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OBJECTIVE@#To construct a recombinant lentiviral expression vector pCDH-Daxx-EGFP to investigate the effect of Daxx on the proliferation of vascular smooth muscle cells (VSMCs).@*METHODS@#The recombinant lentiviral expression vector pCDHDaxx-EGFP was constructed using PCR-based accurate synthesis method. After identification by sequencing and enzyme digestion, the recombinant lentiviral vector was contransfected into 293T cells with lentivirus packaging vector. The recombinant lentivirus particles were collected and purified to infect VSMCs, whose expression of Daxx was detected with Western boltting. The cells infected with the empty vector pCDH-EGFP or pCDH-Daxx-EGFP were incubated in serum-free medium or in the presence of angiotensin Ⅱ (AngⅡ). The cell viability was determined with MTT assay, and the cell cycle changes were analyzed with flow cytometry. The cell migration ability was assessed using a scratch wound healing assay. The expression of p-Akt protein in the cells was detected using Western blotting.@*RESULTS@#Double enzyme digestion and sequencing confirmed successful construction of the recombinant plasmid. Compared with the cells infected with the empty vector, the cells infected with pCDH-Daxx-EGFP exhibited significantly increased expressions of Daxx protein ( < 0.05). AngⅡ treatment of the cells infected with the pCDH-Daxx-EGFP, as compared with the cells infected with the empty vector, significantly lowered the cell viability, S phase cell ratio and cell migration ability ( < 0.05), and significantly decreased the expression level of p-Akt protein ( < 0.05).@*CONCLUSIONS@#We successfully constructed the recombinant lentiviral vector pCDH-Daxx-EGFP and overexpressed Daxx in primary cultured VSMCs using this vector. Daxx overexpression can inhibit AngⅡ-induced proliferation and migration in VSMCs probably by regulating p-Akt protein.
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BACKGROUND: Death domain-associated protein (DAXX), originally identified as a pro-apoptotic protein, is now understood to be either a pro-apoptotic or an anti-apoptotic factor with a chromatin remodeler, depending on the cell type and context. This study evaluated DAXX expression and its clinical implications in squamous cell carcinoma of the esophagus. METHODS: Paraffin-embedded tissues from 60 cases of esophageal squamous carcinoma were analyzed immunohistochemically. An immune reaction with more than 10% of tumor cells was interpreted as positive. Positive reactions were sorted into 2 groups: reactions in 11%–50% of tumor cells and reactions in more than 51% of tumor cells, and the correlations between expression and survival and clinical prognosticators were analyzed. RESULTS: Forty-three of the 60 cases (71.7%) showed strong nuclear DAXX expression, among which 19 cases showed a positive reaction (31.7%) in 11%–50% of tumor cells, and 24 cases (40.0%) showed a positive reaction in more than 51% of tumor cells. A negative reaction was found in 17 cases (28.3%). These patterns of immunostaining were significantly associated with the N stage (p=0.005) and American Joint Committee on Cancer stage (p=0.001), but overall survival showed no significant difference. There were no correlations of DAXX expression with age, gender, or T stage. However, in stage IIB (p=0.046) and stage IV (p=0.014) disease, DAXX expression was significantly correlated with survival. CONCLUSION: This investigation found upregulation of DAXX in esophageal cancer, with a 71.7% expression rate. DAXX immunostaining could be used in clinical practice to predict aggressive tumors with lymph node metastasis in advanced-stage disease, especially in stages IIB and IV.
