High level, tissue-specific expression of a modified calcitonin/calcitonin gene-related peptide promoter in a human medullary thyroid carcinoma cell line
Introduction
Medullary thyroid carcinoma (MTC) is a malignant tumour that arises in the calcitonin-secreting parafollicular C cells of the thyroid. Many patients develop metastatic disease that is poorly responsive to chemotherapy and radiotherapy (Marsh et al., 1995). There is thus a need for an alternative systemic therapy that would enable specific targeting of metastatic deposits of MTC. Targeted gene therapy is a potential new strategy for treatment of this disease.
A number of approaches have been described that target gene expression specifically to tumour cells (Dachs et al., 1997). One approach employs specific promoters to direct the tissue-specific expression of linked therapeutic genes. Targeting at the transcriptional level is facilitated by knowledge of the regulatory sequences responsible for tissue specificity which enable the development of highly selective and efficient expression of a therapeutic gene in target cells.
The CT/CGRP gene is predominantly expressed in thyroid C cells resulting in the production of CT. The promoter is also active in neuronal cells where its expression results in an alternatively spliced product CGRP (Jonas et al., 1985). Given the selective expression of the calcitonin/calcitonin gene-related peptide (CT/CGRP) promoter, we have modified this promoter as the initial step in developing targeted gene therapy for MTC.
Key regulatory elements within the 1.7 kilobase (kb) promoter region, mediating cell specific transcription are the distal cis-acting tissue-specific enhancer elements (TSE) which contain several consensus helix-loop-helix (HLH) motifs and an octamer-binding (OB2) motif (Stolarsky-Fredman et al., 1990, Peleg, 1993, Lanigan and Russo, 1997), and the cAMP induced enhancer comprising CRE and CREL/O sites (cAMP like and octamer elements) within the proximal promoter (De Bustros et al., 1992, Monla et al., 1995). We examined whether the insertion of multiple copies of the TSE adjacent to a minimal human CT/CGRP promoter could be utilized to further enhance CT/CGRP expression in a tissue-specific manner. We demonstrated that the addition of two copies of the TSE to a truncated CT/CGRP promoter not only upregulated the expression, but also increased its tissue specificity for TT cells.
Section snippets
Construction of expression plasmids
Plasmid constructs are shown in Fig. 1. A series of human CT/CGRP promoter luciferase fusion constructs were generated by PCR amplification using the human genomic CT/CGRP gene (kindly provided by Dr R. Gagel and Dr G. Cote, Houston, TX) as the template. Individual TSE units spanning nucleotide positions −1059–−899 were ligated to the truncated promoters. The PCR primers were designed to include restriction enzyme recognition sequences at their 5′ oligonucleotide ends. PCR products were
Expression of the CT/CGRP promoter constructs in the human MTC cell line
Plasmids were constructed in which a one, two or three TSE elements were placed upstream of the CT/CGRP minimal promoter. Furthermore, since sequences between −175 and −129 have been shown to increase the activity of the minimal promoter and to provide for induction by cAMP, we wished to evaluate whether inclusion of these sequences might improve the activity or specificity of the TSE-containing constructs.
The promoter activity of each of the CT/CGRP promoter constructs linked to a luciferase
Discussion
Transcriptional regulation provides a valuable tool for directing selective expression of a potentially therapeutic gene in treatment of disease. However, the use of non modified promoters may be limited by their inability to direct high level, tissue specific expression necessary for effective targeted gene therapy.
We have selected the CT/CGRP promoter as a candidate for directing tissue specific expression in medullary thyroid cancer. Although the full length unmodified CT/CGRP promoter was
Acknowledgements
We are grateful to Peter Brown for helpful discussions. This work was supported by grants from the University of Sydney Cancer Research Fund and the Northern Sydney Area Health Service.
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2002, SurgeryCitation Excerpt :For construction of an Ad vector expressing the dn-RET mutant, a point mutation was introduced at codon 32 into the RET51 cDNA (a gift of M. Billaud) as described.12 Subsequently, the C-cell selective synthetic calcitonin promoter (TSE2.CP1; a gift of M. Messina)15 was cloned into the pShuttle plasmid (pShuttle-TSE2.CP1), followed by insertion of the mutant RET cDNA and Simian Virus 40 poly A (SV40 polyA) from pGL3Basic, resulting in pShuttle-TSE2.CP1-Ret51HSCR32. AdTSE2.CP1-GFP-Luc was generated by cloning the green-fluorescent-protein (GFP) cDNA, encoding the membrane localized enhanced GFP16 together with the internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV), which allows expression as a polycistronic messenger RNA, into pGL3basic.
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