Changes in gene expression during programmed cell death in tomato cell suspensions

To identify genes involved in plant programmed cell death (PCD), changes in gene expression during PCD in a model system of suspension-cultured tomato cells were studied. In this system, cell death is triggered by treatment with camptothecin, an inhibitor of topoisomerase I. Cell death was accompanied by internucleosomal DNA degradation, indicating that the cell death process shares similarities with apoptosis in animals. Tomato homologues of DAD1 and HSR203, two genes that have been implicated in PCD, were isolated. During camptothecin-induced PCD tomato DAD1 mRNA levels roughly halve, while tomato HSR203 mRNA levels increase 5-fold. A differential display approach was used to identify novel genes that show changes in expression levels during camptothecin-induced PCD. This resulted in isolation of two up-regulated (CTU1 and CTU2) and four down-regulated (CTD1, CTD2, CTD4, and CTD5) cDNA clones. CTU1 shows high homology to various gluthatione S-transferases, whereas CTU2 is as yet unidentified. CTD1 is highly similar to Aux/IAA early-auxin-responsive genes. CTD2 corresponds to the tomato RSI-1 gene, CTD4 is an unknown clone, and CTD5 shows limited homology with a proline-rich protein from maize. Addition of the calcium channel blocker lanthanum chloride prevented camptothecin-induced cell death. The effect of lanthanum chloride on camptothecin-induced gene expression was studied to discriminate between putative cell death genes and general stress genes. The possible role of the various predicted gene products in plant PCD is discussed.     

Programmed cell death induced by cadmium stress and/or boron deprivation in tobacco (Nicotiana tabacum L.) cultivar Bright Yellow 2 cells

Cadmium (Cd) is one of the most toxic and widespread heavy metal pollutants in soil. As an essential mineral nutrient, boron (B) plays critical roles in physiological processes of plants. In the present study, programmed cell death (PCD) induced by Cd stress and/or B deprivation was assessed and the underlying mechanisms were clarified in suspension-cultured Nicotiana tabacum L. cultivar Bright Yellow 2 (TBY-2) cells. The PCD in TBY-2 cells was analyzed by Hoechst 33258 staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and then, expression analysis of PCD-related genes was performed using quantitative real-time polymerase chain reaction (qPCR) assays. The production of reactive oxygen species (ROS) was determined using fluorescence microscopy of 2′,7′-dichlorofluorescein diacetate–labeled cells. The levels of lipid peroxides were quantified by the thiobarbituric acid–reactive substances (TBARS) method. Cadmium stress and/or B deprivation treatments induced PCD that was characterized by a significant increase in the percentage of cells stained with Hoechst 33258 or TUNEL-positive cells, and upregulation or downregulation of the expression of PCD-related genes. Treatments with Cd stress and/or B deprivation increased ROS production and the level of lipid peroxides compared to those of the control group. These data showed that in TBY-2 cells Cd stress and/or B deprivation activated ROS signaling pathways, leading to gene expression that was connected with the PCD process.

     

Apoptosis induced by hydrogen peroxide under serum deprivation and its inhibition by antisense c-jun in F-MEL cells

Under serum deprivation F-MEL cells die by apoptosis. We previously showed that apoptosis induced by serum deprivation was suppressed by inhibition of c-jun expression using antisense c-jun transfected cell line, c-junAS. To elucidate the underlying mechanisms we examined the species which is responsible for apoptosis under serum deprivation. When catalase and N-acetyl-L-cysteine (NAC) were included in the medium, cell death under serum deprivation was effectively suppressed in F-MEL cells. Intracellular generation of hydrogen peroxide (H(2)O(2)) was also detected under serum deprivation in parental F-MEL cells, but it was suppressed in c-junAS (+) cells, in which antisense c-jun was expressed and c-Jun protein expression was inhibited as shown by Western blot. When H(2)O(2) was directly applied to F-MEL cells at 3 mM, apoptotic cell death was induced, whereas it was suppressed in c-junAS (+) cells. Induction of apoptosis by H(2)O(2) and its inhibition by antisense c-jun was confirmed by detection of internucleosomal fragmentation of DNA, TdT-mediated dUTP nick end labeling (TUNEL)-positive cells and morphological alteration of nuclei. These results indicate that apoptosis induced by serum deprivation in F-MEL cells is mediated by H(2)O(2) and c-jun expression is essential to apoptosis induced by H(2)O(2) in F-MEL cells.     

