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PubMed2005

Are MTT assays the right tool to analyze drug resistance caused by ABC-transporters in patient samples?

Drug resistance can be caused by ATP-binding-cassette (ABC)-transporters which function as outward pumps for chemotherapeutic drugs. The aim of the present study was to analyze the association between eight ABC-transporters (BCRP, MDR1, SMRP, MRP1, MRP2, MRP3, MRP4, and MRP5) and in vitro drug resistance. Leukemic cells from 52 children with previously untreated acute leukemia (ALL: n=37; AML: n=15) were analysed. The expression of the ABC-transporters was measured by TaqMan real-time PCR. In vitro drug resistance to cytarabine, vincristine, tioguanine, daunorubicin, etoposide, dexamethasone, and prednisone was analysed with methyl-thiazol-tetrazolium (MTT) assays.MDR1 was weakly associated with resistance to vincristine (p<0.05) in AML samples. No other correlation between an ABC-transporter and a higher in vitro drug resistance was found. In vitro drug resistance was not associated with the simultaneous expression of a larger number of ABC-transporters.MTT assays are a widely used and validated method to analyse in vitro drug resistance but they may not be a useful tool to detect resistance which is caused by drug efflux in patient samples. If that is the case, MTT assays and the expression of ABC-transporters could provide complementary information on the drug resistance profile of patients with acute leukemia.

ATP-Binding Cassette Transporters↗

PubMed1985

Immunoperoxidase determination of terminal deoxynucleotidyl transferase in acute leukemia using PAP and ABC methods: experience in 102 cases.

Immunoperoxidase (IP) and immunofluorescence (IF) technics for the detection of terminal deoxynucleotidyl transferase (TdT) were applied to 102 cases of acute leukemia to compare their relative usefulness in the diagnosis and classification of acute leukemia. Two different IP technics were used, peroxidase-antiperoxidase (PAP) on 50 cases, avidin-biotin-peroxidase complex (ABC) on 42 cases, and both PAP and ABC on ten cases. Using 40% TdT+ cells to define positivity, 71 of 102 cases were IP+/IF+, 12 were IP+/IF-, and 19 were IP/IF-. The finding of 12 IP+/IF- cases suggests greater sensitivity of the IP method in detecting TdT+ acute leukemia. This greater sensitivity was demonstrated by both PAP and ABC technics. Direct comparison of ABC and PAP technics in ten cases showed the results by both methods to be similar. In addition, previous reports utilizing the IF method found approximately 10% of cases of AML to have greater than 10% TdT+ cells. The authors' findings for the IF method were similar (15%), but using the IP methods, the authors were able to detect greater than 10% TdT+ cells in 46% of AML cases and greater than 40% TdT+ cells in 31% of AML cases. The authors conclude that the IP technics for the detection of TdT offer several advantages and may be more sensitive relative to the IF method. Further, either the PAP or the ABC method is suitable for routine use in hematopathology laboratories.

Acute Disease↗

PubMed1986

Quantitation of carcinogen-induced DNA damage and repair in human cells with the UVR ABC excision nuclease from Escherichia coli.

Recent studies by others have shown that the endonuclease complex coded for by the uvrA, uvrB and uvrC genes of Escherichia coli (UVR ABC excision nuclease) can incise DNA containing a variety of 'bulk-type' lesions, such as those resulting from u.v. light, (+/-)-7 alpha,8 beta-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE), and N-acetoxy-2-acetylaminofluorene. Using partially purified UVR ABC excision nuclease, we have quantitated the number of endonuclease sensitive sites (ESS) in purified DNA isolated from human fibroblasts treated with u.v. light or BPDE. The number of ESS/10(8) daltons of DNA were calculated from the number average mol. wt. of the DNA as determined by sedimentation in alkaline sucrose gradients. The number of endonuclease sites increased linearly with increasing doses of either u.v. light or BPDE. The UVR ABC excision nuclease was able to incise a majority of the BPDE-DNA adducts. Xeroderma pigmentosum fibroblasts, complementation group A (XP12BE) had 20-25% more ESS at each dose than the BPDE-treated normal (HSBP) cells. Cells treated with 4 microM BPDE and allowed 12 h of incubation to perform excision repair showed removal of 60% of the initial number of ESS from HSBP DNA and 40% of the ESS from XP-A DNA. Beyond 12 h XP12BE cells lost no additional ESS while HSBP cells continued to lose ESS, although at a slower rate, until at 48 h only 22% of the initial ESS remained. In cells treated with 10 J/m2 of u.v. light, the UVR ABC excision nuclease detected 60% of the sites recognized by the pyridimine dimer specific Micrococcus luteus glycosylase/apyrimidinic endonuclease. These results demonstrate the potential use of the UVR ABC excision nuclease in a quantitative assay for determining the number of carcinogen-induced lesions in human DNA.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

