Chapter 12

Maintenance of Genomic Integrity and the Development of Cancer

~ 12.4 – 12.10 ~

12.4 Cell genomes are threatened by errors made 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 error. – incorporation of chemically different nucleotide precursors

2. The nucleotides within DNA molecules undergo chemical changes spontaneously.

3. DNA molecules may be attacked by various mutagenic agents, including endogenous and exogenous agents.

12.5 Cell genomes are under constant attack from endogenous biochemical processes

- Endogenous biochemical processes may make greater contributions to genome mutation than do exogenous mutations.

- DNA molecules are subjected to chemical damage through the actions of hydrogen and hydroxy ions that are present at low concentration (~ 10-7 M) at neutral pH.

Figure 12.11a The Biology of Cancer (© Garland Science 2007)

Spontaneous depurination

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

or ADENINE

Figure 12.11b The Biology of Cancer (© Garland Science 2007)

Base deamination

C→ T transition

C→ T transition

CpG

- 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.

Oxidation

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 radical

reactive oxygen species (ROS)

2. Oxidants arisen as by-products 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).

Figure 12.12 The Biology of Cancer (© Garland Science 2007)

Oxidation of bases in the DNA

ROS ↓

Figure 12.14c The Biology of Cancer (© Garland Science 2007)

Methylation 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.

Sidebar 12.3 Inflammation can have both mitogenic and mutagenic consequences

- The phagocytic cells destroy infected cells in part by

releasing oxidants - nitric oxide (NO), superoxide ion (O2.- ),

hydrogen peroxide (H2O2), and hypochlorite (OCl-).

- These oxidants act as mutagens on the genomes of nearby bystander cells through their ability to generate chemically modified bases via nitration, oxidation, deamination, and halogenation.

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

Figure 12.13 The Biology of Cancer (© Garland Science 2007)

[Sidebar 12.4] Oxidation products in urine provide an estimate of the rate of ongoing damage to the cellular genome

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.

12.6 Cell genomes are under occasional attack from exogenous mutagens 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 – far 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

Figure 12.14a & b The Biology of Cancer (© Garland Science 2007)

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

Products of UV irradiation

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

Figure 12.14c The Biology of Cancer (© Garland Science 2007)

Methylation of bases by alkylating agents

Figure 12.15b The Biology of Cancer (© Garland Science 2007)

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

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

Figure 12.16 The Biology of Cancer (© Garland Science 2007)

Formation of DNA adducts

chemically reactive epoxide group

6

7

ultimate carcinogens can also link to O6 or N7

Figure 12.18b The Biology of Cancer (© Garland Science 2007)

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

Figure 12.19a The Biology of Cancer (© Garland Science 2007)

Heterocyclic amines (HCA) are generated from meats which are cooked at high temperature

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

Figure 12.19b The Biology of Cancer (© Garland Science 2007)

Oxidation of PhIP and the formation of DNA adduct

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

- 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

Figure 12.20 The Biology of Cancer (© Garland Science 2007)

Melanin pigment shields keratinocyte nuclei from UV radiation

supranuclear cap(parasol or sun umbrella)

keratinocyte nucleus

Superoxide dismutases (SOD)

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

Mn 3+ − SOD + O2− → Mn 2+ − SOD + O2

Mn 2+ − SOD + O2− + 2H+ → Mn 3+ − SOD + H2O2

- 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

Figure 12.21 The Biology of Cancer (© Garland Science 2007)

Effect of glutathione-S-transferase (GST)

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.

