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Lecture on 31. May 2006 cancelled!
Tumorbiology SS2006-5
 Regulation of cell growth

Cell cycle: CDK, Cyclins, CKI

Apoptosis

Cell senescence/immortalization

Detection of tumorigenic mutations
Kontrollpunkte des Zellzyklus
G1
S
DNA damaged
G2
DNA replication
complete?
M
Chromosomes attached
DNA
to damage?
spindle fibers
Cyclin-dependent
(CDK) phosphorylate
Cell size belowProtein
threshhold Kinases
level
Cell size below threshhold level
proteins:
Protein
biosynthesis, DNA replication, build-up pf
Unfavorable
environment
spindle apparatus, desintegration of the nucleus, formation of
the nuclear membrane, cytokinesis
G1-Cyclins
S-Cyclins
G2/M-Cyclins
CDK
CDK: (cyclin dependent kinase) Protein-Ser/Thr-Kinase
SP oder TP
Binding of the regulatory subunit cyclin necessary for activation.
G1:
CDK2
CDK4
CDK5
CDK2
Cyclin D
-
p16
Cyclin E
-
p21, p27, p57
S:
CDK2
Cyclin A
G2/M:
cdc2
cdc2
Cyclin A
Cyclin B
Candidate substrates of CDK
Substrate
Result of phosphorylation
G1 --> S/S-Phase
pRB
p53
release of transcription factors
regulation of nuclear localisation
G2 -->M/M-Phase
Tyrosine Kinase
Ser/Thr-Kinase
Histon H1
HMG
Nucleolin
Myosin light chain
Lamine
MAP4
Reorganisation of cytoskeleton
?
Chromosome condensation
Chromosome condensation
Nucleoli desintegration
Delay of cytokinesis
Breakdown of nuclear membrane
Collapse of spindle fibres
Cell death by „suicide“
Todesligand
CD95/Fas
Todesrezeptor
Mitochondrium
Caspase 8
Zymogene
Casp-3
Casp-6
Casp-3
Casp 9
Apaf-1
Limitierte Spaltung von Substraten
Cytochrom C
Apoptose
Death may be signaled by direct ligand-enforced clustering of receptors at the cell surface, which leads to the activation of
the "initiator" caspase-8. This caspase then directly activates the "executioner" caspases 3 and 7 (and possibly 6), which
are predominantly responsible for the limited proteolysis that characterizes apoptotic dismantling of the cell. Alternatively,
irreparable damage to the genome caused by mutagens, pharmaceuticals that inhibit DNA repair, or ionizing radiation leads
to the activation of another initiator, caspase-9. The latter event requires the recruitment of pro-caspase-9 to proteins such
as Apaf-1, which requires the proapoptotic factor cytochrome c (cyto C) to be released from mitochondria. Though other
modulators probably regulate the apoptotic pathway in a cell-specific manner, this framework is considered common to most
mammalian cells.
Telomer: spezifische Sequenzen an den Chromosomenenden
This fluorescence microscope image shows human telomeres highlighed by a fluorescent
probe to the human telomere base sequence. The chromosomes glow blue against the dark
background, while the telomere sequences glow greenish. Centromers are in pink.
15
* TRF length in kb
Hayflick limit:
Most normal somatic cells derived
from adults are limited in the
number of times they can divide.
The number of replicative events
that a cell or cell line can undergo
before replicative arrest is known
as the Hayflick limit, named for
their discoverer, Leonard Hayflick.
Germ line
Telomerase active
10
Somatic cells
Telomerase inactive
5
immortalization
Telomerase active
Tumor cells are telomerase positive
immortalized
(TERT+)
M1 M2
crisis
Hayflick
Limit
* DNA loss per division
TRF: telomeric restriction fragment
Capped chromosome ends due to telomeric repeat
The appropriate response to the uncapping of a telomere is action by telomerase
(primarily) or homologous recombination, protecting and/or elongating the telomere so
that cell cycling can resume. Non-homologous end-joining of telomeres can occur,
fusing them and removing the immediate damage signal, but when cell divisions
resume the fused chromosomes are unstable. If none of these ways of capping occurs,
the response of a normal cell is exit from the cell cycle or, in certain mammalian cells,
cell suicide (apoptosis)
Immortalisation
Telomerase positive cells: divide permanently
(immortalized)
Primary stem cells:  telomerase+  „immortal“
Cell lines and tumor cells (tissue) are telomerase+.
