Preface

Chapter 8 **Telomere Length and Its Relation to Human Health 163**

Chapter 9 **On the Far Side of Telomeres: The Many Roles of Telomerase in the Acquisition and Retention of Cancer Stemness 187**

Vivian F. S. Kahl and Juliana da Silva

**VI** Contents

Kerem Teralı and Açelya Yilmazer

Historically, Hermann Muller in the 1930s was the first researcher to point out that the ends of the chromosomes had unique properties and functions as well. Muller named these chro‐ mosomal ending part "telomeres" (from the Greek words telo, meaning "end" and mere, meaning "part"), based on their terminal position. Furthermore, realizing that the ends of chromosomes were strangely resistant to X-ray-induced chromosomal damage, he hypothe‐ sized that "the terminal gene must have a special function, that of sealing the end of the chromosome, so to speak, and that "for some reason, a chromosome cannot persist indefi‐ nitely without having its ends thus sealed". Nowadays, we know that Muller was only parti‐ ally correct. It is very well know and scientifically accepted that telomeres do indeed play an essential role in stabilizing the ends of chromosomes, but, on the other hand, they do not contain active genes within their DNA sequences. Instead, telomeres possess an array of highly repeated DNA sequences and specific binding proteins that form a unique, peculiar, and functional structure at the end of the chromosome. Thus, telomeres should be consid‐ ered not just a simple end part of a chromosome, but instead a dynamic and functional part of them.

In addition, Barbara McClintock, a brilliant American cytogeneticist who was awarded the 1983 Nobel Prize in Physiology or Medicine, had developed reliable methods for preserving and staining maize chromosomes. McClintock observed that the broken chromosomal ends induced by clastogens were unstable. Furthermore, she noticed that these broken ends fused with any other broken ends when they contacted each other. When the replication process occurred after such contact, the cycle of breakage--fusion could, thus, be repeated. However, it was also noticed that the breakage--fusion cycle was broken when dicentric chromosomes were present, as in embryonic cells, where the broken ends of the chromosomes were some‐ how "healed." Nowadays, the scientific community knows that the ends were healed by the addition of a telomere, a process that is catalyzed by a specific reverse transcriptase enzyme called telomerase. Furthermore, we know that telomerase is active in germ cells, embryonic cells, and some somatic cells, but not in the endosperm cells that McClintock examined in her research work. It is worth mentioning that McClintock not only highlighted that cells do not accept the presence of unprotected chromosome ends, but also that the chromosomal broken ends are quickly fused together by the mechanism of DNA repair. Furthermore, she also observed that telomeres prevent these fusion events from occurring. Despite the magni‐ tude and importance of these observations, 40 years of scientific research would pass by be‐ fore the actual known structure of telomeres was revealed.

In the mid-1970s, Elizabeth Blackburn determined the telomere sequences for the ribosomal DNA minichromosome of the free-living ciliate protozoa Tetrahymena sp. Sequencing ex‐ periments demonstrated that the telomeres of this structure possessed 20-70 tandem copies

of a simple hexanucleotide with the sequence 5'-CCCCAA-3' on one strand and 5'-TTGGGG on the complementary strand. Noteworthy, the GT-rich strand represented the 3'-end of the minichromosomes. Actually, it is well known that these tandem repeats of short GT-rich se‐ quences are characteristic of almost all eukaryotic telomeres. They consist, generally, of a 6-8 bp sequence that is repeated hundreds or thousands of times, varying the actual repeated sequence and the number of repeats among species. In this sense, e.g., human telomeres range in size from 2-50 kb and consist of approximately 300-8,000 precise repeats of the se‐ quence CCCTAA/TTAGGG, whereas telomeres from the budding yeast Saccharomyces cer‐ evisiae are smaller and more heterogeneous in their composition, containing about 60-100 copies of the sequence C1-3A/TG1-3. Although such variations exist, common features of all telomeres are the orientation of the G-rich strand, which makes up the 3'-end of the chromo‐ some, and that the terminal portion of the G-rich strand is single-stranded, generating the so-called "G-tail," with an average length of 50-100 nucleotides in yeast but up to 75-300 nu‐ cleotides in humans.

