Differences in patterns of cell death between ray parenchyma cells and ray tracheids in the conifers <Emphasis Type="Italic">Pinus densiflora</Emphasis> and <Emphasis Type="Italic">Pinus rigida</Emphasis>

Differences in patterns of cell death between ray parenchyma cells and ray tracheids in the conifers Pinus densiflora and Pinus rigida were clarified. Differentiation and cell death of ray tracheids occurred successively and both were related to the distance from the cambium. In this respect, they resembled those of longitudinal tracheids. Thus, the cell death of short-lived ray tracheids could be characterized as time-dependent programmed cell death. In contrast, ray parenchyma cells survived for several years or more, and no successive cell death occurred, even within a single radial line of cells in a ray. Thus, the features of death of the ray parenchyma cells were different from those of ray tracheids. Cell death occurred early in ray parenchyma cells that were in contact with ray tracheids. The initiation of secondary wall thickening occurred earlier in ray parenchyma cells that were in contact with ray tracheids in Pinus densiflora than in others. In addition, localized thickening of secondary walls occurred only in ray parenchyma cells that were in contact with ray tracheids in Pinus rigida. Moreover, no polyphenols were evident in such cells in either species. Therefore, ray parenchyma cells that were in contact with ray tracheids appeared not to play a role in the formation of heartwood extractives. Our observations indicate that short-lived ray tracheids might affect the pattern of differentiation and, thus, the functions of neighboring long-lived ray parenchyma cells in conifers.     

UV-B Irradiated Cell Lines Execute Programmed Cell Death in Various Forms

We investigated changes typical for apoptosis in various cell lines after UV-B irradiation. Using established methods for detection of apoptosis we demonstrate changes of cellular morphology, phosphatidylserine (PS) exposure, ollgonucleosomal DNA fragmentation and generation of hypochrome nuclei. To isolated high-molecular-weight (hmwt) DNA fragments we engaged a new method avoiding pulse field gel electrophoresis. Most UV-B irradiated cell lines showed oligonucleosomal DNA fragmentation, hypochrome nuclei, morphological changes, annexin-V binding and positive TUNEL reaction. However, no oligonucleosomal DNA fragmentation could be detected in Raji and HaCaT cells. Whereas HaCaT cells displayed all other changes typical for apoptosis, Raji cells were TUNEL negative, formed low amounts of hmwt DNA and showed an 'atypically' low hypochrome shift. Nevertheless, UV-B irradiated Raji cells excluded propidium iodide (PI), bound annexin-V and stopped proliferation. This suggests that Raji cells underwent growth arrest with exposure of PS being the only feature of apoptosis. However, in the presence of phagocytes expressing the phosphatidylserine receptor these cells would share the removal pathway with apoptotic cells. Since UV-B induced programmed cell death differs in dependence of cells under investigation, the failure to detect oligonucleosomal DNA fragmentation or chromatin condensation is not suitable to exclude programmed (apoptotic?) cell death.     

拟南芥根毛衰老死亡过程的PCD检测

根毛是植物体吸收养分的重要器官, 自然条件下根毛的寿命很短, 仅能存活2–3周, 随即脱落死亡。以模式植物拟南芥(Arabidopsis thaliana)根毛为材料, 对根毛死亡的细胞学特征进行了报道。结果发现, 根毛衰老死亡后细胞内的原生质体发生了收缩, 并在胞质中观察到凝集物的出现; 通过原位末端标记(TUNEL)检测, 发现幼根上的根毛细胞核DNA发生了片段化。上述结果表明, 拟南芥根毛的衰老死亡很可能是植物体自主调控的程序性细胞死亡(PCD)。另外, 当根毛衰老死亡后,细胞核大多会迁移到靠近根毛基部的位置, 且正常的长管状根毛发生旋转扭曲。     

