agents that target telomerase and telomeres

583

Agents that target telomerase and telomeres Eric Raymond*, Daekyu Sun, Shih-Fong Chen, Bradford Windle and Daniel D Von Hoff

Telomeres are guanine-rich regions that are located at the ends of chromosomes and are essential for preventing aberrant recombination and protecting against exonucleolytic DNA degradation. Telomeres are maintained by telomerase,

an RNA-dependent DNA polymerase. Because telomerase is known to be expressed in tumor cells, which concurrently have short telomeres, and not in most somatic cells, which usually have long telomeres, telomerase and telomere structures have been recently proposed as attractive targets for the discovery of new anticancer agents. The most exciting current strategies are aimed at specifically designing new drugs that target telomerase or telomeres and new models have been formulated to study the biological effects of inhibitors of telomerase and telomeres both in vitro and in vivo.

Addresses Human Telomerase Research Group, Institute for Drug Development - Cancer Therapy and Research Center, 14960 Omicron Drive, San Antonio, TX 78245-3217, USA * Correspondence: Eric Raymond, Translational Research Laboratory, Institute for Drug Development, 14960 Omicron Drive, San Antonio, TX 78245-3217, USA

Current Opinion in Biotechnology 1996, 7:583-591

0 Current Biology Ltd ISSN 0958-1669

Abbreviations AZT azidothymidine AZTTP 3"-azido-3"-deoxythymidine-5"-triphosphate Cl io Chinese hamster ovary ddG dideoxyguanosine TRAP telomeric repeats amplification protocol

Introduction Human telomeres are simple (TTAGGG)n repeated sequences located at the ends of chromosomes [1-3]. This structure capping the chromosome termini is essential to prevent aberrant recombination, protect chromosomes against exonucleolyt ic degradat ion and could be involved in the regulation of genes at distal loci [4,5,6"']. Evidence indicates that telomeric D N A shortens during the growth of various human somatic cells. Senescence of these cells may occur as a result of a checkpoin t arrest in response to the shortened telomeres. T h e loss of telomeric repeats after each cell division may be a biological clock l imit ing the proliferative lifespan of somatic cells [7-13,14°,15°]. To compensa te for the sequence loss that results from incomplete terminal replication, germline cells, immortalized cells and tumor cells express an R N A - d e p e n d e n t D N A polymerase [14°,16-Z1]. This enzyme, known as telomerase, is a r ibonucleoprotein containing a short RNA

motif that serves as a templa te for the synthesis of telomeric D N A [22,23"°,24-26].

Recent reports of te lomcrasc reactivation in a large proport ion of human cancers [27,28] have led to the proposal of te lomerase as a cancer marker [29], a prognostic factor [30,31,32 °] and a promising therapeut ic target for novel anticancer drugs [33-35]. Telomerase has been f requent ly descr ibed as an ideal cancer target because it is activated in most tumor cells (Table 1) that concurrently have short te lomeres and is not expressed in normal somatic cells [33]. Conversely, human germ cells and stem cells that also express telomerase activity have long te lomeres and, therefore, would be affected by telomerase inhibitors later than cancer cells [36-39]. This would lead to maximal ant i tumor activity with minimal toxicity in clinical trials.

Table 1

Telomerase activity in human tumor tissues*.

Tumor type Tested Positive Total %

Hematologic malignancies 57 85 67 Acute myeloid leukemia 8 12 67 Acute lymphoid leukemia 18 25 72 Chronic myeloid leukemia 12 12 1 O0 Chronic lymphoid leukemia (early) 2 14 14 Chronic lymphoid leukemia (late) 5 9 55 Myeloma 1 1 100 Low-grade [ymphoma 2 3 67 High-grade lymphoma 9 9 100

Breast 28 33 85 Prostate 24 30 80 Lung 88 115 76

Non-small cell 74 101 73 Small cell 14 14 100

Colon 22 23 95.6 Ovarian 13 14 93 Head and neck 14 16 87 Kidney 41 55 74 Melanoma 7 7 100 Neuroblastoma 53 76 70 Glioblastoma 6 8 75 Hepatocellular carcinoma 28 33 85 Gastric 56 66 85 Bladder 39 40 97

Total 476 601 79

* This estimation was obtained from results published in the literature combined with the authors' unpublished data.

However, even though the telomeric D N A in tumor cells is shorter than in somatic cells, several cell divisions may be required before compromising te lomere function

584 Pharmaceutical bioteehnology

after treatment with telomerase inhibitors [18,40]. This could be a limiting factor in the therapy of several human cancers exhibiting long telomeres and/or long doubling times. Moreover, the recent characterization of tumor cells maintaining their telomere length without telomerase activity suggests other possible mechanisms to stabilize chromosome termini in human cancer [41°°]. Direct telomere targeting appears to be another exciting therapeutic possibility to destabilize DNA, and to quickly limit the proliferative lifespan of cancer cells. Many telomere-interacting compounds function by nonspecific alkylation or intercalation into telomeric DNA [42]. Recent understanding of the spatial structure of telomeric DNA, folded into G-quartet structures, led to the design of compounds interacting more specifically with the telomere G-quartet, which may influence the extent of telomere elongation in vitro. The ultimate goal will be to define compounds with specific activity on telomerase and telomeres of tumor cells. In this review, we will summarize the recent advances and our progress with telomerase- and telomere-targeting drugs.

Telomerase inhibition measurement in cell-free systems Accurate and quantitative methods are needed in an attempt to characterize the effects of telomerase inhibitors. Basically, a conventional assay for telomerase activity requires only a single-stranded (TTAGGG)n DNA primer mimicking the chromosome end, nucleotides and the telomerase enzyme [43]. However, the telomerasc activity in human tumors has been difficult to establish due to the limited sensitivity of the conventional assay. Thus, Kim et al. [44] developed a procedure consist- ing of a detergent-based extraction and a PCR-based telomerase detection assay designated TRAP (telomeric repeats amplification protocol), which together resulted in an estimated 104-fold improvement in detectability. Interestingly, the primer sequence in this highly sensitive assay differs markedly from the native human telomeric sequence. This method was used to detect telomerase activity in cells representing 18 tissues of different origins. Extracts from 94 out of 94 tumor-derived cell lines, 4 of 6 transformed cell lines, and none of 22 normal somatic cell cultures tested positive for telomerase activity. During the past three years, several additional exciting reports using the TRAP assay have indicated that telomerase is switched on in human tumor cells and, therefore, have suggested that inhibitors of this enzyme might constitute a new class of anticancer drugs for most types of cancers [18,20,21,27,45~49]. However, recent publications describing telomerase activity in Saccharomyces cerevisiae using the TRAP assay or conventional methods have led to opposite results [50-52]. This was also recently illustrated by two articles analyzing the variations of telomerase activity during the cell cycle with the TRAP assay and showing very different results, suggesting that current assay protocols are not always entirely reliable for the description of telomerase properties [53,54].

