DNA damage and cancer

What is DNA damage?

Modification in the molecular structure of genetic material

What are mutations?

Permanent alteration in the genetic sequence

• Point mutations e.g., base substitutions A to G or chromosome level changes (deletions etc).

From DNA damage to cancer

DNA damage can become fixed as a mutation if not repaired or the abnormal cell is not removed from the population (apoptosis).

Carcinogenesis

• A complex, multi-step process, requiring an accumulation of changes

• The simplest view describes carcinogenesis as initiation, promotion and malignant conversion (progression)

• Oncogenes, tumour suppressor genes

DNA sources of damage

• Endogenous

• Exogenous

Spontaneous DNA damage

Deamination:

• Loss of NH₂

• Can lead to a base change

Depurination (G and A):

• Bond linking base to deoxyribose breaks spontaneously → apurinic site (lost base)

Depyrimidination is the equivalent for C and T and is less prevalent than depurination

Endogenous biochemical processes

• Reactive oxygen species (ROS) can lead to oxidative DNA damage

• ROS are by-products of metabolism and inflammation

• Radiolysis of intracellular H₂O → free radicals which recombine to form ROS, e.g., superoxide, hydrogen peroxide

• Strongly implicated in aetiology of cancer

• The majority of studies show elevated levels of oxidatively modified DNA lesions in many tumours

8-oxoguanine frequently mis-pairs with adenine during replication → can lead to mutation

DNA replication errors threaten cell genomes

• Cells can undergo division 50-60 times over an average human lifetime

• Each cell division requires incorporation of ~6 × 10⁹ new nucleotides

• Mutations induced by replication errors are termed mismatched

DNA replication

• DNA polymerase (DNA synthesis enzyme) has a proofreading capacity, detection of mismatched DNA bases

• However, slippage of the proof-reading enzyme, DNA polymerase, along the DNA strand → errors → mutations

• Common in DNA regions carrying repeat sequences

• Mismatch repair (MMR) monitors recently synthesised DNA to detect errors overlooked by proofreading

Ultraviolet (UV) Radiation

• UV radiation frequently forms stable pyrimidine dimers

• Mutagenic properties of pyrimidine dimers demonstrated dramatically by the spectrum of CC and TT mutations in p53

Benzo(a)pyrene

A potent carcinogen

Sources of exposure:

• Cigarette smoke, chargrilled meats, vehicle exhaust fumes

• Metabolised by cytochrome P-450 (CYP) enzymes

• Forms ultimate carcinogen BPDE after activation of Phase I CYP’s

• Smoking accounts for more than ¼ cancer deaths, and nearly 1/5th of all cancer cases

Cellular defences to protect DNA

• Physical barriers

• Detoxification

• DNA repair

• Apoptosis

Physical barriers

• Membranes e.g., higher organisms have a nuclear and mitochondrial membranes to protect DNA

• The stem cells present in the small intestine and colon are protected from the contents of lumen by a mucus barrier secreted by cells in the crypt

Detoxification

ROS and other free radical can be detoxified by:

• Vitamins - E and A, beta-carotene

• Cellular antioxidant e.g., glutathione

• ‘scavenging’ enzymes e.g., superoxide dismutase (SOD), catalase, and peroxidases

• Each daily portion of fruit or veg halves the risk of oral cancer, reduces risk of squamous cell carcinoma of the oesophagus by ~20%, and of stomach cancer by ~30%

DNA repair

(a) Reversal of base damage (direct repair)
(b) Excision of base damage

• Nucleotide excision repair (NER)

• Base excision repair (BER)

• Mismatch repair (MMR)

c) Double strand break (DSB) repair

Reversal of base damage (direct repair)

• Simplest DNA repair mechanism - single enzyme consisting of a single polypeptide chain catalyses a single step reaction

• Normal structure is restored

• E.g., O⁶ - methylguanine DNA methyltransferase (MGMT) transfers methyl group to a cysteine residue on the enzyme

Nucleotide excision repair (NER)

• Broad substrate specificity

• Removes bulky adducts that distort the DNA, e.g., UV light induced photoproducts

