Cell Death Symptoms Research Articles

Curcumin induces cell death without oligonucleosomal DNA fragmentation in quiescent and proliferating human CD8+ cells.

Cytotoxic CD8+ cells play an important role in determining host response to tumor, thus chemotherapy is potentially dangerous as it may lead to T cells depletion. The purpose of this study was to elucidate the propensity of quiescent and proliferating human CD8+ cells to undergo cell death upon treatment with curcumin, a natural dye in Phase I of clinical trials as a prospective chemopreventive agent. We treated human quiescent or proliferating CD8+ cells with 50 microM curcumin or irradiated them with UVC. Cell death symptoms such as decreased cell viability, chromatin condensation, activation of caspase-3 and specific DFF40/CAD endonuclease and oligonucleosomal DNA fragmentation were analyzed using MTT test, microscopic observation, Western blotting and flow cytometry. Curcumin decreased cell viability, activated caspase-3 and decreased the level of DFF45/ICAD, the inhibitor of the DFF40/CAD endonuclease. However, this did not lead to oligonucleosomal DNA degradation. In contrast, UVC-irradiated proliferating, but not quiescent CD8+ cells revealed molecular and morphological changes characteristic for apoptosis, including oligonucleosomal DNA fragmentation. Curcumin can induce cell death in normal human lymphocytes both quiescent and proliferating, without oligonucleosomal DNA degradation which is considered as a main hallmark of apoptotic cell death. Taking into account the role of CD8+ cells in tumor response, their depletion during chemotherapy could be particularly undesirable.

Curcumin Affects Components of the Chromosomal Passenger Complex and Induces Mitotic Catastrophe in Apoptosis-Resistant Bcr-Abl-Expressing Cells

The Bcr-Abl oncoprotein plays a major role in the development and progression of chronic myeloid leukemia and is a determinant of chemotherapy resistance occurring during the blast crisis phase of the disease. The aim of this article was to investigate the possibility of combating the resistance to apoptosis caused by Bcr-Abl by inducing an alternative cell death process. As a model of chronic myeloid leukemia, we employed Bcr-Abl-transfected mouse progenitor 32D cells with low and high Bcr-Abl expression levels corresponding to drug-sensitive and drug-resistant cells, respectively. The drug curcumin (diferuloylmethane), a known potent inducer of cell death in many cancer cells, was investigated for efficacy with Bcr-Abl-expressing cells. Curcumin strongly inhibited cell proliferation and affected cell viability by inducing apoptotic symptoms in all tested cells; however, apoptosis was a relatively late event. G(2)-M cell cycle arrest, together with increased mitotic index and cellular and nuclear morphology resembling those described for mitotic catastrophe, was observed and preceded caspase-3 activation and DNA fragmentation. Mitosis-arrested cells displayed abnormal chromatin organization, multipolar chromosome segregation, aberrant cytokinesis, and multinucleated cells-morphologic changes typical of mitotic catastrophe. We found that the mitotic cell death symptoms correlated with attenuated expression of survivin, a member of the chromosomal passenger complex, and mislocalization of Aurora B, the partner of survivin in the chromosomal passenger complex. Inhibition of survivin expression with small interfering RNA exhibited similar mitotic disturbances, thus implicating survivin as a major, albeit not the only, target for curcumin action. This study shows that curcumin can overcome the broad resistance to cell death caused by expression of Bcr-Abl and suggests that curcumin may be a promising agent for new combination regimens for drug-resistant chronic myeloid leukemia.

Overexpression of Farnesyl Diphosphate Synthase in Arabidopsis Mitochondria Triggers Light-dependent Lesion Formation and Alters Cytokinin Homeostasis

