Cancer Investigation, 18(3), 269-277 (2000)

BASIC SCIENCE REVIEW

Telomerase: Therapeutic Potential in Cancer

Alison J. Davis, M.B.B.S. (Hons), and Lillian L. Siu, M.D.

Department of Medical Oncology and Hematology Princess Margaret Hospital/The Toronto Hospital Toronto, Ontario, Canada

BACKGROUND

Telomeres are the end portions of eukaryotic chromo- somes, characterized by short tandemly repeated DNA sequences i n complex with specific proteins. These ends were first recognized in the 1930s and 1940s by Muller ( I ) and McClintock ( 2 ) as essential structures in the maintenance of chromosomal integrity. Chromosomes lacking telomeres of sufficient length are innately unsta- ble and prone to degradation by exonucleases, fusion with the ends of other chromosomes by ligases, or loss upon cell division (2,3).

More recently, with the advent of molecular biology, it has been possible to elucidate the nature and the dy- namic properties of telomeres. In their pioneering study of the ciliate Tetruhynzenu, Blackburn and Gall (4) deter- mined that Tetruhymena telomeres consist of the DNA sequence S’GGGGTT3’ repeated to a variable degree at the chromosomal termini. Other telomeric repeats have since been identified in a variety of organisms, including humans ( 5 ) , and generally consist of six to eight G-rich base pairs extending 5‘ to 3‘ toward the chromosome end (6). The actual number of tandem sequence repeats per telomere is not fixed but varies between species and be- tween different chromosomes within the same cell of each species (7). Furthermore, there is evidence that te-

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Copyright 0 2000 by Marcel Dekker, Inc.

lomeric length and its regulation vary markedly with the age of the organism (8,9).

With each round of cellular replication there is a net loss of DNA material due in part to the disparate mode of replication between the leading and the lagging strands of DNA. The leading strand replicates in a continuous fashion using DNA polymerase, whereas the lagging strand replicates using discontinuous Okazaki fragments with DNA polymerase filling the gaps between these fragments (Fig. 1). For the lagging strand to begin DNA polymerization in the 5’ to 3‘ direction, opposite to the replication fork, it must first rely on an RNA primer to initiate synthesis of an Okazaki fragment. Throughout the entire length of the lagging strand, except at its extreme 3‘ end, the RNA primers are degraded and replaced by DNA synthesis extending from an upstream primer, re- sulting ultimately in the ligation of Okazaki fragments. At the 3‘ terminus, the RNA primer cannot be replaced, leading to progressive shortening of DNA in the daughter chromosomes after successive cycles of cell division. This phenomenon has been described as the “end replica- tion problem” (10- 14). If left uncorrected, replicative senescence and cell death will eventually occur.

Telomeres attempt to abrogate the end replicative problem by providing a “buffer zone” of expendable noncoding sequences at chromosomal ends. Normal so-

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270 Davis and Siu

5’ * :: leadingsuand 3’

1 - i , lagging strand 5’ -

3 ’ 4 -4 I

-. 5’ :: leading strand 3’ 3

:: lagging sband 5’ 3’ 4

Figure 1. The telomere end replicative problem (see text for details). The removal and lack of replacement of the terminal RNA primer (rectangular box) led to sequence loss at the 3’ chromosomal end.

matic cells with no means of reextending their telomeres will inevitably reach a shortened chromosomal length that signals a halt to cell division. At this time, termed mortality stage 1 (Ml), tumor suppressor proteins such as p53 and retinoblastoma gene product (pRB) can exert their regulatory functions. Somatic cells thereafter be- come senescent, although remain viable at an arrested GO- or G1-like state (15-18). In some cells, the inactiva- tion of p53 and pRB by viral oncoproteins or by muta- tions allow them to bypass the M1 stage and continue to divide, until their telomeric length reaches a critical level known as mortality stage 2 (M2). Elimination of the M1 checkpoint does not confer immortality but instead yields an extended though still finite lifespan. Most cells that enter M2, or crisis, die (19). Those that survive possess a salvage mechanism for the loss of telomeric length and are capable of indefinite proliferation (20).

Telomerase is a specialized polymerase enzyme re- sponsible for the maintenance of telomeres and thereby contributes to cellular immortalization. The presence of telomerase was first demonstrated in Tetruhymenu by Greider and Blackburn (21-23) and has subsequently been isolated in other lower eukaryotes (24-26) and in humans (27,28). Telomerase is a ribonucleoprotein com- plex that uses a short region of its RNA component as a “template domain” for the de novo addition of telomeric DNA repeats to the 3‘ end of chromosomes, via a reverse transcriptase mechanism (22) (Fig. 2). The two polypep- tides that form the protein components of the Tetruhy- menu telomerase enzyme have been isolated (29,30), and more recently their counterparts in mammalian cells have been cloned and characterized (31-33).

