Genetyka Nowotworów

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C A N C E R

G E N E T I C S

ALEKSANDER L. SIEROŃ

KATEDRA I ZAKŁAD

BIOLOGII OGÓLNEJ MOLEKULARNEJ I GENETYKI

ŚLĄSKA AKADEMIA MEDYCZNA

*** KATOWICE 2006***

http://biolmolgen.slam.katowice.pl

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CYCLIN : Cdk

pRB : E2F

ppRB : E2F

E2F

ATP

pRB : E2F

ADP

PHASE G1

PHASE S

CYCLIN

Cdk

PHASE G1

PHASE S

REPAIRE OR

APOPTOSIS

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p53

p21

WAF1/CIP

CYCLIN : Cdk

pRB : E2F

ppRB : E2F

E2F

ATP

pRB : E2F

ADP

PHASE G1

PHASE S

CYCLIN

Cdk

PHASE G1

PHASE S

REPAIRE OR

APOPTOSIS

ERROR

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CYCLIN : Cdk

pRB : E2F

ppRB : E2F

E2F

ATP

pRB : E2F

ADP

PHASE G1

PHASE S

CYCLIN

Cdk

PHASE G1

PHASE S

REPAIRE OR

APOPTOSIS

IF LACK OF p53 ACTIVITY

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RECEPTOR

MITOCHONDRIAL

A P

O P

T O

S I

S

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T

R

A

N

S

M

E

M

B

R

A

N

E

D

O

M

A

IN

EX

TR

AC

EL

LU

LA

R D

OM

AIN

C

Y

T

O

P

L

A

S

M

IC

D

O

M

A

IN

Complex

Complex

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Complex

Complex

E

X

T

R

A

C

E

L

L

U

L

A

R

S

P

A

C

E

C

Y

T

O

P

L

A

S

M

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FasL

Extracellular

Domain

pro-Caspase-8

pro-Caspase-8

pro--Bid

cytochrome c

Caspase-9/cytochrome c

Executing Caspases &
Other Substrates

Mitochondria

p15tBid

Fas/CD95

FADD

FADD

Cell Membrane

Receptor Pathway

Mitochondrial Pathway

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(Cys-Proteinases)

INFLAM

ATION

A P

O P

T O

S I

S

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pro--Bid

pro-Caspase-8

pro-Caspase-8

Cytochrome c

Caspase-9/cytochrome c

Executing Caspases &
Other Substrates

Mitochondria

p15tBid

Extracellular
Domain

FasL

Fas/CD95

Cell Membrane

FADD

FADD

Caspase 8

Receptor Pathway

Mitochondrial Pathway

After Ligand Binding (e.g.: Fas)

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Execyting Caspases
& Other Substrates

Caspase 3

pro-Caspase-8

pro-Caspase-8

pro--Bid

Caspase 8

Cytochrome c

Caspase-9/cytochrome c

Mitochondria

p15tBid

Extracellular Domain

FasL

Fas/CD95

Cell Membrane

FADD

FADD

Receptor Pathway

Mitochondrial Pathway

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MUTATIONS

of

A P O P T O S IS

REGULATORS

DiSTU

rbaNc

e

T

ro

uG

h

&

CELL CYCLE

REGULATORS

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pro-Caspase-8

pro-Caspase-8

Extracellular
Domain

FasL

Fas/CD95

Cell Membrane

FADD

FADD

MUTATIONS

INACTIVE

CASPASE 8

Executing Caspases &
Other Substrates

Caspase 3

Cytochrome c

Caspase-9/cytochrome c

Mitochondria

p15tBid

Nucleus

B

cl

2

MUTATIONS

INACTIVE

RECEPTORS

MUTATIONS INACTIVE LIGANDS

MUTATIONS

INACTIVE

KINASES

=

???N

EOPL

AST

IC T

RAN

SFO

RMA

TIO

N???

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Blocking Apoptosis

(Eight known proteins in 2001)

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pro-Caspase-8

pro-Caspase-8

Extracellular
Domain

FasL

Fas/CD95

Błona komórkowa

FADD

FADD

Nucleus

B

cl

2

MUTATIONS

INACTIVE

CASPASE 8

Executing Caspases &
Other Substrates

Caspase 3

Cytochrome c

Caspase-9/Cytochrome c

p15tBid

Mitochondria

p53

B

ax

MUTATIONS

INACTIVE

RECEPTORS

MUTATIONS INACTIVE LIGANDS

MUTATIONS

INACTIVE

KINASES

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p53

p21

WAF1/CIP

CYCLIN : Cdk

pRB : E2F

ppRB : E2F

E2F

ATP

pRB : E2F

ADP

PHASE G1

PHASE S

CYCLIN

Cdk

PHASE G1

PHASE S

REPAIR OR

APOPTOSIS

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Induction of Apoptosis

(fourteen proteins known in 2001)

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pro-Caspase-8

pro-Caspase-8

Extracellular
Domain

FasL

Fas/CD95

Cell Membrane

FADD

FADD

Nucleus

B

cl

2

MUTATIONS

INACTIVE

CASPASE 8

Mitochondria

p53

B

ax

MU

TA

TI

ON

S

M

U

T

A

T

IO

N

S

!!!N

EOP

LAS

TIC

TR

AN

SFO

RM

ATI

ON

!!!

