Clinical applications of telomerase in cancer treatment

REVIEWS Clinical applications of telomerase in cancer treatment Isabella Faraoni,1 Enzo Bonmassar,1,2 Grazia Graziani1 1

Section of Pharmacology and Medical Oncology, Department of Neuroscience,

University of Rome ‘Tor Vergata’, Rome, Italy, 2Istituto Dermopatico dell’immacolata’ (IDI, IRCCS), Rome, Italy

Abstract Telomerase activity has been found in most cancer cells, but not in the majority of normal differentiated tissues. Therefore, telomerase has been considered a relatively selective and widely expressed tumor marker to be used as a diagnostic tool, and in some cases, as a potential prognostic indicator.Telomerase activity can also be used to evaluate chemosensitivity of neoplastic cells obtained from cancer patients, by measuring residual telomerase activity after drug treatment. Finally, telomerase has been considered to represent a suitable target for designing new anticancer strategies.This review focuses on present and future clinical applications of telomerase studies in cancer management. © 2000 Harcourt Publishers Ltd

INTRODUCTION ince the first report on the detection of telomerase in a human tumor cell line,1 an increasing number of studies has been published that demonstrate high expression of this enzyme preferentially in tumors rather than in normal tissues. Even though the mechanisms that regulate telomerase function and expression have not been completely elucidated, its potential role in medical oncology is now firmly established. In this article recent results that may lead to clinical applications of telomerase research are reviewed and the utility of telomerase in the diagnosis of cancer and its possible prognostic value are discussed. Particular emphasis is given to the use of telomerase for developing novel chemosensitivity assays and to the design of anti-telomerase strategies for the treatment of neoplastic diseases.

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Telomeres The ends of linear chromosomes, including chromosomes of human cells, possess specialized terminal elements termed telomeres, that are composed of DNA and proteins and are essential in maintaining the stability of the eukaryotic genome.2 Telomeres consist of a variably long series of tandemly repeated DNA sequences that in mammalian species are TTAGGG hexamers.They are mostly composed of double-stranded DNA, that at the very end becomes a singlestranded filament, formed by a 3′ short G-rich overhang of about 40 base pairs (bp) in human cells.The total length of telomeres ranges from a limited number of bp up to 16 000

bp. Telomeres do not code for peptide products, but are required to stabilize chromosomes by ‘sealing’ their ends. Indeed, fragmented chromosomes are degraded by nucleases and become sticky, thus undergoing several types of recombination. Furthermore, telomeres protect genome from the loss of significant coding regions that might occur during cell proliferation. Substantial portions of telomeres (25–200 bp) are lost during each cell division without compromising the genome. In normal somatic tissues, cell duplication can go on for up to 100 divisions in the absence of mechanisms capable of restoring telomeric sequences.2 However, reduction beyond a critical length provides a signal for ‘cellular senescence’. It has been proposed that telomere shortening might function as a molecular clock that counts the number of divisions a cell can undergo before entering into senescence phase and eventually die.3 A number of proteins participate in the construction of telomeres. In humans, at least two proteins are involved in maintaining the correct structure of the telomere: the ‘Telomeric-Repeat binding Factor’ (TRF) 1 and 2.4 They carry a C-terminal motif that can bind the double-stranded telomeric repeat sequences. TRF1 binds along the length of the duplex telomeric repeat tracts and regulates telomere length. Its function requires other telomere-associated proteins, such as tankyrase, that catalyzes ADP-ribosylation of TRF1, increasing the accessibility of the telomere to telomerase,5 and the recently characterized protein TIN2.6 TRF2, instead, would play an important role in sequestration of the G-rich overhang thus preventing its recognition as a DNA break.7 The most recent view of telomere structure proposes that mammalian telomere ends are not linear, whereas telomeric sequences fold back to form a unique structure, the ‘t loop’, with the 3′ G strand overhang buried inside the double-stranded DNA at the loop junction. Sequestration of the single-stranded terminus into the duplex telomeric repeat generates a displacement loop (D-loop).TRF2 would stabilize the formation of the D-loop, thus hiding the potentially vulnerable G single-strand terminus8 and preventing an inappropriate DNA damage response. Telomerase Cells that escape limitations to proliferative potential could be able to evade telomere shortening either by activating telomerase or utilizing a telomerase-independent mechanism (Alternative Lengthening of Telomeres, ALT),9 thus restoring telomeric sequences that are lost during cell division. Telomerase is a ribonucleoprotein with reverse-trancriptase activity that uses its own RNA template to add TTAGGG repeats to the 3′ ends of DNA.1 It is a large multi-protein complex with two core elements represented by the ribonucleoprotein component (TR) and the catalytic subunit (TERT). While human TR (hTR) is expressed ubiquitously, human TERT (hTERT) expression strictly correlates with the enzymatic activity. This suggests that regulation of telomerase is mainly under the transcriptional control of hTERT. Telomerase activity has been shown to be undetectable in terminally differentiated cells of most organs.10 Conversely, it is present in most fetal tissues, germinal cells, inflammatory cells, and stem cells of self-renewing tissues (e.g. stem cells in  2000 Harcourt Publishers Ltd Drug Resistance Updates (2000) 3, 161–170 doi: 10.1054/drup.2000.0139, available online at http://www.idealibrary.com on

