Telomeres and Telomerase: A Modern Fountain of Youth?

Interestingness: 3

By João Pedro de Magalhães and Olivier Toussaint, in Rejuvenation Research, July 2004, 7(2): 126-133. doi:10.1089/1549168041553044.

Not as much new material in this short paper as I expected, probably because I've read quite a bit on the topic since this was published. Interesting bits picked out:

• No correlation between maximum number of cell replications (cumulative population doublings) and age (post birth). (I have the vague recollection of some paper saying the opposite)
• No connection between mean telomere length and mammalian aging. (I think they mean across species, the species with the longer telomeres don't live any longer).
• Telomere length in vivo has very high variability

In summary, they think that telomerase might be beneficial for specific diseases but they doubt it'll turn out to be an anti-aging agent.

Telomerase, Telomerase Inhibition, and Cancer

Interestingness: 3

By Ali Ahmed and Trygve O. Tollefsbol, in the Journal of Anti-Aging Medicine, December 2003, 6(4): 315-325. doi:10.1089/109454503323028911.

It didn't turn up to be as interesting as I first thought, but definitely new information regarding telomerase, mostly of the type that I'll forget by tomorrow (ie these genes upregulate this, these downregulate it). In factoid form: telomerase is present in normal human liver cells in an inactive form, c-Myc upregulates telomerase, Mad1 suppresses it.

Telomerase is probably a good thing to test for when looking for cancer since it's very commonly present, ranging from 50-90% of the tests, with the lower numbers mostly seeming from fluids from tests. It is quite rare for it to be expressed in non-cancer cells, outside of the immune system, and even when it is, the numbers are much higher in cancer cells. 

Some numbers from the paper: 90% of bladder cancers, 80% of prostate cancers, 69% of renal cancers, 82% of thyroid cancers, 95% of breast cancers.  Some studies seem to show a correlation between cancer stage and quantity of telomerase. They also mention correlation between telomerase levels in the tumour and mortality and/or recurrence.

It then talks about methods of downregulating telomerase: transfecting with a dominant negative hTERT gene, antisense on the RNA component of telomerase, and immune hammering of telomerase-positive cells. I didn't know dominant negative genes would be easy to make.  They express the usual concerns about what turning off telomerase would do to stem cells and germ cells, but say that both those types are likely to have much longer telomeres than cancer cells.

Adrenocortical Cells Immortalized by Telomerase: Potential Use for Ex Vivo Gene Therapy

Interestingness:2

By Peter J Hornsby, Khan Ozol, and Keyi Yang, in the Journal of Anti-Aging Medicine, Volume 3, Issue 4 2000 (pp 411-417, doi:10.1089/rej.1.2000.3.411.)

Report on injecting bovine adrenocortical cells into rat brains, after immortalisation via telomerisation and addition of SV40 T antigen, which suppresses p53 and retinoblastoma activation. In one experiment, they also added nerve growth factor (NGF) gene to see if it'd produce it.

Results: lots of immune reaction to the foreign tissue, even when using cyclosporin A, an immunosuppressant, so most cells killed quite quickly.

Also, brief review stating that telomerised cells don't induce tumours in nude mice, and look as if they maintain their cell type.

Telomere Length Dynamics in Normal and Malignant Hematopoiesis

Interestingness: 2

By Tim H Brümmendorf, Peter M Lansdorp and Nathalie Rufer, in the Journal of Anti-Aging Medicine, Volume 3, Issue 4, 2000 (pp 397-409, doi:10.1089/rej.1.2000.3.397.)

This seems to be a double-length episode of the previous paper, relating how telomere lengths change in blood cells across time and diseases. One thing it clarified was that they thought the shortening of telomeres in bone marrow donor recipients was due to the repopulation of the bone marrow stem cells from the few donor cells.  Also, they kept on a theme that the telomere lengths of most blood cells are merely reflecting the lengths in the haematopoietic stem cells (HSC aka bone marrow stem cells) which produced them.

