Maintenance of Genomic Integrity Maintenance of Genomic Integrity and the Development of Cancerand the Development of Cancer

Cell genomes are threatened by errors Cell genomes are threatened by errors made during DNA replicationmade during DNA replication

The stability of genome is under constant challenge by a variety of agents and processes:1. The replication of DNA is subjected to a low but

significant level of errors. incorporation of chemically different nucleotide

precursors2. The nucleotides within DNA molecules undergo chemical

changes spontaneously.3. DNA molecules may be attacked by various mutagenic

agents, including endogenous and exogenous agents.

Cell genomes are under constant attack Cell genomes are under constant attack from from endogenousendogenous biochemical processes biochemical processes

Endogenous biochemical processes may make greater contributions to genome mutation than do exogenous mutations. Chemical damage of DNA molecules through the actions

of hydrogen and hydroxy ions that are present at low concentration (~ 10-7 M) at neutral pH.

By some estimates, as many as 10,000 purine bases are lost by depurination each day in a mammalian cell.

or ADENINE

Spontaneous depurinationSpontaneous depurination

C → T transition

C → T transitionCpG

Spontaneous base deaminationSpontaneous base deamination

The deamination of 5-methylcytocine represents a major source of point mutations in human DNA.

By one estimate, 63% of the point mutations in the genomes of tumors of internal organs arise in CpG sequences. Among mutant p53 alleles, about 30% seem to arise from CpG sequences present in the wild-type p53 alleles.

OxidationOxidation

1. Generation of a variety of intermediates as O2 is progressively reduced to H2O in mitochondria:

O2 + e- → O2.- + e- → H2O2 + e- → .OH + e- → H2O

superoxide hydrogen hydroxy ion peroxide radicalreactive oxygen species (ROS)

2. Oxidants arisen as byproducts of various O2-utilizing enzymes, including those in peroxisomes and from spontaneous oxidation of lipids.

3. Inflammation provides an important source of the oxidants,

e.g., NO, O2.- , H2O2 , OCl- (hypochlorite).

ROS ↓

Oxidation of bases in the DNAOxidation of bases in the DNA

Methylation of bases in the DNAMethylation of bases in the DNA

The oxidation, depurination, deamination, and methylation, which together may alter thousands of bases per cell genome each day, greatly exceeds the amount of damage created by exogenous mutagenic agents in most tissues.

Inflammation can have both mitogenic and Inflammation can have both mitogenic and mutagenic consequencesmutagenic consequences

The phagocytic cells destroy infected cells in part by releasing oxidants - NO, O2

.- , H2O2, and OCl-. These oxidants act as mutagens on the genomes of

nearby cells (nitration, oxidation, deamination, and halogenation).

The DNA of inflamed and neoplastic tissues have been found to carry increased concentrations of 8-oxo-dG, one of the primary products of DNA oxidation.

Rat cells suffer about 10-fold more oxidative hits per cell per day in their genomes than do human cells because they have about a 7-fold greater metabolic rate.

Oxidation products in urine provide an estimate Oxidation products in urine provide an estimate of the rate of ongoing damage to the cellular of the rate of ongoing damage to the cellular genomegenome

Cell genomes are under occasional attack from Cell genomes are under occasional attack from exogenous exogenous mutagens and their metabolitesmutagens and their metabolites

X-rays – ionizing radiation generate ionized, chemically reactive molecules create s.s. and d.s. breaks in the double helix

UV radiation – more common source of environmental radiation than X-rays form thymidine dimers

Chemicals – many are electrophilic alkylating agents are mutagens which are capable of

attaching alkyl groups covalently to the DNA bases form DNA adducts

cyclobutane pyrimidine dimers pyrimidine (6-4) pyrimidinone (60% T-T, 30% C-T, 10% C-C dimers)

Products of UV irradiationProducts of UV irradiation

In benign skin lesions and basal cell carcinomas of the skin, many of the mutant p53 alleles carry a dipyrimidine substitution.

