RESUMEN
The mitotic spindle is composed of dynamic microtubules and associated proteins that together direct chromosome movement during mitosis. The spindle plays a vital role in accurate chromosome segregation fidelity and is a therapeutic target in cancer. Nevertheless, the molecular mechanisms by which many spindle-associated proteins function remains unknown. The nucleolar and spindle-associated protein NUSAP1 is a microtubule-binding protein implicated in spindle stability and chromosome segregation. We show here that NUSAP1 localizes to dynamic spindle microtubules in a unique chromosome-centric pattern, in the vicinity of overlapping microtubules, during metaphase and anaphase of mitosis. Mass spectrometry-based analysis of endogenous NUSAP1 interacting proteins uncovered a cell cycle-regulated interaction between the RanBP2-RanGAP1-UBC9 SUMO E3 ligase complex and NUSAP1. Like NUSAP1 depletion, RanBP2 depletion impaired the response of cells to the microtubule poison Taxol. NUSAP1 contains a conserved SAP domain (SAF-A/B, Acinus, and PIAS). SAP domains are common among many other SUMO E3s, and are implicated in substrate recognition and ligase activity. We speculate that NUSAP1 contributes to accurate chromosome segregation by acting as a co-factor for RanBP2-RanGAP1-UBC9 during cell division.
Asunto(s)
Segregación Cromosómica/fisiología , Proteínas Activadoras de GTPasa/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Segregación Cromosómica/efectos de los fármacos , Proteínas Activadoras de GTPasa/genética , Células HeLa , Humanos , Proteínas Asociadas a Microtúbulos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Chaperonas Moleculares/genética , Proteínas de Complejo Poro Nuclear/genética , Paclitaxel/farmacología , Dominios Proteicos , Huso Acromático/genética , Huso Acromático/metabolismo , Enzimas Ubiquitina-Conjugadoras/genéticaRESUMEN
The epithelial cell adhesion molecule (EpCAM) is expressed by a wide range of human carcinomas, making it an attractive diagnostic and therapeutic target in oncology. Its recent identification on cancer stem cells has raised further interest in its use for tumor targeting and therapy. Here, we present the characterization and therapeutic potential of 3-17I, a novel human EpCAM-targeting monoclonal antibody. Strong reaction of 3-17I was observed in all lung, colon, and breast human tumor biopsies evaluated. By flow cytometry and confocal fluorescence microscopy, we demonstrate that 3-17I specifically targets EpCAM-positive cell lines. We also show evidence for mAb-sequestration in endo-/lysosomes, suggesting internalization of 3-17I by receptor-mediated endocytosis. The ribosomal-inactivating toxin saporin was linked to 3-17I, creating the per se non-toxic immunotoxin 3-17I-saporin, a promising candidate for the drug delivery technology photochemical internalization (PCI). PCI is based on a light-controlled destruction of endolysosomal membranes and subsequent cytosolic release of the sequestered payload upon light exposure. EpCAM-positive human cancer cell lines MCF7 (breast), BxPC-3 (pancreas), WiDr (colon), and the EpCAM-negative COLO320DM (colon), were treated with 3-17I-saporin in combination with the clinically relevant photosensitizer TPCS2a (Amphinex), followed by exposure to light. No cytotoxicity was observed after treatment with 3-17I-saporin without light exposure. However, cell viability, proliferation and colony-forming capacity was strongly reduced in a light-dependent manner after PCI of 3-17I. Our results show that 3-17I is an excellent candidate for diagnosis of EpCAM-positive tumors and for development of clinically relevant antibody-drug conjugates, using PCI for the treatment of localized tumors.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/farmacología , Anticuerpos Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias del Colon/tratamiento farmacológico , Inmunotoxinas/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Antineoplásicos/inmunología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/patología , Línea Celular Tumoral , Neoplasias del Colon/inmunología , Neoplasias del Colon/patología , Sistemas de Liberación de Medicamentos/métodos , Femenino , Humanos , Inmunotoxinas/inmunología , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/patología , Fotoquímica/métodos , Proteínas Inactivadoras de Ribosomas Tipo 1/inmunología , SaporinasRESUMEN
Autoimmune diseases are a significant area of unmet medical need in the Western World, but human antibodies are an emerging drug class that could address this demand. Some autoimmune diseases, such as rheumatoid arthritis, are currently benefiting from antibody treatment and new and existing technologies for antibody generation could facilitate the production of effective human antibodies as future drug candidates for other autoimmune diseases. Several methods of generating human antibodies for use as therapeutics have been established, the most commonly used being phage display and transgenic mouse technologies and more recently, cell-free display technologies have also emerged. In this review, we explain the principles behind the various methods of antibody generation and highlight some potential benefits of certain approaches in the context of treatment of autoimmune disease.
