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The human multidrug resistance 1 (MDR1) gene encoding P-glycoprotein is often overexpressed in various human tumors after chemotherapy. During treatment with various chemotherapeutic agents, the MDR1 gene is activated at the transcriptional level and/or amplified, resulting in overexpression. Our previous studies demonstrated that an inverted CCAAT box (Y-box) might be a critical cis-regulatory element regulating UV or drug-induced MDR1 gene expression. We have now established various cell lines from human head and neck cancer KB cells which were stably transfected with the chloramphenicol acetyltransferase (CAT) reporter gene driven by various MDR1 promoter deletion constructs. Transient transfection of antisense YB-1 expression constructs resulted in a decrease of both YB-1 protein levels and DNA binding activity to the inverted CCAAT box, as determined by Western blot and gel mobility shift assays. The limited expression and binding activity due to expression of antisense YB-1 constructs were also observed when cells were treated with UV. CAT activity of constructs containing the Y-box was enhanced after treatment with UV irradiation as well as genotoxic agents such as cisplatin and etoposide. Moreover, this activation was reduced by 50 -80% by transfection of antisense YB-1 expression constructs. In contrast, transfection of antisense YB-1 expression constructs had no effect on CAT activity driven by MDR1 promoter constructs not containing the Y-box. These data indicate that YB-1 is directly involved in MDR1 gene activation in response to genotoxic stress.The overexpression of P-glycoprotein (P-gp) 1 appears to be closely associated with multidrug resistance in human malignancies, suggesting that P-gp is a useful prognostic marker for assessing therapeutic efficacy (1, 2). The human multidrug resistance 1 (MDR1) gene encoding P-gp is highly susceptible to transcriptional activation and gene amplification during the selection of drug-resistant cell lines (3-5). The MDR1 gene is up-regulated in response to ultraviolet light (UV), anticancer agents, serum starvation, heat shock, phosphatase inhibitors, and phorbol ester in cultured human cancer cells (6 -15) and also in some clinical malignancies in vivo after cancer chemotherapy (16,17). Expression of MDR1 gene is enhanced in cultured human cancer cells and in vivo after transient exposure to both P-gp-and non-P-gp-targeted cytotoxic anticancer agents (7-10) and also in some clinical tumors after cancer chemotherapy (16,17).We previously identified the DNA binding protein, which recognized the cis-regulatory element, Y-box, on the MDR1 promoter (6,18,19). The Y-box binding family proteins are widely distributed from bacteria to mammals, and Y-boxes are located on the promoter of many genes such as the major histocompatibility complex class II gene, epidermal growth factor receptor, proliferating cell nuclear antigen, DNA polymerase ␣, thymidine kinase, and topoisomerase II␣ (20, 21). Furthermore, we found that the human Y-box binding protein (YB-1) gene spans ...

