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UCSF s Elizabeth Blackburn on Telomerase and RNAi

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At A Glance

Name: Elizabeth Blackburn

Position: Professor, biochemistry/biophysics, microbiology/immunology, University of California, San Francisco

Background: PhD, molecular biology, University of Cambridge — 1975

MSc, biochemistry, University of Melbourne — 1972

BSc, biochemistry, University of Melbourne — 1970


After completing a postdoctoral stint at Yale University, Elizabeth Blackburn made her way west to hold a number of faculty positions within the University of California school system. During that time, she picked up a number of awards and honors, including the National Academy of Science Award in Molecular Biology, the California Scientist of the Year Award, the American Cancer Society Medal of Honor, the Memorial Sloan-Kettering Cancer Center Katharine Berkan Judd Award Lectureship, and the Harvey Prize.

Her research is currently focused on telomeres and their role in cancer, and she recently co-authored a paper in the Journal of Biological Chemistry detailing the use of RNAi to deplete telomerase RNA.

Blackburn spoke with RNAi News this week about her work.

 

Let's start with an overview of your lab and the focus of your research.

My lab focuses on telomeres and telomerase, and on how they both mechanistically work to protect genetic material in the cell and on the impact that this system … [has] on human cells and disease. We look at it in the context of both healthy cells and in cancer cells.

How did you get involved in that?

Long story. I was very interested in DNA sequences in the days before DNA sequencing was even worked out, and I got interested in looking at the telomeres, which are the ends of the chromosomal DNAs — the long DNAs that make up genetic material. Way back I sequenced the first telomeres and found they had intriguing structures and intriguing molecular properties that eventually prompted us to look for what was then an undiscovered enzyme, telomerase, which I and my graduate student Carol Greider discovered. I've just been fascinated by how this system works and signals cells ever since.

Could you give an overview of [telomerase's] properties and how it relates to cancer?

We discovered way back that telomerase is necessary for cells to keep on multiplying. Since cancer cells have, as one of their notable properties, the ability to keep on multiplying, people looked and quickly found that telomerase was prevalent in high activity in cancer cells. That made total sense given the fact the cancer cells have to keep on multiplying.

This new paper [appearing in the Journal of Biological Chemistry] gets to something quite unexpected because there was no particular reason to think that telomerase would be doing anything else. But what we discovered was, by using RNAi to knock down the telomerase very abruptly in cancer cells, which have become completely habituated to high levels of telomerase, that the cells basically went into shock; they stopped growing, they had a cell death response, and they changed their gene expression pattern in a way that told us that the high levels of telomerase were provoking aspects of cancer … that had to do with malignancy, especially … how well cells metastasize.

Also last year, in work we had going in parallel, we had shown that if you turn telomerase down in a mouse model system — now this wasn't RNAi, it was a different method, but we have repeated it with RNAi — you can get melanoma cells to metastasize less, just by the act of knocking down their telomerase. What this did was it said that telomerase seems to be having effects — in the JBC we particularly showed this — that don't seem to be on the telomere. I call them off-label effects. So, there're other functions being hinted at very strongly. … Telomerase [is] not simply maintaining the telomeres, but [has] other functions as read out by this cellular response to the abrupt knock down of telomerase.

Jumping back, you said that last year's work was done with a different technology?

That's right, it was ribozymes and it was in a mouse model for melanoma. But we subsequently repeated this, and it's not published yet, but the same results do hold with RNAi as an independent method to knock down the level of telomerase.

What led you to switch over to RNAi?

Well, RNAi is just such a fabulous technique. It suddenly lets you do what yeast geneticists could do for years, knock genes out at will. But that was technically so difficult with mammalian cells because of the genome size, and the necessity to target both chromosomal copies of any particular gene.

Were the hairpin siRNAs something you were able to put together on your own?

We did it all ourselves using two published [sources of information]. One is the lentivector system, which is the vector backbone that provides a way of getting a gene into a cell where it integrates. It is very efficient compared with other methods, so, often, you can directly get over 95 percent of cells receiving the gene of interest, and it gets expressed very well and very stably. Into that vector we put a short hairpin RNA somewhat like the kind that Greg Hannon pioneered.

There's a little variant in this story that was very interesting. The part of telomerase that we were targeting was not the protein portion but the essential RNA portion. Telomerase has two parts: the essential protein and the essential RNA. This RNA is not a messenger RNA.

Basically all the siRNA work has largely focused on messenger RNA. So we -meaning my postdoctoral fellow Shang Li — had to go through a lot of promoter and gene-expression cassette systems and work out one that would work for this non-messenger RNA. People hadn't even thought you could target non-mRNA by RNAi and we could, so that was very thrilling. We'd actually published that as more of a side note in a Cancer Research paper last year, but this JBC paper spells out more of the details.

RNAi can be used successful for non-mRNAs and that isn't something that people generally appreciate. So that's cool. Then we bought a couple more oligos of different kinds from the commercial vendors — short-interfering RNAs, the kind you chemically put on the cells.

Back to the focus of your research and telomerase, you found that …

Here was the old idea, which is true: If you just inhibited telomerase activity in a way that stopped the enzyme from doing its reaction, which is to replenish DNA as it wears down off the ends of telomeres, then the telomeres would just erode away. But it would happen over time, over perhaps 10 cell generations or more. So nobody expected that if you quickly knocked telomerase down and looked right away that there'd be a rapid cell response while the telomeres were still long. As far as we can tell, the telomeres were still intact and there was no DNA damage signal, which is one of the down-stream consequences if you muck up a telomere, or uncap it.

That was what was so exciting. It said, 'This is not telomerase acting on-label. This is off-label.' The big difference here was that we were knocking down the total telomerase complex level, and that's different from inhibiting the activity, because when people inhibit the activity — which a number of labs have done successfully — they typically don't deplete the cell of the enzyme. It's just not active, but it's there. So we're thinking there're other activities of some kind, and we do not understand what these are, but you can imagine we're working very hard to figure this out. But we don't understand this activity that doesn't depend on the enzyme making telomeres longer. All we got was the cell telling us that there was something there.

Where does that bring you now?

[Our findings] make telomerase an interesting component of cancer cells because it's one of the most frequent things you find in human cancer cells. It's just so common in most human cancers, and we knew already that telomerase is needed for cancer cells to keep proliferating, but now if that strong up-regulation makes them susceptible to a quick knockdown, as we found, then that's another way of saying, 'Perhaps we could target cancer cells and telomerase in this way.'

There are lots of targets [for cancer therapy], but this one has a particularly attractive feel to it because it's a novel cell response — the cells respond in a way that we hadn't seen before; it's not a DNA damage response.

There are companies looking at telomerase, like Geron …

They are focused on the inhibitor idea, the straightforward idea. There's nothing wrong with that, but it doesn't have this advantage of the quick effect. In the clinic, you don't know what's going to work, of course. But we do know that, at least in the mouse melanoma model we used, the telomerase knockdown agent was delivered systemically and there was less metastasis.

Are you optimistic, then, at the possibility of RNAi being an effective therapeutic modality for cancer?

The answer is that it should be really tried very hard, then the human body will tell us if it will work or not. We get surprises. COX-2 inhibitors: a fabulous class of drugs, but the complex human body says there's a downside. So you do your best, you try and find out everything you can, and then you hope you can make predictions about how something will play out clinically — but we all know that's tricky.

All you can do is that, so I'm always optimistic, but not unrealistic.

Is that something you would get involved in?

Oh yes. We collaborate with our cancer center colleagues a great deal, and are certainly interested in working with the [National Cancer Institute] to see where this will go.

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