DNA Digestion and Chromatin Condensation during Nuclear Death inTetrahymena

DNA fragmentation and nuclear condensation are key features in the regulated cell death of higher animal cells. Nuclear death also occurs as part of a developmentally programmed process during the sexual life cycle of the unicellular organismTetrahymena.We examined the regulation of nuclear death and the relationship between DNA fragmentation and chromatin condensation in this model system. Nuclear death is accompanied by DNA digestion to low-molecular-weight oligonucleosomal-length fragments, in agree- ment with a previous study [17], indicating an endonuclease-like activity typical of apoptosis in higher organisms. Actinomycin D and cycloheximide block DNA digestion as well as nuclear condensation suggesting that nuclear death is under genetic regulation. DNA digestion is completely blocked by aurin, a general nuclease inhibitor. In addition, when DNA fragmentation is blocked, nuclear condensation also fails to occur. Moreover, a kinetic analysis of DNA breakdown, using agarose gels, shows that some DNA digestion occurs before nuclear condensation has taken place. Thus the initiation of DNA digestion may provide conditions necessary for nuclear condensation. Temporary inhibition of nuclear death aborts the death program since after removal of inhibitors cells revert to a vegetative pathway without having eliminated the old or developed the new macronucleus. Zn²⁺and EGTA, both of which inhibit apoptosis in some cell types, fail to prevent nuclear condensation or DNA digestion inTetrahymena,suggesting a requirement here for an endonuclease which is Ca²⁺-independent and Zn²⁺-insensitive. With the TUNEL assay, DNA breakdown is detected exclusively in the condensed macronucleus (and occasional micronuclei identified as degenerating haploid products of meiosis), but not in precondensed macronuclei. These studies show that apoptotic-like DNA fragmentation occurs after condensation of the degenerating macronucleus. However, early DNA digestion may be critical for nuclear condensation and subsequent degeneration.     

Time-course of programmed cell death during leaf senescence in <Emphasis Type="Italic">Eucommia ulmoides</Emphasis>

Leaves of Eucommia ulmoides Oliv. harvested between April to November were examined for programmed cell death (PCD) during growth and senescence. Leaves developed in April, becoming fully expanded in late May, remaining unchanged until November when they started to dehisce. Falling leaves retained a green color. Our results showed that (1) mesophyll cells gradually reduced their nuclei from September to November, (2) positive TUNEL signals appeared on the nuclei from August, (3) ladder-like DNA fragmentation occurred in September and October, and (4) a 20-kDa Ca²⁺-dependent DNase appeared in these same months. In fallen leaves, intact mesophyll cell nuclei could not be detected, but a few cells around the vascular bundle had nuclei. Therefore, (1) programmed cell death (PCD) of leaf cells occurred in the leaves of E. ulmoides, (2) the progress of mesophyll cell PCD lasted for more than 2 months, and (3) PCD of leaf cells was asynchronous in natural senescing leaves. Electronic Publication     

A Bacillus thuringiensis delta-endotoxin induces programmed cell death in mosquito larvae

We present evidence that a delta-endotoxin isolated from Bacillus thuringiensis subsp.israelensis induces programmed cell death in polytene midgut cells of Culex pipiens larvae. After exposure to toxin, polytene nuclei in the anterior region of the larval midgut undergo many of the morphological and physiological changes which are characteristic of apoptosis, including the ability to stain with the vital dye, acridine orange, and fragmentation of nuclear DNA as demonstrated by agarose gel electrophoresis and in situ TUNEL labeling. The temporal sequence of toxin ingestion, acridine orange staining and larval death suggests a cause and effect relationship between programmed cell death and larval death. Amino sugars that interfere with toxicity also interfere with the time course of acridine orange staining of larval polytene nuclei. The toxin first causes programmed cell death of anterior midgut and gastric caeca cells and, subsequently, posterior midgut cells. This pattern is similar to the temporal sequence of larval polytene cell death that occurs during metamorphosis. From the size and distribution of the nuclei that are stained with acridine orange, it appears that only polytene midgut cells are affected by toxin and that the diploid regenerative cell are not affected.     