The control of telomerase activity is highly cell-type

specific. In most normal somatic cells, telomerase activ- ity is repressed and the number of cell divisions is limited by progressive telomeric shrinkage. Germline cells do not suffer telomeric attrition, and telomerase actively sus- tains and replenishes terminal sequences of chromosomes in these tissue types. Telomerase reactivation appears to be associated with the process of cellular immortalization and malignant transformation. Telomerase activity has been detected in about 85% of malignant human tumors, whereas its expression is low or negligible in benign or premalignant tissue specimens (10,34). The absence of telomerase activity in a small percentage of malignant or metastatic tumors raises questions about whether the telomere hypothesis is the sole explanation for cellular senescence and immortalization (35). It is possible that another strategy for unlimited cellular proliferation ex- ists. Alternatively, these cases may reflect false-negative results due to technical artifacts or even the true lack of immortal cells in such tumors.

Measurement of Telomerase Activity and Telomeric Length

Telomerase Activity

Early methods for detecting telomerase activity in- volved a primer extension assay that measures the ability of the cell extract to add telomeric repeats to the 3‘ end of synthetic oligonucleotide primers in vitro (21,22). The incorporation of nucleotides could be distinguished by labeling them with a radioactive material such as 32P. The reaction products were separated on a polyacrylamide gel, which was subsequently exposed to an autoradio- graphic film (1 1). These methods required at least lo7- 1 O8 cells with variable efficiency of telomerase extraction between cell types, and interference by background sig- nals could be problematic in the interpretation of results (10,20,28). Kim et al. (36) optimized telomerase extrac- tion with a more reliable detergent-mediated cell lysis method and improved telomerase measurement via a polymerase chain reaction (PCR)-based assay known as TRAP (telomeric repeat amplification protocol). In this assay, the extension products synthesized by telomerase serve as the templates for PCR amplification. This method is able to detect telomerase with greater speed and with a lO‘-fold enhanced sensitivity.

Despite the improved simplicity and sensitivity pro- vided by the TRAP assay, several limitations still exist (34). The assay is poorly quantitative, with a nonlinear dependence of product synthesis on enzyme content, primer, and nucleotide concentrations (37). Assessments

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Therapeutic Potential of Telornerase 27 1

TTAGGGTTAG rn TTACGG - AATCCCAA UCCCAAU

I ELONGATION

TTAGGGTTAG n GTTAGGGTTAG -3’ AATCCCAA

TRANSLOCATION

AUCCCAAUC 5’

3’

AATCCCAA

AUCCCAAU 5’

3’

AATCCAA

J ELONGATION

Figure 2. Chromosomal DNA binds part of the telomerase region in telomerase RNA. Polymerization occurs to elongate the telomere. The chromosome is then translocated and repositioned to repeat the polymerization step. The steps are reiterated to add multiple copies of the telomeric repeat sequence (in bold). (From Ref. 22.)

of relative levels of telomerase activity are therefore dif- ficult. Some tissue samples contain nonspecific inhibitors of Taq polymerase, the DNA polymerase that extends the reverse primer in the PCR cascade. These inhibitors can produce false-negative results in telomerase-positive samples. Furthermore, the necessity of preparing a cell or tissue extract in the TRAP assay implies that it can be applied to small but not microscopic samples. Finally, because the assay evaluates the biochemical activity of telomerase, a function sensitive to heat, RNase, and pro- tease contamination, frozen rather than fixed or heated specimens are required.

A recently modified protocol, described by Wright et al. (38), which includes a 150-bp internal standard in the assay, allows for better quantitation of telomerase activ-

ity through a comparison of the intensity of the telo- merase ladder signals with that of the internal standard. This internal standard also functions as a control and identifies false-negative results caused by inhibitors of the Taq polymerase.

Both nonamplified and TRAP assays detect telo- merase activity by measuring its ability to extend an oli- gonucleotide primer (39). Cloning of the RNA compo- nent of telomerase (hTR) offers an alternative strategy to measure the enzymatic expression in clinical samples. Analytical techniques such as Northern blots and reverse transcriptase-PCR can be performed on fixed or heated specimens and on frozen samples (27). However, diffi- culties remain regarding accurate quantitation of hTR in malignant cells and distinguishing true signals from

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272 Davis and Siu

background interference. More recently, a radioactive in situ hybridization assay for expression of hTR has been developed (40). This technique allows the visualization and cellular localization of hTR in archival paraffin-em- bedded tissues. malignant cells.

sis yet to be proven. Besides being an attractive therapeu- tic target, telomerase may also function as a useful diag- nostic and prognostic tumor marker because of the differ- ential pattern of its expression between normal and

Telomeric Length Telomerase as a Therapeutic Target

Conventional methods for the measurement of telo- meric length are informative but far from definitive. Shortened chromosomes have a tendency to fuse with one another, forming “telomeric associations” or “di- centric” chromosomes. The occurrence of such structural anomalies bears an uncertain association with telomeric length and may represent an epiphenomenon from unre- lated cellular processes (10). Direct assessment of te- lomeric length involves the digestion of genomic DNA using restriction endonucleases, followed by Southern hybridization with a telomere-specific probe. Because the human telomeric repeats do not contain any known cleav- age sites for restriction enzymes, they are removed to- gether with a subtelomeric region to form a terminal re- striction fragment (TRF) (41). There is substantial heterogeneity between the TRFs of different chromo- somes within a cell due to variation in the length of both the telomeric and subtelomeric components. Therefore, the average TRF length is obtained and this parameter provides a reasonable overall estimate of telomeric length.