Differences in the results could derive from the presence of contaminating competitive RNA-dependent primer enzyme activity, inhibitors in the telomerase extract, a nonlinear dependence of product synthesis on enzyme content, primer and nucleotide concentrations, and on assay-dependent alteration of the processivity [25]. This is particularly important considering that actual information on telomerase activity in most human tumors is only provided by the TRAP assay (Fig. 1). Moreover, this indicates that a careful selection of tools measuring telomerase activity is needed to meet the sensitivity, specificity and quantitative aspects required to analyze the effects of telomerase-targeting drugs in patients. To enhance the sensitivity and to quantify the telomerase activity in vivo, we have developed several improvements on the standard and PeR-based telomerase assay [55,56]. These approaches have led us to a better characterization of the telomerase activity in human and rodent tumor cells. A processive telomerase, capable of adding consecutive 5 ' -TTAGGG-3 ' DNA repeats to an 18 base primer, was detected in the extracts from the 293 cells (transformed human embryonic kidney) and a nonprocessive activity that added only one six-base extension was identified in extracts from Chinese hamster ovary (CliO) cells [57,58]. On the basis of our experience, it appears that a nonampli- fled improvement of standard methods is more reliable for quantifying the effects of telomerase inhibitors in cell-free systems and in cancer cell lines, mainly because of a linear dependence between the substrate concentration and the direct measurement of the enzyme processivity. However, the competitive nonspecific interactions with various contaminating RNA-dependent primer enzymes, RNA or DNA fragments or proteins contained in the telomerase extract remain major concerns in measuring the effects of candidate compounds against telomerase activity in cell-free systems. The purification of human telomerase should allow improvements in the specificity of current telomerase inhibitor evaluation.

In vitro and in vivo models to study drugs that target telomerase and telomeres Several observations suggest that specific models should be considered to study the anticancer effects of telomerase inhibitors. Cytotoxicity and telomere length have been initially selected to evaluate the antiproliferative effects of telomerase- and telomere-targcting agents. Because telomere length is maintained in tumor cells by telomerase, it has been calculated that more than 20 cell-population doublings would be necessary before chromosome instability causes cancer cell death [40]. Traditional methods of evaluating the growth inhibitory activity of anticancer drugs may not be applicable to telomerase inhibitors, which require a very long-term culture before any biological effects are observed. However, because unspecific cytotoxicity is likely to occur before telomere shortening, the cytotoxicity assay remains a very interesting way to determine candidate compounds that target DNA by other mechanisms than telomerase inhibition or telomere

Agents that target telomerase and telomeres Raymond et aL 585

Figure 1

Human HeLa cells cancers Lymphomas

1000 10 000 m (~ cells cells + <7 .~ ne m

1 2 3 4 5

Telomerase activity in human tissue biopsies taken directly from patients. The telomerase activity in detergent extracts was measured using the PCR-based TRAP assay according to the method published by Kim et aL [97]. Telomerase synthesizes telomeric "I-rAGGG repeats onto the oligonucleotide TS (5'-AATCCGTCGAGCAGAG'I-F-3'). Telomerase products are amplified by PCR using the CX 5'-(CCC'I-rA)3CCCTAA-3' and TS primers. HeLa cell extracts were used as positive controls. RNAse-pretreated extracts do not show telomerase activity (third lane). Using the same number of cells (2 lal of extract corresponding to 14-18 lag of protein), important variations in telomerase activity were observed from one tissue biopsy to another (see differences in the signal obtained between lanes 1, 2 and 3 for lymphomas). This may suggest that telomerase is not uniformly expressed by all cancer cells in human tumors but by a specific subset of cells. This may also suggest that the level of telomerase activity varies from one tumor type to another.

interaction. Similarly, telomere-length measurement is subject to important variations due to the heterogeneity of cancer cell populations and will require a long-term culture to observe significant shortening. Thus, new approaches to select candidates have been recently proposed. Several cytogenetic anomalies have been observed in tumor cells in relation to telomeric degradation [17,59]. In a recent report, Reimann et al. [60] observed that metacentric and submetacentric chromosomes frequently observed in malignant tumors were cytogenetic products of head-to- head telomeric fusion. Therefore, immunofluorescence analysis of dicentric chromosomes could be considered as a fast, cheap and attractive endpoint for the study of the biological effects of telomerase- and telomere-targeting drugs.

The antitumor effects of telomerase- and telomere-targeting drugs represent another challenge. Chadeneau et al. [61"'] have evaluated the telomerase activity in malignant tissues in transgenic murine. However, telomerase in mice tumor cells was shown to present nonprocessive characteristics distinct from human telomerase. This could be a limiting factor in the study of telomerase inhibitors in transgenic tumor models [55,57,58,62-65]. Human xenografts in athymic mice would be closer to clinical situations and would allow the selection of

tumors that demonstrate high telomerase activity in vitro. Moreover, considering the expected delay of the antitumor action of drugs targeting telomerase and telomeres, initial debulking chemotherapy could be required. In this case, the human xenograft model, in which chemotherapy has been extensively studied for years, would be preferred.

To select the most appropriate tumors for in vitro and in vivo evaluation of telomerase inhibitors, we have characterized human breast, lymphoma and prostate tumor models with high processive telomerase activity. Human tumor xenografts in athymic mice will allow us to mimic those advanced cancers in which telomerase inhibitors are urgently needed. Considering the delayed effects of telomerase inhibitors, initial debulking chemotherapy with alkylating agents has been considered, immediately followed by a prolonged administration of telomerase inhibitors targeting a minimal residual tumor volume. Hopefully, this approach will be followed by clinical trials in patients with advanced breast cancers or lymphomas after treatment with conventional or high-dose chemotherapy (Fig. 2).

