Embryonic stem cell

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Embryonic_stem_cell 

 

Human embryonic stem cells in cell culture

 

Pluripotent: Embryonic stem cells are able to develop into any type of cell, excepting those of the placenta. Only embryonic stem cells of the morula are totipotent: able to develop into any type of cell, including those of the placenta.

Embryonic stem cells (ES cells or ESCs) are pluripotent stem cells derived from the inner cell mass of a blastocyst, an early-stage pre-implantation embryo. Human embryos reach the blastocyst stage 4–5 days post fertilization, at which time they consist of 50–150 cells. Isolating the embryoblast, or inner cell mass (ICM) results in destruction of the blastocyst, a process which raises ethical issues, including whether or not embryos at the pre-implantation stage have the same moral considerations as embryos in the post-implantation stage of development.

Researchers are currently focusing heavily on the therapeutic potential of embryonic stem cells, with clinical use being the goal for many laboratories. Potential uses include the treatment of diabetes and heart disease. The cells are being studied to be used as clinical therapies, models of genetic disorders, and cellular/DNA repair. However, adverse effects in the research and clinical processes such as tumours and unwanted immune responses have also been reported.

Properties

The transcriptome of embryonic stem cells

Embryonic stem cells (ESCs), derived from the blastocyst stage of early mammalian embryos, are distinguished by their ability to differentiate into any embryonic cell type and by their ability to self-renew. It is these traits that makes them valuable in the scientific and medical fields. ESCs have a normal karyotype, maintain high telomerase activity, and exhibit remarkable long-term proliferative potential.

Pluripotent

Embryonic stem cells of the inner cell mass are pluripotent, meaning they are able to differentiate to generate primitive ectoderm, which ultimately differentiates during gastrulation into all derivatives of the three primary germ layers: ectoderm, endoderm, and mesoderm. These germ layers generate each of the more than 220 cell types in the adult human body. When provided with the appropriate signals, ESCs initially form precursor cells that in subsequently differentiate into the desired cell types. Pluripotency distinguishes embryonic stem cells from adult stem cells, which are multipotent and can only produce a limited number of cell types.

Self renewal and repair of structure

Under defined conditions, embryonic stem cells are capable of self-renewing indefinitely in an undifferentiated state. Self-renewal conditions must prevent the cells from clumping and maintain an environment that supports an unspecialized state. Typically this is done in the lab with media containing serum and leukemia inhibitory factor or serum-free media supplements with two inhibitory drugs ("2i"), the MEK inhibitor PD03259010 and GSK-3 inhibitor CHIR99021.

Growth

ESCs divide very frequently due to a shortened G1 phase in their cell cycle. Rapid cell division allows the cells to quickly grow in number, but not size, which is important for early embryo development. In ESCs, cyclin A and cyclin E proteins involved in the G1/S transition are always expressed at high levels. Cyclin-dependent kinases such as CDK2 that promote cell cycle progression are overactive, in part due to downregulation of their inhibitors. Retinoblastoma proteins that inhibit the transcription factor E2F until the cell is ready to enter S phase are hyperphosphorylated and inactivated in ESCs, leading to continual expression of proliferation genes. These changes result in accelerated cycles of cell division. Although high expression levels of pro-proliferative proteins and a shortened G1 phase have been linked to maintenance of pluripotency, ESCs grown in serum-free 2i conditions do express hypo-phosphorylated active Retinoblastoma proteins and have an elongated G1 phase. Despite this difference in the cell cycle when compared to ESCs grown in media containing serum these cells have similar pluripotent characteristics. Pluripotency factors Oct4 and Nanog play a role in transcriptionally regulating the ESC cell cycle.

Uses

Due to their plasticity and potentially unlimited capacity for self-renewal, embryonic stem cell therapies have been proposed for regenerative medicine and tissue replacement after injury or disease. Pluripotent stem cells have shown promise in treating a number of varying conditions, including but not limited to: spinal cord injuries, age related macular degeneration, diabetes, neurodegenerative disorders (such as Parkinson's disease), AIDS, etc. In addition to their potential in regenerative medicine, embryonic stem cells provide a possible alternative source of tissue/organs which serves as a possible solution to the donor shortage dilemma. There are some ethical controversies surrounding this though (see Ethical debate section below). Aside from these uses, ESCs can also be used for research on early human development, certain genetic disease, and in vitro toxicology testing.

Utilizations

According to a 2002 article in PNAS, "Human embryonic stem cells have the potential to differentiate into various cell types, and, thus, may be useful as a source of cells for transplantation or tissue engineering."

Tissue Engineering

In tissue engineering, the use of stem cells have been recently discovered and are known to be of importance. In order to successfully engineer a tissue, the cells used must be able to perform specific biological function such as secretion of cytokines, signaling molecules, interacting with neighboring cells, and producing an extracellular matrix in the correct organization. Stem cells demonstrates these specific biological functions along with being able to self-renew and differentiate into one or more types of specialized cells. Embryonic stem cells is one of the current sources that are being considered for the use of tissue engineering. The use of human embryonic stem cells have opened many new possibilities for tissue engineering, however, there are many hurdles that must be made before human embryonic stem cell can even be utilized. It is theorized that if embryonic stem cells can be altered to not evoke the immune response when implanted into the patient then this would be a revolutionary step in tissue engineering.

Embryoid bodies 24 hours after formation.

However, embryonic stem cells are not limited to cell/tissue engineering.

Cell replacement therapies

Current research focuses on differentiating ESCs into a variety of cell types for eventual use as cell replacement therapies (CRTs). Some of the cell types that have or are currently being developed include cardiomyocytes (CM), neurons, hepatocytes, bone marrow cells, islet cells and endothelial cells. However, the derivation of such cell types from ESCs is not without obstacles, therefore current research is focused on overcoming these barriers. For example, studies are underway to differentiate ESCs into tissue specific CMs and to eradicate their immature properties that distinguish them from adult CMs.

Clinical potential

• Researchers have differentiated ESCs into dopamine-producing cells with the hope that these neurons could be used in the treatment of Parkinson's disease. ESCs have been differentiated to natural killer (NK) cells and bone tissue.
• Studies involving ESCs are underway to provide an alternative treatment for diabetes. For example, D’Amour et al. were able to differentiate ESCs into insulin producing cells and researchers at Harvard University were able to produce large quantities of pancreatic beta cells from ES.
• An article published in the European Heart Journal describes a translational process of generating human embryonic stem cell-derived cardiac progenitor cells to be used in clinical trials of patients with severe heart failure.

Drug discovery

Besides becoming an important alternative to organ transplants, ESCs are also being used in field of toxicology and as cellular screens to uncover new chemical entities (NCEs) that can be developed as small molecule drugs. Studies have shown that cardiomyocytes derived from ESCs are validated in vitro models to test drug responses and predict toxicity profiles. ES derived cardiomyocytes have been shown to respond to pharmacological stimuli and hence can be used to assess cardiotoxicity like Torsades de Pointes.

ESC-derived hepatocytes are also useful models that could be used in the preclinical stages of drug discovery. However, the development of hepatocytes from ESCs has proven to be challenging and this hinders the ability to test drug metabolism. Therefore, current research is focusing on establishing fully functional ESC-derived hepatocytes with stable phase I and II enzyme activity.

Models of genetic disorder

Several new studies have started to address the concept of modeling genetic disorders with embryonic stem cells. Either by genetically manipulating the cells, or more recently, by deriving diseased cell lines identified by prenatal genetic diagnosis (PGD), modeling genetic disorders is something that has been accomplished with stem cells. This approach may very well prove valuable at studying disorders such as Fragile-X syndrome, Cystic fibrosis, and other genetic maladies that have no reliable model system.

Yury Verlinsky, a Russian-American medical researcher who specialized in embryo and cellular genetics (genetic cytology), developed prenatal diagnosis testing methods to determine genetic and chromosomal disorders a month and a half earlier than standard amniocentesis. The techniques are now used by many pregnant women and prospective parents, especially couples who have a history of genetic abnormalities or where the woman is over the age of 35 (when the risk of genetically related disorders is higher). In addition, by allowing parents to select an embryo without genetic disorders, they have the potential of saving the lives of siblings that already had similar disorders and diseases using cells from the disease free offspring.