MU

TA

TIO

NS

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Complex

Complex

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E

X

T

R

A

C

E

L

L

U

L

A

R

C

Y

T

O

P

L

A

S

M

Complex

Complex

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(Wyniki mutacji)

NO

REKRUTATION

OF

DEATH

COMPLEX

NO

PROTEOLYTIC

ACTIVITY

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Mutations of Apoptosis Inducing Factors

***

***

***

***

***

***

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p53

p21

WAF1/CIP

CYCLIN : Cdk

pRB : E2F

ppRB : E2F

E2F

ATP

pRB : E2F

ADP

PHASE G1

PHASE S

CYCLIN

Cdk

PHASE G1

PHASE S

REPAIR OR

APOPTOSIS

MUTATIONS

U

N

CO

N

TR

O

LE

D

D

IV

IS

II

O

N

S

CU

M

U

LA

TI

O

N

O

F

M

U

TA

TI

O

N

S

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p53

p21

WAF1/CIP

CYCLIN : Cdk

pRB : E2F

ppRB : E2F

E2F

ATP

pRB : E2F

ADP

PHASE G1

PHASE S

CYCLIN

Cdk

PHASE G1

PHASE S

REPAIR OR

APOPTOSIS

MUTATIONS

U

N

CO

N

TR

O

LE

D

D

IV

IS

IO

N

S

CU

M

U

LA

TI

O

N

O

F

M

U

TA

TI

O

N

S

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p53

p21

WAF1/CIP

CYCLIN : Cdk

pRB : E2F

ppRB : E2F

E2F

ATP

pRB : E2F

ADP

PHASE G1

PHASE S

CYCLIN

Cdk

PHASE G1

PHASE S

REPAIR OR

APOPTOSIS

MUTATIONS

U

N

CO

N

TR

O

LE

D

D

IV

IS

IO

N

S

CU

M

U

LA

TI

O

N

O

F

M

U

TA

TI

O

N

S

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FEW

EXAMPLES

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Clasic

Medulloblastoma

Neuroblastic

Medulloblastoma

Desmoplastic

Medulloblastoma

CLASSIFICATION OF

MEDULLOBLASTOMAS

Krynska B., et al.
(1999) PNAS,
96:11519-24

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IMMUNOHISTOCHEMICAL DETECTION

OF JCV T-ANTIGEN IN MEDULLOBLASTOMAS

Magnification x400 (A &
B); x200 (C); x1000
(inserts)

Arrowheads point to
proliferating cells

Arrows indicate cells with
JCV T-antigen

K

ry

ns

ka

B.

,

et

a

l.

(1

9

9

9

)

PN

A

S

,

9

6

:1

15

19

-2

4

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IMMUNOHISTOCHEMICAL ANALYSIS OF

MEDULLOBLASTOMA MARKERS

GFAP

Magnifications x200 (D) &
x400 (E i F).

Synaptophysin

ββββ

tubulin class III

K

ry

ns

ka

B.

,

et

a

l.

(1

9

9

9

)

PN

A

S

,

9

6

:1

15

19

-2

4

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1469

2533

VP

1

47

71

44

26

5

0

1

3

2603

T

1

7

3

b

p

(4

3

0

3

-4

3

2

7

)

(4

2

5

5

-4

2

7

4

)

(4

40

8-

44

27

)

(2

03

9-

20

19

)

(1

8

4

8

-1

8

2

8

)

(1

89

1-

18

72

)

21

2

b

p

(27

97-2

776

)

(2600-2578)

(2668-26

63)

220 bp

PCR primers

Amplified DNA

Probe

JCV

0.6

0.7

0.8

0.9

0.0

0.1

0.2

0.3

0.4

0.5

(Mad-1)

5130 bp

Control Region

5130/0

44

95

2

7

7

4

9

2

5

2

6

88

3

1560

Ag

no

V

P

3

V

P

2

t

Krynska B., et al.
(1999) PNAS,
96:11519-24

JC VIRUS

GENOME

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PCR DETECTION OF JCV DNA

IN MEDULLOBLASTOMAS

Early coding region

N-terminus of T-antigen

Early Coding Region,

C-terminus of T-antigen

Late coding region of VP1

capsid protein

Krynska B., et al.
(1999) PNAS,
96:11519-24

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Krynska B., et al. (1997) JCB, 67:223-30

T ANTIGEN & p53 COMPLEXES

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Krynska B., et al. (1997) JCB, 67:223-30

p21 EXPRESSION

IN THE PRESENCE OF T-ANTIGEN

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K

ry

ns

ka

B.

