Recombinant Galectins of <Emphasis Type="Italic">Haemonchus contortus</Emphasis> Parasite Induces Apoptosis in the Peripheral Blood Lymphocytes of Goat

The effect of Haemonchus contortus galectin peptides rHco-gal-m/f to induce apoptosis in the peripheral blood lymphocytes (PBLCs) of goats was investigated. Analysis of apoptosis was carried out with agarose gel electrophoresis, flow cytometry and transmission electron microscopy. The results indicated that there were visible apoptosis bodies and typical DNA ladders by genomic DNA fragmentation. The quantitative analysis of apoptosis by flow cytometry indicated that rHco-gal-m/f peptides induced apoptosis was time and dose dependent. Ultrastructural studies of the PBLCs revealed that a large number of apoptotic cells were present in galectin-treated cells, which had the typical morphologic changes of apoptosis such as reduction of the cytoplasmic volume, loss of cell surface microvilli, chromatin condensation and fragmentation of the apoptotic cells into small apoptotic bodies.     

VK3诱导悬浮培养的胡萝卜细胞凋亡

Menadione (VK3), a quinone that undergoes redox cycles leading to the formation of superoxide radicals, was found to induce cell death in suspension culture of carrot cells. The effect of menadione was in a dose-dependent manner. 100-800 mumol/L menadione caused 10-33 percent cell death. When concentration of menadione reached 1 mmol/L, 100 percent of cell death was observed. DNA cleavage, a hallmark of apoptosis was further studied. DNA ladders were observed in cells treated with 600 and 800 mumol/L menadione but not with lower concentration treatments where only very low percentage of cell death was found. There was no DNA ladders in the cells treated with 1 mmol/L menadion indicating that necrosis may occur. In situ detection of nuclear DNA fragmentation by TUNEL reaction revealed fragmented nuclear DNA in cells treated with 100-800 mumol/L menadion but not in cells treated with 1 mmol/L menadione.     

细胞色素c能诱导植物细胞编程性死亡

以悬浮培养的胡萝卜（ＤａｕｃｕｓｃａｒｏｔａＬ．）与烟草（ＮｉｃｏｔｉａｎａｔａｂａｃｕｍＬ．ｃｖ．ＢＹ２）细胞原生质体为材料,加入一定浓度的细胞色素ｃ和ｄＡＴＰ。不同取样时间的ＤＡＰＩ荧光染色与电镜超薄切片观察的结果显示染色质发生凝集、趋边化,最终形成凋亡小体。核酸电泳显示ＤＮＡ发生特异降解并形成电泳“阶梯”（ＤＮＡｌａｄｄｅｒ）。用末端脱氧核糖核酸转移酶介导的ｄＵＴＰ切口末端标记方法（ＴＵＮＥＬ）检测发现ＤＮＡ的３＇ＯＨ断端被原位特异标记。以上结果说明：细胞色素ｃ能诱导植物细胞发生典型的凋亡。     

Characterization of rat parotid and submandibular acinar cell apoptosis in primary culture

Summary Apoptosis is a highly organized cellular process that is critical for maintaining glandular homeostasis. We have used primary rat salivary acinar cells from the parotid and submandibular glands to investigate the critical regulatory events involved in apoptosis. Caspase-3 activity, cleavage of caspase substrates, and deoxyribonucleic acid (DNA) fragmentation were assayed in cells treated with etoposide, a DNA-damaging agent, or brefeldin A (BFA), a Golgi toxin. Dose-response studies showed that the sensitivity of both cell types to etoposide and BFA was similar, with 150 μM etoposide or 1.5 μM BFA inducing maximal caspace activation. However, BFA induced a more robust activation of caspase and DNA fragmentation in both cell types. Similar results were observed when the caspase cleavage of poly(adenosine 5′-diphosphate ribose) polymerase and protein kinase C delta were analyzed by Western blot. Analysis of the kinetics of apoptosis showed that caspace-3 activation was maximal at 8 h of etoposide or BFA treatment in the parotid cells and at 8–18 h in the submandibular cells. A similar time course was observed when DNA fragmentation was assayed, although maximal DNA fragmentation in BFA-treated cells was two-to threefold higher than that observed in etoposide-treated cells. Despite slight kinetic differences, it would appear that the apoptotic cascade is very similar in both primary parotid and submandibular acinar cells. Although limited in their long-term stability in culture, the use of primary, nonimmortalized salivary acinar cultures will also permit the use of specific transgenic animals to further characterize the molecular events involved in the regulation of salivary gland acinar cell apoptosis.     

