Transcript DNA repair and Cancer Susceptibility

DNA Repair and
Cancer Susceptibility
Hernan A. Flores-Rozas, PhD
Dyson Pharmacy 221
[email protected]
What we will address in this lecture:
1.
Introduction to cancer and control of cell proliferation.
2.
Oncogenes, tumor suppressors and clonal origin of cancer.
3. Mutations and the genetic origin of cancer.
4. Repair of replication errors.
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. A Hereditary cancer syndrome caused by a defect in DNA repair.
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. What to do with this knowledge.
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Cancer Introduction
•
Cancer can be defined as the
uncontrolled growth and spread of cells
throughout the body
•
Cancer is not a single disease but is
comprised of over 100 different
diseases, defined on the basis of site of
origin, specific characteristics and in
some cases causative molecular events
•
The human body contains trillions of
cells, and the growth and survival of
these cells must be controlled to
maintain normal homeostasis
•
Cancer is a breakdown of the normal
mechanisms controlling cell growth and
survival
Positive signals
proliferation
Negative signals
apoptosis
growth arrest/senescence
differentiation
Cells Enter The Cell Cycle to Proliferate
GO
G1
M
S
G2
Interphase generally lasts at least 12 to 24
hours in mammalian tissue. During this period,
the cell is constantly synthesizing RNA,
producing protein and growing in size. By
studying molecular events in cells, scientists
have determined that interphase can be
divided into 4 steps:
Gap 0 (G0)
Gap 1 (G1)
S (synthesis) phase
Gap 2 (G2)
In Mitosis (M) growth stops and the cell
divides into 2 daughter cells.
Evidence for a Genetic Origin of Cancer
• Many carcinogens are mutagens.
• Susceptibility to certain carcinogens is dependent on the ability of
cellular enzymes to convert them to a mutagenic form.
• Defects in DNA repair increase the probability of cancer.
• Some cancers are inherited (?)
• Malignant tumors contain mutated oncogenes.
• Some tumors have lost or mutated tumor suppressor genes.
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Types of Mutations
 What is a mutation?
 It is an irreversible change on the DNA sequence. In some cases it
affects gene sequences and consequently protein structure and function.
 Sometimes it affects regulatory sequences altering the expression levels.
 There are different types of mutations with different consequences to
the protein sequence and structure:
 Missense mutations: changes in aminoacid sequence.
 Nonsense mutations: early protein termination.
 Insertion/deletions: changes large regions of a protein.
 Translocations: generates protein fusions.
*
Normal Activity
Abnormal Activity
Types of Injuries That Can Result in Mutations
Spontaneous Alterations of and Damage to DNA
- Mismatches (replication errors)
- Spontaneous alterations in the chemistry of DNA
bases
•
Deamination of bases
•
Loss of bases
•
Oxidative damage to DNA
Environmental Damage to DNA
- Physical damage to DNA
•
Ionizing radiation (X-rays)
•
UV radiation (Sun light)
- Chemical damage to DNA
•
Alkylating agents
•
Crosslinking agents
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Mutations that affect Oncogenes and Tumor
Suppressors are Required for Carcinogenesis
Oncogenes
Generated by gain-of-function mutation.
Encode proteins that promote tumor
formation.
Tumor suppressor genes
Encode proteins that inhibit tumor
formation. Loss of function mutation
occurs in tumors
Mutations on these genes provide
selective advantage to the cell.
Oncogenes
Tumor suppressors
Cancers are of Clonal Origin
Both alleles of a tumor suppressor gene (TSG)
need to be inactivated to initiate the
carcinogenesis process
Cancer Risk Increases with Age
- The number of cases
for any given cancer
increase as the
individual age
increases.
- Mathematical analysis
of this data suggests
that 5 to 7 mutations are
necessary for malignant
transformation of a
normal cell.
- This suggests that
mutations accumulate
as we age.
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Lynch Syndrome: Hereditary Nonpolyposis
Colorectal Carcinoma (HNPCC)
When several members of a family are afflicted by a particular type of
cancer, it is highly probable that is has a hereditary component.
One classical example is hereditary colon cancer:
- Dominant mode of transmission.
- Founder mutation mimics sporadic case.
- Early age of onset compared to sporadic cancers.
- Multiple primary tumors.
- High Proportion of colorectal cancer. Also develop tumor of the stomach,
endometrial, small bowel, bladder, ovary, biliary tract, pancreas, sebaceous
skin tumors and gliomas.
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A Brief History of HNPCC
First identification of an HNPCC family was made by renowned pathologist Aldred
Warthin, M.D. in 1985 based on the family of his seamstress who died at an early age of
metastatic endometrial carcinoma. Warthin published a description of this family, which
he called Family G, in 1913.
In 1962, Henry Lynch, M.D., had a patient who was recovering from
delirium tremens. His excessive drinking was caused by his fear of
dying of cancer of the colon just like most members of his family.
Shortly thereafter, he was diagnosed with adrenal cortical carcinoma.
