Ectopically expressed perforin-1 is proapoptotic in tumor cell lines by increasing caspase-3 activity and the nuclear translocation of cytochrome C.

Perforin-1 (PRF), a cytotoxic lymphocyte pore-forming protein, plays an important role in the action of cytotoxic T cells and natural killer cells in that it causes the lysis of abnormal body cells and the elimination of virus-infected cells and tumors. Upon degranulation, PRF inserts itself into the target cell's plasma membrane, forming a pore. The subsequent translocation of pro-apoptotic granzymes (including granzyme B, A, M et al.) into the cytoplasm provides the proteases with access to numerous protein substrates that promote apoptosis after cleavage. These proteases are believed to be the main executioners of target cell apoptosis. Although the PRF and granzyme components are both critical to this process and in some way involved in inducing cell death in target cells, the inhibition of tumor growth could still be efficient in granzyme-deficient mice. It is unclear whether PRF alone can suppress tumors. In this study, we discovered that forced ectopic expression of PRF alone, in the absence of granzymes, could mediate cell death in cancer cells. Notably, transient expression of both full-length and truncated active-form PRF in human Hep G2, SK-BR-3, and HeLa cells was found to induce apparent cell growth inhibition and cell death, as evidenced by chromosome condensation and DNA fragmentation, increased caspase-3 activity, and the release of apoptosis inducing factor (AIF) and cytochrome c from the mitochondria. This PRF-induced cell death could be abrogated by pan-caspase inhibitor (Z-VAD) and mitochondria protector (TAT-BH4). The implication of these results is that ectopically expressed PRF has apoptosis-inducing abilities, and PRF alone is sufficient to induce apoptotic cell death in cells with ectopic expression. Taking this into consideration, our results suggest the possibility of using PRF as a pro-apoptotic gene for tumor therapeutics.

Replication-dependent γ-H2AX formation is involved in docetaxel-induced apoptosis in NSCLC A549 cells.

Docetaxel is a member of the taxane anti-microtubule class of chemotherapeutic agents, which are currently widely used in clinical cancer therapy. However, the anti-tumor mechanisms of docetaxel are not fully understood. Herein we show that docetaxel induces dose-dependent apoptosis in non-small cell lung cancer A549 cells, as detected by Annexin-V positive cells and PARP cleavage, which is via mitochondrial pathway and dependent on caspase-3 activation. Our study on the mechanisms confirms that docetaxel induces dose-dependent accumulation of cells in M phase and acetylation of α-tubulin, marker of tubulin stablization. Furthermore, docetaxel induces replication-dependent γ-H2AX formation which plays a crucial role in docetaxel-triggered apoptosis. The DNA polymerase inhibitor aphidicolin dose-dependently prevents docetaxel-induced γ-H2AX formation, as well as apoptosis. Notably, 0.6 µM APC almost completely blocked docetaxel-induced γ-H2AX formation and apoptosis. In addition, wortmannin pretreatment caused elevated γ-H2AX level, which was accompanied with increased apoptosis. This effect was due to the inhibition of DNA repair process by wortmannin, as down regulation of p21Waf1/Cip1 and DNA repair proteins such as Ku70, Ku80, DNA-PKcs and Rad50, were detected. These data show, for the first time, that the induction of apoptosis by docetaxel requires DNA replication, and replication-mediated DSBs are critical triggers of docetaxel-induced apoptosis.

[Expression and localization of adenovirus-mediated transcriptional factor Runx3 in malignant glioblastoma cells].

