DNA Damage & Carcinogenesis (MSc Cancer, UCL)

UCL Cancer Institute Faculty of Medical Sciences DNA Damage and Carcinogenesis Prof John Hartley [email protected] MSc Cancer 2 Part 1 Spontaneous DNA Damage Learning Outcomes Following this topic, students should be able to: • Distinguish between the two main categories of DNA damage –...

UCL Cancer Institute Faculty of Medical Sciences DNA Damage and Carcinogenesis Prof John Hartley [email protected] MSc Cancer 2 Part 1 Spontaneous DNA Damage Learning Outcomes Following this topic, students should be able to: • Distinguish between the two main categories of DNA damage – spontaneous (endogenous) and environmental (exogenous) • Understand the main types and causes of spontaneous damage to DNA 3 4 DNA • As the genetic material, we assume DNA must be very stable • In reality, it is constantly being damaged and can accumulate damage over its lifetime • It is the only molecule that relies solely on repair of existing molecules Compared to just replacing damaged proteins with new ones 5 The Balance of Life In his 2001 review in Nature Reviews Cancer, Errol Friedberg showed this figure, describing how life requires a finely tuned balance between the avoidance of mutations by DNA repair and other cellular responses to DNA damage that affect genetic stability, and the generation and persistence of mutations to allow genetic diversity. There are phenotypic consequences of the latter, including cancer. evolution Categories of DNA Damage - 1 DNA damage can be broadly subdivided into two main categories: • Spontaneous (endogenous) • Arising during DNA replication, DNA repair and DNA rearrangement • Resulting from alterations in the chemistry of DNA bases rearrangement of the positions of protons and electrons within a – Tautomeric shifts Spontaneous molecule - forming an isomer – Deamination of bases – Depurination, depyrimidination 6 Categories of DNA Damage - 2 • Environmental (exogenous) • Exposure to chemical mutagens e.g. – Alkylating agents – Polycyclic aromatic hydrocarbons – Aflatoxins • Exposure to physical agent mutagens e.g. – Ultraviolet light – Ionising radiation eg x-ray or therapeutic radiotherapy 7 Categories of DNA Damage - Question Which category contributes more to damage of DNA, spontaneous or environmental? Answer: endogenous biochemical processes usually make far greater contributions to genome mutation than do exogenous mutagens. These processes occur at a higher rate - giving higher risk for mutations just due to the number of times the cell is undergoing the process (eg DNA repair, translation etc.) 8 Proofreading by DNA Polymerases Many DNA polymerases have a proofreading ability to minimize base misincorporation 9 Proofreading by DNA Polymerases and cancer incidence • Aspartic acid to alanine change at residue 400 in the proofreading domain of DNA polymerase delta (responsible for the bulk of leading and lagging strand synthesis) • Deaths of the mutant homozygotes were due to malignancies (lymphomas, skin squamous cell carcinomas and lung carcinomas). can copy the DNA but can’t proof read showed the mice dying at an earlier age - because they generated mutations constantly 10 DNA polymerase errors and mismatch repair • DNA polymerases including polymerase delta, can “stutter” or skip a base when copying repeated sequences of DNA in the template strand. • Mismatch repair proteins recognize and repair these mistakes 11 DNA Replication Error Frequency rates for one cycle of replication: • DNA polymerases have an Without proofreading incorporation error frequency of 1 in 105 copied nucleotides • 3’ to 5’ proofreading by polymerases reduces this to 1 in 107 copied nucleotides • The mismatch repair system reduces this to 1 in 109 copied nucleotides A very low mutation rate of 1 nucleotide per billion synthesised! 12 Formation of DNA strand breaks during replication 13 During replication, DNA is vulnerable to breakage in the single strand region near the replication fork • The single strand break is functionally equivalent to a DNA double strand break. • Estimated that 10 double strand breaks are formed per cell during S-phase. Single stranded DNA is susceptible to breaks • Failure to repair could lead to chromosomal breaks and translocations. 