Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan

Large, long-lived species experience more lifetime cell divisions and hence a greater risk of spontaneous tumor formation than smaller, short-lived species. Large, long-lived species are thus expected to evolve more elaborate tumor suppressor systems. In previous work, we showed that telomerase activity coevolves with body mass, but not lifespan, in rodents: telomerase activity is repressed in the somatic tissues of large rodent species but remains active in small ones. Without telomerase activity, the telomeres of replicating cells become progressively shorter until, at some critical length, cells stop dividing. Our findings therefore suggested that repression of telomerase activity mitigates the increased risk of cancer in larger-bodied species but not necessarily longer-lived ones. These findings imply that other tumor suppressor mechanisms must mitigate increased cancer risk in long-lived species. Here, we examined the proliferation of fibroblasts from 15 rodent species with diverse body sizes and lifespans. We show that, consistent with repressed telomerase activity, fibroblasts from large rodents undergo replicative senescence accompanied by telomere shortening and overexpression of p16(Ink4a) and p21(Cip1/Waf1) cycline-dependent kinase inhibitors. Interestingly, small rodents with different lifespans show a striking difference: cells from small shorter-lived species display continuous rapid proliferation, whereas cells from small long-lived species display continuous slow proliferation. We hypothesize that cells of small long-lived rodents, lacking replicative senescence, have evolved alternative tumor-suppressor mechanisms that prevent inappropriate cell division in vivo and slow cell growth in vitro. Thus, large-bodied species and small but long-lived species have evolved distinct tumor suppressor mechanisms.     

Telomere biology in mammalian germ cells and during development

The development of an organism is a strictly regulated program in which controlled gene expression guarantees the establishment of a specific phenotype. The chromosome termini or so-called telomeres preserve the integrity of the genome within developing cells. In the germline, during early development, and in highly proliferative organs, human telomeres are balanced between shortening processes with each cell division and elongation by telomerase, but once terminally differentiated or mature the equilibrium is shifted to gradual shortening by repression of the telomerase enzyme. Telomere length is to a large extent genetically determined and the neonatal telomere length equilibrium is, in fact, a matter of evolution. Gradual telomere shortening in normal human somatic cells during consecutive rounds of replication eventually leads to critically short telomeres that induce replicative senescence in vitro and probably in vivo. Hence, a molecular clock is set during development, which determines the replicative potential of cells during extrauterine life. Telomeres might be directly or indirectly implicated in longevity determination in vivo, and information on telomere length setting in utero and beyond should help elucidate presumed causal connections between early growth and aging disorders later in life. Only limited information exists concerning the mechanisms underlying overall telomere length regulation in the germline and during early development, especially in humans. The intent of this review is to focus on recent advances in our understanding of telomere biology in germline cells as well as during development (pre- and postimplantation periods) in an attempt to summarize our knowledge about telomere length determination and its importance for normal development in utero and the occurrence of the aging and abnormal phenotype later on.     

Telomerase activity in human germline and embryonic tissues and cells

Telomerase is a ribonucleoprotein that synthesizes telomere repeats onto chromosome ends and is involved in maintaining telomere length in germline tissues and in immortal and cancer cells. In the present study, the temporal regulation of expression of telomerase activity was examined in human germline and somatic tissues and cells during development. Telomerase activity was detected in fetal, newborn, and adult testes and ovaries, but not in mature spermatozoa or oocytes. Blastocysts expressed high levels of telomerase activity as did most human somatic tissues at 16–20 weeks of development with the exception of human brain tissue. This activity could no longer be detected in the somatic tissues examined from the neonatal period onward. Neither placenta nor cultured fetal amniocytes contained detectable telomerase activity. Fetal tissues explanted into primary cell culture showed a dramatic decline in telomerase activity which became undetectable after the first passage in vitro. Elucidation of the regulatory pathways involved in the repression of telomerase activity during development may lead to the ability to manipulate telomerase levels and explore the consequences both for cellular aging and for the survival of cancer cells. © 1996 Wiley-Liss, Inc.     

