Written By Vivek Athalye
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written by VIVEK ATHALYE
BIO + MED
Discovering
Cellular
The concept of immortality has intrigued mankind since the very beginning. From the first major literary work, the Epic of Gilgamesh, to the Bible and other religious texts to modern fantasies such as Harry Potter, we find the theme of immortality rooted in our culture and our imagination. However, immortality is not merely the subject of fiction. It exists on a cellular level—when man is formed from the division of embryonic stem cells, and when cancer develops from malignant cells’ uncontrollable growth. One important player in cellular immortality is telomerase.
“I call it the problem of telomerase dark matter. The telomerase complex has a mass from 1 to 2 megadaltons, but the portion of it that we’ve identified weighs only 300 kilodaltons. We’re trying to discover the missing matter.” - Steven Artandi
stanford scientific
Telomerase is a massive enzyme complex that prevents a cell from aging by preserving the integrity of its DNA during cellular division. Since its discovery in 1984, scientists and pharmaceutical companies have been intensely interested in the enzyme because of its implications in both cellular and systemic aging, stem cell proliferation, and cancer. Yet, despite the enzyme’s profound implications for science and medicine, much of telomerase’s mechanisms and composition remain a mystery. Dr. Steven Artandi, Assistant Professor of Medicine at Stanford University and telomerase and cancer researcher, calls it “the problem of telomerase dark matter.” He explains, “The telomerase complex has a mass from 1 to 2 megadaltons, but the portion of it that we’ve identified weighs only 300 kilodaltons. We’re trying to discover the missing matter.” Now scientists in the Stanford University School of Medicine are two steps closer. As published in the March 21, 2008 issue of Cell, Dr. Artandi, graduate student Andrew Venteicher and Dr. Timothy Veenstra, along with NCI Frederick’s Dr. Zhaojing Meng and University of Washington’s Dr. Philip Mason, recently discovered two new proteins that function in the telomerase complex.
Stanford scientists unveil
The two proteins are provocative as they suggest a new level of complexity in telomerase while providing a new direction in which to search for other proteins. Furthermore, their discovery presents new approaches in cancer treatment that could potentially inhibit cancer cells’ fatal immortality.
In Pursuit of Understanding Immortality
According to emotion research, each of us can regulate our emoTo begin to understand cellular immortality, we must first consider cellular division and the end replication problem. Imagine for a moment that you are a DNA polymerase enzyme, a protein with the important job of copying the cell’s genetic material so that when it divides, each new cell can receive an exact copy of the entire genome. You travel along the chromosome, making just one error in every 10,000 nucleotides copied, but as you approach the end, you realize that it is out of reach. Unable to copy these last few bases, you leave the replica chromosome shorter than the original. What is happening, and what will be the consequences for the new cell? Because DNA polymerase can only extend a growing chain, with each cellular division the chromosomes become shorter and shorter as the last bits of information are snipped off. If the chromosome were to end flush with its final gene, this gene would eventually be lost after successive replications, leading to cellular abnormalities, cellular senescence—a state of cellular aging marked by the cell’s inability to divide—or apoptosis, programmed cell death. Evolution has solved this engineering problem with the telomere, a region of nucleotide repeats that do not code for
Immortality two proteins functioning in telomerase required proteins, but serve as buffers that cap the ends of the chromosomes. Now with each division the telomere shortens instead of the essential genes. Not only does this prolong the cell’s lifetime, but it also suggests the specific length of the telomere could function as a genetic clock, regulating the number of times the cell can divide before important genetic information is lost and the cell senesces or dies. Enter telomerase, the biological mechanism of immortality. Telomerase works by maintaining the length of the chromosome, synthesizing the telomeres at the ends where DNA polymerase could not otherwise reach. Telomerase’s mechanism is fascinating—the only reverse transcriptase in human cells, it reads off an RNA template to produce the DNA repeats of the telomere. The expression of telomerase allows a cell to divide indefinitely, hence the idea of cellular immortality. While telomerase is undetectable in normal adult cells, it is naturally expressed in stem cells, progenitor cells, and cells such as immune cells which must divide prolifically. Cancer cells also use telomerase to divide endlessly without check, making cancer the deadly force we know it to be today.
