A Little Telomerase Isn’t Enough

Study Links Length Of Chromosome Ends To A Rare Disease Of Stem Cells

Chromosome ends, or telomeres, are repetitive stretches of DNA that protect chromosomes in much the same way as plastic tips on shoelaces prevent the fabric from fraying. Each time a cell divides, its chromosome ends get a little shorter, and eventually the cell can no longer divide because its critical genetic information is exposed. In stem cells, however, a protein called telomerase normally maintains the telomeres’ length, allowing the cells to divide indefinitely.

Now, the Hopkins researchers report that mice engineered to have just half the normal amount of telomerase can’t maintain their stem cells’ chromosome ends, showing that a little telomerase isn’t enough. In these “half-telomerase” mice, their telomeres shortened over time, bringing an early demise to stem cells that replenish the blood supply, immune system and intestine, the researchers report. Moreover, offspring of these mice bred to have normal levels of telomerase still exhibited early loss of stem cells, the researchers report in the Dec. 16 issue of Cell.

“These offspring have what we have called ‘occult’ genetic disease — their genetic make-up is perfectly normal, but they still have the physical problems of their parents,” says Carol Greider, Ph.D., director and professor of molecular biology and genetics in the Johns Hopkins Institute of Basic Biomedical Sciences. “This phenomenon could complicate the hunt for disease genes.”

Scientists generally figure that inherited disease accompanies an inherited mutation in one or more genes. In the case of the genetically normal offspring of two half-telomerase parents, however, the disease is still present. The problem in these animals turns out to be the animal’s inherited telomere length, not the status of the telomerase gene, says Greider.

“If you were to search for the genetic mutation behind this mouse’s disease, you wouldn’t find it — there isn’t one,” says Greider. “These mice develop disease only because their telomeres are short, and having telomerase doesn’t lengthen them right away.”

http://biosingularity.wordpress.com/2005/12/24/a-little-telomerase-isnt-enough-study-links-length-of-chromosome-ends-to-a-rare-disease-of-stem-cells/

Telomeres

Telomeres, together with the enzyme Telomerase are mentioned a lot lately in association with both aging and with cancer. It seems that their gradual shortening during the lifetime of a cell eventually results in damage to DNA and thus the genes of which they are a part. This has led to a great deal of research into Telomeres and Telomerase, some of which will be outlined in this section of the site. First of all however, let us get some of the basic definitions out of the way.

Definition of a Telomere

A telomere is a region of highly repetitive DNA at the end of a linear chromosome that functions as a disposable buffer. Every time linear eukaryotic chromosomes are replicated during late S-phase the DNA polymerase complex is incapable of replicating all the way to the end of the chromosome; if it were not for telomeres, this would quickly result in the loss of useful genetic information, which is needed to sustain a cell’s activities. http://en.wikipedia.org/wiki/Telomere

Definition of Telomerase

Telomerase is the reverse transcriptase responsible for the extension of telomeric repeat sequences in most species studied. If telomerase activity is diminished or absent, telomeres will shorten. Shortened telomeres appear to lead to cell senescence. Eventually telomeric sequences can shorten to the point where they are not long enough to support the telomere-protein complex protecting the ends and the chromosomes become unstable. These shortened ends become 'sticky' and promote chromosome rearrangements. Some rearrangements may contribute to the development of cancers. http://www.genlink.wustl.edu/teldb/tel.html

Role, Function and Compesition of Telomeres

In the majority of Prokaryotes, the chromosomes are circular. This means that they do not have ends that are prone to damage or ‘premature replication termination.’ Note that a small number of bacteria, Borrelia and Streptomyces for example, do possess linear chromosomes (together with circular plasmids). These chromosomes are very different however, from those which are found in Eukaryotic cells (such as those in humans.

A telomere consists of repeating base sequences (in humans this is a repeating string of TTAGGG bases between 3 to 20 kilobases in length). In addition to the telomere itself, there is a 100-300 kilobase sequence associated with the telomere, which is located between the telomere and the rest of the chromosome.

