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Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-.

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Telomerase and Radiosensitivity of Human Tumors

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Introduction

Telomerase is a ribonucleoprotein complex that elongates telomeres. It is ubiquitous in embryonic tissues but down-regulated in most of the somatic tissues. Biochemical and genetic studies have established an association between telomere maintenance and extended life span through telomerase expression. Telomerase activity is present in unicellular organisms and germ cells, in both situations it is expected to play a role for indefinite cell cycling and protection from shortening of the telomeres. Telomerase consists of proteins complexed with an essential telomerase RNA that is an intrinsic part of the active enzymatic complex. It has been suggested that the telomere-telomerase system represents an adaptation of organisms with prolonged life span to avoid malignant tumors, at the expense of the cellular dysfunction associated with the aged phenotype. An important discovery has been the observation that about 85% tumors are positive for telomerase activity. The observation that telomerase activity is present in most human tumors but absent in most normal nonneoplastic cells has not only made telomerase a superb target for the diagnosis of malignancy, but also for the development of novel therapeutic agents.

Telomerase activation is controlled by cellular proliferation, but it is an early step in the development of many tumors. Normal human somatic cells have a finite life span in vivo as well as in vitro and retire into senescence after a limited number of cell divisions. Cellular senescence is triggered by the activation of two interdependent mechanisms which involve irreversible cell cycle exit and this is indicated by a critical shortening of telomeres. The shortening of telomeres in telomerase-negative somatic cells has been linked with genomic instability and carcinogenesis. Intense research is ongoing to determine the link between ionizing radiation sensitivity and telomerase activity.

Ionizing radiation (IR) is an important therapeutic modality in clinical cancer management, as it plays a key role in the treatment of many tumors. Exposure to IR induces DNA damage, which has been linked with the cell death (if the DNA damage is not repaired), as well as neoplastic transformation (if the DNA damage is misrepaired). In several instances the sensitivity to cell killing by IR have been related to their greatly reduced ability to repair DNA double-strand breaks (DSB). The genes that have been found to be involved in the repair of DNA DSB are XRCC5, XRCC6, XRCC7 and Ku80. In addition, there are several human disorders, whose affected gene functions have been linked with maintenance of genomic stability. The inherited human syndromes associated with sensitivity to IR are ataxia telangiectasia, basal cell nevoid syndrome, Cockayne's syndrome, Down syndrome, Fanconi's anemia, Gardner's syndrome, Nijmegan breakage syndrome and Ushers's syndrome. The gene associated with the ataxia telangiectasia is ATM, which is a protein kinase involved in signal transduction and telomere turnover.1,2 There is increasing evidence that proteins involved in DNA DSB repair may have a role at the telomeres. Yeast telomeres are enriched in yKu80 and Sir3p, which upon induction of DNA DSB are translocated from the telomeres to the sites of DNA damage.3,4 In mammals, Ku binds to telomeres and thus protects from telomere fusions.5,6Mutations in DNA repair proteins like yKu80, yKu70, Mre11, rad50, Xrs2, Sir2, Sir 3 result in altered telomere structure and altered IR sensitivities.7 An association between short telomeres and organismal hypersensitivity to IR has been reported in mTR/ mice.8 This enhanced sensitivity to ionizing radiation has been reported not to be the direct consequence of a defect in a known DNA DSB repair pathway.8

In this chapter, I attempt to summarize information about the relationship between telomerase activity and radiosensitivity in human tumors. The information about the regulation of the telomerase through the cell cycle may prove helpful in designing the therapeutic agents either for its activation in cells where its expression can overcome the senescence and prolong the life of postmitotic cells, or inhibition of telomerase where it is essential for the proliferation of tumor cells and provides a therapeutic advantage in killing them.

Telomerase in Cell Proliferation and Development

Telomerase Function

Eukaryotic DNA polymerases are unable to start de novo, leading to the problem that extreme 3'-terminal sequences will not be replicated. This could lead to loss of the 5' end of one daughter DNA strand. To counter this problem, a special type of DNA polymerase related to reverse transcriptase and called telomerase is found in eukaryotes. Telomerase activity was first reported in vitro in Tetrahymena by a biochemical assay in which a single-stranded telomeric primer d(TTGGGG)4 oligonucleotide was used as substrate for the addition of dTTP and [32P]dGTP.9 The essential factor that influences the correct synthesis of telomeric repeats without a DNA strand was characterized by Greider and Blackburn10 and thus found that telomerase contains an essential RNA component. This RNA contains the sequence that serves as a template for telomere repeat addition. Alteration of a single nucleotide in the CAACCCCAA region of RNA gene of Tetrahymena telomerase leads to change in telomere lengths. These observations confirmed that the telomerase is a reverse transcriptase with an RNA moiety acting as a template and is responsible for the synthesis of telomeres. Telomerase is capable to extend the 3' end of the G-rich strand of the telomeric repeats, and conventional synthesis of the lagging C-rich strand completes the replication of chromosomal ends, thus compensating for the shortening that otherwise occurs. Moyzis et al,11 demonstrated that the vertebrate telomeric DNA repeat sequence is TTAGGG and Morin12 detected telomerase activity in HeLa cells that could extend (TTAGGG)n oligonucleotides in vitro. Realizing the role of telomerase in maintaining telomeres, telomerase activity has been examined in a variety of organisms.

Components of Human Telomerase

Telomerase is composed of an RNA moiety and several proteins. However, the only essential protein for telomerase activity is hTERT. In addition to the RNA and telomerase reverse transcriptase protein (hTERT) component, telomerase complex contains proteins hsp 90 and p23, and three telomerase RNA binding proteins, dyskerin, L22 and hStau, are each associated with telomerase activity in cell extract.13,15 The other protein that interacts with telomerase RNA is TEP1.16 Even though TEP1 is associated with mammalian telomerase RNA and the telomerase catalytic subunit hTERT, mice deficient in mTEP1 show no significant alteration in telomerase activity or telomere length. TEP1 is expressed in many tissues including those that are negative for telomerase activity. These and other reports suggest that all telomerase-associated proteins like TEP1 may not be essential for telomerase activity, but might be important for other cellular functions.

