Abstract
We previously reported that blood TIMP-1 (Tissue Inhibitor of Metalloproteinase), OPG (Osteoprotegerin), and TPO (Thrombopoietin) levels are increased and blood MMP-3, and bone marrow SDF-1 levels are decreased in patients with AMM. Enhanced TGF-ß 1 activity with Transforming Growth Factor Receptor Type II (TGF ß RII) abnormalities is likely to be associated with these findings. It has been previously reported that TGF-ß RII levels are decreased in AMM patients. The current study was undertaken to explore the defects in the TGF-ß1 pathway in this disease. We cloned and sequenced the full length TGF ß RII cDNA from AMM patients and no mutations were detected on sequencing. We then determined the expression levels of TGF ß RII in the CD34+ progenitor cells by Real-Time RT-PCR and found it to be reduced in patients with AMM compared to normal volunteer CD34+ cells used as controls. Hence we cloned the promoter region of TGF ß RII (−1233 to +50) from the genomic DNA of Peripheral Blood Monocytes by PCR, using gene specific primers. On sequencing, no mutations were detected in the promoter regions. The same promoter region when cloned upstream of a luciferase gene, drove the transcription of the reporter gene similar to the levels observed with the promoter from controls. This augmented the absence of any structural abnormalities in the region of the TGF b RII promoter. We then analyzed the methylation status of TGF ß RII promoter using the technique, Sodium Bisulfite-PCR for Sequencing. Genomic DNA was modified using Sodium Bisulfite and the TGF ß RII promoter was amplified by PCR with Primers capable of amplifying both unmethylated and methylated DNA. The modified PCR products were cloned into TOPO TA sequencing vector from Invitrogen, CA and sequenced. The sequencing results revealed absence of any CpG hyper-methylation in the promoter region of TGF ß RII. To account for reduced TGF ß RII expression in the absence of coding region abnormalities, promoter abnormalities or hypermethylation, we determined Histone Deacetylase Activity HDAC activity in the CD34+ cells of AMM patients. There was an indirect correlation between HDAC activity and TGF ß RII mRNA levels implying HDAC mediated silencing of the TGF ß RII promoter in AMM patients.
Transforming Growth Factor Receptor Type I and Transforming Growth Factor 1 Polymorphisms Are Not Associated with Postmenopausal Breast Cancer