We previously observed that glucose deprivation induces cell death in multidrug-resistant human breast carcinoma cells (MCF-7/ADR). As a follow up we wished to test the hypothesis that metabolic oxidative stress was the causative process or at least the link between causative processes behind the cytotoxicity. In the studies described here, we demonstrate that mitogen-activated protein kinase (MAPK) was activated within 3 min of being in glucose-free medium and remained activated for 3 h. Glucose deprivation for 2-4 h also caused oxidative stress as evidenced by a 3-fold greater steady state concentration of oxidized glutathione and a 3-fold increase in pro-oxidant production. Glucose and glutamate treatment rapidly suppressed MAPK activation and rescued cells from cytotoxicity. Glutamate and the peroxide scavenger, pyruvate, rescued the cells from cell killing as well as suppressed pro-oxidant production. In addition the thiol antioxidant, N-acetyl-L-cysteine, rescued cells from glucose deprivation-induced cytotoxicity and suppressed MAPK activation. These results suggest that glucose deprivation-induced cytotoxicity and alterations in MAPK signal transduction are mediated by oxidative stress in MCF-7/ADR. These results also support the speculation that a common mechanism of glucose deprivation-induced cytotoxicity in mammalian cells may involve metabolic oxidative stress.

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BACKGROUND Quantitation of adult erythrocytes (RBC) containing fetal hemoglobin (F cells) is of potential clinical utility in evaluating erythropoietic disorders, such as myelodysplasia and hemoglobinopathies, and in monitoring F‐cell augmenting therapy. F‐cell counting methodologies include fluorescence microscopy and flow cytometry. Previous flow cytometric methods have employed an isotype antibody control to distinguish F cells from non‐F cells. We investigated the feasibility of using the orange autofluorescence signal (FL2) in glutaraldehyde‐fixed RBC to substitute for fluorescein isothiocyanate (FITC)‐labeled isotype control antibody use in F‐cell quantitation. METHODS Our previously published method for fetal red cell detection in fetomaternal hemorrhage was used, employing a FITC‐labeled anti‐hemoglobin F (HbF) monoclonal antibody reagent. Blood samples with varying F‐cell counts were quantitated for F cells using both immunofluorescence microscopy and flow cytometry comparing FITC‐labeled isotype to FL1 thresholding defined by FL2 autofluorescence. RESULTS F cell percentages obtained by using an FL2 defined threshold for FL1 gating correlated well with expected values in diluted blood samples (r2 = 0.994, slope = 1.019, intercept = 0.24), values obtained using an isotype control (r2 = 0.996, slope = 1.012, intercept = −0.17), and microscopic immunofluorescence counts (r2 = 0.989, slope = 0.999, intercept = −0.72). F‐cell quantitation by the isotype control and FL2 autofluorescence methods was also comparable in 40 blood samples (r2 = 0.994, slope = 1.014, intercept = 0.03). Intra‐assay, interobserver, and interinstrument precision with this autofluorescence gating method exhibited low imprecision (coefficient of variation <14%). CONCLUSION This novel method is a more objective and less laborious alternative for F‐cell quantitation by flow cytometry compared to using an isotype control or microscopy, thereby providing a more robust methodology for clinical studies and consideration as a laboratory reference method for F‐cell counting. Cytometry (Comm. Clin. Cytometry) 42:239–246, 2000. © 2000 Wiley‐Liss, Inc.

Background: Quantitation of adult erythrocytes (RBC) containing fetal hemoglobin (F cells) is of potential clinical utility in evaluating erythropoietic disorders, such as myelodysplasia and hemoglobinopathies, and in monitoring F-cell augmenting therapy. F-cell counting methodologies include fluorescence microscopy and flow cytometry. Previous flow cytometric methods have employed an isotype antibody control to distinguish F cells from non-F cells. We investigated the feasibility of using the orange autofluorescence signal (FL2) in glutaraldehyde-fixed RBC to substitute for fluorescein isothiocyanate (FITC)-labeled isotype control antibody use in F-cell quantitation.Methods: Our previously published method for fetal red cell detection in fetomaternal hemorrhage was used, employing a FITC-labeled anti-hemoglobin F (HbF) monoclonal antibody reagent. Blood samples with varying F-cell counts were quantitated for F cells using both immunofluorescence microscopy and flow cytometry comparing FITC-labeled isotype to FL1 thresholding defined by FL2 autofluorescence.Results: F cell percentages obtained by using an FL2 defined threshold for FL1 gating correlated well with expected values in diluted blood samples (r 2 ‫؍‬ 0.994, slope ‫؍‬ 1.019, intercept ‫؍‬ 0.24), values obtained using an isotype control (r 2 ‫؍‬ 0.996, slope ‫؍‬ 1.012, intercept ‫؍‬ ؊0.17), and microscopic immunofluorescence counts (r 2 ‫؍‬ 0.989, slope ‫؍‬ 0.999, intercept ‫؍‬ ؊0.72). F-cell quantitation by the isotype control and FL2 autofluorescence methods was also comparable in 40 blood samples (r 2 ‫؍‬ 0.994, slope ‫؍‬ 1.014, intercept ‫؍‬ 0.03). Intra-assay, interobserver, and interinstrument precision with this autofluorescence gating method exhibited low imprecision (coefficient of variation <14%).Conclusion: This novel method is a more objective and less laborious alternative for F-cell quantitation by flow cytometry compared to using an isotype control or microscopy, thereby providing a more robust methodology for clinical studies and consideration as a laboratory reference method for F-cell counting. Cytometry (Comm. Clin. Cytometry) 42:239 -246, 2000.

