Critical Reviews in Oncology/Hematology 41 (2002) 2940 www.elsevier.

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Telomerase in cancer and aging

Meaghan P. Granger, Woodring E. Wright, Jerry W. Shay *
Department of Cell Biology, The Uni6ersity of Texas Southwestern Medical Center, 5323 Harry Hines Boule6ard, Dallas, TX 75390 -9039, USA Accepted 2 August 2001

Contents
1. Telomere biology . . . . . . . 1.1. Introduction . . . . . . . 1.2. End-replication problem 1.3. Telomere hypothesis . . . 1.4. Telomere conguration . 1.5. Telomerase holoenzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 30 30 30 30 31 32 32 33 33 33 34 35 35 36 36 36 37 37 37 37 38 38 38 38 39 40

2. Aging . . . . . . . . . . . . . . . . . . . . . . 2.1. Senescence and telomerase . . . . . . . 2.2. Genetic disorders and telomeres . . . . 2.3. Hematopoietic system and telomerase. 2.3.1. Stem cells . . . . . . . . . . . . . 2.3.2. Peripheral blood leukocytes . . .

3. Cancer and telomerase . . . . . . . . . . . . . . . . . . 3.1. Survey of telomerase and cancer. . . . . . . . . . 3.2. Role of telomerase in malignant transformation . 3.3. Methods of telomerase acquisition. . . . . . . . . 3.4. Prognostic implications of telomerase detection . 3.5. Residual disease . . . . . . . . . . . . . . . . . . . 3.6. Diagnostic potential . . . . . . . . . . . . . . . . . 3.7. Therapeutic potential . . . . . . . . . . . . . . . . 3.7.1. Telomerase inhibitors . . . . . . . . . . . . 3.7.2. Immunotherapy and telomerase . . . . . . 3.7.3. Chemoprevention and telomerase . . . . .

4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Reviewers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biographies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

* Corresponding author. Tel.: + 1-214-648-3282; fax: + 1-214-648-8694. E -mail addresses: meaghan.granger@utsouthwestern.edu (M.P. Granger), jerry.shay@utsouthwestern.edu (J.W. Shay).

woodring.wright@utsouthwestern.edu

(W.E.

Wright),

1040-8428/02/$ - see front matter 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 1 0 4 0 - 8 4 2 8 ( 0 1 ) 0 0 1 8 8 - 3

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Abstract The telomeretelomerase hypothesis is the science of cellular aging (senescence) and cancer. The ends of chromosomes, telomeres, count the number of divisions a cell can undergo before entering permanent growth arrest. As divisions are being counted, events occur on the cellular and molecular level, which may either delay or hasten this arrest. As humans age, a particular concern is the accumulation of events that lead to the progression of cancer. Telomerase is a mechanism that most normal cells do not possess, but almost all cancer cells acquire, to overcome their mortality and extend their lifespan. This review aims to provide a comprehensive understanding of the role of telomerase in cancer development, progression, diagnosis, and in the future, treatment. The ultimate goal of telomerase research is to use our understanding to develop anti-telomerase therapies, an almost universal tumor target. 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Telomere; Telomerase; Senescence; Replicative aging; Cancer; Immunosenescence; Telomerase inhibitors

1. Telomere biology

1.1. Introduction
After fertilization and mixing of 23 paternal and 23 maternal chromosomes, human life begins as a single cell with 46 chromosomes whose initial function is to divide. Each new generation of daughter cells successively divides until it forms and develops into a complex, differentiated organism. With each division, the genetic code is transferred as our chromosomes are replicated and distributed into the daughter cells. There are many cellular mechanisms in place to ensure that the transfer of information is done in a reliable, accurate, and efcient manner throughout the many duplications required over a human lifetime. Two of the mechanisms central to the subject of this review are the semi-conservative replication of DNA and cellular senescence.

end of the chromosome. Thus, the extreme end of the chromosome is not replicated and the telomeres progressively shorten. This is known as the end-replication problem. Fortunately, this problem does not result in the loss of essential genes in that each of the 46 human chromosomes is capped with long repeats of expendable noncoding DNA bases called telomeres (Fig. 1). Loss of the telomeric DNA continues with successive divisions until the telomeres reach such a critically short length that replication is halted. Human cells are estimated to have the potential to undergo on average 60 70 divisions, and at this point the cells growth arrest and enter senescence [1].

1.3. Telomere hypothesis

The sequence of human telomeres was identied as repeats of 6 base pairs (bp), (TTAGGG)n, by Moyzis in 1988 [2], although the name telomere (telos = end; meros = part) and the observation of the specialized genetic structures at the ends of chromosomes dates back to 1938 [2,3]. Human telomeres may vary with age and cell type and in general range from 6 to 12 kb in length in somatic cells [1]. Approximately 50 100 bp are lost with each cell cycle [4]. The shortening of telomeres is responsible for the counting mechanism that Hayick observed in normal cells in tissue culture in 1961. He found that normal human broblasts predictably entered a period where they ceased replication but continued metabolism (reviewed in [5]). The telomere hypothesis is the idea that progressive telomere shortening is a biologic or mitotic clock of the cell, keeping track of the number of replications a cell has used and indicating the time for permanent growth arrest when some of the telomeres are sufciently short.