By the early 1970s, the mechanism of cellular DNA replication was clarified, and then scien‐ tists realized that this system offered an essential dilemma. Particularly, the ends of chromo‐ somes are supposed to gradually shorten with each cycle of DNA replication. This so-called "end-replication" problem results as a direct consequence of DNA polymerase's properties. It is known that DNA polymerase requires the presence of short RNA primers to begin rep‐ lication. It can then extend the primers in a 5'-to-3'-direction. Accordingly, as the replication fork progresses along the chromosome, one of the two daughter strands is synthesized con‐ tinuously, whereas the other daughter strand is synthesized discontinuously in short frag‐ ments (also known as Okazaki fragments, possessing their own RNA primers), this being the so-called lagging strand. Subsequently, the RNA primers are degraded, with the gaps between the Okazaki fragments filled by the DNA enzymatic apparatus. However, a serious problem arises at the ending part of each chromosome: the DNA repair machinery is not capable of completing the gap left by the terminal RNA primer. As a consequence, the new‐ ly synthesized DNA molecule is shorter than the template (parent) DNA molecule by at least the length of one RNA primer. Without a solution to this end-replication problem, chromosomes would progressively shorten over cell-cycle divisions. We know that the "cleaver" cellular machinery must avoid such a problem, which would bring about cata‐ strophic consequences.

By the mid-1980s, accumulating evidence started to suggest that cells are capable of solving this replication dilemma by lengthening their telomeres. Researchers from the Blackburn and Szostak laboratories were able to demonstrate not only that DNA sequences from Tetra‐ hymena may function as telomeres for linear plasmids introduced into yeast cells, but also that the Tetrahymena telomere sequences were elongated during the process. They also demonstrated, when sequencing the new telomeres, that the elongated telomeres had re‐ peated copies of the yeast TG1-3 repeat sequences rather than the Tetrahymena TTGGGG repeat ones. Accordingly, a new open question arose from these observations: How is it pos‐ sible that yeast cells can elongate telomere sequences from another organism with copies of that organism's own telomere repeat?

Fortunately, the answer to this question was provided by the discovery of the telomerase. Carol Greider, a student in Blackburn's lab, further investigated Tetrahymena extracts able to incorporate nucleotides into a synthetic oligonucleotide that contained four copies of the Tetrahymena telomere repeat sequences. Greider purified an enzyme able to lengthen

telomeres. This enzyme, later called telomerase, turned out to be a highly specialized re‐ verse transcriptase, in other words, an enzyme able to synthesize DNA from an RNA tem‐ plate. The telomerase can also be classified as a ribonucleoprotein, since the RNA template is a constitutional part of the enzyme complex itself. Finally, it can also be stressed that its RNA template includes a base sequence complementary to the telomere repeat unit in the same organism.

of a simple hexanucleotide with the sequence 5'-CCCCAA-3' on one strand and 5'-TTGGGG on the complementary strand. Noteworthy, the GT-rich strand represented the 3'-end of the minichromosomes. Actually, it is well known that these tandem repeats of short GT-rich se‐ quences are characteristic of almost all eukaryotic telomeres. They consist, generally, of a 6-8 bp sequence that is repeated hundreds or thousands of times, varying the actual repeated sequence and the number of repeats among species. In this sense, e.g., human telomeres range in size from 2-50 kb and consist of approximately 300-8,000 precise repeats of the se‐ quence CCCTAA/TTAGGG, whereas telomeres from the budding yeast Saccharomyces cer‐ evisiae are smaller and more heterogeneous in their composition, containing about 60-100 copies of the sequence C1-3A/TG1-3. Although such variations exist, common features of all telomeres are the orientation of the G-rich strand, which makes up the 3'-end of the chromo‐ some, and that the terminal portion of the G-rich strand is single-stranded, generating the so-called "G-tail," with an average length of 50-100 nucleotides in yeast but up to 75-300 nu‐