The disparity between human cell senescence in vitro and lifelong replication in vivo

Cultured human fibroblasts undergo senescence (a loss of replicative capacity) after a uniform, fixed number of approximately 50 population doublings, commonly termed the Hayflick limit. It has been long known from clonal and other quantitative studies, however, that cells decline in replicative capacity from the time of explantation and do so in a stochastic manner, with a half-life of only approximately 8 doublings. The apparent 50-cell doubling limit reflects the expansive propagation of the last surviving clone. The relevance of either figure to survival of cells in the body is questionable, given that stem cells in some renewing tissues undergo >1,000 divisions in a lifetime with no morphological sign of senescence. Oddly enough, these observations have had little if any effect on general acceptance of the Hayflick limit in its original form. The absence of telomerase in cultured human cells and the shortening of telomeres at each population doubling have suggested that telomere length acts as a mitotic clock that accounts for their limited lifespan. This concept assumed an iconic character with the report that ectopic expression of telomerase by a vector greatly extended the lifespan of human cells. That something similar might occur in vivo seemed consistent with initial reports that most human somatic tissues lack telomerase activity. More careful study, however, has revealed telomerase activity in stem cells and some dividing transit cells of many renewing tissues and even in dividing myocytes of repairing cardiac muscle. It now seems likely that telomerase is active in vivo where and when it is needed to maintain tissue integrity. Caution is recommended in applying telomerase inhibition to kill telomerase-expressing cancer cells, because it would probably damage stem cells in essential organs and even increase the likelihood of secondary cancers. The risk may be especially high in sun-exposed skin, where there are usually thousands of p53-mutant clones of keratinocytes predisposed to cancer.     

Telomerase and differentiation in multicellular organisms: turn it off, turn it on, and turn it off again

Telomerase is a ribonucleoprotein complex that catalyses the addition of TTAGGG repeats onto telomeres, repetitive DNA structures found at the ends of linear chromosomes. The majority of human somatic tissues do not display telomerase activity and undergo telomeric shortening with consecutive divisions. This telomeric shortening results in replicative senescence in vitro and likely in vivo. Telomerase activity is present in the vast majority of tumors, preventing telomeric shortening and thereby enabling indefinite cell divisions. Telomerase activity is regulated throughout human development, undergoing silencing in almost all organ systems from embryogenesis onwards. However, regulated telomerase activity is seen in basal/stem cell compartments of highly regenerative tissues, such as those of the immune system, skin, and intestine. Avian species display telomerase repression and telomeric shortening similar to that seen in humans. However, rodents retain telomerase-competency throughout their lifespan and have not been shown to display division-dependent telomere shortening. The regulation of telomerase activity in plants is less well understood, although early indications suggest ubiquitous competency. The aim of this review is to present current data regarding developmental regulation of telomerase in humans, mice, chickens and flowering plants. Differentiation, quiescence and telomerase activity regulation will then be addressed in three human representative tissue systems; blood, skin, and intestine. We will also highlight similarities, differences and misconceptions in the developing field of telomere and telomerase biology.     

Telomere and telomerase in oncology

Telomere and cell replicative senescenceTelomeres, which are located at the end of chro-mosome, are crucial to protect chromosome againstdegeneration, rearrangment and end to end fusion[1].Human telomeres are tandemly repeated units of thehexanucleotide TTAGGG. The estimated length oftelomeric DNA varies from 2 to 20 kilo base pairs,depending on factors such as tissue type and hu-man age. The buck of telomeric DNA is double-stranded, but the end of telomeric DNA consists of3' overhang of…     

Telomerase maintains telomere structure in normal human cells

In normal human cells, telomeres shorten with successive rounds of cell division, and immortalization correlates with stabilization of telomere length. These observations suggest that human cancer cells achieve immortalization in large part through the illegitimate activation of telomerase expression. Here, we demonstrate that the rate-limiting telomerase catalytic subunit hTERT is expressed in cycling primary presenescent human fibroblasts, previously believed to lack hTERT expression and telomerase activity. Disruption of telomerase activity in normal human cells slows cell proliferation, restricts cell lifespan, and alters the maintenance of the 3' single-stranded telomeric overhang without changing the rate of overall telomere shortening. Together, these observations support the view that telomerase and telomere structure are dynamically regulated in normal human cells and that telomere length alone is unlikely to trigger entry into replicative senescence.     

