Arrested development: When cells make mistakes


Editor's Introduction

Requirement for p53 and p21 to sustain G2 arrest after DNA damage

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Our DNA is constantly being damaged by environmental factors like radiation and chemicals, as well by errors that occur during replication. Fortunately, our cells have many tools for repairing this damage. But if the damage is not repaired before the cell divides or its DNA is replicated, the results can be catastrophic, causing cell death or cancer. Many of our cells go through a sequence of events that culminate in cell division (known as the cell cycle). p53 and p21 are two proteins that are responsible for arresting the cell cycle when DNA damage occurs. When these proteins are missing or defective, the cell cannot stop to repair damage prior to replicating or dividing.

This classic 1998 article by Bunz et al. made an important contribution to establishing the roles of these proteins, specifically at the boundary between two phases of the cell cycle: G2 phase and M phase (mitosis).

This link takes you to an online interactive that explores the phases, checkpoints, and protein regulators of the cell cycle. It also explains how mutated versions of these proteins can lead to the development of cancer.

Paper Details

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Original title
Requirement for p53 and p21 to sustain G2 arrest after DNA damage
Original publication date
Vol. 282, Issue 5393, pp. 1497-1501
Issue name


After DNA damage, many cells appear to enter a sustained arrest in the G2 phase of the cell cycle. It is shown here that this arrest could be sustained only when p53 was present in the cell and capable of transcriptionally activating the cyclin-dependent kinase inhibitor p21. After disruption of either the p53 or the p21 gene, γ radiated cells progressed into mitosis and exhibited a G2 DNA content only because of a failure of cytokinesis. Thus, p53 and p21 appear to be essential for maintaining the G2 checkpoint in human cells.

Video. p53 is a recessive tumor suppressor gene that halts the cell cycle when the cell's DNA is damaged. 


DNA damaging agents in the form of γ radiation and chemotherapeutic drugs are the mainstays of most current cancer treatment regimens. This has stimulated much research to understand the cellular responses to DNA damage.

Video. Dr. Sawyers outlines the role of cell division and cell death in cancer and the traditional treatments used to treat the disease. 

After DNA damage, cells arrest at the transition from G1 to S phase (G1-S) or from G2 to M phase (G2-M) of the cell cycle, with DNA complements of 2n or 4n, respectively (1). Arrest at these checkpoints prevents DNA replication and mitosis in the presence of unrepaired chromosomal alterations. The proportion of cells that arrest at G1-S or G2-M depends on cell type, growth conditions, and the checkpoint controls operative in the cell (2). The G1-S arrest results, at least in part, from p53-regulated synthesis of the cell cycle inhibitor p21WAF1/CIP1 (3–5), which leads to inhibition of the cyclin-cdk complexes required for the transition from G1 to S phase. However, arrest in G2 after DNA damage occurs in both murine and human cells in the absence of p21 or p53 (3–6). This arrest is thought to result from activation of a protein kinase, Chk1, that phosphorylates and inhibits the function of the protein phosphatase Cdc25C (7). Inhibition of Cdc25C prevents the removal of inhibitory phosphates from Cdc2, a protein kinase that complexes with mitotic cyclins and is required for mitotic entry (8).

Video. In a lecture in 2003, Dr. Bert Vogelstein, one of the co-authors of this study, introduces p53 and the cellular break it regulates, p21 (WAF1). Mutations in different locations of the p53 gene can cause different types of cancer. 

The integrity of the Chk1-Cdc25C-Cdc2 pathway in p21- or p53-mutant cells would appear to explain the prolonged G2-M arrest that occurs in such cells (3–7). However, there are some features of this arrest that have remained unexplained. Cells with disrupted p21 or p53 that are arrested in G2 can undergo DNA synthesis, in some cases resulting in cells with DNA contents of 8n or higher (9, 10). Such rereplication also occurs in cells blocked in mitosis (11), but it is unclear how this could occur in cells arrested before mitosis, as it is thought that negatively acting factors that prevent DNA synthesis must be degraded during the mitotic phase (12) and that positively-acting “licensing factors” required for the next S phase cannot traverse the nuclear membrane and therefore can enter the nucleus only during mitosis (13).

