Synergistic antitumor activity of triple-regulated oncolytic adenovirus with VSTM1 and daunorubicin in leukemic cells.

V-set and transmembrane domain-containing 1 (VSTM1), which is downregulated in bone marrow cells from leukemia patients, may provide a diagnostic and treatment target. Here, a triple-regulated oncolytic adenovirus was constructed to carry a VSTM1 gene expression cassette, SG611-VSTM1, and contained the E1a gene with a 24-nucleotide deletion within the CR2 region under control of the human telomerase reverse transcriptase promoter, E1b gene directed by the hypoxia response element, and VSTM1 gene controlled by the cytomegalovirus promoter. Real-time quantitative PCR and Western blot analyses showed that SG611-VSTM1 expressed VSTM1 highly efficiently in the human leukemic cell line K562 compared with SG611. In Cell Counting Kit-8 and flow cytometric assays, SG611-VSTM1 exhibited more potent anti-proliferative and pro-apoptotic effects in leukemic cells compared with SG611 and exerted synergistic cytotoxicity with low-dose daunorubicin (DNR) in vitro. In xenograft models, SG611-VSTM1 intratumorally injected at a dose of 1 × 10(9) plaque forming units combined with intraperitoneally injected low-dose DNR displayed significantly stronger antitumor effects than either treatment alone. Histopathologic examination revealed that SG611-VSTM1 induced apoptosis of leukemic cells. These results implicate an important role for VSTM1 in the pathogenesis of leukemia, and SG611-VSTM1 may be a promising agent for enhancing chemosensitivity in leukemia therapy.

[Preparation and identification of the polyclonal antibody against human VSTM1].

AIM: To prepare and characterize the polyclonal antibody against human VSTM1. METHODS: VSTM1 has two main isoforms, VSTM1-v1, a type I transmembrane protein, and VSTM1-v2, a classical secretory protein, lacking only the transmembrane domain compared with VSTM1-v1. Two recombinant prokaryotic proteins of VSTM1-v2, Trx-His-S-VSTM1-v2 and GST-VSTM1-v2, were constructed, expressed, purified, and then used for immunization of New Zealand rabbits to prepare anti-VSTM1 antibody and coupling with CNBr-activated Sepharose 4B to purify the antibody by immunoaffinity chromatography, respectively. ELISA was performed to detect the titers of the antiserums. After purification, the antibody was identified by Western blotting and immunofluorescence cytochemistry. RESULTS: The titers of the antiserums from the two immunized rabbits were both 1:10(6);. Western blotting confirmed that the purified antibody could recognize both the overexpressed and endogenous VSTM1 specifically. Immunofluorescence cytochemistry verified that the antibody could also recognize the overexpressed VSTM1-v1 on the surface of HEK293T cells. CONCLUSION: The rabbit antibody against human VSTM1 of a high titer has been obtained, which can be used for recognizing endogenous VSTM1 in immunofluorescence cytochemistry.

[Preparation and characterization of monoclonal antibodies against CMTM7].

OBJECTIVE: To obtain monoclonal antibodies against CMTM7 (CKLF-like MARVEL transmembrane domain containing 7) for further study of the structure and biological function of CMTM7. METHODS: Three polypeptides were synthesized based on the bioinformatics analysis of the CMTM7 and coupled with keyhole limpet hemocyanin (KLH). Balb/c mice were immunized with these mixed CMTM7 polypeptides. Hybridomas were generated by the fusion of the spleenocytes from these mice with Sp2/0 myeloma cells. Resulting hybridomas producing anti-CMTM7 antibodies were screened by enzymejlinked immunosorbent assay (ELISA). The specificities of these monoclonal antibodies were determined by Western blot, immunofluorescecence and immunocytochemistry (ICC). RESULTS: Two hybridoma cell lines (MC9 and 2C9) stable in secreting anti-CMTM7 monoclonal antibodies (MAbs) were generated. Both of them produced immunoglobulin G1 (IgG1) against CMTM7. 2C9 and MC9 recognized a region between amino acid residues 19-44 and 163-175 of CMTM7, respectively. MC9 antibody could be used for Western blot, immunofluorescecence and immunocytochmistry assay. However, 2C9 antibody could be only used for Western blot. Data obtained from immunofluorescence and ICC indicated that CMTM7 protein expression was upregulated in the early stage of lymphocyte activation treated with phytohemagglutinin (PHA). CONCLUSION: Monoclonal antibodies of high specificity against CMTM7 have been successfully generated, which could be utilized as a useful reagent for the analysis of biochemical, structural, and functional properties of CMTM7.

[Preparation, purification and characterization of the polyclonal antibody against human CMTM4].

