HMG-CoA reductase (HMGR) catalyzes the rate controlling step in cholesterol production. This enzyme is highly expressed in the liver where it is subject to extensive hormonal and dietary regulation. This study was undertaken to examine the occupancy and function of the hepatic HMGR promoter in regards to insulin and sterol regulation. HMGR protein and mRNA are substantially decreased in diabetic animals and rapidly restored by administration of insulin. Nuclear run-on assays revealed that HMGR transcription was substantially reduced in the diabetic rats, and fully restored within two hours after insulin treatment. In vivo footprinting revealed several areas of protein binding as shown by dimethyl sulfate protection or enhancement. The CRE was heavily protected in all conditions - including diabetes, cholesterol feeding, or statin treatment. Striking enhancements in footprints from diabetic animals were observed at -142 and at -161 (in the SRE). Protections at a newly ident ified NF-Y site at -70/-71 were seen in normal animals, and not in diabetics. This proximal NF-Y site was found to be required for efficient HMGR transcription. CREB-1 was able to bind the HMGR CRE in vitro, and to the promoter in vivo. The data supports an essential role for CREB in transcription of hepatic HMGR, and identifies at least two sites where in vivo occupancy is regulated by insulin. The technique of in vivo electroporation was utilized to perform the first functional analysis of the HMGR promoter in live animals. Analysis of a series of deletion constructs showed that deletion of the region containing the cyclic AMP response element (CRE) at -104 to -96 and the newly identified NF-Y site at -70 resulted in marked reduction of promoter activity. Possible sterol regulation of the promoter was investigated by raising tissue cholesterol levels by feeding cholesterol, or by inhibiting cholesterol synthesis with a statin (lovastatin). It was found that HMGR promoter constructs r esponded to lovastatin, in agreement with previous findings in cultured cells. This work sheds light on the regulation of the HMGR promoter in the liver, whose expression is a key determinant of serum cholesterol levels- a major risk factor for cardiovascular disease.

Novel therapies and delivery methods directed against malignancies such as melanoma, and particularly metastatic melanoma, are needed. The HIV-1 accessory protein Vpr (viral protein R) has previously been demonstrated to induce G2 cell cycle arrest as well as in vitro growth inhibition/killing of numerous tumor cell lines. In vivo electroporation has been utilized as an effective delivery method for pharmacologic agents as well as DNA plasmids that express "therapeutic" proteins and has been targeted to various tissues including malignant tumors. In this study, we assessed the ability of electroporation-mediated delivery of Vpr plasmid (pVpr) to induce growth attenuation or complete tumor regression in C57BL/6 mice with subcutaneous B16.F10 melanoma lesions. To assess the administration of intratumoral delivery of pVpr with in vivo electroporation, a range of Vpr plasmid dosages, electroporation parameters, and treatment days were evaluated in a subcutaneous B16 murine melanoma model. pVpr was injected directly into the tumors. Immediately following the injection, the subcutaneous tumors were electroporated. Treatment with 25 microgram or 100 microgram of pVpr plus electroporation on days 0 and 4 resulted in complete tumor regressions with long-term survival in 14.3% and 7.1% of the mice, respectively. In order to optimize the treatment regimen, B16 tumors were treated on days 0, 2, and 4 with 100 microgram pVpr plus electroporation which resulted in 50% of the mice with complete tumor regressions and long-term survival. Additional investigations revealed intratumoral Vpr expression and demonstrated that apoptosis was the mechanism by which Vpr caused tumor regression in vivo. This study confirmed that treatment with 100 microgram of pVpr plus electroporation led to durable complete regressions in established murine melanoma lesions. The pVpr plus electroporation treatment regimen has induced complete regressions in mice as well as resistance to tumor challenge in some of the animals. This is the first comprehensive study demonstrating the ability of Vpr, when delivered as a DNA expression plasmid with in vivo electroporation, to induce complete tumor regressions coupled with long- term survival of mice in a highly aggressive and metastatic solid tumor model.

In this study, we examined the effects of thyroid hormone on regulatory processes of cholesterol metabolism. Specifically, the pathways of cholesterol synthesis and cholesterol efflux were investigated. Hepatic HMG-CoA reductase (HMGR) is the rate-limiting enzyme in cholesterol synthesis. Hypothyroid rats exhibit decreased expression of this gene, which can be induced by subsequent treatment with thyroid hormone. The mechanism of this activation was previously unknown. Utilizing in vivo electroporation, we identified HMGR promoter elements necessary for the induction of HMGR by thyroid hormone. The -316/-321 element, the sterol response element, and nuclear factor-y site were all found to be necessary to induce HMGR promoter activity by thyroid hormone. We used electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) studies to demonstrate increased binding of upstream transcription factor-2 (USF-2) to the -316/-321 element in the HMGR promoter in response to thyroid hormone. Finally, co-electroporation of the wild-type HMGR plasmid with siRNA to USF-2, SREBP-2, or NF-Y nearly abolished the T3 induction as measured by promoter activity. Microarray and real-time PCR analysis demonstrated an induction of the apolipoproteins ApoA-I and ApoA-IV mRNA by T3. Serum levels of ApoA-I and ApoA-IV proteins were induced by T3. We collected serum from rats treated with or without T3 and used these sera in an in vitro macrophage efflux model. We found that T3 promoted cholesterol efflux via the ABCA1 cholesterol transporter and not via the ABCG1 transporter. We propose that the induction of serum ApoA-I and ApoA-IV by thyroid hormone promotes cholesterol efflux via the ABCA1 cholesterol transporter. Hepatic ABCG5 and ABCG8 are cholesterol transporters that promote biliary secretion of cholesterol. We utilized EMSAs to scan the shared ABCG5/G8 rat promoter for a thyroid hormone response element (TRE). We identified a TRß binding site at -392/-376 of the ABCG8 promoter. Collectively, these observations provide new insight into the cholesterol-lowering function of thyroid hormone.