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

Apoptosis is necessary for the development and maturation of Leydig cells. However, increased apoptosis results the decline of testosterone production, which may increase germ cell apoptosis and the possibility of infertility. There are several aspects contributing to Leydig cell apoptosis such as ethane dimethanesulphonate (EDS), glucocorticoid, developmental stage and some hormones including FSH, LH/hCG and testosterone. A number of genes are involved in the regulation of Leydig cells apoptosis. It was reported that SCF/c-kit, Bcl-2 and Bcl-xl inhibited the apoptosis while caspase-3, Fas, Bax and clusterine stimulated it.
Animals ; Apoptosis ; Caspase 3 ; Caspases ; physiology ; Humans ; Leydig Cells ; cytology ; Male ; Proto-Oncogene Proteins c-bcl-2 ; physiology ; Stem Cell Factor ; physiology ; bcl-X Protein

OBJECTIVETo explore the molecular mechanism of apoptosis induced by enterovirus 71 (EV71) infection.

METHODSThe effects of EV71 on Rhabdomyosarcoma (RD) cell viability were detected by CCK8 assay. EV71-induced apoptosis on RD cells were detected by Hoechst 33342 staining and Western blot targeting Caspase 3, 8 and PARP. Bax conformational change was detected by immunoprecipitation with Bax 6A7 antibody.

RESULTSEV71 decreased the viability of RD cells and induces the activation of Caspase 3, 8 and PARP. Bax expression increases in RD cells after EV71 infection, and Bax conformational change also can be detected after EV71 infection.

CONCLUSIONOur study reveals that EV71 induces Caspase-dependent apoptosis by Bax conformational change.

Apoptosis ; Caspases ; physiology ; Cell Line, Tumor ; Enterovirus A, Human ; pathogenicity ; Humans ; Rhabdomyosarcoma ; pathology ; virology ; bcl-2-Associated X Protein ; physiology

The purpose of study was to explore the possible functions of Bcl-xL in the glucosamine sulfate-induced apoptosis of chronic myeloid leukemia K562 cells. Light microscopy and Wright-Giemsa staining were used to investigate the morphologic evidences for apoptosis of K562 cells induced by glucosamine sulfate (GS); immunofluorescence was used to observe the translocation of cathepsin D and cytochrome C during the apoptosis; Western blot was performed to detect the expression of Bcl-xL, Bid, Bax in K562 cells treated by GS. The results showed that many vacuoles were observed in the cytoplasma of the K562 cells treated by GS; fluorescent signals of cathepsin D and cytochrome were fransformed from granules to disperse form by using immunofluorescence; the expression of Bcl-xL was found down-regulated in K562 cells treated by GS, but not in the cells pre-treated with pepstatin A; the significant changes were not detected in expression of Bax and Bid protein before or after apoptosis. It is concluded that Bcl-xL protein may mediate relationship between cathepsin D and mitochondia pathway, Cathepsin D may play an important role in the GS inducing apoptosis of K562 cells through downregulation of Bcl-xL expression.
Apoptosis ; drug effects ; physiology ; BH3 Interacting Domain Death Agonist Protein ; metabolism ; Blotting, Western ; Cathepsin D ; metabolism ; Cytochromes c ; metabolism ; Fluorescent Antibody Technique ; Glucosamine ; pharmacology ; Humans ; K562 Cells ; bcl-2-Associated X Protein ; metabolism ; bcl-X Protein ; metabolism ; physiology

OBJECTIVETo investigate the changes of bcl-2 gene family and the molecular mechanism of neuronal apoptosis following traumatic brain injury (TBI) in rats.

METHODSMale Sprague-Dawley (SD) rats were subjected to lateral fluid percussion brain injury (FPBI) of moderate severity. The bcl-x(L) and bax mRNA expression was detected by reverse transcription polymerase chain reaction (RT-PCR). In addition to morphological evidence of apoptosis, terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labeling (TUNEL) histochemistry was used to identify the DNA fragmentation in situ at both light and electron microscope levels, whereas characteristic internucleosomal DNA fragmentation of apoptosis was demonstrated by DNA gel electrophoresis.

RESULTSThe apoptotic response to trauma was regionally distinct and may be involved in both acute and delayed cell death. The bcl-x(L) mRNA expression of the impact site was significantly lower (67.42%+/-7.54%) than that of the ipsilateral hemisphere at 6 hours after injury (P<0.01). The decrease of bcl-x(L) mRNA expression preceded apoptosis at 24 hours after injury. The bax mRNA expression rose slowly, doubled at 3 days after injury and returned to the sham level slowly.

CONCLUSIONSDecreased expression of bcl-x(L) mRNA and increased expression of bax mRNA coincides with apoptosis following brain injury. The bcl-2 gene family is involved in neuronal apoptosis after TBI, and the changes of mRNA expression of the family members lead the neuronal cells to apoptosis.

