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D W Hedley

Publications and source records attributed to D W Hedley.

At least 55 records · Page 3Linked to original sources
PubMed1994

Constitutive expression of P-glycoprotein as a determinant of loading with fluorescent calcium probes.

Determination of intracellular calcium levels in Chinese hamster ovary (CHO) cells using the fluorescent calcium probe indo-1AM was hindered by the low level of accumulation of indo-1 in these cells. CHO cells are known to express basal levels of the multidrug resistance efflux pump P-glycoprotein (P-gp). Rhodamine-123, which is a known substrate of P-gp, was used to confirm the presence of P-gp in CHO cells. Verapamil and cyclosporin (CsA), both inhibitors of P-gp, enhanced accumulation of indo-1 in these cells and therefore allowed for improved intracellular calcium measurements. P-gp overexpressing colchicine-resistant CHO cells (CHRC5) also displayed enhanced indo-1AM loading with P-gp inhibitors. Nondetectable levels of P-gp activity were found in wild-type CEM-CCRF cells (human T lymphoblasts), and these cells did not show any difference in indo-1AM loading in the presence or absence of P-gp inhibitors. Loading of a second calcium fluorescent probe fluo-3AM was improved in CHO cells by P-gp inhibition, whereas the structurally related pH probe BCECF-AM was minimally affected. Because low levels of P-gp may be expressed by a range of cell lines and normal tissues, it is suggested that this be considered if difficulties are encountered in loading fluorescent calcium probes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

ICI D1694 and idoxuridine: a synergistic antitumor combination.

The cytotoxicity of idoxuridine (IdUrd), a thymidine analogue, and ICI D1694 (D1694), a folate-based thymidylate synthase (TS) inhibitor, were examined individually and in combination in two human tumor cell lines. MGH-U1 bladder cancer and HCT-8 colon cancer cells were grown as monolayer cultures with and without thymidine. The cytotoxicity of these agents alone and in combination were determined using normal human bone marrow colony-forming unit, granulocyte-macrophage (CFU-GM) as a surrogate for myelosuppression in vivo. Thymidylate synthase inhibition, IdUrd incorporation into DNA, and DNA single-strand breaks were measured in each cell line and related to cytotoxicity. The cytotoxicity of a 24-h exposure to IdUrd or D1694 increased with drug concentration in each cell line. The drug concentrations producing 50% and 10% clonogenic survival in MGH-U1 cells, respectively, were 0.006 and 0.009 microM for D1694 and 13.0 and 81.0 microM for IdUrd. Those for HCT-8 cells, respectively, were 0.009 and 0.018 microM for D1694 and 7.5 and 20.5 microM for IdUrd. The cytotoxicity of IdUrd combined with D1694 was synergistic in both MGH-U1 and HCT-8 cells as determined by median-effect analysis. The addition of thymidine at concentrations of 0.1, 0.3, and 1.0 microM to the culture medium did not decrease the cytotoxicity of D1694 in either tumor cell line. TS inhibition using the whole cell assay was observed with only D1694, producing 50% inhibition of TS activity at 0.002 microM for MGH-U1 and 0.007 microM for HCT-8 cells. IdUrd did not inhibit TS activity, nor did it enhance the TS inhibitory effects of D1694. The incorporation of IdUrd into DNA increased with increasing concentrations of D1694. This increased DNA incorporation correlated with the increase in DNA single-strand breaks. DNA single-strand breaks paralleled cytotoxicity. CFU-GM survival, exposed to the same drug concentrations as those used in the tumor cell lines, revealed that the therapeutic index was greater for the combination than for either agent alone. These findings suggest that IdUrd plus D1694 is a promising new drug combination, which may have a favorable therapeutic index in vivo.

Antimetabolites, Antineoplastic↗
PubMed1994

Evaluation of methods for measuring cellular glutathione content using flow cytometry.

The currently available flow cytometric stains for cellular glutathione were evaluated, examining the labelling of both human and rodent cell lines under various conditions of concentration, time, and temperature. Procedures were used that depleted glutathione (GSH) while having a minimal effect on other cellular sulphydryls in order to estimate linearity and the extent of background staining. As previously reported, monochlorobimane was highly specific for GSH in rodent cells but failed to label human cells adequately because of its low affinity for human glutathione S-transferases. Higher concentrations of monochlorobimane achieved more complete labelling of the human cellular GSH pool but gave increased background fluorescence due to non-GSH binding. The analogue monobromobimane, which binds nonenzymatically to sulphydryls, reacted more readily with GSH than with protein sulphydryls and, provided that stain concentration and incubation time were controlled, gave reproducible staining of human cells with approximately 20% of total fluorescence due to background staining. Of the currently available stains for measuring GSH in human cells, monobromobimane is the agent of choice. Mercury orange also binds more readily to GSH than to protein, giving a degree of specificity, and it has the additional advantage of being excited at 488 nm. However, the reproducibility of staining with mercury orange was less consistent than that using monobromobimane, and a higher background fluorescence was seen. Two additional stains, o-phthaldialdehyde and chloromethyl fluorescein, could also be used to label cellular GSH, but both gave an unacceptably high level of background staining. It is recommended that flow cytometric GSH assays should routinely include a sample of cells that have been depleted of GSH in order to determine the extent of background labeling.