As replication of DNA occurs during the lifetime of an organism, the telomeres gradually shorten. This shortening eventually results in damage to the chromosomes themselves and to the genes that they contain. The results of this are either a reduction in the cells ability to express its genes and a descent into cellular senescence (cellular dormancy) or in some cases the over or under-expression of specific genes. When this chromosomal damage leads to either an over or under-expression of certain genes, then cellular functionality can be compromised. In some cases the organisms survival can also be compromised as cellular replication can become unchecked. These cells thus become cancerous and can lead to the death of the organism.

Role, Function and Compesition of Telomeres

During DNA replication, The DNA unzips and a complimentary strand is formed against the unzipped sections. Telomeres shorten during this process due to the ‘lagging strand’ phenomenon.

Basically DNA replication does not begin at the end of the DNA, but in the centre. DNA Helicase unzips the DNA forming Replication bubbles. An RNA Primer or Primase then attaches to each DNA strand and replication begins in the 3-5 direction, thus forming a new strand in the 5 -3 direction.

DNA polymerases move and replicate the DNA in the 3 to the 5 direction (thus making a replica strand in the 5 to 3 direction). Note that 3 refers to the 3 OH group of the sugar and the 5 refers to the 5 phosphate group of the nucleotide.

The leading strand of DNA is the strand that is oriented in the 3-5 direction. The primer of complementary molecules that binds to the first few of the exposed bases ends with a 3 sugar group. The phosphate of a new nucleotide can be attached here by DNA polymerase. DNA polymerase then continues along the strand, synthesising a new strand as it goes. An animation of this process can be seen below, provided by the Nobel e-museum.

http://www.nobel.se/medicine/educational/dna/a/replication/replication_ani.html.

The lagging strand however, faces more problems when it comes to replication. Because DNA polymerase can only attach to the 3 sugar group and thus move in the 3 to the 5 direction, it needs therefore to move in small jumps (in the opposite direction to overall replication) to replicate the lagging strand. See below animation. This is again provided by the Nobel e-museum.

http://www.nobel.se/medicine/educational/dna/a/replication/lagging_ani.html

Note that the small segments of DNA and primers which are produced on the lagging strand are called Okazaki fragments. Another DNA polymerase enzyme is then recruited to remove the primers and to replace them with DNA. Finally DNA Ligase seals the gaps between the Okazaki fragments.

DNA Replication

The problem with Telomere shortening occurs because, in order to change the RNA primers into DNA, there must be another DNA segment in front of the primer. There is a DNA segment ahead of the primer at every section of the strand, except where the last primer attaches (the end of the telomere). This means that this final primer cannot be replaced with DNA. It does get removed by various enzymes however, but in the process, the telomere shortens.

In human blood cells, the range of lengths of the telomeres varies between 8000 base pairs at birth, to 1,500 base pairs in the elderly. During cellular division, an average of 30 to 200 base pairs are removed from the ends of the telomeres.

In normal cases, the cells of a human can divide between 50 to 70 times, with the telomeres shortening with each division, until the cells either commit suicide through a process known as Apoptosis, become Senescent (dormant) or transform to cancerous cells due to genetic damage.

The process of Cellular senescence due to Telomere Shortening

As a cells telomeres shorten during multiple cellular divisions, DNA damage occurs and the cell, recognising this damage shuts itself down. Below is a simplified diagram of how this occurs.

Telomere shortening and aging

DNA damage results in an activation of the p53 gene. p53 then activates p21, which blocks the actions of a number of CDK’s (Cycline Dependent Kinases). Note that CDK’s are involved in the regulation of the cell cycle, transcription and mRNA processing. The blocking of certain CDK’s prevents the phosphorylation of pRb. This lack of hyperphosphorylated pRb results in a failure of expression of several critical genes, which are involved in cellular division. Cellular division then stops.

