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. Author manuscript; available in PMC: 2011 May 1.
Published in final edited form as: Cancer Gene Ther. 2010 Jan 22;17(5):373–374. doi: 10.1038/cgt.2009.90

Neuroblastoma-specific expression of potential therapeutics cannot be achieved using a promoter region of the NCX (TLX2) gene

A Tivnan 1, RL Stallings 1,*
PMCID: PMC2943140  NIHMSID: NIHMS236130  PMID: 20094074

Neuroblastoma is a paediatric solid tumour which arises from precursor cells of the sympathetic nervous system1. Narita et al. published an interesting report that the promoter region of the TLX2 (NCX, Hox11L1, ENX) gene, a member of the HOX11 homeobox gene family, might be used to selectively express potential therapeutic gene sequences in neuroblastoma cells, allowing targeted expression of a suicide gene in cancer cells2. The approach of conditional expression of a gene therapy has been successfully developed and tested using a variety of disease- or cell-specific promoters3, 4. The rationale for using the TLX2 gene promoter to achieve neuroblastoma specific expression was that expression of the endogenous TLX2 gene is primarily confined to neural crest-derived tissues2,5–7.

The NCX1.7-luc vector is a modified pGL2-basic vector containing a 1.7kb region of the NCX promoter upstream of the ATG start codon, which was generated through sequential deletions of genomic DNA of the NCX gene2. This promoter region was activated in neuroblastoma cells and expression was measured as a function of luciferase expression. Notably, Narita et al. demonstrated that transfection of this construct into six neuroblastoma cell lines (SH-SY5Y, NGP, NMB, NBL-S, NLF and SMS-SAN) resulted in luciferase expression, but not in negative controls which included fibroblasts (HFF, MRC-5 and CDC18Co), melanoma (A875), pancreatic cancer (AsPC-1) and lung carcinoma (QG56) cell lines. Our group has identified miRNAs that have anti-proliferative effects when ectopically up-regulated in neuroblastoma cell lines8,9, so we were interested in validating the cell specificity of NCX1.7-luc expression for use in microRNA mediated therapeutics. The NCX1.7-luc vector was transfected into nine neuroblastoma cell lines [Kelly, NGP, IMR-32, SKNBE, SKNAS, LAN-66, SH-SY5Y and SHEP T21N (a non-MYCN amplified cell line containing a tetracycline repressible MYCN expression construct)], and in several putative negative controls, including human embryonic kidney (HEK293) cells, cervical cancer (HeLa) cells, breast cancer (MDA-MB-231 and Hs578T) cells and human astrocytoma glioblastoma (U373MG) cells.

As expected, we confirmed that the NCX1.7-luc vector was expressed in all neuroblastoma cell lines, although expression in one cell line, SK-N-AS was low (Figure 1). Surprisingly, however, NCX1.7-luc expression was also noted in breast cancer (MDA-MB-231 and Hs578T), HeLa and U373MG cell lines to a greater degree than several of the NBL cell lines when compared to luciferase expression in HEK293 transfected cells (Figure 1).

Figure 1.

Figure 1

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Luciferase expression in NCX1.7-luc-transfected cell lines 48 hours after transfection. All data was normalised to pGL2-basic empty vector (encoding the luciferase gene devoid of a promoter region) transfected cells. pMIR-Report vector (Applied Biosystems) was used as a positive control for successful transfection in each of the cell lines tested (data not shown). All expression values are relative to HEK293-transfected cells, set as 1.0.

Consistent with our experimental observations is the fact that endogenous TLX2 gene expression occurs in a number of non-neuroblastoma tissues, as indicated by the Oncogenomics Normal Tissue Database10. These include the heart, bladder, cerebrum, adrenal glands and intestines, a fact which was also reiterated by Borghini et al (2006)5.

In summary, our results indicate that ectopic expression of NCX1.7-luc is not as specific to neuroblastoma cells as previously thought, leading us to conclude that the TLX2 promoter cannot be considered cell or tissue-specific with regard to generating conditional gene therapies for neuroblastoma.

Acknowledgements

The NCX1.7-luc vector was obtained from Dr. Masahiko Hatano, Dept of Biomedical Science, Chiba University, Japan. HeLa cells were kindly donated by Dr. Rowan Higgs, MCT, RCSI. Hs578T cells were obtained from Ms. Leanne Moore, The Ocular Genetics Unit, Trinity College Dublin and U373MG cells were donated by Ms. Elif Dagdan, The Ocular Genetics Unit, Trinity College Dublin. This work was supported by grants from Science Foundation Ireland, Children’s Medical and Research Foundation, and the NIH (5R01CA127496).

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