T e l o m e r a s e inh ib i tors One strategy for identifying telomerase inhibitors is based on the rational design of inhibitors according to

586 Pharmaceutical biotechnology

Figure 2

Clinical trial design

Patients with metastatic breast cancer maximally

debulked with chemotherapy (Tamox,,en +,e,omorase' k N specific inhibitor ,/)

@ 1996 Current Opinion in Biotechnology

Preclinical and clinical trial designs for telomerase- and telomere-interacting agents in human breast cancers. Advanced breast cancers show telomerase activity [32"]. Human breast cancer xenografts in athymic mice allow characterization of the antitumor effects of new compounds that interact with telomerase and telomeres. Advanced and metastatic human breast cancers can be maximally debulked with conventional or high-dose chemotherapy, then randomized to either receive or not receive telomerase inhibitors. Time to recurrence is a major endpoint in clinical trials. About 75% of patients with metastatic breast cancers treated with high-dose chemotherapy are likely to have their cancers recur within the first year of treatment, even when they receive prophylactic therapy with tamoxifen. Hopefully, compounds selected for clinical trials will be telomerase-specific inhibitors at clinically relevant concentrations, active orally to allow prolonged administration, well-tolerated, and not affected by multidrug-resistance mechanisms (i.e. cross the blood-brain barrier). Similar trial designs are under investigation in poor-prognosis lymphomas and hormone-independent prostate cancers.

the continuous improvement in the knowledge of the properties of telomerase or on a concomitant screening of a library of compounds interacting with DNA, RNA and proteins. There are several rational approaches including the following strategies (Table 2).

Nucleoside and non-nucleoside reverse transcriptase inhibitors Because telomerase has some properties similar to retroviral reverse transcriptase, this led to the hypothesis that azidothymidine (AZT) could be used as a telomerase substrate and preferentially incorporated into telomeric DNA by that enzyme [66]. Preferential localization of AZT to the telomeric regions of CHO cell chromosomes has been demonstrated by immunofluorescence using anti-AZT antibodies [67]. Quantitations of preferential

AZT binding to the telomeric fragments were twofold to fourfold higher than in the nontelomeric DNA-enriched fractions. Moreover, the incorporation of AZT into CHO immortalized cells but not into primary fibroblasts that lack telomerase has indirectly reinforced the hypothesis that AZT incorporation may be telomerase-mediated [68].

We therefore tested whether A Z T T P (3'-azido-3'- deoxythymidine-5"-triphosphate) has any effect on telomerase activity and whether AZT has any effect on telomere function in whole CHO and 293 cells. We observed that AZTTP, but not AZT, inhibits the activity of telomerase isolated from CHO and 293 cells. The inhibition of both human and hamster telomerase by A Z T T P was shown to be concentration dependent with 50% inhibition at 500 ~M, which is fourfold lower than the concentration of dATP and d'VFP present in the enzyme reaction. The continuous growth of the human tumor cells in 800!ttM AZT resulted in the progressive shortening of telomeres at a rate of approximately 100 bases per generation, and we observed the expected chromosome end fusions resulting from telomere loss. These data have suggested that high concentrations of AZT are capable of interfering with telomeres in tumor cells [69].

The nucleotide substrate specificity of human telomerase was subsequently studied using various nucleotide analogs showing that modifications to the 5" phosphates, sugar ring structure and base structure resulted in inhibitors of telomerase activity. The effects of 2',3'-dideoxythymidine triphosphate (ddTTP), 3'-fluoro-3"-deoxythymidine- 5'-triphosphate (3 '-F-TTP), and A Z T T P on the DNA primer extension activity were investigated using the processive and the nonprocessive telomerase enzymes showing that A Z T T P inhibited both of them. Unexpect- edly, ddTTP was found to inhibit the human processive, but not the hamster nonprocessive telomerase, whereas 3 ' -F-TTP inhibited the hamster nonprocessive, but had little effect on the human processive, telomerase. The differential effects of compounds with similar structure on various forms of telomerase provide important information in targeting tumor cell telomerase [57]. Similar results

Table 2

Telomeres and te lomerase as targets for new drug discovery.

Agents Targets under investigation Strategy

Telomerase-interact ing compounds

Telomere-interacting compounds

RNA component

Indirect inhibition

Protein component? Telomerase-gene modulation? Alkylation of telomeric DNA repeats G-quartet

The nucleoprotein complex?

Antisense strategy Targeting RNA component with antibiotics Oligonucleotides that mimic telomere sequences Modification of telomere structures Reverse transcriptase inhibitors ? ?

Nucleoside analogs 7-deaza-dGTP Intercalation of chemical into the telomere

G-quartet structure ?

Agents that target telomerase and telomeres Raymond et al. 587

were obtained with the human JY616 and the Jurkat E6-1 cell lines: using a panel of retroviral reverse transcriptase inhibitors, dideoxyguanosine (ddG) was shown to cause reproducible, progressive telomere shortening over several weeks of passaging, after which the telomeres stabilized and remained short. However, the prolonged passaging in ddG caused no observable effects on cell population doubling rates or morphology. AZT caused progressive telomere shortening in some but not all T- and B-cell cultures. Telomerase activity was present in both cell lines and was inhibited in vitro by ddGTP and AZT triphosphate. Prolonged passaging in arabinofuranyl-guanosine, dideoxyinosine (ddI), dideoxyadenosine (ddA), didehydro- thymidine (d4T), or phosphonoformic acid (foscarnet) neither caused reproducible telomere shortening nor decreased cell growth rates or viability. Combining AZT, foscarnet, and/or arabinofuranyl-guanosine with ddG did not significantly augment the effects of ddG alone. Strikingly, with or without inhibitors, telomere lengths were often highly unstable in both cell lines and varied between parallel cell cultures [70°']. On the basis of these results, various nucleoside triphosphates and non-nucleoside reverse transcriptase inhibitors ate under further investigation.