An exciting new method for the measurement of telo- meric length involves quantitative fluorescence in situ hybridization (42). Metaphase chromosomes are hybrid- ized with a synthetic peptide nucleic acid (PNA) telomere probe, which consists of nucleotide bases attached to an uncharged peptide-like backbone. This backbone pro- vides increased stability compared with DNA or RNA probes. Quantitation of the telomere fluorescence inten- sity is feasible using either flow cytometry or image anal- ysis methods. Results of flow fluorescence in situ hybrid- ization measurements have been shown to correlate well with those of conventional telomere length measurements by Southern blot analysis (43).

CLINICAL APPLICATIONS

The strong correlation between telomerase activity, cellular immortalization, and the malignant phenotype has spurred intense research interest in the development of inhibitors of telomerase as anticancer therapies. How- ever, the association remains correlative rather than caus- ative, and whether the suppression of telomerase activity will actually lead to tumor cell death in vivo is a hypothe-

In support of a role for inhibitors of telomerase in can- cer chemotherapy, a growing pool of experimental data provides confirmation that telomerase is required for maintaining telomeres that have reached the critical M2 stage. Telomeric length has been demonstrated to be gen- erally shorter in malignant cells as compared with normal somatic cells, and in studies of ovarian carcinoma and leukemias, a progressive decline in length with increas- ing stage of disease has been observed (44,45). Indeed, if telomerase activation were a late event that prevents malignant cells from entering M2 and renders them im- mortal, telomeres would be of short length. It would be expected therefore that inhibition of telomerase would re- sult in malignant cells reaching M2 earlier than normal cells.

Studies with human fibroblasts (20,46) have shown that their ability to replicate in culture is proportional to the mean TRF length and chromosomes with critically shortened telomeres may require telomerase to prolifer- ate. Furthermore, when antisense transcripts of hTR were introduced into HeLa cells (27), a reduced mean TRF length was observed with induction of a crisis state. These studies suggest that inhibition of telomerase activ- ity may bring about cell death. Ohmura et al. (47) were able to show that cellular senescence could be restored in an immortalized renal cell carcinoma line, RCC23, by reintroducing a normal chromosome 3. The deletion of a gene(s) on chromosome 3 was associated with indefinite growth in this cell line, and the correction of this abnor- mality appeared to result in telomerase inhibition and telomere shortening.

Evaluation of Potential Therapeutic Agents

in Vitl-o

Telomeric length has been used to evaluate the anti- proliferative effects of telomerase- and telomere-tar- geting agents. However, due to the inherent heterogeneity of cancer cell populations and the need for long-term cul- turing of cells to allow sufficient cell division to reach a critical degree of telomere shortening, this method may not be optimal. Reimann et al. (48) observed that meta-

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Therapeutic Potential of Telomerase 213

centric and submetacentric chromosomes often detected in malignant cells were cytogenetic products of telomere fusion between shortened chromosomes. Immunofluo- rescence analysis of dicentric chromosomes might there- fore be a useful end point, although the utility of this method for studying the biological effects of these agents still requires validation.

In Vivo

The transgenic murine model is not ideal for assessing the in vivo response to inhibitors of telomerase because telomerase activity is seen in both normal and malignant cells (49). Athymic mice with human tumor xenografts will probably be a more appropriate model that mimics the clinical situation. Xenograft models from human breast cancer, lymphoma, and prostate cancers with high telomerase activity are being developed for the evalua- tion of candidate telomerase inhibitors (1 0,37).

Clinical Trials

Special consideration is required in designing clinical trials for evaluation of telomerase inhibitors. Telomerase inhibitors will only have a clinical impact after telomeres have been allowed to shorten to a critical level, beyond which point senescence and cell death will occur. The expected long lag time until the onset of antitumor effects by telomerase inhibitors suggests that their long-term ad- ministration after cytotoxic therapy offers the most logi- cal therapeutic approach. This tactic involves the initial debulking by cytotoxic agents to reduce tumor burden to a minimum, at which point the telomerase inhibitor is added to suppress further proliferation of the tumor.

Traditional end points used to monitor the clinical ef- ficacy of cytotoxic therapy, such as tumor shrinkage and duration of response, may not be relevant to telomerase inhibitors or other biological agents (50). The mechanism of action of these agents tends to be the suppression of ongoing tumor proliferation rather than active cell kill. Hence, maintenance of a stable disease status or delay of time to progression may be better indices of their clinical activity.

