Different sublines of Jurkat cells respond with varying susceptibility of internucleosomal DNA degradation to different mediators of apoptosis

Abstract

The ability of two different Jurkat sublines, termed standard and JM, to form DNA ladders was investigated after various apoptotic stimuli. Exposure to a broad spectrum of drugs interfering with signal transduction or cellular metabolism revealed distinct differences between both Jurkat sublines with regard to the pattern of DNA degradation. In standard Jurkat cells, internucleosomal DNA cleavage occurred only after treatment with the protein kinase inhibitor staurosporine. In contrast, the JM subline responded with internucleosomal DNA fragmentation to exposure to gemcitabine, cycloheximide or staurosporine. All drugs induced the formation of DNA fragments of about 50 kb in both sublines, as revealed by pulse field electrophoresis, except H2O2, which caused unspecific DNA degradation. The staurosporine-induced DNA ladder formation was accompanied by an increase in caspase-3 activity in both lines which, however, was considerably lower in Jurkat JM cells after gemcitabine or cycloheximide exposure. When the analysis of internucleosomal DNA degradation was carried out after mycoplasma infection, both Jurkat lines responded with DNA ladder formation after exposure to all drugs used (here only shown for the standard subline). Employing the zymogram technique, nuclease activities of 47 kDa and 54 kDa were detected in culture supernatants, cell homogenates and nuclear extracts only when mycoplasma-infected, whereas the samples obtained from mycoplasma-free sublines were nuclease-negative using this technique, indicating that these endonucleases were of mycoplasmal origin. After drug exposure, the mycoplasmal nucleases must have gained access to the cytoplasm and nuclei of their host cells by an unknown mechanism.

Partitioning apoptosis: A novel form of the execution phase of apoptosis

“…8 The sequence and kinetics of degradative events that contribute to nuclear disassembly during apoptosis may be variable in different cells types and in response to different apoptosis-inducing signals. 31 Thus, the specific effector caspases activated, their kinetics and level of activation, the movement of activated caspases into the nucleus 32 and their specificity for matrix-associated proteins could account for the differences in nuclear structures observed during the classical and partitioning forms of apoptosis. 30 Partitioning apoptosis is distinct from necrosis, which is characterized by a generalized cell swelling due to the accumulation of water and electrolytes, early membrane rupture, and disruption of cellular organelles.…”

Mycoplasma fermentans inhibits tumor necrosis factor α-induced apoptosis in the human myelomonocytic U937 cell line

“…These findings also imply that the presence of mycoplasmas in various cell lines might be affecting the results of apoptosis research. 31 …”

Chromatin breakdown during necrosis by serum Dnase1 and the plasminogen system

“…For the HMW DNA fragmentation analysis by pulsed-field gel electrophoresis (PFGE), MCF-7 cells were treated as described above, scraped off, pelleted, and embedded in prewarmed 1% (w/v) agarose dissolved in 0.25ϫ Tris-borate-EDTA buffer. Subsequently, proteinase K digestion and PFGE were carried out as previously described (26), except that the voltage changed logarithmically from 230 volts to 180 volts over the period of time.…”

“…Isolation of MCF-7 cell nuclei was performed as previously described (26). Cell nuclei (10 5 ) were treated with either 5% (v/v) serum or pure proteins at the concentrations indicated for the cellular DNA degradation assays in 200 l of reaction buffer (10 mM HEPES, 50 mM NaCl, 2 mM MgCl 2 , 2 mM CaCl 2 , 40 mM ␤-glycerophosphate; pH 7.0) at 37°C for various lengths of time.…”