Lynch to compiled the family history of this patient, many of whom
were farmers residing in Missouri, Kansas, and Nebraska and
reported a personal history of cancer, particularly involving the colon
and often with early age of onset. Among the women, CRC was often Henry T. Lynch, M.D.
associated with endometrial and ovarian carcinoma. He referred to
this
familyLynch
as Family
N. for significant NIH grant support, he was turned down because
Although
applied
reviewers did not believe that genetics was the primary cause for cancer in these
families. A common suggestion by the reviewers was to search for environmental
carcinogens, particularly pesticides and herbicides, given the heavy farming background
of many of the families.
In 1993 Fishel and Kolodner (Harvard) and Vogelstein (Johns Hopkins) identified defects
in mismatch repair as the cause of HNPCC.
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Pedigree of a Swedish Lynch Syndrome Family
Circles denote women; squares denote men; filled symbols denote Lynch syndrome; symbols with a dot denote inferred or verified
unaffected mutation carriers; half-filled symbols denote non-Lynch syndrome cancer; plus sign denotes verified mutation; arrow denotes
proband of initial subfamily. BST, benign skin tumor; CC, colon cancer; CxC, cervical cancer; D, died; EC, endometrial cancer; H, healthy; KC,
kidney cancer; OC, ovarian cancer; PC, prostate cancer; RC, rectal cancer; Sa, sarcoma. Numbers denote age of diagnosis or death.
Proband: The family member through whom a family's medical history comes to light.
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DNA Replication Errors
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The Fidelity of DNA Replication is Significantly
Increased by DNA Mismatch Repair
Mechanisms for Insuring Replicative Fidelity
Mechanism
1. Base pairing
2. DNA polymerases
- base selection
- proofreading
3. Accessory proteins
- single strand binding protein
4. Mismatch correction
Error frequency
10-1 to 10-2
10-5 to 10-6
10-7
10-10
"If our strands of DNA were stretched out in a line, the 46 chromosomes making up the human
genome would extend more than six feet [close to two metres]. If the ... length of the 100 trillion cells
could be stretched out, it would be ... over 113 billion miles [182 billion kilometres]. That is enough
material to reach to the sun and back 610 times." [Source: Centre for Integrated Genomics]
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Common Features of the Excision-Type
Repair Pathways
Recognition Step: Altered DNA is
recognized and bound by a specific
damage recognition protein. First step of
the pathway, recruits other components
required for the repair reaction.
Excision: The damaged base(s), and in
some cases additional nucleotides
adjacent to the damage, are excised
(removed) from the DNA by
exonucleases, resulting in a gapped
DNA.
Resynthesis: The gap on the DNA is
refilled by a DNA polymerase using the
complementary strand as the template.
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Mutations on mismatch repair genes that are
associated with HNPCC and sporadic cancers
H 49%
S 85%
H 2%
MLH1
PMS2
H 38%
S 15%
MSH2
MSH6
Base:Base mispair
H: hereditary
S: sporadic
H 9%
MLH1
MSH2
MLH3
MSH3
H 2%
0%
Insertion/Deletion mispair
The germline mutation in Lynch’s Family N was
identified in the year 2000 as a MSH2 R680X
mutation.
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Mismatch Repair in Mammalian Cells
MutSa (Msh2/Msh6) - recognizes mismatch or 1 bp IDL
MutSb (Msh2/Msh3) - recognizes 2-12 bp IDL
MutLa (Mlh1/Pms2) and MutLb (Mlh1/Pms1) couple the binding of
the mismatch bases to the downstream steps
The correct strand to be removed is discriminated by
sensing the replication machinery .
Exonucleases (Exo I and FEN1) degrade the strand containing the
incorrect base, generating a gap.
The replication machinery, including the polymerases Pold and
Pole fill-in the missing fragment of DNA.
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What are the Benefits of Identifying the
Genetic Defects in Cancer
Allows us to provide genetic counseling to patients belonging to afflicted families.
Provides a more accurate diagnosis of the type of cancer.
Suggests more effective therapy, in particular since mutations in certain genes
correlate with the sensitivity of some cancers to smart drugs.
Genomic sequences prone to mutation
t
gtacag ttttt
catgtcaaaaaaaaagtc
1) Mononucleotide runs:
2) Di or trinucleotide repeats:
gc
a a
cg
cca gta cag cag cag
ggt cat gtc gtc gtc gtc gtc
3) Direct repeats (short (4-9bp)
and distant (>30bp))
Molecular Features of HNPCC
Mutations in mismatch repair genes: MSH2, MLH1, MSH6 and PMS2
Immunohistochemical absence of one or more MMR gene products.
Microsatellite instability (MSI-H):
10%–15% of sporadic tumors have MSI
95% of HNPCC tumors have MSI at multiple loci
Electrophoresis:
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Possible organization of proteins at the
replication fork
RPA
PCNA
pol delt a
Helicase
Futile repair pathway (structure model)