Multidrug resistance protein 1 (MRP1/ABCC1) is an ATP-binding cassette transporter that confers resistance to drugs and mediates the transport of organic anions. MRP1 has a core structure of two membrane spanning domains (MSDs) each followed by a nucleotide binding domain. This core structure is preceded by a third MSD with five transmembrane (TM) helices, whereas MSD2 and MSD3 each contain six TM helices. We investigated the consequences of Ala substitution of 18 Pro residues in both the non-membrane and TM regions of MSD2 and MSD3 on MRP1 expression and organic anion transport function. All MRP1-Pro mutants except P1113A were expressed in human embryonic kidney cells at levels comparable with wild-type MRP1. In addition, five mutants containing substitutions of Pro residues in or proximal to the TM helices of MSD2 (TM6-Pro 343 , TM8-Pro 448 , TM10-Pro 557 , and TM11-Pro 595 ) and MSD3 (TM14-Pro 1088 ) exhibited significantly reduced transport of five organic anion substrates. In contrast, mutation of Pro 1150 in the cytoplasmic loop (CL7) linking TM15 to TM16 caused a substantial increase in 17␤-estradiol-17-␤-(D-glucuronide) and methotrexate transport, whereas transport of other organic anions was reduced or unchanged. Significant substrate-specific changes in the ATP dependence of transport and binding by the P1150A mutant were also observed. Our findings demonstrate the importance of TM6, TM8, TM10, TM11, and TM14 in MRP1 transport function and suggest that CL7 may play a differential role in coupling the activity of the nucleotide binding domains to the translocation of different substrates across the membrane.The human MRP1 1 (gene symbol ABCC1) that was originally cloned from a multidrug-resistant small cell lung cancer cell line is an integral membrane protein that belongs to subfamily C of the ABC superfamily of transporter proteins (1-3). The human ABCC subfamily contains nine MRP-related proteins (MRP1-6/ABCC1-6 and MRP7-9/ABCC10 -12), the cystic fibrosis transmembrane conductance regulator (CFTR/ABCC7), and the sulfonylurea receptors SUR1 (ABCC8) and SUR2 (ABCC9) (4, 5). All 12 ABCC members have a core four domain structure consisting of two MSDs (each containing 6 TM helices) and two NBDs configured MSD-NBD1-MSD-NBD2. The core structures of MRP1-3, -6, and -7, as well as SUR1 and SUR2 are connected by a cytoplasmic loop, CL3 (or L o ) to a fifth domain, an NH 2 -terminal MSD consisting of 5 TM helices. This third MSD is the major structural feature that distinguishes these five domain, 17-TM helix proteins from CFTR, MRP4,3,4,6). The precise physiological functions of all ABCC proteins are not yet fully understood, although several are known to be important in certain genetic disorders. For example, mutations in MRP2 (ABCC2) are responsible for Dubin-Johnson syndrome, a form of congenital conjugated hyperbilirubinemia (7), and mutations in CFTR (ABCC7) cause cystic fibrosis (8).Increased expression of MRP1 has been detected in drugresistant tumor cell lines and tumor tissues, and MRP1 has been demo...

The human ATP-binding cassette (ABC) transporter, multidrug resistance protein 1 (MRP1/ABCC1), confers resistance to a broad range of anti-cancer agents and transports a variety of organic anions. At present, essentially no structural data exists for MRP1 that might be used to elucidate its mechanism of transport. Consequently, we have applied a modeling strategy incorporating crystal and indirect structural data from other ABC transporters to construct a model of the transmembrane domains of the core region of MRP1 that includes the amino acid side chains. Three conserved Trp residues and one non-conserved Tyr residue, shown previously to be of functional importance (Koike, K., Oleschuk, C. J., Haimeur, A., Olsen, S. L., Deeley, R. G., and Cole, S. P. C. ) was identified that, with the Trp and Tyr residues, completed a ring or "basket" of aromatic amino acids and, accordingly, we postulated that it would also be of functional importance. To test this idea, MRP1-Phe 594 mutants were expressed in human embryonic kidney cells, and their properties were examined using membrane vesicles. Substitution of Phe 594 with Ala substantially reduced or eliminated the transport of five organic anion substrates by MRP1 and abrogated the binding of leukotriene C 4 . On the other hand, the conservatively substituted F594W and F594Y mutants remained transport competent, although significant substrate-and substitution-specific changes were observed. These studies provide some structural insight into a possible substrate binding/transport site of MRP1 at the beginning of a putative substrate translocation pathway and demonstrate the usefulness of modeling for directing structure-function analyses of this transporter.Chemotherapeutic approaches in cancer treatment are often hindered by the fact that tumor cells can become simultaneously resistant to a number of different anti-cancer agents. The multidrug resistance phenotype can often be attributed to the increased expression of one or more membrane proteins belonging to the ATP-binding cassette (ABC) 1 transporter superfamily, which can actively extrude anti-cancer drugs from the cell. Two of the best characterized examples of these transporters are the 170-kDa P-glycoprotein and the 190-kDa MRP1, encoded by the human ABCB1 and ABCC1 genes, respectively (1, 2). Despite its shared ability with P-glycoprotein to confer resistance to a broad spectrum of anticancer agents, MRP1 is distinct from P-glycoprotein in that it can also transport organic anions, many of which are conjugated to glutathione, glucuronate, or sulfate (1).The 1531-amino acid human MRP1 contains two NBDs and up to 17 TM helices distributed within three TMDs. This contrasts with P-glycoprotein, which contains just 12 TM helices in two TMDs. However, it has been shown that an NH 2 -terminally truncated "core" form of MRP1 in which the extra fivehelix TMD has been removed retains its ability to transport organic anions (3).To understand the MRP1 transport mechanism, high-resolution structures of the protein at each ph...

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