Resistance of mitochondrial DNA to degradation characterizes the apoptotic but not the necrotic mode of human leukemia cell death

Cell death can occur by two basically different processes. The original term, necrosis, is now reserved for the generally destructive series of events which include the release of lysosomal enzymes and loss of cell membrane integrity. In contrast, mild treatment with cell damaging agents, or withdrawal of growth factors, may result in a characteristic form of degradation of cellular DNA which is associated with cell death that has morphology known as apoptosis. In this study human leukemia cells were exposed to agents or conditions previously reported to cause necrosis or apoptosis, monitored by detection of DNA “ladders,” and the integrity of cellular DNA was determined on Southern blots. Nuclear DNA was distinguished from mitochondrial DNA by use of probes specific for nuclear genes or for mitochondrial DNA. When HL60, K562, MOLT4, or U937 cells were exposed to conditions which resulted in necrosis, mitochondrial DNA was damaged at approximately the same rate as nuclear DNA, but in apoptosis mtDNA was not degraded. Thus, the ratio of the relative (to untreated cells) abundance of mitochondrial DNA measured by a probe for 16S mitochondrial ribosomal RNA on Southern blots, to the relative abundance of DNA of any nuclear gene, was 1 or less in necrosis, but rose to values greater than 2 in apoptosis. It is concluded that the comparison of the degree of fragmentation of mitochondrial and nuclear DNA provides a quantitative way of distinguishing necrosis from apoptosis.     

APOPTOTIC‐LIKE CELL DEATH PATHWAY IS INDUCED IN UNICELLULAR CHLOROPHYTE CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE) CELLS FOLLOWING UV IRRADIATION: DETECTION AND FUNCTIONAL ANALYSES1

Chlamydomonas reinhardtii (Ehrenberg) cells exhibited cell death process akin to that of apoptosis when exposed to ultraviolet (UV)‐C irradiation (1–100 J/m²). We observed typical hallmarks of apoptosis including cell shrinkage, associated nuclear morphological changes, flipping of phosphatidylserine, and DNA fragmentation detected by the terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling assay and oligonucleosomal DNA laddering assay. Interestingly, fluorescence imaging of DNA changes in UV‐C exposed cells, following PicoGreen staining, revealed that extra‐nuclear DNA disintegrates before that of nuclear changes, where the latter extensively diffuses out of the nuclear compartment, spreading into the whole cell and reaching the periphery of dying cells. Antibodies against a mammalian caspase‐3 shared epitopes with a protein of 28 kDa; whose pattern of expression correlated with the onset of cell death. Moreover, growth experiments indicate that spent medium recovered from UV‐C exposed cells exhibit a protective effect against cell killing of fresh cultures of C. reinhardtii cells by UV irradiation. The protective effect of UV‐spent medium is not a general growth promotional response on normal cells, but rather, is specific to UV‐exposed cells. We propose a model that C. reinhardtii cells exposed to UV elicit apoptotic‐like changes, which in turn lead to an adaptive response in neighboring cells against fresh rounds of UV exposure, thereby promoting survival of the cell population.     

