Rhythmic cardiac contractions depend on the organized propagation of depolarizing and repolarizing wavefronts. Repolarization is spatially heterogeneous and depends largely on gradients of potassium currents. Gradient disruption in heart disease may underlie susceptibility to fatal arrhythmias, but it is not known how this gradient is established. We show that, in mice lacking the homeodomain transcription factor Irx5, the cardiac repolarization gradient is abolished due to increased Kv4.2 potassium-channel expression in endocardial myocardium, resulting in a selective increase of the major cardiac repolarization current, I(to,f), and increased susceptibility to arrhythmias. Myocardial Irx5 is expressed in a gradient opposite that of Kv4.2, and Irx5 represses Kv4.2 expression by recruiting mBop, a cardiac transcriptional repressor. Thus, an Irx5 repressor gradient negatively regulates potassium-channel-gene expression in the heart, forming an inverse I(to,f) gradient that ensures coordinated cardiac repolarization while also preventing arrhythmias.
The cytotoxicity and tumor-targeting properties of the anti-HER2/neu monoclonal antibody trastuzumab modified with peptides (CGYGPKKKRKVGG) harboring the nuclear localization sequence ([NLS] italicized) of simian virus 40 large T-antigen and radiolabeled with 111 In were evaluated. Methods: Trastuzumab was derivatized with sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) for reaction with NLSpeptides and labeled with 111 In using diethylenetriaminepentaacetic acid (DTPA). The immunoreactivity of 111 In-NLS-trastuzumab was determined by its ability to displace the binding of trastuzumab to SK-BR-3 human breast cancer (BC) cells. Cellular uptake and nuclear localization were evaluated in SK-BR-3, MDA-MB-361, and MDA-MB-231 BC cells, expressing high, intermediate, or very low levels of HER2/neu, respectively, by cell fractionation and confocal microscopy. Biodistribution and nuclear uptake were compared in athymic mice bearing MDA-MB-361 xenografts. The cytotoxicity of 111 In-trastuzumab and 111 In-NLS-trastuzumab was studied by clonogenic assays, and DNA damage was assessed by probing for phosphorylated histone H2AX (gH2AX) foci. Results: The dissociation constant for binding of 111 In-NLS-trastuzumab to SK-BR-3 cells was reduced ,3-fold compared with that of 111 In-trastuzumab, demonstrating relatively preserved receptorbinding affinity. The receptor-mediated internalization of 111 Intrastuzumab in SK-BR-3, MDA-MB-361, and MDA-MB-231 cells increased significantly from 7.2% 6 0.9%, 1.3% 6 0.1%, and 0.2% 6 0.05% to 14.4% 6 1.8%, 6.3% 6 0.2%, and 0.9% 6 0.2% for 111 In-NLS-trastuzumab harboring 6 NLS-peptides, respectively. NLS-trastuzumab localized in the nuclei of BC cells, whereas unmodified trastuzumab remained surface-bound. Conjugation of 111 In-trastuzumab to NLS-peptides did not affect its tissue biodistribution but promoted specific nuclear uptake in MDA-MB-361 xenografts (2.4-2.9 %ID/g [percentage injected dose per gram] for 111 In-NLS-trastuzumab and 1.1 %ID/g for 111 In-trastuzumab). 111 In-NLS-trastuzumab was 5-and 2-fold more potent at killing SK-BR-3 and MDA-MB-361 cells than 111 In-trastuzumab, respectively, whereas toxicity toward MDA-MB-231 cells was minimal. 111 In-NLS-trastuzumab was 6-fold more effective at killing SK-BR-3 cells than unlabeled trastuzumab. The development of recombinant antibodies for cancer therapy has emerged as one of the most promising areas in oncology (1). Trastuzumab (Herceptin; Hoffmann-La Roche), in particular, is a humanized monoclonal antibody (mAb) directed against the human epidermal growth factor receptor-2 (HER2/neu), a transmembrane receptor tyrosine kinase that is overexpressed in 25%-30% of breast cancers (BCs) and distant metastases (2). Trastuzumab shows clinical activity in women with HER2/neu-overexpressing metastatic BC and exhibits synergistic antitumor effects when combined with paclitaxel or anthracyclines, achieving overall response rates of 40%-60% (2). Despite its effectiveness in combination regimens, the response rate...
Our goals in this study were to determine whether 111 In-trastuzumab coupled to peptides harboring nuclear localizing sequences (NLSs) could kill trastuzumab-resistant breast cancer cell lines through the emission of Auger electrons and whether the combination of radiosensitization with methotrexate (MTX) would augment the cytotoxicity of this radiopharmaceutical. Methods: Trastuzumab was derivatized with sulfosuccinimidyl-4-(Nmaleimidomethyl)cyclohexane-1-carboxylate for reaction with NLS peptides and then conjugated with diethylenetriaminepentaacetic acid for labeling with 111 In. HER2 expression was determined by Western blot and by radioligand binding assay using 111 In-trastuzumab in a panel of breast cancer cell lines, including SK-BR-3, MDA-MB-231 and its HER2-transfected subclone (231-H2N), and 2 trastuzumab-resistant variants (TrR1 and TrR2). Nuclear importation of 111 In-NLS-trastuzumab and 111 Intrastuzumab in breast cancer cells was measured by subcellular fractionation, and the clonogenic survival of these cells was determined after incubation with 111 In-NLS-trastuzumab, 111 Intrastuzumab, or trastuzumab (combined with or without MTX). Survival curves were analyzed according to the dose-response model, and the radiation-enhancement ratio was calculated from the survival curve parameters. Results: The expression of HER2 was highest in SK-BR-3 cells (12.6 · 10 5 receptors/cell), compared with 231-H2N and TrR1 cells (6.1 · 10 5 and 5.1 · 10 5 receptors/cell, respectively), and lowest in MDA-MB-231 and TrR2 cells (0.4 · 10 5 and 0.6 · 10 5 receptors/cell, respectively). NLS peptides increased the nuclear uptake of 111 In-trastuzumab in MDA-MB-231, 231-H2N, TrR1, and TrR2 cells from 0.1% 6 0.01%, 2.5% 6 0.2%, 2.8% 6 0.7%, and 0.5% 6 0.1% to 0.5% 6 0.1%, 4.6% 6 0.1%, 5.2% 6 0.6%, and 1.5% 6 0.2%, respectively. The cytotoxicity of 111 In-NLS-trastuzumab on breast cancer cells was directly correlated with the HER2 expression densities of the cells. On a molar concentration basis, the effective concentration required to kill 50% of 231-H2N and TrR1 cells for 111 In-NLS-trastuzumab was 9-to 12-fold lower than for 111 Intrastuzumab and 16-to 77-fold lower than for trastuzumab.MDA-MB-231 and TrR2 cells were less sensitive to 111 In-NLStrastuzumab or 111 In-trastuzumab, and both cell lines were completely insensitive to trastuzumab. The radiation-enhancement ratio induced by MTX for 231-H2N and TrR1 cells after exposure to 111 In-NLS-trastuzumab was 1.42 and 1.68, respectively. Conclusion: Targeted Auger electron radioimmunotherapy with 111 In-NLS-trastuzumab can overcome resistance to trastuzumab, and MTX can potently enhance the sensitivity of HER2-overexpressing breast cancer cells to the lethal Auger electrons emitted by this radiopharmaceutical.
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