https://ris.utwente.nl/ws/files/6782642/JIM1995.pdf

A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V.

Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis.

Induction of Apoptosis by Apo-2 Ligand, a New Member of the Tumor Necrosis Factor Cytokine Family *

Background Loss of myocytes is an important mechanism in the development of cardiac failure of either ischemic or nonischemic origin. However, whether programmed cell death (apoptosis) is implicated in the terminal stages of heart failure is not known. We therefore studied the magnitude of myocyte apoptosis in patients with intractable congestive heart failure. Methods Myocardial samples were obtained from the hearts of 36 patients who underwent cardiac transplantation and from the hearts of 3 patients who died soon after myocardial infarction. Samples from 11 normal hearts were used as controls. Apoptosis was evaluated histochemically, biochemically, and by a combination of histochemical analysis and confocal microscopy. The expression of two proto-oncogenes that influence apoptosis, BCL2 and BAX, was also determined. Results Heart failure was characterized morphologically by a 232-fold increase in myocyte apoptosis and biochemically by DNA laddering (an indicator of apoptosis). The histochemical demonst...

Apoptosis in the Failing Human Heart

The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis.

DNA strand breaks which occur in HL-60 cells as a result of activation of endonuclease during apoptosis induced by cell treatment with the DNA topoisomerase I inhibitor camptothecin and topoisomerase II inhibitors teniposide, 4′-(9-acridinylamino)-3-methanesulfon-m-anisidide, and fostriecin were labeled in situ, in individual fixed and permeabilized cells, with biotinylated dUTP (detected by fluoresceinated avidin), using the terminal deoxynucleotidyl transferase or nick translation assays During the early stage of apoptosis, prior to nuclear fragmentation, the breaks were predominantly localized at the nuclear periphery, close to the nuclear envelope In more advanced stages, all cellular DNA, then localized within the cell as dense, homogeneous granules of a variety of sizes, was strongly labeled, indicating extensive and more uniform distribution of breaks throughout genomic DNA Bivariate analysis of the incorporated biotinylated dUTP and cellular DNA content by flow cytometry made it possible to estimate the kinetics of the labeling reaction and relate DNA breaks to cell position in the cycle The kinetics of biotinylated dUTP incorporation was faster, and the distinction of cells with DNA breaks was more pronounced, using the terminal transferase rather than the nick translation assay Camptothecin, teniposide, and 4′-(9-acridinylamino)-3-methanesulfon-m-anisidide induced DNA breaks preferentially in S-phase cells, having little effect on cells in the G1 phase of the cycle In contrast, fostriecin affected cells indiscriminately, in all phases of the cell cycle The method of detection of DNA strand breaks (3′-hydroxyl termini) individual cells offers several advantages and can be applied to clinical material (tumor biopsies) to study the induction of apoptosis in tumors during treatment, as a possible prognostic marker The protein-associated DNA breaks in the “cleavable” DNA-topoisoerase complexes, which are the primary lesions induced by the inhibitors and precede apoptosis, were not detectable by the present methods

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Detection of DNA Strand Breaks in Individual Apoptotic Cells by the in Situ Terminal Deoxynucleotidyl Transferase and Nick Translation Assays

A Selective Procedure for DNA Extraction from Apoptotic Cells Applicable for Gel Electrophoresis and Flow Cytometry