AIM: To construct the replicative deficient adenovirus Ad-Runx3 expressing Runx3, and to express it in U251 malignant glioblastoma cells. METHODS: The runx3 gene with a flag tag was amplified by PCR using pCMV5-AML2 as a template, and was confirmed by DNA sequencing. The adenovirus shuttle vector pShuttle-CMV-Runx3 was constructed by introducing runx3 DNA fragment into the sites of Kpn I and Xho I of pShuttle-CMV vector. This recombinant plasmid was linearized by PmeI and electronically transfected into BJ5183 cells to get the recombinant adenovirus vector Ad-Runx3. The recombinant adenovirus expressing Runx3 was infected into U251 malignant glioblastoma cells. The expression of exogenous Runx3 was observed by immonoblotting and its localization was detected by immunostaining using anti-Flag tag antibody. RESULTS: The recombinant adenovirus expressing Runx3 with a Flag tag was constructed and infected into U251 glioblastoma cells. The exogenous Runx3 protein was detected only in the nuclei. CONCLUSION: The recombinant adenovirus expressing Runx3 with a Flag tag is constructed successfully, and the Runx3 protein expressed in the nuclei of infected cells. The study laid a foundation for further research of the function of Runx3 in gliocarcinogenesis.

[Comparison of the effect of arsenic sulfide on apoptosis and hTERT-mRNA expression in two leukemia cell lines].

AIM: To explore the different effect and mechanism of arsenic sulfide on telomerase activity and hTERT-mRNA expression in CML cell lines-K562 and APL cell lines-NB4. METHODS: Telomerase activity was determined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA). The expression of hTERT-mRNA was analyzed by semi-quantitative RT-PCR. Flow cytometry was used to analyze the cell cycle and apoptosis. RESULTS: 0.15-0.6 mg/L arsenic sulfide (72 h)can induce apoptosis and inhibit telomerase activity and hTERT-mRNA expression in NB4 cell. The concentration of arsenic sulfide with the same effect on K562 cell was 0.3-3 mg/L. 0.3 mg/L arsenic sulfide (72 h) can cause the proportion of the NB4 cell in G2/M phase increased, but for K562 cell, The concentration of arsenic sulfide was 1.5 mg/L. CONCLUSION: Telomerase system may be one of the pathway for arsenic sulfide inducing apoptosis of NB4 and K562 cell; G2/M phrase arrest may have correlation with decrease of telomerase activity; The sensitivity of NB4 and K562 cell for arsenic sulfide is different, the mechanism of it need to study more.

The proapoptotic activity of C-terminal domain of apoptosis-inducing factor (AIF) is separated from its N-terminal.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that mediates both NADH-oxidizing and caspase-independent apoptosis. Further, the proapoptotic activity of AIF is located in the C-terminus of AIF, although the precise minimum sequence responsible for apoptosis induction remains to be investigated. In the present study, we generated two truncated AIFs, AIFDelta1-480-FLAG, which is a FLAG-tagged C-terminal peptide comprising amino acids from 481 to 613, and AIF360-480 containing amino acids from 360 to 480 of AIF. We used confocal microscopy to demonstrate that both the truncated proteins are expressed and located in the cytoplasm of transfected cells. AIFDelta1-480 but not AIF360-480 induces apoptosis in transfected cells. We also found that the expression of AIFDelta1-480 could initiate the release of cytochrome c from the mitochondria. The suppression of caspase-9 via siRNA blocked the proapoptotic activity of AIFDelta1-480. Therefore, AIFDelta1-480 is sufficient for inducing caspase-9-dependent apoptotic signaling, probably by promoting the release of cytochrome c. At last, we generated a chimeric immuno-AIFDelta1-480 protein, which comprised an HER2 antibody, a Pseudomonas exotoxin A translocation domain and AIFDelta1-480. Human Jurkat cells transfected with the immuno-AIFDeltal-480 gene could express and secrete the chimeric protein, which selectively recognize and kill HER2-overexpressing tumor cells. Our study demonstrates the feasibility of the immuno-AIFDeltal-480 gene as a novel approach to treating HER2-overexpressing cancers.

