Abstract
Deoxyadenosine plus deoxycoformycin (dCf) causes increased DNA breaks in lymphoid cells. This study explored the possible inhibition of repair synthesis of DNA by dAdo plus dCf as a cause of DNA breakage. It was shown that DNA breaks accumulated in a human T-lymphoblast cell line, CCRF-CEM, following incubation with dAdo plus dCf and were not fully repaired 20 h after their removal. Analysis of the density distribution of radiolabeled DNA on alkaline CsCl gradient showed that incubation of CCRF-CEM cells with dAdo plus dCf caused inhibition of semiconservative, but not repair synthesis of DNA. Semiconservative synthesis of DNA was also inhibited in CCRF-CEM nuclei isolated from cells pretreated with dAdo and dCf, suggesting damage to DNA replicative machinery. However, no such inhibition was observed in the nuclei of a similarly treated CCRF-CEM mutant that was deficient in adenosine kinase and deoxycytidine kinase. This suggests that dAdo must be phosphorylated in intact cells to exert its effect. Using [3H]dTTP incorporation in isolated CCRF-CEM nuclei to measure DNA synthesis, it was found that a high concentration (greater than 100 microM) of dATP inhibits semiconservative but not repair synthesis of DNA. The present studies thus indicate that accumulation of DNA strand breaks induced by dAdo plus dCf is not the consequence of inhibition of repair DNA synthesis. This implies the mechanism may involve perturbation of DNA ligation or activation of a certain process which causes DNA strand breaks. In addition, dATP may interfere with some steps of semiconservative DNA synthesis, but not the repair synthesis of DNA.
Highlights
The Effectof Deoxyadenosine Plus Deoxycoformycin on Replicative and Repair Synthesiosf DNA in Human Lymphoblasts and Isolated Nuclei”
No such inhibition wasobserved in the nuclei of a treated CCRF-CEM mutant that was plus dCf [17, 18].Recent studies have shown that treatment of lymphoid cells with dAdo plus dCf leads to accumulation of DNA strand breaks [19, 20]
DNA Breakage-Following incubation of CCRF-CEM cells with 20 PM dAdo plus 2 p~ dCf for 4 h, the relative fluorescence was reduced to 49% of the control value as measured by DNA unwinding technique (Table I)
Summary
Accumulation of dATP-CCRF-CEM lymphoblasts that were treated with 20 PM &do and dCf for 4 h were shown to contain 266 f 19 pmol of dATP/106 cells by HPLC measurements. DNA Breakage-Following incubation of CCRF-CEM cells with 20 PM dAdo plus 2 p~ dCf for 4 h, the relative fluorescence was reduced to 49% of the control value as measured by DNA unwinding technique (Table I). This indicated that there was an increase in the number of DNA strand breaks following dAdotreatment. When cells were incubated with MMS plus dAdo and dCf, the reduced relative fluorescence was still present by 20 h after resuspension in fresh medium (Table I) These findings suggest that DNA strand breaks caused by MMS were rapidly repaired upon the removal of MMS.
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