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.
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
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.
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.
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