1.2. End -replication problem

Semi-conservative replication of DNA is the process of duplicating the original DNA such that the nished products are two double DNA strands, each with one original and one new strand, to be distributed to the daughter cells. Replication begins with the separation of the double-stranded molecule so that the replication of each strand is done individually. As the two strands are separated, new bases must be added in the 5% to 3% direction. That task is straightforward on the leading strand, whose template is of the opposite polarity, and the bases are added in serial fashion. On the opposing lagging strand, replication must be done in segments, called Okazaki fragments, in order to accomplish 5% to 3% addition of bases. A new RNA primer is synthesized and used to initiate the synthesis of each fragment and eventually the fragments are ligated together to create a continuous strand. A problem occurs when the lagging strand, or the backwards strand, nears the end of the chromosome. There is no DNA beyond the end to serve as a template for the next Okazaki fragment to ll in the gap between the last Okazaki fragment and the

1.4. Telomere conguration

The fact that these bases do not code for any genetic information does not diminish their importance. We

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Fig. 1. Metaphase spread of human broblasts visualized in a uorescence microscope. Fluorescence in situ hybridization (FISH) using telomeric probes reveal the red/pink dots at the ends of each chromosome. Each dot identies a telomere and shows the two telomeres per chromosome with a total of 96 telomeres per normal human cell.

now know they are a site of dynamic activity beyond being the biologic timepiece [6]. They have a unique T-looped conguration where the telomere bends back on itself [7]. The overhanging guanine-rich single strand is tucked into the double stranded telomere. This creates a second smaller d-loop by displacing one of the telomere strands. This structure appears to protect the telomeres from end to end fusion with other chromosomes and from cell cycle checkpoints that would otherwise recognize the telomeres as chromosome breaks requiring repair (reviewed in [8]). Proteins that localize specically to telomeric DNA are the duplex telomere binding proteins, TRF1 and TRF2. TRF1 and 2 and their associated proteins have the primary responsibility of stabilizing the complex and forming the t-loop. Some degree of stabilization is intrinsic to the telomere overhang due to the G-rich nature of the TTAGGG repeats that form quadruplex structures. TRF1 is important in intratelomeric coiling [7]. TRF2 also binds along the length of the telomere but appears to be particularly abundant at the base of the t-loop and is important for its stabilization and formation [7]. Their cooperation is similar to two hands tying a knot, the rst hand (TRF1) forms a loop and the second hand (TRF2) tightens the strand and secures it. These duplex telomere DNA binding proteins also have their own associated proteins [9]. Human rap1p is integrated into the t-loop complex and interacts with TRF2, but its specic role in humans is unknown [10].

Tankyrase has the ability to inhibit TRF1, thereby releasing it from the t-complex and allowing telomerase and other enzymes to bind. TIN2 promotes TRF1 function and causes it to bind to the telomere [9]. The DNA damage response complex RAD50/MRE11/ NBS1 also cooperates with TRF2. The MRE11 complex functions conventionally in homologous recombination to repair DNA double strand breaks [11]. At the telomere, however, it is thought to stabilize the d-loop where the single stranded tail invades the duplex telomere. Based on its function in vitro, the role of NBS1 during the S phase may be to unwind the t-loop via a helicase [12].

1.5. Telomerase holoenzyme

Telomerase is a reverse transcriptase enzyme that can add the hexameric repeats, TTAGGG, to chromosome ends, extending and maintaining the length of the telomeres and thereby extending the number of divisions the cell may undergo (Fig. 2) [13]. The holoenzyme is composed of a RNA subunit, hTR, a protein subunit, hTERT, and many associated proteins. The reverse transcriptase complex catalyzes the addition of DNA bases, TTAGGG, to the telomere ends that are complementary to the RNA template sequence of hTR [14,15]. The human holoenzyme requires foldosome proteins p23 and hsp90 to assemble the telomerase components in vivo, which is conrmed in vitro since

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Fig. 2. Telomerase holoenzyme. The telomerase holoenzyme adds telomeric repeats, TTAGGG, in two steps (1) elongation and (2) translocation in succession. The enzyme is composed of two primary parts: hTR is the telomerase functional or template RNA portion, and hTERT is the telomerase reverse transcriptase enzymatic portion. The telomeric end can binds to the template region of hTR and is elongated by the addition of the bases complementary to the template via the catalytic subunit (hTERT). The complex then pauses and translocates and repeats the elongation of the telomere (e.g. the human telomerase complex is processive).

combining recombinant foldosome proteins, hTR, and hTERT is sufcient to reconstitute the holoenzyme [16]. The telomerase gene was recently mapped to 5p15.33 as one of the most distal genes on chromosome 5p. This has raised questions about whether its proximity to the telomere might result in it being regulated by telomere position effect mechanisms [17 19]. The introduction of the catalytic protein (hTERT) component of telomerase into normal broblasts and epithelial cells prevents shortening of the telomeres, and results in immortalization [20]. The key role of telomerase in immortalization is to maintain telomere length, not to produce a net increase in length [15]. Transient expression of a cre -excisable telomerase results in a preferential lengthening of the shortest telomeres and an increase in lifespan proportional to the length of the shortest telomere [21]. Likewise, the inhibition of telomerase in immortalized human cells leads to progressive telomere shortening and cell death [22].

2. Aging

2.1. Senescence and telomerase

Humans are living longer than ever before. Life expectancy at birth was 47.3 years in 1900 compared to 70.8 years in 1970 and 76.5 years in 1997. Centenarians are one of the most rapidly growing segments of the population. By the year 2050, persons greater than 85 years of age are expected to comprise nearly 15% of the entire population [23].

Normal cells have a nite number of divisions they can undergo before entering retirement or replicative senescence. Cells removed from older individuals, in general, divide fewer times in culture when compared to cells obtained from younger patients. Replicative senescence is the process by which cells stop dividing due to a genetically programmed event. Normal cells reach a period of growth arrest termed M1, or mortality stage 1, that is controlled by cell cycle regulatory genes p53/p21 and perhaps p16/Rb. There is speculation that M1 might be initiated by the presence of at least one sufciently short telomere and activation of the DNA damage response, although at this growth point most of the 92 telomeres still have several kilobase pairs of telomeric repeats. Other possibilities include regulation by subtelomeric genes or by transcription factors associated with the telomere [9]. If p53 function is altered or blocked (as with SV40 T antigen or E6/E7 papillomavirus proteins) cells continue to divide with progressive telomere shortening until they reach a second stage known as M2, mortality stage 2. It has been established that telomere shortening controls both M1 and M2 [15,24]. The M2 stage is often referred to as crisis at the point where many telomeres have been critically shortened and can no longer protect the telomeres so that chromosome fusion and breakage cycles occur and the cells eventually undergo apoptosis. In human broblasts in vitro that express viral oncogenes, a small number of cells (1 10 7), are able to escape M2 crisis and immortalize by the acquisition of a method for maintaining stable telomeres. This is accomplished through a reactivation oftelomerase in most cells, but alternative lengthening of telomere mechanisms (ALT) exist that use recombination and copy switching to move DNA from one telomere to another [25]. It is believed that replicative senescence decreases the number of mutations that can occur bylimiting the number of times the cell can divide. Properties of senescence are dependent on the number of cell divisions not time. It entails cells entering an irreversible state incapable of proliferation and with altered function. Cells become growth arrested in G1 and are unable to replicate their DNA [26]. What is the relationship between senescence, aging, cancer, and telomerase? Telomeres shorten in aging cell populations in vitro and in vivo (Fig. 3). Human broblasts from fetal tissues can typically undergo 60 80 population doublings (PDs), whereas young adult cells achieve only 2040 doublings, and older adult cells 1020 doublings before entering senescence. It is important to understand the molecular mechanism regulating senescence in oncology because it is the very cellular outcome we are seeking, for the cancer cells to stop dividing [26].

