Lecture on 31. May 2006 cancelled!

Regulation of cell growth Cell cycle: CDK, Cyclins, CKI Apoptosis Cell senescence/immortalization Detection of tumorigenic mutations Tumorbiology SS2006-5

Kontrollpunkte des Zellzyklus

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-CyclinsS-CyclinsG2/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 CDK4Cyclin Dp16 CDK5 CDK2Cyclin Ep21, p27, p57 S:CDK2Cyclin A G2/M:cdc2Cyclin A cdc2Cyclin B --

Candidate substrates of CDK SubstrateResult of phosphorylation G1 --> S/S-Phase pRBrelease of transcription factors p53regulation of nuclear localisation G2 -->M/M-Phase Tyrosine KinaseReorganisation of cytoskeleton Ser/Thr-Kinase? Histon H1Chromosome condensation HMGChromosome condensation NucleolinNucleoli desintegration Myosin light chainDelay of cytokinesis LamineBreakdown of nuclear membrane MAP4Collapse of spindle fibres

Growth signals Cell proliferation in response to extracellular signals (growth factors, space) Differentiation Cells with defects (genomic integrity, correct differentiation) die Apoptosis Cell death harmless

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-6 Casp-3 Limitierte Spaltung von Substraten ApoptoseApoptose Mitochondrium Apaf-1 Casp 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. HayflickLimit * DNA loss per division TRF: telomeric restriction fragment Germ line Somatic cells immortalization M1M2 * TRF length in kb Telomerase active Telomerase inactive Telomerase active crisis Tumor cells are telomerase positive immortalized (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

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 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.

Regulation of cell growth Cell cycle: CDK, Cyclins, CKI Signal transduction Apoptosis Cell senescence/immortalization Detection of tumorigenic mutations Tumorbiology SS2006-5

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 factorsOncogenes

History of tumor genes 1911 Rous Sarcoma Virus (RSV) wird entdeckt 1970 RSV kodiert ein transformierendes Gen (v-src) 1976v-src stammt von einem zellulären Gen (c-src) 1978src kodiert für eine Proteinkinase 1979chemisch transformierte Zellen enthalten ein aktiviertes Onkogen Ras bindet Guaninnukleotide 1980src-Kinase phosphoryliert Tyrosinreste 1981Virale Insertion aktiviert c-myc-Onkogen 1982Punktmutation aktiviert ras in menschlichem Blasentumor 1983v-sis kodiert für einen Wachstumsfaktor, Onkogene kooperieren zur Zelltransformation 1984v-erb-B kodiert für einen verkürzten Wachstumsfaktorrezeptor 1986Genprodukte von transformierenden Genen der DNA-Viren binden Rb, BCL-2 inhibiert programmierten Zelltod 1989TP53 ist ein Tumorsuppressorgen 1991Rb ist an der Regulation des Zellzyklus beteiligt 1993hereditäres Kolonkarzinom wird durch defekte DNA-Mismatch- Reparaturgene verursacht 1994Brustkrebs-Suszeptibilitätsgen (BRCA-1) wird kloniert 1911 Rous Sarcoma Virus (RSV) wird entdeckt 1970 RSV kodiert ein transformierendes Gen (v-src) 1976v-src stammt von einem zellulären Gen (c-src) 1978src kodiert für eine Proteinkinase 1979chemisch transformierte Zellen enthalten ein aktiviertes Onkogen Ras bindet Guaninnukleotide 1980src-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, 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 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.

Rous sarcoma virus

Mouse Fibroblasten (Bindegewebszellen), hier NIH 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". RSV TS

Envelope proteins (env) Lipid- membrane Capsid proteins (gag) RNA Reverse Transcriptase Integrase Protease (pol) HIV (EM) Schematic Structure of a Retrovirus/Genome Cap (A) n gag polenv RU5 PBS Leader PPT U3R

Cap (A) n gagpolenv PBSPPT U3 R R R Region: A short ( nt) sequence which forms a direct repeat at the both ends of the genome, which is therefore 'terminally redundant'. U5Leader Leader: A relatively long ( 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 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) n gagpolenv RU5U3R Reverse transcription gagpolenv RU5U3 RU5 LTRLTR ABCDEF FEDCBA CATT GTAA AATG TTAC Integration ABCDEF FEDCBA CA GT ABCDEF FEDCBA TG AC Virus-RNA Virus-dsDNA

Cap(A) n gagpolenv RU5U3R 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 c-Src Cellular gene = Proto-Oncogene (c-onc)

