bluuebunny/crossref_metadata_2025_split huggingface dataset:
Experimental and clinical evaluation by flow cytometry for the mechanism of combination therapy (cisplatin and peplomycin)
Authors: Kawamoto Keiji , Wada Yasuhiro , Kumazawa Hirofumi , Yamashita Toshio , Kumazawa Tadami , Matsumura Hiroshi 
Published: August 2005

AbstractPharmacologic effects of cisplatin (CDDP) and peplomycin (PEP) on tumor cell kinetics were studied bath in vitro and in vivo with the aid of flow cytometry (FCM). Double staining with propidium iodide (PI) and fluorescein isothiocyanate (FITC)‐labeled bromodeoxyuridine (BrdU) was used to analyze the cell cycle, and the number of viable cells was determined with fluorescein diacetate (FDA). Effects of combining the 2 agents were also studied to establish the most effective method of combination therapy. Furthermore, these agents were tried clinically on the basis of experimental results. Results showed that CDDP exerted its action at the S and G2M phases in the cell cycle and PEP at the G2M phase. Among the combination regimens in the experiments with CDDP, PEP, and CDDP + PEP as analyzed by FCM, the strongest block on the G4M phase was shown in the one at a 2‐day interval, resulting in the most effective killing of the tumor cells. Clinical trial of the combination therapy showed the same results as the in vitro experiment; the therapy proved useful for improving the patient's clinical condition and the results obtained with CT imaging and pathology.
Chromosome detection by in situ hybridization in cancer cell populations which were flow cytometrically sorted after immunolabeling
Authors: Beek Johan L. M. , Hopman Anton H. N. , Peter Vooijs G. , Ramaekers Frans C. S. 
Published: August 2005

AbstractWe examined the feasibility of performing non‐radioactive in situ hybridization (ISH) in flow cytometrically sorted (tumor) cells with a chromosome #1 specific centromere probe. The study was performed in a model system of HL60 cells mixed with different quantities of HeLa cells. These latter cells were sorted directly onto poly‐l‐lysine coated glass slides on the basis of their keratin content, a cytoskeletal component not present in HL60 cells. Overall morphology of the separated HeLa cells was excellent and, after the ISH procedure, the appropriate number of ISH spots was observed in more than 85% of the sorted cells. This percentage did not differ significantly in cell mixtures with different percentages of HeLa cells (down to 1%). Sorting of HeLa cells in different phases of the cell cycle, and subsequent ISH, revealed the same spot number for chromosome #1 in all cell cycle stages, including mitosis. In the latter phase of the cell cycle we did not find a duplication of the chromosome #1 centromere, not even after sorting of the mitotic cells on the basis of specific labeling with an antibody to mitotin. The early G2 mitotin negative fraction, however, showed a significant percentage of cells with a duplicate spot number, most likely representing a tetraploid cell fraction in this HeLa cell culture. The protocol that evolved from these model studies was applied to cell suspensions of malignant body cavity effusions as well as solid bladder carcinomas. In several of these cases numerical chromosome aberrations could be detected by ISH more evidently after sorting on the basis of keratin labeling.
Flow‐cytometric analysis of the effects of triethylenemelamine on somatic and testicular tissues of the rat
Authors: Bickham John W. , Sawin Virginia L. , Burton David W. , McBee Karen 
Published: August 2005

AbstractThe effects of short‐term (24 h) exposure to triethylenemelamine on cellular DNA in five tissues (bone marrow, spleen, kidney, large intestine, and testis) of the rat were studied using flow cytometry. Mean coefficients of variation of the G1 peaks were increased in both the low and high dosage groups relative to controls. Bone marrow exhibited the highest degree of effect, possibly due to the rapid rate of cell division in that tissue, and spleen was next highest. Thus, hematopoietic tissues are highly responsive to short‐term, acute exposure to this mutagen. The results of the flow‐cytometry assay closely paralleled a simultaneous chromosomal assay conducted on bone marrow of the same rats. These data are interpreted to be consistent with the hypothesis that the observed increase in mean coefficients of variation is due to the clastogenic effects of the mutagen and subsequent unequal distribution of DNA among the daughters of affected cells.
Three probe flow cytometry of a human foam‐cell forming macrophage
Authors: Hassall D. G. 
Published: August 2005

