Elizabeth R. Bergert

In vivo sodium iodide symporter gene therapy of prostate cancer

Radioiodine therapy, the most effective form of systemic radiotherapy available, is currently useful only for thyroid cancer because of thyroid-specific expression of the sodium iodide symporter (NIS). Here we explore the efficacy of a novel form of gene therapy using adenovirus-mediated in vivo NIS gene transfer followed by 131I administration for treatment of prostate cancer. Prostate cancer xenografts in nude mice injected with an adenovirus carrying the NIS gene linked to the cytomegalovirus (CMV) promoter revealed highly active uptake of radioiodine. Following administration of 3 mCi of 131I, we observed an average tumor volume reduction of 84 ± 12%. These results show for the first time that in vivo NIS gene delivery into non-thyroidal tumors is capable of inducing accumulation of therapeutically effective radioiodine doses and might therefore represent an effective and potentially curative therapy for prostate cancer.

In vivo Radioiodide Imaging and Treatment of Breast Cancer Xenografts after MUC1-Driven Expression of the Sodium Iodide Symporter

Purpose: Expression of the sodium iodide symporter (NIS) in the thyroid gland provides for effective imaging and treatment of thyroid cancer using radiolabeled iodide. Transfer of NIS into other tumors would expand the utility of this treatment to tumors of nonthyroid origin. MUC1 is a transmembrane glycoprotein that is overexpressed in many tumor types, including breast, pancreatic, and ovarian. The aim of this study was to create a construct containing NIS under the control of the MUC1 promoter to target expression specifically to MUC1-positive breast cancer cells. Experimental Design: A replication-deficient adenoviral construct was created containing the MUC1 promoter followed by the human NIS gene. Iodide uptake assays, Western blot, and immunohistochemistry were used to confirm NIS expression and function. Breast cancer xenografts in mice were infected with Ad5/MUC1/NIS and then imaged and treated using radioiodide. Results: A 58-fold increase in iodide uptake was observed in infected MUC1-positive T47D cells with no significant increase observed in MUC1-negative MDA-MB-231 cells or in cells infected with the control virus. The in vivo study yielded clear images of Ad/MUC1/NIS-infected tumor xenografts using 123I. Administration of a therapeutic dose of 131I resulted in an 83% reduction in tumor volume, whereas control tumors continued to increase in size (P < 0.01). Conclusions: These results show that the MUC1 promoter is capable of directing efficient and selective expression of the NIS gene in MUC1-positive breast tumor cells. This could potentially have applications for both imaging and therapy in a range of MUC1-positive tumor types.

Radioiodine therapy of colon cancer following tissue-specific sodium iodide symporter gene transfer.

We investigated the feasibility of using radioiodine therapy in colon carcinoma cells (HCT 116) following tumor-specific expression of the human sodium iodide symporter (hNIS) using the carcinoembryonic antigen (CEA) promoter. HCT 116 cells were stably transfected with an expression vector, in which hNIS cDNA has been coupled to a CEA promoter fragment. This promoter is responsible for tissue-specific expression of CEA in gastrointestinal tract epithelium, and has been shown to target therapeutic genes to colorectal cancer cells. Functional NIS expression was confirmed by iodide uptake assay, Western blot analysis, immunostaining and in vitro clonogenic assay. The stably transfected HCT 116 cells concentrated 125I about 10-fold in vitro without evidence of iodide organification. In contrast, transfection of control cancer cells without CEA expression did not result in iodide accumulation. Western blot analysis using a hNIS-specific antibody revealed a band of approximately 90 kDa. In addition, immunostaining of stably transfected HCT 116 cells revealed hNIS-specific membrane-associated immunoreactivity. In an in vitro clonogenic assay approximately 95% of stably transfected HCT 116 cells were killed by exposure to 131I, while only about 5% of NIS-negative control cells were killed. Further, using an adenovirus carrying the NIS gene linked to the CEA promoter, high levels of tumor-specific radioiodide accumulation were induced in HCT 116 cells. In conclusion, a therapeutic effect of 131I has been demonstrated in colon carcinoma cells following induction of tumor-specific iodide uptake activity by CEA promoter-directed NIS expression in vitro. This study demonstrates the potential of NIS as a therapeutic gene allowing radioiodine therapy of colon cancer following tumor-specific NIS gene transfer.

Sodium iodide symporter-mediated radioiodide imaging and therapy of ovarian tumor xenografts in mice

Ovarian cancer represents the fifth leading cause of cancer death among women in the United States, with >16 000 deaths expected this year. This study was carried out to investigate the potential of sodium iodide symporter (NIS)-mediated radioiodide therapy as a novel approach for ovarian cancer treatment. Radioiodide is routinely and effectively used for the treatment of benign and malignant thyroid disease as a result of native thyroidal expression of NIS, which mediates iodide uptake. In vitro gene transfer studies in ovarian cancer cells revealed a 12- and five-fold increase in iodide uptake when transduced with Ad/CMV/NIS or Ad/MUC1/NIS, respectively. Western blot/immunohistochemistry confirmed NIS protein expression. In vivo ovarian tumor xenografts were infected with the adenoviral constructs. 123I imaging revealed a clear image of the CMV/NIS-transduced tumor, with a less intense image apparent following infection with MUC1/NIS. Therapeutic doses of 131I following CMV/NIS infection caused a mean 53% reduction in tumor volume (P<0.0001). MUC1/NIS-transduced tumors did not regress, although at 8 weeks following therapy, tumor volume was significantly less that of control animals (166 versus 332%, respectively, P<0.05). This study represents a promising first step investigating the potential for NIS-mediated radioiodide imaging and therapy of ovarian tumors.