We therefore investigated G2-M arrest in p53-deficient cells in more detail. We initially used a panel of six human colorectal cancer cell lines, three with intact p53 genes and three with mutant genes. After irradiation, most of the cells in each culture were arrested with a DNA complement of 4n and >95% of the cells were in interphase (9, 14). To determine whether any of these cells entered M phase after irradiation, we treated them with nocodazole, a microtubule-disrupting agent that can trap cells in mitosis for several hours. In cells with wild-type p53, the mitotic index was very low after irradiation, as expected for cells truly blocked in G2 (Fig. 1A). Unexpectedly, a large fraction of the p53-mutant cells entered mitosis after irradiation (Fig. 1B).


Fig. 1. Entry of colorectal cancer cell lines into mitosis after γ radiation. Cell lines with endogenous wild-type (A) or mutant (B) p53 genes were treated with nocodazole beginning 30 min after γ radiation (32). At the indicated times, cells were fixed, stained, and examined by fluorescence microscopy to determine the fraction of cells in mitosis (mitotic index). Cell lines were HCT116 (closed triangles), RKO (closed squares), SW48 (closed circles), DLD1 (open circles), HT29 (open diamonds), and Caco2 (open triangles).

Mitotic Index

A characteristic feature of cells that undergo mitosis is the condensation of chromosomes. The mitotic index tells us how many cells in a population are dividing and is often used as a prognostic tool in cancer treatments.

It is calculated by counting the number of cells with condensed chromosomes and dividing that number by the total number of cells observed.

Wild-type cell lines

The three cell lines with normal p53 (HCT116, RKO, and SW48) do not enter mitosis after the cells are subjected to radiation (DNA damage).

Radiation is used to damage the DNA.

Mutant p53 cell lines

The three cell lines with p53 mutations (DLD1, HT29, and Caco2) enter mitosis despite the fact that their DNA was damaged by radiation.


These results imply that p53 is preventing entry into mitosis in the presence of DNA damage.

This is important because if a cell tries to divide despite DNA damage, the daughter cells may inherit mutated, broken, or incomplete DNA. At best, this will result in cell death. However, depending on the damage, the cell could lose control of growth or division, leading to the development of cancer.

These experiments suggested that p53 controls a G2 checkpoint that prevents entry into mitosis after DNA damage. However, this conclusion was tempered by the fact that the six lines, all derived from colorectal cancers, might differ in ways other than p53 status. We therefore disrupted the p53 gene by homologous recombination in one of the wild-type p53-containing cell lines. The HCT116 cell line was chosen because it has apparently intact DNA damage-dependent and spindle-dependent checkpoints, and it is suitable for targeted homologous recombination (4, 9, 15). Two promoterless targeting vectors, each containing a geneticin- or hygromycin-resistance gene in place of genomic p53 sequences, were used to sequentially disrupt the two p53 alleles in HCT116 cells (16). Cells with the desired genotypes (Fig. 2A) were used to test the response to DNA damage. After treatment with γ radiation, parental cells (p53+/+) and those with one of the two p53 alleles disrupted (14) arrested in either G1 or G2, as expected for cells with intact checkpoints (Fig. 2B). The vast majority of cells with both p53 alleles disrupted (p53–/–) appeared to arrest at G2-M, with a DNA content of 4n, whereas a minor fraction had a DNA content of less than 2n, which is indicative of apoptosis (Fig. 2B). A substantial G2 arrest was observed in cells of all genotypes from 24 to 72 hours after 12-gray (Gy) of γ radiation (Fig. 2B). However, morphologic examination revealed mitotic cells in the p53–/– cell population within 24 hours after irradiation, whereas cells with one or two intact copies of p53 remained mitotically inactive for the duration of the experiment (Fig. 2C). All cell types exhibited mitotic arrest in response to nocodazole treatment in the absence of irradiation (Fig. 2D). A nocodazole-trapping experiment, however, confirmed that only p53–/– cells entered mitosis after irradiation (Fig. 2E). Thus, although G2 arrest was initiated after irradiation in all cells tested, this arrest was not sustained in the absence of functional p53 (Figs. 1 and 2E).



