EGF is incomplete mitogen in porcine aortic smooth muscle cells: DNA synthesis without cell division

To characterize growth effects of epidermal growth factor (EGF) in subconfluent quiescent porcine aortic vascular smooth muscle cells (VSMC), we measured DNA and protein synthesis by [³H]thymidine (Thd) and [³⁵S]methionine (Met) incorporation, respectively, and cell proliferation rates over 0-6 days in Dulbecco's modified Eagle's-Ham's F-12 media containing 0.4% fetal calf serum (FCS) and insulin. EGF induced dose- dependent [³H]Thd uptake (P < 0.001); after 10^-9 M EGF, DNA synthesis rate peaked at 24 h, averaging 77% of the response to 10% FCS, and then declined steeply with nadir at 48-60 h. Unexpectedly, EGF failed to induce cell proliferation in the first 4 days, leaving this initial burst of DNA synthesis (12-60 h) uncoupled from cell division. A second lesser but sustained phase of increased DNA synthesis, apparent by day 3-4, was associated with a small increase in cell number on day 6 (P < 0.05). The early unsustained burst of DNA synthesis reflects EGF's potent mitogenic efficacy for DNA synthesis (G₁- to S-phase traversal), probably acting on a subset of cells partially synchronized initially at an EGF-responsive G₀/G₁ locus; the minimal cell division despite brisk DNA synthesis documents EGF's limited efficacy for (or inhibition of) late cell-cycle events required for completion of mitosis. Late cell-cycle processes are thus rate limiting. EGF also increased protein synthetic rate over control (P < 0.03) but to a lesser degree (P < 0.01) than 10% FCS. Indomethacin (10^-6 M) did not alter DNA or proliferative responses to 10^-9 M EGF but transiently augmented EGF-induced protein synthesis (P < 0.025) at 24 h only. We conclude that, in porcine aortic VSMC studied in 0.4% FCS-insulin, EGF is a potent mitogen for DNA synthesis but an incomplete mitogen for the late cell-cycle events required for completion of cell division, a pattern potentially relevant to the phenomenon of polyploidy observed in VSMC hypertrophy in vivo.