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Fig. 3. Telomere hypothesis. (a) With progressive cell divisions, telomeres shorten until they reach a critical shortened length. At this point, they undergo growth arrest or apoptosis (depending on whether other cellular pathways have been altered) unless they are able to maintain their telomeres to allow for subsequent divisions. (b) Stem cells exhibit a slower rate of telomere shortening because of the intrinsic presence of telomerase in these cells indicating that they may undergo more doubling prior to becoming senescent. (c) Committed peripheral blood lymphocytes (PBL, dotted line) are derived from the stem cell compartment and have a telomere length correlating with their age at the time of commitment. PBLs, upon activation, can have a brief period of telomerase upregulation followed by continued telomeric shortening. (d) Stem cell transplant recipients have accelerated telomeric shortening following transplant and then continued shortening at a rate proportional to the donor stem cells. (e) Cancer cells (dashed lines) may develop at any point in normal and hematopoietic cells and, in most cases, have utilized telomerase to maintain their telomeres. (f) All cells have higher rates of telomere loss from birth to 1 year, somewhat less from 1 to 4 years, followed by consistent loss of 50 100 bp/division.

2.2. Genetic disorders and telomeres

Patients with Hutchison Gilford progeria exhibit accelerated aging effects noticeable by age 2 years. They have short stature and abnormal posture and possess the typical aging phenotype of alopecia, joint stiffness, atrophic and wrinkled skin, atherosclerosis, and coronary artery disease including angina pectoris and myocardial infarction [27,28]. Fibroblasts from these patients show shorter telomere lengths than age matched controls and entered senescence in vitro much earlier than the aged matched control cells. When infected with hTERT they immortalize and telomere shortening is prevented without affecting checkpoints, functions, and cellular controls [28]. Similar results are achieved with cultures of skin broblasts from patients with Werners syndrome. These patients have premature aging effects of vascular disease, diabetes mellitus, cataracts, skin atrophy, graying hair, testicular atrophy, and cancer with an average lifespan of 47 years [28,29]. Trisomy 21 is another disorder with features of accelerated aging. Telomeres lengths have been found to shorten in lymphocytes obtained from Downs syndrome patients three times faster than normal individuals [30].

2.3. Hematopoietic system and telomerase 2.3.1. Stem cells Telomerase activity can be detected in both hematopoietic stem cells and in stem cell populations in other tissues such as skin, hair follicles, small intestine crypt cells, and lymphoid cells. Though the hematopoietic cells possess telomerase, they still have telomeres that shorten. Stem cells that are CD34 +/CD38 have shorter telomeres in adults than the same cell type in fetal and newborn tissue [31]. It is believed that the expression of telomerase in stem cells may help slow down, but does not completely prevent telomere attrition in cells that have a high rate of turnover (Fig. 3). Telomerase activity ensures that the stem cell compartment will be able to handle potentially large expansion demands, preserving the ability to maintain and repair the tissues. Though the telomeres still shorten, the time to critically shortened length may be delayed by telomerase [32]. Studies in stem cell transplant patients have shown that stem cells are on average 0.4 kb shorter in the reconstituted recipient when compared concurrently with the donor (Fig. 3). It is likely that the proliferation demands required to reconstitute the entire hematopoietic system

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results in aging of the cells prematurely by : 15 years [33]. Further, this shortening could eventually be sufcient to contribute to genetic instability and account for some of the secondary neoplasms seen in stem cell transplant patients beyond those attributed to alkylating agents and etoposide [31]. It is unknown whether the cumulative dose of stem cells given patients have an effect on this aging [33]. Telomere lengths in aged hematopoietic stem cells have not been shown to reach a critically shortened length leading to complete senescence. However, the cellularity of the bone marrow compartment is reduced by one-third at the age of 70 years [34]. It has been suggested that the replicative stress of shortening telomeres, particularly in lymphocytes, seen in early childhood and in the elderly might be responsible for the coinciding with the bimodal distribution of some hematopoietic disorders [34]. Acute lymphoblastic leukemia peaks in occurrence in children at an average age of 4 years [35] and again in adults after the age of 45 years [36]. The fact that this disorder is extremely rare during the critical years of childbearing and rearing and more common in early childhood and the elderly may be evidence of an evolutionary tumor protective mechanism.