Sidebar 12.5 Inter-individual differences in carcinogen activation seem to contribute to cancer risk and responses to therapy

cytochrome-encoding Cyp1A1

glutathione-S-transferase M1 (GSTM1)

N-acetyltransferase 1 (NAT1) - breast cancer

(help to convert heterocyclic amines into active mutagens)

- lung cancer

Figure 12.22a The Biology of Cancer (© Garland Science 2007)

12.8 Repair enzymes fix DNA that has been altered 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.

12.4 Cell genomes are threatened by errors made 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.

Figure 12.10 The Biology of Cancer (© Garland Science 2007)

- 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

Figure 12.6 (part 1 of 2) The Biology of Cancer (© Garland Science 2007)

Proofreading by DNA polymerases

δ

Figure 12.6 (part 2 of 2) The Biology of Cancer (© Garland Science 2007)

Figure 12.7 The Biology of Cancer (© Garland Science 2007)

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 δ and cancer incidence in mice

Figure 12.8c The Biology of Cancer (© Garland Science 2007)

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

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.

The action of mismatch repair system is critical inregions of the DNA that carry repeated sequences(microsatellite sequence).1. Mononucleotide repeats: AAAAAAA

2. Dinucleotide repeats: AGAGAGAG

3. Repeats of greater sequence complexity

Figure 12.8a The Biology of Cancer (© Garland Science 2007)

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

Figure 12.28 The Biology of Cancer (© Garland Science 2007)

A TGF-β receptor gene affected by microsatellite instability

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).

10 A’s

8 A’s

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 enzymes 2. base-excision repair (BER) 3. nucleotide-excision repair (NER) 4. error-prone repair

Figure 12.22 The Biology of Cancer (© Garland Science 2007)

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

DNA alkyltransferase removes methyl or ethyl adducts from the O6 position of guanine

(ethylnitrosourea)

- 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.

Figure 12.22c The Biology of Cancer (© Garland Science 2007)

Overexpression of MGMT increases the resistance to methylnitrosourea (MNU)-induced mutagenesis in mice

(wild type mice)

(MGMT transgenic mice)

(methylnitrosourea)

Figure 12.23a The Biology of Cancer (© Garland Science 2007)

Base-excision repair (BER)

cleave the glycosyl bondlinking the altered base and the deoxyribose

apurinic/apyrimidinicendonuclease

- 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.

Figure 12.23b The Biology of Cancer (© Garland Science 2007)

Nucleotide-excision repair (NER)

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) 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.

- NER can be divided into 2 subtypes: transcription-coupled repair (TCR) global genomic repair (GGR)

- The p53 activates expression of several genes encoding NER proteins involved in global genomic repair (GGR).

Figure 12.24 The Biology of Cancer (© Garland Science 2007)

Error-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.

12.9 Inherited defects in nucleotide-excision repair (NER) and mismatch repair (MMR) lead to specific cancer susceptibility NER defect: Xeroderma pigmentosum (XP)

MMR defect: Hereditary non-polyposis colon cancer (HNPCC)

Xeroderma pigmentosum (XP) syndrome : - extremely sensitive to UV radiation - show dry, parchment-like skin (xeroderma) and many freckles (“pigmentosum”) - 1,000-fold increased risk of skin cancer and 100,000-fold increased risk of squamous cell carcinoma of the tip of the tongue. - Infants suffer severe burning of the skin after minimal exposure to sunlight. - Skin cancers appear in children with a median age of 8. - inherited defects in NER genes - 8 NER genes (XPA,-B,-C,-D,-E,-F,-G and XPV) have been identified.

Figure 12.25 The Biology of Cancer (© Garland Science 2007)

Hereditary non-polyposis colon cancer (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

Table 12.1 The Biology of Cancer (© Garland Science 2007)

Table 12.2 The Biology of Cancer (© Garland Science 2007)

discovered in sporadic cancers

repeated sequences

12.10 A variety of other DNA repair defects confer 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.

BRCA1 and BRCA2- breast carcinoma 1, 2- two unrelated proteins- mutations predispose to breast, ovarian and other cancers.

Figure 12.34a The Biology of Cancer (© Garland Science 2007)

The BRCA1 and BRCA2 proteins form a large complex with other known DNA repair proteins

- All types of homology-directed repair (HDR) are compromised in cells lacking either BRCA1 or BRCA2 function.

Figure 12.32 The Biology of Cancer (© Garland Science 2007)

during the late S phase and G2 phase of the cell cycle