The Relationship between Telomeres, Telomerase, Cancer & Aging By, Allison Pilgrim

Outline n DNA Replication n Telomeres n Telomerase n Cancer n Telomerase & Cancer n Telomerase & Aging n Future Trends

DNA Replication n Part of understanding the importance of telomeres and telomerase involves the understanding of DNA replication. n Each strand of DNA can act as a template or a mold for the synthesis of a new complimentary strand. n DNA polymerase is the enzyme used to synthesize the new DNA.

DNA Replication Cont’d n DNA polymerase requires the 3’ hydroxyl group in order to add the next nucleotide therefore, DNA synthesis starts at the 3’ end of the template. n The new chain is synthesized in the 5’ to 3’ direction. n Replication forks are formed because of the direction of synthesis.

Replication Fork

n The template for the lagging strand does not have a 3’ hydroxyl group so RNA primers are made by the enzyme primase to provide it. n Okazaki fragments are made. n The RNA primers are removed by the enzyme nuclease. n DNA polymerase fills the gaps. DNA Replication Cont’d

n The problem with DNA replication is that the replication of linear DNA would result in the loss of genetic material at the ends, without a mechanism to add DNA directly to the ends. DNA Replication Cont’d

Telomeres n Telomeres are the genetic material at the ends of chromosomes and are essential for proper chromosome structure and function. n Telomeres are a repeating sequence of base pairs. In humans, and all vertebrates, the sequence is TTAGGG. n The sequences form structures that protect the DNA from attack by nucleases that degrade the ends of DNA molecules in the cell.

n Without these end caps, chromosomes stick to one another, and undergo structural changes. These activities threaten cell survival and the faithful replication of chromosomes. n Chromosomes slowly lose base pairs at the end of their strands during replication. To overcome the end-replication problem, most eucaryotic cells utilize the enzyme telomerase that allows the replication of the lagging strand to be completed. Telomeres

Telomerase n Telomerase is a ribonucleoprotein enzyme capable of extending chromosome ends consisting mainly of a protein. n It also includes a single molecule of RNA that contains the critical nucleotide template for building telomeric subunits.

How Does Telomerase Work? n Telomerase places one strand of DNA on the RNA, positioning itself so that the template lies adjacent to the tip of the chromosome. n Then, the enzyme adds one DNA nucleotide at a time until a full telomeric subunit is formed. n When the subunit is complete, telomerase can attach another by sliding to the new end of the chromosome and repeating the synthetic process.

Role of Telomerase

Telomerase cont’d n Telomerase is synthesized by nearly all organisms with nucleated cells. The precise makeup differs from species to species, but each version possesses a species-specific RNA template. n Lack of telomerase activity in human cells leads to telomere shortening. Eventually this leads to an end that is no longer recognized as a telomere by the cell. n For there to be continued cell division, telomere loss must stop and telomerase must be activated.

Cancer n There are many different types of cancers varying in appearance and locations in the body. The unifying aspect of cancer is uncontrollable growth and immortality. n The tissues expand without limit, compromising the function of organs and threatening the life of the organism.

Cancer cont’d n Cancers arise when a cell acquires multiple genetic mutations that together cause the cell to escape from normal controls on replication and migration. n As the cell and its offspring multiply uncontrollably, they can invade and damage nearby tissue. Through metastasis, cells may invade nearby blood or lymphatic vessels, establishing new malignancies at distant sites.

Telomerase and Cancer n In theory, a lack of telomerase would retard the growth of tumors by causing continually dividing cells to lose their telomeres and to die before they did any damage. n Therefore, the removal of telomerase could be a possible treatment for cancer. n In regard to this theory, many experiments have been conducted to detect telomerase in cancers versus normal somatic tissue.

n In 1994, groups led by Calvin B. Harley and Jerry W. Shay detected telomerase in 90 of 101 human tumor samples representing twelve tumor types. n In the 50 samples of normal somatic tissue representing four tissue types, that were tested, no telomerase was detected. Telomerase and Cancer Experiment

n Titia de Lange and a group led by Nicholas D. Hastie discovered that the telomeres in human tumors were shorter than telomeres in normal surrounding tissue. n Studies were done to explain why the telomeres in the tumors were so small. n Cells were taken from humans and a viral protein was engineered that caused cells to ignore the alarm signals that usually warn them to stop dividing. Telomerase and Cancer - Study

n The treated cells continued to divide long after they would normally enter senescence. n In most of the cells, the telomeres shortened drastically and the cells eventually died. However, some cells persisted and became immortal. n In these immortal cells, the telomeres were maintained at a significantly short length. Telomerase and Cancer - Study

n The outcome of this experiment implies that the reason why telomeres are so short in tumor cells is because the telomerase is activated after the cell has began replicating uncontrollably. n The telomerase stabilizes the incredibly short lengths of the telomeres. n These results coincide with Harley’s theory of the role of telomerase being permissive rather than having an initiative role for cancer. Telomerase and Cancer

Telomeric Repeat Amplification Protocol (TRAP) n There has also been a more recent development of a TRAP (Telomeric Repeat Amplification Protocol) assay that is able to detect the presence and level of telomerase activity in small tissue samples. n This test has allowed the evaluation of telomerase activity in a wide range of cancers. Because of such a large variety of cancers containing telomerase activity, telomerase may be the most widely expressed and specific cancer marker known.