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Faraoni et al. the crypts of the gastrointestinal tract, basal cells of the epidermis, hematopoietic cells, endometrium during the proliferative phase of the menstrual cycle), and transiently in activated lymphocytes.10 Telomerase activity is overexpressed in the vast majority (~85%) of malignant tumors. It has been noted that tumor cells, in spite of possessing short telomeres, do not show loss of average telomere length during cell division.This suggests that telomerase activity may be required for indefinitely proliferating cells to escape from senescence processes. Telomerase activity can be easily measured by the PCRbased Telomeric Repeats Amplification Protocol (TRAP).11 The presence of enzymatic activity is revealed by the ability of cell extract to add telomeric repeats to a substrate primer. At the end of the incubation period, a second primer, complementary to telomeric repeats, is added and PCR amplification of telomerase extension products is carried out.This method allows a semiquantitative analysis of the enzymatic activity and is capable of detecting as low as 2–10 neoplastic cells, even in the context of a heterogeneous cell population.The assay has been further modified by adding an enzyme-linked immunoassay (TRAP-ELISA) to detect the telomeric ladder (Roche Molecular Biochemicals). This procedure appears to be similar to the TRAP assay in terms of specificity and sensitivity, having the advantage of a rapid analysis of a large number of samples. Furthermore, the cloning of hTR and hTERT has allowed measurement of telomerase expression on the basis of transcript12 or protein.13 Therefore, detection of the enzyme at the single-cell level, by in situ hybridization technique and immunohistochemistry, is now feasible. TELOMERASE ACTIVITY FOR CANCER DETECTION The absence or weak expression of telomerase activity in normal tissues and overexpression in tumor cells has suggested telomerase as a molecular marker for cancer detection. In most cases telomerase is useful for distinguishing malignant or premalignant tumors from benign neoplasms.A number of studies suggest that reactivation of telomerase might be an early event in carcinogenesis; for example, in lung14 and breast cancer.10,15 The development of the TRAP assay for telomerase activity in small tissue samples has opened up the possibility of evaluating telomerase expression in almost any type of clinical specimen (reviewed in ref. 16). In particular, telomerase activity has been tested in (Fig. 1): (a) fine needle aspirates from patients with breast carcinoma; (b) urine samples from bladder cancer patients; (c) bronchial washings or pleural effusions in lung cancer; (d) ascites in hepatocellular carcinoma17; (e) pancreatic duct brushing in pancreatic cancer; (f) oral rinses in head and neck squamous cell carcinoma; (g) biopsies in the peritoneal cavity of patients with ovarian carcinoma18; and (h) exfoliated cells from intestinal lavage collected during colonoscopy for colorectal carcinoma. Evaluation of telomerase activity may also be useful for the detection of residual disease after surgery. Recently, measurement of telomerase activity in ‘normal’ epithelium adjacent to resected tumor in patients with oesophageal squamous cell carcinoma, was found to be a highly sensitive method for detecting cancer cell microinvasion.19 162

