p21(Cip1) and p27(Kip1) regulate cell cycle reentry after hypoxic stress but are not necessary for hypoxia-induced arrest

Abstract

We investigated the role of the cyclin-dependent kinase inhibitors p21(Cip1) and p27(Kip1) in cell cycle regulation during hypoxia and reoxygenation. While moderate hypoxia (1 or 0.1% oxygen) does not significantly impair bromodeoxyuridine incorporation, at very low oxygen tensions (0.01% oxygen) DNA replication is rapidly shut down in immortalized mouse embryo fibroblasts. This S-phase arrest is intact in fibroblasts lacking the cyclin kinase inhibitors p21(Cip1) and p27(Kip1), indicating that these molecules are not essential elements of the arrest pathway. Hypoxia-induced arrest is accompanied by dephosphorylation of pRb and inhibition of cyclin-dependent kinase 2, which results in part from inhibitory phosphorylation. Interestingly, cells lacking the retinoblastoma tumor suppressor protein also display arrest under hypoxia, suggesting that pRb is not an essential mediator of this response. Upon reoxygenation, DNA synthesis resumes by 3.5 h and reaches aerobic levels by 6 h. Cells lacking p21, however, resume DNA synthesis more rapidly upon reoxygenation than wild-type cells, suggesting that this inhibitor may play a role in preventing premature reentry into the cell cycle upon cessation of the hypoxic stress. While p27 null cells did not exhibit rapid reentry into the cell cycle, cells lacking both p21 and p27 entered S phase even more aggressively than those lacking p21 alone, revealing a possible secondary role for p27 in this response. Cdk2 activity is also restored more rapidly in the double-knockout cells when returned to normoxia. These studies reveal that restoration of DNA synthesis after hypoxic stress, but not the S phase arrest itself, is regulated by p21 and p27.

FIG. 1

Cell cycle response to moderate and stringent hypoxia in immortalized MEFs. (A) 2D cell cycle diagrams of cells treated with 1, 0.1, or 0.01% oxygen compared to time-matched normoxic controls. The x axis is red propidium iodide fluorescence; the y axis is green fluorescence from FITC-conjugated anti-BrdU antibody. The percentage of cells that are BrdU positive is indicated. (B) Composite data from multiple experiments, indicating the percentage of cells that are BrdU positive after 24 h (12 h in the case of 0.01% oxygen treatment).

FIG. 1

Cell cycle response to moderate and stringent hypoxia in immortalized MEFs. (A) 2D cell cycle diagrams of cells treated with 1, 0.1, or 0.01% oxygen compared to time-matched normoxic controls. The x axis is red propidium iodide fluorescence; the y axis is green fluorescence from FITC-conjugated anti-BrdU antibody. The percentage of cells that are BrdU positive is indicated. (B) Composite data from multiple experiments, indicating the percentage of cells that are BrdU positive after 24 h (12 h in the case of 0.01% oxygen treatment).

FIG. 2

Effect of mild hypoxia on tritiated-thymidine incorporation. Raw counts are plotted for dishes incubated in 2 or 21% oxygen for 24 or 48 h.

FIG. 3

Cell cycle behavior of matched immortalized MEFs during hypoxia. (A) 2D cell cycle diagrams of all four cells lines from a representative experiment. (B) Composite data for wild-type (⧫) and p21^−/− p27^−/− double-knockout (■) cell lines from all experiments; error bars represent the standard error. Cells were placed in an anaerobic chamber for the times indicated, pulse-labeled for 30 min with BrdU under continuous hypoxia, and then collected and prepared for analysis as described in Materials and Methods.

FIG. 3

Cell cycle behavior of matched immortalized MEFs during hypoxia. (A) 2D cell cycle diagrams of all four cells lines from a representative experiment. (B) Composite data for wild-type (⧫) and p21^−/− p27^−/− double-knockout (■) cell lines from all experiments; error bars represent the standard error. Cells were placed in an anaerobic chamber for the times indicated, pulse-labeled for 30 min with BrdU under continuous hypoxia, and then collected and prepared for analysis as described in Materials and Methods.

FIG. 4

(A) Representative Western blots of p21 and p27 in hypoxic cell extracts. (B) Composite Western blot data from multiple experiments. Blots were scanned on a Storm Imager and analyzed with ImageQuant; the numbers given are ratios relative to the control. (C) Western blot of pRb in hypoxia-treated cells; the higher mobility band represents unphosphorylated or hypophosphorylated pRb (pRb) while the lower mobility band contains hyperphosphorylated pRb (ppRb). WT, wild type.

FIG. 5

(A) Cell cycle profiles of Rb^−/− and wild-type fibroblasts during hypoxia. (B) Cell cycle profiles of p130^−/− cells under hypoxia compared to the wild type. Cells were labeled with BrdU for the final 2 h of treatment. WT, wild type.

FIG. 5

(A) Cell cycle profiles of Rb^−/− and wild-type fibroblasts during hypoxia. (B) Cell cycle profiles of p130^−/− cells under hypoxia compared to the wild type. Cells were labeled with BrdU for the final 2 h of treatment. WT, wild type.

FIG. 6

Cdk2 activity regulation in hypoxic cells. (A) Cdk2 activity in hypoxia-treated cells, measured by immune-complex kinase assay. Autoradiograms of phosphorylated Histone H1 were scanned and then analyzed by using ImageQuant, and ³²P incorporation was plotted as a percentage of the control. (B) Histone H1 autoradiogram and Western blots of Cdk2, cyclin E, and cyclin A from a representative time course. (C) Kinase activity and Cdk2 content of cyclin A and cyclin E immunoprecipitates from control and hypoxic cell extracts.

FIG. 7

Inhibitory phosphorylation under hypoxia. (A) Antiphosphotyrosine and anti-Cdk2 Western blots of anti-Cdk2 or mock IPs. (B) Histone H1 kinase activity of Cdk2 complexes treated with GST-CDC25B.

FIG. 8

Cell cycle profiles of matched immortalized MEFs after reoxygenation. (A) 2D cell cycle diagrams from a representative experiment. (B) Composite BrdU incorporation data for wild-type and double-knockout cell lines from all experiments. (C) [³H]thymidine incorporation. After a 12-h hypoxic treatment, cells were returned to atmospheric oxygen and allowed to reenter the cell cycle. Incorporation for each cell line was normalized to a 12-h aerobic control.

FIG. 8

Cell cycle profiles of matched immortalized MEFs after reoxygenation. (A) 2D cell cycle diagrams from a representative experiment. (B) Composite BrdU incorporation data for wild-type and double-knockout cell lines from all experiments. (C) [³H]thymidine incorporation. After a 12-h hypoxic treatment, cells were returned to atmospheric oxygen and allowed to reenter the cell cycle. Incorporation for each cell line was normalized to a 12-h aerobic control.

FIG. 9

Western blots of p21 and p27 from reoxygenated cells (A), along with a graph of average values over all experiments (B). Cells were exposed to hypoxia for 12 h prior to being returned to atmospheric oxygen.

FIG. 10

Cdk2 activity regulation in reoxygenated cells. (A) Autoradiograms of phosphorylated Histone H1 and Western blots of Cdk2, cyclin E, and cyclin A from a representative reoxygenation time course. (B) [³²P]phosphate incorporation into Histone H1 averaged over all experiments. WT, wild type.