Repair of DNA damage

Differentiated somatic cells and ES cells use different strategies for dealing with DNA damage. For instance, human foreskin fibroblasts, one type of somatic cell, use non-homologous end joining (NHEJ), an error prone DNA repair process, as the primary pathway for repairing double-strand breaks (DSBs) during all cell cycle stages. Because of its error-prone nature, NHEJ tends to produce mutations in a cell's clonal descendants.

ES cells use a different strategy to deal with DSBs. Because ES cells give rise to all of the cell types of an organism including the cells of the germ line, mutations arising in ES cells due to faulty DNA repair are a more serious problem than in differentiated somatic cells. Consequently, robust mechanisms are needed in ES cells to repair DNA damages accurately, and if repair fails, to remove those cells with un-repaired DNA damages. Thus, mouse ES cells predominantly use high fidelity homologous recombinational repair (HRR) to repair DSBs. This type of repair depends on the interaction of the two sister chromosomes formed during S phase and present together during the G2 phase of the cell cycle. HRR can accurately repair DSBs in one sister chromosome by using intact information from the other sister chromosome. Cells in the G1 phase of the cell cycle (i.e. after metaphase/cell division but prior the next round of replication) have only one copy of each chromosome (i.e. sister chromosomes aren't present). Mouse ES cells lack a G1 checkpoint and do not undergo cell cycle arrest upon acquiring DNA damage. Rather they undergo programmed cell death (apoptosis) in response to DNA damage. Apoptosis can be used as a fail-safe strategy to remove cells with un-repaired DNA damages in order to avoid mutation and progression to cancer. Consistent with this strategy, mouse ES stem cells have a mutation frequency about 100-fold lower than that of isogenic mouse somatic cells.

Clinical trial

Main article: Human embryonic stem cells clinical trials

On January 23, 2009, Phase I clinical trials for transplantation of oligodendrocytes (a cell type of the brain and spinal cord) derived from human ES cells into spinal cord-injured individuals received approval from the U.S. Food and Drug Administration (FDA), marking it the world's first human ES cell human trial. The study leading to this scientific advancement was conducted by Hans Keirstead and colleagues at the University of California, Irvine and supported by Geron Corporation of Menlo Park, CA, founded by Michael D. West, PhD. A previous experiment had shown an improvement in locomotor recovery in spinal cord-injured rats after a 7-day delayed transplantation of human ES cells that had been pushed into an oligodendrocytic lineage. The phase I clinical study was designed to enroll about eight to ten paraplegics who have had their injuries no longer than two weeks before the trial begins, since the cells must be injected before scar tissue is able to form. The researchers emphasized that the injections were not expected to fully cure the patients and restore all mobility. Based on the results of the rodent trials, researchers speculated that restoration of myelin sheathes and an increase in mobility might occur. This first trial was primarily designed to test the safety of these procedures and if everything went well, it was hoped that it would lead to future studies that involve people with more severe disabilities. The trial was put on hold in August 2009 due to FDA concerns regarding a small number of microscopic cysts found in several treated rat models but the hold was lifted on July 30, 2010.

In October 2010 researchers enrolled and administered ESTs to the first patient at Shepherd Center in Atlanta. The makers of the stem cell therapy, Geron Corporation, estimated that it would take several months for the stem cells to replicate and for the GRNOPC1 therapy to be evaluated for success or failure.

In November 2011 Geron announced it was halting the trial and dropping out of stem cell research for financial reasons, but would continue to monitor existing patients, and was attempting to find a partner that could continue their research. In 2013 BioTime, led by CEO Dr. Michael D. West, acquired all of Geron's stem cell assets, with the stated intention of restarting Geron's embryonic stem cell-based clinical trial for spinal cord injury research.

BioTime company Asterias Biotherapeutics (NYSE MKT: AST) was granted a $14.3 million Strategic Partnership Award by the California Institute for Regenerative Medicine (CIRM) to re-initiate the world's first embryonic stem cell-based human clinical trial, for spinal cord injury. Supported by California public funds, CIRM is the largest funder of stem cell-related research and development in the world.

The award provides funding for Asterias to reinitiate clinical development of AST-OPC1 in subjects with spinal cord injury and to expand clinical testing of escalating doses in the target population intended for future pivotal trials.

AST-OPC1 is a population of cells derived from human embryonic stem cells (hESCs) that contains oligodendrocyte progenitor cells (OPCs). OPCs and their mature derivatives called oligodendrocytes provide critical functional support for nerve cells in the spinal cord and brain. Asterias recently presented the results from phase 1 clinical trial testing of a low dose of AST-OPC1 in patients with neurologically-complete thoracic spinal cord injury. The results showed that AST-OPC1 was successfully delivered to the injured spinal cord site. Patients followed 2–3 years after AST-OPC1 administration showed no evidence of serious adverse events associated with the cells in detailed follow-up assessments including frequent neurological exams and MRIs. Immune monitoring of subjects through one year post-transplantation showed no evidence of antibody-based or cellular immune responses to AST-OPC1. In four of the five subjects, serial MRI scans performed throughout the 2–3 year follow-up period indicate that reduced spinal cord cavitation may have occurred and that AST-OPC1 may have had some positive effects in reducing spinal cord tissue deterioration. There was no unexpected neurological degeneration or improvement in the five subjects in the trial as evaluated by the International Standards for Neurological Classification of Spinal Cord Injury (ISNCSCI) exam.

The Strategic Partnership III grant from CIRM will provide funding to Asterias to support the next clinical trial of AST-OPC1 in subjects with spinal cord injury, and for Asterias’ product development efforts to refine and scale manufacturing methods to support later-stage trials and eventually commercialization. CIRM funding will be conditional on FDA approval for the trial, completion of a definitive agreement between Asterias and CIRM, and Asterias’ continued progress toward the achievement of certain pre-defined project milestones.

Concern and controversy

Adverse effects

The major concern with the possible transplantation of ESC into patients as therapies is their ability to form tumors including teratoma. Safety issues prompted the FDA to place a hold on the first ESC clinical trial, however no tumors were observed.

The main strategy to enhance the safety of ESC for potential clinical use is to differentiate the ESC into specific cell types (e.g. neurons, muscle, liver cells) that have reduced or eliminated ability to cause tumors. Following differentiation, the cells are subjected to sorting by flow cytometry for further purification. ESC are predicted to be inherently safer than IPS cells created with genetically-integrating viral vectors because they are not genetically modified with genes such as c-Myc that are linked to cancer. Nonetheless, ESC express very high levels of the iPS inducing genes and these genes including Myc are essential for ESC self-renewal and pluripotency, and potential strategies to improve safety by eliminating c-Myc expression are unlikely to preserve the cells' "stemness". However, N-myc and L-myc have been identified to induce iPS cells instead of c-myc with similar efficiency. More recent protocols to induce pluripotency bypass these problems completely by using non-integrating RNA viral vectors such as sendai virus or mRNA transfection.

Ethical debate

Main article: Stem cell controversy

Due to the nature of embryonic stem cell research, there are a lot of controversial opinions on the topic. Since harvesting embryonic stem cells necessitates destroying the embryo from which those cells are obtained, the moral status of the embryo comes into question. Some people claim that the 5-day old mass of cells is too young to achieve personhood or that the embryo, if donated from an IVF clinic (which is where labs typically acquire embryos from), would otherwise go to medical waste anyway. Opponents of ESC research claim that an embryo is a human life, therefore destroying it is murder and the embryo must be protected under the same ethical view as a more developed human being.

History

• 1964: Lewis Kleinsmith and G. Barry Pierce Jr. isolated a single type of cell from a teratocarcinoma, a tumor now known from a germ cell. These cells were isolated from the teratocarcinoma replicated and grew in cell culture as a stem cell and are now known as embryonal carcinoma (EC) cells. Although similarities in morphology and differentiating potential (pluripotency) led to the use of EC cells as the in vitro model for early mouse development, EC cells harbor genetic mutations and often abnormal karyotypes that accumulated during the development of the teratocarcinoma. These genetic aberrations further emphasized the need to be able to culture pluripotent cells directly from the inner cell mass.

Martin Evans revealed a new technique for culturing the mouse embryos in the uterus to allow for the derivation of ES cells from these embryos.

