lecture on 31. may 2006 cancelled! regulation of cell growth cell cycle: cdk, cyclins, cki apoptosis...

Lecture on 31. May 2006 cancelled!

Regulation of cell growth Cell cycle: CDK, Cyclins, CKI Apoptosis Cell senescence/immortalization

Detection of tumorigenic mutations

Tumorbiology SS2006-5Tumorbiology SS2006-5

Kontrollpunkte des Zellzyklus

G1G1 SS G2G2 MM

Unfavorable environment

Cell size below threshhold level

DNA damaged

Cell size below threshhold level

DNA replication complete?

DNA damage?Chromosomes attached

to spindle fibers

Cyclin-dependent Protein Kinases (CDK) phosphorylate proteins: Protein biosynthesis, DNA replication, build-up pf spindle apparatus, desintegration of the nucleus, formation of the nuclear membrane, cytokinesis

G1-Cyclins S-Cyclins G2/M-CyclinsCDKCDK

CDK: (cyclin dependent kinase) Protein-Ser/Thr-Kinase

SP oder TP

Binding of the regulatory subunit cyclin necessary for activation.

G1: CDK2CDK4 Cyclin D p16CDK5CDK2 Cyclin E p21, p27, p57

S: CDK2 Cyclin A

G2/M: cdc2 Cyclin Acdc2 Cyclin B

-

-

Candidate substrates of CDKCandidate substrates of CDK

Substrate Result of phosphorylation

G1 --> S/S-PhasepRB release of transcription factorsp53 regulation of nuclear localisation

G2 -->M/M-PhaseTyrosine Kinase Reorganisation of cytoskeletonSer/Thr-Kinase ?Histon H1 Chromosome condensationHMG Chromosome condensationNucleolin Nucleoli desintegrationMyosin light chain Delay of cytokinesisLamine Breakdown of nuclear membraneMAP4 Collapse of spindle fibres

Cell death by „suicide“Cell death by „suicide“

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.

Todesligand

Todesrezeptor

Caspase 8

Zymogene

Casp-6Casp-3

Casp-3

Limitierte Spaltung von SubstratenLimitierte Spaltung von Substraten ApoptoseApoptoseApoptoseApoptose

Mitochondrium

Apaf-1Casp 9 Cytochrom C

CD95/Fas

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.

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.

HayflickHayflickLimitLimit

* DNA loss per divisionTRF: telomeric restriction fragment

5

10

15 Germ line

Somatic cells

immortalization

M1M1 M2M2* T

RF

leng

th in

kb

Telomerase active

Telomerase inactive

Telomerase active

crisisTumor cells are telomerase positiveimmortalized(TERT+)

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)

Capped chromosome ends due to telomeric repeat

ImmortalisationImmortalisation

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 Transformed cells: no contact inhibition (form foci in soft agar) in vitro,establish tumor in immunodeficient mice (nude mice, SCID mice)

Steps in tumorigenesisSteps in tumorigenesis

ImmortalizationCarcinoma in situ - CIN III (HP)Abrupt change from normal to highly dysplastic cells, no cell diferentiation, basal membrane still intact.

Regulation of cell growth Cell cycle: CDK, Cyclins, CKI Signal transduction Apoptosis Cell senescence/immortalization

Detection of tumorigenic mutations

Tumorbiology SS2006-5Tumorbiology SS2006-5

OncogenesOncogenes

Discovery of oncogenesExamples for oncogenes

Dominant functions of oncogenic gene products with regard to the regulation of cell proliferation:

Tyrosine kinasesSignal transduction moleculesTranscription factors

OncogenesOncogenes

History of tumor genes

• 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

Zelltransformation• 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-Mismatch-

Reparaturgene verursacht• 1994 Brustkrebs-Suszeptibilitätsgen (BRCA-1) wird kloniert


Ras bindet Guaninnukleotide• 1980 src-Kinase phosphoryliert Tyrosinreste

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, asearch began for a virus that could cause cancer. To many investigators, the searchseemed 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 study-ing 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 bacte-rial 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.

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

Rous sarcoma virusRous sarcoma virus

Mouse Fibroblasten (Bindegewebszellen), hierNIH 3T3 Zellen, wachsen in der Zellkultur als adhärente Zellen, die Kontaktinhibition zeigen (Bild oben).

3T3-Fibroblasten, die transformiert wurden (Bild unten).

In the late 1950s Temin and Rubin showed that such viruses could be quantitatively studied in cell cultures. Rous sarcoma virus could cause cancer-like foci of "transformation" in a dish of normal chicken cells. Because transformation was stably inherited in infected cells, Temin proposed that RNA tumor viruses converted their genomes into DNA and integrated into the cellular DNA. This heretical proposal went against the "central dogma" of molecular biology that "DNA makes RNA makes protein". However, Temin was eventually proven right when his own lab and David Baltimore independently demon-strated the existence of a viral enzyme called reverse transcriptase that could convert RNA into DNA. Because of this "backwards" flow of information, these viruses then became known as "retroviruses".

RSVRSVTSTS



Envelope proteins(env)

Lipid-membrane

Capsid proteins(gag)

RNA

ReverseTranscriptase

IntegraseProtease

(pol)

HIV (EM)

Schematic Structure of a Retrovirus/Genome

Cap (A)ngag pol envR U5

PBS

Leader

PPT

U3 R



Cap (A)ngag pol env

PBS PPT

U3R R

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 Leader

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.

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.