Path of Discovery: Connecting Pontin and Reptin to Telomerase
Scientists have been able to describe how telomerase functions in maintaining telomeres based on their knowledge of its catalytic core, which has three primary components. The first is TERT, the protein catalytic subunit that synthesizes the telomere. The second is TERC, the RNA subunit that serves as the template for telomere synthesis. The third is dyskerin, a protein that assists in binding TERC to TERT. While these are the main subunits, they only add up to 15-30% of telomerase’s total
composition, leaving a biochemical mystery for scientists to explore. The first obstacle the Stanford group had to overcome was the scant amounts of telomerase available in an organism: normal adult cells do not express telomerase. The Stanford group solved the problem by producing vats of HeLa cells (human cervical cancer cells) that express and harness telomerase in order to exist as an indefinitely dividing malignancy. Even with this vast quantity of cells, the amount of available protein is very small. However, “our research is backed by years of technological advancement,” says Artandi. “With technology such as Dual Affinity Purification and the advances of the Human Genome Project, we are actually able to analyze the small quantities of protein.” Dual Affinity Purification, which was adapted from its normal use in yeast cells, infuses the protein ends with tags containing cleavage sites. By using a specific protease, an enzyme utilized to dissolve specific bonds on a protein’s cleavage sites, the Stanford group was able to cut the proteins into sections only at the locations of the tags. Dr. Artandi said, “These are gentle and very specific steps that allowed us to get a huge fold of enrichment.” Having purified and cut the protein, the group was able to analyze telomerase’s composition using mass spectrometry, a technique that measures the charge-tomass ratio of particles in order to generate a spectrum of the telomerase components. This information can be checked against a database developed by the Human Genome Project to identify the proteins included in the complex. According to the scientists’ publication in Cell, analysis of the band in which “the most prominent TERT-associated proteins”
exist allowed the Stanford group to identify pontin and reptin as proteins within the telomerase complex. The Stanford group then went on to study what physical associations the proteins might have with each subunit of telomerase’s catalytic core. First, the group studied the relation of pontin and reptin to TERT, the telomeresynthesizer. Using purified pontin and reptin the group was able to repeatedly immunoprecipitate a polypeptide with the same mass as TERT, demonstrating a strong interaction between the three proteins. After verifying this association, the group sought to establish a connection between the pontin, reptin, TERC and dyskerin. By removing pontin and reptin from the telomerase complex, subsequent observation of TERC and dyskerin activity clearly showed that pontin and reptin are critical for accumulation of TERC and dyskerin. Finally, by depleting pontin and reptin with shRNA, a short sequence that can be used to silence gene expression through RNA interference, the Stanford group observed telomerase activity fall to a mere 10-20% of its natural activity. These were exciting results demonstrating pontin and reptin’s crucial roles in each aspect of telomerase functioning.
Biogenesis: Pontin, Reptin, and the Telomerase Creation Story
There is more to the story than the strong association of pontin and reptin with telomerase. Indeed, the discovery of pontin and reptin as essential proteins was particularly provocative because they are ATPases, enzymes that catalyze
volume VII
BIO + MED
The Process of DNA Replication 1. Helicase unwinds the complimentary DNA strands. 2. RNA primase attaches RNA nucleotides to the template. The first DNA nucleotides will be added to this primer.
McMaster University History of Health Care in Hamilton Gallery
3. There are two separate elongation processes for the two DNA strand templates. The 5’ to 3’ template is synthesized continuously. The daughter strand that runs from 3’ to 5’ is called the leading strand. 4. The 3’ to 5’ strand is copied in steps, producing non-continuous pieces called Okazaki fragments. 5. Finally, the RNA primers are removed and replaced by DNA. Because of the gap left by the RNA primer, DNA polymerase cannot attach and seal the last section of the lagging strand. This is where the telomere comes into play and explains the problem of telomereshortening.
stanford scientific
the breakdown of the cellular fuel ATP to provide energy for other processes. The question remains: for what process are they required? This question propelled the Stanford group to look more specifically at the functions of pontin and reptin, and the answer was biogenesis. Pontin and reptin are central to the assembly of the core telomerase complex including TERT, TERC, and dyskerin.
