BOD - Cancer overview

Cells replicating normally are mediated by contact inhibition. Thismeans that when they realise there are a lot of cells in that area they stop undergoing mitosis. If a cell is created with an abnormality, it recognises this itself and kills itself off - apoptosis. There is ~100 billion new cells made in the body per day so it is easy to see how an error can occur. Some mutations are minor and will have little effect on the cells structure and function and others are major and trigger apoptosis. On occasion, the cell contains a mutation that will cause a major structural or functional change and will not apoptose. The mutation could prevent apoptosis and/or cause rapid mitosis. This abnormal cell then replicates uncontrolled and forms a neoplasm - group of abnormal cells. Neoplasms do not have to be stationary and can circulate the body. The get larger and noticable and are referred to as tumours. These tumours could have little effect and grow at a similar pace to the surrounding cells and are benign tumours. If one of the cells within the benign tumour develops a mutation tat increase the rate of mitosis. This cells becomes invasive and starts infiltrating surrounding tissue and replicating. I tis likely that thesubsequent cells will also contain mutations that will effect replication and cause further abnormality and an increase in mutations within these cells. This is reffered to a metastasising and is a malignant tumour composed of cancer cells. Cancer essentially is just a by-product of broken DNA affecting apoptosis and mitosis.   

It is used for 2 unrelated concepts in biochemistry: plant physiology and oncology. Normally glucose obtained from the diet moves into the cell via facilitated diffusion and in the cytosol in undergoes glycolosis. This process provides 2 molecules of ATP and 2 molecules of pyruvic acid. This is an anaerobic process. The pyruvic acid is moved into the pyruvate pathway which is dependent on the presence of oxygen. Anumber of products an dby-products are made in this pathway but the molecules of importance are lactate and acetyl CoA. If there is oxygen deficiency in the pathway, it leads to the formation of lactic acid. If there is enough oxygen present, the pathway become aerobic and follows intot he citric acid cycle. The formation of lactic acid is anarobic glycoloysis and produces 2 molecules of ATP but also produces an increase in hydrogen ion which could result in lactic acidosis and an acid-base imbalance. The formation of acetyl CoA leads to activation of the citric acid cycle in the mitochondiral matrix and results in the production of 3 molecules of NADH and 1 molecule of FADH2. The NADH and FADH are then used in the electron transport chain or oxidative phosphorylation. This chain produces 30-32 molecules of ATP for every glucose molecule. Otto Warburg discovered that in cancer cells there is a metabolic shift to using glycolysis more even if oxygen is readily available. Cancer cells make use of anaerobic respiration even when oxygen is present. Cancer cells also have a huge uptake of glucose into the cells. This is the Warburg effect. Cancers cells can be detected by PET scans in which radioactive tracers show in colour huge build ups of glucose in higher metabolic cells like cancer cells. There are a few theories for why this happens. Some are: efficiency; biosynthesis; assured ATP synthesis and increased invasivenesss. Liberty and Locasale 2016 hypothesised that in cancer cells producing lactic acid from glucose is 10-100 times faster than oxidative phosphorylation. It also insures there is always ATP present for cellular activity as tumours grow they may outgrow there supply of oxygen. Glucose is a 6 carbon molecule and can be used for anabolic proliferation an defficiently use all aspects of the glycolysis pathway to make new cancer cells. The increased anaerobic glycolysis causes lactic acidosis which disrupts the stroma of the cells and allows the cancer cells to be more invasive. 

Mortality is the number of deaths recorded in a set population over a set period of time. Cancer research UK statistics. There are around 165,000 cancer deaths in the UK every year, that's around 450 every day (2015-2017). Cancer accounts for more than a quarter (28%) of all deaths in the UK (2017). In females in the UK, there were around 77,700 cancer deaths in 2017. In males in the UK, there were around 88,900 cancer deaths in 2017.

The WHO estimate that 9.6 million deaths were from cancer in 2018, working out at around 1 in every 6 deaths. Around 1 in 3 of these deaths are due to the major risk factors for cancer: high BMI; low fruit and veg intake; lack of exercise; smoking and alcohol consumption. Smoking is the most prevalent risk factor accounting for about 22% of cancer deaths. The most common cancers causing death are: lung (1.76 million); colorectal (862000); stomach (783000); liver (782000) and breast (627000). Infections from helicobactor pylori, HPV, hep B, C and epstein-barr virus increase the risk of cancer and were associated with 15% of cancer diagnoses in 2012.

