Arana F E, Pérez-Victoria J M, Repetto Y, Morello A, Castanys S, Gamarro F
Instituto de Parasitología y Biomedicina López-Neyra, Consejo Superior de Investigaciones Científicas, Granada, Spain.
Biochem Pharmacol. 1998 Nov 1;56(9):1201-8. doi: 10.1016/s0006-2952(98)00129-4.
Clinical resistance to pentavalent antimonials, in the form of pentostam (sodium stibogluconate) or glucantime (N-methylglucamine antimoniate), has long been recognized as a problem in Leishmaniasis. However, the mechanisms of resistance are unclear. We selected in vitro a Leishmania tropica line resistant to 1.2 mg/mL of Sb(V) of glucantime (GLU-R10). The cell line has a stable phenotype for at least 6 months and a resistance index of 1400-fold. The resistant line has no cross-resistance to pentostam or to SbCl3 and SbCl5. The resistance to glucantime was reverted by buthionine sulfoximine (BSO) and chlorambucil (CLB); however, thiol analyses by HPLC of wild-type and GLU-R10 cell lines, in the presence or absence of the drug, showed no differences between these two cell lines. The resistant line had a DNA amplification shown as a circular extrachromosomal element (G-circle) of approximately 22 kb. However, the specific probes for gamma-glutamyl cysteine synthetase, ornithine decarboxylase and trypanothione reductase did not recognize the G-circle amplified in the GLU-R10. The G-circle did not arise from the H region and was not related with P-glycoprotein Pgp-MDR- or Pgp-MRP-like genes. Northern blot analysis of the G-circle showed that a single transcript of approximately 6 kb was overexpressed in the resistant line. Molecular characterization of the G-circle would lead to the determination of the gene(s) involved in resistance to glucantime in Leishmania.
长期以来，临床上对以喷他脒（葡糖酸锑钠）或戊脘脒（甲葡胺锑酸盐）形式存在的五价锑剂产生耐药性一直被认为是利什曼病治疗中的一个问题。然而，耐药机制尚不清楚。我们在体外筛选出了一株对1.2 mg/mL戊脘脒的Sb(V)具有抗性的热带利什曼原虫株系（GLU-R10）。该细胞系具有至少6个月的稳定表型，耐药指数为1400倍。该耐药株系对喷他脒或SbCl3和SbCl5没有交叉耐药性。丁硫氨酸亚砜胺（BSO）和苯丁酸氮芥（CLB）可逆转对戊脘脒的耐药性；然而，通过高效液相色谱法对野生型和GLU-R10细胞系在有或无药物情况下进行的硫醇分析表明，这两种细胞系之间没有差异。该耐药株系存在DNA扩增，表现为一个约22 kb的环状染色体外元件（G环）。然而，γ-谷氨酰半胱氨酸合成酶、鸟氨酸脱羧酶和锥虫硫醇还原酶的特异性探针未能识别在GLU-R10中扩增的G环。G环并非起源于H区域，且与P-糖蛋白Pgp-MDR或Pgp-MRP样基因无关。对G环的Northern印迹分析表明，一个约6 kb的单一转录本在耐药株系中过度表达。对G环进行分子特征分析将有助于确定利什曼原虫中参与对戊脘脒耐药的基因。
