Expression of Platelet Derived Growth Factor Receptor- Beta (PDGFR-β) in phenotypically distinct human neuroblastoma cell lines




Lisa Sarran

Introduction

Neuroblastoma is the most common extracranial childhood cancer and the most common tumor occurring during infancy. It is an embryonal malignancy of the sympathetic nervous system arising from neuroblasts (pluripotent sympathetic cells). In the developing embryo, these cells invaginate, migrate along the neuraxis, and populate the sympathetic ganglia, adrenal medulla, and other sites. The pattern of distribution of these cells correlates with the sites of primary disease presentation. There are three distinct neuroblastoma cell types- I-type stem cells, N-type neuroblastic/ neuroendocrine precursors, and S-type schwannian cell/ melanoblastic precursors (Ross et.al., 2003). The S- type cells form monolayers in culture and show contact inhibition, whereas, the N-type cells grow as mutilayers with focal aggregates in culture (Ross et.al., 2003).
Crucial events for the invasion and spread of tumoral cells are the ability to digest the extracellular matrix, migrate, cross blood vessel walls, and reach the circulation (Kramer et.al., 2001). It is known that many growth factors can produce some of the characteristic features of invasive growth like proliferation, motility, and increased survival. Among these, platelet-derived growth factor (PDGF), a mitogen mostly for cells of mesenchymal origin, was shown to induce chemotaxis and actin reorganization and prevent cells from dying by apoptosis (Heldin et.al., 1998). Like a variety of other neural crest-derived tumor cell lines, neuroblastoma cells express specific transcripts of PDGF genes and both ά and PDGF receptor genes (Matsui et.al., 1993). It is thought that the simultaneous presence of PDGF and its receptor genes contributes to tumor cell growth and motility (Matsui et.al., 1993). The biological behavior of neuroblastoma can vary, in that, some tumors regress spontaneously, whereas others progress despite aggressive treatment. Infants diagnosed during their first year of life have a good prognosis, even in the presence of metastatic disease, whereas older patients with metastatic disease fare poorly, even when treated with aggressive therapy (Ribatti et.al., 2004). Unfortunately, approximately 70-80% of patients older than 1 year are diagnosed with metastatic disease, usually to lymph nodes, liver, bone, and bone marrow (Ribatti et.al., 2004). Fewer than half of these patients are cured, even with the use of high-dose therapy followed by bone marrow or stem cell rescue (Ribatti et.al., 2004).
Many chromosomal and molecular abnormalities have been identified in neuroblastoma. These biologic markers have been evaluated to determine their value in assigning prognosis, and some of these have been incorporated into the strategies used for risk-assignment. The most important of these biologic markers is MYCN. MYCN is an oncogene often expressed in neuroblastoma (Guo et.al., 2000). This gene is amplified in approximately 25% of de novo cases and is more common in patients with advanced-stage disease (Guo et.al., 2000). Patients whose tumors have MYCN amplification tend to have rapid tumor progression and a poor prognosis (Spitz et.al., 2004). Studies have also shown that neuroblastomas contain multiple cell phenotypes and this feature is often used to determine the prognosis of the disease. The phenotypically distinct neuroblastoma cell lines have been reported to show varying levels and differential expression of the MYCN gene. Higher expression of MYCN corresponds to a poorer prognosis. Most N and I type cell lines have higher levels of MYCN and some show overexpression of the MYCN gene. In the S-type cell line MYCN expression is reported to be down regulated (Spengler et.al., 1997). New therapeutic approaches are needed to improve the prognosis of neuroblastoma patients with high risk disease. Receptor tyrosine kinases have been proposed as potential targets for antitumor therapy because tyrosine kinase receptors play a role in angiogenesis, an essential step for tumor growth and metastasis (Beppu et. al., 2004). Anti-angiogenic cancer therapies are attracting increasing attention. Neuroblastoma cells express platelet- derived growth factor (PDGF), stem cell factor (SCF), and vascular endothelial growth factor (VEGF) and their respective receptors, PDGFR, c-Kit, and Flk-1. Interest has predominantly focused on interfering with endothelial mitogens such as VEGF and FGF. However, increasing evidence also implicate PDGF receptor signaling in tumor angiogenesis. The aim of this project is to explore PDGFR as a potential target for antitumor therapy in neuroblastoma patients. By investigating whether or not there are differences in the expression of platelet derived growth factor receptor-beta between phenotypically distinct neuroblastoma cell lines and then determining if these differences can be seen on the protein level we can begin to consider that this gene may play a role in the generation of neuroblastoma.

