OBJECTIVETo investigate the role of antiapoptotic Bcl-x(L) protein in megakaryocyte differentiation and maturation.

METHODSRNA interference was used to block the expression of Bcl-x(L) when K562 cells were induced to differentiate into megakaryocyte (CD61 + cells) by PDBu, and the expression of Bcl-x(L) was evaluated with flow cytometry and reverse transcription polymerase chain reaction (RT-PCR). The CD34 + cell fraction was positively isolated by using the MiniMACS system from normal bone marrow. Immunochemical staining and flow cytometry were used to detect the expression of Bcl-x(L) in the differentiation (CD41 + cells) of CD34 + cells induced by trombopoietin (TPO).

RESULTSAmong K562 cells induced by PDBu, the percentage of CD6L + cells rapidly increased in 24 hours and maintained at a high positive level in 72 hours. When exposured to si-Bcl-x(L), the percentage of CD6 1 + cells only slightly increased in 72 hours. The expression of Bcl-x(L) mRNA was significantly decreased after transfection compared with that of control group, and Bcl-x(L) protein expression decreased correspondingly. After the CD34 + bone marrow cells having been treated with TPO for 5 days to 20 days, the Bcl-x(L)-megakaryocytes increased as the culture time prolonged, and there was a strong expression of Bcl-x(L) in immature megakaryocyte and an obviously decreased expression in degenerating megakaryocytes maturation.

CONCLUSIONSIncreased expression of antiapoptotic Bcl-x(L) may be essential to mature megakaryocyte. The down-regulation of antiapoptotic Bcl-x(L) in mature megakaryocyte may be crucial to platelets formation.

Cell Differentiation ; Humans ; K562 Cells ; Megakaryocytes ; physiology ; RNA Interference ; bcl-X Protein ; biosynthesis ; genetics ; physiology

The efficiency and safe range of Lipofectamine2000 (LF2000)/bcl-xl applied in human keratocytes, the optimal ratio of LF2000/bcl-xl and the bcl-xl gene expression in human keratocytes were investiaged. By using trypan-blue staining, the effects of LF2000 and bcl-xl on the survival rate of the cultured human keratocytes were measured respectively. By using semi-quantitative RT-PCR, the efficiency and the expression of LF2000-mediated bcl-xl transfection into keratocytes were examined. The results showed that the survival rate of human keratocytes had no signficant change in the presence of LF2000 (20 microg/ml) or bcl-xl (10 microg/ml) for 24 h. LF2000 could effectively mediate the transfection of exogenous gene bcl-xl into human keratocytes. The best transfection efficiency could be obtained when the ratio of bcl-xl/LF2000 was 1:8. One day after transfection, the positive cells for bcl-xl could be detectable, and the positive rate reached the peak-on the posttransfection day 3 (48.3%), then gradually decreased. Fifteen days after transfection, there were few positive cells. It was suggested that LF2000 could effectively transfer the exogenous gene bcl-xl into human keratocytes without obvious toxicity during a concentration range. LF2000/bcl-xl may be likely to play an important role in gene therapy of human keratocytes.
Cations/administration & dosage ; Cornea/cytology ; Gene Therapy ; Keratinocytes/cytology ; Keratinocytes/*metabolism ; Liposomes ; Transfection ; bcl-X Protein/biosynthesis ; bcl-X Protein/*genetics

OBJECTIVETo investigate the role of reactive oxygen species (ROS) and ROS-caused mitochondrial transmembrane potential loss in sodium selenite-induced apoptosis in NB4 cells.

METHODSROS production was measured by ROS-specific probe DCFH-DA. Sodium selenite mitochondrial transmembrane potential loss was evaluated by flow cytometry with Rh123 staining. Protein levels of cytochrome C, Bid, Bcl-xl, and Bax were measured by Western blot using protein-specific antibodies. NB4 cells were pre-incubated by MnTmPy or BSO before selenite treatment to further confirm the effects of ROS on NB4 cells.

RESULTS20 micromol/L sodium selenite induced ROS production and mitochondrial transmembrane potential loss in NB4 cells time-dependently. Cytochrome C accumulated in cytoplasm after selenite treatment. Sodium selenite also downregulated Bcl-xl and activated Bax and Bid at protein level. Pretreatment with antioxidant MnTmPy almost fully abrogated the proapoptotic effect of sodium selenite prevented the cleavage of Bid protein and in turn the mitochondrail transmembrane potential loss. On the contrary, pretreatment with BSO intensified the mitochondrail transmembrane potential loss induced by sodium selenite.

CONCLUSIONSSodium selenite may induce apoptosis by inducing ROS production in NB4 cells, which leads to the downregulation of Bcl-xl, upregulation of Bax, and cleavage and activation of Bid. Bax and tBid then agregate on mitochondrial membrane, which in turn causes a decrease of mitochondrial transmembrane potential and release of cytochrome C into cytoplasm.

