Partial correction of chromosome instability in Fanconi anemia by desferrioxamine.
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Anemia, Aplastic↗
Biomedical subjects
H Hoehn
Publications and source records attributed to H Hoehn.
Detection of free radical-induced DNA damage with bromodeoxyuridine/Hoechst flow cytometry: implications for Bloom's syndrome.
The clinical radiosensitizer bromodeoxyuridine (BrdU) was shown to enhance oxygen free radical-mediated growth inhibition. Cells from Bloom's syndrome, a rare autosomal recessive disorder characterized by pre- and post-natal growth deficits, telangiectatic erythema, recurrent respiratory infections and a high incidence of cancer, exhibit in culture a hypersensitivity to BrdU. We analysed disturbed cell kinetics of Bloom's syndrome fibroblasts and permanent B-cell lines with a novel cell kinetic method: BrdU/Hoechst flow cytometry. Fibroblasts show a pattern similar to that of normal cells exposed to a breakdown product of lipid peroxides, whereas B-cells exhibit the cell kinetic disturbance provoked by elevated oxygen concentrations in normal cells. In both cell types the cell kinetic pattern was dependent upon the BrdU concentration in the culture medium. These data suggest an elevated endogenous generation of oxygen free radicals in Bloom's syndrome cells, which may relate to the elevated incidence of malignancies in these patients.
B-Lymphocytes↗
Cell cycle analysis using continuous bromodeoxyuridine labeling and Hoechst 33258-ethidium bromide bivariate flow cytometry.
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Animals↗
The minor-groove binding DNA-ligands netropsin, distamycin A and berenil cause polyploidisation via impairment of the G2 phase of the cell cycle.
Distamycin A, netropsin and berenil are known to cause undercondensation of heterochromatic regions of metaphase chromosomes. These ligands interfere with DNA curvature by binding to the minor groove of the DNA. Whereas the effects of these ligands upon chromatin structure are well established, little is known about their possible interference with cell cycle progression. We show that the presence of these DNA-ligands causes protracted cell growth consisting of a prolongation of the G1 phase of the cell cycle along with arrest in the G2 compartment. Concomitant with these cell kinetic disturbances the DNA ligands cause increased polyploidisation. These observations suggest that the DNA-minor groove may play an important role in progression through the G2 phase and proper mitotic transit.
Amidines↗
Improvement of human lymphocyte proliferation and alteration of IL-2 secretion kinetics by alpha-thioglycerol.
Using a novel flow cytometric assay (BrdU/Hoechst flow cytometry) for the visualization and quantitation of cell activation and cell progression through multiple cell cycles we show that (1) resting mononuclear cells from human venous blood respond to polyclonal activation in a highly asynchronous fashion, as some of these cells enter their first cell cycle as early as 30 and as late as 80 hrs after exposure to the mitogen; (2) reducing agents improve the mitogenic response of polyclonally activated human lymphocytes by increasing the recruitment of non-cycling G0/G1 cells; (3) of 8 reducing agents tested, alpha-thioglycerol proved most effective with respect to enhancing recruitment into the cell cycle; (4) the effective agents needs to be present permanently for maximum response; (5) the growth-promoting effect of alpha-thioglycerol is mediated, but not solely caused by increased IL2 production. The latter conclusion was arrived at by using the IL2-dependent CTLL cell line in conjunction with the BrdU/Hoechst flow technique as a sensitive bioassay system for the growth promoting activity of the cell cycle progression factor IL2; (6) exogenously added IL-2, however, did not improve cell activation towards proliferation. These data indicate that increased IL-2 levels are probably due to increased cell activation rates but are not the primary cause of improvement of human lymphocyte proliferation.
Adult↗
Bromodeoxyuridine amplifies free-radical-mediated DNA damage.