The Apoptotic and Nonapoptotic Nature of the Terminal Differentiation of Erythroid Cells

The morphology of erythroid cells changes dramatically during the course of their terminal differentiation. According to calculations made with cytospin preparations obtained from Syrian hamster yolk-sac-derived erythroid cells, the area of nuclei at day 10 of gestation ranges from 25 to 85 μm²and is reduced to 15–25 μm²on day 13 [K. Morioka and R. Minamikawa-Tachino,Dev. Growth Differ.35, 569–582, 1993]. The DNA and protein contents of each nucleus also decrease during this period. Nonspecific fragmentation of DNA was detected by agarose gel electrophoresis in all samples obtained from day 10 to day 13 of gestation, while distinct ladders of DNA fragments were not detected. DNA fragmentation was also detected by anin situDNA-end labeling (TUNEL) assay. As the terminal differentiation proceeded, gradual decreases in levels of both histone H1 and most nonhistone proteins were observed by SDS–polyacrylamide gel electrophoresis, while levels of core histones appeared to be constant. In particular, lamin B₂was almost completely lost from the nuclear matrix fraction on day 11. These results suggest that the terminal differentiation of erythroid cells and apoptosis might have common mechanisms. However, expansion of the cytoplasm during the terminal differentiation distinguishes these processes. In addition, in the erythroid terminal differentiation, nuclei never form lobules or become fragmented; no apoptotic bodies are formed, occurrence of the apoptosis-like cellular change is not sporadic but rather synchronous, and the process is slow, with at least several days being required for cell death. These characteristics are different from those of typical apoptosis. Thus, the terminal differentiation of nucleated embryonic erythroid cells exhibits both apoptotic and nonapoptotic features.     

斜纹夜蛾核型多角体病毒DNA诱导同源昆虫细胞的凋亡

发现野生型斜纹夜蛾核型多角体病毒（Spodoptera litura nuclear polyhedrosis virus,SpltNPV）DNA转染SL-1细胞能诱导细胞凋亡.SpltNPV-DNA转染其同源细胞系斜纹夜蛾核SL-1细胞6 h后,光镜下即可见细胞膜表面突出或形成小泡,细胞碎裂成凋亡小体,18 h后,细胞100%碎裂成凋亡小体.DAPI荧光染色显示感染细胞核渐呈半月形,直至碎裂被凋亡小体包裹.被转染的SL-1细胞DNA琼脂糖凝胶电泳呈典型梯形谱带.野生型SpltNPV病毒粒子感染的SL-1细胞既无多角体的出现,也无凋亡现象的发生.     

Light-induced and apoptosis-like cell death in the unicellular eukaryote, Blepharisma japonicum

The unicellular eukaryote, Blepharisma japonicum, is a light-sensitive ciliated protozoa. It possesses a photoreceptor pigment called blepharismin that plays critical roles in defensive behavior against predators and step-up photophobic response. In addition, the pigment generates reactive oxygen species such as singlet oxygen and hydroxyl radicals which contribute to photodynamic action. Previous studies reported that intense light (>300 W m⁻²) induced rapid photodynamic killing (necrosis) characterized by cell swelling and plasma efflux, while moderate light (3-30 W m⁻²) only induced pigment extrusion and photooxidation. We have found that moderate light (5 W m⁻²) induced apoptosis-like cell death. Microscopically it was found that >3 h of moderate light irradiation induced macronuclear condensation and plasma efflux without cell swelling. Single cell gel electrophoresis assay showed that DNA fragmentation occurred between 1 and 3 h of irradiation, and the condensed macronuclei contained quite fragmented DNA. Macronuclear DNA extracted from light-irradiated cells contained DNA fragments of 180-200 and 360-400 bp, which were seen as apoptosis ladders.     

Effect of different zinc sources and levels on inhibition of the apoptosis induced by glucocorticoid of thymocytes in vitro

