The Connection Between Cell Fate and Telomere.

Abolition of telomerase activity results in telomere shortening, a process that eventually destabilizes the ends of chromosomes, leading to genomic instability and cell growth arrest or death. Telomere shortening leads to the attainment of the "Hayflick limit", and the transition of cells to state of senescence. If senescence is bypassed, cells undergo crisis through loss of checkpoints. This process causes massive cell death concomitant with further telomere shortening and spontaneous telomere fusions. In functional telomere of mammalian cells, DNA contains double-stranded tandem repeats of TTAGGG. The Shelterin complex, which is composed of six different proteins, is required for the regulation of telomere length and stability in cells. Telomere protection by telomeric repeat binding protein 2 (TRF2) is dependent on DNA damage response (DDR) inhibition via formation of T-loop structures. Many protein kinases contribute to the DDR activated cell cycle checkpoint pathways, and prevent DNA replication until damaged DNA is repaired. Thereby, the connection between cell fate and telomere length-associated telomerase activity is regulated by multiple protein kinase activities. Contrarily, inactivation of DNA damage checkpoint protein kinases in senescent cells can restore cell-cycle progression into S phase. Therefore, telomere-initiated senescence is a DNA damage checkpoint response that is activated with a direct contribution from dysfunctional telomeres. In this review, in addition to the above mentioned, the choice of main repair pathways, which comprise non-homologous end joining and homologous recombination in telomere uncapping telomere dysfunctions, are discussed.

The Hayflick Limit and Age-Related Adaptive Immune Deficiency.

The adaptive immune system (AIS) acquires significant deficiency during chronological ageing, making older individuals more susceptible to infections and less responsive to vaccines compared to younger individuals. At the cellular level, one of the most striking features of this ageing-related immune deficiency is the dramatic loss of T-cell diversity that occurs in elderly humans. After the age of 70 years, there is a sharp decline in the diversity of naïve T cells, including a >10-fold decrease in the CD4+ compartment and a >100-fold decrease in the CD8+ compartment. Such changes are detrimental because the AIS relies on a diverse naïve T-cell pool to respond to novel pathogens. Recent work suggests that this collapse of naïve T-cell diversity results from T cells reaching the Hayflick limit and being eliminated through both antigen-dependent and -independent pathways. The progressive attrition of telomeres is the molecular mechanism that underlies this Hayflick limit. Therefore, we propose that by measuring the telomere lengths of T cells with high resolution, it is possible to develop a unique biomarker of immune deficiency, potentially much better correlated with individual susceptibility to diseases compared to chronological age alone.

Cell Immortalization: In Vivo Molecular Bases and In Vitro Techniques for Obtention.

Somatic human cells can divide a finite number of times, a phenomenon known as the Hayflick limit. It is based on the progressive erosion of the telomeric ends each time the cell completes a replicative cycle. Given this problem, researchers need cell lines that do not enter the senescence phase after a certain number of divisions. In this way, more lasting studies can be carried out over time and avoid the tedious work involved in performing cell passes to fresh media. However, some cells have a high replicative potential, such as embryonic stem cells and cancer cells. To accomplish this, these cells express the enzyme telomerase or activate the mechanisms of alternative telomere elongation, which favors the maintenance of the length of their stable telomeres. Researchers have been able to develop cell immortalization technology by studying the cellular and molecular bases of both mechanisms and the genes involved in the control of the cell cycle. Through it, cells with infinite replicative capacity are obtained. To obtain them, viral oncogenes/oncoproteins, myc genes, ectopic expression of telomerase, and the manipulation of genes that regulate the cell cycle, such as p53 and Rb, have been used.

Influence of telomerase activity and initial distribution on human follicular aging: Moving from a discrete to a continuum model.

