By David N. Leff
Two human genes, BRCA1 and BRCA2, are between them held guilty of causing 90 percent of early onset familial breast and ovarian cancers. Of the two, BRCA2's rap sheet includes mammary carcinoma in males as well as females, plus perhaps a role in prostate cancer instead of ovarian.
Although the two genes are genomically similar - both expressed at high levels in testis, for example - their mutation patterns differ sharply. BRCA2 gene alterations involve twice as many DNA truncations as do BRCA1.
Now, a research paper in the current issue of Cell, dated Jan. 26, 2001, implicates BRCA2 as perp of more unexpected functions than malignancies alone. Its title: "A human BRCA2 complex containing a structural DNA binding component influences cell cycle progression." The article's senior author is biochemist and molecular geneticist Ramin Shiekhattar, a faculty member of the Wistar Institute in Philadelphia.
"This study," Shiekhattar told BioWorld Today, "helps us to understand what BRCA2 is doing, but it also identifies a new, related gene, BRAF35, which sits on the long arm of human chromosome 19, a hot spot for ovarian cancer. The protein it expresses, BRAF35, he explained, "stands for 'BRCA2-Associated Factor,' comprising 35 kiloDaltons in amino-acid length."
Shiekhattar cited three key elements reported in the Cell paper:
"One is that we found BRCA2 to be part of a multi-subunit complex," he enumerated. "The BRAF35 protein is a component of that bigger machinery.
"Two, we showed with great rigor that the machinery has a role in cell-cycle progression, which is completely new for BRCA2. That is, when we neutralized the complex, cells got stuck, and couldn't go through with their mitosis.
"Three, we have identified one of the subunits of the complex, and this BRAF35 protein seemed to be at least a DNA-binding component. This specific type of molecule recognized a form of DNA we call a four-way, cruciform Holliday junction - where its two double-helix strands cross over."
The Wistar co-authors built their approach on prior insights by others into the cancer-causing BRCA2 gene.
Busy-Body Genes Swarm All Over
"Since these genes were found," Shiekhattar observed, "very little information has been gained by just looking at their genomic sequences. For example, BRCA2's amino-acid sequence is very uninformative about its gene function. So the first question for us was: What are these genes - in particular BRCA2 - doing in any cell?
"Because," he pointed out, "these genes are ubiquitous. That is, every cell in your body has that BRCA2. Which leads to the million-dollar question: Why is it, then, that mutations in this gene cause breast and ovarian cancer specifically?
"When scientists a few years ago first knocked out BRCA2 in mouse embryos," Shiekhattar continued, "they recognized that it has a developmental function. You would not expect this from a gene that has not been conserved throughout evolution. BRCA2 is a new gene. The mouse has it, we humans have it - and that's it. If you go to Drosophila or any lower organism, there's no such thing as BRCA2 in their genome.
"Therefore," he observed, "one would not have thought that they would be required for embryonic development or control, because those are machineries that are normally conserved throughout evolution. But that's not the case with BRCA2. If you mutate it or knock it out, the embryo doesn't develop beyond seven gestational days.
"So then," Shiekhattar recalled, "we asked the question: What is the real role that these proteins play in early development? The first clue," he recounted, "came from these same studies that knocked the gene out. What those investigators found was that the embryos were sensitive to ionizing radiation. This suggested that BRCA2 has a role somehow in DNA repair.
"So we took something of a blind approach, biochemically purifying the gene's multi-subunit complex protein from human HeLa cells, to find out what its function is. We are now in the process of analyzing the associated polypeptides, and trying to figure out, one by one, what these other components do. Our hope is that we will find the critical component, which targets the complex to a specific site.
"Our Cell paper," Shiekhattar pointed out, "does not address the role of BRCA2 and BRAF35 in DNA repair. But these two roles - cell cycle progression and DNA repair - are not mutually exclusive. Evolution often finds a way to use such complexes as dual-purpose tools, like an Allen wrench in a Swiss Army knife.
"Our team," he related, "is at the very beginning of this hunt, this journey. And what I think is going to be important for the pharmaceutics is that we find targets that could be therapeutically attacked to either correct or prevent the cancer-causing problem. For example, the DNA-binding component could be a drug target. Small molecules might be discovered that would prevent or stabilize that DNA binding, which is the targeting part of the complex."
Drug Discovery Lurks In Protein Complexes
"The other thing," Shiekhattar continued, "is that once we have found - and I think we have it - the catalytic subunit that governs the activity of this complex, it would be a target of drugs. Once you have an enzyme, that enzyme could be a very profitable drug target. So this is the direction in which we're going. We're trying to biochemically identify the other components of this machinery, and once we do, then these would be real targets for either preventing or treating disease.
"Clearly," Shiekhattar said, "the problem is that we know very little about cancer in general, and very little about these molecules in particular - where they actually work. So one of the things I'm working on is that within the next couple of years we really gain a much better understanding about the role of these things, and the other components associated with them.
"All that said," he concluded, "we may have a breakthrough with one of these critical components which would be the key to the whole thing. That would really be a lucky stroke, and we may already have it. But I don't want to count my chickens before they hatch."