Vesicle-associated membrane protein of Arabidopsis suppresses Bax-induced apoptosis in yeast downstream of oxidative burst

Programmed cell death (PCD) in many systems is controlled by relative amounts of the apoptosis-regulating proteins Bax and Bcl-2 through homo- or heterodimerization. Here we show that Bax-induced PCD of yeast was suppressed by transformation with a vesicle-associated membrane protein from Arabidopsis (AtVAMP), which was isolated by screening a cDNA expression library against sugar-induced cell death in yeast. AtVAMP expression blocked Bax-induced PCD downstream of oxidative burst. AtVAMP also prevented H(2)O(2)-induced apoptosis in yeast and in Arabidopsis cells. Reduced oxidation of lipids and plasma membrane proteins was detected in the AtVAMP-transformed yeast, suggesting improved membrane repair. Inhibition of intracellular vesicle trafficking by brefeldin A induced apoptosis from a sublethal concentration of H(2)O(2). No protection occurred by overexpression of the yeast homolog SCN2. However, efficient suppression of yeast PCD occurred by expression of a chimeric gene, composed of the conserved domains from yeast, fused to the variable N-terminal domain from Arabidopsis, resulting in exchange of the proline-rich N-terminal domain of SCN2 with a proline-poor Arabidopsis sequence. Our results suggest that intracellular vesicle traffic can regulate execution of apoptosis by affecting the rate of membrane recycling and that the proline-rich N-terminal domain of VAMP inhibited this process.     

Detection of apoptosis induced by topoisomerase inhibitors and serum deprivation in syrian hamster embryo cells

The sensitivity of normal diploid Syrian hamster embryo (SHE) cells to apoptosis was tested after treatment with the topoisomerase inhibitors camptothecin and etoposide and after serum withdrawal. Programmed cell death (PCD) was identified through morphological, biochemical, and molecular changes and compared with that of HL60 cell line. The results showed that topoisomerase inhibitors, which were shown to be potent PCD inducers in the HL60 cell line, induced a weaker apoptotic response in SHE cells than after growth factor deprivation. In addition, serum-free medium, which rapidly induced apoptosis in SHE cells, did not affect the HL60 cell line. In both cell types, PCD was expressed by condensed chromatin, fragmented nuclei, and DNA laddering on electrophoretic gels, an indisputable sign of apoptosis. In apoptotic HL60 cells, the cleavage of 113-kDa poly(ADP-ribose)polymerase (PARP) resulted in the so-called apoptotic 89-kDa fragment and was associated with increased caspase-3 activity. In apoptotic SHE cells, PARP degraded early but the degradation profile was not characterized by the appearance of an 89-kDa fragment. Moreover, no activation of caspase-3 was noted. ZnCl(2), which is known to prevent protease activity responsible for apoptosis features, inhibited PARP cleavage and nuclear modifications induced by apoptotic stimuli in both cell types, but with a higher sensitivity in SHE cells. Apoptosis induced by serum deprivation was linked with c-myc negative regulation in SHE cells, but not with p53 protein accumulation, while topoisomerase inhibitors led to p53 stabilization without any change in c-myc expression. Serum-free medium and topoisomerase inhibitors did not modify c-myc expression in the HL60 cell line. The overall results demonstrated that apoptosis, which is a carefully regulated process of cell death, may proceed through mechanisms varying according to cell type or apoptosis inducer. In addition, markers which are generally considered hallmarks of apoptosis may fail to appear in some cell types.     

A tomato metacaspase gene is upregulated during programmed cell death in<Emphasis Type="Italic"> Botrytis cinerea</Emphasis>-infected leaves

Programmed cell death (PCD) in plant cells is often accompanied by biochemical and morphological hallmarks similar to those of animal apoptosis. However, orthologs of animal caspases, cysteinyl aspartate-specific proteases that constitute the core component of animal apoptosis, have not yet been identified in plants. Recent studies have revealed the presence of a family of genes encoding proteins with distant homology to mammalian caspases, designated metacaspases, in the Arabidopsis thaliana genome. Here, we describe the isolation of LeMCA1, a type-II metacaspase cDNA clone from tomato (Lycopersicon esculentum Mill.). BLAST analysis demonstrated that the LeMCA1 gene is located in close vicinity of several genes that have been linked with PCD. Southern analysis indicated the existence of at least one more metacaspase in the tomato genome. LeMCA1 mRNA levels rapidly increased upon infection of tomato leaves with Botrytis cinerea, a fungal pathogen that induces cell death in several plant species. LeMCA1 was not upregulated during chemical-induced PCD in suspension-cultured tomato cells.     

Boron Nutrition of Tobacco BY-2 Cells. V. Oxidative Damage is the Major Cause of Cell Death Induced by Boron Deprivation

Boron (B) is an essential micronutrient for vascular plants.However, it remains unclear how B deficiency leads to variousmetabolic disorders and cell death. To understand this mechanism,we analyzed the physiological changes in suspension-culturedtobacco (Nicotiana tabacum) BY-2 cells upon B deprivation. When3-day-old cells were transferred to B-free medium, cell deathwas detectable as early as 12 h after treatment. The B-deprivedcells accumulated more reactive oxygen species and lipid peroxidesthan control cells, and showed a slight but significant decreasein the cellular ascorbate pool. Supplementing the media withlipophilic antioxidants effectively suppressed the death ofB-deprived cells, suggesting that the oxidative damage is theimmediate and major cause of cell death under B deficiency.Dead cells in B-free culture exhibited a characteristic morphologywith a shrunken cytoplasm, which is often seen in cells undergoingprogrammed cell death (PCD). However, they did not display otherhallmarks of PCD such as internucleosomal DNA fragmentation,decreased ascorbate peroxidase expression and protection fromdeath by cycloheximide. These results suggest that the deathof tobacco cells induced by B deprivation is not likely to bea typical PCD.     

