Hutchinson-Gilford Progeria Syndrome (“HGPS” or “progeria”) is a very rare autosomal dominant disorder which results in premature aging and eventually death. Patients are often very underweight at birth and will display conclusive symptoms of premature aging within 18-24 months, such as reduced body fat, hair loss and aged skin, alongside tissue and organ degeneration. Average lifespan for sufferers of HGPS is thirteen years and cause of death is invariably due to heart failure. Due to the nature of these symptoms, HGPS is referred to as an “accelerated aging disorder”. Currently, only 100 cases of HGPS have been formally documented, around 50 of which are children who are alive today; however it is estimated that a further 150 children worldwide currently suffer from HGPS, but have not yet been formally diagnosed. The identification of the HGPS mutation has only recently been identified and located on codon 608 of the LMNA gene, which codes for four types of lamin proteins: lamin A, lamin C, lamin Aâˆ†10 and lamin Câˆ†2. These, along with the B-type lamin are responsible for keeping the structure of the nucleus together by forming a scaffold which lines and interacts with the nuclear membrane. The LMNA mutation leads to the expression of progerin, rather than lamin A, altering the structure of the nuclear membrane and leading to loss of nuclear integrity, DNA damage and a compromised DNA double-strand-break repair mechanism. This causes a variety of problems relating to normal cellular function as well as resulting in increased apoptosis and significantly decreased proliferation rates. Until recently, medical assistance for children with HGPS only involved treating the symptoms which accompany the disease, rather than the effects of the HGPS mutation; however in August 2009, clinical trials began testing the effects of farnesyltransferase inhibitors (FTIs) on HGPS patients. This was following experimental evidence from Toth et al (2005) who showed that human fibroblast cells expressing progerin had reversed nuclear alterations when treated with farnesyltransferase inhibitors in vitro.
Aging is a normal and natural physiological process which all living things must experience, unless death should occur prematurely. Aging impairs verve and mobility, causes hair loss, weakens bones and inevitably will ultimately lead to death. As humans, the price that must be paid for our unique self-awareness is the knowledge that we will eventually age and die, forcing us to come to terms with our own mortality and compelling us to value youth. Patients suffering from HGPS are not given the opportunity to experience their youth in the same way as most. By 18-24 months their bodies are already showing signs of physiological aging and by 5 years old they will show more signs of age-related disease than many adults in their 70s. In young, healthy humans, cells are able to cope with the assault of DNA damage which all livings organisms will unavoidably encounter over time. Over time, the ability to deal with this damage lessens and the physiological process of aging will gradually occur. HGPS cells are not capable of dealing with this DNA damage to the same extent and so aging happens quickly and prematurely. Despite this, intelligence and cognitive function in children with HGPS are often above average compared to other children within their age bracket (Progeria Research Foundation, 2006).
To understand the cellular and molecular mechanisms of HGPS, it is first important to understand the process of normal aging in healthy humans.
Normal Physiological aging
Aging on an evolutionary level may be described as a progressive decline in fitness (the ability to survive and reproduce) due to a decline in tissue functionality with increasing age (Partridge & Gems, 2002). On a cellular level it may be described as a progressive functional decline and increase in cell mortality (Lombard et al., 2005). Although many theories have been proposed, no conclusive theory has been agreed upon to explain how or why we age.
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Aging is thought to be caused by various genetic and environmental factors. At the cellular level this progressive malfunction of tissue is thought to be due to accumulation of damage by various biomolecules which leads to cell loss or damage (Vijg, 2000). Usually the body has the ability to regenerate these damaged or lost cells through pools of stem cells, however the body does not have the ability to regenerate infinitely and over time this causes a decline in tissue functions typical of aging. This is consistent with the disposable soma theory of aging (Kirkwood & Holliday, 1979), which states that cell repair and maintenance (including DNA repair, defence against oxidants, etc) are costly activities. To work to extend life indefinitely would make little sense, as in the wild many organisms have an extremely high mortality rate. Therefore, animals have evolved in such a way that energy invested in maintaining the soma is limited, so that the animal is kept alive long enough for it to reproduce, but not to keep it alive indefinitely. Past this point, the body’s regeneration mechanisms fail and physiological aging becomes apparent. From an evolutionary point of view, this theory makes more sense than others which are based around aging being a genetically inbuilt process, as this would provide no benefit whatsoever to the individual animal or the species as a whole.
