L9 - genomic integrity of cancer cells Flashcards
(69 cards)
1
Q
what’s the importance of the stem cell lineage?
A
it is the only stable repository of genetic information
2
Q
what are transit-amplifying cells?
A
the descendents of the second daughter cells and will carry out frequent mitosis into highly differentiated cells
3
Q
how much does the stem cell population constitute?
A
0.1 - 1.0% of the total cell population
4
Q
How are intestinal stem cells protected from mutagens?
A
- Shielding from mutagenic contents, carcinogens.
- ISCs are embeded deep within the crypts.
- secretion of thick layer of mucus (mucin). - Flushing of descendent cells (susceptible to mutations) out of the crypts and elimination after 5-7 days.
5
Q
How do stem cells protect their genomes?
A
- relatively infrequent replication
- found in anatomically protected site
- initiate rapid apoptosis
- pump out toxic mutagens via Mdr1
- asymmetric DNA strand allocation
asymmetric DNA strand allocation: retain template strand in SC while replicated strand goes to daughter TA cells
6
Q
what are the 3 major mutagenic processes?
A
- replication of DNA during S phase
- nucleotides undergo chemical changes in the absence of mutagens
- DNA attacked by endogenous physical mutagens (metabolites) and exogenous chemical species (X-ray, UV)
7
Q
Mutation in DNA pol-δ result in:
A
LOF of DNA pol-δ 5’-to-3’ exonuclease proofreading ability
8
Q
what is strand slippage?
A
when parental an nascent strands slip out of proper alignment so DNA pol create errors that result in longer/shorter repeat sequences
9
Q
What is the microsatellite sequence?
A
a tract of highly repetitive genomic DNA which certain DNA motifs (1-more base pairs) are repeated, 5-50 times
10
Q
explain how microsatellite instability comes about
A
occurs due to deffective mismatch repair system so it fails to remove stuttering mistakes made by DNA pol resulting in the expansion/shrinkage of the sequence in progeny cells
11
Q
How do we detect microsatellite instability?
A
- PCR: amplify the microsatellite region
- in normal tissue, the size of PCR products will fit Gaussian distribution.
- Size of the PCR products of microsatellite region from colon tumor samples is significantly
reduced, indicating the shrinkage of microsatellite
12
Q
what are the chances of DNA pol making a mistake?
A
1 in 10^5 nucleotides
13
Q
what are the chances of DNA pol failing to carry out mismatch repair?
A
1 out of 100
14
Q
what is the mutation rate in normal cells in terms of nucleotides?
A
1 nucleotide per 10^9 that are synthesized
15
Q
majority of HNPCC result from what kind of mutations?
HNPCC: hereditary non-polyposis colorectal cancer
A
gern-line mutation in genes encoding 2 important mismatch repair proteins: MSH2 and MLH1
16
Q
describe microsatellite instability in TGF-beta receptor
A
- change in # of adenosine nucleotides in coding region (serine/threonine kinase domain) of TGF-beta receptor cause unexpected stop codon
- when one receptor-coding gene undergoes inactivating mutation, the surviving WT allel is discarded through LOH
17
Q
State some parts of the genome that are prone to instability.
A
- microsatellite region
- replication fork
18
Q
what is the rate of replication fork dsDNA breaks?
A
~10 dsDNA breaks occur per cell genome each time a cell passes through S phase
19
Q
which part of the replication fork is susceptible to dsDNA breaks?
A
single strand DNA at the unwound but not yet replicated portion of the parental DNA is susceptible to inadvertant breakage
20
Q
what are some of the consequence of replication fork dsDNA breaks
A
- chromosomal breaks and translocations
21
Q
how to prevent replication fork dsDNA breaks
A
detecting damage, repair, emitting alarm leading to apoptosis
22
Q
how does DNA being double stranded act as a defence mechanism against mutation?
compare with ssDNA
A
- ssDNA are 100-1000x more vulnerable to oxidative damage than dsDNA
- because ds helix protects nucleotide from chemical attack so it is harder for depurination, depyrimidination and deamination to occur
23
Q
describe depurination
A
- the chemical bond linking a purine base (adenine or
guanine) to deoxyribose breaks spontaneously - As many as 10,000 purine bases are lost by depurination each day in a
mammalian cell (>1017 in a human body)
24
Q
describe depyrimidination
A
occurs at a 20- to 100-fold lower rate but still results
in 500 cytosine and thymine bases loss per day
25
describe deamination
may occur in which the amine groups that protrude from cytosine, adenine and guanine rings are lost
26
what promotes oxidative damage?
some damage occurs through the actions of hydrogen and hydroxyl ions that are present at low concentration (~10-7 M) at neutral pH (H2 O)
27
describe transition mutation
## Footnote
pyrimidine to pyrimidine
Uracil may be read as a thymine during subsequent DNA replication thereby causing a C-T point mutation,
28
Give 3 sources of intracellular metabolites.
