Valutazione del coinvolgimento dell'oncogene c-myc e dei trasportatori abc nella farmacoresistenza dell'osteosarcoma ad alto grado

L’insorgenza di fenomeni coinvolti nello sviluppo della farmacoresistenza costituisce al momento la principale causa di mancata risposta al trattamento chemioterapico nell’osteosarcoma. Questo è in parte dovuto ad una sovraespressione di diversi trasportatori ABC nelle cellule tumorali che causano un aumento dell’efflusso extracellulare del chemioterapico e pertanto una ridotta risposta al trattamento farmacologico. L'oncogene C-MYC è coinvolto nella resistenza al metothrexate, alla doxorubicina e al cisplatino ed è un fattore prognostico avverso, se sovraespresso al momento della diagnosi, in pazienti affetti da osteosarcoma. C-MYC è in grado di regolare l'espressione di diversi trasportatori ABC, probabilmente coinvolti nella resistenza ai farmaci nell’osteosarcoma, e questo potrebbe spiegare l’impatto prognostico avverso dell’oncogene in questo tumore. L’espressione genica di C-MYC e di 16 trasportatori ABC, regolati da C-MYC e / o responsabili dell'efflusso di diversi chemioterapici, è stata valutata su due diverse casistiche cliniche e su un pannello di linee cellulari di osteosarcoma umano mediante real-time PCR. L'espressione della proteina è stata valutata per i 9 trasportatori ABC risultati più rilevanti.Infine l'efficacia in vitro di un inibitore, specifico per ABCB1 e ABCC1, è stata valutata su linee cellulari di osteosarcoma. ABCB1 e ABCC1 sono i trasportatori più espressi nelle linee cellulari di osteosarcoma. ABCB1 è sovraespresso al momento della diagnosi in circa il 40-45% dei pazienti affetti da osteosarcoma e si conferma essere un fattore prognostico avverso se sovraespresso al momento della diagnosi. Pertanto ABCB1 diventa il bersaglio di elezione per lo sviluppo di strategie terapeutiche alternative, nel trattamento dell’osteosarcoma, atte al superamento della farmacoresistenza. L’inibizione dell'attività di tale trasportatore causa un aumento della sensibilità al trattamento chemioterapico nelle linee cellulari di osteosarcoma farmacoresistenti, indicando questo approccio come una possibile strategia per superare il problema della mancata risposta al trattamento farmacologico nei pazienti con osteosarcoma che sovraesprimono ABCB1.

Differential expression of ABC transporters and their regulatory genes during lactation and dry period in bovine mammary tissue

ATP-binding cassette (ABC) transporters play a pivotal role in human physiology, and mutations in these genes often result in severe hereditary diseases. ABC transporters are expressed in the bovine mammary gland but their physiological role in this organ remains elusive. Based on findings in the context of human disorders we speculated that candidate ABC transporters are implicated in lipid and cholesterol transport in the mammary gland. Therefore we investigated the expression pattern of selected genes that are associated with sterol transport in lactating and nonlactating mammary glands of dairy cows. mRNA levels from mammary gland biopsies taken during lactation and in the first and second week of the dry period were analysed using quantitative PCR. Five ABC transporter genes, namely ABCA1, ABCA7, ABCG1, ABCG2 and ABCG5, their regulating genes LXRalpha, PPARgamma, SREBP1 and the milk proteins lactoferrin and alpha-lactalbumin were assessed. A significantly enhanced expression in the dry period was observed for ABCA1 while a significant decrease of expression in this period was detected for ABCA7, ABCG2, SREBP1 and alpha-lactalbumin. ABCG1, ABCG5, LXRalpha, PPARgamma and lactoferrin expression was not altered between lactation and dry period. These results indicate that candidate ABC transporters involved in lipid and cholesterol transport show differential mRNA expression between lactation and the dry period. This may be due to physiological changes in the mammary gland such as immigration of macrophages or the accumulation of fat due to the loss of liquid in the involuting mammary gland. The current mRNA expression analysis of transporters in the mammary gland is the prerequisite for elucidating novel molecular mechanisms underlying cholesterol and lipid transfer into milk.

