Characterization of the programmed cell death induced by metabolic products of Alternaria alternata in tobacco BY-2 cells.

Alternaria alternata has received considerable attention in current literature and most of the studies are focused on its pathogenic effects on plant chloroplasts, but little is known about the characteristics of programmed cell death (PCD) induced by metabolic products (MP) of A. alternata, the effects of the MP on mitochondrial respiration and its relation to PCD. The purpose of this study was to explore the mechanism of MP-induced PCD in non-green tobacco BY-2 cells and to explore the role of mitochondrial inhibitory processes in the PCD of tobacco BY-2 cells. MP treatment led to significant cell death that was proven to be PCD by the concurrent cytoplasm shrinkage, chromatin condensation and DNA laddering observed in the cells. Moreover, MP treatment resulted in the overproduction of reactive oxygen species (ROS), rapid ATP depletion and a respiratory decline in the tobacco BY-2 cells. It was concluded that the direct inhibition of the mitochondrial electron transport chain (ETC), alternative pathway (AOX) capacity and catalase (CAT) activity by the MP might be the main contributors to the MP-induced ROS burst observed in tobacco BY-2 cells. The addition of adenosine together with the MP significantly inhibited ATP depletion without preventing PCD; however, when the cells were treated with the MP plus CAT, ROS overproduction was blocked and PCD did not occur. The data presented here demonstrate that the ROS burst played an important role in MP-induced PCD in the tobacco BY-2 cells.

Different enhancement of senescence induced by metabolic products of Alternaria alternata in tobacco leaves of different ages.

The purpose of this study was to explore the mechanisms by which Alternaria alternata damages tobacco (Nicotiana tabacum) leaves. Treatment with A. alternata metabolic products enhanced senescence in leaves of different ages, as indicated by the significant decrease in chlorophyll, soluble protein, photosynthetic O(2) evolution and catalase (CAT, EC 1.11.1.6) activity as well as an increase in H(2)O(2) content. The induction of senescence by A. alternata metabolic products increased as the age of the leaves increased. A. alternata metabolic products greatly influenced the behavior of photosystem II (PSII) in the leaves: oxygen evolving complex (OEC) activity and electron transport from primary quinone electron acceptor of PS II (Q(A)) to secondary quinone electron acceptor of PS II (Q(B)) were both significantly inhibited. This inhibition also became more pronounced in older leaves. In vitro experiments revealed that, without the influence of natural senescence, the A. alternata metabolic products directly inhibited the activity of a commercial CAT solution and inhibited photosynthetic O(2) evolution, which resulted in excess PSII excitation pressure and an overaccumulation of H(2)O(2) in leaf segments. These results suggest that the significant declines in photosynthesis and CAT activity induced by the metabolic products of A. alternata were important contributors to the overaccumulation of reactive oxygen species (ROS), which accelerated senescence in tobacco leaves. The fact that the enhancement of senescence was getting more pronounced with the age of tobacco leaves might be related to the fact that older leaves already had higher H(2)O(2) levels and less antioxidant activity as reflected in lower CAT activity.

[Effects of transcription factor GATA-2 on transcriptive regulation of iASPP gene].

iASPP can prompt the cell proliferation and inhibit the apoptosis of many cells. There are putative binding sites of transcription factor GATA-2 upstream of iASPP transcription start site. GATA-2 plays an important role in the proliferation and differentiation of hematopoietic stem cells (HSC) and progenitors. This study was aimed to explore the role of GATA-2 protein in iASPP gene transcription. Firstly, the expression of iASPP and GATA-2 protein in some leukemia cell lines was detected by Western blot. Second, The expressive vector of pCMV5-GATA2 and the luciferase reporter vectors containing possible binding sites of GATA-2 were constructed and co-transfected into HEK293 and CV-1 cells. Then the luciferase activity was assayed by luminometer. Also, ChIP assays were performed to further confirm the specific binding of GATA-2 to iASPP promoter. The results showed that GATA-2 was overexpressed in most cell lines with high level of iASPP. GATA-2 exhibited a significant effect on luciferase activity of reporter gene iASPP and in a dose-dependant manner. The relative luciferase activity was up-regulated to about two-fold of the empty vector control when the transfection dose of pCMV5-GATA2 plasmid was increased to 100 ng. While the effect was more significant in CV-1 cells and showed a 6.7-fold increase. The ChIP assay demonstrated the in vivo specific binding of GATA-2 to iASPP. The binding sites of GATA2 were located between nt -361 ∼ -334 in upstream of iASPP gene transcription start site. It is concluded that transcription factor GATA-2 can bind with the cis-regulatory region of the iASPP promoter and up-regulate iASPP expression.

[Detection of heterogeneity and evolution of subclones in t(8;21) AML by QM-FISH].

