[Molecular mechanism of hydroxyurea enhances K562 cell apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand].

OBJECTIVE: To explore the molecular mechanism via which the chemotherapeutic drug hydroxyurea (HU) enhances K562 cell apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). METHODS: Chronic myelogenous leukemia-derived K562 and SVT-35 cells were treated with recombinant soluble TRAIL (rsTRAIL) alone or combined with HU for a time course, and the cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-4-sulfophenyl-2H-tetrazolium-phenazine methosulphate assay. Western blot was performed to analyze the activation of apoptosis-related protein kinases and the expression of apoptosis inhibitor molecules. RESULTS: The survival rates of SVT-35 and K562 cells treated with 1 µg/ml rsTRAIL for 24 hours were 32% and 93%, respectively. HU significantly increased the sensitivity of K562 cells to rsTRAIL cytotoxicity. Combination of rsTRAIL and HU resulted in the phosphorylation of rat sarcoma (RAS), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase and in the significant reduction of apoptosis-inhibited molecule Fas associated death domain protein-like interleukin-1 beta-convening enzyme inhibitory protein and cellular inhibitor of apoptosis protein-1 in K562 cells. CONCLUSIONS: HU enhanced K562 cell sensitivity to rsTRAIL is mediated by Ras-MEK-ERK signaling pathway. Expression of antiapoptotic proteins cellular Fas associated death domain protein-like interleukin-1 beta-convening enzyme inhibitory protein and cellular inhibitor of apoptosis protein-1 is also down-regulated during this process. These results may through light on the therapeutic study of human chronic myelogenous leukemia.

[Construction and stable expression of anti-human tumor necrosis factor-related apoptosis-inducing ligand receptor 2 chimeric antibody in CHO cells].

AIM: To establish an human-mouse chimeric antibody against tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor 2 (death receptor 5, DR5) in an eukaryotic cell line and analyse its tumoricidal activity. METHODS: The cDNAs encoding for the variable regions of heavy chain (V(H);) and light chain (V(L);) of AD5-10 were amplified by PCR and inserted into the human IgG heavy and light chain containing expression vector RpCI-neo, respectively. The recombinant plasmids were co-transfected into HEK293 and/or CHO cells. The production of anti-DR5 human-mouse chimeric antibody (hmAD5-10) and the antibody affinity for DR5 were identified by ELISA and Western blot assay. The tumoricidal activity of hmAD5-10 was demonstrated by MTS assay. The stable expression cells were selected and cultured in serum-free medium. RESULTS: Two stable CHO cells CHO-A5 and CHO-B11 with the chimeric antibody hmAD5-10 expression were established, in which the production of hmAD5-10 were reached at (0.36±0.11) mg/L and (0.16±0.01) mg/L, respectively. The hmAD5-10 secreted from the cells can well bind with DR5 and kill the cultured leukemia SVT35 cells by apoptosis remarkably. CONCLUSION: The human-mouse chimeric antibody hmAD5-10 was successfully expressed in the eukaryotic cells and resulted tumor cell death by apoptosis. This study lays a fundamental basis for the potential application of the recombinant chimeric antibody in cancer therapy.

Structural and functional analysis of the N-terminal region of death receptor 5 / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the structure and function of the N-terminal region (NTR) of death receptor 5 (DR5).</p><p><b>METHODS</b>A series of deletions of the DR5 extracellular domain (DR5-ECD) proteins were expressed in E.coli. and purified by affinity chromatography. The binding ability of these deletant proteins to AD5-10, a mouse anti-human DR5 monoclonal antibody, was evaluated by immunoblotting and ELISA.</p><p><b>RESULTS</b>Recombinant DR5-ECD proteins containing the NTR were recognized and bound by AD5-10, while the other deletant proteins without the NTR failed to interact with AD5-10.</p><p><b>CONCLUSION</b>There is an AD5-10 targeting site in the NTR of DR5, which may play a role in developing novel immunotherapies for cancers.</p>

[Effects of human telomerase reverse transcriptase promoter and survivin promoter in targeted tumor gene therapy].

