Transforming Growth Factor Beta (TGFβ), ubiquitously expressed during embryonic development and in most adult tissues, is a potent growth inhibitor for both tumor and non-tumor cells. TGFβ exerts its biological effects by binding to cell surface receptors; TGFβRI, TGFβRII and TGFβRIII, initiating a signaling cascade, ultimately activating several transcription factors that regulate genes involved in the cell cycle. Disruption of the TGFβ signaling pathway is observed in several human cancers, either by downregulation of either the ligand itself or its receptor expression.
Figure 1-Primers Used in RT-PCR
Figure 2-RT-PCR analysis of TGFβ receptor transcript levels in SK-N-ER and SH-EP1 cell lines. RT-PCR products generated from RNA isolated from each of the cell lines, amplified by primers recognizing GAPDH and TGFβRI (a) and (b) , TGFβRII (c) and (d), TGFβRIII (e) and (f) respectively.
Figure 3-The ClustalW alignment of the partial sequence of the TGFβRI (a), TGFβRII (b), TGFβRIII (c), amplified using specific primers with a segment of Homo sapiens mRNA sequence from NCBI.
The aim of this project was to investigate the mRNA expression levels of the three TGFβ receptor genes in an invasive (SK-N-ER) and a non invasive (SH-EP1) neuroblastoma cell line. The RT-PCR analysis suggested a higher expression for TGFβRII and TGFβRIII mRNA in SH-EP1, as compared to SK-N-ER neuroblastoma cell lines. There appeared to be no significant change in TGFβRI mRNA expression in the two cell lines.
Consistent with results from RT-PCR (Fig. 2) of this study, several other investigators reported decreased expression of TGFβRII and RIII mRNA in highly invasive cancer cells. However, TGFβRI mRNA expression wasn’t significantly different in the two neuroblastoma cell lines.
The ClustalW alignment, of the partial sequence of each of the three TGFβR products from RT-PCR amplified using primers specific to each receptor, confirmed that these primers amplified the desired TGFβ receptor transcript, respectively (Fig. 3).
Based on the obtained data, it cannot be concluded that, the decreased expression of TGFβRII and RIII mRNA in the SK-N-ER neuroblastoma cells has a direct correlation with its invasiveness. There are other factors that may also affect the invasiveness of these cells. Due to the increasing relevance of abberations in the TGFβ dependent pathway in human tumorigenesis, further studies are warranted to study the level and activity of other components, of this pathway particularly SMAD transcription factors. Research in this direction could prove useful to develop molecular therapeutics targeting the TGFβ pathway.
I would like to thank Leleesha Samaraweera and Joseph Frezzo for their invaluable help and guidance with techniques employed throughout this project. I would also like to thank Jinsong Qiu for his advice. Finally, I would like to thank Dr Berish Rubin for providing me with an opportunity to pursue this project.
|This document was last modified 05/09/2007.|
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