Abstract
MET protein, known as hepatocyte growth factor (HGF) receptor, is a member of receptor tyrosine kinase family. It exerts its action through various signal transduction pathways leading to cell proliferation, scattering and acquisition of migratory phenotype. The MET mutations or protein overexpression are believed to play a crucial role in cancer growth and metastasis formation, acting by upregulating proliferation, inhibiting apoptosis and enhancing migrative capacity of the cells. The expression of MET is driven by several regulatory elements. Its promoter is characterized by an extremely high GC content and lack of TATA box sequence. The current knowledge of the mechanism of transcriptional control exerted on the MET promoter and the putative mutations leading to an increased protein expression is very limited.
Therefore, the aim of our project was to investigate and characterize the promoter region.
The first stage of the investigation involved establishing and comparing the promoter sequence from several multiple myeloma cell lines, as well as from CD138+ cells collected from multiple myeloma patients and from healthy individuals. The ulterior phases of the project included examination of the CpG islands methylation pattern in multiple myeloma cell lines. Results were compared to the expression of MET gene in cell lines and healthy individuals. The third aim of our project is to identify and characterize protein factors binding to the promoter sequence in multiple myeloma cell lines.
Experiments were performed by the mean of DNA sequencing of DNA cloned to vector pCR2.1. Methylation analysis required genomic DNA treatment with Sodium Bisulphite and cloned into the pCR2.1 vector and sequenced. “Protein fishing” was performed by the use of cell lysates incubated with biotinylated MET promoter specific probe and streptavidin magnetic beads and separated on magnetic µ‐columns. Proteins were analyzed in one‐ and twodimensional electrophoresis and silver stained.
Promoter sequencing revealed some dispersed and random alterations in myeloma and healthy individuals samples. Observed the most common substitutions −304C>A and +206C>G with higher occurrence in cancer cell lines. Methylation analysis of multiple myeloma cell lines showed lower methylation of MET promoter sequence than control DNA.
“Protein fishing” analysis resulted in differences in bands profile between cytoplasmic and nuclear samples of different cell lines. The dots visualised in 2‐DE electrophoresis, possible transcription factors, are currently under investigation.
The identified mutations, methylation analysis and proteins profile observed so far are to be further investigated to assess their relevance for the MET transcriptional regulation.
Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A209.
- Copyright © 2009, American Association for Cancer Research
Volume 8, Issue 12 Supplement
