In both colorectal cell lines www.selleckchem.com/products/PF-2341066.html (HCT116 and COLO 320), the 6kb promoter fragment induced a 2.5-fold increase of the LUC activity compared to the vector containing 200bp of the promoter (Figure 3B, lane 4 and 3C, lane 4). The low-ERBB2-expressing ovary cancer cells, OVCAR-3, showed an increase of the LUC activity with the promoter size (Figure 3D). This increase was similar to that observed in the colorectal cells (compare Figure 3B�CD). Only the p756-LUC construct increased the LUC activity in ERBB2-overexpressing SK-OV-3 cells (Figure 3E, lane 2). The longer promoter fragments downregulated the transcriptional activity to the basal value (Figure 3E, lanes 3 and 4). It is interesting to note that in this cell line the ERBB2 gene is amplified four-fold and overexpressed more than 60 times (King et al, 1992 and personal results, Table 1).

Figure 3 Luc assays using different ERBB2 promoter constructs. (A) Illustration of the different reporter vectors used. The promoter fragment sizes are indicated in the hatched boxes. The luciferase activity was measured in colon HCT 116 (B), COLO 320 (C) and … DISCUSSION Around 30% of breast cancers overexpress the ERBB2 gene and this is correlated with a poor prognosis. Besides gene amplification, several investigators have described the involvement of ETS family (Scott et al, 2000) and AP-2 family (Bates and Hurst, 1997) transcription factors in the gene overexpression. ERBB2 overexpression has also been reported in cancers of colon (Nakae et al, 1993; Kapitanovic et al, 1994; Maurer et al, 1998), prostate (Ross et al, 1993; Morote et al, 1999), ovary (Fajac et al, 1995) and pancreatic (Yamanaka et al, 1993) origin.

However, the mechanisms leading to increased expression of the gene have not been investigated in these tumours. Gene amplification is rare in these tumours and cannot account for the observed increase in the mRNA or protein levels. To our knowledge, this is the first attempt to understand the molecular mechanisms leading to ERBB2 overexpression in non-breast cancers. First, we assessed p185c-erbB-2 levels by ICC and by Western blotting. One of the main observations of this study is that breast and ovary cancer cell lines were characterised by a membrane homogeneous staining, whereas in the non-breast cancer cells, the staining was cytoplasmic and heterogeneous (Figure 1, Table 1).

Several truncated intracellular p185c-erbB-2 fragments have been described (Scott et al, 1993; Esparis-Ongando et al, 1999; Christianson et al, 1998; Molina et al, 2002). These fragments, which signal actively, might be responsible for the cytoplasmic staining observed in most non-breast cancer cells. However, only the full-length protein was detected by Western blotting in these cells, which does not support the hypothesis that ICC detects Entinostat a truncated cytoplasmic protein.