2.3.2. Peripheral blood leukocytes The aging immune system involves a complex change in the entire system, both constitutionally and functionally. It is clinically apparent that aging individuals are at increased risk for infection, cancer, decreased immunity from previous vaccination, and reactivation of latent disease such as varicella. An overview of the global nature of these changes has recently appeared [37]. T cells, in general, shift from na ve to mature memory types with an increased proportion found in the bone marrow rather than peripheral blood. There are proportionally more CD8 + T-cells than CD4 + . B cells also show increased levels in the bone marrow with overall qualitative defects in antibody production. This is presumed to be from increased somatic mutations affecting Ig-gene rearrangements but is also inuenced by the shift in the T-helper cell population from Th1 to Th2. In contrast to decreased circulation of T and B cells, NK cells are found in increased numbers [37]. Granulocytes show decreased phagocytosis and respiratory burst in aging individuals. Monocytes are more activated, dendritic cells are unchanged, and macrophages increase their production of cytokines. Erythrocytes exhibit a shift in proportions of young to old populations [37]. As in the stem cell compartment, both circulating T and B cells have progressive telomere shortening with age and express low levels of telomerase activity at

rest, but levels transiently increase with stimulation by mitogens (Fig. 3, committed PBL). Interestingly, hTERT (the mRNA component of telomerase) appears to be constant among all lymphocyte stages independent of the level of telomerase activity [38,39]. This is consistent with most normal telomerase positive somatic cell types that still exhibit shortening in spite of the presence of telomerase and could reect alternatively spliced variants of hTERT that are inactive. Several studies have shown that telomere shortening with aging in peripheral blood leukocytes, both T lymphocytes and neutrophils, occurs in at least two phases. First, there is a rapid shortening from birth to 4 years at about 1 kb per year. Next there is a gradual shortening until : 40-years-old of 2050 bp per year and more slowly thereafter (Fig. 3) [34,40]. These reect the complexities due to the presence of telomerase, which may make telomere lengthening and shortening a more dynamic system depending on the hematopoietic requirements of the body. Certainly, the demand for clonal proliferation of a committed lymphocyte may increase or decrease telomere length, but also the telomere length of the originating stem cell can play a role [34]. Replicative senescence is intact in normal T-cells just as in broblasts and other cells. However, the implications of senescence in the immune system are more signicant. Mature T cells are required to give rise to clonal proliferations of cells to respond to foreign antigens upon activation. This cannot occur if the T cells are senescent. Senescence in culture reliably occurs in T lymphocytes, both CD4 + and CD8 + , after 2540 PDs. Thus, each mature T cell is capable of producing : 240 cells, or 1 1012 cells, before senescing [41]. There are signicant functional changes in senescent T cells. The most important being the lack of expression of CD28, which plays a key role in the transduction of IL-2 transcription and receptor expression, cooperation with B cells for antibody production, T cell homing, and signaling the induction of telomerase activity [41]. CD28 is present on 99% of neonatal T cell compared to only 45% of centenarian T cells. Telomeres of CD28 cells are shorter than CD28 + telomeres [41]. The CD28 cells are primarily of the CD8 + subset, which play a pivotal role in cytotoxic functions against cells with endogenously expressed antigens such as virally infected cells and tumor cells. Senescent T cells also acquire resistant to apoptosis that results from an increase in bcl-2 [41]. Telomeric changes in B cells are quite different from T cells. Rather than the steady but slow decline in telomeric length of aging T cells, activated B cells in germinal centers of tonsillar tissue show an increase in

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telomere length from their na ve state. The length then begins to decline once the B cells enter the memory compartment. Telomerase activity is highest in tonsillar B cell germinal centers, which corresponds to the point of longest telomeres. This is possibly a mechanism to protect the telomeres of highly specic B cells from the replicative stress placed on B memory cells [42].

3.1. Sur6ey of telomerase and cancer

An extensive summary of telomerase in human tumors has been surveyed as shown in Fig. 4. Telomerase can be measured by the TRAP assay, which uses PCR to amplify the extension products of the telomerase enzyme. The assay is quite sensitive and can detect as few as 0.01% positive cells. The background tissue in most cases is of normal somatic derivation and does not contribute telomerase activity. However, in cases where the histological environment of the tumor is naturally telomerase expressing (such as intestinal epithelium), a positive result is considered only when telomerase levels are higher than the matched control tissue [43]. The hematopoietic tumors present a unique and difcult assessment since activated lymphocytes have some inherent telomerase activity. Cells from patients with chronic lymphoid leukemia, in the early-stages, have low levels of telomerase that progressively increase

3. Cancer and telomerase Telomerase expression is a hallmark of cancer. Nearly the complete spectrum of human tumors has been shown to be telomerase positive (Fig. 4). In general, malignant tumors are characterized by telomerase expression, indicating the capacity for unlimited proliferation and thus immortality. Most benign tumors are characterized by the absence of telomerase, indicating their limited proliferative capacity, and ultimate senescence.

Fig. 4. Summary of telomerase activity expression in human cancers from a review of the literature. Tumor samples were assayed by the TRAP assay. Percentages in parentheses refer to the number of samples that were telomerase positive compared to matched control tissue. Adapted from Shay and Bacchetti, 1997. Please refer to original article for details and discussion [43]

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over the course of the disease. This increase is accompanied by a net loss in telomeric length [32]. A series of 58 patients showed that high telomerase activity and shorter telomeres had an adverse prognosis [44]. Chronic myeloid leukemia does not show an increase in telomerase activity over peripheral mononuclear cells, however, a shorter TRF length correlates with shorter time to blast crisis phase. Small studies in acute lymphoblastic leukemia have found telomerase activity to be variable [32]. Acute myelogenous leukemia, multiple myeloma, plasma cells leukemia, and nonHodgkins lymphoma all exhibit telomerase positivity [45]. However, Hodgkins lymphoma does not exhibit telomerase activity [46].

