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Heterozygous germline mutations in breast cancer 1 (BRCA1) strongly predispose women to breast cancer. BRCA1 plays an important role in DNA double-strand break (DSB) repair via homologous recombination (HR), which is important for tumor suppression. Although BRCA1-deficient cells are highly sensitive to treatment with DSB-inducing agents through their HR deficiency (HRD), BRCA1-associated tumors display heterogeneous responses to platinum drugs and poly(ADP-ribose) polymerase (PARP) inhibitors in clinical trials. It is unclear whether all pathogenic BRCA1 mutations have similar effects on the response to therapy. Here, we have investigated mammary tumorigenesis and therapy sensitivity in mice carrying the Brca1185stop and Brca15382stop alleles, which respectively mimic the 2 most common BRCA1 founder mutations, BRCA1185delAG and BRCA15382insC. Both the Brca1185stop and Brca15382stop mutations predisposed animals to mammary tumors, but Brca1185stop tumors responded markedly worse to HRD-targeted therapy than did Brca15382stop tumors. Mice expressing Brca1185stop mutations also developed therapy resistance more rapidly than did mice expressing Brca15382stop. We determined that both murine Brca1185stop tumors and human BRCA1185delAG breast cancer cells expressed a really interesting new gene domain-less (RING-less) BRCA1 protein that mediated resistance to HRD-targeted therapies. Together, these results suggest that expression of RING-less BRCA1 may serve as a marker to predict poor response to DSB-inducing therapy in human cancer patients.

Unlabelled: Inhibition of PARP is a promising therapeutic strategy for homologous recombination-deficient tumors, such as BRCA1-associated cancers. We previously reported that BRCA1-deficient mouse mammary tumors may acquire resistance to the clinical PARP inhibitor (PARPi) olaparib through activation of the P-glycoprotein drug efflux transporter. Here, we show that tumor-specific genetic inactivation of P-glycoprotein increases the long-term response of BRCA1-deficient mouse mammary tumors to olaparib, but these tumors eventually developed PARPi resistance. In a fraction of cases, this resistance is caused by partial restoration of homologous recombination due to somatic loss of 53BP1. Importantly, PARPi resistance was minimized by long-term treatment with the novel PARP inhibitor AZD2461, which is a poor P-glycoprotein substrate. Together, our data suggest that restoration of homologous recombination is an important mechanism for PARPi resistance in BRCA1-deficient mammary tumors and that the risk of relapse of BRCA1-deficient tumors can be effectively minimized by using optimized PARP inhibitors., Significance: In this study, we show that loss of 53BP1 causes resistance to PARP inhibition in mouse mammary tumors that are deficient in BRCA1. We hypothesize that low expression or absence of 53BP1 also reduces the response of patients with BRCA1-deficient tumors to PARP inhibitors.

The human multidrug resistance proteins MRP4 and MRP5 are organic anion transporters that have the unusual ability to transport cyclic nucleotides and some nucleoside monophosphate analogs. Base and nucleoside analogs used in the chemotherapy of cancer and viral infections are potential substrates. To assess the possible contribution of MRP4 and MRP5 to resistance against these drugs, we have investigated the transport mediated by MRP4 and MRP5. In cytotoxicity assays, MRP4 conferred resistance to the antiviral agent 9-(2-phosphonomethoxyethyl)adenine (PMEA) and high-performance liquid chromatography analysis showed that, like MRP5, MRP4 transported PMEA in an unmodified form. MRP4 also mediated substantial resistance against other acyclic nucleoside phosphonates, whereas MRP5 did not. Apart from low-level MRP4-mediated cladribine resistance, the cytotoxicity of clinically used anticancer nucleosides was not influenced by overexpression of MRP4 or MRP5. In contrast, MRP5 mediated efflux of the pyrimidine-based antiviral 2',3'-dideoxynucleoside 2',3'-didehydro-2',3'-dideoxythymidine 5'-monophosphate (d4TMP) and its phosphoramidate derivative alaninyl-d4TMP from cells loaded with the 2',3'-didehydro-2',3'-dideoxythymidine prodrugs cyclosaligenyl-d4TMP and aryloxyphosphoramidate d4TMP (So324), respectively. Moreover, only inside-out membrane vesicles derived from MRP5-overexpressing cells accumulated alaninyl-d4TMP. Cellular efflux and vesicular uptake studies were carried out to further compare transport mediated by MRP4 and MRP5 and showed that dipyridamole, dilazep, nitrobenzyl mercaptopurine riboside, sildenafil, trequinsin and MK571 inhibited MRP4 more than MRP5, whereas cyclic nucleotides and monophosphorylated nucleoside analogs were equally poor inhibitors of both pumps. These results strongly suggest that the affinity of MRP4 and MRP5 for nucleotide-based substrates is low.