Activity of Human Telomerase During Development

Telomerase activity in humans has been detected in fetal, newborn, and adult testes and ovaries, but not in mature spermatozoa or oocytes. It has been suggested that telomerase activity remains high in germ cells to ensure normal chromosome length in offspring and decreases in embryonic cells as they differentiate.17,18 Telomerase activity is present in pachytene spermatocytes and round spermatids of the rat but telomerase activity has not been detected in ejaculated human or rat spermatozoa.19,20 Blastocysts express high levels of telomerase activity as do most human somatic tissues at 16-20 weeks of development with the exception of human brain tissue. Telomerase activity could no longer be detected in the somatic tissues examined from the neonatal period onward. Neonatal human somatic tissues have a very little or no detectable levels of telomerase activity. However, when fetal tissues were explanted into primary cell culture, these show a dramatic decline in telomerase activity which became undetectable after the first passage in vitro. Elucidation of the regulatory pathways involved in the repression of telomerase activity during development may lead to the ability to manipulate telomerase levels and explore the consequences both for cellular aging and for the survival of cancer cells.

Normal human somatic cells have a finite life span in vitro and retire into senescence after a predictable number of divisions. Cellular senescence is triggered by the activation of two interdependent mechanisms. One induces irreversible cell cycle exit involving activation of two tumor suppressor genes, p53 and pRb, and the proper time point is indicated by a critical shortening of chromosomal ends due to the endreplication problem of DNA synthesis. Another occurs when telomeres are so short that massive end-fusion events prevent further replication. The development of a malignant cancer cell is only possible when both mechanisms are circumvented. While the hTR subunit of telomerase is present in almost all human cells, this is not the case for a human hTERT which is limited to germ cells and certain stem cells. Thus, telomerase activity has been shown to correlate more closely with the expression status of the telomerase catalytic subunit gene TERT. The human TERT is a polypeptide with a molecular weight of 127 kDa having a unique conserved region called the T-motif.

Telomerase and Cellular Turnover

Telomerase synthesizes telomere repeats onto chromosome ends to overcome the loss of sequences during normal replication. Telomeres shorten in most of the somatic cells because of the lack of telomerase activity. However, in germ and some stem cells, telomeres are maintained as these cells have telomerase activity, and telomere attrition has been proposed as a signal that may determine the replicative life span of cells in culture.21 It has been demonstrated that during the process of immortalization of human cells in vitro, telomerase is activated after crisis. In support of this, Counter et al,22 demonstrated that primary cells transfected with T-antigen initially show telomere shortening and no telomerase activity, subsequently cells go through crisis, at which stage most of the cells die and those cells that continue to grow have telomerase activity and relatively long telomeres. In such situations, it is believed that it took several generations for activation of the telomerase by introduction of the T-antigen. This is in contrast to the situation where primary human cells are transfected with hTERT, and where telomerase activity is seen immediately.

Ectopic hTERT expression has been demonstrated to prevent replicative senescence in several normal cell types including fibroblasts and epithelial cells.23,25 Recent studies demonstrated that TERT exerts its antiapoptotic action at an early stage of the cell death process prior to mitochondrial dysfunction and caspase activation.26 Immortalization of human keratinocytes and mammary epithelial cells in cell culture was also achieved by hTERT expression in conjunction with the cell culture induced loss of p16-dependent cell cycle control, although this data is subject to other interpretations.27,28 These observations support the hypothesis that replicative senescence in humans results from inactivation of telomerase and short telomeres.

It is unclear, whether telomerase is necessary in nonproliferating cells, since telomeres do not continue to shorten in the absence of cell division. Quiescent and differentiated cells may have alternative means of regulating the expression and activity of telomerase to compensate for the lack of cellular proliferation.29,31 It is possible that quiescent cells down-regulate the expression of telomerase by repressing transcription as the kinetics of the loss of activity are not significantly different from the half-life studies.32 The half-life data suggest that telomerase is a rather stable entity. However, differentiating cells appear to down-regulate telomerase within the first 24 hr of being stimulated to differentiation. The regulation of telomerase activity in differentiating cells may be either through direct physical interaction of telomerase with regulatory proteins or degradation of the RNA or protein components.

Several studies indicate that decline in physiological function during aging may be the result of telomere-dependent in vivo replicative senescence among certain cell types. This is evident from the fact that wound healing is less efficient in the elderly animal33 and age-related changes in collagen synthesis have been reported in fibroblasts.34 A small number of genes show consistent age-related changes in their expression both in fibroblasts35 and in tissues.36,37 Interestingly the patterns of gene expression in immortalized hepatoma cells is similar to young but not old hepatocytes,38 which are quiescent telomerase-negative cells with defective telomere status. Several investigations are under way to determine whether alteration in telomere size or structure influences gene expression in human system. As it has been already demonstrated in yeast that nontelomeric DNA created by enzymatic cleavage leads to genomic instability and cell cycle arrest.39 Yeast telomeres are known to exert a position effect on recombination between internal tracts of telomeric DNA.40 It is clear in yeast, that alteration in the telomere chromatin could influence the expression of genes in the subtelomeric region. Recently, Baur et al,41 have demonstrated the presence of telomere position effect on gene expression in human cells. Since human fibroblasts lack detectable levels of telomerase activity and may not be able to maintain the telomeres, it is reasonable to believe that telomerase has a significant role to play in cellular activities and turnover during the organismal development.

