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 mdr1-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 mdr1b 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.
In mice, the mdr1a and mdr1b genes encode drug-transporting proteins that can cause multidrug resistance in tumor cells by lowering intracellular drug levels. These P-glycoproteins are also found in various normal tissues such as the intestine. Because mdr1b P-glycoprotein is not detectable in the intestine, mice with a homozygously disrupted mdr1a gene [mdr1a(؊͞؊) mice] do not contain functional P-glycoprotein in this organ. We have used these mdr1a(؊͞؊) mice to study the effect of gut P-glycoprotein on the pharmacokinetics of paclitaxel. The area under the plasma concentration-time curves was 2-and 6-fold higher in mdr1a(؊͞؊) mice than in wild-type (wt) mice after i.v. and oral drug administration, respectively. Consequently, the oral bioavailability in mice receiving 10 mg paclitaxel per kg body weight increased from only 11% in wt mice to 35% in mdr1a(؊͞؊) mice. The cumulative fecal excretion (0-96 hr) was markedly reduced from 40% (after i.v. administration) and 87% (after oral administration) of the administered dose in wt mice to below 3% in mdr1a(؊͞؊) mice. Biliary excretion was not significantly different in wt and mdr1a(؊͞؊) mice. Interestingly, after i.v. drug administration of paclitaxel (10 mg͞kg) to mice with a cannulated gall bladder, 11% of the dose was recovered within 90 min in the intestinal contents of wt mice vs. <3% in mdr1a(؊͞؊) mice. We conclude that Pglycoprotein limits the oral uptake of paclitaxel and mediates direct excretion of the drug from the systemic circulation into the intestinal lumen.
DNA topoisomerase II inhibitors are a major class of cancer chemotherapeutics, which are thought to eliminate cancer cells by inducing DNA double-strand breaks. Here we identify a novel activity for the anthracycline class of DNA topoisomerase II inhibitors: histone eviction from open chromosomal areas. We show that anthracyclines promote histone eviction irrespective of their ability to induce DNA double-strand breaks. The histone variant H2AX, which is a key component of the DNA damage response, is also evicted by anthracyclines, and H2AX eviction is associated with attenuated DNA repair. Histone eviction deregulates the transcriptome in cancer cells and organs such as the heart, and can drive apoptosis of topoisomerase-negative acute myeloid leukaemia blasts in patients. We define a novel mechanism of action of anthracycline anticancer drugs doxorubicin and daunorubicin on chromatin biology, with important consequences for DNA damage responses, epigenetics, transcription, side effects and cancer therapy.
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...
We have studied in vivo responses of ''spontaneous'' Brca1-and p53-deficient mammary tumors arising in conditional mouse mutants to treatment with doxorubicin, docetaxel, or cisplatin. Like human tumors, the response of individual mouse tumors varies, but eventually they all become resistant to the maximum tolerable dose of doxorubicin or docetaxel. The tumors also respond well to cisplatin but do not become resistant, even after multiple treatments in which tumors appear to regrow from a small fraction of surviving cells. Classical biochemical resistance mechanisms, such as up-regulated drug transporters, appear to be responsible for doxorubicin resistance, rather than alterations in drug-damage effector pathways. Our results underline the promise of these mouse tumors for the study of tumor-initiating cells and of drug therapy of human cancer.multidrug resistance ͉ P-glycoprotein ͉ cancer stem cells
Imatinib mesylate (signal transduction inhibitor 571, Gleevec) is a potent and selective tyrosine kinase inhibitor, which was shown to effectively inhibit platelet-derived growth factor-induced glioblastoma cell growth preclinically. However, in patients, a limited penetration of imatinib into the brain has been reported. Imatinib is transported in vitro and in vivo by P-glycoprotein (P-gp; ABCB1), which thereby limits its distribution into the brain in mice. Previously, imatinib was shown to potently inhibit human breast cancer resistance protein (BCRP; ABCG2). Here, we show that imatinib is efficiently transported by mouse Bcrp1 in transfected Madin-Darby canine kidney strain II (MDCKII) monolayers. Furthermore, we show that the clearance of i.v. imatinib is significantly decreased 1.6-fold in Bcrp1 knockout mice compared with wild-type mice. At t = 2 hours, the brain penetration of i.v. imatinib was significantly 2.5-fold increased in Bcrp1 knockout mice compared with control mice. We tested the hypothesis that P-gp and BCRP inhibitors, such as elacridar and pantoprazole, improve the brain penetration of imatinib. Firstly, we showed in vitro that pantoprazole and elacridar inhibit the Bcrp1-mediated transport of imatinib in MDCKII-Bcrp1 cells. Secondly, we showed that co-administration of pantoprazole or elacridar significantly reduced the clearance of i.v. imatinib in wild-type mice by respectively 1.7-fold and 1.5-fold. Finally, in wild-type mice treated with pantoprazole or elacridar, the brain penetration of i.v. imatinib significantly increased 1.8-fold and 4.2-fold, respectively. Moreover, the brain penetration of p.o. imatinib increased 5.2-fold when pantoprazole was co-administered in wild-type mice. Our results suggest that co-administration of BCRP and Pgp inhibitors may improve delivery of imatinib to malignant gliomas. (Cancer Res 2005; 65(7): 2577-82)
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