Incidence is the frequency of cancer in a set population over a set period of time. Cancer research UK statistics. There are around 367,000 new cancer cases in the UK every year, that's around 1,000 every day (2015-2017). In females in the UK, there were more than 179,000 new cancer cases in 2017. In males in the UK, there were around 187,000 new cancer cases in 2017. Breast, prostate, lung and bowel cancers together accounted for more than half (53%) of all new cancer cases in the UK in 2017. Incidence rates for all cancers combined in the UK are highest in people aged 85 to 89 (2015-2017). Cancer survival rates are recorded at 1, 5 and 10 year milestones. Half (50%) of people diagnosed with cancer in England and Wales survive their disease for ten years or more (2010-11). Cancer survival is higher in women than men. Cancer survival is improving and has doubled in the last 40 years in the UK. Cancer survival is generally higher in people diagnosed aged under 40 years old, with the exception of breast, bowel and prostate cancers, where survival is highest in middle age.

For both men and women in North America Lung cancer was the biggest killer with rates of 28% and 26% respectively. This was followed by prostate cancer (9%) and breast cancer (15%).. Finally colorectal cancer with 9% across both genders. In Europe lung cancer was fatal in 26% of men and 13% of women. 17% of women died of breast cancer and 12% and 13% of men and women respectively died of colorectal cancer. 10% of men died of prostate cancer. In Latin America and the Caribbean: Breast (15%) and Prostate (16%) were fatal, 15% of men and 10% of women diesd of lung cancer and 10% of women died of cervical cancer. 10% of men died of stomach cancer. In Asia: men (25%) and women (15%) died of lung cancer. 15% of men of liver cancer. 13% women of breast cancer and 13% men and (10%) women of stomach cancer.

Lung cancer is one of the top killers in all regions with the exception of sub-saharan africa. The other high mortality cancers are breast, prostate and colorectal. Data from 2014 published in Scientific American. This reflects the difference in income and access to treatment. 

Fertilisation of the egg cell by a single sperm cell occurs and results in the formation of a zygote. The zygote splits into two cells without growing in a process called cleavage. This happen multiple times with the zona pellucida. After several clavages occur the cluster of cells goes froma zygote to a morula. The process now moves from cleavage to blastulation. Compaction begins to occur packing the cells within the morula closer together and the outside layer of cells differentiate to trophoblasts and the inner cells to embryoblasts. The embroblasts form an inner cell mass leaving an empty space within the trophoblasts called the blastocoel. The morula is now a blastocyst. The zona pellucida starts to disintegrate away. This leaves a ring of trophoblasts and the inner cell mass of embryoblasts which have formed another cavity within the clump called the amniotic cavity. The embryoblasts that divided the inner cell mass from the blastocoel have differentiated to form hypoblasts and the cells in the layer above it have become epiblasts. The layers of hypoblasts and epiblasts are called the bilaminar disc. A streak forms on this disc called the primitive streak and marks the start of gastrulation. The primitive streak is the site of epiblast migration into the bilaminar disc. The migrated epiblasts form a new middle layer in the bilaminar disc creating a trilaminar disc. These layers are called the germ layers. From top to bottom, these layers are: ectoderm; mesoderm and endoderm. The next stage in embryogenesis is neurolation. The cells in the centre of the mesoderm differentiate into a notochord. This forms part of the intervertebral discs and can i rare instances form a tumour called a chordoma. Its role in neurolation is to stimulate a change in the above ectoderm. A neural plate is formed where the ectoderm becomes thickened as a result. The neural plate cells begin to dive into the mesoderm, forming a tube structure called the neural tube. Other cells from the ectoderm dive into the mesoderm and form neural crest cells. 

The 3 germ layers are the ectoderm, mesoderm and endoderm. Endodermal cells are responsible for GI tract formation. The lungs develop from the upper GI tract as well as the liver and pancreas. The tube of endodermal cells goes on to form the oeosophagus, stomach, small and large intestines. The mesoderm layer forms the inner layers of skin, muscles and bones. This includes the heart, kidneys, bladder and sexual organs. The ectoderm forms the outer layer of skin, sweat glands, hair and nervous system.