To investigate the role of mitochondrial farnesyl diphosphate synthase (FPS) in plant isoprenoid biosynthesis we characterized transgenic Arabidopsis thaliana plants overexpressing FPS1L isoform. This overexpressed protein was properly targeted to mitochondria yielding a mature and active form of the enzyme of 40 kDa. Leaves from transgenic plants grown under continuous light exhibited symptoms of chlorosis and cell death correlating to H(2)O(2) accumulation, and leaves detached from the same plants displayed accelerated senescence. Overexpression of FPS in mitochondria also led to altered leaf cytokinin profile, with a reduction in the contents of physiologically active trans-zeatin- and isopentenyladenine-type cytokinins and their corresponding riboside monophosphates as well as enhanced levels of cis-zeatin 7-glucoside and storage cytokinin O-glucosides. Overexpression of 3-hydroxy-3-methylglutaryl coenzyme A reductase did not prevent chlorosis in plants overexpressing FPS1L, but did rescue accelerated senescence of detached leaves and restored wild-type levels of cytokinins. We propose that the overexpression of FPS1L leads to an enhanced uptake and metabolism of mevalonic acid-derived isopentenyl diphosphate and/or dimethylallyl diphosphate by mitochondria, thereby altering cytokinin homeostasis and causing a mitochondrial dysfunction that renders plants more sensitive to the oxidative stress induced by continuous light.

Depletion of UDP-d-apiose/UDP-d-xylose Synthases Results in Rhamnogalacturonan-II Deficiency, Cell Wall Thickening, and Cell Death in Higher Plants

D-apiose serves as the binding site for borate cross-linking of rhamnogalacturonan II (RG-II) in the plant cell wall, and biosynthesis of D-apiose involves UDP-D-apiose/UDP-D-xylose synthase catalyzing the conversion of UDP-D-glucuronate to a mixture of UDP-D-apiose and UDP-D-xylose. In this study we have analyzed the cellular effects of depletion of UDP-D-apiose/UDP-D-xylose synthases in plants by using virus-induced gene silencing (VIGS) of NbAXS1 in Nicotiana benthamiana. The recombinant NbAXS1 protein exhibited UDP-D-apiose/UDP-D-xylose synthase activity in vitro. The NbAXS1 gene was expressed in all major plant organs, and an NbAXS1-green fluorescent protein fusion protein was mostly localized in the cytosol. VIGS of NbAXS1 resulted in growth arrest and leaf yellowing. Microscopic studies of the leaf cells of the NbAXS1 VIGS lines revealed cell death symptoms including cell lysis and disintegration of cellular organelles and compartments. The cell death was accompanied by excessive formation of reactive oxygen species and by induction of various protease genes. Furthermore, abnormal wall structure of the affected cells was evident including excessive cell wall thickening and wall gaps. The mutant cell walls contained significantly reduced levels of D-apiose as well as 2-O-methyl-L-fucose and 2-O-methyl-D-xylose, which serve as markers for the RG-II side chains B and A, respectively. These results suggest that VIGS of NbAXS1 caused a severe deficiency in the major side chains of RG-II and that the growth defect and cell death was likely caused by structural alterations in RG-II due to a D-apiose deficiency.

Prohibitin is involved in mitochondrial biogenesis in plants

Prohibitin, which consists of two subunits PHB1 and PHB2, plays a role in cell-cycle progression, senescence, apoptosis, and maintenance of mitochondrial function in mammals and yeast. In this study, we examined the role of prohibitins in plants by using virus-induced gene silencing (VIGS) of two prohibitin subunit genes of Nicotiana benthamiana, designated NbPHB1 and NbPHB2. NbPHB1 and NbPHB2 were targeted to the mitochondria, and their gene expression was suppressed during senescence. VIGS of NbPHB2 caused severe growth inhibition, leaf yellowing and symptoms of cell death, whereas VIGS of NbPHB1 resulted in a milder phenotype. At the cellular level, depletion of these subunits affected mitochondria by severely reducing their number and/or mass, and by causing morphological and physiological abnormalities. Suppression of prohibitin function resulted in a 10- to 20-fold higher production of reactive oxygen species and induced premature leaf senescence. Finally, disruption of prohibitin function rendered the plants more susceptible to various oxidative stress-inducing reagents, including H(2)O(2), paraquat, antimycin A and salicylic acid. These results suggest that prohibitins play a crucial role in mitochondrial biogenesis and protection against stress and senescence in plant cells.

