What is the difference between telomeres and telomerase

Such IR-induced SC enrichment has been implicated in radiotherapy failure, accelerated repopulation, and evasion of tumors to CSC targeted therapies Studies increasingly support SCs as critical considerations in the radiation response, whether associated with treatment of cancer radiotherapy or exposure of normal tissues carcinogenesis as occurs unavoidably in conjunction with radiotherapy and a variety of medical diagnostic procedures, as well as accidentally e.

It is widely viewed that IR-induced enrichment of CSCs results from mobilization and asymmetric division of existing CSCs, which have been shown to be more radioresistant than their more differentiated non-stem cancer cell NSCC counterparts, due not only to their residing in relatively hypoxic niches but also because they possess enhanced DNA repair kinetics, superior endogenous oxidative stress defenses, and slower cell turnover rates Importantly, however, Lagadec et al.

Therefore, enrichment of CSC populations following radiation exposure may arise either by way of mobilization i. Implicit in these observations, is the reality that therapeutic strategies seeking to target CSC populations must address both mobilization and reprograming in order to be effective. Telomerase activity has also been shown to be radiation-inducible in a variety of tumors and cancer cell lines, including mammary carcinoma, acute myeloid leukemia AML , colon carcinoma, squamous cell carcinoma of the oral cavity, lymphoma, and nasopharyngeal carcinoma 28 , 43 — Such observations led us to suspect unappreciated correlations between these processes.

Furthermore, exposures to high LET, high charge, high energy HZE particles, such as those delivered during carbon ion radiotherapy or encountered in the deep space environment, have been shown to invoke very different biological responses than low LET radiations, which may well include IR-induced telomerase activity and subsequent SC enrichment.

However, it is important to note that longer telomeres do not necessarily confer radioresistance. It is also debatable whether such a relationship holds true in telomerase positive cells, as there are reports of no correlation between telomere length and radiosensitivity Other contrasting reports include longer telomeres in irradiated 4 Gy X-rays vs.

Interestingly, low LET X-rays and low energy high LET protons have been shown to induce very different telomeric responses, in that telomeres were shortened 96 h post-X-ray exposure and associated with anaphase bridges and dicentrics, while high LET protons evoked telomere lengthening at 24 and 96 h Considerable controversy and uncertainty surround such results and the processes responsible for them, but accumulating evidence, including much of our own [e.

Here, we provide new insight into the roles of telomeres and telomerase in the radiation response. Specifically, we investigated the influence of dose, dose rate, and radiation quality on IR-induced changes in telomere function, length, and telomerase activity in panels of cancer and non-cancer mammary epithelial and hematopoietic cells.

Depletion of the telomeric end-binding proteins TRF1, TRF2, or POT1 resulted in dysfunctional telomeres that were uncapped as opposed to critically shortened, which 1 increased spontaneous and IR-induced mutation frequencies in a radiation quality-dependent manner, with POT1 depletion being especially effective, and 2 contributed to instability in that they were susceptible to fusion with each other and to IR-induced DSBs, as well as to recombination telomere sister chromatid exchange; T-SCE.

Furthermore, we demonstrate that IR-induced SC enrichment is telomerase dependent, and separate modeling efforts support the necessity of contribution from cellular reprograming for such enrichment manuscript in preparation, Gao et al.

Better understanding of these fundamental processes involving telomeres and telomerase following IR exposure, particularly of different radiation qualities, is vital, as they play potentially critical roles in accelerated tumor repopulation following radiotherapy, as well as IR-induced carcinogenesis following exposure, including those of relevance to astronauts.

The human mammary carcinoma cell line MCF-7 kind gift from L. A human low passage lymphoblastoid cell line LCL, kind gift from A. The WTK1 human lymphoblastoid cell line was derived from the WI-L2 line 69 , and used for mutation analysis, as they are heterozygous at the thymidine kinase TK locus; they also have a single amino acid substitution in codon at TP A variety of control cell lines were included for comparison.

