The expression of Wip1 depends on wtp53 status. Total RNA isolated from WI-38, RKO, U2OS, HCT-116 and Lovo, which all carry wtp53, and from RKO-E6, Saos-2, Calu-6, T98G and HT29, which all harbor mtp53 without (−) or with (+) the treatment of 5-FU respectively was hybridized to the ³²P-labeled Wip1 cDNA or GAPDH cDNA fragment. Wip1 mRNA level is substantially elevated only in cell lines having wtp53 following 5-FU treatment (+). (GAPDH used as RNA loading control). P21/waf1 mRNA expression was detected in the same batch of RNA samples used. The pattern of P21/waf1 mRNA expression is similar to the one of wip1 mRNA expression (data not shown), confirming the activity of wtp53.

The Wip1 induces G₂/M arrest in cells carrying wtp53 but not in cells harbouring mtp53. (A and B) Establishment of conditional Wip1 expression cells. RNA gel blots were prepared using total RNA isolated from un-induced (−Dex) and induced (+Dex) clone U2–15 (A) and TW1F cells (B) derived from U2OS (wtp53) and T98G (mtp53) cell lines respectively, detected by ³²P-labeled Wip1 or GAPDH cDNA fragment. The Wip1 mRNA is increased substantially in cells with the addition of Dex. (GAPDH is loading control). The corresponding levels of Wip1 protein expression were also detected. (C) Wip1 induction of G₂/M arrest in U2–15 cells. Synchronized U2–15 cells (left part) were released and incubated with (+) or without (−) Dex. Cells were collected at the time of 8 h in 2 h interval respectively after starvation synchronization (left part). The fitting curves from Flow Cytometric Analysis showed (right part) are cell cycle profiles for cells tested at 12 and 14 h points. At 14 h Wip1 induction, U2–15 cells could not move from G₂/M into G₁ as they did in the situation of non-induction of Wip1. (D) Induction of Wip1 has no effect on TW1F cells. Synchronized TW1F cells (left part) were released and incubated with (+) or without (−) Dex. Cells were collected at the time of 14 h in 2 h interval respectively. The fitting curves are cell cycle profiles of 18 h and 20 h points (right part). The transition from G₂/M peak to G₁ peak is similarly shown in both Wip1 induced (+) and non-induced (−) TW1F cells. (pMSG-PP2C stable cells were established, and used to conduct a similar cell cycle analysis; but induction of PP2C has no clear effect on cell cycle progression (data not shown), indicating G₂/M arrest triggered by Wip1 is not a general effect of PP2C phosphatase family).

Wip1 induces G₂/M arrest but also decreases cellular apoptosis in Mouse fibroblasts. (A) Mouse fibroblasts were treated with Nocadazole (0.1 µg/ml) for 40 h. The cell cycle profile of these treated cells shows a relatively nice synchronization at G₂ population. (B) Cell cycle analysis on Nocodazole-synchronized clone 23 cells shows a better G₂ arrest triggered by Wip1 in cells carrying functional p53. The cell cycle profiles presented here in DNA content histogram are those of 9 h and 12 h at 32°C (left part) and 37°C (right part). Wip1 induction (+) can increase cell population in G₂ phase and decrease sub-G₁ peak (arrow pointed) at time of 12 h at 32°C, but not at 37°C. (C) Wip1 induction has no effect on synchronized MF-41 cells. (D) Wip1 protein expression in both MF41 cells and clone 23 cells upon the Dex addition. Left part is for cells at 32°C and right part is for cells at 37°C, and both show the correct levels of Wip1 protein expression in cells used for cell cycle analysis, indicating the conditional system remains intact.

