Differential sensitivities of MRP1-overexpressing lung tumor cells to cytotoxic metals
Introduction
The multidrug resistance-associated protein (MRP1) is a 190 Kd membrane protein which belongs to the ATP-binding cassette (ABC) transporter superfamily (Loe et al., 1996a). MRP1 is thought to act as an efflux pump mediating transport of several anionic compounds but also of some cationic drugs which are usually conjugated prior to MRP1-dependent transport or effluxed with glutathione by a co-transport mechanism (Rappa et al., 1997, Priebe et al., 1998). MRP1 is expressed in various normal tissues including lung, intestine and heart, and is thought to participate to some physiological cellular functions (Flens et al., 1996). Indeed, it ensures the transport of endogenous compounds such as leukotriene C4, reduced glutathione, estradiol and bile conjugates (Loe et al., 1996a). MRP1 was initially demonstrated to be overexpressed in tumor cells selected with anticancer drugs (Cole et al., 1992). In such cells, MRP1 confers resistance to multiple classes of chemotherapeutic agents, including anthracyclins, epipodophyllotoxins and some Vinca alkaloids. Increased levels of MRP1 have also been detected in human tumor samples and may be associated with poor responses to chemotherapeutic treatments (Loe et al., 1996a).
In addition to xenobiotics, the heavy metal antimony has been demonstrated to be transported by MRP1. Indeed, we and others have found that MRP1-overexpressing cells poorly accumulated this heavy metal because of enhanced cellular efflux (Chen et al., 1997, Vernhet et al., 1999). Moreover, MRP1-overexpressing cells displayed resistance to antimony which was reversed in the presence of MK571, a known inhibitor of MRP1 function; overexpression of MRP1 thereby appears a way for cells to escape the cytotoxic effects of antimony. In addition, some MRP1-overexpressing cells have been shown to be cross-resistant to arsenical salts, thus suggesting that arsenic may be also a substrate for MRP1, although direct evidence for cellular efflux of this heavy metal in MRP1-positive cells is still lacking (Cole et al., 1994, Chen et al., 1997). Whether other cytotoxic metals such as chromium, aluminium, cobalt and mercury may interact with MRP1 also remains to be determined. The present study was therefore designed to carefully discriminate among various cytotoxic metals, those handled or not by MRP1, using mainly metal toxicity and transport assays in MRP1-overexpressing cells.
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Materials
Cadmium chloride, cobalt nitrate and lanthanum chloride were purchased from Merck (Darmstadt, Germany). Mercuric chloride, aluminium chloride, copper sulphate and potassium chromate were from Prolabo (Paris, France). Calcein acetoxymethyl ester (calcein AM) was obtained from Molecular probes (Eugene, OR, USA) and MK571, a leukotriene D4 receptor antagonist (Jones et al., 1989) which is known to inhibit MRP1 activity (Gekeler et al., 1995) was kindly provided by Dr Ford-Hutchinson (Merck-Frosst,
Cross-resistance to metals of GLC4/Sb30 cells
MRP1-overexpressing GLC4/Sb30 cells, generated by a step-wise selection in antimony, have been previously demonstrated to be 27-fold resistant to antimonial salts (Vernhet et al., 1999). In order to analyse the pattern of cross-resistance to other metals, we first determined sensitivity of GLC4/Sb30 cells to cytotoxic effects of various metal salts using the MTT assay. Table 1 indicates that GLC4/Sb30 cells were cross-resistant to arsenical salts and to mercuric chloride. Interestingly, we
Discussion
We and others have previously demonstrated that antimony is a substrate for the ABC transporter MRP1 in various MRP1-overexpressing cell lines (Cole et al., 1994, Chen et al., 1997, Vernhet et al., 1999). In this study, MRP1-overexpressing antimony-selected GLC4/Sb30 cells were first demonstrated to be highly cross-resistant to arsenical salts and such a resistance was found to be partially reversed by the MRP1 inhibitor MK571. Moreover, we showed that resistance to arsenate was mainly due to a
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