Microcystin-RR-induced apoptosis in tobacco BY-2 cells

Our previous studies showed that microcystin-RR could induce oxidative damage in plant cells as they do with animal cells. However, whether microcystin can induce plant cell apoptosis is still unknown. In this study, the morphological changes of tobacco BY-2 suspension cells exposed to microcystin-RR were observed under light microscopy and transmission electron microscopy, and apoptosis was clearly distinguished by intense perinuclear chromatin margination, condensation of nuclear chromatin after 6d exposure of 50 mg/L (about 50 mu M) microcystin-RR. We also found that microcystin-RR can induce tobacco cell apoptosis in a dose- and time-dependent manner with flow cytometry analysis. Our study provides the first evidence that microcystins can induce plant cell apoptosis. (c) 2006 Elsevier Ltd. All rights reserved.

Characterization and expression analysis of TNF-related apoptosis inducing ligand (TRAIL) in grass carp Ctenopharyngodon idella

TRAIL (Apo2 ligand) described as a type II transmembrame protein belonging to the TNF superfamily can induce apoptotic cell death in a variety of cell types. In the present study, a putative cDNA sequence encoding the 299 amino acids of TRAIL (GC-TRAIL) and its genomic organization were identified in grass carp Ctenopharyngodon idella. The predicted GC-TRAIL sequence showed 44 and 41% identities to chicken and human TRAILs, respectively. In a domain search, a tumor necrosis factor homology domain (THD) was identified in the C-terminal portion of TRAILs. The GC-TRAIL gene consists of five exons, with four intervening introns, spaced over approximately 4 kb of genomic sequence. Analysis of GC-TRAlL promoter region revealed the presence of a number of putative transcription factor binding sites, such as Sp1, NF-kappaB, AP-1, GATA, NFAT, HNF, STAT, P53 and IRFI sequences which are important for the expression of other TNF family members. Phylogenetic analysis placed GC-TRAIL and the putative zebrafish (Danio rerio) TRAIL obtained from searching the zebrafish database into one separate cluster near mammalian TRAIL genes, but apart from the reported zebrafish TRAIL-like protein, indicating that the GC-TRAIL is an authentic fish TRAIL. Expression analysis revealed that GC-TRAIL is expressed in many tissues, such as in gills, liver, trunk kidney, head kidney, intestine and spleen. (c) 2005 Elsevier B.V. All rights reserved.

Induction of apoptosis in a carp leucocyte cell line infected with turbot (Scophthalmus maximus L.) rhabdovirus

A rhabdovirus was observed from the diseased turbot (Scophthalmus maximus L.) with lethal syndrome. In this study, a carp leucocyte (CLC) cell line was used to investigate the infection process and cell death mechanism occurring during the virus infection. Strong cytopathogenic effect (CPE) and the morphological changes, such as extreme chromatin condensation, nucleus fragmentation, and apoptotic body formation, were observed under fluorescence microscopy after DAPI staining in the infected CLC cells. Transmission electron microscopy analysis showed cell shrinkage, plasma membrane blebbing, cytoplasm vacuolization, chromatin condensation, nuclear breakdown and formation of discrete apoptotic bodies. The bullet-shaped nucleocapsids were measured and ranged in size from 110 to 150 nm in length and 40 to 60 nm in diameter. And therefore the virus is called Scophthalmus maximus rhabdovirus (SMRV). Agarose gel electrophoresis analysis of the DNA extracted from infected cells showed typical DNA ladder in the course of SMRV infection. Flow cytometry analysis of SMRV infected CLC cells detected apoptotic peak in the virus infected CLC cells. Virus titre analysis and electron microscopic observation revealed that the virus replication fastigium was earlier than that of the apoptosis occurrence. No apoptosis was observed in the CLC infected with UV-inactivated SMRV. All these supported that SMRV infected CLC cells undergo apoptosis and the virus replication is necessary for apoptosis induction of CLC cells. (C) 2004 Published by Elsevier B.V.