• 1981: Embryonic stem cells (ES cells) were independently first derived from a mouse embryos by two groups. Martin Evans and Matthew Kaufman from the Department of Genetics, University of Cambridge published first in July, revealing a new technique for culturing the mouse embryos in the uterus to allow for an increase in cell number, allowing for the derivation of ES cell from these embryos. Gail R. Martin, from the Department of Anatomy, University of California, San Francisco, published her paper in December and coined the term “Embryonic Stem Cell”. She showed that embryos could be cultured in vitro and that ES cells could be derived from these embryos.
• 1989: Mario R. Cappechi, Martin J. Evans, and Oliver Smithies publish their research that details their isolation and genetic modifications of embryonic stem cells, creating the first "knockout mice". In creating knockout mice, this publication provided scientists with an entirely new way to study disease.
• 1998: A team from the University of Wisconsin, Madison (James A. Thomson, Joseph Itskovitz-Eldor, Sander S. Shapiro, Michelle A. Waknitz, Jennifer J. Swiergiel, Vivienne S. Marshall, and Jeffrey M. Jones) publish a paper titled "Embryonic Stem Cell Lines Derived From Human Blastocysts". The researchers behind this study not only created the first embryonic stem cells, but recognized their pluripotency, as well as their capacity for self-renewal. The abstract of the paper notes the significance of the discovery with regards to the fields of developmental biology and drug discovery.
• 2001: President George W. Bush allows federal funding to support research on roughly 60—at this time, already existing—lines of embryonic stem cells. Seeing as the limited lines that Bush allowed research on had already been established, this law supported embryonic stem cell research without raising any ethical questions that could arise with the creation of new lines under federal budget.
• 2006: Japanese scientists Shinya Yamanaka and Kazutoshi Takashi publish a paper describing the induction of pluripotent stem cells from cultures of adult mouse fibroblasts. Induced pluripotent stem cells (iPSCs) are a huge discovery, as they are seemingly identical to embryonic stem cells and could be used without sparking the same moral controversy.
• January, 2009: The US Food and Drug Administration (FDA) provides approval for Geron Corporation's phase I trial of their human embryonic stem cell-derived treatment for spinal cord injuries. The announcement was met with excitement from the scientific community, but also with wariness from stem cell opposers. The treatment cells were, however, derived from the cell lines approved under George W. Bush's ESC policy.
• March, 2009: Executive Order 13505 is signed by President Barack Obama, removing the restrictions put in place on federal funding for human stem cells by the previous presidential administration. This would allow the National Institutes of Health (NIH) to provide funding for hESC research. The document also states that the NIH must provide revised federal funding guidelines within 120 days of the order's signing.

Techniques and conditions for derivation and culture

Derivation from humans

In vitro fertilization generates multiple embryos. The surplus of embryos is not clinically used or is unsuitable for implantation into the patient, and therefore may be donated by the donor with consent. Human embryonic stem cells can be derived from these donated embryos or additionally they can also be extracted from cloned embryos using a cell from a patient and a donated egg. The inner cell mass (cells of interest), from the blastocyst stage of the embryo, is separated from the trophectoderm, the cells that would differentiate into extra-embryonic tissue. Immunosurgery, the process in which antibodies are bound to the trophectoderm and removed by another solution, and mechanical dissection are performed to achieve separation. The resulting inner cell mass cells are plated onto cells that will supply support. The inner cell mass cells attach and expand further to form a human embryonic cell line, which are undifferentiated. These cells are fed daily and are enzymatically or mechanically separated every four to seven days. For differentiation to occur, the human embryonic stem cell line is removed from the supporting cells to form embryoid bodies, is co-cultured with a serum containing necessary signals, or is grafted in a three-dimensional scaffold to result.

Derivation from other animals

Embryonic stem cells are derived from the inner cell mass of the early embryo, which are harvested from the donor mother animal. Martin Evans and Matthew Kaufman reported a technique that delays embryo implantation, allowing the inner cell mass to increase. This process includes removing the donor mother's ovaries and dosing her with progesterone, changing the hormone environment, which causes the embryos to remain free in the uterus. After 4–6 days of this intrauterine culture, the embryos are harvested and grown in in vitro culture until the inner cell mass forms “egg cylinder-like structures,” which are dissociated into single cells, and plated on fibroblasts treated with mitomycin-c (to prevent fibroblast mitosis). Clonal cell lines are created by growing up a single cell. Evans and Kaufman showed that the cells grown out from these cultures could form teratomas and embryoid bodies, and differentiate in vitro, all of which indicating that the cells are pluripotent.

Gail Martin derived and cultured her ES cells differently. She removed the embryos from the donor mother at approximately 76 hours after copulation and cultured them overnight in a medium containing serum. The following day, she removed the inner cell mass from the late blastocyst using microsurgery. The extracted inner cell mass was cultured on fibroblasts treated with mitomycin-c in a medium containing serum and conditioned by ES cells. After approximately one week, colonies of cells grew out. These cells grew in culture and demonstrated pluripotent characteristics, as demonstrated by the ability to form teratomas, differentiate in vitro, and form embryoid bodies. Martin referred to these cells as ES cells.

It is now known that the feeder cells provide leukemia inhibitory factor (LIF) and serum provides bone morphogenetic proteins (BMPs) that are necessary to prevent ES cells from differentiating. These factors are extremely important for the efficiency of deriving ES cells. Furthermore, it has been demonstrated that different mouse strains have different efficiencies for isolating ES cells. Current uses for mouse ES cells include the generation of transgenic mice, including knockout mice. For human treatment, there is a need for patient specific pluripotent cells. Generation of human ES cells is more difficult and faces ethical issues. So, in addition to human ES cell research, many groups are focused on the generation of induced pluripotent stem cells (iPS cells).

Potential methods for new cell line derivation

On August 23, 2006, the online edition of Nature scientific journal published a letter by Dr. Robert Lanza (medical director of Advanced Cell Technology in Worcester, MA) stating that his team had found a way to extract embryonic stem cells without destroying the actual embryo. This technical achievement would potentially enable scientists to work with new lines of embryonic stem cells derived using public funding in the US, where federal funding was at the time limited to research using embryonic stem cell lines derived prior to August 2001. In March, 2009, the limitation was lifted.

Human embryonic stem cells have also been derived by somatic cell nuclear transfer (SCNT). This approach has also sometimes been referred to as "therapeutic cloning" because SCNT bears similarity to other kinds of cloning in that nuclei are transferred from a somatic cell into an enucleated zygote. However, in this case SCNT was used to produce embryonic stem cell lines in a lab, not living organisms via a pregnancy. The "therapeutic" part of the name is included because of the hope that SCNT produced embryonic stem cells could have clinical utility.

Induced pluripotent stem cells

Main article: Induced pluripotent stem cell

The iPSC technology was pioneered by Shinya Yamanaka’s lab in Kyoto, Japan, who showed in 2006 that the introduction of four specific genes encoding transcription factors could convert adult cells into pluripotent stem cells. He was awarded the 2012 Nobel Prize along with Sir John Gurdon "for the discovery that mature cells can be reprogrammed to become pluripotent."

In 2007 it was shown that pluripotent stem cells highly similar to embryonic stem cells can be generated by the delivery of three genes (Oct4, Sox2, and Klf4) to differentiated cells. The delivery of these genes "reprograms" differentiated cells into pluripotent stem cells, allowing for the generation of pluripotent stem cells without the embryo. Because ethical concerns regarding embryonic stem cells typically are about their derivation from terminated embryos, it is believed that reprogramming to these "induced pluripotent stem cells" (iPS cells) may be less controversial. Both human and mouse cells can be reprogrammed by this methodology, generating both human pluripotent stem cells and mouse pluripotent stem cells without an embryo.

This may enable the generation of patient specific ES cell lines that could potentially be used for cell replacement therapies. In addition, this will allow the generation of ES cell lines from patients with a variety of genetic diseases and will provide invaluable models to study those diseases.

However, as a first indication that the induced pluripotent stem cell (iPS) cell technology can in rapid succession lead to new cures, it was used by a research team headed by Rudolf Jaenisch of the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, to cure mice of sickle cell anemia, as reported by Science journal's online edition on December 6, 2007.

On January 16, 2008, a California-based company, Stemagen, announced that they had created the first mature cloned human embryos from single skin cells taken from adults. These embryos can be harvested for patient matching embryonic stem cells.

Contamination by reagents used in cell culture

The online edition of Nature Medicine published a study on January 24, 2005, which stated that the human embryonic stem cells available for federally funded research are contaminated with non-human molecules from the culture medium used to grow the cells. It is a common technique to use mouse cells and other animal cells to maintain the pluripotency of actively dividing stem cells. The problem was discovered when non-human sialic acid in the growth medium was found to compromise the potential uses of the embryonic stem cells in humans, according to scientists at the University of California, San Diego.