The Cycle of Life: Cell Cycle-Dependent Telomerase Construction
The Stanford group analyzed the patterns of pontin and reptin expression and telomerase production through each phase of the cell cycle. Studies with yeast in 2000 had shown that telomerase lengthens short telomeres in the S Phase of the cell, the phase in which replication of genetic material takes place. In accordance with that study, the Stanford group found that the amount of reptin and pontin bound to TERT peaked during S Phase. This proves pontin and reptin are involved in cell cycle regulation of telomerase, a discovery with significant scientific implications. The study reveals that TERT complexes are dynamic in nature as their function in telomerase depends on the phase of the cell cycle. Furthermore, the results implied that pontin and reptin are required for this cyclical telomerase assembly.
Cancer and Disease: Stopping the Dark Side of Telomerase
Because cancer utilizes telomerase to indefinitely expand its malignancy, scientists and pharmaceutical companies alike have been trying to find a treatment for cancer that blocks telomerase’s activity. However, attempts to identify telomerase inhibitors have failed, “perhaps reflecting difficulty in targeting the enzyme’s reverse transcriptase function,” the Stanford study suggests. But now, having halted telomerase activity in vitro through depletion of pontin and reptin, Dr. Artandi says, “The old approach to treating cancer by inhibiting telomerase’s active site was perhaps phrased incorrectly. Our study suggests a new approach—the blocking of pontin and reptin—for inhibiting telomerase in cancer.” Yet, while the discovery offers promise, there is still a great void in compositional and structural information available regarding telomerase. Targeting telomerase as a cancer treatment at this stage would still involve a fair amount of guesswork. Dr. Artandi says, “I would love to start working on a new cancer treatment, but there is still so much we don’t understand.” The study of telomerase is a vastly important topic as its implications reach even beyond cancer. While one application of understanding telomerase is to steal cancer cells’ immortality, the complex is also implicated with other diseases such as dyskeratosis congenita, aplastic anemia,
Tissue Engineering and Stem Cells: The Power of Telomerase While telomerase may be an aiding factor in cancer development, it is fundamentally essential to our formation and our existence. Embryonic stem cells, the pluripotent cells that divide prolifically and contain the ability to differentiate into any type of cell, express telomerase to form adult humans. Stem cells and progenitor cells are able to regenerate tissue because they both self-renew and differentiate into the appropriate cell types. Scientists have had great interest in taking advantage of these features for treating a myriad of health conditions requiring tissue regeneration such as spinal cord injuries, diabetes, heart disease, Parkinson’s disease, Alzheimer’s disease, and numerous others.
Despite knowing telomerase is expressed in stem cells, its role in these cases is still not understood. Dr. Artandi’s lab, which conducts research on telomerase function in stem cells as well, introduced a new enigma: telomerase action independent of TERC and telomere synthesis. Dr. Artandi’s lab expressed TERT in mouse skin, activating quiescent stem cell populations within the follicles. The results showed that the hair follicles of mice in which TERT was expressed became
active, and the mice displayed vigorous hair growth. Intriguingly, this result was reproducible even without the expression of the RNA subunit TERC. This function of telomerase in stem cells independent of telomere maintenance poses yet another mystery for scientists to understand. Undoubtedly there is still a whole world to explore surrounding the tiny enzyme. Dr. Artandi says, “We are barely scratching the surface.” But given the potential to treat some of the worst diseases and health problems today, it is worth spending a lifetime in pursuit of understanding immortality.
While telomerase may be an aiding factor in cancer development, it is fundamentally essential to our formation and our existence.
VIVEK ATHALYE is a sophomore majoring in Electrical Engineering with an interest in Neuroscience. He takes great pleasure in creative writing, playing tennis, and violin.
To Learn More
For more information, check out Dr. Artandi’s article entitled ‘Telomeres, Telomerase, and Human Disease’ in the September 2006 issue of New England Journal of Medicine, Volume 355: 1195-1197, or his departmental website at http://www.stanford.edu/group/ artandi/.
volume VII
Credit: sxc.com
and pulmonary fibrosis. Thus, the Stanford group’s study suggests “important future studies for these ATPases in human health and disease.”