Toxicity Profile

Telomerase activity is expressed at low levels in a va- riety of normal tissues (5 1 -54), including mononuclear cells of the hematopoietic system, lymph nodes, tonsillar tissue, keratinocytes, intestinal mucosae, and gonadal stem cells. I t is possible therefore that in addition to ex- erting effects on malignant tumors, telomerase inhibitors may induce senescence or cell death in these normal cells

Table 1

Potential Inhibitors of Telomerase arid Their Sites of Activiry

Site of Action Agents

Telomerase interacting RNA component Antisense oligonucleotides

Peptide nucleic acids Aminoglycoside antibodies Telomerase-gene modulation

Protein component ? Telomere interacting G-quartet interactive agents

Reverse-transcriptase inhibitors Unknown Quinolone antibiotics

and tissues. Lymphopenia and immunosuppression may pose a problem, particularly with prolonged administra- tion. Keratinocytes, though normally quiescent, are acti- vated by stimuli such as cutaneous injury and delayed wound healing might occur as a result of telomerase in- hibition. However, it is unlikely that skin aging would be influenced to any appreciable degree, due to the abun- dant supply of integumentary stem cells (55). Gonadal stem cells in humans enter into a prolonged quiescent phase (56) and thus may be spared the side effects of telomerase inhibition. Finally, as for many other biologi- cal agents, unpredictable toxicities might occur, despite preclinical testing, particularly after prolonged adminis- tration.

Potential Agents

Two main classes of agents have been described de- pending on their site of activity, namely the telomerase- interacting compounds and the telomere-interacting com- pounds (Table 1).

Telomerase-Interacting Compounds

Antisense oligonucleotides. Modified deoxyribose oligonucleotides, including phosphorothioate oligonucle- otides, which mimic telomeric sequences, are being eval- uated for their ability to inhibit telomerase activity and tumor growth. When bound to RNA, they mediate cleav- age of the RNA:oligonucleotide hybrid by the cellular endonuclease ribonuclease H (RNase H) and disrupt translation, which is believed to be the most important antiproliferative mechanism of antisense oligonucleo- tides (57). A phosphorothionate hexanucleotide 5‘- d(TTAGGG)-3’ has been shown to inhibit telomerase ac- tivity and induce programmed cell death in a Burkitt’s lymphoma-derived (OMA-BL 1) cell line, whereas oligo- nucleotides with scrambled sequences were inactive.

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274 Davis and Siu

When tested in a xenograft human-nude mouse model, the hexanucleotide caused tumor regression and reduced metastases, compared with the lack of efficacy in the con- trol animals treated with either saline or scrambled oligo- nucleotides (58).

Unmodified oligonucleotides are unstable in the circu- lation, primarily due to attack by 3'-5' exonucleases. Modifications to the phosphodiester backbone, such as the replacement of a nonbridging oxygen with sulfur in phosphorothioate oligonucleotides, improves stability in the circulation. However, significant problems associated with their development as drugs for use in humans re- main. They are highly negatively charged, have very large molecular weights, and are acid labile. These fea- tures necessitate intravenous administration, and even so, passage across cell membranes is probably impaired (57).

In a recent report that examined the combined use of a telomerase antisense oligonucleotide and anticancer agents in several human lung cancer cell lines, the addi- tion of liposomes greatly enhanced the incorporation of the oligonucleotide into tumor cells (59). Furthermore, there was greater inhibition of tumor growth when each chemotherapeutic agent was delivered in conjunction with the antisense oligonucleotide vs. chemotherapy alone.

PNAs are modified oligonucleotides that con- tain a non-ionic backbone in which the deoxyribose link- ages have been replaced by N-(2-amino-ethy1)glycine units. The uncharged nature of PNA internucleotide link- ages increases their affinity and rate of hybridization with targeted nucleic acids and affords greater resistance to degradation by proteases and nucleases. Unlike the phos- phorothionate oligonucleotides, PNA-mediated antisense effects depend on the sterical blocking of RNA pro- cessing, cytoplasmic transport, or translation (60). PNAs recognize the RNA component in telomerase and cause specific inhibition of its activity (61). When the extent of telomerase inhibition by PNAs and phosphorothionate oligonucleotides was compared in JR8 melanoma cell ex- tracts, PNAs inhibited the enzyme activity more effi- ciently and at lower concentrations (62). Unfortunately, clinical utility of PNAs has also been hampered by their poor membrane permeability. Strategies to facilitate cel- lular uptake of PNAs, such as conjugation to carrier mol- ecules or incorporation into liposomes, are under investi- gation (60).

Antibiotics. Aminoglycoside antibiotics, such as neomycin, appear to possess an inhibitory effect against the RNA component of telomerase (63). At high concen- trations, quinolone antibiotics produced growth inhibi-

PNAs.

tion and decreased telomerase activity in human transi- tional carcinoma cell lines by unknown mechanisms (64).