Primary BJ-1 normal human foreskin fibroblasts kind gift from J. Shepherd located at Colorado State University.

Cells were exposed at a dose rate of 2. Unirradiated controls were kept in a separate incubator under identical conditions. Immediately following exposure, cells were shipped overnight back to Colorado State University for processing and analysis. MCF-7 and MCFA cells were seeded in triplicate 48 h prior to irradiation and allowed to incubate under standard culture conditions to ensure that all cultures were in log phase.

Cells were then allowed to incubate for 16 h overnight to allow for repair to occur. Cells were then trypsinized, counted, and plated at the appropriate density in quadruplicate into 60 mm culture dishes. This process was repeated three times to generate the data presented here. Chromosome-orientation fluorescence in situ hybridization CO-FISH was employed to evaluate IR-induced chromosomal instability and performed as previously described 62 , 70 with some modification.

Slides were stained with Hoechst 0. Preparations were examined and images captured and analyzed using a Zeiss Axioskop2 Plus microscope equipped with a Photometrics Coolsnap ES2 camera and running Metavue 7. Telomere sister chromatid exchange was scored as a CO-FISH telomere signal split between the two chromatids of a metaphase chromosome, which were often of unequal intensity due to unequal SCE Two days after irradiation, when phenotypic expression of newly induced mutants was complete, the mutant fractions MFs were determined.

Fresh TFT was added 11 days after plating, and plates were scored for positive or negative wells after 20 days. The MFs were calculated using the Poisson distribution, and statistical analyses were done by t -tests using Sigma Stat 3.

Samples were prepared using standard cytogenetic techniques as described previously with slight modifications 68 , 72 , Fixed cell pellets were then washed and dropped onto glass slides for telomere fluorescence in situ hybridization FISH , which was performed as described previously with modifications Image Z stacks were taken using a Zeiss Axio Imager.

Z2 microscope, with a Coolsnap ES2 camera running Metamorph 7. For each slide, 30—50 images were obtained, each consisting of 22, 0. Metamorph nearest neighbor deconvolution and stack compression functions were applied, followed by image thresholding upper and lower threshold values were held consistent across experiment.

Finally, a region of interest was created for each nucleus, and the intensities of individual telomeres obtained in metamorph. Fluorescence values in each batch of FISH were standardized to the fluorescence intensity of an LY-R mouse lymphoma cell pellet as an internal control.

LY-R cells have long brightly staining telomeres and their use for standardization, which was adapted from Q-FISH 75 , represents a means to accurately compare relative telomere lengths from run to run.

Blinded subjective scoring of blue cells was used to quantify senescent cell fractions. Telomerase activity was evaluated using the telomere repeat amplification protocol TRAP assay originally described by Herbert et al. Protein concentration was determined using the Bradford Assay Biorad. Each well contained between 0. In addition to the treatment samples, a series of controls were included on each plate: 1 no template control with TS primer only, 2 no template control with ACX primer only, 3 no template control with TS and ACX primers used in normalization of samples , 4 heat inactivated control with template protein lysate and TS and ACX primers, and 5 HeLa cell lysate with TS and ACX primers a positive control.

Each sample was run in triplicate on a well plate format allowing for an average Ct to be obtained per sample. Briefly, to calculate the percent relative activity for each sample, first normalize the average sample Ct to the no template control with TS and ACX primers. This is referred to as the delta Ct value. The RTA can be compared between samples assayed across different plates.

Results from two runs were averaged. Thiazolyl blue tetrazolium bromide MTT assay was used to evaluate potential cytotoxic effects of the MST telomerase inhibitor as described previously Cells were incubated in the presence of inhibitor for 48 or 72 h.

At the time of analysis, media was once again removed from wells, and cells were resuspended in fresh media containing 0. After agitation, plates were read on a Modulus Microplate reader Turner Biosystems at nm absorbance.

