TYPES OF GENETIC INSTABILITY IN CANCER Aneuploidy Chromosome breakage Deletions and translocations...
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Transcript of TYPES OF GENETIC INSTABILITY IN CANCER Aneuploidy Chromosome breakage Deletions and translocations...
TYPES OF GENETIC INSTABILITY IN CANCER
Aneuploidy
Chromosome breakageDeletions and translocationsGene Amplification
HSRs - homogeneously staining regionsDMs - double minutes
Elevation of mutation rates
Epigenetic changes"CIMP" (CpG island methylator) phenotype
Chromosomal Instability in Cancer
MSI Negative MSI Positive
APC(D5S346)
BAT25
BAT26
D2S123
Mfd15CA(D17S250)
N T N T
Microsatellite Instability in Cancer
GENETIC BASIS OF INSTABILITY
Mismatch-repair defects
Base-excision repair defect
Cell-cycle checkpoint defects
p53 mutations (Li-Fraumeni syndrome)
Spindle-checkpoint defects (BUB1, MAD2)
Other defects in repair or recombination
Chromosome breakage syndromes
Pathways of DNA repair
O6-alkylguanine DNA alkyltransferase
suicide protein transfers methyl group
Base excision repair
glycosylase, AP endonuclease, DNA polymerase, ligase
Nucleotide excision repair
transcription-coupled and global
Cross-link repair
Fanconi’s anemia genes and BRCA2
Double-strand break repair
Homologous recombination and non-homologous end- joining
Heteroduplex and mismatch repair
Post-replication repair; translesion synthesis by pol eta, zeta, iota or kappa
Familial cancer syndromes with defects in DNA damage response
ataxia telangiectasia (AT), Nijmegen breakage syndrome, AT-like disorder
- cell cycle checkpoints and DNA repair (dsb repair). ATM, NBS1, MRE-11
Fanconi’s anemia (FA) - DNA repair (cross-link repair). BRCA2 and FANC-A, B, C, D1, and E
Hereditary non-polyposis colorectal cancer (HNPCC) – DNA repair (mismatch repair) and cell cycle checkpoints. hMSH2, hMLH1, PMS2, hMSH6
xeroderma pigmentosum (XP) – DNA repair (nucleotide excision repair, translesion synthesis) and transcriptional regulation. XPA, B, C, D, E, F, G and V
Familial breast cancer I – cell cycle checkpoints. BRCA1
Li-Fraumeni syndrome (LFS) - cell cycle checkpoints, DNA repair and transcriptional regulation. p53, Chk2
Bloom’s syndrome, Werner syndrome, Rothmund-Thompson syndrome – DNA repair and cell cycle checkpoints. Blm, Wrn, RecQL
HUMAN MICROSATELLITES
Repeat units of 1 - 5 base pairs
Tracts of ~ 6 - 30 repeat units
Highly interspersed
>100,000 tracts/genome
Many are polymorphic
Lynch & de la Chapelle, New Eng. J. Med. 348:919 (2003)
MISMATCH-REPAIR PROTEINS
Bacteria: MutH, MutS, MutL
Human: hMSH2 hMSH3 = MutS homologues hMSH6
hMLH1 hPMS2
= MutL homologues
mplexMSH2-MSH3 complex MSH2-MSH6 complex
MLH1-PMS2 complex
Familial cancer syndromes
Ataxia telangiectasia - cell cycle checkpoint function, DNA repair. ATM, NBS1, MRE-11
Fanconi’s anemia - DNA repair. BRCA2 and 5 other genes
HNPCC - mismatch repair, hMSH2, hMLH1, PMS2, hMSH6
Xeroderma pigmentosum - nucleotide excision repair and post-replication repair. 8 XP genes
Familial breast cancer I - S and G2 checkpoint responses. BRCA1
Li-Fraumeni syndrome – cell cycle checkpoint function and DNA repair. P53, Chk2
Bloom’s syndrome, Werner syndrome, Rothmund-Thompson syndrome – chromosomal instability. Blm, Wrn, RecQ
Checkpoint genes and cancer
ATM – mutated in ataxia telangiectasia, a familial cancer syndrome; related to DNA-PK and ATR; phosphorylates NBS1, Chk2, p53, Abl, BRCA1
P53 – mutated in Li-Fraumeni syndrome with early onset breast cancer and fibrosarcoma; transactivates p21Waf1, p48/XPE, 14-3-3
BRCA1 – familial breast cancer; interacts with BASC, ATM, ATR, BRCA2, Rad51 and Rad52
Checkpoints and carcinogenesis
Cancer is characterized by enhanced growth and genetic instability
Cell cycle checkpoints slow growth and preserve genetic stability
Defects in cell cycle checkpoint function enhance growth and genetic instability, thereby fueling malignant progression
Inactivation of p53-dependent G1 checkpoint function in diploid human
fibroblasts
HPV16E6 ablates p53 function by ubiquitin-mediated proteolysis