AbstractA human cell line THP‐1 was differentiated into macrophages expressing the scavenger receptor for uptake of modified lipoproteins. The cells were exposed to native low‐density lipoprotein (n‐LDL), acetylated‐low‐density lipoprotein (AcLDL), oxidised‐LDL, or 25‐OH cholesterol, leading to the accumulation of cholesteryl esters within the cells. Harvested macrophages were studied using three separate probes: (1) 1,1′‐dioctadecyl‐3,3,3′,3′‐ tetramethylindocarbocyanine perchlorate (diI‐labelled) LDL to study lipoprotein uptake; (2) the lipophilic fluorescent dye Nile Red to study cholesteryl ester accumulation within the cells; and (3) the polyene antibiotic Filipin III to study free cholesterol homeostasis. Cells were analysed using fluorescence flow cytometry and the three signals analysed separately. THP‐1 macrophages incubated with diI‐labelled modified lipoproteins produced a concentration dependent increase in the fluorescence emissions, consistent with accumulation of the labelled particles. Macrophages exposed to unlabelled modified LDLs were demonstrated, by staining with Nile Red, to accumulate cholesteryl esters within their cytoplasm and to alter their cholesterol content as judged by staining with Filipin. The foam‐cell forming macrophage and its response to modified lipoproteins is considered a key step in the development of atherosclerosis. The use of these three probes during the formation of foamcells in vitro offers a way of studying their behaviour at the single cell level.
DNA stainability in aneuploid breast tumors: Comparison of four DNA fluorochromes differing in binding properties
Authors: Myc Andrzej , Traganos Frank , Lara Jonathan , Melamed Myron R. , Darzynkiewicz Zbigniew 
Published: August 2005

AbstractThe aim of this study was to evaluate whether or not the differences in chromatin structure between diploid stromal cells or lymphocytes, which are often used as DNA ploidy standard, and aneuploid breast tumor cells can significantly affect the estimates of the DNA index of these tumors. To this end, the DNA content estimates of 34 aneuploid breast tumors, differing in size, degree of differentiation, and presence or absence of estrogen and progesterone receptors and metastases, were compared using four common DNA fluorochromes: DAPI, Hoechst 33342, propidium iodide, and acridine orange. These dyes differ in their mode of interaction with DNA (binding to minor groove or intercalation) and for each of them binding to DNA is restricted to a different degree by nuclear proteins. It was expected, therefore, that if differences in chromatin structure play a role in DNA content estimates, the DNA index of the measured tumors may vary depending on the dye. The cell nuclei were isolated from the tumors using a detergent‐based procedure and stained with each of the dyes and the DNA index was estimated using peripheral blood lymphocytes as a DNA content standard. For each of the tumors, the DNA index estimates with all four dyes correlated very well. When the results obtained with individual dyes were compared in pairs, the correlation coefficients (r) of DNA indices were all above 0.96 (correlation at p < 0.001). The best concordance was seen between specimens stained with Hoechst 33342 and DAPI (r = 0.99), and the least between those stained with Hoechst 33342 and propidium iodide (r = 0.96). The data indicate that DNA content analysis of unfixed nuclei, utilizing the above fluorochromes, is not significantly biased by differences in chromatin structure of the measured cells.
DNA ploidy in cell nuclei from paraffin‐embedded material–comparison of results from two laboratories
Authors: Fosså Sophie D. , Berner Aasmund , Wæhre Håkon , Heiden Thomas , Holm Juul Mina E. , Ouden Dies Van Den , Pettersen Erik O. , Wang Naining , Tribukait Bernhard 
Published: August 2005

AbstractIn 49 pairs of contiguous sections from paraffin‐embedded prostatic cancer tissue, the DNA indices (DIs) were determined by flow cytometry (FCM) at 2 different laboratories. In 3 of 45 pairs of evaluable nuclear suspensions, DIs of 1.1 (DNA aneuploid) were found at Laboratory 1, whereas all 3 tumours were classified as DNA diploid at Laboratory 2. In the remaining 42 specimens, the correlation between the DIs was excellent, though the application of strictly defined DNA ploidy ranges led to different DNA ploidy allocation in 3 cases.It is concluded that in 85–90% of the cases, reliable DIs can be obtained by FCM done in paraffin‐embedded material at different laboratories. Slight technical variations aiad interpretation differences may lead to different, ploidy allocation in 10–15% of the cases.
BrdUrd/DNA flow cytometry analyses demonstrates cis‐diamminedichloroplatinum (II)‐induced multiple cell‐cycle modifications on human lung carcinoma cells
Authors: Demarcq Catherine , Bastian Gérard , Remvikos Yorghos 
Published: August 2005