PubMed2000

The role of abacavir (ABC, 1592) in antiretroviral therapy-experienced patients: results from a randomized, double-blind, trial. CNA3002 European Study Team.

OBJECTIVE: To compare the antiviral activity of abacavir (ABC) with stable background therapy (SBG) and SBG alone in antiretroviral therapy-experienced subjects as demonstrated by the proportion of subjects with plasma HIV-1 RNA < or = 400 copies/ml, plasma HIV-1 RNA and CD4 cell count profiles, and safety and tolerance of the two regimens over 16 weeks. DESIGN: One-hundred and eighty-five HIV-1 infected adults, with CD4 cell counts > or = 100 x 10(6)/l and plasma HIV-1 RNA of 400-50,000 copies/ml and who had received SBG therapy for at least 12 weeks, were randomized to receive ABC (300 mg twice daily) or placebo in a double blind, multi-centre study. METHODS: Antiretroviral activity was assessed by measuring changes in plasma HIV-1 RNA levels and CD4 cell counts. Genotypic and phenotypic resistance was determined at baseline and week 16. Evaluation of safety and tolerance was based on clinical adverse events and laboratory analyses. RESULTS: At week 16 significantly more subjects receiving ABC + SBG had plasma HIV-1 RNA < or = 400 copies/ml (36/92, 39%) than subjects receiving SBG alone (7/93, 8%; P < 0.001). A similar response was observed in both the lamivudine naive and lamivudine-experienced subjects. The presence of the M184V mutation did not preclude an antiviral response to ABC; 73% of subjects with the M184V mutation alone experienced a > or = 1.0 log10 copies/ml reduction in plasma HIV-1 RNA or had a value of < or = 400 copies/ml by week 16. CONCLUSIONS: ABC was generally well tolerated and exerted significant antiviral effect when added to combination antiretroviral therapy over 16 weeks.

Adolescent↗

PubMed1991

Radiographic and histologic effects of chondroitinase ABC on normal canine lumbar intervertebral disc.

Twelve dogs were divided into two equal groups and given lumbar intradiscal injections of 10, 50, or 100 U/ml of chondroitinase ABC reconstituted in sodium acetate buffer. Radiographs of the lumbar spine were made before and after surgery in both groups. Additional films were made at 5 days after surgery in Group I and at 7, 14, and 21 days after surgery in Group II. All spaces injected with 50 or 100 U/ml chondroitinase ABC demonstrated significant radiographic narrowing in both groups compared with uninjected control and buffer injected discs (P less than 0.001). Discs injected with 10 U/ml of chondroitinase ABC showed increased narrowing over time from 7 to 21 days (P less than 0.05). A zone of safranin O depletion was present in the ventral anulus fibrosus adjacent to the nucleus pulposus in all treated discs, indicating proteoglycan loss. All histologic effects of chondroitinase ABC were confined to intervertebral disc tissues. Chondroitinase ABC appears to be effective for chemonucleolysis in dogs.

Animals↗

PubMed2000

ABC transporters in cellular lipid trafficking.

ATP-binding cassette (ABC) transporters constitute a group of evolutionary highly conserved cellular transmembrane transport proteins. Recent work has implicated ABC transporters in cellular transmembrane lipid transport and hereditary diseases have been causatively linked to defective ABC transporters translocating lipid compounds. The emerging concept that a defined subset of ABC transporters is intimately involved in cellular lipid trafficking has recently been substantiated convincingly by the finding that ABCA1 plays a central role in the regulation of HDL metabolism and macrophage targeting to the RES or the vascular wall. Differentiation dependent expression of a large number of ABC transporters in monocytes/macrophages and their regulation by sterol flux render these transporter molecules potentially critical players in atherogenesis and other chronic inflammatory diseases.

