p21-Mediated nuclear retention of cyclin B1-Cdk1 in response to genotoxic stress

Abstract

G2 arrest of cells suffering DNA damage in S phase is crucial to avoid their entry into mitosis, with the concomitant risks of oncogenic transformation. According to the current model, signals elicited by DNA damage prevent mitosis by inhibiting both activation and nuclear import of cyclin B1-Cdk1, a master mitotic regulator. We now show that normal human fibroblasts use additional mechanisms to block activation of cyclin B1-Cdk1. In these cells, exposure to nonrepairable DNA damage leads to nuclear accumulation of inactive cyclin B1-Cdk1 complexes. This nuclear retention, which strictly depends on association with endogenous p21, prevents activation of cyclin B1-Cdk1 by Cdc25 and Cdk-activating kinase as well as its recruitment to the centrosome. In p21-deficient normal human fibroblasts and immortal cell lines, cyclin B1 fails to accumulate in the nucleus and could be readily detected at the centrosome in response to DNA damage. Therefore, in normal cells, p21 exerts a dual role in mediating DNA damage-induced cell cycle arrest and exit before mitosis. In addition to blocking pRb phosphorylation, p21 directly prevents mitosis by inactivating and maintaining the inactive state of mitotic cyclin-Cdk complexes. This, with subsequent degradation of mitotic cyclins, further contributes to the establishment of a permanent G2 arrest.

Figure 2.

Increasing association between p21 and cyclin B1-Cdk1 after exposure to DNA damage-inducing drugs. (A) Cell cycle distribution of asynchronously growing untreated NHFs (Cont) and cultures exposed to ICRF-193 or bleomycin for 12 h. DNA content was determined by FACS analysis of propidium iodide-stained cells. The same cell cultures were used for the biochemical analysis described below. (B) Western blot analysis showing cyclin B1, cyclin A, Cdk1, and p21 levels in p21 and cyclin B1 immunoprecipitates (IP) isolated from untreated NHFs (Con) and NHFs exposed to ICRF-193 (ICRF) or bleomycin (Bleo). For all immunoprecipitation experiments, the equivalent amounts of cell extract (150 μg) were used, and the samples were analyzed on the same immunoblots. Numbers 1, 2, and 3 indicate differentially phosphorylated Cdk1 isoforms (see text for the details). (C) Western blot analysis showing cyclin B1 levels after p21 immunodepletion in bleomycin- and ICRF-193-treated NHF. LC, loading control.

Figure 5.

ICRF-193-arrested NHFs expressing HPV16-E7 oncogene accumulate high amounts of cyclin B1 in the nucleus but fail to initiate early mitotic events. (A) Western blot analysis showing the pRb phosphorylation status, and expression of cyclin A, p53, and p21 in asynchronously proliferating (As), serum-deprived (SD), and contact inhibited (CI) wild-type, E6- and E7-expressing NHFs. (B) Colocalization of cyclin B1 and Mpm2 in asynchronous untreated (control) and ICRF-193-treated E7 cells. Cells were fixed in methanol and simultaneously stained with mouse monoclonal anti-Mpm2 (Texas-Red) and with rabbit polyclonal anti-cyclin B1 (fluorescein isothiocyanate, FITC) antibodies. Numbers denote cells arrested in G2 (1) as well as in early (2) and late prophase (3). Arrow depicts the centrosome. Note the centrosomal localization of cyclin B1 in cell 2 exhibiting an elevated Mpm2 signal and nuclear accumulation of cyclin B1 and an accentuated Mpm2 signal in late prophase cell 3 (Figure 3B). (C) Colocalization of cyclin B1 and p21 in untreated (control) and ICRF-193-treated E7 cells. Cells were fixed in methanol and simultaneously stained with mouse monoclonal anti-cyclin B1 (Texas-Red) and with rabbit polyclonal anti-p21 (FITC) antibodies. Numbers denote cells at early (1) and late (2) prophase.

Figure 6.

p21 binds to inactive cyclin B1-Cdk1 complexes. (A) Histone H1 kinase assays and Western blot analysis of cyclin B1 immunoprecipitates isolated from asynchronously growing control (Ct) and ICRF-193-treated E6- and E7-expressing NHFs. The same immunoprecipitates were analyzed by Western blot (W.B.) for the presence of cyclin B1, Cdk1 and p21. Numbers denote hyperphosphorylated (3), partially phosphorylated (2), and hypophosphorylated (1) Cdk1 isoforms (see text for more explanation). Arrows point at hypophosphorylated Cdk1 (isoform 1) that specifically accumulates in ICRF-treated cells. Note the difference in the abundance of this isoform between E6 and E7 cells. (B and C) p21 immunodepletion experiments. The protein extracts prepared from untreated (Ct) and ICRF-193-treated cells were immunodepleted for p21-bound complexes as described in MATERIALS AND METHODS. (B) Western blot analysis showing cyclin B1 and p21 levels in p21 immunoprecipitates. (C) Western blot analysis cyclin B1 immunoprecipitates isolated from mock-treated (-) and p21-depleted cell extracts prepared from the same experiment. Numbers 1, 2, and 3 indicate differentially phosphorylated Cdk1 isoforms. Note that even in untreated E7 cells, a large population of cyclin B1-Cdk1 is bound to p21. In this immunoblot, the cyclin B1 signal (arrow) is obscured by a parasite background band. (D) Histone H1 kinase assays and Western blot analysis of cyclin B1 immunoprecipitates isolated from wild-type NHF synchronized in S, G2/M, and M (+ nocodazole) phases. Numbers 1, 2 and 3 indicate differentially phosphorylated Cdk1 isoforms, whereas the arrow points at the hypophosphorylated and active Cdk1. (E) Western blot analysis comparing cyclin B1 IP isolated from ICRF-193-treated E6 and E7 cultures. Numbers 1, 2, and 3 indicate differentially phosphorylated Cdk1 isoforms. To assess the phosphorylation status of the Cdk1 isoform 1 that accumulates in cyclin B1 IP in ICRF-193-treated E7 cells, immunoblots were probed with an antibody specific for CAK-phosphorylated phospho-threonine 161 (T161-P).

