B/B 06 · Cell cycle checkpoints and p53
A Checkpoint Inhibitor and p53
Use flow cytometry data to reason about the G1 and G2 checkpoints and a cancer-drug strategy.
Passage
The cell cycle has four phases: G1, in which the cell grows and decides whether to divide, S, in which DNA is replicated, G2, in which the cell prepares for division, and M, in which it divides. Checkpoints stop the cycle if something is wrong. In the G1 checkpoint, the tumor suppressor p53 is stabilized after DNA damage and activates the gene for p21, which inhibits cyclin-dependent kinases (CDKs). With CDKs inhibited, the retinoblastoma protein (Rb) stays unphosphorylated and holds back the transcription factors needed to enter S phase. A second checkpoint at the end of G2 relies on a checkpoint kinase and prevents cells with damaged DNA from entering mitosis.
If the damage is too severe to repair, p53 can also trigger apoptosis, the process of programmed cell death. Because many cancers have lost p53 function, their cells may depend on other pathways to survive DNA damage. This idea is the basis for some strategies in which a drug that damages DNA is combined with a drug that blocks a remaining checkpoint, so that cells that lack p53 are pushed into mitosis with damaged DNA and die.
The DNA content of cells can be measured by flow cytometry after the cells are stained with a dye that binds DNA. Cells in G1 have a DNA content of 2N, cells in S phase have between 2N and 4N, and cells in G2 and M have 4N. A researcher studies two lines of cultured cells: one with normal p53 (p53⁺) and one in which both copies of the gene for p53 are deleted (p53⁻). She exposes the cells to a drug that damages DNA, with or without a second drug that inhibits the G2 checkpoint kinase. After 24 hours, the cells are analyzed. Table 1 shows the percentage of cells in each part of the cycle and the percentage of cells that have undergone apoptosis.
For flow cytometry, the cells are fixed in ethanol and treated with an enzyme that destroys RNA, so that the dye binds only to DNA. Thousands of cells are then passed one at a time through a laser beam, and the amount of fluorescence of each cell is recorded and sorted into the 2N, 2N–4N and 4N categories.
Table 1. Cell cycle distribution and apoptosis after 24 hours.
| Cell line and treatment | G1 (2N) | S (2N–4N) | G2/M (4N) | Apoptosis |
|---|---|---|---|---|
| p53⁺, untreated | 55 | 30 | 15 | 2 |
| p53⁺, DNA-damaging drug | 70 | 10 | 20 | 5 |
| p53⁻, untreated | 55 | 30 | 15 | 2 |
| p53⁻, DNA-damaging drug | 25 | 20 | 55 | 4 |
| p53⁺, DNA-damaging drug + checkpoint inhibitor | 68 | 10 | 22 | 6 |
| p53⁻, DNA-damaging drug + checkpoint inhibitor | 35 | 25 | 40 | 50 |
The researcher also measures the amounts of p53 and p21 proteins in the cells before and after treatment with the DNA-damaging drug. Table 2 shows the results, as relative levels.
Table 2. Relative levels of p53 and p21 proteins.
| Cell line | Treatment | p53 | p21 |
|---|---|---|---|
| p53⁺ | None | 1 | 1 |
| p53⁺ | DNA-damaging drug | 10 | 8 |
| p53⁻ | None | 0 | 1 |
| p53⁻ | DNA-damaging drug | 0 | 1 |
Each value in the tables is the mean of three independent cultures. In Table 1, the first three columns are percentages of the cells that are still alive, so each row adds up to 100. The last column is the percentage of all cells in the culture that have undergone apoptosis.
Questions
Question 1 of 6
In p53⁺ cells, the DNA-damaging drug raises the fraction of cells in G1 from 55% to 70%. Which sequence of events best explains this?
Table 2 shows that p53 rises after treatment, and p21 rises with it.
Phosphorylated Rb releases the transcription factors that drive entry into S phase, so it would not hold cells in G1.
The cells with damaged DNA accumulate in G1, and the fraction in G2/M changes only slightly.