BIO + MED
Discovering
Cellular
The concept of immortality has intrigued mankind since the very beginning. From the first major literary work, the Epic of Gilgamesh, to the Bible and other religious texts to modern fantasies such as Harry Potter, we find the theme of immortality rooted in our culture and our imagination. However, immortality is not merely the subject of fiction. It exists on a cellular level—when man is formed from the division of embryonic stem cells, and when cancer develops from malignant cells’ uncontrollable growth. One important player in cellular immortality is telomerase.
“I call it the problem of telomerase dark matter. The telomerase complex has a mass from 1 to 2 megadaltons, but the portion of it that we’ve identified weighs only 300 kilodaltons. We’re trying to discover the missing matter.” - Steven Artandi
stanford scientific
Telomerase is a massive enzyme complex that prevents a cell from aging by preserving the integrity of its DNA during cellular division. Since its discovery in 1984, scientists and pharmaceutical companies have been intensely interested in the enzyme because of its implications in both cellular and systemic aging, stem cell proliferation, and cancer. Yet, despite the enzyme’s profound implications for science and medicine, much of telomerase’s mechanisms and composition remain a mystery. Dr. Steven Artandi, Assistant Professor of Medicine at Stanford University and telomerase and cancer researcher, calls it “the problem of telomerase dark matter.” He explains, “The telomerase complex has a mass from 1 to 2 megadaltons, but the portion of it that we’ve identified weighs only 300 kilodaltons. We’re trying to discover the missing matter.” Now scientists in the Stanford University School of Medicine are two steps closer. As published in the March 21, 2008 issue of Cell, Dr. Artandi, graduate student Andrew Venteicher and Dr. Timothy Veenstra, along with NCI Frederick’s Dr. Zhaojing Meng and University of Washington’s Dr. Philip Mason, recently discovered two new proteins that function in the telomerase complex.
Stanford scientists unveil
The two proteins are provocative as they suggest a new level of complexity in telomerase while providing a new direction in which to search for other proteins. Furthermore, their discovery presents new approaches in cancer treatment that could potentially inhibit cancer cells’ fatal immortality.
In Pursuit of Understanding Immortality
According to emotion research, each of us can regulate our emoTo begin to understand cellular immortality, we must first consider cellular division and the end replication problem. Imagine for a moment that you are a DNA polymerase enzyme, a protein with the important job of copying the cell’s genetic material so that when it divides, each new cell can receive an exact copy of the entire genome. You travel along the chromosome, making just one error in every 10,000 nucleotides copied, but as you approach the end, you realize that it is out of reach. Unable to copy these last few bases, you leave the replica chromosome shorter than the original. What is happening, and what will be the consequences for the new cell? Because DNA polymerase can only extend a growing chain, with each cellular division the chromosomes become shorter and shorter as the last bits of information are snipped off. If the chromosome were to end flush with its final gene, this gene would eventually be lost after successive replications, leading to cellular abnormalities, cellular senescence—a state of cellular aging marked by the cell’s inability to divide—or apoptosis, programmed cell death. Evolution has solved this engineering problem with the telomere, a region of nucleotide repeats that do not code for
Immortality two proteins functioning in telomerase required proteins, but serve as buffers that cap the ends of the chromosomes. Now with each division the telomere shortens instead of the essential genes. Not only does this prolong the cell’s lifetime, but it also suggests the specific length of the telomere could function as a genetic clock, regulating the number of times the cell can divide before important genetic information is lost and the cell senesces or dies. Enter telomerase, the biological mechanism of immortality. Telomerase works by maintaining the length of the chromosome, synthesizing the telomeres at the ends where DNA polymerase could not otherwise reach. Telomerase’s mechanism is fascinating—the only reverse transcriptase in human cells, it reads off an RNA template to produce the DNA repeats of the telomere. The expression of telomerase allows a cell to divide indefinitely, hence the idea of cellular immortality. While telomerase is undetectable in normal adult cells, it is naturally expressed in stem cells, progenitor cells, and cells such as immune cells which must divide prolifically. Cancer cells also use telomerase to divide endlessly without check, making cancer the deadly force we know it to be today.
