ASSOCIATION OF PHYSICIAN OF INDIA

By Prof. J.S.Bajaj
M.D., F.R.C.P. (Ed)., F.R.C.P. [Lond]; F.A.M.S.
D.Sc. [h.c. MGR Med. Univ.]; D.Sc. [h.c. GND Univ.
Hon. D.Sc. [Madras]; D.M. [h.c. Karolinska]
Hon. D.Sc. [Banaras Hindu University]
D.Sc. [h.c. Univ. Health Sc; Andhra]
Honorary President, International Diabetes Federation
President, South East Asia Regional Association for Medical Education
Formerly, Professor and Head, Department of Medicine,
& Presently, Member, Governing body, All-India Institute of Medical Sciences,
New Delhi.
Ernst, Hemingway, the winner of the Nobel Prize for Literature in 1954, and immortalised for posterity by his novel 'For whom the Bell Tolls', provides the inspiration for the choice of the title for the Honour Lecture at the APICON 99. With nearly 40 years of experience in teaching undergraduates and postgraduates, I am almost certain that the majority of those present here, have not heard about telomerase, let alone recognizing it as a quantum leap in cell biology, and realizing its potential for major therapeutic advances in the next century, when we meet again and where I may be invited by the President, Dr. Munjal, to deliver the Millennium Lecture. Muller, in 19381, while working with Drosophila chromosomes, suggested that stabilization of chromosomal ends is by means of specialized structures for which he coined the term telomeres. Since then, thanks to the pioneering work of Nobel laureate Barbara Mclintock2 [of 'jumping genes' fame'] and several others, it is now confirmed that telemeres are essential for chromosomal stability, in addition to participating in nuclear processes such as chromosome positioning in the nucleus, transcription repression, heterochromatin formation, and replication timing. It is their role in cell replication that has provided new insights into cell biology of ageing. In a landmark paper, which I consider as one of the '100 classics' of the present century, Hayflick and Moorehead [1961]3 showed that normal diploid cells, with a few exceptions such as germ cell lineage, certain stem cell lines and cancer cells, have a limited replicative potential, permitting about 50 cell divisions as demonstrated in cultured young skin fibroblasts. Prior to achieving such a maxima, the rate of cell division slows down and the cells begin to show predictable and identifiable morphologic changes, recognized as characteristic of 'senescent cells'. These changes are in turn associated with 'senescence-associated gene expression', which accompany the replicative block in several cell types. It was in the early 1970s that Russian biologist Olovnikov [1973]4, and British investigator Watson [1972]5, independently recognized that a portion of the telomere is 'masked' from DNA polymerase, and therefore is not replicated with each cell division. Thus, a successive shortening of telomere characterizes each cell division, as observed by Harley et al and described in their classic paper in Nature, in 19906. It is now generally accepted, though with some exceptions, that telomere shortening is the clock that times the shift to a 'senescence' pattern of gene expression, and underlies the phenomenon of 'replicative senescence' that forms the basis of "Hayflick limit". Telomerase and Telomere-binding proteins As telomeres cannot be fully replicated by conventional DNA polymerases, such a function is fulfilled by a ribonucleoprotein reverse transcriptase called telomerase. It carries its own template RNA which codes for the DNA repeats. In the human, hRT codes for [TTAGGG]-n 7 [Table 1]. Current models reviewed recently8, suggest that two independent sites on the telomerase enzyme interact with the primer. The template site contains the complementary RNA and aligns the primer 3/ end for elongation, while the anchor site binds to the primer 5/ end of template, thus providing a path for the growing chain to exit the enzyme. Telomerase differs from all the other polymerases in that it utilizes an internal template rather than an external one. This characteristic of the enzyme places specific steric constraints on primer elongation and catalysis. Telomere length regulation is mediated partly by telomere binding proteins [TBPs], which fall into two distinct classes: those that bind the single stranded telomere repeats at the extreme termini, and those bind along the length of the double stranded telomere repeats. The balance between factors involved in telomere shortening [incomplete replication, telomere processing, recombination], telomere stabilization [TBPs, telomere chromatin structure], and telomere lengthening [telomerase, c-strand synthesis, recombination] must be fine tuned through genomic programming and cell signaling pathways9. Using newer techniques of molecular biology, measurements of the length of terminal restriction fragments [TRFs] from highly digested genomic DNA showed that the mean length of TRF decreased in culture human fibroblasts [mean loss 48± 21bp/population doubling], and more importantly, such a decrease was associated with a corresponding decrease in the signal intensity of hybridising probe [TTAGGG]3, including a rate of decrease consistent with the loss of TTAGGG repeats from the ends of TRFs10. Significantly, when cells were maintained for long periods of time in culture without cell division, there was neither any demonstrable decrease in the length of TRFs, nor was any detectable decrease in the intensity of TTAGGG hybridization. These data convincingly show that terminal TTAGGG repeats were specifically lost during ageing in vitro in a replication dependent manner. Finally, a significant correlation was observed between TRF length and the age of donor from whom the skin fibroblasts were obtained for culture. The current model can be summarized as under10: During division of somatic cells, loss of telomeric DNA occurs due to incomplete replication at 5/ end, with or without a specific or non-specific