Damaged DNA Binding Protein 2 Plays a Role in Breast Cancer Cell Growth

Abstract

The Damaged DNA binding protein 2 (DDB2), is involved in nucleotide excision repair as well as in other biological processes in normal cells, including transcription and cell cycle regulation. Loss of DDB2 function may be related to tumor susceptibility. However, hypothesis of this study was that DDB2 could play a role in breast cancer cell growth, resulting in its well known interaction with the proliferative marker E2F1 in breast neoplasia. DDB2 gene was overexpressed in estrogen receptor (ER)-positive (MCF-7 and T47D), but not in ER-negative breast cancer (MDA-MB231 and SKBR3) or normal mammary epithelial cell lines. In addition, DDB2 expression was significantly (3.0-fold) higher in ER-positive than in ER-negative tumor samples (P = 0.0208) from 16 patients with breast carcinoma. Knockdown of DDB2 by small interfering RNA in MCF-7 cells caused a decrease in cancer cell growth and colony formation. Inversely, introduction of the DDB2 gene into MDA-MB231 cells stimulated growth and colony formation. Cell cycle distribution and 5 Bromodeoxyuridine incorporation by flow cytometry analysis showed that the growth-inhibiting effect of DDB2 knockdown was the consequence of a delayed G1/S transition and a slowed progression through the S phase of MCF-7 cells. These results were supported by a strong decrease in the expression of S phase markers (Proliferating Cell Nuclear Antigen, cyclin E and dihydrofolate reductase). These findings demonstrate for the first time that DDB2 can play a role as oncogene and may become a promising candidate as a predictive marker in breast cancer.

DDB2: A Novel Regulator of NF-κB and Breast Tumor Invasion

“…We observed previously that DDB2 expression was inversely correlated with the invasive properties of breast tumor cells, and that a constitutive overexpression of DDB2 in nonmetastatic cells induced cell proliferation by activating the G 1 -S transition phase of the cell cycle (2). In the present study, our findings summarized in Fig.…”

“…DDB2 is overexpressed in nonmetastatic breast tumor cells and plays a role in their proliferation by favoring G 1 -S transition entry and their progression through the S-phase of the cell cycle (2). We reported that DDB2 stimulates the proliferation of nonmetastatic breast tumor cells, at least in part by maintaining a low basal expression of mitochondrial superoxide dismutase (MnSOD) through its binding to a specific and well-characterized DNA sequence in the proximal promoter of the MnSOD gene (3).…”

Identification of DDB2 Protein as a Transcriptional Regulator of Constitutive SOD2 Gene Expression in Human Breast Cancer Cells

“…Expression Vector, RNA Interference, RNA Antisense, and Stable Transfection-The DDB2 pcDNA3(ϩ) expression vector, the pSIREN/U6/DDB2-siRNA vector, and the pcDNA vector containing antisense cDNA of human MnSOD were constructed and used for stable transfection of breast cancer and COS-7 cells, as described elsewhere (14,18).…”

“…Primer sequences (Invitrogen) and the expected product sizes of SOD2, DDB1, DDB2, MDR1, and ␤-actin are detailed in Table 1. PCR analysis was carried out, as described previously (14,18). Ethidium bromide-stained bands resulting from the amplification were visualized by UV transillumination, and the fluorescence intensity was quantified using a Gel Doc 2000 system (Bio-Rad).…”

“…This 48-kDa protein was originally a component of the damage-specific DNA-binding heterodimeric complex DDB, which is involved in nucleotide excision repair of UVinduced DNA damage (17). Recently, we reported that DDB2 is overexpressed in some breast cancer cell lines and plays a role in cell proliferation by controlling the G 1 /S transition of cell cycle (18). Our present results demonstrate that DDB2 regulates negatively the constitutive transcription of the SOD2 gene when it is bound on the proximal promoter and may exert control over proliferation of breast tumor cells through this regulation.…”