Expression of the MAGE Gene Family in Neuroblastoma Cell Lines and the Detection of an Alternatively Spliced Transcript

Xavier Simon


Human neuroblastoma (NB), a solid tumor cancer, is an embryonic cancer arising from neural crest tissue (Ross and Spengler, 2007), more specifically from the postganglionic, sympathetic nervous system (SNS) of embryonic neural crest (Thiele, 1998). Many cases of NB begin in the sympathetic nerve ganglia along the SNS. NB accounts for about 9 % of all childhood cancers (Thiele, 1998), with about 500-700 new cases diagnosed each year.

Biochemical distinctions and differences in malignancy help characterize three different NB cell types: neuroblastic (N); substrate-adherent cells (S); and intermediate type cells (I), which are stem cell precursors to N- and S-type cells. N cells are small, weakly substrate-adherent cells that have cytoplasmic extensions (neurites) and grow as clumps of cells (pseudoganglia) (Ross and Spengler, 2007). S-type cells are substrate-adherent, and form a contact –inhibited monolayer when grown in culture; they are considered non-malignant and unable to form tumors (Ross and Spengler, 2007). I-type cells are considered the most malignant, followed by N-type cells. At its discovery, I-type cells were termed as such because their growth characteristics and morphology resembled an “intermediate” between N- and S-type cells; later research recognized I-type cells to be malignant and most closely resemble cancer stem cells (Ross and Spengler, 2007). Very little is known about what triggers the formation of the actual cancer or what causes the differences in its regression or growth potential.

Melanoma Antigen Gene Family
Human melanoma antigen (MAGE) genes have been shown to be expressed in both normal tissues and in various tumors and tumor related cells. Two types of MAGE genes have been characterized based on their expression: type-I members are silent in all normal tissues except for in the male germ line and placenta while type-II members are expressed ubiquitously in both tumor and normal cells (Figure 1). MAGE-C subfamily members are type-I genes expressed in various tumor types; their proteins are tumor-specific antigens that can be recognized by cytolytic T lymphocytes. MAGE-D subfamily members are type-II members – they do not encode for those tumor-specific antigens seen in type-I MAGE and are also expressed ubiquitously in normal adult tissues. While MAGE genes could be targets for immunotherapy, information on the function and expression pattern of MAGE-C and MAGE–D genes, however, remains incomplete. Analysis of the gene expression of type-I and type-II MAGE genes in various histological tumors may lead to improved diagnoses and the development of MAGE-based anti-cancer immunotherapies.

In this study, the expression levels of two MAGE-C genes, -C1 and –C2, and one MAGE-D2 family gene, were examined in different NB cell lines. MAGE-D2 has three transcript variants (TV), characterized by differences at exon one and intron one between the TVs. RT-PCR using variant-specific primers was performed and the products were analyzed on agarose gels.

To study the expression of MAGE-C1, -C2, -D2 TV1, -D2 TV2, and –D2 TV3, RT-PCR using variant specific primers was performed on mRNA isolated from four neuroblastoma cell lines—LAI-55N, LAI-5S, BE(2)-C, and JMN. RT-PCR was also performed with GAPDH as a loading control to monitor the amount of mRNA in the samples. RT-PCR products were then analyzed on 2% agarose gels. RT-PCR of MAGE-D2 TV3 shows the expression of two splice variants with the MAGE-D2 TV3 primer pair (Fig. 2). Sequencing confirmed the retention of intron 1 in transcript variant 3 (Fig. 3). Band intensities for MAGE-C1, MAGE-C2, MAGE-D2 TV1, MAGE-D2 TV2, and MAGE-D2 TV3 were similar between the cell lines (Fig. 2). Products were confirmed by sequencing and all experiments were replicated at least three times.

