The Cell Counts Backward to Zero

BY RICHARD KESSIN, PH.D. ASSOCIATE DEAN FOR GRADUATE AFFAIRS

REMEMBER THOSE NASA COUNTDOWNS? CHRIS KRAFT WOULD intone 10-9-8 — holding at T minus 7 seconds — guidance systems reporting Go - 6-5-4-3-2-1 - ignition. During the countdown, even before the last 10 seconds, telemetry systems report on the status of the crew capsule, the fuel pumps, the weather, and a thousand programs and devices. A defect in any one of these automatically stops the count. Your cells, every time they divide, go through a similar check list. If you are going to copy 3 billion bits of genetic information in a few minutes and not make any mistakes, all of your systems had better read GO.

From left, Wei Gu, Anatoly Nikolaev, and Chris Brooks
But what is the nature of the cell's check list and what happens if it goes wrong? One element in the central computer, the point at which most information on the state of the cell converges, is a protein poetically named p53 because its mass is 53 kiloDaltons. The fact that p53 is defective in 50 percent of cancers should concentrate everyone's mind. Now imagine that a cell, about to duplicate its DNA and divide, receives a blast of radiation. Depending on the type of radiation, DNA strands are broken or chemically modified. The cell mobilizes astonishing mechanisms to repair the damage. But delay is essential — trying to replicate DNA and permit a cell to divide before the repair is complete would result in genetic disaster. Whole chromosomes could be lost or some pieces of DNA could be brought into juxtaposition with genes that they do not normally regulate, creating a potential for cancer.
When they are irradiated, cells chemically modify p53 and combine it with a number of other proteins. The p53 goes to the nucleus where it stops the cell (holds the count) by turning on genes that delay events until repair is complete. When repair is finished, p53 is inactivated and the cell is free to divide. If the radiation damage is too great to be repaired, p53 activates genes that kill the cell. It is far better for the organism to kill a cell and avoid the risk of letting it turn into a tumor. We do not know how a cell chooses between delay and death.
Wei Gu'95, a Ph.D. graduate in pathology, and his Ph.D. students Anatoly Nikolaev (fourth year) and Christopher Brooks (third year), working in the Institute of Cancer Genetics and the Department of Pathology, have been dissecting the information that flows to p53 to influence a go or no-go decision. This information is astonishingly complex. The p53 protein can be phosphorylated or acetylated. It can be tagged with a small protein called ubiquitin and then degraded. The degradation mechanism is itself under precise control, requiring the binding of a protein called MDM2. As Anatoly Nikolaev and other members of the lab have discovered, the cell also has ways to anchor p53 in the cytoplasm, where it is kept from acting.
As the diagram shows, a rather unusual protein called Parc attaches to p53 and holds it far from its intended site of action in the nucleus. Under these circumstances cells are free to divide, even when the cues coming from their environment say hold the count. There had been suggestions that in unstressed cells, something holds p53 in the cytoplasm and a number of candidate proteins have been proposed, but none had the ring of authenticity. Wei Gu's group used a direct biochemical approach, purifying p53 from the cytoplasms of unstressed cells, and then asking what proteins came along with it. Using sophisticated purification procedures, Parc was identified as the chief binding partner.

Parc got its name because the gene that codes for it is related to a gene called Parkin. Children with mutations in the Parkin gene acquire epilepsy at an early age, but Parc seems to have a different role.
What happens when we manipulate the levels of Parc? When Parc is depleted, p53 goes to the nucleus and causes the cells to die. Overproduction of Parc keeps p53 in the cytoplasm and, as predicted, the cells do not attempt to stop dividing after radiation. It is intriguing that a large percentage of neuroblastomas have an excess of Parc in the cytoplasm of the cells. This abundance of Parc may make the cells resistant to radiation-induced cell death because p53, sequestered in the cytoplasm, cannot keep them from dividing while their DNA is being repaired. If the students deplete the Parc in these neuroblastoma cells, p53 is released, the cells delay their cycle after radiation, and susceptibility to radiation is restored.
How can understanding the many factors that influence p53 help in therapy? The more pathways found, the more opportunity for drug treatments. Are there molecules that disrupt the binding of Parc to p53? They should make cancerous cells more sensitive to radiation. Are there molecules that block degradation of p53? Yes, and they may cause rapidly dividing cells to die. Are there molecules that block export of p53 and keep at work? Perhaps. Most cancer drugs induce p53 because they act to damage DNA. By knowing the pathways that control p53 activation and inactivation, we can improve the efficacy and specificity of these drugs, while reducing unwanted side effects. While these improvements remain in the future, knowing is always better than not knowing.

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