ATP Binding Cassette Transporter 1↗

PubMed2001

The human ATP-binding cassette (ABC) transporter superfamily.

The ATP-binding cassette (ABC) transporter superfamily contains membrane proteins that translocate a variety of substrates across extra- and intra-cellular membranes. Genetic variation in these genes is the cause of or contributor to a wide variety of human disorders with Mendelian and complex inheritance, including cystic fibrosis, neurological disease, retinal degeneration, cholesterol and bile transport defects, anemia, and drug response. Conservation of the ATP-binding domains of these genes has allowed the identification of new members of the superfamily based on nucleotide and protein sequence homology. Phylogenetic analysis is used to divide all 48 known ABC transporters into seven distinct subfamilies of proteins. For each gene, the precise map location on human chromosomes, expression data, and localization within the superfamily has been determined. These data allow predictions to be made as to potential functions or disease phenotypes associated with each protein. In this paper, we review the current state of knowledge on all human ABC genes in inherited disease and drug resistance. In addition, the availability of the complete Drosophila genome sequence allows the comparison of the known human ABC genes with those in the fly genome. The combined data enable an evolutionary analysis of the superfamily. Complete characterization of all ABC from the human genome and from model organisms will lead to important insights into the physiology and the molecular basis of many human disorders.

ATP-Binding Cassette Transporters↗

PubMed1998

A comparative analysis of ABC transporters in complete microbial genomes.

The ABC transporter is a major class of cellular translocation machinery in all bacterial species encoded in the largest set of paralogous genes. The operon structure is frequently found for the genes of three molecular components: the ATP-binding protein, the membrane protein, and the substrate-binding protein. Here, we developed an "ortholog group table" by comparison and classification of known and putative ABC transporters in the complete genomes of seven microorganisms. Our procedure was to first search and classify the most conserved ATP-binding protein components by the sequence similarity and then to classify the entire transporter units by examining the similarity of the other components and the conservation of the operon structure. The resulting 25 ortholog groups of ABC transporters were well correlated with known functions. Through the analysis, we could assign substrate specificity to hypothetical transporters, predict additional transporter operons, and identify novel types of putative transporters. The ortholog group table was also used as a reference data set for functional assignment in four additional genomes. In general, the ABC transporter operons were strongly conserved despite the extensive shuffling of gene locations in bacterial evolution. In Synechocystis, however, the tendency of forming operons was clearly diminished. Our result suggests that the ancestral ABC transporter operons may have arisen early in evolution before the speciation of bacteria and archaea.

ATP-Binding Cassette Transporters↗

PubMed2002

Identification of ABC transporters in vancomycin-resistant Enterococcus faecium as potential targets for antibody therapy.

The occurrence of an outbreak of septicaemias due to vancomycin-resistant Enterococcus faecium (VRE), in Manchester, UK, provided an opportunity to examine the antibody responses in patients infected by the same strain. Immunoblotting sera from 24 cases, six of whom died, showed an immunodominant cluster of antigens at 34, 54 and 97 kDa, with a statistically significant correlate between survival and immunoglobulin G to the 34 and 97 kDa bands (P<0.05). Screening a genomic expression library of VRE with seropositive serum and peritoneal dialysate from a survivor gave a recombinant clone with two contiguous open reading frames, the derived amino acid sequences of which both showed sequence homologue with ABC transporters, with a Walker A and Walker B motif and the signature sequence LSGGQ. The first open reading frame (putative VRE ABC1) showed 57% homologue with YbxA from Bacillus subtilis. A partial sequence (putative VRE ABC2) was also obtained, in the same recombinant clone, of a second ABC transporter with 72% homologue with ybaE from B. subtilis. Affinity selection with the seropositive serum and peritoneal dialysate used to screen the library showed that the eluted antibody bound to the 97, 54, 34 and 30 kDa bands. Direct amino acid sequencing identified this as a possible ABC transporter. Rabbit antiserum against peptides representing Walker A and an area adjacent to the Walker B site cross-reacted with bands at 34, 54, 97, 110 kDa and at 30, 34 and 54 kDa respectively. This therefore appeared to be an immunodominant complex of ABC transporters of which the smallest was the 30 kDa antigen. Epitope mapping of this antigen with seropositive patients' sera delineated three linear epitopes (KVGIV, FGPKNF and RVAI). The Walker A site represented by peptide 1 (GHNGSGKSTLAKTIN), epitope RVAI represented by peptides 2 (MRRVAIAGVLAMPRE) and 3 (ELSGGQMRRVAIAGV), epitope KVGIV represented by peptide 4 (LKPIRKKVGIVFQFP), and recombinant VRE ABC1 and VRE ABC2 expressed in Escherichia coli pBAD were then used to isolate human genetically recombinant antibodies from a phage antibody display library. An assessment of the protective potential of these antibodies was carried out in a mouse model of the infection. This study suggests that an ABC transporter homologue could be a target for antibody therapy against VRE infections.

