title: Specific gene expression and therapy for pancreatic cancer using the cytosine deaminase gene directed by the rat insulin promoter.
abstract: Suicide gene therapy has been shown to be an effective means of destroying pancreatic cancer cells, but cell-specific delivery of the gene is required to limit host toxicity. The objective of this study is to determine whether the rat insulin promoter (RIP) will permit cell-specific gene delivery and subsequent cell death in human pancreatic cancer cells. The RIP DNA was amplified using polymerase chain reaction (PCR), and the purified fragment was inserted into pCR-Blunt II-TOPO plasmid at the SpeI site, which contains the coding sequence of yeast cytosine deaminase (CD). Transfection assays were carried out using both RIP-lacZ and RIP-CD DNA constructs in two human pancreatic cancer cell lines, PANC-1 and MIA PaCa-2. Reporter assays using X-gal staining were performed, and the in vitro cytotoxicity was examined in RIP-CD-transfected cells treated with 5-flucytosine for 5 days. The expression levels of CD protein in the transfected cells were determined 2 days after transfection by Western blot analysis. The expression levels of insulin promoter factor (IPF-1/PDX-1) in these human pancreatic cell lines, as well as in freshly isolated human pancreatic cancer specimens, were determined using in situ immunohistochemistry analysis. After transfection with RIP-lacZ, only PANC-1 cells, but not MIA PaCa-2 cells, were positive for RIP-lacZ expression, indicating that RIP-directed reporter gene expression occurred only in PANC-1 cells. After transfection with RIP-CD and treatment with 5-flucytosine, PANC-1 cells had a significantly increased cell death rate compared with that of MIA PaCa-2 cells, suggesting that RIP-directed suicide gene expression occurred only in PANC-1 cells. Western blot analysis demonstrated that only PANC-1 cells were able to express the CD protein and that significantly increased levels of PDX-1 were found in PANC-1 but not in Mia PaCa-2 cells. In situ immunohistochemical analysis of both cell lines showed that PDX-1 was only expressed in the nuclei of PANC-1 cells and not in MIA PaCa-2 cells. Furthermore, two freshly isolated human pancreatic cancer specimens had significantly increased levels of PDX-1. The RIP is activated in PANC-1 cells, but not in Mia PaCa-2 cells, and the mechanism of activation is via PDX-1. Pancreatic cancer-specific cytotoxicity can be achieved with the use of RIP-CD and 5-flucytosine treatment in vitro. Significantly increased levels of PDX-1 have been found in human pancreatic cancer specimens. These results suggest that RIP could be used for cell-specific suicide gene therapy to target human pancreatic tumors.
authors.affiliation: Michael E DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas 77030, USA.
authors.forename: Xiao-Ping
authors.initials: XP
authors.lastname: Wang
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Yazawa
authors.forename: Jie
authors.initials: J
authors.lastname: Yang
authors.forename: Deborah
authors.initials: D
authors.lastname: Kohn
authors.forename: William E
authors.initials: WE
authors.lastname: Fisher
authors.forename: F Charles
authors.initials: FC
authors.lastname: Brunicardi
mesh_terms: D000963:Antimetabolites / Q000627:therapeutic use; D015153:Blotting, Western; D043525:Cytosine Deaminase / Q000235:genetics*; D005437:Flucytosine / Q000627:therapeutic use; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D007763:Lac Operon; D010190:Pancreatic Neoplasms / Q000628:therapy*; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D000963:Antimetabolites; D018398:Homeodomain Proteins; D015534:Trans-Activators; D014157:Transcription Factors; C084010:pancreatic and duodenal homeobox 1 protein; D005437:Flucytosine; D043525:Cytosine Deaminase