The proapoptotic activity of C-terminal domain of apoptosis-inducing factor (AIF) is separated from its N-terminal

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that mediates both NADH-oxidizing and caspase-independent apoptosis. Further, the proapoptotic activity of AIF is located in the C-terminus of AIF, although the precise minimum sequence responsible for apoptosis induction remains to be investigated. In the present study, we generated two truncated AIFs, AIFΔ1-480-FLAG, which is a FLAG-tagged C-terminal peptide comprising amino acids from 481 to 613, and AIF360-480 containing amino acids from 360 to 480 of AIF. We used confocal microscopy to demonstrate that both the truncated proteins are expressed and located in the cytoplasm of transfected cells. AIFΔ1-480 but not AIF360-480 induces apoptosis in transfected cells. We also found that the expression of AIFΔ1-480 could initiate the release of cytochrome c from the mitochondria. The suppression of caspase-9 via siRNA blocked the proapoptotic activity of AIFΔ1-480. Therefore, AIFΔ 1-480 is sufficient for inducing caspase-9-dependent apoptotic signaling, probably by promoting the release of cytochrome c. At last, we generated a chimeric immuno-AIFΔ 1-480 protein, which comprised an HER2 antibody, a Pseudomonas exotoxin A translocation domain and AIFΔ 1-480. Human Jurkat cells transfected with the immuno-AIFΔl-480 gene could express and secrete the chimeric protein, which selectively recognize and kill HER2-overexpressing tumor cells. Our study demonstrates the feasibility of the immuno-AIFΔl-480 gene as a novel approach to treating HER2-overexpressing cancers.

[Experimental study of IFN-alpha and IFN-gamma on reversing ATRA-resistance in MR2 cell line].

AIM: To explore the possibility and the possible mechanism of reversing ATRA-resistance in MR2 cells by using IFN-alpha and IFN-gamma in combination with all-trans retinoic acid (ATRA). METHODS: After MR2 cells(ATRA-resistance cell line) were treated with IFN-alpha, IFN-gamma and ATRA alone or IFN-alpha and IFN-gamma in combination with ATRA respectively, the cell proliferation was tested by MTT colorimetry, the cell differentiation was tested through light microscope, by NBT test and flow cytometry (FCM). The expression of promyelocytic leukemia (PML) protein was observed by indirect immunofluorescence staining. RESULTS: Both IFN-alpha and IFN-gamma could inhibit the proliferation of MR2 cells. The effects were more obviously in both IFN-alpha+ATRA group and IFN-gamma+ATRA group. But there were no significant difference between either IFN-alpha group and IFN-gamma group or IFN-alpha+ATRA group and IFN-gamma+ATRA group (P>0.05). Both IFN could also induce the differentiation of MR2 cells. The effects of IFN-alpha+ATRA group and IFN-gamma+ATRA group were more obvious. However, the differentiation of MR2 cells induced by IFN-gamma+ATRA group was more higher than that by IFN-alpha+ATRA group (P<0.05). Both IFN could induce the expression of PML protein. CONCLUSION: The reversing effcet of IFN-gamma+ATRA group on ATRA-resistence in MR2 cells are more powerful than that of IFN-alpha+ATRA group, which may be related to the different signal transduction pathway of IFN-alpha and IFN-gamma.

HSP70 gene fused with Hantavirus S segment DNA significantly enhances the DNA vaccine potency against hantaviral nucleocapsid protein in vivo.

Heat shock proteins (HSPs) have been shown to act as adjuvants when coadministered with peptide antigens or given as fusion proteins and enhance the vaccination efficiency. To evaluate the enhancement of the potency of Hantaan virus (HTNV) nucleocapsid protein (NP) immunogenicity by heat shock protein 70 (HSP70), we developed a novel chimeric HTNV S-HSP70 DNA vaccine plasmid by genetically linking HSP70 gene to the full-length HTNV S segment DNA (HTNV S DNA). C57BL/6 mice were immunized with this plasmid followed by a subsequent boost with homologous recombinant protein. The levels of HTNV NP-specific antibody and cellular immune response were measured by use of ELISA, fluorescence activated cell sorter (FACS) analysis, cytotoxicity assay, and IFN-gamma ELISPOT assay. We found that HTNV S-HSP70 DNA vaccination significantly increased the levels of HTNV NP-specific antibody, IgG2a/IgG1 ratio, IFN-gamma producing CD8+ T-cell precursor frequencies, and cytotoxic T lymphocyte (CTL) response when compared with immunization with HTNV S DNA alone or HTNV S DNA physically mixed with HSP70 DNA. By contrast, HSP70 DNA or vector DNA immunization could not induce appreciable levels of specific antibodies and CTL response. Thus, we demonstrate for the first time that HSP70-based HTNV S DNA can induce both humoral and cellular immune response specific for HTNV NP and is a promising candidate DNA vaccine for HTNV infection.

