Table 1

Effects of Cdc25(S216A) overexpression on mitotic and replication checkpoint control. Uninduced cells or cells induced to express either Cdc25C or Cdc25(S216A) were incubated for 8 hours in the presence of nocodazole (for examination of mitotic control) or were incubated for 8 hours in the presence of hydroxyurea followed by an additional 8 hours in the presence of hydroxyurea and nocodazole (for examination of replication checkpoint control). Cells were processed for chromosome spreading as described (8). Mitotic nuclei were identified from a population of 2000 nuclei each in three independent experiments. Data are presented as mean ± SEM.

CellsTotal mitotic nuclei (out of 2000 nuclei)Percent mitotic nucleiNormal mitotic nuclei Abnormal mitotic nuclei
Mitotic control
Uninduced
Cdc25C594 ± 729.7% ± 0.4594 ± 70 ± 0
Cdc25(S216A)584 ± 829.2% ± 0.4584 ± 80 ± 0
Induced
Cdc25C593 ± 529.6% ± 0.3592 ± 50.3 ± 0.3 (0.06% ± 0.06)
Cdc25(S216A)617 ± 230.8% ± 0.1589 ± 527.0 ± 4.0 (4.4%  ± 0.7)
DNA replication checkpoint control
Uninduced
Cdc25C11.0 ± 1.00.5% ± 0.0711.0 ± 10 ± 0
Cdc25(S216A)10.3 ± 0.90.5% ± 0.0410.3 ± 0.90 ± 0
Induced
Cdc25C15.3 ± 1.80.8% ± 0.0910.0 ± 1.55.3 ± 1.8
Cdc25(S216A)76.0 ± 123.8% ± 0.60* 10.7 ± 0.965 ± 11
  • * The percent of Cdc25(S216A) mitotic nuclei are significantly increased compared with Cdc25C (P < 0.01, Student'st test).

  • Percent of abnormal mitotic nuclei. The percent of Cdc25(S216A) abnormal mitotic nuclei are significantly increased compared with Cdc25C (P < 0.005, Student'st test). The fact that only 4.38% of the Cdc25(S216A) mitotic nuclei displayed abnormal chromosomes indicates that Ser216 phosphorylation of Cdc25C is only one mechanism regulating entry into mitosis (28).