Distinct functions and cooperative interaction of the subunits of the transporter associated with antigen processing (TAP)

The ATP-binding cassette (ABC) transporter TAP translocates peptides from the cytosol to awaiting MHC class I molecules in the endoplasmic reticulum. TAP is made up of the TAP1 and TAP2 polypeptides, which each possess a nucleotide binding domain (NBD). However, the role of ATP in peptide binding and translocation is poorly understood. We present biochemical and functional evidence that the NBDs of TAP1 and TAP2 are non-equivalent. Photolabeling experiments with 8-azido-ATP demonstrate a cooperative interaction between the two NBDs that can be stimulated by peptide. The substitution of key lysine residues in the Walker A motifs of TAP1 and TAP2 suggests that TAP1-mediated ATP hydrolysis is not essential for peptide translocation but that TAP2-mediated ATP hydrolysis is critical, not only for translocation, but for peptide binding.

A Saccharomyces cerevisiae homolog of the human adrenoleukodystrophy transporter is a heterodimer of two half ATP-binding cassette transporters.

The adrenoleukodystrophy protein (ALDP) and the 70-kDa peroxisomal membrane protein (PMP70) are half ATP-binding cassette (ABC) transporters in the human peroxisome membrane. ALDP and PMP70 share sequence homology and both are implicated in genetic diseases. PXA1 and YKL741 are Saccharomyces cerevisiae genes that encode homologs of ALDP and PMP70. Pxa1p, a putative ortholog of ALDP, is involved in peroxisomal beta-oxidation of fatty acids while YKL741 is an open reading frame found by the yeast genome sequencing project. Here we designate YKL741 as PXA2 and show that its protein product, Pxa2p, like Pxa1p, is associated with peroxisomes but not required for their assembly. Yeast strains carrying gene disruption of PXA1, PXA2, or both have similar and, in the case of the latter, nonadditive phenotypes. We also find that the stability of Pxa1p, but not Pxa2p, is markedly reduced in the absence of the other. Finally, we find that Pxa1p and Pxa2p coimmuno-precipitate. These genetic and physical data suggest that Pxa1p and Pxa2p heterodimerize to form a complete peroxisomal ABC transporter involved in fatty acid beta-oxidation. This result predicts the presence of similar heterodimeric ABC transporters in the mammalian peroxisome membrane.

Pathogen-responsive expression of a putative ATP-binding cassette transporter gene conferring resistance to the diterpenoid sclareol is regulated by multiple defense signaling pathways in arabidopsis

The ATP-binding cassette (ABC) transporters are encoded by large gene families in plants. Although these proteins are potentially involved in a number of diverse plant processes, currently, very little is known about their actual functions. In this paper, through a cDNA microarray screening of anonymous cDNA clones from a subtractive library, we identified an Arabidopsis gene (AtPDR12) putatively encoding a member of the pleiotropic drug resistance (PDR) subfamily of ABC transporters. AtPDR12 displayed distinct induction profiles after inoculation of plants with compatible and incompatible fungal pathogens and treatments with salicylic acid, ethylene, or methyl jasmonate. Analysis of AtPDR12 expression in a number of Arabidopsis defense signaling mutants further revealed that salicylic acid accumulation, NPR1. function, and sensitivity to jasmonates and ethylene were all required for pathogen-responsive expression of AtPDR12. Germination assays using seeds from an AtPDR12 insertion line in the presence of sclareol resulted in lower germination rates and much stronger inhibition of root elongation in the AtPDR12 insertion line than in wild-type plants. These results suggest that AtPDR12 may be functionally related to the previously identified ABC transporters SpTUR2 and NpABC1, which transport sclareol. Our data also point to a potential role for terpenoids in the Arabidopsis defensive armory.

Phosphate Regulated Proteins Of Xanthomonas Citri Subsp. Citri: A Proteomic Approach.