OBJECTIVE: To investigate the effects of human telomerase reverse transcriptase (hTERT) promoter and survivin promoter in tumor-specific gene therapy. METHODS: hTERT promoter and survivin promoter were obtained by PCR using Jurkat genomic DNA. Recombinant adeno-associated virus (AAV) vectors containing exogenous TRAIL gene and hTERT promoter or survivin promoter were constructed and designated as rAAV-hTERT-TRAIL (h/TRAIL) or rAAV-survivin-TRAIL (s/TRAIL). rAAV particles were obtained after packing and purification and the virus titer was calculated by real-time PCR. Human hepatocellular carcinoma (HCC) cells of the lines SMMC-7721, BEL-7402, HepG2, and Hep3B, and primary human hepatocytes (PHHs) were transfected with h/TRAIL or s/TRAIL. Flow cytometry was used to detect the expression of the reporter gene enhanced green fluorescent protein (EGFP), so as to examine the activity of the two promoters. MTT method was used to detect the activity of the cells. Fifteen BalB/C mice underwent subcutaneous injection of SMMC-7721 cells so as to establish tumor models and then were randomly divided into 3 groups to undergo intra-tumor injection of h/TRAIL, s/TRAIL, or PBS. The growth of tumor was observed for 5 weeks, and then peripheral blood samples were collected to examine the serum AST and ALT levels. TUNEL was used to detect the apoptosis if the tumor cells. RESULTS: All the SMMC-7721, BEL-7402, HepG2, and Hep3B cells driven by both h/TRAIL and s/TRAIL showed EGFP expression, however, no fluorescence could be seen in the PHHs transfected with h/TRAIL and s/ TRAIL. MTT method showed that 72 hours after the transfection of h/TRAIL and s/TRAIL the survival rates of the SMMC-7721, BEL-7402, and HepG2 cells all decreased, however, the survival rate of the Hep3B cells and PHHs did not changed significantly. The size of the subcutaneous tumor of the mice of the h/ TRAIL group was 625 mm3, significantly smaller than that of the PBS group (1500 mm3, P <0.05), however, the tumor size of the s/TRAIL group was 1117 mm3, not significantly different from that of the PBS group (P >0.05). The AST and ALT levels of all mice did not change significantly 5 weeks after the intratumor injection. CONCLUSION: Tumor-specific promoters are promising candidates in targeted tumor gene therapy.

Comparative proteomics analysis of cerebrospinal fluid of patients with Guillain-Barré syndrome.

To better understand the pathophysiologic mechanisms underlying Guillain-Barré syndrome (GBS), Comparative proteomic analysis of cerebrospinal fluid (CSF) between patients with GBS (the experiment group) and control subjects suffering from other neurological disorders (the control group) was carried out using two-dimensional gel electrophoresis (2-DE) technique, in combination with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and database searching to determine abnormal CSF proteins in GBS patients. Image analysis of 2-DE gels silver stained revealed that 10 protein spots showed significant differential expression between the two groups of CSF samples. The expression of cystatin C, transthyretin, apolipoprotein E and heat shock protein 70 were decreased. However, haptoglobin, alpha-1-antitrypsin, apolipoprotein A-IV and neurofilaments were elevated. The subsequent ELISA measured the concentration of cystatin C and confirmed the result of the proteomic analysis. These identified proteins may be involved in the pathophysiological process of GBS and call for further studying the role of these proteins in the pathogenesis of the disease.

[Construction and expression of anti-tumor necrosis factor related apoptosis-inducing ligand receptor death receptor 5 chimeric antibody in eukaryotic cells].