AbstractThe treatment of cultured human cells by cis‐diamminedichloroplatinum (II) (cis‐DDP) has been shown to induce complex modifications in the cell cycle. Using dual parameter DNA/BrdUrd flow cytometric analysis, we were able to monitor the cell cycle traverse of a pulse‐labeled cohort of cells in an asynchronous culture of the A549 cell line (human lung adenocarcinoma). Two major modifications of the cell cycle following cis‐DDP treatment were observed: (1) after 24 h of treatment, the labeling index was significantly increased and was linked with a prolonged S‐phase; the S‐phase delay occurred rapidly after cis‐DDP and was dose dependent but not exposure time dependent; (2) an accumulation of cells at the S/G2 transition with an onset approximately 12 h after cis‐DDP contact, which was found to be dependent on both dose and duration of exposure. The cytokinetic results also predict maximal sensitivity to cis‐DDP for G1 cells and minimal for G2 cells. In our model the late S/G2 accumulation was always preceded by a slowing down of the S‐phase. However, only the former should be the correct indicator of cytotoxicity since it was correlated with cell survival as evidenced by d colony formation assay, under all treatment conditions.
Non‐random distribution of abnormal mitoses in heteroploid cell lines
Authors: Dooley William C. , Allison David C. 
Published: August 2005

AbstractWe have performed absorption‐cytometric DNA measurements of the DNA contents of the G0/G1, G2, metaphase, and telophase cells of the heteroploid MCa‐11 and HL‐60 lines, as well as the WCHE‐5 line which has a narrowly restricted number of chromosomes. We found that morphologically unbalanced mitoses occurred much more frequently in telophase‐cell pairs of the heteroploid MCa‐11 and HL‐60 lines than in those of the chromosomally stable WCHE‐5 line. Furthermore, the morphologically unbalanced mitoses represented unequal segregation of DNA into each of the daughter telophase nuclei. Such mitotic segregation errors (MSE) occurred almost exclusively in telophase cells with DNA contents which were above, or below, the DNA content of the modal telophase population. The net effect of these non‐random, unbalanced divisions of heteroploid cells with non‐modal DNA contents is to produce one daughter cell with a DNA content that tends to return to the modal DNA content peak.
Semi‐automated detection of aberrant chromosomes in bivariate flow karyotypes
Authors: Boschman Gert A. , Manders Erik M. M. , Rens Wim , Slater Rosalyn , Aten Jacob A. 
Published: August 2005

AbstractA method is described that is designed to compare, in a standardized procedure, bivariate flow karyotypes of Hoechst 33258 (HO)/ Chromomycin A3 (CA) stained human chromosomes from cells with aberrations with a reference flow karyotype of normal chromosomes. In addition to uniform normalization of normal and abnormal flow karyotypes, the main purpose is detection of structurally abnormal chromosomes in often complex karyotypes of tumor cells. The method, which has been implemented in a computer program, consists of a comparison of individual chromosome peaks with the positions of peaks in the flow karyotype constituted by normal chromosomes and takes into account the natural variability in base composition of normal chromosomes among healthy individuals. Flowkaryotypes are normalized using an iterative fitting procedure, using corrections for (1) amplification of HO and CA fluorescence, (2) cross‐talk between the fluorescence signals of HO and CA, and (3) offset of the HO and CA origin. Flow karyotypes of two cell lines, one with a simple deletion and the other with more complex karyotypic changes, were analyzed. The results of flow analysis were found to be in general agreement with the cytogenetic analysis of quinacrine banded karyotypes.
Influence of different cell extraction methods on cytometric features
Authors: Thunnissen F. B. J. M. , Perdaen H. , Forrest J. 
Published: August 2005

