed by flow cytometry. Although under most conditions in vitro GSH content is relatively homogeneous, under certain circumstances, such as release from hypoxia, heterogeneity in populational GSH levels was observed. The significance of this heterogeneity is discussed in regard to the induction of gene amplification and drug resistance by transient hypoxia. Numerous subclones of Chinese hamster ovary cells selected by growth in Adriamycin or methotrexate-containing medium express elevated levels of GSH per cell. The method was extended to quantitate the GSH content of cells excised from EMT-6/SF mouse tumors that had been treated in vivo with L-buthionine-S-R-sulfoximine, an inhibitor of GSH synthesis. The bivariate analysis (forward angle light scatter versus monochlorobimane fluorescence) of cells derived from these tumors gave excellent resolution of normal and tumor cells and demonstrated extensive heterogeneity in the tumor cell population with respect to GSH content per cell.

Animals↗
PubMed1986

Cellular glutathione, thermal sensitivity, and thermotolerance in Chinese hamster fibroblasts and their heat-resistant variants.

HA-1 Chinese hamster fibroblasts and two heat-resistant variants, designated 2242 and 3012. have been investigated to determine the role that glutathione (GSH) plays in intrinsic cellular resistance to heat and in the development of thermotolerance. The constitutive levels of GSH did not correlate with intrinsic heat sensitivities, but depletion of GSH sensitized all three cell lines to thermal stress. After heating (43.5 degrees C/2 h), surviving fractions were 1 X 10(-3), 1 X 10(-2), and 8 X 10(-3) for HA-1, 2242, and 3012 cells, respectively. Depletion of cellular GSH with L-buthionine-S, R-sulfoximine to less than 10% of control values sensitized such that the thermal responses of these three cell lines were nearly indistinguishable at 43.5 degrees C. Surviving fractions were 2 X 10(-4), 1 X 10(-4), and 1 X 10(-4) for L-buthionine-S,R-sulfoximine-treated HA-1, 2242, and 3012 cells, respectively, following heating at 43.5 degrees C for 2 h. The development of thermotolerance in HA-1 cells following heat shock (45 degrees C/15 min) was unaffected by the inhibition of GSH synthesis. On the other hand, when GSH levels were maintained at extremely low levels, the development of thermotolerance was inhibited. In addition, following heat shock, cellular GSH was decreased and remained below control levels during the development of thermotolerance.

Animals↗
PubMed1985

A method to measure the duration of DNA synthesis and the potential doubling time from a single sample.

A method is described whereby the DNA synthesis time, Ts, can be calculated using data of a single sample of cells taken several hours after labelling with bromodeoxyuridine (BrdUrd). The method involves a simple calculation using flow cytometry data of BrdUrd incorporation (green fluorescence, FITC-labelled anti-BrdUrd-DNA antibody) and total DNA content (red fluorescence, propidium iodide). The movement of BrdUrd-labelled cells through the S phase can be quantified by measuring their mean red fluorescence relative to that of G1 and G2 cells. Assuming the movement of the labelled cells toward G2 is linear with time, Ts can be calculated by measuring their relative movement at any one time. The method was tested on cells in vitro and on bone marrow and tumor cells in vivo. Reasonable agreement was seen with published estimates of Ts for these tissues.

Animals↗
PubMed1985

The in vitro sensitivity of chronically hypoxic EMT6/SF cells to X-radiation and hypoxic cell radiosensitizers.

We have investigated the effect of extreme, prolonged hypoxia on the radiosensitivity of EMT6/SF cells in vitro. As cells were kept hypoxic for 1-24 h, their radiosensitivity increased, but no further change was noted for hypoxic incubation beyond 24 h. Chronically hypoxic (45 h) cells were more radiosensitive than acutely hypoxic (1 h) cells by a factor of 1.43. When chronically hypoxic cells were re-aerated, the increased radiosensitivity persisted, although it was reduced. Misonidazole (MISO) radiosensitization was equally effective under conditions of acute and chronic hypoxia. In contrast, MISO, SR2555 and SR2508 were more cytotoxic in chronically hypoxic cultures than in acutely hypoxic cells. Measurements suggested that intracellular thiols may play an important role in the effects observed.

