Differential Expression of Neurotrophic Tyrosine Kinase Receptor A (TrkA) in Two Human Neuroblastoma Cell Lines




Lauren Buro

Introduction

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.
Numerous genetic and biological features have been studied in an effort to improve the understanding of neuroblastoma behavior and to identify markers that can improve the prognosis of patients with this cancer. One of these biological features that is being studied is the expression of neurotrophic tyrosine kinase receptor (Trk) family which is a molecule that binds nerve growth factors. Nerve growth factor has different effects on cells depending on the type. For neurons and neuroblastoma cells it plays a role in their differentiation.
The Trk family of receptors consists of three different members, TrkA, TrkB and TrkC. Analysis of the Trk family indicates that these receptors bind neurotrophins differentially. TrkA is the preferred receptor for nerve growth factor, TrkB for brain derived neurotrophic factor and neurotrophin 4/5, and TrkC for neurotrophin 3.
Differential expression of various gene products has been studied in different neuroblastoma lines to try to determine if there is a correlation between gene expression and growth. Neuroblastoma cells that express high levels of TrkA have been shown to have a more favorable prognosis than tumors with low levels of TrkA. Because of the ability of these receptors to bind nerve growth factors and play a role in cell growth differentiation and programmed cell death of neurons in both the peripheral and central nervous system, the differential expression of TrkA in two different neuroblastoma cell lines, LA1-55N and LA1- 5S has been investigated. An unexpected result was obtained, where in LA1-5S, a schwannian/ melanoblastic precursor, showed expression of TrkA. This was unexpected because TrkA is a neuronal marker, and LA1-5S showed expression of this gene even though it is not a neuronal precursor.

Figures


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).


Summary
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.

Full Paper

Acknowledgments

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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