By David N. Leff

"Stop the world — I want to get off!"

At the molecular level, that once-trendy musical aptly describes dividing cells that suddenly decide to step off the cell proliferation carousel. They don't die, but retreat to quiescence, a hibernation-like state in which they await return to activity.

Malignant tumors, of course, are the fastest, most relentless passengers on the cell cycle. Unlike normal, healthy cells, they don't need growth factors in order to divide and multiply.

As molecular and cell biologist Stephen Lee put it, "If you place normal cells in culture, in the presence of growth factor (GF), they'll grow. If you remove those growth factors, they'll stop growing, and go into quiescence. But a tumor cell grows with or without those factors."

Lee continued: "That's the hallmark of tumorigenesis. The vast majority of all tumors in the human population have the characteristic of being able to grow in the absence of GF. And to my knowledge, there is no gene that directly regulates cell cycle exit and quiescence in GF-deficient serum."

Lee is in the cell biology and metabolism branch of the National Institute of Child Health and Human Development within the National Institutes of Health, in Bethesda, Md.

He is co-first author of a paper in the current Proceedings of the National Academy of Sciences, dated Feb. 3, 1998. Its title: "The von Hippel-Lindau tumor suppressor gene is required for cell cycle exit upon serum withdrawal." Its senior author is Richard Klausner, director of the National Cancer Institute.

VHL Disease Rare But Devastating

Von Hippel-Lindau (VHL) syndrome, Lee observed, "is a very infrequent syndrome, because its unfortunate victims have a hard time reproducing." In the U.S., it affects one individual out of about every 36,000 new births.

But VHL, however infrequent, has a high-frequency kicker.

"The gene for the familial type of VHL disease," Lee went on, "is responsible for a slew of tumors in VHL patients, including brain, spinal cord, eye disease — and notably kidney cancer." A VHL patient's kidney may contain up to 600 independent solid tumors, and 1,100 cysts. But that's a lot less than the half of it.

"It just so happens," Lee continued, "that this infrequent syndrome is responsible not only for kidney cancer of VHL patients, but also for the most frequent kind of kidney cancer in the general population — renal clear cell carcinoma. That's the seventh- or eighth-highest cancer killer in the U.S. So now you're looking at a frequency of 25,000 to 30,000 Americans each year, newly diagnosed with a VHL-related kidney cancer. Deaths are estimated at 8,000 to 15,000."

A VHL patient usually develops his or her first tumor around the mean average age of 26. In children, its initial symptom is retinal angioma, striking around age eight. Inheritance is autosomal dominant.

The VHL gene resides on the short arm of human chromosome 3. It expresses a stubby protein of unknown function, 213 amino acids long.

That VHL gene is a promiscuous mutant. "It's at the distal part of chromosome 3," Lee pointed out, "so you often see that part totally deleted. You can have a small truncation, a nonsense mutation, or what is interesting in the VHL case, just missense mutations — and these are found everywhere on the VHL gene. It's not like the p53 tumor suppressor gene," he added, "which has discrete hot spots for its missense mutations."

A year and a half ago, Lee and his co-authors reported that cells lacking the VHL protein grow and eventually die in the absence of GF serum, whereas VHL-positive cells do not die. The team also has shown recently that the VHL protein assembles with three other cellular proteins, including one bearing the name human cullin-2. The researchers discovered that human cullin-2 is similar in sequence to a yeast protein (CDE-53) known to regulate the cell cycle.

"So we said to ourselves," Lee recalled, "maybe VHL has a role in the human cell cycle." As things turned out, there was no maybe about it.

"We came to realize," he continued, "from in vitro experiments, that VHL's role in the cell is to permit normal kidney cells to withdraw from the cell cycle, when they are incubated in the absence of GF. But a kidney tumor cell goes right on growing with or without those factors. However," Lee recounted, "when we introduced over-expressed VHL into those tumor cells, we found that they were able to exit the cycle in the absence of GF and behave like normal cells.

"And that," Lee pointed out, "has a lot of relevance to therapy, because if the only defect in kidney cancer is the lack of VHL, then one can easily understand that reintroducing VHL restores their normal ability to sense GF for proliferation or absence of proliferation."

He went on, "Now the next question is: How does VHL do this? And we're working on that."

In Vivo Trial? 'Any Company Can Do It'

Meanwhile, Lee speculated: "What would happen if you infect nude mice with an adenovirus vector [AV] driving a strongly expressing VHL cDNA? We know its over-expression has no toxic effect, whereas if you overexpress p53 in a nude mouse, every cell that expresses it is going to die. What's more, you wouldn't need to target your VHL delivery package to a specific tumor target; you just need to transfect everything, including the tumors."

Lee made the added point that for such a nude-mouse experiment, "You wouldn't need a fancy human kidney tumor from a fancy patient. You can readily buy VHL-negative cells from the American Type Culture Collection.

"Inject them into your mouse, see tumor formation, and then infect it with an AV vector delivering VHL expression. You would see, perhaps not total tumor inhibition, degradation and disappearance, but maybe relaxation in growth rate."

Lee concluded: "I think that would be a key experiment, and an easy one for any company to do that's set up for this type of research." *

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