However, a study published in the online edition of Lancet Medical Journal on March 8, 2005 detailed information about a new stem cell line that was derived from human embryos under completely cell- and serum-free conditions. After more than 6 months of undifferentiated proliferation, these cells demonstrated the potential to form derivatives of all three embryonic germ layers both in vitro and in teratomas. These properties were also successfully maintained (for more than 30 passages) with the established stem cell lines.

Hayflick limit

From Wikipedia, the free encyclopedia

https://en.wikipedia.org/wiki/Hayflick_limit 

 

Animation of the structure of a section of DNA. The bases lie horizontally between the two spiraling strands. Nitrogen: blue, oxygen: red, carbon: green, hydrogen: white, phosphorus: orange

The Hayflick limit, or Hayflick phenomenon, is the number of times a normal somatic, differentiated human cell population will divide before cell division stops.

The concept of the Hayflick limit was advanced by American anatomist Leonard Hayflick in 1961, at the Wistar Institute in Philadelphia, Pennsylvania, United States. Hayflick demonstrated that a normal human fetal cell population will divide between 40 and 60 times in cell culture before entering a senescence phase. This finding refuted the contention by Alexis Carrel that normal cells are immortal.

Each time a cell undergoes mitosis, the telomeres on the ends of each chromosome shorten slightly. Cell division will cease once telomeres shorten to a critical length. Hayflick interpreted his discovery to be aging at the cellular level. The aging of cell populations appears to correlate with the overall physical aging of an organism.

Macfarlane Burnet coined the name "Hayflick limit" in his book Intrinsic Mutagenesis: A Genetic Approach to Ageing, published in 1974.

History

The belief in cell immortality

Prior to Leonard Hayflick's discovery, it was believed that vertebrate cells had an unlimited potential to replicate. Alexis Carrel, a Nobel prize-winning surgeon, had stated "that all cells explanted in tissue culture are immortal, and that the lack of continuous cell replication was due to ignorance on how best to cultivate the cells". He claimed to have cultivated fibroblasts from the hearts of chickens (which typically live 5 to 10 years) and to have kept the culture growing for 34 years.

However, other scientists have been unable to replicate Carrel's results, and they are suspected to be due to an error in experimental procedure. To provide required nutrients, embryonic stem cells of chickens may have been re-added to the culture daily. This would have easily allowed the cultivation of new, fresh cells in the culture, so there was not an infinite reproduction of the original cells. It has been speculated that Carrel knew about this error, but he never admitted it.

Also, it has been theorized that the cells Carrel used were young enough to contain pluripotent stem cells, which, if supplied with a supporting telomerase-activation nutrient, would have been capable of staving off replicative senescence, or even possibly reversing it. Cultures not containing telomerase-active pluripotent stem cells would have been populated with telomerase-inactive cells, which would have been subject to the 50 ± 10 mitosis event limit until cellular senescence occurs as described in Hayflick's findings.

Experiment and discovery

Hayflick first became suspicious of Carrel's claims while working in a lab at the Wistar Institute. Hayflick noticed that one of his cultures of embryonic human fibroblasts had developed an unusual appearance and that cell division had slowed. Initially, he brushed this aside as an anomaly caused by contamination or technical error. However, he later observed other cell cultures exhibiting similar manifestations. Hayflick checked his research notebook and was surprised to find that the atypical cell cultures had all been cultured to approximately their 40th doubling while younger cultures never exhibited the same problems. Furthermore, conditions were similar between the younger and older cultures he observed — same culture medium, culture containers, and technician. This led him to doubt that the manifestations were due to contamination or technical error.

Hayflick next set out to prove that the cessation of normal cell replicative capacity that he observed was not the result of viral contamination, poor culture conditions or some unknown artifact. Hayflick teamed with Paul Moorhead for the definitive experiment to eliminate these as causative factors. As a skilled cytogeneticist, Moorhead was able to distinguish between male and female cells in culture. The experiment proceeded as follows: Hayflick mixed equal numbers of normal human male fibroblasts that had divided many times (cells at the 40th population doubling) with female fibroblasts that had divided fewer times (cells at the 15th population doubling). Unmixed cell populations were kept as controls. After 20 doublings of the mixed culture, only female cells remained. Cell division ceased in the unmixed control cultures at the anticipated times; When the male control culture stopped dividing, only female cells remained in the mixed culture. This suggested that technical errors or contaminating viruses were unlikely explanations as to why cell division ceased in the older cells, and proved that unless the virus or artifact could distinguish between male and female cells (which it could not) then the cessation of normal cell replication was governed by an internal counting mechanism.

These results disproved Carrel's immortality claims and established the Hayflick limit as a credible biological theory. Unlike Carrel's experiment, Hayflick's have been successfully repeated by other scientists.

Cell phases

Hayflick describes three phases in the life of normal cultured cells. At the start of his experiment he named the primary culture "phase one". Phase two is defined as the period when cells are proliferating; Hayflick called this the time of "luxuriant growth". After months of doubling the cells eventually reach phase three, a phenomenon he named "senescence", where cell replication rate slows before halting altogether.

Telomere length

The typical normal human fetal cell will divide between 50 and 70 times before experiencing senescence. As the cell divides, the telomeres on the ends of chromosomes shorten. The Hayflick limit is the limit on cell replication imposed by the shortening of telomeres with each division. This end stage is known as cellular senescence.

The Hayflick limit has been found to correlate with the length of the telomeric region at the end of chromosomes. During the process of DNA replication of a chromosome, small segments of DNA within each telomere are unable to be copied and are lost. This occurs due to the uneven nature of DNA replication, where leading and lagging strands are not replicated symmetrically. The telomeric region of DNA does not code for any protein; it is simply a repeated code on the end region of linear eukaryotic chromosomes. After many divisions, the telomeres reach a critical length and the cell becomes senescent. It is at this point that a cell has reached its Hayflick limit.

Hayflick was the first to report that only cancer cells are immortal. This could not have been demonstrated until he had demonstrated that normal cells are mortal. Cellular senescence does not occur in most cancer cells due to expression of an enzyme called telomerase. This enzyme extends telomeres, preventing the telomeres of cancer cells from shortening and giving them infinite replicative potential. A proposed treatment for cancer is the usage of telomerase inhibitors that would prevent the restoration of the telomere, allowing the cell to die like other body cells.

Organismal aging

Hayflick suggested that his results in which normal cells have a limited replicative capacity may have significance for understanding human aging at the cellular level.

It has been reported that the limited replicative capability of human fibroblasts observed in cell culture is far greater than the number of replication events experienced by non-stem cells in vivo during a normal postnatal lifespan. In addition, it has been suggested that no inverse correlation exists between the replicative capacity of normal human cell strains and the age of the human donor from which the cells were derived, as previously argued. It is now clear that at least some of these variable results are attributable to the mosaicism of cell replication numbers at different body sites where cells were taken.

Comparisons of different species indicate that cellular replicative capacity may correlate primarily with species body mass, but more likely to species lifespan. Thus, the limited capacity of cells to replicate in culture may be directly relevant to organismal aging.

Cancer and cellular aging

An anomaly to the cellular aging process is oncogenic cells. In oncogenic cells, human telomerase gene hTERT promoter mutation and mutations in genes that are engaged in the alternative lengthening of telomere pathways such as ATRX, DAXX offer maintenance pathways that are used to stretch the telomere length in cells. This gives rise to tumours and may ultimately result in cancer cells. Cancer cells achieve a proliferative immortality by multiplying the silent HERT gene that codes for reverse transcriptase enzyme to elongate the telomere in order to circumvent senescence. In simple words, cancer cells contain large amount of telomerase enzyme to elongate their telomere which prevents their aging and turning them into immortal cells.

See also

• Ageing
• Apoptosis
• Biological immortality
• HeLa cells
• Induced stem cells

DNA damage (naturally occurring)

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https://en.wikipedia.org/wiki/DNA_damage_(naturally_occurring) 

DNA damage is distinctly different from mutation, although both are types of error in DNA. DNA damage is an abnormal chemical structure in DNA, while a mutation is a change in the sequence of base pairs. DNA damages cause changes in the structure of the genetic material and prevents the replication mechanism from functioning and performing properly.

