Analysis of nuclear apoptotic process in a cell-free system

We report an analysis of the apoptotic process of mouse liver nuclei induced in a cell-free carrot cytosol system by cytochrome c. Typical characteristics of apoptosis were observed, such as chromatin condensation, margination, apoptotic bodies and DNA ladders. Furthermore, transmission and scanning electron microscope analysis of the apoptotic nuclei detected chromatin-free nuclear vesicles before apoptotic bodies appeared at a comparatively late phase. When AC-YVAD-CHO, an inhibitor of caspase 6, was introduced into the system, these vesicles and apoptotic bodies disappeared completely within our study sections. We confirmed the results using whole-mount electron microscopy, and found that although the nuclear lamina was destroyed early, the nuclear matrix largely remained intact during the course of apoptosis. The nuclear matrix played an important role in maintaining the integrity of apoptotic cells and connecting the apoptotic bodies and apoptotic nucleus. Received 29 September 2000; revised 10 December 2000; accepted 13 December 2000     

Chromosomal territory segmentation in apoptotic cells

The nuclear architecture of selected chromosomes in apoptotic nuclei of human leukemic cells K-562 and HL-60 was investigated. Etoposide and prolonged confluence were used for the induction of apoptosis. DAPI as well as TUNEL labeling of apoptotic nuclear bodies was combined with visualization of chromosomal territories by the FISH technique. Simultaneous vital staining by annexin V, propidium iodide, and Hoechst 33342 was applied to distinguish apoptotic, necrotic, and intact cell fraction of tested populations. Our FISH analyses revealed that the three-dimensional (3D) structure of apoptotic nuclei as well as the 3D structure of apoptotic bodies is preserved in formaldehyde-fixed cells. High-molecular-weight DNA fragmentation was determined in apoptotic K-562 cells in contrast to oligonucleosomal cleavage observed in apoptotic HL-60 cells. In K-562 populations, chromosomal territories were located separately either in one apoptotic body or underwent disassembly into chromosomal segments dispersed into single and/or several apoptotic bodies. The apoptotic disorganization of chromosomal territories was irregular, leading mainly to chromosomal segments of different sizes and, consequently, chromosomal disassembly was not observed at specific sites. In comparison with the control, an increased number of centromeric FISH signals were observed in prolonged confluence-treated K-562 cells induced to apoptosis. This finding can be explained either as a consequence of apoptosis or by polyploidization. Sequential staining of the same apoptotic nuclei by the FISH and TUNEL techniques revealed that chromosomal territory segmentation precedes the formation of nuclear apoptotic bodies.     

Induction of vanadium accumulation and nuclear sequestration causing cell suicide in human Chang liver cells

Very little is known about the modulation of vanadium accumulation in cells, although this ultratrace element has long been seen as an essential nutrient in lower life forms, but not necessarily in humans where factors modulating cellular uptake of vanadium seem unclear. Using nuclear microscopy, which is capable of the direct evaluation of free and bound (total) elemental concentrations of single cells we show here that an NH₄Cl acidification prepulse causes distinctive accumulation of vanadium (free and bound) in human Chang liver cells, concentrating particularly in the nucleus. Vanadium loaded with acidification but leaked away with realkalinization, suggests proton-dependent loading. Vanadyl(4), the oxidative state of intracellular vanadium ions, is known to be a potent source of hydroxyl free radicals (OH^.). The high oxidative state of nuclei after induction of vanadyl(4) loading was shown by the redox indicator methylene blue, suggesting direct oxidative damage to nuclear DNA. Flow cytometric evaluation of cell cycle phase-specific DNA composition showed degradation of both 2N and 4N DNA phases in G₁, S and G₂/M cell cycle profiles to a solitary 1N DNA peak, in a dose-dependent manner, effective from micromolar vanadyl(4) levels. This trend was reproduced with microccocal nuclease digestion in a time response, supporting the notion of DNA fragmentation effects. Several other approaches confirmed fragmentation occurring in virtually all cells after 4 mM V(4) loading. Ultrastructural profiles showed various stages of autophagic autodigestion and well defined plasma membrane outlines, consistent with programmed cell death but not with necrotic cell death. Direct intranuclear oxidative damage seemed associated with the induction of mass suicide in these human Chang liver cells following vanadium loading and nuclear sequestration.     

