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

Adult ; Female ; Hepatitis C, Chronic ; virology ; Humans ; Liver Cirrhosis ; virology ; Male ; Middle Aged ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics ; Viral Core Proteins ; physiology

The purpose of the present study was to determine the effects of recombinant human interleukin-6 (rhIL-6) on the Bcl-2 and Bax expression and apoptosis after anoxia-reoxygenation in cultured rat hippocampal neurons. The control and rhIL-6 treated hippocampal neurons cultured for 12 d were exposed to anoxia environment (90% N2+10% CO2) for 2 and 4 h and then were reoxygenated for 24 and 72 h. The expression of Bcl-2 and Bax was revealed immunocytochemically using the antiserum against Bcl-2 and Bax. The apoptosis was examined by the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nickel end labeling (TUNEL) method and flow cytometric analysis. The results showed that in cultured hippocampal neurons the Bcl-2 expression decreased while Bax expression and the percentage of apoptotic neurons increased after anoxia-reoxygenation compared with those before anoxia. In comparison with the control, after anoxia-reoxygenation the Bcl-2 expression in hippocampal neurons was higher than that in rhIL-6 group; however the Bax expression and the percentage of the apoptosis were decreased in rhIL-6 group. It is suggested that rhIL-6 may play a role in protecting neurons from the damage induced by anoxia-reoxygenation.
Animals ; Apoptosis ; drug effects ; Cell Hypoxia ; drug effects ; physiology ; Cells, Cultured ; Hippocampus ; cytology ; Interleukin-6 ; pharmacology ; Neurons ; drug effects ; physiology ; Proto-Oncogene Proteins ; biosynthesis ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; Rats ; Rats, Wistar ; Recombinant Proteins ; pharmacology ; bcl-2-Associated X Protein

The cell-surface expression and functional status of the CD95/Fas antigen on primitive hematopoietic progenitors isolated from human cord blood were studied. The CD34(+) cells freshly isolated from cord blood displayed low CD95 expression, combinations of cytokines such as SCF+FL upregulated the expression of CD95 in CD34(+) cells. Tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) further increased the CD95 expression induced by positive cytokines. The functional status of CD95-mediated apoptosis was analysed by incubation of CD34(+) cells in the presence of anti-CD95 monocloned antibody (McAb). The effect of anti-CD95 McAb was measured by viable cells counting, flow cytometry, LTC-IC and CFU-C assays. Viable cells and CFU-C numbers were 31.9 +/- 11.2 and 43 +/- 2.0 respectively, the rate of apoptosis was 42.9 +/- 12.4 in the presence of anti-CD95 McAb and TNF-alpha or IFN-gamma. However, growth factor deprivation or the early-acting cytokine such as SCF and FL cross-linking of CD95 lead to low apoptosis of CD34(+) cells. The correlation of increased intracytoplasmic levels of Bcl-2 and the presence of CD95 on surface of CD34(+) cells suggests that Bcl-2 may be involved in protecting against CD95-mediated apoptosis of cord blood CD34(+) cell.
Antibodies ; immunology ; Antigens, CD34 ; analysis ; Apoptosis ; Female ; Fetal Blood ; metabolism ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; physiology ; fas Receptor ; biosynthesis ; immunology ; physiology

The purpose of the present study was to explore the seizure-induced changes in Bad (Bcl-2-associated death protein), 14-3-3, phosphoBad, Bcl-2 and Bcl-XL expression in the rat model of focal limbic seizure. Unilateral intra-amygdaloid injection of kainic acid (KA) was made to induce seizure. Electroencephalogram (EEG) and regional cerebral flow (r-CBF) were monitored continuously. Diazepam (30 mg/kg) was administered to terminate the seizure. The apoptotic and surviving neurons in the hippocampus were observed by terminal deoxynucleotidyl transferrase-mediated dUTP nick end labeling (TUNEL) and cresyl violet staining, the expression of Bad, 14-3-3, phosphoBad, Bcl-2 and Bcl-XL were detected with immunofluorescence, Western blot and immunoprecipitation. The results showed that TUNEL-positive neurons appeared at 8 h and reached maximum at 24 h following seizure cessation within the ipsilateral CA3 subfield of the hippocampus. Seizure induced the dephosphorylation of Bad and the dissociation of Bad from its chaperone protein 14-3-3 and subsequent dimerization of Bad with Bcl-XL. The expression of phosphoBad decreased and Bcl-2 increased. There was little change in r-CBF after the seizure. These results suggest that seizure leads to a dephosphorylation of Bad and an upregulation of Bcl-2. Dephosphorylation of Bad may be injurious while the upregulation of Bcl-2 may be protective to the brain damage induced by seizures, but not related with r-CBF.
Amygdala ; physiology ; Animals ; Epilepsies, Partial ; chemically induced ; metabolism ; Hippocampus ; metabolism ; Kainic Acid ; Male ; Microinjections ; Phosphorylation ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Rats ; Up-Regulation ; bcl-Associated Death Protein ; metabolism