DNA damage and mutation have different biological consequences. While most DNA damages can undergo DNA repair, such repair is not 100% efficient. Un-repaired DNA damages accumulate in non-replicating cells, such as cells in the brains or muscles of adult mammals, and can cause aging. (Also see DNA damage theory of aging.) In replicating cells, such as cells lining the colon, errors occur upon replication past damages in the template strand of DNA or during repair of DNA damages. These errors can give rise to mutations or epigenetic alterations. Both of these types of alteration can be replicated and passed on to subsequent cell generations. These alterations can change gene function or regulation of gene expression and possibly contribute to progression to cancer.

Throughout the cell cycle there are various checkpoints to ensure the cell is in good condition to progress to mitosis. The three main checkpoints are at G1/s, G2/m, and at the spindle assembly checkpoint regulating progression through anaphase. G1 and G2 checkpoints involve scanning for damaged DNA. During S phase the cell is more vulnerable to DNA damage than any other part of the cell cycle. G2 checkpoint checks for damaged DNA and DNA replication completeness. DNA damage is an alteration in the chemical structure of DNA, such as a break in a strand of DNA, a base missing from the backbone of DNA, or a chemically changed base such as 8-OHdG. DNA damage can occur naturally or via environmental factors. The DNA damage response (DDR) is a complex signal transduction pathway which recognizes when DNA is damaged and initiates the cellular response to the damage.

Types

Damage to DNA that occurs naturally can result from metabolic or hydrolytic processes. Metabolism releases compounds that damage DNA including reactive oxygen species, reactive nitrogen species, reactive carbonyl species, lipid peroxidation products and alkylating agents, among others, while hydrolysis cleaves chemical bonds in DNA. Naturally occurring oxidative DNA damages arise at least 10,000 times per cell per day in humans and as much as 100,000 per cell per day in rats as documented below.

Oxidative DNA damage can produce more than 20 types of altered bases as well as single strand breaks.

Other types of endogeneous DNA damages, given below with their frequencies of occurrence, include depurinations, depyrimidinations, double-strand breaks, O6-methylguanines and cytosine deamination.

DNA can be damaged via environmental factors as well. Environmental agents such as UV light, ionizing radiation, and genotoxic chemicals. Replication forks can be stalled due to damaged DNA and double strand breaks are also a form of DNA damage.

Frequencies

The list below shows some frequencies with which new naturally occurring DNA damages arise per day, due to endogenous cellular processes.

• Oxidative damages
  • Humans, per cell per day
    • 10,000
      11,500
      2,800 specific damages 8-oxoGua, 8-oxodG plus 5-HMUra
      2,800 specific damages 8-oxoGua, 8-oxodG plus 5-HMUra
  • Rats, per cell per day
    • 74,000
      86,000
      100,000
  • Mice, per cell per day
    • 34,000 specific damages 8-oxoGua, 8-oxodG plus 5-HMUra
      47,000 specific damages oxo8dG in mouse liver
      28,000 specific damages 8-oxoGua, 8-oxodG, 5-HMUra
• Depurinations
  • Mammalian cells, per cell per day
    • 2,000 to 10,000
      9,000
      12,000
      13,920
• Depyrimidinations
  • Mammalian cells, per cell per day
    • 600
      696
• Single-strand breaks
  • Mammalian cells, per cell per day
    • 55,200
• Double-strand breaks
  • Human cells, per cell cycle
    • 10
      50
• O6-methylguanines
  • Mammalian cells, per cell per day
    • 3,120
• Cytosine deamination
  • Mammalian cells, per cell per day
    • 192

Another important endogenous DNA damage is M1dG, short for (3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-pyrimido[1,2-a]-purin-10(3H)-one). The excretion in urine (likely reflecting rate of occurrence) of M1dG may be as much as 1,000-fold lower than that of 8-oxodG. However, a more important measure may be the steady-state level in DNA, reflecting both rate of occurrence and rate of DNA repair. The steady-state level of M1dG is higher than that of 8-oxodG. This points out that some DNA damages produced at a low rate may be difficult to repair and remain in DNA at a high steady-state level. Both M1dG and 8-oxodG are mutagenic.

Steady-state levels

Steady-state levels of DNA damages represent the balance between formation and repair. More than 100 types of oxidative DNA damage have been characterized, and 8-oxodG constitutes about 5% of the steady state oxidative damages in DNA. Helbock et al. estimated that there were 24,000 steady state oxidative DNA adducts per cell in young rats and 66,000 adducts per cell in old rats. This reflects the accumulation of DNA damage with age. DNA damage accumulation with age is further described in DNA damage theory of aging.

Swenberg et al. measured average amounts of selected steady state endogenous DNA damages in mammalian cells. The seven most common damages they evaluated are shown in Table 1.

Table 1. Steady-state amounts of endogenous DNA damages

Endogenous lesions	Number per cell
Abasic sites	30,000
N7-(2-hydroxethyl)guanine (7HEG)	3,000
8-hydroxyguanine	2,400
7-(2-oxoethyl)guanine	1,500
Formaldehyde adducts	960
Acrolein-deoxyguanine	120
Malondialdehyde-deoxyguanine	60

Evaluating steady-state damages in specific tissues of the rat, Nakamura and Swenberg indicated that the number of abasic sites varied from about 50,000 per cell in liver, kidney and lung to about 200,000 per cell in the brain.

Biomolecular pathways

Proteins promoting endogenous DNA damage were identified in a 2019 paper as the DNA "damage-up" proteins (DDPs). The DDP mechanisms fall into 3 clusters:

• reactive oxygen increase by transmembrane transporters,
• chromosome loss by replisome binding,
• replication stalling by transcription factors.

The DDP human homologs are over-represented in known cancer drivers, and their RNAs in tumors predict heavy mutagenesis and a poor prognosis.

Repair of damaged DNA

In the presence of DNA damage, the cell can either repair the damage or induce cell death if the damage is beyond repair.

Types

Main article: DNA repair

The seven main types of DNA repair and one pathway of damage tolerance, the lesions they address, and the accuracy of the repair (or tolerance) are shown in this table. For a brief description of the steps in repair see DNA repair mechanisms or see each individual pathway.

Aging and cancer

DNA damage in non-replicating cells, if not repaired and accumulated can lead to aging. DNA damage in replicating cells, if not repaired can lead to either apoptosis or to cancer.

The schematic diagram indicates the roles of insufficient DNA repair in aging and cancer, and the role of apoptosis in cancer prevention. An excess of naturally occurring DNA damage, due to inherited deficiencies in particular DNA repair enzymes, can cause premature aging or increased risk for cancer (see DNA repair-deficiency disorder). On the other hand, the ability to trigger apoptosis in the presence of excess un-repaired DNA damage is critical for prevention of cancer.

Apoptosis and cancer prevention

DNA repair proteins are often activated or induced when DNA has sustained damage. However, excessive DNA damage can initiate apoptosis (i.e., programmed cell death) if the level of DNA damage exceeds the repair capacity. Apoptosis can prevent cells with excess DNA damage from undergoing mutagenesis and progression to cancer.

Inflammation is often caused by infection, such as with hepatitis B virus (HBV), hepatitis C virus (HCV) or Helicobacter pylori. Chronic inflammation is also a central characteristic of obesity. Such inflammation causes oxidative DNA damage. This is due to the induction of reactive oxygen species (ROS) by various intracellular inflammatory mediators. HBV and HCV infections, in particular, cause 10,000-fold and 100,000-fold increases in intracellular ROS production, respectively. Inflammation-induced ROS that cause DNA damage can trigger apoptosis, but may also cause cancer if repair and apoptotic processes are insufficiently protective.

Bile acids, stored in the gall bladder, are released into the small intestine in response to fat in the diet. Higher levels of fat cause greater release. Bile acids cause DNA damage, including oxidative DNA damage, double-strand DNA breaks, aneuploidy and chromosome breakage. High-normal levels of the bile acid deoxycholic acid cause apoptosis in human colon cells, but may also lead to colon cancer if repair and apoptotic defenses are insufficient.

Apoptosis serves as a safeguard mechanism against tumorigenesis. It prevents the increased mutagenesis that excess DNA damage could cause, upon replication.

At least 17 DNA repair proteins, distributed among five DNA repair pathways, have a "dual role" in response to DNA damage. With moderate levels of DNA damage, these proteins initiate or contribute to DNA repair. However, when excessive levels of DNA damage are present, they trigger apoptosis.