Results show the discovery of a new MAGE-D2 variant that retains intron one. The significance of this variant with the retained intron remains unknown. Papageorgio et al. 2007 suggests MAGE-D2 is a potential dissociator of p53, a tumor suppressing gene, but whether or not MAGE genes play a significant role in cell cycle regulation and subsequent cancer prognosis remains to be seen. Subsequent analysis of MAGE-D2 sequencing data –with a focus on splice sites and possible mutations within those sites—may provide further insight.

There were no significant differences observed in expression levels of MAGE-C1 or MAGE-C2 between the three NB cell lines. Further studying of differential expression between different tumor and normal cell lines should be done to conclusively state that there is no difference in gene expression. Studies examining other cell lines could provide clues as to the function of the inclusion of intron one in the MAGE-D2 variant, and could also provide further understanding of the role of the MAGE gene family in neuroblastoma, other tumor cells and in normal tissues. Monte et. al. (2005) write that MAGE-A1, a type-I MAGE, has shown resistance to chemotherapeutic agents, but whether this is universal in all MAGE family genes has yet to be established, and further studies on MAGE type-I and –II genes could be done to aid in the development of possible MAGE-based anti-cancer immunotherapies.

Literature cited
Monte, M., Simonatto, M., Peche, L. Y., Bublik, D.R., Gobessi, S., Pierotti M.A.,
Rodolfo, M., and Schneider, C. (2005). MAGE‐A tumor antigens target p53 transactivation function through histone deacetylase recruitment and confer resistance to chemotherapeutic agents. Proc. Natl. Acad. Sci. 103: 11160‐11605.

Papageorgio, C., Brachmann, R., Zeng, J., Culverhouse, R., Zhang, W., and McLeod, H.
(2007) MAGE-D2: a novel p53-dissociator. Int. J. Oncol. 31(5): 1205-1211.

Ross, R.A., and Spengler, B.A. (2007) Human neuroblastoma stem cells. Semin. Caner.
Biol. 17, 241–247.

Thiele, C. J. (1998). Neuroblastoma cell lines. In J. Masters (Ed.), Human cell culture
(pp. 21–53). Lancaster, UK: Kluwer Academic Publishers.


Figure 1-Human MAGE Gene Family

Figure 2-RT-PCR results showing MAGE-C1, -C2, -D2 TV1, TV 2, and TV 3 expression. GAPDH was used as a loading control

Figure 3-Alternative Splicing in MAGE-D2. (Top) - Design of Alternative Splicing in MAGE-D2 TV3 (shown in red). (Bottom) -Partial BLAST Alignment results of extracted bands showing the retention of intron 1 in the top band


Human neuroblastoma (NB), a solid tumor cancer, arises from neural crest tissue of the developing sympathetic nervous system (SNS). NB accounts for about 9 % of all childhood cancers, with about 500-700 new cases diagnosed each year. Three phenotypically distinct cell types compose NB cell lines: neuroblastic (N), substrate-adherent (S), and intermediate (I). A subfamily of human melanoma antigen (MAGE) genes (MAGE-A,-B-and –C) encodes tumor-specific antigens and MAGE genes have been shown to be expressed in various tumor types. Here, the expression of MAGE-C1, -C2, and –D2 was studied by RT-PCR in the different NB cell lines represented here by LAI-55(N), LAI-5(S), BE(2)-C (I) and JMN (I) cells. RT-PCR yielded an alternatively spliced transcript variant of MAGE-D2. Sequencing verified the transcript variant retained the 79bp intron one found in MAGE-D2 transcript variant three cDNA. No significant differences were observed in the expression levels of MAGE-C1, and –C2, between the cell lines.

Full Paper


I would like to thank Dr. Berish Rubin and the laboratory for Familial Dysautonomia research for their help and guidance. I would also like to thank Alex Bulanov and Xie Xie for their time, assistance, and patience. Additionally, I would like to thank Dr. Robert Ross and the Laboratory of Neurobiology, Department of Biological Sciences, Fordham University, for providing the Neuroblastoma cells.

This document was last modified 05/20/2011.
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