Path of Discovery: Connecting Pontin and Reptin to Telomerase
Scientists have been able to describe how telomerase functions in maintaining telomeres based on their knowledge of its catalytic core, which has three primary components. The first is TERT, the protein catalytic subunit that synthesizes the telomere. The second is TERC, the RNA subunit that serves as the template for telomere synthesis. The third is dyskerin, a protein that assists in binding TERC to TERT. While these are the main subunits, they only add up to 15-30% of telomerase’s total
composition, leaving a biochemical mystery for scientists to explore. The first obstacle the Stanford group had to overcome was the scant amounts of telomerase available in an organism: normal adult cells do not express telomerase. The Stanford group solved the problem by producing vats of HeLa cells (human cervical cancer cells) that express and harness telomerase in order to exist as an indefinitely dividing malignancy. Even with this vast quantity of cells, the amount of available protein is very small. However, “our research is backed by years of technological advancement,” says Artandi. “With technology such as Dual Affinity Purification and the advances of the Human Genome Project, we are actually able to analyze the small quantities of protein.” Dual Affinity Purification, which was adapted from its normal use in yeast cells, infuses the protein ends with tags containing cleavage sites. By using a specific protease, an enzyme utilized to dissolve specific bonds on a protein’s cleavage sites, the Stanford group was able to cut the proteins into sections only at the locations of the tags. Dr. Artandi said, “These are gentle and very specific steps that allowed us to get a huge fold of enrichment.” Having purified and cut the protein, the group was able to analyze telomerase’s composition using mass spectrometry, a technique that measures the charge-tomass ratio of particles in order to generate a spectrum of the telomerase components. This information can be checked against a database developed by the Human Genome Project to identify the proteins included in the complex. According to the scientists’ publication in Cell, analysis of the band in which “the most prominent TERT-associated proteins”
exist allowed the Stanford group to identify pontin and reptin as proteins within the telomerase complex. The Stanford group then went on to study what physical associations the proteins might have with each subunit of telomerase’s catalytic core. First, the group studied the relation of pontin and reptin to TERT, the telomeresynthesizer. Using purified pontin and reptin the group was able to repeatedly immunoprecipitate a polypeptide with the same mass as TERT, demonstrating a strong interaction between the three proteins. After verifying this association, the group sought to establish a connection between the pontin, reptin, TERC and dyskerin. By removing pontin and reptin from the telomerase complex, subsequent observation of TERC and dyskerin activity clearly showed that pontin and reptin are critical for accumulation of TERC and dyskerin. Finally, by depleting pontin and reptin with shRNA, a short sequence that can be used to silence gene expression through RNA interference, the Stanford group observed telomerase activity fall to a mere 10-20% of its natural activity. These were exciting results demonstrating pontin and reptin’s crucial roles in each aspect of telomerase functioning.
Biogenesis: Pontin, Reptin, and the Telomerase Creation Story
There is more to the story than the strong association of pontin and reptin with telomerase. Indeed, the discovery of pontin and reptin as essential proteins was particularly provocative because they are ATPases, enzymes that catalyze
volume VII
BIO + MED
The Process of DNA Replication 1. Helicase unwinds the complimentary DNA strands. 2. RNA primase attaches RNA nucleotides to the template. The first DNA nucleotides will be added to this primer.
McMaster University History of Health Care in Hamilton Gallery
3. There are two separate elongation processes for the two DNA strand templates. The 5’ to 3’ template is synthesized continuously. The daughter strand that runs from 3’ to 5’ is called the leading strand. 4. The 3’ to 5’ strand is copied in steps, producing non-continuous pieces called Okazaki fragments. 5. Finally, the RNA primers are removed and replaced by DNA. Because of the gap left by the RNA primer, DNA polymerase cannot attach and seal the last section of the lagging strand. This is where the telomere comes into play and explains the problem of telomereshortening.
stanford scientific
the breakdown of the cellular fuel ATP to provide energy for other processes. The question remains: for what process are they required? This question propelled the Stanford group to look more specifically at the functions of pontin and reptin, and the answer was biogenesis. Pontin and reptin are central to the assembly of the core telomerase complex including TERT, TERC, and dyskerin.
