bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heterogeneity of chromosome 17 and erbB‐2 gene copy number in primary and metastatic bladder cancer
Authors: Sauter Guido , Moch Holger , Gasser Thomas C. , Mihatsch Michael J. , Waldman Frederic M. 
Published: June 2005

AbstractTo study the relationship of tumor genomic heterogeneity with bladder cancer phenotype and p53 gene alterations, 138 primary bladder tumors were examined by dual labeling fluorescence in situ hybridization (FISH) using probes for chromosome 17 centromere (p17H8) and p53 (17p13.1). The number of different aneusomic populations >5% (and monosomic populations >20%) of cells served as a marker for heterogeneity. Nuclear p53 overexpression and Ki67 labeling index (Ki67 LI) were determined by immunohistochemistry. The number of aneusomic populations was 0 in 53 tumors, 1 in 18, 2 in 47, 3 in 9, and >3 in 11 tumors. Presence of aneusomy was associated with tumor grade and stage (P < 0.0001 each). Ki67 LI was low in disomic tumors (11.0 ± 7.7), higher in tumors with 1–3 aneusomic populations (17.4 ± 11.3), and highest in tumors with >3 aneusomic populations (25.8 ± 10.9; P = 0.02 for >3 vs. 1–3 populations). Aneusomy and heterogeneity were associated with p53 alterations. Aneusomy was seen in 35% of tumors with neither p53 expression nor p53 deletion but in 97% of tumors with both p53 deletion and expression. Nine of 11 tumors with > 3 aneusomic populations exhibited both p53 deletion and overexpression.To study genomic heterogeneity in tumor progression, two recurrences and three metastases of a tumor with known erbB‐2 amplification were examined for centromere 17 and erbB‐2 copy number. A considerable heterogeneity in centromere 17 and erbB‐2 gene copy number was found in both recurrences and metastases, indicating a marked genomic instability in these metastatic cells. These results show that genomic heterogeneity is common in bladder cancer. Highly heterogeneous tumors might represent a particularly aggressive subtype of bladder carcinoma. © 1995 Wiley‐Liss, Inc.
Heterogeneous cell kinetics in tumors analyzed with a simulation model for bromodeoxyuridine single and multiple labeling
Authors: Baisch H. , Otto U. , Hatje U. , Fack H. 
Published: June 2005

AbstractBromodeoxyuridine (BrdUrd) labeling of DNA and flow cytometry measurement of bivariate BrdUrd‐DNA content distributions yield proportions of cells in the cycle phases. After application of BrdUrd, with time, these proportions change according to the cell kinetic parameters of the investigated cell line or tumor. In a previous study of S‐phase transit time using the relative movement method, we obtained better fits with S‐duration distributions rather than constant values (Baisch and Otto: Cell Prolif 26:439–448, 1993). Now, we have developed a simulation model using asymmetric phase duration distributions in all phases of the cell cycle to fit the experimental data after single or multiple BrdUrd labeling. The model includes transit of cells from proliferating to quiescent compartments in all phases. The results yield the phase duration distributions, mean and median percentages of quiescent cells in all phases, growth fraction, and potential doubling time. The model was used to fit data of five renal cell carcinomas xenotransplanted into nude mice that were obtained after single and multiple labeling up to 93 hours. The estimated phase duration distributions varied from narrow to extremely asymmetric. In particular, TG2M duration and asymmetry were nearly as large as those of G1 phase in some tumors. The contribution of inter‐and intratumoral heterogeneity cannot be separated by the simulation model, but evidence of intratumoral heterogeneity is provided by DNA content distributions at extended time spans after BrdUrd labeling. © 1995 Wiley‐Liss, Inc.
Heterogeneity of bromodeoxyuridine sensitivity of cultured cells from melanoma metastases
Authors: Poot Martin , Hiller Karl‐Heinz , Heimpel Simone , Schuster Armin , Köhler Julia , Hoehn Holger 
Published: June 2005

