Changes in elemental content during apoptotic cell death studied by electron probe X-ray microanalysis

Recent data suggest that changes in ionic content, primarily potassium, play a pivotal role in the progression of apoptosis. However, the changes in total element content, i.e., sodium (Na), magnesium (Mg), phosphorous (P), chlorine (Cl), potassium (K), and calcium (Ca), during apoptosis have not been evaluated. Electron probe X-ray microanalysis (EPXMA) was used to measure total element content in U937 cells before and after the induction of apoptosis. As an experimental model we used U937 cells irradiated with ultraviolet (UV) light. Apoptosis was evaluated with phase-contrast microscopy, with scanning and transmission electron microscopy, and with the fluorescent dye bisbenzimide (Hoechst 33342). Plasma membrane permeability as a measure of cell death was determined by trypan blue dye exclusion. To investigate element content with EPXMA, cells were cryoprepared, i.e., cryofixed and freeze-dried, and analyzed as whole cells using a scanning electron microscope. We found that the UV irradiation induced rapid (within 2 h) morphological changes associated with apoptosis, such as plasma membrane blebbing, condensation of the chromatin, and the formation of membrane-bound apoptotic bodies. At this time, 95% of the apoptotic cells excluded trypan blue dye. EPXMA results demonstrated that UV light-irradiated apoptotic cells (cells with membrane-bound apoptotic bodies) had a lower Cl content (P < 0.001) and K content (P < 0.001) and a higher Na content (P < 0.001) in comparison with nonirradiated control cells. Also, P and Ca content was higher in apoptotic cells than in control cells, but this difference did not reach statistical significance. No differences were found in Mg. These data indicated that morphological changes characteristic of apoptotic cell death are related with significant changes in sodium, chlorine, and potassium content. In addition, we demonstrated that these changes in elemental composition were not associated with loss of cell membrane integrity.     

Cell nucleus and DNA fragmentation are not required for apoptosis

Apoptosis is the predominant form of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Activation of an endonuclease which cleaves genomic DNA into internucleosomal DNA fragments is considered to be the hallmark of apoptosis. However, no clear evidence exists that DNA degradation plays a primary and causative role in apoptotic cell death. Here we show that cells enucleated with cytochalasin B still undergo apoptosis induced either by treatment with menadione, an oxidant quinone compound, or by triggering APO-1/Fas, a cell surface molecule involved in physiological cell death. Incubation of enucleated cells with the agonistic monoclonal anti-APO-1 antibody revealed the key morphological features of apoptosis. Moreover, in non-enucleated cells inhibitors of endonuclease blocked DNA fragmentation, but not cell death induced by anti-APO-1. These data suggest that DNA degradation and nuclear signaling are not required for induction of apoptotic cell death.     

Computerized video time-lapse microscopy studies of ionizing radiation-induced rapid-interphase and mitosis-related apoptosis in lymphoid cells

Computerized video time-lapse (CVTL) microscopy of X-irradiated cultures of cells of the murine lymphoma cell lines ST4 and L5178Y-S and the human lymphoid cell line MOLT-4 demonstrated that these cells exhibit a wide disparity in the timing of induction and execution of radiation-induced cell death that included rapid-interphase apoptosis, delayed apoptosis, and postmitotic apoptosis. ST4 cells that received 2.5 or 4 Gy of X radiation underwent rapid-interphase apoptosis within 2 h. Apoptosis commenced with a 10-20-min burst of membrane blebbing followed by swelling for 2-4 h and cell collapse. No apoptotic bodies were formed. After a dose of 1 Gy, approximately 90% of ST4 cells died by rapid-interphase apoptosis, while the remainder completed several rounds of cell division prior to cell death. Postmitotic death of ST4 cells occurred with the same morphological sequence of events as during rapid-interphase apoptosis induced by doses of 1-4 Gy. In contrast, L5178Y-S and MOLT-4 cells that received 4 Gy underwent apoptosis more slowly, with a complex series of events occurring over 30-60 h. Only 3% of L5178Y-S cells and 24% of MOLT-4 cells underwent apoptosis without attempting cell division. The cells became abnormally large during a long G(2)-phase delay, and then most of the cells (76-97%) attempted to divide for the first or second time at approximately 18-30 h postirradiation. However, either mitosis failed or division was aberrant; i.e., the large cells divided into three or four fragments which eventually fused together. This process was followed by several rounds of complex and unpredictable membrane blebbing, gross distortions of shape, fragmentation-refusion events, and formation of apoptotic bodies, after which the cells collapsed at 36-60 h postirradiation.     

