Flow Cytometric FRET Analysis of ErbB Receptor Tyrosine Kinase Interaction

Simone Diermeier‐Daucher1, Gero Brockhoff1

1 Institute of Pathology, University of Regensburg, Regensburg, Germany
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 12.14
DOI:  10.1002/0471142956.cy1214s45
Online Posting Date:  July, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The homologous and heterologous interaction of members of the epidermal growth factor (EGF)–related receptor tyrosine kinase (RTK) family (ErbB or HER family receptors) upon ligand binding is the initial key event in signal transduction by these receptors. In addition to the availability of their respective ligands, the relative expression level of the four ErbB receptors triggers receptor cross‐activation, which determines signal diversification and the cells' biological response. However, the function of ErbB receptors and their ligands appears highly complex, and its impact on cell growth and proliferation of normal and tumor cells is incompletely understood. Flow cytometric fluorescence resonance energy transfer (FRET) measurements facilitate the quantitative analysis of receptor interaction. This unit delineates the cell‐by‐cell analysis of epidermal growth factor receptor (EGFR, ErbB1, HER1) and ErbB2 (HER2) receptor interaction in ErbB2‐overexpressing BT474 and SK‐BR‐3 breast cancer cell lines, using a dual‐laser flow cytometer. ReFlex software–based quantification of energy transfer efficiency (E) directly reflects the amount of receptor interaction. Curr. Protocol. Cytom. 45:12.14.1‐12.14.19. © 2008 by John Wiley & Sons, Inc.

Keywords: receptor‐tyrosine‐kinases; FRET; trastuzumab; pertuzumab; ErbB2 receptor; flow cytometry; breast cancer; receptor interaction; ErbB2 homodimerization

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Assessment of ErbB2‐Receptor Homodimerization
  • Support Protocol 1: FRET Evaluation Using ReFlex Software
  • Basic Protocol 2: Assessment of EGF‐Receptor Homodimerization
  • Basic Protocol 3: Assessment of EGFR/ErbB2 Heterodimerization
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Assessment of ErbB2‐Receptor Homodimerization

  Materials
  • SK‐BR‐3 breast cancer cells (ATCC #HTB‐30D) grown to a subconfluent density in 75‐cm2 cell culture flasks (see appendix 3B for culture of mammalian cells)
  • Complete Dulbecco's modified Eagle medium (DMEM; appendix 3B)
  • Complete DMEM containing 5% fetal bovine serum (FBS; DMEM‐5; appendix 3B)
  • Trastuzumab: (also known as rhuMAb 4D5 and Herceptin) humanized monoclonal antibody acting on the ErbB2 (HER2/neu) receptor
  • Accutase solution (PAA Laboratories)
  • Cyanine 3 (Cy3; donor fluorophore)‐labeled pertuzumab (also known as rhuMAb 2C4 and Omnitarg; humanized antibody targeting the ErbB2‐based signaling pathway; Roche Diagnostics): label with commercially available kits (e.g., Amersham Pharmacia) according to the manufacturer's instructions
  • Cyanine 5 (Cy5; acceptor fluorophore)‐labeled pertuzumab (Roche Diagnostics): label with commercially available kits (e.g., Amersham Pharmacia) according to the manufacturer's instructions
  • PBS/0.1% NaN 3/0.2% BSA: phosphate‐buffered saline (PBS; appendix 2A) containing 0.1% NaN 3 and0.2% bovine serum albumin (BSA); store up to 2 weeks at 4°C
  • PBS/0.1% NaN 3/2% BSA: store up to 2 weeks at 4°C
  • 4% (w/v) paraformaldehyde (PFA; see recipe)
  • 15‐ml centrifuge tubes (e.g., 16 × 125–mm polystyrene round‐bottom tubes, BD)
  • Refrigerated centrifuge, 4°C
  • Water jet pump (e.g., BRAND), optional
  • 5‐ml sample tubes (e.g., 12 × 75–mm polystyrene round‐bottom tubes, BD)
  • Flow cytometer with a dual‐laser excitation (e.g., FACSCalibur, BD): see Table 12.14.2 for required filters
  • Acquisition software that stores data in list‐mode flow cytometry standard (FCS) files (e.g., CellQuest, CellQuest Pro, FACS Diva)
  • ReFlex software (see protocol 2)
    Table 2.4.2   MaterialsDetection Channels on the FACSCalibur for FRET Measurement with the Cy3/Cy5 and PE/Cy5 Donor/Acceptor Dye Pair