The generation of mutant telo- merase RNA may be used to compete with endogenous wild-type RNA for assembly into complexes with telo- merase proteins. These manipulated telomerases might then attach incorrect nucleotide sequences to chromo- somal termini, resulting in unstable and incompetent telomeres. This strategy could be used to directly disrupt the telomere-telomerase interaction and thus induce se- nescence and cell death (22).

Gene modulators.

Telomere-Targeting Agents

Nucleoside and nonnucleoside reverse transcriptase inhibitors. Inhibitors of reverse transcriptase interfere with the polymerase active site of telomerase. Nucleoside analogues such as azidothymidine (AZT) or dideoxygua- nosine (ddG) can be preferentially incorporated into telo- meric DNA by telomerase and result in chain termina- tion. AZT has been shown to cause telomere shortening in Tetrahymena (65) and was subsequently analyzed with ddG for their effects in long-term cultures of two immor- talized human B- and T-cell lines (66). Progressive telomere shortening was observed in all cultures of both cell lines with ddG, with eventual stabilization after sev- eral weeks. AZT caused progressive telomere decline in some but not all cell cultures. The triphosphates of both ddG and AZT inhibited telomerase in both cell lines, but no senescent phenotype was detected even with long- term cultures, suggesting that counteractive regulation may be present to prevent further losses of telomeric DNA. Prolonged passaging in arabinofuranyl-guanosine, dideoxyinosine, dideoxy adenosine, didehydrothymidine, or phosphonoformic acid (foscamet) produced neither telomere shortening nor reduced cell growth or viability.

Whether pharmacologic doses of these agents can achieve biologically relevant concentrations to inhibit telomerase in humans is doubtful. Further research is needed to produce more potent telomere-targeting nucle- oside and nonnucleoside reverse transcriptase inhibitors with tolerable toxicity profiles.

G-quartet interactive agents. During the transloca- tion step of telomere synthesis, telomerase dissociates from the extended DNA and realigns for the next round of elongation. This intermediate step is facilitated by the telomere DNA product forming secondary structures in the form of a tetraplex held together by G-quartets (67). Several G-quartet interactive agents have been identified, such as cationic porphyrins and other compounds that are

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Therapeutic Potential of Telomerase 215

designed using a structure-based approach (68,69). These agents bind to the G-quadruplex and prevent proper sub- strate recognition by telomerase. An alternative approach by Fletcher et al. (70) used 7-deazanucleotides, which were thought to disrupt G-quadruplex formation due to the lack of the 7-nitrogen in these substituted nucleo- tides.

Telomerase as a Diagnostic Marker

Telomerase activity is detected in about 85% of malig- nancies (34) but is virtually absent in normal somatic cells. This differential finding, when combined with the high sensitivity of the TRAP assay, could potentially be used to confirm malignancy in situations where histo- pathologic techniques are suboptimal. Telomerase detec- tion may become a useful adjunct in cases of diagnostic dilemma with indeterminate cytologic or pathologic findings.

Several groups have shown increased levels of telo- merase in preinvasive tumors of the colon (71) and head and neck (72), albeit at lower levels than in the corre- sponding invasive tumors. Early detection or screening might be feasible if noninvasive sensitive techniques are available. Tarin et al. studied an unselected series of pa- tients with bladder, breast, and colon cancers to assess the potential screening validity of telomerase (73-75). Urine samples from patients with and without bladder cancer were obtained. Telomerase was detected in exfoli- ated bladder cells in I6 of 26 patients with bladder cancer (62% sensitivity) and 3 of 83 nonmalignant urine samples (96.4% specificity). In patients with and without breast cancer, fine-needle aspirate samples yielded a sensitivity of 67% and a specificity of 90%. Colonic washings from patients with and without colon cancer resulted in a sensi- tivity of 60% and specificity of 100%. Though these re- sults are very promising, further studies are needed to better define the sensitivity and specificity of telomerase in various tumor types and so clarify its role in screening and early diagnosis.

Telomerase as a Prognostic Marker

Several studies have shown a correlation between telomerase activity and clinical outcome. Patients with neuroblastoma were found to have a poor prognosis when high levels of telomerase were detected, whereas others with low telomerase activity despite being in the meta- static stage have shown spontaneous regression (76). An association between telomerase activity and poor clinical outcome was also found in patients with gastric cancer (77). These findings suggest that the measurement of

telomerase activity may be a useful prognostic indicator and may be helpful in stratifying outcome risks and in determining appropriate therapy for individual patients. Further prospective studies are necessary to better assess its utility as a prognostic marker.