Telomerase Activity and Cell Signaling Proteins

Different reports have appeared concerning the cell cycle regulation of telomerase activity. Some studies have suggested that telomerase activity is regulated at each stage of the cell cycle,42 yet others have found that telomerase activity does not vary significantly at the different stages of the cell cycle.32,43 There is also a controversy whether quiescent cells have telomerase activity. A situation where cells are pushed to the stage of terminal differentiation by the agents like retinoic acid also show down-regulation of the telomerase activity. Cell cycle-dependent telomerase activity has been reported in yeast as single-stranded telomeric DNA appears on telomeres specifically during S phase.44 It has been suggested that telomere replication may serve as a checkpoint for completion of the S phase.45 In the micronucleus of the ciliate Oxytricha nova, the telomeres are bound to a heterochromatic telomere protein that is phosphorylated by cyclin-dependent kinases.46 Further progression through the M phase involves phosphorylation of a set of proteins including histone H1, nuclear lamins, caldesomon and vimentins. It is possible that Cdc2/cyclin A and Cdc2/cyclin B kinases may be required for the regulation of telomerase activity. However this assumption is not yet supported by the experimental evidence as it has been demonstrated that extracts from S-phase cells of Xenopus with no Cdc2 kinase activity and M-phase extracts with high Cdc2 kinase activity both have identical levels of telomerase activity per unit of protein as determined by in vitro assay.47 A similar situation was found when telomerase activity was compared in S and M phase of HeLa or NIH3T3 cells.48 Similar contradictions were reported for quiescent cells. Telomerase activity in lymphocytes was present in S-phase cells but not in quiescent, G0-phase cells.48 Furthermore, in quiescent mouse cells arrested by either serum deprivation or growth to confluency, telomerase activity was reported to be similar to those in exponentially growing cells.47 A decline in telomerase activity was observed in cells whose growth rate is reduced from seven to eight population doublings per week to one to two doublings per week. Thus, telomerase activity correlates with growth rate and is repressed in cells that exit the cell cycle and become quiescent. A different situation was reported by Zhu et al,42 who reported that highest levels of telomerase activity were detected in S-phase cells. Surprisingly they also reported that cells arrested at G2/M phase of the cell cycle were almost devoid of telomerase activity. This group of investigators attempted to demonstrate that the cell cycle blockers e.g., transforming growth factor 1 and various cytotoxic agents, also caused inhibition of telomerase activity. However it is not clear how the cell cycle blockers could down-regulate the telomerase activity. It is possible that if the cell cycle regulators are protein modifiers, like kinases or acetyltransferases, presence or absence of such factors could effect the stability and the enzymatic function of the telomerase. But most probably, the cells that are differentiated or physiologically dead and in such cells telomerase complex is degraded. It will be of great interest to know if any of the cell cycle regulators interact with components of telomerase and thus influence its enzymatic role. This would trigger a new area of research for the identification of new targets for telomerase inhibition.

The regulation of telomerase activity can be divided into two states: an assembly state and a replication state. The former state allows the assembly of RNA component with proteins but this assembly is not sufficient to initiate the synthesis of the telomeres until other critical factors are supplied such as G1/S cyclin/cdk activity. The latter state allows telomere synthesis from preassembled origin of replication complex.

One way to look for the regulation of the components of telomerase is to find a link between the factors that arrest cells in G0 phase. This phase of the cell cycle is achieved when a cell does not traverse G1. In vivo, lymphocytes or epithelial cells which are normally arrested in a quiescent phase can be induced into proliferative phase by agents like mitogenic factors. These cells in G0 phase do not show any telomerase activity. In vitro it is clear that the complete transit across G1 phase is governed by the presence of extracellular growth factors that allows the cell to bypass the restriction point. Biochemical events that are concurrent with passage through this restriction point include the hyperphosphorylation of retinoblastoma (Rb) protein, the functional activation of E2F-1, loss of several cyclin-dependent kinase inhibitors and subsequent accumulation of a certain threshold level of cyclin/cdk activity. It has been demonstrated in the past that forced Rb gene overexpression caused cells to arrest in G1 phase. Rb protein becomes progressively hyperphosphorylated as cells pass from G1 into S phase.49,50 Several proteins that interact with Rb protein have been identified, however, as yet it is not known if the telomerase complex or the components of the telomerase interact with Rb. One of the proteins that interacts with Rb is E2F-1 that is central to how Rb prevents cell proliferation. E2F-1 is particularly important for regulating the G1 to S transition. As a transcriptional factor E2F-1 activates the expression of a variety of genes, many of which encode transcription factors of proteins that are involved in DNA synthesis.

Proliferation Status and Telomerase Activity

The major task of the cell division cycle is to have error free DNA replication in order to segregate the replicated DNA equally to daughter cells during mitosis or meiosis stage 2. Cell cycle transitions are controlled by holoenzymes that contain catalytic (cdk) and regulatory subunits (cyclin). These most likely exist as higher order complexes that include additional proteins. Early embryonic cell cycles exhibit rapidly alternating S and M phases without gap phases between them. Whether the reduced gap phase seen in somatic cell cycle has any link with cellular differentiation is not yet known. It is surprising that many of the cell cycle regulatory genes that are essential for cell cycle control are the proteins whose inactivation does not seem to have any influence on the telomerase activity. Although there may not be a direct correlation between the cell cycle regulatory elements, but it is believed that cells which use Myc or loss of Rb/p53 to circumvent senescence will eventually face frequent genome instability partly due to the loss of the chromosome ends. Such cells require a mechanism to maintain the telomeres. Within this context, whether telomerase is an oncogene or a tumor suppressor has been addressed by several groups. Wood et al,51 examined the effect of hTERT expression on proliferation markers and tumorigenesis in fibroblasts derived from ataxia telangiectasia (AT) and normal individuals, and found that there were no significant differences in the expression of PCNA, an accessory factor of DNA polymerase that reflects the proliferative activity of the cells. However, there was a significant increase in protein levels for ER, PgR, HSP and ErbB3 both in A-T and normal fibroblasts with ectopic expression of hTERT compared to fibroblasts without hTERT expression, suggesting up-regulation of these genes might be required for immortalization, but not for the tumorigenic transformation. The levels of the tumor suppressor genes like Tsg101 and ErbB2 were similar in A-T and control cells with and without ectopic expression of hTERT gene. Furthermore, no tumors were formed by injecting hTERT A-T-cells or hTERT normal cells in Nu/Nu mice. The current data are not conclusive as to whether telomerase is an oncogene or a tumor suppressor. However, there is evidence that some of the oncogenes interact with hTERT and thus regulate the telomerase activity.

Rb Protein

Rb interacts with a family of cell cycle transciptional factors known as E2F. Inactivation of Rb/p16 and p53 allows continued proliferation which may be accompanied by further telomere loss. In such situations stabilization of telomeric repeats may be prerequisite for tumorigenesis. It has been proposed that oncogenes might have a role in maintaining the telomeres and thus might be involved in the activation of the components of telomerase.22,52 Within this context, several laboratories have made an attempt to determine whether expression or inactivation of such oncogenes could activate the telomerase.