14 Rare Tautomeric Forms of DNA Bases - 1 • Each of the common bases in DNA can undergo a transient rearrangement, termed a tautomeric shift. • This alters its base pairing properties. 3 of the bases lie in this configuration: C T G (Except A) A G and C lie in this configuration Changes the ability to H bond Rare Tautomeric Forms of DNA Bases - 2 Normal form • If a base in the template strand exists in its rare form during DNA replication, misincorporation in the daughter strand can result. incorrect bases are incorporated when the base is in a different tautomeric form (A can pair with C and G can pair with T) 15 Deamination of Bases - 1 • The exocyclic amine groups of cytosine, adenine and guanine can be lost. • This deamination results in uracil, hypoxanthine and xanthine, respectively. • Uracil may be read as thymine during replication causing a C-T point mutation known as a transition mutation. 16 Deamination can occur due to fluctuations in pH and temperature Natural base but for RNA Mutagenic - can base pair with C Non natural bases unable to base pair so it arrests DNA synthesis 17 Deamination of Bases - 2 • The bases generated by deamination are all non-natural to DNA and can easily be recognized by DNA repair mechanisms. • If they evade repair, they are potential sources of mutation. can be found in CpG islands Natural base so proves difficult to recognise the mistake • Deamination of 5-methylcytosine yields thymine. • This is a problem for DNA repair since thymine is a normal DNA base. • The T:G base pair could lead to a C-T transition. ? 18 Depurination • The glycosidic bond between a purine base and a deoxyribose group in DNA can break spontaneously. • This is called depurination. • It has been estimated that 10,000 purines can be lost each day in a mammalian cell. • Depyrimidination can occur, but at a much lower rate. For pyrimidines: T and C This happens less as the bond is more stable Purines: G and A Depurination is a loss of A or G DNA becomes an apurinic site Summary • DNA molecules are constantly being damaged. • Spontaneous (endogenous) damage arising during DNA replication, DNA repair and DNA re-arrangement, or resulting from alterations in the chemistry of the DNA bases, is the largest contribution to damage overall. • Spontaneous damage could be as high as 105 lesions per (Damages) cell per day! 19 20 Part 2 Environmental DNA Damage Learning Outcomes Following this topic, students should be able to: • Understand the main types and causes of environmental DNA damage • Understand the biological consequences of DNA alkylation • Understand the role of metabolism in the formation of some carcinogens 21 DNA alkylation Coloured atoms are susceptible to alkylation - could be simple methylation or a more complex addition N3 of A N7 of G Both sites of preferred alkylation • not about accessibility (only applies for a bulky alkylating agent) - it is about the charge that is produced by the intermediate that is + charged 22 • Many sites on DNA (shown in pink and red) are susceptible to alkylation by simple alkylating agents • The guanine-N7 position (in the DNA major groove) and adenine-N3 position (in the DNA minor groove) are the major sites of alkylation Why guanine-N7 and adenine-N3? 23 - Most negative Molecular electrostatic potential maps of the DNA bases show that the guanine-N7 and adenine-N3 positions are the most negative sites on the DNA bases. Makes the alkylating intermediate more attracted to the most negative site Biological consequences of DNA alkylation - 1 24 For simple monofunctional alkylating agents e.g. methylating and ethylating agents: does destabilise the bond between the purine and the making it susceptible to deprivation - but overall is • 7-alkylguanine is the major product backbone, pretty harmless – Base pairing is unaltered – It is a relatively harmless lesion – The glycosidic bond can slowly hydrolyse to give an apurinic site • 3-alkyadenine is formed less frequently – It is in the DNA minor groove inhibit the enzyme - and the cell arrests and if not – It blocks the progress of DNA polymerase Can dealt with, the cell dies – It