Oligonucleotides that mimic telomere sequences Inhibition of telomerase activity with an oligonucleotide that mimics the telomere sequence might result in decreased replicative capacity and perhaps death of tumor cells [23°°,34]. The effects of various oligonucleotides on three different human hematological malignant cell lines and normal human mononuclear cells were studied. Cells were incubated with 1.25 and 2.5 ~tM concentrations of a phosphorothioate oligonucleotide, 5'-d(TTAGGGq~FAGGG)-3", for 24-120h, which led to a significant growth inhibition of OMA-BL1 cells but not of Raji or K562 cell lines or mononuclear cells. The control scrambled sequence did not show significant growth inhibition. OMA-BL1 cell growth was also inhibited by telo-6-ODN 5'-d(TTAGGG)-3', telo-18-ODN (5'-d[TTAGGG]3')3 and telo-24-ODN (5'-d[TTAGGG]-3')4. Truncated pentamer oligonucleotides (5'-d[TTAGG]-3' and 5'-d[TAGGG]-3') did not produce similar inhibitory effects in OMA-BL1 cells and suggest the sequence specificity of oligonucleotide inhibition of telomerase activity. Cytomorphological analysis demonstrated induc- tion of apoptosis following incubation. These results showed that phosphorothioate oligonucleotide-mimicking telomeric sequences are potential therapeutic agents for the elimination of selected tumor cells without the inhibition of normal cells [71]. Other authors have investigated oligonucleotides with sequence antisense to c-myc and c-myb, which contain sequences similar to telomeres, for their potential to inhibit telomcrase. The telomere consensus sequence, 5'-TTAGGG-3', inhibits hematological malignant cell growth, arrests cells in the S-phase of the cell cycle and causes apoptosis. Antisense oligonucleotides that have reported efficacy in

other malignant cells contain sequences similar to telomere sequence (e.g. c-myc, 5'-AACGGTGAGGGGCAT-3'; c-myb, 5'-TATGCTGTGCCGGGGTCTTCGGGC-3"). An inhibition of cell growth, an arrest in the S-phase of the cell cycle, and apoptosis were observed with truncated versions of the oligonucleotides complementary to c-myc and c-myb. In addition, an inhibition of telomerase activity was observed with the nonamer from the antisense c-myc sequence. The nonamer oligonucleotides will not form stable hybrid duplexes at 37°C, indicating that the observations with the truncated oligonucleotides are not due to antisense mechanisms. These studies suggest that embedding a telomere sequence in an antisense oligonucleotide should result in a therapeutic agent with multiple favorable mechanisms of action [72].

Genetic modulation of t e lomerase activity There is recent evidence suggesting that the restoration of defective mechanisms of cell senescence could lead to the inhibition of telomerase activity in cancer. Ohmura et al. [73] have observed that the deletion of a gene(s) on chromosome 3 is common in human renal cell carcinoma and that the reintroduction of a normal chromosome 3 into immortal renal carcinoma cell lines restored the program of cellular senescence [73]. The loss of indefinite growth potential was associated with the loss of telomerase activity and the shortening of telomeres in cells with a normal chromosome 3. However, microcell hybrids that escaped from senescence and microcell hybrids with an introduced chromosome 7 or 11 maintained telomere lengths and telomerase activity similar to those of the parental tumor cells. Thus, the restoration of the cellular senescence program by chromosome 3 is associated with the repression of telomerase function in renal carcinoma cells. However, chromosome 3 did not suppress telomerase in another cancer cell type. This suggests that multiple genetic events could be needed to activate telomerase. Moreover, a more recent report suggests that telomerase by itself could be mutated in a way that will affect its function. Ahmed et al. [74] isolated cells expressing high levels of the mutant telomerase RNA and exhibiting lethal phenotypes that were due to the presence of very short telometes. Further identification and cloning of the human telomcrase gene would provide a new target for modulating telomerase activity in human cancers.

Res is tance to t e l o m e r a s e inh ib i tors There is evidence suggesting that some advanced tumors are not composed of immortalized cells [75]. Therefore, primary resistance to telomerase inhibitors is initially likely to occur in these nonimmortalized telomerase-negative tumor cells [44]. The TRAP assay is able to detect less than one positive cell in one thousand. With this assay, telomerase-negative tumors account for about 15% of human tumors [27]. Moreover, in tumors showing telomerase activity, variations of the signal using a semiquantitative TRAP assay suggest that the number of telomerase-positive cells varies markedly from one tumor to another (see


Figure 3

Structure of G-quartets. The (TTAGGG)n repeats that characterize telomeric DNA can form secondary structures in the form of a tetraplex held together by G-quartets [91]. G-quartet-interacting agents stabilize this structure and inhibit the proper recognition of telomeric DNA by telomerase.

0 0 0 0 0 0 0 0 0 0 0 0 5' T-T-A-G-G-G-T-T-A-G-G-G-T-T-A-G-G-G-T-T-A-G-G-G 3'

[ ] dT

Ak dA

• dG

& G-quartet

Intramotecular fold-back structure

5'

© 1996 Current Opinion in Biotechnology

Fig. 1 and figures in [29,76-79]). Considering that some tumors are composed of telomerase positive and negative cells, initial debulking by conventional therapy would be required in the design of clinical trials with telomerase inhibitors. Other concerns are the secondary mechanisms of resistance, including the multidrug-resistance mechanisms, that could be developed in tumor cells exposed to telomerase inhibitors. Another major problem may be the ability of tumor cells to solve the problem of telomere shortening by mechanisms that do not involve telomerase, for example, by DNA recombination [80]. This was initially proposed in Saccharomyces [81,82] and Drosophila [83,84], and it has been recently suggested in immortalized human tumor cells that maintain their telomere length without telometase activity [41"']. Our experience with cells continuously exposed to AZT has initially shown that telomere shortening leads to chromosome fusion, but subsequently the number of cells with fused chromosomes declines slowly after approximately eight cell generations [85]. Whether this resistance is a mechanism of resistance for AZT and derivatives or is an adaptive mechanism developed by cells to compensate for telometase inhibition remains to be determined.

Telomere-targeting agents The DNA component: G-quartet interactive compounds Because of its unique mode of replication and its special G-rich structure, telomeric DNA has been described as the Achilles heel of chromosomes and, therefore, constitutes a very attractive target in human cancer cells. A large

variety of chemicals may interact with varying degrees of specificity in this region of the chromosome. Direct telomcre-targeting would lead to rapid destabilization of chromosomes and presumably interact with telomerase and various telomerase-independent mechanisms that maintain telomere length in tumor cells.