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Another application of telomerase measurement is in the evaluation of response to chemotherapy or the detection of early tumor recurrence after surgery. In one study, in patients with ovarian carcinoma initially treated with tumor reductive surgery and chemotherapy, and then restaged by laparotomy,18 common tumor markers and telomerase activity were monitored in peritoneal biopsies. The results showed that the sensitivity of TRAP assay was 88% vs 64% of the routine cytological examination. Telomerase activity has also been used as a molecular marker for the detection of circulating cancer cells in the blood of patients with solid tumors. In a recent study with metastatic breast cancer patients, 84% of the patients were positive for telomerase activity in the blood.20 To prevent false positive results due to the presence of activated lymphocytes, immunobead enrichment of epithelial cells was carried out prior to the TRAP assay. Combination of this test with the telomerase-based chemosensitivity assay developed in our laboratory21,22 could provide a new approach to predict tumor responses to medical treatment in patients with metastatic disease (see below). In some cases telomerase activity can also be detected in benign tumors. This might occur when the tumor derives from tissues containing actively regenerating cells or when activated lymphocytes are present. Moreover, it has to be taken into account that about 15% of tumors can be telomerase-negative. In all these cases telomerase assay may give false positive or false negative results. DOES TELOMERASE HAVE PROGNOSTIC VALUE IN CANCER PATIENTS? Although a strong association between high telomerase activity and tumor progression has been documented, its prognostic value in cancer patients is still controversial. High levels of telomerase activity have been clearly associated with poor survival in neuroblastoma, as reported by Hiyama23 and neuroblastomas belonging to the stage IVS and lacking telomerase regressed.23 The prognostic value of telomerase in neuroblastoma has been further confirmed.24,25 Interestingly,these studies showed that telomerase activity helps to define two biologically distinct groups in stage IVS patients: a telomerase-negative one that regresses, and a telomerase-positive one that mimics advanced stage neuroblastoma with poor clinical outcome. A possible role of telomerase in defining a subset of patients at high risk of relapse has been recently demonstrated also for stage I non-small cell lung cancer.26 Moreover, studies on meningioma indicated that, although less than 20% of the cases were telomerase-positive, tumors with detectable activity had a very high probability of recurrence.27 Thus, telomerase expression appears to be a valuable index for identifying subsets of tumors with aggressive behavior in these types of neoplasia. However, for most other tumor types, a number of conflicting results have been published on the prognostic value of telomerase. In the case of breast cancer for example, a recent report indicates that the level of telomerase activity in invasive non-metastatic cancer patients does not predict survival.28 In contrast, another group demonstrated a correlation between high levels of telomerase activity and poor overall

Telomerase and cancer treatment

DIAGNOSIS

NON-INVASIVE

MINIMALLY-INVASIVE

PROCEDURES

PROCEDURES

blood

endoscopic brushing

pap smear

pancreatic juice

oral rinse

fine-needle aspirate

sputum

bone-marrow aspirate

urine

peritoneal fluid tru-cut biopsy

OPERABLE TUMORS

TREATMENT

INOPERABLE TUMORS

SURGERY

DRUG TREATMENT and/or RADIOTHERAPY

staging

Chemosensitivity assay on biopsy

prognosis post-surgery treatment

Chemotherapy and/or

Chemosensitivity assay

radiotherapy +

on biopsy

anti-telomerase treatment

Chemotherapy and/or

Evaluation of tumor response

radiotherapy + anti-telomerase treatment

Evaluation of tumor response

FOLLOW-UP

DETECTION OF TUMOR RECURRENCE (non-invasive or minimally invasive procedures)

Fig. 1 The role of telomerase in cancer treatment.Telomerase activity might be considered a detection tool for the presence of viable cancer cells and a target of tumor-suppressive treatments. See text for details.

survival in breast cancer patients with involvement of axillary lymph nodes.29 Establishing a clear-cut correlation between the level of telomerase activity and prognosis would certainly help to define patients that could benefit from adjuvant therapy. One of the problems encountered in studies that attempt to correlate telomerase levels and clinical outcome is due to the difficulty in quantitating telomerase activity in tumor cells from tissue specimens. The usual method of standardizing the TRAP assay by protein content has a number of limitations, such as the presence of telomerase negative normal

cells or necrotic areas in the tumor samples, that might result in underestimating activity levels in tumors. Thus, in situ techniques to detect telomerase components, in particular hTERT, might be required to quantitate telomerase expression and to better define a relationship, if any, between telomerase activity and cell survival. THE R-TRAP: A NOVEL CHEMOSENSITIVITY ASSAY One particularly interesting application of telomerase activity in cancer treatment has been proposed by our group.21,22 The

