Alpha-difluoromethylornithine resistance in Leishmania donovani is associated with increased ornithine decarboxylase activity.
The promastigote form of Leishmania donovani is sensitive to growth inhibition by DL-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase (ODC), the first enzyme of the polyamine biosynthetic pathway, with an EC50 value of approximately 30 microM. 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 alpha-methylornithine, alpha-monofluoromethyl-3,4-dehydroornithine methyl ester, and delta-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.
Coons
T
T
Department of Biochemistry, Oregon Health Sciences University, Portland 97201-3098.
Hanson
S
S
Bitonti
A J
AJ
McCann
P P
PP
Ullman
B
B
D000345:Affinity Labels; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001119:Arginase / Q000378:metabolism; D015317:Biogenic Polyamines / Q000032:analysis; D004247:DNA / Q000032:analysis; D004262:DNA Restriction Enzymes; D004351:Drug Resistance / Q000235:genetics; D000518:Eflornithine / Q000378:metabolism / Q000494:pharmacology*; D007893:Leishmania donovani / Q000187:drug effects* / Q000201:enzymology; D009154:Mutation; D009955:Ornithine Decarboxylase / Q000378:metabolism*
D000345:Affinity Labels; D000596:Amino Acids; D015317:Biogenic Polyamines; D004247:DNA; D004262:DNA Restriction Enzymes; D001119:Arginase; D009955:Ornithine Decarboxylase; D000518:Eflornithine