erosion of DNA ends. When a critical length is reached on one or more telomeres in a dividing cell, a signaling mechanism [possibly involving the p53 tumour suppressing protein cascade] is triggered which evokes the Hayflick limit: cells stop dividing. It is assumed that such a sequence of events results from the loss of TTAGGG repeats on one or more telomeres. Telomere length is a resultant of a balance between processes that lengthen and those that shorten telomeres. The lengthening of telomeres is dependent on the activity of telomerase, a ribronucleoprotein polymerase, that specifically elongates telomeres. Telomerase is active at some stage of gametogenesis and thus maintains telomere length in germ cells between generations of the organism. During differentiation of most, if not all, somatic tissue, telomerase is repressed. Telomerase activity is absent, in cultured human fibroblasts. Immortalization of a cell involves, among other events, activation of telomerase. Depending upon the telomerase activity in individual clones, telemeres may be essentially of any length, from very short, to very long. Essentially, therefore, it can be hypothesised that telomere loss acts both as a mitotic clock, reflecting the replicative history of normal somatic cells, as well as a genetic times bomb contributing to chromosomal abnormalities in cellular ageing. There are two questions which logically emerge from the above discussion: [I] If telomere ticks as the cellular clock for replicative senescence, can we 'rewind' it so that it can be 'reset' to see yet another 'innings'?; and [ii] what is the relationship between cellular senescence and biological ageing in the organism? As an essential corollary of the second question: what are the clinical implications regarding causes, and potential management, of age-related disorders such as cancer, atherosclerosis and dementias? The year 1998 was remarkable as it provided a definitive answer to the first question. Scientific data affirmed that telomere is the clock of replicative senescence, and it can indeed be reset. This landmark paper of 1998, and indeed of the present decade, by Bodnar et al in Science11, and the affirmation of its published data in a subsequent paper by Vaziri et al in Current Biology12, rekindles the eternal hope that we may yet live to see another day. First, the paper by Andra G. Bodnar and her colleagues wherein a gene for the catalytic component of telomerase was transfacted in the cells undergoing replicative senescence, resulting in extended telomeres. More significantly, this procedure extended also the replicative life span of such cells and gave them a pattern of gene expression typical of young cells. By the time the results were submitted from publication, transfacted cells had already undergone 40% more population doublings, without showing any evidence of slowing their rate of cell division. Thus, telomeres not only shorten with cell ageing, but relengthening the telomeres through telomerase activation appears to reset gene expression as measured by expression of beta-galactosidase [an established biomarker of ageing], cell morphology, and the replicative life span. This leads to the second question which is indeed the ultimate question: will the recent breakthrough provide us with the means for therapeutic modification of the cellular mechanisms underlying age-related diseases, to an as yet 'unparalleled and effective degree'? The possibilities are immense and immediately include effective prevention and treatment of atherosclerosis, immune senescence, and Alzheimer dementia through interventions at a fundamental cellular level. Telomere-Telomerase in Oncology The last five years have witnessed a plethora of publications emphasising telomerase activity in the cancer cells both as a diagnostic indicator and as a prognostic determinant. A special issue of the European Journal of Cancer [Vol 33; No. 5,1997] is a most timely compendium on this subject. Telomerase as a Diagnostic Marker of Malignancy With the recent availability of a highly sensitive TRAP [Telomeric Repeat Amplification Protocol] assay, it has become possible to detect and measure the presence of, and quantify the amount of, telomerase activity from small tissue samples as well as biological fluids. In this assay procedure, telomerase first synthesises extension products which then serve as templates for PCR amplification. More recently, an internal standard to permit semiquantitation of levels of telomerase activity has been incorporated in the assay procedure. With the introduction of a research kit [TRAPeze] for detecting telomerase activity [Oncor, Gaithersburg, Maryland, USA], variability between laboratories is likely to be minimized and external quality control achieved in the near future. A recent publication summarizes the available data [Table II] based on the measurements in tumours of diverse origin and morphology. According to this review, 758 of 895 [85%] of malignant tumours, but none of the 70 normal somatic tissues, expressed telomerase activity13, 14. The tabulated data also reaffirms the presence of such activity in all germ-line tissues tested. It is of considerable significance to note the presence of telomerase in histologically normal tissue adjacent to a malignant tumour. Since the TRAP assay is highly sensitive to detect telomerase activity from as little as 1-10 telomerase expressing cells, such activity may provide an early guidance to the presence of morphologically unidentifiable infiltrating cancer cells in seemingly normal tissue adjacent to a malignant tumour cell mass. Based on the data summarized in the table, the results show a specificity of 91%, sensitivity of 85%, positive predictive value of 93%, and negative predictive value of 81%. Preliminary as the results are, these do indicate the promising potential of telomerase as a useful diagnostic marker of malignancy. As of now, several