U5

Primer Binding Site: 18 nt complementary to the 3' end of the specific tRNA primer used by the virus to begin reverse transcription.

PBS

Polypurine Tract: A short (~10 nt) run of A/G residues responsible for initiating (+)strand synthesis during reverse transcription.

PPT

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.

U3

Cap (A)ngag pol env

R U5 U3 R

Reverse transcription

gag pol env

R U5U3 U3 R U5

LTRLTR LTRLTR

ABCDEF

FEDCBA

CATTGTAA

AATGTTAC

Integration

ABCDEFFEDCBACAGT

ABCDEFFEDCBA TGAC

Virus-RNA

Virus-dsDNA

Cap (A)ngag pol envR U5 U3 R

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 v-Myb of the avian myeloblastosis virus.

RSV: genomic RNA

1 3 4 1 6c-Srcc-Src

Cellular gene = Proto-Oncogene (c-onc)



Oncovirus/OncogeneOncovirus/Oncogene

src

Survival AngiogenesisProliferationMotility/Migration/Invasion

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. In fibroblasts, Src is bound to endosomes, perinuclear membranes, secretory 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.

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 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, 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-Mismatch-


Protein phosphorylation

Serine90 %

COOHH3N+-C-H

CH2OH

COO-H3N+-C-H

H2C-O-P=O OH

O-ATPATP

Threonine10 %

ATPATP

COOHH3N+-C-H

CH2OH

CH3

Tyrosine0.05 %

COOHH3N+-C-H

CH2

OH

COOHH3N+-C-H

CH2

O-P=OO

OH

Y

SH3 SH2 Kinase 19CH3-(CH2)12-CO-c-Src 534 As

Structure of p60src

Aliphatic myristoyl group attached to the N-terminus (-Ser-Gly-NH-CO-(CH2)12-CH3)

Src homology domains (SH): SH1: tyrosine kinaseSH2: binds phoshorylated tyrosine residues (EXXY)SH3: binds proline-rich polypeptide sequences (PXXP)

ATPATP

SH

SH

22

SH

SH

33

P

YY

active proteinactive proteintyrosine kinasetyrosine kinase

YY

Phosphorylation of pp60src at S, T and Y:PKA: protein kinase APKC: protein kinase CCSK: C-terminal src kinase

Protein kinase phosphorylation sites and Protein kinase phosphorylation sites and organization of Src organization of Src (chicken)



Autoinhibition of Src when carboxyterminal Autoinhibition of Src when carboxyterminal tyrosine phosphorylated: interaction with tyrosine phosphorylated: interaction with

internal SH2 comaininternal SH2 comainChicken Chicken Y527Y527, human , human Y530Y530

Why is v-src oncogenic?Why is v-src oncogenic?

SH3 SH2 Kinase 10CH3-(CH2)12-CO-v-Src 526 As

Y

SH3 SH2 Kinase 19CH3-(CH2)12-CO-c-Src 534 As

p60src

Differences: promotercarboyterminus3´-untranslated region

v-Src is oncogenic in vivo and transforms fibroblasts in vitro:

1) Strong constitutive expression from viral promoter/enhancer (LTR).2) v-Src gene product is constitutive active due to the lack of the carboxyterminal tyrosine. p60v-src kann nicht negativ reguliert werden.

Inactive proteinInactive proteintyrosine kinasetyrosine kinase

YP

SH

SH

33S

HS

H22

YY

ATPATP

YP

SH

SH

33S

HS

H22

YY

P

PYY

Y

Active proteinActive proteintyrosine kinasetyrosine kinase

Oncoviruses encode besides the genes for its replication additional sequences which endow them with tumorigenic potential:viral oncogene (v-onc).

Oncogene = DNA sequence with proven tumorigenic potential: in tissue culture, animal models or human cancer.

OncogenesOncogenes

Oncovirus and oncogenes: Act dominantly with

regard to cell proliferation

Additional oncogenic tyrosine kinases

Signal transduction molecules

Transcription factors

OncogenesOncogenes

Geschichte der Tumorgene


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

Zelltransformation

• 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-Mismatch-


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

Wachstumfaktoren und Wachstumsfaktorrezeptoren

PDGFR

PDGFRß

CSF-1R

c-kit

EGFR=HER1

HER2

FGFR1

etc.

NGFR

BDNFR

IR

PDGF-A

PDGF-B

CSF; kit-L

EGF

TGF-ß

FGF-5

aFGF

bFGF

KGF

NGF

BDNF

Neurotrophine

Insulin

IGF-1

TM

Ligand

TK

TK

TK

TK

TK

ZytoplasmaTK

CS

F-1

R: c-

fms

SC

FR

: c-kit

EG

FR

: c-

erbB

HE

R2

: neu

NG

FR

: c-trkB

DN

FR

: c-trkB

IR: c-ro

s

Y YP Y P

Mitogenes SignalMitogenic Signal

RezeptorautophosphorylierungRezeptorautophosphorylierung

SH2P

Y Y

SH3

SH2P

Y

Changed subcellular localization,Changed subcellular localization,Phosphorylation, conformational change Phosphorylation, conformational change Change in protein activity Change in protein activity

SH2-Proteine binden an Tyr P

Signal reception Specific transduction

Signal effect

PP

P

P

P

PP

P P

PP

PLC PI 3´K Adapters

DG IP3 PIP3 AKT Ras

lecture on 31. may 2006 cancelled! regulation of cell growth cell cycle: cdk, cyclins, cki apoptosis...

Documents