Sharpless thought that obesity would accelerate molecular aging, but his lab found only a weak correlation between body mass index (BMI) and p16. “That surprised us because you’d think people who exercise more would have lower BMI, and that’s generally true,” he says. “Also, we knew that eating less is a good way to retard aging in mammals. But as we thought about it, our results made sense because BMI is not a good measure of caloric intake or exercise.” One study volunteer was a football player, very young and fit. But he had a high BMI. “We’ll have to do more studies, but it might be that exercise is good for you even if you don’t become thin by doing it. In other words, you don’t have to exercise to the point that you’re skinny. Just exercise, per se, is a good thing to do.”
I’m thirty-nine years old. But after going to RealAge.com and typing in a bunch of lies — I mean, information — about my diet and habits, it turns out I’m actually thirty-five. My diet is pretty good, the site tells me. And I’m wise for not smoking. Who knew? But my real age would be even lower if I’d spend more time exercising and less time eating cheese-filled pastries. Guilty as charged.
I should eat more fish, broccoli, cranberries, apples — anything high in omega-3 fatty acids, flavonoids, or antioxidants. Green tea is a must, of course. Oh, and tomatoes. I should eat more of them. Or tomato paste, juice, sauce, soup, ketchup. Tomatoes are high in lycopene, the site tells me, which is supposed to prevent prostate cancer.
The site leaves a lot to the imagination. For instance, how does it know how long I’ve not been exercising? What if eating pounds of blueberries every week is actually bad for me? Has anyone studied that? And the site didn’t ask me about my salt intake; I add it to stuff here and there.
Still, people seem to love RealAge.com. A friend bragged that he was really thirty-two, not forty-two. Good for him.
Unfortunately, oncologist Ned Sharpless is old. “I took that test in my thirties and it said I was in my forties,” Sharpless says. “That really ticked me off.”
You see, Sharpless doesn’t eat enough ketchup. “Yeah, it’s good that I don’t smoke and I exercise regularly, but I don’t have enough lycopene in my diet,” he jokes. “I swear that thing is run by the ketchup industry.”
Sharpless says there’s no real proof that lycopene is so beneficial that it adds years to our lives, no proof that it slows the degenerative process of aging. Same with highly touted wonder foods. That said, he’d love to know how old he really is, down to his blood and bones. He’d especially like to know how old his patients really are. Generally speaking, the younger we are the better we respond to treatments such as chemotherapy, radiation, and organ transplant. But that’s not much more specific or scientific than RealAge.com. Wouldn’t it be best if we could peek inside the human body and measure something that tells us how old we really are, or whether our lifestyles and habits hurt or help us?
That’s what Sharpless did. He devised a blood test to measure a protein called p16INK4a, a tumor suppressor that builds up in cells as we age. (We’ll refer to it as p16.) He can tell you how old you really are and — in some cases — why. He’s already found out how old he really is. And someday he might even be able to tell us whether eating blueberries and tomatoes really does add years to our lives.
In UNC’s Lineberger Comprehensive Cancer Center, at the end of a long hallway lined with posters of colorful cells and carcinomas and confusing equations, is a small office where Sharpless spends a lot of time talking with students and postdocs about what makes a good scientist. He’s put a few photos on his walls to make his points. One is of the devil tempting Jesus. “Get thee behind me, Satan,” he tells students. Don’t let the less challenging path tempt you. “There’s a lot of easy stuff we could do that’s not the right stuff to do.” There’s a postcard of George Washington. “When you run a lab, you have to be a good leader,” he tells students. “You have to remember what’s in it for your people.” Next is St. Thomas More. “He was uncompromising. You gotta stick to your principles.” And there’s a large poster of the cover of London Calling where The Clash’s Paul Simonon is destroying his bass guitar. “There are established paradigms in science, and if all you do is work within them, you won’t make progress,” Sharpless says. “Don’t be afraid to do something that someone else says is dumb. Don’t be afraid to break a guitar.” (Also, he really likes that album.)
Scientists have known for several years that p16 increases with age — in rodents, at least. “That was interesting,” Sharpless says, “but no one could say whether the increase was twofold or twentyfold.” And no one knew which cells were important to p16 expression. Not all cells have the protein, and it doesn’t work the same way in all cell types.
Sharpless, curious, decided to break a guitar — he dedicated part of his lab to aging when he arrived at Carolina in 2002. “Not all of my colleagues were enthusiastic,” he says. “At that stage of my career it might have been better to focus solely on cancer.” (His lab does study p16 as it relates to cancer. We’ll get to that.) “But there wasn’t a lot known about p16 in aging. My interest has always been the cell cycle: what makes cells divide. That’s important for cancer, but there’s more to it than cancer.” And, Sharpless says, “my first graduate student, my first postdoc, and my first technician all conspired to study p16 in rodent aging.”