distinct advantages of using telomerase as a diagnostic tool for malignancy are being substantiated. Firstly, telomerase activity is detected in the most common cancers, such as those of the prostate, breast, colon, lung and liver in 85-90% of patients examined. More importantly, such activity is detectable in many instance, at an in situ stage. In lung cancer, telomerase activity has even been detected in preneoplasia of smokers and former smokers. However, it is not so in pancreatic and colon cancer. Nevertheless, even in these cancers it appears in 90-95% of early stage, although not in the stage of preneoplasia. A second advantage of telomerase assay is that it can be detected in nearly every type of clinical specimen including exfoliated cells and FNAC material. Shay and Gazdar [1997] have summarized the current situation15. Table III summarises the available data. Telomerase as a Prognostic determinant in Malignancy In contrast to a well substantiated role of teromease as a diagnostic marker in malignancy, there are early indications from a few published reports as to its possible role in predicting prognosis in a known case of malignancy. High levels of telomerase have been shown to be strongly correlated with poor clinical outcome in neuroblastoma [Hiyama et al, 1995]16. Likewise, clinical outcome and several prognostic indicators of breast cancer do show a statistically significant correlation with the level of telomerase activity, though confirmatory evidence of such observations from other centres is still awaited. The potential value of such a prognostic marker will be in accurately identifying, for example, a population within node negative breast cancer patients, that are likely to experience tumour recurrence and therefore may benefit if a more aggressive adjuvant therapy is planned and offered. Such an approach may be equally valid in several other malignancies. In addition to the clinical usefulness as a diagnostic marker and a prognostic indicator, telomerase expression may offer a potential approach to monitor the effectiveness of cancer therapy. As recent data indicates that the level of telomerase activity in a biopsied specimen is directly proportional to the percentage of cancer cells in the tumour, the level of telomerase expression may find a possible use in determining the number of cancer cells present in a patient undergoing treatment; a natural extension will be to detect the presence of cancer cells in a patients' bone marrow used in autologous transplantation. Telomere targetting in Oncotherapy Based on the premise that telomerase activation is a pre-requisite for oncogenesis and its activity can serve as a molecular biomarker for most of the common human cancers, irrespective of cell or tissue of origin, it is imperative that basic information regarding telomere length homeostasis be applied to explore novel approaches in therapeutic oncology. The possible lines of attack include: [I] research and development of compounds with intrinsic activity as telomerase inhibitors; and [ii] selective destabilization of telomeres in cancer cells through 'poisoning' [analogous to compounds which act as spindle poisons in normal dividing cells]. A small molecule, compound or nucleoside analogue that may integrate specifically at preselected telomere site[s], with or without the aid of telomerase, may be one such therapeutic approach, amongst several others, to target and destabilise the telomeres in mitotic cells. As and when such an approach becomes clinically viable, a distinct advantage will be the assessment of therapeutic response through serial telomere length measurements which would indirectly reflect the maximum replicative capacity of malignant cells at any point in time. A word of caution, however, would be in order. As mentioned earlier, telomerase is also expressed in stem cells and hemotopoietic cells in normal circumstances. Thus the toxicology profile of a telomerase inhibitor would require suitably designed protocols in appropriate animal models [s]. Telomerase and Tankyrase: A Synergistic interaction Human telomere function requires two telomere-specific DNA binding proteins, TRF1 and TRF2. While the TRF2 protects the chromosomal ends, the TRF1 regulates telomere length. Overexpression of TRF1 in a telomerase-expressing cell line leads to progressive telomere shortening, whereas the inhibition of TRF1 increases telomere length. The latest publication [Science, 20 November, 1998]17 opens new vistas to facilitate basic understanding of the mechanism of action of telomerase. A new protein, tankyrase, has been discovered in the human cells. Tankyrase is a poly [ADP-ribose] polymerase and binds to the telomeric protein TRF1 [telomeric repeat factor 1]. It has been suggested that tankyrase facilitates the access of telomerase to chromosome ends by 'stripping' TRF1 [telomere specific DNA binding protein]. If this function of tankyrase is confirmed, it may lead to research and development of a new class of compounds which would up- or down- regulate tankyrase to control cell lifespan. Tankyrase activators could turn on telomerase activity in cells used for gene - or cell-based therapies, extending their lives. In contrast, new anti-cancer agents may be developed which might act by inhibiting tankyrase, thereby blocking telomerase activity, and making cancer cells mortal again. Epilogue In the past year, the first insight into the recognition of double-stranded telomeric DNA has come from both structural and genetic studies on telomere binding proteins and from evolutionary distant species. The probability that ageing and its consequent clinical outcomes may be alterable at the cellular and chromosomal levels, although speculative at the turn of the century, nevertheless has moved into the realm of possibility in the next millennium. A new paradigm putting senescence and malignancy on the two sides of the same coin has emerged.