After damage recognition, NER proceeds in 3 steps:

• Unwinding of DNA around the site of damage (damage recognition)

• Bimodal incision of DNA and recruitment of endonucleases to excise a large DNA fragment surrounding lesion (~30 bases)

• Repair synthesis and ligation

Base excision repair (BER)

• Main guardian against damage generated by cellular metabolism

• Initiated by a specific class of enzymes - DNA glycosylases, each specialised to recognise a limited number of BER substrates

• E.g., identification and removal of 8-oxoG is the role of OGG1

• Glycosylases remove suspect base leaving an abasic site

• Involves excision of a single base or several bases (2-10 bases)

• Filled by DNA polymerase (and other enzymes)

Mismatch repair (MMR)

• The specificity of MMR is primarily for base-base mismatches and insertion/deletion mispairs generated during DNA replication and recombination

• Therefore, identifies intact bases incorporated into the incorrect positions

• Heterodimers MUTS-alpha and MUTS-beta recognise mismatches/small loops generated by insertion/deletion of nucleotides

Double strand break repair

• Ataxia telangiectasia mutated (ATM) is central protein in DSB cascade

• ATM phosphorylates downstream substrates including p53 and BRCA1

Repair is achieved by 2 distinct pathways

• Non-homologous end-joining (NHEJ); no synapsis with undamaged partner molecule, more error prone

• Homologous recombination (HR); retrieves missing genetic information from undamaged homologous chromosomes

Cancer susceptibility syndromes or DNA repair syndromes

• Mutations in the enzymes involved in the repair pathways

• Classification of a number of cancer susceptibility syndromes caused by defects in DNA repair

• The importance of maintaining genome integrity to deter carcinogenesis

• DNA repair plays a crucial role in this process

DNA repair syndromes - XP

• Xeroderma pigmentosum (XP) mutation in any 1 of 8 genes involved in NER

• Extreme sensitivity to UV radiation

• 1000 fold increase in skin cancer

• Skin cancer appears in children with a median age of 8 years

• Squamous cell carcinomas, basal cell carcinomas an lentigo-malignant melanomas are the most frequent tumours

DNA repair syndromes - AT

• Ataxia telangiectasia (AT) mutations in ATM gene involved in DSB repair

• Hallmark of AT phenotype is genetic instability

• Predisposition to malignancy

• Common neoplasms are cancers of immune system, non-Hodgkins lymphomas

• Patient mortality in early adulthood

DNA repair syndrome - HNPCC

• Germline mutations of MMR genes cause susceptibility to Hereditary Nonopolyposis Colon Cancer (HNPCC)

• Cells deficient in MMR have mutation rates 100-1000 times higher than normal

• Syndromes that predisposes to colon cancer

• Affected individuals also develop tumours in endometrium, ovaries, stomach, pancreas, small bowel, and brain

Karotype of cancer cells

• Great majority of solid tumours (>85%) contain chromosome aberrations

• Cancer cell chromosome abnormalities can be structural/numerical

Folicular Non-Hodgkin’s Lymphoma

• A cancer affecting B-lymphocytes

• A very common type of lymphoma, ~25% of all cases. In the US there are ~61,000 new cases if NHL diagnosed annually

• Primarily caused by a translocation between chromosome 14 and 18

• Results in the over-expression of the bcl-2 gene

• Inappropriate recombination by complex machinery involved in immunoglobulin gene arrangements

Numerical changes: Aneuploidy

• = Deviation from a normal (euploid) karyotype

• Chromosomal instability (CIN) leads to aneuploidy

• CIN is a hallmark of most solid tumours

• Chromosome mis-segregation is an important mechanism of tumour adaptation

• Individuals with constitutional global aneuploidy e.g., down syndrome, are found to exhibit increased rates of malignancies

Mechanisms of CIN

• Dozens of gene products are involved in ensuring chromosome segregation fidelity

Defects in multiple mechanisms that lead to errors in chromosome segregation appear in cancer

• Defective centrosome duplication

• Loss of function of cell cycle and spindle assembly checkpoint (SAC) genes e.g., BUB1

• Overly stable attachments of microtubules to chromosomes