DNA damage response

The packaging of eukaryotic DNA into chromatin is a barrier to all DNA-based processes that require enzyme action. For most DNA repair processes, the chromatin must be remodeled. In eukaryotes, ATP-dependent chromatin remodeling complexes and histone-modifying enzymes are two factors that act to accomplish this remodeling process after DNA damage occurs. Further DNA repair steps, involving multiple enzymes, usually follow. Some of the first responses to DNA damage, with their timing, are described below. More complete descriptions of the DNA repair pathways are presented in articles describing each pathway. At least 169 enzymes are involved in DNA repair pathways.

Base excision repair

Main article: Base excision repair

Oxidized bases in DNA are produced in cells treated with Hoechst dye followed by micro-irradiation with 405 nm light. Such oxidized bases can be repaired by base excision repair.

When the 405 nm light is focused along a narrow line within the nucleus of a cell, about 2.5 seconds after irradiation, the chromatin remodeling enzyme Alc1 achieves half-maximum recruitment onto the irradiated micro-line. The line of chromatin that was irradiated then relaxes, expanding side-to-side over the next 60 seconds.

Within 6 seconds of the irradiation with 405 nm light, there is half-maximum recruitment of OGG1 to the irradiated line. OGG1 is an enzyme that removes the oxidative DNA damage 8-oxo-dG from DNA. Removal of 8-oxo-dG, during base excision repair, occurs with a half-life of 11 minutes.

Nucleotide excision repair

Main article: Nucleotide excision repair

Ultraviolet (UV) light induces the formation of DNA damages including pyrimidine dimers (such as thymine dimers) and 6,4 photoproducts. These types of "bulky" damages are repaired by nucleotide excision repair.

After irradiation with UV light, DDB2, in a complex with DDB1, the ubiquitin ligase protein CUL4A and the RING finger protein ROC1, associates with sites of damage within chromatin. Half-maximum association occurs in 40 seconds. PARP1 also associates within this period. The PARP1 protein attaches to both DDB1 and DDB2 and then PARylates (creates a poly-ADP ribose chain) on DDB2 that attracts the DNA remodeling protein ALC1. ALC1 relaxes chromatin at sites of UV damage to DNA. In addition, the ubiquitin E3 ligase complex DDB1-CUL4A carries out ubiquitination of the core histones H2A, H3, and H4, as well as the repair protein XPC, which has been attracted to the site of the DNA damage. XPC, upon ubiquitination, is activated and initiates the nucleotide excision repair pathway. Somewhat later, at 30 minutes after UV damage, the INO80 chromatin remodeling complex is recruited to the site of the DNA damage, and this coincides with the binding of further nucleotide excision repair proteins, including ERCC1.

Homologous recombinational repair

Main article: Homology directed repair

Double-strand breaks (DSBs) at specific sites can be induced by transfecting cells with a plasmid encoding I-SceI endonuclease (a homing endonuclease). Multiple DSBs can be induced by irradiating sensitized cells (labeled with 5'-bromo-2'-deoxyuridine and with Hoechst dye) with 780 nm light. These DSBs can be repaired by the accurate homologous recombinational repair or by the less accurate non-homologous end joining repair pathway. Here we describe the early steps in homologous recombinational repair (HRR).

After treating cells to introduce DSBs, the stress-activated protein kinase, c-Jun N-terminal kinase (JNK), phosphorylates SIRT6 on serine 10. This post-translational modification facilitates the mobilization of SIRT6 to DNA damage sites with half-maximum recruitment in well under a second. SIRT6 at the site is required for efficient recruitment of poly (ADP-ribose) polymerase 1 (PARP1) to a DNA break site and for efficient repair of DSBs. PARP1 protein starts to appear at DSBs in less than a second, with half maximum accumulation within 1.6 seconds after the damage occurs. This then allows half maximum recruitment of the DNA repair enzymes MRE11 within 13 seconds and NBS1 within 28 seconds. MRE11 and NBS1 carry out early steps of the HRR pathway.

γH2AX, the phosphorylated form of H2AX is also involved in early steps of DSB repair. The histone variant H2AX constitutes about 10% of the H2A histones in human chromatin. γH2AX (H2AX phosphorylated on serine 139) can be detected as soon as 20 seconds after irradiation of cells (with DNA double-strand break formation), and half maximum accumulation of γH2AX occurs in one minute. The extent of chromatin with phosphorylated γH2AX is about two million base pairs at the site of a DNA double-strand break. γH2AX does not, itself, cause chromatin decondensation, but within 30 seconds of irradiation, RNF8 protein can be detected in association with γH2AX. RNF8 mediates extensive chromatin decondensation, through its subsequent interaction with CHD4, a component of the nucleosome remodeling and deacetylase complex NuRD.

Pause for DNA repair

After rapid chromatin remodeling, cell cycle checkpoints may be activated to allow DNA repair to be completed before the cell cycle progresses. First, two kinases, ATM and ATR, are activated within 5 or 6 minutes after DNA is damaged. This is followed by phosphorylation of the cell cycle checkpoint protein Chk1, initiating its function, about 10 minutes after DNA is damaged.

Role of oxidative damage to guanine in gene regulation

The DNA damage 8-oxo-dG does not occur randomly in the genome. In mouse embryonic fibroblasts, a 2 to 5-fold enrichment of 8-oxo-dG was found in genetic control regions, including promoters, 5'-untranslated regions and 3'-untranslated regions compared to 8-oxo-dG levels found in gene bodies and in intergenic regions. In rat pulmonary artery endothelial cells, when 22,414 protein-coding genes were examined for locations of 8-oxo-dG, the majority of 8-oxo-dGs (when present) were found in promoter regions rather than within gene bodies. Among hundreds of genes whose expression levels were affected by hypoxia, those with newly acquired promoter 8-oxo-dGs were upregulated, and those genes whose promoters lost 8-oxo-dGs were almost all downregulated.

As reviewed by Wang et al, oxidized guanine appears to have multiple regulatory roles in gene expression. In particular, when oxidative stress produces 8-oxo-dG in the promoter of a gene, the oxidative stress may also inactivate OGG1, an enzyme that targets 8-oxo-dG and normally initiates repair of 8-oxo-dG damage. The inactive OGG1, which no longer excises 8-oxo-dG, nevertheless targets and complexes with 8-oxo-dG, and causes a sharp (~70^o) bend in the DNA. This allows the assembly of a transcriptional initiation complex, up-regulating transcription of the associated gene.

When 8-oxo-dG is formed in a guanine rich, potential G-quadruplex-forming sequence (PQS) in the coding strand of a promoter, active OGG1 excises the 8-oxo-dG and generates an apurinic/apyrimidinic site (AP site). The AP site enables melting of the duplex to unmask the PQS, adopting a G-quadruplex fold (G4 structure/motif) that has a regulatory role in transcription activation.

When 8-oxo-dG is complexed with active OGG1 it may then recruit chromatin remodelers to modulate gene expression. Chromodomain helicase DNA-binding protein 4 (CHD4), a component of the (NuRD) complex, is recruited by OGG1 to oxidative DNA damage sites. CHD4 then attracts DNA and histone methylating enzymes that repress transcription of associated genes.

Role of DNA damage in memory formation

Oxidation of guanine

Oxidation of guanine, particularly within CpG sites, may be especially important in learning and memory. Methylation of cytosines occurs at 60–90% of CpG sites depending on the tissue type. In the mammalian brain, ~62% of CpGs are methylated. Methylation of CpG sites tends to stably silence genes. More than 500 of these CpG sites are de-methylated in neuron DNA during memory formation and memory consolidation in the hippocampus and cingulate cortex regions of the brain. As indicated below, the first step in de-methylation of methylated cytosine at a CpG site is oxidation of the guanine to form 8-oxo-dG.

Role of oxidized guanine in DNA de-methylation

Initiation of DNA demethylation at a CpG site. In adult somatic cells DNA methylation typically occurs in the context of CpG dinucleotides (CpG sites), forming 5-methylcytosine-pG, or 5mCpG. Reactive oxygen species (ROS) may attack guanine at the dinucleotide site, forming 8-hydroxy-2'-deoxyguanosine (8-OHdG), and resulting in a 5mCp-8-OHdG dinucleotide site. The base excision repair enzyme OGG1 targets 8-OHdG and binds to the lesion without immediate excision. OGG1, present at a 5mCp-8-OHdG site recruits TET1 and TET1 oxidizes the 5mC adjacent to the 8-OHdG. This initiates demethylation of 5mC.

