This blog entry reviews some recent research topics related to the molecular biology of telomere length homeostasis and to impacts of telomere lengths and cell maturation on health on aging.
This is the third in a 3-part mini-series of blog posts concerned with telomere length topics. Part1 was concerned with telomere lengths, cancers and disease processes. There I focused on a couple of specific questions: Are shorter telomere lengths predictive of cancers and other disease processes? And, are disease processes or unhealthful body conditions characterized by shorter telomere lengths? Part2 was concerned with lifestyle, dietary, and other factors associated with telomere shortening and lengthening. Both of those blog entries reported on population studies and neither were concerned with the molecular biology of telomere formation and telomere impacts on cells as this blog entry is.
It has long been known that uncapped or too-short telomeres can trigger cell senescence or apoptosis and that, with aging, telomeres tend progressively to become shorter. And some researchers have thought that short telomeres and resulting tissue dysfunctionality might be a main cause of aging. Again, if you are new to the subject of telomeres and telomerase, I suggest you start with reviewing the discussion of the 12th theory of aging covered in my treatise, Telomere Shortening and Damage. Also, you may wish to review some of the previous telomere and telomerase-related blog entries listed in the previous blog entry.
In recent years, there has been an increasing appreciation of the detailed nature of biological changes happening in the process of cell maturation and the negative consequences of too-short telomeres and cell replicative senescence. Further, there has been discovery that, even short of cell senescence, cell maturation is accompanied by drastic changes in cell gene expression and even restructuring of cell chromatin. I review selected publications showing important results from 2008 to the latest publication which appeared a few weeks ago.
Better biomarkers are needed for stem and progenitor cells with DNA or telomere damage
The 2008 publication Determining the influence of telomere dysfunction and DNA damage on stem and progenitor cell aging: what markers can we use? relates “The decline in organ maintenance and function is one of the major problems limiting quality of life during aging. The accumulation of telomere dysfunction and DNA damage appears to be one of the underlying causes. Uncapping of chromosome ends in response to critical telomere shortening limits the proliferative capacity of human cells by activation of DNA damage checkpoints inducing senescence or apoptosis. Telomere shortening occurs in the vast majority of human tissues during aging and in chronic diseases that increase the rate of cell turnover. There is emerging evidence that telomere shortening can limit the maintenance and function of adult stem cells — a cell type of utmost importance for organ maintenance and regeneration. In mouse models, telomere dysfunction leads to a depletion of adult stem cell compartments suggesting that stem cells are very sensitive to DNA damage. Both the rarity of stem and progenitor cells in adult organs and their removal in response to damage make it difficult to assess the impact of telomere dysfunction and DNA damage on stem and progenitor cell aging. Such approaches require the development of sensitive biomarkers recognizing low levels of telomere dysfunction and DNA damage in stem and progenitor cells. “
Circulating proteins are associated with short telomeres and DNA damage
The 2008 publication Proteins induced by telomere dysfunction and DNA damage represent biomarkers of human aging and disease reports the existence of circulating proteins that are created by dysfunctional telomeres and that can serve as biomarkers. “Telomere dysfunction limits the proliferative capacity of human cells by activation of DNA damage responses, inducing senescence or apoptosis. In humans, telomere shortening occurs in the vast majority of tissues during aging, and telomere shortening is accelerated in chronic diseases that increase the rate of cell turnover. Yet, the functional role of telomere dysfunction and DNA damage in human aging and diseases remains under debate. Here, we identified marker proteins (i.e., CRAMP, stathmin, EF-1alpha, and chitinase) that are secreted from telomere-dysfunctional bone-marrow cells of late generation telomerase knockout mice (G4mTerc(-/-)). The expression levels of these proteins increase in blood and in various tissues of aging G4mTerc(-/-) mice but not in aging mice with long telomere reserves. Orthologs of these proteins are up-regulated in late-passage presenescent human fibroblasts and in early passage human cells in response to gamma-irradiation. The study shows that the expression level of these marker proteins increases in the blood plasma of aging humans and shows a further increase in geriatric patients with aging-associated diseases. Moreover, there was a significant increase in the expression of the biomarkers in the blood plasma of patients with chronic diseases that are associated with increased rates of cell turnover and telomere shortening, such as cirrhosis and myelodysplastic syndromes (MDS). Analysis of blinded test samples validated the effectiveness of the biomarkers to discriminate between young and old, and between disease groups (MDS, cirrhosis) and healthy controls. These results support the concept that telomere dysfunction and DNA damage are interconnected pathways that are activated during human aging and disease.”