is the major toxic monofunctional alkylation Biological consequences of DNA alkylation - 2 25 eg methyl • O6-alkylguanine is formed even less frequently than N7Because the O6 is involved in H bonding - when for example, can only form 2 H bonds to C alkylguanine and N3-alkyladenine methylated instead of 3 – The base is locked in the enol tautomeric form – It can base pair with either C or T (see next slide) as T needs only 2 H bonds – It can result in a G to A transition mutation which is critical in carcinogenesis • Although a lethal event can occur once in every few thousand adducts, a mutation can occur once in every 8 adducts O6-methylguanine in DNA Not a problem G:C mismatch highly mutagenic/ carcinogenic O6-MeG : T • O6-MeG : C • O6-methylguanine can base pair with either C or T • Methylnitrosourea (MNU) is highly mutagenic • It can produce mammary carcinomas in rats • This can result from a single base G to A transition in the HRas gene resulting from the formation of O6-methylguanine 26 27 Bifunctional alkylating agents • Bifunctional alkylating agents can react with two nucleophilic Can produce 2 covalent bonds - by centers with the potential to produce crosslinks. producing 2 intermediates (+ charged) • Crosslinks onDNA DNA can be interstrand, intrastrand or DNA-protein adducts formed by bifunctional agents Both react with DNA on the same strand DNA-protein crosslink intrastrand crosslink monoadduct one with DNA and one with water (major adduct interstrand crosslink different strand • A DNA interstrand crosslink, for example between two guanine-N7 positions is highly cytotoxic Inter-strand crosslink can make it hard for the 2 strands to open - as it covalently binds the two strands together via 2 base pairs - the cell is unable to unwind the DNA and replicate A few crosslinks that remain unprepared is enough to kill the cell Metabolism of carcinogens 28 Carcinogens when ingested are not yet carcinogens - but are called procarcinogens ad the process is during metabolism • Chemicals foreign to the body (xenobiotics) can be metabolised by systems intended for detoxification and excretion. • By an accident of chemistry, a reactive electrophile can be produced which can attack DNA A chemically inert, unreactive procarcinogen can therefore be converted into a highly reactive ultimate carcinogen Phase 1 enzymes take the substrate to create a “handle” - it does this by creating reactive intermediates, for the phase 2 enzyme to be able to excrete the product For some chemicals when the reactive intermediate is produced - it can react with the DNA (now it has been converted into a carcinogen) Nitrosamines found in tobacco - 1 • Nitrosamines are formed during the processing, curing and storage of tobacco. • The are present in tobacco smoke and betel quid. • Linked to adenocarcinoma of the lung in humans. • Examples are N’-nitrosonornicotine (NNN) and 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) They are pro-carcinogens 29 30 Nitrosamines found in tobacco - 2 highly reactive intermediates NNN and NNK are extensively metabolized and can produce highly Relationship between DNA reactive species which can react with DNA adduct formation and Metabolism of NNK and NNN tumour incidence in rats treated with NNK Nitrosamines found in tobacco - 3 • There is a clear relationship between DNA adduct formation Metabolism of (O6-methylguanine) and lung and cancer incidence NNK NNN in rats treated with NNK Belinsky et al, Cancer Res., 1990, 50: 3772-3780 31 Rela ad tum B 1 Aflatoxin and liver carcinogenesis - 1 Fungal toxin - made by certain moulds incredibly high incidence of the liver cancer • area is known to be very humid found to have the fungal toxin aflatoxin B1 32 • Within the Jiansu province of eastern China, the incidence of hepatocellular carcinoma is much Liver cancer higher in the very humid Qidong peninsular (arrow). • The fungal toxin aflatoxin B1 is made by moulds that grow on peanuts and grains stored improperly, particularly in humid areas. • Aflatoxin B1 is a potent mutagen. Aflatoxin and liver carcinogenesis - 2 • Activation of aflatoxin B1 by cytochrome P450s results in a highly reactive form. • This may be detoxified by through several routes. • Alternatively, it can react with DNA forming a bulky adduct on the N7 position of guanine. 