3.2. Role of telomerase in malignant transformation

Telomerase expression alone is not the inciting event in the transformation to neoplasia. While introduction and expression of telomerase has been shown to immortalize cells, it does not by itself induce a transformed phenotype [47,48]. In human broblasts, many factors are required to experimentally transform telomerase positive cells, including overexpression of a mutant version of the H-ras oncogene to constitutively activate signal transduction pathways, SV40 large T antigen to block pRb and p53 cell cycle checkpoints, and SV40 small t antigen to inhibit phosphatase activity [15,49]. Human broblast cells that express only hTERT exhibit normal cell cycle activities, maintain contact inhibition, adherence, growth requirements, and maintain normal karyotype [40]. It has been suggested that there are at least six essential alterations necessary for malignancy shared by virtually all types of cancers. They are the generation of self-stimulatory growth signals, insensitivity to inhibitory growth signals, resistance to apoptosis, unlimited potential for proliferation, capacity for angiogenesis, and tissue invasion and metastasis [50]. Thus, there is a diverse system of cellular mechanisms in place to suppress the development of neoplastic cells. It is further postulated that the multiplicity of these defenses explains the relative rarity of human cancer [50]. Indeed, the cancer rate is estimated to be 400 cases per 100 000 individuals for all types, age, sex, and sites. However, when viewed adjusted for age the rate rises sharply. For individuals over age 65 the estimated incidence is 2151 cases per 100 000 [51].

are based on the idea that cancer arises by clonal expansion of proliferating cells, and it is the stem cells of epithelial tissues that constitute the pool of proliferating cells. Alternatively, it might be expected that cancer would arise in differentiated cells rather than stem cells since the mass of most tissues is comprised of differentiated cells. Following a mutation that initiates clonal expansion, the pre-malignant cell accumulates other critical mutations such as p53 resulting in genomic instability and continued cell division and further shortening of telomeres. This repetitive, clonal expansion leads to the acquisition of other mutations, loss of heterozygosity and the ultimate upregulation or reactivation of telomerase. This upregulation or reactivation of telomerase permits the stabilization of the telomeres and an immortal state [52].

3.4. Prognostic implications of telomerase detection

Many studies have been conducted to assess the prognostic implications of telomerase expression. Telomerase activity increases in direct proportion to grade of malignancy in a series of cutaneous melanocytic lesions, from low in benign nevi to very high in melanoma [53]. One of the classic examples of clinical outcome as predicted by telomerase activity is in childhood neuroblastoma. High levels of telomerase are found in advanced, Stage 4 disease that is of very poor prognosis. However, Stage-4s neuroblastoma is a disseminated form of the disease (s is for special) known to present almost exclusively in infancy and often spontaneously regress. These particular tumors have low to absent levels of telomerase activity and very short telomeres suggesting that inability to maintain telomere length could contribute to their regression [54]. These studies also show that a cell does not necessarily need to have telomerase activity to become malignant, but a mechanism must be engaged to maintain telomere stability to confer long-term growth of the tumor. Numerous studies have shown that telomerase activity in breast carcinomas is an adverse prognostic sign as it is in other malignancies. In a retrospective prognostic study of 398 patients with breast carcinoma involving lymph nodes, telomerase activity as analyzed by the TRAP assay was shown to strongly correlate with an aggressive phenotype in terms of the fraction of cells in S-phase, progesterone receptor level, DNA ploidy, and lymph node status. Increased TRAP also indicated decreased disease-free survival (P = 0.041), decreased overall survival (P = 0.009), strong predictor of death (P = 0.027), but was only moderately predictive of relapse (P = 0.08) [55]. Another study examined 125 patients with various stages of breast carcinoma and also found that telomerase activity correlated with stage in a statistically signicant manner (P = 0.02) [56]. Suspi-

3.3. Methods of telomerase acquisition

There is debate among investigators over just how cancer cells acquire telomerase activity. For example, does the neoplasm originate from a telomerase-competent stem cell or is telomerase turned on at some phase in neoplasia? Models for the origin of the former

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cious cytogenetic abnormalities in breast cancer correlate with increased telomerase activity, namely 3q + (site of hTR), 8q + (c-myc), and 17p (p53) [57]. One hundred patients with colorectal cancer were followed for 3 years after surgery. High telomerase activity was found in 44/100 patients at the time of biopsy and correlated with a signicantly (P = 0.01) decreased survival, 81% vs. 43% [58]. A retrospective study in patients with meningioma appeared to predict relapse. In 25 patients that were examined, ve patients had detectable telomerase activity and subsequently relapsed. Twenty-ve patients had no detectable telomerase activity and did not relapse [59]. Glioblastoma is one of the few examples where no correlation has been seen between grade and telomerase activity. TRAP levels have been found to be highly variable both within the same patient and within a series of glioblastomas [60]. These observations of the prognostic utility of telomerase assays have not yet reached the clinic in terms of predicting outcome for patients.

with telomerase activity were ultimately diagnosed as carcinoma and 6/7 without telomerase activity were ultimately diagnosed as benign lesions with a P = 0.0007. The TRAP assay thus has the potential to augment the FNA screening tool in combination with cytology in the early diagnosis of breast cancer [61]. Telomerase activity has all the desired characteristics to be used as a potential cancer-screening tool. It requires a small amount of tissue, can be done on a variety of tissue types or body uids requiring minimal invasiveness, has a sensitive assay, is specic to the malignant state in most instances, and can be done at a low cost [62].

3.7. Therapeutic potential

In most cases, chemotherapy targets dividing cells. It has limited effectiveness in specically targeting cancer cells, even further limitations in eradicating minimal residual disease, and can often be evaded through drug resistance mechanisms. Many would claim that the current miracles of chemotherapy have been exhausted and future therapeutic advances require a more sophisticated armamentarium. Telomerase inhibitors are an attractive weapon against this problem, largely because of the specicity of telomerase activity in tumor cells. This is currently and area of intense investigation worldwide and several classes of potential agents have been developed. A key to understanding the role for this class of agents is that the inhibitory effects are only apparent after the cancer cells shorten their telomeres sufciently through continued proliferation to cause them to enter crisis. Therefore, time to effectiveness in halting tumor growth is dependent on the original length of the telomeres in the cancer cell. Because the cells will continue to proliferate before inhibition is sensed by the cell, they are less likely to be used in up-front therapy and more likely to play a supportive role to control minimal residual disease after initial control is accomplished through conventional surgery, chemotherapy or radiation. Levels of telomerase are detectable in the same regenerative tissues that are vulnerable to the toxic effects of chemotherapy, such as the hematopoietic tissues, germ cells, skin and hair cells, and gastrointestinal cells. However, the effects here are predicted to be minor since the stem cells in these tissues tend to have much longer telomeres compared to cancer cells. As is always the case, there remains the possibility that drug resistance mechanisms would develop [63].