Adducts of Oxylipin Electrophiles to Glutathione Reflect a 13 Specificity of the Downstream Lipoxygenase Pathway in the Tobacco Hypersensitive Response

The response to reactive electrophile species (RES) is now considered as part of the plant response to pathogen and insect attacks. Thanks to a previously established high-performance liquid chromatography tandem mass spectrometry methodology, we have investigated the production of oxylipin RES adducts to glutathione (GSH) during the hypersensitive response (HR) of plants. We have observed that RES conjugation to GSH in tobacco (Nicotiana tabacum) leaves is facile and nonspecific. In cryptogein-elicited tobacco leaves, we show that the oxylipin RES adducts to GSH are produced in correlation with GSH consumption, increase in glutathione S-transferase activity, and the appearance of the cell death symptoms. In this model, the adducts arise mainly from the downstream 13 lipoxygenase (LOX) metabolism, although the induced 9 LOX pathway leads massively to the accumulation of upstream metabolites. The main adducts were obtained from 2-hexenal and 12-oxo-phytodienoic acid. They accumulate transiently as 1-hexanol-3-GSH, a reduced adduct, and 12-oxo-phytodienoic acid-GSH, respectively. RES conjugation does not initiate cell death but explains part of the GSH depletion that accompanies HR cell death. The nature of these GSH conjugates shows the key role played by the 13 LOX pathway in RES signaling in the tobacco HR.

Silencing of NbECR encoding a putative enoyl-CoA reductase results in disorganized membrane structures and epidermal cell ablation in Nicotiana benthamiana

The very long chain fatty acids (VLCFAs) are synthesized by the microsomal fatty acid elongation system in plants. We investigated cellular function of NbECR putatively encoding enoyl-CoA reductase that catalyzes the last step of VLCFA elongation in Nicotiana benthamiana. Virus-induced gene silencing of NbECR produced necrotic lesions with typical cell death symptoms in leaves. In the affected tissues, ablation of the epidermal cell layer preceded disintegration of the whole leaf cell layers, and disorganized cellular membrane structure was evident. The amount of VLCFAs was reduced in the NbECR VIGS lines, suggesting NbECR function in elongation of VLCFAs. The results demonstrate that NbECR encodes a putative enoyl-CoA reductase and that the NbECR activity is essential for membrane biogenesis in N. benthamiana.

Hemagglutinating Virus of Japan–Artificial Viral Envelope Liposome-Mediated Cotransfer of bag-1 and bcl-2 Genes Protects Hepatic Cells Against Ischemic Injury Through BAG-1-Assisted Preferential Enhancement of Bcl-2 Protein Expression

Hepatic injury subsequent to ischemia-reperfusion (I/R) was demonstrated in our previous study to be prevented by hemagglutinating virus of Japan (HVJ)-artificial viral envelope (AVE) liposome-mediated gene transfer of the antiapoptotic gene, human bcl-2 (h-bcl-2). In the present study, we introduced simultaneously both mouse Bcl-2-associated athanogene 1 (m-bag-1) and the h-bcl-2 gene by the same HVJ-AVE liposome transfection method, and found that I/R-induced hepatic injuries such as release of hepatic marker enzymes into blood, cell morphological degeneration, and cellular DNA strand cleavage were suppressed more effectively than by transfection with either gene singly. In addition, the h-Bcl-2 expression level in the ischemic state, but not in the nonischemic state, was markedly higher in h-bcl-2/m-bag-1-cotransfected liver than in h-bcl-2-transfected liver. In contrast, the m-BAG-1 expression level in the ischemic state, but not in the nonischemic state, was only slightly higher in h-bcl-2/m-bag-1-cotransfected liver than in m-bag-1-transfected liver. Thus, with dual gene cotransfer, coexistent Bcl-2 protein exerts no activity to assist a marked enhancement of BAG-1 protein, whereas the function of overexpressed BAG-1 as a Bcl-2-binding protein may lead to the enhancement of efficient expression of h-Bcl-2 in I/R-treated liver as compared with nonischemic liver, which results in repression of diverse I/R-induced cell death symptoms, presumably through the formation of functional complexes of BAG-1 and Bcl-2.