33 Epoxide is added highly mutagenic as it is not a small adduct on the N7 position anymore - it is a bulky addition 34 Percivall Pott • Percival Pott was an English surgeon. • He was the first person to suggest that a cancer might be caused by an environmental agent. • He noted the high incidence of scrotal cancer in chimney sweeps and suggested that this was due to occupational exposure to a component of soot. 35 Polycyclic aromatic hydrocarbons • Polycyclic aromatic hydrocarbons (PAHs) are found in tobacco smoke and polluted environments. • A common PAH is benzo[a]pyrene. • It is extensively metabolized and following two successive oxidation reactions mediated by cytochrome p450s it can form the ultimate carcinogen benzo[a]pyrenediolepoxide (7,8-diol:9,10-epox in figure) which can form adducts on DNA. Metabol benzo-[a The epoxide is highly reactive with DNA - producing this carcinogen Benzo[a]pyrenediolepoxide DNA adduct • The highly reactive benzo[a]pyrenediolepoxide can form bulky adducts on the exocyclic amino group of guanine in the minor groove of DNA these bulky adducts occur mainly in the minor groove of the DNA due to its size • results in a G T transversion 36 Clues to the identity of the mutagenic agent 37 • Point mutations in the p53 alleles of cancer cells can provide clues to the identity of the causative carcinogenic agent. • In the example, the G-to-T transversion increases in proportion in lung cancers from smokers and people exposed to smoky coal emissions. • The G-to-T transversion is the mutation usually induced by benzo[a]pyrene. Hotspots of specific mutations can give clues to the type of mutagen Ultraviolet (UV) radiation 38 UV Light • Ultraviolet light from the sun is the most common source of environmental radiation. • DNA absorbs UV light with a maximum absorbance at 260nm. results in energy changes • Covalent crosslinks can form between adjacent pyrimidine bases in DNA. Ultraviolet radiation spectrum 105 UV photoproducts/day in expo Products of UV irradiation of DNA 39 Cyclobutane pyrimidine dimers are the major photoproducts • More than 60% of the pyrimidine dimers are TT, often called thymine dimers. • The remainder are CT dimers (30%) and CC dimers. • These structures are relatively stable and persist unless they are recognised and repaired. As many as 105 DNA photoproducts per day in exposed kerationcytes There isn’t any extra atoms - just due to the energy change. When there are 2 pyrimidines beside each other, the energy change causes the movement of the C=C to open and one bond goes to the other pyrimidine - producing a dimer • this dimer can distort the DNA but is stable and continues until fixed Photosensitisation 40 inert chemicals can absorb UV and become reactive to react with • DNA damage can result from Some the DNA Photosensitization light being absorbed by a sensitiser molecule which can natural product Flat and planar then transfer energy to the • used as a Monoadduct molecule topical agent bases of DNA. Monoadduct • Psoralens are flat, planar molecules that can intercalate Interstrand into DNA and upon UV photoCross-link activation form covalent adducts to pyrimidine bases, including interstrand crosslinks. Psoralen can slip between the bases, which isn’t a problem when it is inert - but can become activated by the UV which results in binding between the psoralen and the bases can happen twice - forming a cross link which kills the cell Radiation Ionising radiation • Exposure to ionising radiation (IR) can be therapeutic, diagnostic or occupational. • DNA damage can occur by direct or indirect action. • Indirect action, e.g. stripping electrons from water molecules, results in free radicals, generating reactive oxygen species (ROS) which can damage DNA. therapeutic, diagnostic and occupational exposure Energy of the IR causes damage of the DNA F 41 The radicals damage the DNA DN Cro DNA damage produced by ionising radiation Radical Formation Changes to the bases Single-Strand Break re Base Oxidation O Double-Strand Break DNA-Protein Cross-Link 42 • DNA damage, either produced directly or indirectly by IR, can be of many types. • These include DNA single- and double-strand breaks. • Double strand breaks are cytotoxic, difficult to repair and may generate chromosome breaks. 