title: Transcriptional regulation of differentiation, selective toxicity and ATGCAAAT binding of bisbenzimidazole derivatives in human melanoma cells.
abstract: To study the relationship between the structure of minor groove ligands and their affinity for specific DNA sequences that regulate gene transcription, three analogues of the A-T-specific DNA minor groove ligands Hoechst 33258 and Hoechst 33342 were synthesized with 5, 8 or 12 carbons in an aliphatic chain attached to the phenolic oxygen of the molecule. There was a striking bimodal relationship between toxicity to HeLa cells and the lipophilicity of the five analogues, toxicity being low for the compounds with a free hydroxyl (Hoechst 33258) or a 12-carbon substituent, yet high for the 5-carbon analogue. Selective killing of human melanoma cells compared with normal fibroblasts was observed for the Hoechst analogue with a 12-carbon chain attached. Hoechst 33258 itself was selectively toxic for the MM96E melanoma cell line compared with other cell lines, induced a highly dendritic morphology, increased tyrosinase activity and tyrosinase mRNA but decreased the level of gp75 (TRP-1) mRNA; message for a third pigment gene, Pmel-17, was unchanged. Tyrosinase activity was decreased in the resistant A2058 melanoma cell line and transcription was affected to a lesser extent than in MM96E. Expression of gp75 protein and two intermediate filament proteins was inhibited by Hoechst 33258 in MM96E cells. There was no major difference in the amount of 125I-Hoechst 33258 taken up by sensitive and resistant cells. Of the five derivatives studied, the parent drug Hoechst 33258 and the 2-carbon analogue (Hoechst 33342) were found to have the most inhibitory effect on affinity of octamer binding proteins for the ATGCAAAT consensus sequence found in the promoter region of certain genes associated with proliferation and differentiation. In contrast to Distamycin A (also an A-T-specific minor groove ligand), Hoechst 33258 displaced proteins already bound to the octamer motif. The G-C ligand chromomycin A3 exhibited a different spectrum of cell toxicity and tyrosinase stimulation compared with Hoechst 33258. Chromomycin A3 but not Hoechst 33258, strongly inhibited the zinc-dependent transcriptional activity of the sheep metallothionein-Ia promoter in reporter gene assays of transfected cells. Since the six metal-responsive elements of the promoter are GC-rich, this provides independent evidence for the sequence-specificity of transcriptional inactivation by one of these drugs in melanoma cells. Overall, the results suggest that Hoechst 33258 acts by inhibiting the transcription of specific genes, cell lines evidently differing in the accessibility to drugs of certain A-T-rich sequences.
authors.affiliation: Queensland Cancer Fund Unit, Queensland Institute of Medical Research, Herston, Australia.
authors.forename: S S
authors.initials: SS
authors.lastname: Wong
authors.forename: R A
authors.initials: RA
authors.lastname: Sturm
authors.forename: J
authors.initials: J
authors.lastname: Michel
authors.forename: X M
authors.initials: XM
authors.lastname: Zhang
authors.forename: P A
authors.initials: PA
authors.lastname: Danoy
authors.forename: K
authors.initials: K
authors.lastname: McGregor
authors.forename: J J
authors.initials: JJ
authors.lastname: Jacobs
authors.forename: A
authors.initials: A
authors.lastname: Kaushal
authors.forename: Y
authors.initials: Y
authors.lastname: Dong
authors.forename: I S
authors.initials: IS
authors.lastname: Dunn
mesh_terms: D001483:Base Sequence; D001562:Benzimidazoles / Q000494:pharmacology* / Q000633:toxicity; D006690:Bisbenzimidazole / Q000494:pharmacology* / Q000633:toxicity; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D014128:Chromomycin A3 / Q000494:pharmacology; D004273:DNA, Neoplasm / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008545:Melanoma / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000187:drug effects; D010088:Oxidoreductases; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000032:analysis; D014158:Transcription, Genetic / Q000187:drug effects* / Q000235:genetics; D014407:Tumor Cells, Cultured
chemical_list: D001562:Benzimidazoles; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D008562:Membrane Glycoproteins; D011506:Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D014128:Chromomycin A3; D010088:Oxidoreductases; C489799:TYRP1 protein, human; C064056:tyrosinase-related protein-1; D014442:Monophenol Monooxygenase; D006690:Bisbenzimidazole; C017807:bisbenzimide ethoxide trihydrochloride