Oncovirus/Oncogene

src Survival AngiogenesisProliferation Motility/Migration /Invasion p60 src 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) 1976v-src stammt von einem zellulären Gen (c-src) 1978src encodes a protein kinase 1979chemisch transformierte Zellen enthalten ein aktiviertes Onkogen Ras bindet Guaninnukleotide 1980src-kinase phosphorylates tyrosine residues 1981Virale Insertion aktiviert c-myc-Onkogen 1982Punktmutation aktiviert ras in menschlichem Blasentumor 1983v-sis kodiert für einen Wachstumsfaktor, Onkogene kooperieren zur Zelltransformation 1984v-erb-B kodiert für einen verkürzten Wachstumsfaktorrezeptor 1986Genprodukte von transformierenden Genen der DNA-Viren binden Rb, BCL-2 inhibiert programmierten Zelltod 1989TP53 ist ein Tumorsuppressorgen 1991Rb ist an der Regulation des Zellzyklus beteiligt 1993hereditäres Kolonkarzinom wird durch defekte DNA-Mismatch- Reparaturgene verursacht 1994Brustkrebs-Suszeptibilitätsgen (BRCA-1) wird kloniert

Protein phosphorylation Serine 90 % COOH H 3 N + -C-H CH 2 OH COO - H 3 N + -C-H H 2 C-O-P=O OH O-O- ATP Threonine 10 % ATP COOH H 3 N + -C-H CH 2 OH CH 3 Tyrosine 0.05 % COOH H 3 N + -C-H CH 2 OH COOH H 3 N + -C-H CH 2 O-P=O O OH

Y SH3 SH2Kinase 19 CH 3 -(CH 2 ) 12 -CO- c-Src 534 As Structure of p60 src Aliphatic myristoyl group attached to the N-terminus (-Ser-Gly-NH-CO-(CH 2 ) 12 -CH 3 ) Src homology domains (SH): SH1:tyrosine kinase SH2:binds phoshorylated tyrosine residues (EXXY) SH3:binds proline-rich polypeptide sequences (PXXP)

ATP SH2 SH3 P Y active protein tyrosine kinase Y

Phosphorylation of pp60 src at S, T and Y: PKA: protein kinase A PKC: protein kinase C CSK: C-terminal src kinase Protein kinase phosphorylation sites and organization of Src Protein kinase phosphorylation sites and organization of Src (chicken)

Autoinhibition of Src when carboxyterminal tyrosine phosphorylated: interaction with internal SH2 comain Chicken Y527, human Y530 Why is v-src oncogenic?

SH3 SH2 Kinase 10 CH 3 -(CH 2 ) 12 -CO- v-Src 526 As Y SH3 SH2Kinase 19 CH 3 -(CH 2 ) 12 -CO- c-Src 534 As p60 src Differences:promoter carboyterminus 3´-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. p60 v-src kann nicht negativ reguliert werden.

Inactive protein tyrosine kinase Y P SH3 SH2 Y ATP Y PSH3 SH2 Y P PY Y Active protein tyrosine 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.Oncogenes

Oncovirus and oncogenes:Act dominantly with regard to cell proliferation Additional oncogenic tyrosine kinases Signal transduction molecules Transcription factorsOncogenes

Geschichte der Tumorgene 1911 Rous Sarcoma Virus (RSV) wird entdeckt 1970 RSV kodiert ein transformierendes Gen (v-src) 1976v-src stammt von einem zellulären Gen (c-src) 1978src kodiert für eine Proteinkinase 1979chemisch transformierte Zellen enthalten ein aktiviertes Onkogen Ras bindet Guaninnukleotide 1980src-Kinase phosphoryliert Tyrosinreste 1981Virale Insertion aktiviert c-myc-Onkogen 1982Punktmutation aktiviert ras in menschlichem Blasentumor 1983v-sis kodiert für einen Wachstumsfaktor, Onkogene kooperieren zur Zelltransformation 1984v-erb-B kodiert für einen verkürzten Wachstumsfaktorrezeptor 1986Genprodukte von transformierenden Genen der DNA-Viren binden Rb, BCL-2 inhibiert programmierten Zelltod 1989TP53 ist ein Tumorsuppressorgen 1991Rb ist an der Regulation des Zellzyklus beteiligt 1993hereditäres Kolonkarzinom wird durch defekte DNA-Mismatch- Reparaturgene verursacht 1994Brustkrebs-Suszeptibilitätsgen (BRCA-1) wird kloniert

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=HER 1 HER2 FGFR1 etc. NGFR BDNFR IR PDGF-A PDGF-B CSF; kit- L EGF TGF- ß FGF- 5 aFGF bFGF KGF NGF BDN F Neurotrophine Insulin IGF-1 TM Ligand TK Zytoplasma TK CSF-1R: c-fmsSCFR: c-kitEGFR: c-erbBHER2: neuNGFR: c-trkBDNFR: c-trkBIR: c-ros

Y Y P Y P Mitogenes Signal Mitogenic Signal

Rezeptorautophosphorylierung SH2 P Y Y SH3 SH2 P Y Changed subcellular localization, Phosphorylation, conformational change Change in protein activity SH2-Proteine binden an Tyr P

Signal reception Specific transduction Signal effect P P P P P P P P P P P PLC PI 3´KAdapters DGIP3PIP3AKT Ras

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