Animals↗
PubMed1985

Cell cycle kinetics of aerated, hypoxic and re-aerated cells in vitro using flow cytometric determination of cellular DNA and incorporated bromodeoxyuridine.

The effects of extreme hypoxia on cell cycle progression were studied by simultaneous determination of DNA and bromodeoxyuridine (BrdU) contents of individual cells. V79-379A cells were pulse-labelled with BrdU (1 microM, 20 min, 37 degrees C) and then incubated for up to 12 hr in BrdU-free medium under either aerated or extremely hypoxic conditions. After the incubation interval (0-12 hr), the cells were trypsinized and fixed in 50% EtOH. Propidium iodide and a fluorescein-labelled monoclonal antibody to BrdU were then used to quantify DNA content and incorporated BrdU, respectively. Measurements in individual cells were made by simultaneous detection of green and red fluorescence upon excitation at 488 nm using flow cytometry. Bivariate analysis revealed progression of BrdU-labelled cells in aerated cultures out of S phase, into G2 and cell division, with halving of mean fluorescence, and back into S phase by approximately 9 hr after the BrdU pulse. Hypoxia immediately arrested cells in all phases of the cell cycle. Both the DNA distribution and the bivariate profile of cells that were fixed from 2 to 12 hr after induction of hypoxia were identical to the 0 hr controls. The percent of cells with green fluorescence in a mid-S phase window remained 100% and the mean fluorescence of these cells remained at control (0 hr) levels. This indicates that, under hypoxic conditions, cells were moving neither into nor out of S phase. Cultures that had been hypoxic for 12 hr exhibited an increasing rate of BrdU uptake with time after re-aeration. Re-aerated cells were able to complete or initiate DNA synthesis, but their rates of progression through the cell cycle were markedly reduced. A large fraction of cells appeared unable to divide up to 12 hr following release from hypoxia.

Aerobiosis↗
PubMed1985

Effects of hypoxia, pH, and growth stage on cell killing in Chinese hamster V79 cells in vitro by activated cyclophosphamide.

Several factors which influence the sensitivity of Chinese hamster V79 cells to cyclophosphamide (CY) have been studied in vitro in both suspension and monolayer cultures. Activated CY was obtained from the blood of mice 15 to 30 min after i.p. injection of CY (400 mg/kg). At pH 7.4, hypoxia rendered the cells more sensitive to activated CY. At lower values of pH (6.6 and 7.0), there was no difference between the sensitivities of oxic and hypoxic cells, although cells in both conditions were more sensitive to CY than at pH 7.4. Drug sensitivity was markedly affected by the stage of cell growth. Monolayer cultures were most sensitive to CY within a few hours of plating. Cultures then rapidly became less sensitive, with maximum resistance occurring between 24 and 48 h after plating, while the cells were still exhibiting rapid exponential growth. This development of resistance parallelled the formation of small colonies (2 to 4 cells), implying that intercellular contact may confer resistance to killing by activated CY.

Animals↗
PubMed1985

Effects of glutathione depletion by buthionine sulfoximine on radiosensitization by oxygen and misonidazole in vitro.