Despite accumulating evidence that multidrug resistance transporter proteins play a part in drug resistance in some clinical cancers, it remains unclear whether the relatively low levels of multidrug resistance transporter expression found in most untreated tumors could substantially affect their basal sensitivity to antineoplastic drugs. To shed light on this problem, the drug sensitivities of wild-type mouse cell lines were compared with those of lines in which the Mdr1a and Mdr1b genes encoding P-glycoprotein (P-gp) were inactivated and lines in which the Mrp1 gene was inactivated in addition to Mdr1a and Mdr1b. These models permit a clean dissection of the contribution of each transporter to drug resistance at expression levels similar to those in normal tissues and avoid complications that might arise from previous exposure of cell lines to drug selection. For substrate drugs, we found that these contributions can indeed be very substantial. Lines lacking functional P-gp were, on average, markedly more sensitive to paclitaxel (16-fold), anthracyclines (4-fold) and Vinca alkaloids (3-fold). Lines lacking both P-gp and Mrp1 were (compared with wild-type lines) hypersensitive to an even broader array of drugs, including epipodophyllotoxins (4-7-fold), anthracyclines (6-7-fold), camptothecins (3-fold), arsenite (4-fold) and Vinca alkaloids, especially vincristine (28-fold). Thus, even very low levels of P-gp and Mrp1 expression that may be difficult to detect in tumors could significantly affect their innate sensitivity to a wide range of clinically important substrate drugs. An implication is that the use of resistance reversal agents to sensitize drug-naive tumors may be appropriate in more cases than is presently appreciated.

The human multidrug transporter MDR1 P-glycoprotein and the multidrug resistance proteins MRP1 and MRP2 transport a range of cytotoxic drugs, resulting in multidrug resistance in tumour cells. To overcome this form of drug resistance in patients, several inhibitors (reversal agents) of these transporters have been isolated. Using polarized cell lines stably expressing human MDR1, MRP1 or MRP2cDNA, and 2008 ovarian carcinoma cells stably expressing MRP1 cDNA, we have investigated in this study the specificity of the reversal agents V-104 (a pipecolinate derivative), GF120918 (an acridone carboxamide derivative also known as GG918), and Pluronic L61 (a (poly)oxypropethylene and (poly)oxypropylene block copolymer). Transport experiments with cytotoxic drugs with polarized cell lines indicate that all three compounds efficiently inhibit MDR1 Pgp. Furthermore, V-104 partially inhibits daunorubicin transport by MRP1 but not vinblastine transport by MRP2. V-104 reverses etoposide resistance of 2008/MRP1 cells, whereas GF120918 does not reverse resistance due to MRP1. V-104 partially inhibits the export of the organic anion dinitrophenyl S-glutathione by MDCKII-MRP1 but not by MDCKII-MRP2 cells. Unexpectedly, export of the organic anion calcein by MDCKII-MRP1 and MDCKII-MRP2 cells is stimulated by Pluronic L61, probably because it relieves the block on entry of calcein AM into the cell by endogenous MDR1 Pgp.

Two prominent members of the ATP-binding cassette superfamily of transmembrane proteins, multidrug resistance 1 (MDR1) P-glycoprotein and multidrug resistance protein 1 (MRP1), can mediate the cellular extrusion of xenobiotics and (anticancer) drugs from normal and tumor cells. The MRP subfamily consists of at least six members, and here we report the functional characterization of human MRP5. We found resistance against the thiopurine anticancer drugs, 6-mercaptopurine (6-MP) and thioguanine, and the anti-HIV drug 9-(2-phosphonylmethoxyethyl)adenine (PMEA) in MRP5-transfected cells. This resistance is due to an increased extrusion of PMEA and 6-thioinosine monophosphate from the cells that overproduce MRP5. In polarized Madin-Darby canine kidney II (MDCKII) cells transfected with an MRP5 cDNA construct, MRP5 is routed to the basolateral membrane and these cells transport S-(2,4-dinitrophenyl)glutathione and glutathione preferentially toward the basal compartment. Inhibitors of organic anion transport inhibit transport mediated by MRP5. We speculate that MRP5 might play a role in some cases of unexplained resistance to thiopurines in acute lymphoblastic leukemia and/or to antiretroviral nucleoside analogs in HIV-infected patients.

Studies with knockout mice lacking mdr1a P-glycoprotein (P-gp) have previously shown that blood-brain barrier P-gp is important in preventing the accumulation of several drugs in the brain. Asimadoline (EMD 61753) is a peripherally selective kappa-opioid receptor agonist which is under development as a therapeutic analgaesic. From the structural characteristics of this drug and its peripheral selectivity, we hypothesized that it is transported by P-gp. Using a pig-kidney polarized epithelial cell line transfected with mdr cDNAs, we demonstrate that asimadoline is transported by the mouse mdr1a P-gp and the human MDR1 P-gp. Furthermore, we show that in mdr1a/1b double knockout mice, the absence of P-gp leads to a 9 fold increased accumulation of asimadoline in the brain. In line with this accumulation difference, mdr1a/1b (-/-) mice are at least 8 fold more sensitive to the sedative effect of asimadoline than wild-type mice. Interestingly, the oral uptake of asimadoline was not substantially altered in mdr1a/1b (-/-) mice. Our results demonstrate that for some drugs, P-gp in the blood-brain barrier can have a therapeutically beneficial effect by limiting brain penetration, whereas at the same time intestinal P-gp is not a significant impediment to oral uptake of the drug.

