ALEKSANDER L. SIEROC

http://biolmolgen.slam.katowice.pl
C A N C E R
G E N E T I C S
KATEDRA I ZAKA
AD
BIOLOGII OGÓLNEJ MOLEKULARNEJ I GENETYKI
ÅšL
SKA AKADEMIA MEDYCZNA
*** KATOWICE 2006***
CYCLIN Cdk CYCLIN : Cdk
ATP
ADP
pRB : E2F pRB : E2F
ppRB : E2F E2F
PHASE G1 PHASE S
PHASE G1 PHASE S
REPAIRE OR APOPTOSIS
p53
ERROR
p21WAF1/CIP
CYCLIN Cdk CYCLIN : Cdk
ATP
ADP
pRB : E2F pRB : E2F
ppRB : E2F E2F
PHASE G1 PHASE S
PHASE G1 PHASE S
REPAIRE OR APOPTOSIS
IF LACK OF p53 ACTIVITY
CYCLIN Cdk CYCLIN : Cdk
ATP
ADP
pRB : E2F pRB : E2F
ppRB : E2F E2F
PHASE G1 PHASE S
PHASE G1 PHASE S
REPAIRE OR APOPTOSIS
RECEPTOR
MITOCHONDRIAL
A
P
O
P
T
O
S
I
S
Complex
Complex
DOMAIN
CYTOPLASMIC
TRANSMEMBRANE DOMAIN
E
X
T
R
A
C
E
L
L
U
L
A
R
D
O
M
A
I
N
Complex
Complex
EXTRACELLULAR SPACE
CYTOPLASM
Receptor Pathway
FasL
Extracellular
Fas/CD95
Domain
Cell Membrane
Mitochondrial Pathway
Mitochondria
pro--Bid
p15tBid
FADD
FADD
cytochrome c
Caspase-9/cytochrome c
pro-Caspase-8
pro-Caspase-8
Executing Caspases &
Other Substrates
(Cys-Proteinases)
N
O
I
T
A
M
A
L
F
N
I
S
I
S
O
T
P
O
P
A
Receptor Pathway
FasL
After Ligand Binding (e.g.: Fas)
Extracellular
Fas/CD95
Domain
Cell Membrane
Mitochondrial Pathway
FADD
FADD
Mitochondria
pro--Bid
p15tBid
pro-Caspase-8
pro-Caspase-8
Cytochrome c
Caspase-9/cytochrome c
Caspase 8
Executing Caspases &
Other Substrates
Receptor Pathway
FasL
Extracellular Domain
Fas/CD95
Cell Membrane
Mitochondrial Pathway
FADD
FADD
Mitochondria
pro--Bid
p15tBid
pro-Caspase-8
pro-Caspase-8
Cytochrome c
Caspase-9/cytochrome c
Caspase 8
Caspase 3
Execyting Caspases
& Other Substrates
DiSTU
MUTATIONS
of
A P O P T O S IS
REGULATORS
CELL CYCLE
REGULATORS
&
h
r
b
G
a
N
u
c
e
o
r
T
MUTATIONS INACTIVE LIGANDS
FasL
MUTATIONS
Nucleus
Extracellular
INACTIVE
Fas/CD95
Domain
RECEPTORS
Cell Membrane
MUTATIONS
INACTIVE
FADD
FADD
Mitochondria
KINASES
p15tBid
pro-Caspase-8
pro-Caspase-8
Cytochrome c
MUTATIONS
Caspase-9/cytochrome c
INACTIVE
Caspase 3
CASPASE 8
Executing Caspases &
Other Substrates
2
l
c
B
?
?
?
N
E
O
P
L
A
S
T
I
=
C
T
R
A
N
S
F
O
R
M
A
T
I
O
N
?
?
?
Blocking Apoptosis
(Eight known proteins in 2001)
MUTATIONS INACTIVE LIGANDS
FasL
MUTATIONS
Nucleus
Extracellular
INACTIVE
Fas/CD95
Domain p53
RECEPTORS
Błona komórkowa
MUTATIONS
INACTIVE
FADD
FADD
KINASES
Mitochondria
p15tBid
pro-Caspase-8
pro-Caspase-8
Cytochrome c
MUTATIONS
Caspase-9/Cytochrome c
INACTIVE
CASPASE 8
Caspase 3
Executing Caspases &
Other Substrates
2
l
c
B
x
a
B
p53
p21WAF1/CIP
CYCLIN Cdk CYCLIN : Cdk
ATP
ADP
pRB : E2F pRB : E2F
ppRB : E2F E2F
PHASE G1 PHASE S
PHASE G1 PHASE S
REPAIR OR APOPTOSIS
Induction of Apoptosis
(fourteen proteins known in 2001)
FasL
Nucleus
Extracellular
Fas/CD95
Domain p53
Cell Membrane
FADD
FADD
Mitochondria
pro-Caspase-8
pro-Caspase-8
MUTATIONS
INACTIVE
CASPASE 8
S
N
S
O
I
T
A
N
T
U
M
O
I
T
2
l
A
M
c
U
T
B
T
x
A
a
T
I
!