Methylation of bases by alkylating Methylation of bases by alkylating agentsagents

Cytochrome-P450s are involved in the biosynthesis or degradation of steroid hormones, cholesterol, bile acids, and fatty acids. In addition, they aid the oxidation and detoxification of drugs and carcinogens.

a polycyclic aromatic hydro-carbon (PAH) present in coal tar and tobacco smoke

Cytochrome P-450 (CYP) enzymes Cytochrome P-450 (CYP) enzymes oxidize procarcinogens to ultimate oxidize procarcinogens to ultimate carcinogenscarcinogens

chemically reactive epoxide group

6

7

ultimate carcinogens can also link to O6 or N7

Formation of DNA adductsFormation of DNA adducts

Activation of aflatoxin B1 (AFB1) by Activation of aflatoxin B1 (AFB1) by cytochrome P-450cytochrome P-450

2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) is the principal HCA in the human diet.

HHeteroeteroccyclic yclic aamines (HCA) are generated from mines (HCA) are generated from meats which are cooked at high temperaturemeats which are cooked at high temperature

Oxidation of PhIP and the formation of Oxidation of PhIP and the formation of DNA adductDNA adduct

Cells deploy a variety of defenses to Cells deploy a variety of defenses to protect DNA molecules from attack by protect DNA molecules from attack by mutagensmutagens

Physical shield skin and the melanin pigment

detoxifying enzymes superoxide dismutase (SOD) & catalase

free-radical scavengers vitamin C, vitamin E, bilirubin

glutathione-S-transferases (GSTs) reacting with electrophilc compounds

supranuclear cap(parasol or sun umbrella)

keratinocyte nucleus

Melanin pigment shields keratinocyte Melanin pigment shields keratinocyte nuclei from UV radiationnuclei from UV radiation

Superoxide dismutases (SOD)Superoxide dismutases (SOD)

The enzyme superoxide dismutase (SOD) catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide.

It is an important antioxidant defense in nearly all cells exposed to O2.

In humans, 3 forms of SOD are present : SOD1 – Cu-Zn-SOD (in cytoplasm) SOD2 – Mn-SOD (in mitochondria) SOD3 – Cu-Zn-SOD (extracellular)

Mice lacking SOD2 die several days after birth with massive oxidative stress.

Mice lacking SOD1 develop a wide range of pathologies, including hepatocellular carcinoma, an acceleration of age-related muscle mass loss, an earlier incidence of cataracts and a reduced lifespan.

In humans, mutations in SOD1 have been linked to familial amyotrophic lateral sclerosis (ALS), a form of motor neuron disease.

Action of catalase : 2 H2O2 → 2 H2O + O2

a tripeptide

reactive epoxide

90% of human prostate adenocarcinomas show a shutdown of GST-π expression due to methylation of the promoter of the GST gene.

Effect of glutathione-S-transferase (GST)Effect of glutathione-S-transferase (GST)

- lung cancer

Inter-individual differences in carcinogen Inter-individual differences in carcinogen activation seem to contribute to cancer risk and activation seem to contribute to cancer risk and responses to therapyresponses to therapy

cytochrome-encoding Cyp1A1 glutathione-S-transferase M1 (GSTM1) N-acetyltransferase 1 (NAT1) - breast cancer (help to

convert heterocyclic amines into active mutagens)

Repair enzymes fix DNA that has been Repair enzymes fix DNA that has been altered by mutagensaltered by mutagens

If genotoxic chemicals are not intercepted before they attack DNA, mammalian cells have a backup strategy for minimizing the genetic damage caused by these potential carcinogens.

Cell genomes are threatened by errors Cell genomes are threatened by errors made during DNA replicationmade during DNA replication

During DNA replication, the DNA molecules are especially vulnerable to breakage in the single-stranded portions of the molecule near the replication fork that have not been undergone replication

A cell has two major strategies for detecting and removing the miscopied nucleotides arising during DNA replication.

1. Proofreading by DNA polymerases

2. DNA repair by mismatch repair (MMR) enzymes

δ

Proofreading by DNA polymerasesProofreading by DNA polymerases

D400A mutation:

change of the #400 a.a. from D (aspartic acid) to A (alanine) in the proofreading domain of DNA polymerase δ

Deaths of the mutant homozygotes were all due to malignancies.

Proofreading by DNA polymerase Proofreading by DNA polymerase δδ and cancer incidence in miceand cancer incidence in mice

Two components of the MMR apparatus, MutS and MutL, collaborate to remove mismatched DNA:

- MutS scans the DNA for mismatches.

- MutL then scans the DNA for single-strand nicks, which identify the strand that has recently been synthesized.

Mismatch repair (MMR) enzymes detect Mismatch repair (MMR) enzymes detect mistakes in newly synthesized DNA strandmistakes in newly synthesized DNA strand

A defective MMR system will result in the expansion or shrinkage of microsatellite sequences, known as microsatellite instability (MIN).