Effects of subretinal implant materials on the viability, apoptosis and barrier function of cultured RPE cells

Background: Subretinal microphotodiode array (MPDA) is a type of visual prosthesis used for the implantation in the subretinal space of patients with progressive photoreceptor cell loss. The present study aimed to evaluate the effect of materials for MPDA on the viability, apoptosis and barrier function of cultured pig retinal pigment epithelium (RPE) cells.Methods: Primary culture of pig RPE cells was performed and 24 pig eyes were used to start RPE culture. The third passage of the cultures was plated on different materials for MPDA and MPDAs. The tetrazolium dye-reduction assay (MTT) was used to determine RPE cell viability. Flow cytometry was measured to indicate the apoptosis rates of RPE cells on different materials. RPE cells were also cultured on microporous filters, and the transepithelial resistance and permeability of the experimental molecule were measured to determine the barrier function.Results: The data from all the methods indicated no significant difference between the materials groups and the control group, and the materials tested showed good biocompatibility.Conclusions: The materials for MPDA used in the present study had no direct toxicity to the RPE cells and did not release harmful soluble factors that affected the barrier function of RPE in vitro.

potential mechanisms involved in resistant phenotype of MCF-7breast carcinoma cells to ionizing radiation induced apoptosis

In the present study, we investigated the mechanisms of apoptosis resistance and the roles of the phosphorylation of BRCA1, p21, the Bax/Bcl-2 protein ratio and cell cycle arrest in IR-induced apoptosis in MCF-7 cells. X-irradiation, in particular at low dose (1 Gy), but not carbon ion irradiation, had a significant antiproliferative effect on the growth of MCF-7 cells. 1 Gy X-irradiation resulted in G1 and G2 phase arrest, but 4 Gy induced a significant G1 block. In contrast, carbon ion irradiation resulted in a significant accumulation in the G2 phase. Concomitant with the phosphorylation of H2AX induced by DNA damage,carbon ion irradiation resulted in an approximately 1.9–2.8-fold increase in the phosphorylation of BRCA1 on serine residue 1524, significantly greater than that detected for X-irradiation. Carbon ion irradiation caused a dramatic increase in p21 expression and drastic decrease in Bax expression compared with X-irradiation. The data implicated that phosphorylation of BRCA1 on serine residue 1524 might,at least partially, induce p21 expression but repress Bax expression. Together, our results suggested that the phosphorylation of BRCA1 at Ser-1524 might contribute to the G2 phase arrest and might be an upstream signal involved in preventing apoptosis signal via upregulation of p21 and downregulation of the Bax/Bcl-2 ratio.

Cell cycle and apoptosis alteration of human hepatocarcinoma cells by subclinical-dose C-12(6+)-beam irradiation

Objective The purpose of this study is to investigate the effect of subdinical-dose C-12(6+)-beam irradiation on cell cycle and cell apoptosis in hepatocarcinoma cells. Materials and methods The HepG(2) cells were exposed to 0-2.0 Gy of either the C-12(6+) beam or a gamma-ray. Cell survival was detected by clonogenic assay. Cell cycle was determined by flow-cytometry analysis. The apoptosis was monitored by fluorescence microscope with DAPI staining. p53 and p21 expression were detected by Western blot. Results The G(0)/G(1) cells in the irradiated groups were significantly more than those in the control (P<0.05). The C-12(6+)-ion irradiation had a greater effect on the cell cycle of HepG(2) cells (including promoting G(1)-phase and G(2)-phase arrest) than gamma-ray irradiation. The apoptotic cells induced by C-12(6+) beam were significantly more numerous than those induced by gamma-ray (P<0.05). The carbon ions had a stronger effect on p53 and p21 expression than the gamma-ray irradiation. The survival fractions for cells irradiated by C-12(6+) beam were significantly smaller than those irradiated by gamma-ray (P<0.05).

Study on the apoptosis and Bcl-2/Bax expression of human hepatoma SMMC-7721 cells after exposure to heavy-ion and X-ray irradiations