B
O
!
U
!
N
N
S
E
M
O
P
L
A
S
T
I
C
T
R
A
N
S
F
O
R
M
A
T
I
O
N
!
!
!
Complex
Complex
Complex
Complex
EXTRACELLULAR
CYTOPLASM
(Wyniki mutacji)
NO NO
REKRUTATIONPROTEOLYTIC
OF ACTIVITY
DEATH
COMPLEX
Mutations of Apoptosis Inducing Factors
***
***
***
***
***
***
p53
MUTATIONS
p21WAF1/CIP
CYCLIN Cdk CYCLIN : Cdk
ATP
ADP
pRB : E2F pRB : E2F
ppRB : E2F E2F
PHASE G1 PHASE S
PHASE G1 PHASE S
REPAIR OR APOPTOSIS
U
N
C
O
D
N
I
T
V
R
C
I
U
O
S
M
I
O
L
I
E
F
U
O
D
L
M
N
A
S
U
T
T
I
O
A
T
N
I
O
N
S
p53
MUTATIONS
p21WAF1/CIP
CYCLIN Cdk CYCLIN : Cdk
ATP
ADP
pRB : E2F pRB : E2F
ppRB : E2F E2F
PHASE G1 PHASE S
PHASE G1 PHASE S
REPAIR OR APOPTOSIS
U
N
C
O
D
N
I
T
V
R
C
I
U
O
S
M
O
L
I
E
F
O
U
D
L
N
M
A
S
U
T
T
I
O
A
T
N
I
O
N
S
p53
p21WAF1/CIP
CYCLIN Cdk CYCLIN : Cdk
ATP
MUTATIONS
ADP
pRB : E2F pRB : E2F
ppRB : E2F E2F
PHASE G1 PHASE S
PHASE G1 PHASE S
REPAIR OR APOPTOSIS
U
N
C
O
D
N
I
T
V
R
C
I
U
O
S
M
O
L
I
E
F
O
U
D
L
N
M
A
S
U
T
T
I
O
A
T
N
I
O
N
S
FEW
EXAMPLES
CLASSIFICATION OF
MEDULLOBLASTOMAS
Clasic Neuroblastic
Medulloblastoma Medulloblastoma
Krynska B., et al.
Desmoplastic
(1999) PNAS,
Medulloblastoma
96:11519-24
IMMUNOHISTOCHEMICAL DETECTION
OF JCV T-ANTIGEN IN MEDULLOBLASTOMAS
Arrows indicate cells with
JCV T-antigen
Arrowheads point to
proliferating cells
Magnification x400 (A &
B); x200 (C); x1000
(inserts)
Krynska B., et al. (1999) PNAS, 96:11519-24
IMMUNOHISTOCHEMICAL ANALYSIS OF
MEDULLOBLASTOMA MARKERS
Synaptophysin ² tubulin class III
²
²
²
Magnifications x200 (D) &
x400 (E i F).
GFAP
Krynska B., et al. (1999) PNAS, 96:11519-24
5130/0
Control Region
JC VIRUS
GENOME
0.6
0.7
0.5 0.8
JCV
(Mad-1)
0.4 0.9
1469
5130 bp
0.3 0.0
0.2 0.1
2533
2603
(2600-2578)
PCR primers
Krynska B., et al.
Amplified DNA
(1999) PNAS,
Probe
96:11519-24
)
)
7
7
5
2
2
4
0
3
4
4
4
1
-
7
-
7
8
3
7
3
7
0
A
2
0
1
4
g
3
p
4
n
(
o
4
b
(
4
2
4
3
t
2
9
7
6
4
1
6
4
2
4
5
9
)
5
4
3
7
8
8
2
4
-
5
5
V
2
V
P
4
(
P
2
3
1
5
6
0
T
)
8
2
8
1
-
8
1
4
P
8
V
1
(
p
b
2
)
)
1
9
1
2
2
0
7
2
-
8
9
1
3
-
(
0
1
2
2
7
9
(
9
8
7
-
1
2
(
7
7
6
)
2
2
0
b
p
(
2
6
6
8
-
2
6
6
3
)
PCR DETECTION OF JCV DNA
IN MEDULLOBLASTOMAS
Krynska B., et al.