The (TTAGGG)n repeats that characterize telomeric DNA can form a secondary structure in the form of a tetraplex held together by G-quartets (Fig. 3). G-quartets may contribute to the regulation of telomere length in vivo through the direct inhibition of telomerase binding to long telomeric sequences or through the regulation of the dissociation of telomerase from telomere DNA. Several G-quartet-interacting agents developed in Laurence Hurley's group at the University of Texas in Austin [86,87] have shown high affinity for this structure, forming stable complexes that may inhibit the proper recognition of telomeric DNA by telomerase. Some promising compounds have been selected for their ability to inhibit telomerase activity in vitro and are currently under investigation in vivo [86,87]. Another approach developed in our group by Fletcher [88] was the use of telomerase to incorporate 7-deaza-dGTP, a nucleoside analog, into telomeric DNA. This strategy was associated with telometase inhibition and cell-growth inhibition. In this model, telomerase inhibition was thought to result in the inhibition of the translocation step because 7-deaza-dGTP lacks the 7 nitrogen which is essential for the formation of G-quartets.

Agents that target telomerase and telomeres Raymond et a/. 589

The nucleoprotein complex Although telomeres could be considered distinct targets for developing specific inhibitors, the complex of telomerase, telomeres and telomere structural proteins could be considered as a nucleoprotein complex that could be studied in order to understand the biological effects of telomerase- and telomere-targeting compounds [4]. For a long time, an interaction between telomeres and the nuclear matrix has been suggested by the position held by chromosome ends in interphase nuclei. The direct evidence that telomeres are anchored to the nuclear matrix via the (TTAGGG)n repeats was provided by de Lange in 1992 [89]. That study provides further evidence that the T T A G G G sequence by itself maintains the nuclear matrix association, but requires anchorage proteins. Those proteins are likely to provide additional protection against exonucleic degradation of chromosome ends and could be essential for proper chromosome segregation and, therefore, nuclear division [90]. Further characterization of that process would provide interesting new targets for drug development.

Conclusions The past five years have revealed the complexity of the structures and regulation of telomerase and telomeres in human tumor cells. Moreover, information already available allows for clearly defined targets for the development of telomerase- and telomere-interacting agents. New compounds that are currently under preclinical investigation may be utilized in clinical trials in the near future. On the basis of the delayed biological effects of those inhibitors, we have proposed clinical trials in patients with minimal residual disease after debulking high-dose chemotherapy. Our preclinical and clinical data suggest that advanced breast cancers and lymphomas constitute very attractive targets for those new drugs in clinical trials.

Acknowledgements "I'his work was supported by a grant from the National Cooperative I)rug DiscoveR' Group (NCDDG#UI9 CA67760) and a grant from Sanofi-Winthmp Research. Eric Raymond (detached from the Service des Maladies Sanguines lmmunitaires et Tumorales, H6pital Paul Bmussc, Villcjuif, France) is the recipicnt of a grant from the Association pour la Recherche contre le Canccr (ARC). The authors thank Alicc Louise Goodwin for her assistance in preparing the manuscript.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as:

• of special interest • • of outstanding interest

1. Moyzis RK, Buckingham JM, Cram LS, Dani M, Deaven LL, Jones MD, Meyne J, Ratliff RL, Wu J-R: A highly conserved repetitive DNA sequence, (TTAGGG) n, present at the telomeres of human chromosomes. Proc Nat/Acad Sci USA 1988, 85:6622-6626.

2. Meyne J, Ratliff RL, Moyzis RK: Conservation of the human telomere sequence (TTAGGG)n among vertebrates. Proc Nat/ Acad Sci USA 1989, 86:7049-7053.

3. Hanish JP, Yanowitz JL, De Lange T: Stringent sequence requirements for the formation of human telomeres. Proc Nat/ Acad Sci USA 1994, 91:8861-8865.

4. Blackburn EH: Structure and function of telomeres. Nature 1991,350:569-573.

5. Murnane JP, Yu LC: Acquisition of telomere repeat sequences by transfected DNA integrated at the site of a chromosome break. Mol Cell Biol 1993, 13:977-983.

6. Kruk PA, Rampino N J, Bohr VA: DNA damage and repair in • . telomeres: relation to aging. Proc Natl Acad Sci USA 1995,

92:258-262. The authors have established a method for the detection of DNA damage and repair in human telomeres. They observed that ultraviolet light induced pyrimidine dimers in telomeric DNA are repaired more efficiently than inac- tive, noncoding regions. Conservation of telomeric integrity appears to be necessary for the maintenance of chromosomal stability. The results of this study also indicate that the telomere repair process declines with aging.

7. Hastie ND, Dempster M, Dunlop MG, Thompson AM, Green DK, Allshire RC: Telomere reduction in human colorectal carcinoma and aging. Nature 1990, 346:866-868.

8. Wright WE, Shay JW: Telomere positional effects and the regulation of cellular senescence. Trends Genet 1992, 8:193-197.

9. Harley CB, Vaziri H, Counter CM, AIIsopp RC: The telomere hypothesis of cellular aging. Exp Geronto11992, 27:375-382.

10. levy MZ, AIIsopp RC, Futcher AB, Greider CW, Harley CB: Telomere end-replication problem and cell aging. J Me/Biol 1992, 225:951-960.

11. AIIsopp RC, Vaziri H, Patterson C, Goldstein S, Younglai EV, Futcher AB, Greider CW, Harley CB: Telomere length predicts replicative capacity of human fibroblasts. Proc Natl Acad Sci USA 1992, 89:10114-10118.

12. Shay JW, Wright WE, Werbin H: Loss of telomeric DNA during aging may predispose cells to cancer. Int J Cancer 1993, 3:559-563.

13. Varizi H, Sh&chter F, Uchida I, Wei L, Zhu X, Effros R, Cohen D, Harley CB: Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes. Am J Hum Genet 1993, 52:661-667.

14. AIIsopp RC, Chang E, Kashefi-Aazam M, Rogaev El, Piatyszek • MA, Shay JW, Harley CB: Telomere shortening is associated

with cell division in vitro and in vivo. Exp Ceil Res 1995, 220:194-200.

This paper shows that in somatic cells that lack telomerase, telomere shortening is dependent upon cell division. The authors suggest the use of telomere length as a biemarker for replicative capacity.

15. Atlsopp RC, Harley CB: Evidence for a critical telomere • length in senescent human fibroblasts. Exp Cell Res 1995,

219:130-136. This paper supports the existence of a critical telomere length in senescin 9 cells and a causal role of telomere shortening in cell senescence.