Figure 7.

p21 prevents Cdc25-dependent activation of cyclin B1-Cdk1 complexes. Histone H1 kinase assays and Western blot analysis of cyclin B1 immunoprecipitates isolated from p21-deficent cells (HeLa arrested by thymidine block and HPV16-E6-expressing NHFs) (A) and NHFs containing normal (WT) or elevated (HPV16-E7-expressing NHFs) p21 levels (B). The cells were incubated with ICRF-193 (or bleomycin), as described in the legend to Figure 6. Before histone H1 kinase assays (H1K), cyclin B1 immunoprecipitates were treated (+) or not (-) with recombinant Cdc25B as described in MATERIALS AND METHODS. As a control, we also show cyclin B1-Cdk1 complexes from untreated asynchronously growing cells (indicated by asterisk; lanes 3 and 4) that do not contain p21. Lanes 5 and 6 show p21-bound cyclin B1-Cdk1 complexes from ICRF-193-treated cells. Note that these p21 immunoprecipitates contain higher amounts of Cdk1, due to the presence of other mitotic cyclin-Cdk complexes. Immunoblots were first exposed for autoradiography and then incubated with the indicated antibodies to reveal the status of cyclin B1, Cdk1, and p21. Numbers 1, 2, and 3 indicate differentially phosphorylated Cdk1 isoforms. Hyperphosphorylated Cdk1 (isoform 3) is the main Cdc25 target. For better appreciation of different Cdk1 isoforms different ECL exposures are shown (SE, short exposure; LE, long exposure). Autoradiograph B is exposed for longer (24 h) than autoradiograph A (3 h) to visualize a weak cyclin B1-Cdk1 activation in ICRF-193-treated wild-type and E7 cells.

Figure 4.

p21^-/- fibroblasts fail to accumulate nuclear cyclin B1 in response to ICRF-193 or γ-irradiation. (A) Western blot analysis of total cell extracts showing the pRb phosphorylation status, and expression of p53 and p21 in asynchronously proliferating (-) and ICRF-193-treated (+) wild-type (WT) and isogenic p21^-/- NHFs. Two different ECL exposures (SE and LE) were shown to demonstrate the maintenance of pRb hyperphosphorylation in ICRF-193-treated p21^-/- NHF. (B) Western blot and histone H1 kinase analysis of cyclin B1 immunoprecipitates (cB1 IP) isolated from the same cell extracts as described above. Although in ICRF-193-treated p21^-/- NHFs most of cyclin B1 is associated with hyperphosphorylated Cdk1, the persistence of kinase activity indicates inefficient G2 arrest. (C) Cyclin B1 localization in NHF p21^-/- exposed to ICRF-193 and γ-irradiation. Cells also were stained with either anti-p21 (left) or with anti-γ-H2AX antibodies (right) to visualize the foci induced by DNA damage. Arrows point at early mitotic cells showing ongoing DNA condensation.

Figure 1.

Nuclear accumulation of cyclin B1 in the presence of ICRF-193, a drug that specifically provokes G2 arrest. (A) Colocalization of cyclin B1 and p21 in ICRF-193-treated synchronized NHFs. Cells were synchronized as described in MATERIALS AND METHODS, and the drug was added when the majority of the cells had passed the G1/S boundary. Control cells were fixed when most control cells had entered mitosis, whereas ICRF-193-treated cells were fixed 3 h later. Cell cycle profiles of synchronized control and drug-treated cells are shown in Supplementary Figure 1a. Cells were simultaneously stained with a mouse monoclonal anti-cyclin B1 and with a rabbit polyclonal anti-p21. (B) Indirect immunofluorescence analysis comparing subcellular localization of cyclin B1 and Mpm2 signal in control G2/M-enriched cultures and in ICRF-193-arrested NHFs. Cells were fixed in methanol and simultaneously stained with a mouse monoclonal anti-Mpm2 (Texas-Red) and with a rabbit polyclonal anti-cyclin B1 (fluorescein isothiocyanate, FITC). Cells 1-3 depict different G2/M transition stages as judged by cyclin B1 localization, the intensity of the Mpm2 signals, separation of the centrosomes (arrows), and DNA condensation levels (Hoechst). Note that only cell 3 exhibits significant DNA condensation (late prophase), whereas in the cells exhibiting increased Mpm2 staining (1 and 2) no DNA condensation is readily detectable. Arrows point at cyclin B1 localized at the centrosome. Asterisks depict cells at earlier stages of the cell cycle exhibiting a background Mpm2 signal. (C) Colocalization of cyclin B1 and γ-tubulin in control G2/M and ICRF-193-treated NHFs. Cells were fixed in methanol and simultaneously stained with a mouse monoclonal anti-γ-tubulin (Texas-Red) and with a rabbit polyclonal anti-cyclin B1 (FITC). Cells in prophase with visible DNA condensation are depicted by an asterisk (^*). Note the poor separation of centrosomes (arrows) in ICRF-193-arrested cells as compared with control G2/M cells.

