Expression of multidrug transporter MRP4/ABCC4 is a marker of poor prognosis in neuroblastoma and confers resistance to irinotecan in vitro.

Members of the multidrug resistance-associated protein (MRP) family of transporters are believed to contribute to cytotoxic drug resistance and chemotherapy failure. We observed frequent MRP4 overexpression in aggressive primary neuroblastoma, a disease for which we have previously shown MRP1 to be a prognostic indicator. High MRP4 expression correlated with MYCN oncogene amplification and was significantly associated with poor clinical outcome. Although MRP4 is known to transport some nucleoside analogues, it has not previously been associated with resistance to drugs used to treat solid tumors. We now show that it mediates substantial resistance in vitro to the topoisomerase I poison irinotecan/CPT-11 and its active metabolite SN-38. These results suggest that MRP4 will be a useful prognostic marker for neuroblastoma and that clinical trials of irinotecan as a neuroblastoma treatment should monitor MRP4 expression. The same may be true for other tumor types expressing high levels of the transporter.

Mechanism of selectivity of an angiogenesis inhibitor from screening a genome-wide set of Saccharomyces cerevisiae deletion strains.

BACKGROUND: The synthetic tripeptide arsenical 4-(N-(S-glutathionylacetyl)amino) phenylarsenoxide (GSAO) is an angiogenesis inhibitor that targets the mitochondria of actively dividing but not quiescent endothelial cells, arresting their proliferation and causing apoptosis. Normal endothelial cells are much more sensitive to GSAO than tumor cells. To elucidate the mechanism of tumor cell resistance, we identified yeast genes that are necessary for resistance to GSAO. METHODS: We screened a genome-wide set of 4546 Saccharomyces cerevisiae deletion strains to identify GSAO-sensitive strains. We then examined GSAO accumulation in and proliferation activity of endothelial cells (BAECs) and tumor cells treated with GSAO and modulators of pathways and proteins identified in the yeast screen. We also examined GSAO effects on proliferation of mammalian cells transfected with transporter protein constructs. RESULTS: Eighty-eight deletion strains were sensitive to GSAO. The most sensitive strains had deletions of genes whose products are involved in vacuolar function (corresponding to drug transport in mammalian cells) and glutathione synthesis. BAECs were more sensitive to GSAO than tumor cells, and cell sensitivity to GSAO was approximately proportional to cellular glutathione levels. Treatment of BAECs and tumor cells with MK-571, an inhibitor of multidrug resistance-associated protein (MRP), or with buthionine sulfoximine, an inhibitor of glutathione synthesis, increased their sensitivity to GSAO. Mammalian cells transfected with MRP1 or MRP2 were resistant to GSAO, whereas cells transfected with MRP3, MRP4, MRP5, P-glypoprotein, or breast cancer resistance protein were not. CONCLUSIONS: Differences in MRP activity and cellular glutathione levels contribute to the selectivity of GSAO for endothelial versus tumor cells. MRP1 and/or MRP2 may transport GSAO from resistant cells, with glutathione acting as a cotransporter. Genetic screening in yeast is a powerful tool for understanding drug action in mammalian cells.

Modification of in vivo and in vitro T- and B-cell-mediated immune responses by the Pseudomonas aeruginosa quorum-sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone.

N-3-(oxododecanoyl)-L-homoserine lactone (OdDHL), a quorum-sensing molecule of Pseudomonas aeruginosa, plays an important role in the pathogenesis of the organism through its control of virulence factor expression. Several reports have suggested that OdDHL can also directly modulate host immune responses. However, the nature of the modulation is controversial, with different reports suggesting promotion of either humoral (Th2-mediated) or inflammatory (Th1-mediated) responses. This report describes a series of studies which demonstrate for the first time that in vivo administration of OdDHL can modulate the course of an antibody response, with an increase in ovalbumin (OVA)-specific immunogloblulin G1 (IgG1) but not IgG2a in OdDHL-treated OVA-immunized BALB/c mice compared to levels for controls. In vitro stimulation of lymphocytes from both Th1-biased C57Bl/6 and T-cell receptor transgenic mice and Th2-biased BALB/c mice in the presence of OdDHL demonstrated that OdDHL inhibits in vitro cytokine production in response to both mitogen and antigen, with gamma interferon (IFN-gamma) tending to be more inhibited than interleukin-4 (IL-4). In vitro mitogen or antigen restimulation of cells from mice treated with OdDHL in vivo shows effects on cytokine production which depend on the underlying immune bias of the mouse strain used, with a relative increase of IFN-gamma in Th1-biased C57Bl/6 mice and a relative increase of IL-4 in Th2-biased BALB/c mice. Thus, the mode of action of OdDHL on T-cell cytokine production is likely to be a relatively nonspecific one which accentuates an underlying immune response bias rather than one which specifically targets either Th1 or Th2 responses.