3.5. Residual disease

Telomerase may play an important clinical role in assessing the extent of tumor margins. Biopsy specimens from a tumor bed show that telomerase activity was detectable in 10% of tissue areas that were presumed disease-free based on morphologic review. This means that margins that were declared free of tumor may not in actuality be free of tumor. An assay for telomerase could theoretically provide a molecular way of determining margins, and thus identifying patients who are at increased risk for local recurrence [13].

3.6. Diagnostic potential

Telomerase activity has been proposed as an adjunctive diagnostic tool in urinary tract cancers. It is estimated that nearly 50% of bladder cancers are missed on initial cytological survey. The specicity for telomerase activity in cancer cells allows for earlier detection and identication using bladder washings in combination with cytology [60]. Fine-needle aspiration is widely used as a diagnostic tool in breast cancer. A recent prospective blinded study included 617 patients and examined 220 FNA samples by both cytology and telomerase activity in which the diagnosis was later conrmed histologically after surgery [61]. The cytology method alone correctly identied 62 out of 93 tumors (67%) that were initially classied as malignant or probably malignant. The telomerase assays correctly identied 72 of the 93 tumors (77%). When both tests were used together on the FNA samples, 84 of 93 (90%) were correctly identied. Of the cytologically indeterminate FNA samples, 10/17

3.7.1. Telomerase inhibitors The RNA template of the telomerase holoenzyme is a popular target for inhibition research using antisense oligonucleotides that are complementary to this region

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of hTR. Regardless of the conguration of telomerase, the template region of hTR must be accessible to bind to the telomeric repeats, which exposes it to inhibition by antisense approaches. The major challenge for this class of drugs is access and stability how to get the oligonucleotides into the cell and then to the enzyme without being degraded by nucleases. One strategy has been to modify the DNA using sugar-modied RNA, such as 2%-O -methyl RNA and the 2%-methoxyethoxy RNA [63]. In the laboratory, telomerase can be inhibited by the introduction of a dominant-negative hTERT gene into the cell. The gene encodes a point-mutated reverse transcriptase crippled hTERT that inhibits wildtype hTERT both by sequestering the available hTR and by competing with the wild-type hTERT for access to the telomeres. In vitro studies have shown that the introduction of the dominant-negative (DNhTERT) into cancer cells inhibits telomerase and leads to progressive telomere shortening and cell death [64]. Wild-type hTERT, DN-hTERT, and control vectors were introduced into 36 M ovarian carcinoma cell lines in culture. After several PDs, the cells were introduced into nude mice to assess for tumorigenicity. The wild-type and control vector cells produced tumors but the DN-hTERT cells did not. The application of this design may be more feasible as the area of gene therapy progresses. Attention has also been given to the reverse transcriptase inhibitor class of drugs, such as AZT, that have been effective in HIV treatment. Unfortunately, it has not been shown to date that this class of agents promotes shortening of telomeres and senescence or apoptosis of the treated cells [63].

agent. Human mammary epithelial cells from women with Li-Fraumeni syndrome are characterized by a mutation in the p53 tumor suppressor gene that makes it nonfunctional. These cells spontaneously immortalize in culture at a reliable frequency. Using a variety of telomerase inhibitors, such as the 2-O -methyl-RNA antisense oligonuclotide, the dominant negative hTERT, or nontoxic concentrations of other chemotherapeutic agents, the rate of in vitro immortalization was signicantly reduced [67]. Other patients at high risk for spontaneous immortalization could benet from this strategy of chemoprevention including those at high risk for lung cancer from smoking or chemical exposure, patients treated for a primary malignancy with a high probability of recurrence, and those with conditions considered premalignant with a high probability of progression.

4. Conclusion A hypothesis gaining support is that the function of cellular senescence is to restrict the number of mutations that can be accumulated by a pre-malignant cell. If one accepts this hypothesis, then counting cell divisions becomes the distinguishing feature of replicative aging. Determining whether replicative aging has relevance to organismal aging remains a fundamental unresolved issue. However, there is mounting experimental support that restoring mortality by inhibiting telomerase in tumors may be an effective therapy and is an area where great progress is anticipated in the near future. Telomere biology is clearly important in replicative aging and cancer. Cancer cells need a mechanism to maintain telomeres, if they are going to divide indenitely, and telomerase solves this problem. The key is to understand how the telomerase holoenzyme and telomere-complex interact to maintain telomere length. The challenge is to learn how to intervene in these processes and exploit our increasing knowledge of telomere biology for cell and tissue engineering as well as the diagnosis and treatment of malignancies.

3.7.2. Immunotherapy and telomerase Recently, Vonderheide, et al. identied a tumor-associated antigen (TAA) that correlates with hTERT expression in an HLA subset of patients. He generated cytotoxic T lymphocytes and demonstrated hTERT specic cytolysis in many tumor lines that spared telomerase positive peripheral blood CD34 + cells. However, since CD40 + activated B cells were lysed, it is possible that the immune system will not function optimally in a clinical setting if it is forced to rely solely on the interaction of antigen processing cells with cytotoxic T cells without activated B cells in the germinal centers [65]. Other investigations have shown similar results with other hTERT peptides that are able to generate a cytotoxic response against tumor cells but not telomerase-positive CD34 + stem cells [66]. 3.7.3. Chemopre6ention and telomerase Telomerase antisense inhibitors have been recently shown to have potential value as a chemopreventative

Reviewers Joachim Lingner, PhD, Swiss Institute for Experimental Cancer Research (ISREC), 155, ch. des Boveresses, CH-1066 Epalinges, Switzerland. Petra Boukamp, PhD, Deutsches Krebsforschungszentrum (DKFZ), Abteilung B0600/FS2, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. Dr. Go ran Roos, Department of Pathology, Umea University, S-90187 Umea, Sweden.