Cancer will present as the invasion of neoplastic epithelial cells through the basement membrane and into tissues. This will cause a stromal response in the host tissue (desmoplasia) as the formation of connective tissue in reaction to the cancer cells invading. It also triggers an inflammatory response within the tissue and results in the recruitment of inflammatory mediators.. Furthermore, there is angiogenesis or the formation of blood vessels. In cancer of eptithelial cells on a slide, it is very hard to distinguish the basement membrane where the cancer cells have invaded. It is filled with inflammatory cells which when stained appear blue. The inflammatory mediators are present in huge areas of epithelium due to hyperplasia. The connective tissue has grown a thick barrier to protect the rest of the cells from the cancerous epithelial cells that have invaded through the basement membrane. This is very characteristic of squamous cell carcinoma histology. In higher magnification slides, it is important to look for Anaplasia, Bizarre, Chromatin (darker nuclei from more chromatin packed in), Dysplasia, Edges (undefined). If the basement membrane remains intact it is dysplasia and not cancer. 

Eptheilal tissue can be divided into 2 things: the primary epithelium that lines the outer and inner body and glandular epithelium that forms glands and secretes molecules e.g. hormones. The primary epithelium combines with connective tissue to create the epidermis. It also composes the cavities throughout the body e.g. cranial, pericardial etc. The epithelium protects the deeper layers of tissue from injury and infection e.g. the stomach is lined with mucous-secreting epithelial cells to prevent digestion of the stomach lining. Epithelial tissues are avascular (no blood supply) relying on the surrounding connective tissue for this. The type of epithelial cell is determined by the shape of the cells and the quantity of layers they form. There are 3 shapes: squamous (fast diffusion and absorption and thin membrane tissues), cuboidal (absorb nutrient and produce secretions) and columnar (absorb nutrient and produce secretions. Cells take a lot of time, materials and energy to make. Squamous cells are smaller and flatter so have quick turnaround. A simple epithelium has only one layer of cells. Stratified has multiple layers of cells. Pseudostratified has mostly one laye but the cells can be of different shapes and sizes with nuclei in different places. Epithelial tissue regenerates very quickly. All epithelial cells are polar. The upper side is exposed to the outside of the body or the internal cavity. the inner side is attached to th basement membrane. The basement membrane holds everything together and anchors the epithelium to the connective tissue. These are selectively permeable layers to allow for absorption, filtration anf excretion. The glandular epithelium form 2 different types of gland: the endocrine glands and exocrine glands.  

Sarcomas - Mesenchymal tumors. These are the second most common cancer type. They are  derived from mesenchyme and can come from fibroblasts, adipocytes, oesteoblasts and myocytes. Rhabdomyosarcoma is a tumour of striated skeletal muscle for example. These cells all originate from the mesoderm.  Leukaemias and related e.g. lymphomas - These also originate from the mesoderm. An example is acute myeloid leukemia.  Neuroectodermal tumors - These originate from the ectoderm. They are the rarest and the most deadly. They are derived from the cells of the nervous system (peripheral and sensory). An example is a glioblastoma. 

Cancer is a progressive disease. A cell proliferates uncontrollable and cause hyperplasia within the tissue. An abnormal cell appears and this is known as dysplasia. These abnormal cells begin to proliferate and result in the formation of a benign tumour. The tumour becomes malignant once it breaches the basement membrane and starts to invade surrounding tissue. If the abnormal cells remain in the one location this is a primary tissue, if they break off and travel to another location (metastasize) this forms a secondary tumour and is much more difficult to treat. 

Cancer stem cells make up a small amount of cancer cells. The maintain their own numbers through endless proliferation and provide the majority of tumour mass. They do this by producing fast proliferating progenitor cells. The progenitor cells can differentiate into multiple cancer cell types. Cancer stem cells can therefore spread cancer to other parts of the body. They break off of the primary tumour and enter circulation either vascular or lymphatic and travel round the body. They invade new tissue and begin to form another secondary tumour. Cancer stem cells appear resistant to conventional therapies. Currently, therapies destroy the quick proliferating cells that make up the bulk of the tumour but leave the slow dividing cancer stem cells that can just re-grow the tumour. 