A discrete model is proposed for the temporal evolution of a population of cells sorted according to their telomeric length. This model assumes that, during cell division, the distribution of the genetic material to daughter cells is asymmetric, i.e. chromosomes of one daughter cell have the same telomere length as the mother, while in the other daughter cell telomeres are shorter. Telomerase activity and cell death are also taken into account. The continuous model is derived from the discrete model by introducing the generational age as a continuous variable in [0,h], being h the Hayflick limit, i.e. the number of times that a cell can divide before reaching the senescent state. A partial differential equation with boundary conditions is obtained. The solution to this equation depends on the initial telomere length distribution. The initial and boundary value problem is solved exactly when the initial distribution is of exponential type. For other types of initial distributions, a numerical solution is proposed. The model is applied to the human follicular growth from preantral to preovulatory follicle as a case study and the aging rate is studied as a function of telomerase activity, the initial distribution and the Hayflick limit. Young, middle and old cell-aged initial normal distributions are considered. In all cases, when telomerase activity decreases, the population ages and the smaller the h value, the higher the aging rate becomes. However, the influence of these two parameters is different depending on the initial distribution. In conclusion, the worst-case scenario corresponds to an aged initial telomere distribution.

2-adic morphogenesis as a metaphorical model of biological growth.

The article proposes a mathematical model of morphogenesis which is based on 2-adic arithmetic. In this model, the process of morphogenesis is separated from its genetic coding and genetic control, and is considered abstractly as a transformation of complex biomorphic structures resulting from the process of sequential geometric cell division. The concept of cellular structure is introduced and the analogies that exist between the transformation of organisms and the transformation of the corresponding cellular structures generated by numerical series are considered, in particular, an analogy is drawn between the transformation of series depending on a complex parameter and the growth of biological organisms. The article also introduces some mathematical formalism used to compare different morphological pathways.

Role of the Circadian Clock "Death-Loop" in the DNA Damage Response Underpinning Cancer Treatment Resistance.

Here, we review the role of the circadian clock (CC) in the resistance of cancer cells to genotoxic treatments in relation to whole-genome duplication (WGD) and telomere-length regulation. The CC drives the normal cell cycle, tissue differentiation, and reciprocally regulates telomere elongation. However, it is deregulated in embryonic stem cells (ESCs), the early embryo, and cancer. Here, we review the DNA damage response of cancer cells and a similar impact on the cell cycle to that found in ESCs­overcoming G1/S, adapting DNA damage checkpoints, tolerating DNA damage, coupling telomere erosion to accelerated cell senescence, and favouring transition by mitotic slippage into the ploidy cycle (reversible polyploidy). Polyploidy decelerates the CC. We report an intriguing positive correlation between cancer WGD and the deregulation of the CC assessed by bioinformatics on 11 primary cancer datasets (rho = 0.83; p < 0.01). As previously shown, the cancer cells undergoing mitotic slippage cast off telomere fragments with TERT, restore the telomeres by ALT-recombination, and return their depolyploidised offspring to telomerase-dependent regulation. By reversing this polyploidy and the CC "death loop", the mitotic cycle and Hayflick limit count are thus again renewed. Our review and proposed mechanism support a life-cycle concept of cancer and highlight the perspective of cancer treatment by differentiation.

Is There a Positive Side to T Cell Exhaustion?

T cell "exhaustion" describes a state of late-stage differentiation usually associated with active prevention of functionality via ligation of negative signaling receptors on the cell surface, and which can be reversed by blocking these interactions. This contrasts with T cell "senescence," which has been defined as a state that is maintained by intrinsic internal cell signaling (caused by DNA damage or other stresses) and which can be reversed pharmacologically. Interventions to alleviate these two different categories of inhibitory pathways may be desirable in immunotherapy for cancer and possibly certain infectious diseases, but reciprocally inducing and maintaining these states, or some properties thereof, may be beneficial in organ transplantation and autoimmunity. Even under physiological non-pathological conditions, T cell exhaustion and senescence may play a role in the retention of T cell clones required for immunosurveillance, and prevent their loss via elimination at the Hayflick limit. This essay briefly reviews T cell exhaustion in contrast to replicative senescence, and circumstances under which their modulation may be beneficial.

