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The development of multidrug resistance (MDR) in tumour cells to a wide range of anticancer drugs has become a major obstacle in the chemotherapeutic treatment of cancer. Molecular characterization of MDR tumour cells has led to the identification of several cell-based genetic alterations including the overexpression of a membrane protein, P-glycoprotein (P-gp). P-gp is a ATP dependent drug efflux pump and P-gp ATPase activity has been demonstrated to be essential in drug transport. In an effort to understand how P-gp ATPase activity is coupled to drug binding and transport, we examined the effects of N-ethylmaleimide (NEM), a potent inhibitor of P-gp ATPase, on P-gp drug binding and transport. Our results show that short term treatment of MDR cells with NEM led to a concentration-dependent increase in P-gp drug binding and phosphorylation. In addition, NEM increases [3H]-vinblastine accumulation in drug resistant cells but not in sensitive cells. Our study suggests that inhibition of P-gp ATPase activity, and not increased phosphorylation of P-gp by NEM, is responsible for the observed increase in P-gp-drug binding. / Selection of tumour cell lines in vitro has led to multiple cellular changes that may mediate drug resistance to anticancer drugs. The role of other mechanisms, in addition to P-gp and multidrug resistance protein (MRP) in drug resistance, is supported by evidence from studies with tumour cell lines and clinical tumours. In an effort to identify other cellular changes that may be important in tumour drug resistance to anticancer drugs, we have used a differential immunodot blot method to isolate monoclonal antibodies that bind to proteins in drug resistant but not in drug sensitive cells. By using the immunodot blot method, we have isolated a monoclonal antibody (IPM96) which recognized a 40 kDa protein (P-40) in several MDR cell lines. The expression of P-40 is concurrent with the level of drug resistance. Biochemical characterization showed P-40 to be associated with the cell membrane and in the soluble fraction. Molecular cloning of P40 cDNA revealed that P-40 is identical to annexin I, a substrate for the epidermal growth factor receptor tyrosine kinase. The observed increase in P-40 (or annexin I) protein levels in drug resistant cells is due to the elevation of P-40 transcripts. The pharmacological characterization of P-40 cDNA transfectants (P-40-MCF-7) has demonstrated that overexpression of P-40 in drug sensitive cells is capable of conferring drug resistance to adriamycin, actinomycin D, Taxol and cisplatin. Taken together, our study provides convincing evidence that annexin I is important in the development of drug resistance in cancer cells. In addition, it suggests a novel mechanism of drug resistance that is different from the ATP-dependent drug efflux pumps that mediate P-gp- and MRP-associated MDR

Multidrug resistance protein (MRP)1, a member of the ABCC branch of the ATP-binding cassette (ABC) superfamily of transporters, can confer resistance to a broad spectrum of chemotherapeutic agents. In addition to the core functional unit of ABC transporters that consists of two membrane spanning domains (MSD) and two nucleotide binding domains (NBD), MRP1 contains a third MSD (MSD0) resulting in the following arrangement: NH2-MSD-(MSD-NBD)2. In lieu of high-resolution structural information for MRP1, cysteine scanning mutagenesis (CSM) was applied to MRP1 and involves the development of a functional template devoid of cysteines into which paired cysteines can be introduced. Previous attempts to create a functional, cys-less template of MRP1 demonstrated that cysteines in MSD0 were structurally and functional important (1;2). However, given that MRP1 lacking MSD0 remains functional, a partially functional, cys-less MRP1 lacking this domain has been expressed in yeast (3-5). Given these results, with the ultimate goal of applying CSM to MRP1 in its entirety, we investigated the endogenous cysteines within MSD0 and co-expressed MRP1 half-molecules and validated these potential CSM templates by transport and ATP binding/hydrolysis assays. Mutation of cysteines within the core of MRP1 had detrimental effects on MRP1 transport activity and further mutation of cysteines by domain revealed that wild-type activity was retained in an MSD0-less MRP1 dual lacking cysteines in both NBDs. This construct was used for introduction of cysteines on juxtaposed faces of the NBD1:NBD2 heterodimer at positions 775 and 1329; comparable residues in the related Cystic Fibrosis Transmembrane Regulator (CFTR/ABCC7) have been suggested to be evolutionarily coupled and joined by a hydrogen bond, maintained in structures of related proteins (6). Unfortunately, functional assays revealed that introduction of cysteines at these positions greatly reduced transport activity of MRP1 and diminished trapping of nucleotide at both NBDs. Finally, alanine substitution of the seven cysteines in MSD0 was not without effect and cellular trafficking assays, co-expression studies and SDS-PAGE analysis suggested an altered conformation of this domain. In addition, a disulfide pair of Cys7 and Cys32 was suggested by these experiments in MSD0 and further supported by examination of these mutants in full-length MRP1. / Thesis (Master, Pathology & Molecular Medicine) -- Queen's University, 2009-02-17 12:51:38.13

