Trastuzumab is a recombinant antibody drug that is widely used for the treatment of breast cancer.

Although trastuzumab, a recombinant humanized anti-ErbB2 antibody, is widely used in the treatment of breast cancer, neither its mechanism of action, nor the factors leading to resistance are fully understood. We have previously shown that antibody-dependent cellular

No abstract

The role of the expression patterns of proteins involved in oncogenesis can be understood after characterizing their multimolecular interactions. Conventional FRET methods permit the analysis of interaction between two molecular species at the most, which necessitates the introduction of new approaches for studying multicomponent signaling complexes. Flow cytometric as well as microscopic donor (dbFRET) and acceptor (abFRET) photobleaching FRET measurements were performed to determine the association states of ErbB2, b1-integrin, and CD44 receptors. Based on consecutively applied abFRET and dbFRET methods (two-sided FRET), the relationship of b1-integrin-ErbB2 heteroassociation to ErbB2 homoassociation and of b1-integrin-ErbB2 heteroassociation to ErbB2-CD44 heteroassociation was studied by correlating pixel-bypixel FRET values of the corresponding abFRET and dbFRET images in contour plots. Anticorrelation was observed between b1-integrin-ErbB2 heteroassociation and ErbB2 homoassociation on trastuzumab sensitive N87 and SK-BR-3 cells, while modest positive correlation was found between b1-integrin-ErbB2 and ErbB2-CD44 heteroassociation on trastuzumab resistant MKN-7 cells. The FRET efficiency values of b1-integrinErbB2 heteroassociation were markedly higher at the focal adhesion regions on attached cells than those measured by flow cytometry on detached cells. In conclusion, we implemented an experimental set-up termed two-sided FRET for correlating two pairwise interactions of three arbitrarily chosen molecular species. On the basis of our results, we assume that the homoassociation state of ErbB2 is dynamically modulated by its interaction with b1-integrins. ' International Society for Analytical CytologyKey terms receptor tyrosine kinase; ErbB2, b1-integrin; CD44; trastuzumab resistance; laser scanning confocal microscopy; fluorescence resonance energy transfer; tsFRET; dbFRET; abFRET UNDERSTANDING the molecular mechanisms of signal transduction processes requires the elucidation of assembling and/or disassembling of protein complexes including the conformational changes accompanying these processes. One of the best approaches for studying these molecular rearrangements is fluorescence resonance energy transfer (FRET), which is a sensitive method for measuring intra-or intermolecular distances. FRET-based methods are capable of resolving molecular associations and conformational changes in the 1-10 nm range far exceeding the diffraction limit of the conventional microscope. FRET is a nonradiative process in which energy is transferred from the first excited electronic state of a donor molecule (D) to a nearby acceptor molecule (A) in a dipole-dipole interaction under favorable conditions, resulting in the quenching of donor fluorescence accompanied by the increase (also known as sensitization) of acceptor fluorescence (1-3). For mapping the physical associations of various membrane molecules, flow cytometric and microscopic

T‐cell receptors (TCRs) can be genetically modified to improve gene‐engineered T‐cell responses, a strategy considered critical for the success of clinical TCR gene therapy to treat cancers. TCR:ζ, which is a heterodimer of TCRα and β chains each coupled to complete human CD3ζ, overcomes issues of mis‐pairing with endogenous TCR chains, shows high surface expression and mediates antigen‐specific T‐cell functions in vitro. In the current study, we further characterized TCR:ζ in gene‐engineered T cells and assessed whether this receptor is able to interact with surface molecules and drive correct synapse formation in Jurkat T cells. The results showed that TCR:ζ mediates the formation of synaptic areas with antigen‐positive target cells, interacts closely with CD8α and MHC class I (MHCI), and co‐localizes with CD28, CD45 and lipid rafts, similar to WT TCR. TCR:ζ did not closely associate with endogenous CD3ε, despite its co‐presence in immune synapses, and TCR:ζ showed enhanced synaptic accumulation in T cells negative for surface‐expressed TCR molecules. Notably, synaptic TCR:ζ demonstrated lowered densities when compared with TCR in dual TCR T cells, a phenomenon that was related to both extracellular and intracellular CD3ζ domains present in the TCR:ζ molecule and responsible for enlarged synapse areas.