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Faraoni et al. selective presence of telomerase in tumor cells and the availability of an extremely sensitive method for detection of activity (the TRAP assay), prompted us to design an in vitro chemosensitivity assay with telomerase activity as a marker for tumor cell survival. The assay is based on the measurement of residual telomerase activity (R-TRAP) after in vitro exposure of cancer cells to antineoplastic agents. In vitro assays capable of predicting in vivo tumor response to drugs have always been considered of primary interest in medical oncology. Since tumor response is unpredictable in many cases, the possibility of determining drug sensitivity/resistance of tumor cells prior to the start of chemotherapy remains attractive. One of the most challenging technical problems in chemosensitivity testing is the difficulty to test the chemosensitivity of a very limited number of neoplastic cells in a heterogeneous cell population such as that obtained from biopsies or surgical specimens. Chemosensitivity assays based on cell viability or metabolism, do not discriminate between normal and neoplastic cells. In addition, chemosensitivity tests based on the colony formation on soft agar may correlate with the in vivo response of cancer patients to treatment. However, a limited number of tumors are able to grow in soft agar with satisfactory plating efficiency, thus preventing the use of this method in clinical practice on a routine basis. In previous studies on chemosensitivity of human tumor cell lines to a number of anticancer agents by R-TRAP, the killing of target cells was found to be associated with a decline of telomerase activity.21 The decrease of the enzymatic activity paralleled growth inhibition evaluated by cell count or by a chemosensitivity assay based on the cell’s ability to reduce tetrazolium salts (MTT). When a mixed cell population composed of tumor and normal cells was exposed to drug treatment, down-modulation of this enzymatic activity selectively reflected the actual drug sensitivity of the tumor cell population. In contrast, tumor chemosensitivity, evaluated by MTT assay, was largely underestimated when tumor cells were admixed with drug resistant normal cells.22 Therefore, it is conceivable that also in the case of tumor samples taken from patients, the presence of normal cells would not interfere with the interpretation of the results. Thus, R-TRAP provides the unique opportunity to test the effect of antitumor drugs selectivity on target cancer cells. The feasibility and reproducibility of the R-TRAP assay has been demonstrated using cell suspensions derived from the mechanical processing of surgical specimens of solid tumors of different tissue origin.22 The principal steps of the R-TRAP assay are outlined in Figure 2. Fresh tumor specimens are collected soon after surgery and are gently sheared in order to obtain a suspension of single cells. Afterwards, cells are counted, seeded in V-bottomed microplates and treated with graded concentrations of the drugs, including that corresponding to the plasma peak. After 24–48 of culture, cells are directly lysed in the plate and equal volumes of cell extracts are examined for the presence of residual telomerase activity. Several properties of the R-TRAP assay render this method a promising candidate for further development. In particular, (a) the assay is highly sensitivity, so that it can be performed with a limited number of tumor cells. This is of 164

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Shear fresh tumor material

4

Seed 10 viable cells in V-bottomed microplates

Drup treatment in a CO2 incubator (24-48 h of culture)

Direct lysis on plate

TRAP assay

Fig. 2 Flow diagram of the major steps in the R-TRAP assay.

particular value for small tumor biopsies; (b) prolonged tumor cell culture is not required. The assay can be completed within 24–72 h after sample collection; (c) the presence of contaminating normal cells is irrelevant; (d) R-TRAP is reproducible and could possibly be automated. TELOMERASE INHIBITION AS A NOVEL ANTI-CANCER THERAPY Telomerase inhibition in dividing cells is followed by telomere shortening leading eventually to cellular senescene and death. However, the time and the number of cell divisions required to produce detrimental effects on cell growth, makes it unlikely that telomerase inhibitors will be successfully used

Telomerase and cancer treatment Surgery

Radiotherapy

Chemotherapy

Reduction of tumor burden

Telomerase positive tumors

Long-term administration of telomerase inhibitors

¥ Death of residual tumor cells through cell senescence ¥ Growth inhibition of cells that escaped treatment ¥ Prevention of the growth of metastatic foci ¥ Suppression of radiation-induced upregulation of telomerase activity