Stem and progenitor cells are subject to replicative senescence and express very different genes when young and old, but not due to telomere erosion
The 2009 publication Aging and Replicative Senescence Have Related Effects on Human Stem and Progenitor Cells is important in that a) it established that at least some stem cells are subject to replicative senescence, b) gene expression patterns of young and old stem cells vary drastically with age, and that c) telomere erosion does not appear to be responsible for the differences in gene expression of old and younger stem cells. The research looked at the gene-expression effects of replicative senescence on mesenchymal stromal cells (MSC) and human hematopoietic progenitor cells (HPC) and compared these to the gene-expression effects found in in-vivo aging. It found the effects to be similar, suggesting that stem and progenitor cells are subject to replicative senescence, just as other types of body cells are. Further, at least in HPCs, telomere erosion does not appear to be well-correlated with aging.
The publication reports “The regenerative potential diminishes with age and this has been ascribed to functional impairments of adult stem cells. Cells in culture undergo senescence after a certain number of cell divisions whereby the cells enlarge and finally stop proliferation. This observation of replicative senescence has been extrapolated to somatic stem cells in vivo and might reflect the aging process of the whole organism. In this study we have analyzed the effect of aging on gene expression profiles of human mesenchymal stromal cells (MSC) and human hematopoietic progenitor cells (HPC). MSC were isolated from bone marrow of donors between 21 and 92 years old. 67 genes were age-induced and 60 were age-repressed. HPC were isolated from cord blood or from mobilized peripheral blood of donors between 27 and 73 years and 432 genes were age-induced and 495 were age-repressed. The overlap of age-associated differential gene expression in HPC and MSC was moderate. However, it was striking that several age-related gene expression changes in both MSC and HPC were also differentially expressed upon replicative senescence of MSC in vitro. Especially genes involved in genomic integrity and regulation of transcription were age-repressed. Although telomerase activity and telomere length varied in HPC particularly from older donors, an age-dependent decline was not significant arguing against telomere exhaustion as being causal for the aging phenotype. These studies have demonstrated that aging causes gene expression changes in human MSC and HPC that vary between the two different cell types. Changes upon aging of MSC and HPC are related to those of replicative senescence of MSC in vitro and this indicates that our stem and progenitor cells undergo a similar process also in vivo.”
The discussion in this same document related to the impact of telomere lengths on aging is telling. “Progressive shortening of the telomeres or modified telomeric structures have been discussed to be the main trigger for replicative senescence and it has been anticipated that telomere shortening provides an internal clock. With every cell division the number of telomere repeats decreases and this has also been demonstrated for MSC , . The process is counteracted by expression of telomerase in somatic stem cells , . This is in line with our results where telomerase activity was detected in HPC. Vaziri et al. have demonstrated that CD34+CD38âˆ’ HPC from human bone marrow have shorter telomeres than those from fetal liver or cord blood . In this study telomere length decreased only slightly upon aging and we did not detect subpopulations with very short telomeres or signs of telomere dysfunction (uncapped telomeres) in any of the samples.1 Hence, it is unlikely that age-induced gene expression changes in HPC are only due to telomere loss. Though discussed controversially, our data rather support studies by others that telomere shortening may not be the only reason for replicative senescence in hematopoietic cells in vivo , –.” Telomere lengths by themselves are only partial and possibly poor indicators of all the changes that go on in cells with progressive aging and better biomarkers like age-related proteins are becoming available.
Chromatin stress and epigenetic changes triggered by telomeres in the process of cell aging
Another important contribution to the picture of how cell senescence affects aging is provided by the October 2010 publication Reduced histone biosynthesis and chromatin changes arising from a damage signal at telomeres. “During replicative aging of primary cells morphological transformations occur, the expression pattern is altered and chromatin changes globally. Here we show that chronic damage signals, probably caused by telomere processing, affect expression of histones and lead to their depletion. We investigated the abundance and cell cycle expression of histones and histone chaperones and found defects in histone biosynthesis during replicative aging. Simultaneously, epigenetic marks were redistributed across the phases of the cell cycle and the DNA damage response (DDR) machinery was activated. The age-dependent reprogramming affected telomeric chromatin itself, which was progressively destabilized, leading to a boost of the telomere-associated DDR with each successive cell cycle.”