The Plant Gene CCD1 Selectively Blocks Cell Death During the Hypersensitive Response to Cauliflower Mosaic Virus Infection

The P6 protein of Cauliflower mosaic virus (CaMV) W260 elicits a hypersensitive response (HR) on inoculated leaves of Nicotiana edwardsonii. This defense response, common to many plant pathogens, has two key characteristics, cell death within the initially infected tissues and restriction of the pathogen to this area. We present evidence that a plant gene designated CCD1, originally identified in N. bigelovii, can selectively block the cell death pathway during HR, whereas the resistance pathway against W260 remains intact. Suppression of cell death was evident not only macroscopically but also microscopically. The suppression of HR-mediated cell death was specific to CaMV, as Tobacco mosaic virus was able to elicit HR in the plants that contained CCD1. CCD1 also blocks the development of a systemic cell death symptom induced specifically by the P6 protein of W260 in N. clevelandii. Introgression of CCD1 from N. bigelovii into N. clevelandii blocked the development of systemic cell death in response to W260 infection but could not prevent systemic cell death induced by Tomato bushy stunt virus. Thus, CCD1 blocks both local and systemic cell death induced by P6 of W260 but does not act as a general suppressor of cell death induced by other plant viruses. Furthermore, experiments with CCD1 provide further evidence that cell death could be uncoupled from resistance in the HR of Nicotiana edwardsonii to CaMV W260.

P-glycoprotein is not involved in pathway of anti-Fas/Fas-induced apoptosis in KBv200 cells

To verify whether P-glycoprotein (P-gp) could induce cell resistance to apoptosis by inhibiting caspase-8 and caspase-3. Human KB cells, either drug-sensitive or with multidrug resistance (MDR) phenotype caused by overexpression of P-gp (KBv200 cells), were treated with anti-Fas (CH-11 monoclonal antibody) to induce apoptosis. Cytotoxicity was detected by MTT assay. Symptoms of cell death were assessed by morphological observation after Hoechst33258 staining, activation of caspase-8 and caspase-3 was measured by Western blotting. Compared with KB cells, the resistance of KBv200 cells to VCR (vincristine) was about 51-fold higher. Anti-Fas (CH-11) induced cytotoxicity and apoptosis in both sensitive KB cells and MDR phenotype KBv200 cells. The IC50 of CH-11 in KB cells was similar to that in KBv200 cells. CH-11 induced similar apoptotic rates in both KB cells and KBv200 cells, which could be classified as caspase-dependent apoptotic pathway. Verapamil (VRP) did not affect CH-11-mediated apoptosis in KBv200 cells. Expression of P-glycoprotein does not induce resistance to caspase-8 and -3 activation or anti-Fas-induced cell apoptosis.

Induction of programmed cell death in lily by the fungal pathogen Botrytis elliptica

SUMMARY The genus Botrytis contains necrotrophic plant pathogens that have a wide host range (B. cinerea) or are specialized on a single host species, e.g. B. elliptica on lily. In this study, it was found that B. elliptica-induced cell death of lily displays hallmark features of animal programmed cell death or apoptosis including cytoplasmic shrinkage, nuclear DNA fragmentation and the accumulation of NO as well as H(2)O(2). A pharmacological approach showed that B. elliptica-induced cell death could be modulated by serine and cysteine protease inhibitors including one caspase inhibitor. Blocking phosphatase activity stimulated cell death and concomitant lesion formation, suggesting that B. elliptica-induced cell death is mediated by kinase/phosphatase pathways. Blocking Ca(2+) influx restricted cell death. Blocking steps of sphingolipid biosynthesis delayed lily cell death for several days. B. elliptica culture filtrate (CF) was able to induce lily cell death by means of secreted proteins. Induction of cell death is necessary and sufficient for pathogenicity and host specialization because prior infiltration of B. elliptica CF enabled subsequent infection of lily by the otherwise incompatible pathogens B. cinerea and B. tulipae. The secreted B. elliptica proteins also induced cell death in some but not all Arabidopsis accessions and mutants. Arabidopsis accessions that respond to infiltration of B. elliptica CF also display cell death symptoms upon inoculation with B. elliptica conidia.

Expression of antiapoptotic genes bcl-xL and ced-9 in tomato enhances tolerance to viral-induced necrosis and abiotic stress.