Adherent cell lines show contact inhibition.
Transformed cells: no contact inhibition (form foci in
soft agar) in vitro,
establish tumor in immunodeficient
mice (nude mice, SCID mice)
Steps in tumorigenesis
Immortalization
Carcinoma in situ - CIN III (HP)
Abrupt change from normal to
highly dysplastic cells, no cell
diferentiation, basal membrane
still intact.
Tumorbiology SS2006-5






Regulation of cell growth
Cell cycle: CDK, Cyclins, CKI
Signal transduction
Apoptosis
Cell senescence/immortalization
Detection of tumorigenic mutations
Oncogenes
Oncogenes
 Discovery of oncogenes
 Examples for oncogenes
Dominant functions of oncogenic gene products with
regard to the regulation of cell proliferation:
Tyrosine kinases
Signal transduction molecules
Transcription factors
History of tumor genes
••
••
••
••
••
••
•
•
•
•
•
•
•
•
•
1911 Rous
Rous Sarcoma
Sarcoma Virus
Virus (RSV)
(RSV) wird
wird entdeckt
entdeckt
1911
1970 RSV
RSV kodiert
kodiert ein
ein transformierendes
transformierendes Gen
Gen (v-src)
(v-src)
1970
1976 v-src
v-src stammt
stammt von
von einem
einem zellulären
zellulären Gen
Gen (c-src)
(c-src)
1976
1978 src
src kodiert
kodiert für
für eine
eine Proteinkinase
Proteinkinase
1978
1979 chemisch
chemisch transformierte
transformierte Zellen
Zellen enthalten
enthalten ein
ein aktiviertes
aktiviertes Onkogen
Onkogen
1979
Ras bindet
bindet Guaninnukleotide
Guaninnukleotide
Ras
1980 src-Kinase
src-Kinase phosphoryliert
phosphoryliert Tyrosinreste
Tyrosinreste
1980
1981 Virale Insertion aktiviert c-myc-Onkogen
1982 Punktmutation aktiviert ras in menschlichem Blasentumor
1983 v-sis kodiert für einen Wachstumsfaktor, Onkogene kooperieren zur
Zelltransformation
1984 v-erb-B kodiert für einen verkürzten Wachstumsfaktorrezeptor
1986 Genprodukte von transformierenden Genen der DNA-Viren binden Rb, BCL2 inhibiert programmierten Zelltod
1989 TP53 ist ein Tumorsuppressorgen
1991 Rb ist an der Regulation des Zellzyklus beteiligt
1993 hereditäres Kolonkarzinom wird durch defekte DNA-MismatchReparaturgene verursacht
1994 Brustkrebs-Suszeptibilitätsgen (BRCA-1) wird kloniert
Chickens have played a central role
in cancer research. The first tumor
viruses were discovered by Bang and
Ellerman in the early 1900s as
"filterable agents“ (i.e. things that were
smaller than bacteria) which caused
lymphomas in chickens. Shortly
thereafter Rous discovered a virus in
chickens which caused solid tumors
called sarcomas. Both of these
viruses were shown to have RNA
rather than DNA as their genetic
material and therefore became known
as "RNA tumor viruses".
Kochs Postulates (1876) {für ein infektiöses Agens als Ursache}
I. The organism, a germ, should always be found microscopically in the bodies of animals
having the disease and in that disease only; it should occur in such numbers and be
distributed in such a manner as to explain the lesions of the disease.
II. The germ should be obtained from the diseased animal and grown outside the body.
III. The inoculation of these germs, grown in pure cultures, freed by successive
transplantations from the smallest particle of matter taken from the original animal, should
produce the same disease in a susceptible animal.
