To find out if these transcription factors are also involved in www.selleckchem.com/products/Tubacin.html the stimulation of ERBB2 expression in non-breast cancer cells, we compared AP-2 and erbB-2 levels in the cell lines used in this study. AP-2�� levels were estimated by Western blotting (Figure 2A) and AP-2 DNA binding activity was analysed by gel retardation experiments (EMSA) (Figure 2B). BT-474 and ZR-75.1 breast cancer cells were used as AP-2-positive controls and HepG2 hepatocarcinoma cells and MDA-MB-231 breast cancer cells as AP-2-negative controls (Bosher et al, 1996). AP-2 levels were very low in colon cancer cells (Figure 2A, lanes 8�C12). Interestingly, in COLO 205 cells, the AP-2 antibody revealed three intense bands of lower molecular weight (Figure 2A, lane 11).

No DNA binding activity was detected in these cells (Figure 2B, lane 11), suggesting that this might be a false positive signal. Low levels of the 50kDa AP-2 factor were detected in prostate cancer cells (Figure 2A, lanes 5�C7). In the two ovary cancer cell lines, AP-2�� was easily detected (Figure 2A, lanes 13, 14). However, the signal was much less intense than in breast cancer cells overexpressing AP-2 (see for instance Figure 2A, lanes 1 and 3). Finally, in the pancreatic cells, the AP-2�� levels were low to moderate. In these cells, a higher molecular weight band was detected together with the 50kDa protein (Figure 2A, lanes 15�C21). The Western blotting and the EMSA results showed comparable patterns in all the cell lines, indicating that when AP-2 was present, it binds efficiently to DNA.

Figure 2 Distribution of AP-2 transcription factor in non-breast cancer cell lines. (A) Western blot with10��g (control, lanes 1 and 3) or 20��g (lanes 2 and 4�C21) of crude nuclear extracts from breast and non-breast cells. … In conclusion, there was no correlation between AP-2 and erbB-2 mRNA or protein levels in the non-breast cancer cell lines. The most striking discrepancy was observed with HepG2 cells, widely used as AP-2 negative controls, but these cells do express the erbB-2 mRNA and protein. Our results thus suggest that the AP-2 is not involved in ERBB2 overexpression in the non-breast cancer cell lines we tested. ERBB2 promoter activity in non-breast cancer cells In order to better understand the mechanisms leading to ERBB2 overexpression in the non-breast cancer cells, we transfected reporter vectors containing progressive deletions of a 6kb promoter fragment (Figure 3A).

We have shown previously that these promoter fragments are active in breast cancer cells (Grooteclaes et al, 1994). The transfection efficiencies of all the cell lines were estimated by Dacomitinib transfection of an EGFP-expression vector (data not shown). Only the cells having a transfection efficiency of at least 5%, namely colon and ovary cancer cells, were used for further studies.