Dynamic changes of apoptosis-inducing ligands and Th1/Th2 like subpopulations in Hantaan virus-induced hemorrhagic fever with renal syndrome.

The expression of the apoptosis-inducing ligands, TNF-alpha, FasL and TRAIL on peripheral blood mononuclear cells (PBMC) and the levels of their soluble form (TNF-alpha, sFasL and sTRAIL) in plasma from 40 hemorrhagic fever with renal syndrome (HFRS) patients as well as 26 healthy blood donors were determined by flow cytometry (FCM) analysis and sandwich ELISA, respectively. The status of Th1, Th2, Tc1 and Tc2 subsets in PBMC was evaluated by intracellular cytokine staining and FCM. Compared to controls, the expression of membrane bound FasL and TRAIL was up-regulated on surface of PBMC isolated from the HFRS patients, particularly on CD8+ T lymphocytes. The levels of TNF-alpha, sFasL and sTRAIL in plasma from the HFRS patients in the acute phase increase 4.7-fold, 6.0-fold and 1.8-fold, respectively, over those from the healthy donors. The percentage of Th1, Tc1 and Tc2 subsets in PBMC from the patients also increased significantly compared with those from healthy donors. These results indicate that dynamic changes occurred in both the membrane bound and soluble forms of apoptosis-inducing ligands (FasL, TRAIL and TNF-alpha) and proportions of Th1 and CTL in HFRS patients increased. Both factors may play an important role in the etiology of Hantaan virus infection in humans.

[Construction of TRAIL gene eukaryotic expression vector modulated by hTERT gene core promoter and its effect on apoptosis of ovarian cancer cells].

AIM: To construct the TNF-related apoptosis inducing ligand(TRAIL) gene eukaryotic expression vector modulated by human telomerase reserse transcriptase (hTERT) gene core promoter and to study its effect on apoptosis of ovarian cancer cells. METHODS: Genomic RNA was extracted from human placenta tissues and the fragment of TRAIL was obtained by RT-PCR. The amplified gene fragment was subsequently cloned into hTERTpromoter-pIRES2-EGFP vector and CMV promoter-pIRES2-EGFP vector after sequencing. The hTERTpromoter-pIRES2-EGFP-TRAIL and CMV promoter-pIRES2-EGFP-TRAIL eukaryotic expression vectors were constructed respectively. The recombinant plasmids were transfected into ovarian carcinoma cell line, SKOV3, and the levels of mRNA were determined by RT-PCR. The cell cycle and apoptosis rate of SKOV3 cells were determined by FCM. RESULTS: The constructed two recombinant vectors were verified by restriction enzyme digestion analysis and DNA sequencing. After being transfected with two recombinant vectors, the growth of SKOV3 cells was strongly inhibited and apoptotic features appeared. CONCLUSION: The recombinant eukaryotic expression vector has been constructed successfully. TRAIL gene driven by hTERTpromoter can be obviously expressed in ovarian carcinoma SKOV3 cells, suggesting that the specific expression vector modulated by hTERT core gene promoter may be a novel and promising approach to the tumor treatment.

[Expression and function of CD226 on NK cells activated by Superantigens].

