Edited by: Paul Smaglik
Thomas R. Cech
Comments by Thomas R. Cech, professor of biochemistry, biophysics, and genetics at the University of Colorado Health Sciences Center, Boulder
This paper is "a derivative," laughs Thomas Cech. The "real breakthrough" paper that made it (and its companion, see below) possible came months earlier, but has received less citation attention than either of its successors. That earlier paper reported the purification of the telomerase enzyme's catalytic subunit--the first such subunit cloned.1 "That paper was the result of four years in the cold room by Joachim Lingner," Cech recalls, adding that the skill and diligence of the then-University of Colorado at Boulder postdoc beat the odds. "There were industrial companies that had a dozen people working full time to purify the human enzyme and had been unsuccessful. And he did it all by himself." Key to the success of that first cloning was the use of Euplotes, rather than human. The ciliated protozoan has an abundance of short chromosomes and consequently, more telomeres, resulting in a higher concentration of telomerase than human.
Once the team found that subunit (and the yeast homologue, in collaboration with Victoria Lundblad, associate professor of molecular and human genetics, Baylor College of Medicine in Houston), unearthing the human version was comparatively simple. "It started out with a database hit to an expressed sequence tag [EST] from the Washington University EST library." Still, some luck was involved, since finding the homologue depended on the matching genetic material being filed in that database. "We knew that it would eventually show up. We had no idea whether we would have to wait months or years. It turned out to be only months," Cech notes. "There was also some luck that the little piece of expressed sequence tag represented a region that we could cleanly identify as being a telomerase reverse transcriptase."
Cech was excited when this paper resulted in an "avalanche" of related work in other laboratories soon after, perhaps most notably as described in a January 1998 paper that showed normal cells could be immortalized by putting the subunit gene, which came to be called hTERT, into them.2 "This is the key switch component," Cech notes. "If you add this back to cells, you get telomerase working again in the cell, you get the telomeres elongated, and you get this incredible result of indefinite lifespan of normal cells."
Cech notes that the more recent immortalization achievements have eclipsed the earlier two telomerase papers. "That's taken off so much now that the Nakamura et al. paper isn't always cited," he notes. "Maybe that's the highest compliment one can get for one's work. After two years, it's so much part of the common knowledge that it isn't even something one would cite any more."
The next step--finding potential anticancer applications--hasn't moved forward as much as one might have thought, Cech notes. "There still isn't a good pharmaceutical candidate that I know of that's a high-affinity, highly specific telomerase inhibitor that could be used to test the idea that inhibition of telomerase would revert the proliferative propensity of tumor cells." While targeting telomerase may be a "promising" idea, there's no guarantee it will be a panacea. He notes that though 90 percent of tumor types reactivate telomerase, 10 percent don't. "We already know there's likely to be another mechanism that tumor cells might be able to revert into if their telomerase is turned off," Cech concludes.
Robert A. Weinberg
Comments by Robert A. Weinberg, professor of biology, Massachusetts Institute of Technology
The telomerase enzyme is found in vanishingly low amounts in normal cells, but 85 percent of tumor cells express it in readily detectable quantities. Robert A. Weinberg and colleagues sought to better understand the mechanics by which the enzyme contributes to cancer.1 They knew from the work of others that in normal cells, telomeres shrink after each cell cycle, to the point where a cell can no longer divide. But in cancerous ones, telomerase rebuilds the chromosomal ends, enabling the cell and its descendants to proliferate without limit--the phenotype of cell immortality. However, they did not know precisely how the enzyme's component parts contributed to that immortalized growth phenotype, which was presumed to be an essential part of malignant proliferation. At the time, it was known that the enzyme was composed of a reverse-transcriptase-like catalytic subunit and an RNA template subunit.2 Others ruled out the RNA subunit as a key player in cancer formation, because they detected this RNA in significant amounts in normal cells. That left the catalytic subunit as the likely culprit responsible for the sudden appearance of enzyme activity when cells progress to immortal growth.
But they needed more than such circumstantial evidence to confirm the catalytic subunit's complicity in cancer formation. So Weinberg's lab (and Thomas R. Cech's lab, see above) raced to isolate the gene that encodes the catalytic subunit. The Weinberg group developed strong evidence that the gene encoding the catalytic subunit was essential to telomere maintenance in yeast. Next, they hunted for a human homologue, but the Weinberg lab's database search came up empty. Then, in April of 1997, Cech's lab finished the sequence of Euplotes aediculatus. "We looked at that sequence through computer searching and found it to be highly related to the sequence we had been playing with in yeast," Weinberg recalls. "By plugging that sequence into the database, a human expressed sequence tag [EST] came out." The team then studied whether up-regulation of the human homologue, which they named hEST2, could explain the enzyme's abundance in human cancer cells. They found strong evidence supporting this notion. When human cancer cells underwent immortalization, the messenger RNA (mRNA) for the catalytic subunit gene suddenly appeared. When human cancer cells were induced to differentiate, that mRNA disappeared just as quickly. "Simply up-regulating the catalytic subunit gene, which was later renamed hTERT, was sufficient on its own to impart telomerase activity to telomerase-negative cells," Weinberg notes.
Those observations illustrated the power of the subunit. They also underscored the importance of telomerase to cancer. "We believe that telomere maintenance is important for all malignant cells," Weinberg states. "Derepression of that enzyme is a genetic trait that is selected for strongly during tumor progression, because it's essential to enable the tumor cells to proliferate without limit." Understanding its activity further could have important clinical implications, he adds. "In the absence of that acquired immortalized state, we believe--although it's difficult to prove directly--the ability of tumor cells to expand becomes aborted."
Illustrating the immortalization process drew citation attention, Weinberg suspects. "Immortalization is a very specific cancer-cell-associated phenotype," Weinberg notes. "It's never been understood mechanistically at the molecular level." The fact that one enzyme subunit can play such a large role in tumorigenesis is striking, he notes. "A normal cell, could, in principle, have telomerase activity if only it had the catalytic subunit expressed." Experimentally expressing the catalytic subunit gene in normal cells proved quite dramatic. "All of a sudden they go from a telomerase-negative to a telomerase-positive state in one fell swoop."
Subsequently, his team has conducted two sets of experiments that further validate the subunit's importance. In one, they created a dominant-negative telomerase enzyme, which they inserted into tumor cells, causing them to "crash."2 "That validates the telomerase enzyme as an attractive target for antitumor therapy," he explains. "If you had a small-molecular-weight inhibitor of the telomerase enzyme, you might really chance upon a very attractive way of killing certain classes of tumor cells." More recently, Weinberg's team inserted the telomerase gene, along with Ras and other human oncogenes, into normal human fibroblasts and kidney cells and made them tumorigenic.3 "This is actually the first time someone has made a human cancer cell with a defined genetic makeup," Weinberg states. "Until now experimentally, all of the human tumor cells we've had to work with have been from patients, and those cells have an unknown and currently unknowable number of genetic mutations."
The next frontier in telomerase research includes learning what regulates the catalytic subunit's expression, finding potential small-molecular-weight inhibitors, identifying other important subunits, and searching for other proteins that control telomere length. "There are other telomere-associated proteins [that] collaborate to affect telomere length in ways that are very complex and that we don't understand," Weinberg concludes.