Before We Can Add Years to Life, We Must Add Life to Years

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The correlation between telomere length and age is very strong.  Shorter telomeres directly correspond to shorter human life expectancy, but “cause and effect” are still debated. Finding ways to prevent telomere shortening could be an ideal way to address these issues and answer the question once and for all: Does prevention of telomere shortening extend our lifespans? Dr. Laura Briggs of Sierra Sciences presented on the subject of the organization’s research at the Aging ’08 conference in Los Angeles, California in a presentation titled “Before We Can Add Years to Life, We Must Add Life to Years.”

The following transcript of Laura Briggs’ presentation at the Methuselah Foundation conference entitled ”Understanding Aging: Biomedical and Bioengineering Approaches” has not been approved by the speaker.  Video is also available.

 Before We Can Add Years to Life, We Must Add Life to Years

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First, I would like to thank Dr. Aubrey de Grey and his staff for inviting us to speak.  It is truly an honor for myself, as well as Sierra Sciences, to share some of the work we’ve done. I have to thank Jerry Shay for providing most of the introduction, so I will be able to breeze through some of these slides and maybe spend more time on some of our data.

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This is a picture of Woody Brown. He is ninety-five years old and still surfing in Hawaii. Wouldn’t we all like to age as well as he has? When we talk about life extension, of course it is not just about living longer but having increased healthspan.  We have to consider then that almost anything we are going to do to extend the lifespan of our normal cells is potentially also going to expand the lifespan of our cancer cells.  That is something that we are absolutely going to have to deal with.  Based on that, I want to take a short tangent to talk about cancer just a little bit and how it relates to aging and telomeres.

Anything that decreases cell proliferation potentially then is a tumor suppressor pathway.  Replicative senescence or telomere shortening is known to be like a cellular clog that stops cell proliferation once the telomeres get to a critically short length.  As I said previously, we are still going to have to deal with the idea that turning on telomerase in an effort to lengthen telomeres is then going to bring us back to the cancer issue.

The cells we are interested in, these cells really aren’t cancer yet, right?  They maybe are not pre-cancerous cells, but they are cells that lack growth control.  Somewhere along the line they have acquired a mutation that has allowed them to bypass cell cycle regulators.  If we think about a historical timeline of cancer, then eventually, as these cells continue to divide, their telomeres get shorter and shorter, and eventually they are going to become immortal, either through induction of telomerase activity or the ALT pathway.

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When we are talking about extending lifespan and finding ways to turn on telomerase, we are not really interested in these cells—it’s a little bit too late.  In these cells now, we are into curing cancer and fixing things that went wrong. It is actually these cells that we are most concerned with in our field of telomerase activation. We do not really have a good name for these cells, and we found this interesting.  We asked people, What do we really call these cells?  We have not really found a good answer, but they are critically important to anybody that is trying to extend lifespan.

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Another big question that needs to be answered is, How prevalent are these cells?  Is there a whole lot of them? We are going to have to develop means to not only identify these cells, but then deal with them as we are attempting to extend lifespan.  What are some of the solutions then for life extension?

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We heard a little bit last night about this portion of the SENS theory.  One thing we could do is delete hTERT, as well as the ALT pathway.  This would then stop the cancer cells.  What would happen in addition, though, as a result of deleting the hTERT gene, is our telomeres in our stem cells are going to get shorter and shorter.  We are going to have to deal with that issue.  One way would be to infuse the body with stem cells on a regular basis that had these pathways knocked out.  This is a very elegant way, and is just a portion of the SENS idea.

Aging is a hugely complicated process, and multiple facets are going to have to come together for us to actually cure aging.  As parallel efforts, we would suggest cure aging—not necessarily our company, but the community—without inhibiting telomerase.  Then, once we get to that point, we turn telomerase on.  We would suggest too, telomerase induction in a transient environment. It would not be turned on all the time, but a small molecule—one we could take that would turn it on for a few months, extend the telomeres, and then take it away—would be a much safer method than having telomerase turned on all the time in the cell.