Elevated oxygen concentrations and paraquat, a superoxide-generating compound, induce an arrest of cells in the G2 phase of the cell cycle, which can be enhanced by adding bromodeoxyuridine (BrdU) to the culture medium. Experiments with the lipophilic peroxide cumene hydroperoxide and the free-radical scavenger vitamin E demonstrate that the BrdU-dependent G2 arrest is not mediated by lipid peroxidation. The BrdU-dependency of arrest in the G2 phase can be used as a sensitive cell biological assay to detect DNA damage elicited by oxygen free radicals.
Bromodeoxyuridine↗
Continuous bromodeoxyuridine labeling and bivariate ethidium bromide/Hoechst flow cytometry in cell kinetics.
Most techniques of flow cytometric cell cycle analysis are not capable of distinguishing the number of rounds of DNA synthesis that a cell has undergone since the start of an experiment. Continuous labeling with 5-bromodeoxyuridine (BrdUrd) offers such a potential. We illustrate here that the bivariate analysis of non-BrdUrd-quenched ethidium bromide vs. BrdUrd-quenched Hoechst 33258 fluorescence offers a high degree of resolution that enhances the analytical power of the technique, and that this approach can be applied to the analysis of a broad range of human and murine primary cells and established cell lines.
Bisbenzimidazole↗
The effects of bacterial lipopolysaccharide, anti-receptor antibodies and recombinant interferon on mouse B cell cycle progression using 5-bromo-2'-deoxyuridine/Hoechst 33258 dye flow cytometry.
Polyclonal stimulation of resting B cells with anti-antigen receptor antibodies [anti-IgM mu chain antibody (anti-mu)] or with bacterial lipopolysaccharide (LPS) stimulates a proportion of B cells to proliferate. Exposure of resting B cells to both LPS and anti-mu activates a larger population of resting B cells than either alone, suggesting a synergistic effect of these two stimuli. Although recombinant interferon (rIFN) either alone or in combination with anti-mu has no apparent proliferative activity (as measured by [3H]thymidine incorporation), application of 5-bromo-2'-deoxyuridine/Hoechst 33258 dye flow cytometry reveals a distinct effect of rIFN on B cell growth. In the presence of anti-mu plus LPS, rIFN causes the majority of B cells to enter the cell cycle (CC), but a subset of B cells remains in the resting stage. Another subset of B cells has extremely rapid CC transit times, with a CC duration of less than 10 h. These studies show that both anti-mu and LPS are competence factors (which move cells from the G0 phase to the G1 phase). LPS acts also as a CC progression factor, while rIFN is a CC potentiating factor.
Animals↗
Free radical mediated cytotoxicity of desferrioxamine.
Toxic effects of desferrioxamine (DFO) upon cell growth were assayed with continuous bromodeoxyuridine (BrdU) labeling and bivariate ethidium bromide/Hoechst 33258 flow cytometry. At 5% oxygen DFO caused a dose-dependent inhibition of cell growth, which was potentiated at 20% oxygen, and by cumene hydroperoxide but not by paraquat. An irreversible arrest in the G2 phase of the cell cycle was the cell-kinetic mechanism underlying this growth inhibition. The G2 arrest was not dependent upon the BrdU concentration in the medium, thus ruling out a direct attack of a free radical on thymidine residues. The observed cytotoxicity of DFO cautions against its use in the treatment of conditions of elevated oxidative stress.
Bisbenzimidazole↗
Cell kinetic evidence suggests elevated oxidative stress in cultured cells of Bloom's syndrome.
Bromodeoxyuridine/Hoechst flow cytometry was used to analyse disturbed cell proliferation of fibroblasts and lymphoblastoid cells from Bloom's syndrome (BS). Fibroblasts show poor activation, arrest in the G2 phase of the cell cycle along with a prolongation of the G1 phase. This pattern of perturbed cells proliferation is akin to that elicited in normal fibroblasts by 4-hydroxy-nonenal, a breakdown product of lipid peroxides. Treatment with vitamin E improved growth of BS fibroblasts more strongly than growth of normal fibroblasts. Lymphoblastoid cells from BS, to the contrary, experience only a minor arrest in the G2 phase after one round of bromodeoxyuridine incorporation, but are strongly inhibited during and after the second S phase. Thus, their cell cycle arrest is dependent upon BrdU incorporation, as has been found previously in normal cells exposed to elevated concentrations of oxygen or paraquat, a superoxide generating compound. These results suggest that BS cells may suffer from an elevated, endogenous generation of oxygen free radicals.