AIM: To prepare, purify and characterize the polyclonal antibodies against CMTM4. METHODS: Four polypeptides named peptide 1, 2, 3, and 4 were synthesized based on the bioinformatics analysis of the three isoforms of CMTM4, CMTM4-v1, -v2, and -v3, and coupled with keyhole limpet hemocyanin (KLH) for immunization. Among them, peptide 1, 2, and 3, common for CMTM4-v1, v2, and v3, were mixed for immunization to prepare the antibody which can recognize all of the three isoforms (Ab1); while peptide 4, which is specific for CMTM4-v1, was injected separately into New Zealand rabbits to prepare the antibody specifically targeting CMTM4-v1 (Ab2). ELISA assay was used to detect the titers of the antibodies. After purification by immunoaffinity chromatography, Ab1 and Ab2 were identified by Western blot and immunohistochemistry assays. RESULTS: The titers of Ab1 and Ab2 were 1:10(5) and 1:10(6), respectively. Western blot confirmed their high specificity. Ab1 could also be used for immunohistochemistry analysis, but Ab2 could not. Immunohistochemistry analysis with Ab1 demonstrated the high expression level of CMTM4 in human testis, which was consistent with the previous result of Northern blot. Moreover, Western blot and immunohistochemistry analysis verified that Ab1 can also recognize mouse Cmtm4. CONCLUSION: Two specific antibodies against human CMTM4 were obtained, which will be helpful for further study.

[Preparation, identification, and analysis on tissue chips of polyclonal anti-peptide antibody to chemokine-like factor 1].

OBJECTIVE: To prepare the polyclonal anti-peptide antibody against chemokine-like factor1 (CKLF1) and apply it to the expression and functional studies of CKLF1. METHODS: CKLF1 was analyzed with bioinformatics methods. The 16 amino acids sequence peptide was selected from CKLF1 C terminal end. Antibody was raised by immunizing rabbits with the peptide conjugated to keyhole limpet hemocyanin (KLH). RESULTS: A high titer polycolonal antibody was obtained from the rabbit against the peptide. ELISA analysis proved that the titer of rabbit serum against anti-peptide of CKLF1 was up to 10(-4). Western blot analysis revealed that it could react not only with recombinant CKLF1 expressed in a cell-Free Protein Biosynthesis System and Drosophila S2 cells, but also recognize the endogenous CKLFs in the tissue array. Positive staining was detected in the normal bronchial cartilage, gastric mucosa, and gastric smooth muscle tissues. Normal rectum and well-differentiated rectal carcinoma showed strong positive staining, but the poor-differentiated rectal carcinoma samples revealed negative staining. CONCLUSION: The anti-peptide antibody can specifically recognize CKLFs and may be a useful reagent for the detection of CKLF1.

Chemokine-like factor 1, a novel cytokine, contributes to airway damage, remodeling and pulmonary fibrosis.

BACKGROUND: Chemokine-like factor 1 (CKLF1) was recently identified as a novel cytokine. The full-length CKLF1 cDNA contains 530 bp encoding 99 amino acid residues with a CC motif similar to that of other CC family chemokines. Recombinant CKLF1 exhibits chemotactic activity on leucocytes and stimulates proliferation of murine skeletal muscle cells. We questioned whether CKLF1 could be involved in the pathogenesis of inflammation and proliferation in the lung. Therefore we used efficient in vivo gene delivery method to investigate the biological effect of CKLF1 in the murine lung. METHODS: CKLF1-expressing plasmid, pCDI-CKLF1, was constructed and injected into the skeletal muscles followed by electroporation. Lung tissues were obtained at the end of week 1, 2, 3 and 4 respectively after injection. The pathological changes in the lungs were observed by light microscope. RESULTS: A single intramuscular injection of CKLF1 plasmid DNA into BALB/c mice caused dramatic pathological changes in the lungs of treated mice. These changes included peribronchial leukocyte infiltration, epithelial shedding, collagen deposition, proliferation of bronchial smooth muscle cells and fibrosis of the lung. CONCLUSIONS: The sustained morphological abnormalities of the bronchial and bronchiolar wall, the acute pneumonitis and interstitial pulmonary fibrosis induced by CKLF1 were similar to phenomena observed in chronic persistent asthma, acute respiratory distress syndrome and severe acute respiratory syndrome. These data suggest that CKLF1 may play an important role in the pathogenesis of these important diseases and the study also implies that gene electro-transfer in vivo could serve as a valuable approach for evaluating the function of a novel gene in animals.

[A novel cis-acting enhancer element between CKLF and CKLFSF1 genes].

AIM: To explore the roles of the CKLF gene and CKLFSF1 gene sequence (CCS) in transcriptional regulation. METHODS: The target gene fragment was amplified by PCR and then inserted into pGL3-basic and pGL3-SV40 containing luciferase reporter vector gene to construct pGL3-basic-CCS and pGL3-SV40-CCS. Using liposome-mediated method, four recombinant plasmids were respectively transfected into Hela cells. Transient expression was analyzed. RESULTS: The luciferase assay indicated that the no luciferase activity was detected in Hela cells transtected with pGL3-basic and pGL3-basic-CCS. However, the luciferase activity was doubled when pGL3-SV40-CCS was transfected into Hela cells. CONCLUTION: The CCS has no promoter activity, whereas some important cis-acting enhancer elements which modulate its downstream gene expression may exist within this sequence.