ATP-binding cassette (ABC) transporters are transmembrane proteins that transport a wide variety of substrates across intra and extra-cellular membranes. A few examples of endo and xenobiotic substrates are metabolic products, lipids, sterols, and drugs. An important function of ABC transporters involved in export is to prevent intracellular the buildup of toxic products. Several ABC transporters have also been associated with drug resistance upon treatment with chemotherapeutic agents. P-glycoprotein (P-GP) and the multidrug resistant (MRP) transporters of the ABC C family are examples of transporters that confer chemo-resistance. We have studied two unique roles of ABC transporters in the liver and the heart. In the liver, maintenance of bile secretion is important during lactation to ensure proper absorption of nutrients for the offspring. Three main ABC transporters are involved in this process: ABCB11 (transports bile acids), ABCB4 (transporters phospholipids), and ABCG5/ABCG8 (transports cholesterol). In the rat, expression of ABCB11 remains the same as the size of the bile acid pool increases. However, the expression of ABCG5/ABCG8 is abolished, preventing excessive export and loss of cholesterol from the liver. The regulation of these transporters during lactation maintains the production of bile acids from cholesterol by decreasing export while preventing toxicity from bile acids by maintaining bile flow. Another protective role of ABC transporters is seen in oxidative stress-induced toxicity of cardiac tissue following treatment with Doxorubicin (DOX), a drug used in cancer treatment. Multidrug resistance protein 1 (Mrp1) can transport toxic products by conjugation with sulfate, glutathione (GSH) or glucuronide. In Mrp1-/- mice, DOX causes advanced cell damage through intracellular edema and increased apoptotic nuclei. However, P-glycoprotein expression increases upon DOX treatment, potentially compensating for the loss of Mrp1. Mrp1 can also transport GSH, GSH disulfide (GSSG), and products of oxidation, like GSH conjugates. In the absence of Mrp1, GSH levels are increased in the heart, providing protection against oxidative stress. Both of these examples in liver and heart show the diversity of ABC transporters and the role they play in preventing cell toxicity. These studies also provide insight into ways to prevent cell toxicity through manipulation of ABC transport proteins.

Primary brain tumours, and especially the most common form malignant gliomas, usually display a pronounced resistance to other treatment modalities when surgery fails to cure. Growth factors, such as EGF and its receptor, frequently amplified and overexpressed in malignant gliomas, and factors associated with multidrug resistance have been suggested to at least partially explain the poor outcome. The aim of this thesis was to characterise factors in primary brain tumours associated with the development of resistance with focus on the epidermal growth factor receptor (ErbB) family, and multidrug resistance (MDR). Influences of irradiation on the expression and activity of P-glycoprotein (Pgp) in malignant gliomas was evaluated. The effects showed that irradiation increased the efflux activity of Pgp in rat brain vascular endothelial cells, but not in glioma cells. In the intracranial BT4C glioma model, Pgp was detected in the capillary endothelium in the tumour tissue but not in glioma cells. Expression of several factors coupled to MDR (Pgp, MRP1, LRP, and MGMT) in primary brain tumours were analysed and correlated to clinical data. In gliomas, Pgp and MRP1 were predominantly observed in capillary endothelium and in scattered tumour cells, whereas LRP occurred only in tumour cells. In meningiomas, expression of the analysed markers was demonstrated in the capillary endothelium, with a higher expression of Pgp and MRP1 in transitional compared to meningothelial meningiomas. A pronounced expression of MGMT was found independently of the histopathological grade or tumour type. Survival analysis indicated a shorter overall survival for patients suffering from low-grade gliomas with high expression of Pgp. To explore the importance of the epidermal growth factor receptor (EGFR), expression levels of the family members (EGFR, ErbB2-4) were analysed and their relations to various clinical parameters were evaluated in gliomas and meningiomas. In gliomas, the highest EGFR expression was observed in high-grade tumours, while ErbB4 expression was most pronounced in low-grade tumours. In meningiomas, expression of EGFR, ErbB2, and ErbB4 was observed in the majority of the tumours. An intriguing observation in low-grade gliomas was a significantly decreased overall survival for patients with high EGFR protein expression. The effects of different time schedules for administration of the selective EGFR inhibitor ZD1839 in relation to irradiation of glioma cells were analysed. The analyses showed a heterogeneity in the cytotoxic effects of ZD1839 between cell lines, and it was obvious that some of the cell lines showed sensitivity to ZD1839 despite no or low expression of EGFR. The study also demonstrated the importance of timing of ZD1839 administration when this agent is combined with irradiation. In conclusion, in order to enhance the efficacy of radiotherapy by various drugs in malignant gliomas it may be essential to inhibit drug efflux activity in endothelial cells and to deliver drugs in an optimal timing in relation to radiotherapy. The heterogeneity in expression of drug resistance markers, as well as the ErbB family reflects the complexity in classification of primary brain tumours, and indicates that subgroups of patients with low-grade gliomas expressing Pgp and EGFR might benefit from more aggressive and individualised treatment.

