Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:5.99.1.2 (topoisomerase)
 9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. Most of these methods were applied to studies of apoptosis triggered in the human leukemic HL-60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomerase inhibitors or prednisolone. In most cases, apoptosis was selective to cells in a particular phase of the cell cycle: only S-phase HL-60 cells and G0 thymocytes were mainly affected. Necrosis was induced by excessively high concentrations of these drugs. The following cell features were found useful to characterize the mode of cell death: a) Activation of an endonuclease in apoptocic cells resulted in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, led to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content made it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of the apoptotic process. b) Plasma membrane integrity, which is lost in necrotic but not apoptotic cells, was probed by the exclusion of propidium iodide (PI). The combination of PI followed by Hoechst 33342 proved to be an excellent probe to distinguish live, necrotic, early- and late-apoptotic cells. c) Mitochondrial transmembrane potential, assayed by retention of rhodamine 123 was preserved in apoptotic but not necrotic cells. d) The ATP-dependent lysosomal proton pump, tested by the supravital uptake of acridine orange (AO) was also preserved in apoptotic but not necrotic cells. e) Bivariate analysis of cells stained for DNA and protein revealed markedly diminished protein content in apoptotic cells, most likely due to activation of endogenous proteases. Necrotic cells, having leaky membranes, had minimal protein content. f) Staining of RNA allowed for the discrimination of G0 from G1 cells and thus made it possible to reveal that apoptosis was selective to G0 thymocytes. g) The decrease in forward light scatter, paralleled either by no change (HL-60 cells) or an increase (thymocytes) of right angle scatter, were early changes during apoptosis. h) The sensitivity of DNA in situ to denaturation, was increased in apoptotic and necrotic cells. This feature, probed by staining with AO at low pH, provided a sensitive and early assay to discriminate between live, apoptotic and necrotic cells, and to evaluate the cell cycle phase specificity of these processes. i) The in situ nick translation assay employing labeled triphosphonucleotides can be used to reveal DNA strand breaks, to detect the very early stages of apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Features of apoptotic cells measured by flow cytometry. 133 43

Western blotting, indirect immunolocalization, flow cytometry, and a functional assay for drug-induced strand breakage were utilized to examine topoisomerase (topo) II levels during granulocytic maturation in HL-60 human progranulocytic leukemia cells and in samples of normal human marrow. Indirect immunofluorescence revealed that the intensity of the signal for topo II in unsynchronized log phase HL-60 cells varied widely. Indirect immunolabeling combined with propidium iodide staining and two-parameter flow cytometry revealed that topo II levels increased an average of 2-fold as cells progressed from G1 to G2/M. When HL-60 cells were induced to mature toward granulocytes, topo II levels progressively decreased and became undetectable by functional assays, by indirect immunoperoxidase staining, and by Western blotting with an antibody which identified Mr 170,000 and Mr 180,000 forms of topo II. Similar changes were detected during normal granulocytic maturation in human marrow in vivo. Western blotting revealed that levels of the Mr 170,000 (proliferation-associated) isoform of topo II were highest in marrow fractions enriched in progranulocytes and myelocytes, intermediate in unfractionated marrow from normal volunteers, and undetectable in mature granulocytes. The Mr 180,000 topo II polypeptide was also diminished or absent from mature granulocytes. In further experiments, marrow samples from normal volunteers were subjected to flow cytometry after labeling of topo II and various cell surface markers. Levels of the Mr 170,000 topo II polypeptide in CD34-positive cells (multipotent and committed progenitors from several hematopoietic lineages) were indistinguishable from levels observed in the HL-60 leukemia cell line. These results suggest that topo II levels in highly proliferative normal human myeloid cells in vivo approach levels found in corresponding neoplastic cell lines in vitro. Conversely, as the same cells mature into granulocytes in vivo or in vitro, levels of both molecular weight forms of topo II diminish. These results provide a framework for the further investigation of topo II levels and drug sensitivity in human leukemia.
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PMID:Topoisomerase II levels during granulocytic maturation in vitro and in vivo. 164 69