Aging may involve damage to a variety of cellular components; however damage to various DNA and RNA molecules is likely to be a major contributing factor. Despite the cell’s inbuilt mechanisms designed to repair damage, damaged DNA which has not been restored perfectly can lead to mutations with detrimental consequences. Balaban et al. (2005) outlines the potential role for damaged mitochondria DNA in the process of aging, however nuclear DNA is a more likely culprit. Mitochrondial DNA has thousands of copies present within the cell which can be replaced if damaged, whereas there are only two copies of nuclear DNA. Several studies have shown that as age increases, so does the risk of mutation (Martin et al., 1996) and there is substantial evidence to suggest a causal link between damage to nuclear DNA and physiological aging. Sedelnikova et al. (2008) showed that the level of double strand breaks (DSBs) that take place increase with age and older cells are less able to repair these DSBs. Finally, there is the observation that symptoms of progeria are caused by defined mutations in DNA maintenance proteins, which leads to impaired DNA repair mechanisms (Musich & Zou, 2009).
HGPS belongs to a group of disorders known as segmental progeroid syndromes, characterised by early manifestation of features usually associated with normal physiological aging. They are consequently referred to as “accelerated aging disorders”.
HGPS was first described over 120 years ago by Hutchinson (1886) and again later by Gilford (1904). Since its original classification in 1886, just over 100 cases of the disease have been documented. It affects around 1 in 4-8 million newborns all over the world with no preference for gender.
Figure 1 – Locations of children around the world who have been diagnosed with HGPG. There are currently 50 living children diagnosed with HGPS, most of whom reside in affluent Western countries. It is estimated that a further 150 children worldwide suffer from progeria but have not yet been identified due to insufficient means to diagnose in less prosperous countries as well as the rarity and complexity of the disease and the fact that the HGPS gene has only recently been identified. (Adapted from the Progeria Research Foundation, 2006)
Children with progeria are often born appearing healthy, however shortly after birth it becomes apparent that weight and height gain are below that of which is expected of healthy children (Merideth et al., 2008). By 18-24 months of age they begin to display signs of premature aging, which can be seen in figure 2 below.
Figure 2: A 3 and a half year old boy with HGPS showing typical progeroid symptoms. These include stunted growth, loss of hair and body fat, aged skin and unusually prominent eyes. Non-visible symptoms include stiff joints, atherosclerosis., osteoporosis and fatigue.
Impaired growth does not seem to be due to any hormone inbalance/resistance or malnutrition. Cardiovascular problems are generally thought to be caused by loss of smooth muscle cells, disruption of the extracellular matrix and other irregularities in vascular structure. Life span ranges from 8 – 21 years with the average being around 13 years old. Death is almost invariably caused by atherosclerosis (Progeria Research Foundation, 2006).
HGPS has recently been acknowledged as part of a family of diseases known as laminopathies, as it is caused by a dominant mutation on the LMNA gene. Before the identification of the gene responsible for progeria, diagnosis was carried out solely on the symptoms that have been mentioned. Thanks to the recent discovery of the mutated gene (Eriksson et al., 2003), diagnosis can be carried out based on the appearance of this mutation. HGSP is caused by a de novo mutation on the LMNA gene, although other progeroid syndromes may be hereditary.
The LMNA gene codes for the nuclear A-type lamin proteins: lamin A, lamin C, lamin Aâˆ†10 and lamin Câˆ†2 (Fisher et al., 1986). These, along with the B-type lamins are responsible for keeping the structure of the nucleus together by forming a scaffold which lines and interacts with the nuclear membrane. The structure of the nuclear lamina within the nuclear envelope is shown in figure 3.
Figure 3: Structure and function of the nuclear lamina – The lamina exists on the inner nuclear membrane (INM), providing structural support and helping with chromatin organisation as well as binding nuclear pore complexes (NPCs), nuclear proteins (purple) and transcription factors (pink). Barrier to auto integration factor (BAF) is a chromatin-associated protein which binds to the lamina and various nuclear envelope proteins (Coutinho et al. 2009)
Fawcett (1966) deduced that the lamins are components of the nuclear lamina, a layer found between the chromatin and the nuclear envelope initially thought to provide structural support for the nuclear membrane and a location for the chromatin to attach. Recently, further roles of nuclear lamins have been proposed, including DNA synthesis, transcription, apoptosis and assembly of the nuclear envelope.