16. Windle B, McGuire S: Telomeres: the long and the short of it. Proc Am Assoc Cancer Res 1992, 33:594-595.

17. Counter CM, Avilion AA, LeFeuvre CE, Stewart NG, Greider CW, Harley CB, Bacchetti S: Telomere shortening associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J 1992, 11:1921-1929.

18. Harley CB, Kim NW, Prewse KR, Weinrich SL, Hirsch KS, West MD, Bacchetti S, Hirte HW, Counter CM, Greider CW et aL: Telomerase, cell immortality, and cancer. Cold Spring Harb Syrup Ouant B/o/1994, 59:307-315.

19. Counter CM, Botelho FM, Wang P, Harley CB, Bacchetti S: Stabilization of short telomeres and telomerase activity accompany immortalization of Epstein-Barr virus-transformed human B lymphocytes. J V/tel 1994, 68:3410-3414.

20. Counter CM, Leber B, Harley CB, Bacchetti S: Telomere length and telomerase activity in hematological malignancies [abstract]. Proc Am Assoc Cancer Res 1995, 36:555.

21. Shay JW, Piatyszek MA, Word RA, Gazdar AF, Wright WE, Kim NW, Weinrich SL, Prowse KR, Harley CB, Hiyama Eet aL: You have not heard the end of it: telomeres, time, telomerase and tumors [abstract]. Proc Am Assoc Cancer Res 1995, 36:673-674.

22. Romero DP, Blackburn EH: A conserved secondary structure for telomerase RNA. Cell 1991, 67:343-353.

23. Feng J, Funk WD, Wang SS, Weinrich SL, Avilion AA, • • Chiu CP, Adams RR, Chang E, AIIsopp RC, Yu Jet ah The


RNA component of human telomerase. Science 1995, 269:1 236-1241.

In this paper, the cloning of the RNA component of human telomerase is described. The template region of human telomerase encompasses 11 nucleotides (5'-CUAACCCUAAC) complementary to the human telomere sequence (I-rAGGG) n, The authors showed that germ line tissues and tumor cell lines expressed more telomerase than normal somatic cells and tissues, which have no detectable telomerase activity. Moreover, they observed that HeLa cells transfected with an antisense lost telomeric DNA and began to die after 23 to 26 doublings, demonstrating that human telomerase is a critical enzyme for the long-term proliferation of immortal tumor cells.

24. Lingner J, Promisel Cooper J, Cech TR: Telornerase and DNA end replication: no longer a lagging strand problem? Science 1995, 269:1533-1534.

25. Collins K: Structure and function of telomerase. Curr Opin Cell Bio/1996, 8:374-380.

26. Greider CW, Autexier C, Buchkovioh K, Collins K, Blasoo M, Avilion A, Mantell L, Prowse K, Harley C, Funk Wet aL: Telomerase biochemistry and regulation in cellular immortalization [abstract]. Proc Am Assoc Cancer Res 1995, 36:672.

27. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrich SL, Shay JW: Specific association of human telomerase activity with immortal cells and cancer. Science 1994, 266:2011-2015.

26. Bacchetti S, Counter CM: Telomeres and telomerase in human cancer. Int J Cancer 1995, 7:423-432.

29. Sommerfeld H J, Meeker AK, Piatyszek MA, Bova GS, Shay JW, Coffey DS: Telomerase activity: a prevalent marker of malignant human prostate tissue. Cancer Res 1996, 56:218-222.

30. Healy KC: Telomere dynamics and telomerase activation in tumor progression: prospects for prognosis and therapy. Oncol Res 1995, 7:121-130.

31. Hiyama E, Hiyama K, Yokoyama T, Matsuura Y, Piatyszek MA, Shay JW: Correlating telomerase activity levels with human neuroblastoma outcomes. Nat Med 1995, 1:249-255.

32. Hiyama E, Gollahon L, Kataoka T, Kuroi K, Yokoyama 1", Gazdar AF, ,, Hiyama K, Piatyszek MA, Shay JW: Telomerase activity in human

breast tumors. J Natl Cancer/nst 1996, 88:116-1 22. Using a PCR-based telomerase activity assay, the authors examined telomerase activity in 140 breast cancer specimens, four biopsies of phyllodes tumors, 38 biopsies of noncancerous lesions (20 fibroadenomas, 17 fibro- cystic diseases, 1 gynecomastia), 55 biopsies of adjacent noncancerous mammary tissues, and 33 fine needle aspirated breast samples. They detected telomerase activity in 130 of 140 (93%) breast cancers. The activity was detected in more than 95% of advanced stage tumors but in only two (40/o) of 55 adjacent noncancerous tissues. The stage of the disease was correlated with telomerase activity.

33. Morin GB: Is telomerase a universal cancer target? J Nat/ Cancer Inst 1995, 87:859-861.

34. Harley CB, Andrews W, Chiu CP, Feng J, Funk W, Gaeta F, Hirsch K, Kim NW, Kozlowski M, Wang SS et aL: Human telomerase inhibition and cancer [abstract]. Proc Am Assoc Cancer Res 1995, 36:671-672.

35. Parkinson EK: Do telomerase antagonists represent a novel anti-cancer strategy? Br J Cancer 1996, 73:1-4.

36. Counter CM, Gupta J, Harley CB, Leber B, Bacchetti S: Telomerase activity in normal leukocytes and in hematologic malignancies. Blood 1995, 85:2315-2320.

37. Broccoli D, Young JW, De Lange T: Telomerase activity in normal and malignant hematopoietic cells. Proc Nat/Acad Sci USA 1995, 92:9082-9086.

38. Chiu CP, Dragowski V, Kim NW, Thomas TE, Harley CB, Lansdorp PM: Telomerase activity in hematopoietic progenitor cell extracts from adult bone marrow [abstract]. Proc Am Assoc Cancer Res 1995, 36:555.

39. Hiyama K, Hirai Y, Kyoizumi S, Akiyama M, Hiyama E, Piatyszek MA, Shay JW, Ishioka S, Yamakido M: Activation of telomerase in human lymphocytes and hematopoietic progenitor cells. J Immuno11995, 155:3711-3715.

40. Kipling D: Telomerase: immortality enzyme or oncogene? Nat Genet 1995, 9:104-106.

41. Bryan TM, Englezou A, Gupta J, Bacchetti S, Reddel RR: °° Telomere elongation in immortal human cells without

detectable telomerase activity. EMBO J 1995, 14:4240-4248.

