Alpha-difluoromethylornithine resistance in Leishmania donovani is associated with increased ornithine decarboxylase activity

Terry Coons, Sheri Hanson, Alan J. Bitonti, Peter P. McCann, Buddy Ullman

Research output: Contribution to journalArticle

30 Scopus citations

Abstract

The promastigote form of Leishmania donovani is sensitive to growth inhibition by dl-α-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway, with an EC50 value of approximately 30 μM [11]. Exposure of a wild type (DI700) cell population to gradually increasing concentrations of DFMO resulted in the selection of a strain of Leishmania, DFMO-10, which was capable of proliferating in 10 mM DFMO. DFMO-10 cells possessed an EC50 value for DFMO greater than 4 mM, and were cross-resistant to α-methylornithine, α-monofluoromethyl-3,4-dehydroornithine methyl ester, and δ-methyl-acetylenic putrescine, three other inhibitors of ODC activity. DI700 and DFMO-10 cells accumulated and/or transported [3H]DFMO and a spectrum of basic, neutral, and acidic amino acids at comparative rates. However, the DFMO-resistant Leishmania, if suspended in culture medium in the absence of DFMO for several days, expressed up to 15-fold greater levels of ODC activity than did wild-type cells. The overexpressed ODC in mutant cells appeared kinetically normal, since the ODC activities from DI700 and DFMO-10 cells possessed similar apparent Km values for ornithine and were equally sensitive to inactivation by DFMO. Incubation of extracts of DFMO-10 cells, but not of wild-type parental cells, with [3H]DFMO for 1 h resulted in the labeling of a polypeptide, presumably ODC, which migrated with a molecular weight of 76 000 ± 4000 on SDS-gel electrophoretograms. As a consequence of the elevated ODC activities, the levels of putrescine in mutant cells released from DFMO exposure were also elevated by about 15-fold over those of wild-type cells, although spermidine levels in DI700 and DFMO-10 cells were similar. In the absence of prolonged selective pressure, the resistance to DFMO, the ODC activity, and the putrescine levels of DFMO-10 cells all returned to those of wild type cells, indicating that the mutant phenotype of DFMO-selected L. donovani was unstable.

Original languageEnglish (US)
Pages (from-to)77-89
Number of pages13
JournalMolecular and Biochemical Parasitology
Volume39
Issue number1
DOIs
StatePublished - Feb 1990

Keywords

  • Difluoromethylornithine
  • Drug resistance
  • Leishmania
  • Mutant
  • Ornithine decarboxylase
  • Polyamine

ASJC Scopus subject areas

  • Parasitology
  • Molecular Biology

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