AIM: To study the regulation of superantigen in expression and function of CD226 on NK cells. METHODS: Double fluorescent staining and flow cytometry analysis were employed to detect the expression of CD226 on NK cells from human peripheral blood mononuclear cells (PBMC) stimulated by staphylococcus enterotoxin A (SEA) or SEB. (51)Cr release assay was performed to compare the cytotoxicities of NK cells in different activation models. Laser scanning confocal microscope was used to observe the distribution of CD226 on NK cells activated by superantigens at killing stage. RESULTS: In resting NK cells, the percentage of CD56(+) NK cells and CD56(+) CD226(+) cells were 12.3% and 1.4% respectively, and the cytotoxicity of NK cells against K562 cells was (3.2+/-0.2)% at the ratio of 5:1. After PBMC were stimulated by 0.1 microg/mL SEA and SEB for 1 day, the percentage of CD56(+) NK cells was 13.5% and 14.1% respectively, and the percentage of CD56(+) CD226(+) cells were increased. Interestingly, CD226 was mainly expressed on CD56(dim) NK cells. On day 2 after SEA/SEB stimulation, the proportions of CD56(+) CD226(+) cells among CD56(+) cells were 69.1% and 64.3% in SEA and SEB group respectively. On day 3 after SEA/SEB stimulation, the expression of CD226 on NK cells decreased. Furthermore, the cytotoxicity of NK cells stimulated by SEA or SEB for 3 days against K562 cells were much higher than that of the resting NK cells as well as NK cells cultured without SAg for the same culture time (P<0.05), and reached to the peak at day 2 (82.3%+/-6.9% and 80.6%+/-7.5%, respectively). Additionally, we observed that CD226 molecules were colocalized with LFA-1 at the interface of NK cells which contacted K562 cells. CONCLUSION: The cytotoxicity of NK cells enhanced by SEA or SEB may be correlation with the increased expression of CD226 molecule, and CD226 may be involved in synapse formation of NK cells at killing stage.

[Effect of gestrinone on growth and apoptosis in isolated ectopic endometrium cells in vitro].

OBJECTIVE: To investigate the effects of gestrinone on growth and apoptosis, as well as the expression of phosphatase and tension homologue deleted on chromosome 10 (PTEN) in isolated ectopic endometrium cells in vitro and the underlying mechanisms. METHODS: Ectopic endometrium cells were cultured and exposed to gestrinone of different doses of 0, 10(-6) and 10(-4) mol/L respectively. The inhibition of the cells during 48 hours was determined by methylthiazolyl tetrazolium (MTT) assay, and the cell growth curve was made. Gestrinone was administered to the cells and at 24 hours the morphological changes were observed by transmission electron microscopy and the apoptosis rate, cell cycle and PTEN expression were monitored by flow cytometry (FCM) at the same time. RESULTS: Gestrinone at different concentrations could inhibit the growth and proliferation of ectopic endometrium cells in a dose- and time-dependent manner. The inhibition rate of cell growth after exposed to gestrinone for 8, 16, 24, 32, 40 and 48 h was 99.6%, 87.3%, 79.8%, 62.3%, 51.7% and 44.2% in the 10(-6) mol/L group, and 99.2%, 77.1%, 69.6%, 51.1%, 33.7% and 23.6% in the 10(-4) mol/L group (P < 0.05), and cell growth curve was changed accordingly. After 24 hour exposure to gestrinone from 10(-6) to 10(-4) mol/l, apoptotic changes of cells were observed under transmission electron microscope. FCM showed that after the exposure to gestrinone, the apoptotic rate of ectopic endometrium cells was 1.3% in 10(-6) mol/L group and 15.0% in 10(4) mol/L group. It was significantly increased when compared with the 0 mol/L group, the apoptotic rate of which was 0% (P < 0.05). The level of PTEN expression of the ectopic endometrium cells was 60.6% after treated with 0 mol/L gestrinone, while in 10(-6) and 10(-4) mol/l groups the level of PTEN expression was increased to 75.3% and 85.7%, significantly higher than that of the 0 mol/L group (P < 0.05). CONCLUSION: Gestrinone can significantly inhibit the growth and proliferation of ectopic endometrium cells, and this effect was related to increase of PTEN expression.

[Regulation of soluble TRAIL and membrane TRAIL in Jurkat cells by PMA].