(1999) PNAS,
96:11519-24
Early coding region
N-terminus of T-antigen
Early Coding Region, Late coding region of VP1
C-terminus of T-antigen capsid protein
T ANTIGEN & p53 COMPLEXES
Krynska B., et al. (1997) JCB, 67:223-30
p21 EXPRESSION
IN THE PRESENCE OF T-ANTIGEN
Krynska B., et al. (1997) JCB, 67:223-30
EXPRESSION OF CYCLIN E, A, & CDK2
IN THE PRESENCE OF T-ANTIGEN
Krynska B., et al. (1997) JCB, 67:223-30
EXPRESSION OF CYCLIN D, CDK4, & CDK6
IN THE PRESENCE OF T-ANTIGEN
Krynska B., et al. (1997) JCB, 67:223-30
INHIBITION OF APOPTOSIS BY p300
REQUIRES PRESENCE OF CYCLIN D1
COMPLEXES OF T-ANTIGEN & pRB
Krynska B., et al. (1997) JCB, 67:223-30
EXPRESSION OF E2F & PCNA
IN THE PRESENCE OF T-ANTIGEN
PCNA  Proliferation Cells Nuclear Antigen
Krynska B., et al. (1997) JCB, 67:223-30
Rizzo P., et al. (2001) Cancer Biol, 21:63-71
GENOME
SV40
Krynska B., et al.
(1999) PNAS,
PCR A M P L I F I C A T I O N
96:11519-24
Immuno
cyto SV40 JCV
Age chemistry T-Ag T-Ag T-Ag
No. Sex years Diagnosis T-Ag (C-term) (N-term) (C-term) VP-1
1 M 5 Classic N/A - + - +
2 M 7 Desmoplastic N/A + + + +
3 M 6 Desmoplastic N/A - + - +
4 M 4 Classic N/A - + + +
5 M 5 Desmoplastic N/A - + - +
6 F 11 Classic N/A + + - +
7 M 2 Neuroblastic N/A - + + +
8 M 1.5 Desmoplastic - - + - +
9 F 4 Desmoplastic - - + + +
10 M 15 Desmoplastic + + + - -
11 F 42 Desmoplastic - - + - -
12 M 7 Classic - - + + +
13 F 18 Neuroblastic - - + - +
14 F 8 Desmoplastic - - + - +
15 M 7 Classic + - + + +
16 M 9 Classic - + + + +
17 F 3 Desmoplastic + - + + +
18 F 9 Desmoplastic - - - + +
19 M 5 Neuroblastic + - + + +
20 F 2 Classic - - - + +
21 M Newborn Classic - - + + +
22 M 12 Classic - + - - -
23 M 5 Neuroblastic - - + + +
V
C
J
S
&
N
E
0
S
4
E
G
I
V
S
T
T
S
A
N
N
C
E
A
S
%
-
E
2
T
R
2
P
T
U
O
B
A
N
I
Table 2. SV40 DNA detection in human tumors: PCR based
evidence
Tumor type Reference Primer set (amplicon size bp) Positivity %
rate
Pleural Griffiths et al.36 SV.for3/SV.rev (105) 26/26 100
mesothelioma SV.for2/SV.rev (574) 3/26 12
De Luca et al.27 SV.for3/SV.rev (105) 30/35 86
Pass et al.30 SV.for3/SV.rev (105) 30/42 71
Mayall et al.41 SV.for3/SV.rev (105) 5/7 71
Shivapurkar et al.44 SV.for3/SV.rev (105) 61/93 66
Carbone et al.16 SV.for3/SV.rev (105) 29/48 60
PYV.for/PYV.rev (172) 36/48 75
Procopio et al.54 PYV.for/PYV.rev (172) 50/83 60
Ramael et al.103 PYV.for/PYV.rev (172) 14/25 56
SV.for2/SV.rev (574)
Cristaudo et al.48 RA3/RA4 (482) 10/18 56
Galateau-Salle PYV.for/PYV.rev (172) 10/21 48
et al.33
Table 2. SV40 DNA detection in human tumors: PCR based
evidence (cont. 1)
Tumor type Reference Primer set (amplicon size bp) Positivity %
rate
Pleural Dhaene et al.40 SV.for3/SV.rev (105) 13/28 46
mesothelioma Pepper et al.18 SV.for3/SV.rev (105) 4/9 44
PYV.for/SV.rev (172) 6/9 67
Strizzi et al.47 PYV.for/PYV.rev (172) 9/23 39
Mutti et al.100 SV.for3/SV.rev (105) 8/25 32
Strickler and SV.for3/SV.rev (105) 0/50 0
Goedert 23
Mulaterro et al.63 PYV.for/SV.rev (172) 0/12 0