Telomerase and Aging n Leonard Hayflick first described cellular aging in detail in He explained that most normal human cells are mortal because they can divide only a finite number of times. n In culture, cells divide from 20 to 100 times before they stop. The limit of cell reproduction is often referred to as the “Hayflick Limit.” n Since Hayflick’s discoveries many theories have been formed to explain the mechanism behind cell aging.

n The connection between telomeres and aging first emerged in 1986 from observations made by Howard Cooke. n Cooke noticed that the length of telomeres in reproductive cells were longer than telomeres in somatic cells. Since Cooke understood that telomeres shortened each time that a cell divided, he concluded that somatic cells must not make telomerase. Telomerase and Aging

n Cooke’s discovery coincides with one of the theories used to describe aging, the telomere hypothesis. n This theory proposes that as mortal cells divide, they eventually lose parts of their telomeres until they ultimately die. Only cells that can continue to reactivate their telomerase will continue to live. Cells that can not reactivate their telomerase will stop dividing, or senesce. Telomerase and Aging

n In the following model of the telomere hypothesis the terminal restrictive fragment (TRF) is plotted against the age of replication. n An estimate of telomere length is measured by digesting cellular DNA with restriction enzymes having 4-base recognition sites, so that most of the DNA is reduced to short fragments. Because telomere repeats lack restriction sites, they remain as relatively long terminal restrictive fragments. Telomere Hypothesis

hTRT n In 1997, Thomas Cech and colleagues at Geron Corporation isolated the human gene for a catalytic protein called telomerase reverse transcriptase (hTRT). n This gene is only found in immortal cells. The function of the hTRT is to add the repeating sequences to the chromosomes, lengthening the telomeres.

hTRT - Experiment n The group introduced the hTRT into mortal cells. n The outcome of the experiment was the production of active telomerase and the extended life of the originally mortal cells. n These findings show that by reactivating telomerase activity in cells, cellular aging can be bypassed.

Telomerase and Aging - More Experiments n Experiments have been performed on somatic cells from newborns and seventy-year olds. n The cells derived form the newborns were found to divide 80 to 90 times in culture while the cells derived from the seventy-year olds are likely to divide only 20 to 30 times. n When human cells that are normally capable of dividing, stop reproducing they look different and function less efficiently, and eventually they die.

Future Trends n Telomerase has many diagnostic, therapeutic and prognostic potential applications for the future. n One of the potential applications of telomerase is to postpone cellular aging by delaying critical telomere loss therapeutically, through reactivation of telomerase. n If the telomerase is reactivated in cells that have gone into senescence, the cells could continue to divide and bypass cellular aging.

n Telomerase could also be used to slow the rate of telomere loss and as a result extend cell life span. n This could reduce the impact of senescence on the human body as well as on diseases that occur as a result of cell senescence. Future Trends - Aging

n The Geron Company received a patent relating to the RNA component of human telomerase in They believe that by combining the RNA component with the hTRT gene product they could immortalize cells without turning them cancerous. n If this technique works, not only would the possibility to rejuvenate human cells to treat age-related diseases be more feasible, but also other technologies that are currently limited by the mortality of normal human cells. Future Trends - Geron

n There are also applications of telomerase for the treatment of cancer. Inhibition of telomerase could provide a safe and effective way to eliminate cancers, by making immortal cells, mortal. n Since telomerase is present in most cancer types and is not biologically active in most normal cells, telomerase could be the first universal marker for cancer. Future Trends - Cancer

n This unique property of telomerase, makes it possibile for the enzyme to be a target for anticancer drugs. This could allow the drugs to only effect the tumor cells and to ignore the normal cells. n The Geron Company is currently pursuing the identification of telomerase inhibitors as potential lead compounds for preclinical development. Future Trends - Geron

n They are focused on developing a small molecule inhibitor because it has the ability of being ingested orally, and having a low manufacturing cost. n Since the major function of telomerase in cells is the maintenance of telomere length, there could be a relationship between telomere size and telomerase expression. Future Trends - Geron

n This relationship could mean that in the future, telomeres could be used as an alternative therapeutic agent. n The ability to measure the length of telomeres could be an important way of monitoring inhibitor therapy because it could determine the effects of the therapy and how much longer the therapy is needed. Future Trends

n Further more, if the amount of telomerase activity is found to have a correlation to prognosis, the TRAP assay developed for measuring the activity of telomerase, could be used as a prognostic indicator of outcome. Future Trends

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