Apoptotic and non-apoptotic cell death in hormone-dependent glands

The proliferation of cells and cell death are involved in the maintenance of appropriate tissue homeostasis. In the present study, two different mechanisms of cell death were identified in the prostate and pituitary glands when morphological data, fragmentation of DNA, and TUNEL labelling of apoptotic nuclei were compared. Typical cell death by apoptosis was identified by morphological and molecular approaches in the prostate after orchidectomy. By contrast, neither DNA fragmentation nor TUNEL labelling were found in dead cells occurring in the pituitary gland after interruption of lactation. Regressing lactotrophs were characterised by condensation and disruption of the cytoplasmic matrix, but preserved intact nuclei until advanced stages of regression. Degenerating “dark” cells comparable to those described in the pituitary were also seen coexisting with typical apoptosis in the prostate epithelial lining of orchidectomised rats. Both forms of cell death could be clearly differentiated, because dark cells suffer severe alterations of cytoplasmic organelles while maintaining the integrity of the nucleus. In contrast, apoptotic cells present well-preserved cytoplasmic organelles, but grossly disrupted nuclei with fragmentation and condensation of chromatin.     

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     

Structural analysis of K<Superscript>+</Superscript> dependence in <Emphasis Type="SmallCaps">l</Emphasis>-asparaginases from <Emphasis Type="Italic">Lotus japonicus</Emphasis>

Wood is composed of various types of cells and each type of cell has different structural and functional properties. However, the temporal and spatial diversities of cell wall components in the cell wall between different cell types are rarely understood. To extend our understanding of distributional diversities of cell wall components among cells, we investigated the immunolabeling of mannans (O-acetyl-galactoglucomannans, GGMs) and xylans (arabino-4-O-methylglucuronoxylans, AGXs) in ray cells and pits. The labeling of GGMs and AGXs was temporally different in ray cells. GGM labeling began to be detected in ray cells at early stages of S₁ formation in tracheids, whereas AGX labeling began to be detected in ray cells at the S₂ formation stage in tracheids. The occurrence of GGM and AGX labeling in ray cells was also temporally different from that of tracheids. AGX labeling began to be detected much later in ray cells than in tracheids. GGM labeling also began to be detected in ray cells either slightly earlier or later than in tracheids. In pits, GGM labeling was detected in bordered and cross-field pit membranes at early stages of pit formation, but not observed in mature pits, indicating that enzymes capable of GGM degradation may be involved in pit membrane formation. In contrast to GGMs, AGXs were not detected in pit membranes during the entire developmental process of bordered and cross-field pits. AGXs showed structural and depositional variations in pit borders depending on the developmental stage of bordered and cross-field pits.     

AMPA-Induced Excitotoxicity Increases Nuclear Levels of CAD, Endonuclease G, and Acinus and Induces Chromatin Condensation in Rat Hippocampal Pyramidal Neurons

Programmed cell death has been linked to AMPA-receptor-mediated excitotoxicity in pyramidal neurons of the hippocampus. The intent of this study was to investigate the roles of caspase-dependent and independent nuclear death-related factors in mediating AMPA-induced nuclear changes in PyNs by use of immunohistochemistry and transmission electron microscopy (TEM). Data indicate increases in the nuclear levels of caspase-activated acinus and DNase and Endonuclease G (a caspase-independent endonuclease) in CA1 and CA3 PyN nuclei with different temporal patterns following an AMPA-insult. Hoechst staining and TEM confirm AMPA-induced chromatin condensation. The presence of active acinus in nuclei suggests it mediates chromatin condensation. Interestingly, a DNA fragmentation labeling protocol showed that there was no chromatin cleavage up to 90 min after AMPA-insult. Overall, we conclude that: 1) AMPA-induced excitotoxicity increases nuclear immunoreactivity of pro-death enzymes from multiple programmed cell death pathways, 2) differential chromatin condensation patterns occur between CA1 and CA3, and 3) there is no chromatin cleavage within our experimental timeframe. Abbreviations: AIF, apoptosis inducing factor; AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; CAD, caspase-activated DNase; CIP, calf intestinal alkaline phosphatase; EndoG, endonuclease G; ICAD, inhibitor of CAD; NMDA, N-methyl D-aspartate; TdT, terminal deoxynucleotidyl transferase; TEM, transmission electron microscopy; TUNEL, terminal deoxynucleotidyl transferase biotin-UTP nick end labeling     

