RESUMO
BACKGROUND: Epothilones are a novel group of microtubule (mt) targeting cancer drugs that bind to the ß-subunit of the αß-tubulin dimer. Epothilones inhibit cell proliferation and induce cell death by interfering with the normal mt function. In this study, we examined the consequences of altered expression of human ß-tubulin isotypes in terms of the epothilone drug response in human lung and breast cancer cell lines. METHODS: The ß-tubulin isotypes TUBB2A-C, TUBB3 and TUBB were silenced or overexpressed in A549, A549EpoB40 and MCF7 cell lines in the presence or absence of epothilones. The drug effects on cell proliferation, mitosis and mt dynamics were determined using live cell microscopy and immunofluorescence assays. RESULTS: Loss of TUBB3 enhanced the action of epothilones. TUBB3 knockdown increased the severity of drug-induced mitotic defects and resulted in stabilisation of the mt dynamics in cells. Moreover, exogenous expression of TUBB3 in the epothilone resistant cell line conferred the response to drug treatments. In contrast, reduced levels of TUBB2A-C or TUBB had not apparent effect on the cells' response to epothilones. CONCLUSION: Our results show that the expression of TUBB3 contributes to the cellular response to epothilones, putatively by having an impact on the mt dynamics.
Assuntos
Antineoplásicos/farmacologia , Epotilonas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Mitose/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Linhagem Celular Tumoral , Feminino , Inativação Gênica , Humanos , Células MCF-7 , Neoplasias , Fuso Acromático/efeitos dos fármacos , Transfecção , Tubulina (Proteína)/genética , Moduladores de Tubulina/farmacologiaRESUMO
Fibroblast growth factor-8 (FGF-8) is implicated in the development and progression of breast cancer and its levels are frequently elevated in breast tumors. The mechanisms driving FGF-8-mediated tumorigenesis are not well understood. Herein we aimed to identify target genes associated with FGF-8b-mediated breast cancer cell proliferation by carrying out a cDNA microarray analysis of genes expressed in estrogen receptor negative S115 breast cancer cells treated with FGF-8b for various time periods in comparison with those expressed in non-treated cells. Gene and protein expression was validated for selected genes by qPCR and western blotting respectively. Furthermore, using TRANSBIG data, the expression of human orthologs of FGF-8-regulated genes was correlated to the Nottingham prognostic index and estrogen receptor status. The analysis revealed a number of significantly up- and down-regulated genes in response to FGF-8b at all treatment times. The most differentially expressed genes were genes related to cell cycle regulation, mitosis, cancer, and cell death. Several key regulators of early cell cycle progression such as Btg2 and cyclin D1, as well as regulators of mitosis, including cyclin B, Plk1, survivin, and aurora kinase A, were identified as novel targets for FGF-8b, some of which were additionally shown to correlate with prognosis and ER status in human breast cancer. The results suggest that in stimulation of proliferation FGF-8b not only promotes cell cycle progression through the G1 restriction point but also regulates key proteins involved in chromosomal segregation during mitosis and cytokinesis of breast cancer cells.
Assuntos
Neoplasias da Mama/genética , Ciclo Celular/genética , Fator 8 de Crescimento de Fibroblasto/genética , Fator 8 de Crescimento de Fibroblasto/metabolismo , Neoplasias Mamárias Animais/genética , Mitose/genética , Animais , Apoptose/genética , Aurora Quinase A , Aurora Quinases , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proliferação de Células , Ciclina B/genética , Ciclina B/metabolismo , Ciclina D1/genética , Ciclina D1/metabolismo , Feminino , Fator 8 de Crescimento de Fibroblasto/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Proteínas Inibidoras de Apoptose/genética , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias Mamárias Animais/metabolismo , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno , Receptores de Estrogênio/biossíntese , Transdução de Sinais , Survivina , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Quinase 1 Polo-LikeRESUMO
The Ikaros family transcription factor Aiolos is important for B cell function, since B cells of Aiolos-null mutant mice exhibit an activated phenotype, enhanced B-cell receptor (BCR) signalling response and develop a systemic lupus erythematosus (SLE) type autoimmune disease. Aiolos has also been reported to interact with anti-apoptotic Bcl-2 and Bcl-x(L) in T cells, but whether Aiolos regulates cell death has not been studied in B cells. Here we show that the disruption of Aiolos in the DT40 B cell line induces a cell death sensitive phenotype, as the Aiolos(-/-) cells are more prone to apoptosis by nutritional stress, BCR cross-linking, UV- or gamma-irradiation. Furthermore, the Aiolos(-/-) cells have defective Ig gene conversion providing evidence that Aiolos is needed for the somatic diversification of the BCR repertoire. The re-expression of DNA-binding isoform Aio-1 was able to restore the gene conversion defect of the Aiolos-deficient cells, whereas the introduction of dominant negative isofom Aio-2 had no effect on gene conversion, thus demonstrating the functional importance of alternative splicing within Ikaros family. Although the Aiolos(-/-) cells exhibit reduced expression of activation-induced cytidine deaminase (AID), ectopic AID overexpression did not restore the gene conversion defect in the Aiolos(-/-) cells. Our findings indicate that Aiolos may regulate gene conversion in an AID independent manner.