This study is aimed at observing the apoptosis and Bcl-2/Bax gene expression of mammalian cells following heavy-ion and X-ray irradiations. Exponentially growing human hepatoma SMMC-7721 cells cultured in vitro were irradiated with a C-12 ion beam of 50 MeV/u (corresponding to a LET value of 44.56 keV/mu m) from Heavy Ion Research Facility in Lanzhou (HIRFL) at doses varying from 0 to 3 Gy. The X-ray irradiation (8 MV) was performed in the therapy unit of the General Hospital of the Lanzhou Military Area. Survival fractions of irradiated cells at various doses were measured by means of MTT assay. Apoptotic cells after irradiation were analyzed with fluorescence microscope and flow cytometer (FCM). Immuno-histological assay were applied to detect the expression of Bcl-2/Bax genes in the irradiated cells. The survival fraction of SMMC-7721 cells decreased gradually (vs. control p<0.05) with increasing the dose of the carbon ion beam more obviously than X-ray irradiation, and the carbon ion irradiation efficiently induced cell apoptosis and significantly promoted the expression of Bax gene while Bcl-2 gene expression was restrained. High-LET heavy ion beam would induce cell apoptosis effectively than low-LET X-ray, and the apoptosis rate is correlated with the transcription of Bcl-2/Bax and the ratio of Bcl-2/Bax in human hepatoma SMMC-7721 cells after irradiation to heavy ion beam.

Survivin表达在高LET射线诱导细胞凋亡中作用机理的初步研究；A Preliminary Study on Action Mechanisms of Survivin Expression in Cell Apoptosis Induced by High-LET Radiation

先前的研究表明,肿瘤细胞中survivin的高表达与细胞对高传能线密度(LET)射线的辐射抗性相关。研究了survivin表达在高LET射线诱导的细胞凋亡中的作用,发现抑制survivin表达对高LETC离子辐射诱导的Bcl-2和Bax表达没有明显的影响。在高LET射线辐照中,survivin可能通过抑制caspase-3和-9活性的途径,抑制了细胞凋亡。

Hemizygosity for Atm and Brca1 influence the balance between cell transformation and apoptosis

Background: In recent years data from both mouse models and human tumors suggest that loss of one allele of genes involved in DNA repair pathways may play a central role in genomic instability and carcinogenesis. Additionally several examples in mouse models confirmed that loss of one allele of two functionally related genes may have an additive effect on tumor development. To understand some of the mechanisms involved, we examined the role of monoallelic loss or Atm and Brca1 on cell transformation and apoptosis induced by radiation. Methods: Cell transformation and apoptosis were measured in mouse embryo fibroblasts (MEF) and thymocytes respectively. Combinations of wild type and hemizygous genotypes for ATM and BRCA1 were tested in various comparisons. Results: Haploinsufficiency of either ATM or BRCA1 resulted in an increase in the incidence of radiation-induced transformation of MEF and a corresponding decrease in the proportion of thymocytes dying an apoptotic death, compared with cells from wild-type animals. Combined haploinsufficiency for both genes resulted in an even larger effect on apoptosis. Conclusions: Under stress, the efficiency and capacity for DNA repair mediated by the ATM/BRCA1 cell signalling network depends on the expression levels of both proteins.

Targeted RNA interference of cyclin A(2) mediated by functionalized single-walled carbon nanotubes induces proliferation arrest and apoptosis in chronic myelogenous leukemia K562 cells

Cyclin A(2) plays critical role in DNA replication, transcription, and cell cycle regulation. Its overexpression has been detected and related to many types of cancers including leukemia, suggesting that suppression of cyclin A(2) would be an attractive strategy to prevent tumor progression. Herein, we apply functionalized single wall carbon nanotubes (f-SWNTs) to carry small interfering RNA (siRNA) into K562 cells and determine whether inhibition of cyclin A(2) would be a potential therapeutic target for chronic myelogenous leukemia.

Knocking-Down Cyclin A(2) by siRNA Suppresses Apoptosis and Switches Differentiation Pathways in K562 Cells upon Administration with Doxorubicin

Cyclin A(2) is critical for the initiation of DNA replication, transcription and cell cycle regulation. Cumulative evidences indicate that the deregulation of cyclin A(2) is tightly linked to the chromosomal instability, neoplastic transformation and tumor proliferation. Here we report that treatment of chronic myelogenous leukaemia K562 cells with doxorubicin results in an accumulation of cyclin A(2) and follows by induction of apoptotic cell death. To investigate the potential preclinical relevance, K562 cells were transiently transfected with the siRNA targeting cyclin A(2) by functionalized single wall carbon nanotubes. Knocking down the expression of cyclin A(2) in K562 cells suppressed doxorubicin-induced growth arrest and cell apoptosis. Upon administration with doxorubicin, K562 cells with reduced cyclin A(2) showed a significant decrease in erythroid differentiation, and a small fraction of cells were differentiated along megakaryocytic and monocyte-macrophage pathways. The results demonstrate the pro-apoptotic role of cyclin A(2) and suggest that cyclin A(2) is a key regulator of cell differentiation.