Studies have suggested that recombinant tumor necrosis factor-alpha (TNF-alpha) may potentiate the killing of murine tumor cells by drugs targeted at DNA topoisomerase II. We have examined the combined cytotoxic effects of the topoisomerase-targeted drug etoposide and TNF in small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cell lines using clonogenic assays and a novel flow cytometry technique relying on differential uptake of fluorescein diacetate (FDA) and propidium iodide (PI) by viable and nonviable cells. Good correlation of IC50 determinations for etoposide were noted between clonogenic assays and the FDA/PI technique for both classic and variant SCLC cell lines. The effects of etoposide on the classic SCLC line H209 were potentiated by TNF with a decrease in the IC50 from 3.3 microM to 1.0 microM as determined by FDA/PI. Tumor necrosis factor alone had little effect on the growth or cloning efficiency of H209 cells. Tumor necrosis factor alone stimulated the growth and cloning of variant SCLC line N417, but the cytotoxicity of etoposide was not potentiated by TNF in N417 cells. Tumor necrosis factor alone inhibited the growth and cloning of the NSCLC line H125 but exerted a marked protective effect against higher concentrations of etoposide. It appears that the interaction of TNF with etoposide varies between cell lines and between subclasses of human lung cancer.
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PMID:Interaction of recombinant human tumor necrosis factor and etoposide in human lung cancer cell lines. 217 61

FDCP-Mix, a pluripotent murine hemopoietic stem cell line which undergoes typical internucleosomal cleavage of DNA when induced to apoptosis by either drugs or withdrawal of growth factor (interleukin-3) was studied after treatment with the topoisomerase II inhibitor etoposide (0.5-4 microM). An increase in autolytic activity was the major early morphological change within the cytoplasm, with mitochondria as the main target for autolytic digestion. Despite this macroautophagy, thin sections showed a high number of mitochondria, suggesting mitochondrial proliferation as a result of drug treatment. This observation of an increase in the number of mitochondria was confirmed by flow cytometric studies of mitochondrial overall mass. Multiparameter flow cytometry of cells double stained with propidium iodide and nonyl-acridine orange gave an accurate assay for mitochondrial mass in relation to cell cycle stages. The increase in mitochondrial mass was found in all cell cycle stages. The results suggest a drug-induced proliferation of mitochondria separate from the processes involved in the doubling of mitochondrial mass during the cell cycle and a decline of mitochondria in the later stages of apoptosis.
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PMID:Changes of mitochondrial mass in the hemopoietic stem cell line FDCP-mix after treatment with etoposide: a correlative study by multiparameter flow cytometry and confocal and electron microscopy. 749 25

In this study we describe a cytometric method to sort apoptotic cell fractions, suitable for biochemical and morphological analyses. Rat thymocytes were used as a model system, as apoptosis naturally occurs in the thymus, where the negative selection of the T cell repertoire takes place. Massive apoptosis was induced in vitro by the topoisomerase-II inhibitor, etoposide. After etoposide treatment, a large fraction of thymocytes showed the morphological and electrophoretic features of apoptotic cells. Unfixed thymocytes were stained for 30 min with propidium iodide (PI: 50 micrograms/ml containing RNase type A and detergent), and analyzed by flow cytometry. Apoptotic thymocytes typically showed sub-G1 DNA contents. Compared to non-apoptotic thymocytes, sub-G1 cells had lower values of low-angle (FSC) scatter and higher values of right-angle (SSC) scatter, so that, in dual parameter cytograms of FSC versus SSC, two distinct cell populations were apparent, and were separated by flow sorting. The purified cell fractions obtained by this procedure had a very well preserved ultrastructural morphology; both early and late apoptotic stages (until the onset of cytoplasm segmentation) were present in the sorted apoptotic fraction.
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PMID:A single-step staining procedure for the detection and sorting of unfixed apoptotic thymocytes. 751 May 45