Monolayers MCF-7 and MCFA mammary epithelial cells or hematopoietic cells in suspension were dissociated and stained for marker expression. Cells were pelleted, aspirated, resuspended in buffer containing efflux inhibitor, and analyzed on a flow cytometer.

Cells were plated into well plates at limiting dilutions, then allowed to form spheres for up to 10 days with fresh media supplementation every 3 days. Therefore, we can now surmise that the MFs for Fe in the earlier experiments reported here are likely two to threefold higher than shown. This in turn would imply important differences for HZE exposures in the context of telomere deficiencies. Interestingly, telomere deficiencies consistently resulted in higher MF than inhibition of DNA-PKcs kinase activity, a well-characterized contributor to DNA repair, providing additional support for the significance of telomere proteins in the DDR.

Curvilinear dose responses for individual knockdowns were also suggested Figure 1 , indicating intertrack interaction of multiple lesions at higher doses, and likely reflecting additional interactions between dysfunctional telomeres and IR-induced DSBs T-DSB fusions 66 ; such a supposition is supported by increased frequencies of these events at 2 Gy Figure 2. Figure 1. Compromise of telomeric end-capping function elevates spontaneous and radiation-induced mutagenesis.

Figure 2. The contribution of telomere end-capping function to IR-induced chromosomal instability was also evaluated, as per our previous works 61 — 63 , Previous reports have demonstrated elevated telomerase activity following IR exposure; however, results are often conflicting in regard to dose, dose rate, radiation quality, method of telomerase activity measurement, and cell line examined.

Therefore, we sought to more clearly characterize telomerase activity in response to a variety of IR exposures in both tumor and non-tumor cells. Telomerase activity was evaluated relative to the telomerase-positive HeLa cell line Figure 3. Figure 3. Telomerase activity in cancer and non-cancer cell lines.

Data are represented on a log scale, and all values are reported as telomerase activity relative to HeLa cells. Figure 4. In general, cell lines with higher levels of background activity experienced higher, more significant elevation of activity 24—48 h postexposure, the notable exception being normal PBMCs very low telomerase activity; significant IR-induced elevation.

Telomerase activity in irradiated cells is reported relative to unirradiated control samples collected at the same time point. The low passage transformed normal lymphoblastoid cell line LCL very low telomerase activity showed elevated telomerase activity at 24 h post 1 and 4 Gy exposure, but neither rose to the level of significance. Telomerase activity was not significantly elevated at either 1 or 4 Gy total dose, at any dose rate, in any cell line examined Figure 5 A. These results imply that in contrast to acute exposures, elevation of telomerase activity is not triggered by low LET, LDR exposures.

Figure 5. In general, no elevation of telomerase activity was observed, with the exception of WTK1 at 1 Gy, 1. B In contrast, telomerase activity in stimulated PBMCs very low telomerase activity exposed to a total dose of 1 Gy delivered at low dose rate of 4. Data are expressed as telomerase activity relative to unirradiated controls within each group.

Furthermore, histogram analysis of individual telomere lengths demonstrated that IR exposure shortened all of the telomeres in the population i. The observed post-IR telomere shortening also corresponded with a significant increase in senescent cells SA-Beta gal positive at day 5, which remained significantly elevated until at least day 10 Figure 6 C. Furthermore, by day 10 postexposure, telomere length in the surviving cell population began to recover, despite reduced levels of telomerase activity during this same time period.

These results support the notion that following IR exposure, telomerase is acting outside of its canonical role in elongating telomeres. Figure 6. Ionizing radiation-induced telomere shortening in mammary epithelial cells and stimulated PBMCs.

By 10 days, telomere length had increased but still remained significantly shortened compared to unirradiated controls. As normal SC and CSC populations generally possess higher telomerase activity than their non-stem counterparts, and IR-induced enrichment of putative SC populations in mammary carcinoma cells has been reported by multiple groups 23 , 28 , 29 , 31 , 83 , we hypothesized that observations of elevated telomerase activity following IR exposure may result from the enrichment of CSC populations.