Hirvonen et al.37 SV2for/SV.rev (576) 0/49 0
Emri et al.51 SV.for2/SV.rev (574) 0/29 0
Table 2. SV40 DNA detection in human tumors: PCR based
evidence (cont. 2)
Tumor type Reference Primer set (amplicon size bp) Positivity %
rate
Peritoneal Shivapurkar et al.44 SV.for3/SV.rev (105) 6/11 55
mesotheliomas
Brochopulmonary Galateau-Salle PYV.for/PYV.rev (172) 18/63 29
carcinomas et al.33
Shivapurkar et al.44 SV.for3/SV.rev (105) 0/20 0
Procopio et al.34 PYV.for/SV.rev (172) 0/18 0
Carbone et al.16 SV.for3/SV.rev (105) 0/13 0
Pepper et al.18 SV.for3/SV.rev (105) 0/9 0
SV.for3/SV.rev (105)
Choroid plexus Martini et al.20 PYV.for/PYV.rev (172) 5/6 83
tumours Bergsagel et al.14 SV.for3/SV.rev (105) 10/20 50
Huang et al.39 SVTAGP1/SVTAGP3 (158) 6/16 38
Lednicky et al.106 LA1/LA2 (294) 10/13 77
Bergsagel et al.14 SV.for3/SV.rev (105) 10/20 50
Ependymomas Bergsagel et al.14 SV.for3/SV.rev (105) 10/11 91
Lednicky et al.106 LA1/LA2 (294) 3/3 100
Martini et al.20 PYV.for/PYV.rev (172) 8/11 73
Table 2. SV40 DNA detection in human tumors: PCR based
evidence (cont. 3)
Tumor type Reference Primer set Positivity %
(amplicon size bp) rate
Ependymomas Weggen et al.53 SV.for3/SV.rev (105) 1/25 4
Bersagel et al.14 SV.for3/SV.rev (105) 7/11 64
Medulloblastomas Lednicky et al.106 LA1/LA2 (294) 2/6 33
Huang et al.39 SVTAGP1/SVTAGP3 (158) 5/17 29
Weggen et al.53 SV.for3/SV.rev (105) 2/116 2
Krynska et al.102 SV.for3/SV.rev (105) 5/23 22
Astrocytomas Martini et al.20 PYV.for/PYV.rev (172) 8/17 17
Huang et al.39 SVTAGP1/SVTAGP3 (158) 22/50 44
Meningiomas Martini et al.20 PYV.for/PYV.rev (172) 1/7 14
Weggen et al.53 SV.for3/SV.rev (105) 1/131 1
Oligodendroglioma Huang et al.39 SVTAGP1/SVTAGP3 (158) 3/12 25
Martini et al.20 PYV.for/PYV.rev (172) 0/9 0
Glioblastomas Martini et al.52 PYV.for/PYV.rev (172) 10/30 33
Table 2. SV40 DNA detection in human tumors: PCR based
evidence (cont. 4)
Tumor type Reference Primer set Positivity %
(amplicon size bp) rate
Osteosarcomas Lednicky et al.15 SV.for3/SV.rev (105) 5/10 50
TA1/TA2 (441) 5/10 50
Yamamoto et al.50 R1/R2 (315) 21/54 39
Carbone et al.17 SV.for2/SV.rev (574) 40/126 32
Mendoza et al.32 SV.for3/SV.rev (105) 9/35 26
Other bone Carbone et al.17 SV.for2/SV.rev (574) 14/34 32
Tumours Gamberi et al.46 PYV.for/SV.rev (172) 30/107 28
SV.for2/SV.rev (574)
Papillary thyroid Pacini et al.31 SV.for3/SV.rev (105) 3/69 4
carcinomas (PTC) LA1/LA2 (294)
Non-PTC thyroid Pacini et al.31 SV.for3/SV.rev (105) 0/9 0
tumours LA1/LA2 (294)
Non-Hodgkin s Rizzo et al.38 V5/SV6 (172) 3/29 10
lymphomas Martini et al.52 PYV.for/PYV.rev (172) 13/95 14
Table 2. SV40 DNA detection in human tumors: PCR based
evidence (cont. 5)
Tumor type Reference Primer set Positivity %
(amplicon size bp) rate
Hodgkin s Martini et al.52 PYV.for/PYV.rev(172) 8/55 15
lymphomas
AIDS lymphoma Rizzo et al.38 SV5/SV6 (172) 2/5 40
Prostatic tumours Radu et al.68 SV.for3/SV.rev (105) 4/33 12
2948 2953, PNAS, March 5, 2002, vol. 99, no. 5
Annemieke de Vries* ! , Elsa R.
