Insulinoma-induced hypoglycemic death in mice is prevented with beta cell-specific gene therapy.
OBJECTIVE
Tumor-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.


METHODS
Rat 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.


RESULTS
Only NIT-1 cells stained blue after X-gal staining or had detectable levels of beta-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.


CONCLUSIONS
The 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.
Tirone
T A
TA
Departments of Molecular Biology and Center for Gene Therapy, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, USA.
Fagan
S P
SP
Templeton
N S
NS
Wang
X
X
Brunicardi
F C
FC
D000818:Animals; D000998:Antiviral Agents / Q000627:therapeutic use; D004195:Disease Models, Animal; D005260:Female; D015774:Ganciclovir / Q000627:therapeutic use; D015972:Gene Expression Regulation, Neoplastic; D015316:Genetic Therapy; D007340:Insulinoma / Q000235:genetics* / Q000628:therapy*; D007763:Lac Operon; D051379:Mice; D008813:Mice, Inbred ICR; D016513:Mice, SCID; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D010190:Pancreatic Neoplasms / Q000235:genetics* / Q000628:therapy*; D012269:Ribosomal Proteins; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D000998:Antiviral Agents; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D012269:Ribosomal Proteins; C069798:Rps15 protein, mouse; D015774:Ganciclovir