Reversible manipulation of telomerase expression and telomere length. Implications for the ionizing radiation response and replicative senescence of human cells

Most human cells do not express telomerase and irreversibly arrest proliferation after a finite number of divisions (replicative senescence). Several lines of evidence suggest that replicative senescence is caused by short dysfunctional telomeres, which arise when DNA is replicated in the absence of adequate telomerase activity. We describe a method to reversibly bypass replicative senescence and generate mass cultures that have different average telomere lengths. A retrovirus carrying hTERT flanked by excision sites for Cre recombinase rendered normal human fibroblasts telomerase-positive and replicatively immortal. Superinfection with retroviruses carrying wild-type or mutant forms of TIN2, a negative regulator of telomere length, created telomerase-positive, immortal populations with varying average telomere lengths. Subsequent infection with a Cre-expressing retrovirus abolished telomerase activity, creating mortal cells with varying telomere lengths. Using these cell populations, we show that, after hTERT excision, cells senesce with shorter telomeres than parental cells. Moreover, long telomeres, but not telomerase, protected cells from the loss of division potential caused by ionizing radiation. Finally, although telomerase-negative cells with short telomeres senesced after fewer doublings than those with long telomeres, telomere length per se did not correlate with senescence. Our results support a role for telomere structure, rather than length, in replicative senescence.     

Telomere loss: mitotic clock or genetic time bomb?

The Holy Grail of gerontologists investigating cellular senescence is the mechanism responsible for the finite proliferative capacity of somatic cells. In 1973, Olovnikov proposed that cells lose a small amount of DNA following each round of replication due to the inability of DNA polymerase to fully replicate chromosome ends (telomeres) and that eventually a critical deletion causes cell death. Recent observations showing that telomeres of human somatic cells act as a mitotic clock, shortening with age both in vitro and in vivo in a replication dependent manner, support this theory's premise. In addition, since telomeres stabilize chromosome ends against recombination, their loss could explain the increased frequency of dicentric chromosomes observed in late passage (senescent) fibroblasts and provide a checkpoint for regulated cell cycle exit. Sperm telomeres are longer than somatic telomeres and are maintained with age, suggesting that germ line cells may express telomerase, the ribonucleoprotein enzyme known to maintain telomere length in immortal unicellular eukaryotes. As predicted, telomerase activity has been found in immortal, transformed human cells and tumour cell lines, but not in normal somatic cells. Telomerase activation may be a late, obligate event in immortalization since many transformed cells and tumour tissues have critically short telomeres. Thus, telomere length and telomerase activity appear to be markers of the replicative history and proliferative potential of cells; the intriguing possibility remains that telomere loss is a genetic time bomb and hence causally involved in cell senescence and immortalization.     

Changes in Telomerase Activity and Telomere Length during Human T Lymphocyte Senescence

It has been proposed that telomeres shorten with every cell cycle because the normal mechanism of DNA replication cannot replicate the end sequences of the lagging DNA strand. Telomerase, a ribonucleoprotein enzyme that synthesizes telomeric DNA repeats at the DNA 3′ ends of eukaryotic chromosomes, can compensate for such shortening, by extending the template of the lagging strand. Telomerase activity has been identified in human germline cells and in neoplastic immortal somatic cells, but not in most normal somatic cells, which senesce after a certain number of cell divisions. We and others have found that telomerase activity is present in normal human lymphocytes and is upregulated when the cells are activated. But, unlike the immortal cell lines, presence of telomerase activity is not sufficient to make T cells immortal and telomeres from these cells shorten continuously duringin vitroculture. After senescence, telomerase activity, as detected by the TRAP technique, was downregulated. A cytotoxic T lymphocyte (CTL) cell line that was established in the laboratory has very short terminal restriction fragments (TRFs). Telomerase activity in this cell line is induced during activation and this activity is tightly correlated with cell proliferation. The level of telomerase activity in activated peripheral blood T cells, the CTL cell line, and two leukemia cell lines does not correlate with the average TRF length, suggesting that other factors besides telomerase activity are involved in the regulation of telomere length.     

