Objective and Summary Background DataTumor-specific gene therapy can be achieved if a tumor-specific promoter can be identified. In this study the authors investigated the use of the rat insulin promoter (RIP) for insulinoma-specific expression of a reporter gene. Insulinoma-specific cytotoxicity using the suicide gene thymidine kinase (tk) was studied both in vitro and in vivo. RIPtk gene therapy, delivered by a nontoxic, noninflammatory liposomal delivery system, was used in an insulinoma ICR/SCID mouse model to prevent hypoglycemic death. MethodsRat insulin promoter (0.502 kb) was ligated to the reporter gene lacZ and ligated to the tk gene. These two genes were transfected into a mouse insulinoma (NIT) cell line to ascertain insulinoma-specific expression and insulinoma-specific cytotoxicity in vitro. Reverse transcriptase-polymerase chain reaction and electrophoretic mobility-shift assays were performed on NIT-1 cell RNA and nuclear extract, respectively, to determine the transcription factors present and responsible for RIP activation in NIT-1 cells. A mouse insulinoma model was created with NIT-1 cells. These mice were treated with the RIPtk gene, and both blood sugars and animal viability were monitored. ResultsOnly NIT-1 cells stained blue after X-gal staining or had detectable levels of -galactosidase protein. A significant decrease in cell survival was observed in NIT-1 cells transfected with RIPtk in vitro. Messenger RNA for both BETA2 and PDX-1 was found in NIT-1 cells, and a supershift was observed for both BETA2 and PDX-1. Experimental mice treated with the RIPtk gene, delivered by a liposomal gene delivery system, maintained their blood glucose levels, and the animals did not die of hypoglycemia. ConclusionsThe data suggest that the RIP is an insulinoma-specific promoter. An ICR/SCID mouse insulinoma model was used to show that insulinoma-specific cytotoxicity can be accomplished by RIP coupled to a suicide gene in vivo, preventing hypoglycemic death.Insulinomas are the most common of the islet cell tumors. Ninety percent of beta cell tumors are benign, but the complication rate associated with their removal is significant: the fistula rate is 11%.1 Malignant insulinomas have a 63% 5-year recurrence rate, with an average survival of less than 4 years.1 Further, there is no effective medical treatment for the devastating symptoms associated with hyperinsulinemia of insulinoma.With the identification of tissue-specific promoters, tumors can be targeted by gene therapy. The rat insulin promoter (RIP) is an example of a cell-specific promoter that is activated in cells that produce insulin. NIT-1 cells are a mouse beta cell tumor line derived from the nonobese diabetic mouse.2 The purposes of this study were to use RIP to target NIT-1 cells for genetic manipulation and to determine the transcription factors responsible for NIT-1 cellspecific activation of RIP. An ICR/SCID mouse insulinoma model was used to determine whether gene therapy could be effective in vivo to prevent hypoglycemic death in thi...
The formation of a normal pancreas and the activation of insulin production are, in part, dependent on the expression and activation of the pancreatic duodenal homeobox gene 1 (PDX-1). The expression of PDX-1 also has been detected in various human pancreatic ductal adenocarcinoma (PDA) cell lines. This has made it possible to generate a cancer cell-specific gene expression system to treat human pancreatic cancer. In this study, we have developed a cell-specific cytotoxic model of PDA cells using the expression of herpes simplex virus thymidine kinase (TK) under the control of the rat insulin promoter (RIP-TK). We have shown that the cell-specific cytotoxicity in human PDA cells depends on the presence of PDX-1. Our results also demonstrate that in vivo PDA-specific cytotoxicity can be achieved with RIP-TK using an intraperitoneal liposomal gene delivery method followed by a short period of ganciclovir treatment in severe combined immunodeficient (SCID) mice. Furthermore, PDX-1 protein was found in all six freshly isolated human pancreas cancer specimens and two liver metastasis samples that were group-tested, suggesting the feasibility of using RIP-TK gene therapy in humans. This study may provide an alternative strategy for the future treatment of pancreatic cancer.
Somatostatin (SST) peptide is produced by various SST-secreting cells throughout the body and acts as a neurotransmitter or paracrine/autocrine regulator in response to ions, nutrients, peptides hormones and neurotransmitters. SST is also widely distributed in the periphery to regulate the inflammatory and immune cells in response to hormones, growth factors, cytokines and other secretive molecules. SST peptides are considered the most important physiologic regulator of the islet cell, gastrointestinal cell and immune cell functions, and the importance of SST production levels has been implicated in several diseases including diabetes. The expression of SST receptors has also been found in T lymphocytes and primary immunologic organs. Interaction of SST and its receptors is also involved in T-cell proliferation and thymocyte selection. SSTR gene-ablated mice developed diabetes with morphologic, physiologic and immunologic alterations in the endocrine pancreas. Increased levels of mononuclear cell infiltration of the islets are associated with the increased levels of antigen-presenting cells located in the islets and peripancreatic lymph nodes. Increased levels of SST were also found in antigen-presenting cells and are associated with a significant increase of CD8 expression levels on CD4(+)/CD8(+) immature thymocytes. These findings highlight the crucial role of this neuroendocrine peptide and its receptors in regulating autoimmune functions.
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