Figure 1.
hTREX84 is aberrantly expressed in ovarian cancer cells.
A, hTREX84 protein expression in representative ovarian cancer cell lines (OVCAR10, UPN251, UPN275, UPN289), immortal epithelial cell lines (HIO-118, HIO-102, HIO-104, HIO-113), primary epithelial cells (ROE). Protein samples were separated on a SDS-polyacrylamide gel immunoblotted using anti-hTREX84 or ß-actin monoclonal antibodies. B, hTREX84/ß-actin ratio in primary ovarian epithelial cell cultures (epithelial), immortal epithelial cell lines (HIO) and cancer cell lines (cancer).
Figure 2.
Depletion of hTREX84 leads to defects in cellular proliferation of OVCAR10.
A, Analysis of hTREX84 and GAPDH mRNA levels following treatment of cells with siRNA against hTREX84 or control siRNA. B, Analysis of hTREX84 and β-actin protein levels after treatment of cells with siRNA against hTREX84 or control siRNA. C, Analysis of hTREX84 expression following siRNA treatment for 72 hours by immunofluoresence staining in the cells (left, cells transfected with control siRNA; right, cells treated with hTREX84-siRNA). D, Photomicrographs showing the morphology following depletion of hTREX84 (left, tumor cells transfected with control siRNA; right, cells treated with hTREX84-siRNA). E, Cell proliferation assay of tumor cells following depletion of hTREX84. Cell proliferation and apoptosis (data not shown) was examined using Guava ViaCount and Nexin assays respectively. The number of viable cells (x104) are plotted against treatment duration at 24, 48, and 72 hrs after treatment with control siRNA or with hTREX84-siRNA. Shown are the results of three independent experiments. The difference is statistically significant. *, p<0.05; **, p<0.01. F. FACS analysis of the cells following down-regulation of hTREX84 levels. Shown is the percentage of cells in G1, S, G2-M after 72 hour of treatment with either siRNA (left panel) or hTREX84-siRNA (right panel).
Figure 3.
HIO-107 cells were treated with 5-aza-dC to examine changes in DNA methylation in CpG sequences in hTREX84.
A, HIO-107 cells were treated with 5-aza-dC at concentrations of 1, 5, 10, 50 µM respectively for 5 days. RT-PCR show hTREX84 mRNA expression and B, western blot analysis show hTREX84 protein levels. C, D, Quantitative mRNA and Western blot data were calculated from densitometric analysis of bands with the NIH imageJ software, respectively. The values were normalized to β-actin as internal control.
Figure 4.
Sodium bisulfite DNA sequencing of CpG sites in the hTREX84 promoter and exon 1 regions.
A, DNA sequence of hTREX84 regulator regions. CpG sites are shown in green color. Nucleotides are numbered on the right from the AUG translation start code which is underlined. B, Sodium bisulfite sequencing of DNA isolated from untreated (I) and treated (II) cells. The stars indicate CpG sites. C. Sodium bisulfite sequencing of DNA from a normal breast tissue (N) and an invasive ductal carcinoma (T). The stars indicate CpG sites.
Figure 5.
NF-κB activation enhances hTREX84 expression in immortal and/or cancer cells.
A, Schematic diagram of the hTREX84 promoter indicating the conserved NF-κB DNA binding motif. B, ChIP assay of RelA/p65 binding to hTREX84 gene promoter in MDA-MB-231 (lane 1, 2); OVCAR5 (lane 3, 4); OVCAR 10 (lane 5, 6). Cells were cultured for 48 h. ChIP assays were then performed with anti-RelA/p65 antibody. PCR analysis was performed on immunoprecipitation samples without antibody (lane 1, 3, 5), with RelA/p65 antibody (lane 2, 4, 6). C, MCF-10F cells were transiently transfected with a control vector (lane 1) or a RelA/p65 cDNA expression construct for 48 hours. Western blot analysis for RelA/p65, hTREX84 and β-actin. D, Western blot analysis of RelA/p65, hTREX84 and β-actin protein levels after treatment of MDA-MB-231 cells with control siRNA (lane 1) and siRNA against RelA/p65 (lane 2) for 72 hours.
Figure 6.
Determination of hTREX84 promoter activities in MCF-10F cells with hTREX84/pGL3 reporters.
A, DNA sequence of NF-κB1M demonstrating that the first NF-kB binding site is mutated from 5′-GGAAACTCCC-3′ to 5′-CCAAACTCCC-3′. B, DNA sequence of NF-κB2M, demonstrating that the second NF-kB binding site is mutated from 5′-AGGTAATCCA-3′ to 5′-ACCTAATCCA-3′. N5-κB1/2M represented both of the two NF-κB binding sites in hTREX84 promoter region were mutated as described above (sequence not shown). C, Promoter activities among the three reporters constructs containing either a single mutated NF-κB binding sites or both (NF-κB1/2M) as determined by a luciferase assay. 1) Wild type NF-κB binding sites; 2) NF-κB1M; and 3) NF-κB2M; and 4) NF-κB1/2M.
Table 1.
RelA/p65 expression in human normal breast tissue and tumors.
Figure 7.
Immunohistochemical staining of RelA/p65 proteins in representative breast cancer tissue specimens.
A, RelA/p65 was weakly detected in normal breast epithelial cells and positive products were located in the cell cytoplasm. B, Staining for RelA/p65 was detected mainly in the cytoplasm in high differentiated tumors. C, Intense and distinctly granular staining for RelA/p65 was detected in stained nuclei of low differentiated tumor specimens. D, Tumor section evaluated without the primary antibody to serve as a negative control. Magnification 200x.