An alternative approach to the treatment of type I diabetes is the use of genetically altered neoplastic liver cells to synthesize, store and secrete insulin. To try and achieve this goal we modified a human liver cell line, HUH7, by transfecting it with human insulin cDNA under the control of the cytomegalovirus promoter. The HUH7-ins cells created were able to synthesize insulin in a similar manner to that which occurs in pancreatic b cells. They secreted insulin in a regulated manner in response to glucose, calcium and theophylline, the dose-response curve for glucose being near-physiological. Perifusion studies showed that secretion was rapid and tightly controlled. Removal of calcium resulted in loss of glucose stimulation while addition of brefeldin A resulted in a 30% diminution of effect, indicating that constitutive release of insulin occurred to a small extent. Insulin was stored in granules within the cytoplasm. When transplanted into diabetic immunoincompetent mice, the cells synthesized, processed, stored and secreted diarginyl insulin in a rapid regulated manner in response to glucose.Constitutive release of insulin also occurred and was greater than regulated secretion. Blood glucose levels of the mice were normalized but ultimately became subnormal due to continued proliferation of cells. Examination of the HUH7-ins cells as well as the parent cell line for b cell transcription factors showed the presence of NeuroD but not PDX-1. PC1 and PC2 were also present in both cell types. Thus, the parent HUH7 cell line possessed a number of endocrine pancreatic features that reflect the common endodermal ancestry of liver and pancreas, perhaps as a result of ontogenetic regression of the neoplastic liver cell from which the line was derived. Introduction of the insulin gene under the control of the CMV promoter induced changes in these cells to make them function to some extent like pancreatic b cells. Our results support the view that neoplastic liver cells can be induced to become substitute pancreatic b cells and become a therapy for the treatment of type I diabetes.

In order to design a feasible somatic cell gene delivery sysgogues. While glucose responsiveness commenced at a tem for the treatment of type I diabetes, a suitable cell type lower concentration than normal islets, a secretion curve needs to be determined. We have previously shown that approaching normal physiological conditions was generthe stable transfection of the full-length insulin cDNA into ated. Immunoelectron microscopy revealed the presence the human liver cell line, (HEP G2ins) resulted in synthesis, of insulin-containing granules, similar in size and appearstorage and acute regulated release of insulin to analogues ance to those of the normal beta cell. These results demof cAMP, but not to the physiological stimulus glucose. In onstrate that while it is most likely that the HEP G2ins/g attempting to explain the lack of glucose responsiveness cell line predominantly secretes insulin via the constitutive of the HEP G2ins cells we have stably transfected these pathway, significant acute regulated release was seen in cells with the human islet glucose transporter GLUT 2 response to glucose, and thus represents significant pro-(HEP G2ins/g cells). The HEP G2ins/g cell clones exhibit gress in the creation of a genetically engineered 'artificial glucose-stimulated insulin secretion and glucose potentibeta cell' from a human hepatocyte cell line. ation of the secretory response to nonglucose secreta-

We have recently shown a close correlation between expression of the Multidrug Resistance-associated Protein (MRP) gene and the MYCN oncogene and provided evidence that high MRP expression is a powerful independent predictor of poor outcome in neuroblastoma (Norris et al., New Engl. J. Med., 334, 231 ± 238, 1996). The e ect of MYCN down-regulation on MRP expression and response to cytotoxic drugs was investigated in NBL-S neuroblastoma cells transfected wtih MYCN antisense RNA constructs. Concomitant with MYCN down-regulation, the level of MRP expression was decreased in the NBAS-4 and NBAS-5 antisense transfectants. These cells demonstrated signi®cantly increased sensitivity to the high a nity MRP substrates vincristine, doxorubicin, sodium arsenate and potassium antimony tartrate, but not to the poor MRP substrates, taxol or cisplatin. Similarly, transfection of full-length MYCN cDNA into SH-EP neuroblastoma cells resulted in increased MRP expression and signi®cantly increased resistance speci®cally to MRP substrates. The results provide evidence for the MYCN oncogene in¯uencing cytotoxic drug response via regulation of MRP gene expression. Our data also provide a link between the malignant and chemoresistant phenotypes of this childhood malignancy.