Telomerase and Telomere extension

A study was conducted (as so many aging studies are) using the nematode Caenorhabditis elegans (Joeng KS, Song EJ, Lee KJ, Lee J (2004). "Long lifespan in worms with long telomeric DNA". Nature Genetics 36 (6): 607-11.). This study indicates that by lengthening the Telomere, longevity can be increased.

Two distinct groups of Nematodes were engineered. The only difference between the two groups, was the length of the Telomere. The group with the longer telomere’s lived approximately 20 percent longer than the group with the shorter telomeres. Also, it was observed that the Nematode’s with the longer telomeres possessed a greater resistance to the effects of heat exposure.

Telomerase is the natural enzyme which promotes telomere repair. It is however not active in most cells. It certainly is active though in stem cells, germ cells, hair follicles and (worryingly) in 90 percent of cancer cells. Telomerase functions by adding bases to the ends of the telomeres. As a result of this telomerase activity, these cells seem to possess a kind of immortality.

In 1990 a team at Geron Corp in Menlo Park, California led by Serge Lichtsteiner and Andrea Bodnar,in association with the University of texas Southwestern Medical center, managed to activate the production of telomerase in cells that do not usually produce this enzyme. The results were that the telomeres started to lengthen. By the time that they released the results of their study, their cells had divided 20 or more times than would normally be expected before senescence would set in. These cells also seemed to retain their normal gene expression and showed no signs of becoming cancerous.

I will now try to explain in more detail the details and results of the teams study.

Basically, telomerase is present in all cell types, however the human gene for the catalytic protein telomerase transcriptase (hTRT) is present only in immortal cells (such as stem cells or cancerous cells). Scientists from the teams observed that lengthening of telomeres in retinal pigment epithelial cells, foreskin fibroblasts and vascular endothelial cells by introducing the hTRT gene into these cells, results in a resumption of telomerase activity. This resumption resulted in the extended longevity of the cultured cells, as discussed previously.

Varying Telomere decline

One interesting study by Peter Lansdorp of the Terry Fox Laboratory, Canada, observed in human fibroblasts that the rate of telomere shortening as cells divide varies between different telomeres. This variation is between 50 to 150 base pairs per cell division. It is worth noting that the telomeres that are shorter initially. For example in humans the 17p telomere, are not necessarily the ones to be destroyed first! It does seem that it is the shortening of specific telomeres that are linked to a cells decline into senescence, apoptosis or its transformation into a cancer. Martens et al, 1998, observed that the shortening of telomeres 1p, 5p and 22p, but not that of 17p, showed a statistical correlation with the descent into cellular senescence.

Despite all of the above outlined observations, it is a statement of fact that the mean telomere length of a species does not always relate to the longevity of that species. For example Katuo et al, 1999, noted that of all studied primates, humans seem to have both the shortest telomeres and the longest lifespan! Bassham et al, 1998, also observed that the long lived frog Xenopus Laevis displayed a great variation in telomere length and that telomere length could even diminish between parent and offspring, with no detectable consequences.

To summarise, it seems that it has not yet been determined whether the instability of a chromosome and its eventual deterioration is a result of general telomere shortening or the shortening of specific telomeres. It has however been found that by artificially lengthening the telomeres of cells, which do not normally have a mechanism to lengthen their own telomeres, the cells longevity and ability to function normally for longer, does increase. More research therefore needs to be conducted in order to determine the exact significance of telomere length in the aging of an organism and the exact consequences of artificially stimulating telomere repair.

http://www.whyweage.com/node/11

Does the fountain of youth spring from our chromosomes?

