p53 regulates a G2 checkpoint through cyclin B1

Abstract

The p53 tumor suppressor controls multiple cell cycle checkpoints regulating the mammalian response to DNA damage. To identify the mechanism by which p53 regulates G2, we have derived a human ovarian cell that undergoes p53-dependent G2 arrest at 32 degrees C. We have found that p53 prevents G2/M transition by decreasing intracellular levels of cyclin B1 protein and attenuating the activity of the cyclin B1 promoter. Cyclin B1 is the regulatory subunit of the cdc2 kinase and is a protein required for mitotic initiation. The ability of p53 to control mitotic initiation by regulating intracellular cyclin B1 levels suggests that the cyclin B-dependent G2 checkpoint has a role in preventing neoplastic transformation.

Figure 1

Ts-SKOV3 cells undergo G₂ arrest at 32°C. (A) A propidium iodide (PI) DNA-staining profile of Ts-SKOV3 and parental SKOV3 cells at 38°C and after 48 hr at 32°C. At 32°C the majority of Ts-SKOV3 cells are either in G₁ or G₂. A 32°C incubation has no effect on the cell cycle profile of the parental SKOV3 cells. The percentage of cells in each phase of the cell cycle, as determined by the m-cycle program, is indicated. (B) The fraction of Ts-SKOV3 cells in G₂ increases as a function of time at 32°C (●). There is no change in the G₂ fraction in parental SKOV3 cells as a function of time (○). (C) Ts-SKOV3 expresses substantial amounts of the p53^val135 protein at 38°C and 32°C. The parental SKOV3 line expresses no detectable mutant or wild-type p53 protein. (D) MPM-2 reactivity, as measured by ELISA, is reduced in Ts-SKOV3 cells at 32°C relative to 38°C. MPM-2 reactivity in SKOV3 cells is similar between 32°C and 38°C.

Figure 2

G₂ arrest in Ts-SKOV3 cells is associated with a decrease in cyclin B1/cdc2 kinase activity. (A) Cdc2 kinase activity, as measured using Histone H1 as a substrate, as a function of time at 32°C. Cdc2 activity decreases in Ts-SKOV3 (●) but not SKOV3 (○) at 32°C. (B) The decrease in cdc2 kinase activity is not a result of a change in cdc2 protein levels or cdc2 inhibitory tyrosine phosphorylation as determined by Western blotting.

Figure 3

G₂ arrest in Ts-SKOV3 is associated with a decrease in cyclin B1 protein and mRNA. (A) Total cyclin B1 protein levels, as determined by Western blotting, decrease in Ts-SKOV3 cells at 32°C. There is no change in cyclin B1 protein in SKOV3 cells at 32°C. There is a small change in cyclin A protein levels in Ts-SKOV3 at 32°C. (B) Total cyclin B1 mRNA decrease as a function of time in Ts-SKOV3 cells grown at 32°C. Cdc2 mRNA levels are unchanged at 32°C. Glyceraldehyde-3-phosphate dehydrogenase is used as a loading control.

Figure 4

Rescue of p53-mediated G₂ cell cycle arrest by cyclin B1 overexpression. (A) Transient transfection of cyclin B1 (cytomegalovirus promoter) into Ts-SKOV3 cells abolishes the G₂ arrest that occurs at 32°C in control (pCDNA3) transfected cells. Transfection of cdc2 has no effect on the G₂ arrest. Transient transfection of cyclin B1 has no effect on the cell cycle profile of Ts-SKOV3 cells at 38°C. (B) Total protein levels of cyclin B1 and cdc2, as determined by Western blot, for each transfection condition in A. (C) Cyclin B1/cdc2 kinase activity, as determined by Histone H1 phosphorylation, for each condition in A.

Figure 5

p53 decreases the activity of the cyclin B1 promoter. (A) The activity of the cyclin B1 promoter (36) (1,050 bp 5′ of transcription initiation) transiently transfected into Ts-SKOV3 cells is markedly lower at 32°C (solid bars) than at 37°C (open bars) 24 and 48 hr after transfection. Promoter activity is not altered substantially in SKOV3 cells between 37°C (open bars) and 32°C (solid bars). (B) Wild-type p53 can suppress the activity of the cyclin B1 promoter in transient transfection assays in SKOV3, DP16, Saos-2, and A431 cells. Assays were performed at 37°C, and all data points are the mean of duplicate CAT measurements.