In this paper, the authors examined whether telomerase activation is always necessary for immortalization. As expected, the normal human fibroblasts tested were negative for telomerase activity and 13 DNA tumor virus-transformed cell cultures were negative in the pre-crisis stage. Of 35 immortalized cell lines, 20 had telomerase activity. Fifteen cell lines had no detectable telomerase activity but very long and heterogeneous telomeres. Hybrids between telomerase-negative and telomerase-positive cells showed that activation of telomerase is not sufficient for immortalization. This article suggests that the presence of lengthened or stabilized telomeres is necessary for immortalization, and that this may be achieved either by the reactivation of telomerase or by another unidentified mechanism.

42. Burger LM, Double JA, Newell DR: Correlation between cisplatin mediated inhibition of telomerase activity and cell growth in human testicular cancer cell lines [abstract]. Proceedings of the 9th NCI-EORTC Symposium on New Drugs in Cancer Therapy. Edited by Lobbezoo MW. Dordrecht: Kluwer Academic Publishers; 1996:28.

43. Shay JW, Brasiskyte D, Ouellette M, Piatyszek MA, Werbin H, Ying Y, Wright WE: Analysis of telomerase and telomeres. In Methods in Molecular Genetics, edn 5. Edited by Adolph KW. New York: Academic Press; 1994:263-280.

44. Kim NW, Piatyszek MA, Prowse KR, Harley CB, West MD, Ho PL, Coviello GM, Wright WE, Weinrioh SL, Shay JW: Specific association of human telomerase activity with immortal cells and cancer. Science 1994, 266:2011-2015.

45. De Lange T: Activation of telomerase in a human tumor. Proc Nat/Acad Sci USA 1994, 91:2882-2885.

46. Greider CW, Blackburn EH: Telomeres, telomerase and cancer. Sci Am 1996, 274:92-97.

47. De Lange T: Activation of telomerase in a human tumor. Proc Natl Acad Sci USA 1994, 91:2882-2885.

48. Harley CB, Villeponteau B: Telomeres and telomerase in aging and cancer. Curt Opin Genet Dev 1995, 5:249-255.

49. Axelrod N: Of tetomeres and tumors. Nat Ivied 1996, 2:158-159.

50. Cohn M, Blackburn EH: Telomerase in yeast. Science 1995, 269:396-400.

51. Lue NF, Wang JC: ATP-dependent processivity of a telomerase activity from Seccharomyces cerevisiae. J Biol them 1995, 270:21453-21456.

52. Lin J J, Zakian VA: An in vitro assay for Saccharomyces telomerase requires ESTI. Cell 1995, 81:1127-1135.

53. Zhu M, Kumar R, Mandal M, Sharma N, Sharma HW, Dhingra U, Sokoloski JA, Hsiao R, Narayanan R: Cell cycle-dependent modulation of telomerase activity in tumor cells. Proc Nat/Acad Sci USA 1996, 93:6091-6095.

54. Holt SE, Wright WE, Shay JW: Regulation of telomerase activity in immortal cell lines [abstract]. Proc Am Assoc Cancer Res 1996, 37:559.

55. Qiu M, Windle B: Analysis of processive and non-processive telomerase activity using a sensitive and quantitative ligation and PCR-based assay [abstract]. Proc Am Assoc Cancer Res 1996, 37:561.

56. Sun D, Chen S-F, Von Hoff D: A sensitive and rapid assay method of human telomerase using dynabeads [abstract]. In Proceedings of the 9th NCI-EORTC Symposium on New Drugs in Cancer Therapy. Edited by Lobbezoo MW. Dordrecht: Kluwer Academic Publishers; 1996:71.

57. Maine IP, Windle B, Chen SF: In vitro investigations of processive and non-processive telomerase activities [abstract]. Proc Am Assoc Cancer Res 1995, 36:554.

58. Maine IP, Chen SF, Windle B: Interaction between telomeric oligonucleotide primers and CHO non-processive telomerase [abstract]. Proc Am Assoc Cancer Res 1996, 37:561.

59. Park VM, Gustashaw KM, Wathen TM: The presence of interstitial telomeric sequences in constitutional chromosome abnormalities. Am J Hum Genet 1992, 50:914-923.

60. Reimann N, Rogalla P, Kazmierczak B, Bonk U, Nolte I, Grzonka T, Bartnitzke S, Bullerdiek J: Evidence that metacentri¢ and submetacentric chromosomes in canine tumors can result from telomeric fusions. Cytogenet Cell Genet 1994, 67:81-85.

61. Chadeneau C, Siegel P, Harley CB, Muller WJ, Bacchetti S: °° Telomerase activity in normal and malignant murine tissues.

Oncogene 1995, 11:893-898.

Agents that target telomerase and telomeres Raymond et aL 591

This study characterizes an animal model for inhibition studies, and investi- gates telomerase activity during mammary tumorigenesis in transgenic mice overexpressing the neu gene. The authors detected activity in primary mammary tumors and lung metastases but also in normal mammary glands and other organs. Activity was elevated in tumors versus normal tissues and was enhanced by short-term culturing of normal cells. They propose this mouse model of tumorigenesis for experimental systems assessing the outcome of in vivo inhibition of telomerase in malignant and normal cells.

62. Prowse KR, Avilion AA, Greider CW: Identification of a nonprocessive telomerase activity from mouse ceils. Proc Natl Acad Sci USA 1993, 90:1493-1497.

63. Bednarek A, Budunova I, Slaga TJ, Aldaz CM: Increased telomerase activity in mouse skin premalignant progression. Cancer Res 1995, 55:4566-4569.

64. Bacchetti S, Counter CM, Chadeneau C, Gupta J, Harley CB, Leber B, Gallinger S, Hirte HW, Siegel P, Muller WJ: Telomerase activity in human and murine somatic tissues and tumors [abstract]. Proc Am Assoc Cancer Res 1995, 36:674.

65. Burger AM, Bibby MC, Double JA: Telomerase activity in normal and malignant mammalian tissues: feasibility of telomerase as a potential target for cancer chemotherapy [abstract]. Proc Am Assoc Cancer Res 1996, 37:495.

