The fleeting signals of cells

During the rhythmic squeeze of a heartbeat or the powerful contraction of labor, our cells' gap junctions are hard at work.

Though they are merely minuscule protein tunnels that connect cells together, gap junctions play a big role in development, metabolic transport and growth control.

A gap-junction tunnel, at 1.5 to 2 billionths of a meter in diameter, sends and receives ions and small molecules. This flow of molecules and ions can signal the cell, telling it how to operate. Smooth-muscle cells of the uterus, for example, relay the flow of signals to coordinate an effort like a contraction.

In healthy cells, signals flow freely. Yet in tumor tissue, gap junctions usually lose basic functions and prevent molecular flow. Then the tumor cells can become islands of unregulated growth.

"When you get a loss of gap junctions, you affect cell-growth control," said Dr. Paul Lampe of Public Health Sciences.

Because growth control plays an important role in cancer treatment, Lampe studies the cell biology of these gap junctions. He is most interested in the signaling pathways that help build and destroy the gap-junction structures.

Like a construction manager, the signaling-pathway system for gap junctions can control the rate at which the channels are built and even inhibit their assembly. These systems, called kinases, can even cause the channels of the gap junctions to close, in a process called channel gating. Other kinase systems will promote the degradation of the channels, ending the life of the gap junction.

Short-lived junctions

From assembly to destruction, the lives of these gap junctions don't last long. Lampe's research shows that gap junctions have half-lives of one to five hours.

"That is one of the things that's really weird about gap-junction proteins," he said. "They turn over rapidly for an integral membrane protein."

The process of intercellular communication is highly regulated, Lampe said, so the cells expend a lot of energy to constantly produce new gap-junction proteins.

Clicking open a video on his Mac computer screen, Lampe can illustrate the lives of gap junctions in a "family photo album" enabled by time-lapse microscopy. A bright array of colors pops up, displaying an image of tightly packed cells.

Lampe can point out the gap junctions, clumps of small green dots that appear between the red-colored nuclei and the blue DNA. While the nuclei and the DNA remain intact, the small green dots of the gap junctions continually vanish and reappear like fireflies in the night.

Similar techniques also can display how well the gap junctions transport molecules to one another.

Microscopist Theresa Richards demonstrates the imaging technology as she carefully positions a thin glass needle. Puncturing one cell, she slowly injects it with a dye. With time-lapse microscopy, Richards watches the dye spread like a blooming flower as it travels from cell to cell.

In healthy cells, the dye spreads to its neighbors via the gap junctions. In cells treated with tumor promoters, the defunct gap junctions eliminate the spread of dye from the injected cell.

At the molecular level, Lampe's lab hopes to prove which kinase systems are involved in the regulation of the molecules, such as the channel assembly, gating and turnover.

To better understand the roles of the proteins that form gap junctions, Lampe studies genes that encode the gap junctions' proteins, called connexin genes. Researchers are perplexed by the multitude of about 20 connexin family members, Lampe said, so they test the different genes that are expressed in tissues.

Lampe's other research shows that some connexins transfer certain molecules better than others, such as the molecule ATP, commonly described as the metabolic currency of cells. For this reason, different connexins may control the energy status of a cell.

The roles of connexins and gap junctions in cell growth and control aren't Lampe's only interests. His initial study guided him to new collaborations.

"My interest in gap junctions as potential biomarkers to detect cancer has expanded to a search for other potential biomarkers," he said.

Now, the Lampe lab collaborates with several Fred Hutchinson colleagues to study potential biomarkers for colon and breast cancer.

In the epithelium of colon tissue, the cells link closely together to form a U-shaped structure called a crypt. The cells in the bottom of the "U" proliferate and expand upward. As the cells move up, the top layer of cells sloughs off in a process that essentially replaces most of the epithelial cells of the colon within one month.

In collaboration with Dr. John Potter of PHS, Lampe observed that in colon-cancer patients, cells proliferate further up the crypt. Certain proteins may be responsible, as some can control the natural progression of programmed cell death, called apoptosis.

With assistance from pathologists Dr. Dave Myerson and Dr. Peggy Porter and the Pathology shared resource, Lampe's lab has processed more than 3,000 slides of colon crypts and studied about 20,000 images with a specifically designed analysis program.

Team members compare the crypts of cancer patients vs. normal patients. They also evaluate the crypts of patients who exercise or consume soy isoflavones in studies led by Potter and Drs. Anne McTiernan and Johanna Lampe of PHS. These ongoing studies will help researchers determine if diet and exercise have potential benefits to biomarker distribution in the crypts.

"It's a massive amount of data, and we're still collecting it for these studies," Lampe said.

Other areas for potential biomarkers may lie within the blood-serum samples of breast-cancer patients.

Biomarker search

In the search for biomarkers, each sample goes through a detailed process. First, researchers remove the albumin from the blood. Albumin is 60 to 80 percent of the protein and hides other important signals, Lampe said.

The purified samples arrive at Dr. Philip Gafken's lab in Shared Resources. He takes the samples and puts them into a MALDI (matrix-assisted laser desorption/ionization mass spectroscopy) machine, an instrument that determines the molecular-weight distribution of all of the constituents in the serum.

The machine spits out data in the form of a complex spectra, appearing somewhat like a seismograph reading from a massive earthquake.

Dr. Yutaka Yasui of PHS interprets these lines of data by running a computer algorithm to look for outlying data in the spectra. If the outlying data of cancer patients consistently matches, a specific protein is present in cancer patients. Therefore, that protein could become a potential biomarker.

Lampe's lab can draw on nearly 30 freezers packed with samples from breast- and colon-cancer patients. The samples have been collected in a controlled way, Lampe said. They contain all information on patients and their cancer status, and sometimes even samples of patients' blood before and after their cancer.

"We have some incredible, valuable samples," Lampe said.

Although the lab works with leading-edge technology, Lampe said, other people use similar technologies to discover biomarkers.

"I think the real asset we have is the banks of freezers full of carefully collected serum and plasma," he said. "Those are really going to give us great advantages down the line."

[Marita Graube, a graduate in technical communications at the University of Washington, is a writer for Northwest Science and Technology.]