Phosphorothioate oligonucleotides, suramin and heparin inhibit DNA-dependent protein kinase activity

Abstract

Phosphorothioate oligonucleotides and suramin bind to heparin binding proteins including DNA polymerases, and inhibit their functions. In the present study, we report inhibition of DNA-dependent protein kinase activity by phosphorothioate oligonucleotides, suramin and heparin. Inhibitory effect of phosphorothioate oligonucleotides on DNA-dependent protein kinase activity was increased with length and reached a plateau at 36-mer. The base composition of phosphorothioate oligonucleotides did not affect the inhibitory effect. The inhibitory effect by phosphorothioate oligodeoxycytidine 36-mer can be about 200-fold greater than that by the phosphodiester oligodeoxycytidine 36-mer. The inhibitory effect was also observed with purified DNA-dependent protein kinase, which suggests direct interaction between DNA-dependent protein kinase and phosphorothioate oligonucleotides. DNA-dependent protein kinase will have different binding positions for double-stranded DNA and phosphorothioate oligodeoxycytidine 36-mer because they were not competitive in DNA-dependent protein kinase activation. Suramin and heparin inhibited DNA-dependent protein kinase activity with IC(50) of 1.7 microM and 0.27 microg ml(-1) respectively. DNA-dependent protein kinase activities and DNA double-stranded breaks repair in cultured cells were significantly suppressed by the treatment with suramin in vivo. Our present observations suggest that suramin may possibly result in sensitisation of cells to ionising radiation by inactivation of DNA-dependent protein kinase and the impairment of double-stranded breaks repair.

Figure 1

Structure of suramin. The hexasodium salt is shown. The molecular weight of suramin is 1429.

Figure 2

Effect of oligonucleotides on DNA-PK activity. (A) Effect of S-dC₆, S-dC₁₂, S-dC₂₄, S-dC₃₆, S-dC₄₈, and S-dC₆₀ on DAN-PK activity. Whole cell extract prepared from LM217 was used. (B) Effect of S-dC₃₆ on DNA-PK activity. Whole cell extract prepared from T98G was used. (C) Effect of S-dC₃₆, S-dG₃₆, S-dA₃₆, S-dT₃₆, and dC₃₆ on DNA-PK activity. Whole cell extract prepared from LM217 was used. (D) Effect of S-dC₃₆ and dC₃₆ on DNA-PK activity. Purified DNA-PK was used. (E) Effect of HCl-treated S-dC₃₆ and untreated S-dC₃₆ on DNA-PK activity. Whole cell extract prepared from LM217 was used. Salt concentration of untreated S-dC₃₆ was adjusted to that of HCl-treated S-dC₃₆. DNA-PK activities are expressed as values relative to that of control, which is set to a value of 1. The data represents the means±s.d. (n=3).

Figure 3

Dose-response curve after treatment with S-dCn. IC₅₀ was calculated from the data used for Figure 2A.

Figure 4

Effect of dsDNA concentration on inhibition of DNA-PK activity by S-dC₃₆. Concentrations of dsDNA ranged from 0.05 μg ml⁻¹ to 5 μg ml⁻¹. Whole cell extract prepared from LM217 was used. Details are the same as shown in Figure 2. Figure 5. Effect of suramin (A) and heparin (B) on DNA-PK activity. Whole cell extract prepared from LM217 was used. Details are the same as shown in Figure 2.

Figure 5

Effect of suramin (A) and heparin (B) on DNA-PK activity. Whole cell extract prepared from LM217 was used. Details are the same as shown in Figure 2.

Figure 6

Kinetics of DNA double strand breaks after 50 Gy irradiation in exponentially growing LM217 cells in the presence or absence of 1 mM suramin. Cells were treated with suramin 20 h before irradiation and the drug was kept in the medium during the post-irradiation repair period. The data represent the means±s.d. (n=3). *P<0.05.