ABSTRACT
Understanding the mechanisms of multidrug resistance (MDR) could improve clinical drug efficacy. Multidrug resistance is associated with ATP binding cassette (ABC) transporters, but the factors that regulate their expression at clinically relevant drug concentrations are poorly understood. We report that a single-step selection with low doses of anti-cancer agents, similar to concentrations reported in vivo, induces MDR that is mediated exclusively by ABCG2. We selected breast, ovarian and colon cancer cells (MCF-7, IGROV-1 and S-1) after exposure to 14 or 21 nM doxorubicin for only 10 days. We found that these cells overexpress ABCG2 at the mRNA and protein levels. RNA interference analysis confirmed that ABCG2 confers drug resistance. Furthermore, ABCG2 upregulation was facilitated by histone hyperacetylation due to weaker histone deacetylase 1-promoter association, indicating that these epigenetic changes elicit changes in ABCG2 gene expression. These studies indicate that the MDR phenotype arises following low-dose, single-step exposure to doxorubicin, and further suggest that ABCG2 may mediate early stages of MDR development. This is the first report to our knowledge of single-step, low-dose selection leading to overexpression of ABCG2 by epigenetic changes in multiple cancer cell lines.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Antibiotics, Antineoplastic/pharmacology , Doxorubicin/pharmacology , Epigenesis, Genetic , Neoplasm Proteins/metabolism , Neoplasms/drug therapy , Neoplasms/metabolism , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Acetylation , Antineoplastic Agents/pharmacology , Blotting, Western , Breast Neoplasms/drug therapy , Breast Neoplasms/metabolism , Cell Line, Tumor , Chromatin Immunoprecipitation , Colonic Neoplasms/drug therapy , Colonic Neoplasms/metabolism , Drug Resistance, Multiple , Drug Resistance, Neoplasm , Female , Gene Expression Regulation, Neoplastic , Histones/metabolism , Humans , Mitoxantrone/pharmacology , Multidrug Resistance-Associated Proteins/metabolism , Neoplasm Proteins/genetics , Neoplasms/genetics , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , RNA, Messenger/isolation & purification , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Up-RegulationABSTRACT
Restriction fragment length polymorphism (RFLP) was performed on 32 isolates of the pathogenic fungus Paracoccidioides brasiliensis from geographically separated regions of South America. The use of HinfI and HincII gave clear RFLP patterns, for which high discriminatory indices could be calculated. Computational analysis of the RFLP patterns for the 32 isolates suggested that at least five groups of strains existed, each of which was geographically distinct and corresponded closely with present country borders. These results underline the belief that P. brasiliensis infections are acquired from exogenous sources and that this fungus occupies specialist endemic niches within the natural environment.
Subject(s)
Paracoccidioides/genetics , DNA, Fungal/analysis , Deoxyribonucleases, Type II Site-Specific , Humans , Molecular Epidemiology , Paracoccidioides/classification , Paracoccidioidomycosis/epidemiology , Phylogeny , Polymorphism, Restriction Fragment Length , South Africa/epidemiologyABSTRACT
Zygosporium geminatum, isolated as a contaminant in a culture of the mycelial phase of Paracoccidioides brasiliensis, was lethal to the latter organism. Its lytic action was due to exocellular alpha-1,3- and beta-1,3-glucanases which degraded the P. brasiliensis cell wall. The alpha-1,3-glucanase was more active at 30 degrees C and the beta-1,3-glucanase at 23 degrees C, each having pH 6.0 as its optimum.
Subject(s)
Glycoside Hydrolases/metabolism , Mitosporic Fungi/enzymology , Paracoccidioides , beta-Glucosidase/metabolism , Cell Wall , Glucan 1,3-beta-Glucosidase , Kinetics , Mitosporic Fungi/growth & development , Paracoccidioides/growth & development , ThermodynamicsABSTRACT
Randomly amplified polymorphic DNA (RAPD) analysis of 33 Paracoccidioides brasiliensis strains from Argentina, Brazil, Colombia, Peru, and Venezuela produced reproducible amplification products which were sufficiently polymorphic to allow differentiation of the strains. Types generated with five primers (OPG 03, OPG 05, OPG 14, OPG 16, and OPG 18) resulted in a high discriminatory index (0.956). The discriminatory index was slightly reduced (0.940) when only two primers (OPG 3 and OPG 14) were used. A dendrogram based on these results showed a high degree of similarity among the strains, and genetic differences were expressed in clusters related to geographical regions but not to pathological features of the disease. With a few exceptions, strains were sorted into five groups by geographical origin as follows: group I, Venezuelan strains; group II, Brazilian strains; group III, Peruvian strains; group IV, Colombian strains; and group V, Argentinian strains. The group containing the most disparate strains was group V (discriminatory index, 0.633); the discriminatory index for the other four groups was 0.824. The use of primer OPG 18 by itself was sufficient to discriminate species specificity, and the use of primer OPG 14 by itself was sufficient to discriminate among the geographical locations of the strains in the sample. This method may be helpful for epidemiological studies of P. brasiliensis.
Subject(s)
Mycological Typing Techniques , Paracoccidioides/classification , Paracoccidioidomycosis/epidemiology , Random Amplified Polymorphic DNA Technique , DNA, Fungal/analysis , Geography , Humans , Latin America/epidemiology , Paracoccidioides/genetics , Paracoccidioides/isolation & purification , Paracoccidioidomycosis/microbiology , Phylogeny , Species SpecificityABSTRACT
Saperconazole, a new triazole related to itraconazole, was tested against Paracoccidioides brasiliensis and results compared with ketoconazole and itraconazole. The fungus was highly sensitive to the action of these compounds, particularly saperconazole, with minimum inhibitory concentrations and minimum fungicidal concentrations ranging from 10(-7) to 10(-10) M (equivalent to 6.7 x 10(-2)-6.7 x 10(-5) micrograms ml-1 for saperconazole), according to the morphological phase and the antifungal tested. The yeast phase was more sensitive than the mycelial phase to any of the azoles. Morphological changes were observed in the cell membranes, particularly when saperconazole was used as the antifungal agent.