Fig. 3 Role of telomerase suppression in cancer therapy.

as single agents in cancer treatment. Conversely, repression of telomerase would rather represent a novel adjuvant modality of cancer therapy following conventional treatment with surgery, chemotherapy or radiotherapy (Fig. 3). In this setting, anti-telomerase treatment would limit recovery of residual tumor cells through induction of cell senescence and would increase tumor cell killing by subsequent cycles of chemotherapy. Actually, high levels of telomerase expression are associated not only with unlimited proliferative potential, but also with increased resistance to apoptosis.30 Interestingly, it has been recently demonstrated that inhibition of telomerase increases the susceptibility of glioblastoma cells to cisplatin-induced apoptosis, thus suggesting that telomerase inhibition might represent a novel method of chemosensitisation for drug-resistant tumors.31 It has also been proposed that telomerase inhibitors could act as chemopreventive agents in cancer-prone syndromes or in early stage cancer to prevent spreading and overgrowth of metastatic cells.32 Finally, anti-telomerase agents might be used to counteract telomerase up-regulation induced by radiation. In fact, telomerase plays a role in the repair of radiation-induced chromosome breaks.33 This could contribute to enhance the proliferative potential and aggressiveness of cancer cells. It follows that anti-telomerase treatment might increase the efficacy of radiotherapy, by antagonizing possible mechanisms of radioresistance. The relative selectivity of anti-telomerase approaches in cancer treatment seems to rely on two main observations: (a) tumor cells possess telomerase levels higher than those of normal cells, at least in the case of activated lymphocytes, as demonstrated in our laboratory (Franzese O., unpublished observations) and elsewhere34; (b) neoplastic cells exhibit

telomeres that are shorter compared to those of telomerasepositive normal cells. This leads to the hypothesis that antitelomerase therapies would cause growth inhibition and possibly apoptosis of tumor cells, but not damage to normal cells. In the search of potent anti-telomerase agents two main strategies have been pursued so far: (a) the design of molecular and pharmacological modalities to selectively inhibit telomerase components or function, and (b) the large scale screening of compounds either of different structures and properties or selected on the basis of structural similarities with agents endowed with proven anti-telomerase activity. Molecular and pharmacological modalities to selectively inhibit telomerase components or function No data are presently available to establish whether the best strategy for telomerase inhibition would be to target the ribonucleoprotein complex, the multi-stranded structures of telomeric DNA or the binding proteins. Most of these approaches have been recently extensively reviewed by Helder et al. in the February 1999 issue of this journal.35 Herein, an update on the most recent results obtained with well-established anti-telomerase strategies is given and novel possible modalities to inhibit telomerase are presented. Target 1: the ribonucleoprotein complex Most of the approaches aimed at targeting the ribonucleoprotein complex have been directed against hTR. Successful inhibition of hTR has been achieved using a number of different modalities, targeting the template36 or the regulatory regions.37 In the latter case oligonucleotides would alter RNA