The search for immortality has long been a quest of the human spirit. Whether it manifests as a belief in some sort of spiritual afterlife or in prolonging our mortal lives, humanity seems to find the ending of consciousness a horrid thought. Naturally, the mechanisms for why people grow old and die would gain a huge amount of attention by both researchers and the non-scientific community. Many scientists believe the key to mortality has already been found, and it is located at the ends of our chromosomes. Research has discovered that regions of repetitive DNA stretches called telomeres found on the ends of our DNA strands are cut shorter every time they are copied. Eventually the telomeres are worn away and genes near the end of the chromosomes are lost which contain protein instructions the body desperately needs to survive. Some believe that it is this slow yet eventual erosion of the ends of our chromosomes that leads to aging. The discoveries in this area will have a huge impact on more than just showing the way to a possible fountain of youth. Cancer research and cloning may also hinge on developments in the field of telomere research.

Telomeres exist as the body’s way of solving a problem with DNA replication. DNA is replicated by the use of an enzyme called DNA polymerase. DNA polymerase functions to copy our chromosomal DNA, using an existing DNA "parental" strand as a template. The polymerase performs this feat by attaching nucleotides to polymerize a new "daughter" DNA strand in complement to the parental strand. Adenine (A) is added to the new strand complementary to thymine (T) while guanine (G) is added complementary to cytosine (C), and visa versa. There are two main problems with the capabilities of the DNA polymerase. The first is that it can’t start from scratch. There must be a segment of the new strand from which the polymerase can begin attaching new nucleotides. The use of primers easily solves this problem. These primers are RNA fragments that bind by random assortment complementary to sites on the parent strand of DNA, and must be in place before the DNA polymerase can begin copying the parent strand.

The second problem caused by DNA polymerase during replication is much more difficult for cells to surpass. DNA polymerase can only work in a 5’ to 3’ direction. The terms 5’ and 3’ refer to the sugar molecule in the sugar/phosphate DNA backbone. The numbers relate to the carbon in that ring of sugar. 5’ is the fifth carbon in that ring. 3’ is the third. In order for the polymerase to attach a new complementary nucleotide, an alcohol (-OH) group must be available on the 3’ carbon of the sugar molecule. That is the site where the polymerase attaches the phosphate group of the next nucleotide. This phosphate group is subsequently attached to the 5’ carbon of the new nucleotide’s sugar group. The polymerase can therefore only work from the direction of the previously attached 5’ carbon to the 3’ carbon, which has the –OH group available for the attachment of the next nucleotide. The problem with this unidirectional movement lies with the primers, for they can’t stay in the new strand because they are RNA, and not DNA. Removal of these RNA primers is really not a problem when they are located in the middle of the new daughter strand. There will be a 5’ carbon available for a DNA polymerase to fill in the gap that remained after primer removal. However, the problem lies at the beginning each chromosome. A primer was necessary to provide a 5’ carbon for the beginning of synthesis, yet once it is removed, an upstream 5’ carbon is not available from which a polymerase can attach nucleotides and fill in the gap. Therefore, because the nucleotides are are not replaced after removal of the first primer at the beginning of every chromosome, every time the chromosome replicates the daughter strand will be shorter than the parental strand. Studies have shown that the length of a chromosome shortens by about 50 nucleotides every time it replicates. The damage isn’t huge compared to the overall length of a chromosome, but it does mean the chromosome is mortal in that it is slowly being eaten away at the ends with every cell division. If any of these 50 nucleotides contains the instruction to begin the transcription of a gene, that gene and the protein it encodes will never be usable by the body again.

Replication shortens the chromosome

The body’s natural cure to this dilemma is the production of expendable nucleotides at the 3’ end of every chromosome. These "cannon fodder" nucleotides are called telomeres. Telomeres are repetitive hexameric (6 base pair) sequences of DNA. In humans this repeated G-rich sequence is AGGGTT. These sequences are 1000-1700 base pairs long at the beginning of a mammalian life. Cells seldom survive past about 50 divisions in vitro, which most researchers ascribe to the deletion of too many genes in the process of replication.