    Dye Absorption‐maxima (nm) Emission maxima (nm) Excitation wavelength (nm) Detection channel Filter
    PE 480; 565 578 488 FL2 585/42
    Cy3 512; 552 565; 615 488 FL2 585/42
    FRET 488 FL3 670LP
    Cy5 625‐650 670 ∼635 FL4 661/16

NOTE: Protect labeling solutions and labeled samples from light.

Support Protocol 1: FRET Evaluation Using ReFlex Software

  Materials
  • SK‐BR‐3 breast cancer cells (ATCC #HTB‐30D) grown to a subconfluent density in 75‐cm2 cell culture flasks (see appendix 3B for culture of mammalian cells)
  • A431 epidermoid carcinoma cells (ATCC #CRL‐1555) grown to a subconfluent density in 75‐cm2 cell culture flasks (see appendix 3B for culture of mammalian cells)
  • Complete Dulbecco's modified Eagle medium (DMEM; appendix 3B)
  • DMEM containing 5% fetal bovine serum (FBS; complete DMEM‐5; appendix 3B)
  • Accutase solution (PAA Laboratories)
  • Ligand: e.g., 5 nM (30 ng/ml) recombinant human epidermal growth factor (rhEGF)
  • 117 µg/ml phycoerythrin (PE; donor fluorophore)‐labeled anti‐EGFR antibody (clone EGFR.1, IgG2b; BD Biosciences)
  • Cyanine 5 (Cy5; acceptor fluorophore)‐labeled anti‐EGFR antibody: label clone EGFR.1 (BD Biosciences) with Cy5 antibody (see Diermeier et. al., )
  • PBS/0.1% NaN 3/0.2% BSA: phosphate‐buffered saline (PBS; appendix 2A) containing 0.1% NaN 3 and 0.2% bovine serum albumin (BSA); store up to 2 weeks at 4°C
  • PBS/0.1% NaN 3/2% BSA: store up to 2 weeks at 4°C
  • 4% (w/v) paraformaldehyde (PFA; see recipe)
  • 15‐ml centrifuge tubes
  • Refrigerated centrifuge, 4°C
  • Water jet pump (e.g., BRAND), optional
  • 5‐ml sample tubes (e.g., 12 × 75–mm polystyrene round‐bottom tubes, BD)
  • Flow cytometer with a dual‐laser excitation (e.g., FACSCalibur, BD)
  • Acquisition software that stores data in list‐mode flow cytometry standard (FCS) files (e.g., CellQuest, CellQuest Pro, FACS Diva)
  • ReFlex software (see protocol 2)