Figures


Figure 1-Expression of human PDGFR-β and GAPDH transcripts in neuroblastoma. 60 ηg of total RNA from the LAI-55N, LAI-5S , and Hela cell lines was subjected to RT-PCR amplification using primers whose sequences matched a region of human PDGFR-β cDNA. The sequence of the forward primer to PDGFR-β is 5- TCCGATGGAAGGTGATTG -3(position 2047-2063) and the sequence of the reverse primer is 5 TAGATGGGT CCTCCTTTGG -3 (position 2369-2389) accession number NM_002609. These RT-PCR reactions were subjected to 50 cycles of amplification. 2 ηg of total RNA from the LAI-55N, LAI-5S, and Hela cell lines was subjected to RT-PCR amplification using primers whose sequences matched a region of the human GAPDH gene. The sequence of the forward primer to GAPGH correspond to 100-119 and the reverse sequence corresponds to 308-327 of accession number NM_002046. These RT-PCR reactions were subjected to 25 cycles of amplification. Sizes of the RT-PCR products were revealed in relation to a 100 bp ladder.


Figure 2-Sequence analysis of the generated RT-PCR product revealing 100% sequence identity to human PDGFR-β (position 2109-2218 accession number NM_002609. The RT-PCR product was purified, sequenced, and aligned to the cDNA sequence of human PDGFR-β in the GenBank NCBI database using the ClustalW Formatted program..


Figure 3-Expression of PDGFR-β protein (180 kDa glycosylated mature and 165kDa nonglycosylated immature) and GAPDH (35 kDa) proteins in neuroblastoma cell lines. 60 g of protein from the LAI-55N, LAI-5S, and Hela cell lines were resolved on an 8% SDS PAGE gel, transferred to a nitrocellulose membrane, and probed with anti- PDGFR-β and anti-GAPDH antibodies. Sizes of recognized proteins were revealed using a prestained molecular weight marker.


Neuroblastoma cells express specific transcripts of Platelet Derived Growth Factor (PDGF) and both α and β PDGF receptor genes. The presence of PDGFR-β in neuroblastoma was confirmed by sequencing RT-PCR products that were generated using primers whose sequences matched cDNA of human PDGFR-β. There is a difference in the expression of platelet derived growth factor receptor-beta between phenotypically distinct neuroblastoma cell lines. The PDGFR-β transcripts and protein are both expressed at higher levels in the non- malignant S-type cell line compared to the malignant N-type cell line. The S-type phenotype is similar to that of Schwannian precursor cells which are known to upregulate the PDGF receptor. It seems that in neuroblastoma the expression of PDGFR-β is not an indicator of malignancy but is more likely an indicator of phenotype.

Full Paper

Acknowledgments

I would like to especially thank Jingsong Qiu and Brian Fox for their help for the entire duration of this semester. Their endless patience and advice was invaluable. I would also like to thank Dr. Berish Rubin for his guidance and for the opportunity to carry out this project. Additional thanks to Dr. Ross and his lab for providing the cell lines that I used and for their direction with interpreting my results. Also, I very much appreciate my fellow classmates who were an encouraging part of this experience. Last but not least, I would like to thank my husband for his support and patience.


This document was last modified 01/31/2006.
This site is powered by the versatile Zope platform.
This is a project of the Biology Department of Fordham University
Biotechniques.org Home