Fig. 2. Targeted deletion of p53 in a colorectal cell line. (A) Southern blot after Hind III digestion of genomic DNA of selected clones (33). Fragments corresponding to the wild-type allele (wt) (2.5 kb), and neomycin (neo) (3.5 kb) and hygromycin (hyg) (3.7 kb) homologous integrants are shown. (B) Flow cytometric analysis of parental HCT116 cells (p53+/+) and HCT116 cells with targeted deletions of both (p53–/–) p53 alleles at the indicated time points after 12-Gy γ radiation. Flow cytometry was done as described (4). Ten thousand cells were analyzed in each experiment; n represents haploid DNA content and results are plotted on a logarithmic horizontal axis. Mitotic indices of parental cells (closed squares) and cells with targeted deletions of one (half-closed squares) or both (open squares) alleles of p53 are shown at the indicated times after 12-Gy γ radiation alone (C), nocodazole treatment alone (D), or 12-Gy γ radiation followed by nocodazole treatment (E).

Southern blot

This technique allows the researcher to detect specific DNA sequences in a sample. These are the steps:

1. A DNA sequence complementary to the target sequence is designed and labeled with a radioactive probe.

2. Sample DNA is digested by sequence-specific enzymes that cleave the DNA into defined fragments. These fragments are run on a gel to separate them by size.

3. The DNA fragments in the gel are transferred to a membrane, which can then be probed with the radioactive sequence.

4a. If the target DNA is present, the complementary radioactive probe will bind, and a band will appear on a film placed on top of the membrane.

4b. If the sequence is not present, no band will be observed because the probe won't bind.

Here, the authors are showing that their cell line carries the correct p53 allele (normal or deleted). In cases where the p53 gene was deleted, it was replaced with the antibiotic resistance marker (hyg or neo). So each column represents a different cell line, and the band patterns below show what DNA is present in these samples.

Flow cytometry

This technique can be used to detect a variety of different characteristics of individual cells. In this case, the authors were looking at DNA content, determined by using a DNA-binding dye.

The flow cytometer determined whether each individual cell had two sets of chromosomes (2n; before S phase) or four sets (4n; after S phase). This gave the authors a sense of the proportion of cells in different cell cycle phases. Note that as time passes, most of the cells are in G2 phase (4n DNA content) following radiation, whether or not they have functional p53.

The G1 peak disappears only in the p53 deletion graph because p53 blocks cells from entering S phase after radiation.

Radiation only (2C)

In this graph, notice that the cell lines with at least one functional p53 (closed and half-closed squares) do not enter mitosis. p53 must be activating a checkpoint that is blocking mitotic entry. The p53 deletion mutant (open squares) begins to enter mitosis around 24–48 hours postirradiation because it is not blocked from doing so.

Radiation and nocodazole (2E)

Here, nocodazole traps the cells that enter mitosis. Because cells with functional p53 arrest because of DNA damage, they do not enter mitosis. The p53-deficient cells do, however. Even in these cells, though, there is still a delay in mitotic entry. But the delay is not sustained in the absence of p53.

Several potential mechanisms could account for these observations because p53 regulates the expression of many genes, including p21, that can affect the cell cycle (3–5, 17, 18). We examined the possible function of p21 in the maintenance of the G2 arrest by using HCT116 cells in which the p21 genes were disrupted by homologous recombination (4). The p21–/– HCT116 cells arrested with a DNA content of 4n after γ radiation (4), but they continued to enter mitosis (Fig. 3, A and C). A control HCT116 line with targeted disruption of both alleles of the Smad4 gene (19) behaved identically to the parental HCT116 cells (Fig. 3, A to C). The G2 checkpoint was activated after exposure of cells to 2-, 6-, and 12-Gy doses of γ radiation in wild-type, p53-, and p21-deficient cells (Fig. 3, D to F). However, the p53- and p21-deficient cells escaped the G2 arrest (Fig. 3, E and F), whereas the parental cells maintained it after exposure to 6 (Fig. 3D) or 12-Gy doses of γ radiation (Figs. 2E and 3C). The length of the G2 arrest in the p21- or p53-deficient cells depended on the dose of γ radiation, with the higher doses delaying the appearance of mitotic nuclei proportionately (Fig. 3, D to F). Escape from G2 arrest occurred earlier in p21- than in p53-deficient cells (Figs. 2E and 3C). This may be because some p53-independent p21 synthesis occurred after irradiation of p53–/– cells (Fig. 4A). These results are consistent with the fact that p53 is a major, but not sole, transcriptional regulator of p21 in mammalian cells (20).