Oddly, these telomeres are not encoded in the initial DNA resulting from egg fertilization. What this means is that the telomeres must be added later in development. In 1985 Elizabeth Blackburn and Carol Greider discovered a new DNA polymerase which can add telomeres to DNA. This polymerase, called telomerase, is a ribonucleoprotein present in the very early stages of development. Telomerase activity stops in later development, as it is only required to put the telomeres in place once. Ribonucleoproteins contain RNA, which telomerase uses as a template to synthesize the hexameric DNA telomeres. Because telomerase is a polymerase that copies an RNA template (its own) into DNA, it is a reverse transcriptase. A reverse transcriptase is so named because it is capable of writing codes of DNA from an RNA template which is the reverse of transcription. Reverse transcriptases have gained a lot of fame because they are used by retroviruses, notably HIV, for viral replication.

Telomerase binds to the 3’ end of a chromosome and lines its own RNA template so that a few of its RNA base pairs are complementary to that of the strand. Another segment of the ribozyme hangs over the edge providing a template for the synthesis of the telomeres (CCUAAC). Telomerase synthesizes the hexomeric sequence and then translocates to a new 3’ recognition site, which is within the hexanucleotide it just produced, and repeats the procedure. A normal DNA polymerase and primer can then complete the complementary strand’s 5’ end with all of the new hexomeric repeats--all except the last bit of course. The exact details of telomerase function are currently under research, but its currently understood mechanism as a DNA polymerase that carries its own template appears quite unique and phenomenal.

Could the "Fountain of Youth," simply be a shot of telomerase? Some research hints that this might be a good start to combat aging. For example, recent studies have shown that mice deficient in the gene for encoding telomerase RNA (mTR) developed liver cirrhosis sooner and regenerated much slower than normal mice. These same mice also showed improved liver function upon receiving gene delivery of telomerase. In the future, it may be possible to induce telomerase to reset aging cells back to their chromosomal state during a person’s young and vibrant 20’s. However, in most cases the addition of telomerase into somatic cells late in development would be a death sentence. Cell death (apoptosis) is often a good thing in the body. If some cells didn’t die, some tissues would never stop growing. Apoptosis is a crucial tool used by the body to maintain proper development. Certain cells must die at certain times or else the entire organism will perish.

Another aging-related subject that telomere research might prove helpful to is cloning research. Cloning researchers have found that unfortunately the telomeres of cloned animals (such as the famed cloned sheep named "Dolly") are much shorter than a counterpart of the same developmental "age". Even though cloning technology has attained successful birth rates as high as 80%, most of these clones die before even reaching adulthood. Shortened telomeres appear to be the most likely cause of these deaths. Research seeks to uncover a means of safely extending the telomeres of the clones. Some may hope that the solution to the clone problem will eventually bring about a magic youth potion to humanity.

Telomerases might be cancer's Achilles heel

Besides the prevention of age-related health problems, another motivating drive for telomerase research is to develop effective cancer treatments. Scientists are attempting to destroy the telomeres by eradicating telomerase activity in cancer cells. The purpose is to limit the number of divisions possible in these cells. Normal somatic cells have no telomerase present in them because the expression of the telomerase gene is shut down early in life. Because these cells live a long time in the body, the telomeres created early in life are long enough to serve them for the number of divisions they need to make during the lifetime of the organism. However, cancer cells are defined by unbridled cell division, and therefore it is the telomerase which allows cancer cells to continue their unhindered proliferation and subsequent immortality. One of the mutations that leads to a cell becoming cancerous is one that disrupts the cells ability to shut down telomerase expression. Cancer researchers have become very interested in designing drugs that target and inactivate telomerase, for if telomerase could be inactivated this would lead to cancer cells becoming mortal again and stop them in their tracks.

Although using telomere research for finding a treatment for cancer is a popular concept that everyone supports, the idea of significantly extending life is much more controversial. With the population of Earth bulging proudly over 6 billion souls one has to ponder if human immortality would be a blessing at this point in time. Endless life could be to society what cell immortality is to the body.

http://www3.interscience.wiley.com:8100/legacy/college/boyer/0471661791/cutting_edge/telomeres/telomeres.htm

What are telomeres and telomerase?

