tetramethylrhodamine and Breast-Neoplasms

Tetramethylrhodamine (TAMRA)-phenyl azide is a chemical probe used to detect intracellular acrolein directly in live cells. Herein, we demonstrated that TAMRA is the optimum fluorophore for the probe. TAMRA-phenyl azide was used to reveal that high levels of acrolein are generated in a variety of breast cancer cells, regardless of the tumor subtype. These findings corroborate the analysis presented in our previous report, in which TAMRA-phenyl azide was used to label breast cancer tissues resected from breast cancer patients. Because high levels of acrolein were generated in all cancer cell types, we believe that acrolein detection may be useful as a general method for labeling cancerous tissues.

Topics: Acrolein; Azides; Biomarkers; Breast Neoplasms; Cell Line, Tumor; Cycloaddition Reaction; Fluorescent Dyes; Humans; Microscopy, Fluorescence; Oxidative Stress; Rhodamines

The quantitative sensitivity and dynamic range of conventional immunohistochemistry (IHC) with 3,3'-diaminobenzidine (IHC-DAB) used in pathological diagnosis in hospitals are poor, because enzyme activity can affect the IHC-DAB chromogenic reaction. Although fluorescent IHC can effectively increase the quantitative sensitivity of conventional IHC, tissue autofluorescence interferes with the sensitivity. Here, we created new fluorescent nanoparticles called phosphor-integrated dots (PIDs). PIDs have 100-fold greater brightness and a more than 300-fold greater dynamic range than those of commercially available fluorescent nanoparticles, quantum dots, whose fluorescence intensity is comparable to tissue autofluorescence. Additionally, a newly developed image-processing method enabled the calculation of the PID particle number in the obtained image. To quantify the sensitivity of IHC using PIDs (IHC-PIDs), the IHC-PIDs method was compared with fluorescence-activated cell sorting (FACS), a method well suited for evaluating total protein amount, and the two values exhibited strong correlation (R = 0.94). We next applied IHC-PIDs to categorize the response to molecular target-based drug therapy in breast cancer patients. The results suggested that the PID particle number estimated by IHC-PIDs of breast cancer tissues obtained from biopsy before chemotherapy can provide a score for predicting the therapeutic effect of the human epidermal growth factor receptor 2-targeted drug trastuzumab.

Topics: 3,3'-Diaminobenzidine; Antibodies; Antineoplastic Agents, Immunological; Biopsy; Biotin; Breast Neoplasms; Diagnostic Imaging; Female; Fluorescence; Fluorescent Dyes; Gene Expression; Humans; Imides; Immunohistochemistry; Middle Aged; Nanoparticles; Neoplasm Proteins; Particle Size; Perylene; Receptor, ErbB-2; Rhodamines; Streptavidin; Trastuzumab

Heregulin (HRG) belongs to the family of EGFs and activates the receptor proteins ErbB3 and ErbB4 in a variety of cell types to regulate cell fate. The interactions between HRG and ErbB3/B4 are important to the pathological mechanisms underlying schizophrenia and some cancers. Here, we observed the reaction kinetics between fluorescently labeled single HRG molecules and ErbB3/B4 on the surfaces of MCF-7 human breast cancer cells. The equilibrium association and the dissociation from equilibrium were also measured using single-molecule imaging techniques. The unitary association processes mirrored the EGF and ErbB1 interactions in HeLa cells [Teramura Y, et al. (2006) EMBO J 25:4215-4222], suggesting that the predimerization of the receptors, followed by intermediate formation (between the first and second ligand-binding events to a receptor dimer), accelerated the formation of doubly liganded signaling dimers of the receptor molecules. However, the dissociation analysis suggested that the first HRG dissociation from the doubly liganded dimer was rapid, but the second dissociation from the singly liganded dimer was slow. The dissociation rate constant from the liganded monomer was intermediate. The dynamic changes in the association and dissociation kinetics in relation to the dimerization of ErbB displayed negative cooperativity, which resulted in apparent low- and high-affinity sites of HRG association on the cell surface.

