Biological Sciences

New Breast Cancer Treatment Proposed
Radioactive Antibody Targets Tumor Blood Vessels
ORNL Closing In on Obesity Genes

Photograph by Tom Cerniglio

How do energy-related physical and chemical agents—ranging from radiation to fossil fuel pollutants—interact with living organisms and the environment? Can we reduce the risk to human health from exposure to hazardous materials and radionuclides? As we learn where genes sit on chromosomes and how their chemical building blocks are arranged, can we also find out what these genes do? Can we decipher the molecular basis of protein as well as gene function? Can we treat cancer and other diseases using our isotopes and technologies such as the laser?   These are some of the questions being addressed by researchers in ORNL’s Biological and Environmental Research Program, under the sponsorship of the DOE-Energy Research, Office of Biological and Environmental Research (OBER). Our program, which covers a diverse range of basic and applied studies, is one of the broadest biological and environmental multidisciplinary research programs in the nation.   Our biology research calls for expertise in mammalian genetics, molecular genetics, protein engineering, cell biology, carcinogenesis, macromolecular structure, mutagenesis, and risk assessment. We are developing competencies in new areas such as functional genomics and computational biology. For this work, we use our existing facilities such as the Mouse House with its 92,000 mutant mice and the Intel Paragon supercomputers. We don’t keep the answers to our questions—the products of our work—to ourselves. We are proud of our successes in transferring OBER-funded research findings and technological developments to the private sector and the R&D awards we have won.

New Breast Cancer Treatment Proposed

For many breast cancer patients, today’s treatment options can be a nightmare. They must face possible removal of one or both breasts by a surgeon. And they may have to endure side effects from chemotherapy or radiation treatments used to kill cancer cells on the loose.

A new breast cancer treatment concept
shows promise in destroying breast
tumors without surgery
or side effects.

A new treatment for early breast cancer may soon be at hand. ORNL has developed a treatment approach that shows promise in destroying breast tumors without surgery or side effects. This kinder, gentler therapy for breast cancer combines laser light and currently available drugs in a minimally invasive technique that awaits in-vivo testing.

Former ORNL chemist Eric Wachter is part of a team that is testing the medical uses for this two-photon, near-infrared laser. The light from this laser can target and damage cancer cells by activating an ingested drug that concentrates in them. This two-photon laser technique has great potential for curing breast cancer. Photograph by Tom Cerniglio.

When fully developed, the technique will use a focused beam of near-infrared laser light that passes harmlessly through skin and delivers photons in a one-two punch to the target. The beam of light, two photons at a time, is absorbed by the targeted tumor, activating an ingested pharmaceutical agent that is taken up by rapidly proliferating cells like those found in cancerous tumors. The activated agent kills the cancer cells, either by disabling their DNA or destroying their cell walls.

Pinpoint activation of the pharmaceutical agent is confined to a tightly controlled area as a result of the unique physics of the photoactivation process, called simultaneous two-photon excitation. In contrast, other commonly used optical excitation processes can cause undesirable activation, even at low intensities, and can produce damage over far wider areas than is desired.

The laser light can be focused deep within the tumor, and the drug is activated only in this focus. Therefore, in contrast to conventional radiation or chemotherapy, only tumor tissue is affected. Normal tissue is unaffected outside the focus of the beam—even as the light enters and leaves the body.

We have already demonstrated that the technique can selectively kill Salmonella bacteria and human breast cancer cells. The ability of the technique to focus deep within tissue has been shown in a tumor that was removed from a mouse with breast cancer.

A drug that could be safely used with this laser method is 8-MOP, a derivative of psoralen, which has been approved by the Food and Drug Administration for treatment of a number of diseases, including skin cancer and psoriasis. The 8-MOP and other psoralen derivatives are normally activated using ultraviolet light. As a consequence of the low penetration of UV light through skin, such agents are currently only effective for topical applications and treatment of near-surface lesions. Because our simultaneous two-photon excitation process uses near-infrared light (which, unlike ultraviolet light, can penetrate deeply into tissue), drugs like 8-MOP will become useful for treatment of subsurface lesions, such as breast cancer.

We believe that the majority of photoactive pharmaceutical agents will work well with the new activation method. As a result, the technique may be used someday to treat many other diseases, including cancers of the skin and liver, possibly spelling an end to some people’s bad dreams.

The research was supported by ORNL’s Laboratory Directed Research and Development Program.

Correction

In the former version of this article, it was incorrectly stated that ORNL licensed the technology described to a local company, Photogen, Inc.


Radioactive Antibody Targets
Tumor Blood Vessels

If a radioisotope could be delivered specifically to a solid tumor, its radiation should destroy the collection of cancer cells. That was the theory behind radioimmunotherapy in which a radioisotope is coupled with a monoclonal antibody that homes in on a specific biological target, like a guided missile. After 20 years of research, scientists have concluded that radioimmunotherapy is not effective on solid tumors. A large fraction of the antibody never reaches the tumor, and the fraction that does is not uniformly distributed among the cells.

However, recent successes indicate that leukemia and other “liquid tumors” may be treatable by radioimmunotherapy. A number of clinical human trials are under way at Memorial Sloan-Kettering Cancer Center and other U.S. cancer treatment centers. ORNL will be supplying the radioisotope actinium-225 and radionuclide generators needed to produce bismuth-213 for early patient studies.

