I. Drug Target Validation.  As a result of new high-throughput techniques aimed at identifying genes and gene products involved in tumor formation, large amounts of data has been generated in the past few years. The list of potential drug targets and anti-cancer compounds is long. Right now, there is a need to sort through all this data and identify the real drug targets. It is a well known fact that drugs often fail in the clinic. As discussed by Smith (1), this is sometimes due to the lack of a well designed early-stage target validation.

II. Model Systems.  Drug discovery, drug validation, and mechanism-of-action studies can be done in a variety of model systems (2). Computer models can sometimes be a good starting point. However, they need to be supported by in vivo experiments. Cell lines can be a good tool for various kinds of experiments, for example microarray studies and knock-out experiments. Cell lines, however, do not always reflect the biology of cells in a living organism. Therefore, animal models must be used. Whereas the ultimate tests must be performed in mammalian models and with clinical trials, nonmammalian models are important for early-stage drug studies. This is because research in mammalian models is often costly and time-consuming.

III. Zebrafish as a Cancer Model System.  The zebrafish Danio rerio is emerging as a powerful cancer model system (reviewed by Amatruda et al. (3), and Stern and Zon (4)). Zebrafish neoplasms are in many cases similar to human cancers, and important factors involved in human cancers, such as p53, c-Myc, and angiogenesis factors, have been found to play a role in fish tumorigenesis. The main advantages of zebrafish as a model organism are short generation times and transparent embryos that develop outside of the mother's body. In addition, they don't cost much and don't need much space. Zebrafish can be genetically manipulated, both via reverse and forward genetics. Several reverse genetics techniques are applicable in zebrafish. Transgenic fish can be created by injecting DNA constructs into one-cell embryos. The transgene expression can even be visualized in vivo by coupling the transgene to a fluorescent protein tag. Fish with specific gene disruptions can also be isolated with a technique called TILLING, which will be discussed later. Forward genetics techniques that can be used in zebrafish include ENU mutagenesis and insertion mutagenesis. Various phenotype assays, such as apoptosis assays, can also be performed in zebrafish. In addition to these techniques, small molecule screens and genetic modifier screens are relatively easy to perform in zebrafish. A good overview of what has been described above can be found in Figure 1 in a review paper by Stern and Zon (4) and an example of a setup for small molecule screens can be seen in Figure 4 of the same paper.

IV. The Importance of TP53.  As reviewed by Vogelstein et al. (5), the tumor suppressor protein TP53 plays an important role in the cell's response to various stress signals. In a cell which is not under stress, the TP53 levels are kept down by degrading the protein. Activation of the TP53 network results in posttranslational stabilization of the TP53 protein which allows it to transactivate downstream effectors such as p21 and Bax, which then can mediate growth arrest or cell death. The importance of TP53 is underscored by the fact that mutations in the TP53 gene are found in more than half of all human tumors (6).

V. "tp53 Mutant Zebrafish Develop Malignant Peripheral Nerve Sheath Tumors" by Berghmans et al. (7) 

i. Zebrafish as a Model for Studying TP53 Function

Even though TP53 has been extensively studied, a more detailed understanding of its function and downstream pathways is needed. Recently, researchers have started using the zebrafish as a vertebrate model system to study TP53. Transient transfection studies (8) have already shown that tp53 activates the apoptotic response to DNA damage in zebrafish and is regulated by Mdm2. Furthermore, the same study showed that p21 is a downstream target of tp53. A new study by Berghmans et al. (7) goes further and shows that fish with mutated forms of tp53 are prone to cancer development, indicating a tumor suppressor role for zebrafish tp53. The results of these studies establish the zebrafish as a model system relevant to studies of human TP53 function and its role in human cancers.

ii. Isolating Zebrafish Lines with Point Mutations in the Zebrafish tp53 Gene

Berghmans et al. (7) started by isolating zebrafish lines with mutations in the DNA binding domain of tp53. The reason they chose the DNA binding domain is that a large majority of identified tp53 mutations in human tumors are found in the DNA binding domain of the protein. They identified the mutations by using a target-selected mutagenesis strategy called TILLING (targeting induced local lesions in the genomes). This technique is well described by Wienholds et al. (9). In short, 2679 ENU-mutagenized F1 male fish were screened for mutations within exons 4-8 of tp53. These exons encode the DNA binding domain of the protein. Zebrafish lines carrying the identified mutations were then recovered through in vitro fertilization and inbred to homozygosity.

Using the technique described above, they identified five mutations within tp53 (Supporting Table 1). Two of the five mutations, N168K and M214K, are orthologous to TP53 mutations found in human cancers. These mutations represent two classes of TP53 DNA binding domain mutations. The M214K is a DNA contact mutation whereas the N168K mutation is a structural mutation which modifies the protein conformation.

iii. The tp53N168K and tp53M214K Mutations Function in a Dominant-negative Manner

In order to analyze the functional significance of the mutant zebrafish tp53 alleles, the authors carried out in vitro reporter assays. They generated expression vectors which contained the mutant tp53 alleles and transfected them into cells along with a luciferase reporter construct. The luciferase gene was under the control of the p21 response element which contains a tp53 binding site. Whereas a wild-type tp53 protein was able to activate transcription of the luciferase gene, the tp53^N168K and tp53^M214K mutant proteins were unable to perform the same task (Supporting Figure 6). Furthermore, the mutant proteins inhibited the ability of the wild-type tp53 to activate luciferase transcription, which suggests that the mutations function in a dominant-negative manner. This can be easily understood considering the fact that tp53 binds to DNA as a tetramer.