CONCLUSION

The discovery of the telomere-telomerase complex has offered tremendous insight into the cellular processes of replicative senescence and immortalization. Measure- ment of telomeric length and telomerase activity in hu- man tissues is now feasible, and ongoing efforts continue to optimize these techniques. The high prevalence of telomerase activity in malignant cells, in contrast to its virtual absence in normal somatic cells, suggests that inhi- bition may be of therapeutic potential. Telomere- and telo- merase-targeting compounds have generated great inter- est as antitumor agents, but further research and evalua- tion are necessary before their application in the clini- cal setting is possible. Despite progress achieved thus far, many questions remain to be answered. 1s the rela- tionship between telomerase reactivation and cellular im- mortalization causative? How does one explain telo- merase negativity in a small percentage of malignant tumors? Does a tumor-specific telomerase inhibitor exist and, if so, how is it best used in the clinical setting? Can telomerase provide a helpful diagnostic and prognostic tool in cancer patients? It is promising that with time, the list of uncertainties associated with the telomere- telomerase complex will become progressively shorter in length.

Address reprint requests to: Lillian L. Siu, M.D., Department of Medi- cal Oncology and Hematology, Princess Margaret HospitaVThe To- ronto Hospital, 610 University Avenue, Toronto, Ontario, M5G 2M9, Canada. Fax: 416-946-6546: e-mail: lillian.siu@uhn.on.ca

REFERENCES

I .

2.

3.

4.

5 .

Muller HJ: The remalung of chromosomes. The collecting net. Woods Hole 13:181-198, 1938. McClintock B: The stability of broken ends of chromosomes in Zea mays. Genetics 41 :234-282, 1941. Sandell LL, Zakian VA: Loss of a yeast telomere: arrest, recovery, and chromosome loss. Cell 75:729-739. 1993. Blackburn EH, Gall JG: A tandemly repeated sequence at the ter- mini of the extrachromosomal RNA genes in Tetrahymena. J Mol Biol 12033-35, 1978. Moyzis RK, Buckingham JM, Cram LS, et al: A highly conserved repetitive DNA sequence (TTAGGG)n present at the telomeres of human chromosomes. Proc Natl Acad Sci USA 85:6622-6626. 1988.

Can

cer

Inve

st D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

ichi

gan

Uni

vers

ity o

n 11

/03/

14Fo

r pe

rson

al u

se o

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276 Davis and Siu

6. Blackbum, EH: Telomeres: Do the ends justify the means? Cell 37:7-8, 1984.

7. Wellinger RJ, Sen D: The DNA structures at the ends of eukaryo- tic chromosomes. Eur J Cancer 33:735-749, 1997.

8. Harley CB, Vaziri H, Counter CM, et al: The telomere hypothesis of cellular aging. Exp Gerontol 27:375-382, 1992.

9. Frenck RW, Blackburn EH, Shannon KM: The rate of telomere sequence loss in human leukocytes varies with age. Proc Natl Acad Sci USA 95:5607-5610, 1998. Sharma S, Raymond E, Soda H, et al: Preclinical and clinical strat- egies for development of telomerase and telomere inhibitors. Ann Oncol 8:1063-1074, 1997.

11. Rhyu MS: Telomeres, telomerase, and immortality. J Natl Cancer Inst 87:884-894, 1995.

12. Moriu GB: The implications of telomerase biochemistry for hu- man disease. Eur J Cancer 33:750-760, 1997.

13. Watson JD: Origin of concatameric T4 DNA. Nat New Biol239: 197-201, 1972.

14. Hamilton SE, Corey DR: Telomerase: anticancer target or just a fascinating enzyme? Chem Biol 3:863-867, 1996.

15. Wright WE, Shay JW: The two-stage mechanism controlling cel- lular senescence and immortalization. Exp Gerontol 27:383-389. 1992. Shay JW, Wright WE: Mechanisms of escaping human cellular senescence. Radiat Oncol Invest 3:284-289, 1996. Wright WE, Pereira-Smith OM, Shay JW: Reversible cellular se- nescence: implications for a two-stage model for the immortaliza- tion of normal human diploid fibroblasts. Mol Cell Biol9:3088- 3092, 1989. Shay JW, Pereira-Smith OM, Wright WE: A role for both RB and p53 in the regulation of human cellular senescence. Exp Cell Res 196:33-39, 1991. Harley CB, Kim NW, Prowse KR, et al: Telomerase, cell immor- tality and cancer. Cold Spring Harbor Symp Quant Biol 59:307- 315, 1994.

20. Counter CM, Avilion AA, LeFeuvre CE, et al: Telomere shorten- ing associated with chromosome instability is arrested in immortal cells which express telomerase activity. EMBO J 1 1:1921-1929, 1992. Greider C, Blackburn EH: The Telomere terminal transferase of Tetrahymena is a ribonucleoprotein enzyme with two kinds of primer specificity. Cell 513877898, 1987. Greider CW, Blackbum EH: A telomeric sequence in the RNA of Tetrahymena telomerase required for telomere repeat synthesis. Nature 337:331-337, 1989. Greider CW, Blackburn EH: Identification of a specific telomere terminal transferase activity in tetrahymena extracts. Cell 43:405- 413, 1985. Blasco MA, Funk W, Villeponteau B, et al: Functional character- ization and developmental regulation of mouse telomerase RNA. Science 269:1267-1270, 1995. Melek M, Davis BT, Shippen DE: Oligonucleotides complemen- tary to Oxutricha nova telomerase RNA delineate the template domain and uncover a novel mode of primer utilization. Mol Cell Biol 14:7827-7838, 1994.