ATP-Binding Cassette Transporters↗

PubMed1999

Subunit interactions in ABC transporters: towards a functional architecture.

The ABC superfamily is a diverse group of integral membrane proteins involved in the ATP-dependent transport of solutes across biological membranes in both prokaryotes and eukaryotes. Although ABC transporters have been studied for over 30 years, very little is known about the mechanism by which the energy of ATP hydrolysis is used to transport substrate across the membrane. The recent report of the high resolution crystal structure of HisP, the nucleotide-binding subunit of the histidine permease complex of Salmonella typhimurium, represents a significant breakthrough toward the elucidation of the mechanism of solute translocation by ABC transporters. In this review, we use data from the crystallographic structures of HisP and other nucleotide-binding proteins, combined with sequence analysis of a subset of atypical ABC transporters, to argue a new model for the dimerisation of the nucleotide-binding domains that embraces the notion that the C motif from one subunit forms part of the ATP-binding site in the opposite subunit. We incorporate this dimerisation of the ATP-binding domains into our recently reported beta-barrel model for P-glycoprotein and present a general model for the cooperative interaction of the two nucleotide-binding domains and the translocation of mechanical energy to the transmembrane domains in ABC transporters.

ATP-Binding Cassette Transporters↗

PubMed2000

The ATP binding cassette (ABC) transport systems of Mycobacterium tuberculosis.

We have undertaken the inventory and assembly of the typical subunits of the ABC transporters encoded by the complete genome of Mycobacterium tuberculosis. These subunits, i.e. the nucleotide binding domains (NBDs), the membrane-spanning domains (MSDs) and the substrate binding proteins (SBPs), were identified on the basis of their characteristic stretches of amino acids and/or conserved structure. A total of 45 NBDs present in 38 proteins, of 47 MSDs present in 44 proteins and of 15 SBPs were found to be encoded by M. tuberculosis. Analysis of transcriptional clusters and searches of homology between the identified subunits of the transporters and proteins characterized in other organisms allowed the reconstitution of at least 26 complete (including at least one NBD and one MSD) and 11 incomplete ABC transporters. Sixteen of them were unambiguously classified as importers whereas 21 were presumed to be exporters. By searches of homology with already known transporters from other organisms, potential substrates (peptides, macrolides, carbohydrates, multidrugs, antibiotics, iron, anions) could be attributed to 30 of the ABC transporters identified in M. tuberculosis. The ABC transporters have been further classified in nine different sub-families according to a tree obtained from the clustering of their NBDs. Contrary to Escherichia coli and similarly to Bacillus subtilis, there is an equal representation of extruders and importers. Many exporters were found to be potentially implicated in the transport of drugs, probably contributing to the resistance of M. tuberculosis to many antibiotics. Interestingly, a transporter (absent in E. coli and in B. subtilis) potentially implicated in the export of a factor required for the bacterial attachment to the eukaryotic host cells was also identified. In comparison to E. coli and B. subtilis, there is an under-representation of the importers (with the exception of the phosphate importers) in M. tuberculosis. This may reflect the capacity of this bacterium to synthesize many essential compounds and to grow in the presence of few external nutrients. The genes encoding the ABC transporters occupy about 2.5% of the genome of M. tuberculosis.