Hanahan and Weinberg published a paper in 200 detailing the 6 traits all cancers cells have and called it the hallmarks of cancer. 1. Sustaining proliferative signalling - growth factor from neighbouring cells is picked up by cell surface receptors and causes an intracellular cascade to activate, leading to cell growth and division. This ensures co-ordinated proliferation. Cancer cells do not need a growth factor activation and can maintain the intracellular cascade without activation. This is due to a mutation that is passed down to each new cell and will eventually form a tumour. 2. Evading growth suppressors - to maintain homeostasis as well as growth factors there are growth suppressors. These prevent uncontrolled growth in the cells. Cancer cells can ignore these growth suppressor signals and continue to divide. 3. resistance to apoptosis- cancer cells often have another mutation in the signalling pathway that initiates apoptosis. 4. enababling replicatve immortality - cells have a natural expiry date when they initiate cell death. Cancer cells do not have this and can divide indfinitely making them immortal. 5. angiogenesis - cancer cells secret molecules that encorage angiogenesis within a tumour to supply nutrients and resources for proliferation. 6. Activating invasion and metastasis - the cancer cells invade the neighbouring tissue and also break away from the primary tumour and travel to other tissues and begin tumour formation there. 

Retroviruses are not lytic or lysogenic viruses. It is different from other viruses in several ways. It is an enveleoped single-stranded RNA virus. It carries 3 specific proteins inside its envelope. Enveloped viruses can enter cells by receptor-mediated endocytosis or direct fusion. Receptor-mediated endocytosis is basically when the virus tricks the cell into letting it through like any other molecule. The HIV retrovirus for example uses direct fusion. The envelope releases the nucleocapsid into the target cells and the nucleocapsid becomes uncoated. This releases everything that was inside the retrovirus coat. The firt specific protein used is reverse transcriptase which reverse transcribes the RNA to make cDNA. This process is repeated again to creat another cDNA strand. Both cDNA strands can come together and create a double stranded DNA. Integrase is the next specific protein used. It removes the 3 prime ends of the double stranded DNA creating sticky ends  and integrate this new HIV DNA into the host DNA. This is called the pro-virus stage. Retroviruses do not have suppressor genes and are therfore not dormant. This means the viral DNA gets transcribed when the host DNA. is transcribed. The viral RNA is transcriped and moves to the cytosol where the protease (3rd specific protein) is synthesised. The virus can now self-assemble with all its components minus the envelope meaning they are immature viruses. These immature viruses take advantage of the host cell membrane and bud off using the membrane as an envelope. The protease in these immature viruses will cleave the other proteins to ensure the virus and proteins inside it are fully functional before it infects a new host cell. 

Retroviruses can activate proto-oncogenes to oncogenes by other methods. If they insert adjacent to a proto-oncogene then expression of that gene comes under the control of the virus promoter or viral regulatory sequences. This is called insertional mutagenesis and converts the proto-oncogenes to oncogenes. The slide indicates two mechanisms of insertional mutagenesis, one called promoter insertion and one called enhancer insertion. For promoter insertion the proto-oncogene expression is derived from the virus promoter. For enhancer insertion, viral promoter regulatory sequences stimulate the activity of the proto-oncogene gene promoter itself. The consequences in each case are to stimulate constitutive expression of the proto-oncogene, a mutagenic event, converting it to an oncogene. The example shown here is for the proto-oncogene C-myc, but the principle applies to many other proto-oncogenes. 

One popular and successful strategy developed to discover human oncogenes was the NIH3T3 cell transformation assay. This assay takes advantage of the fact that mouse NIH3T3 cells very efficiently take up DNA, a process called DNA transfection, and also that these cells can be transformed by the introduction and expression of an oncogene. Identification of transformed NIH3T3 cells is easy as they grow in a dis-organised fashion called a transformed foci.