Animals↗
PubMed1993

DNA Cytometry Consensus Conference. DNA flow cytometry and breast cancer.

Measurement of cellular DNA content by flow cytometry is capable of detecting aneuploid stemlines, and also of giving an indication of tumor proliferation kinetics by approximating the percentage of cells in S-phase of the replicative cycle. Because it can be applied both to fresh frozen material submitted for steroid hormone receptor analysis and to fixed paraffin-embedded blocks, it is particularly well suited to the study of breast cancer. Despite being a relatively straightforward test which is now widely used in the risk assessment of patients with early breast cancer, in common with many other prognostic markers its precise clinical role remains uncertain. An extensive body of published data has appeared in the last few years, but the results often appear to be inconclusive or contradictory. In order to define the prognostic significance of DNA cytometry in malignant diseases of the breast, large bowel, bladder, prostate, and hematopoietic system, and to clarify some of the technical issues related to clinical laboratory standards and quality controls, a DNA Cytometry Consensus Conference was held in Prout's Neck, Maine, on October 1-4, 1992. This meeting was sponsored by the NCI, the International Society for Analytical Cytology, and industry. The significance of the meeting's conclusions for clinical breast cancer are discussed here. The consensus statement regarding the clinical utility of DNA cytometry in breast cancer, and the Guidelines for the Implementation of Clinical DNA Cytometry which were generated at this meeting, also appear in this issue of Breast Cancer Research and Treatment.

Aneuploidy↗
PubMed1993

Regulation of intracellular pH in subpopulations of cells derived from spheroids and solid tumours.

Solid tumours are known to develop regions of extracellular acidity and survival of tumour cells in such regions depends on membrane-based mechanisms which regulate intracellular pH (pHi). We have therefore developed a method, based on dual staining of cells and flow cytometry, to study the regulation of pHi in subpopulations of tumours and spheroids. The activity of membrane-based pHi regulating transporters was studied in EMT-6 and MGH U1 cells grown in monolayer culture, spheroids, and tumours. pHi was measured with the fluorescent pH probe 2'7'-bis-(2-carboxyethyl)-5-(and-6)carboxyfluorescein, and Hoechst 33342 was used to identify cells from different regions of tumours and spheroids. In monolayer culture, incubation of cells for 18 h at pHe 6.6 led to a 1.3-1.5-fold enhancement in the activity of both the Na+/H+ exchanger and the Na(+)-dependent Cl-@HCO3- exchanger. This effect was prevented by the protein synthesis inhibitor cycloheximide. Cells from the centre of EMT-6 spheroids had increased activity of the Na+/H+ exchanger compared to cells from the periphery, when spheroids were grown in medium at pH 6.6, but not at 7.4. By contrast, in MGH U1 spheroids, cells from the centre had increased activity of the Na+/H+ antiport under both sets of conditions. There was no significant difference in the activity of the Na+/H+ exchanger in cells derived from different subpopulations of EMT-6 tumours or MGH U1 xenografts in nude mice. Although upregulation of Na+/H+ exchange occurs after exposure to acidic conditions in vitro, the microenvironmental conditions found within solid tumours do not appear to cause this effect. Our results suggest the feasibility of pharmacological inhibition of Na+/H+ exchange activity as an approach to therapy directed against nutrient-deprived tumour cells.

Animals↗
PubMed1992

Multifactorial resistance to antineoplastic agents in drug-resistant P388 murine leukemia, Chinese hamster ovary, and human HeLa cells, with emphasis on the role of DNA topoisomerase II.

The role of DNA topoisomerase II in multifactorial resistance to antineoplastic agents is reviewed. We have previously observed that in Adriamycin (ADR) resistant P388 murine leukemia cells, DNA topoisomerase II enzyme content and cleavage and catalytic activities were all reduced and correlated with drug sensitivity. A subsequent study provided evidence for an allelic mutation of the gene for DNA topoisomerase II as a possible molecular mechanism underlying the enzyme alterations. To ascertain how universal were these observations, a study was undertaken of DNA topoisomerase II (topo II) in other cell lines resistant either to ADR or another topo-II-interactive drug, mitoxantrone. In ADR-resistant Chinese hamster ovary (CHO) cells, topo II cleavage and catalytic activities and the gene product were all reduced; however, only cleavage activity correlated with drug sensitivity. No differences were noted between ADR-sensitive and -resistant CHO cells by Northern or Southern blot analysis, raising the possibility that the enzyme in resistant cells may be regulated at a posttranscriptional level. Findings on a gel retardation or immunoblot band depletion assay showed that the enzyme in CHO/ADR-1 cells failed to bind to the DNA-drug-enzyme complex, suggesting a qualitative as well as quantitative enzyme alteration in those cells. Mitoxantrone-resistant HeLa cells (Mito-1) displayed not only a lower level of cleavage activity but also of enzyme content and catalytic activity, relative to the parental drug-sensitive HeLa cells. As with the CHO cells, no differences were noted between mitoxantrone-sensitive and -resistant HeLa cells on Northern and Southern blot analyses, suggesting that enzyme regulation in these resistant cells may also be at a posttranscriptional level. There was no evidence of enzyme binding to DNA-drug-enzyme complex in resistant HeLa/Mito-1 cells, once again suggesting the presence of a qualitative enzyme alteration. The findings in both ADR-resistant CHO cells and mitoxantrone-resistant HeLa cells do not exclude the possibility that subtle changes in the topoisomerase II gene, such as point mutations, may account for these enzyme changes. The apparent qualitative changes observed in enzyme may result from posttranslational modifications such as phosphorylation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Flow cytometric measurement of glutathione content of human cancer biopsies.