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Faraoni et al. secondary structure and interfere with the assembly of the ribonucleoprotein. In most cases, antisense approaches resulted in profound inhibition of human tumor cell growth that paralleled progressive telomere erosion.A variety of antisense oligomers have been tested, such as phosphorothioate oligonucleotides (PS), peptide nucleic acids (PNA)36,38 and 2′O-methyl-RNA oligomer.39 PNAs largely replaced PS, since they are resistant to degradation by proteases or nucleases and bind to targeted RNA with high affinity.The 2′-O-methylRNA oligomers are as potent telomerase inhibitors as PNA, even though they have lower affinity for complementary sequences.39 The absence of correlation between hybridization affinity and potency has been explained with the chemical and sterical similarities of 2′-O-methyl-RNA with DNA, the natural substrate of telomerase. These properties would favor electrostatic interaction with the protein components of telomerase. Moreover, differently to PNA, 2′-O-methyl-RNA oligomers can be easily delivered inside the cells, even though novel protocols for introducing PNA into eukaryotic cells have been recently described.37 Hammerhead ribozymes have been designed to selectively cleave the template region of hTR. Exposure of endometrial carcinoma40 or melanoma41 cells to these molecules resulted in reduction of telomerase activity. Telomere shortening was observed only in the case of endometrial cells. However, in both studies no persistent growth inhibitory effects were observed. It has been recently demonstrated that the efficacy of antisense molecules targeting hTR can be greatly enhanced by linking the antisense oligonucleotide to 2′,5′-oligoadenylate (2–5A), that activates RNAse L and degrades the RNA target.42,43 Surprisingly, treatment of tumor cells with this modified antisense induced profound apoptosis within 5–7 days that could not be due to telomere erosion, since cells would not have undergone enough cell divisions to critically shorten their telomeres.Therefore, these studies suggest that a different modality of antitumor activity might be exerted by telomerase inhibitors and indicate that a prolonged inhibition of the enzyme with gradual telomere erosion might not always be required. Target 2: the hTERT catalytic subunit Few successful modalities of telomerase inhibition that directly target the catalytic component of the enzyme have been described and they rely on the introduction of a dominant negative mutant of hTERT into tumor cells.44,45 Telomerase positive human cancer cell lines of different tissue origin, upon transfection of the mutant gene, underwent progressive telomere shortening during cell division. Interestingly, the authors demonstrated that the replicative capacity of transfected cells correlated with telomere length. Cells with short telomeres rapidly underwent growth arrest and apoptosis, whereas cells with long telomeres continued to proliferate for several cell generations before they stopped dividing. Inhibitors of reverse transcriptase (RT) activity such as nucleoside analogs and their triphosphate derivatives have been also evaluated as potential inhibitors of telomerase. Among these compounds 3′-azido-3′ deoxythymidine (AZT) and dideoxyguanosine (ddG) have been most intensively 166

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studied.The cellular response to these agents was highly variable, possibly depending on the cell ability to uptake the nucleosides and to generate the active triphosphate derivative.46,47 Transient telomere shortening has been observed, that was not always accompanied by growth inhibition. Moreover, in some cases cells resumed proliferating activity and became resistant to further treatment with RT inhibitors.48 Recently, it has been demonstrated that high concentrations of AZT are able to inhibit the growth of breast cancer cell lines and telomerase activity as early as three days after treatment.49 The mechanisms responsible of the prompt appearance of the antitumor effect have not been elucidated yet. However, it cannot be excluded that, due to ability of AZT to be preferentially incorporated in telomeric DNA, altered telomere structure might be sufficient to trigger growth arrest even in the absence of telomere shortening. Target 3: telomeric DNA and telomere-binding proteins An alternative way to interfere with telomerase function is to block the interaction between the enzyme and the telomere. Compounds that bind the telomere by interacting with guanine quadruplex formed by folding of the G-rich overhang, have been shown to inhibit telomerase in cancer cells.50 The so-called G-quartet ligands, are able to stabilize the G-quadruplex structure,thus rendering the telomeric ends inaccessible to telomerase.A number of studies has been published on the ability of compounds such as cationic porphyrin51, acridine,52 anthraquinone53 derivatives to induce reduction of telomerase activity. In the case of cationic porphyrin telomerase inhibition is accompanied by growth arrest.51 However, some concerns have been raised by the ability of G-quartet ligands to bind not only quadruplex but also duplex DNA and RNA with consequent toxicity against normal cells. Telomere damage can also occur after treatment of tumor cells with a low concentration of the antineoplastic agent cisplatin.54 Telomere shortening appeared as early as 24 h after drug exposure. Moreover, p53-dependent apoptosis was observed, presumably triggered by altered telomere structure. In this regard, it has to be noted that the telomeric repeat sequence TTAGGG might represent a good target for platination, since most of the DNA adducts generated by cisplatin are 1,2 intrastrand d(GpG) cross-links. Inhibition of telomeric-repeat binding proteins might represent another possible strategy for limiting the proliferative potential of tumor cells as suggested by data from Karlseder et al.55 Adenovirus-mediated transduction of a variety of tumor cell lines with a dominant negative mutant of TRF2 resulted in rapid induction of apoptosis in cells with intact p53 function.The prompt appearance of the antitumor effect suggests that telomeres lacking TRF2 might resemble damaged DNA and directly trigger cell death. It has been hypothesized that in the absence of TRF2, unmasked telomeres might be recognized as double-stranded breaks and signal the p53-pathway of cell death.55 Other telomereinteracting agents such as tankyrase or TRF1 itself may represent possible targets that merit to be investigated during the search of anti-telomerase strategies. New insights into the mechanisms of telomerase regulation will possibly identify novel targets for anti-telomerase

