Biology of Cancer - Tunghai University

Transcript Biology of Cancer - Tunghai University

Chapter 12 Maintenance of Genomic Integrity and the Development of Cancer

~ 12.4 – 12.10 ~ May 31 & Jun 5, 2007

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.

Spontaneous depurination

ADENINE

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

Figure 12.11a

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Base deamination

C→ T transition

CpG

Figure 12.11b

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- 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 the wild-type

p53

alleles.

p53

alleles, about 30% seem to arise from CpG sequences present in

Oxidation

1. Generation of a variety of intermediates as O 2 is progressively reduced to H 2 O in

mitochondria

: O 2 +

→ O 2

→ H 2 O 2 +

→

OH +

→ H 2 O superoxide hydrogen hydroxy ion peroxide radical

r eactive o xygen s pecies (ROS)

2. Oxidants arisen as by-products of various O 2 utilizing enzymes, including those in

peroxisomes

and from spontaneous oxidation of lipids.

3. Inflammation provides an important source of the oxidants, e.g., NO, O 2

, H 2 O 2 , OCl (hypochlorite).

Oxidation of bases in the DNA ROS ↓

Figure 12.12

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Methylation of bases in the DNA

Figure 12.14c

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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 (O hydrogen peroxide (H 2 O 2 ), 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.

[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. Figure 12.13

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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

Products of UV irradiation

cyclobutane pyrimidine dimers pyrimidine (6-4) pyrimidinone (60% T-T, 30% C-T, 10% C-C dimers) Figure 12.14a & b

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- 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 agents

Figure 12.14c

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

olycyclic

romatic tar and tobacco smoke

ydro carbon (PAH) present in coal 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.

Figure 12.15b

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Formation of DNA adducts chemically reactive epoxide group

6 7

ultimate carcinogens can also link to O 6 or N 7 Figure 12.16

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

Figure 12.18b

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etero

yclic

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

Figure 12.19a

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enyl

midazo [4,5-b]

yridine (PhIP) is the principal HCA in the human diet.

Oxidation of PhIP and the formation of DNA adduct

Figure 12.19b

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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

Melanin pigment shields keratinocyte nuclei from UV radiation

supranuclear cap (parasol or sun umbrella) keratinocyte nucleus Figure 12.20

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Superoxide dismutases

(

SOD

) - The enzyme

superoxide dismutase

the dismutation of

superoxide

into (

SOD

oxygen

) catalyzes and

hydrogen peroxide

. As such, it is an important antioxidant defense in nearly all cells exposed to O 2 .

- 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+ Mn 2+ − SOD + O 2 − − SOD + O 2 − → Mn + 2H + 2+ − SOD + O 2 → Mn 3+ − SOD + H 2 O 2

- 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

myotrophic

ateral

clerosis (ALS), a form of motor neuron disease.

Action of catalase :

2 H 2 O 2 → 2 H 2 O + O 2

Effect of glutathione-S-transferase (GST)

reactive epoxide a tripeptide 90% of human prostate adenocarcinomas show a shutdown of GST-π expression due to

methylation

of the promoter of the

GST

gene.

Figure 12.21

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

cytochrome-encoding

Cyp1A1

lung cancer glutathione-S-transferase M1 (

GSTM1

) N-acetyltransferase 1 (

NAT1

) breast cancer (help to convert heterocyclic amines into active mutagens)

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.

Figure 12.22a

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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

replication.

portions of the molecule near the replication fork that have not been undergone Figure 12.10

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

Proofreading

by DNA polymerases 2.

DNA repair

atch

epair (MMR) enzymes

Proofreading by DNA polymerases δ

Figure 12.6 (part 1 of 2)

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Figure 12.6 (part 2 of 2)

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Proofreading by DNA polymerase δ and cancer incidence in mice

D400A mutation: change of the #400 a.a. from D (aspartic acid) to A (alanine) in the proofreading domain of DNA polymerase

Deaths of the mutant homozygotes were all due to malignancies.

Figure 12.7

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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. Figure 12.8c

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The action of mismatch repair system is critical in regions of the DNA that carry

repeated sequences

(microsatellite sequence).

1. Mononucleotide repeats: AAAAAAA 2. Dinucleotide repeats: AGAGAGAG 3. Repeats of greater sequence complexity A defective MMR system will result in the expansion or shrinkage of microsatellite sequences, known as

m icrosatellite in stability (

MIN).

Figure 12.8a

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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

icrosatellite

stability (MIN).

10 A’s 8 A’s

Figure 12.28

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Cells deploy the very challenging task of restoring normal DNA structure.

a wide variety of enzymes

to accomplish - 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

DNA alkyltransferase removes methyl or ethyl adducts from the O 6 position of guanine

(

thyl

itroso

rea) Figure 12.22

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ethyl

uanine-DNA

ethyl

ransferase or O 6 -

lkyl

uanine DNA alkyl

ransferase (AGT) or DNA alkyltransferase

- 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.

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

(wild type mice) (MGMT transgenic mice) (

ethyl

itroso

rea) Figure 12.22c

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Base-excision repair (BER)

cleave the glycosyl bond linking the altered base and the deoxyribose

urinic/apyrimidinic

ndonuclease Figure 12.23a

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- 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.

Nucleotide-excision repair (NER)

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

PCNA:

roliferation-

ell

uclear

ntigen

RPA:

single-strand DNA-binding protein Figure 12.23b

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- 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:

ranscription-

oupled

epair (TCR)

lobal

enomic

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

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.

Figure 12.24

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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

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ereditary

on-

olyposis

olon

ancer (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

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discovered in sporadic cancers

Table 12.2

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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

BRCA2

allele.

- By some estimates, 70 to 80% of all familial ovarian cancers are due to mutant germline alleles of

BRCA1

BRCA2

BRCA1 and BRCA2

b r east ca rcinoma 1 , 2 - two unrelated proteins - mutations predispose to breast, ovarian and other cancers.

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

Figure 12.34a

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

during the late S phase and G 2 phase of the cell cycle Figure 12.32

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Biology of Cancer - Tunghai University

Transcript Biology of Cancer - Tunghai University

Chapter 12 Maintenance of Genomic Integrity and the Development of Cancer

BRCA1 and BRCA2

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