OBJECTIVE: To construct the human/mouse chimeric antibody of a functional anti-death receptor 5 (DR5) antibody. Methods The viable region of light chain (VL) and viable region of heavy chain (VH) genes of anti-DR5 antibody were amplified and cloned into the light- and heavy-chain expression vectors respectively, then the recombinant plasmids were co-transfected into dihydrofolate reductase(-) Chinese hamster ovary cell (CHO-dhfr(-)) for expression. The positive clone was screened by the two selective genes (neo and dhfr). The humanization and specificity of chimeric antibody was identified by ELISA and Western blotting, and the tumoricidal activity of the expressed chimeric antibody was detected by tetrazolium salt phenazine methosulfate assay. RESULTS: The expression vectors stably expressed chimeric antibody in CHO-dhfr(-). In the cell supernatant of the F4' clone, the human IgG heavy constant region and light constant region were identified. Moreover, the secreted chimeric antibody retained the binding capacity to the antigen (DR5) and decreased the cell viability of Jurkat and HCT116 cells to 73.15% and 77.30% in vitro respectively. CONCLUSION: The human/mouse anti-DR5 chimeric antibody has been successfully expressed in eukaryotic cells and shows tumoricidal activity, which establishes a foundation for the future research of humanized antibody medicine.

Ornithine decarboxylase gene is overexpressed in colorectal carcinoma.

AIM: To investigate the ornithine decarboxylase (ODC) gene expression in colorectal carcinoma, ODC mRNA was assayed by RT-PCR and ODC protein was detected by a monoclonal antibody against fusion of human colon ODC prepared by hybridoma technology. METHODS: Total RNA was extracted from human colorectal cancer tissues and their normal counterpart tissues. ODC mRNA levels were examined by RT-PCR. ODC genes amplified from RT-PCR were cloned into a prokaryotic vector pQE-30. The expressed proteins were purified by chromatography. Anti-ODC mAb was prepared with classical hybridoma techniques and used to determine the ODC expression in colon cancer tissues by immunohistochemical and Western blotting assay. RESULTS: A cell line, which could steadily secrete anti-ODC mAb, was selected through subcloning four times. Western blotting reconfirmed the mAb and ELISA showed that its subtype was IgG2a. RT-PCR showed that the ODC mRNA level increased greatly in colon cancer tissues (P<0.01). Immunohistochemical staining showed that colorectal carcinoma cells expressed a significantly higher level of ODC than normal colorectal mucosa (98.6+/-1.03% vs 5.26+/-5%, P<0.01). CONCLUSION: ODC gene overexpression is significantly related to human colorectal carcinoma. ODC gene expression may be a marker for the gene diagnosis and therapy of colorectal carcinoma.

[Mechanisms of sensitization by 8-chloro-adenosine of human tumor cells to TRAIL-induced apoptosis].

OBJECTIVE: To study the effect of 8-chloro-adenosine (8-Cl-Ado) on the sensitivity of human hepatoma and breast cancer cell lines to TRAIL-induced apoptosis in vitro and its mechanisms. METHODS: Recombinant soluble TRAIL (rsTRAIL) or 8-Cl-Ado was used to treat hepatoma cell line BEL-7402 and breast cancer cell line MCF-7 in vitro. MTT assay was used to evaluate cell viability. The effect of cotreatment with rsTRAIL and 8-Cl-Ado was analyzed. NF-kappaB activity reporter plasmid was designed to measure the activity of transcription factor NF-kappaB. After transient transfection with the reporter plasmid, which contains NF-kappaB-responsive elements, into the cell lines, cells were treated with rsTRAIL and/or 8-Cl-Ado, then the activity of the reporter gene luciferase was determined. Different kinds of caspase inhibitors were used to measure the effect of caspases in the rsTRAIL and/or 8-Cl-Ado induced apoptosis. RESULTS: 8-Cl-Ado could greatly enhance sensitivity of BEL-7402 and MCF-7 cells to reTRAIL. Treatment with 8-Cl-Ado and rsTRAIL inactivated transcription factor NF-kappaB and induced apoptosis in BEL-7402, but not in MCF-7. Caspase family inhibitor could not prevent apoptosis induced by 8-Cl-Ado and rsTRAIL in BEL-7402 cells, however, it could block apoptosis in MCF-7 cells, indicating that two different apoptosis pathways in MCF-7 and BEL-7402 might exist, one was caspase dependent and the other caspase independent. Moreover, all of the inhibitors of caspse-3, -8 and -9 could not block apoptosis induced by the co-treatment. CONCLUSION: 8-chloro-adenosine can enhance the sensitivity of human hepatoma cell line BEL-7402 and breast cancer cell line MCF-7 to rsTRAIL, even though MCF-7 is TRAIL-resistant. 8-Cl-Ado combined with rsTRAIL can trigger different signal pathways in MCF-7 and BEL-7402, which are caspase dependent and independent, respectively.