Counter to our initial hypothesis, IR-induced elevation of telomerase activity preceded the observed enrichment of SC compartments, indicating that they are not directly correlated i. Figure 7. Ionizing radiation-induced enrichment of putative mammary stem cell populations. A Clonogenic survival curves and IR dose response. B Time course evaluation of putative mammary stem cell compartments. C Radiation dose response of putative mammary stem cells 5 days postexposure.

This result indicates that radiation does not induce enrichment of putative SC populations in all cell lines, cancer types, or tissues, which may, at least to some degree, be dependent on background levels. Figure 8. Ionizing radiation-induced enrichment of putative hematopoietic stem cell populations. Figure 9. B MST reduced telomerase activity in a dose-dependent fashion in both cell lines. The results reported here provide valuable insight into the critical roles telomeres and telomerase play in the radiation response and thereby support further exploration for their roles in the context of both radiotherapy and IR-induced carcinogenesis.

Specifically, we have assessed the roles telomeres play in maintaining genomic stability following IR exposure, elaborated on the role telomerase plays in cell survival and repopulation postexposure, and identified a potentially targetable role of telomerase activity in cells exposed to therapeutically relevant doses of low LET radiation.

Of relevance in this regard are reports of decreased expression of TRF2 associated with increased breast cancer malignancy 95 , as well as the demonstration of telomere fusions in early human breast carcinoma Furthermore, our finding of telomere uncapping with POT1 deficiency is consistent with POT1 mutations identified in a subset of patients with chronic lymphocytic leukemia CLL , which were associated with increased levels of chromosomal fusions involving telomeres A particularly relevant recent report specifically associated various POT1 variants with telomere length and radiosensitivity in colon and gastric adenocarcinoma Our results provide additional support for the view that in addition to critical telomere shortening, telomeres rendered dysfunctional by virtue of deficiencies in telomeric proteins and the end-capping failure that ensues, also contribute to the carcinogenic potential of radiation exposure.

These observations are consistent with the proposed roles for POT1 in suppressing ATR at telomeres 90 and facilitating a RPA-to-POT1 switch 91 during replication, and thereby suppressing telomeric recombination, here particularly in response to the complex damage induced by high LET radiation exposure. Taken together with the reported association of POT1 variants with radiosensitivity and colon and gastric adenocarcinoma 98 , our results suggest that heavy ion radiation therapy may be particularly effective in treating these cancers.

Consistent with previous reports, we also demonstrate that telomerase activity is an IR-inducible function. We elaborate that in vitro , this phenomenon appears to be peculiar to cell lines with high background levels of telomerase activity e. Expression of telomerase subunits in MCFA cells was dramatically different in that both hTERT mRNA and hTERC steadily decreased from 1 to 5 days postexposure, a finding consistent with the absence of significant elevation of telomerase activity in this same timeframe.

It is also important to appreciate, however, that although not significant, increases in telomerase activity were observed in the non-tumor MCFA and normal LCL mammary epithelial cell lines following acute IR exposure, indicating that telomerase may indeed be induced, but the low background level of activity in these cell lines may yield such an increase relatively insignificant.

Additionally, evaluation of telomerase activity in stimulated normal human PBMCs low background level revealed a significant increase following an acute dose 1 Gy , 2 days postexposure.

And these red And I'm going to tell you a lot about that telomeric So why are telomeres important? Their role is to cap So that's a simple concept, So when we think Now, the job of a cell is to seal And so a Also, such DNA breaks are subject to Now the telomere protects, it caps, the end How does it do that? The DNA sequences that you They're They're not genes in the sense of In humans for example, Another feature of the chromosomal DNAs And in fact the And that turns out to be important.