Apoptosis ; BH3 Interacting Domain Death Agonist Protein ; biosynthesis ; Cell Line, Tumor ; Cytochromes c ; metabolism ; Humans ; Membrane Potential, Mitochondrial ; drug effects ; Reactive Oxygen Species ; metabolism ; Sodium Selenite ; pharmacology ; bcl-2-Associated X Protein ; biosynthesis ; bcl-X Protein ; biosynthesis

OBJECTIVETo explore the mechanism of reactive oxygen species (ROS) in manganese chloride (MnCl(2))-induced apoptosis in PC12 cells.

METHODSThe model that MnCl(2) induced apoptosis in PC12 cells was established. The apoptotic effect of MnCl(2) on PC12 cells was analyzed with the MTT, the flow cytometry and the DNA fragmentation. The production of ROS and ATP in MnCl(2)-induced apoptosis of PC12 cells was examined. The influence of MnCl(2) on the expression of bcl-xl, bax and the activity of Caspase 3 was also analyzed.

RESULTSMnCl(2) triggered PC12 cells apoptosis in a dose-and time-dependent manner (P < 0.01). The rate of apoptosis was significantly increased (P < 0.01) when MnCl(2) of 2 mmol/L induced PC12 cells for 36 hours. The production of ROS was increased (P < 0.001) and the quantity of ATP was decreased (P < 0.01) in PC12 cells with the same inducement of MnCl(2). The expression of bcl-xl was inhibited and the bax was activated in this process (P < 0.01). Caspase 3 was also activated (P < 0.01).

CONCLUSIONMnCl(2) induces apoptosis of PC12 cells, which is related to the increase of ROS, the inhibition of the mitochondria and the activation of Caspase 3.

Adenosine Triphosphate ; biosynthesis ; Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Chlorides ; toxicity ; DNA Fragmentation ; drug effects ; Manganese Compounds ; PC12 Cells ; Rats ; Reactive Oxygen Species ; metabolism ; bcl-2-Associated X Protein ; biosynthesis ; bcl-X Protein ; biosynthesis

To explore the effects of vascular endothelial growth factor (VEGF) on the mechanisms of CML pathogenesis, the effect of VEGF on K562 cell apoptosis induced by As(2)O(3) was analyzed through morphologic observation, DNA fragmentation agarose gel electrophoresis and DNA ploidy flow cytometry analysis, and the effect of VEGF on the expression of bcl-X(L), Bax and caspase-3 in K562 cells was determined by Western blot, meanwhile the expression difference between bcl-X(L) and Bax mRNA in above conditions was detected by RT-PCR. The results showed that after VEGF added, the apoptosis of K562 cells reduced, however, there was no significant changes in cell cycle distribution (P > 0.05). At the same time, following the increasing of the concentration of VEGF, expression of mRNA and protein of bcl-X(L) was up-regulated and the expression of Bax protein was down-regulated in K562 cells, and the activation of pro-caspase-3 into caspase-3 was inhibited or reduced. It is concluded that VEGF may suppress the apoptosis of K562 cells through its influence on the bcl-X(L)/Bax expression ratio in K562 cells.
Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Blotting, Western ; Chlorides ; pharmacology ; Flow Cytometry ; Humans ; K562 Cells ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Vascular Endothelial Growth Factor A ; pharmacology ; bcl-2-Associated X Protein ; biosynthesis ; genetics ; bcl-X Protein ; biosynthesis ; genetics

This study was purposed to observe the changes of caspase-3 activity during apoptosis of HL-60 cells induced by an iron chelator, DFO (deferoxamine), and to explore the mechanism underlying apoptosis in HL-60 cells. The HL-60 cells treated with DFO were examined by light microscopy, flow cytometry (FCM) and DNA agarose gel electrophoresis; the activity of caspase-3 was determined by cellular immunohistochemistry; the transcription of the apoptotic gene of bax was detected by hybridization in situ. The results showed that the typical morphological character of apoptosis cells, DNA ladder and FCM assay confirmed that DFO could induce the apoptosis in HL-60 cells. The apoptotic rate increased in dose-and time-dependent manner. When cells had been cultivated with 100 micromol DFO for 12 hours, a few caspase-3 positive cells were found. In the process of time, the rate of caspase-3 positive cells was progressively higher than that in control (P < 0.05), while the level of bax transcription was also higher than that in the control. It is concluded the activation of caspase-3 and gene bax may be involved in the apoptosis of HL-60 cells induced by DFO.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Deferoxamine ; pharmacology ; HL-60 Cells ; Humans ; bcl-2-Associated X Protein ; biosynthesis ; genetics

