Human neuroblastoma (HN) is one of the most common solid cancers in infants and children. Tumors contain three distinct cell types: N-type (neuroblastic), S-type (schwannian) and I-type (intermediate phenotype) cells, which can differentiate predictably in response to certain morphogens. The differentiated subtypes possess characteristic tumorigenicities and vary in frequency in different stages of tumor progression. In our current study we investigate the differential expression of two known tumor markers: hTERT (human telomerase reverse transcriptase) and S100P, in RA (retinoic acid), BuDR (5-bromo-2-deoxyuridine) and untreated neuroblastoma BE(2)-C cells. HTERT is the catalytic component of telomerase involved in stabilizing the protective caps of chromosomes. It has an important role in cellular immortalization and tumorigenisis, and is over-expressed in 90% of cancerous cells. HTERT is highly expressed in HN, but the high hTERT expression levels have, as yet, not been linked to the three neuroblastoma cell types. Our studies have shown that hTERT is a marker for the tumorigenic N-type and I-type cells and can possibly indicate the presence of tumorigenic cells in neuroblastoma tissues. S100P is a calcium binding protein that has recently been correlated with tumor proliferation. It is over-expressed in lung, breast, colon and pancreatic cancer and has been implicated with metastasis. It is considered a potential tumor marker and a target for tumor diagnostics and therapy. We demonstrate that S100P may not be a tumor marker in neuroblastoma tissues.
Figure 1-Primer sequences and position on genomic DNA for hTERT (A) and S100P (B). Primer set (A) spanned exons 8 and 9 of the cDNA for hTERT. In between these two exons there is a 2480bp intron in the genomic DNA. The primer set amplifies a 159bp product. Primer set (B) spanned regions of exons 1 and 2 in S100P cDNA, flanking a 2822bp intron in the genomic DNA. The primer amplifies a 240bp product.
Figure 2-RT-PCR detection of hTERT (A) and S100P (B) A) Three samples containing 25ng of RNA isolated from RA treated (N), BuDR treated (S) and untreated (I) BE (2)-C cells was subjected to RT PCR using the respective primer pairs for hTERT (A) and S100P (B) (see Fig. 1) B.) RT-PCR for GAPDH was performed to control for the amount of RNA used.
Figure 3-Sequencing results for the amplified products of h(TERT)(A) and S100P(B).Nucleotide sequence of the amplified products of S100P (B) and hTERT (A) were blasted against NCBIís nucleotide databases. The alignments confirm that the amplified products for hTERT and S100P match their sequences within 99% accuracy.
Figure 4-Treatment induced fold changes in expression of hTERT and S100P. Sigma Gel scanning analysis was used to compare the band intensities. The values were standardized to the GAPDH control and are presented relative to expression levels in untreated cells.
The differential expression of two known tumor markers, hTERT and S100P, was studied in RA treated (N-type), BuDR treated (S-type) and untreated (I-type) neuroblastoma BE(2)-C10 cells, using RT-PCR.
The RT-PCR results for hTERT showed a treatment induced down-regulation. Untreated cells had the highest expression, RA treated cells showed a 1.7 fold expression decrease, and BuDR treated cells lacked hTERT expression.
From our study, hTERT appears to be a potential marker for tumorigenic cell types in neuroblastoma tissues.
The RT-PCR results for S100P showed a treatment induced up-regulation. Untreated BE(2)-C10 cells demonstrated very low expression levels, RA treated cells showed a 35 fold increase in expression levels, and BuDR treated cells showed a 92 fold expression level increase.
From our results, S100P can not be considered a potential tumor marker in neuroblastoma.
Further studies, investigating expression levels of hTERT and S100P in several different neuroblastoma cell lines, need to be conducted to validate the obtained results.
I thank Leleesha Samarawera and Bo Liu for their continuous help and patience and Jinsong Qiu for his positive mentality and late hours. Additional thanks to Dr. Robert A. Ross for providing me with the cell lines and helpful advice. Sincere thanks to Dr. Berish Rubin for pushing me to do my best and providing me with the opportunity to pursue this project.
|This document was last modified 05/14/2008.|
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