Cancer telomeres and white crows.

This mini-review article discusses past and present prostate-focused research on telomere and telomerase biology conducted at Johns Hopkins, through the eyes of a Donald S Coffey trainee. Included are past discoveries of abnormalities in telomere biology in the context of prostate cancer and its pre-malignant precursor prostatic intraepithelial neoplasia (PIN); the finding that telomerase activity is androgen-regulated in the prostate, and the potential role of telomerase in prostate epithelial stem cells. Also reviewed are more recent results showing that in situ telomere length measurements in patient tissue specimens may have utility in risk assessment and as a prognostic biomarker. Highlighted throughout the article are some of the training and mentorship approaches employed by the late Dr. Coffey, former Director of Urologic Research at the Brady Urological Research Institute, which inspired new research ideas, team science, and discovery.

Cancer follows chromosome missegregation when all endogenous repair mechanisms fail.

Almost all solid tumors consist of aneuploid cells with highly abnormal chromosome numbers. Such cancer cells could very well originate from chromosome missegregation which is a disturbingly common phenomenon, happening in 0.01 to 4 percent of cell divisions. Missegregated cells are aneuploid, typically lacking a chromosome or having one in surplus. Missegregated cells have mutation in the gene dose of the perhaps a thousand genes on a chromosome in one step. After missegregation cell division cannot be done right, as at least one daughter cell has a faulty chromosome number. At division in cells with surplus chromosomes the number will tend to increase due to mismatch in the division machinery. The organism has a number of repair mechanisms in place to prevent potential damage of accumulating aneuploidy. The first is the roll-back of the cell division itself, leading to tetraploidy or sometimes two nuclei in one cell; another is the prevention of further divisions. A very important one is induction of apoptosis, the cellular suicide. A special case is the elimination of the nucleus itself in the formation of red blood cells. Many aneuploid cells are probably eliminated by the immune system. A hypothetical mechanism would be the prevention of metastasis. Missegregation increases with age when the chromosomes lose their protective telomere ends at the Hayflick limit after about 50 divisions, and the unraveled chromosomes fuse and break. For cancer to develop all of these repair mechanisms must fail. The hypothesis offers a straightforward rationale for the multiple hit hypothesis of cancer development.

Fundamental mechanisms of telomerase action in yeasts and mammals: understanding telomeres and telomerase in cancer cells.

Aberrant activation of telomerase occurs in 85-90% of all cancers and underpins the ability of cancer cells to bypass their proliferative limit, rendering them immortal. The activity of telomerase is tightly controlled at multiple levels, from transcriptional regulation of the telomerase components to holoenzyme biogenesis and recruitment to the telomere, and finally activation and processivity. However, studies using cancer cell lines and other model systems have begun to reveal features of telomeres and telomerase that are unique to cancer. This review summarizes our current knowledge on the mechanisms of telomerase recruitment and activation using insights from studies in mammals and budding and fission yeasts. Finally, we discuss the differences in telomere homeostasis between normal cells and cancer cells, which may provide a foundation for telomere/telomerase targeted cancer treatments.

The Immortal Senescence.

Activation of oncogenic signaling paradoxically results in the permanent withdrawal from cell cycle and induction of senescence (oncogene-induced senescence (OIS)). OIS is a fail-safe mechanism used by the cells to prevent uncontrolled tumor growth, and, as such, it is considered as the first barrier against cancer. In order to progress, tumor cells thus need to first overcome the senescent phenotype. Despite the increasing attention gained by OIS in the past 20 years, this field is still rather young due to continuous emergence of novel pathways and processes involved in OIS. Among the many factors contributing to incomplete understanding of OIS are the lack of unequivocal markers for senescence and the complexity of the phenotypes revealed by senescent cells in vivo and in vitro. OIS has been shown to play major roles at both the cellular and organismal levels in biological processes ranging from embryonic development to barrier to cancer progression. Here we will briefly outline major advances in methodologies that are being utilized for induction, identification, and characterization of molecular processes in cells undergoing oncogene-induced senescence. The full description of such methodologies is provided in the corresponding chapters of the book.