OBJECTIVE: To explore the heterogeneous subclones in acute myeloid leukemia (AML) with t(8;21) by quantitative multicolor- fluorescence in situ hybridization (QM-FISH), and to figure out whether there is putative ancestral relationship among different subclones. METHODS: Bacterial artificial chromosomes (BAC) clones that contain the targeted genes including AML1, ETO, WT1, p27 and c-kit were searched in the data base UCSC Genome Bioinformatics. Multicolor FISH probes were prepared by linking fluorescein labeled dUTP or dCTP to targeted genes by nick translation. Bone marrow mononuclear cells from t (8;21) AML patients are dropped on to the wet surface of glass slides after hypotonic treatment and fixation. After hybridization, the fluorescence signals were captured by Zeiss fluorescence microscope. The copy number of AML1, ETO, WT1, p27, c- kit and the AML1-ETO fusion gene in AML1-ETO positive cells was counted. The cells with same signals were defined as a subclone. Various subclones were recorded and their proportions were calculated, and their evolutionary relationship was deduced. The subclones in matched primary and relapsed samples were compared, the evolution of dominant clones were figured out and the genomic abnormality that is associated with relapse and drug resistance were speculated. RESULTS: In this study, 36 primary AML with t(8;21) cases and 1 relapsed case paired with the primary case were detected. In these 36 primary cases, 4 cases (11.1%) acquired additional AML1-ETO fusion signal, 3(8.3%) had additional AML1 signal, 4(11.1%) had additional ETO signal, 20(55.6%) had additional WT1 signal, 15(41.7%) had additional p27 signal and 14(38.9%) had additional c-kit signal. In addition, 10(27.8%) displayed AML1 signal deletion, and such an aberration represents statistic significance in male patients. It seems that male patients usually accompany AML1 signal deletion. Of 36 cases, 28(77.8 %) harbored at least 2 subclones (ranged from 2 to 10). According to the genetic signature of subclones, we can assemble a putative ancestral tree, and the genetic architecture is linear or branching. In particular, the clonal architecture of the relapsed sample exhibited significant clonal evolution compared to its paired sample at diagnosis, including proportion changes in dominant clone, subclone disappearance and appearance of new dominant clones. CONCLUSION: Genomic abnormality is very diverse in t(8;21) AML. Subclones have linear or complex branching evolutionary histories, and clonal architecture is dynamic.

[Effect of metformin on acute promyelocytic leukemia cell line NB4 and its mechanism].

The purpose of this study was to investigate the effect and molecular mechanism of metformin (Met) on biological characteristics of acute promyelocytic leukemia (APL) cell line NB4. NB4 cells were treated with various concentrations of Met for different time, MTT method was used to detect cell proliferation, the alteration of cell apoptosis was analyzed by flow cytometry, and the change of cell adhesion ability was examined by cell adhesion assay. NB4 cells were pretreated with U0126, a specific inhibitor for extracellular signal-regulated kinase (ERK) phosphorylation, ERK phosphorylation was assessed by Western blot analysis, apoptosis and cell adhesion ability were evaluated by flow cytometry and cell adhesion test respectively. The results showed that Met could inhibit the cell proliferation, induce the cell apoptosis and increase the ability of cell adhesion. The pretreatment of NB4 cells with 5 µmol/L U0126 could effectively inhibit the phosphorylation of ERK, and reduce cell apoptosis and adhesion induced by 5 mmol/L Met. It is concluded that Met can inhibit the proliferation and promote the apoptosis and adhesion of NB4 cells. MEK/ERK signaling pathway may be one of the molecular mechanisms of metformin on NB4 cells.

[Study on expression of PTEN gene and its pseudogene PTENP1 in acute leukemia and correlation between them].

OBJECTIVE: To investigate the expression of PTEN (phosphatase and tension homology deletion on chromosome 10, PTEN) and its pseudogene PTENP1 in acute leukemia (AL) and correlation between them, and to explore the role of PTENP1 on the PTEN expression in AL cells. METHODS: PTEN and PTENP1 mRNA expression were evaluated in bone marrow (BM) samples from 138 newly diagnosed AL patients and 15 healthy controls by quantitative real-time RT-PCR (qRT-PCR). pCDH1-PTENP1 3'UTR-GFP lentivirus vectors were constructed. 293T cells were transfected by calcium phosphate precipitation to produce retrovirus. HL-60 cell line was infected with the retroviral vectors expressing pCDH1-GFP and pCDH1-PTENP1 3'UTR-GFP respectively. The flow cell sorter was used to sort the HL-60 with GFP positively expressed. The mRNA expression of PTEN and PTENP1 was detected by qRT-PCR, the expression of PTEN protein by western blot, and the impact of PTENP13'UTR on the proliferation of HL-60 cells by MTT assay. RESULTS: AML patients showed significantly lower PTEN and PTENP1 mRNA expression in BM compared to healthy controls. Correlation analysis showed that the expression of PTEN and PTENP1 mRNA were positively correlated (P < 0.05). The 108 cases of PTENP1(+) AML were classified according to the prognostic classification of 2011 NCCN Clinical Practice Guidelines in AML, there was no difference among different subgroups. HL-60 cell line was infected with the retroviral vectors expressing pCDH1-GFP (control group) and pCDH1-PTENP1 3'UTR-GFP respectively. Compared with the control group, PTENP1 mRNA level of HL-60 infected with the retroviral vectors expressing pCDH1-PTENP1 3'UTR-GFP increased significantly, and PTEN mRNA level also increased. While the PTEN protein level and the cell growth rate of the PTENP1 3'UTR group didn't change significantly. CONCLUSION: PTEN and PTENP1 mRNA expression level of BM cells from AL patients is significantly lower. There is a positive correlation between expression of PTEN and PTENP1 mRNA. PTENP1 may regulate the expression of PTEN in mRNA level.