D satellite RNA (satRNA) is a strain of cucumber mosaic virus (CMV) satRNA that induces an epidemic lethal disease in tomato. No natural resistance or tolerance has ever been found. Previously, we demonstrated the involvement of programmed cell death in disease development. Here, transgenic tomato plants expressing animal antiapoptotic genes bcl-xL and ced-9 were generated through agrobacterium-mediated transformation. High expression of bcl-xL or ced-9 affected plant growth and seed development. Inoculation of seedlings with CMV/D satRNA at T(1) and T(2) generations resulted in delayed cell-death symptoms or absence of symptoms. The degree of symptom suppression was correlated with increasing expression levels of the transgenes. Survival rates were compared among inoculated transgenic lines expressing bcl-xL, ced-9, and bcl-xL (G138A), a loss-of-function mutant of bcl-xL. More than 80% of the bcl-xL and ced-9 T(1) transgenic lines showed higher survival rates than the average for bcl-xL (G138A) transgenic lines. Total RNA extracted from surviving plants contained D satRNA, indicating systemic accumulation of D satRNA. Thus, expression of bcl-xL and ced-9 improved tolerance to, rather than resistance to, CMV/D satRNA infection. In addition, expression of bcl-xL and ced-9 specifically abrogated the formation of necrotic lesions, but not other symptoms, in tomato leaves during chilling at 4 degrees C. At 7 degrees C, temperature-induced leaf senescence was dramatically delayed in bcl-xL and ced-9 transgenic plants, and high levels of anthocyanins accumulated, possibly limiting oxidative stress. Hence, expression of these animal antiapoptotic genes improved plant survival under abiotic or biotic stress.

ROP to PA Pathway for ROS Regulation

The response of plants to stressful conditions, such as pathogen infection or wounding, often includes the generation of reactive oxygen species (ROS). Because levels of phosphatidic acid (PA) increase during a number of stress conditions, this lipid has been implicated in regulating ROS production. Park et al. report that the PA-regulated response in Arabidopsis involves a small guanosine triphosphatase called Rho-related small G protein in plants (ROP). Liposomes containing PA induced cell death when infiltrated into isolated Arabidopsis leaves. Exposure to PA metabolites or other phospholipids had no effect. PA treatment of cultured guard cells or addition of purified recombinant ROP2 to leaf extracts boosted ROS production. Leaves from transgenic Arabidopsis plants expressing a constitutively active form of ROP2 were more sensitive to PA exposure, showing earlier symptoms of cell death compared with wild-type leaves. In contrast, less ROS was generated in response to PA in leaves having a dominant-negative form of ROP2. Although the study places ROP2 in the PA-ROS pathway, activated ROP2 by itself did not induce ROS production or cell death. The authors suggest that PA acts downstream of activated ROP and that the direct target PA remains to be determined. J. Park, Y. Gu, Y. Lee, Z. Yang, Y. Lee, Phosphatidic acid induces leaf cell death in Arabidopsis by activating the Rho-related small G protein GTPase-mediated pathway of reactive oxygen species generation. Plant Physiol. 134 , 129-136 (2004). [Abstract] [Full Text]

P-glycoprotein expression does not change the apoptotic pathway induced by curcumin in HL-60 cells.

One of the mechanisms responsible for the multidrug resistance (MDR) phenotype of cancer cells is overexpression of so-called ATP-dependent drug efflux proteins: the 170-kDa P-glycoprotein (P-gp) encoded by the MDR1 gene and the 190-kDa multidrug resistance-associated protein 1 encoded by the MRP1 gene. The purpose of the present study was to verify the hypothesis postulating that P-gp expression, apart from enabling drug efflux, confers on the cells resistance to apoptosis by inhibiting caspase-8 and caspase-3. Human HL-60 cells, either drug-sensitive or with the MDR phenotype caused by overexpression of P-gp (HL-60/Vinc) or MRP1 (HL-60/Adr), were treated with the natural dye curcumin at 50 micro M or with UVC to induce apoptosis. Symptoms of cell death were assessed by morphological observation after Hoechst staining, DNA fragmentation was measured by flow cytometry and the TUNEL method, and caspase-8 and caspase-3 activation and cytochrome c release from mitochondria were measured by Western blotting. Curcumin induced cell death in HL-60 cells, both sensitive and with the MDR phenotype, which could be classified as caspase-3-dependent apoptosis, together with cytochrome c release, activation of caspase-3 and oligonucleosomal DNA fragmentation. No active caspase-8 was detected. Also UVC caused caspase-3 activation in both the sensitive and the MDR HL-60 cells. Our findings show that there was no correlation between P-gp expression and resistance to caspase-3-dependent apoptosis induced by curcumin and UVC, at least in HL-60 cells. However, we cannot exclude the possibility of parallel P-gp expression and caspase-3 inhibition in some other cell lines, as cancer cells can acquire many different apoptosis-resistance mechanisms.