Molecular turnover, the H3.3 dilemma and organismal aging (hypothesis).

The H3.3 histone variant has been a subject of increasing interest in the field of chromatin studies due to its two distinguishing features. First, its incorporation into chromatin is replication independent unlike the replication-coupled deposition of its canonical counterparts H3.1/2. Second, H3.3 has been consistently associated with an active state of chromatin. In accordance, this histone variant should be expected to be causally involved in the regulation of gene expression, or more generally, its incorporation should have downstream consequences for the structure and function of chromatin. This, however, leads to an apparent paradox: In cells that slowly replicate in the organism, H3.3 will accumulate with time, opening the way to aberrant effects on heterochromatin. Here, we review the indications that H3.3 is expected both to be incorporated in the heterochromatin of slowly replicating cells and to retain its functional downstream effects. Implications for organismal aging are discussed.

Acrylamide induces accelerated endothelial aging in a human cell model.

Acrylamide (AAM) has been recently discovered in food as a Maillard reaction product. AAM and glycidamide (GA), its metabolite, have been described as probably carcinogenic to humans. It is widely established that senescence and carcinogenicity are closely related. In vitro, endothelial aging is characterized by replicative senescence in which primary cells in culture lose their ability to divide. Our objective was to assess the effects of AAM and GA on human endothelial cell senescence. Human umbilical vein endothelial cells (HUVECs) cultured in vitro were used as model. HUVECs were cultured over 3 months with AAM or GA (1, 10 or 100 µM) until growth arrest. To analyze senescence, ß-galactosidase activity and telomere length of HUVECs were measured by cytometry and semi-quantitative PCR, respectively. At all tested concentrations, AAM or GA reduced cell population doubling compared to the control condition (p < 0.001). ß-galactosidase activity in endothelial cells was increased when exposed to AAM (≥10 µM) or GA (≥1 µM) (p < 0.05). AAM (≥10 µM) or GA (100 µM) accelerated telomere shortening in HUVECs (p < 0.05). In conclusion, in vitro chronic exposure to AAM or GA at low concentrations induces accelerated senescence. This result suggests that an exposure to AAM might contribute to endothelial aging.

Differences of isolated dental stem cells dependent on donor age and consequences for autologous tooth replacement.

OBJECTIVE: Autologous therapy via stem cell-based tissue regeneration is an aim to rebuild natural teeth. One option is the use of adult stem cells from the dental pulp (DPSCs), which have been shown to differentiate into several types of tissue in vitro and in vivo, especially into tooth-like structures. DPSCs are mainly isolated from the dental pulp of third molars routinely extracted for orthodontic reasons. Due to the extraction of third molars at various phases of life, DPSCs are isolated at different developmental stages of the tooth. DESIGN: The present study addressed the question whether DPSCs from patients of different ages were similar in their growth characteristics with respect to the stage of tooth development. Therefore DPSCs from third molars of 12-30 year-old patients were extracted, and growth characteristics, e.g. doubling time and maximal cell division potential were analysed. In addition, pulp and hard dental material weight were recorded. RESULTS: Irrespective of the age of patients almost all isolated cells reached 40-60 generations with no correlation between maximal cell division potential and patient age. Cells from patients <22 years showed a significantly faster doubling time than the cells from patients ≥22 years. CONCLUSION: The age of patients at the time of stem cell isolation is not a crucial factor concerning maximal cell division potential, but does have an impact on the doubling time. However, differences in individuals regarding growth characteristics were more pronounced than age-dependent differences.