In this assay, human DNA which is suspected of containing an oncogene is extracted from a human tumor. This DNA is then introduced into NIH3T3 cells. Each cell receives a piece of tumor DNA. If cells pick up the piece of tumor DNA containing an oncogene then they will grow abnormally as easily recognisable transformed foci. DNA is then prepared from transformed foci cells and once again this DNA is introduced into NIH3T3 cells. This time the cells receive a mixture of mouse and human DNA. The amount of human DNA fragments going into cells is dramatically less than last time. Again cells picking up the human oncogene form transformed foci. In order to isolate the oncogene, a genomic library is prepared from the transformed cells. To isolate clones that contain the oncogene, the library is screened with a repetitive DNA probe that is specific to human DNA. Any clones isolated contain a small fragment of human DNA which might contain the oncogene. This requires further analysis to confirm. This strategy led to the isolation of a number of oncogenes directly from human tumors.

A normal cell is at rest in its G0 phase. This cell can now enter the cell cycle. The first phase of the the cell cycle is the G1 phase where growth happens. The is where the cell organelle copies itself. The next phase is the S phase or the synthesis phase. This is when the DNA copies itself. The next phase is the G2 phase where the cell grows again and begins preparation for the M phase. The M phase is mitosis. This is where the cell divides inot to identical daughter cells and are ready to re-enter the cell cycle and start the process again or go into the G0 phase until they are required to replicate. This is a continuous cycle so checkpoints are important to mediate this cycle and ensure it runs smoothly. The first of these checkpoints is at the end of the G1 phase. This checks there are no issues in the cells DNA or the cell itself. The second checkpoint is the G2 checkpoint at the end of the G2 phase. This checks for issues in the cell before it enters the mitotic cycle. There is also a checkpoint in the M phase. When a cell enters the cell cycle it starts to make proteins which drive the cell cycle. The proteins are cyclins and CDKs. Early on in the G1 phase CDK 4 and 6 are made and CyclinD will cause a reaction inside the cell. It will cause E2F to detach from the retinablastoma protein. E2F acts as a transcription factor and allows the cell to move into the S phase of the cell cycle. Before the cell progresses, at the end of the G1 phase  another CDK and cyclin are made: CDK 2 and cyclinE. At the end of the S phase another CDK and cyclin are made: CDK 1 and 2 and cyclinA. At the G2 phase another CDK and cyclin are made: CDK1 and cyclin B. These CDK and cyclin molecules allow the cell to move through the cell cycle to the mitosis phase. If the CDKs and cyclins are in short supply the cell doesn't progress intot he next phase. If there is too much CDK and cyclin, the cell cycle is out of control and continues to divide cells. This is one of the mechanisms of cancer. 

Mutations can occur in the DNA. of cells that result in changes. Poin mutations are a single change in a nucleotide. DNA amplification is when a specific gene becmes amplified and it may be an unfavourable gene. Chromosomal rearrangement when the chromosomes attach to each other where they shouldn't. The last is epigenetic modifications: methylation and acetylation which can causes genes to be silenced or over-active. Through these mutations a normal cell can become cancerous. These cells bypass the checkpoints and grow uncontrolled. This happens through activation of oncogenes e.g. Ras and myc and inactivation of tumour suppressor genes e.g. p53, APC and BACA 1 and BACA 2. 

Normally the about to enter the G1 phase contains DNA coding for a Ras gene which makes the Ras protein. This is an intracellular protein that sits just underneath the plasma membrane. This is next to a growth factor receptor. A ligand stimulates this growth factor receptor which in turn activates the Ras protein. The acctivation of the Ras protein causes a cascade of intracellular phosphorylation leading to the activation of a transcription factor. This transcription factor goes to the DNA and reads the protein encoding genes responsible for cell growth. These proteins, specifically, allow the cell to move from the G1 to the S phase (CDK 2 and cyclinE). If there is a mutation in the Ras gene it results in the synthesis of Ras proteins that are already activated. The pathway no longer requires a ligand to bind to the growth factor receptor and trigger the phosphorylation cascade that activates transcription factor and causes the production of CDK2 and cyclinE. This causes over-production of CDK2 and cyclinE. 

The myc gene normally makes proteins important in the processes of cell growth, surviival and activity. If there is a mutation in the myc gene the cell becomes more cancerous. It increases the cell growth, survival and activity. Over-activation of these genes allow the cell to completely skip the checkpoints that mediate the cell cycle. 