Ploidy analysis of hydropic placentas is used in conjunction with morphology and clinical data to classify hydatidiform moles and hydropic abortuses. In most studies, ploidy has been assessed by flow cytometry (FCM). To validate image cytometry (ICM) as a method to determine ploidy in this setting, the authors used both FCM and ICM to study 19 hydropic placentas in which cytogenetic analysis was available. Nuclear suspensions from paraffin-embedded tissue were used for both ICM and FCM. Image cytometry of tissue sections was performed in some cases. Image cytometry and FCM were concordant in all 19 cases, but discordant with cytogenetics in 2 of 19 cases. Two hydropic abortuses (HA) with a diploid karyotype were triploid and tetraploid, respectively, by both ICM and FCM, which suggested that the cultured tissue was not representative. DNA indices were most accurate when an internal diploid control was used as the reference. In ICM, higher resolution was achieved by analyzing cell suspensions rather than tissue sections. This study shows that ICM is a valid method of determining ploidy of hydropic placentas and partial hydatidiform moles in archival tissue.

Gamma radiation activates protooncogenes that are involved in early signal transduction, e.g., Raf-1. Most studies of effects of gamma radiation on lymphocytes deal with regulation of gene expression. However, early surface receptor expression in response to radiation has not been reported. We studied the effect of radiation on lymphocyte CD69 expression and BrdU uptake in the absence or presence of phytohemagglutinin (PHA). Radiation induces CD69 expression on T and B cells in a dose-and time-dependent manner. Four hours after a dose of 906 cGy, approximately 90% of B and 12% of T cells express CD69. CD69 expression diminishes after 6 h and requires de novo protein synthesis and protein phosphorylation. Radiation alone does not stimulate cell proliferation, as measured by BrdU incorporation, at any radiation dose tested. Furthermore, radiation enhances PHA induced CD69 expression at 2 h, but inhibits BrdU incorporation at day 3 in a dose-dependent fashion. CD69 functions as a marker for response to radiation, but unlike antigen or mitogen, radiation-induced CD69 expression does not lead to proliferation.

Gamma radiation activates protooncogenes that are involved in early signal transduction, e.g., Raf‐1. Most studies of effects of gamma radiation on lymphocytes deal with regulation of gene expression. However, early surface receptor expression in response to radiation has not been reported. We studied the effect of radiation on lymphocyte CD69 expression and BrdU uptake in the absence or presence of phytohemagglutinin (PHA). Radiation induces CD69 expression on T and B cells in a dose‐ and time‐dependent manner. Four hours after a dose of 906 cGy, approximately 90% of B and 12% of T cells express CD69. CD69 expression diminishes after 6 h and requires de novo protein synthesis and protein phosphorylation. Radiation alone does not stimulate cell proliferation, as measured by BrdU incorporation, at any radiation dose tested. Furthermore, radiation enhances PHA induced CD69 expression at 2 h, but inhibits BrdU incorporation at day 3 in a dose‐dependent fashion. CD69 functions as a marker for response to radiation, but unlike antigen or mitogen, radiation‐induced CD69 expression does not lead to proliferation. Cytometry 30:304–312, 1997. © 1997 Wiley‐Liss, Inc.

The determination of HLA-B27 (B27) status is important in the diagnosis of ankylosing spondylitis, Reiter's disease, and other arthropathies. Flow cytometric (FCM) typing of B27 is a relatively new method which allows for rapid turnaround time and low cost. However, different leukocyte populations may manifest significant variation in staining intensity with B27 antibodies. Therefore, gating utilizing only light scatter properties of cells may lead to high readings in some B27-negative samples. Fluorescence gating on CD3+ cells were postulated as a means to eliminate these anomalies. Furthermore, quantitative FCM measurements, as afforded through use of molecules of equivalent soluble fluorochrome (MESF) units, can minimize day-to-day and instrument-to-instrument variabilities in fluorescence measurements. We compared CD3 gating to light scatter gating and MESF analysis on 123 specimens in a 4-month period and found: 1) CD3 gating gave the lowest nonspecific B27 antibody binding among B27-negative subjects; 2) there was no significant difference in MESF values between CD3 gating and light scatter gating of B27-positive samples; 3) there was a wide range of B27 antibody binding fluorescence intensities among B27-positive subjects; 4) identification of patients with low B27 expression may require the use of CD3 gating in order to avoid costly confirmation testing; 5) fluorescent standard beads are stable for routine use in a clinical flow cytometry laboratory.