Telomerase and cancer treatment therapies. For instance, the Wilms’ tumor 1 suppressor gene can repress hTERT promoter and telomerase activity,56 whereas c-myc is able to reactivate telomerase function.57 In addition a telomerase repressor, capable of inducing telomere shortening and permanent growth arrest in tumor cells, has been recently identified in chromosome 3.58 Finally, biochemical manipulations aimed at preventing telomerase-substrate interaction could be adopted, e.g. by blocking nuclear translocation of the enzyme. With regard to possible clinical applications of compounds that interfere with telomere function, it will be important to evaluate their potential toxicity against normal cells. In fact, telomeres are essential for DNA replication, protection of chromosome and functional organization of the nucleus, either in normal or neoplastic cells. Screening of compounds The screening of chemical libraries is another approach that yielded a number of inhibitors capable to suppress telomerase activity in cell extracts.59 However, until now their ability to inhibit telomerase activity in vivo has not been evaluated. There is an increasing effort in the development of synthetic compounds on the basis of structural similarity of telomerase inhibitors. A new class of molecules has been recently developed.60 They are characterized by a tetracyclic structural motif similar to 9-hydroxyellipticine,61 an antitumor agent that has been shown to inhibit telomerase.These compounds are capable of inhibiting the activity of the enzyme in cell culture and in a cell-free system as well. MECHANISMS OF TUMOR CELL RESISTANCE TO ANTITELOMERASE AGENTS Possible mechanisms of tumor cell resistance to anti-telomerase therapies can be envisaged. A number of studies indicate that an ALT mechanism may allow maintenance of telomere length in the absence of telomerase.Actually, about 15% of malignant tumors, irrespective of their origin, do not express telomerase. Moreover, skeletal and soft tissue tumors are only occasionally telomerase positive and only at very late stages.62,63 In all these cases cancer cells would be naturally resistant to anti-telomerase agents. In addition, emergence in tumor cells of ALT mechanisms under the selective pressure of telomerase inhibitors has to be considered. Furthermore, cells endowed with defective p53 pathways might not properly respond to telomerase inhibitors. In fact, it has been recently demonstrated that telomerase inhibition in the presence of p53 mutation might promote tumorigenesis.64 Loss of p53 function abrogates the checkpoint triggered by critical telomere shortening, thus enabling cells to continue to grow despite increasing telomere disfunction and genomic instability.64 The consequent ‘genetic catastrophe’ would lead to overgrowth of cells with accumulation of genetic alterations. Therefore, therapies based on telomerase inhibition might be undermined by the appearance of therapy-related tumor variants with highly aggressive phenotype. In addition, mechanisms of resistance to anti-telomerase therapies would likely include pre-existing or acquired mutations that might reduce the affinity of the inhibitor for the target.