Calpain activation in apoptosis

Programmed cell death is an active process wherein the cell initiates a sequence of events culminating in the fragmentation of its DNA, nuclear collapse, and disintegration of the cell into small, membrane-bound apoptotic bodies. Examination of the death program in various models has shown common themes, including a rise in cytoplasmic calcium, cytoskeletal changes, and redistribution of membrane lipids. The calcium-dependent neutral protease calpain has putative roles in cytoskeletal and membrane changes in other cellular processes; this fact led us to test the role of calpain in a well-known model of apoptotic cell death, that of thymocytes after treatment with dexamethasone. Assays for calcium-dependent proteolysis in thymocyte extracts reveal a rise in activity with a peak at about 1 hr of incubation with dexamethasone, falling to background at approximately 2 hr. Western blots indicate autolytic cleavage of the proenzyme precursor to the calpain I isozyme, providing additional evidence for calpain activation. We have also found that apoptosis in thymocytes, whether induced by dexamethasone or by low-level irradiation, is blocked by specific inhibitors of calpain. Apoptosis of metamyelocytes incubated with cycloheximide is also blocked by calpain inhibitors. These studies suggest a required role for calpain in both “induction” and “release” models of apoptotic cell death. © 1994 wiley-Liss, Inc.     

Regulation of programmed death in erythroid progenitor cells by erythropoietin: effects of calcium and of protein and RNA syntheses.

Erythropoietin (EPO) retards DNA breakdown characteristic of programmed cell death (apoptosis) and promotes survival in erythroid progenitor cells. The mechanism by which EPO inhibits programmed death is unknown. In the well-characterized model of glucocorticoid-treated thymocytes, activation of a Ca2+/Mg(2+)-sensitive endonuclease and new protein and RNA syntheses have been found necessary for apoptosis. We examined the effects of EPO on the free intracellular calcium ion concentration ([Ca2+]i), and the roles of Ca2+ and RNA and protein syntheses on DNA cleavage in erythroid progenitor cells. The murine model of erythroid differentiation using Friend leukemia virus-infected proerythroblasts (FVA cells) was used. EPO did not affect the [Ca2+]i in FVA cells. Decreasing [Ca2+]i by extracellular Ca2+ chelation with EGTA facilitated DNA breakdown. Increasing [Ca2+]i with the calcium ionophore 4-bromo-A23187 increased DNA cleavage; however, DNA fragments generated by high [Ca2+]i were much larger than those seen in the absence of EPO or presence of EGTA. Increased [Ca2+]i also inhibited DNA breakdown to small oligonucleosomal fragments characteristic of cells cultured without EPO. However, no concentration of ionophore protected the high molecular weight DNA as did EPO. Cycloheximide inhibited DNA breakdown in a dose dependent manner in cultures lacking EPO, but two other protein synthesis inhibitors, pactamycin and puromycin, did not prevent DNA breakdown. Inhibition of RNA synthesis with actinomycin D did not prevent DNA breakdown. Cells with morphological characteristics similar to those reported in other cells undergoing programmed death accumulated in EPO-derived cultures. These studies demonstrate that although DNA cleavage and morphological changes are common to apoptotic cells, the roles for Ca2+ and protein and RNA syntheses are not universal and suggest that apoptosis can be regulated by different biochemical mechanisms in different cell types.     

The cellular apoptosis of murine BW5147 thymoma during cold shock]

The mode of T-lymphoma cell death induced by cold shock was studied. The rewarming of cells at 37 degrees C following a brief period of cold (0 degrees C) resulted in internucleosomal DNA fragmentation. The cells underwent cold shock-mediated apoptosis only at a reduced (2%) serum concentration. The apoptosis was not blocked by macromolecular synthesis inhibitors such as cycloheximide and antinomycin D, or by Quin-2. EGTA per se was responsible for the initiation of cell death. Colchicine also induced internucleosomal fragmentation of DNA. Our findings suggest that cold shock induced apoptosis is associated with low temperature mediated disruption of microtubules. The role of Ca2+ and growth factors in cold shock induced cell death is discussed.     