The figure in this section shows a CpG site where the cytosine is methylated to form 5-methylcytosine (5mC) and the guanine is oxidized to form 8-oxo-2'-deoxyguanosine (in the figure this is shown in the tautomeric form 8-OHdG). When this structure is formed, the base excision repair enzyme OGG1 targets 8-OHdG and binds to the lesion without immediate excision. OGG1, present at a 5mCp-8-OHdG site recruits TET1, and TET1 oxidizes the 5mC adjacent to the 8-OHdG. This initiates de-methylation of 5mC. TET1 is a key enzyme involved in de-methylating 5mCpG. However, TET1 is only able to act on 5mCpG if the guanine was first oxidized to form 8-hydroxy-2'-deoxyguanosine (8-OHdG or its tautomer 8-oxo-dG), resulting in a 5mCp-8-OHdG dinucleotide (see figure in this section). This initiates the de-methylation pathway on the methylated cytosine, finally resulting in an unmethylated cytosine (see DNA oxidation for further steps in forming unmethylated cytosine).

Altered protein expression in neurons, due to changes in methylation of DNA, (likely controlled by 8-oxo-dG-dependent de-methylation of CpG sites in gene promoters within neuron DNA) has been established as central to memory formation.

Role of double-strand breaks in memory formation

Generation of Neuronal Activity-Related DSBs

Double-stranded breaks (DSBs) in regions of DNA related to neuronal activity are produced by a variety of mechanisms within and around the genome. The enzyme topoisomerase II, or TOPIIβ plays a key role in DSB formation by aiding in the demethylation or loosening of histones wrapped around the double helix to promote transcription. Once the chromatin structure is opened, DSBs are more likely to accumulate, however, this is normally repaired by TOPIIβ through its intrinsic religation ability that rejoins the cleaved DNA ends.

Failure of TOPIIβ to religase can have drastic consequences on protein synthesis, where it is estimated that “blocking TOPIIβ activity alters the expression of nearly one-third of all developmentally regulated genes,” such as neural immediate early genes (IEGs) involved in memory consolidation. Rapid expression of egr-1, c-Fos, and Arc IEGs have been observed in response to increased neuronal activity in the hippocampus region of the brain where memory processing takes place. As a preventative measure against TOPIIβ failure, DSB repair molecules are recruited via two different pathways: non-homologous end joining (NHEJ) pathway factors, which perform a similar religation function to that of TOPIIβ, and the homologous recombination (HR) pathway, which uses the non-broken sister strand as a template to repair the damaged strand of DNA.

Stimulation of neuronal activity, as previously mentioned in IEG expression, is another mechanism through which DSBs are generated. Changes in level of activity have been used in studies as a biomarker to trace the overlap between DSBs and increased histone H3K4 methylation in promoter regions of IEGs. Other studies have indicated that transposable elements (TEs) can cause DSBs through endogenous activity that involves using endonuclease enzymes to insert and cleave target DNA at random sites.

DSBs and Memory Reconsolidation

While accumulation of DSBs generally inhibits long term memory consolidation, the process of reconsolidation, in contrast, is DSB-dependent. Memory reconsolidation involves the modification of existing memories stored in long-term memory. Research involving NPAS4, a gene that regulates neuroplasticity in the hippocampus during contextual learning and memory formation, has revealed a link between deletions in the coding region and impairments in recall of fear memories in transgenic rats. Moreover, the enzyme H3K4me3, which catalyzes the demethylation of the H3K4 histone, was upregulated at the promoter region of the NPAS4 gene during the reconsolidation process, while knockdown (gene knockdown) of the same enzyme impeded reconsolidation. A similar effect was observed in TOPIIβ, where knockdown also impaired the fear memory response in rats, indicating that DSBs, along with the enzymes that regulate them, influence memory formation at multiple stages.

DSBs and Neurodegeneration

Buildup of DSBs more broadly leads to the degeneration of neurons, hindering the function of memory and learning processes. Due to their lack of cell division and high metabolic activity, neurons are especially prone to DNA damage. Additionally, an imbalance of DSBs and DNA repair molecules for neuronal-activity genes has been linked to the development of various human neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), and amyotrophic lateral sclerosis (ALS). In patients with Alzheimer’s disease, DSBs accumulate in neurons at early stages and are the driving force behind memory loss, a key characteristic of the disease. Other external factors that result in increased levels of activity-dependent DSBs in people with AD are oxidative damage to neurons, which can result in more DSBs when multiple lesions occur close to one another. Environmental factors such as viruses and a high-fat diet have also been associated with disrupted function of DNA repair molecules.

One targeted therapy for treating patients with AD has involved suppression of the BRCA1 gene in human brains, initially tested in transgenic mice, where DSB levels were observed to have increased and memory loss had occurred, suggesting that BRCA1 could “serve as a therapeutic target for AD and AD-related dementia.”  Similarly, the protein ATM involved in DNA repair and epigenetic modifications to the genome is positively correlated with neuronal loss in AD brains, indicating the protein is another key component in the intrinsically-linked processes of neurodegeneration, DSB production, and memory formation.

Role of ATR and ATM

Most damage can be repaired without triggering the damage response system, however more complex damage activates ATR and ATM, key protein kinases in the damage response system. DNA damage inhibits M-CDKs which are a key component of progression into Mitosis.

In all eukaryotic cells, ATR and ATM are protein kinases that detect DNA damage. They bind to DNA damaged sites and activate Chk1, Chk2, and, in animal cells, p53. Together, these proteins make up the DNA damage response system. Some DNA damage does not require the recruitment of ATR and ATM, it is only difficult and extensive damage that requires ATR and ATM. ATM and ATR are required for NHEJ, HR, ICL repair, and NER, as well as replication fork stability during unperturbed DNA replication and in response to replication blocks.

ATR is recruited for different forms of damage such as nucleotide damage, stalled replication forks and double strand breaks. ATM is specifically for the damage response to double strand breaks. The MRN complex (composed of Mre11, Rad50, and Nbs1) form immediately at the site of double strand break. This MRN complex recruits ATM to the site of damage. ATR and ATM phosphorylate various proteins that contribute to the damage repair system. The binding of ATR and ATM to damage sites on DNA lead to the recruitment of Chk1 and Chk2. These protein kinases send damage signals to the cell cycle control system to delay the progression of the cell cycle.

Chk1 and Chk2 functions

Chk1 leads to the production of DNA repair enzymes. Chk2 leads to reversible cell cycle arrest. Chk2, as well as ATR/ATM, can activate p53, which leads to permanent cell cycle arrest or apoptosis.

p53 role in DNA damage repair system

When there is too much damage, apoptosis is triggered in order to protect the organism from potentially harmful cells.7 p53, also known as a tumor suppressor gene, is a major regulatory protein in the DNA damage response system which binds directly to the promoters of its target genes. p53 acts primarily at the G1 checkpoint (controlling the G1 to S transition), where it blocks cell cycle progression. Activation of p53 can trigger cell death or permanent cell cycle arrest. p53 can also activate certain repair pathways such was NER.

Regulation of p53

In the absence of DNA damage, p53 is regulated by Mdm2 and constantly degraded. When there is DNA damage, Mdm2 is phosphorylated, most likely caused by ATM. The phosphorylation of Mdm2 leads to a reduction in the activity of Mdm2, thus preventing the degradation of p53. Normal, undamaged cell, usually has low levels of p53 while cells under stress and DNA damage, will have high levels of p53.

p53 serves as transcription factor for bax and p21

p53 serves as a transcription factors for both bax, a proapoptotic protein as well as p21, a CDK inhibitor. CDK Inhibitors result in cell cycle arrest. Arresting the cell provides the cell time to repair the damage, and if the damage is irreparable, p53 recruits bax to trigger apoptosis.

DDR and p53 role in cancer

p53 is a major key player in the growth of cancerous cells. Damaged DNA cells with mutated p53 are at a higher risk of becoming cancerous. Common chemotherapy treatments are genotoxic. These treatments are ineffective in cancer tumor that have mutated p53 since they do not have a functioning p53 to either arrest or kill the damaged cell.

