Yongjing Guo | PubMed Health

PubMed2005

Characterization of MNAR expression.

We have previously demonstrated that modulator of nongenomic action of estrogen receptor (MNAR) integrates action of estrogen receptor alpha (ERalpha), and potentially some other nuclear receptors (NRs), in regulation of Src/Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathway. MNAR is a scaffolding protein that contains 10 LXXLL type motifs that can interact with NRs and 3 PXXP type motifs that can bind to SH3 domains present in kinases and other signaling molecules. Formation of ER-MNAR-cSrc complex leads to activation of Src and downstream Ras/Raf/MAPK pathway. The goal for this study was to compare MNAR expression in various cell lines, to optimize methods that can be used to manipulate its expression and to evaluate MNAR cellular distribution. We found that MNAR is differentially expressed. The highest levels of its expression were found in fast proliferating cells, such as breast adenocarcinoma (MCF-7)-, T cell lymphoma (Jurkat)-, prostate carcinoma (LNCaP)- and osteosarcoma (SaOS2)-derived cell lines. MNAR was undetectable in African green monkey kidney cells (COS-7) and Chinese hamster ovary cells (CHO-K1). We established and optimized a protocol to knockdown MNAR using siRNA and to overexpress it in MCF-7 cells. Exogenously expressed MNAR was found in both cytoplasmic and nuclear fractions, the majority of MNAR, however, was found in the cytoplasmic fraction. Presence of MNAR in the cell nucleus indicates that it may play a role in regulation of gene expression.

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PubMed2002

Regulation of DNA methylation in human breast cancer. Effect on the urokinase-type plasminogen activator gene production and tumor invasion.

Urokinase-type plasminogen activator (uPA) is a member of the serine protease family and can break down various components of the extracellular matrix to promote growth, invasion, and metastasis of several malignancies including breast cancer. In the current study we examined the role that the DNA methylation machinery might be playing in regulating differential uPA gene expression in breast cancer cell lines. uPA mRNA is expressed in the highly invasive, hormone-insensitive human breast cancer cell line MDA-MB-231 but not in hormone-responsive cell line MCF-7. Using methylation-sensitive PCR, we show that 90% of CpG dinucleotides in the uPA promoter are methylated in MCF-7 cells, whereas fully demethylated CpGs were detected in MDA-MB-231 cells. uPA promoter activity, which is directly regulated by the Ets-1 transcription factor, is inhibited by methylation as determined by uPA promoter-luciferase reporter assays. We then tested whether the state of expression and methylation of the uPA promoter correlates with the global level of DNA methyltransferase and demethylase activities in these cell lines. We show that maintenance DNA methyltransferase activity is significantly higher in MCF-7 cells than in MDA-MB-231 cells, whereas demethylase activity is higher in MDA-MB-231 cells. We suggest that the combination of increased DNA methyltransferase activity with reduced demethylase activity contributes to the methylation and silencing of uPA expression in MCF-7 cells. The converse is true in MDA-MB-231 cells, which represents a late stage highly invasive breast cancer. The histone deacetylase inhibitor, Trichostatin A, induces the expression of the uPA gene in MDA-MB-231 cells but not in MCF-7 cells. This supports the hypothesis that DNA methylation is the dominant mechanism involved in the silencing of uPA gene expression. Taken together, these results provide insight into the mechanism regulating the transcription of the uPA gene in the complex multistep process of breast cancer progression.

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