The studies were aimed to detect the cell cycle-associated differences in the susceptibility of HL-60 cells to apoptosis induced by diverse agents Exponentially growing HL-60 cells were treated with the DNA topoisomerase I inhibitor camptothecin; the DNA topoisomerase II inhibitors teniposide, m-AMSA, Mitoxantrone, or Fostriecin; the presumed tyrosine kinase inhibitor genistein; a serine/threonine kinase inhibitor H7; the protein synthesis inhibitor cycloheximide; the DNA replication inhibitor hydroxyurea; the nucleoside antimetabolites 1-beta-D-arabinofuranosylcytosine and 5-azacytidine; and the alkylating agent nitrogen mustard, cisplatin, hyperthermia, and gamma irradiation Endonucleolysis, which accompanied apoptosis induced by these agents, was assessed by two different flow cytometric methods, one based on DNA content measurements following extraction of low molecular weight DNA, and another using exogenous terminal deoxynucleotidyl transferase to label in situ DNA strand breaks Each method allowed for both identification of apoptotic cells and analysis of the cell cycle distribution of the unaffected cell population; the method using terminal transferase also allowed for identification of the cell cycle position of apoptotic cells Confirmed by analysis of DNA degradation by gel electrophoresis and changes in cell morphology, apoptosis was observed as early as 3 h after administration of most drugs and for some drugs was cell cycle phase specific Cells progressing through S phase were selectively susceptible when treated with camptothecin, teniposide, m-AMSA, Mitoxantrone, H7, hydroxyurea, and 1-beta-D-arabinofuranosylcytosine Cells in G2-M preferentially underwent apoptosis in cultures treated with H7 or with gamma-irradiation Cells in G1 phase were preferentially affected by 5-azacytidine, nitrogen mustard, and hyperthermia No significant cell cycle specificity was observed in the case of Fostriecin, genistein, cycloheximide, or cisplatin The cell cycle related difference in susceptibility to apoptosis may be a reflection of both the severity of the lesion induced by a given drug and the ability of the cells to repair that lesion; both can vary depending on the cell cycle phase

https://cancerres.aacrjournals.org/content/canres/53/13/3186.full.pdf

The Cell Cycle Related Differences in Susceptibility of HL-60 Cells to Apoptosis Induced by Various Antitumor Agents

Publisher Summary Two distinct modes of cell death, apoptosis and necrosis, can be recognized based on differences in the morphological, biochemical, and molecular changes of the dying cell. This chapter discusses the assays of cell viability in light of their applicability to differentiate between these two mechanisms. It has been recognized during the past several decades that one of the major features discriminating dead from live cells is loss of the transport function and physical integrity of the plasma membrane. A plethora of assays of cell viability has been developed based on this phenomenon. There are several assays of cell viability based on the functional tests of cell organelles. Chromatin condensation is one of the early events of apoptosis. It has been observed that sensitivity of DNA in situ to denaturation is increased during chromatin condensation, for example, as during mitosis or in G 0 , and that chromatin condensation in apoptotic cells is also accompanied by increased DNA denaturability.

Assays of cell viability: discrimination of cells dying by apoptosis

A new flow cytometric method is described to detect DNA strand breaks associated with apoptosis, by labeling the 3'-OH termini in the breaks with biotinylated dUTP in a reaction employing exogenous terminal deoxynucleotidyl transferase. The method has been applied in studies on leukemic HL-60 and MOLT-4 cell lines to reveal whether it is specific to apoptotic cells, and whether it can be used in the clinic to detect DNA breakage in leukemic cells during chemotherapy. There was labeling of mononuclear cells in peripheral blood of all 11 patients studied during chemotherapy for acute lymphoblastic, acute myelogenous, or chronic myelogenous leukemia (ALL, AML, or CML) in blastic crisis, indicating induced DNA damage; the number of labeled cells increased from 1-8% before treatment up to 80% during the course of treatment. The DNA topoisomerase inhibitors mitoxantrone, VP-16 (etoposide), and m-AMSA (amsacrine) were more effective in inducing DNA breaks than was hydroxyurea or cytosine arabinoside (AraC). Cells with DNA breaks were identified in peripheral blood for up to 5 days following administration of Mitoxantrone and VP-16. In the case of DNA aneuploid leukemias, the DNA breaks were predominant in the aneuploid cell subpopulations, whereas presumably non-neoplastic diploid cells were unlabeled. In one case of ALL there were two distinct subpopulations of aneuploid cells: one responded to the treatment (by DNA breakage) and the other was non-responding. Thus, cells undergoing apoptosis can be detected by this method of labeling DNA strand breaks and the technique is applicable for analysis of response of leukemic cells to chemotherapy. With this method it may be possible to identify tumor cell sensitivity or resistance to particular drugs early in the course of treatment.

Jianping Gong

Papers

Detection of DNA Strand Breaks in Individual Apoptotic Cells by the in Situ Terminal Deoxynucleotidyl Transferase and Nick Translation Assays

A Selective Procedure for DNA Extraction from Apoptotic Cells Applicable for Gel Electrophoresis and Flow Cytometry

The Cell Cycle Related Differences in Susceptibility of HL-60 Cells to Apoptosis Induced by Various Antitumor Agents

Assays of cell viability: discrimination of cells dying by apoptosis

Induction of DNA strand breaks associated with apoptosis during treatment of leukemias.