Structure of specific IC peptide is identified

San Francisco-The discovery of the peptide made only by interstitial cystitis in a patient's bladder epithelium has held out the possibility of a diagnostic test, but it wasn't until University of Maryland researchers recently identified the peptide's exact structure that such a test was feasible. The latest research on antiproliferative factor (APF) not only moves researchers a step closer to a commercial diagnostic test for IC but also to new possibilities for treatment of both IC and bladder cancer and to intriguing questions about a new family of proteins.

Susan Keay, MD, PhD, professor of medicine at the University of Maryland, Baltimore, and collaborators at the National Cancer Institute, uncovered the peptide's structure by harvesting APF from the supernatant of bladder epithelial cell explants from IC patients. The APF was then purified through a five-step process.

The team then deduced the peptide's structure using mass spectrometry, lectin affinity chromatography, and enzymatic digestion. To make sure they had the right structure, they synthesized APF and showed that the synthetic peptide had the same inhibitory activity on cell proliferation that native APF does.

APF turns out to be a very small sialoglycopeptide with nine animo acids and three sugar miolecules attached to it. The amino acids have 100% homology to a transmembrane domain of a much larger protein-"frizzled 8"-whose genetic code contains 4,000 base pairs. "Frizzled" proteins are mainly expressed during embryologic development and appear to be very important in the development of both neurologic and epithelial cells.

APF is the first secreted frizzled protein-related peptide identified that does not contain a binding site for a protein called Wnt that normally binds to the peptide. However, like other secreted frizzled-related peptides, it appears to be important for controlling cell growth, Dr. Keay reported at the AUA annual meeting. (The findings were subsequently published in Proceedings of the National Academy of Sciences 2004; 101:11803-8.) In addition, it is the first one known to contain the portion of the protein that is usually present within the cell membrane.

"So this may help us to understand yet another way how segments of these frizzled related proteins are important in development of these tissues," Dr. Keay said.

The gene for frizzled 8 is expressed in adults in heart, skeletal muscle, kidney, and pancreas tissues. But as far as the research team can tell, it is not expressed in normal adult human bladder cells.

"It certainly could be expressed during a particular developmental stage that no one has yet identified," she said.

Perhaps the frizzled 8 protein is important for bladder cell development and is not regulated normally or is not turned off in patients with IC, Dr. Keay opined.

"Another possibility is that it is never part of bladder cell expression and there's some stimulus-either from outside the cell or from inside the cell-that is causing patients with IC to make this peptide for reasons that we really don't understand," Dr. Keay said. "I think we're going to learn more about bladder epithelial development through this project as well as learning more about IC."

Treatment potential

Another way the team confirmed the structure of APF was by measuring the biologic activity of the synthetic peptide. Like the native peptide, synthetic APF inhibited bladder epithelial cell proliferation. This activity of APF may be responsible for the epithelial thinning and ulceration so commonly seen in IC bladders and may be responsible for the disease process itself.

"So now what we want to look at are things that inhibit APF activity or inhibit its production," Dr. Keay said.

Dr. Keay's collaborators also found that APF also inhibits proliferation of a bladder cancer cell line-T24-almost as strongly as it inhibits the growth of healthy bladder cells. They are beginning to research the potential of the peptide or derivatives of it as an adjunctive cancer treatment.

But closest to clinical application because of this discovery is a diagnostic test for this IC biomarker. Now that Dr. Keay and her team have enough synthetic APF, they can try to make antibodies to it-the basis of a much easier, less labor-intensive assay than they now use.