Neuroblastoma (NB) is the most common extra cranial solid tumor in children. NB is characterized by its heterogeneous nature. There are three distinct NB phenotypes, N, S and I, each correlating with a different level of tumorigenesis. N cells are neuroblastic cells that form slow growing tumors, S cells are substrate adherent cells that express contact inhibition, and do not grow tumors, I cells are intermediary cells that can differentiate into either N or S cells. I cells are the most tumorigenic of the NB phenotypes. The N and I cells possess noradrenergic properties while S cells do not.
Figure 1-Exon arrangement of TH and primer placement. TH is expressed in the N and I cell lines but not in S. Two transcript varients of TH are seen using primer set 1 and 2 representing transcript variant 1 and 2. A third transcript variant is seen using primer set 3 in the N cell line.
Figure 2-There is a potential novel alternative splice site 90bp into exon 8 that would explain the ~400bp band seen in the N cell line using TH primer set 3
Figure 3-Exon arrangement and primer placement for DBH. DBH is expressed in the N and the I cell lines but not the S. The absense of DBH from the N cell line in primer set two may be due to either a mutation in the location where the primer binds, or the complete exclusion of exon 4 in the N cell line.
Using RT-PCR I was able to compare the expression of TH and DBH among three neuroblastoma cell lines, and identify different potential transcript variants-some of which had not been identified before. TH and DBH are expressed in the N and I cell lines but not in the S cell line. This is an expected result because both the N and I phenotypes are able to produce neurotransmitters while the S phenotype is not
There was one set of curious results in both TH and DBH. Using TH primer set three an unexpected band of about 400bp was seen. Sequencing confirmed that this band is TH, yet its size did not match up with the expected size of 476bp. Examining the sequencing results confirmed the existence of exons 9 and 10, but was only able to confirm the 6bp (on the 3' end) of exon 8. 90bp into exon 8 there is a 3' consensus splice sequence that includes a polypyrimidine tract and the splice acceptor sequence CAG. If this sequence is used as a splice sequence approximately 90bp of exon 8 would be excluded from the transcript explaining the ~400bp band observed in the N cell line.
The N cell line may also be expressing a mutation or alternative splice site in exon 4 of DBH, accounting for the absence of DBH in the N cell line using primer set 2. A mutation would have to occur in the location where the primer is designed to anneal. This would prohibit the primer from sitting on the mRNA and keep the message from being amplified. Exclusion of exon 4 would also prohibit the primer from annealing and the message from being amplified. Further tests need to be done in order to identify what was causing the lack of DBH transcript using primer set two, such as doing PCR that uses primer in exon two and six to see if the N cell line produces a smaller than expected product signifying the exclusion of part of the message.
I would like to thank Dr. Rubin for his guidance, patience, time and advice. A special thanks to Bo Liu and Leleesha Samaraweera for dedicating so much of their time to me, and offering me countless amounts of support, advice and expertise. Dr. Ross, for providing the neuroblastoma cells. Lastly, I would like to thank my classmates: Alex, Chong, Heng, and Kate for their constant support and encouragement.
|This document was last modified 05/12/2010.|
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