The expression of hTERT can prolong the life span of the several different cell types. Some of these cells become immortalized whereas other cell types need additional genetic alterations for immortalization. Kiyono et al28 found that ectopic expression of hTERT in some primary epithelial cells is not sufficient to immortalize. They reported that expression of hTERT is a primary event in the process of immortalization which is followed by a second step involving the inactivation of the Rb/p16 pathway. This suggests that Rb/p16 may be indirectly involved in the regulation of the telomerase and this regulation may be at the posttranscriptional levels.

The speculation that Rb/p16 may be involved in the regulation of telomerase came from the studies on the human primary keratinocytes and fibroblasts. Either human papilloma virus (HPV) E6, which targets p53 for degradation or E7 which inactivates Rb protein, can extend the life span of cells. E6 plus E7 both together are more efficient in immortalization of cells. E7 expression resulted in high levels of p1628 and reduced Rb, although some phosphorylated Rb remained. Kiyono et al,28 and others are of the opinion that inactivation of the Rb/p16 pathway is critical for immortalization of epithelial cells. Since most of the immortalized cells have been reported to have telomerase activity, it seems that E7 or E6 cooperates with hTERT protein. This seems to be consistent in certain situations where E7 proteins are mutated and thus are impaired in Rb binding, such cells do not immortalize. However, inactivation of Rb protein does not seem to be essential for the immortalization of all cell types since there are several immortalized cell types that have intact Rb function.

p53 Protein

The direct test whether p53 is involved in the regulation or activation of telomerase came from studies of Kiyono et al, (28) when they demonstrated that 16E68S9A10T (HPV), which does not target p53 for degradation, but was able to induce telomerase activity. However, 16E6146151, which lacks the hDLG binding motif but induces telomerase. Further, 16E6146151 which eliminated p53 did not induce telomerase activity. These studies indicate that oncoproteins are inducers of the telomerase activity without the involvement of p53 protein. It is consistent with the view that telomerase may be required during certain stages of development and this expression may be regulated by oncoproteins which are expressed during the development.

cMyc Protein

Several oncogenes e.g., mdm2, E7, E6, activated Ras (V12), cyclin D1, cdc24A and cdc25C that were ectopically expressed in human mammary epithelial cells (HMEC) failed to induce telomerase activity. Besides these genes, expression of dominant-negative p53 allele or introduction of E6 failed to activate telomerase. However, introduction of c-Myc expression was able to stimulate telomerase activity in HMEC, IMR90 or WI38 cells. The c-myc gene encodes a nuclear protein whose expression is closely associated with the proliferative state of many mesenthymal cells. It induces a quick passage through G1-phase of the cell cycle in order to make cells independent of growth factors and cellular proliferation. It is activated during oncogenesis by proviral insertion, chromosomal translocation and gene amplification. The levels of c-myc are controlled by the action of growth factors. The constitutive expression of c-myc in certain cell types relieves the growth factor-dependent entry into cell cycle. Constitutive expression of cmyc is also able to block the differentiation of several cell types, specifically the cells from hemopoietic lineage. These observations suggest that c-myc plays a role in the regulatory network that control cellular proliferation and differentiation. How c-myc affects the cellular growth-regulatory network in the process of immortalization has been the focus of several research groups. Kinoshita et al,53 have found that E6 can activate the Myc promoter. Wang et al,54 addressed the question whether E6 regulated the telomerase activity through an effect on Myc expression. Surprisingly, expression of E6 in HMECs induced Myc to levels similar to transduction of HMECs with a Myc retrovirus. Interestingly, E6-induced alterations in Myc protein did not reflect changes in the abundance of Myc mRNA. These observations suggested that Myc expression must be controlled posttranscriptionally by E6 in HMECs. The situation in IMR90 cells was different, as Myc levels remained unaltered following expression of E6 and thus it was incapable of activating telomerase. This suggested that E6 may regulate telomerase by other mechanisms, and therefore there may be cell type-specific inhibitors or activators in which E6 regulates telomerase in HMECs by altering the levels of Myc.

c-Abl Protein

The ubiquitously expressed c-Abl protein tyrosine kinase is tightly regulated in cells.55,56 c-Abl is activated by ATM in cells exposed to ionizing radiation and other DNA-damaging agents.57c-Abl-deficient cells are resistant to DNA damage-induced apoptosis.58 Perusal of literature indicates that c-Abl confers growth arrest and proapoptotic responses to DNA damage by mechanisms that depend partly on p53 and its homolog p73.59 It also functions as an upstream effector of the Jun N-terminal kinase/stress-activated protein kinase and p38 mitogen-activated protein kinase pathways.59 Since c-Abl is activated by DNA double strand breaks60 and several proteins involved in the repair of DNA damage also function in telomere control.61,63 It was expected that c-Abl may interact with the telomere maintaining players. Kharbanda et al64 found that c-Abl associates with the catalytic unit of hTERT in human cell lines. They also reported that endogenous hTERT is detectable in anti-c-Abl immunoprecipitates from MCF7 cells stably overexpressing a kinase-inactive form of c-Abl, c-Abl(K-R) and this further confirmed that the association of c-Abl with hTERT was independent of the c-Abl kinase function. Although the binding of c-Abl with hTERT is independent of c-Abl kinase function, but interestingly when lysates from 293T cells transfected with HA-hTERT incubated with glutathione-S-transferase (GST) fusion proteins containingc-Abl (GST-c-Abl) or the Src homology domain of c-Abl (GSTAbl SH3), it was found that hTERT binds to GST-c-Abl and GST-Abl SH3, however there was no detectable binding of hTERT to a GSTGrb2 fusion protein that contained the amino-terminal SH3 domain. Direct interaction of hTERT (amino acids 308-316) and c-Abl SH3 has been established.64 Since c-Abl is a tyrosine kinase, it has been shown that ionizing radiation induces tyrosine phosphorylation of hTERT by a c-Abl-dependent mechanism. The functional significance of the interaction between c-Abl and hTERT was investigated by assay of telomerase activity. The telomerase activity was inhibited in cells expressing wild type c-Abl compared to cells expressing mutant version of c-Abl(K-R).64

The role of regulation of telomerase activity by c-Abl has been further complemented by examining the early-passage MEFs deficient in c-Abl. Such cells have relatively high telomerase activity as well as long telomeres.64 These studies seem to be consistent with yeast, wherein Saccharomyces cerevisiae, the Rap1p protein binds to telomeric DNA and negatively regulates telomere length.65 The function of Rap1p in telomere regulation is mediated by Rap1-interacting factors, known as Rif1 and Rif2.65 Telomere repeat-binding proteins implicated in regulation of telomere length have been identified in the fission yeast Schizosaccharomyces pombe (Taz1p),66 in human cells (hTRF1)67 and in Chinese hamster cells (chTRF1).68 Such genes negatively regulate the telomerase activity by limiting the access of telomerase for the extension of G overhang and thus maintaining the length of the telomeres. However, c-Abl regulates telomerase activity by phosphorylation of hTERT and thus negatively regulates telomere length.