Bisbenzimidazole↗
Bromodeoxyuridine amplifies the inhibitory effect of oxygen on cell proliferation.
The BrdUrd-Hoechst method was used to analyze the interaction of various oxygen concentrations with BrdUrd substituted DNA with respect to cellular proliferation. At oxygen concentrations above 5%, human diploid fibroblast-like cells and amniotic fluid fibroblast-like cells showed reduced proliferation rates, which resulted from an increase in noncycling cells and from a permanent arrest of cells in the G2 phase of the cell cycle. At 35% oxygen the increase in noncyling cell fraction and the permanent arrest in G2 was strongly dependent upon the concentration of BrdUrd. Incorporation of BrdUrd into DNA, therefore, amplifies the adverse effects of increasing oxygen concentrations upon cell proliferation. The mechanism of this amplification might involve a free radical attack on DNA similar to the radiation sensitizing effect of BrdUrd.
Bromodeoxyuridine↗
Disturbance of cell proliferation by two model compounds of lipid peroxidation contradicts causative role in proliferative senescence.
Cumene hydroperoxide (Chp), a lipophilic peroxide, and hydroxy-nonenal (HNE), a breakdown product of lipid peroxides, were used as model compounds to assess the effects of lipid peroxidation upon cell proliferation. Amniotic fluid fibroblastlike (AFFL) cells and human diploid skin-derived (HDFL) cells were cultured with the two model compounds and cell proliferation was assayed via bromodeoxyuridine-Hoechst flow cytometry. At low doses Chp elicited an accumulation of cells in the S and G2 phase, while at higher doses the fraction of nonproliferating cells increased as well. Low doses of HNE caused an accumulation of cells in the G1 and G2 phase, whereas an additional increase of cells in S phase and in the nonproliferating fraction was found at an elevated concentration. A delay of onset of proliferation was obtained with both Chp and HNE. Permanent arrests in the S, G2, and G1 compartment are provoked by Chp only when Chp was applied together with serum. HNE, to the contrary, elicited a permanent arrest in the G2 and the G1 compartment even if added to quiescent cell cultures. Additionally, HNE caused a combination of a prolongation of the G1 phase of the cell cycle and an arrest in this compartment, which is reminiscent of cell differentiation. HDFL cells were much more sensitive toward Chp than were AFFL cells, but both cell types showed similar sensitivities toward HNE. We conclude that lipophilic peroxides exert toxic effects upon cell proliferation distinct from the pattern elicited by aldehydic breakdown products of lipid peroxides. The pattern of cell cycle arrest induced by Chp and HNE makes it unlikely that Chp and HNE, or related products of lipid peroxidation, are responsible for the limitation of the proliferative life span of human fibroblasts in culture.
Aldehydes↗
Characterization of a newly established human urinary bladder cancer cell line (BT-1).
A new human transitonal cell carcinoma cell line has been established in long term tissue culture. The BT-1 cells were derived from a poorly differentiated human bladder cancer. The tumor cell line produces tumors in nude mice. BT-1 has been characterized by cytogenetic and flow cytometric analysis and by isoenzyme typing. As in direct preparations of human bladder tumors, an isochromosome 5p is a consistent marker of the newly established line. The BT-1 cells produce transforming growth factors but do not respond to exogeneous EGF.
Animals↗
BrdU-Hoechst flow cytometry: a unique tool for quantitative cell cycle analysis.