In order to study the potential effects of exogenous WT1 gene isoform on apoptosis in leukemia cell line NB4 and its possible molecular mechanisms, the eukaryotic expression recombinant vector (pCB6(+)/WTA) containing full-length human WT1 isoform (WTA: -17aa/-KTS) cDNA and the vacant vector-alone were introduced into the leukemia cell line NB4 respectively by electroporation. The WTA mRNA and protein in cells were detected by RT-PCR and Western blot. Binding of Annexin V were tested by flow cytometry and agarose gel electrophoresis to verify whether exogenous WTA could induce apoptosis of NB4 cells. Expressions of p21, p53, bcl-2, bcl-XL and c-myc genes were determined by semi-quantitative RT-PCR after introducing recombinant vectors into the NB4 cells. The results showed that in exposure to As(2)O(3) at 0.8 micromol/L for 48 hours, the NB4/WTA cells exhibited the morphological hallmarks of apoptosis, the marked DNA ladder shown by gel electrophoresis, and the enhanced apoptosis rate marked by Annexin V. RT-PCR showed an increase in p21 and c-myc genes expression, a decrease in bcl-2 and a relative constant expression of p53, bcl-XL in NB4/WTA cells. It is concluded that the introduction and expression of exogenous WTA gene can lead to apoptosis of NB4/WTA cells by down-regulating the Bcl-2 gene expression and up-regulating the p21 and c-myc genes expression.
Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Leukemia ; genetics ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Proto-Oncogene Proteins c-myc ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Tumor Suppressor Protein p53 ; genetics ; WT1 Proteins ; genetics ; metabolism ; physiology ; bcl-X Protein ; genetics

To evaluate the expressions of proliferative antigen Ki-67 and apoptosis-antagonizing protein Bcl-2 as well as their clinical significance, immunohistochemistry staining with SAP was used to detect Ki-67 antigen and Bcl-2 protein in 18 cases of children with acute lymphoblastic leukemia (ALL) and 43 cases of adults with ALL. The results showed that the levels of Ki-67 and Bcl-2 expression in children with ALL were lower than that in adults, but only Bcl-2 expression had significant difference. Both in children and in adults, the levels of Ki-67 expression in T-ALL and My(+) ALL were higher than that in B-ALL and null-ALL. The highest complete remission rate (CR) was seen in the group with lower expression of both indexes (Ki-67 and Bcl-2). The lowest CR rate was seen in the group with higher expression of both indexes. It is concluded that the levels of Ki-67 and Bcl-2 expression in children and adults with ALL were closely related with the subtype of ALL and chemotherapeutic effects.
Adolescent ; Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Apoptosis ; physiology ; Child ; Child, Preschool ; Female ; Humans ; Ki-67 Antigen ; biosynthesis ; Male ; Middle Aged ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; drug therapy ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis

delta12-Prostaglandin (PG) J2 is known to elicit an anti-neoplastic effects via apoptosis induction. Previous study showed delta12-PGJ2-induced apoptosis utilized caspase cascade through cytochrome c-dependent pathways in HeLa cells. In this study, the cellular mechanism of delta12-PGJ2- induced apoptosis in HeLa cells, specifically, the role of two mitochondrial factors; bcl-2 and apoptosis-inducing factor (AIF) was investigated. Bcl-2 attenuated delta12-PGJ2-induced caspase activation, loss of mitochondrial transmembrane potential (delta psi m), nuclear fragmentation, DNA laddering, and growth curve inhibition for approximately 24 h, but not for longer time. AIF was not released from mitochondria, even if the delta psi m was dissipated. One of the earliest events observed in delta12-PGJ2-induced apoptotic events was dissipation of delta psi m, the process known to be inhibited by bcl-2. Pre-treatment of z-VAD- fmk, the pan-caspase inhibitor, resulted in the attenuation of delta psi m depolarization in delta12-PGJ2- induced apoptosis. Up-regulation of Sox-4 protein by delta12-PGJ2 was observed in HeLa and bcl-2 overexpressing HeLa B4 cell lines. Bcl-2 overexpression did not attenuate the expression of Sox-4 and its expression coincided with other apoptotic events. These results suggest that delta12-PGJ2 induced Sox-4 expression may activate another upstream caspases excluding the caspase 9-caspase 3 cascade of mitochondrial pathway. These and previous findings together suggest that delta12-PGJ2-induced apoptosis in HeLa cells is caspase-dependent, AIF-independent events which may be affected by Sox-4 protein expression up-regulated by delta12-PGJ2.
Amino Acid Chloromethyl Ketones/pharmacology ; Antineoplastic Agents/*pharmacology ; Apoptosis/drug effects/*physiology ; Caspases/physiology ; Cytochromes c/physiology ; Female ; Flavoproteins/metabolism/*physiology ; Hela Cells ; High Mobility Group Proteins/*physiology ; Humans ; Membrane Proteins/metabolism/*physiology ; Mitochondria/metabolism/physiology ; Prostaglandin D2/*pharmacology ; Protein Transport/physiology ; Proto-Oncogene Proteins c-bcl-2/biosynthesis/*physiology ; Research Support, Non-U.S. Gov't ; Trans-Activation (Genetics) ; Trans-Activators/*physiology

