Variation through the Cell Cycle in the Dose-response of DNA Neutral Filter Elution in X-irradiated Synchronous CHO-cells

Summary

Dose—response curves for DNA neutral (pH 9·6) filter elution were obtained with synchronized CHO cells exposed to X-rays at various phases of the cell cycle. The dose response was similar in synchronized and plateau-phase G₁ cells, as well as in cells that were arrested at the G₁/S border using aphidicolin; it flattened as cells progressed into S phase and reached a minimum in the middle of this phase. An increase in DNA elution dose response, to values only slightly lower than those obtained with G₁ cells, was observed as cells entered G₂ phase. Significant alterations in the sedimentation properties of the DNA during S phase were also observed in Ehrlich ascites tumor cells using the neutral sucrose gradient centrifugation technique. A significant proportion of the DNA from S cells irradiated with 10 Gy sedimented at speeds (350S–700S) well above the maximum sedimentation speed expected for free sedimenting DNA molecules (s_max = 350S), indicating the formation of a DNA complex. DNA from G₁, G₁/S, or G₂ + M cells sedimented as expected for free sedimenting molecules. These results indicate significant alterations in the physicochemical properties of the DNA–probably caused by DNA replication-associated alterations in DNA structure and chromatin conformation–as cells enter S phase, and are invoked to explain the observed variation in DNA elution dose response throughout the cycle. It is proposed that the formation of a complex DNA structure, resistant to the proteolytic enzymes and detergents used, affected the elution characteristics of the DNA and gave rise to the observed curvilinear DNA elution dose-response curves, as well as to the fluctuations in elution characteristics observed throughout the cell cycle.