Abstract
BackgroundThe cellular retinol binding protein I gene (CRBP) is downregulated in a subset of human breast cancers and in MMTV-Myc induced mouse mammary tumors. Functional studies suggest that CRBP downregulation contributes to breast tumor progression. What is the mechanism underlying CRBP downregulation in cancer? Here we investigated the hypothesis that CRBP is epigenetically silenced through DNA hypermethylation in human and mouse breast cancer.ResultsBisulfite sequencing of CRBP in a panel of 6 human breast cancer cell lines demonstrated that, as a rule, CRBP hypermethylation is closely and inversely related to CRBP expression and identified one exception to this rule. Treatment with 5-azacytidine, a DNA methyltransferase inhibitor, led to CRBP reexpression, supporting the hypothesis that CRBP hypermethylation is a proximal cause of CRBP silencing. In some cells CRBP reexpression was potentiated by co-treatment with retinoic acid, an inducer of CRBP, and trichostatin A, a histone deacetylase inhibitor. Southern blot analysis of a small panel of human breast cancer specimens identified one case characterized by extensive CRBP hypermethylation, in association with undetectable CRBP mRNA and protein. Bisulfite sequencing of CRBP in MMTV-Myc and MMTV-Neu/NT mammary tumor cell lines extended the rule of CRBP hypermethylation and silencing (both seen in MMTV-Myc but not MMTV-Neu/NT cells) from human to mouse breast cancer and suggested that CRBP hypermethylation is an oncogene-specific event.ConclusionCRBP hypermethylation appears to be an evolutionarily conserved and principal mechanism of CRBP silencing in breast cancer. Based on the analysis of transgenic mouse mammary tumor cells, we hypothesize that CRBP silencing in human breast cancer may be associated with a specific oncogenic signature.
Highlights
MethodsCells and reagents Human breast cancer cell lines, with the exception of MTSV1-7 cells [16,28], were obtained from the American Type Culture Collection (Rockville, MD) and grown as recommended. 16MB9a and M1011 (MMTV-Myc) and SMF and NAF (MMTV-Neu/NT) mammary tumor cells were cultured as described [24]
The cellular retinol binding protein I gene (CRBP) is downregulated in a subset of human breast cancers and in MMTV-Myc induced mouse mammary tumors
Results hCRBP expression in breast cancer cells varies widely We first analyzed a panel of human breast cancer cell lines for CRBP expression using a semi-quantitative RT-PCR Southern blot protocol
Summary
Cells and reagents Human breast cancer cell lines, with the exception of MTSV1-7 cells [16,28], were obtained from the American Type Culture Collection (Rockville, MD) and grown as recommended. 16MB9a and M1011 (MMTV-Myc) and SMF and NAF (MMTV-Neu/NT) mammary tumor cells were cultured as described [24]. RNA analyses For RT-PCR, 2 μg total RNA (Purescript kit, Gentra, Minneapolis, MN) were reverse transcribed using the Superscript Preamplification System (Gibco BRL Life Technologies, Grand Island, NY). This RNA input was chosen after preliminary experiments demonstrated a dose-dependent increase in CRBP RT-PCR product within http://www.molecular-cancer.com/content/3/1/13 the range of 0.5 to 4 μg total RNA. The PCR products were separated on 1% agarose, stained with ethidium bromide (18S rRNA) or blotted overnight and probed with full length CRBP cDNA or a commercial GAPDH cDNA fragment (Ambion). Northern blot analyses were performed using standard methods and a full length CRBP cDNA probe as described earlier [7]
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