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Telomerase might not be such a bad thing, because we do know there is quite a bit of evidence that telomerase will actually decrease the risk of cancer.  We will hear more about this area as the conference goes on.  One thing that we are pretty confident about is bad things happen when telomeres get short.  Understanding the cause and effect relationships there is something that we still need to work on.

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In cells, in cell culture anyway, preventing telomere shortening will extend the healthspan and lifespan of those cells.  Unfortunately, we do not know if that is going to apply to humans.  Can we extrapolate what we know about cell cultures to humans?  That is a question that has not been answered yet.  That is the fundamental question that Sierra Sciences is attempting to answer.  We would love to get to this point and answer this question.

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There are a number of diseases that could be helped by the prevention of telomere shortening.  With that I will start with what I came here to talk about, which are some of the tools that we are using in the struggle to keep our telomeres long.  Sierra Sciences has been in existence for about nine years.  We have worked really hard trying to figure out all we can about telomerase.  We have spent quite a few years doing telomere promoter analysis.  We spent a couple years trying to find the transcription factors that actually bind to the telomerase promoter in areas we believe is involved with regulation.  Through these efforts, on the side we were always taking anything we saw in the literature, any small molecules or processes, and asked ourselves if this was the answer to our question.

Unfortunately, we were not able to reproduce any of the work that is in any of the published documents.  We could make a lot of HDAC inhibitors work in a transient transfection assay, for instance using the telomerase minimal promoter driving expression of a reporter.  HDAC inhibitors work very well to induce activity of the telomerase promoter in that context.  In terms of the indogenous promoter, we have not been able to do it.

That really set us back.  If you think about a company that is working with normal cells, trying to turn something on that is turned off, and you set up the best controlled experiment you can imagine, what does a negative answer tell you?  Not necessarily a whole lot.  We have a lot of collaborators, and they would say, “How do you know that that normal cell is even telomerase competent?  Maybe telomerase has been turned off so long that you will never be able to turn it on.  Even if you turn on the message, how do you know that the cell is then capable of processing it correctly and ending up with an actual active protein?”  We are very diligent—we don’t take ‘no’ for an answer—and struggled with this for a long time.  We now have been doing a lot of screening. We now have a 300,000 compound library and are searching for compounds that will turn on telomerase.

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This is the result of one of our screens.  It may not look like much, but what we are seeing here is RT-PCR for hTERT, and you see here along the x-axis a series of numbers representing compounds from our compound library.  The blue bars are actually GAP DH, so they are basically our control for the system.  We have a MRC-5, which is a human fetal lung fibroblast cell line that is ectopically expressing telomerase.  You can see we get a whole bunch of message.  The y-axis is showing raw Ct values—and if you are not familiar with RT-PCR, basically Ct is critical threshold.  The more message you have in your sample, the more quickly you will pass a critical threshold.  In this case, we are looking for lower numbers.  The lower the number, the more message we have.

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What really got us excited was this little blip right here.  Actually, we have been teased so many times that we were only cautiously optimistic.  We repeated it a bunch of times, of course, and saw this same thing happen over and over again.  Here we have demonstrated a compound that is capable of inducing telomerase message.

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We have not shown that we have active protein yet.  In order to assay for active telomerase protein, we do a TRAP assay.  This is based on the idea that we can use an artificial telomere.  If telomerase is in the samples, then it will extend the telomeres.  The telomerase then adds telomere repeat regions six bases at a time successively onto the end of the telomere.

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If you run this product on an electrophoresis gel, then what you see is a latter that represents PCR fragments—telomerase created and then amplified by PCR—that are six base pair differences in length.  Here, in this particular gel, we are seeing HeLa, which is a cancer cell line.  We usually put 100% HeLa and 5% HeLa on our gels.  100% just means we had a hundred thousand HeLa cells.  5% means we had five HeLa cells and 95 normal cells that were lysed, so we use it as a quantitative effort.  Here we see MRC5 cells that are negative for telomerase.

