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Identification and functional characterization of breast cancer resistance protein in human bronchial epithelial cells (Calu-3).

ABSTRACT: Breast cancer resistance protein (BCRP), a 72 kDa protein belongs to the subfamily G of the human ATP-binding cassette transporter superfamily. Overexpression of BCRP was found to play a major role in the development of resistance against various chemotherapeutic agents. BCRP plays an important role in absorption, distribution and elimination of several therapeutic agents. BCRP expression and functional activity across human bronchial epithelium and its impact on pulmonary drug accumulation has not been established. The objective of this study was to identify and characterize the BCRP efflux transporter across human bronchial epithelium. Calu-3, a human bronchial epithelial cell line was employed as a model for this study. Reverse transcription-polymerase chain reaction (RT-PCR), Western blot and immunocytochemical studies were performed to identify and characterize the expression of BCRP. RT-PCR studies detected ABCG2 mRNA levels in Calu-3 cells. A strong band for BCRP with a molecular weight of approximately 72 kDa was observed in Western blot analysis. Immunocytochemical studies confirmed the presence of BCRP on the apical membrane of human bronchial epithelium. Functional activity of BCRP was determined by performing uptake of radioactive substrate [3H]-mitoxantrone in the presence and absence of BCRP inhibitors. Uptake of [3H]-mitoxantrone was elevated significantly in the presence of GF120918 and fumitremorgin C. An increase in the accumulation of Hoechst 33342, a fluorescent dye was also detected in the presence of BCRP inhibitors when compared to control. In summary, this study provides evidence for the presence of an ATP dependent, membrane bound efflux transporter BCRP across human bronchial epithelial cell line, Calu-3.

SUBMITTER: Paturi DK

PROVIDER: S-EPMC2830792 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Identification and functional characterization of breast cancer resistance protein in human bronchial epithelial cells (Calu-3).

Paturi Durga Kalyani DK Kwatra Deep D Ananthula Hari Krishna HK Pal Dhananjay D Mitra Ashim K AK

International journal of pharmaceutics 20090925 1-2

Breast cancer resistance protein (BCRP), a 72 kDa protein belongs to the subfamily G of the human ATP-binding cassette transporter superfamily. Overexpression of BCRP was found to play a major role in the development of resistance against various chemotherapeutic agents. BCRP plays an important role in absorption, distribution and elimination of several therapeutic agents. BCRP expression and functional activity across human bronchial epithelium and its impact on pulmonary drug accumulation has ...[more]

PMID: 19782742

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Project description:Aspergillus fumigatus (A. fumigatus) is a ubiquitous fungus which reproduces asexually by releasing abundant airborne conidia (spores), which are easily respirable. In allergic and immunocompromised individuals A. fumigatus can cause a wide spectrum of diseases, including allergic bronchopulmonary aspergillosis, aspergilloma and invasive aspergillosis. Previous studies have demonstrated that A. fumigatus conidia are internalized by macrophages and lung epithelial cells; however the exact transcriptional responses of airway epithelial cells to conidia are currently unknown. Thus, the aim of this study was to determine the transcriptomic response of the human bronchial epithelial cell line (16HBE14o-) following interaction with A. fumigatus conidia. We used fluorescence-activated cell sorting (FACS) to separate 16HBE14o- cells having bound and/or internalized A. fumigatus conidia expressing green fluorescent protein from cells without spores. Total RNA was then isolated and the transcriptome of 16HBE14o- cells was evaluated using Agilent Whole Human Genome microarrays.Immunofluorescent staining and nystatin protection assays demonstrated that 16HBE14o- cells internalized 30-50% of bound conidia within six hrs of co-incubation. After FAC-sorting of the same cell culture to separate cells associated with conidia from those without conidia, genome-wide analysis revealed a set of 889 genes showing differential expression in cells with conidia. Specifically, these 16HBE14o- cells had increased levels of transcripts from genes associated with repair and inflammatory processes (e.g., matrix metalloproteinases, chemokines, and glutathione S-transferase). In addition, the differentially expressed genes were significantly enriched for Gene Ontology terms including: chromatin assembly, G-protein-coupled receptor binding, chemokine activity, and glutathione metabolic process (up-regulated); cell cycle phase, mitosis, and intracellular organelle (down-regulated).We demonstrate a methodology using FACs for analyzing the transcriptome of infected and uninfected cells from the same cell population that will provide a framework for future characterization of the specific interactions between pathogens such as A. fumigatus with human cells derived from individuals with or without underlying disease susceptibility.

Dataset Information

Identification and functional characterization of breast cancer resistance protein in human bronchial epithelial cells (Calu-3).

Publications

Identification and functional characterization of breast cancer resistance protein in human bronchial epithelial cells (Calu-3).

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