[Expression of TRAIL(114-281) mediated by adeno-associated virus and its tumoricidal activity].

OBJECTIVE: To investigate the expression of the soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated by adeno-associated virus (AAV) and its tumoricidal activity in vitro and vivo. METHODS: The recombinant AAV expression vector encoding the extracellular domain (114-281aa peptide, TRAIL(114-281)) of TRAIL was constructed and transfected into human embryotic kidney cells HEK293 for virus package. The human tumor cell lines of T lymphocyte leukemia Jurkat, liver cancer HepG2 and SMMC-7721, and cervical cancer HeLa were transduced by using the recombinant virus particles respectively. The recombinant virus particles were also injected into C57BL/6 mice via the hepatic portal vein or hypodermic, intramuscular, celiac and oral pathways to study the expression of TRAIL(114-281). The recombinant virus titer was determined by real-time PCR. The expression of TRAIL(114-281) was evaluated by ELISA, Western blotting and immunohistochemistry assay respectively. The tumoricidal activity and apoptosis were evaluated by MTT assay. RESULTS: The recombinant AAV encoding for the soluble TRAIL (114 - 281aa) were constructed successfully. The titer of recombinant virus was 7.5 x 10(12) genome particles (Gps)/ml. Transduction of rAAV-TRAIL(114-281) led to high level expression of TRAIL(114-281) and the induction of apoptosis of Jurkat, Hela and SMMC-7721 cancer cells, but not HepG2 cells, in vitro. The recombinant peptide TRAIL(114-281) in trimeric active form was highly and constantly expressed in the hepatocytes and secreted into the serum up to 6 months in the of C57BL/6 mice injected with the recombinant virus particles via the hepatic portal vein. The peptide TRAIL(114-281) in the livers, but not other tissues, were also detected in the mice administrated with rAAV-TRAIL(114-281) particles via subcutaneous, intramuscular, intraceliac or oral pathway. CONCLUSION: The long term, stable and liver-tropism expression of peptide TRAIL(114-281) in mice mediated by rAAV-TRAIL(114-281) provides a prospective novel strategy for tumor gene therapy of numerous cancers, especially liver cancer.

[Chemotherapeutic drugs enhanced rsTRAIL tumoricidal activity].

OBJECTIVE: To explore the role and mechanisms of chemotherapeutic drugs in TRAIL induced cell death. METHODS: Tumoricidal activities of the chemotherapeutic drugs and/or rsTRAIL in 13 strains of tumor cell lines were evaluated by MTS-PMS assay and flow cytometry. DR5 expression in the cells was observed by Western blot. RESULTS: The apoptosis of human promyelocytic leukemia cells HL-60, liver cancer cells BEL-7402, T-acute lymphoblastic leukemia cells Jurkat, and myeloid leukemia cells K562 treated with rsTRAIL at 0.5 microg/ml were 53.20%, 52.20%, 51.54%, 52.70%, and 41.00%, respectively, while that of the embryonal spleen cells 293 was 24.00%. However, the apoptosis percentages of lung cancer cells anti 973, breast cancer cells MCF-7, Chinese hamster ovarian cancer cells COS-7, neuroglialoma cells U251, neuroblastoma cells SH-SY5Y, glioma cells BT-325, rat pheochromocytoma cells PC12, and mouse adrenal epithelial cells NIH3T3 were all less than 10% under the same conditions. The sensitivity of central neuron cells of SH-SY5Y, PC-12, U251, BT3251, and human embryonal spleen cells 293, which were not sensitive to rsTRAIL challenges, increased remarkably after treatment with CHX, CP, and 8-CA at sub-toxic doses plus rsTRAIL at 0.5 microg/ml. The expressions of DR5 were up-regulated and kept pace with the onset of apoptosis in the BEL-7402 liver cancer cells. CONCLUSION: The chemotherapeutic drugs including CHX, CP, and 8-CA at sub-toxic doses can enhance antitumor activity of rsTRAIL.