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References
[1] Meyerson M. Role of telomerase in normal and cancer cells. J Clin Oncol 2000;18:2626 34. [2] 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 1988;85:6622 6. [3] Krupp G, Klapper W, Parwaresch R. Cell proliferation, carcinogenesis, and diverse mechanisms of telomerase regulation. Cell Mol Life Sci 2000;57:464 86. [4] Harley C, Futcher AB, Greider CW. Telomeres shorten during ageing of human broblasts. Nature 1990;346:866 8. [5] Shay JW, Wright WE. Hayick, his limit, and cellular ageing. Nature Rev 2000;1:72 6. [6] Pardue M-L. Telomeres and telomerase: more than the end of the line. Chromasoma 1999;108:73 82. [7] Grifth JD, Comeau L, Roseneld S, et al. Mammalian telomeres end in a large duplex loop. Cell 1999;97:503 14. [8] Shay JW, Wright WE. Telomeres and telomerase in the regulation of human cellular aging. Mol Biol Aging 1999;44:148 58. [9] Shay JW. At the end of the millennium, a view of the end. Nature Genet 1999;23:382 3. [10] Li B, Oestreich S, de Lange T. Identication of human Rap1p: implications for telomere evolution. Cell 2000;101:471 83. [11] Zhou BS, Elledge SJ. The DNA damage response: putting checkpoints in perspective. Nature 2000;408:433 9. [12] Zhu X, Kuster B, Mann M, Petrini JHJ, de Lange T. Cell-cycle regulated association of RAD50/MRE11/NBS1 with TRF2 and human telomeres. Nature Genet 2000;25:347 52. [13] Holt SE, Shay JW. Role of telomerase in cellular proliferation and cancer. J Cell Physiol 1999;180:10 8. [14] Blackburn E. Telomerases. Annu Rev Biochem 1992;61:113 29. [15] Wright WE, Shay JW. Cellular senescence as a tumor-protection mechanism: the essential role of counting. Curr Opin Genet Dev 2001;11:98103. [16] Holt SE, Aisner D, Baur J, et al. Functional requirement of p23 and hsp90 in telomerase complexes. Gene Dev 1999;13:817 26. [17] Shay JW, Wright WE. Implications of mapping the human telomerase gene (hTERT) as the most distal gene on chromosome 5p. Neoplasia 2000;2:195 6. [18] Wright WE, Shay JW. Telomere positional effects and the regulation of cellular senescence. Trends Genet 1992;8:193 7. [19] Baur J, Zou Y, Shay JW, Wright WE. Telomere position effect in human cells. Science 2001;292:2075 7. [20] Ramirez R, Morales CP, Shea-Herbert B, et al. Putative telomere-independent mechanisms of replicative aging eect inadequate growth conditions. Gene Dev 2001;15:398 403. [21] Steinert S, Shay JW, Wright WE. Transient expression of human telomerase extends the lifespan of normal human broblasts. Biochem Biophys Res Comm 2000;273:1095 8. [22] Herbert BS, Pitts AE, Baker SI, et al. Inhibition of telomerase in immortalized human cells leads to progressive telomere shortening and cell death. Proc Natl Acad Sci 1999;96:14276 81. [23] Health, United States, 2000, With Adolescent Health Chartbook. Hyattsville, MD: National Center for Health Statistics, 2000. [24] Bodnar A, Ouellette M, Frolkis M, et al. Extension of life-span by introduction of telomerase into normal human cells. Science 1998;279:349 52. [25] Dunham MA, Neumann AA, Fasching CL, Reddel RR. Telomere maintenance by recombination in human cells. Nature Genet 2000;26:447 50. [26] Campisi J. Replicative senescence: an old lives tale? Cell 1996;84:497 500.

[27] Berhman RE. Nelson Textbook of Pediatrics, 16th Ed. Philadelphia: W.B. Saunders, 2000. [28] Ouellette M, McDaniel LD, Wright WE, Shay JW, Schultz RA. The establishment of telomerase-immortalized cell lines representing human chromosome instability syndromes. Hum Mol Genet 2000;9:403 11. [29] Goldman L. Cecil Textbook of Medicine. Philadelphia: W.B. Saunders, 2000. [30] Vaziri H, Schacter 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 Human Gene 1993;52:661 7. [31] Shay JW. Accelerated telomere shortening in bone-marrow recipients. Lancet 1998;351:153 4. [32] Shay JW, Werbin H, Wright WE. Telomeres and telomerase in human leukemias. Leukemia 1996;10:1255 61. [33] Wynn R, Cross MA, Hatton C, et al. Accelerated telomere shortening in young recipients of allogeneic bone marrow transplants. Lancet 1998;351:178 81. [34] Robertson J, Wynn R. Telomerase activity and telomere length in the hematopoietic system: changes with aging, disease, and therapy. J Anti-Aging Med 2000;3:389 95. [35] Pizzo PA, Poplack DG. Principles and Practice of Pediatric Oncology. New York: Lippincott-Raven, 1997. [36] Abeloff MD, Armitage JO, Fichter AS, Neiderhuber JE. Clinical Oncology, 2nd Ed. London: Churchill Livingstone, 2000. [37] Globerson A, Effros RB. Ageing of lymphocytes and lymphocytes in the aged. Immunol Today 2000;21:515 21. [38] Weng N-P, Hodes RJ. The role of telomerase expression and telomere length maintenance in human and mouse. J Clin Immunol 2000;20:257 66. [39] Liu K, Hodes RJ, Weng N-P. Cutting edge: telomerase activation in human T lymphocytes does not require increase in telomerase reverse transcriptase (hTERT) protein but is associated with hTERT phosphorylation and nuclear translocation. J Immunol 2001;166:4826 30. [40] Zhu J, Wang H, Bishop JM, Blackburn EH. Telomerase extends the lifespan of virus-transformed human cells without net telomere lengthening. Proc Natl Acad Sci 1999;96:3723 8. [41] Effros RB. Replicative senescence in the immune system: impact of the hayick limit of T-cell function in the elderly. Am J Hum Genet 1998;62:1003 7. [42] Broccoli D, Young JW, de Lange T. Telomerase activity in normal and malignant hematopoietic cells. Proc Natl Acad Sci 1995;92:9082 6. [43] Shay JW, Bacchetti S. A survey of telomerase activity in human cancer. Eur J Cancer 1997;33:787 91. [44] Bechter O, Eisterer W, Pall G, Hilbe W, Kuhr T, Thaler J. Telomere length and telomerase activity predict survival in patients with B cell chronic lymphocytic leukemia. Cancer Res 1998;58:4918 22. [45] Xu D, Zheng C, Bergenbrandt S. Telomerase activity in plasma cell dyscrasias. Br J Hematol 2001;84:621 5. [46] Norrback KF, Enblad G, Erlandson M. Telomerase activity in Hodgkins disease. Blood 1998;92:567 73. [47] Morales CP, Holt SE, Ouellette M, et al. Absence of cancer-associated changes in human broblasts immortalized with telomerase. Nature Genet 1999;21:115 8. [48] Jiang XR, Jimenez G, Chang E, et al. Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nature Genet 1999;21:111 4. [49] Hahn WC, Counter CM, Lundberg AS, Bieijersbergen RL, Brooks MW, Weinberg RA. Creation of human tumor cells with dened genetic elements. Nature 1999;400:464 8. [50] Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57 70.