It has been determined that several isoforms of lamins are encoded by three different genes: The LMNA gene encodes lamin A, lamin C, lamin Aâˆ†10 and lamin Câˆ†2 and the LMNB1 gene encodes lamin B1 and LMNB2 encodes B2 and B3 lamins (Stuurman et al., 1998). Whilst every vertebrate cell expresses at least one type of B lamin, lamins encoded by the LMNA gene are only expressed in differentiated tissue. This observations suggest that lamin encoded by LMNA may have specific roles within certain cells. It is possible that the role of these lamins is in fact to induce or maintain differentiation within these cells, although no conclusive evidence has been presented to support this. It is likely, however, that A type lamins have roles related to and dependant on correct chromatin organisation and nuclear structure. This includes reformation of the nuclear envelope post-mitosis, transcription, DNA replication and nuclear positioning (Holaska et al., 2003; Spann., 2002; Moir et al., 2000; Haque et al., 2006). Although studies have shown that LMNA deficient mice develop normally, shortly after birth growth problems occur (Sullivan et al., 1999).
Shortly before cell division in the late prophase stage of the cell cycle, the phosphorylation of lamin subunits takes place, causing the nuclear envelope to break down. A-type lamins are the first to be disassembled, occurring during early prophase, whilst B-type lamins are disassembled during prometaphase (Georgatos et al., 1997). This course of action is thought to be a necessary requirement for the reassembly of the nuclear envelope after cell division have taken place (Burke and Gerace, 1986). However, studies such as those by Newport et al. (1990) have provided evidence against these theories, stating that when these disassembled lamin subunits are imported, they are done so into a formed nuclear envelope with fully functional pores. Perhaps is it possible that a finite number of lamin monomers are used in the reassembly of the nuclear envelope and the rest are transported in at a later stage.
The role of lamins in DNA replication is unclear, however several studies have suggested that Xenopus interphase extracts were depleted of Lamin B3 which resulted in a lack of DNA replication (Newport et al., 1990), however it remained unclear whether this was entirely due to the absence of lamins or if other factors, such as a smaller and more fragile nuclear envelope, contributed. A later study by Moir et al. (2000) provided evidence to suggest that normal nuclear lamin organisation is required for DNA synthesis and that this dependency is completely unrelated to the insufficient formation of the nuclear envelope. It appears that when nuclear lamin organisation is incorrect, the elongation phase of replication fails to take place, probably due to an alteration in distribution of elongation factors, Replication Factor Complex and Proliferating Cell Nuclear Antigen.
The potential role of nuclear lamins in transcription has been put forward by Spann et al. (2002), who disrupted the normal nuclear organisation of nuclear lamins using a dominant negative mutant lamin lacking the NH2- domain. This resulted in the inhibition of RNA polymerase II activity in both mammalian and embryonic Xenopus cells. Notably, RNA polymerases I and III were not affected.
The role of lamins in apoptosis is directly related to the state of the nuclear envelope. Apoptosis is a very precise physiological mechanism for effective destruction of unwanted cells without causing inflammation or distress to other cells, as would occur in necrosis. Lamin degradation is one of the processes that occur during apoptosis, however several studies have suggested nuclear lamins play a role in the induction of apoptosis (Rao et al, 1996; Di Matola et al., 2001). Post mitosis, protein phosphatise 1 (PP1) is dispatched to the nuclear envelope to initiate lamin B reformation. If this process is abolished, lamin B is degraded and apoptotic signals take place. As previously mentioned, expression of lamins A/C is limited to differentiated cells. Due to their role in DNA replication and transcription, several researchers have hypothesised that they play a role in gene expression as well. Gupta and Saumyaa (2008) propose that specialised A/C lamin expression regulates gene expression in such a way which may prevent cell division and cause the cell to undergo terminal differentiation – a form of programmed cell death.
Lamins are also used for chromatin organisation and positioning within the nucleus, so cells with LMNA mutations exhibit a range of problems involving abnormal chromosome organisation. Glass et al (1993) showed that A lamins interact with chromatin by binding histones as well as indirectly through lamin-binding proteins such as LAP2Î± and barrier-ro-autointegration (BAF) (Holaska et al., 2003).