So, As I said, it doesn't encode any protein They bind to These together make some form of higher order We understand a lot Some of the details of the protein-protein So I've just shown you a functional The This kind of dysfunction caused by the The other way that telomeres can become Now indeed, cells have a lot of very And the consequences for the cell is that, usually So this limits cell renewal capability if this happens If by chance the So clearly telomere function is very And in fact, one of the consequences I'm just going to show you a picture.

So here's the two telomeres at the end, here's the So this is now a chromosome that has two Here's another example of such an end-to- This kind of change can happen The other kind of So what's Well, the mechanism of DNA replication, It's very good at faithfully copying almost all the way Now the And this is just a simple consequence of the But the very ends cannot be So what's the consequence of this loss?

But even on a This was a prescient idea because, in fact, this So, shortening of How is this problem solved? Well, it's solved by an So I'd like to introduce you to Telomerase was And let me give you some examples of such Well, the first one was that in the And heterogeneous in this So So here were different Now, a second kind of The Tetrahymena cells go through a lifecycle And It wasn't clear.

The third observation came And these were being propagated And this didn't look Now one could put But what was found was that if one And that didn't look like a reaction one All of these suggested that And that idea, in a conceptual way, was given In such wild-type maize, if a Nobody knew the molecular basis of that. And when All of which, then, And you can see the This experimental system was And indeed that proved to be the case.

Now let We took a mimic of that And I've just And here's its 3' hydroxyl end. We mixed it All of this was hypothetical In fact, in the test tube, a So we get a lot So this is what telomerase did. Now how is it It was adding a given sequence.

The first Every time we see a labeled band, this is And what you can Every six Now, certain clues came. Not any So, two examples of the telomeric These Another interesting Now, we had And the yeast repeats have It's T, G, sometimes one G, sometimes two, So we wondered if the converse And indeed, we did see such addition.

Now we also noticed When we When we had a primer that ended with three And I haven't shown So this suggested that perhaps something Because in other words, if you What So was there something that aligned Remember this So in fact we looked and If you had three Gs, one G was added. And if you had one G, then it would be three Gs, and So in fact, there was something that was The result of all of this kind of And this So what telomerase does is it takes that single- It aligns it on this part of the sequence, and And so now, you end up with So telomerase is a unique polymerase, it's a It's unique because the RNA component is It has to be built in for that And indeed the enzyme is truly a And unlike, for And it does it by this If I've told you about some of the enzymatic Well, the answer came by manipulating telomerase So if you remember, telomerase is adding And they have plenty of telomerase.

Now, we made The telomeric DNA repeat So in other words, From being immortal, they'd become mortal. And all So, the Telomeres are replenished by telomerase And in fact, that continuing So that's what telomerase does for And that's the important message: Telomeres are Now, let's go into a more If we look at a culture So, the cells are growing well Now if we delete one of the genes for And so now they're no longer See, there's very little growth here.

And we call that So taking away telomerase is a bit of a We've Interestingly, a few cells do survive, and in fact, These cells I'll A For at least These are in the They're needed for Similar phenomena have been seen in They've been less well characterized ALT cells. So, without So, that is another way that Now we Although if you look We asked what's So first of all, we quantified very carefully the cell The colony- And we looked at the First of all we looked And this is what's And the most important are You can Now, there are very few cells here actually, but And then the cells start growing So that's the telomeric profile.

Now, This is what's And one asks, does a particular gene, If it becomes lower, it gets greener and greener. Now right away what you can Six- So one can analyze all the So one can Now we found a set of So the Right at the stage of That actually If you remember, I told you that, At senescence, we see a Although telomeres are generally considered to be localized structures at the ends of chromosomes, such sequences are also being identified at internal positions in chromosomes 4.

The length of telomeres also varies among different species. Humans have telomeres 8—14 kilobasepairs kbp long, whereas the mean telomeric repeat lengths in some ciliates are as little as 36 bp, and those in mice may be as much as kbp 5. In human chromosomes, telomeres are adjoined centromerically by a subtelomeric region consisting of degenerated telomeric DNA sequences and unique repeats 6 , and Fig.