In the past two or three years, several research groups have shown that attacking tumor blood vessels with chemicals or antibodies retards tumor growth. But when therapy stops, resident tumor cell reproduction starts again. ORNL researchers have found that radioimmunotherapy that targets blood vessels, not solid tumor cells, can eradicate implanted lung tumors in mice. We are using an ORNL-developed monoclonal antibody that homes in on the inside of blood vessels in lung tumors. Bismuth-213, an alpha-particle emitter with a 45-minute half-life, is carried to the tumor vessels by the monoclonal antibody; our study is the only one that uses radiolabeled antibodies to attack tumor blood vessels. The bismuth-213 used is the final decay product of uranium-233, over a ton of which is stored in ORNL’s Building 3019. Uranium-233 was originally produced at ORNL as part of the molten salt breeder reactor research program. Today ORNL is the western world’s chief source of actinium-225 and bismuth-213.

A large fraction of the bismuth-213-labeled antibody parks in blood vessels in lung tumors and sprays out high-energy alpha particles. This radiation penetrates 6 to 10 tumor cell layers nearby, killing everything in their short path, including tumor cells and cells lining the blood vessels. The damage to blood vessels may reduce the supply of nutrients to the tumor.

In experiments in hundreds of mice with implanted lung tumors, our researchers showed that 100% of the mice treated with appropriate doses survived for 100 days (even though some normal lung cells were damaged by the treatment). The untreated mice died in 10 to 14 days.

Photomicrographs of 5-µm-thick sections from mouse lungs. Panel A: low magnification of untreated lung—solid pink areas are untreated tumor cells. Panel B: higher (400×) magnification of a small tumor (clusters of pink cells) growing around a blood vessel (red area in center). Panel C: tumor after treatment with high dose of a monoclonal antibody to which bismuth-213 is attached. Tumor contains giant cells (dying) and lots of red blood cells from local hemorrhage. Panel D: 40 days after treatment—scarred area of lung where the tumor was growing before treatment. No live cells remain in the tumor area. Untreated animals had all died 20 days earlier.

To attach the bismuth-213 to the antibody, the molecule CHXb-DTPA is used. At one end of the molecule are “arms” that grab and envelop the bismuth-213 to protect it from being dislodged by competing metal ions; a functional group at the other end attaches the bismuth-213 to the antibody. The assembly of this therapeutic agent involves attaching the CHXb-DTPA molecule to the antibody. The altered antibody is then reacted with bismuth-213 from the actinium-225/bismuth-213 generator. At the top of the generator is actinium-225 (which has a half-life of 10 days)—a decay product of thorium-229, which in turn is a decay product of uranium-233. During the decay of actinium-225, bismuth-213 is formed. As often as every 3 hours, bismuth-213 can be “washed” from the generator for reaction with the antibody. The therapeutic agent is then injected into mice.

We have shown that monoclonal
antibodies labeled with radioactive
bismuth-213 destroy implanted
lung tumors in mice.

ORNL researchers are still searching for the best doses and dose schedules as well as for an explanation of why bismuth-213 effectively destroys lung tumors in mice. A treatment like this might be available for humans in 5 years if an analogous antibody is found. In a decade or so, radioisotope-bearing molecular probes that can bind to tumor blood vessels throughout the body may kill cancer cells that have spread from nearly every kind of solid human tumor.

The research was supported by ORNL’s Laboratory Directed Research and Development Program.

ORNL Closing In on Obesity Genes

For some people, being fat may be an unfortunate fate. Those with inherited obesity have a higher risk of suffering from diabetes, heart disease, and stroke. So, research is under way to target “fat” genes.

Most forms of human obesity are the result of a complex interaction of environmental factors and multiple, interacting genes scattered throughout the genome. These genes govern body weight by controlling appetite, metabolism, storage of body fat, and the balance of energy inputs and outputs. So far, five genes that act singly to cause obesity have been isolated in mice and humans and cloned by researchers at various laboratories. But it is estimated that less than 1% of obesity in the human population is controlled by single genes.

The complexities of the gene-environment interactions that cause most obesity make it difficult to investigate the role of genes that influence body weight in humans. Hence, ORNL is trying the promising approach of using mouse models to identify and characterize genetic factors influencing the quantity and distribution of body fat.

ORNL biologists are close to isolating
and cloning a significant obesity
gene in mice.

Although genes thought to influence polygenic obesity in mice and humans have been mapped, none of these genes has yet been isolated. Biologists at ORNL are striving hard to be the first group to isolate and clone one of these polygenic genes in mice.

Dabney Johnson examines an obese mouse that carries a single-gene obesity mutation. Photograph by Tom Cerniglio.

From among the mouse stocks generated in radiation mutagenesis experiments carried out at ORNL since 1949, many mice were observed to have pink coats and pink eyes. The reason: the radiation knocked out DNA from a coat-color gene on mouse chromosome 7 called p (pink-eyed dilution). During such studies, we observed that when a certain defined region containing the coat-color gene and a region of its neighboring DNA on mouse chromosome 7 were deleted, the animals were about 35% fatter than age-matched animal controls. The actual physiological differences between the fat mice and mice of normal weight are being studied by a collaborator at Louisana State University.

By focusing on DNA from the p coat-color mutant mice that also get fat, ORNL researchers defined a small region on mouse chromosome 7 that might contain gene(s) affecting body weight in mice. Recombinant DNA techniques are being used to identify and characterize genes from this small region that might be logical candidates for polygenic fat genes. These findings should provide insights into the body fat regulatory mechanisms in mice and humans. Once polygenic fat genes are cloned and isolated, it may be possible to isolate the associated gene products that undermine control of body weight and use this information to develop an anti-obesity drug. With a little help from genetic studies and drug designers, being fat forever may not have to be some people’s fate.

The research was supported by DOE’s Office of Biological and Environmental Research.

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