While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.

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Inhibition of dThdPase in human colorectal carcinomas might improve prognosis for some patients.

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MRP1 is a 190-kDa membrane glycoprotein that confers multidrug resistance (MDR) to tumor cells. MRP1 is characterized by an N-terminal transmembrane domain (TMD 0 ), which is connected to a P-glycoprotein-like core region (⌬MRP) by a cytoplasmic linker domain zero (L 0 ).It has been demonstrated that GSH plays an important role in MRP1-mediated MDR. However, the mechanism by which GSH mediates MDR and the precise roles of TMD 0 and L 0 are not known. We synthesized [125 I]11-azidophenyl agosterol A ([ 125 I]azidoAG-A), a photoaffinity analog of the MDR-reversing agent, agosterol A (AG-A), to photolabel MRP1, and found that the analog photolabeled the C-proximal molecule of MRP1 (C 932-1531) in a manner that was GSH-dependent. The photolabeling was inhibited by anticancer agents, reversing agents and leukotriene C 4 . Based on photolabeling studies in the presence and absence of GSH using membrane vesicles expressing various truncated, co-expressed, and mutated MRP1s, we found that L 0 is the site on MRP1 that interacts with GSH. This study demonstrated that GSH is required for the binding of an unconjugated agent to MRP1 and suggested that GSH interacts with L 0 of MRP1. The photoanalog of AG-A will be useful for identifying the drug binding site within MRP1, and the role of GSH in transporting substrates by MRP1.Following exposure to a natural product chemotherapeutic agent, tumor cells often acquire resistance to several structurally and functionally unrelated drugs, so-called multidrug resistance (MDR).1 MDR is the major obstacle to successful cancer chemotherapy. Two membrane proteins, P-glycoprotein (Pgp) and the human multidrug resistance protein (MRP1) are frequently overexpressed in MDR cells (for reviews see Refs. 1-4). Although both MRP1 (190 kDa) and P-gp (170 kDa) are members of the family of ATP-binding cassette transporters (5), they share only 15% amino acid sequence identity (6). The amino acid sequence suggests that P-gp consists of two homologous halves and a variable linker region. Each half of the protein has six transmembrane segments and one nucleotide binding domain (NBD). MRP1 differs from P-gp by the presence of an extra N-terminal extension with five transmembrane segments (TMD 0 ), which is connected to the P-gp-like core (⌬MRP) by a cytoplasmic linker domain zero (L 0 ) (7, 8).The precise roles of TMD 0 and L 0 are unknown. Although MRP1 and P-gp both confer multidrug resistance by actively effluxing drugs from the cells (9, 10), there is compelling evidence that they function very differently in drug transport (6, 11). P-gp confers multidrug resistance by directly binding and transporting unmodified drugs (12, 13). MRP1, however, is an active transporter of amphiphilic conjugated organic anions, including a number of compounds conjugated with GSH, glucuronide, and sulfate. To date, leukotriene C 4 (LTC 4 ) is the best substrate for MRP1 (14). It has been reported that GSH plays a critical role in MRP1-mediated MDR (15). Reduction of GSH level in MRP1-expressing cells by buthionine su...