Xanthomonas citri subsp. citri (X. citri) is the causative agent of the citrus canker, a disease that affects several citrus plants in Brazil and across the world. Although many studies have demonstrated the importance of genes for infection and pathogenesis in this bacterium, there are no data related to phosphate uptake and assimilation pathways. To identify the proteins that are involved in the phosphate response, we performed a proteomic analysis of X. citri extracts after growth in three culture media with different phosphate concentrations. Using mass spectrometry and bioinformatics analysis, we showed that X. citri conserved orthologous genes from Pho regulon in Escherichia coli, including the two-component system PhoR/PhoB, ATP binding cassette (ABC transporter) Pst for phosphate uptake, and the alkaline phosphatase PhoA. Analysis performed under phosphate starvation provided evidence of the relevance of the Pst system for phosphate uptake, as well as both periplasmic binding proteins, PhoX and PstS, which were formed in high abundance. The results from this study are the first evidence of the Pho regulon activation in X. citri and bring new insights for studies related to the bacterial metabolism and physiology. Biological significance Using proteomics and bioinformatics analysis we showed for the first time that the phytopathogenic bacterium X. citri conserves a set of proteins that belong to the Pho regulon, which are induced during phosphate starvation. The most relevant in terms of conservation and up-regulation were the periplasmic-binding proteins PstS and PhoX from the ABC transporter PstSBAC for phosphate, the two-component system composed by PhoR/PhoB and the alkaline phosphatase PhoA.

The Relationship between Population Structure and Aluminum Tolerance in Cultivated Sorghum

Background: Acid soils comprise up to 50% of the world's arable lands and in these areas aluminum (Al) toxicity impairs root growth, strongly limiting crop yield. Food security is thereby compromised in many developing countries located in tropical and subtropical regions worldwide. In sorghum, SbMATE, an Al-activated citrate transporter, underlies the Alt(SB) locus on chromosome 3 and confers Al tolerance via Al-activated root citrate release. Methodology: Population structure was studied in 254 sorghum accessions representative of the diversity present in cultivated sorghums. Al tolerance was assessed as the degree of root growth inhibition in nutrient solution containing Al. A genetic analysis based on markers flanking Alt(SB) and SbMATE expression was undertaken to assess a possible role for Alt(SB) in Al tolerant accessions. In addition, the mode of gene action was estimated concerning the Al tolerance trait. Comparisons between models that include population structure were applied to assess the importance of each subpopulation to Al tolerance. Conclusion/Significance: Six subpopulations were revealed featuring specific racial and geographic origins. Al tolerance was found to be rather rare and present primarily in guinea and to lesser extent in caudatum subpopulations. Alt(SB) was found to play a role in Al tolerance in most of the Al tolerant accessions. A striking variation was observed in the mode of gene action for the Al tolerance trait, which ranged from almost complete recessivity to near complete dominance, with a higher frequency of partially recessive sources of Al tolerance. A possible interpretation of our results concerning the origin and evolution of Al tolerance in cultivated sorghum is discussed. This study demonstrates the importance of deeply exploring the crop diversity reservoir both for a comprehensive view of the dynamics underlying the distribution and function of Al tolerance genes and to design efficient molecular breeding strategies aimed at enhancing Al tolerance.

Crystallization, data collection and data processing of maltose-binding protein (MalE) from the phytopathogen Xanthomonas axonopodis pv. citri

Maltose-binding protein is the periplasmic component of the ABC transporter responsible for the uptake of maltose/maltodextrins. The Xanthomonas axonopodis pv. citri maltose-binding protein MalE has been crystallized at 293 Kusing the hanging-drop vapour-diffusion method. The crystal belonged to the primitive hexagonal space group P6(1)22, with unit-cell parameters a = 123.59, b = 123.59, c = 304.20 angstrom, and contained two molecules in the asymetric unit. It diffracted to 2.24 angstrom resolution.

Characterization of Leishmania (Leishmania) amazonensis promastigotes resistant to pentamidine

Pentamidine is a second-line agent used in the treatment of leishmaniasis and its mode of action and mechanism of resistance is not well understood. It was previously demonstrated that transfection of promastigotes and amastigotes with the ABC transporter PRP1 gene confers resistance to pentamidine. To further clarify this point, we generated Leishmania amazonensis mutants resistant to pentamidine. Our results indicated that this ABC transporter is not associated with pentamidine resistance in lines generated by drug pressure through amplification or overexpression mechanisms of PRP1 gene. (C) 2008 Elsevier Inc. All rights reserved.

