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Tag Archives: Biology

The chances of me posting anything of any substance this week is looking decidedly slim due to the fact that I have 4 days left to write a 3000 word essay which constitutes 8% of my entire degree. It’s all about transposons. Turns out our DNA is approximately 45% transposon, which is pretty cool when you think about it. Especially as transposons don’t actually have a role in cell function per se. What are they for? That’s what I’m trying to find out.

I’m afraid this lack of posts is likely to continue for a couple more days. I’ve been spending most of my time writing my dissertation or revising for exams, and have been spending any spare time I have either outside, drinking, or playing Assassins Creed II. I’ve got 4 half-started reviews saved in the drafts sections and will hopefully get time to finish them over the next week.

However, to keep people entertained for now (I say people, I think there’s been maybe 10 views of this all together, but I shall continue to pretend like everyone reads this) I shall post this. A brief overview of Wnt signalling taken from my dissertation. Enjoy.

The Wnt proteins comprise a large, complex and highly conserved family of secreted glyco-lipoproteins which act as signalling proteins and growth factors. Identified by amino acid sequence rather than functional properties, Wnt proteins are found to be involved in a large number of developmental and homeostatic processes, their involvement in stem cell maintenance and differentiation being a recent driving factor towards understanding an already popular field of research. There are currently at least 19 known Wnts which can be classified as either canonical, signalling through inducing the build-up of intracellular β-catenin, and non-canonical Wnts which signal through other β-catenin independent processes (fig. 1)(9). Both pathways rely largely on the frizzled receptor which binds Wnt in the presence of its co-receptors.

Fig 2.1. Diagram illustrating canonical and non-canonical Wnt signalling pathways. Taken from L. Ling et al (2009)(9)

Non-canonical signalling invokes several β-catenin independent pathways via the activation of the frizzled (fz) receptor and co receptors such as Ror2 and Ryk and transduction through diverse mechanisms dependant on either the Dishevelled (Dvl or Dsh) downstream effector, or Ca2+. Dvl can be activated by fz directly or through the induction of heterodimeric G-proteins. Dvl has been shown to activate the small GTPase RhoA and its effector ROK (Rho-associated kinase) in order to regulate actin cytoskeleton. Another downstream effector of Dvl is JNK which can be activated by RhoA to regulate PCP (planar cell polarity) during eye development. Signal transduction through Ca2+ activates nemo-like kinase (NLK) and the nuclear factor of activated T cells (NFAT) with NLK inhibiting canonical Wnt signalling through the phosphorylation of TCF/LEF. (9)

Canonical Wnt signalling is based around the concept of allowing an increase in the concentration of cytoplasmic β-catenin and facilitating its translocation into the nucleus through inhibition of β-catenin degredation which takes place in the absence of Wnt. The canonical Wnt pathway is initiated by the binding of Wnt to frizzled (Fz) and its co-receptor LRP5/6. Fz contains 7 transmembrane regions and an amino-terminal extension rich in cysteine residues (10) and was first discovered through a mutation which disrupted the planar cell polarity in the wing hairs of mutant drosophila, causing them to point in different directions. Low density lipoprotein (LDL) receptor related protein (LRP) is a single pass transmembrane molecule and is also essential for successful Wnt signal transduction. Wnt forms a trimeric complex with Fz and LRP leading to the phosphorylation of the Fz bound protein Dvl, mediated by several protein kinases, and of LRP, allowing the docking of Axin with LRP. In the absence of Wnt, Axin is part of the β-catenin degradation complex, consisting of glycogen synthase kinase-3β (GSK-3β), Axin, Adenomatous Polypopsis coli (APC), and β-catenin. This complex facilitates the phosphorylation of β-catenin by the serine/threonine kinases, casein kinase Iα (CKIα) and allows the phosphorylated β-catenin to be recognised by β-TrCP and targeted for ubiquitonation which leads to degradation by the proteosome. The binding of Axin by phosphorylated LRP5/6 along with the inhibition of GSK-3β by phosphorylated Dsv prevents the formation of the β-catenin degradation complex and facilitates its cytoplasmic accumulation. Axin can also be recruited to Dsv due to the DIX domain which both Axin and Dsv contain. This region is capable of homodimerization in Axin but can also be heterodimerize with the DIX domain of Dsv. The increased stability of β-catenin leads to its nuclear translocation where it converts the TCF/LEF DNA-binding proteins into transcriptional activators through the displacement of Groucho, a protein bound to TCF/LEF causing it to act as a transcriptional repressor, from the proteins and recruitment of the histone acetylase CBP/p300. This results in the transcription of Wnt activated genes normally suppressed by TCF/LEF in the absence of Wnt. (11)(12)(13)(14)

Due to laziness and lack of time for anything I’m going to do a ‘here’s one I made earlier’. This is a brief review of some of the current views on the relationship between DNA damage and ageing I did as an essay last year. It was give a 2.1 so hopefully it’ll be an enjoyable and enlightening read. lol.

