3-Aminobenzamide is lethal to MMS-damaged human fibroblasts primarily during s phase

Abstract

3‐Aminobenzamide (3‐AB) interferes with DNA repair and enhances lethality in growing MMS (methyl methane sulfonate)‐treated human fibroblasts. This sensitivity to 3‐AB disappears slowly; MMS‐treated cells are sensitive to 3‐AB for up to 36 hours (Boorstein and Pardee, 1984). Evidence is now presented that 3‐AB potentiates the effects of MMS primarily during S phase. When cells were synchronized at the G₁/S boundary, released, and then treated with MMS, 3‐AB caused very substantial lethality in only 4 hours, and a 12‐hour treatment gave maximum lethality. These cells also lost sensitivity to 3‐AB within 12 hours of growth minus 3‐AB. In contrast, MMS‐treated quiescent (G₀) cells did not lose sensitivity to 3‐AB nor did 3‐AB cause lethality during G₀. Enhanced lethality occurred when damaged G₀‐arrested cells were subsequently allowed to proceed through S phase in the presence of 3‐AB; this 3‐AB sensitivity was removed only during growth in the absence of 3‐AB. The lethality of 3‐AB to a population of asynchronously cycling cells treated with MMS is thus the summation of effects on the cells as they traverse S phase. Aphidicolin prevenced lethality of 3‐AB to cells released from G₁/S and treated with MMS. It also inhibited the loss of sensitivity to added 3‐AB later. Correlation with the inhibition of DNA synthesis by this drug suggests that DNA synthesis is essential for the lethality enhancement by 3‐AB. Cells treated first with MMS and then with 3‐AB accumulated in G₂. This G₂ arrest depended on S‐phase events and correlated with cell lethality. Cells treated with a nonlethal dose of MMS at the G₁/S boundary were delayed briefly (3 hours) in their passage through S and G₂. These cells, when also exposed to 3‐AB, were delayed 6–9 hours in S and they became arrested in G₂. There was no G₂ arrest when 3‐AB was added only after these cells had reached G₂. Treatment with 3‐AB during S phase thus resulted in both enhanced lethality and G₂ arrest. 3‐AB inhibited repair of DNA single‐strand damage, shown by alkaline elution analysis, in both S‐phase and quiescent cells. Aphidicolin inhibited disappearance of breaks and eliminated the difference between 3‐AB‐treated and untreated cells. Lethality did not correlate well with the measured single‐strand damage. We propose that there is a class of MMS‐induced lesions whose repair occurs during normal replicative DNA synthesis. When this repair is interrupted by 3‐AB, during S phase, sublethal damage is converted to lesions which arrest the cells in G₂ phase and prevent them from forming colonies. DNA synthesis is required to reach the step in DNA repair at which 3‐AB causes lethality.