The overall picture that can be drawn from all these studies is that upon treatment with telomerase-inhibiting agents in most cases a lag phase is observed before occurrence of cell crisis and death, depending of the telomere length of tumor cells. However, with selected inhibitors, rapid appearance of cytotoxic effects occurred, suggesting the existence of telomere-derived signals other than those stemming from critical telomere erosion. Even though it is now clear that telomerase inhibition certainly represents an interesting and promising modality of cancer treatment, demonstration of the efficacy of this therapeutic approach in animal models is still lacking. Thus, at this point in vivo preclinical studies are eagerly awaited. Moreover, possible strategies to overcome tumor cell resistance to antitelomerase treatment need to be investigated. CONCLUDING REMARKS AND OUTLOOK At present the available methods for detecting telomerase activity are expensive and time-consuming. However, it can be envisaged that in the near future analysis of telomerase activity will be more suitable for everyday clinical practice. This will allow to test the enzymatic activity on a routine basis during the treatment of cancer patients from diagnosis to the evaluation of tumor cell response to the treatment (Fig. 1). Measurement of telomerase activity for the detection of cancer cells can be performed in samples obtained from noninvasive diagnostic procedures. A chemosensitivity test can be carried out by evaluating the residual telomerase activity after in vitro exposure of cancer cells to antineoplastic agents. For this purpose, the R-TRAP assay could be performed using cancer cells collected by non-invasive or minimally-invasive diagnostic procedures. However, when the number of cells available is not sufficient, the assay could be performed using cells released by the mechanical dissociation of biopsies of adequate size.The results of the test might be useful either when the patient will be treated with chemotherapy for advanced disease, or when antitumor agents will be used in neo-adjuvant setting before surgery. When surgery is the first therapeutic option, analysis of telomerase activity might have a number of clinical implications. In particular, telomerase activity can be investigated at the margins of surgical resection for the detection of residual disease. In addition, in those tumors for which telomerase has a proven prognostic meaning, quantitative analysis of the levels of activity present in the tumor mass would possibly help to predict the outcome of the disease. Moreover, if treatment with chemotherapy is required after removal of the tumor, chemosensitivity can be tested by the R-TRAP assay using surgical samples. Finally, determination of telomerase activity in various clinical specimens, including peripheral blood, before and after drug treatment, would provide the unique possibility of determining early tumor response in vivo. As a cautionary note for all clinical applications of telomerase assessment, the measurement of the activity in total cell extract might not always reflect the actual cell’s ability to maintain telomere length by re-activation of telomerase. The telomerase activity should instead be tested directly in the nuclei, because factors that control translocation of telomerase to the nuclei may play a role in post-transcriptional regulation of the enzyme. Telomerase-positive tumors that  2000 Harcourt Publishers Ltd Drug Resistance Updates (2000) 3, 161–170

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Faraoni et al. have a defect in nuclear translocation of the enzyme may indeed behave as telomerase-negative tumors. Moreover, association of the enzyme with nuclear chromatin appears to reduce telomerase function.65 In all cases of proven telomerase-positive tumors, telomerase inhibitors might play a potential role after tumor debulking by any other therapeutic modality (Fig. 3). However, despite the interest raised by telomerase inhibitors for cancer treatment, a number of problems still need to be considered. The toxicity of long-term administration of telomerase inhibitors requires to be carefully assessment. It is unlikely that long-term suppression of telomerase activity might spare hematopoietic stem cells, solely on the basis that these cells possess long telomeres. For instance, patients undergoing hematopoietic stem cell transplantation appear to be at high risk of developing therapy-related toxicity. In fact, it has been recently demonstrated that bone-marrow stem cells undergo telomere shortening after transplantation.66 Moreover, it has to be noted that relapsed malignant lymphomas might possess shortened, unchanged or even elongated telomeres, unrelated to the levels of telomerase activity.67 Therefore, in the case of relapsed tumors with long telomeres, treatment with telomerase inhibitor might likely require prolonged administration, that would possibly affect also normal proliferating cells. In this regard it should be noted that all subsets of normal human lymphocytes, including resting cells, express detectable levels of hTERT transcripts. However, the telomerase activity did not strictly correlate with the levels of the hTERT mRNA, suggesting the existence of post-transcriptional mechanisms of regulation of the enzyme.68 Therefore, agents capable of inhibiting hTERT transcription or translation might prevent telomerase activation when required by the cell in a later stage. This could impair, for example, the functional activity of differentiated cells of the immune system. In conclusion, a way is still to go before all information will be available on the possible role of telomerase determination in cancer diagnosis and prognosis, drug sensitivity testing and on the risks and benefits of anti-telomerase therapy for cancer treatment. ACKNOWLEDGEMENTS

The authors are grateful to Dr Pedro M. Lacal for helping us in preparing the figures and to Mrs Barbara Bulgarini for technical assistance. The number of papers published on telomerase largely exceeds those quoted in this mini-review. We apologize for having omitted many important articles from the reference list due to editorial restrictions.

Received 22 March 2000; Revised 6 April 2000 Accepted 13 April 2000 Correspondence to: Grazia Graziani MD, Department of Neuroscience, University of Rome ‘Tor Vergata’,Via di Tor Vergata 135, 00133 Rome, Italy.Tel: +39 0672596338/35; Fax: +39 0672596323; E-mail: [email protected]

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