By David N. Leff

God warned Moses on the mountain-top that He would "visit the iniquity of the fathers upon the children unto the third and fourth generation."

Molecular geneticist Carol Greider visited the absence of a telomerase gene in her knockout mice unto the sixth generation. Only then did she and her collaborators perceive the organ damage wrought on the rodents by the loss of telomerase function.

Their report appears in Nature, dated April 9, 1998, under the title: "Essential role of mouse telomerase in highly proliferative organs." Greider at the Cold Spring Harbor Laboratory, in N.Y., and molecular biologist Ronald DePinho at Albert Einstein College of Medicine, in The Bronx, N.Y., are the article's two senior authors.

Greider discovered the telomerase enzyme a decade ago, while at the University of California at Berkeley.

Telomerase is a DNA polymerase that officiates at the chromosomal ends of every cell of the body. It does so by adding on telomeres — layers of DNA repeat sequences that protect the tips of every chromosome from damage — especially from fusing with other chromosomes. Over the life of a cell, typically after 50 divisions, the absence of telomerase has shortened its chromosomes to the point where they shed the last of their telomeres and die.

But if that cell hangs on to its telomerase enzyme, there's nothing to prevent it from dividing forever. In other words, it becomes immortal, i.e., cancerous.

Late in 1995, Greider reported cloning the gene for mouse telomerase (See BioWorld Today, Sept. 1, 1995, p. 3. ) She and DePinho could then ask: What role does telomerase play in vivo — in a mouse?

"The best way to address that question as geneticists approach it," she told BioWorld Today, "is to get rid of the gene and find out what happens to the knockout mice. That gene encodes multiple holoenzyme components," she pointed out. "We chose to delete the RNA component on human chromosome 3, which is essential for enzyme activity."

It took them about a year to create their first, and still only, knockout mouse lacking telomerase activity, Greider recalled. "Then we had to breed their progeny for six generations to have the telomeres get short enough to see what would happen to the animals. It takes each mouse generation about three months to reach reproductive age."

It took such a biblical succession of progeny, she explained, because "there were rumors going around that the telomerase didn't matter at all, because you could knock out the enzyme and the mouse was fine. But that's only as far," she pointed out, "as when the telomeres are still long. When they get to be short, only in the sixth generation, then we found drastic phenotypic consequences."

Stem-to-Stern Murine Histology

To find these consequences, the paper's co-authors subjected their test knockout mice to whole-body analysis of every organ system.

"We looked at everything," Greider recounted, "with an eye specifically to cell types where there would be more cell division than in other types — for instance, in the blood and germ cells and gut and skin — because those are known to be more proliferative than others. We did a complete histology on all organ systems," she added, "to just look and see if there was anything that would surprise us.

"One of our conclusions," Greider observed, "is that telomerase per se is not needed for anything besides maintaining telomere length. That was not an obvious thing at the outset, because we saw absolutely no effect on the first several generations of animal.

"And the other finding," she continued, "is that telomere length, once it got to be short, in the later generations of mice, had multiple effects, and is clearly required for chromosome structure maintenance, especially in highly proliferative systems."

In the normal, healthy mammalian body, among the most highly proliferative organs are the testes and ovaries.

In the male reproductive system, spermatocytes divide 60 times or more between the initial zygote and mature sperm cell. The co-authors found that testicular weight in their sixth-generation male knockouts was 80 percent less than in either wild-type controls or third-generation rodents. Histologic analysis indicated increased apoptosis (programmed cell death) with each advancing generation.

Similarly, sixth-generation female mice had "minimal to significant reductions" in size and weight of their ovaries, compared to wild-type animals. However, ovaries at both extremes "showed a full spectrum of follicular development," the Nature article reported.

"One of the implications that people have been suggesting," Greider observed, "is designing a drug to treat cancer situations by inhibiting telomerase. Our study," she went on, "addressed that application in two ways:

"First, if you do have a drug that effectively inactivates telomerase, you would expect the tumor cells to shorten, and eventually die. The second point is that there are likely to be very few side effects, because we've shown that in mice, most of the organ systems operate perfectly well in the absence of telomerase.

"At least half a dozen major pharmaceutical companies," Greider observed, "are involved in this telomerase-inhibition activity. And at Geron Corp. [of Menlo Park, Calif.]," she added, "this is what the whole company does."

Tumors Play End-Run Around Telomerase Inhibition

Greider moved recently from Cold Spring Harbor to The Johns Hopkins School of Medicine, in Baltimore, Md. There she is pursuing "a number of different telomerase questions."

"First of all," she said, "not all cells die when their telomeres get to be very short. And this has implications: Some of the late-generation cells can form tumors when you put them into mice. And we know," she added, "from basic research in yeast, that cells can find alternate pathways to maintain telomeres. So, from the practical standpoint, if you're going to inhibit telomerase in cancer, we need to find out what those pathways are in mouse cells, as a way for cancer cells to get around an anti-telomerase kind of approach."

As for practical application of her latest published results, Greider concluded: "People in biotech would call it 'proof-of-principle.' We don't use that terminology in academia. We say that we have made 'a basic discovery.'" *