Animals ; Apoptosis ; physiology ; Brain Injuries ; metabolism ; DNA Fragmentation ; In Situ Nick-End Labeling ; Male ; Proto-Oncogene Proteins ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; bcl-X Protein

OBJECTIVEDuring the critical period of brain development, insufficiency of thyroid hormone results in severe mental retardation and learning deficit. This study was designed to investigate the effects of hypothyroidism on apoptosis and the expression of Bcl-2 and Bax gene in the developing rat hippocampus neurons and to explore the mechanism of brain development regulated by thyroid hormone.

METHODHypothyroidism was induced by administration of propylthiouracil (PTU, 50 mg/d) solution to the dams from gestational day 15 by gavage. Pups from both hypothyroid and control groups were harvested at postnatal day 1 (P1), P5, P10 and P15, respectively. Blood samples were collected at the time of death for the determination of thyroid hormone. Serum free tri-iodothyronine (FT(3)) and free thyroxine (FT(4)) were measured by using chemoluminescence. Hippocampus collected from the control and hypothyroid pups were examined under light and transmissional electron microscopy. Measurement of DNA fragmentation was carried out by agarose gel electrophoresis. The expression of Bcl-2 and Bax protein in the developing rat hippocampus neurons was performed by Western blotting.

RESULTSSignificantly lower circulating FT(4) and FT(3) levels confirmed the hypothyroid status of the experimental pups. The shrunken and contracted degenerations increased in hippocampus neurons of hypothyroid pups under light microscopy. Enhanced apoptotic cells were found in hippocampus neurons of hypothyroid pups under transmission electron microscopy, especially at P10 and P15. Extensive DNA fragmentation was seen throughout development in hippocampus of hypothyroid pups, but not in the euthyroid controls except for basal level at P10. The expression of Bcl-2 in the hippocampus neurons of hypothyroid pups was significantly lower than that of euthyroid controls at all stages of development (P1: 1.95 +/- 0.27 vs. 2.59 +/- 0.19, P < 0.05, P5: 1.86 +/- 0.24 vs. 2.47 +/- 0.17, P < 0.05, P10: 1.29 +/- 0.22 vs. 1.86 +/- 0.28, P < 0.05 and P15: 1.21 +/- 0.27 vs. 2.18 +/- 0.17, P < 0.01, respectively). The relative amount of expression varied significantly with age in the control pups. The level of Bcl-2 was high in hippocampus neurons of euthyroid at P1, P5, and decreased significantly at P10, and showed a trend of recovery at P15. Similar age-related variation in the expression of Bcl-2 gene was observed in the hypothyroid group at P1, P5 and P10, but the level was maintained low at P15. The expression of Bax in the hippocampus neurons of hypothyroid pups was significantly higher than that of control pups at all stages of development (P1: 1.69 +/- 0.14 vs. 1.24 +/- 0.23, P < 0.05, P5: 1.78 +/- 0.16 vs. 1.29 +/- 0.17, P < 0.05, P10: 1.92 +/- 0.18 vs. 1.45 +/- 0.14, P < 0.05 and P15: 1.86 +/- 0.14 vs. 1.51 +/- 0.12, P < 0.05, respectively). The ratio of Bcl-2/Bax in hippocampus neurons of hypothyroid pups was lower than that of age-matched controls (P1: 1.16 +/- 0.17 vs. 2.12 +/- 0.35, P < 0.05, P5: 1.05 +/- 0.16 vs. 1.94 +/- 0.36, P < 0.05, P10: 0.68 +/- 0.17 vs. 1.29 +/- 0.16, P < 0.05 and P15: 0.67 +/- 0.19 vs. 1.45 +/- 0.22, P < 0.01, respectively).

CONCLUSIONThyroid hormone significantly prevents apoptosis of hippocampus neurons. Congenital hypothyroidism increases not only the extent but also the duration of apoptosis by down-regulation of the anti-apoptotic gene Bcl-2 and maintaining a high level of the pro-apoptotic gene Bax.

Animals ; Animals, Newborn ; Apoptosis ; physiology ; Down-Regulation ; Hippocampus ; metabolism ; Hypothyroidism ; physiopathology ; Neurons ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; bcl-2-Associated X Protein ; metabolism