Akt Protein

Akt protein is a serine/threonine kinase, enhances human telomerase activity through phosphorylation of TERT. It was identified at first as an oncogene because of its ability to transform normal cells.69,70 Akt kinase by phosphorylation inactivates proapoptotic proteins (such as BAD protein or caspase9).71,73 The other proteins that are substrates for Akt are caspase-9, H2B histone.74 From amino acid sequences of all reported Akt kinase substrates, including BAD, PEK2, GSK3, H2B and caspase-9, the putative Akt kinase substrate consensus sequence was identified.71,74 Two putative Akt kinase phosphorylation sites (220GARRRGGSAS229) and (817AVRIRGKSYV826) were noticed in human telomerase reverse transcriptase (hTERT) subunit.75 hTERT is an Akt kinase substrate protein based on nonradioactive protein kinase assay with the fluorescent hTERT peptide (817AVRIRGKSYV826). The phosphorylation of hTERT peptide by the human melanoma cell lysate or the activated recombinant Akt kinase proteins was reported in vitro. The up-regulation of hTERT peptide phosphorylation and the telomerase activity was observed after the treatment of the growth factor deprivation or okadaic acid.75 Interestingly, it was shown that Wortmannin down-regulates hTERT peptide phosphorylation and telomerase activity together. However, telomerase activity was enhanced upon pretreatment with Akt kinase in vitro. Thus, these observations suggest that Akt kinase enhances human telomerase activity through phosphorylation of hTERT subunit as one of its substrate proteins.

14-3-3 Proteins

The 14-3-3 family of proteins plays a key regulatory role in signal transduction, checkpoint control, apoptotic, and nutrient-sensing pathways. 14-3-3 proteins act by binding to partner proteins, and this binding often leads to the altered subcellular localization of the partner and thus promotes the cytoplasmic localization of binding partners, which include cell cycle regulatory phosphatase, Cdc25c as well as catalytic subunit of telomerase (TERT). 14-3-3 proteins are highly acidic dimeric intracellular proteins that chiefly bind to phosphoserine motifs. Some well described 14-3-3 binding partners include the protein kinases Raf1, KSR1, Ask1, MEKK1, Bcr, calcium/calmodium kinase, protein kinase C, the protein phosphatase Cdc25C, cCbl, BAD, A20, transcriptional factors like FKKHRL1, MSN2, MSN4, glucocorticoid receptor (GR), Tlx2, NFAT and the telomerase catalytic subunit, TERT.76 One of the isoforms of 14-3-3 protein is 14-3-3σ, which was originally identified as an epithelial-specific marker, HME1, which was down-regulated in a few breast cancer cell lines but not in cancer cell lines derived from other tissue types.77 Recent data indicates that the expression of 14-3-3σ is lost in 94% of breast tumors.78 At the functional level, the 14-3-3σ protein has been implicated in the G2 checkpoint.79 Thus, 14-3-3σ has been implicated in maintaining a post-DNA damage G2 arrest, thereby allowing for DNA repair.80 Such cell cycle checkpoints are considered to be the guardians of genome integrity, with their abrogation contributing to reduce genomic stability.

14-3-3σ is highly specific for stratified epithelia.81,82 Dellambra et al,82 reported that down-regulation of 14-3-3σ is accompanied by the maintenance of telomerase activity and by a strong down-regulation of the p16INK4a tumor suppressor gene. Interestingly, inactivation of 14-3-3σ in keratinocytes leads to maintenance of telomerase activity.82 This maintenance is accompanied by the down-regulation of the p16INK4a tumor suppressor gene in keratinocytes. 14-3-3σ can also act as a p53-regulated inhibitor of the G2/M progression phase of the cell cycle after DNA damage. Since there seems to be consensus that cell cycle, senescence, and cell differentiation can be regulated by common molecular pathways which, however, can act independently.

Dhar et al,80 demonstrated that inactivation of 14-3-3σ gene influences genome integrity and cell survival. Cells with both copies of 14-3-3σ gene inactivated showed frequent losses of telomeric repeat sequences, enhanced frequencies of chromosome end-to-end associations, and terminal nonreciprocal translocations. These phenotypes correlated with a reduction in the amount of G-strand overhangs at the telomeres and an altered nuclear matrix association of telomeres in these cells. Since the p53-mediated G1 checkpoint is operative in 14-3-3σ cells, the chromosomal aberrations observed occurred preferentially in G2 after irradiation with gamma rays, corroborating the role of the 14-3-3σ protein in G2/M progression.80 Dhar et al80 also reported that even in untreated cycling cells, occasional chromosomal breaks or telomere-telomere fusions trigger a G2 checkpoint arrest followed by repair of these aberrant chromosome structures before entering M phase. Since 14-3-3σ cells are defective in maintaining G2 arrest, they enter M phase without repair of the aberrant chromosome structures and undergo cell death during mitosis. Thus, the studies by Dhar et al,80 provided evidence for the correlation among a dysfunctional G2/M checkpoint control, genomic instability, and loss of telomeres in human cells.

Another cell cycle regulatory gene that may have some influence on the regulation of the telomerase activity is Id2. Id proteins inhibit the functions of transcription factors in a dominant-negative manner by suppressing their heterodimerization partners through the HLH domains. Members of the Id family also promote cell proliferation, implying a role in the control of cell differentiation. This is based upon the fact that cell cycle progression induced by Myc oncoprotein requires inactivation of Rb by Id. This is supported by the fact that in neuroblastoma, an embryonal tumor derived from the neural crest, Id2 is overexpressed in cells carrying copies of the N-myc gene. The overexpression of Id2 results from transcriptional activation by oncoproteins of the Myc family. Cell cycle progression induced by Myc oncoproteins require inactivation of Rb by Id2.83 Thus a dual connection links Id2 and Rb: during normal cell cycle, Rb prohibits the action of Id2 on its natural targets, but oncogenic activation of the Myc-Id2 transcriptional pathway overrides the tumor suppressor function of Rb.