By the early 1970s, the mechanism of cellular DNA replication was clarified, and then scien‐ tists realized that this system offered an essential dilemma. Particularly, the ends of chromo‐ somes are supposed to gradually shorten with each cycle of DNA replication. This so-called "end-replication" problem results as a direct consequence of DNA polymerase's properties. It is known that DNA polymerase requires the presence of short RNA primers to begin rep‐ lication. It can then extend the primers in a 5'-to-3'-direction. Accordingly, as the replication fork progresses along the chromosome, one of the two daughter strands is synthesized con‐ tinuously, whereas the other daughter strand is synthesized discontinuously in short frag‐ ments (also known as Okazaki fragments, possessing their own RNA primers), this being the so-called lagging strand. Subsequently, the RNA primers are degraded, with the gaps between the Okazaki fragments filled by the DNA enzymatic apparatus. However, a serious problem arises at the ending part of each chromosome: the DNA repair machinery is not capable of completing the gap left by the terminal RNA primer. As a consequence, the new‐ ly synthesized DNA molecule is shorter than the template (parent) DNA molecule by at least the length of one RNA primer. Without a solution to this end-replication problem, chromosomes would progressively shorten over cell-cycle divisions. We know that the "cleaver" cellular machinery must avoid such a problem, which would bring about cata‐

By the mid-1980s, accumulating evidence started to suggest that cells are capable of solving this replication dilemma by lengthening their telomeres. Researchers from the Blackburn and Szostak laboratories were able to demonstrate not only that DNA sequences from Tetra‐ hymena may function as telomeres for linear plasmids introduced into yeast cells, but also that the Tetrahymena telomere sequences were elongated during the process. They also demonstrated, when sequencing the new telomeres, that the elongated telomeres had re‐ peated copies of the yeast TG1-3 repeat sequences rather than the Tetrahymena TTGGGG repeat ones. Accordingly, a new open question arose from these observations: How is it pos‐ sible that yeast cells can elongate telomere sequences from another organism with copies of

Fortunately, the answer to this question was provided by the discovery of the telomerase. Carol Greider, a student in Blackburn's lab, further investigated Tetrahymena extracts able to incorporate nucleotides into a synthetic oligonucleotide that contained four copies of the Tetrahymena telomere repeat sequences. Greider purified an enzyme able to lengthen

cleotides in humans.

VIII Preface

strophic consequences.

that organism's own telomere repeat?

Telomerase binds to the G-tail of the telomere through the RNA template, and it then cata‐ lyzes the extension of the G-tail. The enzyme is able to repeat this cyclic procedure many times by moving to new binding sites along the newly transcribed G-tail. In this way, in the next cycle of DNA replication, both the DNA polymerase and the DNA repair enzymes, among other enzymes, fill in the other strand. Thus, telomerase is able not only to maintain but also extend the length of telomeres.

Since the discovery of telomerase, it has become evident that this "key molecule" plays a crucial role in the regulation of the length of telomeres. Universally, telomeres are likely to shorten in cells without telomerase activity over time, and cells may stop dividing and be‐ come senescent after their telomeres shorten below a critical length. Some of the first abnor‐ malities appear in reproductive and hematopoietic tissues. Indeed, in most animals, embryonic cells and germ cells possess telomerase activity, but many types of somatic cells do not. The exceptions include highly proliferative cells, such as those in the skin, hemato‐ poietic tissues, and the intestinal epithelium.