Cloning of the mouse sodium iodide symporter.

The iodide-concentrating ability of the thyroid gland is essential to the production of thyroid hormone. We report the nucleotide and amino acid sequence of the mouse sodium iodide symporter (mNIS), which mediates this activity within the thyroid gland. An open reading frame of 1,857 nucleotides codes for a protein of 618 amino acids with 95% identity to rat NIS and 84% identity to human NIS. Transient expression of the mNIS cDNA in Chinese hamster ovary (CHO) cells, a nonthyroid cell line, resulted in sodium-dependent, perchlorate-sensitive iodide uptake. Western blot analysis of membrane preparations of CHO cells transiently transfected with mNIS cDNA showed a band of 90 kd when probed with an antibody directed against rat NIS. mNIS will serve as an important reagent in determining the role of NIS in experimental thyroid diseases and for monitoring the immune response to in animal models of NIS-mediated gene therapy.

Construction of an MUC-1 promoter driven, conditionally replicating adenovirus that expresses the sodium iodide symporter for gene therapy of breast cancer

IntroductionThe sodium iodide symporter (NIS) directs the uptake and concentration of iodide in thyroid cells. This in turn allows radioiodine imaging and therapy for thyroid cancer. To extend the use of NIS-mediated radioiodine therapy to other types of cancer, we successfully transferred and expressed the sodium-iodide symporter (NIS) gene in prostate, colon, and breast cancer cells both in vivo and in vitro by using non-replicating adenoviral vectors.MethodsTo improve virotherapy efficiency, we developed a conditionally replicating adenovirus (CRAd) in which the transcriptional cassette RSV promoter-human NIScDNA-bGH polyA was also inserted at the E3 region. The E1a gene is driven by the tumor-specific promoter MUC-1 in the CRAd Ad5AMUCH_RSV-NIS.ResultsIn vitro infection of the MUC-1-positive breast cell line T47D resulted in virus replication, cytolysis, and release of infective viral particles. Conversely, the MUC-1-negative breast cancer cell line MDA-MB-231 was refractory to the viral cytopathic effect and did not support viral replication. The data indicate that Ad5AMUCH_RSV-NIS activity is stringently restricted to MUC-1-positive cancer cells. Radioiodine uptake was readily measurable in T47 cells infected with Ad5AMUCH_RSV-NIS 24 hours after infection, thus confirming NIS expression before viral-induced cell death.ConclusionsThis construct may allow multimodal therapy, combining virotherapy with radioiodine therapy to be developed as a novel treatment for breast and other MUC1-overexpressing cancers.

The effect of deglycosylated human chorionic gonadotropin on corpora luteal function in healthy women

Fourteen healthy young women were studied through a control and a treatment menstrual cycle in two series of experiments. In the first series, they were given one of four doses of deglycosylated human chorionic gonadotropin (hCG) as a 24-hour infusion during the mid-luteal phase of the cycle. In these studies, there were no significant alterations of the length of the luteal phase of the treatment cycle, and there was no decrease in serum progesterone (P) during the infusion. In fact, serum P increased during the infusion. In the second series of studies, five subjects were given a 48-hour infusion of normal saline during the control cycle, and a 48-hour infusion of deglycosylated alpha-intact beta-hCG during the treatment cycle, both being administered during the mid-luteal phase. Treatment did not alter luteal phase duration and, again, increased serum P. It is concluded that deglycosylated preparations of hCG are not clinically useful as luteinizing hormone antagonists, probably because of residual agonist activity.

Identification of thyroid blocking antibodies and receptor epitopes in autoimmune hypothyroidism by affinity purification using synthetic TSH receptor peptides

UNLABELLED To examine the interaction of immunoglobulins from patients with newly diagnosed hypothyroidism with the TSH receptor (TSHr), we tested protein-A purified IgG in an ELISA assay with a series of peptides representing the entire extracellular domain (ECD) of human TSHr. Antibodies bound, on average, 4.1 peptides (range 0-16) per patient, and antibodies from 26 of 30 patients (86.6%) demonstrated binding to at least one peptide. Six of the 20-mer peptides (61, 151, 181, 301, 361, 376) were most frequently recognized. These were used to construct affinity columns and separate IgGs from 10 patients into bound and unbound fractions. All fractions were tested for their ability to stimulate and inhibit cAMP generation in FRTL-5 cells. Inhibitory IgGs were purified from 9 patients (90%), suggesting that the incidence of blocking antibodies (TBAb) in autoimmune hypothyroidism is higher than previously reported. 7 of 10 patients had antibodies that recognized peptide 361 further supporting the importance of this epitope in TBAb binding. Anti-microsomal and anti-thyroglobulin antibodies did not co-purify with inhibitory antibodies, and were always in the unbound fractions. We found no correlation between the pattern of antibody binding or bioactivity with clinical manifestations of hypothyroidism. CONCLUSIONS (1) The majority of patients with autoimmune hypothyroidism have antibodies against the TSHr-ECD that recognized linear epitopes. Most have antibodies directed at more than one site and the pattern is quite heterogeneous. (2) Six sites (noted above) are most frequently recognized. (3) Inhibitory antibodies are distinct from anti-microsomal and anti-thyroglobulin antibodies.