DNA Damage: A Cause or Consequence of Aging?

There many interacting factors which contribute to the appearance of aging. From the specific death or loss of function of individual cells, to the dysfunction and gradual decline of individual tissues, and eventually the physical and mental ability of a whole organism. However recent research into aging has been focused on our genes, how they are affected by the passage of time, and how this affects the aging process. It is known that there are many external factors that can cause damage to our DNA such as UV light, excessive unhealthy food, and various toxic compounds we encounter in daily life. However this is not all that causes our DNA damage, internal factors such as reactive oxygen species can cause oxidative damage, and telomeres, our own genetic clock, are degraded with every cell division, ultimately resulting in senescence. How do all these factors cause damage to our DNA and how is this related to aging?

DNA damage by reactive oxygen species as a cause of aging.

One of the most popular theories relating DNA damage to aging is the free radical theory. During normal cellular metabolism the mitochondria produce reactive oxygen species (ROS), highly reactive ions or small molecules which can be damaging to certain elements of the cell if left unchecked. The cell produces enzymes to actively scavenge these harmful ROS such as superoxide dismutases or catalases as well as dietary antioxidants such as vitamin C which, under normal circumstances, prevent ROS levels from becoming harmful to the cell. However, in certain situations when the cell is put under stress and mitochondrial activity is increase, such as during strenuous exercise, ROS levels can increase above levels which can be managed by enzymes. ROS levels can also be raised in cells in close proximity to fatty deposits, such as in the brain, due to increased metabolic rates. This increase of ROS is known as oxidative stress and can potentially cause damage to the DNA, cell membranes, and organelles through oxidation. The free radical theory suggests that ROS at normal levels are not scavenged with complete efficiency, and those that are left unchecked cause small amounts of damage to mitochondrial DNA. Combined with the higher levels of ROS involved in oxidative stress, over time this results in a cumulative build up of damage in mtDNA.

Oxidative damage to mtDNA can be in the form of base modifications, abasic sites, and various other lesions (Druzhyna. 2008). One of the more widely studied forms of oxidative damage is 8-oxoGuanine (8-oxoG) which results in a G-C to A-T mutation. Several studies have shown that levels of 8-oxoG damage increase in both mtDNA and nuclear DNA with age. In mtDNA this process becomes a vicious cycle as increased amounts of damage result in less efficient oxidative phosphorylation and an increase in ROS levels, causing further damage. Eventually the accumulation of oxidative damage in the mtDNA results in the cell undergoing apoptosis. Evidence for this was shown in experiments using mice deficient in mtDNA repair mechanisms, the mice expressed a faster aging phenotype than normal mice and were found to have increased numbers of cells undergoing apoptosis, shown by increased levels of cleaved caspase-3 in older tissues (Kujoth et al. 2005). The model proposed from these findings was that the increased numbers of cells undergoing apoptosis was the cause of the tissue dysfunction observed in aging phenotypes. This model would suggest that in this case, DNA damage is a cause of aging.

Cellular senescence as a consequence of aging.

Another important model proposed as a consequence of aging is cellular senescence. Senescence was first described by Hayflick and Moorfield in 1961 when they discovered that normal human fibroblasts had a replication limit in vitro, eventually undergoing morphological changes and an irreversible cell cycle arrest. The cells also undergo changes in genetic expression, with the production of certain proteins being completely stopped and others genes becoming upregulated, including many genes coding for secretory proteins (Jayapalan. 2008).

The most widely understood reason for this was discovered to be the shortening of telomeres due to the end replication problem, a mechanism by which a small amount of DNA (30-50bp) is lost with each round of replication. This kind of telomere dependant senescence is known as ‘intrinsic senescence’ and is regulated by the p53 and p16 enzymes (Itahana et al. 2004). Though the mechanism by which the shortening of telomere length is detected by p53 is still unknown it is thought that the critical shortening of telomeres results in a disruption of the looped structure they form. This results in either the detection of the end of the DNA strand being interpreted by damage repair enzymes as a break in the chromosome, eventually leading to the activation of p53, or the disassociation of telomere associated proteins, a process known as uncapping, which could also be detected by p53 through either phosphorylation or acetylation (Itahana et al. 2004).