Use replication-defective retrovirus to transduce HPV16E6 or dominant-negative p53 alleles
p53 dominant-negative alleles (V143A, H179Q) compete for substrates
Flow cytometric analysis of cell cycle checkpoint response to DNA damage:
normal human fibroblasts
G1 checkpoint G2 checkpoint
DNA/PI
Pho
spho
-his
tone
H3
FIT
C-a
nti-B
rdU
P53-dependent G1 checkpoint function
20
40
60
80
100
120
140
10/2
7/200
0
11/3
/2000
11/1
0/200
0
11/1
7/200
0
11/2
4/200
0
12/1
/2000
12/8
/2000
12/1
5/200
0
12/2
2/200
0
12/2
9/200
0
1/5/2
001
1/12/
2001
1/19/
2001
1/26/
2001
2/2/2
001
2/9/2
001
2/16/
2001
2/23/
2001
3/2/2
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3/9/2
001
3/16/
2001
3/23/
2001
3/30/
2001
4/6/2
001
4/13/
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4/20/
2001
4/27/
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001
5/11/
2001
5/18/
2001
5/25/
2001
6/1/2
001
6/8/2
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6/15/
2001
6/22/
2001
6/29/
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7/6/2
001
7/13/
2001
7/20/
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7/27/
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8/3/2
001
8/10/
2001
8/17/
2001
8/24/
2001
8/31/
2001
9/7/2
001
Weeks in Culture
Cu
mu
lati
ve P
DL
NHF1 HIT/LXIN NHF1 HIT/p53-A143VNHF1 HIT/p53-H179Q NHF1 HIT/E6
Inactivation of p53 extends cellular lifespan but leads to crisis; immortal
lines may emerge from crisis
Chromosomal aberrations in aging E6-expressing fibroblasts
Genetic instability in human fibroblasts
Finite lifespan Extended lifespan Immortal
G1 + G1 -G1 -
G2 + G2 +/- G2 +/-
diploid aneuploid,polyploid
aneuploid
What is the source of polyploidy?
E6-expressing cells start with diploid stable genomes
Attenuation of DNA damage G2 checkpoint function in ataxia telangiectasia is not associated with polyploidy
P53-defective cells are prone to polyploidy when they experience stress on the mitotic spindle
Polyploidization in aging E6-expressing cells
DNA inputs to the G2 checkpoint
Decatenation checkpoint monitors chromatid separation by topoII
Model of ATR/ATM-dependent G2 checkpoints
Catenated Chromatids
ATR
DNA dsb
BRCA1
G2 M
Chk1
Cdc25C
Cyclin B1/Cdk1Activity
Plk1
ATM/ATR
Cyclin B1/Cdk1Localization
Attenuation of decatenation checkpoint in aging E6-expressing cells
Expression of telomerase suppresses chromosomal
destabilization in aging E6-expressing cells
Aberrations per 50 cells• Cell Strain PDL Dicentrics/ Breaks/ %Polyploidy %Evading G2
Rings Exchanges Delay
• F7-neo 10 0 2 8 7 5
30 0 2 4 0, 2
• F7-E6 10 0 1 2 17 5
30 19 6 4 22, 48
58 25 38 33 133 66
• F7-E6-TRT(-) 58 40 18 36 88 11
• F7-E6-TRT(+) 58 0 2 3 9 7
0
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normal
lym
phoblast
s
normal
fibro
blast
s
HeLa
DLD1
HCT116
DU145
HEC59
LoVo
"norm
al" b
reas
t epith
elia
Sum10
2
Sum14
9
Sum44
Sum18
5
normal
bla
dder
epith
eliaRT4
UM-U
C-3
TCC-SUP
J82
T24
per
cen
t o
f ce
lls
evad
ing
G2
del
ay
DNA damage Decatenation
Defective G2 checkpoint function in cancer lines
0
10
20
30
40
50
60
mutation status
per
cen
t ev
adin
g G
2 d
elay
wt N B
Figure 5. DNA damage G2 checkpoint function in normal human melanocytes and melanoma cell lines. Upper left panels: flow cytometric quantification of mitotic cells labeled with Anti-phospho-histone H3 antibody. Upper right panel: percent of cells in mitosis 2 h after 1.5 Gy IR, equivalent to the percent of cells evading IR-induced G2 delay. Bottom left panel: G2 checkpoint function in melanoma lines with wildtypeN-Ras and B-Raf alleles (wt, n=4), with mutant N-Ras (N, n=8) or mutant B-Raf (B, n=6).
Summary
• Cancers display two different types of genetic instability affecting microsatellites and chromosomes
• Microsatellite instability is due to defects in mismatch/heteroduplex repair and increases mutation rates
• Chromosomal instability is due to defects in cell cycle checkpoint function and may be driven by telomere erosion to the point of crisis
• Genetic instability increases the risk that clones activate oncogenes and inactivate tumor suppressor genes