Biochemical changes associated with alpha-difluoromethylornithine uptake and resistance in Trypanosoma brucei

Abstract

Procyclic Trypanosoma brucei grown in semi-defined media are sensitive to alpha-difluoromethylornithine (DFMO) (EC50 100 microM), an inhibitor of ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis. Organisms resistant to 5 mM DFMO (EC50 greater than 20 mM) were obtained by passage in incremental amounts of drug. Resistant and wild-type cells accumulated DFMO by passive diffusion with a consequent decrease in polyamine levels, indicating inhibition of ODC in both cell types. The resistant phenotype was stable in the absence of DFMO, in which state there was no increase in ODC abundance or activity. By kinetic analysis, the ODC of resistant cells appeared normal. In wild-type and resistant cells, [3H]DFMO equally and uniquely affinity-labelled a 50 kDa polypeptide corresponding to the ODC subunit. Levels of ODC and tubulin mRNAs were elevated 4-fold in resistant cells grown in the presence of DFMO, although there was no indication of gene amplification. The intracellular concentration of dihydrotrypanothione (N1,N8-bis(glutathionyl)-spermidine), a redox intermediate unique to kinetoplastids, was unchanged in resistant cells growing in DFMO but was halved in wild-type cells exposed to DFMO for 48 h. The exceptionally elevated levels of ornithine found in DFMO-treated resistant cells most likely play a crucial role in cell survival by maintaining intracellular concentrations of dihydrotrypanothione by competing with DFMO for ODC.