Article
cDNA profiling of breast cancer brain and bone metastases in vitro
cDNA Profil von Mammakarzinom-Hirn- und Knochenmetastasen in-vitro
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Published: | April 23, 2004 |
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Outline
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Objective
Haematogenous metastasis to the bone and brain is an increasingly common complication in cancer. Recently, bone (231-bone) and brain- (231-brain) seeking clones of MDA-MB-231 (231-parental) breast cancer cells have been established in a mouse model. We performed cDNA expression analysis of the different sublines to identify factors related to the metastatic spread to brain and bone.
Methods
Total RNA was isolated from MDA-MB-231 cells (parental) and the two sublines using the Trizol®-method. Reverse transcription labelling was performed with fluorescence markers (Cy5-dUTP and Cy3-dUTP) and followed by hybridization to the 16K gene chip fabricated by the National Human Genome Research Institute, NIH, Bethesda, MD, USA. Hybridizations were performed for (1) 231-parental versus 231-bone as well as (2) 231-parental versus 231-brain cells. For data analysis, a threshold value of 2.5 fold over or under expression was defined to identify significant changes in gene expression. cDNA array results were validated using semiquantative RT-PCR.
Results
113 genes were significantly over- or underexpressed in brain and / or bone-seeking 231-variants in comparison to 231-parental cells. Among these, 10 genes were selected for semi-quantative RT-PCR validation. Finally, three candidate metastasis regulatory genes were identified: (1) metastasis suppressor gene KiSS-1, (2) the tyrosine kinase transmembrane receptor TIE-1 which is supposed to be essential for vascular genesis and (3) PTPRN2, a protein that is associated with CNS and pancreas genesis. Functional experiments further characterized the factors identified.
Conclusions
Gene chips offer expression profiling of cells and tissue specimens on a genome-wide scale. Using the 16K gene chip of the NHGRI, we identified over 100 potential metastasis regulatory genes. RT-PCR data validation identified 3 "hot spot" metastasis regulatory genes, which have to be further examined for their role in breast cancer brain and bone metastases.