Telomerase Activity and Telomere Length in the Haemopoietic System: Changes with Aging, Disease, and Therapy

Interestingness: 3

By JD Robertson and RF Wynn, in the Journal of Anti-Aging Medicine, Volume 3, Issue 4, 2000 (pp 389-395, doi:10.1089/rej.1.2000.3.389)

Review of what was known about telomere length and telomerase in blood cells. Haemopoietic stem cells (HSC) have active telomerase but their telomeres shorten gradually. Same for T-cells. Telomeres in neutrophils also shorten at about the same rate as in T-cells (20-50 base pairs per year), but from what I understood, they don't have active telomerase, so the telomerase is acting as a compensation method for the occasional clonal expansion of T-cells.

Checking for which X chromosome is inactivated, in old women most blood cells have the same one, as if they come from fewer and fewer stem cells.

In people with acute leukemia, telomeres are short and telomerase long, and they suggest this suggests that telomerase activates late in the process of disease. Also short telomeres on aplastic anemia and Fanconi's anemia.  Also shorter telomeres in bone marrow transplant recipients than in donors (about 15 years worth), but I'm not clear if they are saying this is because the transplanted tissue has had to undergo quick replication to refill the recipient's system, or that this was there before.

Cellular Senescence Mechanisms Independent of Telomere Shortening and Telomerase: Other Barriers to Cell Immortalization and Carcinogenesis

Interestingness: 4

By Izumi Horikawa, Toshio Yawata, and J Carl Barrett in the Journal of Anti-Aging Medicine, Volume 3, Issue 4, 2000 (pp 373-382, doi:10.1089/rej.1.2000.3.373.)

 Not all cells given telomerase escape senescence and cells with active telomerase can be made to senesce in lots of ways. The p16^INK4A/RB pathway can trigger senescence as well as the p14^ARF/MDM2/p53 pathway. p53 is probably related to telomeres but other parts probably aren't. By introducing single chromosomes into immortal cancer cell-lines and making them senesce, they infer the existence of other independent pathways of senescence. The mechanisms that trigger senescence are cell-type dependent.

Interesting factoid: mouse cells senesce after much fewer replications (10-20 vs 50-80) even though much longer telomeres.

Telomeres, Telomerase, and Premature Aging

Interestingness: 4

By Corrin V Wallis and Richard GA Faragher in the Journal of Anti-Aging Medicine, Volume 3, Issue 4, 2000.

Nice summary of telomeres, telomerase and also about the relations of telomeres, Werner's syndrome, Hutchinson-Gilford progeria syndrome and aging. Quite a few details of the proteins involved that I wasn't aware of and that I'll forget about in the next hour.

Prevalence of Telomerase in Coronary Artery Atherosclerosis

Summary: Telomerase detected in atherosclerotic plaque tissue, likely to be related to restenosis.

Interestingness: 3

Paper by Madhu Gupta, Marie R Shogreen, Gregory A Braden, Wain L White and David C Sane in the Journal of Anti-Aging Medicine, Volume 3, Issue 1, Spring 2000.


(((
They measured the presence of telomerase in the bits cut out of 23 people that had directional coronary atherectomy (DCA). The bits cut out are blockages of the coronary artery and the DCA cuts it out. They correlated the tissues in which they detected telomerase with those that had restenosis, which is when the blockage/narrowing of the artery reappears.

They detected telomerase in 8 out of the 23 total, in 5 out of the 7 people who later developed restenosis, and on 2 out of the 10 who didn't (p < 0.05). Results were inconclusive for restenosis in the other 6. There was no correlation between what the people had come in for and the presence of telomerase.

They mention that atherosclerotic plaques have a monoclonal population of smooth muscle cells, but I don't know what other type you could have inside one person. They offer three explanations for the 35% detection rate of telomerase, that is, how come it's not 100%:

• that the tissue is maintaining its telomeres by means other than telomeres
• that the tissue is senescent or closer to senescence, with some evidence coming from studies on replicative capacity of muscle cells from plaque-derived tissue compared to healthy arteries. The presence of telomerase would probably be induced by cells having replicated beyond the normal senescent stage by a viral infection or broken tumor-suppressors, and that this would activate telomerase. These cells would then be better able to cause restenosis. I have no idea how much reality there is to that idea of telomerase reactivation.
• that there was no telomerase in the smooth-muscle cells at all, and instead the telomerase was detected from other cells in the tissue cut out. This could be from vascular stem cells, or from non-related cells like endothelial cells, lymphocytes or macrophages.