All chromosomes lose a small amount of telomeric DNA during each cell division Fig. The leading strand is replicated continually. Removal of the terminal RNA primer on the lagging strand leaves a gap that ordinarily is filled in by extension of the next Okazaki fragment. The loss of genomic sequences at each replication cycle can be compensated by addition of terminal sequences through various mechanisms: e.

Moreover, organisms possess the ability to transfer species-specific terminal sequences onto DNA: Shampay et al. The crucial experiments came from Greider and Blackburn 10 11 , who detected in Tetrahymena extracts an activity that added telomeric repeats to single-stranded telomeric DNA oligonucleotide primers; they also found that this process is inactivated by treating the extract with a RNA-degrading enzyme. Therefore, this RNA-dependent activity, named terminal transferase or telomerase, was found to be a ribonucleoprotein complex that utilizes sequences of its own RNA component as a template for the de novo synthesis of telomeric DNA sequences.

Both RNA and several protein components of telomerase are needed for enzymatic activity. Recently, the telomerase RNAs of yeasts and humans have been cloned 12 The telomerase proteins seem to have structural importance by binding dNTPs deoxynucleoside triphosphates or DNA sequences. The basic telomerase reaction mechanisms are primer recognition and base pairing, nucleotide addition, and translocation. Accordingly, Kim et al. Based on identification of telomerase mechanisms and properties, the telomere repeat amplification protocol includes preparation of a protein extract by cell lysis and adding a primer and dNTPs.

If telomerase is active in the extract, it elongates the added template, and the reaction product is amplified by PCR. Because telomerase pauses after synthesis of a set of six nucleotides, amplification products separated on a polyacrylamide gel look like a 6-bp nucleotide ladder. Using this amplification protocol, one can detect 1 telomerase-positive cell among 10 cells. Because the lost sequences of the telomeres at each replication cycle can be resynthesized by telomerase, the enzyme has essential functions for cell immortalization Fig.

More than 20 years ago, Olovnikov showed that the loss of telomere sequences because of the end replication problem might explicate a possible role in regulating cellular lifespan Telomere shortening was ascribed a function, i. According to this telomere theory, a small amount of telomeric DNA is lost with each round of cell division.

When the telomere length is reduced to a critical point, a signal is given to stop further cell division, the hallmark of cellular senescence.

Telomere length and stability in adult somatic cells differ from those in fetal and germline cells. In the somatic tissues of an individual, the telomeres are noticeably shorter than in sperm cells, and lengths decrease with increasing age of the individual.

On the other hand, telomeres in sperm maintain their length independently of increasing individual age In cultured cells, the loss of telomeric DNA depends on the number of cell divisions, and Allsopp et al. In this context, the question arises as to whether one could achieve unlimited replication capacity and immortality in somatic cells if telomere length could be maintained.

Counter et al. The transfected cells divided and entered a point of crisis, in which most of the cells died; only some cells became immortal. During the period of cell division, the telomeres shortened continually and no telomerase activity could be detected. Those cells that survived the crisis point and became immortal had reactivated telomerase and stabilized their telomeres. This means that even somatic cells can gain the ability of endless replication if telomere length is maintained and or the enzyme telomerase is activated.

The stimulus for the induction of M1 may be DNA-damage signals from the altered expression of subtelomeric regulatory genes or from a critical shortened telomere. P53 and the retinoblastoma gene product pRb are involved in the execution of M1.

One hypothesis for the induction of M1 postulates the following: a a single chromosome denuded of telomeric repeats produces a DNA-damage signal, which b induces p53 and p21; c p21 inhibits the cyclin-dependent kinases, which then d are prevented from phosphorylating pRb; e the presence of unphosphorylated pRb coupled with other actions of p53 and p21 results in the M1 arrest If these cell cycle regulators are mutated or blocked, the cells continue to divide and thus the telomeres continue to shorten.