The human multidrug resistance protein MRP1 mediates transport of organic substrates conjugated to glutathione, glucuronide, or sulfate. The naturally occurring prostaglandins A1 and A2 can form two diastereomeric glutathione S-conjugates, and it has been speculated that these might be substrates for MRP1. Here we present evidence that polarized MDCKII cells expressing MRP1 cDNA transport PGA1-GS to the basolateral side of a cell monolayer, in accordance with the lateral localization of human MRP1 in these cells. Furthermore, we show that vesicles made from yeast cells expressing MRP1 cDNA and from mouse erythrocytes (known to contain mrpl) actively accumulate both diastereomers of PGA2-GS with a similar efficiency. Recently, we generated mice with a homozygous mutant mrp1 allele. Uptake of PGA2-GS in vesicles made from erythrocytes of these mice was 3.2 times lower than in wild-type vesicles, but was still significantly above background. This residual transport activity was partly inhibited by methotrexate and cAMP, whereas mrp1-mediated activity was unaffected by these compounds. We conclude that mouse erythrocytes contain at least two transport systems for PGA2-GS. One of these is mrp1; the other one has not been identified yet, but can be inhibited by methotrexate and cAMP.

The mdr1-type P-glycoproteins (P-gps) confer multidrug resistance to cancer cells by active extrusion of a wide range of drugs from the cell. To study their physiological roles, we have generated mice genetically deficient in the mdr1b gene [mdr1b (-/-) mice] and in both the mdr1a and mdr1b genes [mdr1a/1b (-/-) mice]. In spite of the host of functions speculatively attributed to the mdrl-type P-gps, we found no physiological abnormalities in either strain. Viability, fertility, and a range of histological, hematological, serum-chemical, and immunological parameters were not abnormal in mdr1a/1b (-/-) mice. The high level of mdrlb P-gp normally present in the pregnant uterus did not protect fetuses from a drug (digoxin) in the bloodstream of the mother, although the protein did reduce drug accumulation in the adrenal gland and ovaries. Pharmacologically, mdr1a/1b (-/-) mice behaved similarly to the previously analyzed mdr1a (-/-) mice, displaying, for instance, increased brain penetration and reduced elimination of digoxin. However, both mdr1a and mdr1b P-gps contributed to the extrusion of rhodamine from hematopoietic progenitor cells, suggesting a potential role for the endogenous mdr1-type P-gps in protection of bone marrow against cytotoxic anticancer drugs. This, and the normal viability of mdr1a/1b (-/-) mice, has implications for the use of P-gp-blocking agents in cancer and other chemotherapy. mdr1a/1b (-/-) mice should provide a useful model system to further test the pharmacological roles of the drug-transporting P-gps and to analyze the specificity and effectivity of P-gp-blocking drugs.

The human MDR3 (or MDR2) P-glycoprotein is probably involved in the transport of phospholipids from liver hepatocytes into bile (Smit et al. (1993) Cell 75, 451-462). In accordance with this function, MDR3 is highly expressed in human liver, but lower mRNA levels were also found in adrenal, heart, muscle and cells of the B-cell compartment. We have cloned and analyzed the MDR3 promoter region. It is GC-rich, and contains neither a TATA nor a CAAT box, but it does contain multiple putative SP1 binding sites, features also found in so-called housekeeping genes. RNase protection and primer extension analyses indicate that the MDR3 gene has multiple transcription start sites in a GC-rich region with considerable homology to the putative mouse mdr2 promoter. A 3 kb genomic fragment containing the MDR3 start sites directs transcription of a chloramphenicol acetyltransferase (CAT) reporter gene upon transient transfection in the human hepatoma cell line HepG2. This transcription is orientation dependent, and stimulated by a SV40 enhancer, indicating that the 3 kb insert contains the core promoter elements of the MDR3 gene. The promoter region contains several consensus sequences where known or putative liver-specific (C/EBP, HNF5) or lymphoid specific (Pu.1, ets-1) transcription factors may bind.

Amplification and overexpression of the neu gene have been found in several human adenocarcinomas. We have obtained monoclonal antibodies to the human neu protein by immunizing a Balb/c mouse with a Balb/c cell line expressing the human neu gene by transfection. The monoclonal antibodies reacted with neu protein on intact cells by immunofluorescence and immunoprecipitated neu in metabolically labeled cells, also in the presence of tunicamycin. We tested possible down-modulating effects of these monoclonal antibodies on SKBR-3 mammary tumor cells, which express high levels of wild-type human neu protein. We also used NIH3T3 cells transfected with either a normal or a mutated human neu gene, encoding a protein with a valine to glutamic acid substitution in the transmembrane domain. Down-modulation of the normal cell-surface neu protein was inefficient. In contrast, the antibodies induced 50-65% down-modulation in NIH3T3 cells expressing the mutated human neu protein and could inhibit these cells to form colonies in soft agar. We propose that these differences are due to changed aggregation properties of the point-mutated protein.