PubMed · 12642338
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P T Khaw, A P Wells, K S Lim. 2003. Surgery for glaucoma.. https://doi.org/10.1136/bjo.87.4.517
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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.
5-Fluorouracil reduces proliferation rates of fibroblasts, myofibroblast differentiation and contractility of ocular fibroblasts in vitro. This double-blind randomized clinical trial assesses whether intra-operative topical treatment with 5-fluorouracil reduces the recurrence rate after limited excision of Dupuytren's tissue. Patients with two-digit disease were randomized to having 5-fluorouracil (25mg/ml) treatment for 5 minutes on one digit and placebo on the other. Fifteen patients were enrolled with 18 months follow-up. There were no peri-operative complications. Wound healing was not delayed and there was no deterioration in the flexion deformity of the 5-fluorouracil treated digits. Patients were subsequently assessed by joint angle measurement at 3, 6, 12 and 18 months. There was no significant difference between control and 5-fluorouracil treated digits.
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