The M2 mechanism is probably induced when so few telomere repeats remain that the unprotected chromosomal ends block further proliferation. The M2 block might be overcome in some cells by reactivation of telomerase, the repair of chromosome ends, the stabilization of telomere length, and the generation of an immortal cell clone 21 , and Fig.

The findings concerning telomerase activity in various human tissues are in accord with the differences in the telomere length described above: The enzyme is detectable in germline cells, but not in most postnatal somatic tissues Table 1. Telomerase-independent mechanisms may also exist that stabilize telomere length.

Murnane et al. So far, it is not clear whether telomere stabilization in these cells is achieved by the above-mentioned recombination or transposition events, or whether transformed cells may develop alternative mechanisms to circumvent any possible telomerase inhibition.

The present knowledge about the role of telomeres and telomerase in cellular senescence carries scientists a step forward toward understanding the phenomenon of human aging—a process accompanied by an accumulation of various cytogenetic changes and an increasing deficiency in DNA repair mechanisms. Lindsey et al. These premature aging syndromes are characterized in progeria by growth retardation and accelerated degenerative changes of the cutaneous, musculoskeletal, and cardiovascular systems in young patients 25 , and in Werner syndrome, for which recently the a candidate gene has been identified 26 , by an early-onset and accelerated rate of development of major geriatric disorders such as atherosclerosis, diabetes mellitus, osteoporosis, and various neoplasms Recently, Kruk et al.

Possibly this deficiency in telomeric repair is correlated with an age-related increase in genetic instability. The cell cycle includes the orderly sequence of events that ensure the faithful duplication of all the cellular components in their correct sequence and the partitioning of these components into two daughter cells.

Two classes of genes and their protein products are used to accomplish this process: genes whose products are obligatory for progress through the cell cycle phases, and genes whose proteins act as checkpoints for monitoring the efficacy and completion of these obligatory events and stopping the progression through the cell cycle if conditions are not satisfactory. The loss of cell cycle control generally leads to cell death but can also result in abnormal cells that continue to replicate and eventually form a tumor for a review, see The theory of carcinogenesis suggests that unlimited cell proliferation is required for development of malignant disease, and cancer cells must attain immortality for progression to malignant states.

As shown above, shortening of telomeres may contribute to the control of the proliferative capacity of normal cells, and the enzyme telomerase may be essential for unlimited cell proliferation. The length of telomeres in cancer cells depends on a balance between the telomere shortening at each cell cycle and the telomere elongation resulting from telomerase activity.

Tumors with shorter telomeres than in the original tissue have been detected in many cancer types for a review, see In neuroblastoma, endometrial cancer, breast cancer, leukemias, and lung cancer, a correlation between decreasing telomere lengths and an increasing severity of disease has been described 31 32 33 34 Short telomeres seem to be a primary cause for karyotype instability in malignant cells.

According to the above-described theory of telomere dynamics during cell progression, tumor cells with shortened telomeres can be considered to have undergone many cell divisions, with an accumulation of various genetic alterations.

After a point of critical telomere shortening, telomerase might be reactivated to stabilize or elongate the telomeric DNA. Tumors with telomeres just as long as or even longer than in the original tissue seem to be rarer but have been described in some human malignant tissues, e. There are two possible explanations for this phenomenon: Either an activated telomerase has elongated the once-shortened telomeres back to former length, or the tumor cells have not yet undergone enough cell divisions to induce significant shortening of telomeres.

Low amounts of telomerase activity in normal human tissues were found only in hematopoietic progenitor cells and activated T- and B-lymphocytes 41 ; in germ cells, ovaries, and testes 42 ; and in physiologically regenerating epithelial cells Results from examinations of normal tissues and benign cancers as well as malignant primary and metastatic tumors permit several conclusions.