Senescent cells have been found in many niches in vivo, frequently in damaged, diseased, or aging tissues. The many different factors secreted by senescent cells are thought to be highly involved in age related pathologies due to their action on healthy cells. These factors are harmless in small amounts but as the organism ages the number of senescent cells increases, in vivo studies have shown whole cell cultures can eventually become senescent, and this increase in senescent cells results in an increase in secreted factors which can reach damaging concentrations. The proteins secreted include proteases which in high concentrations cause massive disruption of tissues. In some studies senescent cells have been shown to secrete cytokines which stimulate the expression of growth factors in healthy cells, causing increased proliferation and further tissue disruption. They have also been shown to secrete various other degradative enzymes and inflammatory cytokines causing the appearance of aging phenotypes (Jayapalan. 2008). Senescent cells have also been detected in premalignant cancers and have also been shown to cause a progression of premalignant cells to malignant.

Senescent cells are thought to be an evolutionary mechanism developed to suppress the occurrence of tumours and other diseases caused by DNA damage, as the reaction to shortened telomeres reduces the chance of loss of coding DNA from the genome. However, with longer life comes an increased build up of senescent cells. Further support for this theory of senescence being a protective mechanism against DNA damage comes from the telomere independant pathway of senescence, ‘extrinsic senescence’ (Itahana et al. 2004). This can be caused by damage to the DNA unrelated to telomere length, the most common type of damage reacted to being oxidative damage. Cells can undergo senescence by this pathway through other forms of external stress such as through toxins or mutagens.

An important factor in the mechanisms of aging is the action of DNA repair mechanisms. Genetic disorders such as Werner Syndrome (WS) and other progeroid syndromes are caused by the dysfunction of certain DNA repair mechanisms and have been used in some studies to gain an insight into changes in gene expression during aging through comparisons of normal older cells and young cells to WS cells (KJ Kyng et al. 2005). One study in particular by Kyng et al showed varied changes in overall gene expression, but specific DNA repair mechanisms were shown to be downregulated in old and WS cells. The study also showed that WS cells were particularly susceptible to damage by 4NQO which induces bulky DNA adducts, strand breaks, and interestingly due to its proposed involvement in aging, oxidative damage. Other repair mechanisms appeared to be active, such as UV damage response, though it was suggested that this could explain the lack of other age related symptoms such as senility.

One experimental model used to investigate the influence of DNA damage repair on aging is to compare the repair capabilities of species with different lifespans, for example, a mouse to a human. This is done by measuring the number of DNA abnormalities in vivo. Interestingly it has been found that the rate of occurrence of DNA damage in mouse and human T cells is similar, however human cells possess a much higher capacity for repair, so the total amount of DNA damage in the genome accumulates slower. Despite a large amount of data to support the relationship between DNA repair and species longevity there is little conclusive evidence to support it (Vijg. 2008)

Base excision repair (BER) is a DNA repair mechanism important in repairing spontaneous damage and mutagenesis and is one of the main repair pathways. DNA polymerase-β is the main protein involved in this process and is used to analyse BER activity. Several studies have been performed analysing BER activity levels in cells taken from varying ages of both mice and humans however the results have been said to be unreliable in some cases, due to their variation. Despite their variation though, studies by Cabelof et al and Intano et al both showed that BER activity reduces with age in various tissues and later, more accurate studies have confirmed this (Xu et al. 2008). Similar studies in humans have yielded results in agreement with the results obtained from mice. Several interesting studies into diet have revealed that reduced calorie intake causes an increase in the activity of BER in mice, reversing the age related decline. However, the age related conditions which cause the decline of BER activity are still unknown. Nucleotide excision repair (NER) is a DNA repair mechanism involved in repairing helix disrupting damage. NER is especially important in repairing UV damage to the genome. Several age related phenotypes can be seen in people with NER defects, including rapid photoaging of the skin and eyes, the early onset of neurodegeneration, reduced proliferation of hematopoietic stem cells, and an increased risk of cancer are some of the most serious (Neidernhofer. 2008).

These are just a few of the many factors that contribute to both DNA damage and the aging process. It is unclear with many of them as to whether or not they are a causative factor in aging or simply a consequence. The accumulation of DNA damage with time can be seen as a cause of the aging phenotype, but whether or not it is a cause or consequence of aging could depend on how you view age. If you consider aging as the change in mental and physical ability, then it would be easy to class DNA damage as a cause of this. However it is also possible to view aging simply as the passage through time, which would lead to think of the accumulation of DNA as a consequence of time, and therefore aging. As well as these two views there is also the issue of cellular senescence as a consequence of telomere degradation. This could also easily be seen as both a cause and consequence of aging, senescence being a consequence of repeated cell divisions, but proteins secreted by senescent cells being a cause of many of the physical changes associated with aging. Extensive research into various mechanisms of DNA damage and their relationship to aging is still being carried out. Comparative studies between species with different longevities are still being carried out using new techniques in comparative genomics. Still very little is known about the mechanisms by which DNA damage repair pathways become downregulated with age, whether it’s due to damage to promoters as a consequence of age, or a programmed change in cellular function.