Expression of the mexCD-oprJ multidrug efflux operon is enhanced by the presence of membrane damaging agents [e.g., the biocide chlorhexidine (Chx)] or mutations in the nfxB gene encoding a repressor of efflux gene expression, both dependent on the AlgU envelope stress response sigma factor. Details of mexCD-oprJ regulation are, however, lacking. In examining the mexCD-oprJ locus, a gene, PA4596, encoding a homologue of NfxB (61% identity) was identified downstream of oprJ, a location conserved in all sequenced Pseudomonas aeruginosa isolates and in Pseudomonas putida. Opposite to mexCD-oprJ, PA4596 expression was reduced by Chx exposure, as assessed using RT-PCR; although like mexCD-oprJ, this was AlgU-dependent (i.e., lost in a ΔalgU strain). Deletion of PA4596 had no impact on Chx resistance indicating that it is not required for Chx-inducible mexCD-oprJ expression/ MexCD-OprJ-dependent Chx resistance. In contrast, mexCD-oprJ expression and the attendant multidrug resistance of nfxB deletion mutants were compromised upon deletion of PA4596, indicating that PA4596 plays a positive role in mexCD-oprJ expression in such mutants. Consistent with this, PA4596 expression increased in nfxB deletion and missense mutants in parallel with mexCD-oprJ. Intriguingly, mexCD-oprJ expression and multidrug resistance were observed in a mutant lacking an nfxB mutation (demonstrating an NfxB-like phenotype) and in an nfxB missense mutant and these were not compromised upon deletion of PA4596. Thus, mexCD-oprJ hyperexpression can be both PA4596-dependent and -independent. A bacterial 2-hybrid assay revealed a PA4596-PA4596 interaction, consistent with the protein forming dimers as NfxB has been shown to do. Two-hybrid assays also demonstrated that NfxB and PA4596 interact. While the functional significance of this remains to be elucidated, it is consistent with their common role in regulating mexCD-oprJ expression and is suggestive of a complex and possibly novel regulatory mechanism. These data highlight the complexity of mexCD-oprJ regulation and the apparently multiple pathways to efflux gene expression, suggestive of multiple roles for this efflux system in P. aeruginosa independent of antimicrobial efflux. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-08-18 14:25:18.107