They also mention that the telomerase could be driving the hyperplasia not by replication but by stopping apoptosis.

)))


Abstract follows:

Telomerase is an essential enzyme for maintaining the telomeres of chromosomes and thereby enhancing the sustained replication of cells. Because atherosclerosis and restenosis are characterized by cellular proliferation, we determined whether telomerase enzyme activity was present in coronary artery tissue from 23 patients undergoing directional coronary atherectomy. Telomerase activity was determined from detergent lysates of the atherectomy tissue using an enzyme-linked immunoadsorbent assay (ELISA)-based modification of the Telomere Repeat Amplification Protocol. The presence of telomerase activity was correlated with the occurrence of coronary artery restenosis. Eight of the 23 samples (35%) were positive for telomerase. Seventeen of the 23 patients had adequate clinical follow-up to judge restenosis status. Of these, 7 had restenosis and 5 of these 7 had detectable telomerase. Of the 10 patients without restenosis, 8 were telomerase negative (p <= 0.05). We have shown, for the first time, that telomerase is found in 35% of atherosclerotic tissues. There was a strong trend toward an association between telomerase presence and restenosis in patients for whom follow-up data were available. The presence of telomerase in atherosclerotic tissue may enable a robust, sustained cellular proliferation in response to vascular injury that culminates in restenosis.

Importance of T-Cell Replicative Senescence for the Adoptive Immunotherapy of Cancer in Humans?

Summary: Review of replication of T-cells in vitro

Interestingness: 3

Paper by Graham Pawelec in the Journal of Anti-Aging Medicine, Volume 2, Issue 2, Summer 1999.

(((
This is another paper on T-cell senescence (previous one here: http://readingrejuvenationresearch.blogspot.com/2010/06/immunosenescence-analysis-and-genetic.html). It focuses on in-vitro studies, saying they are clinically important since that is how immune therapies will likely work best (eg training T-cells on tumor cells outside the body and then reinserting them) to work around the low immune responses of old people. I think their main area of investigation is trying to optimise the conditions under which T-cells replicate the longest.

It says the average number of population doublings (PD) of a T-cell in vitro before it becomes senescent, when externally stimulated, is 17, but 33 for cells that manage to get "established" (ie they get to a million cells). Seems like a very arbitrary cutoff but it better matches the numbers in the previous paper (25-40). The longest living ones reach 80 PDs on average and their record is around 170. They don't know why the large variability exists. The age of the person they were taking from doesn't seem to be one of the important variables. Longevity of CD34+ stem cells differentiated in vitro is no different to that of mature CD3+ cells.

They then switch to the link between telomeres and senescence. Fibroblast telomere length is directly proportional to replicative capacity. They say that this might apply to lymphocytes since the telomere lengths of human blood cells ex vivo are related to donor age, and the rate of telomere shortening with each doubling is about the same as for fibroblasts (120 bp per cell doubling). To me this would contradict what they said before that the replicative longevity was not related to the age of the donor, unless they mean blood cells other than T-cells.

In experiments by other people (Weng, Levine, June, et al) they found that CD4+ memory cells have shorter telomeres than naive cells, and that the difference is independent of the age of the donor. Telomere length decreases during autocrine replication of both of these and naive cells have higher replicative longevity than memory cells. The authors of this paper say this might not give the same results if externally stimulated replication was being used, since this can go on for way longer than the capacity for the cells to secrete interleukin-2, which triggers replication under autocrine replication, and that it doesn't necessarily follow that telomere length is the determining cause of senescence. Telomerase activity is upregulated in T cells when stimulated with CD3 and CD28 simultaneously but this might not happen optimally under various experimental setups, and might not happen optimally in-vivo due to decreased expression of CD28 with age. This, they say, might be the driving mechanism to senescence.

From small experiments they ran on oldish (<35 PD) and older (>43 PD) CD4+ cells, they noticed an upregulation of three mitotic inhibitors (p16-INK4alpha, p21-WAF, and p27-kip1) which suggest that upregulation of mitotic inhibitors might be an alternative hypothesis as the cause of senescence.
)))


Abstract follows:

Replicative senescence may compromise T cell-dependent immune responses to intermittent or chronic antigenic stimulation. While the impact of senescence in vivo remains hard to ascertain, clonal cultures of T cells in vitro provide models for longitudinal studies of aging in well-defined populations. Functional and phenotypic studies as well as investigations into average and maximal longevity of T cells can be performed conveniently with these cloned cells (the former in fact only with cloned cells). Many of the age-associated alterations observed during culture in vitro have also been noted ex vivo in T cells from the elderly.