IV. The germs should be found in the diseased areas so produced in the animal.
A Solution–55 Years Later:
After microbiologists established the existence of viruses at the turn of the century, a
search began for a virus that could cause cancer. To many investigators, the search
seemed foolhardy because cancer did not appear to be an infectious disease.
Nevertheless, one virus did emerge as an apparent cause of a type of cancer.
In 1911, an American physician, Francis Peyton Rous, was studying chickens that had a tumor of the connective tissues called a
sarcoma. Rous decided to test the tumor for virus content, and
he mashed up a section of tissue and passed it through a bacterial filter. To his astonishment, the clear filtrate caused tumors in
healthy chickens. Rous did not refer to the infecting material as a
virus, but others gradually did, and for many decades thereafter, the "Rous sarcoma
virus" remained as a clear-cut example of a cancer-causing virus. The virus soon
became an important tool of cancer researchers. In 1966, Rous was awarded the
Nobel Prize in Physiology or Medicine, 55 years after his discovery. At that time he
was 87 years old.
Rous sarcoma virus
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Mouse Fibroblasten (Bindegewebszellen), hier
NIH
3T3late
Zellen,
in der
Zellkultur
In the
1950swachsen
Temin and
Rubin
showed als
that
such viruses
could die
be quantitatively
studiedzeigen
in cell
adhärente
Zellen,
Kontaktinhibition
cultures.
Rous sarcoma virus could cause cancer(Bild
oben).
like foci of "transformation" in a dish of normal
chicken cells. Because transformation was stably
inherited in infected cells, Temin proposed
that
TS
RSV
RNA tumor viruses converted their genomes into
DNA and integrated into the cellular DNA. This
heretical proposal went against the "central
QuickTime™
a
dogma" of molecular biology
thatand"DNA
makes
TIFF (LZW) decompressor
are
needed
to
see
this
picture.
RNA makes protein". However,
Temin was eventually proven right when his own
lab and David Baltimore independently demonstrated the existence of a viral enzyme called
reverse transcriptase that could convert RNA into
DNA. Because of this "backwards" flow of
3T3-Fibroblasten,
die transformiert
information, these viruses
then becamewurden
known as
"retroviruses".
(Bild
unten).
Schematic Structure of a Retrovirus/Genome
Envelope proteins
(env)
HIV (EM)
Lipidmembrane
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
RNA
Capsid proteins
(gag)
Reverse
Transcriptase
Integrase
Protease
(pol)
R U5
Leader
U3
gag
Cap
PBS
pol
R
(A)n
env
PPT
R
U5
Leader
Cap
U3
gag
PBS
pol
R
(A)n
env
PPT
R Region: A short (18-250 nt) sequence which forms a direct repeat at the both ends of
the genome, which is therefore 'terminally redundant'.
U5: A unique, non-coding region of 75-250 nt which is the first part of the genome to be
reverse transcribed, forming the 3‘ end of the provirus genome.
Primer Binding Site: 18 nt complementary to the 3' end of the specific tRNA primer used by
the virus to begin reverse transcription.
Leader: A relatively long (90-500 nt) non-translated region downstream of the transcription
start site and therefore present at the 5' end of all virus mRNAs.
Polypurine Tract: A short (~10 nt) run of A/G residues responsible for initiating (+)strand
synthesis during reverse transcription.
U3: A unique non-coding region of 200-1,200 nt which forms the 5' end of the provirus
after reverse transcription; contains the promoter elements responsible for transcription of
the provirus.
R U5
Virus-RNA
Cap
U3 R
gag
pol
(A)n
env
Reverse transcription
U3 R U5
AATG
TTAC
gag
pol
LTR
LTR
Integration
ABCDEF
FEDCBA
ABCDEFTG
AC
CATT
GTAA
env
CA ABCDEF
GT
FEDCBA
FEDCBA
Virus-dsDNA
U3 R U5
RSV: genomic RNA
R U5
Cap
U3
gag
pol
env
R
(A)n
v-src
Evidence from several laboratories in the 1970s demonstrated that Rous sarcoma virus had an "extra"
gene which was not required for viral growth, but was required for oncogenic transformation. Such genes
became known as "viral oncogenes". Perhaps the biggest surprise came in the mid-1970s when Stehelin,
Varmus, Bishop, and Vogt demonstrated that the viral oncogene of Rous sarcoma virus (v-Src) had
actually been captured from a normal cellular "proto-oncogene" (c-Src). Furthermore, a closely related
gene was also found in humans. Other viral oncogenes of cellular origin were then identified including vMyb of the avian myeloblastosis virus.
