Checkpoints in Mammalian Cells

1
Checkpoints in Mammalian Cells

• DNA Damage Checkpoint
• Spindle Checkpoint

2
Genetic Instability in Human Cancers

• Mutation
• Chromosome Number
• Chromosome Translocations
• Gene Amplification

3
Genetic Instability in Human Cancers
2 bp deletion in colorectal cancer cell line
(MMR-)
Mutation in p53 gene in XP patient
Translocation of chr 1 (red) to chr 17 (yellow)
in neuroblastoma
Loss of chr 3 (red) and chr 7 (yellow) in CRC
cells
Amplification of N-myc (yellow) in neuroblastoma
4
Mutation Rate in CIN vs MIN Lines
5
Reduction to Homozygosity in CIN vs MIN Lines
6
Checkpoints - A Signal Transduction Pathway

• Sensors
• Signal Transducers
• Targets

7
DNA Damage Checkpoint Pathway in S. pombe
ATM/ATR
hChk2
hChk1
14-3-3
Human Homologs
8
Players in DNA Damage Checkpoint and Repair
9
DNA Damage Checkpoint Pathway Signal?
Single stranded DNA bound by RPA recruits
ATR-ATRIP and Rad17 complex. Colocalization of
the two complexes leads to initiation of the
checkpoint signal.
10
Ataxia Talangiectasis

• Cerebellar Ataxia
• Talangiectasia
• Reduced cellular immunity abnormal thymus
  reduced Ig
• T-cell lymphomas and leukemias predisposition
  to breast cancer
• Radiation hypersensitivity
• Hypogonadism
• Defective G1/S and G2/M arrest
• Mutated ATM

11
ATM Functions

• ATM-/- Cell Lines
• G1/S, InterS andG2/M Checkpoint deficient
• Radiation hypersensitive
• Checkpoint and repair functions genetically
  separable
• ATM-/- Mice
• Similar phenotypes to AT humans
• Reduced CNS apoptosis following g irradiation
• ATM Kinase
• Upstream of BRCA1, BRCA2, p53, Chk1, NBS
• Phosphorylates Brca1, p53, Chk1, Chk2 in response
  to IR
• Specific for (S/T)Q

12
Focus Formation Following Ionizing Radiation
Ionizing radiation induces immediate activation
of ATM (1981S phosphorylation) and modification
of histone H2AX at numerous foci, followed by
accumulation of ATM at the foci.
13
Histone H2AX Deficiency Causes Tumors
H2Ax/ p53/
H2Ax/ p53-/-
H2Ax/D p53-/-
H2AxD/D p53-/-
Mice lacking one of both copies of H2Ax rapidly
develop B and T cell lymphomas and solid tumors
in the absence of p53 function, much more slowly
in the presence of p53.
14
Focus Formation Following Ionizing Radiation
H2AX activation and Nbs recruitment to foci are
independent of 53BP.
15
ATR Functions

• ATR/- Mice
• Small increase in tumor incidence
• Small decrease in survival
• ATR-/- Mice
• Preimplantation embryonic lethals
• Blastocyst cells die during proliferation and
  suffer chromosomal fragmentation
• ATR Kinase
• Required for checkpoint responses to UV, HU and
  IR
• Phosphorylates Chk1 (Ser345), p53 (Ser15) and
  Brca1

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Chromosome Fragmentation in ATR-/- Cells
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Chk1 Functions

• Chk1/- Mice
• Increased tumor incidence in WNT-1 background
• Chk1-/- Mice
• Peri-implantation embryonic lethals
• Blastocyst cells die from apopotosis during
  proliferation
• ES cells defective in G2/M arrest in response to
  IR
• Chk1 Kinase
• Phosphorylated in response to UV, HU and IR
• Phosphorylation dependent on ATR

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ATR/Chk1 and ATM/Chk2 Pathways
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Focus Formation Following Ionizing Radiation
p53BP1 and activated Chk2 are recruited to
IR-induced foci. Activation and recruitment of
Chk2 depends on p53BP.
20
ATM/Chk2 Activation of E2F-1 Promotes Apoptosis
21
Activation of E2F-1 Promotes Apoptosis
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ATR/Chk1 and ATM/Chk2 Pathways in G1

• Phosphorylation of p53 S15 enhances
  transcriptional activation while phosphorylation
  of S20 ablates MDM2 binding.
• Phosphorylation of Cdc25A induces its
  degradation and prevents activation of Cdk2 and
  Cdk4