1. Reactive oxygen species (ROS) like superoxide ion, HO and hydroxyl radical, leaking out of mitochondria into cytosol
2. Peroxisome from spontaneous oxidation of lipids
3. Inflammation providing oxidants favouring mutagenesis and carcinogenesis
29
What do intracellular metabolites target?
- bases within DNA (depurination, depyrimidination)
- single or double strand breaks
- Abasic sites
- Protein-DNA crosslinging
30
How does DNA oxidation occur?
- formation of 8-oxo-dG can readily pair with dA so it creates a danger of mutation
- during DNA replication, G:C pair could be turned into T:A pair due to G—>T (transversion; purine —> pyrimidine)
31
32
What are examples of exogenous mutagenic factors?
- UV rays
- X-rays
33
How does UV cause damage to DNA
- ionizing radiation strip electrons from water molecules and generates ROS that create ss/ds breaks in DNA double helix
- frequently result in formation of pyrimidine dimers (covalent bond) between 2 adjacent pyrimidines (T and C) in the same DNA strand
- dimers are very stable
34
35
What is the percentage of pyrimidine dimers formed?
Mammals: >60% are TT, 30% are CT and 10% CC
36
What is the most common type of mutation caused by UV radiation
- CC —> TT substitution, arising from CC dimers in cancer because TT is easy to repair
- UV-induced DNA lesion result in C-to-T transition mutations
37
Example of how significant UV is in causing mutation
Squamous cell skin carcinomas double with each 10-degree decline in latitude, peak at equator (strongest UV exposure)
38
Endogenous agents example
Alkylating agents
- electrophilic
- attach alkyl groups covalently to the DNA bases: destabalizes its covalaent bond to deoxyribose —> loss of purine/pyrimidine from DNA or alkylated bases may be misread during DNA replication
39
Hwo does P450 metabolism generate mutagen
- when P450 oxidizes and detoxifies polycyclic hydrocarbons
- creates chemicals highly reactive with DNA, causing mutations
- pro-carcinogens converted into highly reactive ultimate carcinogens forming covalent bonds with various bases, termed DNA adduct
40
How do heterocyclic amines generate DNA mutations
- CYPs oxidization for detoxification
- some oxidize exocyclic amine groups to form active compounds reactive with DNA and proteins (carcinogenic) in the liver which is circulated to other organs
41
What is AFB1
- Aflatoxin B1 exposure result in 3x higher risk of hepatocellular carcinoma (HCC)
- AFB1 is made by molds growing on peanuts and grains that are not stored properly
42
How does AFB1 cause DNA mutations?
- Activation of AFB1 by CYPs —> attack guanine anad formation of DNA adduct, G-to-T mutation
- codon 249 of p53, AGG is frequently mutated
to AGT in about 50% of hepatoma patients
- addition of active form of AFB1-8,9-oxidare to dG —> G-to-T transversion mutation of codon 249 of p53 (AGG —> AGT)
43
Alcohol act as a DNA adduct?
- increase risk of oesophageal cancer
44
Explain physical shielding of keratinocyte
- melanosome carries melanin (pigment) are transferred to keratinocyte in basal layer of epidermis
- Melanosomes are assembled into tiny sub umbrellas (supranuclear cap) sitting above the nuclei and shielding from UVB radiation
- decreases UV induced DNA damage by 4x
45
Explain how enzymes to detoxify carcinogens are an important defense system
Glutathione S-transferase (GST) uses the sulfhydryl (-SH) group of glutathione (GSH) to detoxify electrophilic carcinogens before they react with DNA
46
How could GST be lost
- GST-π is greatly lost in PIN (prostatic intraepithelial neoplasia)
- The reduction of GST expression in
prostate cancer is mostly due to the
hyper-methylation of the promoter of
GSTP gene
47
Explain de-alkylating enzymes as a defense system
-O6 position is vulnerable to alkylation by agents like ENU (ethylnitrosourea)
- MGMT (O6-alkylguanine-DNA methyltransferase) is a dealkylating enzyme that flips the alkylated guanine for repair
48
Explain the enzymes involved in base and nucleotide excision repair
BER (base-excision repair) fix leions by endogenous sources: DNA glycosylase cleaves chemically altered base, AP endonuclease cleaves deoxyribosylphosphate, B+DNA ligase carry out short patch repair, flap endonuclease and polymerases carry out long patch repair
NER (nucleotide-excision repair) fix lesions by exogenous sources: enzyme cleaves DNA fragment ~24 nucleotides (nt) on 5’ side and 5nt on 3’ side of adduct in DNA that is experiencing helix distortion, fill in gap by polymerases, PCNA and RPA
49
What inherited condition leads to. A 2000x increase risk of squamous cell carcinoma?
