Indian J Physiol Pharmacol1996; 40(3): 199-204

REVIEW ARTICLE

THE ROLE OF TELOMERES AND TELOMERASE INHUMAN CANCER

SIVAPRAKASAM BALASUBRAMANIAN AN;!) NEETA SINGH*

Department of Biochemistry,All India Institute of Medical Sciences,New Delhi - 110029

( Received on March I, 1996 )

Abstract; Human cancers/malignant transformation of normal cellsoccur from multiple independent genetic changes/mutations that cansubvert the normal growth controls of cells, leading to distinctphenotypic changes and immortalization. Normal human somatic cellshave a limited proliferative capacity both in vitro and in vivo andundergo senescence. Recent studies have implicated telomeres andtelomerase in the regulation of lifespan of cells. Telomeres are thestretches of DNA consisting of tandem repeats of nucleotide sequencesthat cap chromosomes and prevent its degradation and play a roie,both in normal control of cell proliferation and abnormal growth ofcancers. They are highly conserved during evolution. Telomerase, thenovel reverse transcriptase enzyme that synthesizes telomeric DNA isrepressed in most human somatic cells, it results in telomere shorteningwith each cell division, leading to a process thought to contribute tosenescence. Recent research proposes that activation of telomerase isimportant for cells to proliferate indefinitely and that all humancancer cells require activation of this enzyme to maintain telomericDNA, to overcome cellular senescence and to attain immortality. Thustelomeres and telomerase offer potential for diagnostics, cancer therapyas well as for understanding the process of aging.

Key. words; telomerescancer

telomerase senescenceimmortality

INTRODUCTION

Normal human somatic cells have limitedproliferative capacity both in culture and invivo. This phenomenon, termed replicativesenescence has often been used as a model forcellular aging (1, 2). However, the progressionof normal human cells to tumors involves theescape from limitations on proliferations imposedby cellular senescence. Transformation in vitroconfers an extended lifespan to cens, buttransformed cells eventually undergo aproliferative crisis accompanied by cell death,

"Corresponding Author

from which rare immortal clones emerge (3).Circumstantial evidence suggests thatacquisition of extended proliferative capacity,and even of immortality, can also occur in vivoduring the development of tumors (4).Immortalization of human cells may result inpart from chromosomal or chromatindestabilization i.e. either spontaneously orinduced by carcinogens, physical agents or DNAtumor viruses (5). Recent studies haveimplicated telomeres and telomerase in theregulation of cellular lifespan and have proposedthat activation of the enzyme telomerase and

200 Balasubramanian and Neeta Singh

stabilization of telomeres are necessary forhuman cells to become immortal, or capable ofproliferating indefinitely (6-8). Hence, in thisreview, we emphasize the role of telomeres andtelomerase in cancer and immortality.

TELOMERES

Eukaryotic chromosomal ends consist ofspecialized nucleoprotein structures calledtelomeres. Telomere is a Greek work (telosmeans end; meros means part) coined by thenobel laureate Hermann J. Muller (9) in 1930s.He was the first Scientist to recognize thatchromosome ends, or telomeres, are essentialto maintain chromosomal integrity. Thereafter,investigators (IO) began to deci her its structureand confirmed that though the telomere carriedno genes, it is vital for chromosomal survival.The telomeric DNA is highly conserved in allwell-characterized eukaryotic nuclearchromosomes and is quite different from thetermini of linear viral, extracellular plasmid,or mitochondrial DNA (11).

The telomeric DNA is formed of simpletandemly repeated nucleotide units with aG-rich strand oriented 5' to 3' toward thechromosomal end and sometimes protrudes as3' overhangs. Although telomeric sequences canvary from species to species, a given organismhas a characteristic repeat at all telomeres(2). In humans, these sequences arepredominantly composed of TTAGGG repeatsrepeated 800-3000 times, making up a total of5 to 15 kilobase pairs (13, 14). Proximal to theessential telomeric repeats, some chromosomalends harbor additional common elements canedsubtelomeric repeats or telomere-associatedsequences (15, 16). Unlike the telomeric repeats,these sequence are not conserved and theirfunction remains unclear (17). In humans, themajority and most distal repeats are of theform TTAGGG, although variant forms such asTTGGGG and TGAGGG exist subterminally(13, 18). Initial work with ciliates and yeastsuggested that telomeric DNA associates withspecific proteins to form a telomeric

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nucleoprotein complex (19). Binding of thenecessary protein may rely on the sequence ofthe repeat as the alteration of the telomericsequence in both human and Tetrahymena cellscauses formation of incomplete telomeres(20-22). Human and other telomeres have beendemonstrated to associate with nuclear matrixprotein fraction, which may indude nuclearenvelope and nuclear matrix (23).

