Fulltext38 - Flow Cytometric FRET Analysis of erbB Receptor Interaction on a Cell-by-Cell Basis SIMONE DIERMEIER-DAUCHER,a MAX HASMANN,b a b AND

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Flow Cytometric FRET Analysis of erbB Receptor Interaction on a Cell-by-Cell Basis S IMONE D IERMEIER -D AUCHER , a M AX H ASMANN , b AND G ERO B ROCKHOFF a a Institute of Pathology, University of Regensburg, Regensburg, Germany b Roche Diagnostics GmbH, Pharma Research Penzberg, Penzberg, Germany Lateralinteractionofc-erbBfamilyreceptorsresultingindimerformationisthekeyeventinitiating signal transduction. Consequently cross-activation and intracellular signaling is triggered with immediate impact on cell proliferation, migration, cell survival, and differentiation. In order to elucidate the connection of signal input (receptor activation) and signal output (altered cellular behavior) we dynamically assessed cell proliferation of BT474 and SK-BR-3 breast cancer cell lines. We quantitated c-erbB2 receptor homodimerization upon treatment with the therapeutic monoclonal anti-c-erbB2 antibodies trastuzumab (Herceptin r ) and pertuzumab by flow cytometric FRET (FCET) measurements on a cell-by-cell basis and calculated the extent of antibody-induced cell cycle exit. The results confirm that trastuzumab does not decrease c-erbB2 homodimers despite its strong potency to drive c-erbB2-overexpressing cells into quiescence. Pertuzumab, however, is able to prevent c-erbB2 homodimerization and thereby enhance the antiproliferative effect of trastuzumab when administered in combination. Key words: FRET; FCET; trastuzumab; pertuzumab; flow cytometry; c-erbB2 homodimerization; receptor interaction; breast cancer Introduction The interaction of transmembrane growth factor receptors like the erbB family of receptor-tyrosine- kinases (RTKs) can be estimated by labeling the extra- cellular domains with fluorochrome-conjugated mon- oclonal antibodies and quantification of fluorescence resonance energy transfer (FRET). FRET measure- ments can be performed by spectrofluorometry and image or flow cytometry. 1–3 Fluorescence microscopy is the method of choice to investigate c-erbB receptor interaction on a subcellular level. In contrast, eval- uation with flow cytometric FRET (FCET) provides the advantage of generating data on a cell-by-cell ba- sis with excellent statistical accuracy by analyzing a large number of cells with the potential to discover cellular heterogeneity. 4 As outlined elsewhere 5 FCET measurements on a cell-by-cell basis require two-laser excitation equipment to reliably calculate the energy transfer efficiency. Four closely related c-erbB receptors play a key role in tissue differentiation and maintenance by Address for correspondence: Simone Diermeier-Daucher, Institute of Pathology, University of Regensburg, Franz-Josef-Strauss Allee 11, 93053 Regensburg. Voice: + 49-0-941-944-6707; fax: + 49-0-941-944-6602. [email protected]
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This note was uploaded on 07/11/2010 for the course SPECTOGRAP 545 taught by Professor Gdf during the Spring '10 term at AIB College of Business.

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Fulltext38 - Flow Cytometric FRET Analysis of erbB Receptor Interaction on a Cell-by-Cell Basis SIMONE DIERMEIER-DAUCHER,a MAX HASMANN,b a b AND

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