Flores*, Barbara Miranda* , Harn-Mei
Hsieh*, Conny Th. M. van Oostrom ! ,
Julien Sage*, and Tyler Jacks*
Howard Hughes Medical Institute,
Massachusetts Institute of Technology,
Cambridge, MA
and ! National Institute of Public Health,
Laboratory of Health Effects Research, 3720 BA
Bilthoven, The Netherlands
Targeted point mutations of p53 lead to
dominant-negative inhibition of wild-type
p53 function
ONE ALLELE MUTATION OF p53 GENE
Mutation
Homologous recombination
Mutation
Mutation
de Vries A. et al. (2002) PNAS, 99:2948-53.
EFFECT OF ONE ALLELE MUTATION OF p53 GENE
ON EMBRYONIC CELLS SURVIVAL
TWO p53 ALLELES
INACTIVE
HETEROZYGOUS
p53 MUTATION
ONE p53 ALLELE
INACTIVE
NORMAL/
WILDE p53 GENE
de Vries A. et al.
(2002) PNAS, 99:2948-53.
EFFECT OF ONE ALLELE p53 GENE MUTATION
ON THYMUS CELLS SURVIVAL
p53 DEPENDENT APOPTOSIS p53 INDEPENDENT
APOPTOSIS
de Vries A. et al.
(2002) PNAS, 99:2948-53.
1 5, Nature Genetics, FEBRUARY 11, 2002, online publication
Ronit I. Yarden1, Sherly Prado-Reoyo1,
Magda Sgagias2, Kenneth H. Cowan2 &
Lowrence C. Brody1
1 Genome Technology Branch, National Human
Genome Research Institute, National Institutes
of Health, Bethesda, MD
2 Eppley Institute of Cancer Research,
University of Nebraska Medical Center, NA
BRCA1 regulates the G2/M checkpoint by
activating Chk1 kinase upon DNA damage
DNA Damage
ATM/ATR
ATM, ATR and hCds1/Chk2
Are DNA Damage Response
Proteins Changing
BRCA phosphorylation
?
hCds1/Chk2 BRCA1 Chk1  A Regulatory Kinase
Wee1 Cdc25C
Kinase
Cdc2/cyclin B
G2 M
DNA Damage
ATM/ATR
ATM, ATR and hCds1/Chk2
Are DNA Damage Response
Proteins Changing
BRCA phosphorylation
?
hCds1/Chk2 BRCA1 Chk1 - A Regulatory Kinase
Wee1 Cdc25C
Kinase
Cdc2/cyclin B
UNCONTROLED PROLIFERATION
G2 M
Y
T
I
V
I
T
C
A
1
A
C
R
B
F
O
K
C
A
L
Lindenboim L., et al. Cell Death and
Differentiation (2005) 12, 713 723
Figure 6 A model depicting the
apoptotic effects of Bcl-xS in MEFs.
Expression
of Bcl-xS in MEFs induces exposure
of NT of Bak, leading to its
activation. The
activated Bak can induce three
pathways: a major pathway leading to
cytochrome c release, which in turn
causes apoptosome activation and cell
death in a caspase-dependent manner;
a second pathway, which leads to
Apaf-
1- and caspase-9-independent cell
death; and a third pathway, which
induces the
exposure of Bax NT, both in a
caspase-9-dependent and -
independent manner,
causing its activation. The first and
the second pathways contribute to the
death
process. The role of the third pathway
is not yet known.
CANCER TRANSFORMATION
Normal Epithelium
Uncontroled divisions
Progresive Cumulation
Early Changes
of Gene Damage
Precancer
Late Changes
CANCER
Cancer In Situ
Malignant Form (Invasive)
Metastasis
L
O
R
T
N
O
C
L
A
I
C
)
O
S
S
I
S
F
O
O
T
P
S
O
S
P
O
A
L
(
MAJOR CONCLUSION
DNA repair disorders may
complicate,
if the disorder causes the patient to be unusually
sensitive to the therapy:
Cancer genetics
Cancer genetics
1. cancer chemotherapy
&
2. radiation therapy
THANK YOU
FOR YOUR ATTENTION