43 Oxidation of bases in DNA • ROS produced by IR (or more commonly by endogenous biochemical processes in cells) can result in the oxidation of DNA bases. • A frequent oxidation reaction involves deoxyguaninosine which is oxidized to 8-oxo-deoxyguanosine (8-oxo-dG). • 8-oxo-dG can mispair with deoxyadenosine, which can lead to a G to T transversion. can happen spontaneously but also by radiation to the cell Will now base pair with another purine - resulting in a mismatch Summary 44 • DNA damage can be caused by many different types and classes of environmental chemical agent, either directly, or following metabolism to the ultimate carcinogen • Physical agents including ionising radiation, and in particular ultraviolet light, are important sources of environmental DNA damage. 45 Part 3 The Multistage Model of Carcinogenesis Learning Outcomes Following this topic, students should be able to: • Understand the concept of the multistage model of carcinogenesis 46 Epidemiology 47 • Cancer is largely a disease of old age • Most human cancers develop over many decades • Logarithmic increase best explained by need for several mutations to accumulate • Evidence for a multistage model of carcinogenesis • Cancer incidence is increasing due to increased longevity Most cancers develop over many decades Smoking and Lung Cancer When comparing the annual global consumption of cigarettes (red curve) with the recorded and predicted annual worldwide mortality from lung cancer, there is an approximate 30-year lag. 48 Cancer incidence and carcinogen exposure 49 The cumulative exposure to a carcinogen, rather than age at first exposure, determines the likelihood of developing cancer • The left graph shows the cumulative risk of developing mesothelioma among insulation workers exposed to asbestos. • The right graph shown the cumulative risk of developing skin tumours in mice after administration of a carcinogen mesothelioma - caused by exposure to asbestos Histopathological evidence • Histology provides evidence of multistep tumourigenesis. • Colon cancer is the classic example where the various types of abnormal tissue can be arranged in succession of increasing abnormality. 50 Multi-step tumourigenesis in different sites • Like colon cancer, the pathogenesis of other carcinomas follows similar mechanisms in other epithelial tissues. • Nomenclatures differ, but multi-step tumourigenesis progresses along similar paths, involving similar histological entities. 51 Colon cancer progression • The steps in colon cancer development can be rationalised by an ordered succession of genetic changes as cells progress toward malignancy. • An example of such changes is shown below. 52 Key experiment - done around 30 years ago - performed at the national cancer institute in the USA 53 Inducing skin carcinomas in mice - 1 • 7,12-dimethylbenz[a]anthracene (DMBA) is a highly carcinogenic component of tar (similar to benzo[a]pyrene). • Daily painting of DMBA on a patch of mouse skin results in skin cancer after several months. Tumour initiator • 12-O-tetradecanoylphorbol-13-acetate (TPA), also known as phorbol-12-myristate13-acetate (PMA) is an irritant extracted carcinogenic - it is a from the seeds of the croton plant. not mitogenic agent • It activates protein kinase C-a in cells. tumour promoter Highlighting that cancer can arise form the right level of tumour initiator with a tumour promoter such as these Inducing skin carcinomas in mice - 2 54 Experimental protocols involving ‘initiator’ DMBA and ‘promoter’ TPA were very revealing about the mechanisms of skin cancer induction • A single painting of DMBA (A), or multiple paintings with TPA (B), on the skin caused no change after 3 months. • However, multiple paintings with TPA after a single painting of DMBA (on the same area of skin) resulted in papillomas Pre-cancerous lesion after 4 to 8 weeks (C). 