26. Singer MS, Gottschling D E Template RNA component of Saacharomyces cerevisiae telomerase. Science 266:404-409, 1994. Feng J , Funk W, Wang S, et al: The RNA component of human telomerase. Science 269: 1236- 1241, 1995. Morin GB: The human telomere terminal transferase enzyme is

10.

16.

17.

18.

19.

21.

22.

23.

24.

25.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

a ribonucleoprotein that synthesizes TTAGGG repeats. Cell 59: 521-529, 1989. Collins K, Kobayashi R, Greider CW: Purification of Tetrahy- mena telomerase and cloning of genes encoding the two protein components of the enzyme. Cell 81 :677-686, 1995. Greider CW, Autexier C, Buchkovich K, et al: Telomerase bio- chemistry and regulation in cellular immortalization. Proc Am Assoc Cancer Res 36672. 1995. Harrington L, McPhail T, Mar V, et al: A mammalian telomerase- associated protein. Science 275:973-977, 1997. Harrington L, Zhou W, McPhail T, et al: Human telomerase con- tains evolutionarily conserved catalytic and structural subunits. Genes Dev 11:3109-3115, 1997. Nakayama J, Saito M, Nakamura H. et al: TLPl: a gene encoding a protein component of mammalian telomerase is a novel member of WD repeats family. Cell 88:875-884, 1997. Shay JW, Wright WE: Telomerase activity in human cancer. Curr Opin Oncol 8:66-71, 1996. Bryan TM, Englezou A, Gupta J. et al: Telomere elongation in immortal human cells without detectable telomerase activity. EMBO J 14:4240-4248, 1995. Kim NW, Piatyszek MA, Prowse KR, et al: Specific association of human telomerase activity with immortal cells and cancer. Sci- ence 266:2011-2015, 1994. Raymond E, Sun D, Chen S-F, et al: Agents that target telomerase and telomeres. Curr Opin Biotech 7333-591, 1996. Wright WE, Shay JW, Piatyszek MA: Modifications of a te- lomeric repeat amplification protocol (TRAP) result in increased reliability, linearity and sensitivity. Nucleic Acids Res 23:3794- 3795, 1995. Kim NW: Clinical implications of telomerase in cancer. Eur J Cancer 33:781-786, 1997. Yashima K, Piatyszek MA, Saboorian HM. et al: Telomerase activity and in situ telomerase RNA expression in malignant and non-malignant lymph nodes. J Clin Pathol 50:110-117. 1997. Bacchetti S : Telomere maintenance in tumour cells. Cancer Surv 28: 197-21 6, 1996. Lansdorp PM, Venvoerd NP. van de Rijke FM, et al: Heterogene- ity in telomere length of human chromosomes. Hum Mol Genet 5:685-691, 1996. Rufer N, Dragowska W, Thombury G, et al: Telomere length dy- namics in human lymphocyte subpopulations measured by flow cytometry. Nat Biotech 16:743-747, 1998. Counter C, Hirte H, Bacchetti S, et al: Telomerase activity in hu- man ovarian carcinoma. Proc Natl Acad Sci USA 91:2900-2904, 1994. Counter CM, Gupta J, Harley CB, et al: Telomerase activity in normal leukocytes and in hematologic malignancies. Blood 85: 2315-2320, 1995. Allsopp RC, Harley CB: Evidence for a critical telomere length in senescent human fibroblasts. Exp Cell Res 219:130-136, 1995. Ohmura H, Tahara H, Suzuki M, et al: Restoration of the cellular senescence program and repression of telomerase by human chro- mosome 3. Jpn J Cancer Res 86, 899-904, 1995. Reimann N, Rogalla P, Kazmierczak B, et al: Evidence that meta- centric and submetacentric chromosomes in canine tumors can result from telomeric fusions. Cytogenet Cell Genet 67:8 1-85, 1994. Chadeneau C, Siege1 P, Harley CB, et al: Telomerase activity in normal and malignant murine tissues. Oncogene 1 1 :893-898, 1995.

Can

cer

Inve

st D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

ichi

gan

Uni

vers

ity o

n 11

/03/

14Fo

r pe

rson

al u

se o

nly.

Therapeutic Potential of Telomerase

so.

51.

52.

53.

54.

55.

56.

57.

58.

59.

60.

61.

62.

63.

64.