Induction of Wip1 increases the level of inhibitory tyrosine phosphorylation on Cdc2 protein. (A) The levels of nuclear cyclin B and cdc2 proteins are the same between the Wip1 induced and uninduced cells at time of 12 h. The western blot prepared from the nuclear fraction of Wip1 induced (+) and uninduced (−) U2–15 cells at time of 12 h, was blotted by cyclinB and cdc2 antibodies, respectively. There is no detectable difference in both protein levels of Cdc2 and cyclin B between lane (−) and lane (+). Cytoplasmic levels of both proteins were also the same between Wip1 induced and uninduced cells (data not shown) (the stained blot for the control of protein loading). (B) The level of inhibitory tyrosine phosphorylation on Cdc2 is increased when wip1 is induced. The immunoprecipitate from the total soluble protein of uninduced (lane⁻) and induced (lane⁺) U2–15 cells at time of 12 h using anti Cdc2 antibody was detected by anti-phosphotyrosine-15 monoclonal antibody. There is an increased level of inhibitory phosphotyrosine on Cdc2 protein in Lane (+). The same blot shown in the top part of (B) was stripped and detected using anti-Cdc2 antibody. There is no detectable difference in Cdc2 protein level between lane (−) and lane (+) (the middle part of B). The IP input control is provided (the bottom part of B).

The Wip1 is induced by an elevated steady-state level of wtp53. (A) Autoradiogram of RNA gel blots using either Wip1 cDNA or GAPDH as probe. Lanes: marked (−), GM47.23 cells without Dex (control, no induction of wtp53); Marked (+), GM47.23 cells with Dex (for the induction of wtp53). (GAPDH used as a RNA loading control). The Wip1 mRNA can be detected in GM cells with the wtp53 induction. (B) Western blots analysis of p53 and p21 proteins in both GM and its parental cell line T98G cells. The p21 is only detected in GM cells with the addition of Dex (+), indicating the activity of wtp53.

Wip1 induction induces G₂/M arrest in un-synchronized mouse cells carrying functional p53. (A) The Wip1 mRNA expression in mouse fibroblast (p53^−/−) derivative cells (MF-41). RNA gel blot was prepared using total RNA isolated from the induced (+) and un-induced (−) No.41 clone, which is named MF41, and detected by ³²P-Wip1 cDNA fragment. The Wip1 signal was increased in lane marked (+). (B) Western blot was prepared using 50 µg/lane of cell soluble protein from mouse fibroblast (MF) (p53^+/+). MF 41 (wip1⁺, p53^−/−) and clone 23 derived from MF41 (tsp53⁺; wip1⁺), detected with primary antibodies against p53 and actin respectively. The p53 protein signal is shown in MEF and clone 23, but not in MF41 cells. (The actin signal used as loading control). (C and D) Asynchronized MF-41 and clone 23 cells were collected after O/N induction (+) or non-induction (−) of Wip1 at both 32°C (wtp53) and 37°C (mtp53). The cell cycle profiles presented in DNA content histogram show that the Wip1 induction can clearly increase cell population in G₂ phase when functional p53 is present (at 32°C). (Red arrow head marks a higher G_2% of cells compared with other cells tested).

Induction of Wip1 inhibits Cdc2 kinase activity. (A) Wip1 modulates the kinase activity of Cdc2/cyclinB. Immune complexes from the total soluble protein of 12 h un-induced (Lane⁻) and induced (lane+) U2–15 cells using anti-cdc2 monoclonal antibodies were mixed with histone H1 and γ-³²P-ATP at 30°C for 30 min. Autoradiogram (top part) shows much less phosphorylated histone H1 in Lane⁺ than in Lane⁻. [Wb (middle part) for the control of cdc2 protein used in the assay and Coomassie blue-stained blot (bottom part) for H1 loading control]. (B and C) Cdc2 kinase activity is decreased upon Wip1 induction in mouse cells when functional p53 is restored. Similar to the experiment conducted in (A), the corresponding mouse cells used for cell cycle analysis showing a nice G₂/M arrest were used for preparing cell lysates, and subsequently Cdc2 IP and kinase assay were performed. The decreased Cdc2 kinase activity was found when Wip1 was induced in clone 23 cells (B) but not in MF-41 cells (C) at 32°C.