Tumour suppressor genes prevent mutated cells from enterring the cell cycle and proliferating out of control. For example, if a cell gets stopped and the G2 checkpoint because the DNA is abnormal or damaged, it will not be let through into the next phase. Normally in a cell with damaged or abnormal DNA the cell itself will produce the p53 protein. p53 can act like a transcription factor. It reads the DNA and created proteins responsible for cell arrests like p21. p21 is important because it blocks the CDKs and cyclins that mediate the cell cycle and prevent the cell cycle from continuing. p53 also makes proteins that are responsible for cell repair. This means that if the cell is arrested by p21 it could possibly repair itself and continue on its way through the cell cycle. If the cell is unable to repair its own DNA, p53 is also responsivle for making proteins that control apoptosis. If the p53 protein is mutated, it cannot make proteins for apoptosis, repair or arrest. 

The cell cycle has 2 main phases the interphase and the M phase. Mitosis occurs in the M phase and results in the production of 2 daughter cells. The first phase of mitosis is prophase. In prophase the centromeres begin to form the mitotic spindle. There will be 46 chromosomes with 92 chromatids present in prophase. The centromere holds a pair of chromatids together. In prophase the chromosomes become compact as they condense making them visible. The cytoskeleton is dissassembled. The nuclear membrane dstarts to degrade and the nuclear envelope is dispersed. The chromosomes are now free. Prophase then moves to metaphase. The early stage of metaphase is prometaphase. The centrosomes that are forming the mitotic spindle provide microtubules that will connect to the centromere of the chromosome. During prometaphase, the chromosomal micortubules attach to kinetochores of the chromosome. This moves the chromosomes to the middle of the cell. The genetic material itself remains unchanged. The late stage of metaphase is then entered. The chromosomes will be alligned in the centre of the cell and the centrosomes are on opposite sides of the cell forming a huge mitotic spindle using the microtubules. The genetic material is still unchanged. The cell now enters anaphase. The centrosomes pull the chromosomes apart using the microtubules separating the chromatids to each pole. There is now 92 chromosomes with 92 chromatids within the cell. The last phase of mitosis is telopahse. The chromosomes are pulled closer to the poles and a membrane begins to form around them. The nuclear envelope assembles and the organelles reform. The final phase in M phase is cytokinesis. This is where the cell separates into to daughter cells with 46 chromosomes each and 46 chromatids. After cytokinesis the cells can go into the G0 phase or go straight into the G1 phase and begin the cell cycle again. 

In a normal colon, in the colon crypts stem cells will migrate upwards to provide new colon cells. In normal colon cells there is a balance between histone methylation and histone acetylation. Acetylation provides increased access for transcription factors to the DNA. Methylation means there is less access for transcription factors to the DNA. If there is a lot of methylation, some genes may not get activated. The enzyme histone deactylase removes the acetyl groups present on the histone proteins and results in decreased access for the transcription factors to the DNA. On DNA there are promoter regions and non-promotor regions. Promotor regions intitiate the transcription of genes and synthesis of RNA. Non-promoter regions do not contain functional genes. In normal colon cells there is about 70-80% methylation in non-promoter regions. In promoter regions genes are normally not methylated to allow gene activation. In 80% of colon carcinogenesis there is a APC mutation. APC is a tumour suppressor gene as it normally encodes for proteins that mediate cell adhesion and transcription. An APC mutation can result in a stem cell in the colon becoming a potental cancer cell. As this stem cell moves upward from the crypt of the colon and begins to divide uncontrolled it will produce a benign growth (polyp). However, with further mutations in the stem cells. There is activation of the k-ras oncogene in 50-60% of colon cancers. There are also mutations in other tumour suppressor genes. The k-ras gene normally controls cell division and a mutation causes cell proliferation and makes it the k-ras oncogene. The abnormal cells will proliferate unchecked and create an adenoma which is a much larger benign growth. As this tissue is now larger and requiring more resources to support proliferation, angiogenesis occurs. As this process of adenoma development occurs there can also be a mismatch of repair gene inactivation and hypermethylation of the DNA resulting in a mutation rate 100-fold higher than normal. A mutation in the p53 gene is usually a late occurrence in colon carcinogenesis. This mutation causes inhibition of cell arrest, repair and apoptosis. The cancer cell can divide uncontrolled withour being apoptosed. The growth will now cave and destroy the crypt structure within the colon resulting in a malignant growth called a carcinoma. At this stage in colon carcinogenesis there is more histone methylation than acetylation. This means there is less access for transcription factors. This is because histone deacetylase is more active in colon cancer cells. There is also more methylation in the promotor regions of the DNA. There is usually hyper-methylation especially in tumour suppressor genes and DNA repair genes. There is also a decrease in methylation in non-promoter regions of the DNA. The hypermethylation on the promoter region will cause silencing of genes mediating apoptosis, DNA repair and the control of the cell cycle. 

