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Figure 2 | Molecular Cancer

Figure 2

From: Aberrant CBFA2T3B gene promoter methylation in breast tumors

Figure 2

CBFA2T3 gene expression levels, 5-Aza-dC re-expression and promoter activity. (A) CBFA2T3 gene expression levels were assayed using real-time RT-PCR. Several breast tumor cell line and normal tissue sample expression levels are shown. The y-axis represents mRNA mlcls expressed per 104 cells shown on a log scale (mean ± SD, n = 6). Fold changes in expression relative to normal breast expression are shown above each sample. The white diamonds and white squares represent expression levels of the housekeeping genes cyclophilin A (CYPA) and ATPase coupling factor 6 subunit (ATP5A), respectively. CBFA2T3B and CBFA2T3 expressed at endogenously low yet aberrant levels in breast tumor cell lines. Using normal breast as a reference, CBFA2T3B (600 mRNA mlcls per 104 cells) and CBFA2T3 (1,800) were low compared to ATP5A (600,000) and CYPA (1,600,000). CBFA2T3 expression ranged 30,000-fold from 4 to 120,000 mRNA mlcls per 104cells in MDA-MB-231 and BT-483, respectively. In contrast, CYPA and ATP5A expression ranged 2-fold and 20-fold, respectively (100–200 and 15–300 mRNA mlcls per cell). Expression levels were also examined in several primary breast tumor samples for which total RNA was available (see Figure 6). (B) CBFA2T3 re-expression levels were examined in MDA-MB-231 cells using 5-Aza-dC. Fold changes in CBFA2T3B, CBFA2T3 and SYK expression levels are shown upon exposure to 5-Aza-dC relative to control untreated cells. > 100-fold re-expression was detected in CBFA2T3 and CBFA2T3B. 5-Aza-dC had no affect on CBFA2T3A expression (data not shown). > 1,000-fold re-expression was also detected in SYK, a control gene known to be hypermethylated and down-regulated in MDA-MB-231 cells. (C) CBFA2T3B promoter activity was assayed using CAT ELISA. Promoter constructs labeled A to D (see Figure 1B) were inserted upstream of the CAT reporter in pBLCAT3 (Boehringer Mannheim). pBLCAT2 driven by the tyrosine kinase promoter was used as a positive control. 2.0 × 106 293T cells were transfected in triplicate using Lipofectamine 2000 (Invitrogen) with 1.5 μg of construct and control vector and 0.3 μg of the internal pSVβ-galactosidase control vector (Stratagene). Cells were lysed after 24 h and CAT concentrations determined using ELISA. Of the four constructs labeled A to D, the 1-kb B construct promoted a 30-fold increase in CAT expression (mean ± SD are triplicates, n = 3).

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