Fig. 3. Mitotic entry after γ radiation. Mitotic indices of parental HCT116 cells (closed circles), cells with targeted deletions of both p21 alleles (open squares), and cells with targeted deletions of both Smad4 alleles (closed triangles) after 12-Gy γ radiation (A), after nocodazole treatment alone (B), or after treatment with both 12-Gy γ radiation and nocodazole (C) (32). Response of wild-type HCT116 (D), p53-deficient (E), and p21-deficient derivatives (F) to lower doses of γ radiation in the presence of nocodazole. (G) Primary human fetal fibroblasts and their p21- and p53-deficient derivatives were treated with 12-Gy γ radiation plus nocodazole and were analyzed at the indicated times. Note that the scale on the y axis in (G) is different than in (D) to (F).

Nocodazole trapping p21 cells

These figures show a new control cell line (triangles) that has a deletion of the Smad4 gene. These look similar to the unmodified control (circles), which shows that deletion of a gene that is involved in a different pathway does not have an effect here.

In contrast to the two control lines, the p21 cells are entering mitosis despite DNA damage, which suggests that p21 is partially responsible for arrest at the G2-M checkpoint in cells with functional p21.

p21 deficient cells

Now compare panel F (p21 deficient) with panels D (normal) and E (p53 deficient). There are two important points here. First, notice that more than half the cells enter mitosis by 24 hours after radiation, regardless of the dose of radiation. Second, although there is a slight delay in mitotic entry, it is much less pronounced than in panels D and E.

This suggests that p21 is driving the arrest of the cell cycle prior to mitotic entry and that it can at least initiate arrest in the absence of p53.

Irradiated fibroblasts

Here (panel G), the authors used a different type of cell to make sure that they still saw the same effects. Importantly, this cell line is noncancerous.

It confirms that their results are not unique to cancer cells (or the HCT116 line), because these fibroblasts are noncancerous used in the rest of Figure 3. Deleting p53 or p21 in fibroblasts still results in abnormal mitotic entry after irradiation.


Fig. 4. Protein expression and kinase activity after γ radiation. (A) Expression of p53 and p21 in parental HCT116 cells (+/+) and HCT116 cells with targeted deletion of one (+/–) or both (–/–) p53 alleles before and 36 hours after 12-Gy γ radiation assessed by immunoblot analysis (34). (B) Analysis of in vitro cyclin B1–associated histone H1 kinase activity in parental HCT116 cells (+/+) and in HCT116 cells with targeted deletions of both p21 alleles (–/–) at the indicated times after γ radiation. Whole cell extracts were immunoprecipitated with antibodies to cyclin B1 and assayed for kinase activity as described (35).

Immunoblot/Western blot

This technique is used to analyze protein levels in a given sample. Recall that the Southern blot (Fig 2a) detects levels of DNA.

The principle behind the western blot is the same, except it uses an antibody against the protein of interest rather than a complementary strand of DNA, as in the southern blot.

Panel A

The top box in panel A shows the levels of p53 in the three different cell lines either left untreated (γ-) or subjected to radiation (γ+). Notice that there are no bands in the lanes corresponding to the p53 deletion cell line (last 2 lanes).

The bottom box shows p21 expression in the same cell lines. Notice that there is no p21 expression in untreated cells, and relatively little p21 expressed in the p53 deletion cell line (last lane). This is because p21 expression is largely dependent on p53 activation, which is triggered by DNA damage. However, the small amount of p21 in the last lane suggests that p21 can still be induced by something other than p53 responding to DNA damage.

Panel B

Here, the authors compared cells with and without functional p21, trying to see whether there was a difference in the activity of a key cell cycle regulation complex, cyclin B1-cdc2.

The bands shown in both boxes represent a phosphorylated substrate, histone H1. The authors extracted cyclin B1 from these two cell lines and incubated it with histone H1 and radioactively labeled phosphate. If cyclin B1-cdc2 (a cyclin-kinase complex) is active, the radioactive phosphate is transfered to histone H1, and it will show up on the blot.

In cells with functional p21, the kinase is inactivated after radiation, and so the top box shows very little activity. When p21 is deleted, the kinase activity initially decreases because the cell cycle is arrested. But the arrest is not sustained, so in the bottom box, you see phosphorylation of the substrate after 24 hours.


