Fig. 4

Luteolin and quercetin induce cleavage of kDNA minicircles. (A) L. donovani AG83 promastigotes (5 × 10⁷ cells/ml) were treated with 0.5% DMSO or the flavonoids for 30 min and then lysed with SDS. SDS lysate was then digested with Proteinase K, RNase A and RNase T1. After phenol extraction and ethanol precipitation, the DNA was resolved in a 2% agarose gel containing 0.5 µg/ml EtBr, transferred to Hybond N membrane and probed with ³²P labeled kDNA. Lane 1, DNA from control cells treated with 1X PBS; lane 2, from control cells treated with 0.5% DMSO; lanes 3–8, from cells treated with etoposide (100 µM), quercetin (50 µM), luteolin (20 µM), rutin (100 µM), flavone A (100 µM) and isoorientin (100 µM), respectively. II, minicircles containing nicks or gaps; III, linearized minicircles; I, covalently closed minicircles. (B) L. donovani AG83 promastigotes (5 × 10⁷ cells/ml) were treated with increasing concentrations of quercetin. Linearization of kDNA minicircles were monitored as described in Panel A. Lane 1, DNA purified from control cells treated with 0.5% DMSO; lane 2, from cells treated with 100 µM etoposide; lanes 3–7, from cells treated with increasing concentrations of quercetin at 25, 50, 100, 150 and 200 µM respectively. (C) same as Panel B, excepting that the L. donovani promastigotes were treated with increasing concentrations of luteolin. Lane 1, DNA from cells treated with 0.5% DMSO; lanes 2–6, from cells treated with 5, 10, 25, 50 and 100 µM luteolin respectively; lane 7, from cells treated with 10 µM mAMSA. (D) Characterization of quercetin induced linearized minicircle DNA molecules. The DNA was processed in the same way as in Panel A, except proteinase K treatment was omitted as indicated. Lane 1, DNA purified from cells treated with 100 µM quercetin; lane 2, DNA from cell lysate not digested with proteinase K prior to extractions with phenol and phenol-chloroform-isoamyl alcohol (25:24:1); lane 3, 100 ng of purified DNA from quercetin-treated cells, digested with exonuclease III (10 units); lane 4, 100 ng of purified DNA from quercetin-treated cells, digested with 10 units of λ exonuclease.