By Dean A. Haycock

Special to BioWorld Today

The now-familiar exercise of cataloging genes may make it seem as if unexpected findings play a smaller role in biological research than they once did. Despite this impression, properly prepared researchers still are managing to ¿stumble¿ upon more intriguing observations than those they set out to make. A discovery made about the function of Id proteins is a recent example.

Id genes encode proteins that appear to act as ¿breaks¿ during development. Id stands for ¿inhibitor of differentiation.¿ Their presence stops young, undifferentiated cells from differentiating prematurely into ¿adult,¿ fully equipped neurons. Id proteins do it by preventing transcription factors ¿ proteins that control the expression of genes ¿ from binding to DNA.

To study this aspect of Id protein function, researchers at the Memorial Sloan-Kettering Cancer Center in New York bred double knockout mice lacking both copies of the Id3 gene and one copy of the Id1 gene. (The researchers found that mice with absolutely no Id3 or Id1 genes did not survive, so one gene was left intact.)

In these animals, cells destined to become neurons ¿ neuroblasts ¿ prematurely withdrew from the cell cycle and began to express features characteristic of mature neurons. But they also displayed malformations in the blood vessels in their forebrains. And they showed a noticeable lack of branching and spouting of blood vessels into a part of the embryo called the neuroectoderm, which goes on to develop into the central nervous system and into crucial components of the peripheral nervous system. This observation, described in today¿s issue of Nature, comes at a time when angiogenesis inhibitors are considered by many to be among the most exciting potential new anticancer therapies.

¿We were basically trying to figure out how these proteins work. We stumbled upon this effect on angiogenesis and then recognized that it might be important in terms of tumor growth and metastases. It really was an amazing example of translational,¿ if you will, research,¿ co-author Robert Benezra, an associate member at the Memorial Sloan-Kettering Cancer Center, told BioWorld Today. Benezra and his colleagues discuss their results in their article, ¿Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumor xenografts.¿

Since the formation of new blood vessels in the brain and in tumors involves the infiltration of tissue by endothelial cells, the researchers decided to see how tumors transferred from humans to the mice thrived in the Id3/Id1-deficient animals. The results were striking. Three different tumor xenografts flourished in wild type, Id3/Id1-equipped mice but failed to ¿take¿ in the knockouts. Furthermore, the tumors that did hang on had poor vascularization and showed evidence of extensive cancer cell death.

For the authors, Ids represent novel and attractive targets. Based on their expression patterns, there is a good chance that drugs that interact specifically with them may not be as toxic as other drugs. This is because the Id proteins are not expressed at high levels in normal adults. Analysis of tissue from human brain tumors, however, revealed significant expression of Id1 and Id3 in blood vessels.

A major advantage of targeting Id, in Benezra¿s view, is that it represents a known anti-angiogenic target. ¿We understand how this protein works, so we might be able to more effectively design drugs because we know essentially what the target is,¿ he said. ¿With some of the other [anti-angiogenic] compounds, very little is known about how they are working. For angiostatin and endostatin, people really don¿t have an idea of how these compounds are working . . . so I think we have a chance of designing a drug that is highly specific.¿

The researchers have already identified a lead compound and are working to develop it with Angiogenex, a company in Seattle. ¿They want to take this lead compound and improve it based on its ability to interact specifically with Id,¿ Benezra said. He described the lead¿s specificity as ¿fairly good,¿ and said their goal is to make it 100-fold better before testing it in animal models.

¿Right now we have shown that the loss of Id function affects essentially any tumor type that we put into a xenograft model,¿ Benezra said. ¿So we can go straight with the xenograft model, but we are hoping that the loss of Id function will also affect animals genetically prone to develop cancer. If it inhibits some and not others, then obviously we would go after those.¿

There also is much to understand about the basic biology of Id proteins. For instance, the researchers would like to understand the interactions of Id proteins and certain important markers of activated angiogenic endothelial cells such as integrin and an enzyme it binds, the matrix metalloproteinase type-2 protein. ¿We would like to understand mechanistically how Id is talking¿ to those proteins,¿ Benezra told BioWorld Today. ¿Now, it could be direct or indirect. That is one thing we would like to figure out. In addition, we would like to understand how the other angiogenesis inhibitors were working vis-a-vis Id ¿ that is, are they affecting the levels of Id expression? We are looking at that now even in human samples by in situ analysis.¿

Other, ongoing experiments involve the breeding of animals that have lost all copies of all three Id genes, Id1, Id2 and Id3. ¿Obviously, that is a relatively rare event. But we are trying to look at the embryos and see whether or not there are other defects in these animals as well,¿ Benezra explained. ¿We want to test whether or not the loss of the last remaining Id allele ¿ remember, to carry the adults into the tumor studies we have to have three out of four alleles missing ¿ we would like to make sure that if you knock out that last allele, that the animal is okay. We suspect it will be because the levels of expression of Id in adults is very, very low. But we have to prove it.¿

Funds for these studies came from the Children¿s Brain Tumor Foundation, the National Institutes of Health, and the National Science Foundation.