Telomeres appear to carry out at least threefunctions: (i) They protect the ends of doublestranded DNA from degradation, fusion andrecombination (10), (ii) Since the telomeres arelocated at the nuclear periphery, they mayplaya role in attaching the ends of chromosomesto nuclear membrane (24), (iii) Telomeres mayprovide a solution to the end replication problem(25); since all known polymerases require primerand ynthesize DNA in the 5' to 3' direction,the 3' ends of linear DNA pose a problem to thereplication machinery. Telomeric repeats maytemporarily nullify this trends by providing acushion of extendable non coding sequence atthe chromosomal ends.

Telomere length plays an important role incellular aging (6, 26), and immortalization ofcells has been suggested by a number ofinvestigators (27, 28). Somatic cells(lymphocytes) telomeres appear to besignificantly shorter than spenuline (sperm)telomeres from the same person (13, 14). It isnow known that in most tissues, chromosomesloose their telomeric (TTAGGG) repeats witheach cell division. The rate of telomere lossmay vary from cell type to cell type. Forexample, skin and lymphocytes loose 15-40 bpper year of their telomeric DNA, whereas thetelomeres of the fibroblasts, embryonic kidneycells, mammary epithelium and cervial cells inculture loose 50-200 bp of their DNA perpopulation doubling and eventually stopdividing at a senescent stage (28-32). Based onthis and other evidence, it has been proposedthat decay of telomeric DNA represents amolecular clock that counts cell division andlimits the replication of primary cells (28).

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Sperm telomeres on the other hand increase inlength with age, indicating that telomeres areactively maintained and even increased in lengthin germ cells (6). The correct sequence oftelomeric repeat is required for its function,since addition of telomeric DNA harboring amutant telomeric sequence to the ends of theendogenous Tetrahymena telomeres led totelomere instability and death (20).

Pathak et al (33, 34) have observed thatonly certain chromosomes (Nos. 1, 2, 3, 4, 6, 7,10 12 14) are involved in their telomericDNA a~sociations, indicating that their telomericDNA is lost more often than that of otherchromosomes that remain mtact. Harley et al(29) reported that a loss of 2 kb from meantelomeric length may imply a large increase inthe proportion of cells missing TTAGGG fromat least one telomere, since each cell contains92 telomeres and the distribution of telomerelength is wide. The loss of even a single telomerecould render the chromosome unstable. Thiscould aid in the end-to-end fusion betweensister chromatids or with another chromosome,in the form of a dicentric or ring chromosomein anaphase abnormality, and in other types ofinstability included in the breakage-fusionbridge-cycle (10). Thus, the decreased length oftelomere can lead to chromosomal inst bilityand genetic changes of possible significance fortumor development (26, 29, 30). Furthermore,reductions in telomere length have beenobserved in different human cancers such ascolorectal carcinoma (30), childhood leukemia(35), endometrial adenocarcinoma (36),neuroblastoma (37), ovarian carcinoma (38) andrenal carcinoma (39) and this may represent acommon pathway in cancer development. Theshortening of tel orne res could lead to asuccession of events: chromosomal instability,additional genetic changes, increasedprolliferation, reactivation of telomerase, andultimately cancer development (7, 40-42).

The telomere hypothesis suggests that lossof telomeres could act as a mitotic clock,reflecting the replicative history of normalsomatic cells, and as a genetic time bomb,

Role of Telomeres and Telomerase in Human Cancer 201

contributing to chromosomal abnormalities incell transformation. Further, immortalizationof a cell involves activation of telomerase,probably at or near criSIS. Chromo~omal

abberations initiated by critical shortenmg oftelomeres contributes to mutation andimmortalization (6, 26).

TELOMERASE

Telomerase is a ribonucleoprotein enzymecapable of extending ends of chromosomes witha specific telomeric sequence by using a portionof its internal RNA component as· the template(43). Greider and Blackburn (44) first identifiedand characterized the activity of telomerase inthe ciliate Tetrahymena. A similar biochemicalactivity was characterized in the Immortalhuman HeLa cell line by Morin (45). The cloningof the 159 nucleotide RNA component ofTetrahymena telomerase (46) clarified severalaspects of the mechanism of action of this novelRNA polymerase and identified the regions frompositions 43 to 51 within RNA as having thesequence 5'CAACCCCAA 3'. This sequence waslater confirmed by site-specific mutations inthis region, which yield telomerase that nowsynthesized the repeats containing thecorresponding change in the cell (20). Greiderand Blackburn (46) recognised that the templateRNA contains approximately one or one and ahalf times the complement of the GGGGTTrepeat and speculated that the surplus seq~e~ce