The study was aimed to explore the apoptosis effect of ouabain on Jurkat cells and its mechanism. MTT method was used to observe the inhibitory effect of ouabain on Jurkat cell proliferation. Apoptosis was detected by using flow cytometry (FCM) and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling reaction method (TUNEL). The protein expressions of Bax, Bcl-2 and active subunits of caspase-3 were measured by Western blot. Activities of caspase-3 were determined by colorimetry method. The results showed that ouabain could induce apoptosis of Jurkat cells, the expression of Bax protein in process of cell apoptosis, caspase-3 activity of Jurkat cells were remarkably enhanced after ouabain treatment. It is concluded that ouabain may induce apoptosis of Jurkat cells due to the activation of caspase-3 resulting from regulation of Bax protein and Bcl-2 gene expressions.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cell Proliferation ; drug effects ; Genes, bcl-2 ; genetics ; Humans ; Jurkat Cells ; Ouabain ; pharmacology ; bcl-2-Associated X Protein ; biosynthesis ; genetics

The study was purposed to explore the effect of adriamycin (ADM) on K562 cells in vitro and the mechanism of expression changes of relevant apoptotic genes and oncogene Gfi-1. The apoptosis was assayed by flow cytometry (FCM) and the DNA electrophoresis; the expression changes of Gfi-1, Bcl-2, bax mRNA and protein were detected by RT-PCR and FCM after K562 cells were treated with different concentrations of ADM for 24 hours. The results showed that when K562 cells were treated with 0 - 2.0 mg/L ADM for 24 hours, the typical apoptotic DNA electrophoresis band of K562 cells were observed with the dose increasing. When concentration of ADM was 0.5 and 2.0 mg/L, the expression of Gfi-1 decreased and the expression of bax increased; when concentration of ADM was 0.5 - 2.0 mg/L, the expression of Bcl-2 was not found to be significantly changed, the levels of Bcl-2 mRNA and protein were of no statistical difference. When dose of ADM was higher than 2.0 mg/L, the percentage of apoptotic K562 cells decreased with cell necrosis. It is concluded that at certain range of concentration, apoptosis or necrosis of K562 cells can be induced by ADM, the percentage of apoptosis, the changes of expression of Bcl-2, bax and Gfi-1 depend on the dose of ADM. The mechanism of apoptosis in K562 cells induced by ADM may be related to suppression of Gfi-1 oncogene and activation of expression of bax gene.
Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; DNA-Binding Proteins ; biosynthesis ; genetics ; Doxorubicin ; pharmacology ; Humans ; K562 Cells ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics ; bcl-2-Associated X Protein ; metabolism

In order to explore whether the member of Bcl-2 gene family, for example, Bcl-2 and Bax, are induced after cerebral ischemia, and whether expression of genes can be modulated by calcium-antagonist, the rat cerebral ischemic models were made by occluding left middle cerebral artery. The expression of Bcl-2 and Bax mRNA was measured by RT-PCR method. After middle cerebral artery occlusion (MCAO), the expression of both Bcl-2 and Bax mRNA were induced. Level of Bcl-2 mRNA increased steadily and level of Bax mRNA increased gradually at first, reached a peak after 24 h, then decreased slowly. After administration of nimodipine, Bcl-2 mRNA was up-regulated in the hippocampus 6 and 24 h after ischemia, while Bax mRNA was down-regulated 6 and 24 h after ischemia. Focal cerebral ischemia can induce proto-oncogenes to express, which was associated with apoptosis. Calcium-antagonist can up-regulate Bcl-2 mRNA and down-regulate Bax mRNA. The increased ratio of Bcl-2 and Bax mRNA may contribute to the anti-apoptic effect of nimodipine. The study indicates that pharmacological modulation of Bcl-2 family member expression could become a new strategy to manage neuronal damage.
Animals ; Apoptosis ; drug effects ; Brain Ischemia ; metabolism ; Calcium Channel Blockers ; pharmacology ; Nimodipine ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; bcl-2-Associated X Protein ; biosynthesis ; genetics

OBJECTIVETo explore the effects of hyperbaric oxygen (HBO) treatment on the neuronal apoptosis at an earlier stage and the expressions of Cytochrome C (Cyt C), Bcl-2 (B-cell lymphoma-2 family) and Bax (Bcl-2 associated X protein) in rat brain tissues after traumatic brain injury (TBI).

METHODSForty adult rats were divided into two groups, i.e., Group A (the rats with untreated TBI) and Group B (rats with HBO treatment after TBI). Sections of brain tissues of these two groups were then detected at 3, 6, 12, 24, 72 hours after TBI by immunohistochemistry and electronmicroscope, respectively.

RESULTSHBO treatment could up-regulate the expression of Bcl-2 within 72 hours, reduce the release of Cyt C from mitochondria, attenuate the formation of dimeric Bax and alleviate the mitochondrial edema within 24 hours after TBI.

CONCLUSIONSHBO treatment can alleviate neuronal apoptosis after TBI by reducing the release of Cyt C and the dimers of Bax and up-regulating the expression of Bcl-2.

Analysis of Variance ; Animals ; Apoptosis ; physiology ; Brain Injuries ; pathology ; therapy ; Cytochromes c ; biosynthesis ; Disease Models, Animal ; Hyperbaric Oxygenation ; Immunohistochemistry ; Male ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; biosynthesis