Any technique with "immuno" in the name (immunoblot, immunoprecipitation, immunostaining) means that an antibody is used to detect a protein of interest.

Antibodies are proteins made by the immune system that bind to very specific protein sequences. They can be used in various techniques to identify the location or quantity of the protein they target. In this figure, the authors use antibodies specific to p53, p21 and cyclin B1.

The most likely biochemical explanation for the entry into mitosis in the absence of p21 was lack of inhibition of the principal mitotic cyclin B1-cdc2 complex by p21 (21). The activity of this complex decreased within 12 hours after γ radiation in both cell types (Fig. 4B), which probably reflects activation of a checkpoint mechanism. This inhibition of cyclin B1-cdc2 kinase activity was not sustained in the absence of p21, as substantially increased activity was observed beginning 24 hours after DNA damage in p21-deficient cells (Fig. 4B).

To determine whether p21 and p53 are required to sustain the G2 arrest in cells other than colorectal cancer or epithelial cells, we disrupted the p53 gene by homologous recombination in normal human fibroblasts (22). Nocodazole trapping was then used to monitor the escape from G2 in parental fibroblasts and in a clone derived from the same fibroblasts in which the p21 genes had been disrupted by gene targeting (5). Again, the parental cells entered a sustained G2 arrest while a substantial fraction of both p21- and p53-deficient fibroblasts escaped G2 and entered mitosis (Fig. 3G).

Cells without p53 or p21 apparently proceed into mitosis after γ radiation but have a 4n DNA content (Fig. 2B) rather than the 2n DNA content expected for cells that had gone through mitosis. To investigate this contradictory result further, we stably transfected cells with a histone H2B–green fluorescent protein fusion vector to allow real-time visualization of the mitotic process (23). Time lapse experiments in the absence of nocodazole showed that 90 ± 6% of the p21–/–cells entered mitosis within 36 hours after 12-Gy γ radiation compared with less than 2% of the parental cells. The first stages of mitosis after irradiation of p21–/– (or p53–/–) cells were indistinguishable from those in cells growing under normal conditions (Fig. 5A). After anaphase, however, the irradiated p53–/– and p21–/– cells never completed cytokinesis (Fig. 5B). These cells eventually flattened and the chromosomes decondensed, and >95% of the cells were found to contain abnormally shaped, multilobulated nuclei (Fig. 5, C, D, and E). A subset of these cells subsequently underwent programmed cell death. Staining with an antibody to the centrosome-specific γ-tubulin revealed that these cells always contained at least three centrosomes or pairs of centrosomes located in a cleft that likely was a remnant of the cleavage furrow associated with the failure of cytokinesis (Fig. 5, D and E). A large number of centrosomes, also observed in mouse cells that lack p53 (24), reflected the centrosome duplication that accompanies DNA synthesis (25) and was consistent with the fact that p53–/– and p21–/– cells often reenter S phase after irradiation, becoming tetraploid or octaploid (9,10).



Fig. 5. Fluorescence microscopy of p21–/– cells.Clones stably expressing the histone H2B/GFP fusion protein were isolated and observed by time-lapse microscopy under bright-field (upper) and fluorescence (lower) illumination (23). (A) A p21–/– cell undergoing mitosis in the absence of radiation. The prometaphase-to-telophase transition time was 66 min. (B) An irradiated p21–/– cell. The prometaphase-to-telophase transition time was 114 min. Forty- eight hours after 12-Gy γ radiation, wild-type (C), p21–/– (D), and p53–/– (E) cells were fixed and stained with 4′,6-diamidino-2-phenylindole (left, blue) or immunostained with antibodies specific for the γ-tubulin component of centrosomes (36) (right, green). The nucleus in (D) was bilobed, with connections between the lobes visible in a different focal plane from the one shown.

Fluorescence microscopy

This technique uses fluorescence (either fluorescent proteins like GFP or antibodies tagged with fluorescent dyes) to analyze the location of particular structures.

In panels A and B, the authors fused GFP to a histone protein, H2B, which is found in the nucleus. They then inserted this fusion protein into cells. Now, the histone fluoresces green, allowing researchers to track the nucleus during mitosis.

Impaired cytokinesis

Both panel A and B show a p21-deficient cell dividing, but only the cell in panel B has been subjected to radiation.