Apoptotic cell death induces temperature-sensitive lethality in hybrid seedlings and calli derived from the cross of Nicotiana suaveolens×N. tabacum

Hybrid lethality expressed in the interspecific hybrid of Nicotiana suaveolens Lehm. ×N. tabacum L. cv. Hicks-2 is one of the mechanisms for reproductive isolation and it is temperature-sensitive. Apoptotic changes were detected in the cells of hybrid seedlings and calli expressing lethality at 28 °C but not under high-temperature conditions (36 °C), when the lethality is suppressed. Condensation of chromatin, fragmentation of nuclei and cytoplasmic reduction are the cytological changes associated with apoptosis leading to hybrid lethality. Fragmentation of nuclei was correlated with the lethal symptoms in both hybrid seedlings and calli, as confirmed by fluorimetry of the nuclear DNA using laser scanning cytometry. Agarose gel analysis of DNA extracted from hybrid seedlings and calli showing lethal symptoms revealed a specific ladder pattern suggesting nucleosomal fragmentation which is one of the biochemical changes of apoptosis. In-situ detection using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) showed that this process occurred in distinct stages on each organ of hybrid seedlings and centripetally in hybrid calli. From these results, we confirmed that cell death inducing hybrid lethality was indeed apoptosis. Received: 23 December 1999 / Accepted: 5 April 2000     

Starvation induces apoptosis in the midgut nidi of <Emphasis Type="Italic">Periplaneta americana</Emphasis>: a histochemical and ultrastructural study

The effects of starvation on cell death in the midgut of Periplaneta americana were studied histochemically and ultrastructurally. TUNEL assays showed that cell death began to increase in the columnar cells and nidi, the nests of stem cells and newborn cells from 2 weeks of starvation. A significant increase in cell death occurred in the nidi after 4 weeks of starvation. Cockroaches starved for 4 weeks showed active-caspase-3-like immuno-reactivity both in the columnar cells and nidi, whereas control cockroaches that were fed for 4 weeks showed this reactivity only in the apical cytoplasm of columnar cells. Electron microscopy revealed no chromatin condensation in the nucleus of columnar cells of cockroaches, whether fed or starved for 4 weeks. Starved cockroaches exhibited many small vacuoles in the cytoplasm of some columnar cells and “floating” organelles including nuclei in the lumen. A 4-week starvation induced the appearance of cytoplasmic fragmentation and secondary lysosomes in the nidi. Each fragment contained nuclear derivatives with condensed chromatin, i.e. apoptotic bodies. Mitotic cells were found in some, but not all nidi, even within the same starved sample. Fragmentation was not observed in the nidi of control cockroaches. Thus, starvation increases cell death not only in the columnar cells, but also in the nidi. The cell death in the nidi is presumably apoptosis executed by caspase 3.     

Leaf‐shape remodeling: programmed cell death in fistular leaves of Allium fistulosum

Some species of Allium in Liliaceae have fistular leaves. The fistular lamina of Allium fistulosum undergoes a process from solid to hollow during development. The aims were to reveal the process of fistular leaf formation involved in programmed cell death (PCD) and to compare the cytological events in the execution of cell death to those in the unusual leaf perforations or plant aerenchyma formation. In this study, light and transmission electron microscopy were used to characterize the development of fistular leaves and cytological events. Terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling (TUNEL) assays and gel electrophoresis were used to determine nuclear DNA cleavage during the PCD. The cavity arises in the leaf blade by degradation of specialized cells, the designated pre‐cavity cells, in the center of the leaves. Nuclei of cells within the pre‐cavity site become TUNEL‐positive, indicating that DNA cleavage is an early event. Gel electrophoresis revealed that DNA internucleosomal cleavage occurred resulting in a characteristic DNA ladder. Ultrastructural analysis of cells at the different stages showed disrupted vacuoles, misshapen nuclei with condensed chromatin, degraded cytoplasm and organelles and emergence of secondary vacuoles. The cell walls degraded last, and residue of degraded cell walls aggregated together. These results revealed that PCD plays a critical role in the development of A. fistulosum fistular leaves. The continuous cavity in A. fistulosum leaves resemble the aerenchyma in the pith of some gramineous plants to improve gas exchange.     