In 2005 the americans launched a similar project to CGP called TCGA. This projects ultimate goal is to determine the molecular signatures of all 200 or so cancers ie to determine all the genetic abnormalities present in all cancers. Just like CGP, a pilot or feasibility study was initially been launched. The fate of the project overall was dependent on the success of the pilot study. The project has two components, firstly to focus on large scale genomic changes in cancer. Cancers are examined for the presence of chromosome rearrangements, changes in gene copy numbers and epigenetic changes. Epigenetic changes are chemical changes in nucleotides such as methylation that result in a change in expression of associated genes, but are not due to mutations. The regions identified by these analyses will subsequently be targeted for sequencing. Also, like CGP this study targets sequencing of gene families already associated with cancer. The pilot study was a 3 year multi-million dollar programme focusing on 3 cancers. These cancers were selected for several reasons. One, they have an incidence of 210,000 US citizens annually, two they all have a poor prognosis and three there are already available high quality collections of tissue samples derived from patients with these cancers. The ultimate goal of this project, like CGP, is to identify new targets for therapy that might improve treatment of cancer. Eligible patients are be asked to donate a small portion of tumour tissue that has been removed as part of their cancer treatment. Several methods will be used to analyse the genetic material and the results will be publicly available, preserving patient anonymity. Tissue samples are catalogued and stored along with patient records. Genomes are characterised by a variety of technologies to identify regions to be targeted for sequencing by genome sequencing centres. The information generated is entered into public data bases. TCGA will describe genetic fingerprints of particular cancers. Researchers will evaluate how the information can be used in cancer treatment. The ultimate goal is to provide clinicians with better ways of treating the disease.

A very ambitious project was launched in the UK in 2001, called the cancer genome project. The basis of the project is that the previous 20 years had taught us that mutations in specific genes are the drivers for cancer development. We now have a draft blueprint of humans as a result of sequencing of the human genome. This provides a resource for the sequence of normal genes and therefore should facilitate the identification of abnormal genes. Establishing this sequence required the development of high throughput technologies for rapidly and efficiently sequencing large pieces of DNA. In addition, the project required the development of bioinformatics to compose, organise and store vast amounts of sequence information. The cancer genome project aims to take advantage of the high throughput and bioinformatics technologies as well as the sequence itself. The project seeks to identify gene mutations present in human cancers. These mutations are somatically acquired, are not present in normal tissue but only present in the tumor DNA of individuals. The study focuses primarily on the identification of small intragenic mutations, ie mutations within the coding region of genes as this is where the majority of mutations of oncogenes and TSG’s have been found previously. In addition to coding sequence, splice junction sequence is also examined as such mutations would cause changes which prevent correct mRNA processing occurring. Such mutations can result in the production of truncated proteins, similar to those generated by nonsense mutations found in coding regions of some tumour suppressor genes. The ambitious aim of the project is to determine the sequence of all human genes from both tumor DNA and normal DNA from the same individual. This strategy can be undertaken for all human tumor types. The results of this analysis are shared by making it publicly available. The data base created for this purpose is called COSMIC, which is a catalogue of somatic mutations in cancer. All genes sequenced in each type of cancer can be viewed in this data base, whether mutated or not. COSMIC also contains already available data obtained from previously published literature. The same strategy being applied here can be applied to the detection of germline mutations in other human genetic diseases. The strategy is very simple, but this should not disguise the fact that this is an enormously ambitious project. The available sequence of the human genome can be used to design primers for the PCR amplification of all human genes. The primers are designed to flank genes and splice junctions. An example of the strategy for one gene is shown in the diagram. Primers are used in separate reactions to amplify DNA fragments from tumor DNA and normal DNA of the same individual. The DNA is sequenced and then compared for mutations. If a mutation is identified, it must be confirmed. Initially by repeating the process on the same tumorr and normal DNA samples and subsequently repeating the process on other samples of the same tumorr type. This will determine if the mutation is common to a particular cancer.

