19.7: p53 Protein Mediates Normal Cell Cycle Control

Cancerous growth can result if a normal dividing cell suffers a somatic mutation that disrupts normal cell-cycle control. The overexpression of cdk, for example, or higher cyclin levels in daughter cells that never drop; such cells would never stop cycling.

CHALLENGE

What is a somatic mutation? What does it mean to say that certain cancers run in families or that specific genes are associated with a particular cancer?

Other possibilities include a cell in \(\rm G_0\) that is stimulated to begin cycling again by an inappropriate encounter with a hormone or other signal. If undetected, these anomalies can transform normal cells to cancer cells. This is apparently the case for a mutation in the human p53 gene that causes Li-Fraumeni syndrome. The symptoms include a high incidence of early-age bone, blood, and breast cancers (see Li-Fraumeni Syndrome for more). How the p53 protein in regulating cancer cell formation was shown using knock-out mice.

We noted earlier the use of knock-out mutations in studies of specific gene function. A protocol for making knock-out mutations in mice is shown below in Figure 19.18.

Fig, 19.18: How to make knock-out mutant mice: Specific deoxynucleotides in a cloned gene of interest are altered by site-directed mutagenesis. The altered gene is inserted into embryonic stem cells, where it recombines with and replaces the homologous gene already in the chromosome. Recombinant clones are selected and reinjected into embryos, which are then incubated in the uteri of “foster mother” mice. Knockout mice in the newborn litter can be selected and studied for the effects of removing a gene.

For demonstrating how knock-out organisms can be genetically engineered, M. R. Capecchi, M. J. Evans, and O. Smithies shared the 2007 Nobel Prize in Physiology or Medicine. (Read all about it at The Knockout Nobel Prize.)

The role of p53 protein in regulating cancer-cell formation in mice was shown in a knock-out experiment, when the normal mouse p53 genes were replaced with a pair of mutant p53 genes that were unable to make functional p53 protein. The resulting knock-out mice developed tumors and cancers at a much higher rate than normal mice. In general, many knock-out organisms have been created to study the effects of loss-of-function mutations in vivo (i.e., in whole animals). Using similar strategies, knock-in protocols can insert normal genes into an organism lacking the genes, allowing study of the effects of gain-of-function mutations.

19.7.1 p53 is a DNA-Binding Protein

If a \(\rm G_0\) cell is stimulated to start cycling again by an inappropriate encounter with a hormone or other signal, it may be transformed into a cancer cell. The job of the p53 protein is to detect such anomalies and to enable dividing cells to repair the damage before they proceed through the next cell-cycle checkpoint. Failing that, the p53 protein signals apoptosis of the cell.

p53 is a DNA-binding, gene-regulatory protein. Figure 19.19 (below) shows the structure of the p53 protein and how it binds to the DNA double helix.

Figure 19.19: Structure of the p53 gene-regulatory protein bound to DNA. p53 was originally called a tumor suppressor protein because when mutated, tumors arose.

Mutations in the gene for the p53 protein in humans (where it is called TP53) are associated with many human cancers. Cultured cells of mutagenized p53 genes also exhibit key characteristics of cancer cells, including uncontrolled replication and cell proliferation and the suppression of apoptosis.

19.7.2 How p53 Works to Salvage Cells

p53 protein is normally bound to an active Mdm2 protein. The p53 must separate from Mdm2 for p53 to enable cell progress through checkpoints. Physical or chemical stress on dividing cells, such as DNA damage during cell growth, can activate an ATM kinase that will phosphorylate p53 and Mdm2, causing their dissociation p53. The same kinase also phosphorylates Chk2. ATM-kinase-initiated events are detailed in Figure 19.20.

19.20: Normal functions of the p53 gene-regulatory protein

Each of the proteins and enzymes phosphorylated by the ATM kinase has a role in cell-cycle checkpoint function and cell-cycle arrest while errors are corrected:

1. Now separated from Mdm2, active pospho-p53 upregulates several genes, including the p21 gene.
2. The p21 protein binds to cdks; cyclins can’t bind p21-cdks.
3. Active phospho-Chk2 catalyzes cyclin phosphorylation; phospho-cyclins can’t bind to p21- cdks.
4. The inability of cyclins to bind cdks specifically blocks the cell cycle between the \(\rm G_1\) and S phases and the \(\rm G_2\)-to-M phases.

Figure 19.21 illustrates the kinase-mediated events at cell-cycle checkpoints.

19.21: Role of the p53 in decision-making at cell-cycle checkpoints.

The cell cycle will remain arrested while the cell attempts to finish the essential biochemical activities necessary to correct stress-induced or other physical or chemical aberrations, before moving on to the next phase of the cycle. If DNA repairs or other corrections are successful, the cell can progress to the next phase. But what happens if repairs are unsuccessful?

19.7.3 How p53 Works When Cells Can’t Be Saved

If DNA repair or other corrections fail, the damaged cell is routed to apoptosis. In this case, proteasomes target the Chk2-cyclin complex for degradation. Likewise, any p53 remaining bound to unphosphorylated Mdm2 is also targeted for proteasome destruction. The result is that the cell, unable to correct effects of stress or chemical damage or to repair DNA damage, is now a target for apoptosis. Levels and activity of p53 (and the other proteins) control the amount of p53 protein available to respond to cell-cycling anomalies. When phosphorylated, active p53 rapidly arrests the cell cycle and turns on genes for proteins needed for DNA repair and for apoptosis (just in case repair efforts fail!). Figure 19.22 summarizes these and related p53 interactions and their consequences.

19.22: A: Inactive p53 & active Mdm2 bind, head for proteasome, & [p53] decreases; B: Active Mdm2 & p14ARF bind, both are sequestered in the nucleolus, increasing available [p53]; C. Inactive Mdm2 can’t bind p53, increasing available p53. D. If ATM kinase is active, available p53 is phosphorylated & active p53 turns of genes for DNA repair, apoptosis & protein factors that arrest cell cycling at G1 or G2; E. Phosphorylated Chk2 activity causes cyclin inactivation, sending it to the proteasome, blocking cell cycling.

To sum up, p53 suppresses malignant tumor growth by either of two methods:

• by allowing DNA or other cellular repair, after which p53 and other proteins are inactivated and/or destroyed, and the cell cycle can resume.
• by upregulating apoptosis genes in case repairs aren’t successful leading to apoptosis, thereby killing off damaged cells and blocking tumorigenesis.

It should be clear now why a mutant p53 that reduces or eliminates p21-protein production, or that blocks essential DNA-repair, will instead allow damaged cells to enter S, transforming them into cancer cells.

In a new twist, few whales or elephants die from cancer compared to humans, even though they have thousands of times more cells. At least for elephants, one reason may be that they have as many as twenty copies (40 alleles) of their p53 genes! Thus, mutation of one p53 allele may have little effect if the tumor-repressing effects of the remaining p53 genes prevail ( Whales and Elephants Don’t Get Cancer!). Now it turns out that duplication of more than a few tumor suppressor genes not only protect large mammals from cancer, but originated in small animals, perhaps facilitating the evolution of large ones ( Elephantine Evolution).

CHALLENGE

How might p53 function in the rapid seasonal growth of deer antlers? (See Ruminant Headgear & Fast Antler Regeneration).