To better understand telomeres and telomerase, let's first review some basic principles of biology and genetics. The human body is an organism formed by adding many organ systems together. Those organ systems are made of individual organs. Each organ contains tissues designed for specific functions like absorption and secretion. Tissues are made of cells that have joined together to perform those special functions. Each cell is then made of smaller components called organelles, one of which is called the nucleus. The nucleus contains structures called chromosomes that are actually "packages" of all the genetic information that is passed from parents to their children. The genetic information, or "genes" are really just a series of base pairs called Adenine (A), Guanine (G), Cytosine (C), and Thymine (T). These base pairs make up our cellular alphabet and create the sequences, or instructions needed to form our bodies. In order to grow and age, our bodies must duplicate their cells. This process is called mitosis. Mitosis is a process that allows one "parent" cell to divide into two new "daughter" cells. During mitosis, cells make copies of their genetic material. Half of the genetic material goes to each new daughter cell. To make sure that information is successfully passed from one generation to the next, each chromosome has a special protective cap called a telomere located at the end of it's "arms". Telomeres are controlled by the presence of the enzyme telomerase. Now that we have covered some basics, let's explore telomeres, telomerase, and their importance to you!

A telomere is a repeating DNA sequence (TTAGGG) at the end of the body's chromosomes. The telomere can reach a length of 15,000 base pairs. Telomeres function by preventing chromosomes from losing base pair sequences at their ends. They also stop chromosomes from fusing to each other. However, each time a cell divides, some of the telomere is lost (usually 25-200 base pairs per division). When the telomere becomes too short, the chromosome reaches a "critical length" and can no longer replicate. This means that a cell becomes "old" and dies by a process called apoptosis. Telomere activity is controlled by two mechanisms: erosion and addition. Erosion, as mentioned, occurs each time a cell divides. Addition is determined by the activity of telomerase.

Telomerase, also called telomere terminal transferase, is an enzyme made of protein and RNA subunits that elongates chromosomes by adding TTAGGG sequences to the end of existing chromosomes. Telomerase is found in fetal tissues, adult germ cells, and also tumor cells. Telomerase activity is regulated during development and has a very low, almost undetectable activity in somatic (body) cells. Because these somatic cells do not regularly use telomerase, they age. The result of aging cells is an aging body. If telomerase is activated in a cell, the cell will continue to grow and divide. This "immortal cell" theory is important in two areas of research: aging and cancer.

Cellular aging, or senescence, is the process by which a cell becomes old and dies. It is due to the shortening of chromosomal telomeres to the point that the chromosome reaches a critical length. Cellular aging is analogous to a wind up clock. If the clock stays wound, a cell becomes immortal and constantly produces new cells. If the clock winds down, the cell stops producing new cells and dies. Our cells are constantly aging. Being able to make the body's cells live forever certainly creates some exciting possibilities. Telomerase research could therefore yield important discoveries related to the aging process.

Cancer cells are a type of malignant cell. The malignant cells multiply until they form a tumor that grows uncontrollably. Telomerase has been detected in human cancer cells and is found to be 10-20 times more active than in normal body cells. This provides a selective growth advantage to many types of tumors. If telomerase activity was to be turned off, then telomeres in cancer cells would shorten, just like they do in normal body cells. This would prevent the cancer cells from dividing uncontrollably in their early stages of development. In the event that a tumor has already thoroughly developed, it may be removed and anti-telomerase therapy could be administered to prevent relapse. In essence, preventing telomerase from performing its function would change cancer cells from "immortal" to "mortal".

Knowing what we have just learned about telomeres and telomerase, it can be said that scientists are on the verge of discovering many of telomerase's secrets. In the future, their research in the area of telomerase could uncover valuable information to combat aging, fight cancer, and even improve the quality of medical treatment in other areas such as skin grafts for burn victims bone marrow transplants, and heart disease. Who knows how far this could go?

http://www4.utsouthwestern.edu/cellbio/shay-wright/intro/facts/sw_facts.html