The objective of this dissertation is to study the mechanism by which HIV protease inhibitors enter into the central nervous system (CNS) and breast milk of rats, and what effects MDR modulators have on the distribution and metabolism of HIV protease inhibitors. The transporter P-glycoprotein (P-gp) has been shown to limit the distribution of HIV protease inhibitors into the CNS of rodents. This thesis examined the effects of GF120918, an MDR modulator, on the CNS distribution of amprenavir, an HIV protease inhibitor, in rats. GF120918 significantly increased the unbound CNS concentrations of amprenavir without altering the unbound blood concentrations of amprenavir. The results of these studies show that GF120918 can inhibit P-gp at the blood brain barrier (BBB) to increase the unbound CNS concentration of amprenavir and potentially other HIV protease inhibitors. Many first generation MDR modulators inhibited both P-gp transport and CYP3A metabolism. Therefore, a principal goal of this thesis was to determine if GF120918 could selectively inhibit P-gp transport without inhibiting CYP3A metabolism. Using in vitro (human) and in vivo (rat) studies, GF120918 selectively inhibited P-gp at the BBB without inhibiting CYP3A metabolism. The transporter MRP1 has been shown to both transport HIV protease inhibitors and expressed in the CNS. Studies contained in the thesis have shown that mrp1 is not localized to the BBB of rats, therefore, mrp1 is unlikely to play a significant role in the distribution of HIV protease inhibitors into the CNS of rats. The distribution of nelfinavir, an HIV protease inhibitor, into rat breast milk was studied in the thesis as a first approach in understanding the extent to which HIV protease inhibitors can accumulate into milk. The concentration of nelfinavir in rat milk was approximately half that of plasma. P-gp protein expression was detected in lactating rat mammary tissue. However, GF120918 showed no effect on the distribution of nelfinavir into rat milk suggesting that P-gp does not play a significant role in the distribution of HIV protease inhibitors into milk.

Multidrug resistance-associated protein 1 (MRP1) is a member of the adenosine 5’-triphosphate (ATP)-binding cassette (ABC) transporters. MRP1 actively effluxes a variety of endogenous and exogenous substrates from cells, ultimately, working to remove these compounds from the body. MRP1 was initially discovered based on its ability to confer resistance against a variety of chemotherapeutics when overexpressed in cancer cells lines. MRP1 function is important for a number of physiological processes, including regulating cellular and extracellular levels of the anti-inflammatory leukotriene C4 (LTC4) and the antioxidant glutathione (GSH). Our studies have focused on the role of MRP1 in regulating hematopoietic stem cell (HSC) self-renewal and differentiation and the role of CK2 as a regulator of MRP1 function. Reactive Oxygen Species (ROS) cellular levels are tightly regulated and fluctuations in ROS levels affect many cellular processes, including the self-renewal and differentiation of hematopoietic stem cells and kinase signaling pathways. MRP1 regulates ROS through the transport of reduced and oxidized GSH. MRP1 is highly expressed in HSCs, therefore we hypothesized that MRP1 regulates ROS levels in HSCs via efflux of GSH. We have shown that MRP1 regulates HSC self-renewal by modulating cellular ROS via the efflux of GSH. The decrease in ROS results in downregulation of p38 activity and altered expression of a number of redox response genes. CK2 is a master regulator of the cell and controls cell growth, proliferation, death and survival. Yeast studies from our lab using Ycf1p (a homologue of MRP1) and Cka1p (a homologue of CK2) have found that Cka1p regulates Ycf1p function. This result suggests that CK2 regulates MRP1 function via phosphorylation. We have found that CK2 does regulate MRP1 function via phosphorylation of the N-terminal extension at Thr249. Using A549, H460, and HeLa cancer cell lines, we found that inhibition of CK2 with tetrabromobenzimidazole (TBBz) reduces MRP1 function and increases cellular toxicity to known MRP1 substrates.