AIM: To investigate the regulation of soluble and membrane bound TNF-related apoptosis-inducing ligand (TRAIL) in Jurkt cells by phorbol myristic acctate (PMA), and the cytotoxicity of the two forms of TRAIL. METHODS: Jurkat cells were cultured in the presence of 40 ng/mL PMA for different time. The production of sTRAIL was determined by ELISA, and expression of mTRAIL was analyzed by indirect fluorescence staining and flow cytometry analysis. The cytotoxicites of sTRAIL and mTRAIL were detected by (51)Cr release assay, in which DR4/DR5-expressing Raji cells were employed as target cells. RESULTS: The expression of both sTRAIL and mTRAIL in Jurkat cells were upregulated by PMA. The level of sTRAIL in supernatant from PMA-stimulated Jurkat cell culture increased and reached peak at 48 hours after PMA treatment, whereas expression peak of mTRAIL was at 60 hours. Both sTRAIL and mTRAIL exhibited cytotoxicity against Raji cells. CONCLUSION: PMA, a PKC activator, can upregulate the expression of both sTRAIL and mTRAIL in Jurkat cells, and the two forms of TRAIL have cytotoxic activity.

[The effects of rBCG expressing Der p2 in the form of lipoprotein on murine immune response].

AIM: To investigate the effects of rBCG vaccination containing foreign antigen Der p2 in the form of lipoprotein on murine immune response. METHODS: 6 to 8 weeks old and newborn BALB/c mice were vaccined intraperitoneally with 10(6) CFU rBCG or BCG. At the same time, the control group was injected with saline. Six weeks later, all animals were injected with Der p2 (20 microg). After two weeks later, the concentrations of IL-4 and IFN-gamma in the serum and splenocyte culture supernatant (STLCS) were determined by ELISA, and Th subgroups were determined by double fluorescent staining and flow cytometry. RESULTS: After vaccination, the serum and STLCS from both rBCG-immunized and BCG-immunized group of adult and newborn BALB/c mice had significantly higher level of IFN-gamma and lower level of IL-4 than those from control groups. Besides, there was the larger percentage of CD4 (+) IFN-gamma (+) cells in spleen from rBCG-vaccined and BCG-vaccined mice than that from control group. However, the percentage of CD4 (+) IL-4 (+) cells in spleen cells from rBCG-vaccined and BCG-vaccined group was lower than that from control group. Moreover, the level of IFN-gamma in STLCS from rBCG-immunized was significantly higher, compared with that from BCG-immunized mice. At the same time, the percentage of CD4 (+) IFN-gamma (+) cells in spleen from rBCG-vaccined mice was larger than that from BCG-vaccined group. CONCLUSION: Both rBCG and BCG could stimulate Th1 predominant immune response, when injected intraperitoneally into adult or newborn BALB/c mice, The Der p2 expressed on the cell wall of BCG can work as the component of BCG and be recognized by the immune system of mice, therefore stimulates Der p2-specific Th1 predominant immune response. These data indicate that recombinant BCG-expressing antigens can be used as the antigen-specific vaccines against allergic diseases by regulating the balance of Th1/Th2.

Effects of STI571 and p27 gene clone on proliferation and apoptosis of K562 cells.

AIM: To investigate the combined effect of STI571 and p27 gene clone on the regulation of proliferation, cell cycle and apoptosis of K562 cell line. METHODS: p27 gene was obtained by RT-PCR, and its sequence was approved to be correct. Then p27-pcDNA3.1 vector was constructed and transfected into K562 cell line. p27-pcDNA3.1-K562 cell clone was screened by G418 after transfection, p27 protein was identified by Western blot. MTT was used to detect the survival rate of the cell. Flow cytometry was used to detect cell cycle and apoptosis index. RESULTS: The expression of p27 protein could be detected by Western blot in p27-pcDNA3.1-K562 cells. A strong inhibition of cell proliferation was observed in p27-pcDNA3.1-K562 cells as compared with that of the control (pcDNA3.1-K562 cells). The cells at G0/G1 phase were significantly increased, and cells at S phase were greatly declined. The apoptosis index was increased greatly after p27-pcDNA3.1-K562 cells were treated with STI571, and survival rate of the cell was markedly declined (0.35-0.58, P<0.05-0.048 vs STI571-K562 cell, 0.35-0.72, P<0.01-0.001 vs p27-K562 cell). CONCLUSION: p27 and STI571 have a synergistic action on inhibition of proliferation and induction of apoptosis on K562 cells.