Notice how the cell in panel A completely separates into two daughter cells. This is the process of cytokinesis, which occurs during mitosis. In panel B, the radiated cell cannot complete cytokinesis and remains a single cell. This is likely because unrepaired DNA damage is preventing the chromosomes from separating properly.


(Full name: 4',6-diamidino-2-phenylindole) This is a dye that glows blue when it is bound to DNA. It is commonly used to stain the nucleus of cells in microscopy.

Panels C, D, and E show DAPI staining on the left side. The pictures on the right show the exact same cells, but with a green fluorescent stain.

Centrosome immunostaining

Here, the left panels show the nuclei of each cell type in blue, and the right panels show γ-tubulin, a component of centrosomes.

Centrosomes are a key part of cell division, because they facilitate the even separation of the chromosomes. Normally, a cell has a single centrosome that is duplicated along with the DNA. In mitosis, each centrosome moves to one of the two daughter cells, along with the duplicated chromosomes.

Notice how each separate cell in panel C has a single centrosome. In panels D and E, the nuclei have not completely separated, and there are multiple centrosomes. These cells lack p21 (D) or p53 (E). We know that this deficiency can result in incomplete cytokinesis (see panel B), but the extra centrosomes indicate that multiple rounds of DNA replication have occurred.

These results demonstrate that induced expression of p21 and p53 is essential to sustain the G2 checkpoint after DNA damage in human cells. Although most research on p53- and p21-regulated checkpoints has focused on the G1-S transition, several previous observations are consistent with an important role for these genes in G2-M (26–29). The p21 protein is synthesized in G2 (27, 28), promotes a pause in late G2 under normal growth conditions (26, 28), and, when expressed exogenously, causes cells to arrest in G2 (29). In contrast, neither p53 nor p21 appears to play a major role in the spindle checkpoint because p53–/– and p21–/– cells respond normally to microtubule disruption (Figs. 2D and 3B). It is not yet clear whether cells with heavily damaged DNA fail to undergo cytokinesis because of a cytokinesis checkpoint (18, 30) or because of a simple mechanical problem.

Although p53 mutations provide cells with a selective growth advantage, such mutations burden them with a significant checkpoint deficit; they cannot respond normally to DNA-damaging agents and enter mitosis and subsequently replicate their genomes in the presence of DNA damage. Such checkpoint defects (31) may be exploited to treat the many cancers with abnormalities of p53 function.

Video. Cancer research is a rapidly evolving field. As of spring 2013, 15 years after this paper was published, 140 genes had been found to be involved in cancer, half of them affecting the cell cycle. Knowing the genes can help us limit the drugs needed to treat cancer. To learn more, watch the complete lecture From Cancer Genomics to Cancer Drugs"

References and Notes

  1. M. B. Kastan, O. Onyekwere, D. Sidransky, B. Vogelstein, R. W. Craig, Cancer Res. 51, 6304 (1991); A. Maity, W. G. McKenna, R. J. Muschel, Radiother. Oncol. 31, 1 (1994); L. S. Cox and D. P. Lane, Bioessays 17, 501 (1995).

  2. L. H. Hartwell and M. B. Kastan, Science 266, 1821 (1994); A. B. Niculescu et al., Mol. Cell. Biol. 18, 629 (1998).

  3. C. Deng, P. Zhang, J. W. Harper, S. J. Elledge, P. Leder, Cell 82, 675 (1995); J. Brugarolas et al., Nature 377, 552 (1995).

  4. T. Waldman, K. W. Kinzler, B. Vogelstein, Cancer Res. 55, 5187 (1995).

  5. J. P. Brown, W. Wei, J. M. Sedivy, Science 277, 831 (1997).

  6. S. N. Powell et al., Cancer Res. 55, 1643 (1995); B. B. Wang, K. J. Hayenga, D. G. Payan, J. M. Fisher, Biochem. J. 314, 313 (1996); S. Fan et al., Cancer Res. 55, 1649 (1995); K. J. Russell et al., ibid. 55, 1639 (1995).

  7. Y. Peng et al., Science 277, 1501 (1997); Y. Sanchez et al., ibid., p. 1497; P. Nurse, Cell 91, 865 (1997).

  8. M. J. Solomon, T. Lee, M. W. Kirschner, Mol. Biol. Cell. 3, 13 (1992); C. Smythe and J. W. Newport, Cell 68, 787 (1992); C. Jessus and D. Beach, ibid. p. 323; P. Nurse, Nature 344, 503 (1990).