c-Src
1
3
4
Cellular gene = Proto-Oncogene (c-onc)
1
6
Oncovirus/Oncogene
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
p60src
Src is expressed ubiquitously in vertebrate cells; however, brain, osteoclasts, and
platelets express 5- to 200-fold higher levels of this protein than most other cells.
src perinuclear membranes, secretory
In fibroblasts, Src is bound to endosomes,
vesicles, and the cytoplasmic face of the plasma membrane where it can interact
with a variety of growth factor and integrin receptors. The expression of high
levels of Src in platelets (anucleate cells) and in neurons (which are postmitotic)
indicates that Src participates in processes other than cell division.
Survival
Angiogenesis
Proliferation
Motility/Migration
/Invasion
Geschichte der Tumorgene
•
•
•
1911 Rous Sarcoma Virus (RSV) wird entdeckt
1970 RSV kodiert ein transformierendes Gen (v-src)
1976 v-src stammt von einem zellulären Gen (c-src)
•
1978 src encodes a protein kinase
•
1979 chemisch transformierte Zellen enthalten ein aktiviertes Onkogen
Ras bindet Guaninnukleotide
•
1980 src-kinase phosphorylates tyrosine residues
•
•
•
•
•
1981
1982
1983
1984
1986
•
•
•
1989
1991
1993
•
1994
Virale Insertion aktiviert c-myc-Onkogen
Punktmutation aktiviert ras in menschlichem Blasentumor
v-sis kodiert für einen Wachstumsfaktor, Onkogene kooperieren zur Zelltransformation
v-erb-B kodiert für einen verkürzten Wachstumsfaktorrezeptor
Genprodukte von transformierenden Genen der DNA-Viren binden Rb, BCL-2 inhibiert
programmierten Zelltod
TP53 ist ein Tumorsuppressorgen
Rb ist an der Regulation des Zellzyklus beteiligt
hereditäres Kolonkarzinom wird durch defekte DNA-MismatchReparaturgene verursacht
Brustkrebs-Suszeptibilitätsgen (BRCA-1) wird kloniert
Protein phosphorylation
Serine
90 %
Threonine
10 %
COOH
H3N+-C-H
CH2OH
ATP
COOH3N+-C-H OH2C-O-P=O
OH
COOH
H3N+-C-H
CH2OH
CH3
Tyrosine
0.05 %
COOH
H3N+-C-H
CH2
OH
ATP
COOH
H3N+-C-H
CH2
O
O-P=O
OH
Structure of p60src
c-Src
CH3-(CH2)12-CO-
SH3
SH2
Kinase
19
534 As
Y
Aliphatic myristoyl group attached to the N-terminus
(-Ser-Gly-NH-CO-(CH2)12-CH3)
Src homology domains (SH):
SH1:
tyrosine kinase
SH2:
binds phoshorylated tyrosine residues (EXXY)
SH3:
binds proline-rich polypeptide sequences (PXXP)
SH3
ATP
SH2
Y
Y P
active protein
tyrosine kinase
Protein kinase phosphorylation sites and organization of Src
(chicken)
Phosphorylation of pp60src at S, T and Y:
PKA: protein kinase A
PKC: protein kinase C
CSK: C-terminal src kinase
Autoinhibition of Src when carboxyterminal tyrosine
phosphorylated: interaction with internal SH2 comain
Chicken Y527, human Y530
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Why is v-src oncogenic?
p60src
v-Src
CH3-(CH2)12-CO-
SH3
c-Src
CH3-(CH2)12-CO-
SH3 SH2
SH2
Kinase
Kinase
10
526 As
19
534 As
Y
Differences:
promoter
carboyterminus
3´-untranslated region
v-Src is oncogenic in vivo and transforms fibroblasts in vitro:
1)
2)
Strong constitutive expression from viral promoter/enhancer (LTR).
v-Src gene product is constitutive active due to the lack of the carboxyterminal tyrosine.
p60v-src kann nicht negativ reguliert werden.