66. Olivero OA, Gomez DE, Kassim A, Windle BE: Preferential incorporation of 3'-azido-2",3'dideoxythymidine (AZT) into telomeric DNA of Chinese hamster ovary (CHO) cells [abstract]. Proc Am Assoc Cancer Res 1994, 35:340.

67 Olivero OA, Poirier MC: Z-DNA configuration in chinese hamster ovary cells as a preferential target for 3'azido-2'- 3'-dideoxythymidine (AZT) incorporation [abstract]. Proc Am Assoc Cancer Res 1992, 33:171.

68. Olivero OA, Gomez DE, Kassim A, Windle BE: Telomeric DNA of CHO cells target for AZT (3'-azido-2",3'-dideoxythymidine) incorporation through telomerase [abstract]. Proc Am Assoc Cancer Res 1995, 36:556.

69. Chen SF, Maine IP, Windle B: Effect of 3'-azidothymidine triphosphate (AZTTP) and 3'-azidothymidine (AZT) on telomerase activity and telomere function [abstract]. Prec Am Assoc Cancer Res 1995, 36:554.

70. Strahl C, Blackburn EH: Effects of reverse transcriptase *. inhibitors on telomere length and telomerase activity in two

immortalized human cell lines. Mo/Cell Bio/1996, 16:53-65. This study analyzes telomere maintenance in two immortalized human cell lines and determines whether known inhibitors of retroviral reverse transcrip- tases perturb telomere lengths and growth rates of cells in culture.

71. Joshi SS, Palen BN, Mata JE, Iversen PL: Growth inhibitory effects of telomere mimic-oligonucleotides on human leukemia/lymphoma cells in vitro [abstract]. Proc Am Assoc Cancer Res 1995, 36:441.

72. Iversen PL, Pirruccello S J, Mata JE, Joshi SS: Antisense oligonucleotides which demonstrate tumor specific action also inhibit telomerase in vitro [abstract]. Proc Am Assoc Cancer Res 1995, 36:412.

73. Ohmura H, Tahara H, Suzuki M, Ide T, Shimizu M, Yoshida MA, Tahara E, Shay JW, Barrett JC, Oshimura M: Restoration of the cellular senescence program and repression of telomerase by human chromosome 3. Jpn J Cancer Res 1995, 86:899-904.

74. Ahmed SC: Telomeric nucleic acids: C-strand structure and a telomerase RNA mutant [abstract]. Diss Abstr/nt [B] 1995, 56:623.

75. Edington KG, Loughran OP, Berry I J, Parkinson EK: Cellular immortality: a late event in the progression of human

squamous cell carcinoma of the head and neck associated with p53 alteration and a high frequency of allele loss. Mo/ Carcinog 1995, 13:254-265.

76. Chadeneau C, Hay K, Hirte HW, Gallinger S, Bacchetti S: Telomerase activity associated with acquisition of malignancy in human colorectal cancer. Cancer Res 1995, 55:2533-2536.

77 Hiyama, E, Yokoyama 1", Tatsumoto N, Hiyama K, Imamura Y, Murakami Y, Kodama T, Piatyszek MA, Shay JW, Matsuura Y: Telomerase activity in gastric cancer. Cancer Res 1995, 55:3258-3262.

78. Hiyama K, Hiyama E, Ishioka S, Yamakido M, Inai K, Gazdar AF, Piatyszek MA, Shay JW: Telomerase activity in small-cell and non-small-cell lung cancers. J Natl Cancer Inst 1995, 87:895-902.

79. Lin Y, Miyamamoto H, Fujinami K, Uemura H, Hosaka M, Iwasaki Y, Kubota Y: Telomerase activity in human bladder cancer. Clin C Res 1996, 2:929-932.

80. Ezzell C: The telomerase-cancer connection: will exceptions make a new rule? J NIH Res 1995, 7:41-45.

81. Wang S, Zakian VA: Telomere-telomere recombination provides an express pathway for telomere acquisition. Nature 1990, 345:456-458.

82. Lundblad V, Blackburn EH: An alternative pathway for yeast telomere maintenance rescues est- senescence. Cell 1993, 73:347-360.

83. Levis RW, Ganesan R, Houtchens K, Tolar LA, Sheen FM: Transposons in place of telomeric repeats at a Drosophila telomere. Cell 1993, 75:1083-1093.

84. Biessmann H, Mason JM, Ferry K, D'Hulst M, Valgeirdottir K, Traverse KL, Pardue ML: Addition of telomere-associated HRT DNA sequences "heals" broken chromosome ends in Drosophila. Cell 1990, 61:663-673.

85. Windle B, Maine IP, Wajima M, Chen SF: Nucleotide specificity and inhibition of telomerase, and cellular resistance to telomerase inhibition [abstract]. Proc Am Assoc Cancer Res 1995, 36:556.

86. Thompson B, Salazar M, Sun D, Jenkins T, Neidle S, Hurley L: Telomerase inhibition by G-quartet interactive compounds [abstract]. In Proceedings of the 9th NCI-EORTC Symposium on New Drugs in Cancer Therapy. Edited by Lobbezoo MW. Amsterdam: Kluwer Academic Publishers; 1996:71.

87. Salazar M, Thompson B, Kerwin SM, Hurley L: Unwinding of telomeric hybrids is facilitated by G-quartet formation [abstract]. In Proceedings of the 9th NCI-EORTC Symposium on New Drugs in Cancer Therapy. Edited by Lobbezoo W. Dordrecht: Kluwer Academic Publishers; 1996:71.

88. Fletcher TM, Chen S-F: The effect of 7-deaza-2'- deoxyguanosine-5'-triphosphate and 7-deaza-2"- deoxyadenosine-5'-triphosphate on telomerase activity [abstract]. In Proceedings of the 9th NCI-EORTC Symposium on New Drugs in Cancer Therapy. Edited by Lobbezoo MW. Dordrecht: Kluwer Academic Publishers; 1996:71.

89. De Lange T: Human telomeres are attached to the nuclear matrix. EMBO J 1992, 11:717-724.

90. Chong L, Van Steensel B, Broccoli D, Erdjument-Bromage H, Hanish J, Tempst P, De Lange T: A human telomeric protein. Science 1995, 270:1663-1667.

91. Zahler AM, Williamson JR, Cech TR, Prescott DM: Inhibition of telomerase by G-quartet DNA structures. Nature 1991, 350:718-720.

agents that target telomerase and telomeres

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