The other gene product that has been reported to influence telomerase activity is PTEN. Restoration of wild-type PTEN expression leads to apoptosis, induces differentiation, and reduces telomerase activity in human glioma cells.84

As outlined earlier, several laboratories are attempting to determine whether telomerase is a tumor suppressor or an oncogene. With this regard ectopic expression of hTERT has been demonstrated to prevent replicative senescence in several normal cell types including fibroblasts and epithelial cells.23,24,54 Recently it has been demonstrated that TERT may exert its antiapoptotic action at early stage of the cell death process prior to mitochondrial dysfunction and caspase activation.26 Ectopic expression of hTERT in human keratinocytes and mammary epithelial cells has been reported to induce loss of p16-dependent cell cycle control and thus leads to immortalization; however, this data is subject to other interpretations.27,28 These studies support the hypothesis that replicative senescence results from telomere shortening. It has been proposed that telomere shortening during replicative aging of some cells finally generates antiproliferative signals which accumulate p53 protein accompanied by the G1 arrest, frequently observed in senescent cells. However in the presence of T antigen or E6, known to bind p53 protein, telomere shortening signals are bypassed, leading to extension of the life span, which may or may not be accompanied by the genomic instability. One way to prevent the antiproliferative signal is to maintain the telomere length equilibrium. It is possible that expression of telomerase prevents the antiproliferative signal generated by telomere shortening at senescence. Vaziri et al85 suggested that prevention of the p53-mediated antiproliferative signal in response to telomere shortening allows cells to divide further. The extension of the life span by SV40 large T antigen, however, relies on inactivation of p53 and pRb proteins, and telomere shortening continues to persist until crisis. This suggested that extended growth of cells by forced telomerase expression may thus not interfere with the p53-dependent signaling pathway, and therefore cells immortalized with ectopic expression of hTERT retain the genomic integrity.

ATM Protein

Replicative senescence can be accelerated by specific mutations that cause some human diseases. Among them is Ataxia telangiectasia (AT) which is a rare autosomal recessive disorder characterized by progressive cerebellar degeneration, premature aging, growth retardation, specific immunodeficiencies, genomic instability and gonadal atrophy.86,88A-T patients have an increased sensitivity to ionizing radiation and an elevated incidence of cancer. Cells derived from A-T patients show higher frequency of chromosome end-to-end associations, suggesting defects with their telomeres.89,91The gene mutated in AT, ATM, encodes a protein kinase that is distantly related to the yeast MEC1 and TEL1 proteins that function in maintaining the integrity of telomeres.92 ATM has been shown to be active throughout the cell cycle and phosphorylates a number of nuclear proteins, including the nuclear c-Abl tyrosine kinase,93,95 the tumor suppressor protein p53,96,97 the breast cancer susceptibility gene product BRCA1,98 the human checkpoint kinase hCds1/chk2,99,102 and the Nijmegen Breakage Syndrome gene product NBS1.103 This network of ATM-regulated phosphorylation events regulates cell cycle checkpoints and contributes to the processing of DNA double strand breaks, produced by ionizing radiation.104,105 Determination of the precise mechanism by which ATM may regulate the structure and function of telomeres is currently under intensive investigation. The downstream targets like c-Abl have been shown to negatively regulate the telomerase activity. Lymphoblastoid cells derived from A-T individuals have been shown to have relatively higher telomerase activity as compared to the normal controls.89 Recent studies have demonstrated that expression of the catalytic subunit of telomerase extends the lifespan of fibroblasts derived from A-T individuals without changing their phenotypic properties.51 The parental A-T primary fibroblasts exhibited the hallmark characteristics of senescence quite early during culture including the increase in size, appearance of SAGal staining and cessation of DNA replication. Though expression of hTERT extended the life span of A-T cells, however occasional appearance of SAGal cells was still observed. DNA-damaging agents (ionizing radiation, bleomycin) are known to induce SAGal positivity.106 It has been suggested that the occasional SA-β-Gal (+) cell in the hTERT cells reflects the consequences of DNA damage (chromosome end-to-end associations) events in the A-T cells. The presence of end-to-end chromosome associations suggested that such cells could have still altered telomere nuclear matrix interactions as has been reported previously,91 and such altered telomere interactions may influence the senescence signaling mechanism. Alternatively, though hTERT lengthened the mean telomere length in A-T cells, it may be possible that a separate triggering event occurs in conjunction with the loss of ATM expression leading to the low level but continual entry into senescence of the hTERT+ A-T cell population. These hTERT+ A-T cells continue to grow slowly as compared to normal human fibroblasts expressing hTERT. These studies suggested that though hTERT can extend the life span, the overall growth properties of the cells are not affected. These studies also suggested that the additional loss of ATM signaling with telomerase expression is still not sufficient to transform cells and that further genetic changes are necessary for malignant transformation.

Telomerase Activity and Ionizing Radiosensitivity

Tumor growth is the result of a balance between cell production from division and various types of cell loss. The main factor that influences the growth rate of a tumor is the cell cycle of the proliferative cells in the population.Tumors of the same histological type arising in different patients differ widely in growth rate. It is generally found that irradiation causes an elongation of the generation cycle of tumor cells and shortening of the cell cycle of normal cells, although there are exceptions to this rule. The probable consequence of this change in cell cycle is that normal tissues do not respond by speeding up the progress of cells through their cell cycle for some time after the delivery of the dose of radiation. If this time is comparable to the overall time of a fractionated course of radiotherapy, then the preirradiation cell cycle times of the normal and malignant tissues are relevant to a discussion of dose-time relationships in radiotherapy and to the use of cycle-specific drugs in combination with radiotherapy.