First, they measured RNA molecules for the p16 gene to figure out which cells in rodents expressed p16. And one of their first findings was that rodents with low p16 were younger and fitter.
In one experiment, Sharpless restricted the diets of rats and then studied different organs to see what happened. He got a provocative result: caloric restriction didn’t do much to p16 levels in some organs, but p16 expression in the kidneys and in a few other tissues was almost completely stifled as the rats aged. And rats don’t need a lot of p16 in their kidneys: they don’t usually die of kidney cancer; they die of kidney failure.
Sharpless knew from other studies that calorically restricted rodents seemed much younger than normal rats. The dieting rodents performed better in dozens of ways, including on the classic maze test. They had better brain function, heart function, muscle and kidney and immune functions. “They live longer, more robust lives,” he says. And now he had proof that they are molecularly younger — at least in some tissues, including kidney tissue.
In later experiments, Sharpless used RNA-based tests to show that p16 doesn’t increase in every kind of cell as mice age. “It does go up in lots of cells, but we found it doesn’t do much in many of those cells,” Sharpless says. “We think p16 is unimportant in some cells. But in a few cells, it’s very important.”
For instance, he found that the protein hinders the division of pancreatic beta cells, which produce insulin in rodents — and in humans. If we can’t make enough insulin, we get type 2 diabetes. Previous to this work, diabetes researchers had thought that beta cells didn’t divide — or if they did, that they didn’t replicate fast enough to matter. “That’s what we were taught in medical school,” Sharpless says. “Turns out that’s wrong.” Sharpless and Harvard scientist Susan Bonner-Weir showed that beta cells divide, and p16 stops that division. High levels of p16 are not good for beta cells, which rarely turn into cancer and have to divide so that the pancreas can produce enough insulin.
So what does p16 do in humans? It’s not easy to harvest cells from human organs. But in 2006 Sharpless worked with hematologist David Scadden to find that p16 hindered division in hematopoietic stem cells as rodents got older, a clear sign that p16 levels correlated to the aging process. So Sharpless thought he’d study human hematopoietic stem cells. But he ran into trouble: you have to extract those cells from bone marrow. And that hurts. A lot.
“When we tried to get this study off the ground, I pitched the idea to cancer doctors,” Sharpless says. “They said, ‘This is awful. You gotta do three bone marrows? Who’s gonna do that? Why can’t you just test the blood?’” Sharpless thought, “Oh, you stupid clinicians. We smart scientists have elegant reasons for why we need to use marrow.”
Sharpless (who, by the way, is also a clinician) contacted anyone in the country he thought could help him find bone marrow donors. Over three years colleagues in Seattle gathered eighteen people — too few to conduct a full study. But his lab did find that human stem cells always have low levels of p16, no matter how old or young someone is, and that makes the protein harder to study. It would be better to study a line of cells with a wide range of p16 expression.
“We gave up,” Sharpless says. Well, they retreated. His lab decided to heed the advice of the stupid clinicians and use T cells in blood. Sharpless’s team set up a blood-drawing station in the lobby of UNC Memorial Hospital and paid volunteers ten dollars for a sample of blood and answers to a few questions.
This is where I first heard of Sharpless’s research. His team took my blood while I filled out a questionnaire about my general health, disease history, and exercise and smoking habits. The idea, according to a white-coated lab tech, was to see whether certain behaviors help keep people molecularly younger. Sounded interesting.
“I really didn’t think this would work,” Sharpless says. “As a mouse geneticist, I thought these questionnaire-based studies were just the softest stuff you could imagine. If I asked you how much you exercise, would you answer that question the same today as you would six months from now? It just didn’t seem very precise.”
For my part, I’m confident I gave him good data: I’ve been consistently lazy and a devout nonsmoker for a good decade.
Team Sharpless collected two hundred blood samples and used a common technique — polymerase chain reaction — to analyze the p16 gene in T cells. To Sharpless’s surprise, the results were robust. P16 expression in T cells increased exponentially with age, and T cells allowed for a much purer measurement of age than did hematopoietic stem cells. Smokers who didn’t exercise had higher levels of p16. Sharpless says doctors have known that longtime smokers seemed physiologically older — they looked older than their stated age. It seemed as if smokers were molecularly older. “And now we have proof of that,” he says.
Now, the elephant on the page: if p16 is a tumor suppressor, and smoking increases p16, how does smoking cause tumors? Why doesn’t all that p16 wipe out the cancer? “When I give talks I get that question every time,” Sharpless says.