Sulfated fucan oligosaccharides elicit defense responses in tobacco and local and systemic resistance against tobacco mosaic virus.

Sulfated fucans are common structural components of the cell walls of marine brown algae. Using a fucan-degrading hydrolase isolated from a marine bacterium, we prepared sulfated fucan oligosaccharides made of mono- and disulfated fucose units alternatively bound by alpha-1,4 and alpha-1,3 glycosidic linkages, respectively. Here, we report on the elicitor activity of such fucan oligosaccharide preparations in tobacco. In suspension cell cultures, oligofucans at the dose of 200 microg ml(-1) rapidly induced a marked alkalinization of the extracellular medium and the release of hydrogen peroxide. This was followed within a few hours by a strong stimulation of phenylalanine ammonia-lyase and lipoxygenase activities. Tobacco leaves treated with oligofucans locally accumulated salicylic acid (SA) and the phytoalexin scopoletin and expressed several pathogenesis-related (PR) proteins, but they displayed no symptoms of cell death. Fucan oligosaccharides also induced the systemic accumulation of SA and the acidic PR protein PR-1, two markers of systemic acquired resistance (SAR). Consistently, fucan oligosaccharides strongly stimulated both local and systemic resistance to tobacco mosaic virus (TMV). The use of transgenic plants unable to accumulate SA indicated that, as in the SAR primed by TMV, SA is required for the establishment of oligofucan-induced resistance.

Poster Sessions BP01: Energy Metabolism. NMR investigation of metabolic alterations in the brain of thiamine‐deficient rats

Thiamine deficiency (TD) results in region‐selective impairment of brain metabolism. Since thiamine is a cofactor for enzymes involved in glucose metabolism, 1H and 13C‐NMR was used to investigate metabolic fluxes through the major pathways of glucose metabolism in vulnerable (medial thalamus, MT; inferior colliculus, IC) and nonvulnerable brain structures of rats made thiamine deficient following treatment with the central thiamine antagonist pyrithiamine vs. pair‐fed controls. Symptomatic stages of TD resulted in decreased glutamate and GABA in MT an IC confirming previous biochemical studies. 13C‐isotopomer analysis revealed decreased de novo synthesis of [4–13C]glutamate (30%p < 0.02) and [2–13C]GABA (60%p < 0.01) in MT and IC consistent with decreased activities of pyruvate‐ and α‐ketoglutarate dehydrogenases. These changes were accompanied by decreased consumption of glucose and increased synthesis of lactate from [1–13C]glucose confirming decreased mitochondrial metabolism. Accumulation of glyceraldehyde‐3‐phosphate suggested inhibition of glucose flux through the thiamine‐deficient enzyme transketolase. Onset of symptoms of TD and significant cell death was accompanied by decreased neuronal marker molecules NAA and NAAG in MT. Focal lactate accumulation resulting from decreased activities of mitochondrial thiamine‐dependent enzymes appears to play a key role in the pathogenesis of selective neuronal cell death in TD. [funded by CIHR Canada].

Resistance to fungal pathogens triggered by the Cf9‐Avr9 response in tomato and oilseed rape in the absence of hypersensitive cell death

summary In tomato and related species, the Cf9 resistance gene induces hypersensitive cell death and activates downstream defence pathways upon recognition of the Avr9 elicitor. We investigated whether the Cf9-Avr9 response without hypersensitive cell death symptoms increases resistance to several fungi. A low Avr9 dose that does not cause hypersensitive cell death was injected in Cf9 tomato and transgenic Cf9 oilseed rape plants. Subsequently, the injected leaves were infected with different fungal pathogens. The disease development of Botrytis cinerea was delayed in Cf9 tomato when the pathogen was inoculated on, or around, the Avr9 injection site. Disease development of Leptosphaeria maculans and Sclerotinia sclerotiorum was delayed on Cf9 oilseed rape plant parts located around the Avr9 injection site. Disease development of Oidium lycopersicum in Cf9 tomato or Erysiphe polygoni in Cf9 oilseed rape was not restricted on leaves injected with Avr9. The Avr9 injection induced systemic resistance to L. maculans and E. polygoni in Cf9 oilseed rape. F(1)(Cf9xAvr9) oilseed rape plants, obtained from crosses of transgenic Cf9x transgenic Avr9 oilseed rape, exhibited higher levels of resistance to L. maculans and E. polygoni but not to S. sclerotiorum, than wild-type plants. F(1)(Cf9xAvr9) plants treated with benzothiadiazole (BTH) did not show elevated levels of expression of some pathogenesis-related genes but developed higher levels of resistance to L. maculans than BTH-treated wild-type plants. This report demonstrates that the hypersensitive cell death which is associated with the Cf9-Avr9 response is not required for quantitative disease resistance.