This experiment was conducted to investigate the effects of zinc sulfate and zinc methionine (Zn-Met) and their levels on apoptosis induced by glucocorticoid of thymocytes and the possible mechanism. Dexamethasone was used to make the apoptosis model of thymocytes; zinc sulfate and zinc methionine were supplemented to the medium at levels of 0,50, 100, 500, and 1000 μM. The activity of cells,Cu,Zn superoxide dismutase (Cu,Zn-SOD), DNA ladder pattern, intracellular calcium concentration, and the percentage of apoptosis nuclei were determined. Both ZnSO₄ and Zn-Met could modulate apoptosis; they inhibited apoptosis and decreased DNA fragmentation. The regulation was concentration dependent. At levels of 50 and 100 μM, the effect of Zn-Met on inhibiting apoptosis was less efficient than that of ZnSO₄ (p<0.05), but the activity of the cells cultured with Zn-Met was higher than those cultured with ZnSO₄; they showed no difference in modulating apoptosis when added at levels of 500 and 1000 μM to the medium (p>0.05). Intracellular calcium concentrations of cells cultured with Zn-Met were higher than those cultured with ZnSO₄ at the same levels. Zinc supplementation decreased the concentration of intracellular calcium significantly (p<0.05) and increased the activity of Cu,Zn-SOD in the extract of the cells (p<0.05). Both zinc sulfate and Zn-Met could modulate apoptosis of thymocytes induced by glucocorticoid; the mechanism might involve the exchange of intracellular calcium, the redox of cells, and the two forms of zinc might go different ways in the regulations.     

The nephrotoxin ochratoxin A induces apoptosis in cultured human proximal tubule cells

To test the apoptotic potential of the nephrotoxic mycotoxin ochratoxin A (OTA), we exposed human proximal tubule-derived cells (IHKE cells) for various times to OTA concentrations close to those occurring during dietary exposure (from 2 to 100 nmol/L) and investigated caspase 3 activation, chromatin condensation, and DNA fragmentation. OTA induced a time- and concentration-dependent activation of caspase 3: concentrations as low as 5 nmol/L OTA caused a slight but significant increase in caspase 3 activity after 7 days of OTA exposure. Exposure to 10 nmol/L OTA for 72 or 24 h led to a significantly increased activity of caspase 3 in human proximal tubule-derived cells. Radical scavengers such as N-acetylcysteine had no effect on OTA-induced caspase 3 activation. Chelation of intracellular calcium with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethylester) (BAPTA-AM) also showed no effect. Exposure to 30 nmol/L or more OTA led to DNA fragmentation and chromatin condensation in IHKE cells. Cultured renal epithelial MDCK-C7 and MDCK-C11 or OK cells also showed increased caspase 3 activity after OTA exposure. We conclude that exposure to low OTA concentrations can lead to direct or indirect caspase 3 activation and subsequently to apoptosis in cultured human proximal tubule cells and in other renal epithelial cell lines of different origins. This revised version was published online in July 2006 with corrections to the Cover Date.     

Apoptosis detected in hybrids between Nicotiana glutinosa and N. repanda expressing lethality

Hybrid lethality is one of the mechanisms for reproductive isolation. Apoptotic features were detected in the cells of hybrid seedlings of Nicotiana glutinosa L. ×N. repanda Willd. Condensation of chromatin and fragmentation of nuclei were observed in the leaf protoplasts isolated from hybrid seedlings expressing this lethality. Fragmentation of nuclei was correlated with the progression of lethal symptoms, as confirmed by fluorimetry of the nuclear DNA using laser scanning cytometry. Agarose gel analysis of DNA extracted from hybrid leaves showing lethality revealed a specific ladder pattern suggesting nucleosomal fragmentation associated with nuclear fragmentation. In-situ detection of DNA fragmentation using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) showed that this process occurred in all leaf cells. This is the first evidence that apoptosis can induce suicide of hybrid plants, thus leading to reproductive isolation. Received: 18 June 1999 / Accepted: 20 July 1999     

Apoptosis: A Functional Paradigm for Programmed Plant Cell Death Induced by a Host-Selective Phytotoxin and Invoked during Development