AbstractThe purpose of this study was to investigate the influence of different cell extraction procedures on relative nuclear DNA content (IOD), nuclear area, and texture features of Feulgen‐stained nuclei. In imprints and smears of fineneedle aspirates and suspensions from one human liver specimen, 50 diploid, 50 tetraploid, and 25 octaploid nuclei were measured from each slide. In addition, for DNA measurements, the progressive mean of IOD and tetraploid/diploid and octaploid/diploid ratios was calculated. The results show that the progressive mean of the IOD is constant after measuring 25–30 nuclei. For the three type of specimens, the IOD of diploid nuclei varied slightly. The average coefficient of variation was about 5% for the fineneedle aspirates, imprints, and suspensions. For all tissue sampling methods, the 99% confidence limits of the tetraploid/diploid ratio and octaploid/diploid ratio were within the range of 1.9–2.1 and 3.7–4.3, respectively. The nuclear area and most of the texture features showed a significant difference (p < 0.01) between the three sampling methods in all nuclear populations. In conclusion, different tissue sampling methods have little or no effect on DNA‐related IOD measurements, whereas the outcome of nuclear area and texture features is very dependent of the cell extraction procedure.
Effect of ionic strength in immunocytochemical detection of the proliferation associated nuclear antigens p120, PCNA, and the protein reacting with Ki‐67 antibody
Authors: Bruno Silvia , Gorczyca Wojciech , Darzynkiewicz Zbigniew 
Published: August 2005

AbstractThis study was aimed at revealing whether or not ionic interactions between the epitope of the antigen detected by Ki‐67 antibody, or the proliferation‐associated proteins PCNA or p120, and neighboring cellular constituents impede detectability of these antigens in HL‐60 cells by indirect immunofluorescence assay. To this end, the ionic strength (NaCl concentration) of the solutions in which cells were suspended during their fixation with 0.5% paraformaldehyde was increased, to up to 1.65 M NaCl, to weaken the intra‐ and/or intermolecular ionic interactions during the process of crosslinking, and the cells were then immunostained. Fluorescence of cells reacting with Ki‐67 antibody was maximally increased after their treatment with 1.15 M NaCl; the average increase was nearly 110% above the level seen with the standard methodology utilizing 0.15 M NaCl. The increase was greater for cells in the G1 phase of the cell cycle compared to cells in S or G2. Fluorescence of cells stained with the PCNA antibody was maximally enhanced after cell treatment with 0.65 M NaCl. The enhancement, however, varied depending on the source of the antibody; it was nearly 200% in the case of the antibody provided by Boehringer and over 100% by DAKO. Detection of the nucleolar antigen p 120 was not significantly affected by 0.65–1.65 M NaCl. The data indicate that ionic interactions between cellular constituents indeed play a role in masking the epitope of PCNA and the antigen detected by Ki‐67. These results also suggest that to enhance the detectability of the intracellular proteins, the current immunocytochemical methods should be modified and customized for each particular antigen in terms of selection of the optimal ionic strength during the fixation procedure.
NADH‐dependent dehydrogenase activity estimation by flow cytometric analysis of 3‐(4,5‐dimethylthiazolyl‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction
Authors: Huet Olivier , Petit Jean Michel , Ratinaud Marie Hélène , Julien Raymond 
Published: August 2005

AbstractMTT reduction is usually analysed by colorimetric assay to study mitochondrial dehydrogenase activity as a test of cytotoxicity. This enzymatic reaction produces dark‐blue granules of formazan, which increase cell refringency. In this work, we define the conditions for MTT use in quantitative flow cytometric analysis. MTT reduction provides a nonfluorescent dye usable by this technique to study an intracellular NADH‐dependent dehydrogenase activity in vital cells. We observe that formazan production increases asymptotically with cell concentration and that this temperature‐dependent Michaelis enzymatic reduction is produced essentially by mitochondrial dehydrogenases. In isolated mitochondria from rat hepatocytes and in whole L1210 murine leukemiia cells, the Michaelis constants (KM) observed in the presence of respiratory substrates were, respectively, 10 μM And 500 μM. The inhibition of mitochoAldrial protein synthesis by chloramphenicol, which induces a rise of MTT reduction due to the correlative stimulation of glycolysis (Pasteur effect), is a limit of the MTT assay as a cytotoxicity test.
Method to improve the sensitivity of flow cytometric membrane potential measurements in mouse spinal cord cells
Authors: Seamer Larry C. , Mandler Raul N. 
Published: August 2005

