By David N. Leff
Cell biologists speeding efforts toward clinical payoffs from telomerase-based therapies encountered a yellow light last week. A one-page article in Nature dated June 15, 2000, carried the warning title: "Risky immortalization by telomerase."
Its senior author is cancer biologist David Beach, of the Wolfson Institute for Biomedical Research at University College in London. Beach, long a senior staff scientist at the Cold Spring Harbor Laboratory in New York, is a co-founder of Mitotix Inc. and is president of Genetica Inc., both in Cambridge, Mass.
His caveat anent the unique promise of telomerase in prolonging the lives of cells, Beach told BioWorld Today, "is that although telomerase can immortalize certain cells, you cannot presume that their continued proliferation in culture can occur without further genetic changes.
"The promise of telomerase," he recalled, "was as a strategy for growing the large cell numbers required for cell-based therapies. You would introduce the catalytic subunit of the telomerase enzyme into cells, allow them to proliferate indefinitely without undergoing oncogenic transformation, then return them to the patient. And what we have showed in one particular cell line is that, in fact, you do not indefinitely protect them from oncogenic changes."
Then Beach switched his yellow light to green. "The idea," he continued, "which still remains a good one, and will come to fruition eventually, is to amplify certain human cells, and there are some fairly obvious ones. The most exciting potential is probably the pancreatic islet cells, which secrete insulin. Stem cells of the immune system can propagate them in vitro, and either do something to them genetically or just keep them as they are and simply expand their population. Then the clinician would transplant them back into the diabetic patient without the need to do immunosuppression.
"The promise," Beach went on, "is that these cells will not be on their way to becoming tumor cells. What we're saying is that if you're not careful, they may well have begun to move down the pathway of becoming tumorigenic."
How Telomerase Does Its Thing
To determine whether permanent telomerase expression was necessary to enable mammary epithelial cells to continue growing beyond their usual senescence point, Beach and his co-authors carried out an in vitro experiment with an unexpected kicker. Their inquiry started from the generally accepted concept that during gestation, the telomerase enzyme outfits the tips of every embryonic chromosome with a layer of protective DNA caps called telomeres. After birth, the newborn's cells shed one of these telomeric caps every time the cell divides. In human cells, 50 such doublings - the so-called Hayflick limit - demarcates the cell's life span and the onset of its senescence. (See BioWorld Today, May 8, 2000, p. 1.)
"We took some cells from the mammary epithelium of the breast," Beach recounted. "They usually undergo 55 to 60 population doublings before they stop growing or senesce."
To determine whether permanent telomerase expression was necessary to enable those mammary cells to continue growing beyond that senescence point, the co-authors delivered the human gene for their telomerase catalytic unit via a retroviral vector. That package is called TERT - "telomerase reverse transcriptase" (previously known as EST -"ever-shortening telomeres"). TERT activates telomerase. (See BioWorld Today, March 31, 1999, p. 1.)
"Those mammary epithelial cells," Beach pointed out, "are a particularly important model because it is those cells in a real person that give rise to the majority of breast cancers. We introduced the catalytic subunit of the telomerase, thereby extending the telomeres that capped their chromosomes. Most cells in our culture continued to proliferate considerably longer than they would normally have done - up to 250 population doublings.
"Then," he went on, "we removed the exogenous TERT to see what would happen, using a genetic trick to get the thing back out. To our considerable surprise, the enzyme activity remained high, even though we had excised the genetic construct that was turning it on, and the cells persisted through another 20 doublings. So we said maybe some further genetic changes occurred in the cells, and one such change must have turned on the endogenous telomerase enzyme. There's only one gene at the moment that we know does that. It's the myc oncogene, which regulates telomerase expression.
"So we looked at the level of Myc protein in these cells," Beach related, "and lo and behold it was very clear that Myc had become overexpressed two- to three-fold. And their doublings increased between 107 and 135. Activation by myc oncogenes happens in a wide variety of tumor types. It occurs in some 70,000 fatal cancers per year in the U.S. So there's little question that although these mammary cells were not directly tumorigenic as they are, they had definitely taken a step in the pathway to tumorigenesis."
How Myc Cleans Cells' Clock
"We don't know the presumed mechanism by which the myc oncogene took over from the telomerase catalytic subunit. Myc is a very unstable protein, and all we showed is that the abundance of the protein had gone up to tumor levels."
Beach makes the point that "the use of TERT for expansion of normal human cells for therapeutic purposes must be approached with caution. I think it means that when you come to do the clinical quality control of these cells, when it comes to persuading the FDA or whoever, you are going to have to monitor certain genetic loci that we know predispose to tumorigenesis.
"You are not increasing the rate of mutation by doing this," he added, "but you are selecting for cells that have mutated. And we know where on the human genome these oncogenic loci are - like the ink-4 locus, like the c-myc, like the p53. And I think those will have to be monitored in the next polymerase chain reaction or antibody protocols. That's not terribly difficult - to test the integrity of these few key genetic loci."
Beach recommends that once such testing turns up an oncogene on the premises, "you have to find a different protocol for propagating these cells - a truly genoprotective protocol. What we're saying is that telomerase alone isn't enough. Our work here in London, at Cold Spring Harbor and Genetica," he concluded, "is very much focused on trying to find this protocol right now."