Defect in 1 of the 8 genes leading to xeroderma pigmentation (XP): extreme sunlight sensitivity)
50
Mutations in normal cells vs in MLH1/MSH2 mutant cells
Normal: alkylating agent induced mutation —> G2/M checkpoint apoptosis
Mutated: cell advances and avoids apoptosis, endometrial carcinoma tissue usually lack expression of MLH1 - heavily methylated
51
BRCA1 mutations
Carriers of germline mutations in BRCA1/2 alleles have increased 50-70% risk of developing breast cancer before 70 y/o
BRCA1 associated with replication fork —> dsDNA damage because it is a scaffold to assemble MMR complex for homolgy-directed repair (HR)
52
53
Homology directed repair vs non-homologous end joining
1: late S and G2 phase
- dsDNA break, resection by exonuclease
- base-pairing with unwound DNA of sister chromatid
- strand extension
- extended strands disengages and pair
- restore WT helix
2: G1 phase
- dsNDA break, resection of single strands by exonuclease
- DNA strands brought together, some base pairing
- strands filled in but several base pairs present in original sequence are missing
54
Why is PARP important in the HR pathway
PARP1 generates a chain of poly-ADP that may recruit a set of repair enzyme and ADP-ribosylate them to complete BER.
55
Why is the BCR-ABL1 fusion gene significant in cancer treatment?
It serves as a diagnostic marker for chronic myeloid leukemia (CML) and is a target for specific therapies that inhibit its activity.
56
What is the Philadelphia chromosome?
A specific genetic abnormality found in chromosome 22 of leukemia cells, particularly in chronic myeloid leukemia (CML).
57
What does the notation t(9;22)(q34;q11) represent in the fusion gene BCR-ABL1?
It describes a translocation between chromosome 9 and chromosome 22, indicating the specific locations of the exchange.
The formation of the BCR-ABL1 fusion gene combines portions of the BCR gene from chromosome 22 and the ABL1 gene from chromosome 9.
58
What type of protein does the BCR-ABL1 fusion gene encode? What is the result?
A constitutively active tyrosine kinase that promotes uncontrolled cell division.
It disrupts genomic stability and interferes with signaling pathways, leading to uncontrolled cell proliferation.
59
What is Gleevec used to treat and how does it work?
Treat chronic myelogenous leukemia (CML) by inhibiting the kinase activity of BCR-Abl fusion protein.
60
What type of interactions and where does Gleevec make with the Abl kinase?
Gleevec forms multiple hydrogen bonds with the catalytic cleft of Abl kinase beterrn its N- and C-terminal lobes.
61
What is TMPRSS2 in relation to ERG translocation in prostate cancer?
- Binding of Androgen receptor to TMPRSS2 promotes expression of ERG gene
- Over expression of ERG leads to elevated level of ERG target genes
62
What is karyotype instability?
The continuation of the instability existed in vivo during tumorigenesis.
63
How do CIN and MIN contribute to cancer progression?
They remodel the cellular genome in a way that favors evolution toward neoplasia, particularly aiding in metastasis.
64
What does CIN and MIN stand for?
Chromosomal instability
Microsatallite instability
65
Why do tumor cells need increased mutability in their genomes?
Increased mutability allows tumor cells to adapt and evolve, enhancing their survival and ability to metastasize.
66
Are CIN and MIN mutually exclusive states in cancer?
No, they are not always mutually exclusive; some tumors can exhibit both types of instability simultaneously.
67
What role do unbalanced mitogenic signals play in cancer cells?
They cause uncoordinated firing of replication origins and frequent replication fork collapse.
68
What happens in the genes when replication forks collapse in cancer cells?
The repair of these collapsed replication forks is often imperfect, leading to genomic instability.
This result in local amplifications and deletions in genomes of solid tumors.
69
What is chromothripsis?
chromosome shatters and then is reassembled in a chaotic manner, leading to multiple rearrangements within a small region of the genome. This can result in complex structural variations.