Antifungal Resistance Mechanisms in Dermatophytes

Although fungi do not cause outbreaks or pandemics, the incidence of severe systemic fungal infections has increased significantly, mainly because of the explosive growth in the number of patients with compromised immune system. Thus, drug resistance in pathogenic fungi, including dermatophytes, is gaining importance. The molecular aspects involved in the resistance of dermatophytes to marketed antifungals and other cytotoxic drugs, such as modifications of target enzymes, over-expression of genes encoding ATP-binding cassette (ABC) transporters and stress-response-related proteins are reviewed. Emphasis is placed on the mechanisms used by dermatophytes to overcome the inhibitory action of terbinafine and survival in the host environment. The relevance of identifying new molecular targets, of expanding the understanding about the molecular mechanisms of resistance and of using this information to design new drugs or to modify those that have become ineffective is also discussed.

Isolation of transcripts overexpressed in the human pathogen Trichophyton rubrum grown in lipid as carbon source

Trichophyton rubrum is the most common etiological agent of human dermatophytosis. Despite the incidence and medical importance of this dermatophyte, little is known about the mechanisms of host invasion and pathogenicity. Host invasion depends on the adaptive cellular responses of the pathogen that allow it to penetrate the skin layers, which are mainly composed of proteins and lipids. In this study, we used suppression subtractive hybridization to identify transcripts over-expressed in T rubrum cultured in lipid as carbon source. Among the subtractive cDNA clones isolated, 85 clones were positively screened by cDNA array dot blotting and were sequenced. The putative proteins encoded by the isolated transcripts showed similarities to fungal proteins involved in metabolism, signaling, defense, and virulence, such as the MDR/ABC transporter, glucan 1,3-beta-glucosidase, chitin synthase B, copper-sulfate-regulated protein, and serine/threonine phosphatase (calcineurin A). These results provide the first molecular insight into the genes differentially expressed during the adaptation of T. rubrum to a lipidic carbon source.

Accumulation of manganese in Neisseria gonorrhoeae correlates with resistance to oxidative killing by superoxide anion and is independent of superoxide dismutase activity

As a facultative aerobe with a high iron requirement and a highly active aerobic respiratory chain, Neisseria gonorrhoeae requires defence systems to respond to toxic oxygen species such as superoxide. It has been shown that supplementation of media with 100 muM Mn(II) considerably enhanced the resistance of this bacterium to oxidative killing by superoxide. This protection was not associated with the superoxide dismutase enzymes of N. gonorrhoeae. In contrast to previous studies, which suggested that some strains of N. gonorrhoeae might not contain a superoxide dismutase, we identified a sodB gene by genome analysis and confirmed its presence in all strains examined by Southern blotting, but found no evidence for sodA or sodC. A sodB mutant showed very similar susceptibility to superoxide killing to that of wild-type cells, indicating that the Fe-dependent SOD B did not have a major role in resistance to oxidative killing under the conditions tested. The absence of a sodA gene indicated that the Mn-dependent protection against oxidative killing was independent of Mn-dependent SOD A. As a sodB mutant also showed Mn-dependent resistance to oxidative killing, then it is concluded that this resistance is independent of superoxide dismutase enzymes. Resistance to oxidative killing was correlated with accumulation of Mn(II) by the bacterium. We hypothesize that this bacterium uses Mn(II) as a chemical quenching agent in a similar way to the already established process in Lactobacillus plantarum. A search for putative Mn(II) uptake systems identified an ABC cassette-type system (MntABC) with a periplasmic-binding protein (MntC). An mntC mutant was shown to have lowered accumulation of Mn(II) and was also highly susceptible to oxidative killing, even in the presence of added Mn(II). Taken together, these data show that N. gonorrhoeae possesses a Mn(II) uptake system that is critical for resistance to oxidative stress.

Functional sphere profiling reveals the complexity of neuroblastoma tumor-initiating cell model.

Neuroblastoma (NB) is a neural crest-derived childhood tumor characterized by a remarkable phenotypic diversity, ranging from spontaneous regression to fatal metastatic disease. Although the cancer stem cell (CSC) model provides a trail to characterize the cells responsible for tumor onset, the NB tumor-initiating cell (TIC) has not been identified. In this study, the relevance of the CSC model in NB was investigated by taking advantage of typical functional stem cell characteristics. A predictive association was established between self-renewal, as assessed by serial sphere formation, and clinical aggressiveness in primary tumors. Moreover, cell subsets gradually selected during serial sphere culture harbored increased in vivo tumorigenicity, only highlighted in an orthotopic microenvironment. A microarray time course analysis of serial spheres passages from metastatic cells allowed us to specifically "profile" the NB stem cell-like phenotype and to identify CD133, ABC transporter, and WNT and NOTCH genes as spheres markers. On the basis of combined sphere markers expression, at least two distinct tumorigenic cell subpopulations were identified, also shown to preexist in primary NB. However, sphere markers-mediated cell sorting of parental tumor failed to recapitulate the TIC phenotype in the orthotopic model, highlighting the complexity of the CSC model. Our data support the NB stem-like cells as a dynamic and heterogeneous cell population strongly dependent on microenvironmental signals and add novel candidate genes as potential therapeutic targets in the control of high-risk NB.

