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. 1999 Mar 2;96(5):2147-52.
doi: 10.1073/pnas.96.5.2147.

p53 regulates a G2 checkpoint through cyclin B1

Affiliations

p53 regulates a G2 checkpoint through cyclin B1

S A Innocente et al. Proc Natl Acad Sci U S A. .

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.

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Figures

Figure 1
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 p53val135 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
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
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
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
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.

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