enables telomerase to hybridize to an eXIstingtelomeric repeat via several bases and to extendthe repeat using the remaining bases as thetemplate. Recent mutational analyses using invitro reconstituted enzyme suggest that thetemplating RNA bases lie at the 5' end of the 9nucleotide stretch, whereas the nucleotides atthe 3' end of this region sen e for alignment,allowing correct positioning of the 3' end of thetelomeric DNA at the beginning of eachelongation cycle (47). U has been proposed thatsynthesis of the complementary C-rich sequencestrand is carried out by primase-polymerase­mediated discontinuous synthesis, typical ofsemi-conservative DNA replication mechanisms,using the extended G-rich strand as a template

202 Balasubramanian and Neeta Singh

(48). Isolation of protein components of thetelomerase enzyme has proved more elusive.Recently, Collins et al (49) described thepurification of Tetrahymena telomerase andcloning of the two genes encoding the twoprotein components of the enzyme.

In normal human tissues, telomeraseactivity is only observed in germ cells and someactivity is also detected in normal bone marrow,peripheral blood leukocytes and hematopoieticprogenitor cells (50, 51). All other human tissuesappear not to show evidence of activetelomerase, even after screening with recentlydeveloped, very sensitive peR-mediatedtelomeric repeat amplification protocol (TRAP)method (52). Telomerase activity was firstdemonstrated in ovarian carcinoma (38) andhas now been found in approximately 90% oftumors as observed from more than 100 primarybiopsies from over a dozen different tumortypes, but was absent in 50 normal somatictissues (52). Recent studies correlate thereactivation of telomerase with progression ofvarious human malignant tumors (53-58).

Hiyama et al (58) reported thatneuroblastomas with high telomerase activityhad other genetic changes like N-mycamplification and an unfavourable prognosiswhereas tumors with low telomerase activitywere devoid of such genetic changes and wereassociated with a favourable prognosis.Bednareck et al (59) have observed that theprogressive increase in the telomerase activityis associated with the increased level of genomicinstability and the phenotypic progression ofskin premalignant papillomas into malignantones.

The expression of telomerase and ensuingstabilization of telomeres appear to beconcomittantwith the attainment of immortalityin human tumor cells (7, 52). Thus te omeraseactivity appears to be repressed in somatic cellsand tissues but is reactivated in immortal cellsand human cancers, an indication that in almostall instances tumor growth is maintained byimmortal cells. Moreover, it was hypothesized

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that in normal tissues, telomerase could bephysiologically repressed to reduce the chancesof cancerous growth (8).

BIOLOGICAL SIGNIFICANCE OF TELOMERES

AND TELOMERASE.If telomere loss and telomerase activation

are casually involved in cellularimmortalization, which in turn contributes tocancer, then telomeres and telomerase presentexciting new targets for drug discovery anddiagnostics. Inhibition of telomerase couldprovide a safe and effective therapy for cancer.The strategy taken by Greider and Blackburn(60) for obstructing telomerase RNA activitythrough an antisense oligonucleotide targetedto the template region, is one approach thatcould be applied to the human telomerase RNA.Another inhibition strategy with precedent inbasic research is generation of mutant telomericRNA which elongates the wrong sequence ofchromosome termini, resulting in telomeres thatfail to stabilize chromosomes and thatconsequently induce senescence in ciliates (20).The protein components of the telomerase wouldpresent another viable target for inhibition ifthe human telomerase protein could beidentified.

Studies of subtelomeric DNA may uncovernew DNA markers associated with disease­causing genes, for example, the gene responsiblefor Huntington's disease. It will also help ininvestigation into the causes of cellular aging.An effective inhibitor of telomerase mightinduce prompt senescence in rapidly dividingtumor with small telomeres. The drugs aimedat telomerase inhibition could provide a therapywith relatively little side effects as the studiessuggest that hematopoietic stem cells may alsohave low levels of telomerase activity, inaddition to the sperm and oocytes. However,some studies propose that stabilization oftelomeric length appears to be maintained bysome other mechanisms, in addition totelomerase (61, 62). Hence, the elucidation ofthe dynamics of telomere maintenance andfactors which modulate telomere loss and re-

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aquisition of telomerase activity will haveprofound implications towards understandingthe fate of a cell. Finding a specific telomeraseinhibitor and the cloning of the humantelomerase are awaited to gather furtherinsights.

Role of Telomeres and Te!omer~~(' III Human Cancer 203

ACKNOWLEDGEMENTS

The financial assistance (ResearchAssociateship) provided by Council of Scientificand Industrial Research, New Delhi, isgratefully acknowledged by S. Balasubramanian.

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