Genetic lesions that disable key regulators of G1 phase progression in mammalian cells are present in most human cancers and such cells are mostly positive for telomerase activity. Mitogen-dependent cdk4 and cdk6 phosphorylate Rb protein, that help to cancel Rb's growth-inhibitory effects and enables E2F transcription factors to activate genes required for entry into the S phase, may be involved in the upregulation of telomerase. Disruption of the Rb pathway by overexpression of cyclin D-dependent kinases or through loss of p16 (INK4a), an inhibitor of the cyclin D-dependent kinases may be linked with the regulatory role for the telomerase function. Evidence is accumulating that the reduction in levels of p27(Kip1) and increased expression of cyclin E correlate with the telomerase activity. Whether there is any correlation between the abnormal growth signal and the activation of the telomerase is yet an open area of investigation. It will be worthwhile to test whether ARF tumor suppressor, encoded by an alternative reading frame of the INK4aARF locus, senses mitogenic current flowing through the Rb pathway and is induced by abnormal growth-promoting signals that could have any potential link with the regulation of the telomerase either at the transcriptional or post-translational level.

There is evidence that telomerase activity is enhanced when cells are challenged with DNA-damaging agents. However, it is known that ARF is not directly activated by signals that damage DNA. Inactivation of the ARF does not only dampen the p53 response to abnormal mitogenic signals but also renders tumor cells resistant to treatment by cytotoxic agents and such tumor cells have been found to have relatively higher telomerase activity, indicating that inactivation of the ARF might play a role in the function of telomerase.

The most compelling data that supports the view that telomere loss eventually restrains the proliferation of human cells arose from human tumors and immortal human tumor cell lines. Telomere shortening has been believed to restrict the number of cell divisions and this restraint is overcome by immortalization which is accompanied by the activation of telomerase or by an alternative mechanism in order to maintain the telomeres. Telomere loss and activation of telomerase is as frequent in tumors as are mutations in the Rb and p53 pathways. It has been suggested that telomerase activation is the post consequences of rapid proliferation because of the fact that stem cells have much lower telomerase activity than tumors, and many proliferating cells e.g., fibroblasts lack telomerase.

Several classes of human cells are now known to count divisions by monitoring the progressive attrition of telomeres, leading to the activation of a p53-p21 WAF-dependent G1 checkpoint. Ectopic expression of hTERT has been shown to prevent senescence in several cell types and offers the potential for interventions in the aging process based on tissue engineering, gene therapy or homeografts. However, this telomere-driven senescence mechanism seems to be absent from rodents, which use telomere-independent means (perhaps based upon p14arf) to count divisions. Similar senescence pathways are now being reported in humans, and this, coupled with the demonstration of tissue-specific telomeric loss rates, has the potential to render strategies based on the use of telomerase dependent on the characteristics of the target tissue.

Telomerase Activity as a Measure for Monitoring the Radiocurability of Tumors

Radiotherapy plays a key role in the treatment of many tumors and therefore determination of the surviving fraction of tumor cells after radiation treatment becomes critical to judge the success of radiotherapy. Although several biomarkers have been used in the past to determine the residual disease,107,108 there is no universal marker that could be used for most types of tumor. Since telomerase activity is present in almost all carcinomas73 and the level of telomerase activity correlates with the stage of carcinogenesis,109 determination of telomerase activity has a great potential for use in clinical diagnosis as well as to follow the recurrence of various malignancies after radiotherapy. As telomerase activity is frequently associated with the malignant phenotype, Sawant et al,110 established a correlation between ionizing radiation induced cell death and telomerase activity.

Since cells in an actively growing tumor are distributed in different cell cycle stages, Sawant et al,110 determined whether the telomerase activity in exponentially growing cells is affected by ionizing radiation. Cell cycle phase distribution in growing HeLa cells was evaluated by flow cytometry. The exponential HeLa population contains about 40% of G1phase, 50% of S-phase and about 10% G2/M phase cells. Exponentially growing HeLa cells were treated with graded doses of gamma rays, incubated at 37°C for different time periods and analyzed for telomerase activity. Sawant et al110 reported no difference in telomerase activity among treated and untreated exponentially growing cells immediately after radiation treatment, but, unlike plateau phase cells, there was a two-to-threefold increase in telomerase activity in cells collected 72 h postirradiation. Samples collected 120 h and later, however, showed a decrease in telomerase activity in treated cells compared to the untreated cells. In contrast to the initial increased telomerase activity in irradiated HeLa cells, treatment of exponentially growing RKO cells showed no such increase in the telomerase activity but showed a dose- and time-dependent difference between irradiated and control cells.

In vitro studies demonstrated a link between the cell kill and telomerase activity.110 Furthermore similar results were found when the telomerase activity in tumors in nude mice were determined before and after irradiation. The irradiated tumors showed a decrease in the telomerase activity. In tumors destined not to recur, telomerase activity was zero, whereas in tumors that were to relapse, telomerase activity never reached zero.

Influence of Ionizing Radiation on hTERT Expression

Sawant et al,110 and others have reported telomerase activity increased after gamma ray treatment. Whether the enhancement of the telomerase activity is due to transcriptional activation of hTERT or the postmodification of TERT has been a subject of great debate. However, Sawant et al110 examined the mRNA levels of hTERT in HeLa cells after radiation treatment. Cells were irradiated with 10 Gy and mRNA levels of hTERT were examined up to 96 h post-irradiation. It was reported that no change in levels of hTERT mRNA was observed. In view of the correlation between the expression status of hTERT and telomerase activity,111,112 possible reasons for the increase of telomerase activity in the absence of corresponding increase in the hTERT mRNA levels could be due to a reduced degradation of the telomerase enzyme postirradiation or release of telomerase component due to structural change/s brought about by ionizing radiations. It is known that ionizing radiation causes DNA-DNA as well as DNA protein crosslinks,113 and also releases soluble proteins bound to the chromatin. Thus the increase in telomerase activity in some cell types could be due to release of chromatin-bound telomerase by ionizing radiation.

Both in vitro and in vivo studies showed that important information regarding the viable residual tumor cell burden can be gained by comparing the telomerase activity in samples collected postirradiation. The current data suggest that the telomerase activity varies among different telomerase-positive cell lines. A link has been established between cell kill and telomerase activity after radiation treatment. Reduction in the telomerase activity in vivo and in vitro experiments seen after radiation treatment parallels cell kill. These findings imply the clinical potential of telomerase enzyme as a marker of efficacy of a radiation therapy protocol.