Basic Protocol 2: Assessment of EGF‐Receptor Homodimerization

  Materials
  • SK‐BR‐3 breast cancer cells (ATCC #HTB‐30D) grown to a subconfluent density in 75‐cm2 cell culture flasks (see appendix 3B for culture of mammalian cells)
  • Complete Dulbecco's modified Eagle medium (DMEM; appendix 3B)
  • Complete DMEM containing 5% fetal bovine serum (FBS; DMEM‐5; appendix 3B)
  • Therapeutic antibody (e.g., trastuzumab, also known as Herceptin)
  • Accutase solution (PAA Laboratories)
  • Cyanine 3 (Cy3; donor fluorophore)‐labeled pertuzumab (also known as rhuMAb 2C4 and Omnitarg; humanized antibody targeting the HER2‐based signaling pathway; Roche Diagnostics): label with commercially available kits (e.g., Amersham Pharmacia) according to the manufacturer's instructions
  • Cyanine 5 (Cy5; acceptor fluorophore)‐labeled pertuzumab (Roche Diagnostics): label with commercially available kits (e.g., Amersham Pharmacia) according to the manufacturer's instructions
  • Goat anti‐mouse F(ab) against the H + L chain of IgG (Dianova)
  • Unlabeled anti‐EGFR antibody (clone EGFR.1, IgG2b; BD Biosciences)
  • Cy3‐conjugated goat anti‐mouse F(ab) against the H + L chain of IgG (Dianova)
  • PBS/0.1% NaN 3/0.2% BSA
  • PBS/0.1% NaN 3/2% BSA
  • 4% (w/v) paraformaldehyde (PFA; see recipe)
  • 5‐ml centrifuge tubes
  • Refrigerated centrifuge, 4°C
  • Water jet pump (BRAND), optional
  • 5‐ml sample tubes (e.g., 12 × 75–mm polystyrene round‐bottom tubes, BD)
  • Flow cytometer with a dual‐laser excitation (e.g., FACSCalibur)
  • Acquisition software that stores data in list‐mode flow cytometry standard (FCS) files (e.g., CellQuest, CellQuest Pro, FACS Diva)
  • ReFlex software (see protocol 2)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Adams, C.W., Allison, D.E., Flagella, K., Presta, L., Clarke, J., Dybdal, N., McKeever, K., and Sliwkowski, M.X. 2006. Humanization of a recombinant monoclonal antibody to produce a therapeutic HER dimerization inhibitor, pertuzumab. Cancer Immunol. Immunother. 55:717‐727.
   Barok, M., Isola, J., Palyi‐Krekk, Z., Nagy, P., Juhasz, I., Vereb, G., Kauraniemi, P., Kapanen, A., Tanner, M., Vereb, G., and Szöllősi, J. 2007. Trastuzumab causes antibody‐dependent cellular cytotoxicity‐mediated growth inhibition of submacroscopic JIMT‐1 breast cancer xenografts despite intrinsic drug resistance. Mol. Cancer Ther. 6:2065‐2072.
   Brockhoff, G., Heiss, P., Schlegel, J., Hofstaedter, F., and Knuechel, R. 2001. Epidermal growth factor receptor, c‐erbB2 and c‐erbB3 receptor interaction, and related cell cycle kinetics of SK‐BR‐3 and BT474 breast carcinoma cells Cytometry 44:338‐348.
   Chow, N.H., Liu, H.S., Yang, H.B., Chan, S.H., and Su, I.J. 1997. Expression patterns of erbB receptor family in normal urothelium and transitional cell carcinoma. An immunohistochemical study. Virchows Arch. 430:461‐466.
   Diermeier, S., Horváth, G., Knuechel‐Clarke, R., Hofstaedter, F., Szöllősi, J., and Brockhoff, G. 2005. Epidermal growth factor receptor coexpression modulates susceptibility to Herceptin in HER2/neu overexpressing breast cancer cells via specific erbB‐receptor interaction and activation. Exp. Cell Res. 304:604‐619.
   Horváth, G., Petrás, M., Szentesi, G., Fábián, A., Park, J.W., Vereb, G., and Szöllősi, J. 2005. Selecting the right fluorophores and flow cytometer for fluorescence resonance energy transfer measurements. Cytometry A 65:148‐157.
   Jones, R.B., Gordus, A., Krall, J.A., and Macbeath, G. 2006. A quantitative protein interaction network for the ErbB receptors using protein microarrays. Nature 439:168‐174.
   Laskin, J.J. and Sandler, A.B. 2004. Epidermal growth factor receptor: A promising target in solid tumours. Cancer Treat. Rev. 30:1‐17.
   Nagy, P., Bene, L., Balazs, M., Hyun, W.C., Lockett, S.J., Chiang, N.Y., Waldman, F., Feuerstein, B.G., Damjanovich, S., and Szöllősi, J. 1998. EGF‐induced redistribution of erbB2 on breast tumor cells: Flow and image cytometric energy transfer measurements. Cytometry 32:120‐131.
