title: Evaluation of cell death in EBV-transformed lymphocytes using agarose gel electrophoresis, light microscopy and electron microscopy. II. Induction of non-classic apoptosis ("para-apoptosis") by tritiated thymidine.
abstract: There is an extensive literature dating back to the late 1950's, on the damaging biological effects of radiolabeling DNA in vivo. Nonetheless, tritiated thymidine has often been used to label DNA in studies of programmed cell death (apoptosis). In the present study, we have investigated the effects of incorporation of tritiated thymidine into the DNA of an Epstein-Barr virus-transformed cell line (NC-37) in the absence of any other apoptosis-inducing agent. Cells were incubated in media containing 1-20 microCi/ml [methyl-3H]-thymidine ([3H]-TdR). At each concentration of tritiated thymidine used, cell proliferation ceased within 12 hours of incubation. The mode of cell death caused by tritiated thymidine incorporation was evaluated using DNA degradation patterns and cellular morphology. DNA degradation, in the absence of a "ladder" pattern, was shown by agarose gel electrophoresis. Electron microscopy was used as the "gold standard" to evaluate the specific morphologic type of cell death that accompanied the DNA degradation. Although some of the features of apoptosis were present, the cells lacked the early margination of the chromatin within an intact nucleus and surface blebbing leading to apoptotic body formation, two characteristic morphological features of apoptosis. We, therefore, coined the term "para-apoptosis" to be more precise about the morphologic type of cell death. The percent of para-apoptotic cells was quantitated by light microscopy using whole mount preparations (cytospins). The morphologic criteria of chromatin condensation, nuclear fragmentation, increase in cell density and cytoplasmic vacuolization were used for the evaluation of para-apoptosis by light microscopy of cytospin preparations. In the absence of tritiated thymidine, < 2% of the cells became apoptotic/para-apoptotic after 43 hours of incubation. However, at all concentrations of tritiated thymidine used in the incubation medium (1-20 microCi/ml), the number of para-apoptotic cells increased. In addition, we detected perturbations in the timing of the cell cycle of the surviving cells and an increase in the number of micronuclei after only one division cycle. The induction of para-apoptosis and micronuclei formation represent two distinct modes of cell death caused by tritiated thymidine incorporation. These studies emphasize the necessity for morphological examination in characterizing the induction of cell death in a new experimental system.
authors.affiliation: Department of Pathology, University of Arizona, Tucson 85724, USA.
authors.forename: E
authors.initials: E
authors.lastname: Asher
authors.forename: C M
authors.initials: CM
authors.lastname: Payne
authors.forename: C
authors.initials: C
authors.lastname: Bernstein
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D001692:Biological Transport; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral; D004247:DNA / Q000032:analysis* / Q000096:biosynthesis / Q000302:isolation & purification; D004249:DNA Damage; D004587:Electrophoresis, Agar Gel; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D008853:Microscopy; D008854:Microscopy, Electron; D013936:Thymidine / Q000378:metabolism / Q000494:pharmacology*; D013997:Time Factors; D014316:Tritium
chemical_list: D014316:Tritium; D004247:DNA; D013936:Thymidine