We analysed the possible involvement of AP-2 in the increased ERB

We analysed the possible involvement of AP-2 in the increased ERBB2 expression, because of the well-characterised role of http://www.selleckchem.com/products/ganetespib-sta-9090.html this transcription factor in breast cancer cells. Indeed, the 500bp proximal ERBB2 promoter contains two AP-2 binding sites, which contribute to the gene overexpression. The first site is located 215bp upstream from the transcription initiation site (Bosher et al, 1995), whereas the second lies 501bp upstream from the transcription start site (Grooteclaes et al, 1999; Vernimmen et al, 2003). Nuclear AP-2�� levels and AP-2 DNA binding activities were considerably lower in prostate, colon, ovary and pancreatic cell lines than in breast cancer cells. Since AP-2 could be cytoplasmic rather than nuclear (see below), we also analysed whole-cell extracts by Western blotting.

We did not observe any difference between nuclear and total AP-2 levels, indicating that the low levels of the nuclear factor is not the consequence of its cytoplasmic retention (data not shown). Besides the full-length 50kDa protein, we detected lower and higher molecular weight proteins as well. The lower molecular weight bands might result from the proteolysis of the transcription factor. Higher molecular weight immunoreactive forms were observed mainly in the pancreatic cells (Figure 2A) and might represent the recently described, 60kDa sumolated form of the protein. The transcriptional activity of these sumolated factors is reduced (Eloranta and Hurst, 2002). AP-2�� levels were not correlated with ERBB2 expression in non-breast cancer cell lines.

This indicates that AP-2 is not involved in ERBB2 gene overexpression. In other cancers, the role of AP-2 has been shown to vary according to the tumour type. Some authors described the loss of AP-2�� expression early in the development of prostate adenocarcinoma (Ruiz et al, 2001). Moreover, AP-2B, a dominant-negative variant of AP-2��, was detected by RT�CPCR in LNCaP cells (Buettner et al, 1993). Others did detect AP-2�� by immunohistochemistry in some prostate cancer specimens (Lipponen et al, 2000). In primary colorectal (Ropponen et al, 2001) and ovarian cancers (Anttila et al, 2000), AP-2�� was detected in the nucleus or the cytoplasm of tumour cells. However, the presence of the transcription factor in the cytoplasm precludes its function in transcription.

Interestingly, high AP-2�� mRNA levels were detected in some tumours which were negative for the protein (Karjalainen et al, 2000). To analyse the molecular mechanisms leading to ERBB2 overexpression in non-breast cancer cell lines, we transfected four GSK-3 LUCIFERASE reporter vectors containing 200bp�C6kb fragments of the ERBB2 promoter in colon and ovary cell lines. The relative transcriptional activity of each reporter was compared to the luciferase activity induced by the p255-LUC vector, considered as equal to one (Grooteclaes et al, 1994).

Leave a Reply

Your email address will not be published. Required fields are marked *

*

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>