Effect of Curcumin on the Apoptosis of Rodent and Human Nonproliferating and Proliferating Lymphoid Cells

Curcumin, a major active component of turmeric, has been recognized as an anticarcinogenic agent because of its propensity to induce apoptosis in vivo and in vitro. Previously, we showed that curcumin protects cells against oligonucleosomal DNA fragmentation and induces a novel apoptosis-like pathway in Jurkat cells (Piwocka et al. Exp Cell Res 249, 299-307, 1999). Here, we have studied the ability of curcumin to induce cell death in other human and rodent transformed as well as normal cells. Normal cells were quiescent or stimulated to proliferate. We showed that 50 μM pigment is able to induce cell death in all studied cells, but cell death symptoms varied for different cells. All the cells died as assessed by the TdT-mediated UTP nick end labeling method or trypan blue exclusion test. No one type of cells showed oligonucleosomal DNA fragmentation (DNA "ladder") due to curcumin action, although in HL-60 cells, we were able to observe sub-G1 formation and caspase-3 activation. Together, these data showed that curcumin induces cell death in all tested cells that can be classified as apoptosis-like, and only in HL-60 cells can it be recognized as classical apoptosis.

Early Symptom Development and Histological Changes Associated with Migration of Bursaphelenchus xylophilus in Seedling Tissues of Pinus thunbergii.

In order to clarify the mechanism of pine wilt caused by the pinewood nematode (PWN), Bursaphelenchus xylophilus, nematode migration in tissues and disease symptoms in Pinus thunbergii seedlings were investigated. One-year-old seedlings were inoculated with different pathogenic isolates of PWN under two different temperatures. At an early stage of symptom development, a virulent isolate of PWN multiplied in both bark and xylem and was distributed in cortical resin canals, cortical tissue, and xylem resin canals at 30°C. Cell death and disease symptoms developed in both bark and xylem. The virulent isolate of PWN at 25°C and the avirulent isolate of PWN at 30°C were distributed mainly in cortical resin canals, but rarely in xylem resin canals and cortical tissue. Disease symptoms and cell death occurred in cortical resin canals and rarely occurred in other tissues. These results demonstrated that the virulent isolate of PWN at low temperature and avirulent nematodes could not easily migrate to xylem resin canals and cortical tissue. It was shown that cell death and early symptom development coincided with PWN migration and, therefore, PWN invasion induces cell death and early symptom development.

Cellular responses to postsegregational killing by restriction-modification genes.

Plasmids that carry one of several type II restriction modification gene complexes are known to show increased stability. The underlying mechanism was proposed to be the lethal attack by restriction enzyme at chromosomal recognition sites in cells that had lost the restriction modification gene complex. In order to examine bacterial responses to this postsegregational cell killing, we analyzed the cellular processes following loss of the EcoRI restriction modification gene complex carried by a temperature-sensitive plasmid in an Escherichia coli strain that is wild type with respect to DNA repair. A shift to the nonpermissive temperature blocked plasmid replication, reduced the increase in viable cell counts and resulted in loss of cell viability. Many cells formed long filaments, some of which were multinucleated and others anucleated. In a mutant defective in RecBCD exonuclease/recombinase, these cell death symptoms were more severe and cleaved chromosomes accumulated. Growth inhibition was also more severe in recA, ruvAB, ruvC, recG, and recN mutants. The cells induced the SOS response in a RecBC-dependent manner. These observations strongly suggest that bacterial cells die as a result of chromosome cleavage after loss of a restriction modification gene complex and that the bacterial RecBCD/RecA machinery helps the cells to survive, at least to some extent, by repairing the cleaved chromosomes. These and previous results have led us to hypothesize that the RecBCD/Chi/RecA system serves to destroy restricted "nonself" DNA and repair restricted "self" DNA.