Von Hoff DD: There are no bad anticancer agents, only bad clini- cal trial designs. Twenty-first Richard and Hinda Rozenthal Foun- dation Award Lecture. Clin Cancer Res 4: 1079-1086. 1998. Norrback K-F. Roos G: Teloineres and telomerase in normal and malignant haematopoietic cells. Eur J Cancer 33:774-780. 1997. Holt SE, Wright WE, Shay JW: Multiple pathways for the regula- tion of telomerase activity. Eur J Cancer 33:761-766. 1997. Shay JW, Bacchetti S: A survey of telomerase activity in human cancer. Eur J Cancer 33:787-791. 1997. Wright WE, Piatyszek MA, Rainey WE, et al: Telomerase activity in human germline and embryonic tissues and cells. Dev Genet 18:173-179, 1996. West MD: The cellular and molecular biology of skin aging. Arch Dermatol 130:87-95, 1994. Potten CS: Cell cycles in cell hierarchies. Int J Radiat Biol Relat Stud Phys Chem Med 49:257-278, 1986. Boral AL, Dessain S, Chabner BA: Clinical evaluation of biologi- cally targeted drugs: obstacles and opportunities. Cancer Cheino- ther Pharmacol 42(Suppl):S3-S21, 1998. Mata JE, Joshi SS, Palen B, et al: A hexameric phosphorothioate oligonucleotide telomerase inhibitor arrests growth of Burkitts’ lymphoma cells in vitro and in vivo. Toxicol Appl Phartnacol 144: 189-197. 1997. Kato H, Araya S. Hirose K. et al: In vitro tumour growth inhibi- tion by telomerase antisense and anticancer agents. Proc Am Assoc Cancer Res 39:416. 1998 (abstract 2833). Knudsen H, Nielson PE: Application of peptide nucleic acid in cancer therapy. Anti Cancer Drugs 8.1 13-1 18, 1997. Norton JC, Piatyszek MA, Wright WE, et al: Inhibition of human telomerase activity by peptide nucleic acids. Nat Biotechnol 14: 615-619. 1996. Zaffaroni N, Villa R, Folini M, et al: In vitro inhibition of human melanoma telomerase by peptide nucleic acids (PNAsj. Proc Am Assoc Cancer Res 39:304, 1998 (abstract 2078). Mazumder A, Pommier Y: Telomerase inhibition by RNA-di- rected antibiotics. Proc Am Assoc Cancer Res 37:396, 1996 (ab- stract 2702). Yamakuchi M, Nakata M, Kawahara K, et al: New quinolones, ofloxacin and levofloxacin inhibit telomerase activity in transi- tional cell carcinoma lines. Cancer Lett 119:213-219, 1997.

65. Strahl C, Blackburn EH: The effects of nucleoside analogs on

66.

67.

68.

69.

70.

71.

72.

73

14

75

76

77

211

telomerase and telomeres in Terrahymena. Nucleic Acids Res 22: 893-900, 1994. Strahl C, Blackburn E: Effects of reverse transcripase inhibitors on telomere length and telomerase activity in two immortali7ed human cell lines. Mol Cell Biol 16:53-65, 1996. Salazar M, Thompson BD. Kenvin SM. et al: Thermally induced DNA.RNA hybrid to G-quadruplex transitions: possible implica- tions for telomere synthesis by teloinerase. Biochemistry 35: 16110-16115, 1996. Wheelhouse RT, Sun D. Hurley LH: Non-nucleotide telomerase inhibitors: structure-based design of G-quadruplex interactive agents. Proc Am Assoc Cancer Res 38:637, I997 (abstract 4279). Wheelhouse RT, Han FX, Han H. et al: The interaction of‘ te- lomerase-inhibitory porphyrins with G-quddIUpkX DNA. Proc Am Assoc Cancer Res 39:430. 1998 (abstract 2925). Fletcher TM, Salazar M, Chen SF: Human telornerase inhibition by 7-deaza-2’-deoxypurine nucleoside triphosphates. Biocheniis- try 35:15611-15617, 1996. Tahara H, Kuniyasu H. Yoko7aki H. et al: Telomerase activity i n preneoplastic and neoplastic gastric and colorectal lesions. Clin Cancer Res 1:1245-1251. 1995. Califano J, Ahrendt SA. Meininger G, et al: Detection of te- lomerase activity in oral rinses from head and neck squamouh cell carcinoma patients. Cancer Res 565720-5722, 1996. Yoshida K, Sugino T. Goodison S. Tarin D. et al: Detection of telomerase activity in exfoliated cancer cells in colonic luininal washings and its related clinical implications. Br J Cancer 75: 548-553, 1997. Yoshida K, Sugino T, Tahara H. Tarin D. et al: Telomerase activ- ity in bladder carcinoma and its implication for noninvasive diag- nosis by detection of exfoliated cancer cells in urine. Cancer 79: 362-369. 1997. Sugino T. Yoshida K, Bolodeoku J, Tarin D, et al: Teloinerase activity in human breast cancer and benign breast lesions: diag- nostic applications in clinical specimens, including tine needle as- pirates. Int J Cancer 69:301-306, 1996. Hiyama E. Hiyama K, Yokoyama T, et al: Correlating telomerase activity levels with human neuroblastoma outcomes. Nat Med 1 : 249-255, 1995. Tahara E. Semba S, Tahard H: Molecular biological observations in gastric cancer. Semin Oncol 23:307-315, 1996.

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