title: Dendritic cell transfected with secondary lymphoid-tissue chemokine and/or interleukin-2 gene-enhanced cytotoxicity of T-lymphocyte in human bladder tumor cell S in vitro.
abstract: OBJECTIVE
  The aim of this study was to investigate whether dendritic cells (DCs) transfected with human secondary lymphoid-tissue chemokine (hSLC) and human interleukin-2 (hIL-2) genes are capable of improving DC's proliferation and to produce a marked antitumor effect in vitro combined with T-lymphocyte (TC).
  
  
  METHODS
  SLC gene primer was designed based on the corresponding gene sequence in GenBank. The Kpn I site was introduced into the upstream of the primer and Xho I site into the downstream. The SLC gene was amplified with the template of pET32a(+)-SLC by polymerase chain reaction. SLC was cloned into pBudCE4.1/IL-2 (TRAIL was cut from pBudCE4.1/TRAIL- IL-2 before) to construct recombinant plasmid pBudCE4.1/SLC-IL-2(PSI). DCs were transfected with pBudCE4.1/SLC-IL-2 by gene electric transfection. Protein expression was determined with Western blot and enzyme-linked immunosorbent assays. Cytotoxicity of TC and DC against the human bladder tumor cell were examined by chromium release assay. Flow cytometric analyses were performed to determine the apoptosis of tumor cells and the percentage of Treg.
  
  
  RESULTS
  A high level of expression of SLC and IL-2 was observed in DCs transfected with SLC and IL-2 genes. The mean production of IL-2 was 19.8 +/- 2.5, 511.10 +/- 52.36, and 541.3 +/- 62.04 ng/10(6) cells/24 hours in the DC/vector, DC/IL-2, and DC/SLC-IL-2, respectively. The mean SLC production was 29.8 +/- 4.43, 506.10 +/- 42.36, and 567.34 +/- 52.05 ngs/10(6)cells/24 hours in the DC/ vector, DC/SLC, and DC/SLC-IL-2, respectively. Cytotoxicity to bladder cancer cells was increased. The mean cytotoxicity (the effector/target ratio, 40:1) of TC-DC/parental, TC-DC/IL-2, TC-DC/SLC, and TC-DC/SLC-IL-2(TDSI) to the human bladder cancer cells was 32.1 +/- 5.5%, 63.5 +/- 6.6%, 78.1 +/- 9.63%, respectively. The apoptotsis rate of bladder cancer cells treated with TDSI was 18.6% by flow cytometry. Treg cells' percentage was very small in the DC medium.
  
  
  CONCLUSIONS
  SLC and IL-2 were produced by autocrine in DCs transfected with SLC and IL-2 genes. DC/SLC-IL-2 can promote DC proliferation, while TC-DC/SLC-IL-2 and TC-DC/SLC could strongly enhance significant cytotoxicity against bladder cancer cell that was induced by the coculture of DCs (transfected with SLC and IL-2) and TC.
authors.affiliation: Institute of Urology, Lanzhou University Second Hospital, Lanzhou University Clinical Medical College, Lanzhou, China.
authors.forename: Yong-Gang
authors.initials: YG
authors.lastname: Li
authors.forename: Zhi-Ping
authors.initials: ZP
authors.lastname: Wang
authors.forename: Jun-Qiang
authors.initials: JQ
authors.lastname: Tian
authors.forename: Bin-Qiang
authors.initials: BQ
authors.lastname: Tian
authors.forename: Ronald
authors.initials: R
authors.lastname: Rodrigues
authors.forename: Pan-Feng
authors.initials: PF
authors.lastname: Shang
authors.forename: Tao
authors.initials: T
authors.lastname: Zhang
mesh_terms: D017209:Apoptosis; D019898:Autocrine Communication; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D054421:Chemokine CCL21 / Q000096:biosynthesis* / Q000235:genetics; D018920:Coculture Techniques; D003602:Cytotoxicity, Immunologic; D003713:Dendritic Cells / Q000276:immunology / Q000637:transplantation*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D016219:Immunotherapy, Adoptive; D007376:Interleukin-2 / Q000096:biosynthesis* / Q000235:genetics; D016133:Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000276:immunology*; D050378:T-Lymphocytes, Regulatory / Q000276:immunology; D013997:Time Factors; D014162:Transfection; D001749:Urinary Bladder Neoplasms / Q000276:immunology / Q000473:pathology / Q000628:therapy*
chemical_list: C515478:CCL21 protein, human; D054421:Chemokine CCL21; C508594:IL2 protein, human; D007376:Interleukin-2