Moreover, under circumstances where large numbers of antigen- and function-specific T cells may be required, for example for adoptive immunotherapy, the in vitro longevity of the cells may be critically important to successful outcome. These considerations are discussed in the following commentary in the context of immunotherapy of cancer.

Immunosenescence: Analysis and Genetic Modulation of Replicative Senescence in T Cells

Summary: T-cell senescence might be an important part of aging.

Interestingness: 4

Paper by Rita B Effros and Hector F Valenzuela in the Journal of Anti-Aging Medicine, Volume 1, Issue 4, Winter 1998.

(((These people want to try out upregulating telomerase in old T-cells)))

A major part of the function of T- and B-lymphocytes is based on replication. When differentiating from hematopoietic stem cells, the molecules or particles which the future mature lymphocytes are sensitive to (aka antigens) are encoded by a few sequences of DNA which are combined in random fashion. This makes the system potentially sensitive to hundreds of millions of different antigens. When an antigen is bound by a lymphocyte, the lymphocyte starts replicating, making identical clones (ie their receptor which sensed the antigen is not modified). When the antigen is no longer found in the environment, most of the lymphocytes disappear, but a few memory lymphocytes with the particular receptor remain so that the system can be revved up faster the next time that specific antigen is in the system.

Senescent T-cells can be generated in vitro by repeatedly exposing them to interleukin-2 (IL-2), a T-cell specific growth factor. After about 25-40 replications, they become senescent (ie they stop replicating). Fibroblasts (connective tissue cells) also become senescent after about 21 replications.

The receptor CD28 is not expressed in 95% of CD8+ senescent T-cells, and in all CD4+ senescent T-cells. Without CD28 costimulation, antigen binding doesn't lead to cell replication. CD28 signal transduction upregulates IL-2. It is also hypothesised to upregulate telomerase activity. Telomerase is very active in lymphocytes under certain conditions: in developing T-cells in the thymus and in lymphoid organs, when stimulated with mitogens (particles that upregulate replication), or by combination of antibodies to CD3 and CD28. When CD28 binding is inhibited, telomerase remains inactive even if there is strong stimulation of its T-cell antigen receptor (TCR). Even though telomerase is sometimes active in T-cells, senescent T-cells have short telomeres typical of other senescent cells. When split into CD28+ and CD28- T-cells, CD28- cells have shorter telomeres and lower replicative capacity when stimulated.

In vivo, CD28- T-cells are 1% fraction of neonates' total T-cells, 30% of (average) 78 year olds, 40% of people over 100, and 50% of HIV patients. Telomere lengths also shorten in peripheral blood lymphocytes as age increases. This loss of CD28 and shortening of telomeres is more pronounced in CD8+ cells, which specialise in anti-viral and anti-tumor activity, than in CD4+ cells (((doesn't this contradict the earlier numbers of 95% in CD8 and all in CD4?))). This could be due to infections by viruses that do not disappear (eg Epstein-Barr, varicella) or by repeated infections (eg influenza).

As people age, memory T-cells become a larger fraction of all T-cells. Senescence is also more common among memory cells. Non-senescent T-cells in old people respond to activation as strongly as those in young people.

During normal immune system activity, once the antigen dissapears from the system, most T-cells die by apoptosis. Senescent cells respond to apoptotic signals much less strongly, especially among CD8+ cells. These leftovers memory T-cells could be crowding out the production of new more useful T-cells. In calorie restricted mice, apoptotic response is maintained at youthful levels.