Rice et al. (1986) have described a flow cytometric method where the non-fluorescent probe monochlorobimane (mBCl) forms a fluorescent adduct with cellular glutathione (GSH) under the action of glutathione-S-transferase. We show here that for EMT6 carcinosarcoma cells there is a close correlation between mean cell fluorescence, expressed as a ratio to that of fluorescence calibration beads, and biochemically determined GSH content over the range 0.2-2.0 fmol cell-1. Single cell suspensions from 14 human cancers were prepared by 23-gauge needle aspiration or mechanical disaggregation of surgical specimens, stained using mBCl and examined by flow cytometry. There was a wide range in individual cell fluorescence, which in contrast to EMT6 cells was not strongly correlated with Coulter volume. By comparing tumour cell fluorescence to that of calibration beads, and assuming that the relationship with GSH content for EMT6 holds for other cells, a mean GSH content of 0.95 fmol cell-1 was derived for nine carcinomas, and 0.21 fmol cell-1 for five non-Hodgkin's lymphomas. Although this semi-quantitation needs further validation, the method used here is rapid, gives an indication of heterogeneity of tumour cell GSH content, and can be applied to fine needle biopsy samples. It therefore shows promise as a means for studying prospectively the relationship of GSH content to clinical drug and radiation sensitivity, and for monitoring the effects of agents such as buthionine sulphoximine which are intended to improve treatment results through tumour cell GSH depletion.

Biopsy, Needle↗
PubMed1989

Flow cytometry using paraffin-embedded tissue: five years on.

The use of paraffin-embedded tissue for flow cytometry is reviewed. A number of technical modifications of the original 1983 method have been described, aimed at improving the accuracy of DNA measurements by minimizing cell debris or reducing coefficients of variation, and at simplifying sample preparation. Over 100 clinical studies have now been reported, mainly assessing the effect of DNA index on prognosis, and those published up until mid-1988 are summarized in an appendix. More recently there have been developments in the use of monoclonal antibodies to measure oncogene products or proliferation markers in addition to DNA content. Detailed clinical evaluation and standardization of these more sophisticated methods is still some way ahead, but as was the case with DNA index, the use of archival material from patients whose outcome is already known should speed this process.

Animals↗
PubMed1989

Flow cytometric measurement of intracellular pH in B16 tumors: intercell variance and effects of pretreatment with glucose.

Flow cytometry was used to measure cytoplasmic pH (pHi) of B16 melanoma cells taken from tumor-bearing animals. We used a ratiometric method to allow measurements on an individual cell basis which were independent of cellular content of the pH indicator BCECF. In order to "freeze" any intercell variance which may have existed within the tumor mass, tumors were mechanically disaggregated in bicarbonate-free medium containing 0.5 mM amiloride at 4 degrees C and loaded with BCECF in choline chloride-based Earle's solution at 37 degrees C. Studies using cells grown in vitro showed that this protocol prevented acid load recovery during the 30-min period typically required between tumor excision and pHi measurement. A calibration curve was obtained by resuspending BCECF-stained cells in a range of buffers containing the proton ionophore nigericin. The range of values for individual cells was estimated by comparing the coefficient of variation of the test sample with that obtained when nigericin was used to reduce all cells to the pHi of the calibration buffer. The average value for mean tumor cell pH was 7.32 +/- 0.05 SD. Pretreatment of animals with intraperitoneal glucose for one hour resulted in an average for mean pHi of 7.17 +/- 0.17 SD. Mean coefficient of variation was 8.7%, and in the presence of nigericin, 8.1%. These values indicate a variance in measured pHi of approximately +/- 0.4 pH units, but most of this results from experimental error rather than true intercell pHi variance. The method used here is capable of detecting reduction in mean tumour pHi caused by ip glucose, but incapable of precise estimation of individual cell values. Despite these uncertainties, the results suggest that the range of pHi within B 16 tumors is small.

Amiloride↗