This same publication puts forward an important new concept: “We propose a mechanism in which changes in the structural and epigenetic integrity of telomeres affect core histones and their chaperones, enforcing a self-perpetuating pathway of global epigenetic changes that ultimately leads to senescence.” Histones, we recall, are the spindles around which DNA is wrapped, and chaperones are “are proteins that assist the non-covalent folding or unfolding and the assembly or disassembly of other macromolecular structures.” in this case the chaperones are ones that assist folding of DNA in histone structures, HSP90 being a main one discussed below.
A recent Science Daily article contains information from an interview with one off the publication’s authors and explains the importance of the research behind thepublication. “In a study published in the Oct. 3, 2010, issue of Nature Structural and Molecular Biology, a team led by Jan Karlseder, Ph.D., at the Salk Institute for Biological Studies reports that as cells count down to senescence and telomeres wear down, their DNA undergoes massive changes in the way it is packaged. These changes likely trigger what we call “aging”. — “Prior to this study we knew that telomeres get shorter and shorter as a cell divides and that when they reach a critical length, cells stop dividing or die,” said Karlseder, an associate professor in the Molecular and Cell Biology Laboratory. “Something must translate the local signal at chromosome ends into a huge signal felt throughout the nucleus. But there was a big gap in between.” — Karlseder and postdoctoral fellow Roddy O’Sullivan, Ph.D., began to close the gap by comparing levels of proteins called histones in young cells-cells that had divided 30 times-with “late middle-aged” cells, which had divided 75 times and were on the downward slide to senescence, which occurs at 85 divisions. Histone proteins bind linear DNA strands and compress them into nuclear complexes, collectively referred to as chromatin. — Karlseder and O’Sullivan found that aging cells simply made less histone protein than do young cells. “We were surprised to find that histone levels decreased as cells aged,” said O’Sullivan, the study’s first author. “These proteins are required throughout the genome, and therefore any event that disrupts this production line affects the stability of the entire genome.” — The team then undertook exhaustive “time-lapse” comparisons of histones in young versus aging cells and confirmed that marked differences in the abundance and variety of histones were evident at every step as cells moved through cell division. — O’Sullivan calls the “default” histone pattern displayed by young cells “happy, healthy chromatin.” By contrast, he says, aging cells appear to undergo stress as they duplicate their chromosomes in preparation for cell division and have difficulty restoring a “healthy” chromatin pattern once division is complete. — Comparisons of histone patterns in cells taken from human subjects-a 9- versus a 92-year-old-dramatically mirrored histone trends seen in cell lines. “These key experiments suggest that what we observe in cultured cells in a laboratory setting actually occurs and is relevant to aging in a population,” says Karlseder. The initiation of diseases associated with aging, such as cancer, is largely attributed to DNA, or genetic, damage. But this study suggests that aging itself is infinitely complex: that progressive telomere shortening hastens chromosomal aging by changing the way genes entwine with histones, so-called “epigenetic” changes. How DNA interacts with histones has enormous impact on whether genes are expressed-hence the current intense interest in the relationship of the epigenomic landscape to disease states. — Rescue experiments in which the team cosmetically enhanced aging cells confirmed that signals emitted by eroding telomeres drove epigenetic changes. When aging cells were engineered to express telomerase, the enzyme that restores and extends stubby telomeres, those rejuvenated cells showed histone levels reminiscent of “happy, healthy chromatin,” and a partial return to a youthful chromatin profile.”
Role of chaperone proteins in telomere activation
The biology of telomere shortening and lengthening is a very complex topic . “As telomere dysfunction has been associated with ageing and developing cancer, understanding the exact mechanisms regulating telomere structure and function is essential for the prevention and treatment of human cancers and age-related diseases. The mechanisms by which cells maintain telomere lengthening involve either telomerase or the alternative lengthening of the telomere pathway, although specific mechanisms of the latter and the relationship between the two are as yet unknown. Many cellular factors directly (TRF1/TRF2) and indirectly (shelterin-complex, PinX, Apollo and tankyrase) interact with telomeres, and their interplay influences telomere structure and function(ref).” . I focus on one special topic here, the role of chaperone proteins HSP90 and p23.
Back in 1999, the publication Functional requirement of p23 and Hsp90 in telomerase complexes reported “Here we show that assembly of active telomerase from in vitro-synthesized components requires the contribution of proteins present in reticulocyte extracts. We have identified the molecular chaperones p23 and Hsp90 as proteins that bind to the catalytic subunit of telomerase. Blockade of this interaction inhibits assembly of active telomerase in vitro. Also, a significant fraction of active telomerase from cell extracts is associated with p23 and Hsp90. Consistent with in vitro results, inhibition of Hsp90 function in cells blocks assembly of active telomerase.”
