Figure 1.
The binding of actinomycin D to DNA.
(A) The chemical structure of actinomycin D (ACTD). (B) Magnified side stereoview of the ACTD-TGCA sequence complex interface (ACTD as a ball-and-stick representation and DNA as a skeletal representation) (PDB:1MNV). The phenoxazone ring is intercalated individually into the GC step.
Figure 2.
The effects of spermine on the DNA-binding affinity of actinomycin D.
Sensorgrams of the interaction between an immobilized hairpin duplex and the target ACTD (6 µM) in the presence of various concentrations of spermine (SPM).
Table 1.
Numerical values of the SPR-derived association rate constants, dissociation rate constants, and association equilibrium constants (ka, kd, and Ka) for immobilized hairpin DNA upon binding to ACTD in the presence of various concentrations of spermine.
Figure 3.
Effects of spermine on the stabilization of actinomycin D on DNA duplexes.
(A) Melting temperatures (Tm in °C) of DNA in the presence of spermine (SPM) at various concentrations without (filled column) or with (open column) ACTD. The DNA sequence was d(TTTGCAAA). (B) Gibb's free energy change, ΔG, for the formation of DNA duplexes in the presence of spermine (SPM) at various concentrations with (open column) or without (filled column) ACTD. Each value was averaged from three separate experimental sets.
Table 2.
The thermodynamic measurements ΔG, ΔH, and TΔS (in kJ/mol) derived from thermal denaturation for the formation of duplex DNA in the presence of spermine at various concentrations.
Table 3.
The thermodynamic measurements ΔG, ΔH, and TΔS (in kJ/mol) derived from thermal denaturation for the formation of duplex DNA with ACTD binding in the presence of spermine at various concentrations.
Figure 4.
Spermine attenuates the inhibition of transcription by actinomycin D in vitro.
(A) The effect of ACTD on T7 RNA polymerase activity in the presence of various concentrations of spermine (SPM). (B) Quantification of the percentage of RNA polymerase activity relative to the control treated with or without ACTD in the presence of various concentrations of spermine (SPM) (0, 0.1, and 0.2 mM). The data represent the mean values ±SDs from three separate experiments.
Figure 5.
Spermine attenuates the inhibition of DNA replication by actinomycin D in vitro.
(A) Effect of ACTD on E. coli DNA polymerase I activity in the presence of various concentrations of spermine (SPM). (B) Quantification of the percent RNA polymerase activity relative to the control treated with or without ACTD in the presence of various concentrations of spermine (SPM) (0, 1, 5, and 10 mM). The data represent the mean values ±SDs from three separate experiments.
Figure 6.
Polyamine depletion enhances the inhibition of transcription by actinomycin D within cells.
The effects of ACTD at 3 µM on the fold change of c-myc gene expression in (A) HeLa, (B) A549, and (C) MCF7 cells by RT-PCR with and without a 24 h MGBG pretreatment versus control samples. β-actin was used as the internal control. The data represent the mean values ±SDs from three separate experiments (*p<0.05).
Figure 7.
Polyamine depletion enhances the inhibition of DNA replication by actinomycin D within cells.
The effects of ACTD at 0.5 µM on the relative BrdU incorporation in in (A) HeLa, (B) A549, and (C) MCF7 cells after a 24 h MGBG pretreatment versus control samples. The data represent the mean values ±SDs from three separate experiments (*p<0.05).
Figure 8.
Polyamine depletion enhances the inhibition of cell viability by actinomycin D.
The effects of ACTD at various concentrations on the viability of in (A) HeLa, (B) A549, and (C) MCF7 cells with or without a 24 h pretreatment with 2 µM MGBG. The data represent the mean values ±SDs from three separate experiments (*p<0.05).