Ultrastructural identification of apoptotic nuclei using the TUNEL technique

Summary We describe an ultrastructural adaptation of the method of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) for the identification of DNA fragmentation. Thin sections of tissue embedded in hydrophilic resin were nick end labelled with biotinylated dUTP which was subsequently labelled with avidin conjugated to gold particles. The technique was validated by labelling the nuclei of L929-8 cells treated with tumour necrosis factor α. These cells are known to respond to treatment with the factor by undergoing apoptosis. The method was then used on tissue from the chick embryo which is known to be undergoing programmed cell death. This tissue was from the neural tube and the posterior necrotic zone of the limb bud, where cells can be identified as undergoing apoptosis based on the morphology of their nuclei. The method specifically labelled heterochromatin adjacent to the nuclear envelope as well as the associated with the nucleolus of cells from regions of the embryo where programmed cell death was expected. In addition to labelling the nuclei of cells that were clearly undergoing apoptosis, the method also identified nuclei of apparently normal cells. This method, used in conjunction with corroborating techniques, provides a means for the early detection of cells undergoing DNA fragmentation, before the onset of gross apoptotic morphology, and in cells that do not show classical apoptotic characteristics.     

The positional distribution of cell death of ray parenchyma in a conifer, Abies sachalinensis

We monitored the distribution of death of secondary xylem cells in a conifer, Abies sachalinensis. The cell death of tracheids, which are tracheary elements, occurred successively and was related to the distance from cambium. Thus, it resembled programmed cell death. By contrast, the death of long-lived ray parenchyma cells had the following features: (1) ray parenchyma cells remained alive for several years or more; (2) in many cases, no successive cell death occurred even within a given radial cell line of a ray; and (3) the timing of cell death differed among upper and lower radial cell lines and other lines of cells within a ray. These results indicate that the death of long-lived ray parenchyma cells involves a different process from the death of tracheids. The initiation of secondary wall formation and the lignification of ray parenchyma cells in the current year's annual ring were delayed in the upper and lower radial cell lines of a ray. In addition, the density of distribution and orientation of cortical microtubules in such cells were different from those in cells in other radial lines. Ray parenchyma cells in the previous year's annual ring within the upper and lower radial cell lines of a ray contained many starch grains. Our results indicate that positional information is an important factor in the control of the pattern of differentiation and, thus, of the functions of ray parenchyma cells that are derived from the same cambial ray cells.     

The nucellus degenerates by a process of programmed cell death during the early stages of wheat grain development

The nucellus, which is the maternal tissue of the wheat grain, degenerates during the early stages of development. We have investigated whether or not this degenerative process may be considered as programmed cell death (PCD). The analysis of DNA of tissues dissected from developing wheat (Triticum aestivum L. cv Chinese Spring) grains at 5-20 days post anthesis (dpa) showed the presence of DNA laddering, which is indicative of internucleosomal fragmentation of nuclear DNA, in maternal tissues but not in the endosperm. The TUNEL assay showed in-situ internucleosomal fragmentation of DNA in nuclei of parenchymal and epidermal cells of the nucellus, as well as in the pericarp, during the early stages of grain development (5 dpa). Furthermore, internucleosomal fragmentation of nuclear DNA was observed in nucellar projection cells in the middle stages of grain development (13-18 dpa), thus showing a process of PCD in these maternal tissues. Electron-transmission microscopy analysis allowed the morphology of PCD to be characterized in this plant tissue. Initially, fragmentation of the cytoplasm was observed, the nuclear envelope appeared dilated and to be forming vacuoles, and the content of heterochromatin increased. A progressive degradation of the cytosolic contents and organelles was observed, and the plasma membrane was disrupted. However, the Golgi apparatus remained intact and apparently functional even in the final stages of cell death.     

