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DURHAM, N.C. - Duke Comprehensive Cancer Center researchers and others report using heat to selectively turn on a therapeutic gene, significantly delaying tumor growth in mice.

While others have recently reported successful heat-dependent gene expression in cell cultures, the Duke experiment represents the first demonstration of a therapeutic benefit of heat-induced genes in an animal model, the scientists said. The results of the study are published in the July 1 issue of Cancer Research, the journal of the American Association for Cancer Research.

Led by principal investigator Chuan-Yuan Li, the scientists used a "heat-shock" gene promoter - a tiny piece of DNA that only stimulates gene expression when the cell becomes too hot. Normally the promoter stimulates a gene that produces a protein that protects the cell from heat damage. However, by linking the heat-induced promoter to a therapeutic gene for interleukin-12 (IL-12) instead of its normal gene, and packaging the new IL-12 gene into a virus delivery system, the researchers were able to create a combination that proved to be a functional heat-dependent gene therapy against cancer.

"We have shown that, with hyperthermia, the heat shock promoter can increase IL-12 expression 300 to 400 times over baseline in vivo, without systemic toxicity to the mice," said Mark Dewhirst, co-author and director of the Hyperthermia Program at Duke University Medical Center. "This is the first use of heat-induced gene therapy in animal studies."

While any cell can take up the modified virus and incorporate the virus's DNA into its genome, only the heated cells actually translate the therapeutic gene's instructions into the protein IL-12. IL-12 is an "anti-angiogenesis" protein, which limits new blood vessel formation, and it also helps alert the immune system to foreign proteins, as might be found in tumor cells. Temperatures needed to turn on this gene therapy product can be achieved in mice and humans, with little danger of heat damage to normal tissues, the researchers said.

In laboratory studies with cells, the scientists showed that heating cancer cells infected with the modified virus caused increased production - a minimum of 13,600 times more than baseline - of the attached IL-12 gene. The researchers also investigated a heat-inducible version of the tumor necrosis factor alpha (TNF-) gene in laboratory studies, but not in mice. This gene was expressed 680,000 times more with heat than without in cell studies.

"We're using the heat shock promoter to selectively turn on a therapeutic gene that is added to the cell's DNA," explained Dewhirst, who is also a professor of radiation oncology.

Animal studies with heat-dependent IL-12 gene therapy showed that tumors in treated mice had lost some of their blood supply, killing the interior of the tumor and slowing its growth, the researchers reported.

The research was funded by the National Cancer Institute, Celsion Corp. and the German Research Council.

In gene therapy, a virus is used to deliver the therapeutic gene to the cells. Viruses normally force infected cells to express the viruses' genes. Gene therapy takes advantage of this ability. A gene with therapeutic potential is substituted into the virus' DNA, and infected host cells then take up and express the therapeutic gene rather than the virus's own genes.

There are two basic strategies for delivering therapeutic genes only to the tissues that need them. One is to inject the modified virus into an artery or vein that immediately enters the target, such as the liver. Another possibility - the one in use for heat-induced gene therapy - is to use external physical agents to turn on the gene only in selected areas.

Ionizing radiation has been used to selectively turn on gene therapy, but it has the drawback of being a cancer-causing agent itself and has not produced the gene expression levels attainable with the heat-induced promoter, the scientists said. For their studies, the Duke scientists selectively targeted the therapeutic gene using hyperthermia, a method that has demonstrated no long-term side effects in more than 20 years of human clinical trials.

"The lack of side effects from hyperthermia indicates that it might be a useful method to help target gene therapy in diseases other than cancer," said Li, assistant professor of radiation oncology. "However, some areas of the body remain difficult to heat reliably, even though technological developments in recent years have expanded the areas we can heat."

Indeed, many of those technological developments have come from the Hyperthermia Program at Duke. The ability to measure the temperature of internal tissues without using an invasive thermometer is one of the research areas that promises to revolutionize the use of hyperthermia, said Dewhirst.

"Non-invasively measuring temperature will allow us to spatially control heating in real time, as it's needed," Dewhirst explained. "We've shown initial feasibility for tumors in the limbs, and now we are trying to apply the technology to other areas of the body and make the improvements needed to move it into clinical trials."

The Hyperthermia Program recently received an $18 million, 5-year program project grant from the National Institutes of Health to begin its 14th year of continuous funding. Among the major projects to be supported by the NIH grant will be to further develop both heat-induced gene therapy and non-invasive thermometry.

Co-authors on the paper are lead author Qian Huang, Jim Hu, Frank Lohr, Li Zhang, Rod Braun and Jennifer Lanzen, all of the department of radiation oncology at Duke, and John Little of the Harvard School of Public Health.