ATP-Binding Cassette Transporters↗

PubMed2005

Complete inventory of ABC proteins in human pathogenic yeast, Candida albicans.

The recent completion of the sequencing project of the opportunistic human pathogenic yeast, Candida albicans (http://www.ncbi.nlm.nih.gov/), led us to analyze and classify its ATP-binding cassette (ABC) proteins, which constitute one of the largest superfamilies of proteins. Some of its members are multidrug transporters responsible for the commonly encountered problem of antifungal resistance. TBLASTN searches together with domain analysis identified 81 nucleotide-binding domains, which belong to 51 different putative open reading frames. Considering that each allelic pair represents a single ABC protein of the Candida genome, the total number of putative members of this superfamily is 28. Domain organization, sequence-based analysis and self-organizing map-based clustering led to the classification of Candida ABC proteins into 6 distinct subfamilies. Each subfamily from C. albicans has an equivalent in Saccharomyces cerevisiae suggesting a close evolutionary relationship between the two yeasts. Our searches also led to the identification of a new motif to each subfamily in Candida that could be used to identify sequences from the corresponding subfamily in other organisms. It is hoped that the inventory of Candida ABC transporters thus created will provide new insights into the role of ABC proteins in antifungal resistance as well as help in the functional characterization of the superfamily of these proteins.

ATP-Binding Cassette Transporters↗

PubMed1986

Suppression of nonspecific binding of avidin-biotin complex (ABC) to proteins electroblotted to nitrocellulose paper.

Nitrocellulose blots of cell extracts reacted in sequence with biotinylated lectins and horseradish peroxidase-labeled avidin-biotin complex (ABC) often show considerable nonspecific staining of protein bands. Experiments were performed to determine which of the components of the ABC were responsible for this and whether or not the nature and ionic strength of the buffer used could alter this binding. Furthermore, as powdered non-fat milk has been proposed as a possible blocking agent for nonspecific binding of ABC, we sought to determine if it would adequately block that binding in our system. The initial experiments showed that nonspecific binding of ABC to proteins transferred to nitrocellulose membranes was due to the avidin component of the ABC; little, if any, binding was seen if biotin alone was incubated with these blots. The spurious binding was shown to be primarily due to the high affinity of avidin to proteins electroblotted to nitrocellulose, when incubated in low-salt buffers. Low-fat milk added to the buffer reduced overall nonspecific reactivity but produced additional artifacts in the form of bands that were not seen in other preparations. Nonspecific avidin binding to proteins transferred to nitrocellulose can therefore be effectively reduced by adding extra salt to buffers, whereas the addition of non-fat dry milk does not seem suitable for this purpose.

Animals↗

PubMed1995

The SA/rABC technique: a new ABC procedure for detection of antigens at increased sensitivity.

Since its introduction, the avidin-biotin-peroxidase (ABC) complex has become an invaluable detection system for a wide variety of bioanalytical applications. In these techniques, avidin and biotin-peroxidase are mixed at a pre-determined ratio so that the soluble ABC complex retains biotin binding sites. Consequently, the complex contains an excess of avidin over biotinylated peroxidase residues. On theoretical considerations, however, an ABC complex designed for maximal signal intensity must consist of an excess of peroxidase over avidin molecules. Therefore, ABC complexes with reversed molar ratios of biotinylated peroxidase to avidin (rABC complexes) were prepared and an intermediate streptavidin step was introduced to bind the rABC complexes to biotinylated IgG molecules. The signal generating power of this new streptavidin-rABC sequence proved superior to that of the conventional ABC technique in ELISA assays and in immunostaining of tissue sections.

Animals↗

PubMed2004

The ABC transporter gene family of Caenorhabditis elegans has implications for the evolutionary dynamics of multidrug resistance in eukaryotes.

