Abstract
Alkylating agents are generally highly reactive with DNA but demonstrate limited DNA sequence selectivity. In contrast, synthetic pyrrole-imidazole polyamides recognize specific DNA sequences with high affinity but are unable to permanently damage DNA. An eight-ring hairpin polyamide conjugated to the alkylating moiety cyclopropylpyrroloindole, related to the natural product CC-1065, affords a conjugate 1-CBI (polyamide 1-CBI (1-(chloromethyl)-5-hydroxyl-1,2-dihydro-3H-benz[e]indole) conjugate), which binds to specific sequences in the minor groove of DNA and alkylates a single adenine flanking the polyamide binding site. In this study, we show that 1-CBI alkylates DNA in both plasmid and intracellular minichromosomal form and inhibits DNA replication under both cell-free and cellular conditions. In addition, it inhibits cell growth and arrests cells in the G2/M phase of the cell cycle.
Highlights
Polyamides are synthetic DNA binding molecules designed to recognize and bind to specific sequences in the minor groove of the double helix with affinities comparable with those of DNA-binding proteins [1, 2]
Inhibition of SV40 DNA Replication in BSC-1 Cells—Since it was found that 1-CBI inhibited cell-free SV40 replication, we examined whether similar activity can be observed in the intact cells
Naked DNA was separated on two-dimensional gel electrophoresis and analyzed as described under “Experimental Procedures.” 1-CBI treatment caused a concentration-dependent decrease in intracellular SV40 replication intermediates detected in the form of bubble arc, as indicated by the arrows in Fig. 5; the bubble arc intensity was reduced at 0.1 M and diminished completely at 3–10 M agent concentration
Summary
Reagents and Cell Cultures—1-CBI, a hairpin polyamide-seco-CBI conjugate (see Fig. 1), was synthesized as described previously [17]. [␥-32P]ATP and [␣-32P]dATP were obtained from Amersham Biosciences. BSC-1 cells (African green monkey kidney cells) were grown in minimal essential medium (MEM) with 8% fetal calf serum and 2% fetal bovine serum (MEM-10). Human 293 cells (transformed embryonic kidney cells) were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (DMEM-10). SV40 DNA Forms Conversion Assay—Naked SV40 DNA or SV40 minichromosomes (150 ng, isolated as described elsewhere [20, 21]) was incubated at 37 °C with 1-CBI in 20 l of TE buffer for up to 16 h
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