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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