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).
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 µg 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.
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.
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