40

M.P. Granger et al. / Critical Re6iews in Oncology /Hematology 41 (2002) 2940

[51] Ries L, Eisner M, Kosary C, Hankey BF, Miller BA, Clegg L, Edwards BK, editors. SEER Cancer Statistics Review, 1973 1998, Table II-3. Bethesda, MD: National Cancer Institute, 2001:137. [52] Shay JW, Wright WE. The reactivation of telomerase activity in cancer progression. Trends Genet 1996;12:129 31. [53] Miracco C, Pacenti L, Santopietro R, Laurini L, Biagioli M, Luzi P. Evaluation of telomerase activity in cutaneous melanocytic proliferations. Hum Pathol 2000;31:1018 21. [54] Shay JW. Telomerase in cancer: diagnostic, prognostic, and therapeutic implications. Cancer J Sci Am 1998;Suppl 1:S26 34. [55] Clark GM, Osborne CK, Levitt D, Wu F, Kim NW. Telomerase activity and survival of patients with node-positive breast cancer. J Natl Cancer Inst 1997;89:1874 81. [56] Hiyama E, Gollahon L, Kataoka T, et al. Telomerase activity in human breast tumours. J Natl Cancer Inst 1996;88:116 22. [57] Herbert B, Wright WE, Shay JW. Telomerase and breast cancer. Breast Cancer Res 2001;3. [58] Tatsumoto N, Hiyama E, Murakami Y, et al. High telomerase activity is an independent prognostic indicator of poor outcome in colorectal carcinoma. Clin Cancer Res 2000;6:2696 701. [59] Langford LA, Piatyszek MA, Xu R, Schold SC, Shay JW. Telomerase activity: a prognostic indicator in ordinary meningiomas. Hum Pathol 1997;28:416 20. [60] Kleinschmidt-Demasters BK, Evans LC, Bobak JB, et al. Quantitative telomerase expression in glioblastomas shows regional variation and down-regulation with therapy but no correlation with patient outcome. Hum Pathol 2000;31:905 13. [61] Hiyama E, Saeki T, Hiyama K, et al. Telomerase activity as a marker of breast carcinoma in ne-needle aspirated samples. Cancer Cytopathol 2000;90:235 8. [62] Shay JW, Gazdar AF. Telomerase in the early detection of cancer. J Clin Pathol 1997;50:106 9. [63] White LK, Wright WE, Shay JW. Telomerase inhibitors. Trends Biotechnol 2001;19:114 20. [64] Hahn WC. Inhibition of telomerase limits the growth of human cancer cells. Nature Med 1999;5:1164 70. [65] Vonderheide RH, Hahn WC, Schultze JL, Nadler LM. The telomerase catalytic subunit is a widely expressed tumor-associated antigen recognized by cytotoxic T lymphocytes. Immunity 1999;10:673 9. [66] Minev B. Cytotoxic T cell immunity against telomerase reverse transcriptase in humans. Proc Natl Acad Sci 2000;97:4796 801. [67] Herbert BS, Wright AC, Passons CM, et al. Effects of chemopreventive and antitelomerase agents on the spontaneous immortalization of breast epithelial cells. J Natl Cancer Inst 2001;93:3945.

Biographies Meaghan P. Granger, MD is a clinical fellow in pediatric Hematology and Oncology at UT Southwestern Medical Center. She received her MD from the University of Arkansas and completed her residency in pediatrics at Vanderbilt University. She is currently conducting telomerase research in the laboratory of Jerry W. Shay, PhD and Woodring E. Wright, MD/ PhD in the Department of Cell Biology. Woodring E. Wright received his BA from Harvard College and then completed his MD/PhD at Stanford University in California, where earned his PhD in the laboratory of Leonard Hayick. He pursued postdoctoral studies at the Pasteur Institute in Paris with Franc ois Gros and then joined the faculty of Southwestern Medical Center where he is currently a professor of Cell Biology. Jerry W. Shay earned his BA and MA at the University of Texas at Austin and his PhD at the University of Kansas at Lawrence. He did his postdoctoral work at the University of Colorado in Boulder with Keith Porter and David Prescott before moving to Dallas where he is currently a professor of Cell Biology at the University of Texas Southwestern Medical Center in Dallas and an Ellison Medical Foundation Senior Scholar. In 1985, Shay and Wright began what has become a very close and productive collaboration. This led to the development of the two-stage model for cellular senescence for which they shared the Allied Signal Award for research on aging in 1995 and in 2001 the American Aging Association Hayick Award. They are both members of Gerons scientic advisory board and have over 15 patents allowed on their telomere and telomerase-based research. Both have served on the Scientic Research Board of the American Foundation for Aging Research.