gms | German Medical Science

27. Deutscher Krebskongress

Deutsche Krebsgesellschaft e. V.

22. - 26.03.2006, Berlin

Reverse phase protein arrays (RPPA) for the quantitative analysis of protein expression in breast cancer

Meeting Abstract

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  • corresponding author presenting/speaker Christian Löbke - Deutsches Krebsforschungszentrum Heidelberg, Deutschland
  • Ulrike Korf - Deutsches Krebsforschungszentrum Heidelberg

27. Deutscher Krebskongress. Berlin, 22.-26.03.2006. Düsseldorf, Köln: German Medical Science; 2006. DocPE089

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Veröffentlicht: 20. März 2006

© 2006 Löbke et al.
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Reverse phase protein arrays (RPPA) for the quantitative analysis of protein expression in breast cancer. Aim of this study is the analysis of deregulated protein expression and signaling pathways in human breast cancer with particular attention to estrogen signaling. Different types of breast cancer are discriminated by their estrogene receptor (ER) status. This analysis is usually based on expression profiling data and or on histological examination. We have extended this approach to the analysis of the breast cancer proteome in different ER-positive and ER-negative breast cancer cell lines. In contrast to previous studies, we will initially characterize the ER receptor status, e.g. for the expression of ER-alpha and/or ER-beta, for all cell lines used in this study. In order to select suitable candidate proteins for the characterization of activated signaling pathways, we have chosen proteins which play hub roles in breast cancer. The step from analyzing RNA expression levels to proteome analysis allows us also to detect aberrant protein phosphorylation patterns. Thus, certain candidate proteins, e.g. ERK1/2, AKT1-3, NFkB and BCL-2 will be characterized for their activation state. In addition, based on the results of two in house cDNA-microarry screens, further candidate proteins were selected based on their potential role in regulating transcription or proliferation. This cDNA-screens discriminate ER positive and ER negative expression status and were performed with the whole human genome, human-Unigene-Set PZPD 3,1, with 37.500 clones. For the candidate proteins we will compare this RNA expression profiling changes with those observable on the protein level. Technically, the RPPA microarray chip consists of micro-dots of immobilised cellular protein extracts which are derived from human breast cancer cell cultures. Each microarray is incubated with specific antibodies (AB) directed against a breast-cancer relevant protein. This method allows direct comparison and expression quantification of different cell cultures on a single chip. The successful application of this method requires highly specific and well-characterised antibodies. The specificity of all ABs is verified by Western Blotting with different ER-positive and -negative human breast cancer cell lines. Antibodies against novel proteins will be produced and tested for their specificity with the help of recombinant proteins. This approach allows us to analyse the outcome of deregulation of selected pathways in breast cancer, and the identification of novel molecular markers. In the future we will extend this study to the analysis of patient-derived tumor samples.

Figure 1 [Fig. 1]