May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
ABCG2 Confers Drug Resistance in Corneal Epithelial Cells in vitro
Author Affiliations & Notes
  • M. Kubota
    Ophthalmology, Keio University School of Medicine, Shinjuku-ku, Japan
  • M. Kawashima
    Ophthalmology, Keio University School of Medicine, Shinjuku-ku, Japan
  • H. Miyashita
    Ophthalmology, Keio University School of Medicine, Shinjuku-ku, Japan
  • K. Tsubota
    Ophthalmology, Keio University School of Medicine, Shinjuku-ku, Japan
    Ophthalmology, Tokyo Dental College, Ichikawa, Japan
  • S. Shimmura
    Ophthalmology, Keio University School of Medicine, Shinjuku-ku, Japan
    Ophthalmology, Tokyo Dental College, Ichikawa, Japan
  • Department of Ophthalmology, Tokyo Dental College
    Ophthalmology, Keio University School of Medicine, Shinjuku-ku, Japan
  • Footnotes
    Commercial Relationships  M. Kubota, None; M. Kawashima, None; H. Miyashita, None; K. Tsubota, None; S. Shimmura, None.
  • Footnotes
    Support  Grant-in-aid to Keio University from the Global Center of Excellence (COE) program
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 4523. doi:https://doi.org/
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      M. Kubota, M. Kawashima, H. Miyashita, K. Tsubota, S. Shimmura, Department of Ophthalmology, Tokyo Dental College; ABCG2 Confers Drug Resistance in Corneal Epithelial Cells in vitro. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4523. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : ABCG2 is a member of the ATP-binding cassette (ABC) transporter family, and is involved in the induction of drug resistance in tumor cells. ABCG2 is also expressed in various stem/progenitor cells, including the corneal epithelium. We used cultured corneal epithelial cells to investigate whether ABCG2 expressing cells were resistant to the antineoplastic agent mitoxantrone.

Methods: : SV40-immortalized human corneal epithelial cells (HCEC) cultured in SHEM medium were analyzed by flow cytometry to determine the presence of side population (SP) cells based on the ability of ABCG2 to efflux Hoechst 33342 dye. Expression of ABCG2 was also examined by RT-PCR. A cytotoxicity assay was performed in semi-confluent HCEC cultures treated with mitoxantrone (0 to 1 µM). Cell viability was analyzed by calcein AM/PI double staining using a fluorescent microscope. The effect of reserpine (15µM), an inhibitor of ABCG2, was also observed in mitoxantrone-treated HCEC.

Results: : Flow cytometry revealed that HCEC contained a high ratio of SP cells with Hoechst 33342 effluxing ability. The presence of ABCG2 was also confirmed by RT-PCR. HCECs were resistant to mitoxantrone (>90% viability) up to a dose of 10 nM, however, higher doses were toxic in a dose-dependent manner. Co-culture with reserpine inhibited resistance to mitoxantrone at doses as low as 0.01 nM, with a statistically higher cell death ratio at concentrations of 0.1 nM or higher. Reserpine alone did not show cell toxicity at the concentration used.

Conclusions: : ABCG2 induced drug resistance in cultured corneal epithelial cells, and may play a role in the protection of progenitor cells against pharmacological stress.

Keywords: cornea: epithelium • apoptosis/cell death • drug toxicity/drug effects 
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