RESUMEN
Understanding the mechanisms of multidrug resistance (MDR) could improve clinical drug efficacy. Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood. We report that a single-step selection with low doses of anti-cancer agents, similar to concentrations reported in vivo, induces MDR that is mediated exclusively by ABCG2. We selected breast, ovarian and colon cancer cells (MCF-7, IGROV-1 and S-1) after exposure to 14 or 21 nM doxorubicin for only 10 days. We found that these cells overexpress ABCG2 at the mRNA and protein levels. RNA interference analysis confirmed that ABCG2 confers drug resistance. Furthermore, ABCG2 upregulation was facilitated by histone hyperacetylation due to weaker histone deacetylase 1-promoter association, indicating that these epigenetic changes elicit changes in ABCG2 gene expression. These studies indicate that the MDR phenotype arises following low-dose, single-step exposure to doxorubicin, and further suggest that ABCG2 may mediate early stages of MDR development. This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Antibióticos Antineoplásicos/farmacología , Doxorrubicina/farmacología , Epigénesis Genética , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2 , Transportadoras de Casetes de Unión a ATP/genética , Acetilación , Antineoplásicos/farmacología , Western Blotting , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/metabolismo , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Femenino , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Humanos , Mitoxantrona/farmacología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/genética , Neoplasias/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , ARN Mensajero/aislamiento & purificación , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
The effects of human papillomavirus type 18 (HPV-18) E6 and E7 proteins on global patterns of host gene expression in primary human keratinocytes grown in organotypic raft culture system were assessed. Primary human keratinocytes were infected with retroviruses that express the wild-type HPV-18 E6 and E7 genes from the native differentiation-dependent HPV enhancer-promoter. Total RNA was isolated from raft cultures and used to generate probes for querying Affymetrix U95A microarrays, which contain >12,500 human gene sequences. Quadruplicate arrays of each E6/E7-transduced and empty vector-transduced samples were analyzed by 16 pairwise comparisons. Transcripts altered in > or =12 comparisons were selected for further analysis. With this approach, HPV-18 E6/E7 expression significantly altered the expression of 1,381 genes. A large increase in transcripts associated with DNA and RNA metabolism was observed, with major increases noted for transcription factors, splicing factors, and DNA replication elements, among others. Multiple genes associated with protein translation were downregulated. In addition, major alterations were found in transcripts associated with the cell cycle and cell differentiation. Our study provides a systematic description of transcript changes brought about by HPV-18 E6/E7 in a physiologically relevant model and should furnish a solid source of information to guide future studies.