AbstractContinuously growing cell cultures, testing positive for tyrosinase activity, were derived from two brain and three lymph‐node metastases of five patients with malignant melanoma. These cell cultures were analyzed regarding their proliferation rate with continuous bromodeoxyuridine (BrdUrd) labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry. Melanoma cell cultures are more sensitive toward BrdUrd in comparison to human diploid fibroblast cultures: 50% growth inhibition at 360 ± 130 μM BrdUrd (range: 130–520; n = 11) vs. 650 ± 50 μM BrdUrd (n = 3) for fibroblasts. Moreover, BrdUrd sensitivity in melanoma cells is oxygen dependent: 50% growth inhibition at 200 ± 55 μM (range: 65–400 μM) for 20% oxygen vs. 360 ± 130 μM BrdUrd for 5% oxygen. The cell cycle kinetic mechanism of BrdUrd‐induced growth inhibition is accumulation of cells in the G2 phase. Cultures from a single metastasis showed up to a 3‐fold variation in BrdUrd sensitivity. In one of the brain metastases two populations of different ploidy level (pseudotriploid vs. pseudotetraploid) and BrdUrd sensitivity could be resolved. Thus, continuous BrdUrd labeling followed by bivariate Hoechst 33258/ethidium bromide flow cytometry is a powerful tool to detect heterogeneity in proliferative capacity and drug sensitivity of cell populations within one tumor biopsy. © 1995 Wiley‐Liss, Inc.
Flow cytometric determination of glutathione in clinical samples
Authors: Chow Sue , Hedley David 
Published: June 2005

AbstractGlutathione‐based processes are believed to be important determinants of resistance to cancer chemotherapy, and measurement of glutathione (GSH) in tumor tissue is therefore of clinical relevance. Flow cytometric methods have been developed for measuring cellular GSH content that appear to correlate well with biochemical determinations. These flow cytometric techniques are rapid, allow tumor cells to be distinguished from stromal elements, and show considerable intratumoral heterogeneity in human tumor cell GSH content. Although large prospective studies are required to determine the correlations between GSH content and treatment outcome in cancer patients, this approach is a powerful alternative to standard biochemical assays for GSH. © 1995 Wiley‐Liss, Inc.
Use of a multiparametric panel to target subpopulations in a heterogeneous solid tumor model for improved analytical accuracy
Authors: O'Rrien Melissa C. , Gupta Ravinder K. , Lee Song Y. , Bolton Wade E. 
Published: June 2005

AbstractThe exclusion of non‐tumor and dead cells from the analysis of live tumor cells can significantly improve the accuracy of prognostic indicators such as proliferative and DNA indexes. To target live breast tumor cells in a heterogeneous breast tumor model, we have designed a panel consisting of the DNA‐specific dye DAPI and epithelial tissue‐specific (cytokeratin), tumor‐associated (MC5), proliferation‐associated (proliferating cell nuclear antigen), and viability‐associated (tubulin) markers. The breast tumor model consisted of a mixture of equal numbers of live and dead MDA‐MB‐175‐VII (breast tumor) cells, live CEM (leukemic) cells, and live peripheral blood mononuclear cells.Targeting the live MDA cells in the mixture by gating on tubulin, cytokeratin, and MC5 resulted in a sevenfold increase in PCNA positivity (from 3% ungated to 22.3%), a 60% decrease in the %S‐phase fraction (from 37.2% ungated to 15%), and elimination of extraneous hypodiploid and diploid components, enriching the tetraploid MDAs. These results are consistent with those obtained for unmixed live MDA cells. The combined utilization of this panel and “cumulative” electronic gating of the targeted population increases the number of relevant parameters that can be analyzed per sample and the accuracy of the resultant data. © 1995 Wiley‐Liss; Inc.
Cytogenetic heterogeneity and histologic tumor growth patterns in prostatic cancer
Authors: Alers J. C. , Krijtenburg P. J. , Vissers C. J. , Bosman F. T. , Van Der Kwast Th. H. , Van Dekken H. 
Published: June 2005

AbstractTwenty‐five prostatic adenocarcinomas were studied for the presence of intratumoral cytogenetic heterogeneity by interphase in situ hybridization (ISH) to routinely processed tissue sections. ISH with a chromosome Y‐specific repetitive DNA probe provided a model to investigate patterns of chromosomal heterogeneity within and between different pathological grades. The Gleason grading system was used, since it is based on a detailed classification of growth patterns. Heterogeneity with respect to ploidy of the tumor was examined by ISH with a repetitive DNA probe specific for chromosome 1. The ploidy status of these cancers was confirmed by DNA flow cytometry (P < 0.001). Cytogenetic heterogeneity at the (Y) chromosomal level was observed between Gleason areas, within one area, and even within single tumor glands. The different patterns of chromosomal heterogeneity were seen in all tumor grades and stages. Differences in ploidy status were also found following the aforementioned histological patterns, again, in all grades and stages. Intraglandular heterogeneity was most frequently seen. No correlation was found between cytogenetic heterogeneity and proliferative activity (Kl‐67 immunostaining). In contrast to current views on clonality, suggesting regional separation of subclones with different DNA content, this study demonstrates that these subclones can be interspersed. © 1995 Wiley‐Liss, Inc.
Flow cytometer and imaging device used in combination
Authors: Kubota Fumio , Kusuzawa Hideo , Kosaka Tokihiro , Nakamoto Hiroyuki 
Published: June 2005