Why the answer is what it is
Table 2 shows that p53 rises tenfold and p21 eightfold in p53⁺ cells after the drug. p21 inhibits CDKs, so Rb stays unphosphorylated and the cells pause before S phase, which fits the rise in G1 and the fall in S phase.
Review: DNA Replication, Repair & the Cell Cycle · Gene Expression & Regulation · Molecular & Cell Biology
Question 2 of 6
DNA damage causes p53⁻ cells to accumulate in G2/M (55%). What does this show?
The p53⁻ cells reach G2 and arrest there. It is entry to S phase from G1 that p53 controls.
If they had no checkpoints, they would be spread as in untreated cells. Instead, many accumulate with a 4N DNA content.
The fraction in S phase falls from 30% to 20%, which does not suggest that cycling has sped up.
Why the answer is what it is
With no p21 induction, p53⁻ cells pass through G1 and replicate their damaged DNA. They then stop at the G2 checkpoint, which is independent of p53, and show a 4N DNA content.
Review: DNA Replication, Repair & the Cell Cycle · Gene Expression & Regulation · Molecular & Cell Biology
Question 3 of 6
Why does the checkpoint inhibitor cause so much apoptosis in the p53⁻ cells (50%) but not in the p53⁺ cells (6%)?
The p53⁻ cells have no p53 to activate, as Table 2 shows.
The p53⁺ cells show a G1 arrest in response to the drug. They are protected by that arrest, not by faster repair.
The inhibitor acts on the G2 checkpoint kinase, so it affects cells at the end of G2.
Why the answer is what it is
Without p53, the cells lack their G1 checkpoint. If the G2 checkpoint is also blocked, cells with damaged DNA enter mitosis and die. The p53⁺ cells are still arrested in G1, so the inhibitor has little effect.
Review: DNA Replication, Repair & the Cell Cycle · Gene Expression & Regulation · Molecular & Cell Biology
Question 4 of 6
How does the DNA content of a cell in G2 compare with that of a cell in G1?
A cell in G2 has one nucleus. The DNA content goes from 2N to 4N, a doubling.
The number of chromosomes (centromeres) is the same, but each chromosome now has two sister chromatids, so the amount of DNA has doubled.
DNA is not degraded in G2.
Why the answer is what it is
During S phase, the DNA is replicated, so a cell in G2 has twice as much DNA (4N) as a cell in G1 (2N), although the number of chromosomes is unchanged.
Review: DNA Replication, Repair & the Cell Cycle · Gene Expression & Regulation · Molecular & Cell Biology
Question 5 of 6
Why does the fraction of cells in S phase fall from 30% to 10% in p53⁺ cells after the drug?
The data do not show faster replication. They show cells piling up in G1 and a fall in new entry into S phase.
The 4N fraction rises only a little (15% to 20%), so this cannot account for the fall in S phase.
Nothing in the passage suggests this, and the dye is used in all samples.
Why the answer is what it is
The G1 arrest stops new cells from entering S phase. The cells already in S phase finish and move on, and so the fraction of cells in S phase falls.
Review: DNA Replication, Repair & the Cell Cycle · Gene Expression & Regulation · Molecular & Cell Biology
Question 6 of 6
The p53⁻ cells have both copies of the p53 gene deleted. Which statement about p53 is most consistent with the data and with what is known about cancer?
The p53⁻ cells replicate DNA (they have cells in S and G2/M).
Oncogenes promote growth. A gene whose product stops the cycle after damage and whose loss favors tumors is a tumor suppressor.
In Table 1, the p53⁻ cells undergo apoptosis (50%) when the second checkpoint is blocked.
Why the answer is what it is
p53 works as a brake after DNA damage. Cells without it fail the G1 checkpoint, continue to replicate damaged DNA, and can pass on mutations, which is why loss of p53 is common in cancers.
Review: DNA Replication, Repair & the Cell Cycle · Gene Expression & Regulation · Molecular & Cell Biology
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