[Apoptosis-inducing ligand TRAIL can be recruited to lipid rafts].

AIM: To investigate the relationship between tumor necrosis factor-related apoptosis-inducing ligand(TRAIL) and cell membrane microdomain lipid rafts. METHODS: The expression of TRAIL on K562 cells was detected by indirect immunofluorescence staining and flow cytometry. The lipid rafts on K562 cells were detected with FITC-labeled cholera toxin B subunit (CTx-FITC) which bound to the GM1 glycosphingolipid in lipid rafts. The lipid rafts were then cross-linked with an anti-FITC mAb after binding with CTx-FITC. The localization of TRAIL on K562 cells was analyzed with rabbit anti-TRAIL antibody, Cy3-labeled goat-anti-rabbit IgG (Cy3-labeled GAR) and laser scanning confocal microscope. RESULTS: The cross-linking of lipid rafts appeared at 20 minutes, reached peak at 30 minutes, and weakened around 40 minutes. After the cells were labeled with CTx-FITC and anti-FITC mAb TRAIL was aggregated in the cross-linked lipid rafts. CONCLUSION: The anti-FITC mAb could be used for cross-linking of lipid rafts. TRAIL can be recruited to lipid rafts.

[Preparation and characterization of nanoemulsion].

AIM: To prepare nanoemulsion-encapsulated BSA-FITC (NEBSA-FITC), study its characteristics, and measure its uptake by dendritic cells (DCs) and peritoneal macrophages. METHODS: NEBSA-FITC was prepared by a method of interfacial polymerization.The encapsulation rate, drug-carrying capacity and stability of the nanoemulsion were determined by Sephadex-G100 chromatography. The shape and size of NEBSA-FITC were observed under electron microscope. The uptake of NEBSA-FITC by DCs and macrophage cells was detected by FACS and laser confocal microscopy. RESULTS: The mean size of NEBSA-FITC was (25+/-10) nm. The encapsulation rate was 91%, the drug-carrying capacity was 0.091 g/L and NEBSA-FITC had a good stability. The FACS analysis showed that DCs and macrophage cells could take in more NEBSA-FITC than free BSA. The observation under laser confocal microscope found that NEBSA-FITC was located in the cytoplasm of DCs. CONCLUSION: Nanoemulsion can be efficiently taken by DCs and macrophage cells, and therefore may be promising efficient carrier of APCs-targeted antitumor vaccine.

Expression pattern of epithelial cell adhesion molecule on normal and malignant colon tissues.

AIM: To investigate the expression pattern of epithelial cell adhesion molecule (Ep-CAM) on normal and malignant colon tissues to evaluate its diagnostic and therapeutic significance. METHODS: cDNA encoding Ep-CAM extracellular domain was cloned by reverse transcription-polymerase chain reaction (RT-PCR) from excised malignant colon tissues and inserted into a glutathione S-transferase (GST)-tagged vector. Ep-CAM-GST fusion protein was induced by isopropyl-beta-D-thiogalactopyranoside (IPTG) and purified with glutathione-sepharose. The Ep-CAM-GST fusion protein was mixed with Freund's adjuvant and Balb/c mice were immunized with it. Sp2/0 myeloma cells were fused with the spleen cells of the immunized mice. After having selected by indirect ELISA, the anti-Ep-CAM monoclonal antibodies (MAbs) were generated and the corresponding ascites were obtained. Finally, the human colon carcinoma tissue array prepared from seventy individual patients was stained with the anti-Ep-CAM MAbs. RESULTS: The isolated Ep-CAM cDNA sequence was identical to the data in GenBank. The expressed fusion protein was almost soluble and had a molecular weight (MW) of 53 ku. Four MAbs against Ep-CAM were obtained and designated as FMU-Ep1, FMU-Ep2, FMU-Ep3 and FMU-Ep4 respectively. Among them, FMU-Ep4 could recognize the natural Ep-CAM on Colo205 and SW480 cells, and all of them could be used for immunohistochemical staining of tissue sections. It was found that Ep-CAM was distributed differently in normal and various malignant colon tissues, including squamous cell carcinoma, signet-ring cell carcinoma and adenocarcinoma. In normal colon gland epithelia, Ep-CAM antigen was mainly distributed on the basolateral membrane and in the region between the basolateral membrane and the cytoplastic part near the nuclei, whereas the expression pattern of colon malignancies was mainly on the whole surface of epithelia and the expression was much higher than the normal colon tissues. The staining pattern of tissue array showed in adenocarcinoma and papillary adenocarcinoma, and the expression of Ep-CAM was increased from grade I to grade III. CONCLUSION: MAbs against Ep-CAM might be useful for research on the structure and function of Ep-CAM and may have diagnostic and therapeutic value to various colon carcinomas.