1014. Adenovirus-Mediated MUC1-Driven Expression of the Sodium Iodide Symporter (NIS) in Pancreatic Tumors Provides for In Vivo Radioiodide Imaging and Therapy

Top of pageAbstract Pancreatic cancer is the fourth leading cause of cancer death in the United States. It is highly aggressive with no effective chemotherapy available for metastatic disease. The Sodium Iodide Symporter (NIS) is a transmembrane glycoprotein responsible for uptake of iodide into cells. The presence of NIS in thyroid cells has permitted non-invasive diagnostic imaging and ablation of thyroid tumors using radiolabeled iodide. A previous study from this laboratory reported adenovirus-mediated MUC1-driven expression of the NIS gene in breast cancer cells. The use of the MUC1 promoter to drive NIS expression resulted in selective tumor specific expression of the protein. MUC1 is overexpressed in many tumor types including breast, pancreatic, lung, prostate and ovarian. The incidence of MUC1 overexpression in pancreatic tumors has been reported to be as high as 90%. In this study the replication-deficient Ad5-MUC1-NIS construct was used to infect MUC1-positive pancreatic cancer cells both in vitro and in vivo, to investigate the potential for radioiodide imaging and ablation of this disease. In vitro studies revealed a 43-fold increase in iodide uptake in NIS transduced cells compared to controls. Western blot and immunohistochemical analysis confirmed NIS protein expression and membrane localization. MUC1 negative pancreatic cells transduced with the construct did not express NIS, which confirmed the specificity of the construct. In vivo studies were carried out in tumor-bearing nude mice. Intratumoral injection of virus was followed 4 days later by intraperitoneal injection of 0.5mCi 123I. Serial images were then taken of the animals from 1-48 hrs following iodide administration. This revealed clear images of the NIS transduced tumors, with optimal uptake observed 1hr after iodide administration. Tumors infected with a promoterless virus did not concentrate iodide. Studies investigating the potential for treatment of pancreatic cancer using therapeutic doses of 131I are ongoing. The ability to target NIS expression to pancreatic cancer, which has such limited treatment options, may be highly beneficial. This study is an important initial step investigating the potential for use of radiolabeled iodide as a systemic therapeutic agent in pancreatic cancer treatment. The results presented here clearly demonstrate that the application of this virus is not limited to one tumor type, but may provide for imaging and therapy of a range of MUC1-positive malignancies.

29. In Vivo Imaging and Therapy of Breast Cancer Xenografts Following MUC1 Promoter-Driven Expression of the Sodium Iodide Symporter (NIS)

The sodium iodide symporter (NIS) is a transmembrane glycoprotein responsible for the uptake of iodide into cells. Robust NIS expression in the thyroid gland provides for effective imaging and non-invasive treatment of thyroid disease using radioactive iodide. Gene therapy involving selective induction of NIS expression in other tumors offers the possibility of imaging and treating a variety of tumor types with radioactive iodide. We have recently reported in vitro, plasmid-mediated expression of NIS under the control of the MUC1 promoter in a variety of tumor cell lines. Since MUC1 is overexpressed in many tumor types, particularly breast cancer, this promoter provides the ability to selectively target tumor tissue. In this study, a replication-deficient adenoviral construct containing the MUC1-NIS insert was used to induce iodide uptake in breast cancer xenografts. Subcutaneous T47D breast cancer xenografts were established on both flanks of athymic nude mice. When they had reached an appropriate volume, intratumoral injections of Ad5/MUC1/NIS or control virus were administered. Mice were then given an intra-peritoneal injection of either 0.5mCi 123I (imaging) or 3mCi 131I (therapy). Scanning with a gamma camera revealed clear images of iodide uptake in the mouse stomach, thyroid and bladder, as well as at the site of the tumors injected with Ad5/MUC1/NIS. Tumors injected with the promoter-less Ad5/NIS did not take up iodide. Mice treated with 131I showed a marked regression in tumor size within one week of iodide administration, with tumor volume reduced by 50% within three weeks. Tumors that had been injected with control virus did not express NIS, and retained their original volume or increased in size. These preliminary in vivo experiments demonstrate that the Ad5/MUC1/NIS construct is capable of inducing robust NIS expression and iodide uptake in MUC1 positive breast tumor xenografts. This may have potential applications for the imaging and treatment of a variety of MUC1-positive tumor types.