   Nagy, P., Jenei, A., Kirsch, A.K., Szöllősi, J., Damjanovich, S., and Jovin, T.M. 1999. Activation‐dependent clustering of the erbB2 receptor tyrosine kinase detected by scanning near‐field optical microscopy. J. Cell Sci. 112:1733‐1741.
   Rueckert, S., Ruehl, I., Kahlert, S., Konecny, G., and Untch, M. 2005. A monoclonal antibody as an effective therapeutic agent in breast cancer: Trastuzumab. Expert Opin. Biol. Ther. 5:853‐866.
   Schmid, J.A. and Sitte, H.H. 2003. Fluorescence resonance energy transfer in the study of cancer pathways. Curr. Opin. Oncol. 15:55‐64.
   Schulze, W., Deng, L., and Mann, M. 2005. Phosphoryrosine interactome of the erbB‐receptor kinase family. Mol. Syst. Biol. 1:2005.0008 [http://www.nature.com/msb/journal/v1/n1/full/msb4100012.html].
   Sebestyen, Z., Nagy, P., Horváth, G., Vamosi, G., Debets, R., Gratama, J.W., Alexander, D.R., and Szöllősi, J. 2002. Long wavelength fluorophores and cell‐by‐cell correction for autofluorescence significantly improves the accuracy of flow cytometric energy transfer measurements on a dual‐laser benchtop flow cytometer. Cytometry 48:124‐135.
   Sinev, M., Landsmann, P., Sineva, E., Ittah, V., and Haas, E. 2000. Design consideration and probes for fluorescence resonance energy transfer studies. Bioconjug. Chem. 11:352‐362.
   Slamon, D.J., Clark, G.M., Wong, S.G., Levin, W.J., Ullrich, A., and McGuire, W.L. 1987. Human breast cancer: Correlation of relapse and survival with amplification of the HER‐2/neu oncogene. Science 235:177‐182.
   Smith, B.L., Chin, D., Maltzman, W., Crosby, K., Hortobagyi, G.N., and Bacus, S.S. 2004. The efficacy of Herceptin therapies is influenced by the expression of other erbB receptors, their ligands and the activation of downstream signalling proteins. Br. J. Cancer 91:1190‐1194.
   Spector, N., Xia, W., El‐Hariry, I., Yarden, Y., and Bacus, S. 2007. HER2 therapy. Small molecule HER‐2 tyrosine kinase inhibitors. Breast Cancer Res. 9:205.
   Szentesi, G., Vereb, G., Horváth, G., Bodnar, A., Fabian, A., Matko, J., Gaspar, R., Damjanovich, S., Matyus, L., and Jenei, A. 2005. Computer program for analyzing donor photobleaching FRET image series. Cytometry A 67:119‐128.
   Szöllősi, J., Tron, L., Damjanovich, S., Helliwell, S.H., Arndt‐Jovin, D., and Jovin, T.M. 1984. Fluorescence energy transfer measurements on cell surfaces: A critical comparison of steady‐state fluorimetric and flow cytometric methods. Cytometry 5:210‐216.
   Szöllősi, J., Damjanovich, S., Mulhern, S.A., and Tron, L. 1987. Fluorescence energy transfer and membrane potential measurements monitor dynamic properties of cell membranes: A critical review. Prog. Biophys. Mol. Biol. 49:65‐87.
   Tron, L., Szöllősi, J., Damjanovich, S., Helliwell, S.H., Arndt‐Jovin, D.J., Szöllősi, J., Tron, L., Damjanovich, S., Helliwell, S.H., Arndt‐Jovin, D., and Jovin, T.M. 1984. Fluorescence energy transfer measurements on cell surfaces: A critical comparison of steady‐state fluorimetric and flow cytometric methods. Cytometry 5:210‐216.
   Jovin, T.M. 1984. Flow cytometric measurement of fluorescence resonance energy transfer on cell surfaces. Quantitative evaluation of the transfer efficiency on a cell‐by‐cellbasis. Biophys. J. 45:939‐946.
   Wong, S.F. 2005. Cetuximab: An epidermal growth factor receptor monoclonal antibody for the treatment of colorectal cancer. Clin. Ther. 27:684‐694.
   Yarden, Y. and Sliwkowski, M.X. 2001. Untangling the ErbB signalling network. Nat. Rev. Mol. Cell. Biol. 2:127‐137.
   Zaczek, A., Brandt, B., and Bielawski, K.P. 2005. The diverse signaling network of EGFR, HER2, HER3 and HER4 tyrosine kinase receptors and the consequences for therapeutic approaches. Histol. Histopathol. 20;1005‐1015.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library