Human neuroblastoma is the most common extracranial solid tumor in the pediatric population. Tumors arise from the developing neural crest along its migratory pathways and can be characterized clinically by their location, age at diagnosis, metastasis and degree of cellular maturation and heterogeneity. Studies have shown that there are three different cell types found in neuroblastoma. These three different cell types have been characterized into I-type stem cells, N-type neuroblastic/ neuroendocrine precursors, and S-type schwannian/ melanoblastic precursors. The I-type cells show features common to both N and S type cells. The N-type cells adhere tightly to other cells forming structures like pseudoganglia whereas the S type cells adhere tightly to the substrate. The presence of these cell phenotypes are often used for the prognosis of the disease. The I-type and N-type cell lines are malignant but the S-type cell lines are not malignant.
Figure 1- a) RT-PCR examining TrkA in LA1-55N and LA1-5S, using primer set 1 (248-688, exon 1 and exon 6, NM_002529, first three lanes) and primer set 2 (1337-1567, exon 11 and exon 12, NM_002529, last three lanes). 8 ul of the product was loaded onto the 1% Agarose gel. b) RT-PCR examining GAPDH mRNA (100-327, NM_002046) in LA1-55N and LA1-5S used as a RNA loading control. Amplification was preformed for 26 cycles and 8 ul of the product was loaded onto the gel.
Figure 2- Sequencing generated from PCR products A, B and C was then compared to the cDNA of TrkA (MN_002529). Blast analysis was performed on PCR products A, B and C. a) PCR product A matched against the cDNA sequence of TrkA in the region where the primer set 1 was designed. b) PCR product B sequence matched the exon and intron sequence of the TrkA gene in the region where primer set 2 was designed. c) PCR product C sequence matched to the cDNA sequence of the TrkA gene, where primer set 2 is designed.
Figure 3- Western Blot analysis of the protein extracts of the LA1-55N and LA1-5S cell lines was performed using antibodies to TrkA (a) and GAPDH (b).
Based upon the results obtained from the RT-PCR reaction (Figure 1a) and the sequencing reaction (Figure 2), the LA1-5S cell line shows higher expression of TrkA than does the LA1-55N cell line. This was a somewhat unexpected result, because LA1-5S is a schwannian/ melanoblastic precursor and TrkA is a neuronal marker; yet LA1-5S showed expression of this gene even though it is not a neuronal precursor. The LA1-5S cell line also expressed the TrkA protein, while the LA1-55N cell line did not (Figure 3a), which is consistent with the RT-PCR results. High TrkA levels are associated with a better prognosis for people suffering from neuroblastoma. Therefore, it was not unexpected that the LA1-55N, even though it is a neuronal cell line, did not express TrkA because TrkA is generally expressed in less malignant cell lines, and LA1-55N is highly tumorogenic. Since LA1-5S expressing TrkA is somewhat counterintuitive, further research is needed to determine if this is an isolated event or if TrkA expression occurs in other S-type cell lines. Also, further investigation is required to determine if other N-type cell lines of varying malignancy express TrkA.
I would like to thank Jinsong Qiu and Brian Fox for their patience, continuous help and encouragement. I would also like to thank to Dr Robert A Ross for the RNA samples and his helpful advice. I am grateful to Dr Sylvia Anderson for ordering the primers for my project. Sincere thanks to Dr Berish Rubin for his guidance and the use of his laboratory for this project.
|This document was last modified 01/31/2006.|
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