Telomerase Activity and Genomic Stability

The question whether telomerase activity influences genomic stability, was addressed by Vaziri et al,85 by analyzing chromosomal aberrations employing spectral karyotyping in isogenic cells with and without hTERT expression. This analysis used colored fluorescent chromosome-specific paints that provide a complete analysis of the human chromosomal complement. Thus, chromosomal rearrangements can be identified by the juxtaposition of different colors along a single chromosome. The frequency of aberrations found in cells with hTERT were comparable to parental young early-passage cells without ectopic expression of hTERT. Furthermore, Wood et al51 investigated the DNA and chromosome damage and radiation sensitivity in isogenic cells with and without hTERT expression (young early-passage parental cells). Neither the G1 nor G2 type of chromosome damage was different among cells with and without hTERT, suggesting that hTERT does not seem to be involved in repair process of the chromosome damage.51 The levels of chromosome aberrations in A-T cell lines were higher than the normal cells, indicating that the defective chromosome repair is not corrected in A-T cells by the ectopic hTERT expression. Although one cannot rule out the possibility that telomerase is involved in the addition of (TTAGGG)n repeats to sites of double-strand breaks, one cannot detect a measurable physiological difference between the isogenic cells with and without telomerase in response to ionizing radiation. These studies suggested that hTERT expression has minimum influence on the cell cycle check point functions.

Biochemical and genetic studies have established an association between telomere maintenance and extended life span mediated through the expression of the hTERT. Ectopic expression of TERT prevents replicative senescence in several cell types including fibroblasts and epithelial cells.23,24,51,114 It may also exert anti-apoptotic action at early stage of the cell death process prior to mitochondrial dysfunction and caspase activation.26 Choi et al,115 demonstrated that telomerase expression suppresses senescence-associated genes in Werner syndrome cells but it is not known if such suppression correlates with genomic integrity. Since accumulation of DNA damage has been implicated as a potential molecular mechanism regulating cellular senescence, recent studies revealed that ectopic expression of hTERT protein in normal cells influences expression of a subset of genes, reduces spontaneous chromosome damage in G1 cells, and enhances the kinetics of DNA repair.116 These changes were accompanied with increases in ATP levels.116 However, TERT does not influence meiotic recombination in vivo or DNA end rejoining in vitro. It is proposed that hTERT protein through its interaction with telomeres, generates a signal to enhance repair of DNA damage and regulate the expression of a variety of genes required for extended life span.116

Wood et al51 addressed the question whether expression of hTERT influences the cell survival after treatment with ionizing radiation. The presence of hTERT slightly improved the survival of some cell lines. The change in clonogenicity in the irradiated hTERT-expressing cells probably reflects the reduction of the signal from too-short telomeres rather than any fundamental change in the damage-response pathway. These results demonstrate that expression of telomerase does not significantly influence the radiosensitivity of fibroblasts derived from A-T patient.

Several investigators have made an attempt to determine whether telomerase activity correlates with apoptosis or cell growth or survival. Zhang et al117 reported that telomerase activity is not related to apoptosis in leukemia cell lines. However, Kanaya et al,118 demonstrated that adenovirus expression of p53 represses telomerase activity through down-regulation of human telomerase reverse transcriptase transcription. It has been shown that retinoic acid extends the in vitro life span of normal human oral keratinocytes by decreasing p16(INK4A) expression and maintaining telomerase activity.119 Inhibition of telomerase limits the growth of human cancer cells.120 Sawant et al110 found ionizing radiation treatment to decrease the telomerase activity in a dose-dependent manner, which correlated with cell death in in vitro tests as well as during tumor regression in nude mice.

Telomere Dysfunction and Ionizing Radiation Sensitivity

Tumor cells exhibit a diverse set of phenotypic abnormalities, including loss of differentiation, increased mortality and enhanced drug resistance. However, one phenotypic abnormality that is virtually pathognomic of all cancer cells is the telomerase activity. Whether there is a direct correlation between the levels of telomerase and ionizing radiosensitivity for tumor cell kill has not been yet addressed. However, it has been demonstrated by several groups that ectopic expression of the TERT does not change significantly the radiosensitivity of the cells, suggesting that TERT levels itself may have minimum role in the ionizing radiosensitivity.

At present the information about correlation between the telomerase activity and human tumor cell radiosensitivity is minimal. Animal models have shown that telomere dysfunction impairs DNA repair and thus enhances the sensitivity to ionizing radiation induced cell kill. Ionizing radiation may not be a very potent inducer of second malignant tumors, at least in most cases. This prediction is derived from the localized nature of the exposure during clinical radiotherapy, in which the dose to normal tissues is minimized, and from the fact that radiation tends to be cytotoxic rather than mutagenic. Unlike for chemical agents, radiation can be easily detected, and the absorbed dose to critical tissues is precisely measured. The sensitivity of many target tissues to radiation-induced cancer is known. Ionizing radiation is an important therapeutic modality in clinical cancer management. Proliferating cells exhibit enhanced sensitivity to cell kill by gamma radiation, possibly because of the reproductive cell death. Studies on the telomerase-deficient mouse have revealed that although the loss of telomerase activity per se and no discernible impact on the response to ionizing radiation, however late generation telomerase deficient (Terc/) mice with dysfunctional telomeres imparted radiosensitivity syndrome associated with enhanced mortality.121 Interestingly, the gastrointestinal crypt stem cells and primary thymocytes showed increased rates of apoptosis, and mouse embryonic fibroblasts exhibited diminished dose-dependent clonogenic survival. An intimate relationship for ionizing radiation response was observed in such cells for the dysfunctional telomere, DNA breaks repair and cell survival.

Enormous progress has been made in the past several years in our understanding of the gene products governing the response of mammalian cells to ionizing radiation. Many of these are potential targets for enhancing the effectiveness of radiotherapy. However, a major barrier to such efforts is the requirement for a preferential effect on tumor vs. normal cells. Such a requirement can only come about by exploiting a known difference between tumor and normal cells. The differences in telomerase activity between tumor and normal cells could be exploited with fractionated radiotherapy. Telomerase inhibitors could be used to preferentially enhance the response of tumor cells to radiotherapy.

Acknowledgments

I am grateful to the members of my laboratory for the comments on this manuscript. The research in my laboratory is supported by grants from National Institute of Health and A-T Children's Project.

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