AbstractThis report presents a flow cytometer devised by the authors and equipped with a CCD camera to obtain images of selected particles. Each image is linked to a point in the scattergram from which it can be retrieved from memory. The system uses an argon ion laser for flow‐cytometric analysis and a diode‐pumped pulse laser (532nm) to generate CCD images. Aggregated platelets in a blood sample and casts in a urine sample can be detected by this system. A fast flow rate is suitable for efficient flowcytometric detection, and image resolution is sufficiently adequate for recognizing aggregated platelets and casts. Flow‐cytometric and morphological data are used together to distinguish different types of cells in the same region on the scattergram and identify cells present in extremely small numbers among the majority. © 1995 Wiley‐Liss, Inc.
Novel flow‐cytometric method for separating cell types in differentiated F9 embryoid bodies
Authors: Burdsal Carol A. , Pedersen Roger A. , Hyun William C. , Latimer Jean J. 
Published: June 2005

AbstractThe differentiation of F9 teratocarcinoma cells mimics the formation of a mouse embryonic tissue, the primitive endoderm. In vitro, small aggregates of F9 cells, termed embryoid bodies, differentiate in response to retinoic acid and develop a surface epithelium that is characterized by the production of α‐fetoprotein. In the present study, cellular autofluorescence profiles obtained by fluorescence‐activated cell sorting demonstrated that undifferentiated embryoid bodies were composed of a single type of cell. In contrast, retinoic acid‐induced embryoid bodies were composed of two cell types: a major population displaying autofluorescence levels similar to those of cells from undifferentiated embryoid bodies and a second population displaying higher autofluorescence. RNA analyses demonstrated that the transcription of α‐fetoprotein was associated only with the more highly autofluorescent population, indicating that flow cytometry provides a novel mechanism for the separation of undifferentiated cells from differentiated endoderm cells in F9 embryoid bodies. © 1995 Wiley‐Liss, Inc.
Factors affecting flow cytometric detection of apoptotic nuclei by DNA analysis
Authors: Elstein Kenneth H. , Thomas David J. , Zucker Robert M. 
Published: June 2005

AbstractApoptotic thymocyte nuclei normally appear on a flow cytometric DNA histogram as a subdiploid peak. We observed that addition of a specific RNase A preparation to the detergent‐based lysing buffer increased the fluorescence of toxicant‐induced apoptotic nuclei to the level of untreated diploid unclei. The chelating agent EDTA partially inhibited the RNase effect, suggesting contaminating divalent cations may have been involved. Moreover, spectrofluorometric analysis revealed that addition of RNase or divalent cations decreased the amount of DNA present in the lysate. This suggested that the upscale fluorescence shift was due to a decrease in the ability of the lysing buffer to extract DNA, possibly as a result of cation‐induced chromatin condensation, rather than increased accessibility of fluorochrome binding sites due to apoptotic degeneration. Moreover, during a 16‐h culture, we observed a similar, but time‐dependent, upscale shift in the fluorescence of thymocytes undergoing apoptosis either spontaneously or as a result of exposure to 1 μM tributyltin methoxide (TBT), 2% ethanol, 2% methanol, or 1 μM dexamethasone phosphate (DEX). This commonality of effect suggests that a similar magnitude of chromatin reorganization occurs in apoptotic cells in prolonged culture regardless of the method of apoptotic induction. These findings should alert investigators to potential inaccuracies in the flow cytometric quantitation of apoptosis in in vitro systems employing prolonged toxicant exposures or complex lysing cocktails that may contain active contaminants. © 1995 Wiley‐Liss, Inc.*
Flow cytometric detection of human immunodeficiency virus type 1 proviral DNA by the polymerase chain reaction incorporating digoxigenin‐ or fluorescein‐labeled dUTP
Authors: Yang Gang , Olson John C. , Pu Ruilian , Vyas Girish N. 
Published: June 2005

