DNA Synthesis in Nuclei Isolated from Daudi B Cells: A Model to Study the Antiproliferative Mechanisms of Interferon-α

Abstract

To study the antiproliferative response of B cells to interferon-α (IFN-α) at the molecular level, we developed a cell-free system to assess DNA synthesis in nuclei isolated from IFN-sensitive Daudi B lymphoblastoid cells. [³H]dTTP incorporation in isolated nuclei was shown to be representative of replicative DNA synthesis by evidence that (i) incorporation was dependent on ATP and all four nucleoside precursors, (ii) incorporation was inhibited >97% by aphidicolin, a specific inhibitor of DNA polymerase α and δ, and (iii) the DNase I-sensitive product banded in neutral CsCl at a density indicative of replicative DNA. This cell-free model was used in conjunction with flow cytometric cell cycle analysis to determine the effect of IFN-α on DNA synthesis in Daudi cells. The addition of IFN-α to an IFN-growth sensitive Daudi subclone in G₀/early G₁ inhibited the initiation of DNA synthesis, assessed in isolated nuclei, and prevented the progression of cells into S phase. IFN-α failed to inhibit DNA synthesis or cell cycle progression when added to IFN-sensitive Daudi cells in late G₁/early S phase or to an IFN-resistant Daudi subclone. These studies suggest that IFN-α inhibits DNA replication and cellular proliferation in Daudi B cells by interfering with G₁ cell cycle events.