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This is our 57,684th compound in our library.  On the 57,684th compound we tested, we finally saw a little blip.  We call this compound C0057684, but because that is a mouthful, I am going to say “57684.”  When we exposed normal cells to our compound 57684, and this is an experiment done in triplicate, this is the first time we have ever seen something actually turn on telomerase activity in a normal cell.  This was a huge breakthrough for our company.  It validated what we had been doing all along, all of our screens.

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What I want to do now is show you some of the work, and it’s fairly new, investigating this compound.  We have done some dose curves.  It is dose responsive. This is mRNA relative to HeLa.  Everyone seems to know HeLa and telomerase, so we had pretty much done that.  Across the x-axis you see increasing concentrations of 57684 and you can see a concentration-dependent increase in telomerase message.  When we then run trap assays—and I have truncated the work here, I have started with just the 15 micromolar—we start to see a hint, and then increasing amounts.

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We have looked at a time course to see how quickly does it turn on.   Here we have added a compound to normal cells and then assayed for activity various hours later.  You can start to see it at 24 hours, it peaks at 48, and by 96 hours we have pretty much lost the activity.  We have also done some extinction work, where we look to see how long telomerase hangs around after we have removed the compound.  Here you can see that 48 hours after we have removed the compound we are seeing a significant reduction in telomerase activity.

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We have tested this in other cell lines.  Maybe it was a fluke, right?  These are two other fetal lung fiberblasts, MRC9 as well as IMR90.  They both respond to the compound.  We have tried foreskin fiberblasts.  Again, they respond to the compound. We have looked at keratinocytes, and this is a phenomenon that we have seen in other cell lines.  These are adult keratinocytes and neonatal keratinocytes, and the younger cells seem to respond more strongly to the compound than older cells.  We have also looked at some confluent versus non-confluent cells and see a similar phenomenon.  It seems to suggest that for this compound to work, the cells need to be actively dividing.

Because we were unable to repeat other labs’ work on other occasions, we felt really strongly that we would in no way say we had a compound that worked until we had independent verification.  We were fortunate enough to have Jerry Shay and Woody Wright’s lab test it for us.  They have been able to repeat the results in different cell lines.  Judy Campisi has also repeated it for us, and Richard Allsopp at the University of Hawaii.  We are thankful for their participation in this effort, and now we feel pretty confident that we can say compound 57684 does induce hTERT expression.

Another compound we have had the privilege to work with, and actually it is a neutraceutical, is a compound called TA-65.  TA-65 is derived from the astragalus root.  It is a compound that was identified by Geron Corporation and is now being commercially distributed by TA Sciences.

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This is TA-65 in keratinocytes.  You can see, while not strong, telomerase activity in response to TA-65.  We now have two compounds that we can use that seem to act a little bit differently too.  TA-65 is not as concentration responsive as 57684 and seems to express telomerase at lower levels.  We have asked ourselves about immortal cells, and old cell lines respond to 57684 with increased message.  We are still figuring out exactly how to do TRAP on these cells.   TA-65 does not seem to affect cancer cells.  That probably is good news.

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We have done TRAP assays on HeLa cells.  You can see 50% and 70% relateive to the 57684.  It is not only increasing message but it is increasing active telomerase compound.  We have looked at a few other positive telomerase cell lines.  In most cases we see increases in expression.  Maybe the downside of 57684 is it is mildly cytotoxic.   48 hours after treatment with DMSO, we see confluent image.  We see here that with 57684, we really have not had a lot of cell growth.  We do not see dead cells—we just see reduced number of cells.  This is concentration dependent.

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We wondered if these cells were actually alive or not.  We did some dead cell assays.  In this particular image we see light microscopy.  You can see here, we see all live cells. It looks like we are not killing the cells.  One thing we also know is that when we take the compound away, the cells recover and start to grow again.  TA-65 on the other hand is not toxic at all to the cell at these concentrations.

