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ONCOLOGY Zebrafish Lights Path of LeukemiaMay Facilitate Gene and Drug Screens for Effects on Cancer HMS researchers have created the first transgenic zebrafish model of human cancer, which should help to identify genes that block or promote the carcinogenic process--and potentially help find new drugs to treat the disease.
The fluorescence above illuminates leukemia arising in the thymus of a 22-day-old transgenic zebrafish. The fish expresses green fluorescent protein fused to the mouse cMyc gene within lymphoblasts. These cells are spreading around the expanded thymus. (Image courtesy of David Langenau) The transgenic fish, which develops T cell leukemia, was made by fusing the mouse c-myc oncogene to a zebrafish promoter that is active in lymphocytes. The work is described in a Feb. 7 Science article by senior author A. Thomas Look, HMS professor of pediatrics at the Dana-Farber Cancer Institute, with first author and HMS graduate student David Langenau and colleagues at Children's Hospital, Brigham and Women's Hospital, and other institutions.
Over the past three years, Look's and Zon's labs have been collaborating on the development of the zebrafish system as a cancer model. Zon's lab has been undertaking genetic screens to find new cancer genes and is examining the use of chemical genetics to look for suppressors of cancer. The collaborators plan to apply this approach to Look's leukemia-prone fish, with the hope of finding leads for therapeutics. In the current study, said Look, "We adopted a strategy that was pioneered in the mouse--to overexpress the myc gene by linking it to a promoter that's highly expressed in lymphocytes, making a transgenic animal in which the oncogene will be expressed in cells susceptible to leukemogenesis." Zon added, "Given the zebrafish genetics, it should be possible to find modifier genes that reduce the rate of leukemia in these transgenic lines." How the Model WorksThe myc gene is one of the best-known human oncogenes. Removing its normal regulation leads to cancer, probably by promoting proliferation. In humans, chromosomal translocations of myc cause both Burkitt's lymphoma, a B cell malignancy, and acute T cell leukemia. When it migrates to the locus of an immunoglobulin gene, the result is Burkitt's; when translocated into a T cell receptor gene, it gives rise to T cell leukemia.
David Langenau, a graduate student in the laboratory of Thomas Look, displays a tank of zebrafish, which Look's research team has used to create a model for screening potential treatments for human T cell leukemia. Shown above are (l to r) Adolfo Ferrando, Look, Langenau, and John Kanki. (Photo by Steve Gilbert) Another advantage of zebrafish for research is circadian control of their behavior. Kept in the dark overnight, the fish will begin to breed as soon as the lights are turned on in the morning. This on-demand breeding allowed Langenau to harvest embryos immediately after fertilization, at the one-cell stage. To make the transgenic fish, he attached murine myc (mMyc) to the zebrafish zRag2 promoter, which targets gene expression to early developing B and T cells. He then injected the zRag2-mMyc transgene into the one-celled embryos. These grew up to become genetic mosaics, with some cells incorporating the transgene into their DNA. About one in 20 fish--those that incorporated the transgene into their lymphoid cell lineage--developed tumors originating in the thymus, where T cells differentiate, after an average of 44 days. "We found that the fish that had incorporated this transgene into the lymphoid lineage developed T cell leukemia, in contrast to mice, which generally develop B cell leukemia," said Look. "We're not sure why, but we're delighted with the specificity." The tumors quickly spread throughout the bodies of the fish. To see whether leukemic cells were transplantable, the researchers injected the zRag2-mMyc transgene into fertilized eggs from fish carrying a gene for green fluorescent protein (GFP). When these eggs matured into adults, their lymphoblasts were injected into healthy zebrafish, and the transplanted GFP-expressing leukemia cells could readily be monitored through the translucent skin of the zebrafish. The Visible Fish"One of the powers of the zebrafish is that they're virtually transparent, so we're able to track where the disease originates and where it spreads," said Langenau. In the next round of studies, the researchers hope GFP will show tumors regressing as well as progressing. Within a year, they plan to develop a genomewide modifier screen to find genes that affect the development of leukemia. In addition to this screen, the team is planning to develop a direct screening technique for potential drugs."What we're gearing up to do," said Langenau, "is to take a fish that has leukemia, drop it into water containing unknown compounds, and see if the leukemia regresses by tracking GFP." Currently, since the fish rapidly develop leukemia, they are unable to breed normally. So the researchers must take sperm from killed leukemic males to perform in vitro fertilization on eggs harvested from healthy females. But Langenau hopes to eliminate the need for the IVF procedure by using a hormonal on-off switch that has been used successfully in mice. "We are working to develop alternative approaches in which we fuse the estrogen receptor to the myc gene so myc can be turned on selectively by estrogen," he said. If it works, myc could be switched on and off simply by adding an estrogenic compound to the water the fish swim in. --Tom Reynolds |