,

et

a

l.

(1

9

9

7

)

J

C

B,

6

7

:2

2

3

-3

0

EXPRESSION OF CYCLIN E, A, & CDK2

IN THE PRESENCE OF T-ANTIGEN

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K

ry

ns

ka

B.

,

et

a

l.

(1

9

9

7

)

J

C

B,

6

7

:2

2

3

-3

0

EXPRESSION OF CYCLIN D, CDK4, & CDK6

IN THE PRESENCE OF T-ANTIGEN

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INHIBITION OF APOPTOSIS BY p300

REQUIRES PRESENCE OF CYCLIN D1

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COMPLEXES OF T-ANTIGEN & pRB

Krynska B., et al. (1997) JCB, 67:223-30

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EXPRESSION OF E2F & PCNA

IN THE PRESENCE OF T-ANTIGEN

Krynska B., et al. (1997) JCB, 67:223-30

PCNA – Proliferation Cells Nuclear Antigen

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R

iz

zo

P.

,

et

a

l.

(2

0

0

1)

C

an

ce

r

Bi

ol

,

2

1:

6

3

-7

1

SV40

GENOME

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PCR A M P L I F I C A T I O N

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

-

-

+

+

+

Krynska B., et al.
(1999) PNAS,
96:11519-24

SV

40

&

JC

V

T-

AN

TI

GE

NS

PR

ES

EN

T

IN

AB

OU

T 2

2%

CA

SE

S

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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

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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

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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

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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

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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

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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

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2948–2953, PNAS, March 5, 2002, vol. 99, no. 5

Targeted point mutations of p53 lead to

dominant-negative inhibition of wild-type

p53 function

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

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Mutation

Mutation

Mutation

Homologous recombination

ONE ALLELE MUTATION OF p53 GENE

de Vries A. et al. (2002) PNAS, 99:2948-53.

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EFFECT OF ONE ALLELE MUTATION OF p53 GENE

ON EMBRYONIC CELLS SURVIVAL

NORMAL/

WILDE p53 GENE

ONE p53 ALLELE

INACTIVE

HETEROZYGOUS

p53 MUTATION

de Vries A. et al.
(2002) PNAS, 99:2948-53.

TWO p53 ALLELES

INACTIVE

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p53 DEPENDENT APOPTOSIS

p53 INDEPENDENT

APOPTOSIS

EFFECT OF ONE ALLELE p53 GENE MUTATION

ON THYMUS CELLS SURVIVAL

d

e

V

ri

e

s

A

.

e

t

a

l.

(2

0

0

2

)

PN

A

S

,

9

9

:2

9

4

8

-

5

3

.

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1–5, Nature Genetics, FEBRUARY 11, 2002, online publication

BRCA1 regulates the G2/M checkpoint by

activating Chk1 kinase upon DNA damage

Ronit I. Yarden

1

, Sherly Prado-Reoyo

1

,

Magda Sgagias

2

, Kenneth H. Cowan

2

&

Lowrence C. Brody

1

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

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DNA Damage

ATM/ATR

Chk1 –

A Regulatory Kinase

hCds1/Chk2

?

Cdc25C

Wee1

Kinase

Cdc2/cyclin B

G2

M

ATM, ATR and hCds1/Chk2
Are DNA Damage Response
Proteins Changing
BRCA phosphorylation

BRCA1

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DNA Damage

ATM/ATR

BRCA1

Chk1 -

A Regulatory Kinase

hCds1/Chk2

?

Cdc25C

Wee1

Kinase

Cdc2/cyclin B

G2

M

ATM, ATR and hCds1/Chk2
Are DNA Damage Response
Proteins Changing
BRCA phosphorylation

UNCONTROLED PROLIFERATION

LA

C

K

O

F

B

R

C

A

1

A

C

T

IV

IT

Y

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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.

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Uncontroled divisions

Early Changes

Late Changes

CANCER

Cancer In Situ

Malignant Form (Invasive)

Normal Epithelium

Precancer

Metastasis

Progresive Cumulation

of Gene Damage

CANCER TRANSFORMATION

LOS

S O

F SO

CIA

L CO

NTR

OL

(APO

PTO

SIS

)

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DNA repair disorders may

complicate,

if the disorder causes the patient to be unusually

sensitive to the therapy:

1. cancer chemotherapy

&

2. radiation therapy

MAJOR CONCLUSION

Cancer genetics

Cancer genetics

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THANK YOU

FOR YOUR ATTENTION


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