55 Inducing skin carcinomas in mice - 3 • If the area of skin painted by DMBA and TPA is not the same, no papillomas appear (D). • If TPA painting is halted soon after papillomas appear they can regress (E). • However, if TPA painting is continued for several months, some papillomas will persist (F). reversible Doesn’t reverse even after stopping the promoter painting • If a papilloma is then treated again with DMBA, it can progress to a carcinoma, even in the absence of further TPA treatment (G). Initiation and promotion of skin carcinomas in mice • The experimental observations described can be rationalised as shown here. when you paint with the initiator - the cells have the mutagen not yet understood why the initiator cells respond more the initiator cells are growing and dividing at a higher rate 56 The three stages in tumourigenesis • Initiation – Can result from a single exposure to a carcinogen and is irreversible • Promotion – Results from repeated application and can be reversible • Progression – May result from further mutagen exposure or further stimulation 57 Genes and proteins involved in mouse skin carcinogenesis • Initiation and promotion can be understood at the genetic level. • This involves mutation of the ras proto-oncogene and the p53 tumour suppressor gene by the initiating agent. • Repeated stimulation is provided by the promoting agent to allow the initiated cell to proliferate. The combination of the mutated ras and p53 gene is what finally causes the carcinoma 58 Tumour initiating agents • These are mutagenic agents causing DNA damage leading to mutation • If not repaired the mutation may be replicated and passed on to daughter cells. • Examples are DMBA, aflatoxin, benzo[a]pyrene, UV, X-rays. • Irreversible, a single exposure may suffice. • Effects are additive. • May act as a complete carcinogen causing promotion and progression through additional mutation. 59 Tumour promoting agents These are mitogenic agents, not mutagenic. Stimulate proliferation of mutated cells. Must follow mutagenic agent and requires prolonged exposure. Establishes clone of initiated cells. Under appropriate conditions mutated cells gain a selective growth advantage. • Examples include phorbol esters, hormones, dioxins, barbiturates. • Effect is reversible at early stages. • Not additive. • • • • • 60 Progression • • • • • Stage of increasing genetic instability. Increased susceptibility to further mutations. Subclones with malignant attributes. Both further mutation and proliferation may be involved. Initiators are effective progressors. 61 Summary • The process of tumour formation is complex and many lines of evidence point to a multistage model for carcinogenesis. • Three steps in tumourigenesis are initiation, promotion and progression. 62 63 Part 4 Measuring DNA Damage Learning Outcomes Following this topic, students should be able to: • Describe the single cell gel electrophoresis (comet) assay and its uses. 64 Single Cell Gel Electrophoresis (Comet) Assay What does it measure? It is a sensitive method to measure DNA damage and repair at an individual cell level its the only measure than is very sensitive to Environmental exposure 65 Application of the Comet Assay • The assay has applications in: – Genetic toxicology – Human biomonitoring – Molecular epidemiology – Ecotoxicology – DNA damage and repair studies – Drug monitoring 66 67 What are its advantages? • The assay is: – Simple – Sensitive – Versatile – Quick – Cheap • It provides individual cell data • It requires only a small sample size Allows you to investigate heterogeneity 68 What does it measure? • The comet assay is a method for measuring DNA strand breaks. • By altering the assay conditions it can measure: although not a big problem for the cell - can be indicative of damage – DNA single strand breaks (alkaline pH) – DNA double strand breaks (‘neutral’ pH) – DNA alkali labile sites (alkaline pH) – DNA base modifications (lesion-specific enzymes) – DNA interstrand cross-links (alkaline pH/irradiation) • Other applcations include comet FISH, measurement of apoptosis and detection of specific adducts with antibodies 69 What types of cells can be used? • Any DNA-containing cells can be used provided a single cell suspension can be obtained. • Types of cells commonly studied are: So not red