AbstractSerological assays are routinely used in the laboratory diagnosis of human immunodeficiency virus type‐1 (HIV‐1) infection, but the polymerase chain reaction (PCR) is ultimately the most sensitive and direct method for establishing definitive diagnosis. As an alternative to the conventional radioactive PCR procedure we have developed and evaluated a pair of rapid nonradioisotopic flow cytometric detection methods. Using heminested PCR we directly incorporated fluorescein‐12‐dUTP (fluo‐dUTP) or digoxigenin‐11‐dUTP (dig‐dUTP) into the PCR‐amplicons. The labeled amplicons were hybridized with biotinylated antisense and sense probes, followed by capture of the hybrid DNA using streptavidin‐coated beads which were finally analyzed in a flow cytometer by (1) direct detection of the fluorescence intensity of the amplicons incorporating fluo‐dUTP and (2) immunodetection of the amplicons incorporating dig‐dUTP by anti‐digoxigenin IgG labeled with fluorescein isothiocyanate (FITC). Although both assays were functionally comparable with radiolabeled probe in reliably detecting as low as five copies of HIV‐1 proviral DNA sequences, the immunodetection of dig‐dUTP consistently yielded higher mean channel fluorescence and gave a stable signal over an extended period of 12–14 weeks. In testing a panel of 20 pedigreed PBMC specimens from blood donors with or without HIV‐1 infection, the results of both flow cytometric assays were identical with those of the conventional radioactive procedure. Therefore, we conclude that the dig‐dUTP incorporation in amplicons, hybridization with a pair of sense‐antisense biotinylated probes and immunodetection of hybrids by flow cytometric analyses is the nonisotopic method of choice for PCR‐diagnosis of HIV‐1 infection. © 1995 Wiley‐Liss, Inc.
Application of flow cytometry in toxinology: Pathophysiology of human polymorphonuclear leukocytes damaged by a pore‐forming toxin from Staphylococcus aureus
Authors: Meunier O. , Falkenrodt A. , Monteil H. , Colin D. A. 
Published: June 2005

AbstractThe pore‐forming activity of leukocidin (PVL) secreted by Staphylococcus aureus has been investigated on human white cells by flow cytometry techniques. This two‐component toxin induced morphological modifications of neutrophils and monocytes as detected by forward light scattering measurements, but was inactive on lymphocytes. These modifications were the consequence of pore formation through the cell membrane leading to its permeabilization. In the absence of calcium, PVL formed pores large enough to allow ethidium ions to penetrate the cells and become fluorescent by intercalating nucleic acids. In the presence of calcium, the pores were too small for ethidium entry but allowed an influx of calcium as shown by the increase in fluorescence of Fluo‐3 loaded in the cells. This increase in intracellular calcium concentration induced the activation of neutrophils by PVL as shown by the liberation of their granule content measured by a decrease in side light scattering. Furthermore, ethidium fluorescence was used to discriminate the cells sensitive to PVL, and the analysis of differentiated HL‐60 cells and cells obtained from a case of chronic myeloid leukemia led to the conclusion that myeloid cells become sensitive to PVL during differentiation to the metamyelocyte stage. © 1995 Wiley‐Liss, Inc.
BPV E1 protein alters the kinetics of cell cycle entry of serum starved mouse fibroblasts
Authors: Belyavskyi Michail , Miller Jane , Belyavskaya Elena , Wilson Van 
Published: June 2005

AbstractA stable bovine papillomavirus E1 expressing cell line (C2E1) was used to investigate the effects of E1 protein on the requirement for growth factors during serum‐induced reentry from quiescence to proliferation. Flow cytometric bivariate DNA/PCNA analysis was utilized to study the expression of proliferating cell nuclear antigen (PCNA) concomitant with this transition. C2E1 cells, unlike the control cells (CNEO), were able to reenter the cell cycle when stimulated with low serum (1%). Stimulation with 10% serum revealed that C2E1 cells entered the first cell cycle faster than CNEO, indicating that E1 protein decreased the time of progression from G0 stage upon serum activation. It was also shown that PCNA expression started earlier in C2E1 cells than in CNEO cells after quiescent cells were stimulated with 10% serum. Addition of 1% serum was able to induce PCNA expression in C2E1 but not in CNEO cells in the first 24 h after stimulation. Using Triton X‐100 treatment, it was found that the distribution between bound and unbound forms of PCNA was altered in E1‐expressing cells compared to CNEO cells. Based on these results, it is suggested that E1 might possess mitogen‐like properties. © 1995 Wiley‐Liss, Inc.
Identification of resting cells by dual‐parameter flow cytometry of statin expression and DNA content
Authors: Pellicciari C. , Mangiarotti R. , Bottone M. G. , Danova M. , Wang E. 
Published: June 2005