Divergent functions of three Candida albicans zinc-cluster transcription factors (CTA4, ASG1 and CTF1) complementing pleiotropic drug resistance in Saccharomyces cerevisiae.

One of the mediators of pleiotropic drug resistance in Saccharomyces cerevisiae is the ABC-transporter gene PDR5. This gene is regulated by at least two transcription factors with Zn(2)-Cys(6) finger DNA-binding motifs, Pdr1p and Pdr3p. In this work, we searched for functional homologues of these transcription factors in Candida albicans. A C. albicans gene library was screened in a S. cerevisiae mutant lacking PDR1 and PDR3 and clones resistant to azole antifungals were isolated. From these clones, three genes responsible for azole resistance were identified. These genes (CTA4, ASG1 and CTF1) encode proteins with Zn(2)-Cys(6)-type zinc finger motifs in their N-terminal domains. The C. albicans genes expressed in S. cerevisiae could activate the transcription of a PDR5-lacZ reporter system and this reporter activity was PDRE-dependent. They could also confer resistance to azoles in a S. cerevisiae strain lacking PDR1, PDR3 and PDR5, suggesting that CTA4-, ASG1- and CTF1-dependent azole resistance can be caused by genes other than PDR5 in S. cerevisiae. Deletion of CTA4, ASG1 and CTF1 in C. albicans had no effect on fluconazole susceptibility and did not alter the expression of the ABC-transporter genes CDR1 and CDR2 or the major facilitator gene MDR1, which encode multidrug transporters known as mediators of azole resistance in C. albicans. However, additional phenotypic screening tests on the C. albicans mutants revealed that the presence of ASG1 was necessary to sustain growth on non-fermentative carbon sources (sodium acetate, acetic acid, ethanol). In conclusion, C. albicans possesses functional homologues of the S. cerevisiae Pdr1p and Pdr3p transcription factors; however, their properties in C. albicans have been rewired to other functions.

Selected ABCB1, ABCB4 and ABCC2 Polymorphisms Do Not Enhance the Risk of Drug-Induced Hepatotoxicity in a Spanish Cohort.

BACKGROUND AND AIMS Flawed ABC transporter functions may contribute to increased risk of drug-induced liver injury (DILI). We aimed to analyse the influence of genetic variations in ABC transporters on the risk of DILI development and clinical presentations in a large Spanish DILI cohort. METHODS A total of ten polymorphisms in ABCB1 (1236T>C, 2677G>T,A, 3435T>C), ABCB4 (1954A>G) and ABCC2 (-1774G>del, -1549A>G, -24C>T, 1249G>A, 3972C>T and 4544G>A) were genotyped using Taqman 5' allelic discrimination assays or sequencing in 141 Spanish DILI patients and 161 controls. The influence of specific genotypes, alleles and haplotypes on the risk of DILI development and clinical presentations was analysed. RESULTS None of the individual polymorphisms or haplotypes was found to be associated with DILI development. Carriers homozygous for the ABCC2 -1774del allele were however only found in DILI patients. Hence, this genotype could potentially be associated with increased risk, though its low frequency in our Spanish cohort prevented a final conclusion. Furthermore, carriers homozygous for the ABCC2 -1774G/-1549A/-24T/1249G/3972T/4544G haplotype were found to have a higher propensity for total bilirubin elevations when developing DILI. CONCLUSIONS Our findings do not support a role for the analysed polymorphisms in the ABCB1, ABCB4 and ABCC2 transporter genes in DILI development in Spanish patients. The ABCC2 -1774deldel genotype was however restricted to DILI cases and could potentially contribute to enhanced DILI susceptibility.