23
Stalled Replication Forks Elicit Three Responses
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ATR/Chk1 and ATM/Chk2 Pathways in S
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ATR/Chk1 and ATM/Chk2 Pathways in G2
G2/M DNA Damage Checkpoint works primarily
through Cdc25C rather than Cdc25A
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Regulation of Cdks by Tyr-15 Phosphorylation
Active
Nim1
Cdc2
Plk
Myt1
Myt1
Cdc25
Cdc25
Wee1
Wee1
Cdc2
PP2A?
Inactive
27
Regulation of Cdks by Tyr-15 Phosphorylation
Cdc25
Active
14-3-3
Cdk1
Plk
Nim1
Inactive
Cdc25C
Cdc25C
Cdc25C
Myt1
Myt1
Wee1
Wee1
Cdk1
Chk1
PP2A?
Inactive
ATM
Chk1
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A Second Pathway for Checkpoint Arrest in G2
14-3-3s Blocks Nuclear Import of Cdk1/CycB
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Dual Pathways for DNA Damage Checkpoint
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p53 and Checkpoints

• p53 induces p21CIP, 14-3-3s, Bax, Puma
• Transient arrest in G2 following DNA damage does
  not require p53
• Prolonged arrest or apoptosis following DNA
  damage requires p53
• Transient arrest in G1 following IR requires p53

31
ATM/ATR Modification of p53
32
BRCA1/BRCA2

• 10 of breast cancers associated with familial
  BRCA1/2 mutations
• Somatic mutations of BRAC1 not associated with
  sporadic breast cancer
• BRCA1/BRCA2 physically associated with hRad51
• BRCA1 phosphorylated by ATM in response to g
  irradiation
• Maximally expressed post S-phase
• BRCA1 regulates transcription

33
BRCA1 Recruitment to Foci Requires 53BP
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BRCA1 is Required for G2/M But Not G1/S Checkpoint
Mouse cells carrying BRCA1 exon 11 deletion
Normal G1/S arrest in response to g irradiation
No G2/M arrest in response to g irradiation
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Functions of BRCA1/2 in DNA Damage Response
36
BRCA1 Mutants Defective in DNA Recombination
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Multiple Functions of BRCA1
The BASC Complex
Double Strand Break Repair
Rad50
Mre11
Nbs1
Mismatch Repair
BRCA1
BLM
MSH2
ATM
MSH6
MLH1
Rfc p40
Rfc p140
Rfc p37
DNA Damage Repair Signaling
38
Multiple Functions of BRCA1
39
BRCA2 Associates with Rad51
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DS DNA Repair Pathways

• Non-Homologous End Joining (NHEJ)
• Primarily active during G1
• Requires DNA-PKcs, Ku70, Ku80
• Homologous Recombination (Sister Chromatid)
• Primarily active during G2
• Requires Rad51 and Rad52 epistasis group
• Both Require ATM

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DNA PK

• Required for DSB repair and V(D)J joining
• Heterotrimer composed of DNA-PKcs, Ku70, Ku80
• DNA-PKcs encoded by scid
• Ku80-/- and scid cell lines hypersensitive to
  g-irradiation
• Ku80-/- mice
• Impaired T and B lymphocytes
• Ku70-/- mice
• Impaired B but not T lymphocytes
• T cell tumors (like ATM-/- and p53-/- but unlike
  Ku80-/- or scid mice)

42
Spindle Checkpoint Pathway
43
Mitotic Checkpoint Defects in CIN Lines
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Mutations in Spindle Checkpoint Genes in Cancer

• 2/19 Colorectal cancer CIN cell lines had
  mutations in hBub1
• Missense mutation (S492Y)
• Deletion of codons 76-141 followed by frameshift
• G A transition at splice site consensus
• But, second hBub1 allele in both cell lines is
  wild type
• 40 of lung cancer cells are spindle checkpoint
  deficient
• 1/49 lung cancer cells lines had mutation in
  hMad1
• Missense mutation (T299A)

45
Spindle Checkpoint and Apoptosis
Cells synchronized at S released into nocodazole
containing media either with an active (Tet) or
inactive (Tet-) spindle checkpoint
Cells entering G1 with an active spindle
ckeckpoint signal undergo apoptosis