Induction of programmed cell death in a dorsal root ganglia × neuroblastoma cell line

Growth factor-dependent neurons die when they are deproved of their specific growth factor. This “programmed” cell death (PCD) requires macromolecular synthesis and is distinct from necrotic cell death. To investigate the mechanisms involved in neuronal PCD, we have studied the sequence of events that occur when a neuronal cell line (F-11: Mouse neuroblastoma X rat dorsal root ganglia) is deprived of serum in a manner analogous to growth factor deprivation from neurons. Protein synthesis was inhibited within the first 8 h of serum deprivation, while DNA cleavage into nucleosome ladders was prominent by 24 h. The DNA cleavage could be inhibited by cycloheximide, consistent with a requirement for protein synthesis. In contrast, mitochondrial function was not compromised by serum deprivation. Rather, the cells appeared to be metabolically activated after serum removal as shown by an increased reduction of MTT by mitochondrial dehydrogenases and an increase in cellular autofluorescence, which is thought to be due to elevated levels of NADH and flavoproteins. Assessment of cell viability by propidium iodide staining showed no indication of cell death within 24 h. After 48 h of serum deprivation, cells decreased in size and increased propidium iodide uptake. Thus, serum deprivation activates PCD in F-11 cells and may be a useful model to study the intracellular events responsible for PCD. © 1993 John Wiley & Sons, Inc.     

Growth arrest of melanoma cells is differentially regulated by contact inhibition and serum deprivation.

Both growth-factor deprivation and contact inhibition suppress cell growth; however, the mechanisms by which they inhibit cell proliferation may not be identical. The function of antiproliferative genes and the induction of programmed cell death are among the potential differences between these growth-arrest mechanisms. Specifically, an inverse relation between the expression of cyclin-dependent kinase inhibitors (CDKIs) and the susceptibility to apoptosis has been reported. To test this relation, we examined the features of growth arrest in a canine melanoma cell line, TLM1. Both contact inhibition and serum deprivation halted cell-cycle progression of TLM1 cells in the G1 phase. Prolonged growth arrest of the cells without restimulation resulted in apoptosis; conversely, the cells reentered the cell cycle after release from contact inhibition or on restimulation with serum. Cell-to-cell contact, but not serum deprivation, led to the expression of p53 and p21/Waf-1. The expression of p21/Waf-1 did not prevent apoptosis. Moreover, the ectopic overexpression of CDKIs increased apoptosis. These results support the premise that growth arrest induced by contact inhibition and serum deprivation are mediated through distinct mechanisms. Furthermore, CDKIs are not universal inhibitors of apoptosis, and in some cases, they may initiate or enhance the apoptotic program.     

Identification of a novel kinase-like gene induced during neuronal cell death.

When deprived of neurotrophic factors, neuronal cells undergo a form of programmed cell death that involves a cascade of gene expression. To better understand this cascade, we screened the genes induced during programmed cell death evoked in neuronal PC6-3 cells by NGF-depletion and discovered a novel gene, NIPK (Neuronal cell death Inducible Putative Kinase), that contains a kinase-like domain. Expression of NIPK was also induced in cultured sympathetic neurons by NGF deprivation and in cortical neurons exposed to the Ca2+ ionophore, A23187. In contrast, NIPK was not induced during non-neuronal cell death evoked by serum or growth factor deprivation, or by treatment with methyl methanesulfonate, an agent that causes cell death by damaging DNA. Taken together, these findings suggest that NIPK is involved in programmed cell death via a pathway that is present in neurons but is absent in non-neurons.     

Autophagy and apoptosis in Chinese hamster ovary cell culture

Upon nutrient deprivation during Chinese hamster ovary (CHO) cell culture for foreign protein production, cells are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. However, only apoptosis has drawn attention in the field of CHO cell culture. Numerous studies on engineering genes or supplementing essential nutrients or chemical additives to culture media to overcome cell death induced by various stimuli have been limited to apoptosis. Recently, autophagic morphologies were demonstrated by the processing of LC3 into the 16 kDa LC3-II form, and the accumulation of multiple autophagosomes in CHO cell culture. Therefore, it seems worthwhile to revisit the issue of cell death in CHO cell culture with the concept of autophagy in mind, in order to achieve a maximum production of foreign proteins by protecting cells from both types of PCD.     

Genomic profiling of cortical neurons following exposure to beta-amyloid

In vitro and in vivo studies have shown that beta-amyloid peptide induces neuronal cell death. To explore the molecular basis underlying beta-amyloid-induced toxicity, we analyzed gene expression profiles of cultured rat cortical neurons treated for 24 and 48 h with synthetic beta-amyloid peptide. From the 8740 genes interrogated by oligonucleotide microarray analysis, 241 genes were found to be differentially expressed and segregated into distinct clusters. Functional clustering based on gene ontologies showed coordinated expression of genes with common biological functions and metabolic pathways. The comparison with genes differentially expressed in cerebellar granule neurons following serum and potassium deprivation indicates the existence of common regulatory mechanisms underlying neuronal cell death. Our results offer a genomic view of the changes that accompany beta-amyloid-induced neurodegeneration.