The CGP observation that BRAF is involved in melanoma initiated major activity in this area around the world. We now know that essentially two pathways are disrupted in melanoma, the MAP Kinase pathway and the activities of the tumor suppressors p53 and pRB. Nearly all melanomas have mutations in either, NRAS, BRAF or BRAF and PTEN. RAS regulates cell proliferation through the MAP kinase pathway and survival through the AKT pathway. The AKT pathway is also regulated by the tumorr suppressor protein PTEN. If NRAS is mutated then BRAF is not and vice-versa, showing the mutations are mutually exclusive. Mutations are also found in the CDKN2A (Ink4) locus that contains both the p16 and ARF genes. These normally regulate pRB and p53. The CDKN2A mutations are very common but the most common are BRAF. This molecular knowledge of the abnormalities in malignant melanoma, stemming largely from CGP, has resulted in the development of several drugs that are currently in clinical trials, all in the space of 5 years. Sorafenib is a kinase inhibitor which can inhibit BRAF and is being used in combination with chemotherapy in clinical trials for the treatment of malignant melanoma.

The oncogene identified in RSV was called src, derived from sarcoma. Like most oncogenes and other genes it has a three-letter acronym. The oncogene originally discovered was found in the retrovirus and this version of the gene was called v-src for viral src. A normal version of this same gene was discovered in normal chicken cells. The normal cellular version was called c-src for cellular src. There are orthologs of the normal version of this gene conserved in other species as well, including humans. Both v-src and c-src encode a cytoplasmic protein tyrosine kinase. They share a kinase catalytic domain and other SH2/SH3 domains that are required for interaction with other cytoplasmic proteins that are substrates for phosphorylation. V-src is a mutated version of C-src and the major difference between the two proteins is that V-src lacks a c-terminal region that normally regulates catalytic activity. C-src normally has a role in stimulating cell proliferation and it does this in a manner that is regulated. V-src also stimulates cell proliferation but this activity is no longer regulated. V-src constantly phosphorylates many cell proteins, including those which are not normally substrates for the normal C-src kinase, and this results in stimulation of cell division.

This example led to the discovery of an oncogene called V-abl. The normal cellular counterpart of this gene is called c-abl.

Mice were infected with a replication competent non-acutely transforming retrovirus called Murine Leukaemia Virus. This virus has three genes normally found in retroviruses which encode a DNA polymerase (reverse transcriptase) and two viral structural proteins called group specific antigen or gag and an envelope protein called env. When the virus infects a mouse it replicates. In some cases and after a long lag phase the mice develop a tumor. In these studies virus was re-isolated from the tumor and used to infect another mouse. On this occasion the recipient mouse rapidly developed a tumor. The virus had changed from a non-acute transforming virus to an acutely transforming retrovirus, ie one that causes tumors very quickly. This is a very rare event. Molecular analysis of the re-isolated virus showed that some viral genes of the original murine leukaemia virus had been partially or completely deleted and replaced by new sequences. The new sequence was the V-abl gene that the virus had picked up by recombination with the C-abl gene located in infected mouse cells. V-abl encodes a deregulated version of C-abl. Again this gene encodes a tyrosine kinase and it phosphorylates cellular protein substrates that stimulate cell proliferation.

A model of how this recombination event of retroviral transduction might occur is also shown in the slide. When retroviral RNA enters an infected target cell it is immediately converted to double stranded DNA. This DNA then inserts essentially randomly into the genome. This event introduces a mutation. Very rarely the insertion occurs adjacent to a gene that can positively stimulate cell proliferation (proto-oncogene). Again, very rarely deletions can occur between part of the viral sequences and the adjacent gene resulting in creation of a fusion gene whose transcription is now under the control of the very strong virus promoter. Fusion gene transcripts are produced that are processed by the deletion of introns. The mature transcript might rarely get the opportunity to recombine with the transcripts of another normal retrovirus transcript in the same cell. This recombination event creates a new viral transcript that has some virus and cellular genes. In this example the cellular gene is v-abl. The new viral transcript is now a substrate for packaging into an infectious virus particle which when released from the cell is able to infect and transform other cells