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Maintenance of Genomic Integrity
and the Development of Cancer
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.
2.
3.
The replication of DNA is subjected to a low but
significant level of errors.

incorporation of chemically different nucleotide
precursors
The nucleotides within DNA molecules undergo chemical
changes spontaneously.
DNA molecules may be attacked by various mutagenic
agents, including endogenous and exogenous agents.
Cell genomes are under constant attack
from endogenous 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.
Spontaneous depurination
or ADENINE
By some estimates, as many as 10,000 purine
bases are lost by depurination each day in a
mammalian cell.
Spontaneous 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
ion
hydrogen
peroxide
hydroxy
radical
reactive 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).
Oxidation of bases in the DNA
ROS
↓
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.
Inflammation can have both mitogenic and
mutagenic 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 8oxo-dG, one of the primary products of DNA
oxidation.
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.
Cell genomes are under occasional attack from
exogenous mutagens and their metabolites

X-rays – ionizing radiation



UV radiation – more common source of
environmental radiation than X-rays


generate ionized, chemically reactive molecules
create s.s. and d.s. breaks in the double helix
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
(60% T-T, 30% C-T, 10% C-C dimers)
pyrimidine (6-4) pyrimidinone

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
Cytochrome P-450 (CYP) enzymes
oxidize procarcinogens to ultimate
carcinogens
a polycyclic aromatic hydrocarbon (PAH) present in coal
tar and tobacco smoke
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.
Formation of DNA adducts
chemically reactive
epoxide group
6
7
ultimate carcinogens can also
link to O6 or N7
Activation of aflatoxin B1 (AFB1) by
cytochrome P-450
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.
Oxidation of PhIP and the formation of
DNA adduct

Cells deploy a variety of defenses to
protect DNA molecules from attack by
mutagens
Physical shield


detoxifying enzymes


superoxide dismutase (SOD) & catalase
free-radical scavengers


skin and the melanin pigment
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
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
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.
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)
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.
Cell genomes are threatened by errors
made during DNA replication

During DNA replication, the DNA molecules are
especially vulnerable to breakage in the singlestranded 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
Proofreading by DNA polymerases
δ
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 δ
Deaths of the mutant
homozygotes were all
due to malignancies.
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 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 microsatellite
instability (MIN).
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).

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
DNA alkyltransferase removes methyl or ethyl
adducts from the O6 position of guanine
(ethylnitrosourea)
O6-methylguanine-DNA methyltransferase
or O6-alkylguanine DNA alkyltransferase (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.
Base-excision repair (BER)
cleave the glycosyl bond
linking the altered base
and the deoxyribose
apurinic/apyrimidinic
endonuclease

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: proliferationcell nuclear antigen
RPA: single-strand
DNA-binding protein



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 helixdistorting 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 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
(NER) and mismatch repair (MMR) lead to
specific cancer susceptibility

NER defect: Xeroderma pigmentosum (XP)

MMR defect: Hereditary non-polyposis colon cancer
(HNPCC)
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
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.