When we looked at some other toxicity indicators, in this case this is measuring ATP levels, these are fiberblast cell lines, and we see a reduction in ATP levels with an increase in concentration.  I don’t know if this is representing any toxic effects on metabolism, because we have less cells at these higher concentrations as well.  I do not think it is reflecting effects in metabolism—I think it is reflecting effects in cell number.  Interestingly enough, when we look at cancer cells, we see a much stronger response.

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Of course, as soon as we showed this data to Dr. Shay, he said it must be a DNA damage response.  Indeed, it may well be.  We do not really have a clear answer to this.  We have work in progress with Dr. Shay’s lab on Gamma H2AX, although it is not very large scale and the results are inconclusive.

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We had never seen in the literature any proof that DNA damaging agents actually turn on telomerase.  There is what we would call circumstantial evidence, but nowhere did we find TRAP assays where we saw increases in telomerase activity.  We stopped and asked ourselves whether a DNA damaging agent will actually turn on telomerase.  We used an etoposide here, a widely recognized DNA damaging agent, and asked ourselves if it turns on telomerase.    Here you can see hTERT message relative to HeLa.  We compared 57684 to etoposide at two concentrations.  30 micromolar seems to be the concentration most widely used in the literature for studying etoposide as a DNA damaging agent.

You can see that we do see some increase in message with the etoposide.  At 30 micromolar, it does not seem to be as responsive, though 57684 does increase much more than the etoposide.  We have hypothesized that maybe the answer to this is that 30 micromolar etoposide is really toxic to the cells, and so most of these cells are dying.  We need to further investigate those results.

We get really excited when we see a compound so we can hit it with everything we’ve got.  We have done some DNA array analysis on these compounds, and maybe another unfortunate result for 57684 is that it is affecting a tremendous amount of genes, way too many for us to be thinking that this is the compound.  1700 of the genes have been upregulated twofold or more.  We are still in the process of analyzing these and have about three different companies looking at this for us.  It seems so far that the vast majority of these, the most significant genes affected are cell cycle control genes.  There are some DNA damage response genes that are upregulated, but they are at much lower levels.

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TA-65 is not showing these kinds of results.  It seems to be very specific and is only increasing a handful of genes.  We have done some extension of lifespan studies.  It is probably premature a little bit, especially given the fact that the compound affects cell proliferation rates.  We have done them anyway.

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You can see here that continuously exposed normal cells at all concentrations that we have tested are not allowed to grow.  Conversely, when we did a 96-hour treatment and then took it away, then followed these cells over time, we see a very modest increase in cell proliferation.  Really not much to write home about at this point.  We are continuing these studies and have a lot of different scenarios we are working on.

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What is next for us?  We are focusing on screening more than we are focusing on characterizing 57684.  We are going to look at other cell types.  We are interested in synergy—maybe TA-65 and 57684 mixture, or maybe HDAC inhibitors with 57684 to see if we can loosen up the chromatin a little bit.    We have been working on some mechanisms of action and structural activity relationships, an attempt to develop more active and less toxic compounds, and of course replicative lifespans.

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Here is RT-PCR of normal cells.  Here we have analogs of  57684.  We have about fifteen or sixteen in our compound library right now.  You can see that some of the analogs result in a response, while others do not.  We have an opportunity to look at structural activity relationships based on this, and when we do TRAP it follows the message really quickly.  We continued to screen, and I want to say that these tools are freely available.

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We are a small company and have an enormous amount of work to do, so we are really interested in collaborations.  We are looking for researchers not only as collaborators, but also for people coming to work for us.  If you have any interest in that, please talk to myself or some of the other Sierra Sciences people.  I wanted to acknowledge Sierra Science’s staff.  There is not any one person who does everything—it is just a huge collaborative effort.  Thank you.

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