Y
Y
Inactive protein
tyrosine kinase
SH2
SH2
P
SH3
SH3
ATP
P
Y
Y
P
Y P
Y
Active protein
tyrosine kinase
Oncogenes
Oncoviruses
Oncogene
encode besides the genes for its replication
additional sequences which endow them with
tumorigenic potential:
viral oncogene (v-onc).
= DNA sequence with proven tumorigenic potential:
in tissue culture, animal models or human cancer.
Oncogenes
Oncovirus and oncogenes:
cell
Act dominantly
with regard to
proliferation
Additional oncogenic tyrosine kinases
Signal transduction molecules
Transcription factors
Geschichte der Tumorgene
•
•
•
•
•
•
•
•
•
1911 Rous Sarcoma Virus (RSV) wird entdeckt
1970 RSV kodiert ein transformierendes Gen (v-src)
1976 v-src stammt von einem zellulären Gen (c-src)
1978 src kodiert für eine Proteinkinase
1979 chemisch transformierte Zellen enthalten ein aktiviertes Onkogen
Ras bindet Guaninnukleotide
1980 src-Kinase phosphoryliert Tyrosinreste
1981 Virale Insertion aktiviert c-myc-Onkogen
1982 Punktmutation aktiviert ras in menschlichem Blasentumor
1983 v-sis kodiert für einen Wachstumsfaktor, Onkogene kooperieren zur
•
1984 v-erb-B kodiert für einen verkürzten Wachstumsfaktorrezeptor
•
1986 Genprodukte von transformierenden Genen der DNA-Viren binden Rb, BCL-2 inhibiert
programmierten Zelltod
1989 TP53 ist ein Tumorsuppressorgen
1991 Rb ist an der Regulation des Zellzyklus beteiligt
1993 hereditäres Kolonkarzinom wird durch defekte DNA-MismatchReparaturgene verursacht
1994 Brustkrebs-Suszeptibilitätsgen (BRCA-1) wird kloniert
•
•
•
•
Zelltransformation
Wachstumfaktoren und Wachstumsfaktorrezeptoren
PDGF-A
PDGF-B
CSF; kitL
Insulin
FGF5
EGF
TGFß
aFGF Neurotrophine
NGF
bFGF
BDN
KGF
F
IGF-1
Ligand
c-kit
TK
TK
TK
TK
EGFR=HER
NGFR
1
FGFR1
BDNFR
HER2
etc.
IR: c-ros
CSF-1R
NGFR: c-trk
BDNFR: c-trkB
PDGFRß
EGFR: c-erbB
HER2: neu
CSF-1R: c-fms
SCFR: c-kit
PDGFRa
TK
TK
TM
IR
Zytoplasma
PDGF: Thrombozytenwachstumsfaktor (platelet derived growth
factor): Thrombozyten, Tumorzelllinien, Endothel,
Makrophagen, Zytotrophoblast
PDGFR: auf Bindegewebszellen
EGF: epidermaler WF: Speicheldrüse, Thrombozyten, etc.
EGFR: epidermale Zellen
CSF-1: koloniestimulierender Faktor-1 (colony stimulating factor):
Fibroblasten
CSF-1R: Makrophagen, Placenta, hämatopoetische Zellen
SCF: Stammzellfaktor: Knochenmark-Stromazellen, T-Zellen,
Fibroblasten, Leber, stimuliert die Hämatopoese,
Melanogenese, Gametogenese
P Y
Y P
Y
Mitogenes
Signal
Mitogenic Signal
SH2
P Y
Y P
Y
SH2
SH3
Rezeptorautophosphorylierung
SH2-Proteine
binden an Tyr P
Changed subcellular localization,
Phosphorylation, conformational change
Change in protein activity
Specific
transduction
Signal reception
PLCg
DG
PIP3
P
P
PI 3´K
IP3
P
P
P
P
AKT
Signal effect
Adapters
Ras
P
P
P
P
ENDE