Our view of the telomere has matured considerably since Blackburn and Gall provided the first information about its molecular composition. Furthermore, two new landmarks can be included in the thrilling and revolutionary history of telomeres/telomerase. The 2009 Nobel Prize in Physiology or Medicine was awarded jointly to Elizabeth H. Blackburn, Carol W. Greider, and Jack W. Szostak for the discovery of how chromosomes are protected by telo‐ meres and the enzyme telomerase and how the enzyme impacts telomere length. A 2010 study by Mariela Jaskelioff and collaborators from Harvard Medical School showed telo‐ mere shortening to be a root cause of cellular aging.

This book, Telomere - A Complex End of a Chromosome , is organized into nine chapters containing the latest aspects of the current knowledge about the structure of telomeres and the crucial role that telomerase plays not only in maintaining chromosomal stability but also in relation to cell immortality, cell instability, and cancer biology. This book begins with two general chapters related to the structural and functional aspects of telomeres. The first chap‐ ter focuses on the structural and functional aspects of telomeric proteins and their impor‐ tance in human diseases. The second chapter clearly reviews the mechanisms involved in post-transplant complications, including acute cellular rejection, chronic rejection, and chronic hepatitis of unknown cause by ageing and senescence due to telomere shortening. Then, this book includes seven chapters describing different aspects related to telomeres, telomere length, telomerase, and their relation to human health. The first reviews the cur‐ rent knowledge about the function of human telomeres and telomerase and their relevance in genomic instability in cancer, focusing on specific results for ovarian cancer. The second focuses on the telomerase enzyme, which maintains telomere length, as the major factor re‐ sponsible for evading cell death and highlights how telomere length maintenance and telo‐ merase expression put together are prerequisite for immortality, an essential character for cancer cells, and that understanding the mechanism of telomere and telomerase functions

may pave the way for eradicating diseases like cancer. The third provides a summary of the current knowledge about telomere instability induced by anticancer drugs on mammalian cells. The fourth provides a broad evaluation of the associated mechanisms between human health and occupational exposure to different xenobiotics and telomere length, including re‐ cent findings and future perspectives. The fifth discusses the pathophysiological role of shortened telomere length in metabolic and endocrine diseases and the significance of cellu‐ lar senescence. The sixth provides a review of the process by which, in a pathological con‐ text, extratelomeric effects of telomerase are related to the emergence and persistence of the cancer stem cell phenotype. Furthermore, given the common conception of cancer stemness as a major contributor to therapy resistance and tumour relapse, a more complete annota‐ tion of biological mechanisms for its regulation by telomerase will provide the opportunity to develop telomerase-targeted anticancer therapies that kill or differentiate cancer stem cells effectively. Finally, the last chapter reviews hereditary diseases caused by the presence of shortened telomeres, collectively named telomeropathies or telomere biology disorders. In these diseases, cell proliferation is impaired, which results in premature aging and dys‐ function of highly proliferative tissues. Among these diseases are dyskeratosis congenita, Hoyeraal--Hreidarsson syndrome, Revesz syndrome, Coats plus syndromes, aplastic ane‐ mia, idiopathic pulmonary fibrosis, and nonalcholic, noninfectious liver disease, in which mutations present in the genes coding for the components of the telomerase and shelterin complexes and other proteins involved in telomere replication are the cause of these disease.

Many researchers have contributed to the publication of this book. Given the fast pace of new scientific publications shedding light on the matter, this book will probably be outdated very soon. We regard this as a positive and healthy fact. The editors hope that this book will continue to meet the expectations and needs of all interested in the telomere/telomerase sci‐ entific field. We now appreciate that these unusual complexes of DNA and proteins we all know as "telomeres" are dynamic and key structures that depend on telomerase and other cellular factors for continuance. Regulation of telomere activity is a dynamic area of current research, and new insights into telomeres and their role in aging and cancer, among other biological functions and pathologies, appear regularly in the scientific world. However, one fact is more than understandable in this difficult biological conundrum: the end of the telo‐ mere story is far from being totally unraveled.

**Prof. Marcelo L. Larramendy, Ph.D.**

Principal Researcher CONICET School of Natural Sciences and Museum National University of La Plata (UNLP) La Plata, Argentina