The 2001 publication Stable association of hsp90 and p23, but Not hsp70, with active human telomerase reported “We have previously found two additional components of the telomerase holoenzyme, the chaperones p23 and heat shock protein (hsp) 90, both of which are required for efficient telomerase assembly in vitro and in vivo. Both hsp90 and p23 bind specifically to hTERT and influence its proper assembly with the template RNA, hTR. We report here that the hsp70 chaperone also associates with hTERT in the absence of hTR and dissociates when telomerase is folded into its active state, similar to what occurs with other chaperone targets. Our data also indicate that hsp90 and p23 remain associated with functional telomerase complexes, which differs from other hsp90-folded enzymes that require only a transient hsp90.p23 binding. Our data suggest that components of the hsp90 chaperone complex, while required for telomerase assembly, remain associated with active enzyme, which may ultimately provide critical insight into the biochemical properties of telomerase assembly.”
About five years ago it was noticed that inhibition of the heat shock protein HSP90 in human tumor cells led to telomere erosion in those cells. As reported in the 2006 publication Induction of nitric oxide synthase-dependent telomere shortening after functional inhibition of Hsp90 in human tumor cells, “ — we observe significant DNA damage assessed by telomere dysfunction, although in the absence of a classical DNA damage response. Overall, our data suggest a novel mechanism whereby inhibition of Hsp90 disrupts free radical homeostasis and contributes directly to telomere erosion, further implicating Hsp90 as a potential therapeutic target for cancer cells.”
As time progressed, interest grew in the possibility of control of cancers via HSP90 modulation such as outlined in the 2009 publication To fold or not to fold: modulation and consequences of Hsp90 inhibition. HSP90 affects folding in additional protein substrates besides hTERT, including for example proteins having to do with neural differentiation(ref). “Many of the Hsp90-dependent client proteins are associated with cellular growth and survival and, consequently, inhibition of Hsp90 represents a promising approach for the treatment of cancer. Conversely, stimulation of heat-shock protein levels has potential therapeutic applications for the treatment of neurodegenerative diseases that result from misfolded and aggregated proteins. — Hsp90 modulation exhibits the potential to treat unrelated disease states, from cancer to neurodegenerative diseases, and, thus, to fold or not to fold, becomes a question of great value.”
As if the situation was not already complicated enough, a substance called CHIP is involved along with HSP90 and p23 in telomere length regulation. An October 19 2010 publication CHIP promotes hTERT degradation and negatively regulates telomerase activity relates “The maintenance of eukaryotic telomeres requires telomerase, which is minimally comprised of a telomerase reverse transcriptase (TERT) and an associated RNA component (TERC). Telomerase activity is tightly regulated by expression of hTERT at both the transcriptional and posttranslational levels. The Hsp90 and p23 molecular chaperones have been shown to associate with hTERT for the assembly of active telomerase. Here we show that CHIP (C terminus of Hsc70-interacting protein) physically associates with hTERT in the cytoplasm and regulates the cellular abundance of hTERT through an ubiquitin-mediated degradation. Overexpression of CHIP prevents nuclear translocation of hTERT and promotes hTERT degradation in the cytoplasm, thereby inhibiting telomerase activity. In contrast, knockdown of endogenous CHIP results in the stabilization of cytoplasmic hTERT. However, it does not affect the level of nuclear hTERT and has no effect on telomerase activity and telomere length. We further show that the binding of CHIP and Hsp70 to hTERT inhibits nuclear translocation of hTERT by dissociating p23. However, Hsp90 binding to hTERT was not affected by CHIP overexpression. These results suggest that CHIP can remodel the hTERT-chaperone complexes. Finally, the amount of hTERT associated with CHIP peaks in G2/M phases but decreases during S phase, suggesting a cell cycle-dependent regulation of hTERT.”