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Carcinoma de Células Escamosas , Cromatina , Neoplasias Esofágicas , Esófago , Articulaciones , Ganglios Linfáticos , Metástasis de la Neoplasia , Regulación hacia ArribaRESUMEN
The effects of two different histone deacetylase (HDAC) inhibitors, sodium butyrate (NAB) and trichostatin A (TSA),on apoptosis of human leukemic cells in vitro and the molecular mechanisms were investigated. The experiments were divided up 5 groups: control group, NaB group, TSA group,NaB+Z-VAD-FMK group and TSA+Z-VAD-FMK group. The apoptosis rate was determined by mor-phological analysis and flow cytomytry. The expression of Daxx, Bcl-2, and Bcl-xl proteins was de-tected by Western blot. NaB and TSA could induce the apoptosis of HL-60 and K562 cells, and Z-VAD-FMK caused a marked decrease in apoptosis induced by HDAC inhibitors. HDAC inhibitors could down-regulate the expression of Daxx protein, but had no significant influence on the expression of Bcl-2 and Bcl-xl proteins. The results suggested that NaB and TSA induce distinct caspase-dependent apoptosis of human leukemic cells through down-regulating the expression of Daxx protein in vitro.
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In order to study the effects and the possible mechanisms of Daxx overexpressed in HepG2 to hydrogen peroxide treatment, and to search new targets for cancer chemotherapy, HepG2cells were transfected using lipofectamine 2000, and selected by treatment with G418. Stable cell lines were confirmed by reverse transeriptase polymerase chain reaction (RT-PCR) targeting vector gene. Experiments include the following groups: (1) control group (non-transfected cells); (2) transfected with empty vector (HepG2/GFP cells); and (3) transfected with pEGFP-C1-Daxx (HepG2/GFP-Daxx cells). After incubation with hydrogen peroxide (H2O2) for 24 h, cellular viability was analyzed by MTT, and cellular apoptosis was measured by flow cytometric analysis. Gene expression at protein level was detected by Western blot. The RT-PCR results showed that Daxx RNA in cells transfected with pEGFP-C1-Daxx was increased significantly compared with that in the HepG2/GFP cells. Fluorescence microscopy revealed that Daxx protein was localized in the nuclei. Hydrogen peroxide was used to induce apoptosis of HepG2 cells and observed that the hydrogen peroxide decreased the viability of HepG2 cells in concentration-dependent pattern. The IC50 values in three groups (Normal cells, HepG2/GFP cells and HepG2/GFP-Daxx cells) were 0.72, 0.76, and 0.49 mmol/L respectively. The apoptotic ratio was significantly higher in HepG2/GFP-Daxx cells as compared to the other two groups. HepG2/GFP-Daxx cell incubated with hydrogen peroxide, showed a significant increase in the activation of caspase-3 and JNK as compare with the other groups. Over-expression of Daxx facilitated HepG2 cells apoptosis induced by hydrogen peroxide. Furthermore, there may be a synergetic relation with apoptosis and increase of JNK activity.
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Objective:To find out the role of JNK/SAPK signaling-transduction pathway in the effect of elemene against hepatocarcinoma, offering the clue to ilustrate the molecular mechanisms of antitumor effects of elemene. Methods: The detection of the distribution of elemene in Hca-F cells was detected by gas chomatography and apoptotic changes in elemene treated. SMMC7721 cells were examined by TEM. After elemene treatment, the activation of JNK/SAPK in HepG2 cellls and the DAXX gene expression in SMMC7721 cells were detected by Western blotting and RT-PCR respectively. Results: Gas chomatography showed that elemene was detected at 8. 42 minute. The SMMMC7721 cells treated by elemene for 3 hours began to show typical apoptotic changes . The JNK/SAPK activity in HepG2 cell treated with heat shock was the highest of all groups and the group treated with elemene was the next and the control group is the lowest one. There was no DAXX gene expression in SMMC7721 cells treated with elemene. Conclusion: Elemene can diffuse into cells. Tumor cell apoptosis treated with elemene may be induced by JNK/SAPK activating and DAXX signal pathway may not play key role in JNK/SAPK activation induced by elemene.
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Daxx is found in the nucleus where it localizes to PML oncogenic domains (PODs). Its multiple domains can interact proteins involved in transcriptional regulation and apoptotic signal transduction. In addition, Daxx is associated with viral infection、tumorigenesis and embryonic development.