Alpha-difluoromethylornithine-resistant cell lines obtained after one-step selection of Leishmania mexicana promastigote cultures.

SANCHEZ CP; MUCCI J; GONZALEZ NS; OCHOA A; ZAKIN MM; ALGRANATI ID

BIOCHEMICAL JOURNAL

PORTLAND PRESS LTD

Año: 1997 vol. 324 p. 847 - 853

0264-6021

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.