[Effect of a hypothetical gene Af116609 on multi-drug resistance of gastric cancer cells].

OBJECTIVE: To investigate the effect of gene Af116609 on gastric cancer multi-drug resistance (MDR) by introducing it into gastric cancer multi-drug resistant (MDR) cell line SGC7901/VCR. METHODS: Gene Af116609 was cloned from SGC7901/VCR by RT-PCR and its differential expression between gastric cancer MDR cells and its parental cells was displayed by Northern blot. The gene was introduced to gastric cancer cells by transfection of recombinant eukaryotic expression vector by electroporation. MTT assay in vitro was applied to investigate its effect on multi-drug resistance phenotype of gastric cancer cells. RESULTS: The full length CDS of gene Af116609, as long as 327 bp, was cloned from gastric cancer MDR cell line SGC7901/VCR and its sequence was coincident with the hypothetical gene Af116609 in GenBank. It was overexpressed in MDR cells than its parental cells at mRNA level. In the MTT assay in vitro, the drug sensitive cells transfected with sense eukaryotic expression vector showed upregulated targeted gene, with increased resistance to vincristine, 5-fliorouracil and arabinoside, and decreased resistance to adriamycin, but no influence on resistance to methotrexate. However, the drug resistant cells transfected with anti-sense eukaryotic expression vector, showed down regulated targeted gene, with less resistance to all the five anticancer drugs to different degrees. CONCLUSION: Gene Af116609 is related to MDR phenotype of gastric cancer cells and may become a candidate molecular target to reverse the MDR of gastric cancer.

[Expression of GST-PRL-2 fusion protein in prokaryotic cells and preparation of Hen egg yolk immunoglobulin (IgY) against PRL-2].

AIM: To express phosphatase 2 of regenerating liver (PRL-2) fusion protein in E.coli and to prepare specific hen egg yolk immunoglobulin(IgY) against PRL-2. METHODS: A full-length human PRL-2 gene coding sequence was cloned into expression vectors pGEX-4T-2 and pET21a, then transformed into E.coli. Fusion protein GST-PRL-2 was expressed in E.coli via IPTG induction. The expressed proteins were purified from lysates through Glutathione Sepharose 4B and the Ni-NTA agarose columns, respectively. Egg-laying hens were immunized using the purified GST-PRL-2 with Freund's complete or incomplete adjuvant. The specificity of the resulting antibody was identified by Western blot. RESULTS: A high level of expression of target protein was detected by Western blot after IPTG induction and purified protein was obtained through Glutathione Sepharose 4B and the Ni-NTA affinity chromatography agarose columns, respectively. Western blot analysis showed that the anti-PRL-2 polyclonal antibody can recognize 6 x His-PRL-2 fusion protein. CONCLUSION: The hen egg yolk immunoglobulin(IgY) against PRL-2 expressed in E.coli has good specificity which provides an useful reagent for the detection of PRL-2.