Nuclear fragmentation and DNA degradation during programmed cell death in petals of morning glory (Ipomoea nil)

We studied DNA degradation and nuclear fragmentation during programmed cell death (PCD) in petals of Ipomoea nil (L.) Roth flowers. The DNA degradation, as observed on agarose gels, showed a large increase. Using DAPI, which stains DNA, and flow cytometry for DAPI fluorescence, we found that the number of DNA masses per petal at least doubled. This indicated chromatin fragmentation, either inside or outside the nucleus. Staining with the cationic lipophilic fluoroprobe DiOC₆ indicated that each DNA mass had an external membrane. Fluorescence microscopy of the nuclei and DNA masses revealed an initial decrease in diameter together with chromatin condensation. The diameters of these condensed nuclei were about 70% of original. Two populations of nuclear diameter, one with an average diameter about half of the other, were observed at initial stages of nuclear fragmentation. The diameter of the DNA masses then gradually decreased further. The smallest observed DNA masses had a diameter less than 10% of that of the original nucleus. Cycloheximide treatment arrested the cytometrically determined changes in DNA fluorescence, indicating protein synthesis requirement. Ethylene inhibitors (AVG and 1-MCP) had no effect on the cytometrically determined DNA changes, suggesting that these processes are not controlled by endogenous ethylene.     

Anti-apoptotic action of insulin-like growth factor-I during human preimplantation embryo development

Insulin-like growth factor I (IGF-I) has been shown to increase the proportion of embryos forming blastocysts and the number of inner cell mass cells in human and other mammalian preimplantation embryos. Here we examined whether the increased cell number resulted from increased cell division or decreased cell death. Normally fertilized, Day 2 human embryos of good morphology were cultured to Day 6 in glucose-free Earle's balanced salt solution supplemented with 1 mM glutamine, with (n = 42) and without (n = 45) 1.7 nM IGF-I. Apoptotic cells in Day 6 blastocysts were identified using terminal deoxynucleotidyl dUTP terminal transferase (TUNEL) labeling to detect DNA fragmentation and 4'-6-diamidino-2-phenylindole (DAPI) counterstain to evaluate nuclear morphology. The number of nuclei and extent of DNA and nuclear fragmentation was assessed using laser scanning confocal microscopy. IGF-I significantly increased the proportion of embryos developing to the blastocyst stage from 49% (control) to 74% (+IGF-I) (P < 0.05). IGF-I also significantly decreased the mean proportion of apoptotic nuclei from 16.3 +/- 2.9% (-IGF-I) to 8.7 +/- 1.4% (+IGF-I) (P < 0.05). The total number of cells remained similar between both groups (61.7 +/- 4.6 with IGF-I; 54.5 +/- 5.1 without IGF-I). The increased number of blastocysts combined with reduced cell death suggests that IGF-I is rescuing embryos in vitro which would otherwise arrest and acting as a survival factor during preimplantation human development.     

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.     

Lack of synchrony among multiple nuclei induces partial DNA fragmentation in V79 cells polyploidized by demecolcine

Abstract. The nuclear morphology of polyploidized cells was examined in V79 Chinese hamster cells polyploidized by demecolcine or K-252a, inhibitors of spindle fibre formation and protein kinases, respectively. A variety of nuclear morphologies, including multinuclei, were observed in V79 cells polyploidized by demecolcine but not by K-252a, which produced mononuclear cells. A lack of synchrony in the nuclear cycle was observed among nuclei in multinuclear polyploidized cells. Partial DNA fragmentation, defined as DNA fragmentation of a nucleus in a multinuclear cell, was detected using the TUNEL method in V79 cells polyploidized by demecolcine but not by K-252a. Apoptosis occurred earlier in cell populations treated with demecolcine than in these treated with K-252a once the drugs were removed from the medium, suggesting that polyploidized cells with separate nuclei tend to apoptose earlier than those with mononuclei.