Figure 3.

NHF expressing the HPV-16 E6 oncogene undergo early mitotic events after exposure to genotoxic stress. (A) Colocalization of cyclin B1 and γ-tubulin in control G2/M-enriched and ICRF-193-treated E6 expressing fibroblasts. Cells were synchronized as specified in the legend of Figure 1, and their cell cycle profile is shown in Supplementary Figure 1B. Note a strong cyclin B1 signal at the centrosomes (arrows) in ICRF-193-treated cells, similar to one observed in early prophase cells. In control G2/M cells, “P” denotes the cells in advanced prophase showing DNA condensation and exhibiting accumulation of cyclin B1 in the nucleus. Cells were simultaneously stained with mouse monoclonal anti-γ-tubulin (Texas-Red) and with rabbit polyclonal anti-cyclin B1 (fluorescein isothiocyanate, FITC). (B) Statistical analysis of nuclear localization of cyclin B1 in untreated (G2/M transition) and ICRF-193-treated wild-type (WT) and E6-expressing NHFs. Cells expressing cyclin B1 were scored for localization in the cytoplasm (C), both cytoplasm and nucleus (CN), and predominantly in the nucleus (N). Black color indicates the population of cells exhibiting a visible DNA condensation pattern (visualized by Hoechst staining). The graph represents an average of at least two independent experiments. (C) Recruitment of activated cyclin B1-Cdk1 at the centrosomes in NHFs expressing E6 but not in wild-type NHFs in the presence of ICRF-193. Double immunofluorescence using rabbit polyclonal anti-phospho-serine 133 cyclin B1 (PS133) and mouse monoclonal anti-γ-tubulin antibodies. Micrographs show untreated cells at G2/M transition (E6) and cells exposed to ICRF-193 (WT and E6). (D) Colocalization of cyclin B1 and Mpm2 in untreated (G2/M) and ICRF-193-treated E6 cells. In control G2/M cells, numbers 1-4 depict different G2-prophase stages as judged by cyclin B1 localization, the intensity of the Mpm2 signal, the separation of the centrosomes (arrows), and DNA condensation levels (Hoechst). According to these criteria, cell 1 is in late G2, cells 2 and 3 in early prophase, and cell 4 in midprophase (cyclin B1 is nuclear and DNA is condensed). According to the same criteria, ICRF-193-arrested cells are in early prophase, exhibiting an increased Mpm2 signal and cyclin B1 localized at the centrosome (arrows; cf. also Figure 2A).

Figure 8.

Role of p21 in establishing irreversible cell cycle arrest in G2 after genotoxic stress. In response to DNA damage, G2 arrest is initiated by Chk1/2-dependent inactivation of Cdc25 family phosphatases (Cdc 25A, B, C), thus preventing activation of cyclin B1-Cdk1. The maintenance of G2 arrest is assured by p21-dependent inactivation of mitotic Cdks (cyclin B1-Cdk1 and cyclin A-Cdk1) and their eventual degradation. Cyclin B1-Cdk1 complexes are maintained in an inactive state by association with p21, which provokes nuclear retention of cyclin B1-Cdk1 and prevents their activation at the centrosome and, possibly, in the cytoplasm and nucleus. By binding to cyclin B1-Cdk1, p21 prevents both activation by CAK-dependent phosphorylation of Thr161 (white circle) and Cdc25-dependent dephosphorylation of Thr14 and Tyr15 (black circles). For the sake of simplicity, Wee1 and CAK, which are predominantly in the nucleus, are placed in cytoplasm. Additionally, p21 inactivates cyclin-Cdk complexes (such as cyclin A-Cdk2) responsible for phosphorylation of pocket proteins (PP; pRB, p107, and p130) during S/G2/M progression. Subsequent accumulation of active pocket proteins blocks expression of genes involved in G2/M progression (Ren et al., 2002; Baus et al., 2003). Inactivation of either p53 or pocket proteins by expression of viral oncogenes (such as HPV16-E6 and HPV16-E7), does not interfere with the cell cycle arrest in G2 but compromises the irreversible cell cycle exit. Black and white circles denote inactivating and activating phosphorylations (P), respectively.