Topics: Breast Neoplasms; Cell Membrane; Dimerization; ErbB Receptors; Female; Fluorescent Dyes; HeLa Cells; Humans; Kinetics; Ligands; Microscopy, Fluorescence; Models, Chemical; Neuregulin-1; Protein Binding; Receptor, ErbB-3; Receptor, ErbB-4; Receptors, Cell Surface; Rhodamines; Signal Transduction

The detection of estrogen receptors (ERs) by immunohistochemistry (IHC) using 3,3'-diaminobenzidine (DAB) is slightly weak as a prognostic marker, but it is essential to the application of endocrine therapy, such as antiestrogen tamoxifen-based therapy. IHC using DAB is a poor quantitative method because horseradish peroxidase (HRP) activity depends on reaction time, temperature and substrate concentration. However, IHC using fluorescent material provides an effective method to quantitatively use IHC because the signal intensity is proportional to the intensity of the photon excitation energy. However, the high level of autofluorescence has impeded the development of quantitative IHC using fluorescence. We developed organic fluorescent material (tetramethylrhodamine)-assembled nanoparticles for IHC. Tissue autofluorescence is comparable to the fluorescence intensity of quantum dots, which are the most representative fluorescent nanoparticles. The fluorescent intensity of our novel nanoparticles was 10.2-fold greater than quantum dots, and they did not bind non-specifically to breast cancer tissues due to the polyethylene glycol chain that coated their surfaces. Therefore, the fluorescent intensity of our nanoparticles significantly exceeded autofluorescence, which produced a significantly higher signal-to-noise ratio on IHC-imaged cancer tissues than previous methods. Moreover, immunostaining data from our nanoparticle fluorescent IHC and IHC with DAB were compared in the same region of adjacent tissues sections to quantitatively examine the two methods. The results demonstrated that our nanoparticle staining analyzed a wide range of ER expression levels with higher accuracy and quantitative sensitivity than DAB staining. This enhancement in the diagnostic accuracy and sensitivity for ERs using our immunostaining method will improve the prediction of responses to therapies that target ERs and progesterone receptors that are induced by a downstream ER signal.

Topics: Breast Neoplasms; Female; Fluorescence; Fluorescent Dyes; Humans; Immunohistochemistry; Nanoparticles; Receptors, Estrogen; Rhodamines; Staining and Labeling; Streptavidin

Estrogenic hormones are classically thought to exert their effects by binding to nuclear estrogen receptors and altering target gene transcription, but estrogens can also have nongenomic effects through rapid activation of membrane-initiated kinase cascades. The development of ligands that selectively activate only the nongenomic pathways would provide useful tools to investigate the significance of these pathways. We have prepared large, abiotic, nondegradable poly(amido)amine dendrimer macromolecules that are conjugated to multiple estrogen molecules through chemically robust linkages. Because of their charge and size, these estrogen-dendrimer conjugates (EDCs) remain outside the nucleus. They stimulate ERK, Shc, and Src phosphorylation in MCF-7 breast cancer cells at low concentrations, yet they are very ineffective in stimulating transcription of endogenous estrogen target genes, being approximately 10,000-fold less potent than estradiol in genomic actions. In contrast to estradiol, EDC was not effective in stimulating breast cancer cell proliferation. Because these EDC ligands activate nongenomic activity at concentrations at which they do not alter the transcription of estrogen target genes, they should be useful in studying extranuclear initiated pathways of estrogen action in a variety of target cells.

Topics: Adaptor Proteins, Signal Transducing; Binding, Competitive; Breast Neoplasms; Cell Membrane; Cell Proliferation; Cytoplasm; Dendrimers; Dose-Response Relationship, Drug; Estradiol; Estrogens; Extracellular Matrix; Extracellular Signal-Regulated MAP Kinases; Gene Expression Regulation; Genome; Humans; Phosphorylation; Polyamines; Receptors, Estrogen; Rhodamines; Shc Signaling Adaptor Proteins; Signal Transduction; Src Homology 2 Domain-Containing, Transforming Protein 1; src-Family Kinases; Tumor Cells, Cultured