RESUMO
Centrosome morphology and number are frequently deregulated in cancer cells. Here, to identify factors that are functionally relevant for centrosome abnormalities in cancer cells, we established a protein-interaction network around 23 centrosomal and cell-cycle regulatory proteins, selecting the interacting proteins that are deregulated in cancer for further studies. One of these components, LGALS3BP, is a centriole- and basal body-associated protein with a dual role, triggering centrosome hypertrophy when overexpressed and causing accumulation of centriolar substructures when downregulated. The cancer cell line SK-BR-3 that overexpresses LGALS3BP exhibits hypertrophic centrosomes, whereas in seminoma tissues with low expression of LGALS3BP, supernumerary centriole-like structures are present. Centrosome hypertrophy is reversed by depleting LGALS3BP in cells endogenously overexpressing this protein, supporting a direct role in centrosome aberration. We propose that LGALS3BP suppresses assembly of centriolar substructures, and when depleted, causes accumulation of centriolar complexes comprising CPAP, acetylated tubulin and centrin.
Assuntos
Antígenos de Neoplasias/metabolismo , Biomarcadores Tumorais/metabolismo , Proteínas de Transporte/metabolismo , Centríolos/metabolismo , Centríolos/patologia , Glicoproteínas/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Centríolos/ultraestrutura , Cromatografia de Afinidade , Proteínas da Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glicoproteínas/genética , Células HEK293 , Humanos , Hipertrofia , Masculino , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Neoplasias/genética , Mapas de Interação de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Seminoma/genética , Seminoma/patologia , Fuso Acromático/metabolismo , Fuso Acromático/ultraestruturaRESUMO
Large-scale screens in mammalian cells demand for flexible high-throughput screening platforms that allow to analyze cellular traits on a genome-wide level or to identify small-molecule inhibitors (SMIs) from complex compound libraries. In this study we developed and tested high-density cell arrays made out of polydimethylsiloxane (PDMS) that support cell growth directly on standard glass microscope objective slides. We analyzed the effect of 3 reference inhibitors (MLN-8054, VX-680, and flavopiridol) and 4 exploratory, cell permeable small-molecule kinase inhibitors (two benzothiophene-based and two 4-amino-6-arylpyrimidine-based compounds) on different cell lines, using prototype 5 × 5 and 9 × 9 array carpets. We found that high-density PDMS cell arrays support growth of a broad range of cell types, are well suited for compound screens, and are compatible with high-content screening platforms. This novel array format is of particular advantage for compound screening to identify SMIs, when a combination of flexibility with respect to culture volume, well density, and high-resolution imaging is required. In addition, we demonstrated the suitability of this format for reverse transfection and siRNA experiments.