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Application of the Cre/loxP System to Enhance Thyroid-Targeted Expression of Sodium/Iodide Symporter

Biochemistry Program (X.L., K.-Y.R., S.M.J.), Department of Physiology and Cell Biology (X.L., K.-Y.R., J.-Y.C., S.M.J.), Department of Pediatrics, College of Medicine (T.J.S.), Department of Internal Medicine and Radiology, Divisions of Endocrinology, Diabetes, and Metabolism, and Nuclear Medicine (R.T.K.); Center for Health Outcome Policy Evaluation Studies (E.L.M.), Ohio State University, Columbus, Ohio 43210; Department of Pathology (A.H.F.), University of Massachusetts, Worcester, Massachusetts 01655; Department of Internal Medicine, University of Florida (E.L.M.), Gainesville, Florida 32608-4653; Department of Biological Science (K.-Y.R.), Stanford University, Stanford, California 94305; and Department of Biochemistry (J.-Y.C.), School of Dentistry, Kyungpook National University, Daegu, 700-422, Republic of Korea

Address all correspondence and requests for reprints to: Sissy M. Jhiang, Ph.D., The Ohio State University, Department of Physiology and Cell Biology, 304 Hamilton Hall, 1645 Neil Avenue, Columbus, Ohio 43210. E-mail: Jhiang.1{at}osu.edu.

Radioiodide uptake activity mediated by the human Na⁺/I^– symporter (hNIS) in thyroid follicular cells is the basis for effective ¹³¹I therapy in thyroid cancer. However, radioiodide therapy is not effective in patients with thyroid cancer displaying low or absent hNIS expression. This study assessed the Cre/loxP system for enhancing thyroid-targeted hNIS expression driven by the thyroglobulin (Tg) promoter. The following three recombinant adenoviruses (rAd) were constructed: rAd-Tg-hNIS drives hNIS expression by the Tg promoter; rAd-Tg-Cre drives Cre expression by the Tg promoter; and rAd-CMV-loxP-hNIS drives hNIS expression by the cytomegalovirus (CMV) promoter after Cre-mediated excision of an intervening loxP-GFP-Zeo-loxP. Immortalized normal and malignant rat thyroid cell lines and primary cultures of normal human thyroid and human follicular adenoma cells were investigated. We found that the relative promoter activity of Tg vs. CMV is critical for the efficacy of the Cre/loxP system. In cells with weak Tg promoter activity, coinfection of rAd-Tg-Cre and rAd-CMV-loxP-hNIS induced higher hNIS expression than single infection of rAd-Tg-hNIS. Finally, Tg promoter activity was partially restored in malignant thyroid cells by forced expression of the paired domain-containing transcription factor (Pax-8), allowing the Cre/loxP system to mildly enhance radioiodide uptake.

This work was supported in part by Department of Defense Prostate Cancer Research Program DAMD 17-02-0119 (to S.M.J.).

Abbreviations: CMV, Cytomegalovirus; DTC, differentiated thyroid carcinoma; FBS, fetal bovine serum; HBSS, Hanks’ balanced salt solution; MOI, multiplicity of infection; NIS, Na⁺/I^– symporter; rAd, recombinant adenovirus; RAIU, radioiodide uptake; Tg, thyroglobulin.

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