Dynamics of telomere erosion in transformed and non-transformed avian cells in vitro

Although vertebrate telomeres are highly conserved, telomere dynamics and telomerase profiles vary among species. The objective of the present study was to examine telomerase activity and telomere length profiles of transformed and non-transformed avian cells in vitro. Non-transformed chicken embryo fibroblasts (CEFs) showed little or no telomerase activity from the earliest passages through senescence. Unexpectedly, a single culture of particularly long-lived senescent CEFs showed telomerase activity after over 250 days in culture. Transformed avian lines (six chicken, two quail and one turkey) and tumor samples (two chicken) exhibited telomerase activity. Telomere length profiles of non-transformed CEF cultures derived from individual embryos of an inbred line (UCD 003) exhibited cycles of shortening and lengthening with a substantial net loss of telomeric DNA by senescence. The telomere length profiles of several transformed cell lines resembled telomere length profiles of senescent CEFs in that they exhibited little of the typical smear of terminal restriction fragments (TRFs) suggesting that these transformed cells may possess a reduced amount of telomeric DNA. These results show that avian telomerase activity profiles are consistent with the telomerase activity profiles of human primary and transformed cells. Further, monitoring of telomere lengths of primary cells provides evidence for a dynamic series of changes over the lifespan of any specific cell culture ultimately resulting in net telomeric DNA loss by senescence.     

Telomere Length Dynamics in Telomerase-Positive Immortal Human Cell Populations

It has been proposed that the progressive shortening of telomeres in somatic cells eventually results in senescence. Previous experiments have demonstrated that many immortal cell lines have acquired telomerase activity leading to stabilization of telomere length. Telomere dynamics and telomerase activity were examined in the telomerase-positive immortal cell lines HeLa and 293 and subclones derived from them. A mass culture of HeLa cells had a stable mean telomere length over 60 population doublings (PD)in vitro.Subclones of this culture, however, had a range of mean telomere lengths indicating that telomeric heterogeneity exists within a population with a stable mean telomere length. Some of the subclones lacked detectable telomerase activity soon after isolation but regained it by PD 18, suggesting that at least some of the variation in telomere length can be attributed to variations in telomerase activity levels. 293 subclones also varied in telomere length and telomerase activity. Some telomerase-positive 293 subclones contained long telomeres that gradually shortened, demonstrating that factors other than telomerase also act to modulate telomere length. Fluctuations in telomere length in telomerase-positive immortalized cells may contribute to chromosomal instability and clonal evolution.     

Cell senescence and human aging: where's the link?

Normal human cells will not divide forever in culture. After a defined number of passages, every culture enters a viable non-dividing state termed senescence. This led to the proposal that the progressive accumulation of senescent cells contributes to (but does not exclusively cause) the aging process. Data now suggest that cell senescence, like apoptosis, occurs as an anti-cancer mechanism. In vivo, extremely low rates of division occur over very long periods of time. Thus, by the end of its lifespan, an organism has performed a surprisingly large amount of cell turnover. In a normal culture the fraction of senescent cells increases smoothly with proliferation in vitro. This process can be accurately modelled by the progressive loss of telomeric sequence (proposed as a counting mechanism for senescence). Telomere length is usually maintained by the enzyme telomerase (repressed in many human tissues). Re-introduction of telomerase prevents the onset of senescence. However, data suggest that a separate telomere-independent pathway exists. Evidence that this pathway is present in humans is now emerging.     

Telomerase: Structure,functions, and activity regulation

Telomerase is the enzyme responsible for maintenance of the length of telomeres by addition of guanine-rich repetitive sequences. Telomerase activity is exhibited in gametes and stem and tumor cells. In human somatic cells proliferation potential is strictly limited and senescence follows approximately 50–70 cell divisions. In most tumor cells, on the contrary, replication potential is unlimited. The key role in this process of the system of the telomere length maintenance with involvement of telomerase is still poorly studied. No doubt, DNA polymerase is not capable to completely copy DNA at the very ends of chromosomes; therefore, approximately 50 nucleotides are lost during each cell cycle, which results in gradual telomere length shortening. Critically short telomeres cause senescence, following crisis, and cell death. However, in tumor cells the system of telomere length maintenance is activated. Besides catalytic telomere elongation, independent telomerase functions can be also involved in cell cycle regulation. Inhibition of the telomerase catalytic function and resulting cessation of telomere length maintenance will help in restriction of tumor cell replication potential. On the other hand, formation of temporarily active enzyme via its intracellular activation or due to stimulation of expression of telomerase components will result in telomerase activation and telomere elongation that can be used for correction of degenerative changes. Data on telomerase structure and function are summarized in this review, and they are compared for evolutionarily remote organisms. Problems of telomerase activity measurement and modulation by enzyme inhibitors or activators are considered as well.