Effects of araC and aphidicolin on DNA chain elongation rate in HeLa S3 cells

Summary The effects of araC and aphidicolin on DNA chain elongation rate were tested. The rate was markedly reduced at low concentrations. Total DNA synthesis was more inhibited, indicating a role of DNA polymerasea in replicon initiation.     

Effects of araC and aphidicolin on DNA chain elongation rate in HeLa S3 cells

The effects of araC and aphidicolin on DNA chain elongation rate were tested. The rate was markedly reduced at low concentrations. Total DNA synthesis was more inhibited, indicating a role of DNA polymerase a in replicon initiation.     

Poliovirus production in HeLa cells in presence of colchicine

Zusammenfassung Die Vermehrung des Poliovirus in HeLa Zellen war in Gegenwart oder Abwesenheit starker Colchicindosengleich hoch. Die biochemischen Prozesse bei der Spindelbildung sind scheinbar unabhängig von denen der Biosynthese eines RNA-Virus.     

Chromosomal accumulation of3H-estradiol in dividing ovarial granulosa cells and ovarial squash preparations

Zusammenfassung ³H-Oestradiol wurde Mäusen injiziert und nachher autoradiographisch verfolgt, wobei Ag-Körnchen über den Chromosomen der Granulosazellen des Ovariums festgestellt werden konnten.     

Calcium and magnesium deficiency-induced atrophy of muscle and calcium accumulation in the spinal cord

After severe dietary calcium-magnesium deficiency in rats, succinic dehydrogenase and acetylcholinesterase enzyme activity of gastrocnemius muscle showed a neurogenic atrophy. This alteration was associated with a high concentration of calcium in the spinal cord.     

Influence of ionizing radiation on protein production by HeLa cells in culture

Zusammenfassung Bei der Züchtung von HeLa-Krebszellen in tritiumhaltiger Nährlösung während 48 h wurde festgestellt, dass die Mitoserate durch viel kleinere Strahlendosen nachweisbar herabgesetzt wird als die radiochemisch gemessene Rate der Proteinsynthese.     

Mitochondria and calpains mediate caspase-dependent apoptosis induced by doxycycline in HeLa cells

Doxycycline (Dc) has been demonstrated to inhibit cell growth and induce apoptosis in tumor cells, although its mechanism of action is not fully understood. The present study demonstrates that apoptosis can be induced in HeLa cells. Western blot data demonstrated that cytochrome c (Cyt c), Smac (the second mitochondria-derived activator of caspase), calpain I, caspase-9, −3 and −8 were involved in the apoptotic process, while the pan caspase inhibitor zVAD-fmk almost completely inhibited Dc-induced apoptosis. We further demonstrated that the release of mitochondrial proteins and the activation of calpains occurred upstream of the caspase cascade, in which caspase-9 was activated in response to the release of Cyt c, that caspase-8 activation was caspase and calpain dependent, and that caspase-3 was activated mainly by caspase-8 and −9. Caspase-8 played important roles in the activation of caspase-3 and induction of apoptosis, whereas the role of the caspase-9 was limited. Received 26 November 2005; received after revision 14 February 2006; accepted 1 March 2006     

Distribution of vitamin D3: Evidence of accumulation in renal proximal tubuli and thyroid parafollicular cells

Zusammenfassung Mit Hilfe der Ganzkörper-Autoradiographietechnik konnte nach Zufuhr von Vitamin D₃-4-¹⁴C an Mäusen der Nachweis einer radioaktiven Ansammlung in gewissen Gebieten (Labyrinth) der Nierenrinde sowie der Thyroidea erbracht werden, die möglicherweise mit parafollikulären Zellen identisch ist.