Cellular and molecular mechanisms of HGPS
The most commonly reported mutation responsible for causing HGPS is LMNA codon 608 in exon 11 (c. 1824 C>T). Although the LMNA gene encodes both A and C type lamins, only A lamins are affected as exon 11 is not present in lamin Cs. Whilst this point substitution does not result in an amino acid change (G608G ) it partially activates a cryptic splice site, resulting in the deletion of ~50 amino acids near the carboxyl terminal in lamin A (LAâˆ†50) but maintaining the CAAX site. Amongst the deleted amino acids is the ZMPSTE24 cleavage site, which is necessary for the maturation of lamin A. This results in farnesylation and carboxymethylation of lamin A, resulting in “progerin” (Capell et al., 2005). This cryptic splice site is only partially activated and it is estimated that only 10-50% of splices mRNA in transcribed. Since the second LMNA allele is normal, there is still some presence of wild-type lamin A although it is present in much lower levels. Whilst most HGPA patients are heterozygous for LMNA p.G608G, mutations have been reported on other location on the LMNA gene, such as one patient with a p. E145K mutation and another with 471C and R527C mutations (Goldman et al., 2002). These mutations have lead to various laminopathies very similar to HGPS (often referred to as atypical HGPS) however the pathophysiological manifestation of these diseases is probably different as they do not result in the production of progerin.
HGPS cells are significantly larger than normal cells and usually have large cytoplasmic vacuoles, an abnormally shaped and sized nucleus, often distorted with chromatin extrusion (De Sandreâ€Giovannoli et al., 2003). Goldman et al. (2004) also report evidence of misshapen nuclear envelopes and abnormally thick lamina, both of which are associated with other mutations on the LMNA gene. Not surprisingly, these structural abnormalities cause a variety of cellular dysfunctions including loss of structural nuclear integrity and certain mitotic problems such as irregular chromosome separation, delays in cytokinesis and nuclear assembly and binucleation (Dechat et al., 2007). The loss of nuclear integrity can have a detrimental effect of the function of the cell, however it does not remove all structural ability of the nucleus and cell, it only reduces it. This means that the tissues most likely to be affected by a comprised nucleus are those which are consistently subjected to mechanical stress, such as blood vessels. Indeed, vasculature in HGPS patients is often severely compromised and death is almost always caused by heart failure.
HGPS cells have abnormal chromosome organization in interphase nuclei and can show a loss of peripheral heterochromatin, possibly due to various epigenetic changes. These include upregulation of genes such as H3K9me3 and H4K20me3, both of which are involved in the definition of constitutive heterochromatin (Columbaro, 2005). The H3K27me3, responsible for the definition of facultative heterochromatin, is downregulated, possible due to a reduction in the expression of the histone methyltransferase enhancer EZH2 used for H3K27 trimethylation (Schumaker et al., 2006). Evidence has suggested that these changes in heterochromatin due to altered gene expression may often result in further changes in gene expression. An interesting line of investigation to follow would be to monitor the expression of various other genes in HGPS cells. It is important to understand if HGPS enhances or inhibits the expression of various other genes and, if so, what these genes are and whether these genes play a part in the HGPS phenotype. Since lamin A only appears in differentiated cells, it is possible that one of the roles of lamin A is to maintain differentiation of the cells by securing tissue-specific gene expression. This could mean that the premature aging phenotype is not actually related to normal physiological ageing, but occurs due to certain tissues being unable to perform their designated function due to incorrect gene expression, leading to cell death, tissue degeneration and organ failure.
Several studies have indicated that HGPS cells show increased damage to DNA. This was demonstrated by Bridger and Kill (2004) whose experiments showed that HGPS cells were unable to proliferate sufficiently when kept in culture and showed increased rates of apoptosis as well as demonstrating early signs of cellular aging. All of these afflictions are caused by damage to nuclear DNA. Liu et al (2006) also demonstrated that HGPS cells have increased activity in their DNA repair pathways, strongly implying that DNA damage has occurred. The most compelling evidence to suggest DNA damage as a likely culprit for progeroid symptoms is presented in a study by Liu et al. (2005), which showed that HGPS cells have a double strand break (DSB) repair defect. These cells show less concentrated levels of DSB repair factors such as Rad50 and Rad51 as well as damage signaling molecules such as 53BP1. With this evidence, the conclusion can be drawn that Lamin A in necessary for complete DNA repair to take place when necessary, and also that DSB repair is diminished in HGPS cells. DSBs are the most dangerous type of DNA damage that can potentially occur as a result of the constant attack from various agents, as they can potentially result in loss or confusion of genetic information or cell death. To some extent, DSBs occur naturally within the body during normal processes such as meiosis (Keeney & Neale, 2006), or during replication when the separated stands encounter blocking lesions. The level of DSBs that occur during these processes can be increased by UV radiation, ionizing radiation, various chemical agents and the presence of free radicals. When a DSB occurs, the cells defense mechanisms commands that the cell cycle halts and DSB repair mechanisms activate. DNA DSB repair mechanisms are thought to occur as one of two processes: Non-homologous end joining (NHEJ) and homologous recombination (HR). Although HR is thought to be considerably more accurate than NHEJ, neither mechanism will produce perfect results. Evidence has been presented to imply that as we age we become increasingly dependent on HR to repair DSBs as activity of NHEJ and all other mechanisms gradually decrease (Johnson-Schlitz & Engels, 2006).