BACKGROUND: Many drugs of natural origin are hydrophobic and can pass through cell membranes. Hydrophobic molecules must be susceptible to active efflux systems if they are to be maintained at lower concentrations in cells than in their environment. Multi-drug resistance (MDR), often mediated by intrinsic membrane proteins that couple energy to drug efflux, provides this function. All eukaryotic genomes encode several gene families capable of encoding MDR functions, among which the ABC transporters are the largest. The number of candidate MDR genes means that study of the drug-resistance properties of an organism cannot be effectively carried out without taking a genomic perspective. RESULTS: We have annotated sequences for all 60 ABC transporters from the Caenorhabditis elegans genome, and performed a phylogenetic analysis of these along with the 49 human, 30 yeast, and 57 fly ABC transporters currently available in GenBank. Classification according to a unified nomenclature is presented. Comparison between genomes reveals much gene duplication and loss, and surprisingly little orthology among analogous genes. Proteins capable of conferring MDR are found in several distinct subfamilies and are likely to have arisen independently multiple times. CONCLUSIONS: ABC transporter evolution fits a pattern expected from a process termed 'dynamic-coherence'. This is an unusual result for such a highly conserved gene family as this one, present in all domains of cellular life. Mechanistically, this may result from the broad substrate specificity of some ABC proteins, which both reduces selection against gene loss, and leads to the facile sorting of functions among paralogs following gene duplication.

ATP-Binding Cassette Transporters↗

PubMed2005

Mechanisms of resistance to anticancer drugs: the role of the polymorphic ABC transporters ABCB1 and ABCG2.

ATP-binding cassette (ABC) genes play a role in the resistance of malignant cells to anticancer agents. The ABC gene products, including ABCB1 (P-glycoprotein) and ABCG2 (breast cancer-resistance protein [BCRP], mitoxantrone-resistance protein [MXR], or ABC transporter in placenta [ABCP]), are also known to influence oral absorption and disposition of a wide variety of drugs. As a result, the expression levels of these proteins in humans have important consequences for an individual's susceptibility to certain drug-induced side effects, interactions, and treatment efficacy. Naturally occurring variants in ABC transporter genes have been identified that might affect the function and expression of the protein. This review focuses on recent advances in the pharmacogenetics of the ABC transporters ABCB1 and ABCG2, and discusses potential implications of genetic variants for the chemotherapeutic treatment of cancer.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2005

Adventures with ABC-proteins: highly conserved ATP-dependent transporters.

The general properties of ABC transporters, from bacteria to humans, including a brief history of their initial discovery, are considered. ABC transporters, one of the largest protein super families and vital for human health, are in toto responsible for the transport of an enormous range of molecules from ions (CFTR) or anti-tumour drugs (Pgp/MDR) to large polypeptides. Nevertheless, all ABC transporters are powered by a conserved ATPase the ABC or NBD domain, using in all probability the same basic mechanism of action for the hydrolysis of ATP and its coupling to the transport process. Based on recent high resolution structures of several NBDs and an intact transporter, a model of how dimers of these important proteins function will be discussed, with particular attention to HlyB, the ABC transporter from E. coli.

ATP-Binding Cassette Transporters↗

PubMed2005

[Reliability and validity of a new assessment of the burden on caregivers (ABC-16)].

The aim of this study was to evaluate the reliability and validity of a newly developed self-administered assessment of the burden on caregivers (ABC-16) in 82 caregivers (21 men: 61 women, mean age 61 +/- 13 years) including 51 family members caring for patients after stroke with physical impairments, and 31 family members caring for patents with chronic schizophrenia. The ABC-16 consists of 16 items and is designed to cover 4 domains (burden of troubles with care receivers, burden of loss of social life, financial burden, burden of impairment of caregiver's health). The internal consistency was high (alpha=0.854) and significant correlations among the four dimensions were found (P < 0.0001). Factorial analysis showed 5 latent factors and a multiple regression analysis showed the strong correlations between the ABC-16 and care giving during the night (P = 0.0045), which strongly suggested excellent validation. The mean and standard deviation of the total score of the ABC-16 was 14.66 +/- 7.87 (burden of troubles with care receivers; 4.10 +/- 2.36, burden of loss of social life; 4.21 +/- 2.54, financial burden; 2.93 +/- 2.65, burden of impairment of caregiver's health; 3.43 +/- 2.32). The results indicated that the ABC-16 was a tool assess care burden with high reliability and validity, and was carried out easily and quickly by all participants without complex explains.

Adult↗