La surexpression de pompes d'efflux par les cellules cancéreuses permet l'élimination d'agents cytotoxiques, induisant alors une résistance à la chimiothérapie. Trois transporteurs ABC sont principalement impliqués dans cette résistance : P-gp/ABCB1, MRP1-ABCC1 et BCRP/ABCG2. La surexpression des ces transporteurs peut également être le "talon d'Achille" des cellules cancéreuses résistantes en les sensibilisant à certains composés. Ce phénomène, appelé sensibilité collatérale, pourrait constituer un nouvel outil thérapeutique conter les les cancers intrinséquement ou rendus résistants en éliminant sélectivement les cellules cancéreuse résistances. Ainsi, le S-vérapamil provoque la mort sélective par apoptose des cellules surexprimant suite à l'extrusion rapide et massive du glutathion 5GSH) intracellulaire par MRP1. Nous avons démontré que le vérapamil est capable de dépléter s"lectivement de leur contenu en GSH les tumeurs de cancer du poumon H69AR, MRP1 positives et résistantes, dès 3 heures d'exposition aiguë. Le vérapamil étant fortemnt carditoxique, nous avons développé de nouveaux agents de sensibilité collatérale, plus sélectif que le vérapamil, comme le xanthone 9, le flavonoïde 36 et le dimère de flavonoïde 4e. Enfin, grâce à l'étude de chimères MRP1/MRP2, nous avons démontré que la région comprenant les boucles L0 et L1-TM12 pourrait constituter les sites modualteurs et substrat du GSH sur MRP1.

Streptococcus pneumoniae (S. pneumoniae) is a Gram-positive, encapsulated bacterium capable of causing significant morbidity and mortality throughout the world. A hallmark of S. pneumoniae infection is infiltration of neutrophils (PMNs) that assist in controlling the spread infection but may also contribute to pathology. Paradoxically, studies have shown that limiting PMN infiltration into the lumen of the lung during infection actually betters clinical outcome in experimental S. pneumoniae infection. The final step in PMN luminal trafficking is a Hepoxilin A3 (HXA3)-dependent migration across the pulmonary epithelium. HXA3 is a PMN chemoattractant that forms gradients along the polarized epithelial face, drawing PMNs from the basolateral to the apical surface during proinflammatory responses. HXA3 requires assistance of an integral- membrane protein transporter to escape the cell and form the gradient. The pulmonary HXA3 transporter is currently unidentified. In this work, we identify the pulmonary HXA3 transporter as the ATP-Binding Cassette Transporter (ABC transporter) Multi-drug Resistance Associated Protein 2 (ABCC2, MRP2). We demonstrate that MRP1 and MRP2 are divergent ABC- transporters that control transepithelial PMN migration through efflux of a distinct anti-inflammatory substance and the pro-inflammatory HXA3 in the context of Streptococcus pneumoniae infection. Enrichment of MRP2 on the plasma membrane requires detection of the bacterial virulence factors pneumolysin (PLY) and hydrogen peroxide. PLY and hydrogen peroxide not only coordinate MRP2 apical membrane enrichment but also influence HXA3-dependent PMN transepithelial migration. They influence migration through stimulation of epithelial intracellular calcium increases that are crucial for HXA3 production as well as MRP2 translocation to the plasma membrane. PLY and hydrogen peroxide are not sufficient in their signaling alone, however, and require at least one additional bacterial signal to induce HXA3/MRP2 proinflammatory activities.

L'acquisition du phénotype de résistance des cancers est souvent corrélée à l'expression de transporteurs membranaires appartenant à la superfamille des transporteurs ABC (« ATP-Binding Cassette »). Un de ces transporteurs, MRP1 (« Multidrug Resistance Protein 1 »), permet l'efflux de nombreux substrats, de type anioniques, conjugués au GSH, glucuronates ou sulfates, ou en co-transport avec le GSH.<br />Dans un premier temps, ce travail a porté sur l'étude des domaines de fixation des nucléotides isolés. Une étude biochimique a montré leur caractère fonctionnel asymétrique concernant les nucléotides, prouvant que seul la séquence primaire de ces NBD est responsable de cette fonctionnalité différentielle. L'étude de la fixation de substrats sur les NBD, a montré que ceux-ci pourraient, de part leur proximité avec les domaines transmembranaires, avoir un rôle dans la fixation des substrats.<br />La deuxième étape de ce travail a concerné la caractérisation de l'activité des énantiomères du vérapamil. Les résultats ont montré que le S-vérapamil est l'isomère responsable de la stimulation du transport du GSH, conduisant à la mort des cellules surexprimant MRP1. Le R-vérapamil est caractérisé comme un inhibiteur de MRP1. Ces résultats ont des répercussions importantes en terme de thérapie.<br />Deux études préliminaires de relation structure/fonction ont été menées en ce qui concerne des dérivés du vérapamil et des dérivés de flavonoïdes, afin de trouver des molécules plus efficaces contre MRP1.