Many of the changes to the nuclear membrane are not a direct result of insufficient lamin A, but instead are caused by the presence of Progerin, an abridged version of lamin A, and its accumulation of the INM. This accumulation results in alterations in the structure of the nuclear lamina. Liu et al. (2005) suggests that the inefficient DNA repair mechanisms which occur in HGPS cells are in fact due to raised levels of Progerin at the INM. Fitting with this theory, experiments designed to reduce the levels of Progerin have successfully managed improve the cellular phenotype of HGPS cells. Scaffidi and Mistelli (2005) proposed inhibiting the production of Progerin whilst leaving lamin A unaffected by using specific antisense morpholinos which inhibit splicing at the deviant site. Another approach is to treat HGPS cells with farnesly transferase inhibitors (FTIs) which, as the name suggests, inhibit farnesyl transferase, so preventing the modification of Progerin to the farnseylated kind seen in HGPS cells. Liu et al., (2006) have shown that this approach does not only improve the cellular and nuclear abnormalities of HGPS cells, but can actually improve the health of HGPS mice. However, FTIs are not specific to Progerin as antisense morpolinos are, and lamin A is likely to be inhibited as well as the modification of Progerin. It is also possible that several other unrelated proteins would be affected by this approach; however these negative side-effects have not yet been documented.
Several potential cellular and molecular mechanisms which may contribute to the HGPS phenotype are described below in figure 4.
Figure 4 – Various cellular and molecular mechanisms which may contribute to the HGPS phenotype. Progerin becomes trapped within the nuclear membrane as a result of permanent farnesylation. Alteration of the normal lamina structure causes vulnerability to mechanical stress and some nuclear blebbing. Other consequences involve disruption of protein interactions, disorganization/loss of heterochromatin and disrupted interactions with RNA polymerase II, RNA splicing factors and transcription factors, causing misregulation of gene expression. (Coutinho et al. 2009)
The way these cellular abnormalities cause the general HGPS phenotype are not yet understood and currently can only be speculated on. As further research is carried out regarding HGPS, the cause of premature aging throughout the whole body may become more transparent.
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Due to the rarity and complexity of HGPS, there is currently no known cure; however, there are a variety of treatments aimed at alleviating the symptoms which are associated with this disease (Progeria Research Foundation, 2006). It is recommended that patients suffering from HGPS have a regular and nutritional diet as well as supplementary vitamin tablets (in normal doses). Dental problems which occur as a result of HGPS are treated with fluoride supplements, and aspirin should be administered on occasion in order to reduce the risk of heart attacks and strokes, which are invariably the main causes of death in HGPS children. In the case of heart-related problems such as angina, drugs like nitroglycerin may be used, although the dose should be measured carefully based on weight and height as use of anesthetics on children can be potentially dangerous. There is little that can be done to reduce the effect of reduced bone mass except for vitamin and calcium supplements, so children should be accompanied at all times as they are at significantly greater risk of fracturing bones. The hip bones are particularly susceptible to dislocation due to coxa valga. Certain surgical procedures can take place to reduce the risk of this, as well as physical therapy in order to keep all joints as mobile as possible. The use of growth hormones has been suggested as a potential treatment for HGPS children; however the long term effects of this have not been shown to be significant.
Currently there are several areas of research into possible clinical therapeutic treatments for progeria. Wang et al. (2008) and Liu et al (2006) demonstrated the use of farnesyltransferase inhibitors (FTIs) which appear to reduce the nuclear abnormalities present in HGPS cells, reducing the severity of symptoms on HGPS mice, improving their general health. Subjects showed improved cardiovascular health, a reduction in the number of bone fractures and improved survival and growth rates. This has been substantiated with evidence from Toth et al (2005) who showed that human fibroblast cells expressing progerin had reversed nuclear alterations when treated with FTIs in vitro. Clinical trials testing the effect of FTIs in children with HGPS began in August 2009 and ended the following December. Results from these trials are still being awaited.