The host-selective AAL toxins secreted by Alternaria alternata f sp lycopersici are primary chemical determinants in the Alternaria stem canker disease of tomato. The AAL toxins are members of a new class of sphinganine analog mycotoxins that cause cell death in both animals and plants. Here, we report detection of stereotypic hallmarks of apoptosis during cell death induced by these toxins in tomato. DNA ladders were observed during cell death in toxin-treated tomato protoplasts and leaflets. The intensity of the DNA ladders was enhanced by Ca2+ and inhibited by Zn2+. The progressive delineation of fragmented DNA into distinct bodies, coincident with the appearance of DNA ladders, also was observed during death of toxin-treated tomato protoplasts. In situ analysis of cells dying during development in both onion root caps and tomato leaf tracheary elements revealed DNA fragmentation localized to the dying cells as well as the additional formation of apoptotic-like bodies in sloughing root cap cells. We conclude that the fundamental elements of apoptosis, as characterized in animals, are conserved in plants. The apoptotic process may be expressed during some developmental transitions and is the functional process by which symptomatic lesions are formed in the Alternaria stem canker disease of tomato. Sphinganine analog mycotoxins may be used to characterize further signaling pathways leading to apoptosis in plants.     

Mitochondrion-mediated apoptosis induced by Rana grylio virus infection in fish cells

A fish cell line, fathead minnow (FHM) cell, was used to investigate the alteration of mitochondrial dynamics and the mechanism of apoptosis under Rana grylio virus (RGV) infection. Microscopy observations, flow-cytometry analysis and molecular marker detection revealed the apoptotic fate of the RGV-infected cells. Some typical apoptotic characteristics, such as chromatin condensation, DNA fragmentation and mitochondrial fragmentation, were observed, and significantly morphological changes of mitochondria, including size, shape, internal structure and distribution, were revealed. The mitochondria in RGV-infected cells were aggregated around the viromatrix, and the aggregation could be blocked by colchicine. Moreover, the Δψm collapse was induced, and caspase-9 and caspase-3 were activated in the RGV-infected cells. In addition, NF-κB activation and intracellular Ca²⁺ increase were also detected at different times after infection. The data revealed the detailed dynamics of mitochondrion-mediated apoptosis induced by an iridovirus, and provided the first report on mitochondrial fragmentation during virus-induced apoptosis in fish cells.     

A DEVD-Inhibited Caspase Other than CPP32 Is Involved in the Commitment of Cerebellar Granule Neurons to Apoptosis Induced by K+ Deprivation

Abstract: Cultured cerebellar granule neurons undergo apoptosis when switched from a medium containing depolarizing levels of K⁺ (25 mM KCI) to medium containing lower levels of K⁺ (5 mM KCI). We used this paradigm to investigate the role of caspases in the death process. Two broad-spectrum caspase inhibitors, tert-butoxycarbonyl-Asp·(O-methyl)·fluoromethyl ketone and benzyloxycarbonyl-Val-Ala-Asp·fluoromethyl ketone, significantly reduced cell death (90 and 60%, respectively) at relatively low concentrations (10–25 µM), suggesting that caspase activation is involved in the apoptotic process. DNA fragmentation, a hallmark of apoptosis, was also reduced by these caspase inhibitors, suggesting that caspase activation occurred upstream of DNA cleavage in the sequence of events leading to cell death. As a step toward identifying the caspase(s) involved, the effects of N-acetyl Tyr-Val-Ala-Asp·chloromethyl ketone (YVAD·cmk), an interleukin-1β converting enzyme-preferring inhibitor, and N-acetyl Asp-Glu-Val-Asp·fluoromethyl ketone (DEVD·fmk), a CPP32-preferring inhibitor, were also evaluated. YVAD·cmk provided only modest (<20%) protection and only at the highest concentration (100 µM) tested, suggesting that interleukin-1β converting enzyme and/or closely related caspases were not involved. In comparison, DEVD·fmk inhibited cell death by up to 50%. Western blot analyses, however, failed to detect an increase in processing/activation of CPP32 or in the proteolysis of a CPP32 substrate, poly(ADP-ribose) polymerase, during the induction of apoptosis in granule neurons. Similarly, the levels of Nedd2, a caspase that is highly expressed in the brain and that is partially inhibited by DEVD·fmk, also remained unaffected in apoptotic neurons undergoing apoptosis. These results suggest that a DEVD-sensitive caspase other than CPP32 or Nedd2 mediates the induction of apoptosis in K⁺-deprived granule neurons.     

