[CANCER RESEARCH 60, 4623– 4628, August 15, 2000] The DNA of Annexin V-binding Apoptotic Cells Is Highly Fragmented 1 Zsolt Bacso ´, 2 Richard B. Everson, and James F. Eliason 3 Barbara Ann Karmanos Cancer Institute, Wayne State School of Medicine Detroit, Michigan 48201 ABSTRACT Jurkat leukemia cells induced to undergo apoptosis by treatment with an antibody against the Fas receptor have two annexin V (AV)-binding subpopulations: (a) single-positive cells that bind AV but not propidium iodide (PI); and (b) double-positive cells that bind AV and PI. The single-positive population is thought to represent an early stage of apo- ptosis. We have examined the relationship between AV binding and a classical characteristic of apoptosis, DNA fragmentation. Time course studies with Jurkat cells treated for 1, 2, or 4 h with anti-Fas indicated that the proportion of AV-binding cells was increased after 2 h. A signif- icant increase in DNA fragmentation was observed only at 4 h as mea- sured by the mean tail moment determined with the alkaline single cell gel electrophoresis (comet) assay. This correlation suggests a temporal rela- tionship between the two parameters, but does not provide direct evidence of what happens in individual cells. We developed a method to measure fluorescent markers of cellular structure or function with a laser scanning cytometer and then perform the comet assay on the same cells. Cells in each AV-binding subpopulation were re-examined before and after elec- trophoresis. Most AV  /PI  cells had no DNA damage, although a few cells showed a pattern of damage characteristic for apoptosis. Double-positive cells all had damaged DNA; approximately half had the apoptotic pattern, and the rest had a pattern typical for necrosis. Nearly all of the single- positive cells had damaged DNA with the apoptotic pattern. Both AV- positive populations contained cells with little or no detectable DNA after electrophoresis, indicating that the DNA was highly fragmented. These results indicate that AV binding is an excellent marker for apoptotic cells, but that these cells already have fragmented DNA. INTRODUCTION Programmed cell death (apoptosis) is an important process in normal development and in tissue homeostasis, as well as a key mechanism by which anticancer therapies exert their cytotoxic effects. Agents like anticancer drugs and ionizing radiation that damage DNA induce apoptosis through a p53-dependent pathway (1). Binding of p53 and other nuclear proteins to the sites of damage in the DNA appears to trigger the apoptotic process. Another mechanism that can induce apoptosis involves the interaction of proteins such as Fas (CD95) or tumor necrosis factor with their receptors on the surface of cells. Signaling from these so-called death receptors starts the apo- ptotic process (2). Regardless of how the apoptotic process has been initiated, by intrinsic signals or extrinsic signals involving death receptors, a hall- mark of apoptosis is fragmentation of the DNA. Two main steps have been identified for apoptotic DNA fragmentation (3). The first in- volves formation of high molecular size DNA fragments of 50 –300 kb. This process is widely observed in different cells and is propa- gated through single- and double-strand breaks in the DNA. The second step generates small, 200 –300-bp DNA fragments. These small fragments lead to DNA ladder formation classically associated with apoptosis, although it can be absent in some cell types (4). Another characteristic of cells undergoing apoptosis is the capacity to bind the protein AV. 4 AV binds to PS, which is normally located on the inner leaflet of the plasma membrane, but it is externalized to the outer leaflet during apoptosis. PI staining is widely used to discriminate living cells, which exclude this DNA dye, from dead cells, which are permeable to it. In populations of cells undergoing apoptosis, there are some cells that bind AV but are not stained with PI. This “single-positive” population is thought to represent cells in an early stage of apoptosis because the cells apparently exclude PI and because it appears earlier than DNA ladders can be seen (5). The Fas-induced apoptotic pathway in Jurkat cells is one of the best-examined models of apoptosis. As in other apoptotic pathways, the activation of cysteine-dependent aspartate-directed proteases, i.e., caspases, is crucial (2). The initial events may be reversible, but in turn, initiate irreversible processes belonging to the “execution phase,” such as externalization of phosphatidylserine in the plasma membrane and fragmentation of nuclear DNA. DNA fragmentation related to apoptosis is usually measured using mass biochemical methods. With heterogeneous cell populations, these techniques will miss contributions from small subpopulations. The comet assay, on the other hand, measures DNA fragmentation on a single cell level, allowing analysis of subpopulations of cells. Our immediate aim was to develop a method to measure the relationship between nuclear DNA fragmentation and membrane PS externaliza- tion, two hallmarks of apoptotic cell death on a cell-by-cell basis. We show that there is a high degree of DNA fragmentation in Jurkat cells that stain with AV after apoptosis is induced by treatment with an antibody against Fas. MATERIALS AND METHODS Cell Culture. Jurkat T lymphocytic leukemia cells (American Type Cul- ture Collection, Manassas, VA) were kept in continuous logarithmic growth by passaging them at a concentration of 2.5  10 5 cells/ml every other day in RPMI 1640 medium supplemented with 10% fetal bovine serum (Life Tech- nologies, Inc., Grand Island, NY), 10 mM HEPES (Sigma, St. Louis, MO), and 50 g/ml gentamicin (Life Technologies, Inc.). Induction of Apoptosis. Cells were subcultured 1 day before each exper- iment was performed. Apoptosis was induced by transfer into fresh culture medium containing 2 g/ml of a monoclonal antibody against human Fas (CD95; mouse IgG1, clone DX2.1; R&D Systems, Minneapolis, MN) and 2 g/ml of protein G (Sigma). The cells were incubated at 37°C for various periods of time. In some experiments, control cells were incubated with protein G alone. AV and PI Staining. After incubation with anti-Fas and or protein G, cells were harvested by centrifugation for 5 min at 200  g and were resuspended in AB buffer (140 mM NaCl and 2.5 mM CaCl 2 ). Aliquots containing 1  10 5 cells in 100 l of buffer were stained with 10 l of PI (50 g/ml) solution and with 5 l of FITC-conjugated AV (17.6 g/ml; PharMingen, San Diego, CA) for 5 min on 37°C. After staining, 400 l of AB buffer were added to the cells, and samples were stored on ice until data acquisition. Measurements were completed within 1 h. Flow Cytometry. Flow cytometric analysis was performed using a FAC- Scan flow cytometer (Becton Dickinson, San Jose, CA). Photomultiplier Received 2/22/00; accepted 6/19/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by a Virtual Discovery Grant from the Karmanos Cancer Institute. 2 Present address: Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen, Debrecen, Hungary H-4012. E-mail: bacso@jaguar.dote.hu. 3 To whom requests for reprints should be addressed, at Barbara Ann Karmanos Cancer Institute, HWCRC 724, Detroit, MI 48201. Phone: (313) 966-7858; Fax: (313) 966-7558; E-mail: eliasonj@karmanos.org. 4 The abbreviations used are: AV, annexin V; PI, propidium iodide; PS, phosphati- dylserine; LMP, low melting point; LSC, laser scanning cytometer. 4623 Research. on October 22, 2021. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from