Most of the breakthrough discoveries regarding HGPS have been made within the last few years. These include the location of the HGPS mutation, the nature of the disease and potential clinical therapies which are aimed at preventing the HGPS phenotype on a cellular and molecular level instead of simply treating the symptoms. There are several ways that the effects of the point mutation on the LMNA gene on chromosome 1 could lead to the pathogenesis of HGPS and it is likely that the combined affects of this mutation results in the HGPS phenotype. Compromised nuclear integrity may lead to reduced structural support for the cell, so those tissues under constant mechanical pressure such as the vasculature will suffer more greatly than other tissues.
Another possible cause of the HGPS phenotype is the accumulation of DNA damage. This is a logical conclusion to draw, as the mechanism of normal human aging is thought to occur in this way. HGPS cells have insufficient DNA DSB repair mechanisms and so the phenotype of premature ageing in children with progeria is simply due to the magnification of one of the factors that causes aging in healthy humans. The pool of stem cells that healthy humans rely upon to counter increased apoptosis as a result of DNA damage would be under more pressure to proliferate in HGPS children, perhaps exhausting supplies and causing tissue degeneration. It is also possible that the stem cells themselves are affected by the HGPS mutation, causing a decline in proliferative ability. The possibility that the HGPS mutation may lead to up or down regulation of other genes is not one that should be ignored. If this is the case, the vast variety of symptoms which accompany HGPS may be explained by the altered levels of expression of other genes.
Until more is understood about Hutchinson-Gilford progeria syndrome, it is impossible to conclusively explain the extraordinary symptoms of this disease. Perhaps the results of the recent clinical trials will shed more light on how the alteration of two proteins due to a single point mutation can cause a child to show such drastic physiological aging.
Experimental data analysis
Programmed cell death (PCD or “apoptosis”) is a necessary part of complex life in all sorts of multicellular organisms. In humans, it is not only essential during embryonic development (preventing all manner of deformities) but also consistently through life. Efficient apoptosis prevents a vast number of diseases by ensuring that any unnecessary or potentially harmful cells are destroyed safely, without harmful effects on neighbouring cells, which happens in necrosis. Apoptosis only causes a diseased state when its rate of action exceeds or falls short of that which is necessary to keep an individual healthy. Insufficient apoptosis is well known culprit for the development of cancers, when tumorous cells which should have been erased are allowed to proliferate and develop into tumours. Another well known example of diseases due to lack of apoptosis is the vast number of immunodeficiency diseases caused by self-targeting T and B lymphocytes. These cells should have been removed through apoptotic signals, but when these signals fail these self-targeting lymphocytes are allowed to survive and will target the body’s own tissues. Increased rates of apoptosis throughout the body are commonly observed in many diseases, for example, HGPS. Abnormalities either within or external to the cell cause apoptotic signals to occur, resulting in PCD of cells which would otherwise not have been destroyed.
PLAC8 (placenta-specific 8) is a gene which encodes a small, highly conserved protein known as onzin. Experimental evidence has been presented to demonstrate that under expression of endogenous onzin results in reduced cell proliferation, whilst over expression results in an increased cell count (Rogulski et al, 2005). This data suggests that onzin has a negative effect on the rate of apoptosis. Li et al. (2006) suggest that expression of onzin within a cell protects it from apoptotic signals and that when onzin levels are depleted the cell becomes sensitive to apoptotic assault.
To test the effects of onzin on the rate of apoptosis, CEM-C7 T-leukemic cells were transfected with either an expression construct containing PLAC8 or pcDNA3, where the pcDNA3 vector acted as the control. Cultures of these cells where then exposed to a range of apoptosis-inducing agents: Fas, Dexamethasone (dex), cisplatin, butyric acid, okadaic acid and UV exposure. This was in order to determine whether expression of the PLAC8 gene effectively reduces the rate of induced apoptosis. Cell counts were taken after 24, 48 and 72 hours.
The significance of the difference between cell counts in PLAC8 and pcDNA3 cultures when exposed to all apoptosis inducing agents was determined using a two-sample T-test. The samples used in each test were the apoptosis-inducing agent transfected with PLAC8 and the same apoptosis-inducing agent transfected with pcDNA3. The results from these two-sample T-tests are shown below in table I.
p-value (24 hours)
p-value (48 hours)
p-value (72 hours)
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