Buthionine sulfoximine (BSO) has been used to deplete glutathione (GSH) in V79-379A cells in vitro, and the effect on the efficiency of oxygen and misonidazole (MISO) as radiosensitizers has been determined. Treatment with 50 or 500 microM BSO caused a rapid decline in GSH content to less than 5% of control values after 10 hr of exposure (t1/2 = 1.6 hr). Removal of BSO resulted in a rapid regeneration of GSH after 50 microM BSO, but little regeneration was observed over the subsequent 10-hr period after 500 microM. Treatment with either of these two concentrations of BSO for up to 14 hr did not affect cell growth or viability. Cells irradiated in monolayer on glass had an oxygen enhancement ratio (OER) of 3.1. After 10-14 hr pretreatment with 50 microM BSO, washed cells were radiosensitized by GSH depletion at all oxygen tensions tested. The OER was reduced to 2.6, due to greater radiosensitization of hypoxic cells than aerated ones by GSH depletion. GSH depletion had the effect of shifting the enhancement ratio vs pO2 curve to lower oxygen tensions, making oxygen appear more efficient by a factor of approximately 2, based on the pO2 required to give an OER of 2.0. In similar experiments performed with MISO, an enhancement ratio of 2.0 could be achieved with 0.2 mM MISO in anoxic BSO-pretreated cells, compared to 2.7 mM MISO in non-BSO-treated cells. Thus MISO appeared to be more efficient in GSH-depleted cells by a factor of 13.5. These apparent increases in radiosensitizer efficiency in GSH-depleted cells could be explained on the basis of radiosensitization of hypoxic cells by GSH depletion alone (ER = 1.29-1.41). The effect of GSH depletion was approximately equal at all sensitizer concentrations tested, except at high oxygen tensions, where the effect was insignificantly small. These results are consistent with hypoxic cell radiosensitization by GSH depletion and by MISO or oxygen acting by separate mechanisms.

Animals↗
PubMed1984

Glutathione depletion in tissues after administration of buthionine sulphoximine.

Buthionine sulphoximine (BSO) an inhibitor of glutathione (GSH) biosynthesis, was administered to mice in single and repeated doses of 0.5, 1 and 5 mmol kg-1 (i.p.). The resultant pattern of GSH depletion was studied in liver, kidney, skeletal muscle and three types of murine tumor. Liver and kidney exhibited a rapid depletion to GSH levels of ca. 20% of controls after single doses of 1-5 mmol kg-1 BSO. Muscle was depleted to a similar level, but at a slower rate after a single dose. All three tumors required repeated administration of BSO over several days to obtain a similar degree of depletion to that shown in the other tissues.

Animals↗
PubMed1983

Effects of extreme hypoxia on the growth and viability of EMT6/SF mouse tumor cells in vitro.

The purpose of this study was to characterize a model system in which to study hypoxic cell biology in vitro as a function of time under extremely hypoxic conditions. EMT6/SF cells that were maintained at 37 degrees under hypoxic conditions showed no increase in cell number for up to 70 hr. The mitotic index of hypoxic cultures was less than 0.1%, compared to 2.3 to 3.0% in aerated cultures. The plating efficiency of hypoxic cells decreased with time to 20 to 30% of control values by 70 hr. Aerated cultures consumed glucose more rapidly than did hypoxic ones, due to increasing cell number in air. But, on a per cell basis, hypoxic and aerated cells consumed glucose at equal rates (congruent to 1.2 X 10(-4) micrograms/cell/hr). Virtually 100% of the glucose consumed was converted into lactic acid in both aerated and hypoxic cultures. The labeling index and rate of incorporation of [3H]thymidine decreased exponentially with time in hypoxia. However, the percentage of cells with S-phase DNA content remained nearly constant for up to 72 hr. The rate of protein synthesis was suppressed in hypoxic cultures to between 20 and 50% of control (aerated) rates. When cultures were reaerated following 45 hr of hypoxia, congruent to 12 hr was required for resumption of DNA synthesis and cell division. The application of this system to further study of hypoxic cell biology is discussed.

Animals↗
PubMedDate not supplied

Protection against cis-dichlorodiammine Pt (II) cytotoxicity in vitro by cysteamine.

EMT6/SF cells exposed to cis-dichlorodiammine Pt(II) (DDP) for 1 hr in the presence of 10 mM cysteamine (MEA) survived better than cells treated with DDP alone (DMF-3.1). MEA added to cultures after removal of DDP also protected, even when the two exposures were separated by as long as 4 hr. Protection by MEA was concentration dependent: no significant protection was observed below 1 mM and maximum protection required 5 mM. When cells were incubated with 10 mM MEA immediately after removal of DDP, survival increased as a function of the length of exposure to MEA, reaching a maximum at 2 hr.

Animals↗