Abstract follows:

Immunosenescence, which constitutes one of the most dramatic physiologic changes associated with aging, may account for the increased susceptibility to infections and the high incidence of cancer in the elderly. A novel facet of T-cell biology has been recently identified that may exert a considerable impact on immune control over infections and cancer during aging. Cell culture studies have shown that after repeated rounds of antigen-driven proliferation, T lymphocytes eventually reach replicative senescence, an irreversible nonproliferative state associated with the loss of expression of a critical T-cell signaling molecule. Identification of this unique, cell-specific marker of senescence has facilitated the documentation and analysis of replicative senescence within the immune system in vivo during aging. This article summarizes the features of T-cell replicative senescence and highlights several genetic strategies that may reverse the process. The ability to manipulate T-cell replicative senescence may ultimately provide a fresh therapeutic approach to extend the years of immunologie "coverage" in the elderly.

Extension of Life Span in Normal Human Cells by Telomerase Activation: A Revolution in Cultural Senescence

Summary: Expressing the protein related to telomerase extends the telomeres and replicative lifespan of human cells.

Interestingness: 2 (but much higher back then)

Paper by Homayoun Vaziri in the Journal of Anti-Aging Medicine, Volume 1, Issue 2, Spring 1998.

(((This paper also feels old nowadays. Telomerase hype killed the excitement)))

(((I won't skip the introduction this time))) The bits at the end of the chromosomes are called telomeres. With each division of the cell, these are shortened. When they are too short, the theory goes, the cell decides to stop dividing, so that the real DNA doesn't get damaged. As evidence, tumours and other immortal cell lines maintain long telomeres somehow, and the correlation between telomere length and replicative lifespan of human fibroblasts is high. If we could activate the system that lengthens telomeres (called telomerase) the hypothesis could be tested in a causative manner.

So that's what they tried and that's what they got: activating hTERT (human telomerase reverse transcriptase) in human cells lengthens telomeres and enhances the replicative lifespan of the cell. (((That's really it for the paper. The rest is fluff because otherwise this would be too short))) It later cautions though, that this can't rule out that hTERT might be extending the replicative lifespan of the cell by a different mechanism than extension of the telomeres (ie that even though it extends telomeres, this might not be what is extending the lifespan, but some other unrelated function of hTERT is).

The rest of the paper is about how this can be used.

For research, once a cell with properties that are wanted are found or created, they can be made to express hTERT leading to an (((infinitely??))) replicative cell line. Same thing for gene therapy, introduce a cell that expresses the protein you want, add hTERT, and it will last longer. As an example of gene therapy, it gives Duchenne muscular dystrophy, even though it gives good reasons why this most likely wouldn't help (((maybe they were running trials for it at the time))). That it might help with HIV, on the hunch that the short telomeres on CD8+CD28- T-cells indicate immunosenescence, even though it might raise the likelihood of leukemia and lymphoma. Similarly for cancer, that it could prevent a hypothetical immunosenescence after chemotherapy, by extracting CD34- cells and introducing hTERT into them and reinserting the cells into the body.

It finishes by suggesting that it is p53 that acts as the detector of short telomeres that triggers senescence in the cell. (((There's a diagram of proposed gene activation in the paper that would be laborious to describe)))

Abstract follows:

Normal human cells have a limited life span in culture, exhaust their replicative potential after a fixed number of doublings, and enter a phase of cell cycle arrest termed "senescence." Senescent cells are metabolically active cells, known to up-regulate several cyclin-dependent kinase inhibitors and to be arrested primarily at the G1 phase of cell cycle. Telomere loss due to incomplete replication of the ends in normal somatic cells is thought to be the signal which initiates the senescence cascade. Lack of telomere maintenance in somatic cells may be caused by the absence or the low enzymatic activity of telomerase, the enzyme responsible for synthesis of telomeric DNA that counteracts the end-replication problem. Previous attempts to increase the life span of human cells involved inactivation of tumor suppressor genes such as p53 were not a viable method of life span extension because of significant risk of genomic instability. Extension of the life span of normal cells with minimal risk of genetic instability may be achieved by manipulation of the most upstream signals that initiate the senescence cascade. We and others have recently shown that reactivation of telomerase in normal human cells leads to restoration of the length of telomeric DNA and to a highly significant increase in cellular life span. These data provide strong evidence consistent with the telomere hypothesis and indicate that elongation of telomere length by genetic manipulation might render normal human cells virtually immortal. These findings indicate that telomere shortening and senescence act as a tumor suppressor mechanism and establish a solid genetic link between telomeres, cellular aging, and immortalization.