OBJECTIVETo observe the effect of KLF6 on prostate cancer cell line PC-3 by transgenic method.

METHODSWe obtained KLF6 cDNA by RT-PCR method from the liver cell, transfected plasmid pEGFP-C, recombinated with KLF6 into PC-3 cells, and used them as a transfection group and a control group. MTT, flow cytometer and immunocytochemical methods were used to observe the effect of anti-oncogene wild type KLF6 on prostate cancer cell line PC-3 by transgenic method for 48 hours.

RESULTSAfter transfected into PC-3 cells, KLF6 enhanced growth suppression, (30.0 +/- 5.4)% in the transfection group and 0% in the control, P < 0.01, apoptosis, (24.3 +/- 2.3)% in the transfection group and (5.2 +/- 0.7)% in the control, P < 0.01, the down-regulation of the expression of cyclin D1, (25.3 +/- 3.7)% in the transfection group and (38.5 +/- 4.6)% in the control, P < 0.05 and Bcl-2, (18.7 +/- 3.2)% in the transfection group, and (41.8 +/- 5.9)% in the control, P < 0.01 in PC-3 cells. It also decreased the ratio of the cell phase G2/M, increased the ratio of G0/G1 from (58.6 +/- 7.3)% in the control to (80.0 +/- 9.8)% in the transfection group, P < 0.05.

CONCLUSIONPC-3 cells transfected with wild type KLF6 can enhance its growth suppression and apoptosis. It shows great potential for the gene therapy of androgen-independent carcinoma of the prostate.

Apoptosis ; physiology ; Cell Cycle ; physiology ; Cell Line, Tumor ; Cyclin D1 ; biosynthesis ; Down-Regulation ; Flow Cytometry ; Humans ; Immunohistochemistry ; Kruppel-Like Factor 6 ; Kruppel-Like Transcription Factors ; genetics ; physiology ; Male ; Prostatic Neoplasms ; metabolism ; pathology ; Proto-Oncogene Proteins ; genetics ; physiology ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection

BACKGROUNDUncontrolled cell division is one of the hallmarks of tumor growth. Researches have been focused on numerous molecules involved in this process. Cyclins are critical regulatory proteins of cell cycle progression and/or transcription. The present study aimed to investigate the anti-proliferative effect of cyclin L2, and to define its growth regulatory mechanisms using human lung adenocarcinoma cell line A549.

METHODSHuman cyclin L2 was transfected into human lung adenocarcinoma cells (A549 cell), and was expressed in a mammalian expression vector pcDNA3.1. The effects and mechanisms of the cyclin L2 in cell growth, cell cycle analysis and apoptosis were studied by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), flow cytometry or Western blot, respectively.

RESULTSOverexpression of cyclin L2 inhibited the growth of A549 cells. Cell cycle analysis in cells transfected with pCCNL2 revealed an increment in proportion in G0/G1 phase ((68.07 +/- 4.2)%) in contrast to (60.39 +/- 2.82)% of the cells transfected with mock vector. Apoptosis occurred in (7.25 +/- 0.98)% cells transfected with pCCNL2, as compared with (1.25 +/- 0.21)% of the mock vector control group. Cyclin L2-induced-G0/G1 arrest and apoptosis involved upregulation of caspase-3 and downregulation of Bcl-2 and survivin.

CONCLUSIONThe results indicate that overexpression of cyclin L2 protein may promote efficient growth inhibition of human lung adenocarcinoma cells by inducing G0/G1 cell cycle arrest and apoptosis.

Apoptosis ; Caspase 3 ; biosynthesis ; Cell Cycle ; Cell Line, Tumor ; Cyclins ; genetics ; physiology ; Humans ; Lung Neoplasms ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; antagonists & inhibitors ; Transcription Factors ; genetics ; physiology ; Transfection