A major problem for life

One indication that DNA damages are a major problem for life is that DNA repair processes, to cope with DNA damages, have been found in all cellular organisms in which DNA repair has been investigated. For example, in bacteria, a regulatory network aimed at repairing DNA damages (called the SOS response in Escherichia coli) has been found in many bacterial species. E. coli RecA, a key enzyme in the SOS response pathway, is the defining member of a ubiquitous class of DNA strand-exchange proteins that are essential for homologous recombination, a pathway that maintains genomic integrity by repairing broken DNA. Genes homologous to RecA and to other central genes in the SOS response pathway are found in almost all the bacterial genomes sequenced to date, covering a large number of phyla, suggesting both an ancient origin and a widespread occurrence of recombinational repair of DNA damage. Eukaryotic recombinases that are homologues of RecA are also widespread in eukaryotic organisms. For example, in fission yeast and humans, RecA homologues promote duplex-duplex DNA-strand exchange needed for repair of many types of DNA lesions.

Another indication that DNA damages are a major problem for life is that cells make large investments in DNA repair processes. As pointed out by Hoeijmakers, repairing just one double-strand break could require more than 10,000 ATP molecules, as used in signaling the presence of the damage, the generation of repair foci, and the formation (in humans) of the RAD51 nucleofilament (an intermediate in homologous recombinational repair). (RAD51 is a homologue of bacterial RecA.) If the structural modification occurs during the G1 phase of DNA replication, the G1-S checkpoint arrests or postpones the furtherance of the cell cycle before the product enters the S phase.

Consequences

Differentiated somatic cells of adult mammals generally replicate infrequently or not at all. Such cells, including, for example, brain neurons and muscle myocytes, have little or no cell turnover. Non-replicating cells do not generally generate mutations due to DNA damage-induced errors of replication. These non-replicating cells do not commonly give rise to cancer, but they do accumulate DNA damages with time that likely contribute to aging (see DNA damage theory of aging). In a non-replicating cell, a single-strand break or other type of damage in the transcribed strand of DNA can block RNA polymerase II-catalysed transcription. This would interfere with the synthesis of the protein coded for by the gene in which the blockage occurred.

Brasnjevic et al. summarized the evidence showing that single-strand breaks accumulate with age in the brain (though accumulation differed in different regions of the brain) and that single-strand breaks are the most frequent steady-state DNA damages in the brain. As discussed above, these accumulated single-strand breaks would be expected to block transcription of genes. Consistent with this, as reviewed by Hetman et al., 182 genes were identified and shown to have reduced transcription in the brains of individuals older than 72 years, compared to transcription in the brains of those less than 43 years old. When 40 particular proteins were evaluated in a muscle of rats, the majority of the proteins showed significant decreases during aging from 18 months (mature rat) to 30 months (aged rat) of age.

Another type of DNA damage, the double-strand break, was shown to cause cell death (loss of cells) through apoptosis. This type of DNA damage would not accumulate with age, since once a cell was lost through apoptosis, its double-strand damage would be lost with it. Thus, damaged DNA segments undermine the DNA replication machinery because these altered sequences of DNA cannot be utilized as true templates to produce copies of one's genetic material.

RAD genes and the cell cycle response to DNA damage in Saccharomyces cerevisiae

When DNA is damaged, the cell responds in various ways to fix the damage and minimize the effects on the cell. One such response, specifically in eukaryotic cells, is to delay cell division—the cell becomes arrested for some time in the G2 phase before progressing through the rest of the cell cycle. Various studies have been conducted to elucidate the purpose of this G2 arrest that is induced by DNA damage. Researchers have found that cells that are prematurely forced out of the delay have lower cell viability and higher rates of damaged chromosomes compared with cells that are able to undergo a full G2 arrest, suggesting that the purpose of the delay is to give the cell time to repair damaged chromosomes before continuing with the cell cycle. This ensures the proper functioning of mitosis.

Various species of animals exhibit similar mechanisms of cellular delay in response to DNA damage, which can be caused by exposure to x-irradiation. The budding yeast Saccharomyces cerevisiae has specifically been studied because progression through the cell cycle can be followed via nuclear morphology with ease. By studying Saccharomyces cerevisiae, researchers have been able to learn more about radiation-sensitive (RAD) genes, and the effect that RAD mutations may have on the typical cellular DNA damaged-induced delay response. Specifically, the RAD9 gene plays a crucial role in detecting DNA damage and arresting the cell in G2 until the damage is repaired.

Through extensive experiments, researchers have been able to illuminate the role that the RAD genes play in delaying cell division in response to DNA damage. When wild-type, growing cells are exposed to various levels of x-irradiation over a given time frame, and then analyzed with a microcolony assay, differences in the cell cycle response can be observed based on which genes are mutated in the cells. For instance, while unirradiated cells will progress normally through the cell cycle, cells that are exposed to x-irradiation either permanently arrest (become inviable) or delay in the G2 phase before continuing to divide in mitosis, further corroborating the idea that the G2 delay is crucial for DNA repair. However, rad strains, which are deficient in DNA repair, exhibit a markedly different response. For instance, rad52 cells, which cannot repair double-stranded DNA breaks, tend to permanently arrest in G2 when exposed to even very low levels of x-irradiation, and rarely end up progressing through the later stages of the cell cycle. This is because the cells cannot repair DNA damage and thus do not enter mitosis. Various other rad mutants exhibit similar responses when exposed to x-irradiation.

However, the rad9 strain exhibits an entirely different effect. These cells fail to delay in the G2 phase when exposed to x-irradiation, and end up progressing through the cell cycle unperturbed, before dying. This suggests that the RAD9 gene, unlike the other RAD genes, plays a crucial role in initiating G2 arrest. To further investigate these findings, the cell cycles of double mutant strains have been analyzed. A mutant rad52 rad9 strain—which is both defective in DNA repair and G2 arrest—fails to undergo cell cycle arrest when exposed to x-irradiation. This suggests that even if DNA damage cannot be repaired, if RAD9 is not present, the cell cycle will not delay. Thus, unrepaired DNA damage is the signal that tells RAD9 to halt division and arrest the cell cycle in G2. Furthermore, there is a dose-dependent response; as the levels of x-irradiation—and subsequent DNA damage—increase, more cells, regardless of the mutations they have, become arrested in G2.

Another, and perhaps more helpful way to visualize this effect is to look at photomicroscopy slides. Initially, slides of RAD+ and rad9 haploid cells in the exponential phase of growth show simple, single cells, that are indistinguishable from each other. However, the slides look much different after being exposed to x-irradiation for 10 hours. The RAD+ slides now show RAD+ cells existing primarily as two-budded microcolonies, suggesting that cell division has been arrested. In contrast, the rad9 slides show the rad9 cells existing primarily as 3 to 8 budded colonies, and they appear smaller than the RAD+ cells. This is further evidence that the mutant RAD cells continued to divide and are deficient in G2 arrest.

However, there is evidence that although the RAD9 gene is necessary to induce G2 arrest in response to DNA damage, giving the cell time to repair the damage, it does not actually play a direct role in repairing DNA. When rad9 cells are artificially arrested in G2 with MBC, a microtubule poison that prevents cellular division, and then treated with x-irradiation, the cells are able to repair their DNA and eventually progress through the cell cycle, dividing into viable cells. Thus, the RAD9 gene plays no role in actually repairing damaged DNA—it simply senses damaged DNA and responds by delaying cell division. The delay, then, is mediated by a control mechanism, rather than the physical damaged DNA.

On the other hand, it is possible that there are backup mechanisms that fill the role of RAD9 when it is not present. In fact, some studies have found that RAD9 does indeed play a critical role in DNA repair. In one study, rad9 mutant and normal cells in the exponential phase of growth were exposed to UV-irradiation and synchronized in specific phases of the cell cycle. After being incubated to permit DNA repair, the extent of pyrimidine dimerization (which is indicative of DNA damage) was assessed using sensitive primer extension techniques. It was found that the removal of DNA photolesions was much less efficient in rad9 mutant cells than normal cells, providing evidence that RAD9 is involved in DNA repair. Thus, the role of RAD9 in repairing DNA damage remains unclear.

Regardless, it is clear that RAD9 is necessary to sense DNA damage and halt cell division. RAD9 has been suggested to possess 3’ to 5’ exonuclease activity, which is perhaps why it plays a role in detecting DNA damage. When DNA is damaged, it is hypothesized that RAD9 forms a complex with RAD1 and HUS1, and this complex is recruited to sites of DNA damage. It is in this way that RAD9 is able to exert its effects.

Although the function of RAD9 has primarily been studied in the budding yeast Saccharomyces cerevisiae, many of the cell cycle control mechanisms are similar between species. Thus, we can conclude that RAD9 likely plays a critical role in the DNA damage response in humans as well.