The Cycle of Life: Cell Cycle-Dependent Telomerase Construction
The Stanford group analyzed the patterns of pontin and reptin expression and telomerase production through each phase of the cell cycle. Studies with yeast in 2000 had shown that telomerase lengthens short telomeres in the S Phase of the cell, the phase in which replication of genetic material takes place. In accordance with that study, the Stanford group found that the amount of reptin and pontin bound to TERT peaked during S Phase. This proves pontin and reptin are involved in cell cycle regulation of telomerase, a discovery with significant scientific implications. The study reveals that TERT complexes are dynamic in nature as their function in telomerase depends on the phase of the cell cycle. Furthermore, the results implied that pontin and reptin are required for this cyclical telomerase assembly.
Cancer and Disease: Stopping the Dark Side of Telomerase
Because cancer utilizes telomerase to indefinitely expand its malignancy, scientists and pharmaceutical companies alike have been trying to find a treatment for cancer that blocks telomerase’s activity. However, attempts to identify telomerase inhibitors have failed, “perhaps reflecting difficulty in targeting the enzyme’s reverse transcriptase function,” the Stanford study suggests. But now, having halted telomerase activity in vitro through depletion of pontin and reptin, Dr. Artandi says, “The old approach to treating cancer by inhibiting telomerase’s active site was perhaps phrased incorrectly. Our study suggests a new approach—the blocking of pontin and reptin—for inhibiting telomerase in cancer.” Yet, while the discovery offers promise, there is still a great void in compositional and structural information available regarding telomerase. Targeting telomerase as a cancer treatment at this stage would still involve a fair amount of guesswork. Dr. Artandi says, “I would love to start working on a new cancer treatment, but there is still so much we don’t understand.” The study of telomerase is a vastly important topic as its implications reach even beyond cancer. While one application of understanding telomerase is to steal cancer cells’ immortality, the complex is also implicated with other diseases such as dyskeratosis congenita, aplastic anemia,
Tissue Engineering and Stem Cells: The Power of Telomerase While telomerase may be an aiding factor in cancer development, it is fundamentally essential to our formation and our existence. Embryonic stem cells, the pluripotent cells that divide prolifically and contain the ability to differentiate into any type of cell, express telomerase to form adult humans. Stem cells and progenitor cells are able to regenerate tissue because they both self-renew and differentiate into the appropriate cell types. Scientists have had great interest in taking advantage of these features for treating a myriad of health conditions requiring tissue regeneration such as spinal cord injuries, diabetes, heart disease, Parkinson’s disease, Alzheimer’s disease, and numerous others.
Despite knowing telomerase is expressed in stem cells, its role in these cases is still not understood. Dr. Artandi’s lab, which conducts research on telomerase function in stem cells as well, introduced a new enigma: telomerase action independent of TERC and telomere synthesis. Dr. Artandi’s lab expressed TERT in mouse skin, activating quiescent stem cell populations within the follicles. The results showed that the hair follicles of mice in which TERT was expressed became
active, and the mice displayed vigorous hair growth. Intriguingly, this result was reproducible even without the expression of the RNA subunit TERC. This function of telomerase in stem cells independent of telomere maintenance poses yet another mystery for scientists to understand. Undoubtedly there is still a whole world to explore surrounding the tiny enzyme. Dr. Artandi says, “We are barely scratching the surface.” But given the potential to treat some of the worst diseases and health problems today, it is worth spending a lifetime in pursuit of understanding immortality.
While telomerase may be an aiding factor in cancer development, it is fundamentally essential to our formation and our existence.
VIVEK ATHALYE is a sophomore majoring in Electrical Engineering with an interest in Neuroscience. He takes great pleasure in creative writing, playing tennis, and violin.
To Learn More
For more information, check out Dr. Artandi’s article entitled ‘Telomeres, Telomerase, and Human Disease’ in the September 2006 issue of New England Journal of Medicine, Volume 355: 1195-1197, or his departmental website at http://www.stanford.edu/group/ artandi/.
volume VII
Credit: sxc.com
and pulmonary fibrosis. Thus, the Stanford group’s study suggests “important future studies for these ATPases in human health and disease.”
