Apoptosis In Animal Cells Research Articles

Pathogen-induced programmed cell death in tobacco.

Sacrificing an infected cell or cells in order to prevent systemic spread of a pathogen appears to be a conserved strategy in both plants and animals. We studied some of the morphological and biochemical events that accompany programmed cell death during the hypersensitive response of tobacco plants infected with tobacco mosaic virus. Certain aspects of this cell death process appeared to be similar to those that take place during apoptosis in animal cells. These included condensation and vacuolization of the cytoplasm and cleavage of nuclear DNA to 50 kb fragments. In contrast, internucleosomal fragmentation, condensation of chromatin at the nuclear periphery and apoptotic bodies were not observed in tobacco plants during tobacco mosaic virus-induced hypersensitive response. A unique aspect of programmed cell death during the hypersensitive response of tobacco to tobacco mosaic virus involved an increase in the amount of monomeric chloroplast DNA. Morphological changes to the chloroplast and cytosol of tobacco cells and increase in monomeric chloroplast DNA occurred prior to gross changes in nuclear morphology and significant chromatin cleavage. Our findings suggest that certain aspects of programmed cell death may have been conserved during the evolution of plants and animals.

Programmed cell death in the root cortex of soybean root necrosis mutants.

The soybean root necrosis (rn) mutation causes a progressive browning of the root soon after germination that is associated with accumulation of phytoalexins and pathogenesis-related proteins and an increased tolerance to root-borne infection by the fungal pathogen, Phytophthora sojae. Grafting and decapitation experiments indicate that the rn phenotype is root-autonomous at the macroscopic level. However, the onset and severity of browning was modulated in intact plants by exposure to light, as was the extent of lateral root formation, suggesting that both lateral roots and the rn phenotype could be directly or indirectly controlled by similar shoot-derived factors. Browning first occurs in differentiated inner cortical cells adjacent to the stele and is preceded by a wave of autofluorescence that emanates from cortical cells opposite the xylem poles and spreads across the cortex. Before any visible changes in autofluorescence or browning, fragmented DNA was detected by TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling) in small clusters of inner cortical cells that subsequently could be distinguished cytologically from neighboring cells throughout rn root development. Inner cortical cells overlying lateral root primordia in either Rn or rn plants also were stained by TUNEL. Features commonly observed in animal cell apoptosis were confirmed by electron microscopy but, surprisingly, cells with a necrotic morphology were detected alongside apoptotic cells in the cortex of rn roots when TUNEL-positive cells were first observed. The two morphologies may represent different stages of a common pathway for programmed cell death (pcd) in plant roots, or two separate pathways of pcd could be involved. The phenotype of rn plants suggests that the Rn gene could either negatively regulate cortical cell death or be required for cortical cell survival. The possibility of a mechanistic link between cortical cell death in rn plants and during lateral root emergence is discussed.

Bax- and Bak-induced cell death in the fission yeast Schizosaccharomyces pombe.

The effects of the expression of the human Bcl-2 family proteins Bax, Bak, Bcl-2, and Bcl-XL were examined in the fission yeast Schizosaccharomyces pombe and compared with Bax-induced cell death in mammalian cells. Expression of the proapoptotic proteins Bax and Bak conferred a lethal phenotype in this yeast, which was strongly suppressed by coexpression of the anti-apoptotic protein Bcl-XL. Bcl-2 also partially abrogated Bax-mediated cytotoxicity in S. pombe, whereas a mutant of Bcl-2 (Gly145Ala) that fails to heterodimerize with Bax or block apoptosis in mammalian cells was inactive. However, other features distinguished Bax- and Bak-induced death in S. pombe from animal cell apoptosis. Electron microscopic analysis of S. pombe cells dying in response to Bax or Bak expression demonstrated massive cytosolic vacuolization and multifocal nuclear chromatin condensation, thus distinguishing this form of cell death from the classical morphological features of apoptosis seen in animal cells. Unlike Bax-induced apoptosis in 293 cells that led to the induction of interleukin-1 beta-converting enzyme (ICE)/CED-3-like protease activity, Bax- and Bak-induced cell death in S. pombe was accompanied neither by internucleosomal DNA fragmentation nor by activation of proteases with specificities similar to the ICE/CED-3 family. In addition, the baculovirus protease inhibitor p35, which is a potent inhibitor of ICE/CED-3 family proteases and a blocker of apoptosis in animal cells, failed to prevent cell death induction by Bax or Bak in fission yeast, whereas p35 inhibited Bax-induced cell death in mammalian cells. Taken together, these findings suggest that Bcl-2 family proteins may retain an evolutionarily conserved ability to regulate cell survival and death but also indicate differences in the downstream events that are activated by overexpression of Bax or Bak in divergent cell types.