  9. T. Waldman, C. Lengauer, K. W. Kinzler, B. Vogelstein, Nature 381, 713 (1996).

  10. P. L. Olive, J. P. Banath, R. E. Durand, Radiat. Res. 146, 595 (1996).

  11. S. H. Khan and G. M. Wahl, Cancer Res. 58, 396 (1998); J. S. Lanni and T. Jacks, Mol. Cell. Biol. 18, 1055 (1998).

  12. T. J. McGarry and M. W. Kirschner, Cell 93, 1043 (1998).

  13. T. T. Su, P. J. Follette, P. H. O’Farrell, ibid. 81, 825 (1995); R. A. Laskey, D. Gorlich, M. A. Madine, J. P. S. Makkerh, P. Romanowski, Exp. Cell. Res. 229, 204 (1996).

  14. F. Bunz, C. Lengauer, K. W. Kinzler, B. Vogelstein, unpublished data.

  15. D. P. Cahill et al., Nature 392, 300 (1998).

  16. The targeting vectors used in this experiment were constructed so that the first codons of the drug resistance markers replaced the first codon of the p53 gene within exon 2. Transfection was done with LipofectAmine (Life Technologies) and subconfluent monolayers of cells. The LipofectAmine-DNA mixture was removed 3 hours after transfection and cells were grown in complete medium for 18 hours before they were replated in selective medium. After transfection with the neomycin-resistance vector, resistant colonies were grown in the presence of geneticin (0.4 mg/ml) (Gibco). One geneticin-resistant clone of 600 assayed was found to harbor a homologous recombinant, and this clone was expanded and transfected with the hygromycin B–resistance vector. Colonies were selected in hygromycin B (0.1 mg/ml). One clone of 940 assayed had a homologous recombinant of the second p53 allele.

  17. A. J. Levine, Cell 88, 323 (1997); L. J. Ko and C. Prives, Genes Dev. 10, 1054 (1996); R. Hansen and M. Oren, Curr. Opin. Genet. Dev. 7, 461 (1997).

  18. H. Hermeking et al., Mol. Cell 1, 3 (1997).

  19. S. Zhou et al., Proc. Natl. Acad. Sci. U.S.A. 95, 2412 (1998).

  20. P. Michieli et al., Cancer Res. 54, 3391 (1994); K. F. Macleod et al., Genes Dev. 9, 935 (1995); A. Moustakas and D. Kardassis, Proc. Natl. Acad. Sci. U.S.A. 95, 6733 (1998).

  21. Y. Xiong et al., Nature 366, 701 (1993); J. W. Harper et al., Mol. Biol. Cell 6, 387 (1995).

  22. Lung-derived human fetal fibroblasts (cell strain LF-1) were electroporated with the neomycin-resistant p53 targeting vector (Fig. 2A) and grown under G418 selection (5). Southern blotting (DNA) analysis revealed that several resultant clones had undergone the desired recombination event. In three independent experiments, the efficiency of targeting was 5, 6, and 31%, respectively. Subclones from heterozygous cells were then serially cultured; 30% of them had an increased life span (an additional 30 population doublings before senescence). Southern blotting indicated that all such long-lived clones had sustained a loss of the remaining wild-type p53 gene. The p53-deficient clones used for nocodazole trapping experiments were in the early phases of life span extension.

  23. Wild-type, p21 / , and p53 / cells were transfected with a vector, pBOSH2BGFP-N1, that encodes H2B/GFP ( T. Kanda, K. F. Sullivan, G. M. Wahl, Curr. Biol. 8, 377 [1998]). Colonies with stable expression of this fusion protein were identified by fluorescence microscopy and expanded into cell lines. Normal mitoses were observed by time-lapse videomicroscopy and both fluorescent and bright-field images were acquired every 6 min with the MetaMorph software package (Universal Imaging).