Functional characterization of DNase X, a novel endonuclease expressed in muscle cells

The activation of endonucleases resulting in the degradation of genomic DNA is one of the most characteristic changes in apoptosis. Here, we report the characterization of a novel endonuclease, termed DNase X due to its X-chromosomal localization. The active nuclease is a 35 kDa protein with 39% identity to DNase I. When incubated with isolated nuclei, recombinant DNase X was capable of triggering DNA degradation at internucleosomal sites. Similarly to DNase I, the nuclease activity of DNase X was dependent on Ca(2+) and Mg(2+) and inhibited by Zn(2+) ions or chelators of bivalent cations. Overexpression of DNase X caused internucleosomal DNA degradation and induction of cell death associated with increased caspase activation. Despite the presence of two potential caspase cleavage sites, DNase X was processed neither in vitro nor in vivo by different caspases. Interestingly, after initiation of apoptosis DNase X was translocated from the cytoplasm to the nuclear compartment and aggregated as a detergent-insoluble complex. Abundant expression of DNase X mRNA was detected in heart and skeletal muscle cells, suggesting that DNase X may be involved in apoptotic or other biological events in muscle tissues.     

Farnesylpyridinium, an analog of isoprenoid farnesol, induces apoptosis but suppresses apoptotic body formation in human promyelocytic leukemia cells

1-Farnesylpyridinium (FPy), an analog of isoprenoid farnesol, initially induced morphological changes similar to those of typical apoptosis in human leukemia HL-60 cells but FPy-treated cells were characterized by the absolute absence of final apoptotic events such as fragmentation into apoptotic bodies. FPy-induced cell death was considered to be apoptotic on the basis of the induction of DNA fragmentation and the protection against these events by the coaddition of a pan-caspase inhibitor. The increase in the cytoplasmic cytochrome c level supported the possibility that FPy-treated cells should have the ability to complete the entire apoptotic process ending in cell fragmentation and apoptotic body formation. At concentrations too low to induce apoptosis, FPy could suppress the induction of apoptotic body formation in HL-60 cells by typical inducers of apoptosis such as actinomycin D or anisomycin. FPy exhibited a cytochalasin-like effect on spatial arrangement of actin filament independent of its apoptosis-inducing activity.     

Thapsigargin increases apoptotic cell death in human hepatoma BEL—7404 cells

Effects of thapsigargin,an inhibitor of Ca^2 -ATPase in surface of endoplasmic reticulum,on apoptotic cell death were studied in human hepatoma cells of BEL-7404 cell line by using both flow cytometry and electron microscopy.Propidium iodide staining and flow cytometry revealed that in the serum-free condition,thapsigargin increased the rate of apoptosis of BEL-7404 cells in a dose-dependent manner.Prolongation of the period of serum-free condition enhanced the apoptosis induced by thapsigargin treatment.Morphological observation with electron microscope further demonstrated that chromatin condensation and fragmentation,apoptotic bodies existed in TG-treated cells,supporting that thapsigargin is a potent activator of apoptosis in the cells.     

Rapid turnover of endogenous endonuclease activity in thymocytes: effects of inhibitors of macromolecular synthesis

Previous work has shown that inhibitors of protein or mRNA synthesis block endonuclease activation in thymocytes undergoing programmed cell death. In the present study we used isolated nuclei to investigate the effects of cycloheximide and actinomycin D, inhibitors of protein and mRNA synthesis, respectively, on endogenous endonuclease activity in thymocytes. We observed a rapid loss of Ca2(+)-dependent endonuclease activity in nuclei isolated from thymocytes treated with these inhibitors. In contrast, pretreatment of cells with antipain and leupeptin, inhibitors of proteases, prevented the depletion of endonuclease activity in the nuclei, suggesting that proteolysis was involved. The effects of cycloheximide and actinomycin D were mimicked by incubating thymocytes with treatments known to exert their effects via activation of protein kinase C. Our results suggest that endonuclease activity in thymocyte nuclei undergoes rapid, spontaneous turnover. Agents interfering with macromolecular synthesis may therefore block DNA fragmentation in thymocytes by depleting nuclei of endogenous endonuclease activity.