Cloning and expression of ornithine decarboxylase gene from human colorectal carcinoma.

AIM: To construct and express ODC recombinant gene for further exploring its potential use in early diagnosis of colorectal carcinoma. METHODS: Total RNA was extracted from colon cancer tissues and amplified by reverse-transcription PCR with two primers, which span the whole coding region of ODC. The synthesized ODC cDNA was cloned into vector pQE-30 at restriction sites BamH I and Sal I which constituted recombinant expression plasmid pQE30-ODC. The sequence of inserted fragment was confirmed by DNA sequencing, the fusion protein including 6His-tag was facilitated for purification by Ni-NTA chromatographic column. RESULTS: ODC expression vector was constructed and confirmed with restriction enzyme digestion and subsequent DNA sequencing. The DNA sequence matching on NCBI Blast showed 99 % affinity. The vector was transformed into E. coli M15 and expressed. The expressed ODC protein was verified with Western blotting. CONCLUSION: The ODC prokaryote expression vector is constructed and thus greatly facilitates to study the role of ODC in colorectal carcinoma.

[Synergetic role of CD3epsilon and 5-fluorouracil in T Lymphocyte apoptosis and P53 expression].

OBJECTIVE: To investigate the relationship between apoptosis induced by CD3epsilon and 5-fluorouracil (5-FU), and study the P53 expression in the apoptosis process provide a novel insight and useful information of the apoptosis signaling pathway induced by CD3epsilon and/or 5-FU, and an important implication for the treatment of T-lymphocyte leukemia. METHODS: The viabilities of Jurkat T lymphocytes (JK), TJK [JK with over-expression of the CD8epsilon chimeria molecule] and T3JK [JK with over-expression of the CD8epsilon (Y170F/Y181F) mutation molecule] cells were cultured and treated with pre-coated anti-CD8 mAb (200 micro g/ml) and/or 5-FU (2.5 micro g/ml) were detected with MTS assay and the apoptosis percentages were calculated. Western blot was used to detect P53 expression. To confirm the role of P53 in 5-FU-treated T lymphocytes, pCMV-p53 plasmid with wild type or mutant p53 were co-transfected transiently with pEGFP-c1 into TJK and T3JK cells, respectively. RESULTS: CD3epsilon or 5-FU induced apoptosis of TJK with increase of P53 expression. Co-treatment with CD3epsilon specific antibody and 5-FU elevated the apoptotic rates and P53 expression in TJK cells remarkably. The cells transfected with wild-type p53 exhibited more sensitivity to 5-FU than that transfected with mutant p53. CONCLUSION: Co-treatment of CD3epsilon and 5-FU increases the apoptosis and p53 expression, suggesting that there is a synergetic role of CD3epsilon and 5-FU on T lymphocytes.

[The primary study on a novel protein binding to the death domain of the death receptor 4].

OBJECTIVE: To clone and identify novel proteins binding to the death domain of the death receptor 4 (DR4). METHODS: The yeast two-hybrid system was used for this study. Automatic sequencing was carried out for DNA sequencing. The sequence homology and the functional domains were analyzed by BLAST and the ScanProsite Tool softwares, respectively. Co-immunoprecipitate method was used to confirm human formyl peptide receptor-like 1 (FPRL1) binding specifically with DR4CD (the cytoplasmic domain of DR4) in HEK293T cells. RESULTS: Two positive clones, named as pADB1 and pADB2, were obtained. BLAST searching showed that the homology of the insert sequence of pADB1 with the mRNA of FPRL1 was 97%. The insert of pADB2 shared no homology with any known peptides in GeneBank. Co-immunoprecipitate analysis further confirmed that FPRL1 could bind to DR4CD in vivo specifically. CONCLUSIONS: FPRL1 may associate with DR4CD in vivo specifically. The functional studies of FPRL1 in signaling pathway mediated by TNF-related apoptosis inducing ligand (TRAIL) are in active progress in our laboratory.