AbstractWe have developers a technique to improve the sensitivity of relative membrane potential measurements in mouse spinal cord cells using the fluorescent, anionic, voltage sensitive dye, DiBa‐C4(3) (Oxonol) and flow cytometry. In order to attribute cellular fluorescence primarily to membrane potential, signal variability due to cell size and shape was reduced by dividing the log fluorescence signal from each cell by either its log forward angle light scatter or log side scatter signals. The use of these ratios in place of log oxonol fluorescence reduced the coefficient of variation of the distributions while leaving the changes in mean fluorescence largely unaffected. Kolmogorov‐Smirnov analysis of pre‐ vs. postkainate stimulation (an excitatory amino acid) showed improved sensitivity of the assay with the use of this ratio technique.
Quantification by laser scan microscopy of intracellular doxorubicin distribution
Authors: de Lange J. H. M. , Schipper N. W. , Schuurhuis G. J. , ten Kate T. K. , Van Heijningen Th. H. M. , Pinedo H. M. , Lankelma J. , Baak J. P. A. 
Published: August 2005

AbstractChanges in intracellular drug localization accompany doxorubicin resistance in multidrug resistant tumor cells. The purpose of this study was to develop a method to quantify these changes and so detect different levels of resistance. Tumor cells were incubated with the fluorescent anthracycline doxorubicin (excitation at 480 nm; emission maximum at 560–590 nm) and were quantified using laser scanning microscopy. The fluorescent mode was used to record the intracellular drug distribution, whereas the absorption mode was used to define the nuclear and cytoplasmic boundaries. The cell compartments were delineated interactively on an image processing system and the ratio nuclear fluorescence/cytoplasmic fluorescence (N/C ratio) was determined. N/C ratios were: 1.8 in the Chinese hamster ovarian cell line AUXB1 and 0.1 in its MDR subline CHRC5; 3.8 in the human squamous lung cancer cell line SW‐1573 and 1.8 and 0.4 in its MDR sublines SW‐1573/2R120 and SW‐1573/2R160, respectively; and 3.6 in the human myeloma cell line 8226/S and 2.1 and 1.0 in its MDR sublines 8226/Dox4 and 8226/Dox40, respectively.The doxorubicin distribution was independent of the doicorubicin concentration within a range from 1–32 μM. Furthermore, the progressive mean of the nuclear/cytoplasmic: doxorubicin fluorescence ratio showed that a minimal sample size of 30 cells is necessary for reliable results. The results of two independent assessments showed a high reproducibility (r = 0.97). Thus, with the method described in this paper, it is possible to detect relatively low levels of doxorubicin resistance (factor 8). © 1992 Wiley‐Liss, Inc.
Spontaneous evolution of nuclear size and DNA content of human non‐small‐cell‐lung cancers grafted onto nude mice
Authors: Kruczynski Anna , Pauwels Olivier , Wright Michel , Delsol Georges , Kiss Robert 
Published: August 2005

AbstractWe monitored the biological evolution of four human non‐small‐cell lung cancers labeled KLX6, KLX7, KLX9, and KLX14 that had been grafted onto nude mice. This monitoring was carried out by means of digital cell image analysis which assessed morphometric (nuclear area, NA) and densitometric (nuclear DNA content, DI) features on Feulganstained nuclei from imprint smears. In each of the four models, the biological evolution of the NA and DI was characterized through their progression during serial passaging onto nude mice. The results show that of the four lung cancer models analyzed, one (KLX6) remained definitively stable with respect to its DNA content (DI assessments), while the other three, i.e., KLX7, KLX9, and KLX14, varied significantly. As assessed by the morphometric parameter, i.e., the NA, the four xenografted lines also varied significantly over serial passaging. In conclusion, we show that digital cell image analyses of Feulgen‐stained cell nuclei including morphoinetric and densitometric parameters are a powerful tool for monitoring the biological evolution of human lung cancers grafted onto nude mice. We think therefore that the ploidy level of a tumor might be dependent upon its age and/or its related clinical stage. © 1992 Wiley‐Liss, Inc.
Methods for topographical analyses of intra‐nuclear BrdUrd‐tagged fluorescence
Authors: Usson Yves , Humbert Catherine 
Published: August 2005

AbstractThe observation of BrdUrd staining in the nuclei of cells from exponentially growing populations reveals different typical replicating patterns. We propose a methodological approach to order and characterize BrdUrd intranuclear distributions. First, visual ordering of the patterns is assessed using a spectral analysis coupled to a k‐nearest neighbors clustering technique. Subsequently, nine topographical features are introduced to characterize the spatial distribution of BrdUrd‐tagged fluorescence in the nuclei of proliferating cells. These topographical features are based on a structural approach. The localization of fluorescence spots is expressed in terms of the normalized distance from the nuclear border and its standard deviation. These topographical features are simple to calculate and easy to relate to visual experience. © 1992 Wiley‐Liss, Inc.