Emory Medicine, Spring 1995

Insights into Cancer: Exploring Cell Biology

by Holly Korschun

Ever since William Roentgen asked his wife to place her hand on a photographic plate behind a cathode ray tube and inadvertently took the world's first x-ray, people have been exploring the uses of radiation, often cautiously and sometimes carelessly. Although it is used to treat half of all cancer patients, radiation is now well known as both a healer and a killer.

Just down the street from Grady Memorial Hospital in the Loughlin Radiation Oncology Center, Emory scientists are studying both the damaging and the healing effects of radiation.

The Loughlin Center, which opened in 1991, houses facilities on its ground floor for radiation therapy treatments. Upstairs is Emory's new Division of Cancer Biology, where a small staff of biochemists study characteristics of both normal and cancer cells in search of ways of circumventing the cancer process. Under the direction of biochemist Paul Doetsch, these researchers are working to understand, among other things, how the body repairs radiation damage to cells and how radiation therapy can be improved to kill cancer cells more effectively.

Emory's new Division of Cancer Biology is located in the recently constructed Loughlin Radiation Oncology Center, near Grady Hospital.

Biochemist Paul Doetsch holds two grants from the National Cancer Institute in the field of DNA repair mechanisms and one grant in this field from the American Cancer Society. Proudly enthusiastic about the Loughlin laboratories and their state-of-the-art equipment, he displays the same sort of affection for the shiny new tissue culture incubators, ultricentrifuges, high-pressure liquid chromatograph, and polymerase chain reaction equipment that others might show for the refrigerator, gleaming kitchen cabinets, and convertible stove top in their new dream home.

Dr. Doetsch's specialty is the biological effects of ionizing and nonionizing radiation and chemical carcinogens, with particular emphasis on those cellular enzymes whose job is to reverse many different types of genetic damage caused by these environmental hazards.

Although our cells are well equipped to handle small genetic insults from radiation, says Dr. Doetsch, problems arise when cells are subjected to either single large doses or years of cumulative exposure, or when key repair enzymes are missing or defective.

DNA repair is a multi-step pathway designed to return damaged DNA back to its normal pristine state. Although there are several different types of DNA repair, Dr. Doetsch's interest is in base excision repair, accomplished by a team of repair enzymes, each assigned a different piece of the job. The first group of enzymes recognizes the genetic damage and signals a second group to make a tiny cut in the damaged strand of DNA. Yet another enzyme group removes the defective strand before a wrap-up team fills in the gap with the correct genetic information, efficiently copied from the undamaged part of the DNA. Finally, the strand is sealed back together.

The initial steps of recognition and cutting are performed by specialized enzymes which deal only with specific types of genetic damage. Some teams repair chemical damage, while others work with radiation damage. Still others address the daily damage caused by oxygen free radicals that is the result of normal cellular function.

Each type of damaging agent has its own unique effect on DNA, says Dr. Doetsch. Chemical carcinogens enter the cell's nucleus, bond with a component of DNA, and cause bulky formations that hang from the DNA strand. Ionizing radiation reacts with the cell's water molecules, turning them into oxygen free radicals, which damage the DNA. Nonionizing radiation, such as ultraviolet light, damages DNA through a photochemical process.

"The beauty of base excision repair," says Dr. Doetsch, "is that after the initial steps of recognition and cutting, all different kinds of repair are handled in the same way by the same kinds of enzymes."

The beauty of excision repair

Dr. Paul Doetsch specializes in DNA repair enzymes, which work to reverse many different types of genetic damage to the cell.

Ironically, the genes encoding the enzymes that repair DNA often are called "cancer genes." But it is actually the lack of these enzymes, or a defect in their function, that can lead to cancer. Unrepaired damage can either block cell replication or cause a mutation.

Although it takes multiple mutational "hits" to turn a normal cell into a cancer cell, if the level of an important repair enzyme is low at an early stage in the cell's life, or if the enzyme isn't working properly, the cell is potentially at high risk for cancer later on.

In a recent issue of the journal Nature, Dr. Doetsch reported on an enzyme he and his research team found in yeast that repairs ultraviolet-induced genetic damage. Dr. Doetsch believes the study of the YPDE enzyme and its relationship to human enzymes may eventually lead to genetic testing for sunlight sensitivity and ways of beefing up the DNA repair process.

Two biotechnology firms, along with scientists at the University of Vermont, Dana Farber Cancer Institute, and Johns Hopkins School of Medicine, recently announced the discovery of another DNA repair enzyme in humans which is the equivalent of an enzyme originally found in yeast and bacteria. These researchers found that a particular DNA repair gene with a defective version of this enzyme is linked to hereditary nonpolyposis colon cancer and perhaps other types of colon cancer as well. "Any of the DNA repair enzymes that we are working on at Emory are candidates for doing the same kinds of things," says Dr. Doetsch, "not necessarily in colon cancer, but in other human cancers."