Unlike other techniques, flow cytometric analysis of BrdU-quenched 33258 Hoechst fluorescence may be used to measure cell activation and the G1, S, and G2/M compartment distributions in each of three successive cell cycles after growth stimulation of human peripheral blood lymphocytes. Cell cycle kinetic curves can be constructed from the BrdU-Hoechst flow data which allow the simultaneous assessment of growth fraction, lag-time, compartment exit rate, compartment duration, and compartment arrest. Applications of this new versatile technique include the evaluation of drug and growth factor effects, cell aging, and diagnosis in medicine and immunology.
Benzimidazoles↗
The phytohemagglutinin response of human peripheral blood lymphocytes as a function of donor age: a re-examination using BrdU-Hoechst flow cytometry.
Mononuclear cells were isolated from peripheral blood by a standard Ficoll-Hypaque technique from 127 healthy donors, ranging in age from newborns to 86 years of age. As a measure of their in vitro growth response, the fraction of non-cycling cells was determined at 48 and 72 h after phytohemagglutinin (PHA) exposure by means of BrdU-Hoechst flow cytometry. This technique provides an optimal assay system for the non-cycling cell fraction, since all cycling cells will have incorporated BrdU thereby quenching the fluorescence of the Hoechst 33258 fluorochrome. Lymphocytes from prepubertal donors showed significantly decreased non-cycling cell fractions, as did lymphocytes from an additional group of 14 adults with hypogonadism due to the 45, XO condition (Turner-Syndrome). Much to our surprise, we found no definitive correlation between donor age and the non-cycling fraction of cells from the adult lymphocyte donors. Nor did we find any age-related increase in the variance of the non-cycling cell fraction. These observations suggest that the previously reported age-related decline in the PHA response of human PBL may reflect an increasing delay, rather than an overall diminution, of the PHA response as a function of donor age.
Adolescent↗
Fanconi anemia mutation causes cellular susceptibility to ambient oxygen.
The gene defect causing the Fanconi anemia (FA) phenotype appears to be expressed at the cellular level, since FA fibroblasts show a protracted course of explant outgrowth, a diminished in vitro life span, and very poor cloning. We show that exposure of FA fibroblasts to hypoxic (5% v/v oxygen) culture conditions restores their growth in vitro to near normal. Exposure to elevated oxygen tension (35% v/v) causes accumulations of FA cells in the S and G2/M phases of the cell cycle that are in significant excess of those seen in heterozygote and control strains. In the absence of evidence for defective cytoplasmatic radical scavenging systems, these observations suggest increased nuclear susceptibility to ambient oxygen as cause of the FA cellular phenotype.
Adolescent↗
Screening test for ataxia telangiectasia.
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Ataxia Telangiectasia↗
Interphase cell flow cytometry as a means of monitoring genomic size in normal and neoplastoid cell cultures.
The DNA specific fluorescence of mass cultures and clones derived from human skin and bladder tumor tissue was assayed by flow cytometry. In order to detect and quantitate small fluorescence intensity changes, cytogenetically defined triploid or diploid human fibroblast strains were cocultivated, harvested, and stained with the cell strain of unknown karyotype. The triploid standard (derived from human abortus tissue) proved chromosomally unstable at high passage level. Fifteen male, female, and 45,X strains displayed target-to-standard cell fluorescence ratios commensurate with their respective chromosome constitutions. Interstrain variation was highest among the 45,X strains, although mosaicism could not be detected by conventional cytogenetics. Interclonal fluorescence variation was two- to ten-fold higher among the tumor-derived clones tested. Chromosome counts and subcloning experiments indicate that this increased fluorescence variation is due to genome size variation. The clonal evolution of genome size differences was observed in subclones of chromosomally divergent parental clones. These observations suggest that well controlled flow cytometry can adequately resolve subtle degrees of genome size variation in cultivated human cells. The technique is especially suited for monitoring genome size changes in cultivated tumor cells.
Cell Separation↗