Pseudomonas aeruginosa is a common soil organism as well as an opportunistic human pathogen. Treatment of P. aeruginosa infections is often complicated by innate resistance to a variety of antimicrobials mediated by multidrug efflux systems. The MexAB-OprM efflux system is constitutively expressed in wildtype strains and contributes to innate antimicrobial resistance, while hyperexpression of the system results in acquired high levels of resistance. MexAB-OprM is hyperexpressed in nalC mutants containing mutations in the gene encoding NalC, a repressor of a two-gene operon, PA3720-armR. armR encodes a protein modulator of MexR, a repressor of mexAB-oprM expression. Previous reports showed that genes encoding the MexAB-OprM efflux system are upregulated in response to pentachlorophenol (PCP), a phenolic compound that is a common environmental contaminant. Induction of mexAB-oprM and PA3720-armR by PCP was confirmed using RT-PCR, and MexAB-OprM was shown to be involved in PCP resistance. An electromobility shift assay (EMSA) showed that PCP interacts with NalC, interfering with its binding to the PA3720-armR promoter region and thereby promoting PA3720-armR expression. Nonetheless, the increase in ArmR did not drive mexAB-oprM expression suggesting that PCP induction of this efflux operon occurred via a different mechanism. A direct PCP-MexR interaction could not be demonstrated using an EMSA. PCP exposure did, however, reduce expression of nalD, encoding a second repressor of mexAB-oprM, which might explain the PCP-promoted increase in mexAB-oprM expression. PCP is unlikely to be the intended inducer(s)/substrate(s) for this system but probably resembles these. Several compounds related to PCP were tested for an interaction with NalC but all were negative in EMSAs. Plants produce a variety of phenolic compounds, which are often antimicrobial and, so, root extracts of various plants were tested for an ability to interact with NalC and interfere with promoter binding. Extracts from Boehmeria tricuspis, Uncaria tomentosa and Ixiolirion tataricum were shown to interact with NalC, suggesting that plant compounds may be the intended inducers/substrates for NalC/MexAB-OprM. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2010-09-10 10:35:16.271

Over-expression of either P-gp1 and/or MRP1 in tumor cell lines confers resistance to structurally diverse anti-cancer drugs. Although the role of these two proteins in clinical drug resistance remains to be confirmed, the use of Pgp1-specific inhibitors in combination with standard anti-cancer drugs have demonstrated significant improvement in clinical response. However, evidence exists that reversal of P-gp1 alone is not sufficient. Therefore, while no drugs are currently available that can efficiently reverse MRP1 drug efflux in tumor cells, there is an urgent need to develop MRP1-specific blockers. In an effort to gain a better understanding of MRP1-drug interactions and to identify sequences within MRP1 that interact directly with drugs, we developed two structurally diverse photosensitive drug analogues, a quinoline-based compound (IACI) and a xanthone-derivative (IAARh123). Both compounds photolabeled MRP1 and showed a direct and specific interaction with the protein at physiologically relevant sites. Initial mapping of photolabeled sequences in MRP1 (Chapters 2 and 3), identified multiple IACI- or IAARh123-photolabeled peptides (∼4--7 kDa) derived from both the N-terminal (MSD0+MSD1+NBD 1) and C-terminal (MSD2+NBD2) domains of MRP1. A subsequent study (Chapter 4), using MRP1 variants with hemagglutinin (HA) epitopes inserted at eight different locations, led to a higher resolution mapping of the previously identified IACI- or IAARh123-labeled peptides. Specifically, two photolabeled peptides (∼6--7 kDa), derived from variants with insertions at positions 574 and 1222, were immunoprecipitated with anti-HA monoclonal antibody. Based on the location of the HA epitopes in the latter variants together with molecular masses of the two peptides, the photolabeled amino acid residues were localized to MRP1 sequences encoding transmembranes 10 and 11 of MSD1 and transmembranes 16 and 17 of MSD 2. Interestingly, the same sequences were photolabeled with both

Specific bacterial commensals demonstrating multidrug resistance (MDR) are opportunistic pathogens for immunocompromised patients, including Corynebacterium species (spp.). Severe infections due to MDR corynebacteria are being increasingly reported where several MDR phenotypes have been described. One such phenotype, the macrolide-lincosamide-streptogramin B phenotype (MLSB), is characterized by high-level resistance to macrolides, lincosamides, and streptogramin B. Resistance is thought to be attributable to acquisition of the ermX gene, a methyltransferase that alters the ribosomal macrolide binding site. Until recently, ermX had been reported in only six Corynebacterium spp. We have observed other corynebacteria can also display high-level resistance to MLSB antimicrobials and are ermX positive. Hypotheses being tested include: 1) high-level macrolide and lincosamide resistance in Corynebacterium spp. is caused by acquiring ermX; 2) distribution of ermX is more widespread than previously published; 3) ermX is associated with transposon Tn5432; 4) multidrug resistance has spread to Canadian C. afermentans and C. aurimucosum strains.