AbstractStatin, a 57‐kDa nuclear protein, has been recognized as a unique marker of quiescent (G0) cells; specific monoclonal antibodies (MoAb) against statin have been produced and used to label resting cells in tissue sections and in cultured cells. We present an improved method for the identification of G0 cells by dual‐parameter flow cytometry of statin expression and DNA content. The appropriate technical conditions were set up by using resting and cycling human fibroblasts as a model cell system. Several fixatives proved to be suitable for the immunocytochemical detection of statin; among them, 70% ethanol was selected because this fixation procedure is suitable for DNA staining with intercalating dyes and is routinely used for the immunolabeling of proliferation markers (such as proliferating cell nuclear antigen (PCNA) and Ki‐67) and of bromodeoxyuridine (BrdUrd) incorporation. Following cell permeabilization with detergent, exposure to the antistatin antibody (S‐44), and indirect fluorescein isothiocyanate immunolabeling, cells were counterstained for DNA with propidium iodide and analyzed by dual‐parameter flow cytometry. In cells from several animal sources (rat thymocytes and C6 glioma cells, mouse 3T3 cells, and human MCF‐7 cells), under different experimental conditions, the expression of statin was found to correlate inversely with that of PCNA and Ki‐67, and with the BrdUrd labeling index. In dual‐parameter flow scattergrams, G0 (statin positive) cells can be discriminated from the potentially cycling (statin negative) G1 cells, i.e., within a cell fraction having the same DNA content. This approach can be envisaged as a powerful tool both for monitoring changes in the resting cell fraction and for Investigating the process of G0–G1 transition in unperturbed and drug‐treated cell populations. © 1995 Wiley‐Liss, Inc.
Simultaneous triple staining for hypoxia, proliferation, and DNA content in murine tumours
Authors: Webster Lynne , Hodgkiss Richard J. , Wilson George D. 
Published: June 2005

AbstractHypoxia and proliferation rate are two important biological factors influencing the outcome of radiotherapy regimes for solid tumours. Hypoxic cells are more resistant to radiation than aerobic cells and a rapidly dividing tumour may repopulate faster during treatment. Clinical trials are underway to assess the importance of both these parameters. In this article we describe a method to simultaneously measure hypoxia and proliferation using multiparameter flow cytometry. Hypoxic cells were detected using a bioreductively bound marker with an immuno‐recognisable side‐chain, NITP and proliferation was measured by bromodeoxyuridine (BrdUrd) incorporation. These parameters were related to cell cycle position by measuring total DNA content with 7‐aminoactinomycin D. The data were analysed using single laser excitation on a bench top flow cytometer. Simultaneous measurement of the three parameters shows the presence of cells which have incorporated BrdUrd and are also hypoxic by the criterion of NITP binding. The murine SaF tumour has a relatively constant aneuploid labelling index of 24%. However, the level of aneuploid hypoxia was variable ranging from 0.8 to 40.9% with a mean value of 15.6%. Within the BrdUrd labelled population there is a range of hypoxia from 2.8 to 28.5% (mean 15.1%); this represents 0.7 to 6.6% of the total tumour population. There are approximately twice as many oxygenated cells than hypoxic cells actively in the cell cycle. In vivo tumours contain cells with S phase DNA content which do not incorporate BrdUrd. This cell population has equivalent proportions of hypoxic and oxic cells. However, there are up to 12‐fold more hypoxic cells in the unlabelled S than the BrdUrd labelled population. These data show that proliferation and hypoxia can be measured simultaneously using flow cytometry and the technique may form the basis of a predictive assay for these two important biological determinants of radiotherapy outcome. © 1995 Wiley‐Liss, Inc.
Isolation of full‐size mRNA from ethanol‐fixed cells after cellular immunofluorescence staining and fluorescence‐activated cell sorting (FACS)
Authors: Esser Charlotte , Göttlinger Christoph , Kremer Joachim , Hundeiker Claudia , Radbruch Andreas 
Published: June 2005

AbstractPreparation of intact, full‐size RNA from tissues or cells requires stringent precautions against ubiquitous and rather stable RNases. Fluorescence‐activated cell sorting (FACS) usually aims at the isolation of cells according to cell surface markers on living cells, from which RNA can be obtained by standard protocols. The separation of cells according to intracellular immunofluorescence markers, such as intranuclear, intracytoplasmic, or secreted molecules, requires permeation of the cell membrane for the staining antibodies, which is usually achieved by fixation. However, commonly used fixatives such as ethanol, methanol, or formaldehyde do not inactivate RNases completely, thereby hampering the analysis of complete RNA molecules from fixed cells. We report isolation of intact, fullsize RNA suitable for Northern blotting from cells that were fixed by 95% ethanol/5% acetic acid containing RNase inhibitors, stained intracellularly, and sorted by FACS. © 1995 Wiley‐Liss, Inc.