OBJECTIVE: To investigate the process of apoptosis in lungs and liver induced by crushing hindlimbs of rat, and study the mechanism of crush injury. METHODS: The rat experimental model of hindlimbs crush injury was established. The cell apoptosis in lungs and liver was detected by TUNEL assay, and the expression of Bax, Bcl-2 and caspase-3 apoptin was examined by immunohistochemistry. RESULTS: Compared with the control group, the partial muscle injury of rat's hindlimbs was more serious with more apoptosis observed in lungs and liver (P < 0.05). The expression of Bax was up-regulated and Bcl-2 was down-regulated, whereas caspase-3 expression was activated (P < 0.05). CONCLUSION The cell apoptosis has increased significantly in lungs and liver after crush injury of hindlimbs in rat. The correlation factor released during tissue injury may mediate apoptosis process.
Animals ; Apoptosis/physiology* ; Caspase 3/metabolism* ; Genes, bcl-2 ; Hindlimb/injuries* ; Immunohistochemistry ; Liver/physiopathology* ; Lung/physiopathology* ; Rats ; Up-Regulation ; bcl-2-Associated X Protein

OBJECTIVETo investigate apoptosis in XG-7, a human myeloma cell line, induced by IL-6 deprivation and the function of three anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-kappa(L), Mcl-1) in the apoptotic process.

METHODSApoptosis in XG-7 myeloma cells induced by IL-6 withdrawal was determined by flow cytometry with propidium iodide (PI) nuclear staining. Expressions of three Bcl-2 proteins in XG-7 cells were monitored by immunoblotting assay.

RESULTSIn the absence of IL-6 for a certain time, a significant percentage of apoptiotic XG-7 cells can be observed, as well as down-regulated expression of one of the three anti-apoptotic proteins (Mcl-1) in XG-7 cells. IL-6 re-stimulation in XG-7 cells following cytokine removal up-regulated the expression of Mcl-1 and inhibited cell apoptosis.

CONCLUSIONMcl-1,instead of Bcl-2 and Bcl-kappa(L), plays an important role in IL-6 deprivation induced apoptosis in XG-7 human myeloma cells.

Apoptosis ; Humans ; Interleukin-6 ; physiology ; Multiple Myeloma ; pathology ; Myeloid Cell Leukemia Sequence 1 Protein ; Neoplasm Proteins ; analysis ; physiology ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Tumor Cells, Cultured ; bcl-X Protein

Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality globally. It accounts for the majority of primary liver cancer cases. Amyloid precursor protein (APP), a cell membrane protein, plays a vital role in the pathogenesis of Alzheimer's disease, and has been found to be implicated in tumor growth and metastasis. Therefore, to understand the relationship between APP and 5-fluorouracil (5-FU) resistance in liver cancer, Cell Counting Kit-8, apoptosis and cell cycle assays, western blotting, and reverse transcription-quantitative polymerase chain reaction (qPCR) analysis were performed. The results demonstrated that APP expression in Bel7402-5-FU cells was significantly up-regulated, as compared with that in Bel7402 cells. Through successful construction of APP-silenced (siAPP) and overexpressed (OE) Bel7402 cell lines, data revealed that the Bel7402-APP^751-OE cell line was insensitive, while the Bel7402-siAPP cell line was sensitive to 5-FU in comparison to the matched control group. Furthermore, APP overexpression decreased, while APP silencing increased 5-FU-induced apoptosis in Bel7402 cells. Mechanistically, APP overexpression and silencing can regulate the mitochondrial apoptotic pathway and the expression of apoptotic suppressor genes (B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl)). Taken together, these results preliminarily revealed that APP overexpression contributes to the resistance of liver cancer cells to 5-FU, providing a new perspective for drug resistance.
Amyloid beta-Protein Precursor/physiology* ; Apoptosis/drug effects* ; Carcinoma, Hepatocellular/drug therapy* ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; Fluorouracil/pharmacology* ; Humans ; Liver Neoplasms/drug therapy* ; Mitochondria/physiology* ; Proto-Oncogene Proteins c-bcl-2/genetics* ; bcl-X Protein/genetics*

AIMTo explore the mechanisms of hypoxic preconditioning on protecting cultured astrocytes from hypoxia injury.

METHODSCultured astrocytes were divided randomly into several groups: control(C), hypoxia(H) and hypoxic preconditioning (HP). Cells MTT metabolic activity, qualitation of apoptosis and modality to explore the protection effects of hypoxic preconditioning. Immunocytochemistry of Bcl-2 and Bax to explore the mechanisms of hypoxic preconditioning on protecting astrocytes from hypoxia.

RESULTSCompared with H group there was marked increase of MTT metabolic activity in HP48 and HP72 groups. Immunocytochemistry of Bcl-2 and Bax showed that compared with H group, expression of Bcl-2 was increased in HP group, while expression of Bax was decreased in HP group.

CONCLUSIONHypoxic preconditioning can protect astrocytes from hypoxia. One possible mechanism maybe concerned with inhibition of Bax and maintain of Bcl-2 to depress apoptosis procedure.

Adaptation, Physiological ; physiology ; Animals ; Animals, Newborn ; Apoptosis ; physiology ; Astrocytes ; cytology ; physiology ; Cell Hypoxia ; Cells, Cultured ; Ischemic Preconditioning ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; metabolism