Let’s see if we can explain it. When a carcinogen such as cigarette smoke enters the body, it can damage cells and alter DNA. “Say you have ten cells with equal risk of becoming cancer,” Sharpless says. “Eight cells take in a carcinogen, but not enough of it, and spit it out. Nothing happens to those cells; their p16 stays the same. One of the cells takes in the carcinogen, is really damaged, and p16 is activated. P16 stops that cell from dividing. It’s in jail, never to be heard from again.” The tumor suppressor is working, and p16 increases manyfold. “That tenth cell takes in the carcinogen and DNA is damaged, but p16 is not activated for one of two reasons — the DNA damage is not the kind that activates p16, or the carcinogen mutates the p16 gene so that the protein is not induced. In either event, that cell is significantly damaged and well on its way to becoming cancer.”
So that’s how carcinogens and p16 interact. And that’s why so many cancer researchers, including Sharpless, are interested in p16 — it’s one of the few proteins that keep cells from becoming cancer. It interacts with kinases called CDK4 and CDK6, which act like gas pedals — they speed up cell division. Many kinds of cancer hijack CDK4 to cause damaged cells to divide like mad. P16 acts like the brakes: it interacts with CDK4 to stifle cell division. Because of this, over the past decade big pharma has created drugs that mimic p16 to target CDK4 and CDK6. The drugs were supposed to act like an emergency brake, but they didn’t work nearly as well as everyone had hoped. Sharpless created a mouse cancer model to find out why.
“There are so many things that could go wrong with any drug,” Sharpless says. “Maybe it’s not hitting the target. Maybe mice can’t absorb the pill. Maybe the pill gets metabolized really quickly into something else and is inactivated. Well, we found that the p16-like drugs were hitting their target really nicely and just beautifully stopping the proliferation of a few mouse cells that were CDK4 and 6 dependent.” But he found that most cells can divide without those kinases. “A lot of cancer cells use any kinase available to them; they’re promiscuous,” he says. “They have ways of turning on other kinases, such as CDK2.” Scientists have developed drugs that target all three CDKs, but they’re miserably toxic, Sharpless says, because shutting down all three kinases means stopping all cell division. And humans need for some kinds of cells to divide. Blood and gut cells, for instance. “Every week you make a whole new intestinal lining with new cells,” Sharpless says. “If you can’t do that, it’s really unpleasant.”
P16 drugs — several companies have slightly different versions — are not the panacea researchers had hoped. But many scientists are still searching for cancers that would respond to p16 compounds. Sharpless, though, took a different tack. He wanted to know whether the p16 drugs he had been working with shut down cell division in normal tissues. They did, and in a big way.
“The big kahuna cells we could make stop dividing were the hematopoietic stem cells,” Sharpless says. They’re in bone marrow. They’re like blood cell matriarchs, he says, and their progeny include white blood cells, which get whacked during chemotherapy. “Protecting white blood cells is a twenty-billion-dollar-a-year business in oncology.”
But it wasn’t such a big deal to use the p16-like drugs to protect hematopoietic stem cells, because most of them are out of the division cycle at any given time. Radiation and chemo, for example, hurt cells that replicate a lot. The cells responsible for forming hair are some of the fastest-growing cells in the human body; that’s why chemo and radiation often cause hair to fall out. Sharpless knew that the big kahuna cell’s immediate progeny — multipotent progenitor and common myeloid progenitor cells — divide fast. But he thought they would use a different kinase to proliferate, not CDK4. If he was right, then p16-like drugs would not protect those progeny cells from chemo or radiation.
“I was wrong,” Sharpless says. “And that’s good. Turns out, the first couple progeny cells depend on the CDK4 kinase, too.”
In his experiment Sharpless gave mice the drug, gave them radiation treatment, and watched what happened. The result astounded even Sharpless.
“If you don’t give the mouse the drug, it dies of radiation sickness,” he says. “If you give it the drug, it lives; it has nearly complete protection. You never ever get an experiment like this. It worked the first time and it’s worked every time since. Young mice, old mice, mice with genetic deficiencies that make them more or less susceptible to radiation. It always works.”
It also worked up to twenty hours after the mice had been exposed to radiation.
Sharpless’s lab put the drug up against many common chemotherapies, and the drug protected mice against all of them. The idea is that doctors could give the drug to cancer patients. Chemo or radiation would attack the cancer cells while the p16 drug protected bone marrow.
Sharpless started a company called G-Zero Therapeutics that’s developing a class of p16 compounds for human use. The company has received more than one million dollars in funding, mostly from the federal government, which is interested in drugs that protect against radiation poisoning. And G-Zero is also developing a p16 diagnostic test to pinpoint people’s molecular age. One day, Sharpless thinks, you’ll be able to go to your doctor, consult on why it might be good to test for your real age, and find out what exactly is going on in your T cells.