Biochemical, ultrastructural and molecular characterization of the triphenyltin acetate (TPTA)-induced apoptosis in primary cultures of mouse thymocytes

Triphenyltin acetate (TPTA), a triorganotin compound used in agriculture as a biocide, is immunotoxic in vivo and in vitro. The present study was undertaken to ascertain whether apoptosis might play a role in the TPTA toxicity in vitro. Mouse thymocyte primary cultures were exposed to 0, 4 and 8 μmol/L TPTA; methyl prednisolone (1 μmol/L) was used as a positive control. Cell aliquots were harvested after 0, 1, 2, 4, and 8 h and the presence of early or late apoptotic phenomena was checked by (a) morphological investigations; (b) spectrophotometric quantification of fragmented DNA and agarose gel electrophoresis; (c) cell flow cytofluorometry, using an annexin V-FITC kit; and (d) detection of in situ apoptosis by a colorimetric detection kit (Titer-Tacs). TPTA cytotoxicity was also evaluated using the trypan blue dye exclusion test. Morphological investigation indicated apoptosis and/or necrosis. After 8 h of incubation, cells exposed to 4 μmol/L TPTA showed an increase in DNA fragmentation (on electrophoresis), which was confirmed by spectrophotometry (p < 0.05). Flow cytofluorometry pointed out an early (p < 0.05) increase of annexin V-positive (apoptotic) cells in TPTA-exposed flasks, whereas at least partly contradictory, results were obtained with the Titer-Tacs kit. Overall, these results provide evidence that TPTA, at low concentrations (4 μmol/L) induces early and late apoptotic phenomena, whereas cells exposed to the highest concentrations (8 μmol/L) are likely to undergo necrosis rather than apoptosis.     

The effect of melittin on proliferation and death of thymocytes

The effect of melittin, an activator of phospholipase A₂, on proliferation and death of rat thymocytes in a broad concentration range was studied. Cell proliferation was estimated by the accumulation of colchicin metaphases, necrotic death was determined from lysis and staining of cells with trypan blue, and apoptosis was assessed from the type of DNA fragmentation, the amount of fragmented DNA, and the percentage of cells with subdiploid DNA. It was shown that low melittin concentrations (below 5 μg/ml) stimulate thymocyte proliferation. At high melittin concentrations, thymocytes die by the primary necrosis type. Throughout the concentration range studied, melittin does not produce apoptosis in thymocytes. Conversely, high melittin concentrations even inhibit thymocyte apoptosis in the control and after irradiation. An inhibitor of RNA synthesis actinomycin D does not affect thymocyte death in the presence of melittin. It is concluded that the activation of phospholipase A₂ can induce necrosis but not apoptosis and thus is not a necessary step in the signaling cascade that initiates apoptosis in thymocytes.     

Is Dopamine-Induced Inhibition of Adenylate Cyclase Involved in the Autoreceptor-Mediated Negative Control of Tyrosine Hydroxylase in Striatal Dopaminergic Terminals?

Abstract The mechanism of the negative control of tyrosine hydroxylase (TH) activity induced by the stimulation of presynaptic 3,4-dihydroxyphenylethylamine (dopamine, DA) autoreceptors was investigated using rat striatal slices and synaptosomes incubated under control ([K⁺] = 4.8 mM) or depolarizing ([K⁺] = 60 mM) conditions. The stimulation of DA autoreceptors by 7-hydroxy-2-(di-n-propylamino) tetralin (1 μM 7-OH-DPAT) produced a significant decrease in TH activity extracted from striatal slices maintained under control conditions. This effect was associated with the complete conversion of TH into an enzyme form with a low affinity for its pterin cofactor (K_m～0.80 mM). Furthermore, compared to TH extracted from control tissues, that from 7-OH-DPAT-exposed striatal slices was more sensitive to the stimulatdry effects of exogenous heparin and cyclic AMP-dependent phosphorylation. Such changes were opposite to those induced by incubating striatal slices with the adenylate cyclase activator forskolin. Indeed, forskolin treatment completely converted TH into an enzyme form with a high affinity for its pterin cofactor (K_m～0.16 mM). Such conversion was associated with a shift in the optimal pH for TH activity from 5.8 (control) to 7.2 (forskolin). Under depolarizing conditions, the blockade by (—)-sulpiride of the stimulation of DA autoreceptors by endogenous DA was associated with a marked activation of TH. Modifications of enzymatic characteristics triggered by (—)-sulpiride were then similar to those induced by forskolin treatment. These data suggest that presynaptic DA autoreceptors modulate the activity of TH by controlling the degree of cyclic AMP-dependent phosphorylation of the enzyme. The blockade by Pertussis toxin of the 7-OH-DPAT-induced inhibition of TH activity is coherent with a possible negative coupling of presynaptic DA autoreceptors (closely related to the D₂ type) with adenylate cyclase. Such negative coupling would account for the reduction of TH activity when presynaptic DA autoreceptors are stimulated.     

