DNA damage cell checkpoint activities are altered in monocrotaline pyrrole-induced cell cycle arrest in human pulmonary artery endothelial cells

Monocrotaline pyrrole (MCTP) causes cyto- and karyomegaly and persistent ce ll cycle arrest in the G2 stage of the cell cycle in cultured bovine pulmon ary artery endothelial cells. To better characterize the cell cycle regulat ory mechanisms of this process as well as determine whether this process wo uld occur in cells of human origin, we treated human pulmonary artery endot helial cell (HPAEC) cultures with MCTP and determined, by Row cytometry, th e expression of cyclin B1 and p53 in conjunction with DNA content. We also validated by Western blots that the persistence of cdc2 in its inactivated phosphorylated state, previously described in bovine cell cultures, occurre d in HPAEC, Alterations in p53, cyclin A, cyclin B1, and cdc25c expression were also examined in Western blots of treated HPAEC extracts, The response of HPAEC to MCTP was compared with that of adriamycin and nocodazole, agen ts known to cause cell cycle alterations. Results of these experiments demo nstrate that HPAEC treated with MCTP develop a population of cells in G2 th at has increased cyclin B1 expression. These cells express increased amount s of cdc2 but not cdc25c, The ratio of inactive triphosphorylated cdc2 to t he active monophosphorylated form increased moderately from control culture s in contrast to predominance of the active form in nocodazole-treated cult ures. In addition, a second population of cells expressing cyclin B1 had co ntinued incorporation of BrdU and DNA content consistent with 8 N chromosom es. A similar 8 N cell population was evident in nocodazole-treated cells b ut these cells had both cyclin B1 positive and negative components. Compare d with adriamycin, a known inducer of p53, MCTP-treatcd HPAEC expressed p53 only at high concentrations and p53 expression was not coordinated with G2 arrest or polyploidy, We conclude that HPAEC treated with low concentratio ns of MCTP develop G2 arrest in association with persistent cyclin B1 expre ssion, failure to completely activate cdc2, and continued DNA synthesis thr ough a pathway that is unrelated to altered expression of p53. (C) 2000 Aca demic Press.