A series of analogs based on a novel template, 11-aza-(20S)-camptothecin, were obtained from total synthesis and tested as potential anticancer drugs in the topoisomerase I enzyme cleavable complex assay. The parent compound 11-aza-(20S)-camptothecin (8) was derived from a Friedlander condensation between the known aminopyridine derivative 3-(3-amino-4-picolylidene)-p-toluidine and optically active tricyclic ketone 7. Compound 8 had activity approximately twice that of (20S)-camptothecin in the calf thymus topoisomerase I cleavable complex assay. Compounds were prepared wherein the 11-aza nitrogen atom was quaternized as either the corresponding N-oxide or methyl iodide. Compounds with quaternized N-11 showed improved water solubility and were equipotent to the clinically investigated camptothecin analog topotecan in the cleavable complex assay. These compounds were evaluated in vivo in nude mice bearing HT-29 human colon carcinoma xenografts. The analog 11-aza-(20S)-camptothecin 11-N-oxide was found to significantly retard tumor growth when compared to untreated controls. Finally, 7,10-disubstituted 11-azacamptothecin analogs were synthesized using Pd(0) coupling reactions of 10-bromo-7-alkyl-11-aza-(20S)-camptothecins 19 and 20, which in turn were available from a Friedlander condensation of the novel bromopyridine derivatives 17a and 17b with 7. Among the 10-substituted series, a number of analogs displayed extremely high in vitro potency against topoisomerase I and improved aqueous solubility. A significant number of the compounds were found to be active in whole cell cytotoxicity assays and several were evaluated in nude mice bearing the HT-29 tumor xenografts. The most effective of these proved to be (S)-11-aza-7-ethyl-10-(aminohydroximinomethyl)camptothecin trifluoracetic acid salt (27), a potent topoisomerase I inhibitor which demonstrated excellent efficacy in both short term and in extended in vivo assays. A comparison between in vitro enzyme data and in vivo data from nude mouse studies in other compounds in this series revealed a poor overall correlation between topoisomerase inhibition in vitro and antitumor efficacy in vivo.
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PMID:Synthesis, topoisomerase I inhibitory activity, and in vivo evaluation of 11-azacamptothecin analogs. 770 14

Little is known about the regulation of apoptosis in fibroblasts although several model systems including serum deprivation and treatment with staurosporine or topoisomerase inhibitors have been used to induce apoptosis in vitro. To validate a reproducible in vitro model for the study of apoptosis in fibroblasts, we cultured density-inhibited monolayer cultures of Balb/c 3T3 fibroblasts in Dulbecco's modified essential medium plus 15% fetal calf serum and then withdrew serum. Time-lapse video microscopy demonstrated that within minutes of serum withdrawal, cells lost substrate attachment and floated to the top of the liquid growth medium. There was a time-dependent increase in the number of non-adherent cells. Some of these cells regained attachment and spread momentarily, but they eventually rounded up and lost attachment permanently. In contrast to serum-containing cultures in which similar morphological changes were followed by mitosis, in serum-free cultures repeated attempts at mitosis were followed by permanent attachment loss and presumably cell death. To assess whether all the non-adherent cells were in fact dead, the percentages of cells that continued to proliferate upon return to serum-supplemented conditions was computed. After various periods of serum starvation a decreasing proportion (approx. 75% at 30 minutes; < 2% at 24 hours) of the non-adherent cells could be rescued by addition of serum. Transmission electron microscopy of cells 3 hours after serum withdrawal showed that the majority (approximately 60%) of non-adherent cells exhibited marked intranuclear chromatin condensation but maintained integrity of cell and nuclear membranes and cell organelles, morphological changes consistent with those of apoptotic cell death. Scanning electron microscopy of cultures 3 hours following serum withdrawal showed rounded cells with marked surface blebbing. Fluorescence and confocal microscopy revealed increased intensity of nuclear staining with DAPI while actin filaments became indistinct or collapsed around the nucleus. After cycloheximide treatment to inhibit protein synthesis, there was no reduction of apoptosis. Gel electrophoresis of DNA from both control and 3 hour-serum-deprived cells showed intact DNA with no oligonucleosomal length fragmentation. After serum withdrawal, intracellular calcium was reduced by about 32% over 5 minutes as measured by fura2 ratio fluorimetry in single cells. Serum-starved cells showed a time-dependent shrinkage in mean cell diameter compared to trypsinized, adherent control cells (at 0 hours, mean diameter = 18.0 microns--viable; at 4 hours, mean diameter = 15.5 microns--apoptotic). Flow cytometric analysis showed increased propidium iodide staining and reduced fluorescein diacetate uptake over 3 hours, changes that were contemporaneous with the reduction of cell diameter.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Serum deprivation induces apoptotic cell death in a subset of Balb/c 3T3 fibroblasts. 792 26

