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

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

doi:10.1016/0166-6851(90)90010-j

Cited by 39 publications

(16 citation statements)

References 45 publications

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“…Such cross-resistance was also not seen in the present study. Finally, even though the erythrocytic stages of malarial parasites lack the enzymes for the salvage of preformed pyrimidines (29,30) (2,5,6,9,13,18). One characteristic of such resistance is that, in the absence of drug pressure, the parasites readily loose the self-replicating extrachromosomal DNA and the associated drug resistance phenotype.…”

Section: Discussionmentioning

confidence: 99%

Selection and characterization of 5-fluoroorotate-resistant Plasmodium falciparum

Rathod

Khosla

Gassis

et al. 1994

Antimicrob Agents Chemother

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Previous studies have shown that 100 nM 5-fluoroorotate (5-FO) is sufficient to block the in vitro proliferation of Plasmodium fakciparum without causing toxicity to mammalian cells. In anticipation of potential drug resistance, a study was undertaken to identify P. falciparum cells that would proliferate in the presence of 5-FO. About 3 x 106 UV-irradiated as well as nonirradiated parasites were subjected to a one-step selection with 100 nM 5-FO both in the absence and in the presence of preformed pyrimidines (uracil, uridine, thymine, and thymidine). The P. falciparum cells that emerged after 3 weeks were cloned, and the 90% inhibitory concentration of 5-FO for the cloned cells was found to be 100-to 400-fold greater than that for the parent cell line. Two clones that were further characterized retained resistance to 5-FO even after prolonged propagation in culture without drug pressure. Since the mutants were not cross-resistant to 5-fluorouracil or to dihydrofolate reductase inhibitors, it was unlikely that alteration of thymidylate synthase or overproduction of the bifunctional dihydrofolate reductase-thymidylate synthase was responsible for 5-FO resistance. Similarly, resistance was not due to the expression of a pyrimidine salvage pathway since the cells were not pyrimidine auxotrophs, they did not show increased utilization of pyrimidine nucleosides, and they did not show increased susceptibility to 5-fluoropyrimidine nucleosides. However, the mutant parasites did show as much as a 40-fold lower level of utilization of exogenous radioactive orotate, suggesting that the decreased susceptibility to 5-FO was due to decreased transport or decreased activation of this compound to fluorinated nucleotides. When the selection experiments were repeated, without mutagenesis, in the presence of l0-7 M 5-FO with fewer than 106 parasites or in the presence of more than 10-7 M 5-FO with more than 108 parasites, viable mutants could not be recovered from the cultures. The implications of these findings for the in vivo use of 5-FO for malaria chemotherapy are discussed.Malaria continues to be one of the prevalent causes of morbidity and mortality around the world (19). Inhibitors of de novo pyrimidine metabolism may be suitable as antimalarial agents since the blood-stage forms of Plasmodium spp. lack enzymes that can be used to salvage preformed pyrimidines (29,30). In the past it has been demonstrated that a combination of 5-fluoroorotate (5-FO) and uridine allowed for a 1,000-fold difference in the 50% inhibitory concentrations (IC50s) for malarial and mammalian cells in culture (27). The nanomolar concentrations of 5-FO that caused the inhibition of parasite proliferation also inactivated malarial thymidylate synthase activity and triggered cell death (28, 32).It was shown that a combination of 5-FO and uridine was effective not only in vitro but also in vivo; mice infected with a lethal strain of Plasmodium yoelii could be cured with a combination of 5- FO and uridine (12,26). However, the concentrations of 5-FO in...