  24. K. Fukasawa, T. Choi, R. Kuriyama, S. Rulong, G. F. Vande Woude, Science 271, 1744 (1996).

  25. A. Paoletti and M. Bornens, Prog. Cell Cycle Res. 3, 285 (1997).

  26. R. S. Paules et al., Cancer Res 55, 1763 (1995); T. M. Guadagno and J.W. Newport, Cell 84, 73 (1996); M. L. Agarwal, A. Agarwal, W. R. Taylor, G. R. Stark, Proc. Natl. Acad. Sci. U.S.A. 92, 8493 (1995); N. Stewart, G. G. Hicks, F. Paraskeva, M. Mowat, Oncogene 10, 109 (1995).

  27. Y. Li, C. W. Jenkins, M. A. Nichols, Y. Xiong, Oncogene 9, 2261 (1994).

  28. V. Dulic, G. H. Stein, D. F. Far, S. I. Reed, Mol. Cell. Biol. 18, 546 (1998); S. Bates, K. M. Ryan, A. C. Phillips, K. H. Vousden, Oncogene 17, 1691 (1998).

  29. A. B. Niculescu et al., Mol. Cell. Biol. 18, 629 (1998).

  30. L. Muhua, N. R. Adames, M. D. Murphy, C. R. Shields, J. A. Cooper, Nature 393, 487 (1998).

  31. L. Hartwell, P. Szankasi, C. J. Roberts, A. W. Murray, S. H. Friend, Science 278, 1064 (1997).

  32. Cells were grown as monolayers in T-25 flasks and exposed to a 137Cs source (GammaCell 40) for a total dose of 12 Gy delivered over 14.5 min. Where indicated, cells were exposed to lower doses for proportionally shorter time periods. It was important that the irradiation of cells occurred when they were 70% confluent, as very sparse or completely confluent cells responded differently than subconfluent cells to radiation. Mi- totic trapping experiments were done by adding no- codazole to the culture medium (0.2 g/ml). Cells were collected by incubation with trypsin containing EDTA, centrifuged, and fixed in a solution containing 3.7% formaldehyde, 0.5% Nonidet P-40, and Hoescht 33258 (10 g/ml) in phosphate-buffered saline. Nuclei were visualized by fluorescence microscopy. Nuclei with condensed, evenly staining chromosomes were scored as mitotic. At least 300 cells were counted for each determination.

  33. Genomic DNA was purified from cell lysates with the QiaAMP spin blood kit (Qiagen) and used as a substrate for polymerase chain reaction and Southern blot assessment of targeting vector integration.

  34. Equal numbers of cells were collected, lysed in Laemmli sample buffer, and subjected to electrophoresis and protein immunoblotting. Filters were probed with antibodies to p53 (pAb 1801) and p21 {EA10 [W. S. El-Deiry et al., Cancer Res. 55, 2910 (1995)]}. Signals were visualized with enhanced chemilunescence (Pierce).

  35. Modified from K. Kaufmann et al., Cell Growth. Diff. 8, 1105 (1997). In brief, extracts for in vitro kinase assays were prepared by lysis of washed, centrifuged cells in 50 mM tris-HCl (pH 7.5), 0.5% Nonidet P-40, 10% (v/v) glycerol, 100 mM sodium chloride, 10 mM sodium orthophosphate, 5 mM-glycerophosphate, 50 mM sodium fluoride, 0.3 mM sodium orthovanadate, 1 mM dithiothreitol, and 1 complete protease inhibitor cocktail (Boehringer Manheim) for 30 min at 4°C. Kinase complexes were immunoprecipitated by adding monoclonal antibody to cyclin B1 (150 ng, Santa Cruz) and protein A–Sepharose (Life Technologies). Immune complexes were washed with lysis buffer and incubated in 25 l of a solution containing 20 mM tris-HCl (pH 7.5), 7.5 mM magnesium chloride, 1 mM dithiothreitol, 50 M adenosine triphos- phate (ATP), 20 Ci of [ -3 2 P]ATP (6000 Ci/ mmol), and 1 g of histone H1 protein (Boehringer-Manheim) for 30 min at 30°C. After addition of 25 l of 2 sample buffer and SDS–polyacryl- amide gel electrophoresis, 32P-labeled histone H1 was visualized by autoradiography.

  36. Cells were fixed in methanol at 80°C and stained with antibody totubulin (Sigma) and a fluorescently labeled secondary antibody to mouse immunoglobulin G (Molecular Probes).

  37. Supported by the Clayton Fund and NIH grants CA 43460, CA 57345, CA 62924, and GM 41690. We thank G. Wahl for the H2B-GFP fusion vector.