Rhein induces apoptosis and cell cycle arrest in human hepatocellular carcinoma BEL-7402 cells

Rhein, an anthraquinone derivative of rhubarb, inhibits the proliferation of various human cancer cells. In this paper, we focused on studying the effects of rhein on human hepatocelluar carcinoma BEL-7402 cells and further understanding the underlying molecular mechanism in an effort to make the potential development of rhein in the treatment of cancers. Using MTT assay and flow cytometry, we demonstrate a critical role of rhein in the suppression of BEL-7402 cell proliferation in a concentration- and time-dependent manner. The increase of apoptosis rate was observed after incubation of BEL-7402 cells with rhein at 50-200 mu M for 48 hours, and the cells exhibit typical apoptotic features including cellular morphological change and chromatin condensation. Moreover, rhein-induced cell cycle S-phase arrest. Additionally, after rhein treatment, expression levels of c-Myc gene were decreased, while those of caspase-3 gene were increased in a dose-dependent manner by using real-time PCR assay. The results demonstrate for the first time that cell cycle S-phase arrest is one of the mechanisms of rhein in inhibition of BEL-7402 cells. Rhein plays its role by inducing cell cycle arrest via downregulation of oncogene c-Myc and apoptosis through the caspase-dependent pathway. It is expected that rhein will be effective and useful as a new agent in hepatocelluar carcinoma treatment in the future.

Emodin-induced apoptosis in human breast cancer BCap-37 cells through the mitochondrial signaling pathway

Emodin, a natural anthraquinone compound isolated from the rhizome of rhubarb, is reported to suppress the growth of tumor in many clinical situations. In this study, we focused on the effect of emodin in human breast cancer BCap-37 cells and further understand the underlying molecular mechanism in treating breast cancer. Using MTT assay and flow cytometry, we demonstrated the critical role of emodin in the suppression of the proliferation of BCap-37 cells based on a concentration- and time-dependent manner. The increase of apoptotic rate was also observed after incubation of BCap-37 cells on emodin at 20 mu M and 50 mu M for 48 h. The cells exhibited typical apoptotic features including cellular morphological change, chromatin condensation and membrane blebbing. The results of the study further showed that Bcl-2 level decreased, while Bax and cytosolic cytochrome c levels in sample cells increased after the emodin treatment by using Western blot. The decline in the Bcl-2/Bax ratio and the increase of cytosolic cytochrome c concentration were consistent with the increase of the apoptotic ratio. The results strongly suggest that the disruption of the mitochondrial signaling pathway was involved in emodin-induced apoptosis in BCap-37 cells.

Endothelial cells release phenotypically and quantitatively distinct microparticles in activation and apoptosis

Background: Endothelial cells (EC) shed endothelial microparticles (EMP) in activation and apoptosis. Objectives: We compared the antigenic expression of EMP species released during activation as compared to apoptosis, in three cell lines. Methods: EC from renal and brain microvascular (MiVEC) and coronary macrovascular (MaVEC) origin were incubated with TNF-alpha to induce activation, or deprived of growth factors to induce apoptosis. Antigens expressed on EMP and EC were assayed flow cytometrically and included constitutive markers (CD31, CD51/61, CD105), inducible markers (CD54, CD62E and CD106), and annexin V binding. Results: It was found that in apoptosis, constitutive markers in EMP were markedly increased (CD31>CD105), with a concomitant decrease in expression in EC. Annexin V EC surface binding and annexin V+ EMP were more sharply increased in apoptosis than in activation. In contrast, in activation, inducible markers in EMP were markedly increased in both EMP and EC (CD62E>CD54>CD 106). Coronary MaVEC released significantly less EMP than MiVEC. Conclusion: EC release qualitatively and quantitatively distinct EMP during activation compared to apoptosis. Analysis of EMP phenotypic signatures may provide clinically useful information on the status of the endothelium. (C) 2003 Elsevier Science Ltd. All rights reserved.