Enhanced activity of antisense phosphorothioate oligos against Leishmania amastigotes: augmented uptake of oligo, ribonuclease H activation, and efficient target intervention under altered growth conditions

Citation
M. Mishra et al., Enhanced activity of antisense phosphorothioate oligos against Leishmania amastigotes: augmented uptake of oligo, ribonuclease H activation, and efficient target intervention under altered growth conditions, BIOCH PHARM, 62(5), 2001, pp. 569-580
Citations number
46
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Pharmacology & Toxicology
Journal title
BIOCHEMICAL PHARMACOLOGY
ISSN journal
0006-2952 → ACNP
Volume
62
Issue
5
Year of publication
2001
Pages
569 - 580
Database
ISI
SICI code
0006-2952(20010901)62:5<569:EAOAPO>2.0.ZU;2-O
Abstract
Leishmania, a parasitic protozoan, infects human macrophages, often causing severe morbidity and mortality. The pathogenic form of this parasite, the amastigote, lives inside the acidic phagolysosomes of infected macrophages. In our attempt to develop anti-miniexon phosphorothioate oligodeoxyribonuc leotides (S-oligos) as an alternative chemotherapy against Leishmania, we f ound that intracellular as well as 'axenic' amastigotes were more susceptib le to these S-oligos than were the cultured promastigotes. Lower pH (4.5) a nd elevated temperature (35 degrees) of the medium were among the direct en hancing factors for killing. Addition of the cationic polypeptide poly-1-ly sine (PLL) to the growth medium further enhanced the killing effect of the S-oligo at pH 4.5. The enhancement of specific ablation of mRNA expression was directly correlated to the increased leishmanicidal activity of the S-o ligo. This was shown by the increased inhibition of luciferase activity exp ressed in transgenic Leishmania amazonensis promastigotes by anti-miniexon S-oligo or anti-luciferase S-oligo at acidic pHs and in the presence of PLL . The leishmanicidal effects of S-oligos at acidic pH and in the presence o f PLL were related to increased uptake of the S-oligos under these conditio ns. The rate of S-oligo uptake was enhanced up to 15-fold at pH 4.5. The ad dition of PLL to the assay medium at acidic pH further enhanced the uptake of S-oligo up to 80-fold. RNase H is known to accentuate the antisense acti on of S-oligos. We found that at an elevated temperature RNase H activity i n Leishmania cell extracts increased about 5-fold. Thus, enhanced uptake of S-oligos at the acidic pH of macrophage phagolysosomes and activation of R Nase H may explain the efficient killing of the parasite in macrophages, bo th in tissue culture and in the animal model, by antisense miniexon oligonu cleotide/PLL, when targeted directly to the parasite-containing phagolysoso mes. (C) 2001 Elsevier Science Inc. All rights reserved.