In vitro transcription of DNA containing 2-chloro-2'-deoxyadenosine monophosphate.
2-Chloro-2'-deoxyadenosine (cladribine [CldAdo]) represents one of the most promising therapeutic agents for the treatment of pediatric leukemias and adult hairy cell leukemia. We examined whether CldAdo incorporation into DNA inhibited subsequent transcription in vitro using purified phage RNA polymerases. Control (Ade-containing) and 2-chloroadenine (ClAde)-substituted DNA strands that contained a RNA polymerase promoter sequence were synthesized by a modified asymmetric polymerase chain reaction. Complementary (+) and (-) strands were annealed, incubated with phage RNA polymerase, and analyzed with denaturing PAGE. When ClAde was present in both strands, the yield of full-length transcripts (approximately equal to 100 bases) was reduced by approximately equal to 90% relative to control DNA. Transcription was also reduced to a slightly lesser degree when substitutions occurred in only one of two strands. The observed low transcript levels on ClAde-containing DNA were due in part to the presence of the analogue within the promoter region. With gel shift binding assays, we demonstrated that RNA polymerase did not bind as well to ClAde-containing promoters. Polymerase/DNA complex formation was decreased by approximately equal to 80% compared with that on control unsubstituted promoters. In addition, on binding to the substituted promoter, RNA polymerase had an altered conformation that led to enhanced proteolytic clipping by endoproteinase Glu-C. Transcript sequence analysis indicated that SP6 RNA polymerase read through template ClAde residues with no apparent misincorporation into RNA. Our results provide insight into a novel effect of this nucleoside analogue that may explain its cytotoxicity in nondividing cells.
D000970:Antineoplastic Agents / Q000494:pharmacology*; D001483:Base Sequence; D017338:Cladribine / Q000494:pharmacology*; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic / Q000187:drug effects*
D000970:Antineoplastic Agents; D017338:Cladribine