Specific bacterial commensals demonstrating multidrug resistance (MDR) are opportunistic pathogens for immunocompromised patients, including Corynebacterium species (spp.). Severe infections due to MDR corynebacteria are being increasingly reported where several MDR phenotypes have been described. One such phenotype, the macrolide-lincosamide-streptogramin B phenotype (MLSB), is characterized by high-level resistance to macrolides, lincosamides, and streptogramin B. Resistance is thought to be attributable to acquisition of the ermX gene, a methyltransferase that alters the ribosomal macrolide binding site. Until recently, ermX had been reported in only six Corynebacterium spp. We have observed other corynebacteria can also display high-level resistance to MLSB antimicrobials and are ermX positive. Hypotheses being tested include: 1) high-level macrolide and lincosamide resistance in Corynebacterium spp. is caused by acquiring ermX; 2) distribution of ermX is more widespread than previously published; 3) ermX is associated with transposon Tn5432; 4) multidrug resistance has spread to Canadian C. afermentans and C. aurimucosum strains.

The phenomenon of multidrug resistance (MDR) in cancer cells is generally associated with P-glycoprotein (P-gp) expression and presents an obstacle to successful chemotherapy. Attempts to overcome P-gp-associated MDR using P-gp modulators, such as verapamil, have been hindered by their intrinsic in vivo toxicity. In 1991, however, Scala et al. demonstrated the alteration of P-gp function by interferon-alpha (IFN-α) in vitro at non-toxic in vivo concentrations, suggesting a basis for the use of IFN-α clinically in patients exhibiting P-gp-associated MDR. Drug resistance in B-CLL has been linked to the phenomenon of MDR, however, publications regarding this have been conflicting. The contrasting results prompted further investigation of the role of P-gp-associated anthracycline resistance and, using isolated β-lymphocytes from B-CLL patients, this investigation examined P-gp expression, function and IFN-α modulation in vitro. Optimum conditions for in vitro analysis of P-gp-associated anthracycline resistance were determined by examining the stability of the anthracycline, daunorubicin, in varying cell culture conditions. The resulting system balanced conditions affecting drug stability with those affecting cell survival. While other investigations have neglected the issue of drug stability, this study demonstrates that the instability of daunorubicin may be a critical variable determining the outcome of drug sensitivity studies. In RPMI + 2mM L-glutamine and 10% (v/v) FBS, loss of drug concentration is due to both adsorption and degradation and these experiments show that the presumed availability of drug may be over-estimated in in vitro studies. Furthermore, the degradation products might interfere with P-gp function and modulation. MDRl gene mRNA was detected in the B-cells of forty-three out of fifty B-CLL patients analysed, whereas P-gp expression, as measured by flow cytometry, resulted in only sixteen patients out of fifty-five being classed as positive (> 10% increase in staining as compared to the control). P-gp functionality and modulation studies on the B-cells of eleven patients confirmed the existence of an efflux mechanism with identical characteristics to P-gp using verapamil, the dye rhodamine 123 (rho123) and daunorubicin. Four patients were classed as functional low expressers (functional P-gp with low P-gp expression (7-10% increase in staining)), six were classed as functional high expressors (functional P-gp with high P-gp expression (20-57% increase in staining)) and one as a non-functional high expressor (non-functional P-gp with high P-gp expression (13.4% increase in staining)). Verapamil modulated rho123 efflux in all ten patients classed as P-gp functional expressors, and daunorubicin efflux in eight of these patients. However, IFN-α modulated rho123 and daunorubicin efflux in only two and one patients, respectively, even at concentrations higher than 500I.U./ml. In contrast to Scala et al. (1991), this finding suggests that at a well tolerated concentration IFN-α may not be suitable for use as a P-gp modulating agent in vivo in B-CLL, although conclusive evidence would require a larger study.