TERRA RNA binding
The 2009 publication TERRA RNA Binding to TRF2 Facilitates Heterochromatin Formation and ORC Recruitment at Telomeres is one of several recent publications illustrating the wheels-within-wheels complexity of telomere formation and maintenance. “Telomere-repeat-encoding RNA (referred to as TERRA) has been identified as a potential component of yeast and mammalian telomeres. We show here that TERRA RNA interacts with several telomere-associated proteins, including telomere repeat factors 1 (TRF1) and 2 (TRF2), subunits of the origin recognition complex (ORC), heterochromatin protein 1 (HP1), histone H3 trimethyl K9 (H3 K9me3), and members of the DNA-damage-sensing pathway. siRNA depletion of TERRA caused an increase in telomere dysfunction-induced foci, aberrations in metaphase telomeres, and a loss of histone H3 K9me3 and ORC at telomere repeat DNA. Previous studies found that TRF2 amino-terminal GAR domain recruited ORC to telomeres. We now show that TERRA RNA can interact directly with the TRF2 GAR and ORC1 to form a stable ternary complex. We conclude that TERRA facilitates TRF2 interaction with ORC and plays a central role in telomere structural maintenance and heterochromatin formation.”
Mechanism of action of curcumin on cancers
For those of you who are supplement buffs and take curcumin, the molecular mechanisms described above provide a neat explanation for a long-observed effect, the anti-cancer actions of curcumin. The title of the 2010 publication Curcumin inhibits nuclear localization of telomerase by dissociating the Hsp90 co-chaperone p23 from hTERT contains the main message. “Here we demonstrate that curcumin inhibits telomerase activity in a time- and dose-dependent manner by decreasing the level of hTERT expression. Following curcumin treatment, we observed a clear accumulation of hTERT in the cytoplasmic compartment of the cell. The curcumin-induced cytoplasmic retention of hTERT could be due to failure of nuclear import, and the resulting cytoplasmic hTERT protein was rapidly ubiquitinated and degraded by the proteasome. We also report that curcumin treatment results in a substantial decrease in association of p23 and hTERT but does not affect the Hsp90 binding to hTERT. In contrast, the treatment of the Hsp90 inhibitor geldanamycin promotes dissociation of both Hsp90 and p23 proteins from hTERT. Taken together, these results demonstrate that the interaction of the Hsp90-p23 complex with hTERT is critical for regulation of the nuclear localization of telomerase, and that down-regulation of hTERT by curcumin involves dissociating the binding of hTERT with p23. Thus, inhibition of nuclear translocation of hTERT by curcumin may provide new perspectives for regulation of telomerase activity during tumorigenic progression.”
I am pleased to have this explanation as an addendum of the discussion in my blog entry Curcumin, cancer and longevity. But I am not happy with at least one implication of the explanation which is that taking curcumin supplements may result in having shorter telomeres than otherwise. I am uneasily left with a couple of key questions:
· Is downregulation of hTERT the primary mechanism through which curcumin combats cancers? The 2009 publication Curcumin and Cancer Cells: How Many Ways Can Curry Kill Tumor Cells Selectively? States “How curcumin kills tumor cells is the focus of this review. We show that curcumin modulates growth of tumor cells through regulation of multiple cell signaling pathways including cell proliferation pathway (cyclin D1, c-myc), cell survival pathway (Bcl-2, Bcl-xL, cFLIP, XIAP, c-IAP1), caspase activation pathway (caspase-8, 3, 9), tumor suppressor pathway (p53, p21) death receptor pathway (DR4, DR5), mitochondrial pathways, and protein kinase pathway (JNK, Akt, and AMPK). How curcumin selectively kills tumor cells, and not normal cells, is also described in detail.” How important is hTERT downregulation compared to those other pathways when it comes to curcumin controlling cancers? Is the inhibition of hTERT an upstream or downstream event?
· Does taking curcumin as a supplement not only help ward off cancers but also inhibit telomerase expression and therefore results in my telomeres being shorter than what they would be? In other words does taking curcumin as a supplement have both an anti-cancer and pro-aging effect? If so, what is the detailed nature of the tradeoff?
Wrapping it all up
Some key observations based on the research cited above:
· Aging and eventual senescence associated with cell replication applies to certain (I suspect, all) adult stem and progenitor cells as well as to normal functional somatic cells.
· As cells pass through their lifecycle of successive divisions they progressively change in multiple ways: a) in gene expression profiles, b) in the expression of multiple age-related proteins, c) in the abundance and variety of histones, d) in telomere lengths, and e) in possible other epigenetic markers.
· I remind readers of a November 2009 Blog entry breakthrough telomere research finding that links telomere shortening to another of the key theories of aging, Programmed Epigenomic Changes. “Telomeric shortening at some point induces DNA damage which lets loose signaling which changes the epigenome disrupting epigenetic silencing and resulting in pro-aging global DNA expression.”