Received for publication October 14, 2004.
Revised January 14, 2005.
Accepted for publication January 18, 2005.

Cytoplasmic Confinement of Breast Cancer Resistance Protein (BCRP/ABCG2) as a Novel Mechanism of Adaptation to Short-Term Folate Deprivation

Abstract

The unique capability of breast cancer resistance protein (BCRP/ABCG2) to export mono-, di- and triglutamates of folates should limit cellular proliferation under conditions of folate deprivation, particularly upon BCRP overexpression. Here we explored the mode of adaptation of BCRP-overexpressing cells to short-term folate deprivation. MCF-7/MR cells grown in high folate medium (2.3 µM folic acid) containing mitoxantrone had 70% of their overexpressed BCRP in the plasma membrane and only 30% in the cytoplasm. In contrast, cells grown for two weeks in folic acid-free medium followed by an adaptation week in low folate medium (1 nM folic acid) had 86% of BCRP in the cytoplasm and only 14% in the plasma membrane. Unlike BCRP, various transmembrane proteins retained their normal plasma membrane localization in folate-deprived cells. Folate deprivation was also associated with a 3-fold decrease in BCRP and multidrug resistance protein 1 (MRP1/ABCC1) levels. Confocal microscopy with folate-deprived cells revealed that cytoplasmic BCRP co-localized with calnexin, an established endoplasmic reticulum resident. Consistently, the loss of BCRP from the plasma membrane in folate-deprived cells resulted in a 4.5-fold increase in [³H]folic acid accumulation relative to MCF7/MR cells. Hence, cellular adaptation to short-term folate deprivation results in a selective confinement of BCRP to the cytoplasm along with a moderate decrease in BCRP and MRP1 levels aimed at preserving the poor intracellular folate pools. These results constitute a novel mechanism of cellular adaptation to short-term folate deprivation and provide further support to the possible role of BCRP in the maintenance of cellular folate homeostasis.

Key words: MDR/p-Glycoprotein, Fluorescence techniques, Immunocytochemistry, Resistance, Membrane targets