Expression of NCAM1 and CDH2 mRNA and the Presence of Alternatively Spliced NCAM1 mRNA in Neuroblastoma Cell Lines




Alex Bulanov

Introduction

Neuroblastoma makes up approximately 9% of all childhood cancers, occurring once out of every 8,000 live births. In children under 5 years old, it typically presents as tumors in the abdomen, commonly with involvement of the sympathetic ganglia of the paraspinal region or the adrenal gland. In infants less than one year old, the tumors are generally in the thoracic region. Neuroblastoma has the highest rate of spontaneous regression of all tumors, where widely disseminated tumors disappear with minimal treatment. Neuroblastoma is thought to arise from the postganglionic sympathetic neuroblasts of the neural crest. The neuroblastoma population is comprised of three cell types that can exhibit a neuroblastic (N), a flat or substrate adherent (S), or an intermediate (I) phenotype of which KCN-83n, SH-EP1, and BE(2)-C are the representatives, respectively. Of the three, the I cells exhibit the most stem cell features.
The expression levels of mRNA encoding two proteins were studied by RT-PCR in the three cell lines: Neural Cell Adhesion Molecule (NCAM1) and N-cadherin (CDH2).
Neural Cell Adhesion Molecule 1, also CD56, is a calcium independent cell-adhesion glycoprotein from the immunoglobulin (Ig) superfamily, located on chromosome 11q23.1. It binds homophillically in a cis and trans fashion. NCAM1 has five Ig-fold like domains and two fibronectin type III domains. NCAM1 has three observed transcript variants (TV). Transcript variant 1 makes an 180kDa protein with a long cytoplasmic domain and contains 20 exons. Transcript variant 2 makes a 140kDa protein with a short cytoplasmic domain; it is missing the 30bp exon 9 and thus contains 19 exons. Transcript variant 3 makes a 120kDa glycophosphatidylinositol (GPI)-anchored protein which is missing exons 9, 18, 19, 20 but has three additional exons between exons 14 and 15, and an early polyadenylylation with a novel exon 20, resulting in a transcript with 20 exons. Shifts in expression are recorded from the adult 120 kDa isoform to the embryonic 140 and 180 kDa isoforms in various tumors.
NCAM1 has been shown to be upregulated at the synapse during learning, where it will facilitate the release, repositioning and expansion of the synaptic complex. NCAM1 also functions in neuron-neuron adhesion, neurite fasciculation, neuronal pathfinding, and neurite outgrowth.

N-cadherin (CDH2) is a classic cadherin - a calcium dependent glycoprotein that functions in the adherens junctions in the cell. It is located on chromosome 18q11.2, contains 16 exons, and has no known splice variants. It meditates strictly homotypic adhesion first in a cis-dimerization then a trans tetramer adhering two cells with a so called “cadherin zipper”. CDH2 contains five extracellular cadherin repeats, a transmembrane region, and a highly conserved cytoplasmic tail where it interacts β-catenin, γ-catenin and p120ctn to form a cytoplasmic cell adhesion complex where it links to the actin cytoskeleton.
N-Cadherin is essential in synaptic buton - dendritic spine association. It also functions in gastrulation and the establishment of left-right asymmetry. It is described as a mesenchymal cadherin as it tends to be expressed in migratory cells, as opposed to the epithelial cadherins. In fact, a cadherin switch mechanism has been proposed that would explain metastasis as the switching from a pro-adhesive E-cadherin to a pro-migratory N-cadherin expression.
NCAM1 and CDH2 are both highly expressed in the advancing axonal growth cones and the cellular substrates the cone contacts. NCAM1 and CDH2 can associate with and stimulate fibroblast growth factor receptors (FGFRs), inducing signal transduction cascades that affect neurite outgrowth, cell adhesion, motility, and invasion. CDH2 prevents the internalization of ligand bound FGFR increasing signal while NCAM1 directly activates FGFR bay assembling a a classical signaling complex, including FRS-2, c-Src, cortactin and phospholipase Cγ.
NCAM1 and CDH2 are intertwined with neuronal attachment, growth and migration so a differential expression between the three cell lines is expected.

Figures


Figure 1-(Top) - Comparison of the three transcript variants of NCAM1 and the placement of primers (Bottom) RT-PCR results showing differential expression of NCAM1 with more in N than S


Figure 2-Alternative Splicing in NCAM1 (Top) – schematic of alternative splicing of NCAM1 (Bottom)- BLAST results of cut out bands in primer pair N2F/R showing the splicing out of exon 9 in the bottom band


Figure 3-(Top) -The placement of primers in CDH2 (Bottom) RT-PCR results showing no significant difference in expression of CDH2 between the cell lines


Figure 4


NCAM1 showed evidence for differential expression between the different cell lines with lowest expression in the S line and the highest in the N line. There were also two isoforms expressed in all three cell lines encoded by transcript variants one and two. This was confirmed by sequencing of the bands that showed the splicing out of exon 9 (30bp) in the bottom band. It appears that transcript variant 3 is not synthesized in these cells, as there was no RT-PCR product in primer pair three that reflected its presence. As NCAM is used for neurite adhesion, it is logical that the N line, which produces the most neurites, would express the highest levels of this transcript. Further analysis of additional cell lines would be required to substantiate the reduced presence of NCAM1 in S cell lines.
There were no significant differences observed in expression levels of CDH2 between the three cell lines.

Full Paper

Acknowledgments

I would like to thank Dr. Berish Rubin for his guidance and time. I would also like to express my appreciation to Leleesha
Samarawera and Bo Liu for their unflagging patience and support in spite of all we put them through. Also to Dr Ross for providing the neuroblastoma cells.


This document was last modified 05/12/2010.
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