As in most normal tissues, telomerase activity is not expressed in somatic tissues adjacent to the tumor tissue. Accordingly, telomerase activity has proved to be a reliable marker for detecting tumor cells in resection margins. In benign and premalignant tumors, including breast fibrocystic disease and fibroadenomas, benign prostatic hyperplasia, colorectal adenomas, anaplastic astrocytomas, and benign meningiomas and leiomyomas, in general no telomerase activity was detected; however, it was found in malignant tumor stages 44 45 46 In this way, telomerase activity is associated with the acquisition of malignancy.

The detection of telomerase activity at preneoplastic or benign growth stages may signify disease progression and be of diagnostic value. For example, telomerase activity has been found in some cases of benign prostate hyperplasia and of benign giant tumors of the bone 45 48 —all tissues that may progress to malignant tumors. As shown by Hiyama et al. Certain tumor types, such as neuroblastoma, display a lower telomerase activity in early-stage cancers, whereas expression in late-stage cases is higher Neuroblastomas of a special stage stage IV , which had short telomeres and no or weak telomerase activity, tended to regress spontaneously 49 —possible proof of a correlation between an enzyme activity too weak to remain in an immortal tumor status and a favorable outcome for the patient.

Another example of telomerase activity in cancer diagnosis and as a prognostic indicator of clinical outcome is the results found in gastric cancers. Hiyama et al. Although a reliable tumor marker, telomerase activity is not an all-or-none phenomenon. To understand the regulation of telomerase during tumorigenesis, Greider et al. Further prospective and retrospective clinical studies must be carried out to assess the validity of telomere dynamics and telomerase as a diagnostic or prognostic marker in many cancer types.

These findings suggest that reactivation of telomerase may be an obligate event in cell immortalization and in most instances of tumorigenesis. Any kind of inhibition of the enzyme should therefore lead to resumption of telomere shortening and might activate the cellular senescence pathway.

The fact that telomerase is absent and not required in most human somatic tissues but is necessary for tumor growth should make this enzyme an ideal target for anticancer therapy.

One of the greatest challenges in cancer therapy is to achieve a high therapeutic effect by maximizing the desired reactions and minimizing the undesired side-effects.

Several conditions must be successfully fulfilled to achieve a beneficial antitumor effect in vivo:. One of the most hopeful approaches to achieving these goals is oligonucleotide-based gene therapy.

This high degree of specificity has made antisense constructs attractive candidates for therapeutic agents Because the antisense sequences require chemical modifications to avoid destruction by nucleases and to form complexes for better delivery into the cell, peptide nucleic acids PNAs have been designed—with a charge-neutral, pseudo-peptide backbone of N - 2-aminoethyl glycine units instead of a negatively charged deoxyribose-phosphate backbone Recently, Norton et al.

These molecules seem to be very specific and efficient inhibitors of telomerase in vitro. Further experiments with cell cultures and tumor models in mice will continue this path of investigation and could justify the optimism surrounding the telomerase hypothesis and its exploitation as a novel anti-cancer therapy. Several important unanswered questions remain.

A possible therapeutic approach of telomerase to cancer patients would appear to be less toxic than conventional chemotherapy, which affects all dividing cells and has undesirable side effects. However, some normal somatic cells are telomerase-positive at baseline: human hematopoietic progenitor cells, germ cells progenitors of sperm and oocytes , and activated T-and B-lymphocytes. Another problem is in the variability of telomere lengths among tumors. Telomerase inhibition seems to be useful only in malignant cells with short telomeres; tumor cells with long telomeres would require a prolonged treatment, with possible toxic side-effects.

One should also consider that alternative pathways besides telomerase may exist for regulating telomere length. Many other issues remain to be resolved. Are telomeric RNA and proteins regulated at the transcriptional or posttranscriptional level? Does regulation of the RNA or of the protein components determine activity?

Do internal cellular mechanisms exist to repress telomerase?