Apoptotic Cell Death and Cellular Surface Negative Charge Increase in Maize Roots Exposed to Cytotoxic Stresses

Maize root meristematic tissues were exposed to cytotoxic reagents,the RNA-synthesis inhibitor Actinomycin D (ActD), the protein-synthesisinhibitor cycloheximide (CHX) and the mitosis inhibitor colchicine(COL). Morphological and biochemical evidence of specific apoptoticnuclei and chromosomes in individual treated cells was identifiedusing a simple and highly efficient chromosome spreading-basedTUNEL assay, DNA laddering and DNA gel blotting. All of thesedrugs induced DNA cleavage, dose-dependent oligomeric ladders,and characteristic nuclear and chromosomal condensations. Resultsfrom DNA gel blotting showed that DNA ladders could be inducedby exposure to 0.1 mg l^-1ActD, 100 mg l^-1CHX and 500 mg l^-1COLfor 6 h, 6 h and 12 h respectively. The sequence of changesin single cells was studied in detail. DNA cleavage was foundto occur before condensation and disorganization of the nucleus,followed by deformation and condensation of metaphase chromosomes,and marginalization of chromatin. Finally, nucleoli disappearedand fragmentation of the nucleus occurred. Meanwhile, changesin the outer surface charge of apoptotic cells were assessedby electrophoresis. Results indicated quantitatively that thesurface negative charge increased during these apoptotic processes.Our results also showed that the apoptotic pathway induced byeach of these drugs could be reversed before serious cleavageof DNA into oligonucleosomal fragments and universal chromatincondensation. Copyright 2001 Annals of Botany Company Cytotoxin, chromosome spreading, apoptosis, cell electrophoresis     

A Novel Protein Expressed in Mammalian Cells Undergoing Apoptosis

Human and rodent cells undergoing apoptosis were observed to express high levels of a novel 45,000 M_r protein. The protein, which we have termed apoptosis specific protein (ASP), was found in Burkitt lymphoma (BL) cells and in adenovirus-transformed human and rat embryo cells induced into apoptosis by a variety of stimuli, including serum deprivation, exposure to the Ca²⁺ ionophore, ionomycin, treatment with inhibitors of macromolecular synthesis (cycloheximide and actinomycin D), and cold shock. In BL cells treated with apoptotic stimuli, expression of the oncoprotein Bcl-2 was found to both protect from apoptosis and prevent expression of ASP. ASP was not detected either in viable cells or in cells dying passively by necrosis. Laser scanning confocal microscopy showed high levels of ASP in the cytoplasm of cells displaying the chromatin condensation and fragmentation patterns typical of apoptosis. Retention of ASP was observed even when DNA was no longer detectable, and two-color immunofluorescence staining indicated that the protein primarily colocalized with, but was clearly distinct from, nonmuscle actin. These findings, together with the observation that biochemical extraction of ASP was only possible under conditions which caused solubilization of the cytoskeleton, lead us to conclude that ASP forms part of, or at least strongly associates with, a modified cytoskeleton unique to cells undergoing apoptosis. While elucidation of its function will require further work, ASP constitutes a powerful marker for the diagnosis and quantitation of apoptosis in vivo and in vitro.