blood cells – White blood cells – Buccal cells – In vitro cultured cells – Spermatozoa What are the main steps in the assay? Single cell suspension Unwind/relax DNA pH differs Also degrades all the RNA Suspend in a thin agarose gel on a microscope slide Lyse to remove cellular of everything else but the proteins and lipids rid nucleic acid Stained with ethidium bromide Electrophoresis in an alkaline solution Visualisation and image analysis 70 Neutralisation From the running buffer Stain DNA with fluorescent dye What does the end result look like? Can be quantified by using the amount of fluorescent in the breaks 71 • Smaller fragments of DNA create the comet ‘tail’ having moved away from the high molecular weight DNA in the comet ‘head’. • The example shown is of cells that have been irradiated with X-rays to introduce random DNA strand breaks when the DNA has no breaks - it will try to move to the + side of the electrode but because it is such a heavy molecule and is suspended in the solution, it will not move so when movement is seen - there is some breaks that have been able to move (small fragments move more vs big) How do we quantitate the DNA damage? - 1 • Imaging software determines the size and fluorescence of the ‘head’ and the ‘tail’ of each of the cell comets. very sensitive linear relationship between the strand breaks and the tail movement 72 How do we quantitate the DNA damage? - 2 • Measurements include: – Tail length – % DNA in the tail – Tail moment (% DNA in tail x tail length) – Olive tail moment (% DNA in tail x distance between the means of head and tail distributions The bigger the number - the more DNA damage is present 73 Comet assay result examples 70 60 don’t have any damage - but there is background damage Frequency 50 40 30 20 10 0 0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9-10 10-11 11-12 12-13 13-14 14-15 15-16 16-17 Olive tail moment (µm) 70 60 All the cells have strand breaks Frequency 50 40 30 20 10 0 0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9-10 10-11 11-12 12-13 13-14 14-15 15-16 16-17 Olive tail moment (µm) 50 45 Frequency 40 some cells are damaged but not others 35 30 25 20 15 10 5 0 0-1 1-2 2-3 3-4 4-5 5-6 6-7 7-8 8-9 9-10 10-11 11-12 12-13 13-14 14-15 15-16 16-17 Olive tail moment (µm) Can be measured over time - to track cell DNA repair 1. A population of cells showing a background level of DNA strand breaks. 2. The same cells following irradiation with 12Gy X-rays to introduce a fixed level of random DNA strand breaks. 3. Cells exposed to a mutagen that has caused DNA strand breaks in a sub-set of cells showing heterogeneity of response. 74 Radiation response 75 Comet assay radiation response 25 70 60 20 Tail Moment % DNA 50 40 30 15 10 20 5 R2 = 0.9852 R2 = 0.9584 10 0 0 0 5 10 15 20 Gy % DNA in the tail % DNA in Tail 25 0 5 10 15 Gy tail moment Tail Moment 20 25 How do we measure DNA interstrand cross-links? Single cell suspension Suspend in a thin agarose gel on a microscope slide Irradiate cells Unwind/relax DNA Electrophoresis Visualisation and image analysis Lyse to remove cellular proteins and lipids Neutralisation Stain DNA with fluorescent dye 76 Measurement of DNA interstrand cross-links - 1 a) Untreated cells, no irradiation b) Untreated cells + 12Gy X-rays c) DNA cross-linking agent treated cells, no irradiation d) DNA cross-linking agent treated cells + 12Gy X-rays DNA interstrand crosslinks retard the migration of irradiated DNA fragments and is quantitated as the % decrease in tail moment strands of DNA are covalently linked so they can’t move as far 77 Measurement of DNA interstrand cross-links - 2 100 75 % decrease in tail 50 moment 25 0 0 25 50 75 100 Dose of cross-linking drug chlorambucil (µM) There is a linear relationship between the dose of a DNA interstrand cross-linking agent and the % decrease in tail moment measured by the comet assay in irradiated cells. 78 Summary • The single cell gel electrophoresis (comet) assay is a sensitive assay to measure DNA strand breaks at a single cell level. • The assay can be adapted to measure many different types of DNA damage including DNA single and double strand breaks and DNA interstrand cross-links. 79