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Section: Discussionmentioning

confidence: 99%

Selection and characterization of 5-fluoroorotate-resistant Plasmodium falciparum

Rathod

Khosla

Gassis

et al. 1994

Antimicrob Agents Chemother

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“…3; n ϭ 9) (38,39). Incubation in DFMO was ultimately lethal for promastigote cultures (data not shown).…”

Section: Inducible Resistance To Hydrogenmentioning

confidence: 99%

Inducible Resistance to Oxidant Stress in the Protozoan Leishmania chagasi

Miller¹,

McGowan²,

Gantt³

et al. 2000

Journal of Biological Chemistry

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Leishmania sp. protozoa are introduced into a mammalian skin by a sandfly vector, whereupon they encounter increased temperature and toxic oxidants generated during phagocytosis. We studied the effects of 37°C "heat shock" or sublethal menadione, which generates superoxide and hydrogen peroxide, on Leishmania chagasi virulence. Both heat and menadione caused parasites to become more resistant to H 2 O 2 -mediated toxicity. Peroxide resistance was also induced as promastigotes developed in culture from logarithmic to their virulent stationary phase form. Peroxide resistance was not associated with an increase in reduced thiols (trypanothione and glutathione) or increased activity of ornithine decarboxylase, which is rate-limiting in trypanothione synthesis. Membrane lipophosphoglycan increased in size as parasites developed to stationary phase but not after environmental exposures. Instead, parasites underwent a heat shock response upon exposure to heat or sublethal menadione, detected by increased levels of HSP70. Transfection of promastigotes with L. chagasi HSP70 caused a heat-inducible increase in resistance to peroxide, implying it is involved in antioxidant defense. We conclude that leishmania have redundant mechanisms for resisting toxic oxi-

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“…The development of drug-resistant cell lines in vitro has greatly facilitated studies on the molecular basis of chemoresistance. The main molecular mechanisms involved in drug resistance are changes in membrane permeability, resulting in decreased influx or increased efflux of the molecule (11) or quantitative or qualitative modifications of the target, or both (10,12,23). These studies have mainly been carried out with the promastigote form, which is not the parasite stage naturally in contact with drugs in patients.…”

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confidence: 99%

In vitro life cycle of pentamidine-resistant amastigotes: stability of the chemoresistant phenotypes is dependent on the level of resistance induced

Sereno¹,

Lemesre²

1997

Antimicrob Agents Chemother

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Using a continuous drug pressure protocol, we induced pentamidine resistance in an active and dividing population of amastigote forms of Leishmania mexicana. We selected in vitro two clones with different levels of resistance to pentamidine, with clone LmPENT5 being resistant to 5 M pentamidine, while clone LmPENT20 was resistant to 20 M pentamidine. Resistance indexes (50% inhibitory concentration [IC 50 ] after drug presure/IC 50 before drug pressure) of 2 (LmPENT5) and 6 (LmPENT20) were determined after drug selection. Both resistant clones expressed significant cross-resistance to diminazene aceturate and primaquine. Pentamidine resistance was not reversed by verapamil, a calcium channel blocker known to reverse multidrug resistance (A. J. Bitonti, et al., Science 242:1301-1303, 1988; A. R. C. Safa et al., J. Biol. Chem. 262:7884-7888, 1987). No difference in the in vitro infectivity for resident mouse macrophages was observed between the wildtype clone (clone LmWT) and pentamidine-resistant clones. During in vitro infectivity experiments, when the life cycle was performed starting from the intramacrophagic amastigote stage, the drug resistance of the resulting LmPENT20 amastigotes was preserved even if the intermediate promastigote stage could not be considered resistant to 20 M pentamidine. In the same way, when a complete developmental sequence of L. mexicana was achieved axenically by manipulation of appropriate culture conditions, the resulting axenically grown LmPENT20 amastigotes remained pentamidine resistant, whereas LmPENT5 amastigotes lost their ability to resist pentamidine, with IC 50 s and index of resistance values close to those for the LmWT clone. These results strongly indicate that the level of pentamidine tolerated by resistant amastigotes after the life cycle was dependent on the induced level of resistance. This fact could be significant in the in vivo transmission of drugresistant parasites by Phlebotominae. Particular attention should be given to the finding that the emergence of parasite resistance is a potential risk of the use of inadequate doses as therapy in humans.

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

Cited by 39 publications

References 45 publications

Selection and characterization of 5-fluoroorotate-resistant Plasmodium falciparum

Selection and characterization of 5-fluoroorotate-resistant Plasmodium falciparum

Inducible Resistance to Oxidant Stress in the Protozoan Leishmania chagasi

In vitro life cycle of pentamidine-resistant amastigotes: stability of the chemoresistant phenotypes is dependent on the level of resistance induced

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