DNA topoisomerase I inhibitor camptothecin (CAM), topoisomerase II inhibitors teniposide (TN) and amsacrine (m-AMSA) induce apoptosis of HL-60 cells. One of the early events of apoptosis is DNA degradation, which occurs as a result of activation of the specific endonuclease. DNA strand breaks generated during this process were revealed, in the present study, by the in situ nick translation assay which was adapted to flow cytometry. In this assay, the incorporation of biotinylated dUTP by apoptotic cells was detected by the use of fluorescinated avidin, whereas simultaneous staining of DNA with propidium iodide made it possible to correlate the appearance of DNA strand breaks with cell position in the cell cycle. The breaks were detected as early as 90 min after the initial cell contact with CAM, and they were limited to cells in the S phase of the cell cycle. At that early stage of apoptosis DNA was not yet extractable from the cells; the loss of DNA from S-phase cells could not be seen, by flow cytometry, during the initial 2 h of incubation with CAM. DNA strand breaks induced by TN and m-AMSA also occurred preferentially in S-phase cells. The data indicate that DNA strand breaks resulting from activation of endonuclease in HL-60 cells treated with DNA topoisomerase I or II inhibitors can be conveniently measured using the in situ nick translation assay. This assay has certain advantages over other methods of identification of apoptotic cells by flow cytometry, such as providing direct evidence of DNA damage and offering the opportunity to correlate DNA damage with cell position in the cell cycle. The method may be of interest in clinical oncology where testing tumor response (by DNA degradation) to DNA topoisomerase inhibitors or other treatments may be of prognostic value.
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PMID:Apoptosis of S-phase HL-60 cells induced by DNA topoisomerase inhibitors: detection of DNA strand breaks by flow cytometry using the in situ nick translation assay. 838 87

The human histiocytic lymphoma line HL-60 has served as a model of myeloid cell differentiation and can be induced to differentiate along the neutrophil or monocytic lineage, depending on the external stimulus. The nondifferentiated cell line retains a premyeloid leukemic phenotype and is capable of anchorage-independent growth and proliferation. The role of apoptosis in the regulation of immunologic and inflammatory events associated with homeostasis and disease has been most intensively studied in lymphocytes. In the present study, nondifferentiated HL-60 has served as a model for studying myeloid cell apoptosis by investigating apoptotic changes induced by camptothecin, a DNA topoisomerase inhibitor, as well as physiologic stimuli, including ceramide analogs and a monoclonal antibody against the Fas antigen. Multiparameter flow cytometry was used to evaluate apoptosis by measuring changes in both side scatter and propidium iodide staining. The appearance of apoptotic cells was confirmed biochemically by measuring DNA endonuclease activity by both enzyme-linked immunosorbent assay quantitation and DNA ladder formation on agarose gels and morphologically with the detection of micronuclei by confocal laser microscopy. These studies demonstrate that HL-60 can serve as an in vitro model for the detection of physiologic and pharmacologic apoptotic stimuli and for understanding the early and late cellular changes associated with induction of the apoptotic program.
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PMID:In vitro detection of apoptotic stimuli by use of the HL-60 myeloid leukemic cell line. 854 40

Programmed cell death or apoptosis is characterized by typical morphological alterations. By transmission electron microscopy, apoptotic cells are identified by condensation of the chromatin in tight apposition to the nuclear envelope, alteration of the nuclear envelope and fragmentation of the nucleus, whereas integrity of the plasma membrane and organelles is preserved. Conversely cells undergoing necrosis display an early desintegration of cytoplasmic membrane and swelling of mitochondria. In this study we assessed by flow cytometry the sequential alterations of forward angle light scatter, 90 degrees light scatter, and fluorescence associated with fluorescein diacetate, rhodamine 123, and propidium iodide in two human B cell lines undergoing apoptosis induced by the topoisomerase II inhibitor VP-16. The kinetics of these modifications were compared to those of cells undergoing necrosis induced by sodium azide. At the same time intervals, cells were examined by transmission electron microscopy and by UV microscopy after staining with Hoechst 33342. We report that sequential changes in light scatters and fluorescein diacetate are similar in cells undergoing apoptosis or necrosis, whereas apoptosis is characterized by a slightly delayed decrease of mitochondrial activity as assessed by rhodamine 123 staining. Surprisingly a part of cells undergoing apoptosis displayed an early uptake of propidium iodide followed by a condensation and then a fragmentation of their nuclei. It is concluded that uptake of propidium iodide is a very early marker of cell death which does not discriminate between necrosis and apoptosis. Along with biochemical criteria, nuclear morphology revealed by staining with Hoechst 33342 would seem to be of the most simple and most discriminative assay of apoptosis.
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PMID:Kinetics of plasma membrane and mitochondrial alterations in cells undergoing apoptosis. 858 50


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