After analyzing two hundred blood samples, Sharpless’s lab found that twenty-year-olds have an average p16 level of 4.5, as measured by using an RNA analysis technique and fancy math. Of the study participants — all of whom had no preexisting diseases or conditions and were not on medication — a twenty-one-year-old had the lowest p16; it was 3. The highest was 9 — a fifty-eight-year-old smoker who didn’t exercise. That numerical difference of 6 doesn’t seem like much, but on this scale an increase from 5 to 6, for instance, means that the amount of p16 has doubled. So that number 9 represents a real age much higher than the participant’s birth certificate age of fifty-eight. That person’s real age is probably in the eighties.
Sharpless’s blood test is simple; he could tap your arm and give you results within a week. UNC patented the process and is working with G-Zero to commercialize it. But would you really want to know your molecular age? If you already know that smoking isn’t so good for you, do you really need to know that, as a longtime smoker, you’re really sixty instead of fifty? Maybe, maybe not. But doctors would love to know.
Two different groups of European scientists cited Sharpless’s research in their papers on p16 in human kidneys and transplantation. They used a slightly different method, but their test accurately predicted which donors had provided kidneys with the best graft-survival rates. Measuring p16 was better than knowing chronological age when it came to predicting which grafts would be most successful.
A p16 test could predict how patients would handle surgery or how well wounds would heal. Doctors could use Sharpless’s test to see if his p16 drug protects bone marrow in cancer patients who receive chemotherapy or radiation. “P16 goes up when hematopoietic stem cells are damaged,” Sharpless says. “You would be able to see how much older, in molecular terms, someone is after chemotherapy.”
But Sharpless warns that seeking to know your p16 should not be for kicks and giggles. The test could give you really bad news; you’d need to be prepared. Still, Sharpless says, if patients really want to know their molecular age, doctors should let them know.
“You could use your p16 level as evidence that your healthy lifestyle is paying off or not,” he says. “Maybe you’d change behaviors. I think that’s a good reason to do it. A bad reason would be using it as a means for employment or insurance discrimination. Someone could say, ‘Well, you no longer can get health insurance because you’re molecularly old.’”
During Sharpless’s study, the white coats who drew my blood also took a sample from Sharpless and his dad. Both Sharpless men had low p16 for their age. Ned’s was 5; Dad’s was 7. That meant Sharpless was really twenty-eight instead of forty-one. (He’s now forty-three.) His dad was sixty-five instead of eighty-one. “I was very proud about that,” he says. “I was going around the lab, all sanctimonious, telling everyone, ‘That’s the jogging and the nonsmoking. The Sharpless lifestyle is paying off!’”
Unfortunately for the Sharplesses, the lab was not done studying the blood samples. They found a common single-nucleotide polymorphism in the genome that affects p16 levels. Depending on your DNA, you could simply make less p16 than other people. And it turns out that the Sharpless men have the genotype with less p16 in T cells. “I had no right to brag,” he admits. When accounting for genotype, Sharpless was really thirty-six. Still, he gets to relive part of his thirties, and that’s not so bad.
Sharpless is not sure how important p16 genotyping is with regard to aging. “That’s a question we’re trying to figure out now,” he says. “Should you be interested in your total p16 or your p16 normalized for your genotype?” Probably both.
Having a low p16 level in tissues aside from blood is not always a good thing. For one, it means you’re more likely to get atherosclerosis — hardening of the arteries.
But Sharpless isn’t sure whether his own p16 levels are low everywhere. Logic tells him that having a low p16 level in T cells is a good thing. “I think you’d have better immune function because your lymphocytes would work better,” he says. “But that’s pure speculation. We have no data to back that up.” To figure that out, Sharpless says he’d have to test T cells in a lot of people — say, a thousand — and follow them for years to see how they age. “I predict high p16 will show bad outcomes,” he says, no matter the genotypes.
The Sharpless lab is still honing the blood test, collecting more samples from volunteers, and measuring p16 in other tissues. He says he could expand his research to figure out whether other behaviors such as sunbathing or inhaling secondhand smoke are linked to p16 and aging. He could study eating habits. Do blueberries and tomatoes really keep us young? What about red wine or salmon? Bee pollen? Green tea? Dark chocolate donuts filled with grape jelly?
“The best I can tell, lycopene and antioxidants don’t do anything,” he says. Not as antiaging agents, anyway. Resveratrol, the stuff in red grapes that gives red wine such a good reputation, might have beneficial antidiabetes properties. But Sharpless says other research questioned the antiaging properties of resveratrol compounds.
“You know, one day I want to write Ned Sharpless’s Guide to Healthy Aging,” he says. “It will be one page — don’t smoke, avoid carcinogens like tobacco, stay thin, and exercise is probably good. That’s it. Everything else is controversial.”