"If we find out that particular enzymes are important early on in preventing a cell from becoming cancerous, we could potentially devise specific genetic tests to see whether people have inherited mutations in the genes that encode those enzymes," he adds. "We could also develop new therapies by exploiting enzyme defects in cancer cells to kill them."

Missing and mutant repair genes

Most research on the failure of DNA repair mechanisms has focused on the cell replication process, where "spelling" errors can occur in copying DNA before cells divide. If the proofreading enzymes responsible for catching these mistakes are defective or missing, the theory goes, a mutation may result when the cell divides.

But what about those cells that are terminally differentiated and no longer dividing, which account for most of the cells in the human body? Dr. Doetsch has discovered a new explanation for how potential cancer-causing genetic damage is expressed in these cells. His research, published last year in Proceedings of the National Academy of Sciences and funded by the National Cancer Institute, focuses on the transcription and translation process by which nondividing cells make proteins to maintain normal cellular functions. First, the cell makes an RNA copy of the DNA molecule and then makes that RNA copy into a protein. If the RNA-making machinery reads a damaged and uncorrected bit of DNA, it may make a mutant protein. And if that protein happens to be one with an important role in maintaining normal cellular function, serious consequences could result.

Dr. Doetsch was surprised to discover just how often mistakes were made during RNA synthesis. Although the end-product of such mistakes - a mutant protein - is the same as in faulty DNA replication, the cause is quite different. "Until now, virtually nothing has been known about this new pathway," says Dr. Doetsch, "yet it underscores a different way of thinking about how mutant proteins may arise."

A new theory of mutation

An investigator who works with Dr. Doetsch in the Division of Cancer Biology, biochemist Davis Chen, is studying a particular DNA repair enzyme called AP endonuclease (Ape) and its role in tumor cells and in radiation therapy.

For some unknown reason, tumor cells exposed to the ionizing radiation used in cancer therapy are able to elevate their level of Ape enzyme, giving them an extra boost in being able to repair damage and making them more resistant to radiation than normal cells. And Dr. Chen has found that the Ape enzyme has an even more devious role - it activates the oncogenes responsible for turning normal cells into tumor cells in the first place.

He is developing a drug he hopes will inhibit the level of Ape and make tumor cells more susceptible to the damaging effects of radiation therapy. He also is in the early stages of using gene therapy in vitro to block the expression of the gene that encodes Ape. After cancer patients have left the Loughlin Center in the evening, he goes downstairs and uses the facility's linear accelerator to test tumor cells. He has found that by lowering their levels of Ape, he can make these cells much more sensitive to the effects of radiation.

Ape may play a particularly important role in breast cancer. Breast tumor cells have been found to have a tenfold increase in Ape activity over normal breast cells. Dr. Chen is studying oxidative damage, which he believes may affect the tumor suppressor cells which regulate Ape.

While this issue was in press, Dr. Chen was killed in a tragic accident (see related article). Dr. Chen's laboratory work is being continued by Dr. Doetsch.

Subverting survival instincts in tumor cells

The research of Dr. Davis Chen is aimed at inhibiting an enzyme that helps cancer cells resist the damaging effects of radiation therapy.

Biochemist Vicky Stevens also focuses on breast cancer in her work in the Division of Cancer Biology. She is seeking enzymatic markers that normal cells might shed from their surface when they are transformed into tumor cells. In particular, she hopes to identify markers that might help detect breast cancer from a simple test of blood or urine.

Her target is the GPI anchor - a unique combination of lipid and carbohydrates used to attach proteins to the surface of cells. More than 100 proteins are attached to cells through this mechanism.

"We don't know why Mother Nature has gone to the trouble of making this mechanism for attaching proteins to the cell surface," she explains, "but because of its structure, there are special ways in which the cell can cleave that anchor to release a protein. It turns out that in certain diseases, particularly cancer, the proteins normally attached to cells by the GPI anchor are found in serum or other extra-cellular fluids and can be used as tumor markers. We believe that cancer may cause the GPI anchor mechanism to go awry and release these proteins."

According to Dr. Stevens, the standard methodology used to find tumor markers over the past ten to 20 years, which involves injecting tumor cells into laboratory animals and screening the resulting antibodies, has not revealed useful antigens for a number of cancers, including breast cancer. "Because the GPI anchor is associated with several potential markers, we would like to look for GPI anchor proteins that normally are attached to cells but are absent in corresponding tumor cells," she explains. "Right now, we have four or five candidates. The next step will be to see whether we can find these proteins in serum or blood."

Seeking markers for breast cancer

In her research, Dr. Vicky Stevens studies a cellular anchoring mechanism used to attach certain proteins to the cell surface. In cases of cancer, these proteins can be found in the blood or urine and thus may serve as tumor markers.

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