Identification, characterization, and purification of a tobacco endonuclease activity induced upon hypersensitive response cell death.

Programmed cell death (pcd) is activated during the hypersensitive response (HR) of plants to avirulent pathogens. We have recently shown that, similar to pcd in animal cells, nuclei of cells undergoing HR cell death contain fragmented nuclear DNA (nDNA). Here, we report that cell death occurring during the HR is accompanied by an increase in the activity of several deoxyribonucleases. Induction of nuclease activities was coordinated with cell death and may account for the degradation of nDNA during the HR. HR-associated nuclease activities were not induced during senescence, following necrotic cell death resulting from abiotic stress, or in response to induction of plant defense mechanisms by salicylic acid. HR-associated nuclease activities were stimulated by Ca2+ and inhibited by EGTA, EDTA, and Zn2+. At least one of the HR-associated nuclease activities was detected in nuclei purified from leaves undergoing pcd. A nuclease with an electrophoretic mobility similar to that of the nuclease activity found in nuclei isolated from leaves undergoing HR cell death was purified. Our findings are in accordance with some of the biochemical events that occur during pcd in animal cells. However, further analysis of the pattern of nDNA fragmentation and the corresponding structural changes that occur in the nuclei of tobacco cells undergoing HR cell death revealed that these features may have differences from those that take place during apoptosis in animal cells.

Purification of an Interleukin-1β Converting Enzyme-related Cysteine Protease That Cleaves Sterol Regulatory Element-binding Proteins between the Leucine Zipper and Transmembrane Domains

We describe the characterization and purification of a protease that cleaves sterol regulatory element-binding protein-1 (SREBP-1) and SREBP-2 in vitro. Cleavage occurs between the basic helix-loop-helix-leucine zipper and the first transmembrane domain of each SREBP. This is the region in which the SREBPs are cleaved physiologically by a sterol-regulated protease that releases an NH2-terminal fragment that activates transcription of the genes for the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl CoA synthase. The cleavage enzyme, designated SREBP cleavage activity (SCA), belongs to a new class of cysteine proteases of the interleukin-1 beta-converting enzyme (ICE) family, all of which cleave at aspartic acid residues. Like ICE, SCA was inactive in cytosol, and it was activated in vitro by incubation at 30 degrees C. SCA was resistant to inhibitors of serine, aspartyl, and metalloproteases, but it was sensitive to N-ethylmaleimide. The enzyme cleaved SREBP-1 and SREBP-2 between the Asp and Ser of a conserved sequence (S/DEPDSP). The activity was blocked by a tetrapeptide aldehyde, Ac-Asp-Glu-Ala-Asp-aldehyde (Ac-DEAD-CHO). A purified preparation of SCA from hamster liver contained a prominent 20-kDa polypeptide that could be labeled with [14C]iodoacetic acid. Labeling was blocked by Ac-DEAD-CHO. Partial amino acid sequence of this polypeptide revealed that it was the hamster equivalent of human CPP32, a putative protease whose cDNA was recently identified by virtue of sequence homology to ICE. CPP32 and ICE have been implicated in apoptosis in animal cells. Whether SCA/CPP32 participates in vivo in the sterol-regulated activation of SREBP, or whether it activates SREBPs during apoptosis, remains to be determined.

In Situ Detection of nDNA Fragmentation during the Differentiation of Tracheary Elements in Higher Plants.

Programmed cell death (pcd) is thought to occur during the autolysis of xylem vessels. Although several ultrastructural aspects of this differentiation process have been characterized, certain key aspects of this process remain unsolved. Here we demonstrate in pea (Pisum sativum) that nuclei of vessel elements undergoing pcd contain fragmented nDNA. This finding may provide evidence for the activation of a DNA degradation mechanism prior to the final disruption of the nucleus that occurs during the autolysis stage of this differentiation process. In situ detection of DNA fragmentation in nuclei of vessel elements undergoing pcd may therefore suggest that this death process involves the activation of a mechanism for DNA degradation, similar to that activated during apoptosis in animal cells. In addition, this differentiation process may serve as a useful positive control for the in situ detection of pcd in other developmental pathways and during the hypersensitive response of plants to avirulent pathogens.