The action of mismatch repair system is critical in regions of the DNA that carry repeated sequences (microsatellite sequence).1. Mononucleotide repeats: AAAAAAA2. Dinucleotide repeats: AGAGAGAG3. Repeats of greater sequence complexity

The type II TGF-β receptor is frequently inactivated in human colon cancers, which carry defects in mismatch repair genes and exhibit microsatellite instability (MIN).

A TGF-A TGF-ββ receptor gene affected by receptor gene affected by microsatellite instabilitymicrosatellite instability

Cells deploy a wide variety of enzymes to accomplish the

very challenging task of restoring normal DNA structure. Mismatch repair (MMR) enzymes largely focused on

detecting nucleotides of normal structure that have been incorporated into the wrong positions.

Other repair mechanisms detect nucleotides of abnormal chemical structure.1. dealkylating enzymes2. base-excision repair (BER)3. nucleotide-excision repair (NER)4. error-prone repair

O6-methylguanine-DNA methyltransferaseor O6-alkylguanine DNA alkyltransferase (AGT)or DNA alkyltransferase

(ethylnitrosourea)

DNA alkyltransferase removes methyl or ethyl DNA alkyltransferase removes methyl or ethyl adducts from the Oadducts from the O66 position of guanine position of guanine

The MGMT gene is silenced by promoter methylation in 40% of gliomas and colorectal tumors, and in 25% of non-small-cell carcinomas, lymphomas, and head and neck carcinomas.

The loss of this DNA repair function in certain tissues favors increased rates of mutation and hence accelerated tumor progression.

cleave the glycosyl bondlinking the altered base and the deoxyribose

apurinic/apyrimidinicendonuclease

Base-excision repair (BER)Base-excision repair (BER)

Base-excision repair (BER) tends to repair lesions in the DNA that derive from endogenous sources such as the reactive oxygen species (ROS) and depurination events.

BER seems to concentrate on fixing lesions that do not create structural distortions of the DNA double helix.

For example, when U is mistakenly incorporated into the DNA, it is removed by the enzyme uracil DNA-glycosylase and soon replaced with a C.

PCNA: proliferation-cell nuclear antigen

RPA: single-strand DNA-binding protein

NER is accomplished by a large multiprotein complex composed of almost ~20’s subunits.

Nucleotide-excision repair (NER)Nucleotide-excision repair (NER)

Nucleotide-excision repair (NER) focuses largely on repairing the lesions created by exogenous agents, such as UV photons and chemical carcinogens.

NER enzymes can recognize and remove helix-distorting alterations (e.g., bulky base adducts) created by polycyclic aromatic hydrocarbons (PAH), heterocyclic amines, aflatoxin B1, and pyrimidine dimers formed by UV radiation.

For example, following exposure to UV radiation, cultured human cells can repair ~ 80% of their pyrimidine dimers in 24 hrs.

Error-prone repairError-prone repair

Error-prone DNA synthesis occurs when a DNA replication fork is advancing during replication and encounters a still-unrepaired DNA lesion.

The replication apparatus must “guess” which of the 4 nucleotides is appropriate for incorporation.

Inherited defects in nucleotide-excision repair Inherited defects in nucleotide-excision repair (NER) and mismatch repair (MMR) lead to (NER) and mismatch repair (MMR) lead to specific cancer susceptibilityspecific cancer susceptibility

NER defect: Xeroderma pigmentosum (XP) MMR defect: Hereditary non-polyposis colon cancer

(HNPCC)

HHereditary ereditary nnon-on-ppolyposis olyposis ccolon olon ccancer ancer (HNPCC) : (HNPCC) :

a familial cancer syndrome comprising 2 to 3% of all colon cancer cases Some HNPCC patients have increased

susceptibility to endometrial, stomach, ovarian, and urinary tract carcinoma in addition to colon carcinomas.

germline mutations in the genes encoding mismatch repair (MMR) proteins

A variety of other DNA repair defects A variety of other DNA repair defects confer increased cancer susceptibilityconfer increased cancer susceptibility

Almost 50% of all identified familial breast cancers involve germline transmission of a mutant BRCA1 or BRCA2 allele.

By some estimates, 70 to 80% of all familial ovarian cancers are due to mutant germline alleles of BRCA1 or BRCA2.