α-Difluoromethylornithine-resistant cell lines obtained after one-step selection of Leishmania mexicana promastigote cultures
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-α-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase (ODC). Here we report that DEMO-re...
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todo:paper_02646021_v324_n3_p847_Sanchez2023-10-03T15:12:57Z α-Difluoromethylornithine-resistant cell lines obtained after one-step selection of Leishmania mexicana promastigote cultures Sánchez, C.P. Mucci, J. González, N.S. Ochoa, A. Zakin, M.M. Algranati, I.D. antitrypanosomal agent eflornithine messenger RNA ornithine ornithine decarboxylase ornithine decarboxylase inhibitor article binding affinity cell culture cell selection controlled study drug resistance drug uptake enzyme activity enzyme inhibitor interaction enzyme specificity gene amplification Leishmania mexicana nonhuman priority journal promastigote Leishmania mexicana 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-α-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase (ODC). Here we report that DEMO-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°C. The uptake of DEMO 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 DEMO. An 80-fold amplification of the ODC gene and a corresponding increase in its transcripts have been detected in both DEMO-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. Fil:Sánchez, C.P. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:González, N.S. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Ochoa, A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Zakin, M.M. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Algranati, I.D. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. JOUR info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by/2.5/ar http://hdl.handle.net/20.500.12110/paper_02646021_v324_n3_p847_Sanchez |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
antitrypanosomal agent eflornithine messenger RNA ornithine ornithine decarboxylase ornithine decarboxylase inhibitor article binding affinity cell culture cell selection controlled study drug resistance drug uptake enzyme activity enzyme inhibitor interaction enzyme specificity gene amplification Leishmania mexicana nonhuman priority journal promastigote Leishmania mexicana |
| spellingShingle |
antitrypanosomal agent eflornithine messenger RNA ornithine ornithine decarboxylase ornithine decarboxylase inhibitor article binding affinity cell culture cell selection controlled study drug resistance drug uptake enzyme activity enzyme inhibitor interaction enzyme specificity gene amplification Leishmania mexicana nonhuman priority journal promastigote Leishmania mexicana Sánchez, C.P. Mucci, J. González, N.S. Ochoa, A. Zakin, M.M. Algranati, I.D. α-Difluoromethylornithine-resistant cell lines obtained after one-step selection of Leishmania mexicana promastigote cultures |
| topic_facet |
antitrypanosomal agent eflornithine messenger RNA ornithine ornithine decarboxylase ornithine decarboxylase inhibitor article binding affinity cell culture cell selection controlled study drug resistance drug uptake enzyme activity enzyme inhibitor interaction enzyme specificity gene amplification Leishmania mexicana nonhuman priority journal promastigote Leishmania mexicana |
| description |
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-α-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase (ODC). Here we report that DEMO-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°C. The uptake of DEMO 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 DEMO. An 80-fold amplification of the ODC gene and a corresponding increase in its transcripts have been detected in both DEMO-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. |
| format |
JOUR |
| author |
Sánchez, C.P. Mucci, J. González, N.S. Ochoa, A. Zakin, M.M. Algranati, I.D. |
| author_facet |
Sánchez, C.P. Mucci, J. González, N.S. Ochoa, A. Zakin, M.M. Algranati, I.D. |
| author_sort |
Sánchez, C.P. |
| title |
α-Difluoromethylornithine-resistant cell lines obtained after one-step selection of Leishmania mexicana promastigote cultures |
| title_short |
α-Difluoromethylornithine-resistant cell lines obtained after one-step selection of Leishmania mexicana promastigote cultures |
| title_full |
α-Difluoromethylornithine-resistant cell lines obtained after one-step selection of Leishmania mexicana promastigote cultures |
| title_fullStr |
α-Difluoromethylornithine-resistant cell lines obtained after one-step selection of Leishmania mexicana promastigote cultures |
| title_full_unstemmed |
α-Difluoromethylornithine-resistant cell lines obtained after one-step selection of Leishmania mexicana promastigote cultures |
| title_sort |
α-difluoromethylornithine-resistant cell lines obtained after one-step selection of leishmania mexicana promastigote cultures |
| url |
http://hdl.handle.net/20.500.12110/paper_02646021_v324_n3_p847_Sanchez |
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