TRANSCRIPTIONAL REGULATION OF DIFFERENTIATION, SELECTIVE TOXICITY ANDATGCAAAT BINDING OF BISBENZIMIDAZOLE DERIVATIVES IN HUMAN-MELANOMA CELLS

To study the relationship between the structure of minor groove ligand s and their affinity for specific DNA sequences that regulate gene tra nscription, three analogues of the A-T-specific DNA minor groove ligan ds Hoechst 33258 and Hoechst 33342 were synthesized with 5, 8 or 12 ca rbons in an aliphatic chain attached to the phenolic oxygen of the mol ecule. There was a striking bimodal relationship between toxicity to H eLa cells and the lipophilicity of the five analogues, toxicity being low for the compounds with a free hydroxyl (Hoechst 33258) or a 12-car bon 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 332 58 itself was selectively toxic for the MM96E melanoma cell line compa red with other cell Lines, induced a highly dendritic morphology, incr eased tyrosinase activity and tyrosinase mRNA but decreased the level of gp75 (TRP-1) mRNA; message for a third pigment gene, Pmel-17, was u nchanged. Tyrosinase activity was decreased in the resistant A2058 mel anoma cell line and transcription was affected to a lesser extent than in MM96E. Expression of gp75 protein and two intermediate filament pr oteins was inhibited by Hoechst 33258 in MM96E cells. There was no maj or difference in the amount of I-125-Hoechst 33258 taken up by sensiti ve and resistant cells. Of the five derivatives studied, the parent dr ug Hoechst 33258 and the 2-carbon analogue (Hoechst 33342) were found to have the most inhibitory effect on affinity of octamer binding prot eins 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. T he G-C ligand chromomycin A(3) exhibited a different spectrum of cell toxicity and tyrosinase stimulation compared with Hoechst 33258. Chrom omycin A(3) but not Hoechst 33258, strongly inhibited the zinc-depende nt transcriptional activity of the sheep metallothionein-Ia promoter i n reporter gene assays of transfected cells. Since the six metal-respo nsive 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 ge nes, cell lines evidently differing in the accessibility to drugs of c ertain A-T-rich sequences.