SummaryA 12 kDa cysteine-rich protein is secreted by Fusarium oxysporum f. sp. lycopersici during colonization of tomato xylem vessels. Peptide sequences obtained with mass spectrometry allowed identification of the coding sequence. The gene encodes a 32 kDa protein, designated Six1 for secreted in xylem 1. The central part of Six1 corresponds to the 12 kDa protein found in xylem sap of infected plants. A mutant that had gained virulence on a tomato line with the I-3 resistance gene was found to have lost the SIX1 gene along with neighbouring sequences. Transformation of this mutant with SIX1 restored avirulence on the I-3 line. Conversely, deletion of the SIX1 gene in a wildtype strain results in breaking of I-3 -mediated resistance. These results suggest that I-3 -mediated resistance is based on recognition of Six1 secreted in xylem vessels.

Multidrug-resistance-associated protein [6][7][8] and is able to decrease cellular drug levels against a concentration gradient (4, 6). However, recent work has also indicated interesting differences between MRP and Pgp. Increased cellular MRP levels are associated with increased reduced glutathione (GSH) S-conjugate carrier (GS-X pump) activity in isolated plasma membrane vesicles (9-11). This suggests that MRP is a GS-X pump (12) present in many, if not all, mammalian cells (9-13). These pumps transport substrates containing a large hydrophobic moiety and at least two negative charges (12, 13), as present in drug GSH Sconjugates. Moreover, recent studies indicate that GS-X pumps are also involved in the export of cisplatin (14-16) and arsenite (11). Indeed, some cell lines overexpressing MRP are moderately resistant to arsenite (4, 11). These results link MRP to older experiments in which resistance to anthracyclines was found to correlate with increased levels of cellular GSH, GSH synthesis, or GSH Stransferases (17)(18)(19). This link is supported by the strong decrease in drug resistance in two MDR lung carcinoma cell lines that overexpress MRP by DL-buthionine (S,R)-sulfoximine (BSO) (20)(21)(22), an inhibitor of y-glutamylcysteine synthetase, the enzyme that catalyzes the first step in GSH synthesis (23). The interpretation of these inhibitor experiments is not unambiguous, however. In both cell lines, other resistance mechanisms (e.g., alterations in topoisomerase II) contribute to resistance, and it is not clear whether the GSH depletion in these cells does not result in membrane damagee.g., by lipid peroxidation. Damage of the plasma membrane could increase drug influx and, hence, decrease resistance. To test whether GSH is specifically required for MDR caused by MRP but not by Pgp, we have analyzed the effects of BSO treatment on lung cancer cells transfected with an expression vector containing either MRP cDNA or MDR1 cDNA. MATERIALS AND METHODSCell Lines. S1(MRP) was obtained after transfection of non-small cell lung cancer SW-1573/S1 cells with an expression vector containing MRP cDNA and a neomycin-resistance marker gene (pRc/RSV-MRP), followed by selection with geneticin (G418) (6). Sl(MDR1) was previously named S1(1.1) (24) and was obtained after transfection of S1 cells with the expression vector pJ3fl (25) containing MDR1 cDNA, followed by selection with 10 nM vincristine. GLC4/ADR is a MRP-overexpressing subline of the non-small cell lung cancer cell line GLC4 and was obtained by selection with doxorubicin (20,21).Clonogenic Survival Assay. In six-well dishes, 400 cells per well were seeded and incubated in medium with or without 25 ,uM BSO for 24 hr prior to incubation with increasing concentrations of drug. After 1 hr, drug was removed, the wells were rinsed with phosphate-buffered saline, and drug-free medium without BSO was added. Seven days after the start of the experiment, the percentage of cells that were able to produce a colony of >50 cells was used as a measure of cell sur...

Purpose Resistance to antiangiogenic tyrosine kinase inhibitors such as sunitinib is an important clinical problem, but its underlying mechanisms are largely unknown. We analyzed tumor sunitinib levels in mice and patients and studied sensitivity and resistance mechanisms to sunitinib. Experimental Design Intratumoral and plasma sunitinib concentrations in mice and patients were determined. Sunitinib exposure on tumor cell proliferation was examined. Resistant tumor cells were derived by continuous exposure and studied for alterations in intracellular sunitinib accumulation and activity. Results Intratumoral concentrations of sunitinib in mice and patients were 10.9 ± 0.5 and 9.5 ± 2.4 μmol/L, respectively, whereas plasma concentrations were 10-fold lower, 1.0 ± 0.1 and 0.3 ± 0.1 μmol/L, respectively. Sunitinib inhibited tumor cell growth at clinically relevant concentrations in vitro, with IC50 values of 1.4 to 2.3 μmol/L. Continuous exposure to sunitinib resulted in resistance of 786-O renal and HT-29 colon cancer cells. Fluorescent microscopy revealed intracellular sunitinib distribution to acidic lysosomes, which were significantly higher expressed in resistant cells. A 1.7- to 2.5-fold higher sunitinib concentration in resistant cells was measured because of increased lysosomal sequestration. Despite the higher intracellular sunitinib accumulation, levels of the key signaling p-Akt and p-ERK 1/2 were unaffected and comparable with untreated parental cells, indicating reduced effectiveness of sunitinib. Conclusion We report that sunitinib inhibits tumor cell proliferation at clinically relevant concentrations and found lysosomal sequestration to be a novel mechanism of sunitinib resistance. This finding warrants clinical evaluation whether targeting lysosomal function will overcome sunitinib resistance.