title: Alpha-difluoromethylornithine-resistant cell lines obtained after one-step selection of Leishmania mexicana promastigote cultures.
abstract: Proliferation of Leishmania mexicana promastigotes in synthetic medium can be blocked by the depletion of intracellular polyamine pools induced by the presence of D,L-alpha-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase (ODC). Here we report that DFMO-resistant cell lines growing normally at DFMO levels of 10 mM have been obtained from non-proliferating cultures after a single-step selection in the presence of high concentrations of the drug. The DFMO-resistant promastigotes underwent a morphological transformation into an 'amastigote-like' form after incubation for several hours at gradually increasing temperatures up to 35 degrees C. The uptake of DFMO was not significantly altered in the drug-resistant cell lines but in both cases (promastigote and 'amastigote-like' forms) the ODC specific activity was increased approx. 15-fold over the normal enzymic levels found in the wild-type Leishmania. The enzyme affinities for its substrate and for DFMO gave very similar values in the drug-resistant promastigotes and the wild-type parasites. In contrast, ODC from the 'amastigote-like' Leishmania showed a higher affinity for ornithine and a decreased capacity for the binding of DFMO. An 80-fold amplification of the ODC gene and a corresponding increase in its transcripts have been detected in both DFMO-resistant Leishmania cell lines. The drug-resistant phenotypes with their characteristic morphologies, the increased levels of ODC activity and the amplification of the ODC gene have been stable for at least 6 months in the absence of selective pressure.
authors.affiliation: Instituto de Investigaciones Bioquímicas 'Fundación Campomar', Facultad de Ciencias Exactas y Naturales, University of Buenos Aires, Patricias Argentinas 435,1405 Buenos Aires, Argentina.
authors.forename: C P
authors.initials: CP
authors.lastname: Sánchez
authors.forename: J
authors.initials: J
authors.lastname: Mucci
authors.forename: N S
authors.initials: NS
authors.lastname: González
authors.forename: A
authors.initials: A
authors.lastname: Ochoa
authors.forename: M M
authors.initials: MM
authors.lastname: Zakin
authors.forename: I D
authors.initials: ID
authors.lastname: Algranati
mesh_terms: D000818:Animals; D004351:Drug Resistance; D000518:Eflornithine / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005784:Gene Amplification; D007894:Leishmania mexicana / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D009955:Ornithine Decarboxylase / Q000235:genetics; D065108:Ornithine Decarboxylase Inhibitors; D010641:Phenotype; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D065108:Ornithine Decarboxylase Inhibitors; D012333:RNA, Messenger; D009955:Ornithine Decarboxylase; D000518:Eflornithine