iv. Apoptotic Response in Embryos with tp53 Mutations

tp53 is known to play a role in mediating apoptosis in response to DNA damage in zebrafish. In order to examine the effects of the N168K and M214K mutations on the apoptotic response in vivo, the authors treated homozygous and heterozygous embryos with gamma irradiation and performed TUNEL apoptosis assays. The wild-type embryos showed widespread apoptosis in the brain and the spinal cord. However, apoptosis was suppressed in the mutant embryos (Figure 1). In the case of the N168K mutants, the suppression was only seen when the embryos were raised at 37 イ (Supporting Figure 7), indicating that this is a temperature-sensitive mutation.

v. Cell-Cycle Checkpoint Response in Embryos with the p53M214K Mutation

An important role for TP53 in mammalian cells is to arrest the cell cycle at the G₁-phase checkpoint in response to DNA damage. In order to assess the role of zebrafish tp53 in cell-cycle checkpoint control, wild-type and M214K mutant embryos were treated with gamma irradiation and subjected to DNA content analysis. In the wild-type embryos, cells were retained in G₁-phase at early time-points, suggesting a G₁ checkpoint arrest (Figure 2). However, in the mutant embryos, S-phase cells started accumulating much earlier than in the wild-type, indicating a defective G₁ checkpoint.

vi. Effects of the tp53M214K Mutation on the Expression of Downstream Effectors

Among the downstream effectors of tp53 function are mdm2, p21, and bax. The authors tested the effects of the tp53^M214K mutation on the expression of these factors. In addition, they tested the expression of tp53 itself. They treated embryos with gamma irradiation and then performed single-embryo RT-PCR analyses. Upregulation of all of the factors mentioned above was observed in wild-type embryos. However, no upregulation was seen in homozygous mutant embryos (Figure 3). Similar results were seen in the tp53^N168K mutant line at 37 イ.

vii. Tumor Incidence and Tumorigenesis Features in tp53M214K Fish

Mutations orthologous to the tp53^M214K and tp53^N168K mutations have been found in human cancers. To test if zebrafish harboring the tp53^M214K and tp53^N168K mutations are more susceptible to forming tumors than wild-type fish, the authors monitored tumorigenesis in wild-type and mutant fish. As expected, no tumorigenesis was observed in tp53^N168K mutated fish when they were raised at 28 イ. However, the tp53^M214K showed a tumor incidence significantly higher than their wild-type siblings (Figure 4). Microscopic analysis revealed that most of the fish that had developed tumors, had malignant peripheral nerve sheath tumors (MPNST), either in their eyes or their abdominal cavities (Figure 5). It would have been interesting to monitor tumorigenesis in the tp53^N168K mutant line at 37 イ.

VI. Summary and Future Directions.  The results from the study by Berghmans et al. are summarized in the table below.

In short, this study shows that the ability to arrest the cell cycle and induce apoptosis is lost in tp53^M214K and tp53^N168K mutated fish, and that tp53^M214K mutated fish are prone to developing tumors. Even though the tumor spectrum observed in the tp53 mutant zebrafish is very different from that seen in humans and mice, these findings further establish the zebrafish as a relevant model for studying the role of tp53 in tumor development. The mutant zebrafish lines generated in this study provide a model for MPNS tumors, as well as a platform to study tp53 pathways. The next set of experments could for example include modifier screens and small molecule screens in the mutant zebrafish lines. Considering that it took a relatively long time for tumors to develop in the mutant fish lines, it would be worth investigating if this process can be sped up by exposing the fish to carcinogens.

VII. References

1.  Smith, C. 2003. Drug target validation. Hitting the target. Nature 422: 341-347.
2.  Maiolatesi, T.M. and Brenner, C. 2004. Chemical and genetic methods to validate targets in nonmammalian organisms. In: Oncogenomics. Molecular approaches to cancer. Editors: Brenner, C. and Duggan, D. John Wiley and Sons, Inc.
3.  Amatruda, J.F., Shepard, J.L., Stern, H.M., and Zon, L.I. 2002. Zebrafish as a cancer model system. Cancer Cell 1: 229-231.
4.  Stern, H.M. and Zon, L.I. 2003. Cancer genetics and drug discovery in the zebrafish. Nature Reviews Cancer 3: 1-7.
5.  Vogelstein, B. et al. 2000. Surfing the p53 network. Nature 408: 307-310.
6.  Vousden, K.H. and Lu, X. 2002. Live or let die: The cell's response to p53. Nature Reviews Cancer 2: 594-604.
7.  Berghmans, S. et al. 2005. tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors. PNAS 102: 407-412.
8.  Langheinrich, U. et al. 2002. Zebrafish as a model organism for the identification and characterization of drugs and genes affecting p53 signaling. Current Biology 12: 2023-2028.
9.  Wienholds, E., Schulte-Merker, S., Walderich, B., and Plasterk, R.H. 2002. Target-selected inactivation of the zebrafish rag1 gene. Science 297: 99-102.