The experiments reported herein were designed to test
The experiments reported herein were designed to test whether co-expression of MRP and GSTP1-1 offers a level of protection from certain cytotoxic drugs not achieved by the expression of either protein alone. This was accomplished by stable transfection with expression vectors into MCF7/WT cells, which express negligible MRP and no GSTP1-1, and into MCF7/VP cells, MDR variants that express high level MRP but no GSTP1-1 . We examined the cytotoxicities of several drugs towards parental (MCF7/WT and MCF7/VP) and GSTP1-1-transfected derivative MCF7 cells. The drugs were chosen on the basis of their ability to form glutathione conjugates either spontaneously or in GST-catalyzed reactions. They included ethacrynic acid, three oxazaphosphorines (maf, 4-OH-CP, and 4-OOH-CP), and cisplatin. The MCF7 clone pairs were matched for similar levels of GSTP1-1 expression. Ethacrynic figure 1 and the oxazaphosphorines were chosen because either the parent compound (ethacrynic acid) or some of the metabolites (oxazaphosphorines) are known to be substrates of GSTP1-1 , , , . Cisplatin was chosen because it can form glutathione conjugates that are transported by membrane efflux pumps related to MRP , , , . While GSTP1-1 has not been shown to be directly involved in metabolic transformation of cisplatin, results from some laboratories have implicated increased GSTP1-1 in the development of cisplatin resistance , .
Materials and method
Discussion The studies presented in this report tested the postulate that combined expression of GSTP1-1 and MRP can confer cellular resistance to some cytotoxic compounds, including anticancer agents, that is greater than the cellular resistance conferred by either protein alone. This hypothesis is based upon observations that GSTs can catalyze the conjugation with glutathione of some of these toxins and that MRP-containing membrane vesicles can support GS-X transport 3, 15, 16, 17, 18, 19. The studies herein provide insights that could not be drawn from prior in vitro, cell-free studies alone. Indeed, as has been shown by previous transfection studies, the demonstration that a cytotoxic compound is a potential substrate of a GST isozyme in vitro does not necessarily mean that the GST isozyme will confer resistance to the cytotoxin in intact cells 7, 8, 9. Furthermore, the finding that a particular glutathione-toxin conjugate is an in vitro substrate of MRP-associated transport using membrane vesicles is not proof that MRP can confer cellular resistance to this toxin. For example, intracellular conditions may influence substrate availability or modulate enzyme kinetic parameters. In intact cells, the efficiency of glutathione–xenobiotic conjugate formation may be insufficient to influence cytotoxicity significantly. Alternatively, for some compounds, GST-dependent rate enhancement of conjugate formation may be relatively low. Here, non-enzymatic glutathione conjugation may predominate in cells, rendering GST levels irrelevant to detoxification. Finally, the activity of the MRP transporter may differ considerably between isolated membrane preparations and intact cells for a variety of reasons, including altered co-factor levels, alternative pathways of glutathione-conjugate processing and metabolism, or other factors that may be difficult to anticipate in cell-free studies alone. Indeed, we have shown that the apparent K of MRP towards S-(2,4-dinitrophenyl)-glutathione in intact MCF7/VP cells was considerably different from that determined using isolated membrane vesicles (Diah S and Morrow C, unpublished results). Moreover, if the toxin–glutathione conjugate is both stable and non-toxic, MRP-mediated efflux may offer no additional protection from cytotoxicity than that conferred by increased conjugation alone (e.g. by increased GST levels). Hence, the use of intact cells with defined alterations in the levels of MRP and GSTP1-1 provides both novel and complementary information to in vitro biochemical studies.